RESEARCH ARTICLE

Dexamethasone-induced cellular tension requires aSGK1-stimulated Sec5–GEF-H1 interactionHong-Ling Wang1, Chih-Hsuan Yang2, Hsiao-Hui Lee3, Jean-Cheng Kuo1, Sung-Sik Hur4, Shu Chien4,Oscar Kuang-Sheng Lee5, Shih-Chieh Hung5 and Zee-Fen Chang1,2,*

ABSTRACTDexamethasone, a synthetic glucocorticoid, is often used to induceosteoblast commitment of mesenchymal stem cells (MSCs), and thisprocess requires RhoA-dependent cellular tension. The underlyingmechanism is unclear. In this study, we show that dexamethasonestimulates expression of fibronectin and integrin α5 (ITGA5),accompanied by an increase in the interaction of GEF-H1 (alsoknown as ARHGEF2) with Sec5 (also known as EXOC2), amicrotubule (MT)-regulated RhoA activator and a component ofthe exocyst, respectively. Disruption of this interaction abolishesdexamethasone-induced cellular tension and GEF-H1 targeting tofocal adhesion sites at the cell periphery without affectingdexamethasone-induced levels of ITGA5 and fibronectin, and theextracellular deposition of fibronectin at adhesion sites is specificallyinhibited. We demonstrate that dexamethasone stimulates theexpression of serum-glucocorticoid-induced protein kinase 1(SGK1), which is necessary and sufficient for the induction ofthe Sec5–GEF-H1 interaction. Given the function of SGK1 insuppressing MT growth, our data suggest that the induction ofSGK1 through treatment with dexamethasone alters MT dynamicsto increase Sec5–GEF-H1 interactions, which promote GEF-H1targeting to adhesion sites. This mechanism is essential for theformation of fibronectin fibrils and their attachment to integrins atadhesion sites in order to generate cellular tension.

KEY WORDS: Cellular tension, GEF-H1, Glucocorticoid-mediatedosteogenesis, SGK1, Sec5

INTRODUCTIONGlucocorticoids mediate a wide array of physiological processesthrough their binding to the glucocorticoid receptor, which exertsbiological effects through transcriptional processes or non-genomicactions. It is known that endogenous glucocorticoids are requiredfor osteogenesis in mice (Bellows et al., 1987; Sher et al., 2004).Understanding the mechanism of glucocorticoid-receptor-mediated osteogenesis is important for developmental biology andregeneration medicine. Human bone-marrow-derived mesenchymalstem cells (hBMSCs) can be induced into osteoblast differentiationin tissue culture by dexamethasone, a man-made steroid ligand ofthe intracellular glucocorticoid receptor (Shih et al., 2011; Lo et al.,

2012). Mesenchymal stem cell (MSC) differentiation intoosteoblasts requires tension force through RhoA signaling,thereby promoting the gene expression of phenotypic osteoblastmarkers (McBeath et al., 2004; Engler et al., 2006). However, themechanism by which RhoA signaling is activated throughglucocorticoid receptor signaling is unclear. In this study, weinvestigated how dexamethasone acts to increase RhoA-dependentcellular tension.

Among RhoA activators, GEF-H1 (also known as ARHGEF2) isunique in its association with microtubules (MTs), where its activityin RhoA activation is inhibited by phosphorylation (Krendel et al.,2002; Meiri et al., 2009). Upon MT depolymerization, GEF-H1 isreleased and becomes activated. Therefore, GEF-H1 activates RhoAsignaling in a spatiotemporal manner that is dependent on MTdynamics. In vascular endothelial cells, GEF-H1 has been shown tomediate mechanical-stretch-induced endothelial cell permeabilityowing to MT destabilization (Birukova et al., 2010). Using integrin-based stretching, it has been reported that GEF-H1 contributes toRhoA activation to control the physical force through a feedbackmechanism (Guilluy et al., 2011). We have recently shown thatdexamethasone is able to increase the amount of GEF-H1 in focaladhesion sites, where it interacts with myosin-II heavy chain B toconfer stress-fiber polarization (Huang et al., 2014). In thisstudy, we further determined the molecular mechanism of howdexamethasone stimulates the function of GEF-H1 to increaseadhesion-mediated cellular tension.

A recent study has reported that GEF-H1 interacts with Sec5(also known as EXOC2), a component of the exocyst, to increaseexocytosis through RhoA activation (Pathak et al., 2012). Here,we have shown that treatment with dexamethasone stimulatesthe Sec5–GEF-H1 interaction, which is necessary to promote thelocalization of GEF-H1 to focal adhesions and to increase theextracellular deposition of fibronectin fibrils of hBMSCs. Wefurther found that dexamethasone-induced expression of serum-glucocorticoid-induced protein kinase 1 (SGK1) is necessary andsufficient to increase the Sec5–GEF-H1 interaction. It has beenreported previously that SGK1 increases MT depolymerization,through which neurite formation is promoted (Yang et al., 2006).In this study, we found that increased expression of SGK1decreased the density of MT growth in the cell periphery. Giventhe sequestration of GEF-H1 through MT binding, we proposethat glucocorticoid receptor activation upregulates SGK1 to causeMT instability, through which GEF-H1 is released from MTsto increase the interaction with Sec5 in the cell periphery. BecauseSec5 is a component of the exocyst, we further propose that thispathway mediates GEF-H1 targeting to focal adhesion sites,which is essential for transducing inside–out signals in orderto promote fibril formation through the binding of integrins tofibronectin, thereby regulating adhesion to generate cellulartension.Received 12 February 2015; Accepted 1 September 2015

1Institute of Biochemistry and Molecular Biology, National Yang-Ming University,Taipei 11221, Taiwan. 2Institute of Biochemistry and Molecular Biology, NationalTaiwan University, Taipei 11221, Taiwan. 3Department of Life Sciences and Instituteof Genome Sciences, National Yang-Ming University, Taipei 11221, Taiwan.4Department of Bioengineering and Institute of Engineering in Medicine, Universityof California at San Diego, La Jolla, CA 92093, USA. 5Institute of Clinical Medicine,National Yang-Ming University, Taipei 11221, Taiwan.

*Author for correspondence (zfchang@ym.edu.tw)

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RESULTSDexamethasone treatment upregulates adhesion-mediatingmolecules in hBMSCs and stimulates traction forceTo determine the effects of dexamethasone on adhesion-mediatedcell tension, we first tested the influence of dexamethasone oncell adhesion, stress fibers and spreading. The results showedthat dexamethasone treatment expanded the hBMSC-3A6 cell-spreading area within 6 h, with concurrent increases in the intensity

of F-actin staining and the number of focal adhesions (Fig. 1A,B). Itis known that clustering of integrin α5β1 heterodimers (composedof the subunits ITGA5 and ITGB1) mediates hBMSC adhesion tofibronectin and spreading. The expression levels of ITGA5 andfibronectin were increased upon treatment with dexamethasone in atime-dependent manner (Fig. 1C), whereas the level of ITGB1remained constant. Moreover, the extracellular deposition offibronectin colocalized with active ITGB1, as indicated by

Fig. 1. Dexamethasone increases adhesion-mediated tension. (A) hBMSC-3A6 cells were serum-starved for 48 h and treated with dexamethasone (Dex,0.1 µM) and vehicle (ethanol) for 6 h, followed by immunofluorescence staining of paxillin and F-actin. Representative images are shown. (B) Quantificationof immunofluorescence intensity of F-actin, the number of paxillin foci and cell area was performed and calculated relative to EtOH treatment (mean±s.e.m.,≥50 cells for each condition from three independent experiments, ***P<0.001). (C) Western blot analysis of ITGA5, ITGB1 and fibronectin (FN) from hBMSC-3A6cells in response to dexamethasone treatment. β-tub, β-tubulin. (D) Immunofluorescence staining images of active ITGB1 with the 9EG7 antibody (green) andfibronectin (red) on the surface of hBMSC-3A6 cells after dexamethasone treatment. The intensities of ITGB1 and fibronectin immunofluorescence staining weremeasured and calculated relative to the 0 h time point (mean±s.e.m., ≥25 cells for each condition from two independent experiments, *P<0.05, ***P<0.001).(E) hBMSCs seeded onto fibronectin-coated polyacrylamide gel were serum-deprived for 30 h and then treated with or without dexamethasone (0.1 μM) for 18 h.A set of representative images of maps of traction stress magnitude (Tmag; Pa, Pascal) and DIC images are shown. Cell spreading area (μm2) and net contractilemoment (pNm) of each cell were calculated and are represented by box and whisker diagrams (the box represents 25th to 75th percentile, the line representsmedian and whiskers represent minima to maxima) [non-treated (NT), n=16; dexamethasone-treated, n=13, ***P<0.001]. Scale bars: 20 μm.

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staining with the 9EG7 antibody, on the cell surface, which becamemore pronounced after dexamethasone treatment, indicating acorrelation of the synthesis and export of fibronectin to formadhesion complexes with fibrils on the cell surface (Fig. 1D).Furthermore, primary hBMSCs were plated onto compliantpolyacrylamide gel that had been coated with fibronectin tomeasure intracellular tension. After incubation of cells in serum-free medium for 30 h, the cells were treated with and without0.1 μM dexamethasone for 18 h, and we then measured the cell-spreading area and traction stress using traction-force microscopy(TFM) (Hur et al., 2009). The measurement of total intracellulartraction stress and digital computation showed that dexamethasonetreatment caused an enlargement of the cell-spreading area and asignificant increase in net contractile moment, which indicatescellular contractile strength (Fig. 1E). Because the compliant gelused for this traction-force analysis is much softer than coverslips,the increase in cellular tension on gel as a result of dexamethasonetreatment is slower than that on coverslips in stress fiber analyses.

Dexamethasone stimulates the MT-regulated GEF-H1–Sec5interactionWe found that the induction of stress fibers after 6 h ofdexamethasone treatment was abolished by either 1 h of co-treatment with Taxol, which stabilizes MTs (Fig. 2A). BecauseGEF-H1 is an MT-regulated RhoA activator, we further found that

GEF-H1 knockdown decreased dexamethasone-induced F-actinformation (Fig. 2B). Thus, dexamethasone-induced cellular tensionrequires MT dynamics and GEF-H1–RhoA signaling. It is knownthat dexamethasone is also used to induce adipogenic differentiationof MSCs, which downregulates RhoA-mediated contractility. It hasbeen shown that the inhibitory phosphorylation of GEF-H1 atresidue S885 promotes the binding of 14-3-3, which then enhanceslocalization of GEF-H1 to MTs (Zenke et al., 2004; Meiri et al.,2009). We found that treatment of hBMSCs with an adipogenesis-inducing cocktail markedly increased phosphorylation at residueS885 of GEF-H1, whereas an osteogenesis-inducing cocktail hadno effect (Fig. 2C). It is possible that an additional agent, such as 3-isobutyl-1-methylxanthine (IBMX) – an activator of protein kinaseA – in the adipogenesis cocktail is sufficient to increase S885phosphorylation, thereby suppressing GEF-H1-dependent cellulartension. We then asked whether dexamethasone treatmentstimulates GEF-H1 activity. However, the conventional GEF-H1activity assay using GST-RhoAG17A beads in a pulldown assayfailed to reveal any alterations in GEF-H1 activity upondexamethasone treatment (data not shown). It is possible thatdexamethasone only increases the local activation of GEF-H1,which is not revealed in a total GEF-H1 activity assay. We thenanalyzed the subcellular changes in GEF-H1 activity by assessingits spatial interaction with RhoA. The signal in a proximity ligationassay (PLA) for the GEF-H1–RhoA interaction was significantly

Fig. 2. Dexamethasone-induced tension requires microtubule instability and GEF-H1. (A) hBMSC-3A6 cells that had been dexamethasone (Dex)-treatedfor 5 h, followed by another 1 h of incubation with or without Taxol, were subjected to immunofluorescence staining of paxillin (green), F-actin (red) and DNA(blue). The relative intensity of F-actin staining was determined (right). Data represent mean±s.e.m., ≥50 cells for each condition from three independentexperiments, ***P<0.001. (B) Cells were infected with lentivirus encoding shRNAs against LacZ (shLacZ) or GEF-H1 (shGEF-H1) followed by serum starvation for48 h. Cells were treated with dexamethasone for 6 h, as described in the legend to Fig. 1A. Immunofluorescence staining images of paxillin, F-actin and DNA areshown. Immunofluorescence intensities of F-actin were quantified and calculated relative to those of control cells. Data represent mean±s.e.m.,≥50 cells for eachcondition from three independent experiments, ***P<0.001. (C) Cells were treated with an osteogenesis-inducing cocktail (osteo; 0.1 µM Dex, 50 µg/ml ascorbicacid, 10 mM β-glycerophosphate) and adipogenesis-inducing cocktail (adipo; 0.1 µM Dex, 50 µg/ml ascorbic acid, 50 µM indomethacin, 10 µg/ml insulin, 450 µMIBMX) in serum-free (SF) medium for 6 h and analyzed by western blotting using antibodies against GEF-H1 phosphorylated at residue S885 (pS885 GEF-H1),total GEF-H1 and β-tubulin (β-tub). The relative ratio of the intensity of pS885 GEF-H1 to total GEF-H1 from two independent experiments is shown belowthe blots. (D) Cells that had been treated as described in A were fixed for PLA analysis of the endogenous GEF-H1–RhoA interaction, as described in Materialsand Methods. Following ligation and polymerization reactions, cells were stained with Hoechst 33342 and PLA probe. The cell edge is indicated by the outline inwhite. PLA dots were quantified. Data represent mean±s.e.m., ≥50 cells for each condition from three independent experiments, ***P<0.001. Scale bars: 20 μm.

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increased by dexamethasone treatment (Fig. 2D). Taxol treatmentabolished this interaction signal, indicating the requirement of MTdynamics for GEF-H1–RhoA interaction. Knockdown of RhoA orGEF-H1 abolished the PLA signal, indicating the specificity of theinteraction signal of PLA (Fig. S1A). Dexamethasone is a class ofsteroid hormone that binds to the glucocorticoid receptor and is thentranslocated to nucleus, where the glucocorticoid receptor regulatesthe transcription of specific genes. The dexamethasone-inducedGEF-H1–RhoA signal and stress fiber formation were abolished byblocking either general transcription or glucocorticoid receptorfunction with actinomycin D and RU486, respectively (Fig. S1B),indicating the involvement of glucocorticoid-receptor-dependentgene transcription.It is well established that the exocyst is involved in the

adhesion process (Spiczka and Yeaman, 2008; Thapa et al., 2012)and that GEF-H1 interacts with the exocyst through a directinteraction with Sec5 (Pathak et al., 2012). We found thatknockdown of Sec5 also reduced the amount of dexamethasone-induced F-actin (Fig. 3A). We then tested whether dexamethasonetreatment increases the interaction of Sec5 with GEF-H1. By usingco-immunoprecipitation, we were unable to detect the effect ofdexamethasone on Sec5–GEF-H1 complex formation (data notshown). It is possible that their interaction is transient and spatiallycontrolled, which cannot be detected by co-immunoprecipitation.We then analyzed their interaction at subcellular locations byperforming the PLA. The results of the PLA clearly showed thatdexamethasone treatment significantly increased the number ofSec5–GEF-H1 interactions (Fig. 3B). The specificity of the PLAwas confirmed by using knockdown experiments (Fig. S2). Taxolco-treatment also abolished the dexamethasone-induced Sec5–GEF-H1 interaction signal (Fig. 3B).To strengthen the in vivo findings of an interaction between

GEF-H1 and Sec5 in response to dexamethasone treatment, weused an in vivo proximity protein labeling system, in which GEF-H1 is fused with APEX, an engineered ascorbate peroxidase thatcatalyzes biotin-phenol to short-lived phenoxyl radicals (<1 ms),dependent on H2O2 (Rhee et al., 2013). The radicals can thencovalently react with electron-rich amino acids to biotinylateneighboring and interacting proteins. To this end, hBMSC-3A6cells were transfected with cDNA-GEF-H1-GFP-APEX andtreated with dexamethasone for 6 h. To trigger GEF-H1–GFP–APEX-mediated biotin labeling, cells were incubated in mediumto which biotin-phenol had been added and then exposed tohydrogen peroxide for 5 min. After termination of labeling byusing PBS containing an antioxidant, cells were lysed, andendogenous Sec5 that had been biotinylated by GEF-H1–GFP–APEX as a result of their interaction was enriched usingstreptavidin beads. The western blot analysis showedbiotinylation of Sec5 by GEF-H1–GFP–APEX in an H2O2-dependent manner. Owing to the low transfection efficiency ofhBMSC-3A6 cells, the basal signal for Sec5 pulldown was low.However, dexamethasone treatment clearly increased the pulldownamount of Sec5 (Fig. 3C).It is known that GEF-H1 interacts with Sec5 through the

sequence spanning amino acids 119–236 of GEF-H1. Therefore,overexpression of a polypeptide containing this sequence disruptsthe interaction (Pathak et al., 2012). Therefore, we generated a cell-permeable hemagglutinin (HA)-tagged TAT-fusion polypeptide,TAT–HA–GEF-H1a.a.119–236, which was delivered into the cellduring dexamethasone treatment. Treatment with this peptidereduced both the PLA signal of GEF-H1 and Sec5, and the GEF-H1–GFP–APEX-mediated Sec5 biotinylation (Fig. 3D,E).

Consistently, treatment of cellswith the TAT–HA–GEF-H1a.a.119–236

polypeptide inhibited the dexamethasone-induced stress fiberformation and GEF-H1–RhoA interaction signaling (Fig. 3F),indicating that the Sec5–GEF-H1 interaction is crucial for theseresponses to dexamethasone.

Disruption of the Sec5–GEF-H1 interaction reducesdexamethasone-induced fibronectin deposition and GEF-H1targeting to focal adhesionsDelivery of TAT–HA–GEF-H1a.a.119–236 to cells attenuateddexamethasone-induced extracellular fibronectin fibril assemblyand ITGA5 adhesion formation after 6 h (Fig. 4A,B), whereas theaddition of TAT–HA–GEF-H1a.a.119–236 on its own had no effect(Fig. S3). Of note, the expression levels of fibronectin and ITGA5were unaffected by TAT–HA–GEF-H1a.a.119–236 (Fig. 4C). Treatingcells with TAT–HA–C3, an inhibitor of RhoA (Aepfelbacheret al., 1996), also did not affect dexamethasone-induced ITGA5expression (Fig. 4D). Although this treatment did not significantlyreduce the amount of ITGA5 on the cell surface, we noticed thatdexamethasone-induced ITGA5 fibril formation on the cell surfacewas clearly diminished. These results suggest that the Sec5–GEF-H1 interaction is essential specifically for extracellular fibronectinfibril formation on the cell surface, which is a process associatedwith the clustering of active integrin.

GEF-H1 has been identified as a component in the focal adhesioncomplex and as having a functional interaction with myosin-IIheavy chain B in order to regulate anisotropic stress-fiber orientation(Huang et al., 2014). The visualization of the distribution ofenhanced green fluorescent protein (EGFP)–GEF-H1 by using totalinternal reflection fluorescence (TIRF) microscopy showed thedynamic accumulation of GEF-H1 at focal adhesion sites afterdexamethasone treatment (Fig. 5A). Our western blot analysis offocal adhesion complexes, which had been isolated by usinghydrodynamic force, showed that dexamethasone treatment causeda substantial increase in the amount of GEF-H1 in focal adhesioncomplexes (Fig. 5B,C), and this was accompanied by increases inITGA5, paxillin, zyxin, vinculin and α-actinin. Co-treatment withTaxol prevented the increase of GEF-H1 in the focal adhesionfraction but had little effect on the increases of ITGA5, paxillin,zyxin, vinculin and α-actinin. These findings indicate that theincrease of GEF-H1 in focal adhesions is uniquely sensitive to MTdynamics. We then tested whether the Sec5–GEF-H1 interactionplays an essential role in determining GEF-H1 distribution in focaladhesions in response to dexamethasone stimulation. To this end,we tested the effect of the TAT–HA–GEF-H1a.a.119–236 peptide onthe focal adhesion targeting of GEF-H1, indicated by paxillinstaining, and EGFP–GEF-H1 distribution during dexamethasonetreatment. It should be mentioned that EGFP–GEF-H1 by itselfcaused enlarged focal adhesions at the cell periphery region. WithTAT–HA–GEF-H1a.a.119–236 polypeptide co-treatment, the distancebetween the boundary of EGFP–GEF-H1 and the cell edge wasclearly increased, and the colocalization of EGFP–GEF-H1 withfocal adhesions was decreased (Fig. 5D,E). It is likely that theinteraction with Sec5 is required for the trafficking of GEF-H1 to thecell periphery and to focal adhesions.

Dexamethasone upregulates the expression of SGK1, whichis necessary and sufficient for the Sec5–GEF-H1 interaction,dependent on MT instabilityNext, we wanted to understand which glucocorticoid-receptor-regulated gene is responsible for the upregulation of the Sec5–GEF-H1 interaction signal. Aforementioned experiments suggested that

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Fig. 3. Dexamethasone-promoted Sec5–GEF-H1 interaction is required for dexamethasone-induced stress fiber formation. (A) hBMSC-3A6 cells wereinfected with lentivirus encoding shRNAs against LacZ (shLacZ) or Sec5 (shSec5) followed by serum starvation for 48 h. Cells were then treated withdexamethasone (Dex) for immunofluorescence staining. Immunofluorescence images of paxillin (green), F-actin (red) and DNA (blue) are shown.Immunofluorescence intensities of F-actin were determined and calculated relative to those of the shLacZ control. Data represent mean±s.e.m., ≥100 cells foreach condition from three independent experiments, ***P<0.001. (B) Cells were treated with dexamethasone (6 h) in the absence or presence of Taxol (1 h) andwere fixed for PLA analysis of the Sec5–GEF-H1 interaction (GEF-H1/Sec5). Following ligation and polymerization reactions, cells were stained with Hoechst33342 and PLA probes. The cell edge is outlined in white. PLA dots were quantified. Data represent mean±s.e.m., ≥50 cells for each condition from threeindependent experiments, ***P<0.001. (C) After 24 h of serum starvation, cells were transfected with GFP–APEX and GEF-H1–APEX overnight, followed bytreatment with biotin-phenol, hydrogen peroxide and dexamethasone, as indicated for APEX-mediated biotinylation that is described in Materials and Methods.In vivo biotinylated proteins were enriched using streptavidin (SA) beads and analyzed by western blotting. β-tub, β-tubulin. (D) Cells were treated withdexamethasone (6 h) in the absence or presence of TAT–HA–GEF-H1aa119–236 and then fixed for PLA analysis of the Sec5–GEF-H1 interaction, as describedabove. (E) Cells were transfected with the expression vector encoding GEF-H1–GFP–APEX and treated with dexamethasone in the presence and absence of theTAT–HA–GEF-H1aa119–236 peptide for APEX-mediated biotinylation. Sec5 pull down with streptavidin beads was analyzed by western blotting. (F) Cells weretreated with dexamethasone (6 h) in the absence or presence of TAT–HA–GEF-H1aa119–236 and were fixed for PLA analysis of the GEF-H1–RhoA interaction.Cells were stained with F-actin and Hoechst 33342. PLA dots and the immunofluorescence intensity of F-actin were quantified. Data represent mean±s.e.m.,≥50cells for each condition from three independent experiments, ***P<0.001. Scale bars: 20 μm.

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MT instability is required for the dexamethasone-inducedSec5–GEF-H1 signal. We then searched for a glucocorticoid-receptor-regulated gene that destabilizes MTs. Serum- andglucocorticoid-induced protein kinase 1 (SGK1), which belongsto the AGC subfamily of Ser/Thr protein kinases, is rapidly inducedby glucocorticoids (Webster et al., 1993). By using real-time (RT)-PCR and western blot analysis, we showed that dexamethasonetreatment increased SGK1 mRNA and protein expression at 1 h(Fig. 6A). Knockdown of SGK1 abolished the dexamethasone-induced Sec5–GEF-H1 interaction signal and stress fiber formation(Fig. 6B).We co-treated cells with an SGK1 inhibitor, G650, whichalso strongly suppressed the dexamethasone-induced Sec5–GEF-H1 interaction signal, as well as stress fiber formation (Fig. 6C),indicating the contribution of its kinase activity. Consistently,inhibition of SGK prevented dexamethasone-induced extracellularfibronectin fibril assembly (Fig. 6D). By contrast, overexpression ofSGK1 stimulated the interaction of GEF-H1 and Sec5, asdexamethasone treatment did (Fig. 6E). In conclusion,glucocorticoid-receptor-induced SGK1 upregulation is necessaryand sufficient to increase the GEF-H1–Sec5 interaction signal.However, SGK1 overexpression on its own was unable to increasethe levels of ITGA5, fibronectin on the cell surface and stressfibers (data not shown). This is reasonable because the levels offibronectin and ITGA5 expression were not induced by SGK1.We further investigated the role ofMTs in the SGK1-inducedGEF-

H1–Sec5 interaction. Taxol treatment, which stabilizes MTs,prevented the SGK1-induced GEF-H1–Sec5 interaction signal(Fig. 7A), indicating the requirement of MT dynamics. MT

depolymerization by using nocodazole treatment restored thesesignals in cells that had been co-treated with dexamethasone andG650 (Fig. S4A). Thus, MT depolymerization bypasses therequirement of SGK1 activity for the GEF-H1–Sec5 interactionand stress fiber formation. Accordingly, we speculate that theglucocorticoid-receptor-induced SGK1 regulates MT stability inMSCs, thereby increasing the localized release of GEF-H1 forinteraction with Sec5. To test this hypothesis, we first treated cellswith nocodazole to depolymerize MTs, after which nocodazole waswashed out in order to observe the effect of glucocorticoid-receptor-induced SGK1 on MT re-growth. After nocodazole washout for10 min, MT re-growth established a MT network pattern in controlcells. As a comparison, dexamethasone-treated cells exhibited slowerMT re-growth, which was reversed by either RU486-mediatedglucocorticoid receptor inhibition or G650 co-treatment (Fig. 7B).Knockdown of SGK1 also rescued MT re-growth in dexamethasone-treated cells (Fig. S4B). Reciprocally, overexpression of EGFP–SGK1 by itself caused a lower amount ofMT fiber spreading over thecell periphery (Fig. S4C). Taking these results together, we proposethat dexamethasone stimulates glucocorticoid-receptor-mediatedtranscription of SGK1, which has a negative effect on MTpolymerization and stability, thereby increasing the localizedrelease of GEF-H1 for interaction with Sec5 and allowing targetingof GEF-H1 to focal adhesions. Together with glucocorticoidreceptor-induced expression of ITGA5 and fibronectin, this iscrucial for extracellular fibril formation, involving integrin andfibronectin, thus generating adhesion-mediated cellular tension(Fig. 7C).

Fig. 4. The Sec5–GEF-H1 interaction is required for dexamethasone-induced fibronectin deposition. (A) hBMSC-3A6 cells were treated withdexamethasone (Dex; 6 h and 16 h) in the absence or presence of TAT–HA–GEF-H1aa119–236; immunofluorescence staining of ITGA5 and fibronectin (FN) onthe cell surface was then performed. Scale bar: 20 μm. (B) The relative intensities of fibronectin and ITGA5 (integrin α5) staining were determined. Mean±s.e.m.,≥50 cells for each condition from two independent experiments. ***P<0.001. (C) Western blot analysis of ITGA5 and fibronectin in cell lysates. (D) Cells weretreated with dexamethasone for 6 h with and without TAT–HA–C3 (10 µg/ml); lysates were analyzed by western blotting.

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Fig. 5. The involvement of the GEF-H1–Sec5 interaction in dexamethasone-induced GEF-H1 subcellular distribution. (A) Serum-starved hBMSC-3A6cells were transfected with the expression vectors encoding EGFP–GEF-H1 and mApple–Paxillin (mApple-PXN) before dexamethasone (Dex; 0.1 µM)treatment. Images obtained by using time-lapse TIRF microscopy of EGFP–GEF-H1 accumulation in focal adhesions are shown. (B,C) Cells were treated, asindicated, for 6 h. Focal adhesion complexes were isolated by using the hypotonic method, as described in Materials and Methods. (B) The focal adhesion (FA)fraction and whole cell lysates were analyzed by western blotting (left) and silver staining (right). NT, non-treated. (C) Intensities of the staining of focal adhesioncomponents were determined by using densitometry analysis and normalized to the intensity of silver staining of the corresponding area in the parallel gel. Datarepresent mean±s.e.m. from three independent experiments. *P<0.05, **P<0.01. (D,E) Cells were transfected with pEGFP-GEF-H1, and were treated withdexamethasone (0.1 µM) in the presence or absence of TAT–HA–GEF-H1aa119–236. (D) Cells were fixed and stained for paxillin and imaged by using confocalmicroscopy (ZEISS LSM 700). (E) The area between the edge of the cell periphery and the GFP–GEF-H1 boundary (left panel), and the colocalization of EGFP–GFP-H1 with paxillin (right panel) were measured and analyzed (n=3, 15 cells in each experiment). *P<0.05, ***P<0.001. In parallel, peptide treatment wasconfirmed by western blotting. β-tub, β-tubulin. Scale bars: 2 µm (A, D magnified images); 20 μm (D top images).

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DISCUSSIONGlucocorticoid receptor signaling has been long recognized as achemical factor for inducing many important physiologicalprocesses. In this study, we investigated dexamethasone-inducedcellular tension in bone MSCs. Our results highlight the linkbetween glucocorticoid receptor signaling and the GEF-H1–exocystpathway. Most importantly, we have identified SGK1 expression asthe key player that mediates the dexamethasone-induced GEF-H1–exocyst pathway through control of MTs.

The exocyst complex is essential for tethering the traffickingvesicle to the plasma membrane before fusion for exocytosis(Pfeffer, 1999). It is known that RalA signaling controls exocytosisby activating Sec5 to stimulate exocyst complex formation(Moskalenko et al., 2002). Disruption of the RalA interactionwith Sec5 inhibits protein targeting to the basolateral domain inpolarized epithelial cells (Moskalenko et al., 2002). It has beenshown that RalA regulates the interaction of Sec5 with paxillin totarget the exocyst to the focal adhesion complex (Spiczka and

Fig. 6. Dexamethasone stimulates SGK1 expression to increase stress fibers and GEF-H1–Sec5 interaction. (A) hBMSC-3A6 cells treated withdexamethasone (0.1 μM) were harvested at the indicated times for western blot (bottom) and RT-PCR (top) analysis of SGK1. (B) Cells were infected withlentivirus carrying shRNA against LacZ (shLacZ) or SGK1 (shSGK1) and then serum-starved for 48 h. Cells were treated with dexamethasone (Dex; 0.1 μM, 6 h)and then fixed for PLA analysis of the GEF-H1–Sec5 interaction. Following ligation and polymerization reactions, cells were stained with Hoechst 33342, the PLAprobe and phalloidin. The cell edge is outlined in white. Data represent mean±s.e.m., ≥50 cells for each condition from three independent experiments,***P<0.001. (C) Cells were treated with dexamethasone (6 h) in the absence or presence of G650, and then fixed for PLA analysis as described above. Datarepresent mean±s.e.m., ≥50 cells for each condition from three independent experiments, ***P<0.001. (D) Cells were treated with dexamethasone (6 h) in theabsence or presence of G650. Cells were fixed for immunofluorescence staining of the cell surface for fibronectin (FN) and IGTA5 (left) and harvested for westernblotting (right panel). (E) Cells that had been transfected to express EGFP or EGFP–SGK1 were fixed for PLA analysis of the endogenous GEF-H1–Sec5interaction, as described above. PLA dots were quantified. Data represent mean±s.e.m., ≥50 cells for each condition from three independent experiments,***P<0.001. Scale bars: 20 μm.

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Yeaman, 2008). Therefore, RalA-regulated exocyst targeting mightplay a crucial role in molecular targeting for the organization ofpolarity, adhesion and migration. It has been reported that RalAregulates the direct interaction between Sec5 and GEF-H1 in orderto activate RhoA signaling for the assembly, stability andlocalization of the exocyst; these findings pinpoint the importanceof GEF-H1 in exocyst-mediated exocytosis (Pathak et al., 2012).Here, our data add to the knowledge of the function of this pathwayin bringing GEF-H1 to focal adhesion sites in order to establish theinside–out signal that is essential for fibril formation through thebinding of integrin and fibronectin on the cell surface. Here, itshould be emphasized that the expression levels of ITGA5 andfibronectin are increased by dexamethasone treatment, independentof the Sec5–GEF-H1 interaction and RhoA activation. However,disruption of this interaction inhibits fibronectin extracellular

deposition without affecting the ITGA5 that is expressed on thecell surface or the secretion of fibronectin. Therefore, the Sec5–GEF-H1 interaction is not required for the export of ITGA5 andfibronectin, rather it is required for the deposition of fibronectinfibrils and integrin clustering.

It is well known that the cellular GEF-H1 involved in RhoAactivation is mainly upregulated through MT disassembly.Similarly, the Sec5–GEF-H1 interaction is stimulated bytreatment with nocodazole and inhibited by Taxol. Thus, therelease of GEF-H1 uponMT disassembly is sufficient to activate theSec5–GEF-H1–RhoA axis. In agreement with this notion,nocodazole treatment also induces osteoblast differentiation (Zhaoet al., 2009). Then, the question is whether dexamethasonetreatment stimulates MT disassembly, by which the GEF-H1–exocyst signal is promoted. By using immunofluorescence staining

Fig. 7. The relationship between MT instability and the SGK1-induced GEF-H1–Sec5 interaction. (A) Serum-starved hBMSC-3A6 cells that had beentransfected with EGFP-SGK1were treated with Taxol (10 nM) for 1 h and then fixed for PLA analysis of Sec5–GEF-H1 interaction. PLA dots were quantified. Datarepresent mean±s.e.m., ≥50 cells for each condition from three independent experiments, ***P<0.001. (B) Cells were treated with dexamethasone (0.1 μM),RU486 (10 μM) and G650 (1 μM), as indicated, for 4 h followed by addition of nocodazole (0.2 μg/ml) for 2 h, which was washed out for the MT regrowth assay.After CSK buffer wash, cells were fixed and immunostained for α-tubulin. The fluorescence intensity of α-tubulin in each cell was quantified. Data representmean±s.e.m., ≥50 cells for each condition from three independent experiments, ***P<0.001. (C) A proposed model for dexamethasone-induced cellular tractionforce. Dexamethasone stimulates glucocorticoid receptor (GR)-mediated transcription of ITGA5, fibronectin and SGK1, through which the spatial interaction ofGEF-H1 with the Sec5-containing exocyst complex is promoted, dependent on MT instability. As a result, the subsequent exocyst-dependent GEF-H1–RhoAsignal, focal adhesion (FA) targeting of GEF-H1 and adhesion-associated fibronectin (FN) fibril formation are increased to stimulate cellular tension.

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of MTs, we did not find a clear change in the pattern of MTs indexamethasone-treated cells. However, from the nocodazolewashout experiment, we did find that dexamethasone treatmentcaused a delay in MT re-growth and that this effect requires thefunction of SGK1. Consistent with this finding, SGK1 is alsoessential for the dexamethasone-induced GEF-H1–Sec5 interaction.Accordingly, we propose that glucocorticoid receptor signalingstimulates the expression of SGK1, the kinase function of which hasa negative effect on MT growth. In turn, the amount of GEF-H1 thatis free fromMT sequestration is increased for interaction with Sec5.It remains an open question as to how the kinase function of SGK1regulates MTs in hBMSCs. Overall, our data shed new light on therole of dexamethasone-induced SGK1 in facilitating the GEF-H1–exocyst interaction, which cooperates with the induction offibronectin and ITGA5 expression to establish organization ofadhesions for force generation.

MATERIALS AND METHODSCell culture and transfectionPrimary hBMSCs were isolated from bone marrow, as previously described,with Institutional Review Board approval and informed consent (Lee et al.,2004a,b; Shih et al., 2011). These cells were maintained in MesenPRORS™ medium (Gibco) with 2 mM of L-glutamine. The hBMSC-3A6 cellline was generated and immortalized, as described previously (Hung et al.,2004; Tsai et al., 2010). The cells were maintained in low glucoseDulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with10% fetal bovine serum (FBS), 100 U/ml penicillin and 10 μg/mlstreptomycin. hBMSC-3A6 cells were used for all transient transfectionswith expression vector and Turbofect (Thermo Scientific). HEK293T cellswere co-transfected with 5 μg of pLKO or pLenti6 vectors encoding smallhairpin (sh)RNAs against GEF-H1 or Sec5 together with lentivirus packageplasmids containing 4 μg of pCMV delta 8.91 and 1 μg of pMD.G. Culturemedia were refreshed at 6 h post-transfection and lentivirus-containingmedia were collected and filtered for infection.

Analysis of cellular traction force on polyacrylamide gels withTFMElastic polyacrylamide gels with a Young’s elastic modulus E of 21.5 kPa(10% acrylamide and 0.2% bis-acrylamide) were prepared based on themethod developed byWang and Pelham (1998). Red-fluorescent (excitation580, emission 605) polystyrene beads (0.2 μm diameter, Invitrogen) wereadded to the polyacrylamide solution before polymerization. Gels weretypically 30–40 μm thick and covalently cross-linked with fibronectin(fibronectin, 20 μg/ml) on the surface of gel. Cells were allowed to attachonto the fibronectin-conjugated substrate overnight in cell culturemedium and then incubated with serum-free medium for treatment withdexamethasone. Cell outlines were determined from differential interferencecontrast microscopy (DIC) images for cell area analysis. The substratedeformation field was obtained from the lateral displacements of fluorescentbeads embedded in the gel. The lateral traction stresses exerted by the cellswere determined from the measured substrate deformation after solving theequation of static equilibrium for an elastic substrate (Del Alamo et al.,2007; Hur et al., 2009, 2012). The net contractile moment (M) wascalculated from the measured traction stresses by summing the diagonalcomponents of the shear moment matrix in its principal form (Mrot).M=trace (Mrot)=Mxx

rot+Myyrot; where Mxx

rot and Myyrot represent the total

contribution of cell-substrate contraction in the x and y directions (Butleret al., 2002). The procedure was performed using programs written inMATLAB (MathWorks, Natick, MA) (Lee et al., 2013). The finite analysiswas performed with ABAQUS (Dassault System̀es) (Del Alamo et al.,2007; Meili et al., 2010).

Immunofluorescence stainingCells were plated onto fibronectin-coated glass-coverslips. Cells were fixedwith 4% paraformaldehyde in PBS for 15 min at 37°C. Cells werepermeabilized with 0.3% Triton-X100 for 5 min and blocked with 5%

normal goat serum for 1 h at room temperature. Cells were incubated withprimary antibody overnight at 4°C and secondary antibody for 1 h at roomtemperature (anti-paxillin, 1:400, Becton Dickinson; anti-α-tubulin,1:1000, Sigma-Aldrich; goat anti-mouse AlexaFluor568, 1:400; goatanti-mouse AlexaFluor633, 1:400; phalloidin–AlexaFluor488 or –AlexFluor568, 1:200, Invitrogen). For extracellular fibronectin andITGA5 staining, cells were plated onto glass coverslips withoutfibronectin coating. Cells were fixed, blocked and stained as describedabove without permeabilization (anti-fibronectin, 1:400, GeneTex,GTX112794; ITGA5, clone mAb11, a gift from Kenneth Yamada,National Institutes of Health, Bethesda, MD). For MT quantification,cells were treated with 0.2 ng/ml nocodazole for 2 h. After being washedwith ice-cold PBS twice, MTs were allowed to re-grow in serum-freemedium for 10 min or 20 min at 37°C. Cells were incubated with CSKbuffer containing 80 mM PIPES pH 6.8, 1 mMMgCl2, 4 mM EGTA, 0.5%Triton X-100 and 10 nM Taxol for 30 s, and were then washed with PBSonce. After fixation with cold methanol for 10 min at −20°C, cells wereblocked and stained for α-tubulin, as described above. Images wereobtained by using fluorescence microscopy (Carl Zeiss, AxioObserver A1)and confocal laser scanning microscopy (Carl Zeiss, LSM700). AxioVisionRel. 4.8 and ZEN 2009 light Edition (Carl Zeiss) instruments were used forimage quantification and analysis. Statistical analyses were calculated byusing unpaired two-tailed Student’s t-test.

TIRF microscopySerum-starved hBMSCs were co-transfected with EGFP–GEF-H1 andmApple–paxillin. Cells were re-plated onto a 10 μg/ml fibronectin-coatedstandard circular glass coverslip (0.17 mm, n=1.515), which was thenplaced into a live-cell imaging chamber. Dexamethasone (0.1 μM) wasadded into the culture after conditional equilibrium for 1 h. Time-lapserecording was performed by using a total internal reflection fluorescence(TIRF) microscopy (AxioObserver Z1, Carl Zeiss) with an acousto-optictunable filter (AOTF) laser control and α Plan-Apochromat Oil DIC lens(100×, NA=1.46). Images were captured using an electron-multiplyingCCD (EMCCD, Evolve, Photometrics) camera operated throughAxioVision (Zeiss) image software.

Focal adhesion isolationFocal adhesion fractionation was achieved as described previously (Kuoet al., 2011). hBMSC-3A6 cells were plated onto 100-mm-dishes (around80% confluence) coated with 10 μg/ml of fibronectin and were washed withPBS once, and then the cells were subjected to hypotonic shock inlow-ionic-strength buffer with 2.5 mM tri-ethanolamine (TEA) (pH 7.0)for 3 min at room temperature. Hydrodynamic force was pulsed byusing a Waterpik instrument (setting ‘3.5’, Interplak dental water jetWJ6RW, Conair), which was filled with PBS containing PMSF, NaF andNa3VO4. The focal adhesions that remained on the dish were harvested foranalysis.

Construction of pTAT-HA-GEF-H1a.a.119–236 and purification ofTAT-fusion proteinsA PCR-amplified DNA fragment covering the GEF-H1a.a.119–236 sequencewas ligated into the pTAT-HA vector by using the XhoI and EcoRI cuttingsites. BL21 pLysS was transformed with pTAT-HA-GEF-H1a.a.119–236 andpTAT-HA-C3plasmid for peptide purification. ForTAT–HA–GEF-H1a.a.119–236

purification, BL21 pLysS were grown at 37°C to OD600=0.6 followed byincubation with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for4 h at 30°C. Bacteria were centrifuged and lysed in lysis buffer containing20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 5 mM imidazole, 0.1% TritonX-100 and 6 M urea. After centrifugation, cell lysates were incubated withnickel beads (GE Healthcare) for 1 h at 4°C. Peptide-coated beads werewashed with wash buffer containing 20 mM Tris-HCl, pH 7.5, 500 mMNaCl and 20 mM imidazole three times. Nickel-bead-bound peptides wereeluted with a buffer containing 20 mM Tris-HCl, pH 7.5, 500 mM NaCl,1 M imidazole and 6 M urea. The eluted solution was dialyzed with a500-fold volume of PBS for 6–8 h twice and concentrated by using aCentricon instrument (Merck Millipore). Purification of TAT–HA–C3 wassimilar to that described above.

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In situ proximity ligation and in vivoAPEX-mediated biotinylationassaysAn in situ PLA kit was obtained from Sigma-Aldrich (catalog numberDUO92008). Briefly, cells were fixed with 4% paraformaldehyde at 37°Cfor 15 min and were permeabilized in 0.3% Triton X-100 for 5 min.Following blocking, cells were stained using anti-RhoA (1:300, Santa Cruz,catalog number sc-418) or anti-Sec5 (1:600, Proteintech group, catalognumber 66011) antibodies with an anti-GEF-H1 antibody (1:600, GeneTex,GTX125893) overnight at 4°C. After incubation with the primaryantibodies, cells were washed with TBST with 0.1% Triton X-100 for5 min twice and then incubated with the PLA anti-mouse PLUS probe(Sigma-Aldrich, catalog number DUO92001) and anti-rabbit MINUS probe(Sigma-Aldrich, catalog number DUO92005) for 1 h at 37°C. Cells werewashed with TBST for 5 min twice and subjected to the ligation reaction for30 min at 37°C, followed by the amplification reaction for 100 min at 37°C.Afterwards, Hoechst 33342 and AlexaFluor488-conjugated phalloidin(Invitrogen, A12379) were added for DNA and F-actin staining,respectively. Images were acquired by using fluorescence microscopy(Carl Zeiss, AxioObserver A1). The PLA foci were counted by usingImageJ. Over 50 cells were examined for each experiment.

APEX-mediated biotinylation was performed as described previously(Rhee et al., 2013). Cells were transfected with GFP–APEX and GEF-H1–GFP–APEX overnight. To initiate APEX-mediated biotinylation, 500 μMofbiotin-phenol was added to the medium for 30 min before adding 5 mMhydrogen peroxide for 5 min. Cells were washed with quencher solution(10 mM sodium azide, 10 mM sodium ascorbate, 5 mM Trolox in PBS) andlysedwithRIPA buffer (50 mMTris HCl, pH 7.4, 150 mMNaCl, 0.1%SDS,0.1% DOC, 1% Triton X-100, 1 mM PMSF, 10 mM sodium azide, 10 mMsodium ascorbate, 5 mM Trolox). Afterwards, cell lysates were dialyzed in abuffer (50 mMTris HCl, pH 7.4, 150 mMNaCl, 0.1% SDS, 0.1%DOC, 1%Triton X-100) to reduce the biotin-phenol and were subjected to incubationwith streptavidin beads for 1.5 h and analysis by western blotting.

Statistical analysisStatistical significance was determined by using unpaired two-tailedStudent’s t-test.

AcknowledgementsWe thank Eminy H. Y. Lee (Academia Sinica, Taipei, Taiwan) for providing the SGK1expression vector and Kenneth Yamada (National Institutes of Health, Bethesda,MD) for providing the antibody against ITGA5 (mAb11).

Competing interestsThe authors declare no competing or financial interests.

Author contributionsH.-L.W. and C.-H.Y. performed most of the experiments. H.-H.L. performed the TFMexperiment. S.-S.H. analyzed TFM data; S.C. provided technical expertise for theTFM experiment. J.-C.K. performed the TIRF experiment. O.K.-S.L. and S.-C.H.provided primary cells and the hBMSC cell line. H.L.-W., C.H.-Y. and Z.F.-C.designed the experiments and wrote the manuscript, which was commented on byall authors.

FundingThis research was supported by the UST-UCSD International Center of Excellencein Advanced Bio-engineering, sponsored by Taiwan Ministry of Science andTechnology I-RiCE Program [grant number MOST103-2911-I-009-101].

Supplementary informationSupplementary information available online athttp://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.169961/-/DC1

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