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Mesodermal Deletion of Transforming Growth Factor-β Receptor II Disrupts Lung Epithelial Morphogenesis

Mesodermal Deletion of Transforming Growth Factor-β Receptor II Disrupts Lung Epithelial... THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 52, pp. 36257–36264, December 26, 2008 © 2008 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Mesodermal Deletion of Transforming Growth Factor- Receptor II Disrupts Lung Epithelial Morphogenesis CROSS-TALK BETWEEN TGF- AND SONIC HEDGEHOG PATHWAYS Received for publication, September 2, 2008, and in revised form, October 22, 2008 Published, JBC Papers in Press, November 6, 2008, DOI 10.1074/jbc.M806786200 ‡ ‡ § ‡ ‡ ¶ ‡ Min Li , Changgong Li , Yi-hsin Liu , Yiming Xing , Lingyan Hu , Zea Borok , Kenny Y.-C. Kwong , ‡1 and Parviz Minoo ‡ § ¶ From the Division of Neonatology, Department of Pediatrics, the Department of Ophthalmology, and the Will Rogers Institute Pulmonary Research Center, Department of Medicine, University of Southern California Schools of Medicine, Los Angeles, California 90033 In vertebrates, Sonic hedgehog (Shh) and transforming receptors. The binding of TGF- ligand to the receptor com- growth factor- (TGF-) signaling pathways occur in an over- plex induces phosphorylation of type I by the type II recep- lapping manner in many morphogenetic processes. In vitro data tors and activates Smads, a family of transcriptional factors indicate that the two pathways may interact. Whether such that act as intracellular effectors of TGF- signaling (1). interactions occur during embryonic development remains Smad2 and/or Smad3 are phosphorylated in their C-termi- unknown. Using embryonic lung morphogenesis as a model, we nal domain upon stimulation by either activin or TGF- (2). generated transgenic mice in which exon 2 of the TRII gene, Phosphorylation of Smad2 and Smad3 is accompanied by which encodes the type II TGF- receptor, was deleted via a their association with Smad4 and translocation of the het- mesodermal-specific Cre. Mesodermal-specific deletion of eromeric complex to the nucleus where they affect transcrip- TRII (TRII ) resulted in embryonic lethality. The lungs tion of target genes through interaction with promoter-spe- showed abnormalities in both number and shape of cartilage in cific transcriptional factors or by direct DNA binding (3). trachea and bronchi. In the lung parenchyma, where epithelial- Genetic manipulations of endogenous TGF- signaling have mesenchymal interactions are critical for normal development, revealed their important functions in vertebrate development. deletion of mesenchymal TRII caused abnormalities in epithe- Targeted deletion of each of the three ligand isoforms causes lial morphogenesis. Failure in normal epithelial branching severe abnormalities in morphogenesis of various organs morphogenesis in the TRII lungs caused cystic airway mal- including the lung. Tgf-2-null mice exhibit perinatal mortality formations. Interruption of the TRII locus in the lung mes- and a wide range of developmental abnormalities that include enchyme increased mRNA for Patched and Gli-1, two down- cardiac, lung, craniofacial, limb, spinal column, eye, inner ear, stream targets of Shh signaling, without alterations in Shh and urogenital defects (4). Tgf- 3 mutants die as neonates due ligand levels produced in the epithelium. Therefore, we con- to abnormal lung development and cleft palate (5). Targeted clude that TRII-mediated signaling in the lung mesenchyme disruption of the Tgf-1 gene results in abnormalities in the modulates transduction of Shh signaling that originates from lung, manifested as dilation of the airways (6). Mice with tar- the epithelium. To our knowledge, this is the first in vivo geted deletion of Smad3 are viable, but develop lung abnormal- evidence for a reciprocal and novel mode of cross-communi- ities akin to emphysema (7). Little is known about the role of cation between Shh and TGF- pathways during embryonic other components of the TGF- pathway and interactions with development. other signaling molecules in the lung. Embryonic lung development represents a useful model in which to study complex tissue interactions in organ develop- Transforming growth factor- (TGF-) ligands are multi- ment. Lung morphogenesis is strictly dependent on cross-talk functional signaling proteins that exhibit a wide range of bio- between two distinct tissues, the endodermal-derived epithe- logical activities including regulation of cell proliferation and lium and the mesodermal-derived lung mesenchyme (8). A differentiation. TGF- initiates its cellular actions by interact- major signaling pathway in this communication is Shh, the ver- ing with a heteromeric complex of transmembrane serine/thre- tebrate homologue of Drosophila hh, which is highly expressed onine kinase receptors, the type I (TRI) and type II (TRII) by embryonic lung epithelium. Patched (Ptc), the receptor for Shh is expressed by the lung mesenchyme, the site of highly * This work was supported, in whole or in part, by NHLBI, National Institutes of focalized Fgf10 production. The role of Fgf10 in directing epi- Health. This work was also supported by the Hastings Foundation. The thelial morphogenesis is central to lung development (9–12). In costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertise- Fgf10(/) embryos, the lung tissue below the main stem ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. bronchi is entirely absent (13, 14). Shh interacts with the Ptc/ To whom correspondence should be addressed: General Laboratories Bldg. Smoothened (Smo) complex on the mesenchymal cell mem- 1801 E. Marengo St., Rm. 1G1, Los Angeles, CA 90033. Tel.: 323-226-4340; Fax: 323-226-5049; E-mail: [email protected]. brane and activates Gli-3, a 190-kDa transcription factor (15, The abbreviations used are: TGF, transforming growth factor; X-gal, 5-bro- 16). Activated Gli-3 binds directly to the Gli-1 promoter and mo-4-chloro-3-indolyl--D-galactopyranoside; Shh, Sonic hedgehog; induces its transcription in response to Shh (17). Gli-1 is a zinc PBSMC, parabronchial smooth muscle cells; GAPDH, glyceraldehyde-3- phosphate dehydrogenase. transcription factor that activates the transcription of Ptc (18). DECEMBER 26, 2008• VOLUME 283 • NUMBER 52 JOURNAL OF BIOLOGICAL CHEMISTRY 36257 This is an Open Access article under the CC BY license. TGF- Sonic Hedgehog Cross-talk Thus, increased transcription of Gli-1 & Ptc are reliable mark- branes were probed with antibodies to TRII (Abcam) and ana- ers of Shh pathway activation. All three Gli family members are lyzed with the ECL Western blot analysis system as described expressed in and are important for lung development (18, 19). by the manufacturer (GE Health, Memphis, TN). The currently accepted model is that Shh both stimulates and In Situ Hybridization—Whole mount and section in situ restricts the level and spatial distribution of Fgf10 expression hybridization were performed as previously described (27, 28). during lung morphogenesis. Consistent with this concept, dele- The digoxigenin-labeled RNA antisense and sense probes were tion of Shh leads to diffused, but expanded Fgf10 mRNA prepared from following cDNA templates: a 0.4-kb fragment of throughout the mesenchyme as a consequence of which air- SpC, a 1.4-kb fragment of Nkx2.1 (29), a 0.5-kb fragment of ways develop into large cystic structures (20). Precisely how Foxj1, a 0.6-kb fragment of Shh (Dr. Andrew P. McMahon, Har- Shh controls Fgf10 gene expression has hitherto remained vard University), a 0.7-kb fragment of Ptc (Dr. Matthew P. unknown. Scott, Stanford University), a 0.7-kb fragment of Gli-1, a 0.7-kb Because of its established role as a negative regulator of lung fragment of Foxf1 (Dr. Peter Carlsson, Go¨teborg University), branching morphogenesis (21) TGF- is a potential mediator in a 0.4-kb fragment of Fgf10, a 1.3-kb fragment of Tbx4, and a epithelial-mesenchymal cross-talk during lung development. 1-kb fragment of Tbx5(Dr. Virginia Papaioannou, Princeton Conventional deletion of TRII lead to early embryonic lethal- University). ity, and therefore was not informative for lung development RNA Extraction, Polymerase Chain Reaction (PCR), and (22). In the present study, we used a mesodermal-specific cre- Northern Blotting—Total RNA was isolated from embryonic loxP system to delete exon 2 in the TRII locus in the lung lungs and MRC5 cells (ATCC) by using TRIzol (Invitrogen). mesenchyme. The lungs of TRII mouse fetuses are abnor- Superscript First-Strand Synthesis System kit (Invitrogen) was mal with evidence of cystic airway malformations associated used to generate cDNA. Quantification of the selected genes by with alterations in Fgf10 gene expression, likely due to interrup- real-time PCR was performed using a LightCycler (Roche tion of normal epithelial-mesenchymal cross-talk. Inactivation Applied Sciences) as previously described (30). Sequence of the of TRII and hence the specific TGF- signaling pathway medi- primers were as follows: Mouse TrII:5-ATG CAT CCA TCC ated through its normal activity in the lung mesenchyme results ACG TAA G-3(forward), 5-GAC ACG GTA GCA GTA GAA in alterations in Ptc and Gli mRNAs in the mutant lungs indi- GA-3 (reverse); human TrII:5-CAC GTT CAG AAG TCG cating interference with Shh signaling. Thus, TGF- signaling, GAT GT-3(forward), 5-CAT CAG AGC TAC AGG AAC mediated via TRII can modulate mesenchymal reception of AC-3(reverse); Mouse TrII(qPCR): 5-CAT GAA AGA CAG Shh signaling, which originates from the epithelium, indicating TGT GCT GAG A-3(forward), 5-CTC ACA CAC GAT CTG cross-communication between the two signaling pathways GAT GC-3(reverse); Mouse Foxf1:5-AGC ATC TCC ACG during embryonic lung morphogenesis. CAC TCC-3(forward), 5-TGT GAG TGA TAC CGA GGG ATG-3(reverse); Mouse Tbx4:5-GCA TGA GAA GGA GCT MATERIALS AND METHODS GTG G-3(forward), 5-TTA CCT TGT AGC TGG GGA fl/fl Animals—Dermo1-cre, Rosa26-lacZ, and TRII mice ACA-3(reverse); Human PAI-1:5-AAC GGC CAG TGG were generated and genotyped as previously described (23–25) AAG ACT C-3(forward), 5-GGG CGT GGT GAA CTC AGT and maintained on C57BL/6 genetic background. Dermo1-cre; AT-3(reverse); Human Ptc:5-AAC AAA AAT TCA ACC Rosa26-lacZ mice were generated by crossing Dermo1- and AAA CCT C-3(forward), 5-TGT CCT CGT TCC AGT TGA fl/fl Rosa26-lacZ mice. To generate TRII ; Dermo1-cre embryos TGT G-3(reverse); Human Gli-1:5-CAG GGA GGA AAG / fl/ (TRII ), TRII ; Derom1-cre mice were crossed with CAG ACT GA-3(forward), 5-ACT GCT GCA GGA TGA fl/fl TRII mice. CTG G-3(reverse); Human Fgf10:5-CGG GAC CAA GAA Detection of -Galactosidase (LacZ) Activity—LacZ activity GGA GAA CT-3(forward), 5-ACG GCA ACA ACT CCG was determined by X-gal staining as described (26). Whole ATT-3(reverse); Human GAPDH:5-GAA GGT GAA GGT mount staining was performed for embryonic lungs. Lungs of CGG AGT C-3(forward), 5-GAA GAT GGT GAT GGG ATT E15.5 and older were fixed and sectioned by cryostat, and the TC-3(reverse). Ten micrograms of total RNA were electro- frozen sections were stained for LacZ activity. phoresed in 1% RNA formaldehyde-agarose gel and blotted. Immunohistochemistry—For immunohistochemistry, sam- Blots were hybridized with probes specific for TRII, Shh, Ptc, ples were fixed in 4% paraformaldehyde and processed into Gli-1, Fgf10, Gapdh, and then autoradiographed or measured serial paraffin sections using routine procedures. Immuno- with Kodak Molecular Imaging Software Ver 4.0 to determine staining were performed as previously described (27). Primary the quantity. P-labeled probes were synthesized from the fol- antibodies that were used are: -SMA (Sigma), PAI-1 (Abcam lowing cDNA fragments: the TRII probe was from a 0.4-kb Cambridge, MA), PECAM (BD Pharmingen, San Diego, CA), PCR product of TRII coding region. The Shh probe was from Flk1 (Cell Signaling Technology, Beverly, MA), -Tubulin (Bio- a 0.6-kb cDNA (Dr. Brigid L. M. Hogan, Vanderbilt University). genix, San Ramon, CA), Collagen1 (Abcam, Cambridge, MA), The Ptc probe was from a 0.7-kb cDNA (Dr. Matthew P. Scott, and TRII (Abcam Cambridge, MA). Stanford University), The Gli-1 probe was from a 0.7-kb cDNA. Western Blot Analysis—Protein extracts were prepared from The Fgf10 probe was from a 0.4-kb cDNA. The GAPDH probe fl/fl / E15.5 Dermo1-cre; TRII and TRII lungs in RIPA buffer was as reported before (31). (Sigma) from homogenizer, and then separated on 4–12% Cell Culture and TGF- Treatment—Human pulmonary NuPAGE gels (Invitrogen). Proteins were then transferred onto mesenchymal cell line MRC5 (ATCC, Manassas, VA) was Immobilon-P transfer membrane (Millipore Corp.). Mem- maintained in EMEM medium (ATCC), containing 10% fetal 36258 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283 • NUMBER 52 •DECEMBER 26, 2008 TGF- Sonic Hedgehog Cross-talk fl/fl erozygotes to generate TRII ; Dermo1-cre progeny. The lat- ter genotype is referred to simply as TRII . To validate Dermo1-cre-induced recombination and deletion of exon 2 within the TRII gene, we used three approaches. First, we uti- lized lung DNA with PCR primers that could distinguish between deleted and intact TRII alleles. The deletion in TRII gene was verified by genomic PCR analysis shown in Fig. 2B, panel b. Second, we measured TRII protein content by West- ern blot analysis in protein extracts of total lung tissue from control and TRII embryos. These studies showed greater than 60% decrease in total lung TRII protein, the remainder indicating non-mesenchymal (e.g. epithelial and endothelial) TRII protein (Fig. 2B, panels c and d). In contrast, the protein FIGURE 1. TRII expression in the murine lung. A and B show immunolocal- level of TRI and phosphorylated Smad2 (p-Smad2) remained ization of TRII protein in lungs from E15.5 and E18.5 embryos, respectively. the same. Therefore mesenchymal deletion of TRII in the lung The arrow shows TRII protein localized in the subepithelial layer of proximal airways. The arrowhead points to endothelial localized TRII around the does not alter the level of phosphorylated Smad2 and TRI. blood vessel. B shows immunolocalized TRII distributed throughout the Finally, functional deletion of TRII was verified by immuno- parenchyma of E18.5 lungs, with highest expression in the interstitium, including cells of mesenchymal origin. C, semi-quantitative RT-PCR of TRII histochemistry using antibodies to Plasminogen Activator mRNA during murine lung morphogenesis. E, embryonic day; PN, postnatal Inhibitor (PAI-1), which is a downstream target of TGF- sig- day. D, TRII mRNA is also detectable in both human (A549 and H449) and naling. In the control lungs, diffuse PAI-1 was detected murine (MLE15) transformed epithelial cell lines. MRC5 is a fetal human mes- enchymal cell line. Scale bar, 100 m. throughout both the epithelium and the mesenchyme, but par- ticularly in the progenitor of parabronchial smooth muscle cells (PBSMC) in the subepithelial mesoderm (Fig. 2C, panel b, bovine serum and 1% penicillin-streptomycin. MRC5 cells arrows). In contrast, PAI-1 in the TRII lungs was found were treated with recombinant TGF- (R&D systems, Min- neapolis, MN) at 200 ng/ml for 2 h and then collected for only in the epithelium. As expected, there was virtually no RNA extraction. PAI-1 staining in the mesoderm, nor in the PBSMC progenitors in TRII lungs (Fig. 2C, panel d, arrowheads). RESULTS Ultrastructure and Cellular Differentiation in TRII TRII Expression during Lung Development—TRII has been Lungs—Dermo1-cre-induced deletion of exon 2 within the reported to be expressed in both the epithelium and the mes- TRII gene was embryonically lethal by day 16–17 of gestation. enchyme of the lung (32). To elucidate the expression pattern of E16–17 fetuses developed gross abnormalities in multiple TRII in the murine lung, we used semi-quantitative RT-PCR organs that caused fetal demise. We therefore, collected and and immunohistochemistry. Expression of TRII mRNA can characterized lungs from TRII E15.5 fetuses. Gross mor- be detected by RT-PCR throughout lung development (Fig. 1 C). phological assessment of the proximal lung structure showed a Commercially available antibody (Abcam) detects TRII pro- readily discernible phenotype manifested as disorganized for- tein in mesenchymal cells localized around the airways (Fig. 1, mation of tracheal cartilage (Fig. 3N). Both the number as well arrows) and blood vessels (Fig. 1, arrowhead) in E15.5 lungs as the shape of the tracheal cartilage was altered. The first and (Fig. 1A) and throughout the mesenchyme in E18 lungs (Fig. 1B). the second generation bronchi in the mutant lungs were devoid TRII mRNA is clearly expressed in human lung epithelial car- of cartilage altogether (not shown). cinoma, H441 and A549 cell lines as well as mouse-immortal- Mesodermal deletion of TRII also impacted the shape and ized epithelial MLE15 cells (Fig. 1D). the size of the various lung lobes as shown in Fig. 3. However, Recombination Mediated by Dermo1-cre in the Lung the overall process of lobation and the number of lobes were Mesenchyme—Recombination driven by Dermo1-cre in the normal (Fig. 3, H–L). Histological assessment showed a distinct lung was analyzed by crossing with the reporter mouse strain phenotype in TRII lungs, characterized by the presence of Rosa26-LacZ. Double transgenic, Rosa26-LacZ; Dermo1-cre large, dilated airways, lined with columnar epithelial cells in the fetuses were identified by PCR genotyping, and the lungs were proximal lung (Fig. 4A). excised and stained for LacZ. As shown in Fig. 2A, expression of The epithelial cells throughout the TRII lungs showed Dermo1-cre resulted in nearly 100% recombination of the loxP expression of NKX2.1, a transcription factor associated with sites in Rosa26-lacZ lung mesodermal cells (Fig. 2A, panels onset of lung epithelial morphogenesis and normal lung struc- a–f). This recombination was entirely mesenchymal-specific in tural development(Fig. 4B, panels a and d) (33). Transcripts for that no epithelial cells showed LacZ staining (Fig. 2A, panels a, Surfactant Protein C, SPC, a marker of distal epithelial cells and b, d, and f, arrows). a target of NKX2.1 was also expressed in the mutant lungs. Also, Inactivation of TRII by Dermo1-cre-driven Recombination— differentiation of ciliated cells as evidenced by expression of Conventional deletion of TRII led to early embryonic lethality Foxj1 appeared to be normal in the absence of epithelial TRII (22). Thus, we used a conditional Cre-LoxP approach to specif- activity (Fig. 4B). The normal level and distribution of Surfac- ically delete exon 2 in the TRII locus in mesodermal lineages, tant Protein B, SPB, expressed in proximal and distal, differen- fl/fl including those of the lung. Accordingly, we crossed TRII tiated epithelium, as well as -tubulin, an established marker of females with Dermo1-cre male mice and backcrossed the het- airway ciliated cells were also observed by immunohistochem- DECEMBER 26, 2008• VOLUME 283 • NUMBER 52 JOURNAL OF BIOLOGICAL CHEMISTRY 36259 TGF- Sonic Hedgehog Cross-talk (Fig. 4C, panels e and f). Collagen production is also under TGF- control (6). Both in the wild-type control and the mutant lungs, colla- gen Type I was localized to the extracellular matrix surrounding both the distal and the proximal air- ways (Fig. 4C, panels g and h). No drastic alteration in collagen type I was observed in TRII lungs sug- gesting that either TGF- is signal- ing through utilization of type I receptor (homotetramer) or that collagen production and deposition may not be entirely TGF-- dependent. Expression of Fgf10 in TRII Lungs—Whole mount and section in situ hybridization were per- formed for a number of genes with established roles during lung mor- phogenesis. Because the shape and size of the airways can be regulated by FGF10 activity and its expression domain, we investigated the expres- FIGURE 2. Generation and validation of TRII conditional knock-out alleles. A, mesenchymal specific recombi- / sion pattern of Fgf10 in TRII nation induced by Dermo1-cre as determined by LacZ assay in murine lungs from various stages of embryonic lungs. Whole mount in situ hybrid- development. Panels a,c,andearewholemountlungtissue. Panels b,d,andfarefrozensaggitalsectionsofthelungs in panels a, c, and e. Arrows in panels a, b, d, and f show the absence of LacZ in airway epithelial cells. Scale bar,50m. ization revealed increased expres- B, evidence for deletion of TRII gene by Dermo1-cre. Panel a, map of the mouse TRII locus showing the relative sion, and expansion of the Fgf10 position of the lox-P (flox) sequences and the primers used in identifying various genotypes. Primers for Cre were used as described by Yu et al. (23). Panel b, PCR analysis of TRII gene using lung-extracted DNA and the primers P1, domain in the mutant embryonic fl/ P2, and P3, as shown in panel a. Lane 1, TRII lungs, showing two bands corresponding to the wild type (Wt) and lungs (Fig. 5A, panels e and f). A the allele carrying the lox-P insertion. The absence of a PCR product using P1/P3 primers is due to the large distance fl/fl clearer demonstration of this alter- betweenthetwoprimers(absenceofdeletion). Lane 2,TRII lungsshowingasinglebandcorrespondingtofloxed allele. Lane 3, TRII lungs. Deletion of Exon 2 brings the sequences recognized by P1 and P3 primers sufficiently ation was observed by in situ close to allow amplification of a PCR product (deletion). Lane 4, TRII lungs. Panel c, Western blot analysis of TRII, hybridization experiments on tissue fl/fl / TRI, and p-Smad2 in total lung protein from TRII (lane 1) and TRII (lane 2) mice. Panel d, densitometric sections (Figure 5A, compare panels quantification of the Western blot shown in panel c. C, immunohistochemical analysis for PAI-1 in TRII and fl/fl TRII lungs. Arrowheadsin panel dshowthereducedexpressionofPAI-1inthePBSMCsofmutantlungscompared c and g). In the control lungs, Fgf10 with arrows in panel b (control). m, mesenchyme; e, epithelium. Scale bar, 100m for panels a and c,23m for panels mRNA was localized to mesenchy- b and d. mal cells adjacent to the growing istry (data not show). Collectively, these data indicate that the tip of the peripheral airways (Fig. 5A, panels c and d) consist- absence of TRII in the lung mesenchyme does not alter lung ent with previously reported results (12). In the mutant epithelial cell identity and differentiation. lungs, however, we found an expanded Fgf10 expression Expression of TGF- Targets in TRII Lungs—In wild- domain and likely increased mRNA in the peripheral mesen- type embryonic lungs, -smooth muscle actin-positive cells are chyme adjacent to the branching airways (Fig. 5A, panels found as rings of smooth muscle progenitor cells surrounding e– h). This alteration in Fgf10 expression domain may the columnar epithelium of the proximal airways (Fig. 4C, panel explain the phenotype of the TRII lungs in which air- a). In contrast, we found reduced -SMA-positive cells sur- ways are significantly dilated. Two transcription factors, rounding the dilated airways in the mutant lungs (Fig. 4C, panel Foxf1 and Tbx4 have been found to stimulate Fgf10 expres- b). This is the same layer of smooth muscle cells in which PAI-1 sion in the lung mesenchyme (35, 36). Consistent with this was found to be abundantly expressed in the wild-type lung and finding, in situ hybridization on tissue sections (Fig. 5B, pan- drastically reduced in TRII lungs (Fig. 2C). Immunohisto- els a, b, d, and e) and real-time PCR (Fig. 5C) revealed chemistry was also performed to determine potential changes increased transcripts for both in TRII lungs compared in the expression or spatial localization of other mesenchymal with controls. Expression of another Tbx gene, Tbx5 genes including collagen type I and platelet/endothelial cell remained unchanged (Fig. 5B, panels c and f). These data adhesion molecule (PECAM) known to be modulated by support the finding that Fgf10 is both increased and TGF- (6, 34). Reduced levels of PECAM were found in the expanded in its expression domain in TRII lungs. mutant lungs (Fig. 4C, compare panels c and d). Another Cross-talk between TGF- and Shh Pathways—Shh is marker of endothelial cell differentiation, Flk1 appeared to have thought to negatively regulate Fgf10 magnitude and distribu- the same level and distribution in the mutant lungs as wild type tion, thereby, controlling lung branching morphogenesis (20, 36260 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283 • NUMBER 52 •DECEMBER 26, 2008 TGF- Sonic Hedgehog Cross-talk / fl/fl FIGURE 3. Gross morphology of TRII and TRII trachea and lungs from E15.5 embryos. B and N, tracheas. Arrows show abnormal number and shape of tracheal cartilage. C and H, right apical lobes. D and I, right middle lobes. E and J, right caudal lobes. F and K, accessory lobes. G and L, left lobes. Scale bar, 1.0 mm for A and M; 0.2 mm for B and N; 0.8 mm for C–F, H–K, and 1.2 mm for G and L. FIGURE 5. In situ hybridization analysis of Fgf10 and its upstream tran- scriptional regulators in TRII and control lungs. A, whole mount (pan- els a, b, e, and f) and section (panels c, d, g, and h) in situ hybridization. Note expansion and increased Fgf10 expression in TRII (panels e– h) compared fl/fl with TRII (panels a– d) lungs. Scale bar, 2.2 mm (panels a and c);1mm (panels b and d); 100 m(panels e– h). B, section in situ hybridization for Foxf1 (panels a and d), Tbx5 (panels b and e), and Tbx4 (panels c and f)in TRII and fl/fl TRII (control) E15.5 lungs. Scale bar, 100 m. C, quantification of Foxf1 and Tbx4 mRNA by real-time PCR. Values are fold induction or repression, com- fl/fl pared with TRII controls (arbitrarily adjusted to 1). p value, 0.004. stream targets Ptc and Gli-1 in the mutant lungs compared with fl/fl the TRII controls. Whole mount and tissue section in situ hybridization showed similar levels and spatial localization of Shh mRNA in the control and mutant lungs (Fig. 6, A, D, G, and J). In contrast, there was a significant increase in the level of mRNA for both Ptc (Fig. 6, B, E, H, and K) and Gli-1 (C, F, I, and L)inthe TRII mutant lungs compared with the control. To validate the above in situ hybridization results, we used North- ern blot analysis as shown in Fig. 7. Transcripts for TRII were decreased by nearly 80% in TRII lung tissue, compared FIGURE 4. H & E staining and immunohistochemical analysis for lung with controls. Other changes included a 1.8-fold increase in developmental markers in TRII and control lungs. A, gross histology fl/fl / Ptc and Gli-1 mRNA and a 1.5-fold increase in Fgf10. The mag- of lungs from TRII (panels a and b) and TRII (panels c and d) E15.5 embryos. Saggital sections of lungs were analyzed byH&E staining. MB, nitude of Shh remained nearly the same in the mutant and con- mainstem bronchus. Asterisks show dilated proximal airways. Panels b and d trol lungs confirming the in situ hybridization findings. are high magnification of areas within dotted squares in panels a and c, respec- Because deletion of TRII occurs specifically in the lung mes- tively. Scale bar, 330 m for panels a and c; 100 m for panels b and d. B, cell differentiation in TRII embryonic lungs. In situ hybridization for Nkx2.1 enchyme, one potential hypothesis is that TGF- signaling (panels a and d), SpC (panels b and e), and Foxj1 (panels c and f). Asterisks, through TRII normally represses Gli-1 and Ptc mRNA levels dilated airways. Scale bar, 100 m. C, immunohistochemical analysis for -SMA (panels a and b), PECAM (panels c and d), Flk1 (panels e and f), and in the lung mesenchymal cell layer. To determine the validity of fl/fl / Collagen1 (panels g and h) in E15.5 TRII and TRII lungs. Arrowheads in this hypothesis, we used cultured MRC5 cells, which are panel b show reduced expression of -SMA surrounding the dilated airways derived from normal lung mesenchymal cells of a 14-week-old of mutant lungs compared with arrows in panel a (control). Scale bar, 100 m. male fetus, and assessed the impact of exogenous TGF- treat- 37). The mechanistic connection between Fgf10 and Shh ment on Gli-1 and Ptc mRNA levels by real-time PCR. In sup- remained unknown. We therefore examined Shh signaling by port of our in vivo observations, the results clearly showed that investigating the level and distribution of Shh and its down- TGF- represses steady state levels of both Gli-1 and Ptc DECEMBER 26, 2008• VOLUME 283 • NUMBER 52 JOURNAL OF BIOLOGICAL CHEMISTRY 36261 TGF- Sonic Hedgehog Cross-talk FIGURE 9. A hypothetical model for TGF-Shh cross-talk in the devel- oping lung. Shh is made in the lung epithelium. The Shh receptor complex, Ptc/smoothened is expressed on the cell surface of lung mesenchymal cells. Activation of Shh pathway leads to increased Gli-3 activity, which in turn stimulates Gli-1 and Ptc transcription. TGF- ligand acts through TRII and as yet unknown intracellular mediators (e.g. Smads) to interfere with Shh signal transduction in mesenchymal cells, evidenced by reductions in both Ptc and Gli-1 observed in this study. FIGURE 6. Whole mount and section in situ hybridization for components results validate our in vivo-based conclusions and support a fl/fl / of the Shh pathway in TRII and TRII E12.5 (A–F) and E15.5 (G–L) model for interactions between TGF- and Shh signaling path- lungs. Abundance of mRNA for both Ptc and Gli-1 is increased in the mutant lungs (E, F, K, and L) compared with the control lungs (B, C, H, and I). Little if any ways (Fig. 9). change is detectable in the epithelial-localized Shh (A, D, G, and J). Scale bar, 2.2 mm (A, B, E, F, I, J); 100 m(C, D, G, H, K, L). DISCUSSION The purpose of the current study was to examine the conse- quences of interruption in mesenchymal TRII-mediated TGF- signaling on lung morphogenesis. To this end, we gen- erated mice carrying mesodermal-specific deletion of the TRII exon 3 by Dermo1-cre-driven recombination. TRII fetuses died in utero due to multi-organ abnormalities. Examination of embryonic lungs revealed that mesenchymal abrogation of endogenous TGF- signaling caused abnormalities in epithelial morphogenesis, indicating disruption of normal epithelial- mesenchymal communication that is central to lung develop- ment. Analysis of key mediators of this cross-talk showed alter- FIGURE 7. Quantification of mRNA changes in TRII lungs. Total RNA ations in components of the Shh pathway. Both Ptc and Gli-1 as / fl/fl from E15.5 TRII and TRII (control) embryonic lungs was used for well as the transcription factor Foxf1 were increased in the mes- Northern blot analysis (A), and the results were quantified by densitometry and normalized by GAPDH (B). Values are fold induction or repression, com- enchyme of the mutant lungs. Shh mRNA in the lung epithe- fl/fl pared with TRII controls (arbitrarily adjusted to 1). lium was unchanged. Thus, abrogation of TRII-mediated sig- naling in the lung mesenchyme results in increased expression of Shh downstream targets (i.e. Shh signaling). To our knowl- edge, this is the first demonstration of cross-communication between the TGF- and the Shh pathways during vertebrate embryonic development. At least two major phenotypic abnormalities were readily observable in TRII mutant lungs. First, the trachea of the mutant lungs showed abnormalities in both structure and num- ber of cartilage. Both TGF- and Shh are known to be involved in the induction of early cartilaginous differentiation of mesen- chymal cells in the limb and in the spine. In vitro, treatment of human bone marrow-derived mesenchymal stem cells (MSCs) FIGURE 8. In vitro verification of the in vivo evidence that TGF- is a neg- with either TGF- or recombinant Shh induced expression of ative regulator of Shh pathway. MRC5 cells were treated with either 200 cartilage markers aggrecan, Sox9, CEP-68, and collagen type II ng/ml of TGF-1 or equivalent bovine serum albumin (control). Real-time and X (38). Thus, the abnormalities observed in tracheal carti- PCR-quantified mRNA for PAI-1 (positive control), Ptc and Gli-1. Values are fold induction or repression, compared with controls (adjusted to 1). lage formation in TRII lungs can be explained as either a direct result of mesenchymal TRII deletion or indirect effect mRNAs by nearly 50% (Fig. 8). Fgf10 has been proposed to be a of interruption in Shh signaling. target of Shh signaling in the lung (36) although this regulation Mesodermal inactivation of TRII also caused abnormal epi- has not been experimentally explored or demonstrated. In thelial morphogenesis manifested as cystic, dilated bronchi MRC5 cells, TGF- treatment repressed Fgf10 mRNA consist- (Figure 4A). Lung branching morphogenesis is strictly depend- ent with our in vivo observations in Fig. 5A. Thus, these in vitro ent on epithelial-mesenchymal communication between the 36262 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283 • NUMBER 52 •DECEMBER 26, 2008 TGF- Sonic Hedgehog Cross-talk foregut endoderm and the mesodermal-derived splanchnic been examined in in vitro settings. Shh promotes motility and mesenchyme. The lung mesenchyme, via Fgf10 provides invasiveness of gastric cancer cells through TGF--mediated instructional signaling that directs epithelial morphogenesis. activation of the ALK5-Smad3 pathway (45). In contrast to our Targeted deletion of Fgf10 results in profoundly abnormal findings, TGF- was shown to induce Gli-1 and Gli-2 expres- lungs that lack structures distal to the main stem bronchi (13, sion in various human cell lines (46). Also, in transgenic mice 14). Dynamic changes in the magnitude and spatial distribution overexpressing TGF-1 in the skin, Gli-1 and Gli-2 were ele- of Fgf10 are critical to normal branching morphogenesis and vated in a Smad3-dependent manner (46). This apparent dis- are thought to be controlled by diffusible signals originating crepancy may suggest both cell type (skin versus lung) and dose- from the epithelial cells themselves. Shh is expressed in the dependent specificity of TGF- effect on adult and embryonic distal epithelium, from where it activates signaling in the cor- tissue. The relationship between TGF- and Shh during orga- responding mesenchyme via its receptor, the patched (Ptch1)/ nogenesis, the focus of the present study had remained smoothened (Smo) complex and their transcriptional effectors unknown. In particular whether the inhibition of Fgf10 in the Gli-1, Gli-2, and Gli-3 (9, 11). Results from both in vitro and in lung mesenchyme in response to TGF- involves the Shh path- vivo studies indicate that Shh negatively regulates Fgf10 pro- way remained unknown. We found increased expression of Ptc duction in the lung mesenchyme (20, 37). Thus, a current and Gli-1, as well as the transcription factors Tbx4 and Foxf1 in model of embryonic lung development is centered on epithelial the mesenchyme of TRII mutant lungs, suggesting that endog- Shh as playing a dual role in both activating and limiting mes- enous TGF- signaling via the type II receptor in the wild-type enchymal signaling thereby “fine-tuning” the process of lung regulates Shh signal transduction in the mesenchyme. In branching morphogenesis. In Shh(/) lungs, Fgf10 mRNA is previous studies, we found that Wnt5a alters Shh signaling by found diffusely throughout the mesenchyme, as a consequence modulating Shh mRNA in the lung epithelium (30). In contrast, of which the airways develop into large cystic structures and increased Shh signaling in the mesenchyme in response to branching morphogenesis is severely disrupted (20). Although TRII inactivation occurrs in the absence of changes in epithe- not as severe, the phenotypically abnormal, cystic bronchi lial Shh mRNA (Figs. 6 and 7). Therefore, TGF- does not observed in TRII mutant lungs are similar to those observed in directly interfere with Shh originating from the lung epithe- Shh(/) lungs (Fig. 4A). In situ hybridization revealed a clear lium, but dampens its transduction within the target tissue, the expansion of the Fgf10 domain in E15.5 TRII lungs (Fig. lung mesenchyme. This interference by TGF- may provide a 5A). Consistent with these results, Foxf1 and Tbx4, two tran- potential mechanism by which Shh limits Fgf10 expression scription factors that stimulate Fgf10 production in the lung domain in the lung mesenchyme during branching morpho- mesenchyme (35, 36), were also increased in the TRII lungs genesis. In vitro studies using MRC5 cells, showed that treat- (Fig. 5, B and C). ment with TGF- reduces the steady state level of Fgf10, con- An abnormal characteristic of the cystic bronchial airways in comitant with decreases in Ptc and Gli-1 mRNAs (Fig. 8) TRII lungs was the absence of PBSMCs (Fig. 4C). During thereby validating the in vivo observations. Based on the collec- development and in adult tissues, mesenchymal cells serve as tive in vivo and in vitro findings, a hypothetical model depicting precursors to diverse cell lineages, including PBSMCs. The the mechanisms of TGF-Shh cross-talk is proposed in Fig. 9. function of TGF- in promoting myofibroblast differentiation In the wild-type lung, TGF- fine-tunes Shh signaling through is well recognized (39, 40). Therefore, the absence of PBSMCs modulation of its downstream targets, Gli-1 and Ptc, which in may be a direct impact of mesenchymal TRII inactivation, turn may control the magnitude and spatial distribution Fgf10. suggesting that TGF- signaling via the Type II receptor is In this manner, TGF- signaling via TRII participates in the required for PBSMC differentiation. The relatively high level of response of mesenchymal cells to Shh signaling that originates endogenous TRII protein and the TGF- target, PAI-1 in the from the lung epithelium. PBSMCs (Figs. 1 and 2C) and drastic reduction of PAI-1 in Acknowledgments—We thank Dr. Yang Chai for providing the response to mesodermal deletion of TRII in these cells in the homozygous TRII mutant mice, Dr. Andrew P. McMahon for Shh mutant lungs (Fig. 2C, panel d) supports the above hypothesis. cDNA, Dr. Matthew P. Scott for Ptc cDNA, Dr. Peter Carlsson for In addition however, we showed previously that PBSMC pro- Foxf1 cDNA, and Dr. Virginia Papaioannou for Tbx4 and Tbx5 genitors express Fgf10, and the transcription factor Pitx2, both cDNA. of which may be important in maintaining their undifferenti- ated status (41). These proliferating cells cease to express Fgf10 before onset of differentiation into PBSMCs (42). Thus, REFERENCES increased levels, or spatial expansion of Fgf10 distribution in 1. Massague, J. (1998) Annu. Rev. Biochem. 67, 753–791 TRII mutants, may provide another, alternative mechanistic 2. Souchelnytskyi, S., Tamaki, K., Engstrom, U., Wernstedt, C., ten Dijke, P., explanation for the absence of PBSMCs in the dilated bronchial and Heldin, C. H. (1997) J. Biol. Chem. 272, 28107–28115 airways; high levels of Fgf10 may inhibit PBSMC differentiation. 3. Wrana, J. L. (2000) Sci STKE 2000, 2000, RE1 4. Sanford, L. P., Ormsby, I., Gittenberger-de Groot, A. C., Sariola, H., Fried- TGF- signaling is inhibitory to branching morphogenesis man, R., Boivin, G. P., Cardell, E. L., and Doetschman, T. (1997) Develop- (21). Activation of TGF- in mesenchymal cells markedly ment 124, 2659–2670 inhibits Fgf10 expression (37, 43, 44). Positive transcriptional 5. Kaartinen, V., Voncken, J. W., Shuler, C., Warburton, D., Bu, D., Heis- regulators of Fgf10, Tbx4, and Foxf1 are controlled by Shh sig- terkamp, N., and Groffen, J. (1995) Nat. Genet. 11, 415–421 naling that emanates from the branching epithelium. Recently, 6. Zhou, L., Dey, C. R., Wert, S. E., and Whitsett, J. A. (1996) Dev. Biol. 175, potential interactions between Shh and TGF- pathways have 227–238 DECEMBER 26, 2008• VOLUME 283 • NUMBER 52 JOURNAL OF BIOLOGICAL CHEMISTRY 36263 TGF- Sonic Hedgehog Cross-talk 7. Chen, H., Sun, J., Buckley, S., Chen, C., Warburton, D., Wang, X. F., and 28. Li, M., Wu, X., Zhuang, F., Jiang, S., Jiang, M., and Liu, Y. H. (2003) Dev. Shi, W. (2005) Am. J. Physiol. Lung Cell Mol. Physiol. 288, L683–L691 Dyn. 228, 273–280 8. Shannon, J. M., and Hyatt, B. A. (2004) Annu. Rev. Physiol. 66, 625–645 29. Minoo, P., Hu, L., Xing, Y., Zhu, N. L., Chen, H., Li, M., Borok, Z., and Li, 9. Tabin, C. J., and McMahon, A. P. (1997) Trends Cell Biol. 7, 442–446 C. (2007) Mol. Cell. Biol. 27, 2155–2165 10. Miller, L. A., Wert, S. E., and Whitsett, J. A. (2001) J. Histochem. Cytochem. 30. Li, C., Hu, L., Xiao, J., Chen, H., Li, J. T., Bellusci, S., Delanghe, S., and 49, 1593–1604 Minoo, P. (2005) Dev. Biol. 287, 86–97 11. Bellusci, S., Furuta, Y., Rush, M. G., Henderson, R., Winnier, G., and 31. Hamdan, H., Liu, H., Li, C., Jones, C., Lee, M., deLemos, R., and Minoo, P. Hogan, B. L. (1997) Development 124, 53–63 (1998) Biochim. Biophys. Acta 1396, 336–348 12. Bellusci, S., Grindley, J., Emoto, H., Itoh, N., and Hogan, B. L. (1997) 32. Zhao, Y., and Shah, D. U. (2000) Exp. Mol. Pathol. 69, 67–78 Development 124, 4867–4878 33. Minoo, P., Su, G., Drum, H., Bringas, P., and Kimura, S. (1999) Dev. Biol. 13. Sekine, K., Ohuchi, H., Fujiwara, M., Yamasaki, M., Yoshizawa, T., Sato, 209, 60–71 T., Yagishita, N., Matsui, D., Koga, Y., Itoh, N., and Kato, S. (1999) Nat. 34. Zeng, X., Gray, M., Stahlman, M. T., and Whitsett, J. A. (2001) Dev. Dyn. Genet. 21, 138–141 221, 289–301 14. Min, H., Danilenko, D. M., Scully, S. A., Bolon, B., Ring, B. D., Tarpley, J. E., 35. del Moral, P. M., De Langhe, S. P., Sala, F. G., Veltmaat, J. M., Tefft, D., DeRose, M., and Simonet, W. S. (1998) Genes Dev. 12, 3156–3161 Wang, K., Warburton, D., and Bellusci, S. (2006) Dev. Biol. 293, 77–89 15. Ingham, P. W. (1995) Curr. Opin. Genet. Dev. 5, 492–498 36. Cardoso, W. V., and Lu, J. (2006) Development 133, 1611–1624 16. McMahon, A. P. (2000) Cell 100, 185–188 37. Lebeche, D., Malpel, S., and Cardoso, W. V. (1999) Mech. Dev. 86, 17. Dai, P., Akimaru, H., Tanaka, Y., Maekawa, T., Nakafuku, M., and Ishii, S. 125–136 (1999) J. Biol. Chem. 274, 8143–8152 38. Warzecha, J., Gottig, S., Bruning, C., Lindhorst, E., Arabmothlagh, M., and 18. Grindley, J. C., Bellusci, S., Perkins, D., and Hogan, B. L. (1997) Dev. Biol. Kurth, A. (2006) J. Orthop. Sci. 11, 491–496 188, 337–348 39. Cohen, P., Rajah, R., Rosenbloom, J., and Herrick, D. J. (2000) Am. J. 19. Motoyama, J., Liu, J., Mo, R., Ding, Q., Post, M., and Hui, C. C. (1998) Nat. Physiol. Lung Cell Mol. Physiol. 278, L545–L551 Genet. 20, 54–57 40. Black, P. N., Young, P. G., and Skinner, S. J. (1996) Am. J. Physiol. 271, 20. Pepicelli, C. V., Lewis, P. M., and McMahon, A. P. (1998) Curr. Biol. 8, L910–L917 1083–1086 41. De Langhe, S. P., Carraro, G., Tefft, D., Li, C., Xu, X., Chai, Y., Minoo, P., 21. Xing, Y., Li, C., Hu, L., Tiozzo, C., Li, M., Chai, Y., Bellusci, S., Anderson, S., Hajihosseini, M. K., Drouin, J., Kaartinen, V., and Bellusci, S. (2008) PLoS and Minoo, P. (2008) Dev. Biol. 320, 340–350 ONE 3, e1516 22. Oshima, M., Oshima, H., and Taketo, M. M. (1996) Dev. Biol. 179, 42. Mailleux, A. A., Kelly, R., Veltmaat, J. M., De Langhe, S. P., Zaffran, S., 297–302 Thiery, J. P., and Bellusci, S. (2005) Development 132, 2157–2166 23. Yu, K., Xu, J., Liu, Z., Sosic, D., Shao, J., Olson, E. N., Towler, D. A., and 43. Beer, H. D., Florence, C., Dammeier, J., McGuire, L., Werner, S., and Duan, Ornitz, D. M. (2003) Development 130, 3063–3074 D. R. (1997) Oncogene 15, 2211–2218 24. Soriano, P. (1999) Nat. Genet. 21, 70–71 44. Tomlinson, D. C., Grindley, J. C., and Thomson, A. A. (2004) Endocrinol- 25. Chytil, A., Magnuson, M. A., Wright, C. V., and Moses, H. L. (2002) Gen- ogy 145, 1988–1995 esis 32, 73–75 45. Yoo, Y. A., Kang, M. H., Kim, J. S., and Oh, S. C. (2008) Carcinogenesis 29, 26. Pan, Q., Li, C., Xiao, J., Kimura, S., Rubenstein, J., Puelles, L., and Minoo, P. 480–490 (2004) Gene 331, 73–82 46. Dennler, S., Andre, J., Alexaki, I., Li, A., Magnaldo, T., ten Dijke, P., Wang, 27. Li, C., Xiao, J., Hormi, K., Borok, Z., and Minoo, P. (2002) Dev. Biol. 248, 68–81 X. J., Verrecchia, F., and Mauviel, A. (2007) Cancer Res. 67, 6981–6986 36264 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283 • NUMBER 52 •DECEMBER 26, 2008 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

Mesodermal Deletion of Transforming Growth Factor-β Receptor II Disrupts Lung Epithelial Morphogenesis

Li, Min; Li, Changgong; Liu, Yi-hsin; Xing, Yiming; Hu, Lingyan; Borok, Zea; Kwong, Kenny Y.-C.; Minoo, Parviz
Journal of Biological Chemistry – Dec 1, 2008
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Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 52, pp. 36257–36264, December 26, 2008 © 2008 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Mesodermal Deletion of Transforming Growth Factor- Receptor II Disrupts Lung Epithelial Morphogenesis CROSS-TALK BETWEEN TGF- AND SONIC HEDGEHOG PATHWAYS Received for publication, September 2, 2008, and in revised form, October 22, 2008 Published, JBC Papers in Press, November 6, 2008, DOI 10.1074/jbc.M806786200 ‡ ‡ § ‡ ‡ ¶ ‡ Min Li , Changgong Li , Yi-hsin Liu , Yiming Xing , Lingyan Hu , Zea Borok , Kenny Y.-C. Kwong , ‡1 and Parviz Minoo ‡ § ¶ From the Division of Neonatology, Department of Pediatrics, the Department of Ophthalmology, and the Will Rogers Institute Pulmonary Research Center, Department of Medicine, University of Southern California Schools of Medicine, Los Angeles, California 90033 In vertebrates, Sonic hedgehog (Shh) and transforming receptors. The binding of TGF- ligand to the receptor com- growth factor- (TGF-) signaling pathways occur in an over- plex induces phosphorylation of type I by the type II recep- lapping manner in many morphogenetic processes. In vitro data tors and activates Smads, a family of transcriptional factors indicate that the two pathways may interact. Whether such that act as intracellular effectors of TGF- signaling (1). interactions occur during embryonic development remains Smad2 and/or Smad3 are phosphorylated in their C-termi- unknown. Using embryonic lung morphogenesis as a model, we nal domain upon stimulation by either activin or TGF- (2). generated transgenic mice in which exon 2 of the TRII gene, Phosphorylation of Smad2 and Smad3 is accompanied by which encodes the type II TGF- receptor, was deleted via a their association with Smad4 and translocation of the het- mesodermal-specific Cre. Mesodermal-specific deletion of eromeric complex to the nucleus where they affect transcrip- TRII (TRII ) resulted in embryonic lethality. The lungs tion of target genes through interaction with promoter-spe- showed abnormalities in both number and shape of cartilage in cific transcriptional factors or by direct DNA binding (3). trachea and bronchi. In the lung parenchyma, where epithelial- Genetic manipulations of endogenous TGF- signaling have mesenchymal interactions are critical for normal development, revealed their important functions in vertebrate development. deletion of mesenchymal TRII caused abnormalities in epithe- Targeted deletion of each of the three ligand isoforms causes lial morphogenesis. Failure in normal epithelial branching severe abnormalities in morphogenesis of various organs morphogenesis in the TRII lungs caused cystic airway mal- including the lung. Tgf-2-null mice exhibit perinatal mortality formations. Interruption of the TRII locus in the lung mes- and a wide range of developmental abnormalities that include enchyme increased mRNA for Patched and Gli-1, two down- cardiac, lung, craniofacial, limb, spinal column, eye, inner ear, stream targets of Shh signaling, without alterations in Shh and urogenital defects (4). Tgf- 3 mutants die as neonates due ligand levels produced in the epithelium. Therefore, we con- to abnormal lung development and cleft palate (5). Targeted clude that TRII-mediated signaling in the lung mesenchyme disruption of the Tgf-1 gene results in abnormalities in the modulates transduction of Shh signaling that originates from lung, manifested as dilation of the airways (6). Mice with tar- the epithelium. To our knowledge, this is the first in vivo geted deletion of Smad3 are viable, but develop lung abnormal- evidence for a reciprocal and novel mode of cross-communi- ities akin to emphysema (7). Little is known about the role of cation between Shh and TGF- pathways during embryonic other components of the TGF- pathway and interactions with development. other signaling molecules in the lung. Embryonic lung development represents a useful model in which to study complex tissue interactions in organ develop- Transforming growth factor- (TGF-) ligands are multi- ment. Lung morphogenesis is strictly dependent on cross-talk functional signaling proteins that exhibit a wide range of bio- between two distinct tissues, the endodermal-derived epithe- logical activities including regulation of cell proliferation and lium and the mesodermal-derived lung mesenchyme (8). A differentiation. TGF- initiates its cellular actions by interact- major signaling pathway in this communication is Shh, the ver- ing with a heteromeric complex of transmembrane serine/thre- tebrate homologue of Drosophila hh, which is highly expressed onine kinase receptors, the type I (TRI) and type II (TRII) by embryonic lung epithelium. Patched (Ptc), the receptor for Shh is expressed by the lung mesenchyme, the site of highly * This work was supported, in whole or in part, by NHLBI, National Institutes of focalized Fgf10 production. The role of Fgf10 in directing epi- Health. This work was also supported by the Hastings Foundation. The thelial morphogenesis is central to lung development (9–12). In costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertise- Fgf10(/) embryos, the lung tissue below the main stem ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. bronchi is entirely absent (13, 14). Shh interacts with the Ptc/ To whom correspondence should be addressed: General Laboratories Bldg. Smoothened (Smo) complex on the mesenchymal cell mem- 1801 E. Marengo St., Rm. 1G1, Los Angeles, CA 90033. Tel.: 323-226-4340; Fax: 323-226-5049; E-mail: [email protected]. brane and activates Gli-3, a 190-kDa transcription factor (15, The abbreviations used are: TGF, transforming growth factor; X-gal, 5-bro- 16). Activated Gli-3 binds directly to the Gli-1 promoter and mo-4-chloro-3-indolyl--D-galactopyranoside; Shh, Sonic hedgehog; induces its transcription in response to Shh (17). Gli-1 is a zinc PBSMC, parabronchial smooth muscle cells; GAPDH, glyceraldehyde-3- phosphate dehydrogenase. transcription factor that activates the transcription of Ptc (18). DECEMBER 26, 2008• VOLUME 283 • NUMBER 52 JOURNAL OF BIOLOGICAL CHEMISTRY 36257 This is an Open Access article under the CC BY license. TGF- Sonic Hedgehog Cross-talk Thus, increased transcription of Gli-1 & Ptc are reliable mark- branes were probed with antibodies to TRII (Abcam) and ana- ers of Shh pathway activation. All three Gli family members are lyzed with the ECL Western blot analysis system as described expressed in and are important for lung development (18, 19). by the manufacturer (GE Health, Memphis, TN). The currently accepted model is that Shh both stimulates and In Situ Hybridization—Whole mount and section in situ restricts the level and spatial distribution of Fgf10 expression hybridization were performed as previously described (27, 28). during lung morphogenesis. Consistent with this concept, dele- The digoxigenin-labeled RNA antisense and sense probes were tion of Shh leads to diffused, but expanded Fgf10 mRNA prepared from following cDNA templates: a 0.4-kb fragment of throughout the mesenchyme as a consequence of which air- SpC, a 1.4-kb fragment of Nkx2.1 (29), a 0.5-kb fragment of ways develop into large cystic structures (20). Precisely how Foxj1, a 0.6-kb fragment of Shh (Dr. Andrew P. McMahon, Har- Shh controls Fgf10 gene expression has hitherto remained vard University), a 0.7-kb fragment of Ptc (Dr. Matthew P. unknown. Scott, Stanford University), a 0.7-kb fragment of Gli-1, a 0.7-kb Because of its established role as a negative regulator of lung fragment of Foxf1 (Dr. Peter Carlsson, Go¨teborg University), branching morphogenesis (21) TGF- is a potential mediator in a 0.4-kb fragment of Fgf10, a 1.3-kb fragment of Tbx4, and a epithelial-mesenchymal cross-talk during lung development. 1-kb fragment of Tbx5(Dr. Virginia Papaioannou, Princeton Conventional deletion of TRII lead to early embryonic lethal- University). ity, and therefore was not informative for lung development RNA Extraction, Polymerase Chain Reaction (PCR), and (22). In the present study, we used a mesodermal-specific cre- Northern Blotting—Total RNA was isolated from embryonic loxP system to delete exon 2 in the TRII locus in the lung lungs and MRC5 cells (ATCC) by using TRIzol (Invitrogen). mesenchyme. The lungs of TRII mouse fetuses are abnor- Superscript First-Strand Synthesis System kit (Invitrogen) was mal with evidence of cystic airway malformations associated used to generate cDNA. Quantification of the selected genes by with alterations in Fgf10 gene expression, likely due to interrup- real-time PCR was performed using a LightCycler (Roche tion of normal epithelial-mesenchymal cross-talk. Inactivation Applied Sciences) as previously described (30). Sequence of the of TRII and hence the specific TGF- signaling pathway medi- primers were as follows: Mouse TrII:5-ATG CAT CCA TCC ated through its normal activity in the lung mesenchyme results ACG TAA G-3(forward), 5-GAC ACG GTA GCA GTA GAA in alterations in Ptc and Gli mRNAs in the mutant lungs indi- GA-3 (reverse); human TrII:5-CAC GTT CAG AAG TCG cating interference with Shh signaling. Thus, TGF- signaling, GAT GT-3(forward), 5-CAT CAG AGC TAC AGG AAC mediated via TRII can modulate mesenchymal reception of AC-3(reverse); Mouse TrII(qPCR): 5-CAT GAA AGA CAG Shh signaling, which originates from the epithelium, indicating TGT GCT GAG A-3(forward), 5-CTC ACA CAC GAT CTG cross-communication between the two signaling pathways GAT GC-3(reverse); Mouse Foxf1:5-AGC ATC TCC ACG during embryonic lung morphogenesis. CAC TCC-3(forward), 5-TGT GAG TGA TAC CGA GGG ATG-3(reverse); Mouse Tbx4:5-GCA TGA GAA GGA GCT MATERIALS AND METHODS GTG G-3(forward), 5-TTA CCT TGT AGC TGG GGA fl/fl Animals—Dermo1-cre, Rosa26-lacZ, and TRII mice ACA-3(reverse); Human PAI-1:5-AAC GGC CAG TGG were generated and genotyped as previously described (23–25) AAG ACT C-3(forward), 5-GGG CGT GGT GAA CTC AGT and maintained on C57BL/6 genetic background. Dermo1-cre; AT-3(reverse); Human Ptc:5-AAC AAA AAT TCA ACC Rosa26-lacZ mice were generated by crossing Dermo1- and AAA CCT C-3(forward), 5-TGT CCT CGT TCC AGT TGA fl/fl Rosa26-lacZ mice. To generate TRII ; Dermo1-cre embryos TGT G-3(reverse); Human Gli-1:5-CAG GGA GGA AAG / fl/ (TRII ), TRII ; Derom1-cre mice were crossed with CAG ACT GA-3(forward), 5-ACT GCT GCA GGA TGA fl/fl TRII mice. CTG G-3(reverse); Human Fgf10:5-CGG GAC CAA GAA Detection of -Galactosidase (LacZ) Activity—LacZ activity GGA GAA CT-3(forward), 5-ACG GCA ACA ACT CCG was determined by X-gal staining as described (26). Whole ATT-3(reverse); Human GAPDH:5-GAA GGT GAA GGT mount staining was performed for embryonic lungs. Lungs of CGG AGT C-3(forward), 5-GAA GAT GGT GAT GGG ATT E15.5 and older were fixed and sectioned by cryostat, and the TC-3(reverse). Ten micrograms of total RNA were electro- frozen sections were stained for LacZ activity. phoresed in 1% RNA formaldehyde-agarose gel and blotted. Immunohistochemistry—For immunohistochemistry, sam- Blots were hybridized with probes specific for TRII, Shh, Ptc, ples were fixed in 4% paraformaldehyde and processed into Gli-1, Fgf10, Gapdh, and then autoradiographed or measured serial paraffin sections using routine procedures. Immuno- with Kodak Molecular Imaging Software Ver 4.0 to determine staining were performed as previously described (27). Primary the quantity. P-labeled probes were synthesized from the fol- antibodies that were used are: -SMA (Sigma), PAI-1 (Abcam lowing cDNA fragments: the TRII probe was from a 0.4-kb Cambridge, MA), PECAM (BD Pharmingen, San Diego, CA), PCR product of TRII coding region. The Shh probe was from Flk1 (Cell Signaling Technology, Beverly, MA), -Tubulin (Bio- a 0.6-kb cDNA (Dr. Brigid L. M. Hogan, Vanderbilt University). genix, San Ramon, CA), Collagen1 (Abcam, Cambridge, MA), The Ptc probe was from a 0.7-kb cDNA (Dr. Matthew P. Scott, and TRII (Abcam Cambridge, MA). Stanford University), The Gli-1 probe was from a 0.7-kb cDNA. Western Blot Analysis—Protein extracts were prepared from The Fgf10 probe was from a 0.4-kb cDNA. The GAPDH probe fl/fl / E15.5 Dermo1-cre; TRII and TRII lungs in RIPA buffer was as reported before (31). (Sigma) from homogenizer, and then separated on 4–12% Cell Culture and TGF- Treatment—Human pulmonary NuPAGE gels (Invitrogen). Proteins were then transferred onto mesenchymal cell line MRC5 (ATCC, Manassas, VA) was Immobilon-P transfer membrane (Millipore Corp.). Mem- maintained in EMEM medium (ATCC), containing 10% fetal 36258 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283 • NUMBER 52 •DECEMBER 26, 2008 TGF- Sonic Hedgehog Cross-talk fl/fl erozygotes to generate TRII ; Dermo1-cre progeny. The lat- ter genotype is referred to simply as TRII . To validate Dermo1-cre-induced recombination and deletion of exon 2 within the TRII gene, we used three approaches. First, we uti- lized lung DNA with PCR primers that could distinguish between deleted and intact TRII alleles. The deletion in TRII gene was verified by genomic PCR analysis shown in Fig. 2B, panel b. Second, we measured TRII protein content by West- ern blot analysis in protein extracts of total lung tissue from control and TRII embryos. These studies showed greater than 60% decrease in total lung TRII protein, the remainder indicating non-mesenchymal (e.g. epithelial and endothelial) TRII protein (Fig. 2B, panels c and d). In contrast, the protein FIGURE 1. TRII expression in the murine lung. A and B show immunolocal- level of TRI and phosphorylated Smad2 (p-Smad2) remained ization of TRII protein in lungs from E15.5 and E18.5 embryos, respectively. the same. Therefore mesenchymal deletion of TRII in the lung The arrow shows TRII protein localized in the subepithelial layer of proximal airways. The arrowhead points to endothelial localized TRII around the does not alter the level of phosphorylated Smad2 and TRI. blood vessel. B shows immunolocalized TRII distributed throughout the Finally, functional deletion of TRII was verified by immuno- parenchyma of E18.5 lungs, with highest expression in the interstitium, including cells of mesenchymal origin. C, semi-quantitative RT-PCR of TRII histochemistry using antibodies to Plasminogen Activator mRNA during murine lung morphogenesis. E, embryonic day; PN, postnatal Inhibitor (PAI-1), which is a downstream target of TGF- sig- day. D, TRII mRNA is also detectable in both human (A549 and H449) and naling. In the control lungs, diffuse PAI-1 was detected murine (MLE15) transformed epithelial cell lines. MRC5 is a fetal human mes- enchymal cell line. Scale bar, 100 m. throughout both the epithelium and the mesenchyme, but par- ticularly in the progenitor of parabronchial smooth muscle cells (PBSMC) in the subepithelial mesoderm (Fig. 2C, panel b, bovine serum and 1% penicillin-streptomycin. MRC5 cells arrows). In contrast, PAI-1 in the TRII lungs was found were treated with recombinant TGF- (R&D systems, Min- neapolis, MN) at 200 ng/ml for 2 h and then collected for only in the epithelium. As expected, there was virtually no RNA extraction. PAI-1 staining in the mesoderm, nor in the PBSMC progenitors in TRII lungs (Fig. 2C, panel d, arrowheads). RESULTS Ultrastructure and Cellular Differentiation in TRII TRII Expression during Lung Development—TRII has been Lungs—Dermo1-cre-induced deletion of exon 2 within the reported to be expressed in both the epithelium and the mes- TRII gene was embryonically lethal by day 16–17 of gestation. enchyme of the lung (32). To elucidate the expression pattern of E16–17 fetuses developed gross abnormalities in multiple TRII in the murine lung, we used semi-quantitative RT-PCR organs that caused fetal demise. We therefore, collected and and immunohistochemistry. Expression of TRII mRNA can characterized lungs from TRII E15.5 fetuses. Gross mor- be detected by RT-PCR throughout lung development (Fig. 1 C). phological assessment of the proximal lung structure showed a Commercially available antibody (Abcam) detects TRII pro- readily discernible phenotype manifested as disorganized for- tein in mesenchymal cells localized around the airways (Fig. 1, mation of tracheal cartilage (Fig. 3N). Both the number as well arrows) and blood vessels (Fig. 1, arrowhead) in E15.5 lungs as the shape of the tracheal cartilage was altered. The first and (Fig. 1A) and throughout the mesenchyme in E18 lungs (Fig. 1B). the second generation bronchi in the mutant lungs were devoid TRII mRNA is clearly expressed in human lung epithelial car- of cartilage altogether (not shown). cinoma, H441 and A549 cell lines as well as mouse-immortal- Mesodermal deletion of TRII also impacted the shape and ized epithelial MLE15 cells (Fig. 1D). the size of the various lung lobes as shown in Fig. 3. However, Recombination Mediated by Dermo1-cre in the Lung the overall process of lobation and the number of lobes were Mesenchyme—Recombination driven by Dermo1-cre in the normal (Fig. 3, H–L). Histological assessment showed a distinct lung was analyzed by crossing with the reporter mouse strain phenotype in TRII lungs, characterized by the presence of Rosa26-LacZ. Double transgenic, Rosa26-LacZ; Dermo1-cre large, dilated airways, lined with columnar epithelial cells in the fetuses were identified by PCR genotyping, and the lungs were proximal lung (Fig. 4A). excised and stained for LacZ. As shown in Fig. 2A, expression of The epithelial cells throughout the TRII lungs showed Dermo1-cre resulted in nearly 100% recombination of the loxP expression of NKX2.1, a transcription factor associated with sites in Rosa26-lacZ lung mesodermal cells (Fig. 2A, panels onset of lung epithelial morphogenesis and normal lung struc- a–f). This recombination was entirely mesenchymal-specific in tural development(Fig. 4B, panels a and d) (33). Transcripts for that no epithelial cells showed LacZ staining (Fig. 2A, panels a, Surfactant Protein C, SPC, a marker of distal epithelial cells and b, d, and f, arrows). a target of NKX2.1 was also expressed in the mutant lungs. Also, Inactivation of TRII by Dermo1-cre-driven Recombination— differentiation of ciliated cells as evidenced by expression of Conventional deletion of TRII led to early embryonic lethality Foxj1 appeared to be normal in the absence of epithelial TRII (22). Thus, we used a conditional Cre-LoxP approach to specif- activity (Fig. 4B). The normal level and distribution of Surfac- ically delete exon 2 in the TRII locus in mesodermal lineages, tant Protein B, SPB, expressed in proximal and distal, differen- fl/fl including those of the lung. Accordingly, we crossed TRII tiated epithelium, as well as -tubulin, an established marker of females with Dermo1-cre male mice and backcrossed the het- airway ciliated cells were also observed by immunohistochem- DECEMBER 26, 2008• VOLUME 283 • NUMBER 52 JOURNAL OF BIOLOGICAL CHEMISTRY 36259 TGF- Sonic Hedgehog Cross-talk (Fig. 4C, panels e and f). Collagen production is also under TGF- control (6). Both in the wild-type control and the mutant lungs, colla- gen Type I was localized to the extracellular matrix surrounding both the distal and the proximal air- ways (Fig. 4C, panels g and h). No drastic alteration in collagen type I was observed in TRII lungs sug- gesting that either TGF- is signal- ing through utilization of type I receptor (homotetramer) or that collagen production and deposition may not be entirely TGF-- dependent. Expression of Fgf10 in TRII Lungs—Whole mount and section in situ hybridization were per- formed for a number of genes with established roles during lung mor- phogenesis. Because the shape and size of the airways can be regulated by FGF10 activity and its expression domain, we investigated the expres- FIGURE 2. Generation and validation of TRII conditional knock-out alleles. A, mesenchymal specific recombi- / sion pattern of Fgf10 in TRII nation induced by Dermo1-cre as determined by LacZ assay in murine lungs from various stages of embryonic lungs. Whole mount in situ hybrid- development. Panels a,c,andearewholemountlungtissue. Panels b,d,andfarefrozensaggitalsectionsofthelungs in panels a, c, and e. Arrows in panels a, b, d, and f show the absence of LacZ in airway epithelial cells. Scale bar,50m. ization revealed increased expres- B, evidence for deletion of TRII gene by Dermo1-cre. Panel a, map of the mouse TRII locus showing the relative sion, and expansion of the Fgf10 position of the lox-P (flox) sequences and the primers used in identifying various genotypes. Primers for Cre were used as described by Yu et al. (23). Panel b, PCR analysis of TRII gene using lung-extracted DNA and the primers P1, domain in the mutant embryonic fl/ P2, and P3, as shown in panel a. Lane 1, TRII lungs, showing two bands corresponding to the wild type (Wt) and lungs (Fig. 5A, panels e and f). A the allele carrying the lox-P insertion. The absence of a PCR product using P1/P3 primers is due to the large distance fl/fl clearer demonstration of this alter- betweenthetwoprimers(absenceofdeletion). Lane 2,TRII lungsshowingasinglebandcorrespondingtofloxed allele. Lane 3, TRII lungs. Deletion of Exon 2 brings the sequences recognized by P1 and P3 primers sufficiently ation was observed by in situ close to allow amplification of a PCR product (deletion). Lane 4, TRII lungs. Panel c, Western blot analysis of TRII, hybridization experiments on tissue fl/fl / TRI, and p-Smad2 in total lung protein from TRII (lane 1) and TRII (lane 2) mice. Panel d, densitometric sections (Figure 5A, compare panels quantification of the Western blot shown in panel c. C, immunohistochemical analysis for PAI-1 in TRII and fl/fl TRII lungs. Arrowheadsin panel dshowthereducedexpressionofPAI-1inthePBSMCsofmutantlungscompared c and g). In the control lungs, Fgf10 with arrows in panel b (control). m, mesenchyme; e, epithelium. Scale bar, 100m for panels a and c,23m for panels mRNA was localized to mesenchy- b and d. mal cells adjacent to the growing istry (data not show). Collectively, these data indicate that the tip of the peripheral airways (Fig. 5A, panels c and d) consist- absence of TRII in the lung mesenchyme does not alter lung ent with previously reported results (12). In the mutant epithelial cell identity and differentiation. lungs, however, we found an expanded Fgf10 expression Expression of TGF- Targets in TRII Lungs—In wild- domain and likely increased mRNA in the peripheral mesen- type embryonic lungs, -smooth muscle actin-positive cells are chyme adjacent to the branching airways (Fig. 5A, panels found as rings of smooth muscle progenitor cells surrounding e– h). This alteration in Fgf10 expression domain may the columnar epithelium of the proximal airways (Fig. 4C, panel explain the phenotype of the TRII lungs in which air- a). In contrast, we found reduced -SMA-positive cells sur- ways are significantly dilated. Two transcription factors, rounding the dilated airways in the mutant lungs (Fig. 4C, panel Foxf1 and Tbx4 have been found to stimulate Fgf10 expres- b). This is the same layer of smooth muscle cells in which PAI-1 sion in the lung mesenchyme (35, 36). Consistent with this was found to be abundantly expressed in the wild-type lung and finding, in situ hybridization on tissue sections (Fig. 5B, pan- drastically reduced in TRII lungs (Fig. 2C). Immunohisto- els a, b, d, and e) and real-time PCR (Fig. 5C) revealed chemistry was also performed to determine potential changes increased transcripts for both in TRII lungs compared in the expression or spatial localization of other mesenchymal with controls. Expression of another Tbx gene, Tbx5 genes including collagen type I and platelet/endothelial cell remained unchanged (Fig. 5B, panels c and f). These data adhesion molecule (PECAM) known to be modulated by support the finding that Fgf10 is both increased and TGF- (6, 34). Reduced levels of PECAM were found in the expanded in its expression domain in TRII lungs. mutant lungs (Fig. 4C, compare panels c and d). Another Cross-talk between TGF- and Shh Pathways—Shh is marker of endothelial cell differentiation, Flk1 appeared to have thought to negatively regulate Fgf10 magnitude and distribu- the same level and distribution in the mutant lungs as wild type tion, thereby, controlling lung branching morphogenesis (20, 36260 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283 • NUMBER 52 •DECEMBER 26, 2008 TGF- Sonic Hedgehog Cross-talk / fl/fl FIGURE 3. Gross morphology of TRII and TRII trachea and lungs from E15.5 embryos. B and N, tracheas. Arrows show abnormal number and shape of tracheal cartilage. C and H, right apical lobes. D and I, right middle lobes. E and J, right caudal lobes. F and K, accessory lobes. G and L, left lobes. Scale bar, 1.0 mm for A and M; 0.2 mm for B and N; 0.8 mm for C–F, H–K, and 1.2 mm for G and L. FIGURE 5. In situ hybridization analysis of Fgf10 and its upstream tran- scriptional regulators in TRII and control lungs. A, whole mount (pan- els a, b, e, and f) and section (panels c, d, g, and h) in situ hybridization. Note expansion and increased Fgf10 expression in TRII (panels e– h) compared fl/fl with TRII (panels a– d) lungs. Scale bar, 2.2 mm (panels a and c);1mm (panels b and d); 100 m(panels e– h). B, section in situ hybridization for Foxf1 (panels a and d), Tbx5 (panels b and e), and Tbx4 (panels c and f)in TRII and fl/fl TRII (control) E15.5 lungs. Scale bar, 100 m. C, quantification of Foxf1 and Tbx4 mRNA by real-time PCR. Values are fold induction or repression, com- fl/fl pared with TRII controls (arbitrarily adjusted to 1). p value, 0.004. stream targets Ptc and Gli-1 in the mutant lungs compared with fl/fl the TRII controls. Whole mount and tissue section in situ hybridization showed similar levels and spatial localization of Shh mRNA in the control and mutant lungs (Fig. 6, A, D, G, and J). In contrast, there was a significant increase in the level of mRNA for both Ptc (Fig. 6, B, E, H, and K) and Gli-1 (C, F, I, and L)inthe TRII mutant lungs compared with the control. To validate the above in situ hybridization results, we used North- ern blot analysis as shown in Fig. 7. Transcripts for TRII were decreased by nearly 80% in TRII lung tissue, compared FIGURE 4. H & E staining and immunohistochemical analysis for lung with controls. Other changes included a 1.8-fold increase in developmental markers in TRII and control lungs. A, gross histology fl/fl / Ptc and Gli-1 mRNA and a 1.5-fold increase in Fgf10. The mag- of lungs from TRII (panels a and b) and TRII (panels c and d) E15.5 embryos. Saggital sections of lungs were analyzed byH&E staining. MB, nitude of Shh remained nearly the same in the mutant and con- mainstem bronchus. Asterisks show dilated proximal airways. Panels b and d trol lungs confirming the in situ hybridization findings. are high magnification of areas within dotted squares in panels a and c, respec- Because deletion of TRII occurs specifically in the lung mes- tively. Scale bar, 330 m for panels a and c; 100 m for panels b and d. B, cell differentiation in TRII embryonic lungs. In situ hybridization for Nkx2.1 enchyme, one potential hypothesis is that TGF- signaling (panels a and d), SpC (panels b and e), and Foxj1 (panels c and f). Asterisks, through TRII normally represses Gli-1 and Ptc mRNA levels dilated airways. Scale bar, 100 m. C, immunohistochemical analysis for -SMA (panels a and b), PECAM (panels c and d), Flk1 (panels e and f), and in the lung mesenchymal cell layer. To determine the validity of fl/fl / Collagen1 (panels g and h) in E15.5 TRII and TRII lungs. Arrowheads in this hypothesis, we used cultured MRC5 cells, which are panel b show reduced expression of -SMA surrounding the dilated airways derived from normal lung mesenchymal cells of a 14-week-old of mutant lungs compared with arrows in panel a (control). Scale bar, 100 m. male fetus, and assessed the impact of exogenous TGF- treat- 37). The mechanistic connection between Fgf10 and Shh ment on Gli-1 and Ptc mRNA levels by real-time PCR. In sup- remained unknown. We therefore examined Shh signaling by port of our in vivo observations, the results clearly showed that investigating the level and distribution of Shh and its down- TGF- represses steady state levels of both Gli-1 and Ptc DECEMBER 26, 2008• VOLUME 283 • NUMBER 52 JOURNAL OF BIOLOGICAL CHEMISTRY 36261 TGF- Sonic Hedgehog Cross-talk FIGURE 9. A hypothetical model for TGF-Shh cross-talk in the devel- oping lung. Shh is made in the lung epithelium. The Shh receptor complex, Ptc/smoothened is expressed on the cell surface of lung mesenchymal cells. Activation of Shh pathway leads to increased Gli-3 activity, which in turn stimulates Gli-1 and Ptc transcription. TGF- ligand acts through TRII and as yet unknown intracellular mediators (e.g. Smads) to interfere with Shh signal transduction in mesenchymal cells, evidenced by reductions in both Ptc and Gli-1 observed in this study. FIGURE 6. Whole mount and section in situ hybridization for components results validate our in vivo-based conclusions and support a fl/fl / of the Shh pathway in TRII and TRII E12.5 (A–F) and E15.5 (G–L) model for interactions between TGF- and Shh signaling path- lungs. Abundance of mRNA for both Ptc and Gli-1 is increased in the mutant lungs (E, F, K, and L) compared with the control lungs (B, C, H, and I). Little if any ways (Fig. 9). change is detectable in the epithelial-localized Shh (A, D, G, and J). Scale bar, 2.2 mm (A, B, E, F, I, J); 100 m(C, D, G, H, K, L). DISCUSSION The purpose of the current study was to examine the conse- quences of interruption in mesenchymal TRII-mediated TGF- signaling on lung morphogenesis. To this end, we gen- erated mice carrying mesodermal-specific deletion of the TRII exon 3 by Dermo1-cre-driven recombination. TRII fetuses died in utero due to multi-organ abnormalities. Examination of embryonic lungs revealed that mesenchymal abrogation of endogenous TGF- signaling caused abnormalities in epithelial morphogenesis, indicating disruption of normal epithelial- mesenchymal communication that is central to lung develop- ment. Analysis of key mediators of this cross-talk showed alter- FIGURE 7. Quantification of mRNA changes in TRII lungs. Total RNA ations in components of the Shh pathway. Both Ptc and Gli-1 as / fl/fl from E15.5 TRII and TRII (control) embryonic lungs was used for well as the transcription factor Foxf1 were increased in the mes- Northern blot analysis (A), and the results were quantified by densitometry and normalized by GAPDH (B). Values are fold induction or repression, com- enchyme of the mutant lungs. Shh mRNA in the lung epithe- fl/fl pared with TRII controls (arbitrarily adjusted to 1). lium was unchanged. Thus, abrogation of TRII-mediated sig- naling in the lung mesenchyme results in increased expression of Shh downstream targets (i.e. Shh signaling). To our knowl- edge, this is the first demonstration of cross-communication between the TGF- and the Shh pathways during vertebrate embryonic development. At least two major phenotypic abnormalities were readily observable in TRII mutant lungs. First, the trachea of the mutant lungs showed abnormalities in both structure and num- ber of cartilage. Both TGF- and Shh are known to be involved in the induction of early cartilaginous differentiation of mesen- chymal cells in the limb and in the spine. In vitro, treatment of human bone marrow-derived mesenchymal stem cells (MSCs) FIGURE 8. In vitro verification of the in vivo evidence that TGF- is a neg- with either TGF- or recombinant Shh induced expression of ative regulator of Shh pathway. MRC5 cells were treated with either 200 cartilage markers aggrecan, Sox9, CEP-68, and collagen type II ng/ml of TGF-1 or equivalent bovine serum albumin (control). Real-time and X (38). Thus, the abnormalities observed in tracheal carti- PCR-quantified mRNA for PAI-1 (positive control), Ptc and Gli-1. Values are fold induction or repression, compared with controls (adjusted to 1). lage formation in TRII lungs can be explained as either a direct result of mesenchymal TRII deletion or indirect effect mRNAs by nearly 50% (Fig. 8). Fgf10 has been proposed to be a of interruption in Shh signaling. target of Shh signaling in the lung (36) although this regulation Mesodermal inactivation of TRII also caused abnormal epi- has not been experimentally explored or demonstrated. In thelial morphogenesis manifested as cystic, dilated bronchi MRC5 cells, TGF- treatment repressed Fgf10 mRNA consist- (Figure 4A). Lung branching morphogenesis is strictly depend- ent with our in vivo observations in Fig. 5A. Thus, these in vitro ent on epithelial-mesenchymal communication between the 36262 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283 • NUMBER 52 •DECEMBER 26, 2008 TGF- Sonic Hedgehog Cross-talk foregut endoderm and the mesodermal-derived splanchnic been examined in in vitro settings. Shh promotes motility and mesenchyme. The lung mesenchyme, via Fgf10 provides invasiveness of gastric cancer cells through TGF--mediated instructional signaling that directs epithelial morphogenesis. activation of the ALK5-Smad3 pathway (45). In contrast to our Targeted deletion of Fgf10 results in profoundly abnormal findings, TGF- was shown to induce Gli-1 and Gli-2 expres- lungs that lack structures distal to the main stem bronchi (13, sion in various human cell lines (46). Also, in transgenic mice 14). Dynamic changes in the magnitude and spatial distribution overexpressing TGF-1 in the skin, Gli-1 and Gli-2 were ele- of Fgf10 are critical to normal branching morphogenesis and vated in a Smad3-dependent manner (46). This apparent dis- are thought to be controlled by diffusible signals originating crepancy may suggest both cell type (skin versus lung) and dose- from the epithelial cells themselves. Shh is expressed in the dependent specificity of TGF- effect on adult and embryonic distal epithelium, from where it activates signaling in the cor- tissue. The relationship between TGF- and Shh during orga- responding mesenchyme via its receptor, the patched (Ptch1)/ nogenesis, the focus of the present study had remained smoothened (Smo) complex and their transcriptional effectors unknown. In particular whether the inhibition of Fgf10 in the Gli-1, Gli-2, and Gli-3 (9, 11). Results from both in vitro and in lung mesenchyme in response to TGF- involves the Shh path- vivo studies indicate that Shh negatively regulates Fgf10 pro- way remained unknown. We found increased expression of Ptc duction in the lung mesenchyme (20, 37). Thus, a current and Gli-1, as well as the transcription factors Tbx4 and Foxf1 in model of embryonic lung development is centered on epithelial the mesenchyme of TRII mutant lungs, suggesting that endog- Shh as playing a dual role in both activating and limiting mes- enous TGF- signaling via the type II receptor in the wild-type enchymal signaling thereby “fine-tuning” the process of lung regulates Shh signal transduction in the mesenchyme. In branching morphogenesis. In Shh(/) lungs, Fgf10 mRNA is previous studies, we found that Wnt5a alters Shh signaling by found diffusely throughout the mesenchyme, as a consequence modulating Shh mRNA in the lung epithelium (30). In contrast, of which the airways develop into large cystic structures and increased Shh signaling in the mesenchyme in response to branching morphogenesis is severely disrupted (20). Although TRII inactivation occurrs in the absence of changes in epithe- not as severe, the phenotypically abnormal, cystic bronchi lial Shh mRNA (Figs. 6 and 7). Therefore, TGF- does not observed in TRII mutant lungs are similar to those observed in directly interfere with Shh originating from the lung epithe- Shh(/) lungs (Fig. 4A). In situ hybridization revealed a clear lium, but dampens its transduction within the target tissue, the expansion of the Fgf10 domain in E15.5 TRII lungs (Fig. lung mesenchyme. This interference by TGF- may provide a 5A). Consistent with these results, Foxf1 and Tbx4, two tran- potential mechanism by which Shh limits Fgf10 expression scription factors that stimulate Fgf10 production in the lung domain in the lung mesenchyme during branching morpho- mesenchyme (35, 36), were also increased in the TRII lungs genesis. In vitro studies using MRC5 cells, showed that treat- (Fig. 5, B and C). ment with TGF- reduces the steady state level of Fgf10, con- An abnormal characteristic of the cystic bronchial airways in comitant with decreases in Ptc and Gli-1 mRNAs (Fig. 8) TRII lungs was the absence of PBSMCs (Fig. 4C). During thereby validating the in vivo observations. Based on the collec- development and in adult tissues, mesenchymal cells serve as tive in vivo and in vitro findings, a hypothetical model depicting precursors to diverse cell lineages, including PBSMCs. The the mechanisms of TGF-Shh cross-talk is proposed in Fig. 9. function of TGF- in promoting myofibroblast differentiation In the wild-type lung, TGF- fine-tunes Shh signaling through is well recognized (39, 40). Therefore, the absence of PBSMCs modulation of its downstream targets, Gli-1 and Ptc, which in may be a direct impact of mesenchymal TRII inactivation, turn may control the magnitude and spatial distribution Fgf10. suggesting that TGF- signaling via the Type II receptor is In this manner, TGF- signaling via TRII participates in the required for PBSMC differentiation. The relatively high level of response of mesenchymal cells to Shh signaling that originates endogenous TRII protein and the TGF- target, PAI-1 in the from the lung epithelium. PBSMCs (Figs. 1 and 2C) and drastic reduction of PAI-1 in Acknowledgments—We thank Dr. Yang Chai for providing the response to mesodermal deletion of TRII in these cells in the homozygous TRII mutant mice, Dr. Andrew P. McMahon for Shh mutant lungs (Fig. 2C, panel d) supports the above hypothesis. cDNA, Dr. Matthew P. Scott for Ptc cDNA, Dr. Peter Carlsson for In addition however, we showed previously that PBSMC pro- Foxf1 cDNA, and Dr. Virginia Papaioannou for Tbx4 and Tbx5 genitors express Fgf10, and the transcription factor Pitx2, both cDNA. of which may be important in maintaining their undifferenti- ated status (41). These proliferating cells cease to express Fgf10 before onset of differentiation into PBSMCs (42). Thus, REFERENCES increased levels, or spatial expansion of Fgf10 distribution in 1. Massague, J. (1998) Annu. Rev. Biochem. 67, 753–791 TRII mutants, may provide another, alternative mechanistic 2. Souchelnytskyi, S., Tamaki, K., Engstrom, U., Wernstedt, C., ten Dijke, P., explanation for the absence of PBSMCs in the dilated bronchial and Heldin, C. H. (1997) J. Biol. Chem. 272, 28107–28115 airways; high levels of Fgf10 may inhibit PBSMC differentiation. 3. Wrana, J. L. (2000) Sci STKE 2000, 2000, RE1 4. Sanford, L. P., Ormsby, I., Gittenberger-de Groot, A. C., Sariola, H., Fried- TGF- signaling is inhibitory to branching morphogenesis man, R., Boivin, G. P., Cardell, E. L., and Doetschman, T. (1997) Develop- (21). Activation of TGF- in mesenchymal cells markedly ment 124, 2659–2670 inhibits Fgf10 expression (37, 43, 44). Positive transcriptional 5. Kaartinen, V., Voncken, J. W., Shuler, C., Warburton, D., Bu, D., Heis- regulators of Fgf10, Tbx4, and Foxf1 are controlled by Shh sig- terkamp, N., and Groffen, J. (1995) Nat. Genet. 11, 415–421 naling that emanates from the branching epithelium. Recently, 6. Zhou, L., Dey, C. R., Wert, S. E., and Whitsett, J. A. (1996) Dev. Biol. 175, potential interactions between Shh and TGF- pathways have 227–238 DECEMBER 26, 2008• VOLUME 283 • NUMBER 52 JOURNAL OF BIOLOGICAL CHEMISTRY 36263 TGF- Sonic Hedgehog Cross-talk 7. Chen, H., Sun, J., Buckley, S., Chen, C., Warburton, D., Wang, X. F., and 28. Li, M., Wu, X., Zhuang, F., Jiang, S., Jiang, M., and Liu, Y. H. (2003) Dev. Shi, W. (2005) Am. J. Physiol. Lung Cell Mol. Physiol. 288, L683–L691 Dyn. 228, 273–280 8. Shannon, J. M., and Hyatt, B. A. (2004) Annu. Rev. Physiol. 66, 625–645 29. Minoo, P., Hu, L., Xing, Y., Zhu, N. L., Chen, H., Li, M., Borok, Z., and Li, 9. Tabin, C. J., and McMahon, A. P. (1997) Trends Cell Biol. 7, 442–446 C. (2007) Mol. Cell. Biol. 27, 2155–2165 10. Miller, L. A., Wert, S. E., and Whitsett, J. A. (2001) J. Histochem. Cytochem. 30. Li, C., Hu, L., Xiao, J., Chen, H., Li, J. T., Bellusci, S., Delanghe, S., and 49, 1593–1604 Minoo, P. (2005) Dev. Biol. 287, 86–97 11. Bellusci, S., Furuta, Y., Rush, M. G., Henderson, R., Winnier, G., and 31. Hamdan, H., Liu, H., Li, C., Jones, C., Lee, M., deLemos, R., and Minoo, P. Hogan, B. L. (1997) Development 124, 53–63 (1998) Biochim. Biophys. Acta 1396, 336–348 12. Bellusci, S., Grindley, J., Emoto, H., Itoh, N., and Hogan, B. L. (1997) 32. Zhao, Y., and Shah, D. U. (2000) Exp. Mol. Pathol. 69, 67–78 Development 124, 4867–4878 33. Minoo, P., Su, G., Drum, H., Bringas, P., and Kimura, S. (1999) Dev. Biol. 13. Sekine, K., Ohuchi, H., Fujiwara, M., Yamasaki, M., Yoshizawa, T., Sato, 209, 60–71 T., Yagishita, N., Matsui, D., Koga, Y., Itoh, N., and Kato, S. (1999) Nat. 34. Zeng, X., Gray, M., Stahlman, M. T., and Whitsett, J. A. (2001) Dev. Dyn. Genet. 21, 138–141 221, 289–301 14. Min, H., Danilenko, D. M., Scully, S. A., Bolon, B., Ring, B. D., Tarpley, J. E., 35. del Moral, P. M., De Langhe, S. P., Sala, F. G., Veltmaat, J. M., Tefft, D., DeRose, M., and Simonet, W. S. (1998) Genes Dev. 12, 3156–3161 Wang, K., Warburton, D., and Bellusci, S. (2006) Dev. Biol. 293, 77–89 15. Ingham, P. W. (1995) Curr. Opin. Genet. Dev. 5, 492–498 36. Cardoso, W. V., and Lu, J. (2006) Development 133, 1611–1624 16. McMahon, A. P. (2000) Cell 100, 185–188 37. Lebeche, D., Malpel, S., and Cardoso, W. V. (1999) Mech. Dev. 86, 17. Dai, P., Akimaru, H., Tanaka, Y., Maekawa, T., Nakafuku, M., and Ishii, S. 125–136 (1999) J. Biol. Chem. 274, 8143–8152 38. Warzecha, J., Gottig, S., Bruning, C., Lindhorst, E., Arabmothlagh, M., and 18. Grindley, J. C., Bellusci, S., Perkins, D., and Hogan, B. L. (1997) Dev. Biol. Kurth, A. (2006) J. Orthop. Sci. 11, 491–496 188, 337–348 39. Cohen, P., Rajah, R., Rosenbloom, J., and Herrick, D. J. (2000) Am. J. 19. Motoyama, J., Liu, J., Mo, R., Ding, Q., Post, M., and Hui, C. C. (1998) Nat. Physiol. Lung Cell Mol. Physiol. 278, L545–L551 Genet. 20, 54–57 40. Black, P. N., Young, P. G., and Skinner, S. J. (1996) Am. J. Physiol. 271, 20. Pepicelli, C. V., Lewis, P. M., and McMahon, A. P. (1998) Curr. Biol. 8, L910–L917 1083–1086 41. De Langhe, S. P., Carraro, G., Tefft, D., Li, C., Xu, X., Chai, Y., Minoo, P., 21. Xing, Y., Li, C., Hu, L., Tiozzo, C., Li, M., Chai, Y., Bellusci, S., Anderson, S., Hajihosseini, M. K., Drouin, J., Kaartinen, V., and Bellusci, S. (2008) PLoS and Minoo, P. (2008) Dev. Biol. 320, 340–350 ONE 3, e1516 22. Oshima, M., Oshima, H., and Taketo, M. M. (1996) Dev. Biol. 179, 42. Mailleux, A. A., Kelly, R., Veltmaat, J. M., De Langhe, S. P., Zaffran, S., 297–302 Thiery, J. P., and Bellusci, S. (2005) Development 132, 2157–2166 23. Yu, K., Xu, J., Liu, Z., Sosic, D., Shao, J., Olson, E. N., Towler, D. A., and 43. Beer, H. D., Florence, C., Dammeier, J., McGuire, L., Werner, S., and Duan, Ornitz, D. M. (2003) Development 130, 3063–3074 D. R. (1997) Oncogene 15, 2211–2218 24. Soriano, P. (1999) Nat. Genet. 21, 70–71 44. Tomlinson, D. C., Grindley, J. C., and Thomson, A. A. (2004) Endocrinol- 25. Chytil, A., Magnuson, M. A., Wright, C. V., and Moses, H. L. (2002) Gen- ogy 145, 1988–1995 esis 32, 73–75 45. Yoo, Y. A., Kang, M. H., Kim, J. S., and Oh, S. C. (2008) Carcinogenesis 29, 26. Pan, Q., Li, C., Xiao, J., Kimura, S., Rubenstein, J., Puelles, L., and Minoo, P. 480–490 (2004) Gene 331, 73–82 46. Dennler, S., Andre, J., Alexaki, I., Li, A., Magnaldo, T., ten Dijke, P., Wang, 27. Li, C., Xiao, J., Hormi, K., Borok, Z., and Minoo, P. (2002) Dev. Biol. 248, 68–81 X. J., Verrecchia, F., and Mauviel, A. (2007) Cancer Res. 67, 6981–6986 36264 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283 • NUMBER 52 •DECEMBER 26, 2008

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Published: Dec 1, 2008

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