RhoGDIβ Inhibits Bone Morphogenetic Protein 4 (BMP4)-induced Adipocyte Lineage Commitment and Favors Smooth Muscle-like Cell Differentiation*

Background: Bone morphogenetic protein 4 (BMP4) can induce C3H10T1/2 mesenchymal stem cell commitment into the adipocyte lineage. Results: Overexpression of RhoGDIβ in C3H10T1/2 cells prevented BMP4-induced adipogenic commitment, whereas it facilitated expression of smooth muscle-like cell-specific markers. Conclusion: RhoGDIβ plays opposing roles in committing C3H10T1/2 cells to adipocytes and smooth muscle-like cells. Significance: This is a first report implicating a role of RhoGDIβ in C3H10T1/2 cells fate decisions. The integration of signals involved in deciding the fate of mesenchymal stem cells is largely unknown. We used proteomics profiling to identify RhoGDIβ, an inhibitor of the small G-protein Rho family, as a component that regulates commitment of C3H10T1/2 mesenchymal stem cells to the adipocyte or smooth muscle cell lineage in response to bone morphogenetic protein 4 (BMP4). RhoGDIβ is notably down-regulated during BMP4-induced adipocytic lineage commitment of C3H10T1/2 mesenchymal stem cells, and this involves the cytoskeleton-associated protein lysyl oxidase. Excess RhoGDIβ completely prevents BMP4-induced commitment to the adipocyte lineage and simultaneously stimulates smooth muscle cell commitment by suppressing the activation of Rac1. Overexpression of RhoGDIβ induces stress fibers of F-actin by a process involving phosphomyosin light chain, indicating that cytoskeletal tension regulated by RhoGDIβ contributes to determining adipocyte versus myocyte commitment. Furthermore, the overexpression of RacV12 (constitutively active form of Rac1) totally rescues the inhibition of adipocyte commitment by RhoGDIβ, simultaneously preventing formation of the smooth muscle-like phenotype and disrupting the stress fibers in cells overexpressing RhoGDIβ. Collectively, these results indicate that RhoGDIβ functions as a novel BMP4 signaling target that regulates adipogenesis and myogensis.

The integration of signals involved in deciding the fate of mesenchymal stem cells is largely unknown. We used proteomics profiling to identify RhoGDI␤, an inhibitor of the small G-protein Rho family, as a component that regulates commitment of C3H10T1/2 mesenchymal stem cells to the adipocyte or smooth muscle cell lineage in response to bone morphogenetic protein 4 (BMP4). RhoGDI␤ is notably down-regulated during BMP4-induced adipocytic lineage commitment of C3H10T1/2 mesenchymal stem cells, and this involves the cytoskeleton-associated protein lysyl oxidase. Excess RhoGDI␤ completely prevents BMP4-induced commitment to the adipocyte lineage and simultaneously stimulates smooth muscle cell commitment by suppressing the activation of Rac1. Overexpression of RhoGDI␤ induces stress fibers of F-actin by a process involving phosphomyosin light chain, indicating that cytoskeletal tension regulated by RhoGDI␤ contributes to determining adipocyte versus myocyte commitment. Furthermore, the overexpression of RacV12 (constitutively active form of Rac1) totally rescues the inhibition of adipocyte commitment by RhoGDI␤, simultaneously preventing formation of the smooth muscle-like phenotype and disrupting the stress fibers in cells overexpressing RhoGDI␤. Collectively, these results indicate that RhoGDI␤ functions as a novel BMP4 signaling target that regulates adipogenesis and myogensis.
The C3H10T1/2 cell line, derived from C3H mouse embryos (13), behaves similarly to mesenchymal stem cells (14). BMP4 (bone morphogenetic protein 4) induces nearly complete commitment of C3H10T1/2 cells to the adipocyte lineage (15)(16)(17)(18). However, C3H10T1/2 cells can also undergo commitment toward smooth muscle cells (SMCs) under different inducing conditions (19 -21), suggesting that adipocytes and SMCs share a common mesenchymal origin. Indeed, recent research has demonstrated that a subset of beige adipocytes has a smooth muscle-like origin, and vascular smooth muscle cells can be converted into UCP1-positive adipocytes via ectopic expression of PRDM16 (22). These data indicate the possibility of a direct cell fate switch between adipocytes and SMCs.
Several studies have noted that changes in cell shape and cytoskeletal tension can influence the fate of mesenchymal progenitor cells (23,24). We recently reported that three cytoskeleton-associated proteins including Lox (lysyl oxidase) were remarkably up-regulated concomitantly with notable F-actin (filamentous actin) disruption during BMP4-induced adipocyte lineage commitment (15)(16)(17)(18). Knockdown of Lox reorganized the F-actin into stress fibers and totally inhibited commitment to the adipocyte lineage, suggesting that a Loxmediated cytoskeleton change is indispensable for such com-mitment under the influence of BMP4 (15). Interestingly, changes in cytoskeletal tension can also regulate SMC differentiation. It has been demonstrated that inhibition of actin polymerization significantly decreased the expression of SMC differentiation marker genes. In contrast, increased actin polymerization improved the expression of those genes (25). Because inhibition of Lox promotes actin stress fiber formation (15,26), we hypothesized that Lox regulates adipocyte and SMC fates via regulation of actin filament formation.
Rho GTPases (Rho, Rac, and Cdc42) are known to regulate the assembly and organization of F-actin in response to extracellular cues (27)(28)(29)(30). The RhoGDIs (Rho GDP dissociation inhibitors) inhibit the dissociation of GDP from Rho and GTP hydrolysis on Rho proteins (31). The mammalian RhoGDI family has three members: RhoGDI␣, RhoGDI␤, and RhoGDI␥. The Rho GDP dissociation inhibitor ␤ gene is commonly referred to as ARHGDIB, but is also known as LyGDI, GDI-D4, RhoGDI2, or RhoGDI␤. In this study, using iTRAQ-based proteomics profiling, we indentify RhoGDI␤ as a potential target of the BMP4-Lox signaling axis, regulating commitment of C3H10T1/2 cells to either adipocytes or smooth muscle-like cells by reorganizing actin filament formation in a Rac1-dependent manner.

EXPERIMENTAL PROCEDURES
Cell Culture and Induction of Commitment/Differentiation-To induce lineage commitment, C3H10T1/2 stem cells were plated at low density and cultured in DMEM containing 10% calf serum without or with purified recombinant BMP4 (10 ng/ml). To induce adipocyte differentiation, 2-day post-confluent cells (day 0) were fed with DMEM containing 10% fetal bovine serum (FBS), 1 g/ml of insulin, 1 M dexamethasone, and 0.5 mM 3-isobutyl-1-methylxanthine for 2 days and then given DMEM with 10% FBS and 1 g/ml of insulin for another 2 days, after which they were cultured in DMEM with 10% FBS. To induce SMC differentiation, 2-day postconfluent cells were fed with DMEM containing 2% horse serum for 7 days.
Oil Red O Staining-C3H10T1/2 stem cells were induced to adipocyte differentiation as described above. On day 8, the cells were washed three times with PBS (phosphate-buffered saline) and then fixed for 10 min with 3.7% formaldehyde. Oil Red O (0.5% in isopropyl alcohol) was diluted with water (3:2), filtered through a 0.45-m filter, and incubated with the fixed cells for 1 h at room temperature. The cells were then washed with water and the stained fat droplets in the adipocytes were visualized by light microscopy and photographed.
Construction of Expression Plasmids and Generation of Retrovirus-cDNA for RhoGDI␤ was generated by PCR using the following primers: 5Ј-GGAAGATCTGCCACCATGACG-GAGAAGGATGCA-3Ј (forward); 5Ј-CCGGTTAACTCATT-CTGTCCAATCCTTC-3Ј (reverse). The PCR product was cloned into a MSCV retroviral vector with BglII and HpaI. A constitutively active mutant of Rac1 (RacV12) was provided by Dr. Debbie C. Thurmond (Indiana University School of Medicine). RacV12 cDNAs were subcloned into MSCV retroviral vectors with BglII and XhoI. 293T cells cultured in serum-free DMEM were transfected with MSCV or recombinant plasmid and Ecopac plasmids at 95% confluence. Fresh medium containing 10% calf serum was given 4 -6 h after transfection and the viral medium was collected at 48 -72 h. C3H10T1/2 cells were infected with viruses at 20 -30% confluence with Polybrene (8 g/ml).
RNA Interference-Stealth siRNA duplexes specific for Lox were designed and synthesized by Invitrogen. The sequence for successful RNAi knockdown was GCGGAUGUCAGAGAC-UAUGACCACA. Stealth siRNA negative control duplexes with a similar GC content were used as control. C3H10T1/2 stem cells were transfected at 30 -50% confluence with siRNA duplexes using Lipofectamine RNAi MAX according to the manufacturer's instructions (Invitrogen).
F-actin Staining-C3H10T1/2 cells were plated on coverslips and treated as described above; 2-day post-confluent cells were washed three times with PBS and fixed in 4% (w/v) formaldehyde for 10 min at room temperature. F-actin was stained with TRITC-conjugated phalloidin (Molecular Probes, Eugene OR) for 30 min at room temperature. Nuclei were counterstained with DAPI. Images were captured with a Leica confocal microscope.
GST-PAK-PBD Binding Assays-The activation of Rac1 (Rac1-GTP) was determined by a pulldown assay using a commercially available kit according to the manufacturers' instruc-tions (Cytoskeleton). Briefly, 2-day post-confluent C3H10T1/2 cells were washed with ice-cold PBS and lysed. The lysates were clarified by centrifugation at 10,000 ϫ g at 4°C for 1 min and incubated with GST-PAK-PBD-agarose beads at 4°C for 1 h. The beads were washed and eluted. To detect GTP-bound Rac1, eluted agarose-bound proteins were separated by SDS- PAGE, and Western blotting was performed using the antibody against Rac1.
Sample Preparation and iTRAQ Labeling-Total protein was extracted from C3H10T1/2 cells (control, BMP4-treated and LOX knockdown cells) on day 0 using lysis buffer (8 M urea, 2 M thiourea, 2% CHAPS, 60 mM DTT) containing complete protease inhibitor mixture (Roche Applied Science). A total of 100 g of protein from each group was precipitated overnight with 6 volumes of acetone at 4°C and the pellets were resuspended in dissolution buffer containing 20 l of 500 mM triethylammonium bicarbonate and 1 l of 2% SDS. Subsequently, the resuspended proteins were reduced with 2 l of 50 mM tris-2-(carboxyethyl)phosphine at 60°C for 1 h and then alkylated with 1 l of 200 mM methyl methanethiosulfonate in isopropyl alcohol at room temperature for 10 min, followed by digestion with 10 g of sequencing grade trypsin (Applied Biosystems) for 16 h at 37°C. Peptide samples were labeled with iTRAQ tags (isobaric tags for relative and absolute quantitation) at room temperature for 1 h as follows: iTRAQ113 for control C3H10T1/2 cells, iTRAQ114 for BMP4-treated cells, and iTRAQ116 for LOX knockdown cells. Then all the labeled peptides were dried and analyzed by reverse-phase liquid chromatography followed by tandem mass spectrometry (LC-MS/MS).
Statistical Analysis-Values are expressed as mean Ϯ S.D. of at least three independent experiments. The p values were determined by Student's t test, with p Ͻ 0.05 considered significant.

Proteomics Profiling Identified RhoGDI␤ as a Muscular
Development-related Protein-A newly developed iTRAQ technique was used to compare protein expression profiles among control C3H10T1/2 cells, C3H10T1/2 cells treated with BMP4, and Lox knockdown cells treated with BMP4. We took the cut-offs for all iTRAQ ratios as 1.2-fold changes, that is, ratios of Ͼ1.2 or Ͻ0.80, to classify proteins as up-or downregulated, respectively. We were interested in proteins downregulated by BMP4 that were elevated when Lox was knocked down, and proteins up-regulated by BMP4 that were downregulated by knockdown of Lox. According to these criteria, 93 differentially expressed proteins were screened from the iTRAQ experiments (Fig. 1A). Forty were up-regulated in BMP4-treated cells and down-regulated again when Lox was knocked down (Table 1). Fifty-three were down-regulated in BMP4-treated cells and elevated by knockdown of Lox (Table  2). These differentially expressed proteins were analyzed with

up-regulated in BMP4-treated cells and down-regulated in those cells when Lox is knocked down
iTRAQ was used to compare protein expression profiles among MSC controls, MSCs treated with BMP4 only and MSCs treated with BMP4 and Lox RNAi. The change cut-off was set at 1.2-fold for all iTRAQ ratios: the ratios Ͼ1.2 and Ͻ0.80 were used to classify proteins as up-or down-regulated, respectively. Forty proteins were identified as upregulated in BMP4-treated cells and down-regulated when LOX was knocked down. ingenuity pathways analysis software. Detailed information is presented in Fig. 1B. This complex gene network consists of six major subnetworks. Five proteins were linked to skeletal and muscular system development and function (Fig. 1, B and C). Among these muscular development-related proteins, RhoGDI␤ (ARHGDIB) was down-regulated in C3H10T1/2 cells treated with BMP4 and selected for further investigation on the basis of its involvement in cytoskeletal rearrangement (32)(33)(34).

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Down-regulation of RhoGDI␤ Is Required for BMP4-induced Adipocyte Lineage Commitment-The expression of RhoGDI␤ in BMP4-induced committed preadipocytes was confirmed by Western blotting. In line with the proteomics analysis, total cellular RhoGDI␤ protein was decreased in C3H10T1/2 cells treated with BMP4 ( Fig. 2A). To investigate whether RhoGDI␤ is a downstream target of Lox, Lox was knocked down using siRNA in C3H10T1/2 cells treated with BMP4. Expression of RhoGDI␤ inhibited by BMP4 was recovered when Lox was knocked down (Fig. 2B). Consistent with the protein level, quantitative RT-PCR also demonstrated that RhoGDI␤ mRNA expression was down-regulated after BMP4 treatment (Fig.  2C). The mRNA level of RhoGDI␤ inhibited by BMP4 was recovered when Lox was knocked down; the expression of another RhoGDI family member, RhoGDI␥, was also decreased by BMP4, and this inhibition was also recovered when Lox was knocked down. The expression of RhoGDI␣ was not affected in both BMP4-treated cells and Lox-knocked down cells (Fig. 2C).
To test whether the down-regulation of RhoGDI␤ is required for BMP4-induced adipocyte lineage commitment, RhoGDI␤ was overexpressed in C3H10T1/2 cells using a retroviral system (MSCV). The expression of RhoGDI␤ protein was remarkably increased in RhoGDI␤ overexpressing cells than in the control

down-regulated in BMP4-treated cells and up-regulated in those cells when Lox is knocked down
Fifty-three proteins were identified as down-regulated in BMP4-treated cells and up-regulated when LOX was knocked down by iTRAQ. cells infected with empty MSCV, as confirmed by Western blotting (Fig. 3A). After reaching post-confluence, the cells were given a standard adipocyte differentiation protocol (MDI); this forced expression of RhoGDI␤ totally inhibited acquisition of the adipocyte phenotype, as indicated by decreased expression of the adipocyte-specific protein 422/aP2 (Fig. 3B) and lower accumulation of cytoplasmic triglyceride staining with Oil Red O (Fig. 3C). RhoGDI␤ Inhibits BMP4-induced Adipocyte Lineage Commitment in a Rac1-dependent Manner-RhoGDI␤, an inhibitor of the small G-protein Rho family, prevents activation of Rac1/2, RhoA, and Cdc42. To investigate whether Rac1 mediates the action of RhoGDI␤ in adipocyte commitment, we examined the activation of Rac1 during BMP4-induced adipocyte lineage commitment by measuring its binding to the GTPase-binding domain of p21-activated kinase (PAK-PBD) (35). GST-PAK-PBD-bound active Rac1 was detected by Western blotting using the anti-Rac1 antibody. Binding of Rac1 to GST-PAK-PBD was significantly increased after BMP4 treatment (Fig. 4A), indicating that Rac1 is significantly activated in BMP4-induced committed preadipocytes. To examine the contribution of RhoGDI␤ to BMP4-induced activation of Rac1 further, RhoGDI␤ was overexpressed in C3H10T1/2 cells with or without BMP4 treatment, and then the binding of Rac1 to GST-PAK-PBD was examined. Rac1 binding was significantly decreased in RhoGDI␤-overexpressing cells treated with or without BMP4 (Fig. 4B). These results demonstrated that the inhibitory effect of RhoGDI␤ on BMP4-induced adipocyte commitment is due at least in part to inactivation of Rac1.

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Constitutively Active Rac1 Rescues the Inhibition of RhoGDI␤ for Adipocyte Lineage Commitment-Because RhoGDI␤ inhibits BMP4-induced activation of Rac1 in committed preadi-  pocytes, we next investigated whether Rac1 could rescue the inhibition of RhoGDI␤ for adipocyte lineage commitment. A constitutively active Rac1 mutant (RacV12) and RhoGDI␤ were co-overexpressed in C3H10T1/2cells with or without BMP4 treatment. Rac1 binding to GST-PAK-PBD was also examined. As illustrated in Fig. 4C, Rac1 activity in C3H10T1/2 cells overexpressing RhoGDI␤ with RacV12 was dramatically greater than that in cells overexpressing RhoGDI␤ alone. Consistent with these results, this forced expression of RacV12 totally rescued the inhibition of the adipocyte phenotype by RhoGDI␤, as indicated by increased expression of the adipocyte-specific protein 422/aP2 (Fig. 4D) and greater accumulation of cytoplasmic triglyceride staining with Oil Red O (Fig. 4E). These findings demonstrated that the inhibitory effect of RhoGDI␤ on BMP4induced adipocyte commitment is due to the inactivation of Rac1.
RhoGDI␤ Facilitates Smooth Muscle Cell-like Phenotype-C3H10T1/2 cells can undergo commitment and differentiate into multiple mesodermal cell types (14,36), but inducers of differentiation along one lineage often inhibit differentiation of alternative lineages. For instance, Med23 deficiency facilitates SMC differentiation but represses adipocyte differentiation (20). Because muscle development-related RhoGDI␤ suppresses BMP4-induced adipocyte lineage commitment, we speculated that it may favor the commitment to the SMC lineage. To test this hypothesis, C3H10T1/2 cells were infected with MSCV or RhoGDI␤ and treated with or without BMP4 until post-confluence, then cultured in SMC differentiation medium for 7 days before the SMC differentiation markers were examined. Quantitative RT-PCR demonstrated that overexpression of RhoGDI␤ induced the expression of multiple early and mid smooth muscle cell marker genes, e.g. Acta2, Calponin1, Sm22␣, and Sm22␤, even in the presence of BMP4 (Fig. 5A). Furthermore, expression of smMHC, a late marker of SMC differentiation, was also significantly induced (Fig. 5A). Similarly, Western blotting demonstrated increased protein levels of Calponin1 and Acta2 (Fig. 5B) in cells expressing RhoGDI␤, whereas overexpression of constitutively active Rac1 decreased the expression of smooth muscle genes in cells overexpressing RhoGDI␤ (Fig. 5). These results demonstrated that RhoGDI␤ represses BMP4induced adipocytic lineage commitment and favors smooth muscle-like cells differentiation. (15), F-actin filaments in uncommitted post-confluent C3H10T1/2 cells took the form of stress fibers, forming long linear bundles, whereas such F-actin fibers were significantly decreased after BMP4 treatment (Fig. 6A). Overexpression of RhoGDI␤ almost totally rescued the F-actin stress fibers that were disrupted during adipocyte lineage commitment induced by BMP4 (Fig. 6B). In contrast, RhoGDI␤-induced F-actin stress fibers were disrupted again in cells overexpressing RacV12, which became similar in structure to committed preadipocytes induced by BMP4 (Fig. 6B). These findings indicated that RhoGDI␤ facilitates the smooth muscle cell fate through F-actin reorganization.

RhoGDI␤ Favors Smooth Muscle-like Cell Fate via Phospho-MLC-dependent Actin Reorganization-In line with our previous studies
Both MLC and cofilin are related to the dynamics of F-actin. MLC, when phosphorylated (Thr 18 /Ser 19 ), is thought to promote the assembly of filaments (37). Cofilin is a ubiquitous actin-binding factor required for reorganizing actin filaments. Phosphorylation of cofilin at a single site (Ser 3 ) inhibits its actin-depolymerizing activity (38). We therefore examined whether RhoGDI␤ affected the phosphorylation of MLC or cofilin. Our results demonstrated that both phospho-MLC (Thr 18 /Ser 19 ) and phospho-cofilin (Ser 3 ) were lower in BMP4treated cells than control cells (Fig. 6C). We then reasoned that if the decrease in phospho-MLC or phospho-cofilin is a consequence of high levels of active Rac1, then overexpression of RhoGDI␤ should reset phospho-cofilin or phospho-MLC to higher levels. Our data demonstrated that overexpression of RhoGDI␤ drastically increased the levels of phospho-MLC (Thr 18 /Ser 19 ) and phospho-cofilin (Ser 3 ) in cells with or without BMP4 treatment (Fig. 6D). However, overexpression of constitutively active Rac1 only decreased the amount of phospho-MLC in cells overexpressing RhoGDI␤ (Fig. 6D), whereas phosphorylated cofilin was not affected. These results indi- cated that RhoGDI␤ reorganizes F-actin and promotes smooth muscle-like cell lineage commitment by increasing phospho-MLC.

DISCUSSION
MSCs are progenitors capable of differentiating into multiple cell types including adipocytes, osteoblasts, chondrocytes, tenocytes, skeletal myocytes, and visceral stromal cells (39 -44). The decision made by a mesenchymal stem cell to commit to a particular lineage is highly context-dependent and involves the integration of multiple extracellular signals to drive the outcome. Our previous findings demonstrated that BMP4 induces nearly complete commitment of C3H10T1/2 cells to the adipocyte lineage (16,17) and disrupts the formation of filamen-  tous actin (15). Both BMP4-induced adipocyte lineage commitment and actin reorganization are regulated by Lox (15,16). However, the molecular mechanisms by which Lox regulates the changes in the cytoskeleton are not fully understood. In this study, RhoGDI␤ was identified by proteomics profiling as a potential target downstream of the BMP4-Lox signaling axis.
Rho family GTPases are important in many cellular functions (45). RhoGDIs, potent negative regulators of Rho family GTPases, are characterized by their ability to prevent nucleotide exchange and membrane association (31). Within the RhoGDI family members, RhoGDI␣ is ubiquitously expressed, whereas RhoGDI␤ and RhoGDI␥ expression is tissue-specific (31). However, a recent report that RhoGDI␤ mRNA has a widespread tissue distribution (46) indicates a potential role in other tissues. Accumulating evidence indicates that Rho activity is important for normal muscle development. In this study, we demonstrated the importance of RhoGDI␤ in regulating MSCs differentiation into smooth muscle-like cells or adipocytes. We found that RhoGDI␤ was down-regulated by BMP4, and inhibition of Lox up-regulated it during adipocyte lineage commitment (Fig. 2). Overexpression of RhoGDI␤ completely prevented BMP4-induced adipocyte lineage commitment while simultaneously stimulating smooth muscle-like cell differentiation (Figs. 3 and 5). Thus, RhoGDI␤ appears to have opposing roles in adipogenesis and myogenesis. Previous studies by other groups demonstrated that the major Rho inhibitory protein p190-B RhoGAP can also direct the adipogenesis-myogenesis fate decision (47). In contrast, cells that exhibit excessive Rho activity are defective for adipogenesis and undergo myogenesis in response to insulin-like growth factor-1 exposure (47). However, the two studies yielded opposite conclusions about the determination of adipogenesis and myogenesis fates by GTPase inhibitors. This controversy is probably attributable to the different cell models and inducers used.
The commitment of MSCs to different lineages is regulated by many local tissue microenvironmental cues. Cell shape can drive MSC differentiation into different lineages in response to the same soluble factor. Recently, it was reported that cell shape regulates the commitment of human mesenchymal stem cells (hMSCs) to an adipocyte or osteoblast fate (23). Interestingly, a change of cell shape also implements a switch between chondrogenic and smooth muscle cell fates (24), suggesting a common molecular mechanism controlling lineage commitment. It is well established that RhoGTPases regulate cell shape through modulating the cytoskeleton (45). In the present study F-actin reorganization were observed during adipogenenic or myogenenic commitment. F-actin stress fibers were significantly decreased in BMP4-induced committed preadipocytes (15) (Fig. 6, A and B). The overexpression of RhoGDI␤ almost totally rescued the formation of F-actin stress fibers, which were disrupted during BMP4-induced adipocyte lineage commitment (Fig. 6B). These findings indicate that RhoGDI␤ could regulate adipogenesis versus myogenesis via cytoskeletal reorganization.
The small Rho GTPase family members known as Rho, Rac, and Cdc42 were initially linked to changes in the filamentous actin system involving the formation of stress fibers. Because Rho family small GTPases have well established roles in cytoskeletal remodeling, it is not surprising that Rho GTPases are involved in mediating the signals from cytoskeletal changes to determination of cell fate. Rho GTPases cycle between an inactive (GDP bound) state located in the cytosol and an active (GTP bound) state located on the membrane (48). RhoGDIs inhibit Rho GTPases by direct interaction and by maintaining Rho proteins in the inactive state in the cytoplasm and restraining them from the activation site on the membrane (31). A previous study demonstrated that LOX facilitates the formation of the p130 (Cas)/Crk/DOCK180 signaling complex that promotes Rac activation (26). Rac and Cdc42 activity and actin stress fibers also decreased with the reduction in LOX activity, indicating that Rac activation and actin stress fibers are associated with Lox. This study provides evidence that Rac1 is activated in response to BMP4 treatment, and overexpression of RhoGDI␤ (Lox downstream target) prevents BMP4-induced activation of Rac1 and related cytoskeletal reorganization and adipocyte lineage commitment (Figs. 3, 4, and 6), simultaneously promoting smooth muscle-like cell commitment (Fig. 5). Moreover, forced expression of RacV12 totally rescued the inhibition of the adipocyte phenotype by RhoGDI␤ and disrupted the stress fibers in cells overexpressing RhoGDI␤ (Figs. 4, C-E, and 6), simultaneously preventing the formation of the smooth muscle-like phenotype (Fig. 5). Because RhoGDIs are thought to be common inhibitors of all Rho family functions, it is possible that Rho and Cdc42 also contribute to RhoGDI␤mediated cell fate determination.
In summary, we have identified a novel role of RhoGDI␤ in regulating smooth muscle-like cell and adipocyte fate determination. Down-regulation of RhoGDI␤ by the BMP4-Lox signaling axis is required for Rac1-mediated disruption of filamentous actin and adipocyte lineage commitment. Overexpression of RhoGDI␤ inhibits the BMP4-induced activation of Rac1, resulting in the formation of stress fibers and smooth musclelike commitment by increasing phospho-MLC (Fig. 7). Our results suggest a novel role of RhoGDI␤ linking adipogenesis and myogenesis.