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Tyrosine phosphorylation controls Runx2‐mediated subnuclear targeting of YAP to repress transcription

Tyrosine phosphorylation controls Runx2‐mediated subnuclear targeting of YAP to repress... The EMBO Journal (2004) 23, 790–799 & 2004 European Molecular Biology Organization All Rights Reserved 0261-4189/04 | | THE THE www.embojournal.org EMB EMB EMBO O O JO JOU URN R NAL AL Tyrosine phosphorylation controls Runx2- mediated subnuclear targeting of YAP to repress transcription 1 1 1995a, b; Zeng et al, 1997; Davie and Chadee, 1998; McNeil Sayyed K Zaidi , Andrew J Sullivan , 1 2 et al, 1998; Stenoien et al, 1998; Zeng et al, 1998; Mancini Ricardo Medina , Yoshiaki Ito , Andre J 1 1 1 et al, 1999; DeFranco and Guerrero, 2000; Stenoien et al, van Wijnen , Janet L Stein , Jane B Lian 1, 2000; Zaidi et al, 2001). Proteins that transduce signals to the and Gary S Stein * nucleus contain both nuclear import and export signals, and Department of Cell Biology and Cancer Center, University of their nucleo-cytoplasmic shuttling is controlled by physiolo- Massachusetts Medical School, Worcester, MA, USA, and Institute of gical cues. Within the nucleus, these proteins interact with Molecular and Cell Biology, 30 Medical Drive, Singapore transcription factors and modulate their ability to regulate gene expression (Massague, 1998; Akiyama, 2000; Finidori, Src/Yes tyrosine kinase signaling contributes to the reg- 2000; Ihle, 2001). Our studies focus on the mechanisms by ulation of bone homeostasis and inhibits osteoblast activ- which combinatorial activities of transcription factors and ity. Here we show that the endogenous Yes-associated signaling molecules are integrated at specific subnuclear sites protein (YAP), a mediator of Src/Yes signaling, interacts for physiological control of tissue formation. with the native Runx2 protein, an osteoblast-related tran- Bone formation requires transcriptional mechanisms for scription factor, and suppresses Runx2 transcriptional sequential induction and repression of genes that support activity in a dose-dependent manner. Runx2, through its progressive osteoblast phenotype development. The Runx PY motif, recruits YAP to subnuclear domains in situ and transcription factors and their co-regulators control cellular to the osteocalcin (OC) gene promoter in vivo. Inhibition of differentiation and lineage commitment (Ito, 1999; Src/Yes kinase blocks tyrosine phosphorylation of YAP Westendorf and Hiebert, 1999; Li et al, 2002) by influencing and dissociates endogenous Runx2–YAP complexes. the functional architecture of target gene promoters (Stein Consequently, recruitment of the YAP co-repressor to sub- et al, 2000). Runx proteins are directed to subnuclear do- nuclear domains is abrogated and expression of the mains through the C-terminal NMTS and interact with DNA endogenous OC gene is induced. Our results suggest that through the N-terminal runt homology domain (Ogawa et al, Src/Yes signals are integrated through organization of 1993; Zeng et al, 1997, 1998; Tang et al, 1999; Zaidi et al, Runx2–YAP transcriptional complexes at subnuclear 2001). The Runx2 family member is essential for osteoblast sites to attenuate skeletal gene expression. maturation in vivo and is associated with cleidocranial dys- The EMBO Journal (2004) 23, 790–799. doi:10.1038/ plasia (Komori et al, 1997; Mundlos et al, 1997; Otto et al, sj.emboj.7600073; Published online 12 February 2004 1997). In vivo genetic evidence indicates that interference Subject Categories: chromatin & transcription; signal with subnuclear targeting and associated co-regulatory func- transduction tions of Runx2 can account for this block in bone formation Keywords: Cbfa1; nuclear matrix; osteoblasts; osteocalcin; (Choi et al, 2001). Src signaling Runx2 is a target of several extracellular signals that regulate skeletal formation and homeostasis. The C-terminus of Runx2, which includes the NMTS, interacts with proteins involved in the TGFb/BMP (i.e., Smads), the transducin-like Introduction enhancer (TLE)/groucho and the Src/Yes tyrosine kinase (e.g., the Yes-associated protein, YAP) signaling pathways The spatial distribution of binding sites for transcription (Hanai et al, 1999; Yagi et al, 1999; Zhang and Derynck, factors, regulation of their binding to cognate sites by chro- 1999; Javed et al, 2000; Zaidi et al, 2002). Src family tyrosine matin, and the distribution of regulatory factors and their co- kinases are activated by a variety of extracellular stimuli, regulators in distinct subnuclear domains together contribute and broadly control cell cycle regulation, cell migration, cell to the control of gene expression (Berezney and Jeon, 1995; metabolism and survival, as well as cell proliferation and Stein et al, 2000). Transcription factors possess intrinsic differentiation (Thomas and Brugge, 1997; Schlessinger, nuclear import signals that localize these proteins to the 2000). Src homology (SH) domains present in these kinases nucleus. Importantly, several transcription factors have an mediate interactions with downstream signaling proteins additional protein module, the nuclear matrix-targeting signal such as YAP. YAP, in turn, contains a WW domain in the (NMTS), that directs these factors to subnuclear sites where N-terminus (Sudol, 1994) that recognizes a proline-rich motif activation and repression take place (van Steensel et al, (PPxY) present in a broad range of proteins including Runx *Corresponding author. Department of Cell Biology, 55 Lake Avenue factors (Sudol et al, 1995; Sudol and Hunter, 2000). North, Worcester, MA 01655-0106, USA. Tel.: þ 1 508 856 5625; The significance of Src/Yes tyrosine kinase signaling in Fax: þ 1 508-856-6800; E-mail: [email protected] bone development is suggested by the osteopetrotic pheno- type of Src null mice (Soriano et al, 1991) and the accelerated Received: 20 June 2003; accepted: 12 December 2003; Published differentiation of Src-deficient osteoblasts (Marzia et al, online: 12 February 2004 790 The EMBO Journal VOL 23 NO 4 2004 &2004 European Molecular Biology Organization | | Src/Yes signaling suppresses Runx2 activity in osteoblasts SK Zaidi et al 2000). These results indicate that Src signaling inhibits matrix-intermediate filament (NM-IF) fraction in osseous osteoblast differentiation, but the underlying molecular me- cells that express endogenous Runx proteins (Figure 2B, chanisms are not known. Here we show that Src signaling in bottom right panel); however, it is not detected in the NM- osteoblasts function through YAP to inhibit Runx2 activity. In IF preparations of HeLa cells, which do not express any of the response to Src/Yes signaling, YAP is phosphorylated and three Runx proteins (Armesilla et al, 1996) (Figure 1B, bot- recruited by Runx2 to subnuclear sites and chromatin of the tom left panel). We have previously shown that Runx pro- bone-specific OC gene, resulting in its repression. Thus, teins contain a conserved NMTS that targets Runx to punctate Runx2–YAP interactions integrate Src signals at architectu- subnuclear sites (Zeng et al, 1997, 1998; Tang et al, 1999; rally associated subnuclear sites, where regulatory complexes Zaidi et al, 2001). As our data with nonosseous cells suggest assemble to control osteoblast gene expression. that YAP lacks an intrinsic subnuclear targeting signal, we immunostained NM-IF preparations of ROS 17/2.8 cells for both endogenous YAP and Runx2. As shown in Figure 1C, YAP and Runx2 colocalize at punctate foci in the NM-IF Results preparations of osseous cells. Hence, YAP and Runx2 proteins Endogenous Runx2 and YAP proteins interact in vivo interact in vivo and associate in situ at subnuclear sites in and associate in situ at subnuclear sites in osteoblasts osteoblasts. Importantly, YAP association with the nuclear Direct interaction between YAP and Runx protein segments matrix in osseous but not nonosseous cells may depend on has been documented in a cell-free system (Yagi et al, 1999). the presence of Runx2. We therefore determined whether endogenous Runx2 and YAP interact in osteoblasts. Co-immunoprecipitation assays show that endogenous YAP and Runx2 proteins form a The PY motif of Runx2 is required for targeting of YAP complex in vivo in osteoblastic ROS 17/2.8 cells (Figure 1A). to subnuclear domains in situ and its recruitment These findings indicate that Runx2 and YAP interact under to chromatin in vivo physiological conditions. The absence of YAP from the nuclear matrix of nonosseous YAP interacts with Src family kinases at the plasma mem- cells suggests that the interaction of YAP with Runx2 is brane, with 14-3-3 proteins in the cytoplasm and with tran- required for its subnuclear targeting. Therefore, we intro- scription factors in the nucleus (Mohler et al, 1999; Yagi et al, duced a point mutation into the PY motif of Runx2 (Runx2 1999; Strano et al, 2001; Vassilev et al, 2001; Basu et al, 2003). Y433A; Figure 2A, top panel), a mutation that has been We therefore assessed the subcellular localization of YAP in reported to disrupt Runx2–YAP interaction in vitro (Yagi osseous (ROS 17/2.8) and nonosseous (HeLa) cells. et al, 1999). As seen for endogenous proteins (Figure 1), Endogenous YAP is present in the cytoplasm, but is predo- exogenously expressed YAP and Runx2 form precipitable minantly nuclear in both HeLa and ROS 17/2.8 cells complexes in osseous cells (Figure 2A, bottom panel), but (Figure 1B, top panels). YAP associates with the nuclear the Y433A mutation abrogates this interaction (Figure 2A). Figure 1 Endogenous YAP and Runx2 proteins interact in vivo and co-localize in situ in osseous cells. (A) Endogenous Runx2 was immunoprecipitated from ROS 17/2.8 cells with a rabbit polyclonal antibody (1:2000) raised against the Runx2 C-terminus (Zhang et al, 2000). A rabbit polyclonal antibody was used to detect endogenous YAP (top panel). Normal goat IgG was used as a control. The middle panel shows efficient immunoprecipitation of endogenous Runx2. The bottom panel shows the expression of endogenous YAP in ROS 17/2.8 cells (20% of total input). (B) In situ immunofluorescence of whole cell (WC) and nuclear matrix-intermediate filament (NM-IF) preparations was performed to assess the nucleo-cytoplasmic distribution and subnuclear localization of endogenous YAP in nonosseous (HeLa) and osseous (ROS 17/2.8) cells. YAP is predominantly nuclear in both HeLa and ROS 17/2.8 cells (top panels), but is only associated with the nuclear matrix ROS 17/2.8 cells (bottom panels). (C) Same as (B), using deconvoluted images. The merged image reveals that endogenous YAP resides in Runx2 containing subnuclear foci in ROS 17/2.8 cells (bar¼ 10 mm). &2004 European Molecular Biology Organization The EMBO Journal VOL 23 NO 4 2004 791 | | Src/Yes signaling suppresses Runx2 activity in osteoblasts SK Zaidi et al Figure 2 The PY motif of Runx2 is required for interaction with YAP and its recruitment to subnuclear sites as well as target gene promoters in vivo.(A) ROS 17/2.8 cells were cotransfected with Xpress-YAP- and HA-tagged wild-type or the Y433A mutant of Runx2 (numbering according to the mouse MASNS/Runx2 isoform). After 24 h of transfection, proteins were precipitated using a monoclonal antibody against the HA tag (2 mg) and separated by SDS–PAGE. A mouse monoclonal antibody (1:5000) was used to detect XPR-YAP. The blots were stripped and were incubated with HA antibody (1:3000) to assess the expression of Runx2 (wild type or mutant) proteins or with XPR antibody to assess the expression of exogenous YAP. (B) HeLa cells, transfected with XPR-YAP (0.5 mg), were processed for WC and NM-IF preparations. YAP is present both in the cytoplasm and nucleus (right panel), but neither exogenous nor endogenous YAP is found in the nuclear matrix of HeLa cells (left panel). (C) Runx2 does not alter the subcellular localization of YAP in HeLa cells cotransfected with XPR-YAP and HA-Runx2 (wild type and Y433A) (WC, top panels). YAP is associated with the nuclear matrix when co-expressed with wild-type Runx2 (middle panel), but not if the interaction of Runx2 with YAP is disrupted (Y433A mutant, bottom panel) (bar¼ 10 mm). (D) Chromatin immunoprecipitation assay was performed using ROS 17/2.8 cells transfected with indicated tagged expression constructs. Purified immunoprecipitated DNA was amplified with primers spanning the Runx-binding sites B and C in rat osteocalcin promoter as described. OC-derived DNA was observed in chromatin immunoprecipitations with the HA and Xpress antibodies from cells expressing both HA-tagged Runx2 and Xpress-tagged YAP after 21 cycles of PCR amplification. Control lanes () from untransfected cells show that antibodies against HA or XPR tags do not nonspecifically precipitate chromatin (lanes 2 and 3). A low level of OC-derived DNA was detected from cells expressing YAP alone after 25 cycles of PCR amplification due to endogenous Runx2 (data not shown). The unrelated myogenin promoter does not exhibit signals (bottom panel). Thus, the interaction of YAP with Runx2 inside the cell complex to subnuclear domains requires an intact PY motif requires the PY motif of Runx2. and the functional NMTS of Runx2. As YAP lacks an intrinsic subnuclear targeting signal, it As YAP lacks DNA-binding activity, we postulated that may require Runx2 for subnuclear trafficking in osseous cells Runx2 may recruit YAP to promoters of target genes. (Figure 1B). We directly tested whether Runx2 mediates YAP Chromatin immunoprecipitation (ChIP) assays were per- intranuclear targeting. Runx2 (wild-type or the Y433A mu- formed to assess the in vivo association of YAP with the tant) and YAP were expressed in nonosseous cells that lack osteocalcin promoter. YAP alone is unable to bind to the endogenous Runx proteins, and their subnuclear localization promoter region of the OC gene (Figure 2D). However, when was assessed using immunofluorescence microscopy (Figure co-expressed with Runx2, YAP is specifically recruited to the 2B and C). Wild-type Runx2 and the Y433A mutant are OC promoter (see control lanes in upper panel) and not to the exclusively nuclear and exhibit a punctate subnuclear dis- myogenin gene promoter, which is not a Runx-responsive tribution (Figure 2C, left panels). Exogenously expressed YAP gene (Figure 2D, bottom panel). Hence, the interaction with is present in both the cytoplasm and the nucleus, and is not Runx2 results in targeting of YAP to subnuclear domains detected in nuclear matrix preparations (Figure 2B; see also in situ and its recruitment to a target gene promoter in vivo. Figure 1). When co-expressed with wild-type Runx2, YAP shows a discrete subnuclear distribution and the two proteins YAP suppression of Runx2-mediated osteocalcin are colocalized (Figure 2C, middle panels). However, co- activation is relieved by a dominant-negative (DN) expression of the Runx2 Y433A mutant with YAP fails to inhibitor of Src tyrosine kinase target YAP to the nuclear matrix (Figure 2C, bottom panels). The functional consequences of the Runx2–YAP interaction at Similarly, mutations that compromise subnuclear targeting of subnuclear sites were examined by monitoring YAP- and Runx2 also fail to target YAP to the nuclear matrix (Zaidi et al, Runx2-mediated OC promoter activity. Runx2 activates the 2002). Although Runx2 recruits YAP to nuclear matrix-asso- OC promoter from three- to four-fold in nonosseous cells, ciated subnuclear foci, it does not alter the nucleo-cytoplas- while YAP alone has no effect on the basal promoter activity mic distribution of YAP (Figure 2C, top panels). Collectively, (Figure 3A). YAP suppresses Runx2-enhanced OC promoter these results demonstrate that while the nuclear import of activity in a dose-dependent manner (Figure 3A, center YAP is independent of Runx2, targeting of the Runx2–YAP lanes). Strikingly, expression of the Runx2 Y433A mutant 792 The EMBO Journal VOL 23 NO 4 2004 &2004 European Molecular Biology Organization | | Src/Yes signaling suppresses Runx2 activity in osteoblasts SK Zaidi et al Figure 3 YAP suppresses Runx2-mediated activation of the rat osteocalcin promoter. (A) The rOC 208 CAT reporter was transfected in HeLa cells along with the indicated Runx2 constructs (250 ng) and increasing concentrations (0 (first bar of each group), 250, 500 ng and 1 mg) of full- length YAP. Expression of YAP alone does not affect basal promoter activity. Runx2 activates osteocalcin promoter activity, which is suppressed in a dose-dependent manner by increasing concentrations of YAP. The Runx2 Y433A mutant, that does not interact with YAP, shows a 3–4-fold higher activity compared to wild-type Runx2. (B) YAP suppresses the activity of Runx2 in osteoblastic ROS 17/2.8 cells. In contrast to HeLa cells, wild-type YAP slightly enhances basal promoter activity (first group), and the Runx2 Y433A mutant moderately activates rOC 208 due to endogenous Runx2 in ROS 17/2.8 cells. The graphs represent at least three independent experiments (n¼ 6 each); error bars¼ standard error of mean. (C) The rOC 208/CAT reporter was transfected with 250 ng each of Runx2 and YAP. Runx2 activates osteocalcin promoter activity, which is suppressed by YAP in osteoblastic MC3T3 cells, premyoblast C2C12 cells and fibroblast NIH3T3 cells. The Runx2 Y433A mutant which does not interact with YAP shows higher activity compared to wild-type Runx2 in C2C12 and NIH 3T3 cells. This effect, however, is not apparent in MC3T3 cells, probably because of higher levels of endogenous Runx2 protein. (D) Runx2–YAP complex regulates the activity of multiple Runx target promoters. ROS 17/2.8 cells were transfected with the indicated reporter and expression constructs. Cells were harvested 30 h after the transfection and subjected to luciferase reporter assay. The luciferase activity is expressed as fold activation (6  OSE-Luc and TGFbRI-Luc promoter) or fold suppression (in case of Runx2-Luc and p21-Luc promoters). The open squares in promoter constructs represent functional Runx sites. that does not interact with YAP dramatically increases activa- exogenously expressed YAP suppresses Runx2-mediated OC tion of the OC promoter (15-fold) compared to wild-type gene transcription on the native OC gene promoter (Figure 3), Runx2 (Figure 3A, last group). As expected, increasing con- suggesting that the activity of YAP depends on the promoter centrations of YAP do not alter the activity of the Y433A context. mutant, which cannot bind YAP (Figure 2A). Thus, disruption We tested whether the activity of the Runx2–YAP complex of the PY motif allows Runx2 to function unabated by the is promoter context dependent, by examining the regulation presence of endogenous YAP in HeLa cells. In ROS 17/2.8 of various Runx target gene promoters by the Runx2–YAP osteoblastic cells, exogenous YAP expression slightly in- complex. We used luciferase reporters driven by multimer- creases OC promoter activity and suppresses Runx2-mediated ized Runx-binding sites (6X OSE-Luc; activated by Runx2), OC activation (Figure 3B). In addition, the Runx2 Y433A the TGFb receptor type I promoter (TGFbRI-Luc; activated by mutant shows a relatively moderate activation of the OC Runx2), the Runx2 promoter (Runx2-Luc; suppressed by promoter in ROS 17/2.8 compared to HeLa cells, presumably Runx2) and the p21 promoter (p21-Luc; suppressed by due to the presence of endogenous Runx2. Similar results Runx2). Our results show that the co-expression of YAP were obtained in mouse osteoblastic MC3T3 cells, premyo- does not alter Runx2 activity on the 6X OSE-Luc; however, blast C2C12 cells and NIH3T3 fibroblasts (Figure 3C), de- it moderately suppresses Runx2 activity on a natural TGFbRI monstrating that the activity of the Runx2–YAP complex is promoter (Figure 3D, upper panels). YAP co-expression re- independent of cell type. Interestingly, when YAP is expressed sults in increased suppression of the Runx2 promoter by as a Gal4 fusion protein in a heterologous Gal4 system, we Runx2, but does not affect Runx2 suppression of the p21 observe strong transcriptional activation, as has been pre- promoter (Figure 3D, bottom panels), which is known to viously reported (data not shown; Yagi et al, 1999]. However, involve HDAC6 (Westendorf et al, 2002). We conclude that &2004 European Molecular Biology Organization The EMBO Journal VOL 23 NO 4 2004 793 | | Src/Yes signaling suppresses Runx2 activity in osteoblasts SK Zaidi et al Figure 5 Activated Src family kinases contribute to the YAP–Runx2 Figure 4 A DN inhibitor of Src tyrosine kinase relieves YAP- interaction. (A) ROS 17/2.8 cells, transiently transfected with XPR- mediated suppression of Runx2 activity. ROS 17/2.8 cells were YAP and antibodies, were used to immunoprecipitate endogenous transfected with 1 mg rOC 208/CAT reporter along with 250 ng of Yes or Src tyrosine kinases. Association of YAP with these kinases Runx2 and/or YAP expression constructs. Two different concentra- was assessed by western blotting using HRP-conjugated anti-XPR tions of Src DN construct (200 and 800 ng) were used (bars 2 and 3, antibody. (B) ROS 17/2.8 cells, expressing indicated constructs, respectively, of each group). Cells were harvested 24–30 h after were treated with 5 mM of PP2 for 1 h and processed for immuno- transfection and were subjected to CAT reporter assay. The graph precipitation. The Runx2–YAP interaction is completely abrogated represents results obtained from three independent experiments in the presence of PP2 (panel 1). Panel 2 shows the efficiency (n¼ 6 each). of Runx2 immunoprecipitation, while panel 3 shows comparable levels of YAP overexpression in all lanes. (C) As in (B), but antibodies were used to immunoprecipitate endogenous Runx2. The effect of Src DN on the YAP–Runx2 interaction was assessed the activity of the Runx2–YAP complex is promoter context by western blotting using the HRP-conjugated anti-XPR antibody dependent. (panel 1). A mouse monoclonal antibody against Runx2 was used to assess the efficiency of immunoprecipitation of the endogenous YAP is a substrate of the Src/Yes tyrosine kinases (Sudol, Runx2 protein (panel 2). Expression of Src DN and XPR-YAP was 1994). To investigate whether YAP-mediated suppression of confirmed with Src and HRP-Xpress antibodies, respectively (panels Runx2 activity involves Src signaling, we used a DN inhibitor 3 and 4). In each immunoprecipitation, appropriate normal IgG was of Src tyrosine kinase (Src K295R/Y527F or Src DN), which is used as a control. kinase inactive (Mukhopadhyay et al, 1995). This variant constitutively binds to cellular proteins that interact with activated Src under physiological conditions. Expression of associated with both Yes and Src tyrosine kinases in osteo- Src DN increases the basal activity of the osteocalcin gene blasts (Figure 5A). We therefore assessed whether the sup- promoter in ROS 17/2.8 cells in a dose-dependent manner pressor function of YAP on the OC promoter may involve Src/ (Figure 4). The Src DN has a similar effect when co-expressed Yes signaling using the inhibitor PP2 (Hanke et al, 1996). PP2 with Runx2 in nonosseous cells such as HeLa, which express is a broad inhibitor of the Src tyrosine kinase family, and has high endogenous levels of YAP and Src family members (data been reported to inhibit the activity of at least four Src family not shown). This increased basal OC transcription in the members, including Src, Yes, Fyn and Hck (Hanke et al, presence of the Src DN may result from inhibition of interac- 1996). Endogenous Runx2 was immunoprecipitated from tion between endogenous YAP and Src proteins. In addition, untreated (control) or PP2-treated (5 mM for 1 h) ROS 17/2.8 YAP-mediated suppression of Runx2 activity is relieved in the cells expressing XPR-YAP. Runx2 is associated with exogen- presence of the Src DN (Figure 4). Thus, YAP strongly blocks ously expressed YAP in control cells (Figure 5B, lane 3), but Runx2-mediated transcriptional activation of OC, and this not in the presence of PP2 (Figure 5B, lane 4). To address suppression may involve Src tyrosine kinase activity. We more specifically the involvement of Src tyrosine kinase in propose that the PY motif of Runx2 is an essential protein regulating interaction between Runx2 and YAP, we examined module that mediates the suppression of OC promoter activ- the Runx2–YAP interaction in the presence of Src DN (Src ity in response to Src signaling. K295R/Y527F). Forced expression of Src DN significantly reduces (2–3-fold) interaction between Runx2 and YAP (com- pare lanes 3 and 4; Figure 5C); residual interactions between Inhibition of the Src tyrosine kinase family abrogates Runx2 and YAP may occur through signaling pathways Runx2–YAP interaction and induces expression involving other Src family members. Taken together, the of the endogenous osteocalcin gene findings with PP2 and Src DN indicate that the Src family As YAP is an in vitro substrate of the Src/Yes tyrosine kinases tyrosine kinase signaling regulates the Runx2–YAP interac- (Sudol, 1994), we examined whether YAP interacts with Yes tion. and Src tyrosine kinases in vivo in osteoblasts. Endogenous Elevated osteocalcin mRNA in Src-deficient osteoblasts Src or Yes proteins were immunoprecipitated from ROS (Marzia et al, 2000) suggests an inhibitory role for Src 17/2.8 cells expressing XPR-YAP. We find that YAP is indeed 794 The EMBO Journal VOL 23 NO 4 2004 &2004 European Molecular Biology Organization | | Src/Yes signaling suppresses Runx2 activity in osteoblasts SK Zaidi et al Figure 6 Inhibition of endogenous Src signaling or YAP activity induces endogenous osteocalcin gene transcription. (A) Total cel- lular RNA from ROS 17/2.8 cells was analyzed by northern blotting and treated with indicated concentrations of PP2 for 1 h. Endogenous osteocalcin gene transcription is induced in a dose- dependent manner when the activity of Src family members is inhibited by PP2 (upper panel). 18S RNA was used as a loading control (bottom panel). (B) Activity of Src tyrosine kinase or YAP Figure 7 Tyrosine phosphorylation of YAP regulates its interaction was selectively inhibited by overexpressing DN inhibitors (Src DN with Runx2 and subsequent subnuclear trafficking. (A) HeLa cells, for Src tyrosine kinase and YAP DN for YAP). Total cellular RNA was co-expressing YAP and Runx2, were transfected with Src DN or subjected to northern blot analysis from ROS 17/2.8 cells over- treated with PP2 (5 mM) for 1 h and WC or NM-IF preparations and expressing the indicated plasmids. As shown in the top panel, the in situ immunofluorescence microscopy. Inhibition of either Src DN inhibitor of Src (lane 2) or YAP (lane 4) induces endogenous tyrosine kinase (þ Src DN) or its family members (þ PP2) does not osteocalcin gene transcription. (C) Densitometric analysis reveals alter subcellular localization of YAP. Subnuclear trafficking of YAP is the extent to which endogenous osteocalcin is induced under each severely compromised when the kinase activity of Src alone or its experimental condition analyzed. (The bar graph represents the family members is inhibited. (B) To directly assess the effects of Src ratio of densitometric units of osteocalcin and 18S transcripts.) DN or PP2 on tyrosine phosphorylation of endogenous YAP and its interaction with native Runx2, endogenous YAP was immunopreci- pitated from ROS 17/2.8 cells. The immunoprecipitates were re- solved by SDS–PAGE and subjected to western blot analysis. The tyrosine kinase in OC gene expression. We therefore ad- tyrosine phosphorylation of YAP was determined by a mouse monoclonal phospho-specific antibody (Py; 1:2000) raised against dressed the involvement of the Src-YAP signaling pathway phospho-tyrosine (top panel). The blot was stripped and re-probed in the regulation of the endogenous osteocalcin gene in with a mouse monoclonal antibody against Runx2 to assess the osteoblast-like ROS 17/2.8 cells using PP2 and Src DN, as presence of endogenous Runx2 in the immunoprecipitates (middle well as a previously characterized DN inhibitor of YAP (Yagi panel). The efficiency of YAP immunoprecipitation was assessed by a YAP rabbit polyclonal antibody (bottom panel). et al, 1999). Inhibition of Src family members with various concentrations of PP2 results in a dose-dependent increase in endogenous OC expression as measured by mRNA levels (Figure 6A; quantitated in Figure 6C). More importantly, kinase activity by the Src DN or PP2 does not alter the nucleo- endogenous OC gene expression was also upregulated 2–3- cytoplasmic distribution of YAP. However, Runx2-mediated fold when the activity of endogenous Src tyrosine kinase subnuclear targeting of YAP is severely compromised in the (Figure 6B, Src DN lanes) or YAP (Figure 6B, YAP DN lanes; presence of the Src DN, that is, only 20–25% of cells in the also see Figure 6C for quantitation) was blocked. Our results NM-IF preparation are positive for YAP signal (Figure 7A, demonstrate that YAP suppresses Runx2-mediated expression middle panel). As YAP and Runx2 do not interact in the of the endogenous OC gene in response to Src tyrosine presence of PP2 (Figure 5B), YAP is absent in the NM-IF kinases. preparation of PP2-treated cells, even though Runx2 associa- tion with the nuclear matrix is unaltered (Figure 7A, right panel and data not shown). Thus, subnuclear targeting, but Tyrosine phosphorylation of YAP in response to the not nucleo-cytoplasmic distribution, of YAP requires acti- activity of Src family members regulates its interaction with Runx2 and subsequent subnuclear targeting vated Src tyrosine kinases. The molecular mechanism(s) underlying Src-YAP-mediated To further explore the role of Src signaling in the Runx2– suppression of Runx2 activity on the endogenous OC promo- YAP interaction and control of OC gene expression, we assessed the tyrosine phosphorylation status of endogenous ter was further examined by in situ immunofluorescence YAP in osteoblasts upon inhibition of Src signaling. microscopy of nonosseous cells co-expressing Runx2 and Expression of Src DN significantly decreases tyrosine YAP in the presence of PP2 or Src DN. Inhibition of tyrosine &2004 European Molecular Biology Organization The EMBO Journal VOL 23 NO 4 2004 795 | | Src/Yes signaling suppresses Runx2 activity in osteoblasts SK Zaidi et al phosphorylation of YAP (Figure 7B, upper panel) and reduces examined previously (Yagi et al, 1999). Our studies on the its interaction with the native Runx2 protein (Figure 7B, native OC gene promoter establish that YAP suppresses middle panel). In addition, treatment of cells with PP2 Runx2-dependent transcriptional activation. Of interest, a abrogates YAP tyrosine phosphorylation and its interaction multimerized Runx2 element does not respond to YAP repres- with Runx2 (Figure 7B, right lanes). Taken together, our sion, supporting the concept that the formation of multimeric results indicate that Src-related tyrosine phosphorylation of regulatory complexes at Runx sites in native promoters is YAP is required for its interaction with Runx2, and its target- influenced by surrounding sequences (Canon and Banerjee, ing to Runx subnuclear sites. 2003). As Runx2–YAP may be part of a larger regulatory complex, we propose that the functional outcome of YAP activity depends on specific protein–protein interactions that Discussion are promoter context dependent, as is the case for other co- In this study, we have shown that YAP, a downstream target regulators (Lemon and Tjian, 2000). of Src tyrosine kinases, functions to suppress the activity of Runx2, a transcription factor required for osteoblast matura- Regulatory aspects of Runx2-mediated recruitment of tion. We find that YAP interacts with both Src and Yes YAP to subnuclear domains and the OC gene promoter tyrosine kinases in osteoblasts. Tyrosine phosphorylation of Runx transcription factors play a pivotal role in the main- endogenous YAP promotes its interaction with Runx2. Runx2 tenance of different levels of nuclear organization (Stein et al, then recruits YAP to the bone-specific osteocalcin promoter 2000, 2003). Intact Runx-binding sites are required for tran- and to subnuclear sites where Runx2 resides to suppress scription, steroid hormone responsiveness and for the main- osteocalcin promoter activity. Interference with the Src- tenance of open chromatin structure of the rat OC gene YAP–Runx2 pathway at any level results in inhibition of promoter (Javed et al, 1999). In addition, Runx factors YAP tyrosine phosphorylation, disruption of the Runx2–YAP possess a specific intranuclear targeting signal (the NMTS) interaction, block in recruitment of YAP to subnuclear sites that directs these factors to distinct subnuclear domains and and induction of osteocalcin gene expression. We conclude supports transcriptional control of target genes (Zeng et al, that Runx2–YAP interactions are obligatory for integration of 1997, 1998; Tang et al, 1999; Stein et al, 2000; Choi et al, Src signals at architecturally associated sites where regulatory 2001; Zaidi et al, 2001). The NMTS overlaps with interacting complexes assemble to control osteoblast gene expression. domains of several coregulators of Runx function, including YAP and Smads (Hanai et al, 1999; Yagi et al, 1999). Smads, Physiological significance of Src/Yes signaling to Runx2 which transduce BMP signals, require the subnuclear target- activity ing signal of Runx transcription factors to assemble as distinct Negative regulation of Runx2 by Src/Yes kinases may be subnuclear complexes at sites of active transcription to important for maintenance of bone homeostasis. Bone remo- regulate gene expression (Zaidi et al, 2002). Here, we show deling is a dynamic process that requires resorption and that YAP must bind to Runx2 to associate with nuclear formation of bone tissue to ensure the continuous renewal substructures and the promoter regions of target genes. of bone throughout life. This process is regulated by the Taken together, these studies suggest that Runx2 functions physiological balance between the activities of bone-forming as a multifunctional organizer of regulatory complexes at osteoblasts and bone-resorbing osteoclasts in response to subnuclear sites. several physiological cues. Like vitamin D and bone morpho- Our studies also suggest differences in the importance of genetic proteins (BMPs), Src/Yes signaling coordinates both serine and tyrosine phosphorylation in defining subcellular osteoblast and osteoclast activities for bone turnover and subnuclear distribution of YAP. The Akt-kinase-mediated (Katagiri et al, 1990; Soriano et al, 1991; Boyce et al, 1992; serine phosphorylation of YAP disrupts its interaction with Horne et al, 1992; Suda et al, 1992; Van Leeuwen et al, 2001). 14-3-3 proteins, thus resulting in cytosolic localization of YAP The molecular mechanisms involving Src/Yes tyrosine ki- (Basu et al, 2003). We find that tyrosine phosphorylation of nases in osteoclast differentiation are well studied, but a YAP in response to Src/Yes signals does not alter its nucleo- role for this pathway and its downstream signaling molecules cytoplasmic distribution, but is required for interaction of such as YAP in osteoblasts has only recently been suggested. YAP with Runx2 and its consequent subnuclear trafficking. Osteoblasts isolated from Src mice exhibit accelerated Taken together, these observations support a novel mechan- differentiation and elevated levels of OC mRNA and other ism by which regulatory complexes spatially and temporally osteoblast markers, suggesting that c-Src negativity regulates assemble within the nucleus in response to extracellular osteoblast maturation (Thomas and Lafage-Proust, 1999; signals, leading to activation or suppression of target genes. Marzia et al, 2000). We find that several osteoblast-related We propose that the dynamic presence of coregulatory pro- genes are regulated by the Runx2–YAP complex. Thus, YAP teins in Runx subnuclear domains provides a mechanism for modulation of Runx2-mediated gene expression provides a positive and negative transcriptional control of Runx-depen- molecular mechanism for Src signaling in the control of dent promoters within the three-dimensional context of nu- osteoblast differentiation. clear architecture in response to physiological signals to Our studies provide evidence for the novel concept that ensure proper bone formation. YAP can function as a suppressor of gene activation. The carboxy-terminus of YAP contains a strong activation domain Materials and methods as assessed by heterologous gene transcription assays (Yagi et al, 1999; Strano et al, 2001; Vassilev et al, 2001), and YAP Reporter assays enhances the Runx-mediated activation of the IgaC promoter. Rat osteosarcoma ROS 17/2.8 and human cervical carcinoma HeLa However, the effect of YAP on the OC promoter has not been cells were plated at a density of 0.0410 cells/ml. Transient 796 The EMBO Journal VOL 23 NO 4 2004 &2004 European Molecular Biology Organization | | Src/Yes signaling suppresses Runx2 activity in osteoblasts SK Zaidi et al transfections were initiated 24 h later using either Superfect (Qiagen Proteins were separated on 10% SDS–polyacrylamide gel and Inc., Valencia, CA, USA) or Fugene (Roche Applied Science, processed for western blotting using appropriate horseradish Indianapolis, IN, USA) with the indicated expression constructs peroxidase (HRP)-conjugated secondary antibodies (1:2000). The and 1 mgof 208 OC-CAT (chloramphenicol acetyl transferase), signal was visualized using the enhanced chemiluminescence kit spanning one Runx-binding site (Site C) of the rat osteocalcin (Amersham Pharmacia Biotech Inc., Piscataway, NJ, USA) accord- promoter. RSV-Luc was included as a control for transfection ing to the manufacturer’s instructions. efficiency. After 24 h, cells were processed for CAT assay and quantitated using a Storm PhosphorImager (Molecular Dynamics, Chromatin immunoprecipitation Sunnyvale, CA, USA) as described (Javed et al, 1999). Luciferase ROS 17/2.8 cells transfected with HA-Runx2 and/or XPR-YAP by activity was assessed with a Luciferase Assay Kit (Promega, Fugene transfection reagent were crosslinked with 1.1% formalde- Madison, WI, USA) in the same cell lysate and used to normalize hyde in 1 PBS at room temperature for 10 min. Cells were scraped the CAT values. Graphs are representative of three independent in 500 ml of lysis buffer (150 mM NaCl, 50 mM Tris–HCl (pH 8.0), TM experiments (n¼ 6 in each experiment) with different DNA 1% NP-40, 25 mM MG132, 1  Complete protease inhibitor preparations. cocktail). The cell lysate was then sonicated to generate 500– 1000 bp long fragments. Cell lysate was microfuged at 14 000 r.p.m. Expression constructs for 15 min at 41C and the supernatant was precleared with 40 ml The following constructs have been previously reported: HA-Runx2 protein A/G plus agarose beads, 2 mg of sheared salmon sperm DNA (Zaidi et al, 2001), Xpress (XPR)-tagged full-length YAP (1–472) and and 2 mg appropriate normal IgG for 1 h at 20 r.p.m. The precleared its DN inhibitor (1–301; YAP DN hereafter) (Yagi et al, 1999), and rat supernatant was incubated with 2 mg antibody for 1 h at 41C with osteocalcin with one Runx-binding site (rOC 208-CAT; Heinrichs rotation. The immuno-complexes were collected by adding 40 mlof et al, 1993). The Y433A mutant of Runx2 was generated by PCR- protein A/G plus agarose beads and rotating at 41C at 30 r.p.m. for based site-directed mutagenesis. Src DN (Src K295R Y527F) was another 1 h, following centrifugation by extensive washing in purchased from Upstate Biotechnology (Lake Placid, NY, USA; multiple buffers as described in Shen et al (2002). Upon reversal of Mukhopadhyay et al, 1995). crosslinking, DNA was purified using the QIAGEN PCR Purification kit (Qiagen Inc., Valencia, CA, USA), stored in 50 ml, and 1 mlwas In situ immunofluorescence and digital microscopy used for PCR. The following primer sets were used to amplify HeLa or ROS 17/2.8 cells (untransfected or transfected with 0.5 mg promoter regions: OC promoter 5 -GGT GAA TGA GGA CAT TAC of indicated expression constructs) were extracted for in situ TGA CCG CTCC and 5 -CCA AAG GAT GCT GTG GTT GGT GAC, and 0 0 0 nuclear matrix preparation 24 h after transfection and were myogenin promoter 5 -ACC CCT TTC TTG TTC CCT TCC and 5 - processed for digital microscopy as described before (Javed et al, CTC CCG CAG CCC CTC AC. The PCR program used was: 2 min 2000). HA-Runx2 was detected by a rabbit polyclonal antibody 951C denaturing step, then 30 s 951C, 30 s 601C, 45 s 721C for 21 against HA-tag at a dilution of 1:3000 (Santa Cruz Biotechnology cycles, and finally 5 min 721C elongation step. Inc., Santa Cruz, CA, USA). XPR-YAP was detected with mouse monoclonal antibodies at a dilution of 1:1000. Rabbit polyclonal Northern blotting antibodies raised against Runx2 (Oncogene Research Products, La Total cellular RNA was isolated by the Trizol method (Invitrogen Jolla, CA, USA) or YAP (Santa Cruz Biotechnology Inc., Santa Cruz, Corporation, Carlsbad, CA, USA) from ROS 17/2.8 cells (untreated CA, USA) were used to detect endogenous proteins at a dilution of or treated for 1 h with PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl) 1:200. The secondary antibodies used were either Alexa 488 anti- pyrazolo [3,4-d] pyrimidine); Calbiochem-Novabiochem Corp., La rabbit or Alexa 568 anti-mouse (Molecular Probes, Eugene, OR, Jolla, CA, USA). The probes used to detect OC mRNA and 18S rRNA USA) at a dilution of 1:800. have been described (Gutierrez et al, 2002). To determine the role of Src tyrosine kinase and YAP, ROS 17/2.8 cells were transfected with Co-immunoprecipitation and western blotting 10 mg of the Src DN or the YAP DN 36 h prior to RNA isolation using ROS 17/2.8 cells were transfected with expression constructs for Fugene transfection reagent. HA-tagged wild-type or mutant Runx2 proteins and XPR-YAP. Cells were prepared 36 h after transfection in a buffer containing 20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 0.5% Triton X-100, 1% aprotinin Acknowledgements and 1 mM phenylmethylsulfonyl fluoride (PMSF). Lysates were incubated with specific antibodies for 1 h, followed by incubation This work was supported by NIH grants DE12528, POI AR48818 and with protein A/G plus-sepharose beads. Immunoprecipitates were AR39588 POI CA82834. The contents of this manuscript are solely boiled in the SDS sample buffer (100 mM Tris–HCl (pH 6.8), 5% the responsibility of the authors and do not necessarily represent glycerol, 2% SDS, 100 mM DTTand 0.002% bromophenol blue) and the official views of the National Institutes of Health. We thank Judy subjected to western blot analysis. The following antibodies (2 mgin Rask and Karen Concaugh for editorial assistance, Daniel Young for each case) were used for immunoprecipitation: rabbit polyclonal helpful comments on the manuscript and Dr William Horne antibodies against Runx2, YAP and Src tyrosine kinase, and mouse (Department of Cell Biology, Yale University School of Medicine, monoclonal antibody against Yes tyrosine kinase (Santa Cruz New Haven, CT, USA) for providing reagents and for critical Biotechnology, Santa Cruz, CA, USA). suggestions. References Akiyama T (2000) Wnt/beta-catenin signaling. 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Proc Natl Acad Sci USA 97: (2002) Integration of Runx and Smad regulatory signals at 10549–10554 &2004 European Molecular Biology Organization The EMBO Journal VOL 23 NO 4 2004 799 | | http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The EMBO Journal Springer Journals

Tyrosine phosphorylation controls Runx2‐mediated subnuclear targeting of YAP to repress transcription

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Abstract

The EMBO Journal (2004) 23, 790–799 & 2004 European Molecular Biology Organization All Rights Reserved 0261-4189/04 | | THE THE www.embojournal.org EMB EMB EMBO O O JO JOU URN R NAL AL Tyrosine phosphorylation controls Runx2- mediated subnuclear targeting of YAP to repress transcription 1 1 1995a, b; Zeng et al, 1997; Davie and Chadee, 1998; McNeil Sayyed K Zaidi , Andrew J Sullivan , 1 2 et al, 1998; Stenoien et al, 1998; Zeng et al, 1998; Mancini Ricardo Medina , Yoshiaki Ito , Andre J 1 1 1 et al, 1999; DeFranco and Guerrero, 2000; Stenoien et al, van Wijnen , Janet L Stein , Jane B Lian 1, 2000; Zaidi et al, 2001). Proteins that transduce signals to the and Gary S Stein * nucleus contain both nuclear import and export signals, and Department of Cell Biology and Cancer Center, University of their nucleo-cytoplasmic shuttling is controlled by physiolo- Massachusetts Medical School, Worcester, MA, USA, and Institute of gical cues. Within the nucleus, these proteins interact with Molecular and Cell Biology, 30 Medical Drive, Singapore transcription factors and modulate their ability to regulate gene expression (Massague, 1998; Akiyama, 2000; Finidori, Src/Yes tyrosine kinase signaling contributes to the reg- 2000; Ihle, 2001). Our studies focus on the mechanisms by ulation of bone homeostasis and inhibits osteoblast activ- which combinatorial activities of transcription factors and ity. Here we show that the endogenous Yes-associated signaling molecules are integrated at specific subnuclear sites protein (YAP), a mediator of Src/Yes signaling, interacts for physiological control of tissue formation. with the native Runx2 protein, an osteoblast-related tran- Bone formation requires transcriptional mechanisms for scription factor, and suppresses Runx2 transcriptional sequential induction and repression of genes that support activity in a dose-dependent manner. Runx2, through its progressive osteoblast phenotype development. The Runx PY motif, recruits YAP to subnuclear domains in situ and transcription factors and their co-regulators control cellular to the osteocalcin (OC) gene promoter in vivo. Inhibition of differentiation and lineage commitment (Ito, 1999; Src/Yes kinase blocks tyrosine phosphorylation of YAP Westendorf and Hiebert, 1999; Li et al, 2002) by influencing and dissociates endogenous Runx2–YAP complexes. the functional architecture of target gene promoters (Stein Consequently, recruitment of the YAP co-repressor to sub- et al, 2000). Runx proteins are directed to subnuclear do- nuclear domains is abrogated and expression of the mains through the C-terminal NMTS and interact with DNA endogenous OC gene is induced. Our results suggest that through the N-terminal runt homology domain (Ogawa et al, Src/Yes signals are integrated through organization of 1993; Zeng et al, 1997, 1998; Tang et al, 1999; Zaidi et al, Runx2–YAP transcriptional complexes at subnuclear 2001). The Runx2 family member is essential for osteoblast sites to attenuate skeletal gene expression. maturation in vivo and is associated with cleidocranial dys- The EMBO Journal (2004) 23, 790–799. doi:10.1038/ plasia (Komori et al, 1997; Mundlos et al, 1997; Otto et al, sj.emboj.7600073; Published online 12 February 2004 1997). In vivo genetic evidence indicates that interference Subject Categories: chromatin & transcription; signal with subnuclear targeting and associated co-regulatory func- transduction tions of Runx2 can account for this block in bone formation Keywords: Cbfa1; nuclear matrix; osteoblasts; osteocalcin; (Choi et al, 2001). Src signaling Runx2 is a target of several extracellular signals that regulate skeletal formation and homeostasis. The C-terminus of Runx2, which includes the NMTS, interacts with proteins involved in the TGFb/BMP (i.e., Smads), the transducin-like Introduction enhancer (TLE)/groucho and the Src/Yes tyrosine kinase (e.g., the Yes-associated protein, YAP) signaling pathways The spatial distribution of binding sites for transcription (Hanai et al, 1999; Yagi et al, 1999; Zhang and Derynck, factors, regulation of their binding to cognate sites by chro- 1999; Javed et al, 2000; Zaidi et al, 2002). Src family tyrosine matin, and the distribution of regulatory factors and their co- kinases are activated by a variety of extracellular stimuli, regulators in distinct subnuclear domains together contribute and broadly control cell cycle regulation, cell migration, cell to the control of gene expression (Berezney and Jeon, 1995; metabolism and survival, as well as cell proliferation and Stein et al, 2000). Transcription factors possess intrinsic differentiation (Thomas and Brugge, 1997; Schlessinger, nuclear import signals that localize these proteins to the 2000). Src homology (SH) domains present in these kinases nucleus. Importantly, several transcription factors have an mediate interactions with downstream signaling proteins additional protein module, the nuclear matrix-targeting signal such as YAP. YAP, in turn, contains a WW domain in the (NMTS), that directs these factors to subnuclear sites where N-terminus (Sudol, 1994) that recognizes a proline-rich motif activation and repression take place (van Steensel et al, (PPxY) present in a broad range of proteins including Runx *Corresponding author. Department of Cell Biology, 55 Lake Avenue factors (Sudol et al, 1995; Sudol and Hunter, 2000). North, Worcester, MA 01655-0106, USA. Tel.: þ 1 508 856 5625; The significance of Src/Yes tyrosine kinase signaling in Fax: þ 1 508-856-6800; E-mail: [email protected] bone development is suggested by the osteopetrotic pheno- type of Src null mice (Soriano et al, 1991) and the accelerated Received: 20 June 2003; accepted: 12 December 2003; Published differentiation of Src-deficient osteoblasts (Marzia et al, online: 12 February 2004 790 The EMBO Journal VOL 23 NO 4 2004 &2004 European Molecular Biology Organization | | Src/Yes signaling suppresses Runx2 activity in osteoblasts SK Zaidi et al 2000). These results indicate that Src signaling inhibits matrix-intermediate filament (NM-IF) fraction in osseous osteoblast differentiation, but the underlying molecular me- cells that express endogenous Runx proteins (Figure 2B, chanisms are not known. Here we show that Src signaling in bottom right panel); however, it is not detected in the NM- osteoblasts function through YAP to inhibit Runx2 activity. In IF preparations of HeLa cells, which do not express any of the response to Src/Yes signaling, YAP is phosphorylated and three Runx proteins (Armesilla et al, 1996) (Figure 1B, bot- recruited by Runx2 to subnuclear sites and chromatin of the tom left panel). We have previously shown that Runx pro- bone-specific OC gene, resulting in its repression. Thus, teins contain a conserved NMTS that targets Runx to punctate Runx2–YAP interactions integrate Src signals at architectu- subnuclear sites (Zeng et al, 1997, 1998; Tang et al, 1999; rally associated subnuclear sites, where regulatory complexes Zaidi et al, 2001). As our data with nonosseous cells suggest assemble to control osteoblast gene expression. that YAP lacks an intrinsic subnuclear targeting signal, we immunostained NM-IF preparations of ROS 17/2.8 cells for both endogenous YAP and Runx2. As shown in Figure 1C, YAP and Runx2 colocalize at punctate foci in the NM-IF Results preparations of osseous cells. Hence, YAP and Runx2 proteins Endogenous Runx2 and YAP proteins interact in vivo interact in vivo and associate in situ at subnuclear sites in and associate in situ at subnuclear sites in osteoblasts osteoblasts. Importantly, YAP association with the nuclear Direct interaction between YAP and Runx protein segments matrix in osseous but not nonosseous cells may depend on has been documented in a cell-free system (Yagi et al, 1999). the presence of Runx2. We therefore determined whether endogenous Runx2 and YAP interact in osteoblasts. Co-immunoprecipitation assays show that endogenous YAP and Runx2 proteins form a The PY motif of Runx2 is required for targeting of YAP complex in vivo in osteoblastic ROS 17/2.8 cells (Figure 1A). to subnuclear domains in situ and its recruitment These findings indicate that Runx2 and YAP interact under to chromatin in vivo physiological conditions. The absence of YAP from the nuclear matrix of nonosseous YAP interacts with Src family kinases at the plasma mem- cells suggests that the interaction of YAP with Runx2 is brane, with 14-3-3 proteins in the cytoplasm and with tran- required for its subnuclear targeting. Therefore, we intro- scription factors in the nucleus (Mohler et al, 1999; Yagi et al, duced a point mutation into the PY motif of Runx2 (Runx2 1999; Strano et al, 2001; Vassilev et al, 2001; Basu et al, 2003). Y433A; Figure 2A, top panel), a mutation that has been We therefore assessed the subcellular localization of YAP in reported to disrupt Runx2–YAP interaction in vitro (Yagi osseous (ROS 17/2.8) and nonosseous (HeLa) cells. et al, 1999). As seen for endogenous proteins (Figure 1), Endogenous YAP is present in the cytoplasm, but is predo- exogenously expressed YAP and Runx2 form precipitable minantly nuclear in both HeLa and ROS 17/2.8 cells complexes in osseous cells (Figure 2A, bottom panel), but (Figure 1B, top panels). YAP associates with the nuclear the Y433A mutation abrogates this interaction (Figure 2A). Figure 1 Endogenous YAP and Runx2 proteins interact in vivo and co-localize in situ in osseous cells. (A) Endogenous Runx2 was immunoprecipitated from ROS 17/2.8 cells with a rabbit polyclonal antibody (1:2000) raised against the Runx2 C-terminus (Zhang et al, 2000). A rabbit polyclonal antibody was used to detect endogenous YAP (top panel). Normal goat IgG was used as a control. The middle panel shows efficient immunoprecipitation of endogenous Runx2. The bottom panel shows the expression of endogenous YAP in ROS 17/2.8 cells (20% of total input). (B) In situ immunofluorescence of whole cell (WC) and nuclear matrix-intermediate filament (NM-IF) preparations was performed to assess the nucleo-cytoplasmic distribution and subnuclear localization of endogenous YAP in nonosseous (HeLa) and osseous (ROS 17/2.8) cells. YAP is predominantly nuclear in both HeLa and ROS 17/2.8 cells (top panels), but is only associated with the nuclear matrix ROS 17/2.8 cells (bottom panels). (C) Same as (B), using deconvoluted images. The merged image reveals that endogenous YAP resides in Runx2 containing subnuclear foci in ROS 17/2.8 cells (bar¼ 10 mm). &2004 European Molecular Biology Organization The EMBO Journal VOL 23 NO 4 2004 791 | | Src/Yes signaling suppresses Runx2 activity in osteoblasts SK Zaidi et al Figure 2 The PY motif of Runx2 is required for interaction with YAP and its recruitment to subnuclear sites as well as target gene promoters in vivo.(A) ROS 17/2.8 cells were cotransfected with Xpress-YAP- and HA-tagged wild-type or the Y433A mutant of Runx2 (numbering according to the mouse MASNS/Runx2 isoform). After 24 h of transfection, proteins were precipitated using a monoclonal antibody against the HA tag (2 mg) and separated by SDS–PAGE. A mouse monoclonal antibody (1:5000) was used to detect XPR-YAP. The blots were stripped and were incubated with HA antibody (1:3000) to assess the expression of Runx2 (wild type or mutant) proteins or with XPR antibody to assess the expression of exogenous YAP. (B) HeLa cells, transfected with XPR-YAP (0.5 mg), were processed for WC and NM-IF preparations. YAP is present both in the cytoplasm and nucleus (right panel), but neither exogenous nor endogenous YAP is found in the nuclear matrix of HeLa cells (left panel). (C) Runx2 does not alter the subcellular localization of YAP in HeLa cells cotransfected with XPR-YAP and HA-Runx2 (wild type and Y433A) (WC, top panels). YAP is associated with the nuclear matrix when co-expressed with wild-type Runx2 (middle panel), but not if the interaction of Runx2 with YAP is disrupted (Y433A mutant, bottom panel) (bar¼ 10 mm). (D) Chromatin immunoprecipitation assay was performed using ROS 17/2.8 cells transfected with indicated tagged expression constructs. Purified immunoprecipitated DNA was amplified with primers spanning the Runx-binding sites B and C in rat osteocalcin promoter as described. OC-derived DNA was observed in chromatin immunoprecipitations with the HA and Xpress antibodies from cells expressing both HA-tagged Runx2 and Xpress-tagged YAP after 21 cycles of PCR amplification. Control lanes () from untransfected cells show that antibodies against HA or XPR tags do not nonspecifically precipitate chromatin (lanes 2 and 3). A low level of OC-derived DNA was detected from cells expressing YAP alone after 25 cycles of PCR amplification due to endogenous Runx2 (data not shown). The unrelated myogenin promoter does not exhibit signals (bottom panel). Thus, the interaction of YAP with Runx2 inside the cell complex to subnuclear domains requires an intact PY motif requires the PY motif of Runx2. and the functional NMTS of Runx2. As YAP lacks an intrinsic subnuclear targeting signal, it As YAP lacks DNA-binding activity, we postulated that may require Runx2 for subnuclear trafficking in osseous cells Runx2 may recruit YAP to promoters of target genes. (Figure 1B). We directly tested whether Runx2 mediates YAP Chromatin immunoprecipitation (ChIP) assays were per- intranuclear targeting. Runx2 (wild-type or the Y433A mu- formed to assess the in vivo association of YAP with the tant) and YAP were expressed in nonosseous cells that lack osteocalcin promoter. YAP alone is unable to bind to the endogenous Runx proteins, and their subnuclear localization promoter region of the OC gene (Figure 2D). However, when was assessed using immunofluorescence microscopy (Figure co-expressed with Runx2, YAP is specifically recruited to the 2B and C). Wild-type Runx2 and the Y433A mutant are OC promoter (see control lanes in upper panel) and not to the exclusively nuclear and exhibit a punctate subnuclear dis- myogenin gene promoter, which is not a Runx-responsive tribution (Figure 2C, left panels). Exogenously expressed YAP gene (Figure 2D, bottom panel). Hence, the interaction with is present in both the cytoplasm and the nucleus, and is not Runx2 results in targeting of YAP to subnuclear domains detected in nuclear matrix preparations (Figure 2B; see also in situ and its recruitment to a target gene promoter in vivo. Figure 1). When co-expressed with wild-type Runx2, YAP shows a discrete subnuclear distribution and the two proteins YAP suppression of Runx2-mediated osteocalcin are colocalized (Figure 2C, middle panels). However, co- activation is relieved by a dominant-negative (DN) expression of the Runx2 Y433A mutant with YAP fails to inhibitor of Src tyrosine kinase target YAP to the nuclear matrix (Figure 2C, bottom panels). The functional consequences of the Runx2–YAP interaction at Similarly, mutations that compromise subnuclear targeting of subnuclear sites were examined by monitoring YAP- and Runx2 also fail to target YAP to the nuclear matrix (Zaidi et al, Runx2-mediated OC promoter activity. Runx2 activates the 2002). Although Runx2 recruits YAP to nuclear matrix-asso- OC promoter from three- to four-fold in nonosseous cells, ciated subnuclear foci, it does not alter the nucleo-cytoplas- while YAP alone has no effect on the basal promoter activity mic distribution of YAP (Figure 2C, top panels). Collectively, (Figure 3A). YAP suppresses Runx2-enhanced OC promoter these results demonstrate that while the nuclear import of activity in a dose-dependent manner (Figure 3A, center YAP is independent of Runx2, targeting of the Runx2–YAP lanes). Strikingly, expression of the Runx2 Y433A mutant 792 The EMBO Journal VOL 23 NO 4 2004 &2004 European Molecular Biology Organization | | Src/Yes signaling suppresses Runx2 activity in osteoblasts SK Zaidi et al Figure 3 YAP suppresses Runx2-mediated activation of the rat osteocalcin promoter. (A) The rOC 208 CAT reporter was transfected in HeLa cells along with the indicated Runx2 constructs (250 ng) and increasing concentrations (0 (first bar of each group), 250, 500 ng and 1 mg) of full- length YAP. Expression of YAP alone does not affect basal promoter activity. Runx2 activates osteocalcin promoter activity, which is suppressed in a dose-dependent manner by increasing concentrations of YAP. The Runx2 Y433A mutant, that does not interact with YAP, shows a 3–4-fold higher activity compared to wild-type Runx2. (B) YAP suppresses the activity of Runx2 in osteoblastic ROS 17/2.8 cells. In contrast to HeLa cells, wild-type YAP slightly enhances basal promoter activity (first group), and the Runx2 Y433A mutant moderately activates rOC 208 due to endogenous Runx2 in ROS 17/2.8 cells. The graphs represent at least three independent experiments (n¼ 6 each); error bars¼ standard error of mean. (C) The rOC 208/CAT reporter was transfected with 250 ng each of Runx2 and YAP. Runx2 activates osteocalcin promoter activity, which is suppressed by YAP in osteoblastic MC3T3 cells, premyoblast C2C12 cells and fibroblast NIH3T3 cells. The Runx2 Y433A mutant which does not interact with YAP shows higher activity compared to wild-type Runx2 in C2C12 and NIH 3T3 cells. This effect, however, is not apparent in MC3T3 cells, probably because of higher levels of endogenous Runx2 protein. (D) Runx2–YAP complex regulates the activity of multiple Runx target promoters. ROS 17/2.8 cells were transfected with the indicated reporter and expression constructs. Cells were harvested 30 h after the transfection and subjected to luciferase reporter assay. The luciferase activity is expressed as fold activation (6  OSE-Luc and TGFbRI-Luc promoter) or fold suppression (in case of Runx2-Luc and p21-Luc promoters). The open squares in promoter constructs represent functional Runx sites. that does not interact with YAP dramatically increases activa- exogenously expressed YAP suppresses Runx2-mediated OC tion of the OC promoter (15-fold) compared to wild-type gene transcription on the native OC gene promoter (Figure 3), Runx2 (Figure 3A, last group). As expected, increasing con- suggesting that the activity of YAP depends on the promoter centrations of YAP do not alter the activity of the Y433A context. mutant, which cannot bind YAP (Figure 2A). Thus, disruption We tested whether the activity of the Runx2–YAP complex of the PY motif allows Runx2 to function unabated by the is promoter context dependent, by examining the regulation presence of endogenous YAP in HeLa cells. In ROS 17/2.8 of various Runx target gene promoters by the Runx2–YAP osteoblastic cells, exogenous YAP expression slightly in- complex. We used luciferase reporters driven by multimer- creases OC promoter activity and suppresses Runx2-mediated ized Runx-binding sites (6X OSE-Luc; activated by Runx2), OC activation (Figure 3B). In addition, the Runx2 Y433A the TGFb receptor type I promoter (TGFbRI-Luc; activated by mutant shows a relatively moderate activation of the OC Runx2), the Runx2 promoter (Runx2-Luc; suppressed by promoter in ROS 17/2.8 compared to HeLa cells, presumably Runx2) and the p21 promoter (p21-Luc; suppressed by due to the presence of endogenous Runx2. Similar results Runx2). Our results show that the co-expression of YAP were obtained in mouse osteoblastic MC3T3 cells, premyo- does not alter Runx2 activity on the 6X OSE-Luc; however, blast C2C12 cells and NIH3T3 fibroblasts (Figure 3C), de- it moderately suppresses Runx2 activity on a natural TGFbRI monstrating that the activity of the Runx2–YAP complex is promoter (Figure 3D, upper panels). YAP co-expression re- independent of cell type. Interestingly, when YAP is expressed sults in increased suppression of the Runx2 promoter by as a Gal4 fusion protein in a heterologous Gal4 system, we Runx2, but does not affect Runx2 suppression of the p21 observe strong transcriptional activation, as has been pre- promoter (Figure 3D, bottom panels), which is known to viously reported (data not shown; Yagi et al, 1999]. However, involve HDAC6 (Westendorf et al, 2002). We conclude that &2004 European Molecular Biology Organization The EMBO Journal VOL 23 NO 4 2004 793 | | Src/Yes signaling suppresses Runx2 activity in osteoblasts SK Zaidi et al Figure 5 Activated Src family kinases contribute to the YAP–Runx2 Figure 4 A DN inhibitor of Src tyrosine kinase relieves YAP- interaction. (A) ROS 17/2.8 cells, transiently transfected with XPR- mediated suppression of Runx2 activity. ROS 17/2.8 cells were YAP and antibodies, were used to immunoprecipitate endogenous transfected with 1 mg rOC 208/CAT reporter along with 250 ng of Yes or Src tyrosine kinases. Association of YAP with these kinases Runx2 and/or YAP expression constructs. Two different concentra- was assessed by western blotting using HRP-conjugated anti-XPR tions of Src DN construct (200 and 800 ng) were used (bars 2 and 3, antibody. (B) ROS 17/2.8 cells, expressing indicated constructs, respectively, of each group). Cells were harvested 24–30 h after were treated with 5 mM of PP2 for 1 h and processed for immuno- transfection and were subjected to CAT reporter assay. The graph precipitation. The Runx2–YAP interaction is completely abrogated represents results obtained from three independent experiments in the presence of PP2 (panel 1). Panel 2 shows the efficiency (n¼ 6 each). of Runx2 immunoprecipitation, while panel 3 shows comparable levels of YAP overexpression in all lanes. (C) As in (B), but antibodies were used to immunoprecipitate endogenous Runx2. The effect of Src DN on the YAP–Runx2 interaction was assessed the activity of the Runx2–YAP complex is promoter context by western blotting using the HRP-conjugated anti-XPR antibody dependent. (panel 1). A mouse monoclonal antibody against Runx2 was used to assess the efficiency of immunoprecipitation of the endogenous YAP is a substrate of the Src/Yes tyrosine kinases (Sudol, Runx2 protein (panel 2). Expression of Src DN and XPR-YAP was 1994). To investigate whether YAP-mediated suppression of confirmed with Src and HRP-Xpress antibodies, respectively (panels Runx2 activity involves Src signaling, we used a DN inhibitor 3 and 4). In each immunoprecipitation, appropriate normal IgG was of Src tyrosine kinase (Src K295R/Y527F or Src DN), which is used as a control. kinase inactive (Mukhopadhyay et al, 1995). This variant constitutively binds to cellular proteins that interact with activated Src under physiological conditions. Expression of associated with both Yes and Src tyrosine kinases in osteo- Src DN increases the basal activity of the osteocalcin gene blasts (Figure 5A). We therefore assessed whether the sup- promoter in ROS 17/2.8 cells in a dose-dependent manner pressor function of YAP on the OC promoter may involve Src/ (Figure 4). The Src DN has a similar effect when co-expressed Yes signaling using the inhibitor PP2 (Hanke et al, 1996). PP2 with Runx2 in nonosseous cells such as HeLa, which express is a broad inhibitor of the Src tyrosine kinase family, and has high endogenous levels of YAP and Src family members (data been reported to inhibit the activity of at least four Src family not shown). This increased basal OC transcription in the members, including Src, Yes, Fyn and Hck (Hanke et al, presence of the Src DN may result from inhibition of interac- 1996). Endogenous Runx2 was immunoprecipitated from tion between endogenous YAP and Src proteins. In addition, untreated (control) or PP2-treated (5 mM for 1 h) ROS 17/2.8 YAP-mediated suppression of Runx2 activity is relieved in the cells expressing XPR-YAP. Runx2 is associated with exogen- presence of the Src DN (Figure 4). Thus, YAP strongly blocks ously expressed YAP in control cells (Figure 5B, lane 3), but Runx2-mediated transcriptional activation of OC, and this not in the presence of PP2 (Figure 5B, lane 4). To address suppression may involve Src tyrosine kinase activity. We more specifically the involvement of Src tyrosine kinase in propose that the PY motif of Runx2 is an essential protein regulating interaction between Runx2 and YAP, we examined module that mediates the suppression of OC promoter activ- the Runx2–YAP interaction in the presence of Src DN (Src ity in response to Src signaling. K295R/Y527F). Forced expression of Src DN significantly reduces (2–3-fold) interaction between Runx2 and YAP (com- pare lanes 3 and 4; Figure 5C); residual interactions between Inhibition of the Src tyrosine kinase family abrogates Runx2 and YAP may occur through signaling pathways Runx2–YAP interaction and induces expression involving other Src family members. Taken together, the of the endogenous osteocalcin gene findings with PP2 and Src DN indicate that the Src family As YAP is an in vitro substrate of the Src/Yes tyrosine kinases tyrosine kinase signaling regulates the Runx2–YAP interac- (Sudol, 1994), we examined whether YAP interacts with Yes tion. and Src tyrosine kinases in vivo in osteoblasts. Endogenous Elevated osteocalcin mRNA in Src-deficient osteoblasts Src or Yes proteins were immunoprecipitated from ROS (Marzia et al, 2000) suggests an inhibitory role for Src 17/2.8 cells expressing XPR-YAP. We find that YAP is indeed 794 The EMBO Journal VOL 23 NO 4 2004 &2004 European Molecular Biology Organization | | Src/Yes signaling suppresses Runx2 activity in osteoblasts SK Zaidi et al Figure 6 Inhibition of endogenous Src signaling or YAP activity induces endogenous osteocalcin gene transcription. (A) Total cel- lular RNA from ROS 17/2.8 cells was analyzed by northern blotting and treated with indicated concentrations of PP2 for 1 h. Endogenous osteocalcin gene transcription is induced in a dose- dependent manner when the activity of Src family members is inhibited by PP2 (upper panel). 18S RNA was used as a loading control (bottom panel). (B) Activity of Src tyrosine kinase or YAP Figure 7 Tyrosine phosphorylation of YAP regulates its interaction was selectively inhibited by overexpressing DN inhibitors (Src DN with Runx2 and subsequent subnuclear trafficking. (A) HeLa cells, for Src tyrosine kinase and YAP DN for YAP). Total cellular RNA was co-expressing YAP and Runx2, were transfected with Src DN or subjected to northern blot analysis from ROS 17/2.8 cells over- treated with PP2 (5 mM) for 1 h and WC or NM-IF preparations and expressing the indicated plasmids. As shown in the top panel, the in situ immunofluorescence microscopy. Inhibition of either Src DN inhibitor of Src (lane 2) or YAP (lane 4) induces endogenous tyrosine kinase (þ Src DN) or its family members (þ PP2) does not osteocalcin gene transcription. (C) Densitometric analysis reveals alter subcellular localization of YAP. Subnuclear trafficking of YAP is the extent to which endogenous osteocalcin is induced under each severely compromised when the kinase activity of Src alone or its experimental condition analyzed. (The bar graph represents the family members is inhibited. (B) To directly assess the effects of Src ratio of densitometric units of osteocalcin and 18S transcripts.) DN or PP2 on tyrosine phosphorylation of endogenous YAP and its interaction with native Runx2, endogenous YAP was immunopreci- pitated from ROS 17/2.8 cells. The immunoprecipitates were re- solved by SDS–PAGE and subjected to western blot analysis. The tyrosine kinase in OC gene expression. We therefore ad- tyrosine phosphorylation of YAP was determined by a mouse monoclonal phospho-specific antibody (Py; 1:2000) raised against dressed the involvement of the Src-YAP signaling pathway phospho-tyrosine (top panel). The blot was stripped and re-probed in the regulation of the endogenous osteocalcin gene in with a mouse monoclonal antibody against Runx2 to assess the osteoblast-like ROS 17/2.8 cells using PP2 and Src DN, as presence of endogenous Runx2 in the immunoprecipitates (middle well as a previously characterized DN inhibitor of YAP (Yagi panel). The efficiency of YAP immunoprecipitation was assessed by a YAP rabbit polyclonal antibody (bottom panel). et al, 1999). Inhibition of Src family members with various concentrations of PP2 results in a dose-dependent increase in endogenous OC expression as measured by mRNA levels (Figure 6A; quantitated in Figure 6C). More importantly, kinase activity by the Src DN or PP2 does not alter the nucleo- endogenous OC gene expression was also upregulated 2–3- cytoplasmic distribution of YAP. However, Runx2-mediated fold when the activity of endogenous Src tyrosine kinase subnuclear targeting of YAP is severely compromised in the (Figure 6B, Src DN lanes) or YAP (Figure 6B, YAP DN lanes; presence of the Src DN, that is, only 20–25% of cells in the also see Figure 6C for quantitation) was blocked. Our results NM-IF preparation are positive for YAP signal (Figure 7A, demonstrate that YAP suppresses Runx2-mediated expression middle panel). As YAP and Runx2 do not interact in the of the endogenous OC gene in response to Src tyrosine presence of PP2 (Figure 5B), YAP is absent in the NM-IF kinases. preparation of PP2-treated cells, even though Runx2 associa- tion with the nuclear matrix is unaltered (Figure 7A, right panel and data not shown). Thus, subnuclear targeting, but Tyrosine phosphorylation of YAP in response to the not nucleo-cytoplasmic distribution, of YAP requires acti- activity of Src family members regulates its interaction with Runx2 and subsequent subnuclear targeting vated Src tyrosine kinases. The molecular mechanism(s) underlying Src-YAP-mediated To further explore the role of Src signaling in the Runx2– suppression of Runx2 activity on the endogenous OC promo- YAP interaction and control of OC gene expression, we assessed the tyrosine phosphorylation status of endogenous ter was further examined by in situ immunofluorescence YAP in osteoblasts upon inhibition of Src signaling. microscopy of nonosseous cells co-expressing Runx2 and Expression of Src DN significantly decreases tyrosine YAP in the presence of PP2 or Src DN. Inhibition of tyrosine &2004 European Molecular Biology Organization The EMBO Journal VOL 23 NO 4 2004 795 | | Src/Yes signaling suppresses Runx2 activity in osteoblasts SK Zaidi et al phosphorylation of YAP (Figure 7B, upper panel) and reduces examined previously (Yagi et al, 1999). Our studies on the its interaction with the native Runx2 protein (Figure 7B, native OC gene promoter establish that YAP suppresses middle panel). In addition, treatment of cells with PP2 Runx2-dependent transcriptional activation. Of interest, a abrogates YAP tyrosine phosphorylation and its interaction multimerized Runx2 element does not respond to YAP repres- with Runx2 (Figure 7B, right lanes). Taken together, our sion, supporting the concept that the formation of multimeric results indicate that Src-related tyrosine phosphorylation of regulatory complexes at Runx sites in native promoters is YAP is required for its interaction with Runx2, and its target- influenced by surrounding sequences (Canon and Banerjee, ing to Runx subnuclear sites. 2003). As Runx2–YAP may be part of a larger regulatory complex, we propose that the functional outcome of YAP activity depends on specific protein–protein interactions that Discussion are promoter context dependent, as is the case for other co- In this study, we have shown that YAP, a downstream target regulators (Lemon and Tjian, 2000). of Src tyrosine kinases, functions to suppress the activity of Runx2, a transcription factor required for osteoblast matura- Regulatory aspects of Runx2-mediated recruitment of tion. We find that YAP interacts with both Src and Yes YAP to subnuclear domains and the OC gene promoter tyrosine kinases in osteoblasts. Tyrosine phosphorylation of Runx transcription factors play a pivotal role in the main- endogenous YAP promotes its interaction with Runx2. Runx2 tenance of different levels of nuclear organization (Stein et al, then recruits YAP to the bone-specific osteocalcin promoter 2000, 2003). Intact Runx-binding sites are required for tran- and to subnuclear sites where Runx2 resides to suppress scription, steroid hormone responsiveness and for the main- osteocalcin promoter activity. Interference with the Src- tenance of open chromatin structure of the rat OC gene YAP–Runx2 pathway at any level results in inhibition of promoter (Javed et al, 1999). In addition, Runx factors YAP tyrosine phosphorylation, disruption of the Runx2–YAP possess a specific intranuclear targeting signal (the NMTS) interaction, block in recruitment of YAP to subnuclear sites that directs these factors to distinct subnuclear domains and and induction of osteocalcin gene expression. We conclude supports transcriptional control of target genes (Zeng et al, that Runx2–YAP interactions are obligatory for integration of 1997, 1998; Tang et al, 1999; Stein et al, 2000; Choi et al, Src signals at architecturally associated sites where regulatory 2001; Zaidi et al, 2001). The NMTS overlaps with interacting complexes assemble to control osteoblast gene expression. domains of several coregulators of Runx function, including YAP and Smads (Hanai et al, 1999; Yagi et al, 1999). Smads, Physiological significance of Src/Yes signaling to Runx2 which transduce BMP signals, require the subnuclear target- activity ing signal of Runx transcription factors to assemble as distinct Negative regulation of Runx2 by Src/Yes kinases may be subnuclear complexes at sites of active transcription to important for maintenance of bone homeostasis. Bone remo- regulate gene expression (Zaidi et al, 2002). Here, we show deling is a dynamic process that requires resorption and that YAP must bind to Runx2 to associate with nuclear formation of bone tissue to ensure the continuous renewal substructures and the promoter regions of target genes. of bone throughout life. This process is regulated by the Taken together, these studies suggest that Runx2 functions physiological balance between the activities of bone-forming as a multifunctional organizer of regulatory complexes at osteoblasts and bone-resorbing osteoclasts in response to subnuclear sites. several physiological cues. Like vitamin D and bone morpho- Our studies also suggest differences in the importance of genetic proteins (BMPs), Src/Yes signaling coordinates both serine and tyrosine phosphorylation in defining subcellular osteoblast and osteoclast activities for bone turnover and subnuclear distribution of YAP. The Akt-kinase-mediated (Katagiri et al, 1990; Soriano et al, 1991; Boyce et al, 1992; serine phosphorylation of YAP disrupts its interaction with Horne et al, 1992; Suda et al, 1992; Van Leeuwen et al, 2001). 14-3-3 proteins, thus resulting in cytosolic localization of YAP The molecular mechanisms involving Src/Yes tyrosine ki- (Basu et al, 2003). We find that tyrosine phosphorylation of nases in osteoclast differentiation are well studied, but a YAP in response to Src/Yes signals does not alter its nucleo- role for this pathway and its downstream signaling molecules cytoplasmic distribution, but is required for interaction of such as YAP in osteoblasts has only recently been suggested. YAP with Runx2 and its consequent subnuclear trafficking. Osteoblasts isolated from Src mice exhibit accelerated Taken together, these observations support a novel mechan- differentiation and elevated levels of OC mRNA and other ism by which regulatory complexes spatially and temporally osteoblast markers, suggesting that c-Src negativity regulates assemble within the nucleus in response to extracellular osteoblast maturation (Thomas and Lafage-Proust, 1999; signals, leading to activation or suppression of target genes. Marzia et al, 2000). We find that several osteoblast-related We propose that the dynamic presence of coregulatory pro- genes are regulated by the Runx2–YAP complex. Thus, YAP teins in Runx subnuclear domains provides a mechanism for modulation of Runx2-mediated gene expression provides a positive and negative transcriptional control of Runx-depen- molecular mechanism for Src signaling in the control of dent promoters within the three-dimensional context of nu- osteoblast differentiation. clear architecture in response to physiological signals to Our studies provide evidence for the novel concept that ensure proper bone formation. YAP can function as a suppressor of gene activation. The carboxy-terminus of YAP contains a strong activation domain Materials and methods as assessed by heterologous gene transcription assays (Yagi et al, 1999; Strano et al, 2001; Vassilev et al, 2001), and YAP Reporter assays enhances the Runx-mediated activation of the IgaC promoter. Rat osteosarcoma ROS 17/2.8 and human cervical carcinoma HeLa However, the effect of YAP on the OC promoter has not been cells were plated at a density of 0.0410 cells/ml. Transient 796 The EMBO Journal VOL 23 NO 4 2004 &2004 European Molecular Biology Organization | | Src/Yes signaling suppresses Runx2 activity in osteoblasts SK Zaidi et al transfections were initiated 24 h later using either Superfect (Qiagen Proteins were separated on 10% SDS–polyacrylamide gel and Inc., Valencia, CA, USA) or Fugene (Roche Applied Science, processed for western blotting using appropriate horseradish Indianapolis, IN, USA) with the indicated expression constructs peroxidase (HRP)-conjugated secondary antibodies (1:2000). The and 1 mgof 208 OC-CAT (chloramphenicol acetyl transferase), signal was visualized using the enhanced chemiluminescence kit spanning one Runx-binding site (Site C) of the rat osteocalcin (Amersham Pharmacia Biotech Inc., Piscataway, NJ, USA) accord- promoter. RSV-Luc was included as a control for transfection ing to the manufacturer’s instructions. efficiency. After 24 h, cells were processed for CAT assay and quantitated using a Storm PhosphorImager (Molecular Dynamics, Chromatin immunoprecipitation Sunnyvale, CA, USA) as described (Javed et al, 1999). Luciferase ROS 17/2.8 cells transfected with HA-Runx2 and/or XPR-YAP by activity was assessed with a Luciferase Assay Kit (Promega, Fugene transfection reagent were crosslinked with 1.1% formalde- Madison, WI, USA) in the same cell lysate and used to normalize hyde in 1 PBS at room temperature for 10 min. Cells were scraped the CAT values. Graphs are representative of three independent in 500 ml of lysis buffer (150 mM NaCl, 50 mM Tris–HCl (pH 8.0), TM experiments (n¼ 6 in each experiment) with different DNA 1% NP-40, 25 mM MG132, 1  Complete protease inhibitor preparations. cocktail). The cell lysate was then sonicated to generate 500– 1000 bp long fragments. Cell lysate was microfuged at 14 000 r.p.m. Expression constructs for 15 min at 41C and the supernatant was precleared with 40 ml The following constructs have been previously reported: HA-Runx2 protein A/G plus agarose beads, 2 mg of sheared salmon sperm DNA (Zaidi et al, 2001), Xpress (XPR)-tagged full-length YAP (1–472) and and 2 mg appropriate normal IgG for 1 h at 20 r.p.m. The precleared its DN inhibitor (1–301; YAP DN hereafter) (Yagi et al, 1999), and rat supernatant was incubated with 2 mg antibody for 1 h at 41C with osteocalcin with one Runx-binding site (rOC 208-CAT; Heinrichs rotation. The immuno-complexes were collected by adding 40 mlof et al, 1993). The Y433A mutant of Runx2 was generated by PCR- protein A/G plus agarose beads and rotating at 41C at 30 r.p.m. for based site-directed mutagenesis. Src DN (Src K295R Y527F) was another 1 h, following centrifugation by extensive washing in purchased from Upstate Biotechnology (Lake Placid, NY, USA; multiple buffers as described in Shen et al (2002). Upon reversal of Mukhopadhyay et al, 1995). crosslinking, DNA was purified using the QIAGEN PCR Purification kit (Qiagen Inc., Valencia, CA, USA), stored in 50 ml, and 1 mlwas In situ immunofluorescence and digital microscopy used for PCR. The following primer sets were used to amplify HeLa or ROS 17/2.8 cells (untransfected or transfected with 0.5 mg promoter regions: OC promoter 5 -GGT GAA TGA GGA CAT TAC of indicated expression constructs) were extracted for in situ TGA CCG CTCC and 5 -CCA AAG GAT GCT GTG GTT GGT GAC, and 0 0 0 nuclear matrix preparation 24 h after transfection and were myogenin promoter 5 -ACC CCT TTC TTG TTC CCT TCC and 5 - processed for digital microscopy as described before (Javed et al, CTC CCG CAG CCC CTC AC. The PCR program used was: 2 min 2000). HA-Runx2 was detected by a rabbit polyclonal antibody 951C denaturing step, then 30 s 951C, 30 s 601C, 45 s 721C for 21 against HA-tag at a dilution of 1:3000 (Santa Cruz Biotechnology cycles, and finally 5 min 721C elongation step. Inc., Santa Cruz, CA, USA). XPR-YAP was detected with mouse monoclonal antibodies at a dilution of 1:1000. Rabbit polyclonal Northern blotting antibodies raised against Runx2 (Oncogene Research Products, La Total cellular RNA was isolated by the Trizol method (Invitrogen Jolla, CA, USA) or YAP (Santa Cruz Biotechnology Inc., Santa Cruz, Corporation, Carlsbad, CA, USA) from ROS 17/2.8 cells (untreated CA, USA) were used to detect endogenous proteins at a dilution of or treated for 1 h with PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl) 1:200. The secondary antibodies used were either Alexa 488 anti- pyrazolo [3,4-d] pyrimidine); Calbiochem-Novabiochem Corp., La rabbit or Alexa 568 anti-mouse (Molecular Probes, Eugene, OR, Jolla, CA, USA). The probes used to detect OC mRNA and 18S rRNA USA) at a dilution of 1:800. have been described (Gutierrez et al, 2002). To determine the role of Src tyrosine kinase and YAP, ROS 17/2.8 cells were transfected with Co-immunoprecipitation and western blotting 10 mg of the Src DN or the YAP DN 36 h prior to RNA isolation using ROS 17/2.8 cells were transfected with expression constructs for Fugene transfection reagent. HA-tagged wild-type or mutant Runx2 proteins and XPR-YAP. Cells were prepared 36 h after transfection in a buffer containing 20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 0.5% Triton X-100, 1% aprotinin Acknowledgements and 1 mM phenylmethylsulfonyl fluoride (PMSF). Lysates were incubated with specific antibodies for 1 h, followed by incubation This work was supported by NIH grants DE12528, POI AR48818 and with protein A/G plus-sepharose beads. Immunoprecipitates were AR39588 POI CA82834. 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Proc Natl Acad Sci USA 97: (2002) Integration of Runx and Smad regulatory signals at 10549–10554 &2004 European Molecular Biology Organization The EMBO Journal VOL 23 NO 4 2004 799 | |

Journal

The EMBO Journal – Springer Journals

Published: Feb 25, 2004

Keywords: Cbfa1; nuclear matrix; osteoblasts; osteocalcin; Src signaling

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