Lakshmi S. Chaturvedi, 1,2,3 Harold M. Marsh, 2,3 and Marc D. Basson 1,3 1

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1 Am J Physiol Cell Physiol 301: C1224 C1238, First published August 17, 2011; doi: /ajpcell Role of RhoA and its effectors ROCK and mdia1 in the modulation of deformation-induced FAK, ERK, p38, and MLC motogenic signals in human Caco-2 intestinal epithelial cells Lakshmi S. Chaturvedi, 1,2,3 Harold M. Marsh, 2,3 and Marc D. Basson 1,3 1 Department of Surgery, Michigan State University, Lansing; 2 Department of Anesthesiology, Wayne State University, Detroit; and the 3 John D. Dingell Veterans Affairs Medical Center, Detroit, Michigan Submitted 22 December 2010; accepted in final form 11 August 2011 Chaturvedi LS, Marsh HM, Basson MD. Role of RhoA and its effectors ROCK and mdia1 in the modulation of deformation-induced FAK, ERK, p38, and MLC motogenic signals in human Caco-2 intestinal epithelial cells. Am J Physiol Cell Physiol 301: C1224 C1238, First published August 17, 2011; doi: /ajpcell Repetitive deformation enhances intestinal epithelial migration across tissue fibronectin. We evaluated the contribution of RhoA and its effectors Rho-associated kinase (ROK/ROCK) and mammalian diaphanous formins (mdia1) to deformation-induced intestinal epithelial motility across fibronectin and the responsible focal adhesion kinase (FAK), extracellular signal-regulated kinase (ERK), p38, and myosin light chain (MLC) signaling. We reduced RhoA, ROCK1, ROCK2, and mdia1 by smart-pool double-stranded short-interfering RNAs (sirna) and pharmacologically inhibited RhoA, ROCK, and FAK in human Caco-2 intestinal epithelial monolayers on fibronectin-coated membranes subjected to 10% repetitive deformation at 10 cycles/min. Migration was measured by wound closure. Stimulation of migration by deformation was prevented by exoenzyme C3, Y27632, or selective RhoA, ROCK1, and ROCK2 or mdia1 sirnas. RhoA, ROCK inhibition, or RhoA, ROCK1, ROCK2, mdia1, and FAK reduction by sirna blocked deformation-induced nuclear ERK phosphorylation without preventing ERK phosphorylation in the cytoplasmic protein fraction. Furthermore, RhoA, ROCK inhibition or RhoA, ROCK1, ROCK2, and mdia1 reduction by sirna also blocked strain-induced FAK-Tyr 925, p38, and MLC phosphorylation. These results suggest that RhoA, ROCK, mdia1, FAK, ERK, p38, and MLC all mediate the stimulation of intestinal epithelial migration by repetitive deformation. This pathway may be an important target for interventions to promote mechanotransduced mucosal healing during inflammation. RhoA; Rho-associated kinase; mammalian diaphanous formins; epithelial cells; strain; intestine; mechanotransduction; signaling THE INTESTINAL MUCOSA IS SUBJECT to repetitive deformation from diverse physical forces. Peristalsis (38), villus motility (97), and interaction between the mucosa and relatively noncompressible luminal contents (79) all repetitively deform the gut mucosa in complex ways. Epithelial migration is essential to gut development and during different pathological situations such as wound healing (20, 46). We have previously demonstrated that repetitive deformation promotes Caco-2 and intestinal epithelial cell (IEC)-6 wound closure on tissue fibronectin but not on a collagen I extracellular matrix, and intestinal epithelial proliferation on collagen substrates, but not on fibronectin (104). Fibronectin is deposited in tissue during chronic inflammation such as Crohn s disease (92). Thus, Address for reprint requests and other correspondence: M. D. Basson, Dept. of Surgery, College of Human Medicine, Michigan State Univ., 1200 East Michigan Ave., Suite 655, Lansing, MI ( marc.basson@hc.msu. edu). C1224 repetitive deformation may normally support mucosal proliferation on a collagen-rich basement membrane but may promote migration and wound healing when tissue fibronectin is increased by the inflammation that accompanies mucosal injury (32). Stimulation of intestinal epithelial wound closure across tissue fibronectin by repetitive deformation requires focal adhesion kinase (FAK), phosphatidylinositol 3-kinase, protein kinase B, glycogen synthase kinase, extracellular signal-regulated kinase (ERK), p38, and myosin light chain (MLC) (11, 28, 104). ERK and p38 activation in this setting requires FAK but is independent of Src (11, 28). In particular, ERK is required within the nucleus for deformation to promote epithelial motility. (30). However, the upstream mediators that activate this pathway are unclear. Epithelial cell sheet migration involves F-actin restructuring through RhoA guanosine-5=triphosphatases (60, 78). RhoA and its effectors Rho-associated kinases (ROCK1 or ROK and ROCK2 or ROK ) and mdia1 (mammalian diaphanous formins) influence motility in various cells (34, 35, 85), including epithelial cells in response to other stimuli (7, 36, 42, 57, 60, 62, 76, 99). FAK, ERK, and p38 activation depend on activation of the RhoA/Rho-associated coiled coil-containing protein kinase (ROCK) signaling pathway in other settings (3, 10, 24, 37, 41, 44, 47, 83, 87, 88). Furthermore, ROCK activation by RhoA blocks the activity of MLC phosphatase, increasing phosphorylated MLC and cell contractility (80). However, their roles in the modulation of epithelial motility by repetitive deformation are unknown. We studied the contribution of RhoA and its effectors ROCK and mdia1 to deformation-induced intestinal epithelial motility and FAK, ERK, p38, and MLC signaling in Caco-2 human intestinal epithelial cells on tissue fibronectin. In particular, we sought to evaluate the role of RhoA and its effectors in the regulation of strain-induced activation of FAK, ERK, p38, and MLC and cell migration in human Caco-2 intestinal epithelial cells. We used the Flexercell apparatus (Flexcell, McKeesport, PA) to rhythmically deform Caco-2 cell monolayers cultured on tissue fibronectin-coated flexiblebottomed wells at an average 10% repetitive deformation at 10 cycles/min (5), similar in magnitude and frequency to the irregular repetitive deformation that the mucosa experiences in vivo during peristalsis or villous motility (97). We characterized RhoA and ROCK activation and RhoA and ERK translocation in response to repetitive deformation in Caco-2 intestinal epithelial cells and used pharmacological antagonists and smart-pool double-stranded short-interfering RNAs (sirnas) to trace a mechanotransduced pathway that links these signals to a novel motogenic cascade.

2 RhoA MEDIATES THE MOTOGENIC EFFECTS OF STRAIN IN Caco-2 CELLS C1225 EXPERIMENTAL PROCEDURES Materials. Dulbecco s modified Eagle s medium (DMEM), oligofectamine, lipofectamine, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal antibody, phosphospecific polyclonal antibody to FAK at Tyr(P) 397 and Plus Reagent were from Invitrogen (Carlsbad, CA), Western blot stripping reagent was from Chemicon International (Temecula, CA), human transferrin was from Roche Applied Science (Indianapolis, IN), and the FAK inhibitor 1,2,4,5-benzenetetraamine tetrahydrochloride (Y15), trypsin, and horseradish peroxidase-conjugated rabbit anti-mouse IgG were from Sigma (St. Louis, MO). Phosphospecific polyclonal antibodies to FAK at Tyr(P) 925, p44/p42 (phospho-erk1/2)-thr(p) 202/ Tyr(P) 204, p38 (phospho-p38)- Thr(P) 180 /Thr(P) 182, pmlc2 (phospho-mlc2)-ser(p) 19, rabbit polyclonal antibody to p44/42 (total ERK1/2), p38 (total p38), ROCK1/ ROK- (C8F7), RhoA-GDI, histone-2a, and horseradish peroxidaseconjugated anti-rabbit and anti-mouse IgG were from Cell Signaling (Beverly, MA). Phosphospecific rabbit polyclonal antibody to myosin phosphatase target subunit 1 [MYPT1-Thr(P) 696 ] and rabbit polyclonal antibody to MYPT1 and ROK- /ROCK2 (clone A9W4), RhoA activation assay kit, and monoclonal antibody to FAK (clone 4.47) were from Millipore (Temecula, CA). Inhibitors of RhoA (exoenzyme C3) and ROCK [(R)-( )-trans-n-(4-pyridyl)-4-(1-aminoethyl)cyclohexanecarboxamide, 2HCl, Y27632] and mouse monoclonal anti- tubulin antibody (DM1A) were from EMD Biosciences (San Diego, CA). Rabbit polyclonal E-cadherin (H-108) antibody was from Santa Cruz Biotechnology (Santa Cruz, CA), mouse monoclonal GAPDH was from Meridian Life Science (Saco, ME), and a nuclear/cytosol fractionation kit was from BioVision Research Products (Mountain View, CA). sirnas to human RhoA, ROCK1, ROCK2, mdia1, FAK, and control nontargeting sirna 1 (NT1 sirna) were from Dharmacon (Lafayette, CO). Cell culture. The Caco-2 BBE intestinal epithelial cells we studied were a subclone of the original Caco-2 cell line selected for its ability to differentiate in culture as indicated by apical brush-border formation and expression of brush-border enzymes (72). Caco-2 cells were originally derived from a human colon carcinoma but are well differentiated, resemble nonmalignant intestinal epithelial cells, and are a common model for the study of nonmalignant intestinal epithelial biology (1, 18, 31, 59, 106). Caco-2 cells respond to strain similarly to nonmalignant rat IEC-6 cells (11, 13, 29, 100, 101, 104) as well as to nonmalignant primary intestinal epithelial cells (103). Caco-2 cells were cultured as previously described (13). Mechanical strain. Caco-2 cells plated on elastomer membranes (Flexcell International, Hillsborough, NC) coated with tissue fibronectin (104) were exposed to continuous cycles of average 10% strain/relaxation (Flexcell 4000; Flexcell) at 10 cycles/min. Signal inhibition. Exoenzyme C3 and Y27632 were dissolved in sterile double-distilled water, separated into aliquots, stored at 20 C, and diluted in culture medium before use. Caco-2 cells were pretreated with exoenzyme C3 (2.5 g/ml) and Y27632 (10 M) for 60 min before strain. For sirna studies, Caco-2 cells were plated at 50% confluence on Flex I six-well plates precoated with tissue fibronectin 1 day before transfection. sirnas were combined with Plus reagent in Opti-MEM as described previously (12). Migration studies. Motility was assayed by closure of small circular wounds as previously described (11, 53, 64, 104). Western blots. Confluent cells on tissue fibronectin were changed to serum-free media for 24 h, subjected to strain, and lysed as previously described (13). Blots were probed with appropriate primary and secondary antibodies and detected by ECL (Amersham Biosciences, Piscataway, NJ) with a Kodak Image Station 440CF Phosphoimager (Kodak Scientific Imaging Systems, Rochester, NY). RhoA GTPase activity assays. RhoA activity was assessed by pull-down assay as per the manufacturer (Millipore). Active RhoA levels were determined by glutathione S-transferase-conjugated Rhotekin binding domain 7 89 residues (GST-Rhotekin) pull-down assays. RhoA translocation. The RhoA translocation assay was performed as described previously with slight modifications (13, 51). An equal amount of cell lysates (5 10 g protein) of cytosolic and membrane fractions was loaded per well on a 12.5% SDS-polyacrylamide gel, transferred to nitrocellulose, and immunoblotted with monoclonal anti-rhoa antibody to assess the amounts of RhoA in each fraction. The blots were also probed with RhoA-GDI and E-cadherin as protein-loading controls for cytosolic and membrane fractions, respectively. Nuclear/cytosol fractionation. Nuclear and cytoplasmic protein fractions were prepared using a fractionation kit as per the manufacturer (BioVision Research Products). Briefly, cells were grown to confluence and were serum-starved for 24 h before unstimulated cells at time 0 were compared with cells stimulated by strain for 1 h. Statistical analysis. All studies were done independently at least three times unless indicated otherwise. Data were expressed as means SE and analyzed using paired or unpaired t-tests with Bonferroni corrections as appropriate, seeking 95% confidence. RESULTS Strain activates RhoA. To confirm that RhoA is activated in response to repetitive deformation, we exposed cells to repetitive deformation for 0, 2, 5, and 15 min and measured active RhoA (GTP-bound) by pull-down assay. Small guanine nucleotide-binding proteins cycle between a GDP-bound inactive form and a GTP-bound active form and interact with their effector proteins preferentially in the GTP-bound form (93). RhoA activity was increased fold (n 4, P 0.05, Fig. 1A) as early as 5 min after strain initiation and appeared to increase fold compared with unstretched control further at 15 min after initiation (n 4, P 0.05, Fig. 1A). Strain induces RhoA translocation. We also assessed translocation in cells exposed to cyclic strain for 15 min. Strain stimulated RhoA translocation from a cytosol-enriched fraction to a membrane-enriched fraction (Fig. 1B, top). Blots were reprobed with antibodies to the cytosol-abundant RhoA-GDI (Fig. 1B, bottom) and the membrane-abundant E-cadherin (Fig. 1B, bottom) to confirm fraction enrichment and equal loading. Strain increased the ratio of membrane to cytosolic RhoA (132 8%, n 4, P 0.05, Fig. 1B), consistent with our observations of RhoA activation. Strain induces myosin phosphatase target subunit-1 phosphorylation. On activation, ROCK phosphorylates the substrate myosin phosphatase target subunit-1 (MYPT-1) at Thr 696 (45), also called the myosin-binding subunit of myosin phosphatase. Deformation increased MYPT1 phosphorylation at Thr fold compared with unstretched intestinal epithelial cells (n 5, P 0.05, Fig. 1C). RhoA is essential for the stimulation of migration by cyclic strain. We next sought to determine whether RhoA was required for deformation to stimulate wound closure. We made uniform circular wounds in confluent monolayers of Caco-2 cells and treated the monolayers with the RhoA inhibitor exoenzyme C3 (2.5 g/ml) or a DMEM vehicle control for 60 min before repetitive deformation for 24 h. Control cells were treated similarly but without strain. Wounds in DMEM-treated Caco-2 monolayers closed more rapidly in response to deformation than DMEM-treated static controls (n 8, P 0.05, Fig. 2A). Exoenzyme C3 prevented strain stimulation of motility without affecting basal migration (Fig. 2A). In parallel

3 C1226 RhoA MEDIATES THE MOTOGENIC EFFECTS OF STRAIN IN Caco-2 CELLS Fig. 1. Repetitive deformation activates RhoA and Rho-kinase (ROCK) across fibronectin in Caco-2 cells. A: RhoA is rapidly activated by cyclic strain. The effect of cyclic strain on RhoA activation was quantitated by pull-down assays of the glutathione S-transferase-conjugated Rhotekin binding domain 7 89 amino acid residues (GST-Rhotekin) to specifically precipitate active RhoA as described under EXPERIMENTAL PROCEDURES. Total RhoA (t- RhoA) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as controls; representative blots are shown above the bars summarizing densitometric analysis (n 4, *P 0.05). B: cyclic strain also stimulates RhoA translocation from cytosol to the cell membrane. Rho-GDI and E-cadherin served as controls for cytosolic and membrane fractions, respectively. Typical blots are shown on top, and densitometric analysis is shown on bottom (n 4, *P 0.05). C: cyclic strain stimulates ROCK activity. Phosphorylation of myosin phosphatase target subunit 1 (MYPT1, Thr 696 ) was determined by Western blot analysis using a phosphospecific antibody (p-mypt1). Total MYPT1 (tmypt1) served as control. Typical blots are shown on top, and densitometric analysis is shown on bottom (n 5, *P 0.05). studies, we transfected Caco-2 cells with sirna to RhoA or a control NT1 sirna sequence for h until the cells had achieved confluence and then assessed migration with and without strain for 24 h. sirna to RhoA reduced total RhoA protein % (Fig. 2B, top, typical blots). NT1-transfected cells exhibited similarly increased migration in response to deformation compared with cells without strain. We observed a trend toward increased basal motility with RhoA reduction by sirna, but this did not achieve statistical significance (P 0.085). However, the motogenic effect of strain was completely blocked in cells transfected with sirna to reduce RhoA (n 10, P 0.05, Fig. 2B). These results suggest that RhoA is necessary for the strain-induced motogenic response in Caco-2 intestinal epithelial cells. RhoA is required for strain-induced FAK phosphorylation. Next we sought to link the RhoA requirement to the FAK phosphorylation previously shown to be required for strain to stimulate motility (11, 28). Both exoenzyme C3 treatment and RhoA reduction by sirna blocked deformationinduced FAK phosphorylation at Tyr 925 (n 5, P 0.05 for each, Fig. 3, A and B). RhoA is essential for strain-induced p38 MAPK phosphorylation. We have previously shown that p38 is required for deformation-induced migration (28). We next investigated the modulation of p38 activation by RhoA in response to repetitive deformation. Both exoenzyme C3 treatment and RhoA reduction by sirna blocked deformation-induced p38 phosphorylation after the initiation of cyclic strain (n 5, P 0.05 for each, Fig. 3, C and D). mdia1 is required for the stimulation of migration by cyclic strain. We next sought to determine whether an effector of RhoA-GTPase diaphanous (mdia1) is required for the effect of deformation on wound closure. Cells transfected with sirna targeted to mdia1 exhibited a % mdia1 reduction, using GAPDH as a protein-loading control (Fig. 4A, top, typical blots). NT1-transfected cells migrated more rapidly in response to deformation than cells not subjected to strain. This motogenic effect was blocked in cells transfected with sirna to reduce mdia1 (n 10, P 0.05, Fig. 5A). However, mdia1 reduction significantly increased basal migration in the absence of deformation (n 10, P 0.05, Fig. 5A). These results suggested that mdia1 activity stimulates basal migration in the

4 RhoA MEDIATES THE MOTOGENIC EFFECTS OF STRAIN IN Caco-2 CELLS Fig. 2. Role of RhoA in deformation-induced migration across fibronectin in Caco-2 cells. A: RhoA inhibition by exoenzyme C3 inhibits deformationinduced migration. Circular wounds were made in monolayers of cells treated with complete DMEM (vehicle control) or pretreatment for 60 min with the RhoA inhibitor exoenzyme C3 (2.5 g/ml), and these wounds were photographed. Cells were then cultured under static conditions (open bars) or conditions of repetitive deformation (filled bars) for 24 h before the holes were measured again, and wound closure was calculated as described under EXPER- IMENTAL PROCEDURES. RhoA inhibitor exoenzyme C3 prevented the stimulation of migration by strain. Values are means SE; n 8 from 1 of 3 similar experiments. *P B: RhoA reduction by smart-pool double-stranded short-interfering RNAs (sirna) inhibits deformation-induced migration. RhoA sirna transfection achieved 70% knockdown as shown by immunoblot on top. Circular wounds were made in monolayers of cells transfected with either nontargeting sirna 1 (NT1) or sirna targeted to RhoA, and these wounds were photographed. Cells were then cultured under static conditions (open bars) or conditions of repetitive deformation (filled bars) for 24 h before the holes were measured again, and wound closure was calculated. Deformation stimulated wound closure in cells transfected with the NT1 sequence but not in cells in which RhoA had been reduced. Values are means SE; n 10 from 1 of 3 similar experiments. *P absence of repetitive deformation and mediates the stimulation of migration by repetitive deformation in Caco-2 intestinal epithelial cells. mdia1 is required for strain-induced FAK and p38 phosphorylation. We have previously shown that repetitive deformation stimulates p38 phosphorylation in intestinal epithelial C1227 cells cultured on fibronectin and that deformation-induced motility requires FAK, MLC, and p38 (28). Because mdia1 appeared required for strain-stimulated migration, we therefore evaluated whether mdia1 is important in FAK phosphorylation at Tyr 925 and p38 phosphorylation in response to deformation. mdia1 reduction by sirna blocked deformation-induced FAK phosphorylation (n 5, P 0.05 for each, Fig. 4B) as well as deformation-induced p38 phosphorylation (n 5, P 0.05 for each, Fig. 4C). ROCK is essential for the stimulation of migration by cyclic strain. We next sought to determine whether ROCK is required for strain to stimulate motility. Wounds in DMEM-treated Caco-2 monolayers closed more rapidly in response to strain than DMEM-treated static controls (n 10, P 0.05, Fig. 5A). ROCK inhibition by Y27632 increased basal migration in the absence of repetitive strain (n 10, P 0.05, Fig. 5A) but prevented strain stimulation of motility (n 10, P 0.05, Fig. 5A). sirna targeted to either ROCK1 or ROCK2 reduced their respective targets by and 65 5%, using GAPDH as a protein-loading control without affecting protein levels of the opposite isoform (Fig. 5B, top, typical blots). As expected, NT1-transfected cells exhibited increased migration in response to deformation compared with cells transfected with sirna to reduce ROCK1 or ROCK2 (n 10, P 0.05, Fig. 5B). Furthermore, either ROCK1 or ROCK2 reduction increased basal migration (n 10, P 0.05, Fig. 5B) inthe absence of strain. These results suggested that ROCK1 and ROCK2 each promote basal motility and mediate the stimulation of Caco-2 motility by strain. ROCK is required for strain-induced FAK and p38 phosphorylation. Because ROCK was required for strain-stimulated migration, we sought to link it to FAK and p38 activation. Either treating with Rho-kinase inhibitor (Y27632) or reducing ROCK1 or ROCK2 by sirna blocked deformationinduced tyrosine phosphorylation of FAK at Ty r925 (n 5, P 0.05 for each, Fig. 6, A C) and p38 phosphorylation (n 5, P 0.05 for each, Fig. 6, D F) after strain initiation. RhoA and ROCK both are required for strain-induced MYPT1 phosphorylation. Next we sought to link RhoA to ROCK activation. Either Y27632 treatment or ROCK1 or ROCK2 reduction by sirna blocked strain-induced MYPT-1 phosphorylation [n 5, P 0.05 each, Supplemental Fig. S1, A and B (Supplemental data for this article may be found on the Am J Physiol: Cell Physiol website.)]. Furthermore, either exoenzyme C3 or RhoA reduction by sirna completely blocked strain-induced MYPT1 phosphorylation (n 5, P 0.05 for each, Supplemental Fig. S1, C and D). These results indicate that repetitive deformation leads to RhoA/ROCKdependent Thr 696 phosphorylation of MYPT1 and suggest that RhoA directly mediates ROCK signaling in the strain-induced motogenic response in intestinal epithelial cells. RhoA, ROCK, mdia1, and FAK are required for straininduced ERK phosphorylation in the nucleus but not in the cytosol. Because RhoA, ROCK, mdia1, and FAK seem required for strain-stimulated migration, we sought to link these signals to the nuclear ERK activation previously reported required for strain to stimulate motility (11, 28). Both exoenzyme C3 treatment and RhoA reduction by sirna blocked deformation-induced ERK phosphorylation in the nuclear fraction after the initiation of cyclic strain (n 5, P 0.05 each, Fig. 7, A and B). Interestingly, neither treating the cells with

5 C1228 RhoA MEDIATES THE MOTOGENIC EFFECTS OF STRAIN IN Caco-2 CELLS Fig. 3. RhoA inhibition or reduction inhibits deformation-induced focal adhesion kinase (FAK)-Tyr 925, p38 phosphorylation. A F: effects of exoenzyme C3 (2.5 g/ml) or RhoA reduction by sirna on the phosphorylation of FAK at Tyr 925 (A and B) or p38 (C and D) were assessed by Western blot analysis of lysates from cells subjected to cyclic strain for 15 min. Total FAK (t-fak) and total p38 (t-p38) served as a protein-loading control. Top: typical blots; bottom, densitometric analyses. A: cyclic strain stimulates FAK-Tyr 925 phosphorylation in vehicle-treated cells (n 5, *P 0.05). Exoenzyme C3 completely blocked strain-induced FAK-Tyr 925 phosphorylation (n 5, *P 0.05). B: cyclic strain stimulates FAK-Tyr 925 phosphorylation in NT1-treated cells (n 5, *P 0.05). RhoAreduced cells blocked strain-induced FAK- Tyr 925 phosphorylation (n 5, *P 0.05). C: cyclic strain stimulates p38 phosphorylation in vehicle-treated cells (n 5, *P 0.05). RhoA inhibition with exoenzyme C3 (exo-c3) completely blocked strain-induced p38 phosphorylation (n 5, *P 0.05). D: cyclic strain stimulates p38 phosphorylation in NT1-treated cells (n 5, *P 0.05). RhoA-reduced cells blocked strain-induced p38 phosphorylation (n 5, *P 0.05). exoenzyme C3 nor reducing RhoA by sirna blocked the deformation-induced increase in ERK phosphorylation in the cytoplasmic protein fraction (n 5, P 0.05 for each, Supplemental Fig. S2, A and B). In fact, RhoA inhibition by exoenzyme C3 increased basal cytoplasmic ERK phosphorylation (n 5, P 0.05, Supplemental Fig. S2A), but RhoA reduction by sirna did not seem to alter basal cytoplasmic ERK phosphorylation (Supplemental Fig. S2B). Similarly, mdia1 reduction by sirna blocked deformation-induced ERK phosphorylation in the nuclear fraction after the initiation of cyclic strain (n 5, P 0.05 each, Fig. 7C). However, reducing mdia1 by sirna did not block the strain-induced cytoplasmic ERK phosphorylation in either (n 5, P 0.05 for each, Supplemental Fig. S2C). ROCK inhibition with Y27632 and ROCK1, ROCK2, and FAK reduction by sirna blocked strain-induced ERK phosphorylation in the nuclear fraction (n 5, P 0.05 for each, Fig. 7, D-F). However, neither Y27632 treatment nor ROCK or FAK reduction by sirna blocked strain-induced ERK phosphorylation in the cytosolic fraction (n 5 each, Supplemental Fig. S2, D F). RhoA, ROCK, and mdia1 are required for strain-induced MLC phosphorylation. MLC phosphorylation accompanies strain-stimulated migration (104). However, the upstream regulators of this effect were not known. Inhibiting either RhoA by exoenzyme C3 or ROCK by Y27632 blocked strain-induced MLC phosphorylation (n 5, P 0.05 each, Fig. 8, A and B). ROCK inhibition by Y27632 also reduced basal MLC phosphorylation (n 5, P 0.05, Fig. 8B), but RhoA inhibition by C3 did not. Furthermore, reducing RhoA, ROCK1, or ROCK2 and mdia1 by specific sirna blocked strain-induced MLC phosphorylation (n 5, P 0.05 each, Fig. 8, C-E). This suggests that RhoA-ROCK/mDia1 signaling is essential for modulation of strain-induced MLC phosphorylation. FAK inhibition does not prevent strain-induced RhoA activation. RhoA blockade by exoenzyme C3 or sirna prevents FAK activation as above. However, in some experimen-

6 RhoA MEDIATES THE MOTOGENIC EFFECTS OF STRAIN IN Caco-2 CELLS C1229 tal conditions, FAK can also modulate RhoA (66, 73). To elucidate this relationship further, we inhibited FAK with 10 M Y15 for 60 min in confluent serum-starved Caco-2 cells then exposed to repetitive deformation for 15 min. Control cells were either untreated or pretreated with Y15 but not exposed to strain. The inhibition of strain stimulation of FAK activation by Y15 (n 4, P 0.05, Fig. 9A) did not prevent strain activation of RhoA (Fig. 9B). This suggests that RhoA acts upstream of FAK in mechanotransduced signaling in human intestinal epithelial cells. DISCUSSION The intestinal epithelial lining is constantly wounded during normal function and must be repaired regularly (55). The gut mucosa is repetitively deformed by several factors, including peristalsis (38), shear stress from endoluminal chyme (55), and villous motility (97). Increasing evidence suggests that repetitive deformation engendered by peristalsis, villous motility, and interaction with luminal chyme may be trophic for the gut mucosa in normal function but that the intestinal epithelial Fig. 4. Role of mammalian diaphanous formins (mdia1) in deformation-induced migration across fibronectin. A: mdia1 reduction by sirna inhibits deformationinduced migration. mdia1 sirna transfection achieved 75% knockdown as shown by immunoblot on top. Circular wounds were made in monolayers of cells transfected with either NT1 or sirna targeted to mdia1, and these wounds were photographed. Cells were then cultured under static conditions (open bars) or conditions of repetitive deformation (filled bars) for 24 h before the holes were measured again, and wound closure was calculated. Deformation stimulated wound closure in cells transfected with the NT1 sequence compared with cells not subjected to strain. mdia1- reduced cells increased basal wound closure (n 10 from 1 of 3 similar experiments. #P 0.05), but deformation reversed the motogenic effects (n 10 from 1 of 3 similar experiments, *P 0.05). Values are means SE. B: cyclic strain stimulates FAK-Tyr 925 phosphorylation in NT1-treated cells (n 5, *P 0.05). mdia1-reduced cells blocked strain-induced FAK-Tyr 925 phosphorylation (n 5, *P 0.05). C: cyclic strain stimulates p38 phosphorylation in NT1- treated cells (n 5, *P 0.05). mdia1-reduced cells blocked strain-induced p38 phosphorylation (n 5, *P 0.05). response to repetitive deformation may be altered by inflammatory or other states in which plasma or tissue fibronectin levels are increased (reviewed in Ref. 4). Fibronectin is deposited in tissue in settings of chronic inflammation such as Crohn s disease (92) and increased in the plasma in other conditions where gut motility is altered, such as sepsis (48). In vitro, repetitive deformation stimulates Caco-2 and IEC-6 migration across a fibronectin matrix but inhibits migration across a collagen I matrix (104), suggesting that repetitive deformation stimulates the intestinal mucosa in a complex fashion depending on fibronectin levels. The induction of migration in primary colonic lamina propria fibroblasts isolated from patients with inflamed Crohn s disease is also fibronectindependent (8). Although the stimulation of wound closure by repetitive deformation described here in vitro may seem modest, these results are highly statistically significant. Such apparently small changes may be important in an intricately and tightly regulated biological system that is experiencing constant mucosal injury and where the mucosal barrier represents an

7 C1230 RhoA MEDIATES THE MOTOGENIC EFFECTS OF STRAIN IN Caco-2 CELLS Fig. 5. Role of ROCK in deformation-induced migration. A: ROCK inhibition by Y23766 inhibits deformation-induced migration. Circular wounds were made in monolayers of cells and treated with complete DMEM (vehicle control) or pretreatment for 60 min with the ROCK inhibitor Y23766 (10 M), and these wounds were photographed. Cells were then cultured under static conditions (open bars) or conditions of repetitive deformation (filled bars) for 24 h before the holes were measured again, and wound closure was calculated as descried under EXPERIMENTAL PROCEDURES. ROCK inhibitor Y23766 increased basal migration (n 10 from 1 of 3 similar experiments, #P 0.05), but deformation reversed the motogenic effects (n 10 from 1 of 3 similar experiments, *P 0.05). Values are means SE. B: ROCK1 (RC1) or ROCK2 (RC2) reduction by sirnas inhibits deformation-induced migration. ROCK1 or ROCK2 sirna transfection achieved 65 70% knock down as shown by immunoblot on top. Circular wounds were made in monolayers of cells transfected with either NT1 or sirna targeted to ROCK1 or ROCK2, and these wounds were photographed. Cells were then cultured under static conditions (open bars) or conditions of repetitive deformation (filled bars) for 24 h before the holes were measured again, and wound closure was calculated. Deformation stimulated wound closure in cells transfected with the NT1 sequence compared with cells not subjected to strain (n 10 from 1 of 3 similar experiments, *P 0.05). Either ROCK1- or ROCK2-reduced cells increased basal (n 10 from 1 of 3 similar experiments, #P 0.05) wound closure, but deformation reversed the motogenic effects (n 10 from 1 of 3 similar experiments, *P 0.05). Values are means SE. equilibrium between injury and healing. We (6, 12 14, 28, 29) and others (2, 9, 50) have studied changes in cell migration, proliferation, spreading, or signaling in intestinal epithelial cells of similar magnitudes in response to other stimuli. For instance, Buffin-Meyer (9) studied a 15% stimulation of Caco-2 intestinal epithelial wound closure by the 2 -adrenoreceptor agonist 5-bromo-6-(2-imidazolin-2-ylamino)quinoxaline. Such differences in the speed of gut epithelial restitution could determine whether a mucosal wound heals or whether the mucosal barrier breaks down in vivo. Furthermore, strain effects are amplitude-dependent (5). The mucosa in vivo experiences 10% deformation (25), and would likely therefore exhibit greater effects of strain in vivo than those seen here, but in vitro strain amplitude is limited by the experimental apparatus. These studies were aimed at elucidation of the upstream intracellular signaling pathways that mediate the motogenic effects of repetitive mechanical deformation in Caco-2 intestinal epithelial cells. We recently demonstrated that a novel Src-independent FAK-Tyr 925 phosphorylation event is required for motogenic ERK and p38 signaling in response to cyclic strain in intestinal epithelial cells (11, 28). Previously, we reported that phosphorylated ERK is localized to both the lamellopodial edge of the migrating front and the nucleus in migrating Caco-2 cells subjected to cyclic strain on a fibronectin substrate (104). Here we show that RhoA-ROCK/mDia1- FAK-Tyr 925 mediates deformation-induced ERK in the nucleus on a fibronectin substrate that mediates the deformationinduced intestinal migration. We also demonstrate that RhoA, ROCK, and mdia1 mediate this motogenic response by a novel FAK-Tyr 925 -p38- or FAK-Tyr 925 -MLC-dependent signaling pathway that mediates the deformation-induced intestinal migration (Fig. 10). Thus, our findings demonstrate that RhoA GTPase and its effectors ROCK and mdia1 act as upstream regulators of FAK, ERK, p38, and MLC to mediate deformation-induced motility in the human intestinal epithelial cells. Together with these previous studies, our present observations delineate the mechanism by which RhoA and its downstream effectors ROCK and mdia1 regulate a complex cascade of motogenic signals in intestinal epithelial cells subjected to repetitive deformation. Although it is difficult to isolate the effects of increased repetitive deformation in vivo, RhoA seems activated in the intestine of patients with Crohn s disease or ulcerative colitis in whom bowel contractility and pressures are increased (43, 81) and also in the colon of rats with chemical colitis. (81). RhoA and its effectors ROCK and mdia1 could therefore play an important role in the maintenance of epithelial integrity and mucosal restitution in the setting of chronic inflammatory mucosal injury/disease conditions. Rho family GTPases regulate actin dynamics and help coordinate the cellular responses required for motility (22). RhoA triggers many target signaling molecules. Two immediate RhoA targets, ROCK and the formin homology protein mdia1, mediate RhoA effects on matrix adhesions and the cytoskeleton. Appropriately balanced ROCK and mdia1 activities suffice to induce stress fiber and focal contact formation indistinguishable from that induced by activated RhoA (95). Our results suggest that RhoA is activated and translocates to the membrane in response to strain to stimulate intestinal epithelial motility. Some literature may seem inconsistent with the model suggested here. Osada et al. (67) described inhibition of migration and proliferation and selective suppression of RhoA expression at cells at the wound edge in response to repetitive strain at 5 cycles/min in rat gastric epithelial RGM1 cells cultured on collagen type I. Although these authors investigated RhoA immunoreactivity by staining monolayers, they did not study RhoA activation. Moreover, Desai et al. (19) recently reported

8 RhoA MEDIATES THE MOTOGENIC EFFECTS OF STRAIN IN Caco-2 CELLS C1231 Downloaded from Fig. 6. ROCK inhibition or reduction inhibits deformation-induced FAK-Tyr 925 and p38 phosphorylation. A F: effects of Y23762 (10 M), ROCK1 (RC1), or ROCK2 (RC2) reduction by sirna on the phosphorylation of FAK at Tyr 925 (A C) or p38 (D F) were assessed by Western blot analysis of lysates from cells subjected to cyclic strain for 15 min. Total FAK (t-fak) and total p38 (t-p38) served as a protein-loading control. Top, typical blots; bottom, densitometric analyses. Values are means SE. A: cyclic strain stimulates FAK-Tyr 925 phosphorylation in vehicle-treated cells (n 5, *P 0.05). Y27632 completely blocked strain-induced FAK-Tyr 925 phosphorylation (n 5, *P 0.05). B and C: cyclic strain stimulates FAK-Tyr 925 phosphorylation in NT1-treated cells (n 5, *P 0.05). ROCK1- or ROCK2-reduced cells blocked strain-induced FAK-Tyr 925 phosphorylation (n 5, *P 0.05). D: cyclic strain stimulates p38 phosphorylation in vehicle-treated cells (n 5, *P 0.05). RhoA inhibition with exoenzyme C3 completely blocked strain-induced p38 phosphorylation (n 5, *P 0.05). E and F: cyclic strain stimulates p38 phosphorylation in NT1-treated cells (n 5, *P 0.05). ROCK1- or ROCK2-reduced cells blocked strain-induced p38 phosphorylation (n 5, *P 0.05). by on November 23, 2017 that 20% cyclic strain at 30 cycles/min inhibits migration in human airway epithelial cells cultured on collagen IV or laminin-5. The difference between the anti-migratory responses to strain in RGM1 cells described by Osada et al. (67) and Desai et al. (19) and the motogenic RhoA-mediated effect of strain that we have observed in intestinal Caco-2 and IEC-6 epithelial cells (11, 104) could reflect fundamental differences in the response of gastric, airway, and intestinal epithelial cells to repetitive deformation or could reflect differences in responses to different strain frequencies, since cyclic strain responses can vary with frequency (5). However, Osada and Desai studied cells on collagen I, collagen IV, and laminin while we studied Caco-2 cells on tissue fibronectin here. We have previously reported that mechanical deformation stimulates intestinal epithelial wound closure across tissue fibronectin substrates, but deformation stimulates proliferation and inhibits wound closure on collagen substrates (103, 104). This is important because tissue fibronectin is deposited into tissues at increased levels in settings of injury or inflammation when wound healing becomes important (23, 92). Thus, the responses studied here may be more relevant to the effects of deformation on wound healing in injured tissue in vivo than those of Osada et al. (67) and Desai et al. (19) on matrix substrates devoid of fibronectin. Interestingly, Putnam et al. (74) reported that 10% tensile strain inhibits RhoA association with the membrane in rat vascular myocytes on plasma fibronectin but that 10% compressive strain increases RhoA association with the membrane in the same system. Thus, differences in cell type and strain parameters may also affect results. The apparent lack of effect of RhoA inhibition on basal migration and the stimulation of motility in response to ROCK

9 C1232 RhoA MEDIATES THE MOTOGENIC EFFECTS OF STRAIN IN Caco-2 CELLS Downloaded from Fig. 7. RhoA, ROCK inhibition, or RhoA, ROCK1, ROCK2, mdia1, and FAK reduction inhibits deformation-induced nuclear extracellular signal-regulated kinase (ERK) phosphorylation. A F: effects of exoenzyme C3 (2.5 g/ml), ROCK inhibitor Y27632 (10 M) or RhoA, ROCK1, ROCK2, mdia1, and FAK reduction by sirna on the nuclear ERK phosphorylation (A F) were assessed by Western blot analysis of nuclear fraction lysates from cells subjected to cyclic strain for 60 min. Total total-erk (t-erk) and histone (H2A) served as a protein-loading control. Top, typical blots; bottom, densitometric analyses. Values are means SE. A: cyclic strain stimulates ERK phosphorylation in vehicle-treated cells (n 5, *P 0.05). RhoA inhibition with exoenzyme C3 blocks strain-induced nuclear ERK phosphorylation (n 5, *P 0.05). B: cyclic strain stimulates ERK phosphorylation in NT1-treated cells (n 5, *P 0.05). RhoA-reduced cells block strain-stimulated nuclear ERK phosphorylation (n 5, *P 0.05). C: cyclic strain stimulates ERK phosphorylation in NT1-treated cells (n 5, *P 0.05). mdia1-reduced cells block strain-stimulated nuclear ERK phosphorylation (n 5, *P 0.05). D: cyclic strain stimulates ERK phosphorylation in vehicle-treated cells (n 5, *P 0.05). ROCK inhibition with Y27632 blocks strain-induced nuclear ERK phosphorylation (n 5, *P 0.05). E: cyclic strain stimulates ERK phosphorylation in NT1-treated cells (n 5, *P 0.05). ROCK1- or ROCK2-reduced cells block strain-stimulated nuclear ERK phosphorylation (n 5, *P 0.05). F: cyclic strain stimulates ERK phosphorylation in NT1-treated cells (n 5, *P 0.05). FAK-reduced cells block strain-stimulated nuclear ERK phosphorylation (n 5, *P 0.05). by on November 23, 2017 or mdia1 blockade would seem to contrast with the prevention of the motogenic effects of deformation that we observed when these signals were blocked in intestinal epithelial cells migrating across tissue fibronectin. RhoA is required for cell motility in response to some stimuli (3, 26, 65), including strain (98) in other cells, and RhoA-dependent, ROCK-dependent signaling is required for epithelial migration across nondeforming substrates (62, 68, 76), including intestinal IEC-6 and T84 cells (60). Others have also observed that inhibiting RhoA or ROCK can stimulate basal migration (15, 40, 58, 77, 86, 89) while blocking the stimulation of migration by some other stimulus in other cells (36, 39, 94). In our hands, silencing mdia1 also enhanced basal migration across tissue fibronectin similarly to ROCK inhibition or reduction while preventing strain stimulation of migration. The apparent contrast between the lack of effect of RhoA inhibition on basal motility and the stimulation of motility by ROCK or mdia1 inhibition on one hand with the ability of RhoA, ROCK, or mdia1 inhibition to prevent the stimulation of migration by strain on the other may reflect a complex balance between ROCK, mdia1, and other RhoA-mediated downstream signals. Comparison of our basal results without strain suggests that the inhibition or reduction of ROCK1/2 and mdia1 may paradoxically increase migration by a different pathway independent of a FAK, ERK, p38, and MLC signaling cascade. How this occurs awaits further study.

10 RhoA MEDIATES THE MOTOGENIC EFFECTS OF STRAIN IN Caco-2 CELLS C1233 Downloaded from Fig. 8. RhoA, ROCK inhibition, or RhoA, ROCK1, ROCK2, and mdia1 reduction inhibits deformation-induced myosin light-chain (MLC) phosphorylation. A E: effects of exoenzyme C3 (2.5 g/ml), ROCK inhibitor Y27632 (10 M), or RhoA, ROCK1, ROCK2, and mdia1 reduction by sirna on the phosphorylation of MLC at Ser 19 were assessed by Western blot analysis of lysates from cells subjected to cyclic strain for 60 min. Total MLC (t-mlc) served as a protein-loading control. Top, typical blots; bottom, densitometric analyses. Values are means SE. A: cyclic strain stimulates MLC-Ser 19 phosphorylation in vehicle-treated cells (n 5, *P 0.05). Exoenzyme C3 blocked strain-induced MLC-Ser 19 phosphorylation (n 5, *P 0.05). B: cyclic strain stimulates MLC-Ser 19 phosphorylation in vehicle-treated cells (n 5, *P 0.05). Y23762 completely blocked strain-induced MLC-Ser 19 phosphorylation (n 5, *P 0.05) and also significantly inhibited basal MLC phosphorylation (n 5, #P 0.05). C: cyclic strain stimulates MLC-Ser 19 phosphorylation in NT1-treated cells (n 5, *P 0.05). RhoA-reduced cells blocked strain-induced MLC-Ser 19 phosphorylation (n 5, *P 0.05). D: cyclic strain stimulates MLC-Ser 19 phosphorylation in NT1-treated cells (n 5, *P 0.05). ROCK1- or ROCK2-reduced cells blocked strain-induced MLC-Ser 19 phosphorylation without affecting basal MLC phosphorylation (n 5, *P 0.05). E: cyclic strain stimulates MLC-Ser 19 phosphorylation in NT1-treated cells (n 5, *P 0.05). mdia1-reduced cells blocked strain-induced MLC-Ser 19 phosphorylation (n 5, *P 0.05). by on November 23, 2017 mdia1 belongs to the formin protein family that facilitates assembly of specific cellular actin-based structures. Rho GTPases activate formins through direct binding and disruption of an autoinhibitory mechanism mediated by regulatory domains that flank the actin/microtubule-binding formin homology-2 domain (33). mdia1 has recently been shown to influence migration in other cell types in response to other stimuli (82, 96). Although mdia1 is important in microtubule stabilization, polarization, cell adhesion, and actin polymerization (96, 102), its role in mediating mechanical force signaling has not been described previously. Our results suggest that mdia1 modulates deformation-induced motility, FAK, ERK, p38, and MLC phosphorylation in human intestinal epithelial cells. Palazzo et al. (69) concluded that mdia1 acts downstream of both Rho and FAK.

11 C1234 RhoA MEDIATES THE MOTOGENIC EFFECTS OF STRAIN IN Caco-2 CELLS Fig. 9. FAK inhibition does not prevent deformation-induced RhoA activity. A: effect of FAK inhibitor 1,2,4,5-benzenetetraamine tetrahydrochloride (Y15, 10 M) on the phosphorylation of FAK at Tyr 397 was assessed by Western blot analysis of lysates from cells subjected to cyclic strain for 15 min. Total FAK (t-fak) served as a protein-loading control. Top, typical blots; bottom, densitometric analyses. Values are means SE. Cyclic strain stimulates FAK-Tyr 397 phosphorylation in vehicle-treated cells (n 4, *P 0.05). Y15 completely blocked strain-induced FAK-Tyr 397 phosphorylation (n 4, *P 0.05; NS, nonsignificant, P 0.05). B: effect of FAK inhibitor Y15 (10 M) on deformation-induced activity of RhoA was assessed by pull-down assays of the GST-Rhotekin to specifically precipitate active RhoA as described in EXPERIMENTAL PROCEDURES. In parallel, total RhoA was assessed in the cell lysates for protein-loading control; representative blots are shown on top summarizing densitometric analysis (n 4, *P 0.05). Fig. 10. Proposed signal pathway by which repetitive deformation may induce intestinal epithelial cell migration. The cartoon summarizes a proposed signaling pathway for the stimulation of intestinal epithelial cell migration across fibronectin by repetitive deformation. The scheme is based on the results from our previous studies on the same system, some of which we have reconfirmed here (broken lines), taken together with entirely new results from the present study (solid lines). However, in our system, mdia1 modulates FAK phosphorylation, suggesting that mdia1 may act upstream of FAK in mediating physical force effects. Our results further delineate a dual and synergistic mechanism by which RhoA and ROCK regulate intestinal epithelial motility in response to deformation. RhoA and ROCK not only inhibit myosin phosphatase target 1 via phosphorylation of MYPT1 at Thr 696 but also phosphorylate MLC itself directly at Ser 18 /Thr 19 in our system, consistent with previous observations in response to other agonists in the absence of deformation (49, 84, 91). We have previously shown that myosin 2 is critical for the modulation of deformation-induced migration because inhibiting either nonmuscle myosin 2 with blebbistatin (28) or MLCK with HL-7 (104) not only inhibits but actually reverses the stimulation of migration by repetitive deformation. Two mechanisms have been postulated for regulation of myosin 2. One is Ca 2 -calmodulin dependent activation of MLCK, which phosphorylates MLCs, and the other is Ca 2 - independent activation of myosin 2 by RhoA activation of ROCK and direct phosphorylation of myosin regulatory light chains (45). Taken together, our present and previous results suggest that repetitive deformation may stimulate the phosphorylation of MLC to regulate the motogenic response via both RhoA-ROCK- and MLCK-dependent mechanisms. The small GTPase Rho acts on two effectors, ROCK and mdia1, and induces stress fibers and focal adhesions. How-

12 RhoA MEDIATES THE MOTOGENIC EFFECTS OF STRAIN IN Caco-2 CELLS ever, how ROCK and mdia1 individually regulate downstream signals and the dynamics of these structures remains poorly understood. ROCK and mdia mediate Rho action on the actin cytoskeleton; mdia produces actin filaments by nucleation and polymerization, and ROCK activates myosin to cross-link to actin for induction of actomyosin bundles and contractility. Thus cooperation of mdia and ROCK is required for assembly of actomyosin bundles such as stress fibers and contractile rings (63). Furthermore, Tsuji et al. (90) described Rhodependent Rac activation signaling that is mediated by mdia1 through Cas phosphorylation blockade by ROCK. mdia has been potentially linked to Rac activation and membrane ruffle formation through c-src-induced phosphorylation of focal adhesion proteins, and ROCK antagonizes the action of mdia (90). Our present results suggest that both ROCK and mdia are required for deformation-associated downstream signaling and motility. There could also be cross talk between these molecules or some other interacting regulatory mechanism as yet undefined. We have previously described a novel Src-independent FAK phosphorylation at Tyr 925 in response to deformation in intestinal epithelial cells across tissue fibronectin (11, 28). Our current data strongly suggest that RhoA-ROCK-mDia1 are required upstream modulators of this deformation-induced FAK-Tyr 925 phosphorylation because inhibiting RhoA or ROCK or silencing RhoA, ROCK1, ROCK2, or mdia1 prevented FAK-Tyr 925 phosphorylation in response to deformation. Others have reported that strain-induced FAK phosphorylation at Tyr 397 is mediated by RhoA/ROCK signaling in neonatal rat ventricular myocytes cells (88), but the mechanisms responsible for FAK-Tyr 925 phosphorylation in response to strain have not been addressed. This FAK-Tyr 925 phosphorylation is both critical for strain to stimulate intestinal epithelial migration and governed independently of FAK-Tyr 397 phosphorylation (11). These results contrast with the observation that ROCK inhibition does not inhibit overall FAK tyrosine phosphorylation of FAK in response to platelet-derived growth factor (75), but the phosphorylation of individual tyrosines within FAK was not addressed in that previous study, which considered a different stimulus in different cells. The relationship between FAK and RhoA is likely to vary with the stimulus. FAK can modulate RhoA in response to ephrina1 (71), since FAK activity can phosphorylate p190rhoagap and thus increase RhoA activity (73), suggesting that FAK can also regulate RhoA and thus influence cell-cell contact formation. In contrast, our findings suggest that intestinal epithelial cells respond to repetitive deformation by upstream RhoA activation that initiates a motogenic mechanotransduction signaling cascade in intestinal epithelial cells that leads to downstream FAK phosphorylation. It is becoming increasingly clear that conventional signal pathways associated with focal adhesion complex signaling can be bidirectional (21). We previously reported that p38 activation occurs downstream of FAK in response to cyclic strain (28). Blocking RhoA, ROCK1, ROCK2, or mdia1 inhibits strain activation of p38. Thus, RhoA, ROCK, and mdia1 are upstream modulators of p38 as well as FAK. Similar to our findings, RhoA regulates cyclic stretch-induced hypertrophy by modulating p38 in neonatal cardiomyocytes (70). In contrast, p38 is an upstream C1235 regulator of RhoA in MDA-MB-435 cells stimulated by arachidonic acid (27). Although strain changes nuclear phosphorylated ERK, total ERK did not change either in the cytosol or nucleus. Others have made similar observations. For instance, Mebratu et al. (56) reported modulation in phospho-erk in the cytosolic and nuclear fractions in human airway epithelial cells as well as mouse airway epithelial cells responding to IFN- in the presence or absence of BH3 protein (Bik/Blk/Nbk), but total ERK protein levels in the cytosolic and nuclear fractions did not change measurably in their studies (56). Nagasaka et al. (61) described similar results in human embryonic kidney 293 cells in the presence or absence of cell polarity regulator. Such results suggest that various stimuli can affect the activity of ERK either in the cytosolic or nuclear compartment without affecting the total ERK. Neither paper commented on the failure to measure a change in total ERK in either fraction in parallel to changes in phosphorylated ERK in their results. ERK is first phosphorylated in the cytoplasm and then translocates to the nucleus (54). Upon nuclear translocation, phosphorylated ERK in turn phosphorylates transcription factors, including Elk-1, signal transducer and activator of transcription 3, and c-myc. To act on such nuclear downstream targets, ERK must remain phosphorylated within the nucleus. Because dephosphorylation prevails in the nucleus, the maintenance of nuclear activity requires continuous shuttling of activated protein from the cytoplasm (54). Different explanations could be offered for the apparent disparity between changes in phosphorylated ERK and changes in total ERK. Relatively little ERK may actually be phosphorylated in either the cytosol or nucleus so that changes in phosphorylated ERK do not result in measurable changes in total ERK. Alternatively, it could be that we are not actually observing a change in the translocation of phosphorylated ERK into the nucleus but rather an alteration in the regulation of ERK dephosphorylation within the nucleus and/or transport back out of the nucleus that might change nuclear phosphorylated ERK without measurable changes in total nuclear ERK. ERK activation downstream of FAK is also required for strain to stimulate migration across fibronectin (11, 28). Interestingly, inhibiting or silencing RhoA, ROCK, ROCK1, ROCK2, mdia1, or FAK prevented deformation-induced ERK phosphorylation in the nucleus but not in the cytoplasm. In kidney and endothelial cells, a large portion of phosphorylated ERK stimulated via stromal cell-derived factor 1 is similarly found in the nucleus, resulting in increased Elk activation as well as increased cell migration, and both ERK and Elk nuclear accumulation were blocked by Rho inhibition (105). Furthermore, Liu et al. (52) also reported that inhibiting RhoA or ROCK did not prevent cytosolic ERK activation by serotonin in smooth muscle cells but did prevent ERK translocation to the nucleus and its activation there. In fact, we demonstrated that phosphorylated ERK is localized to the lamellipodial edge of the migrating front and the nucleus in Caco-2 cells on fibronectin subjected to strain (104), and we recently reported that inhibiting only nuclear ERK prevents the stimulation of migration by strain while inhibiting cytoplasmic ERK does not (30). Furthermore, blocking the transcription factor Elk also blocks strain-induced migration on fibronectin (30). Elk is a downstream nuclear effector of ERK. Previous (30, 104) observations therefore suggest that the stimulation of intestinal