ROCK1 Feedback Regulation of the Upstream Small GTPase RhoA

Abstract

Rho-associated coiled-coil containing protein kinase 1 (ROCK1) is a key downstream effector of the small GTPase RhoA. Targeting ROCK1 has shown promising clinical potential in cancer, cardioprotection, hypertension, diabetes, neuronal regeneration, and stem cell biology. General working hypothesis in previous studies has centered on the function of ROCK1 as a downstream sequence in the RhoA signaling pathway. In this study, the effects of the direct inhibition of ROCK1 on the activity of upstream RhoA and Rac1 were examined using a combined pharmacological and genetic approach. We report an intriguing mechanism by which the inhibition of ROCK1 indirectly diminishes the activity of upstream RhoA through the stimulation of Tiam1-induced Rac1 activity. This novel feedback mechanism, in which ROCK1 mediates upstream Rac1 and RhoA activity, offers considerable insight into the diverse effects of ROCK1 on the functional balance of the Rho family of small GTPases, which regulates actin cytoskeleton reorganization processes and the resulting overall behavior of cells.

Introduction

Actin cytoskeleton reorganization in eukaryotic cells induces morphological change, which is the basis for alterations in cellular functions such as cell proliferation, adhesion and motility. Dynamic changes in the actin cytoskeleton are tightly regulated by the Rho family of small GTPases, which includes the well-characterized RhoA, Rac1, and Cdc42 [1–3]. Rho GTPases are aptly termed “molecular switches” and their GTP-bound forms are considered “active,” leading to activation of a large number of downstream effectors. These effectors trigger signaling cascades that acutely affect cell physiology. Upon GTP hydrolysis, the Rho GTPase is rendered into “inactive” GDP-bound forms. While GTPases possess intrinsic GTP hydrolytic activity, the catalysis of this dephosphorylation from GTP-bound to GDP-bound states is governed by GTPase activating proteins (GAPs). GDP-bound Rho GTPases are subsequently reactivated through the dissociation of GDP and concomitant binding of GTP; this process is catalyzed by guanine nucleotide exchange factors (GEFs) but inhibited by guanine nucleotide dissociation inhibitors (GDIs).

Our current understanding of RhoA, Rac1, and Cdc42 posits that these Rho GTPases play distinct roles in the regulation of cytoskeleton rearrangement. Briefly and simplistically, RhoA controls actin stability, actin-myosin association and actin polymerization; Rac1 stimulates cell membrane ruffling and filopodial extensions; and Cdc42 governs cell polarity [4]. The activity/inactivity of Rho GTPases must be synchronized and harmonized for proper function of the cell. To avoid being idiomatically “pulled in all directions,” the cell must be able to balance the disparate roles of these small GTPases. Balancing the expression, translocation, and activity of these small GTPases are critical for the regulation of cytoskeletal processes ranging from stress fiber formation to membrane ruffling, and cell shape and function [5–7].

The crosstalk between signaling pathways plays a critical role in the inter-regulation and the balance of small Rho GTPase activity. Several GAPs, GEFs, and GDIs regulate the GTP/GDP switching of multiple Rho GTPases [8–10]. Of particular importance in this study are Rac1-GEFs, T-lymphoma invasion and metastasis -1 and -2 (Tiam1 and Tiam2/STEF), which play important regulatory roles in Rac1 activation [9,11–13]. Tiam1 and Tiam2 activate Rac1 [14,15] by stimulating GDP-GTP exchange activity that leads to activated Rac1. Consequently, the Tiam1/Tiam2-Rac1 signaling pathways regulate cell morphology and motility involved in physiological and pathophysiological processes. Interestingly, it has been demonstrated that Tiam1/Tiam2 enhance Rac1 activity and are involved in the suppression of RhoA activity in N1E-115 cells [16].

In prostate carcinoma PC-3 cells, RhoA is a critical endogenous promoter of cell invasion and migration [17–19]. Inhibition of RhoA or its major downstream effector, Rho-associated coiled-coil containing kinase 1 (ROCK1), diminishes motility of prostate carcinoma cells [17,20]. Despite that both RhoA inhibition and ROCK1 inhibition result in diminished cell motility, our laboratory along with others have observed that these two distinct inhibition steps produce markedly opposing changes in cell morphology [20,21]. The inhibition of RhoA induces cell rounding while the direct inhibition of ROCK1 induces neuron-like outgrowths. The latter observed morphological characteristics mimic the effects of activated Rac1, which is known to promote neurite outgrowth [22]. As such, these results suggest that the direct inhibition of the ROCK1 pathway possesses unique function(s) beyond the broader inhibition of RhoA signaling cascades. In this study, we demonstrate the pivotal role of ROCK1 in both RhoA and Rac1 signaling pathways. ROCK1 regulates RhoA and Rac1 activity through its association with Tiam1, a Rac1 GEF as a critical intermediate step in an inter-pathway feedback mechanism. This study presents a revised understanding of how ROCK1 significantly contributes to the regulation of cytoskeleton and cell function by the Rho family of small GTPases.

Materials and Methods

Materials

PC-3 (CRL-1435) cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). RPMI Medium 1640, Dulbecco’s Modified Essential Medium (DMEM), and Penicillin/Streptomycin, GAPDH primary antibody, secondary antibodies conjugated with horseradish peroxidase (HRP), and SimplyBlue, Coomassie G-250, stain were obtained from Invitrogen (Carlsbad, CA, USA). Fetal Bovine Serum was obtained from Hyclone Laboratories, Inc. (South Logan, UT, USA). Y-27632 (trans-4-[(1R)-1-Aminoethyl]-N-4-pyridinylcyclohexanecarboxamide dihydrochloride) was obtained from EMD Chemicals (Gibbstown, NJ, USA). Fasudil monohydrochloride salt (HA-1077, 5-(1,4-diazepane-1-sulfonyl)isoquinoline) was obtained from LC Laboratories (Woburn, MA, USA). NSC23766 was obtained from Tocris Bioscience (Ellisville, MO, USA). ADP-ribosyltransferase C3 from Clostridium botulinum (C3 exoenzyme) was obtained from Sigma (St. Louis, MO, USA). Rac1 siRNA (target sequence 5’-GUGAUUUCAUAGCGAGUUU-3’), ROCK1 siRNA (target sequence 5’-GCAAAUCAGUCUUUCCGGA-3’), and a non-targeting control siRNA were obtained from Thermo Scientific/Dharmacon (Lafayette, CO, USA). Tiam1 and Tiam2 siRNA pools including primary antibodies against Tiam1 and Tiam2 raised in rabbit were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). RhoA and Rac1 activation (pull down and G-LISA) assay kits were obtained from Cytoskeleton, Inc. (Denver, CO, USA). ROCK1 primary antibody was obtained from Abcam (Cambridge, MA, USA). Primary antibody against phosphorylated Tiam1 (p-Tiam1, Tyr 384) raised in rabbit was obtained from Abcam Inc. (Cambridge, MA, USA). Enhanced chemiluminescence (ECL) for Western blotting detection and bicinchoninic acid (BCA) protein determination assay kits were obtained from Pierce (Rockville, IL, USA). SDS-PAGE ReadyGels™ and Mini-PROTEAN TGX gels were obtained from Bio Rad (Hercules, CA, USA). Distilled, deionized water was used for all experiments.

Cell Culture

PC-3 cells were grown in RPMI 1640 medium supplemented with 10 % fetal bovine serum, 1 % L-glutamine, and 1 % penicillin/streptomycin according to ATCC guidelines. Cells were grown in an incubator at 37° C and 5% CO₂. Cells were used up to10 passages.

Determination of activated GTPases by GTPase activation assay (Pull-down)

PC-3 cells were grown to 60 % confluency, treated accordingly, and pull-down assays for active (GTP-bound) RhoA and Rac1 were performed according to the provided protocols. Cell lysate fractions were collected after each drug treatment (5 min, otherwise indicated) using lysis buffer containing protease inhibitors. Immediately, the cell lysate protein concentration was determined using the BCA protein assay. The remaining lysate was immediately frozen with liquid nitrogen and stored at −80° C. A total of 500 µg of protein (adjusted to identical volumes) from the cell lysate and 20 µg of pull-down beads (GST-tagged, active GTPase protein binding domains) were used. The pull-down was performed on a rocker at 4° C for 1 h. The beads were collected and the protein was released from the beads by resuspension and boiling in Laemmli sample buffer (63 mM Tris pH 6.8, 2% SDS, 5% β-mercaptoethanol, 10% glycerol, 0.05% bromophenol blue) for 5 min. The sample was then subjected to Western blot analysis using a 12 % SDS gel. Primary antibody against RhoA or Rac1 (1:250) and the secondary antibody (1:10,000) were used for detection of the immunoreactive bands. Western blot analysis for total Rac1 and total RhoA (20 µg of cell lysate) was performed in conjunction for normalization of GTP-Rac1 or GTP-RhoA. NIH Image J software was used to perform the densitometric analysis of all Western blots.

Determination of activated GTPases by G-LISA™

G-LISA™ assays for active RhoA and Rac1 were performed using the reagents and protocols provided in each assay kit. It is an alternative assay for fast measurement of GTP-bound Rac1 and GTP-bound RhoA; likewise, all cell culture procedures for the pull-down assay were followed. The assay is conveniently used for a large number of samples. The colorimetric G-LISA™ RhoA activation assays were measured in 96-well plates by an ELX800 microplate reader (Bio-Tek Instruments, Inc.). The luminometric G-LISA™ Rac1 activation assays were measured in 96-well plates by a CytoFluor microplate reader (AB Applied Biosystems). The activity of Rac1 or RhoA was averaged and normalized to the untreated control samples Relative GTPase Activity).

siRNA transfections

Knockdown transfections of Rac1, ROCK1, Tiam1, and Tiam2 were performed on PC-3 cells grown to 40% confluency in 6-well plates. All siRNA including non-targeting controls were transfected at a final concentration of 10 nM in DMEM. After 7 h, the transfection medium was removed and replaced with RPMI 1640 growth medium supplemented with 10% FBS. At 48 h post-transfection, the cells were used for pull-down or GLISA activation assays. A non-targeting, siRNA transfection (siControl) and a mock transfection (Mock) were used as additional experimental controls. The siRNA transfection efficiency was determined using Western blotting for the protein of interest. The same blots were re-probed for GAPDH as a protein control.

Measurement of p-Tiam1 (Tyr 384)

PC-3 cells were treated with fasudil (25 µM) at 37°C for different times. Cells were lysed in lysis buffer (25 mM Tris-HCl, 150 mM NaCl, 5 mM MgCl₂, 5% glycerol, 1% NP-40, 1 mM DTT and pH 7.5) supplemented with phosphatase inhibitors and protease inhibitors. The samples were centrifuged at 800g at 4�C for 15 min to remove particulates. Then, the supernatant was used for Western blot analysis for p-Tiam1 (Tyr 384) on 4–15% Mini-PROTEAN TGX gels and transferred to a nitrocellulose membrane. The p-Tiam1 was incubated at room temperature for 1 h with anti-p-Tiam1 (Tyr 384) primary antibody (1:500 dilution), followed by the secondary antibody-horse radish peroxidase, and detected by chemiluminescence on the film. The nitrocellulose membrane was then re-probed with total Tiam1 antibody (1:1,000 dilution) for loading control and normalization.

Pharmacological treatments of cells

Prior to any treatment, cells were washed twice with warm PBS. Y-27632 and fasudil were applied to the cells at a final concentration of 25 µM. After 5 min at 37° C, the cells were lysed and analyzed for small GTPase activity. NSC23766 was applied at a final concentration of 100 µM for 12 h at 37° C as previously described [23]. C3 exoenzyme was applied at a final concentration of 1 µg/mL for 24 h at 37° C.

Cell Morphology Studies

Cells were grown and treated with pharmacological agents as described above. Images were taken 5 h at 37° C post-treatment with fasudil and Y-27632. For NSC23766 and C3 exoenzyme, cells were pre-treated for 12h and 24 h at 37° C, respectively. To better visualize cell morphology, cells were fixed with cold methanol/ethanol (1:1) then stained with SimplyBlue Coomassie G-250 [24] followed by successive washes with cold PBS. Microscopic images were taken with a Photometrics Sensys CCD camera and processed with Photometrics Image ProPlus software.

Results

Different effects of inhibition of RhoA, ROCK1 and Rac1 on cell morphology

PC-3 cells were used as a cell model in this study because RhoA is a master regulator of motility in these cells [18,19,25–29]. Cells were treated with either C3 exoenzyme (ADP ribosyltransferase isolated from Clostridium botulinum) to directly inhibit RhoA or fasudil (HA-1077) and Y-27632 to directly inhibit the kinase function of ROCK1 [30]. Inhibiting either RhoA or ROCK1 resulted in differences in cell morphology (Fig. 1). Cells treated with C3 exoenzyme became smaller and more rounded than the control cells (Fig. 1panel b). In contrast, cells treated with fasudil and Y-27632 developed multiple slender outgrowths resembling filopodial extensions (Fig. 1, panel c&d), which corroborate previous results [20]. ROCK1 inhibition induces the formation of cell membrane protrusions, which is similar to a morphological feature induced by the activation of the Rac1 signaling pathway [4,17]. Therefore, the initial cell morphological observations suggest that the direct inhibition of ROCK1 may increase Rac1 activity.

Fig. 1.

Morphology of PC-3 cells treated with RhoA, Rac1 and ROCK1 inhibitors. Cells were treated at 37°C for 5 h for fasudil and Y-27632, 12 h for NSC23766, and 24 h for C3 exoenzyme. These results are representative of five independent experiments. (a) Untreated control cells (UT). (b) Cells treated with C3 exoenzyme (1 µg/mL), a RhoA inhibitor. (c) Cells treated with Y-27632 (25 µM), a ROCK1 inhibitor. (d) PC-3 cells treated with fasudil (25µM), a ROCK1 inhibitor. (e) Cells treated with NSC23766 (100 µM), a Rac1 inhibitor. (f) Cells pre-treated with NSC23766 (100 µM) and then fasudil (25 µM).

This hypothesis is further supported by the treatment of PC-3 cells with NSC23766 (NSC), a drug that inhibits Rac1 activity by blocking the interaction between Rac1 and Rac1 GEF, Tiam1 but has no effect on Cdc42 and RhoA [23]. NSC23766 induced rounding and smaller cell size (Fig. 1, panel e) and diminished the ability of fasudil to induce the formation of neuron-like outgrowths (Fig. 1, panel f), suggesting the roles of Rac1 and Rac1-GEFs in the change in cell morphology by the direct inhibition of ROCK1.

Effects of direct inhibition of ROCK1 on Rac1 and RhoA activity

The effects of the direct inhibition of ROCK1 on Rac1 and RhoA activity were determined using two distinct approaches. Levels of GTP-Rac1 and GTP-RhoA were measured by GTPase activation assays (GTP-bound protein pull down assay or G-LISA™). First, a pharmacological approach was used to inhibit the kinase activity of ROCK1. Treatment of PC-3 cells with fasudil (F) increased GTP-Rac1 and decreased GTP-RhoA as compared to the untreated, control cells (UT) (Fig. 2A). The averages of GTP-RhoA and GTP-Rac1 were normalized to total GTPases (Fig. 2B) and indicate a significant decrease of GTP-RhoA (top panel) and an increase of GTP-Rac1 (bottom panel) by fasudil treatment. Second, a genetic manipulation via siRNA transfection was used to knockdown the protein expression of ROCK1. Similar to the results of fasudil treatments, transfection with ROCK1 siRNA (siROCK1) increased the levels of GTP-Rac1 and decreased the levels of GTP-RhoA as compared with the control samples, which included no transfection (NT), mock transfection (M), and non-targeting, negative control siRNA (siCtrl) (Fig. 2C). Again, the averages of GTP-RhoA and GTP-Rac1 were normalized to total GTPases (Fig. 2D) and indicate a significant decrease of GTP-RhoA (top panel) and an increase of GTP-Rac1 (bottom panel) by ROCK1 siRNA, similar to the results obtained from the inhibition of ROCK1 by fasudil. In some Western blots, the band intensity of GTP-RhoA appears to be slightly different from the control. This discrepancy is likely due to small, unintended effects from transfection agents. However, the average of the ratios of GTP-RhoA to total RhoA for the mock transfection is not significantly different from the control averages.

Fig. 2.

Effects of ROCK1 inhibition on Rac1 and RhoA activity. (A) Examples of Western blots of GTP-RhoA, total RhoA, GTP-Rac1, and total Rac1 in untreated control PC-3 cells (UT) and fasudil (F, 25 µM), a ROCK1 inhibitor, at 37°C for 5 min. Also shown are the Western blots of GTP-RhoA and GTP-Rac1 in cells treated with NSC23766 (NSC, 100 µM), a Rac1 activation inhibitor, at 37°C for 12 h and NSC23766 followed by fasudil for 5 min (NSC+F). (B) Average of densitometric analysis of Western blots GTP-RhoA and GTP-Rac1 from Panel A as normalized to total GTPases (n = 4 independent experiments; mean ± s.e.m.; *, significantly lower than control; #, significantly higher than control with p < 0.05). (C) Examples of Western blots of GTP-RhoA and GTP-Rac1 in PC-3 cells with no transfection (NT), cells with mock transfection (M), a non-targeting, negative siRNA (siCtrl) and ROCK1 siRNA (siROCK1). (D) Average of densitometric analysis of Western blots GTP-RhoA and GTP-Rac1 from Panel C as normalized to total GTPases (n = 3 independent experiments; mean ± s.e.m.; *, significantly lower than control; #, significantly higher than control, p < 0.05).

Role of Rac1 in the upstream ROCK1-mediated RhoA activity

To determine whether Rac1 plays a role in the upstream inhibition of RhoA by ROCK1, Rac1 activation was inhibited by treatment of cells with NSC23766 (NSC) [23]. NSC23766 decreased GTP-Rac1 and increased GTP-RhoA (Fig. 2A). Treatment of cells with NSC23766 abolished the ability of fasudil to increase GTP-Rac1 or decrease GTP-RhoA (Fig. 2A). The averages of GTP-RhoA and GTP-Rac1 were normalized to total GTPases (Fig. 2B) and indicate a significant increase of GTP-RhoA (top panel) and a decrease of GTP-Rac1 (bottom panel) by NSC23766. Furthermore, NSC23766 abolished the effects of fasudil on Rac1 and RhoA activity.

Transfection of PC-3 cells with Rac1 siRNA decreased Rac1 protein (Fig. 3A). However, Rac1 siRNA (siRac1) and siRNA with subsequent fasudil treatment (siRac1 + F) did not significantly change GTP-RhoA (Fig. 3B). In contrast, fasudil alone reduced GTP-RhoA in all control samples (Fig. 3A), consistent with our results from cells treated with fasudil (Fig. 2A). The average RhoA activity indicates that the results from Rac1 siRNA were similar to the experiments with NSC23766, and Rac1 siRNA also abolished the ability of fasudil to reduce the RhoA activity (Fig. 3B). These results suggest the role of Rac1 as an intermediary in the upstream ROCK1-inhibited RhoA activity.

Fig. 3.

Rac1 is required for the ROCK1 inhibition of RhoA activity. PC-3 cells with no transfection (NT), mock transfection (M), a non-targeting, negative control siRNA (siCtrl), and Rac1 siRNA (siRac1). Then, cells were treated with vehicle or fasudil (25 µM) at 37°C for 5 min (designated as +F). (A) Examples of Western blots of GTP-RhoA, total RhoA, Rac1 and GAPDH from two independent experiments. (B) Average of RhoA activity from G-LISA™ analysis from 3 separate experiments of 4 samples for each treatment as in Panel A. The GTP-RhoA in siCtrl and siCtrl+F samples were not included in the Western blots; however, RhoA activity in these samples was measured by G-LISA. The RhoA activity was normalized to the untreated control cells. The results are mean ± s.e.m.; , not significantly different; *, significantly lower than control, p < 0.05.

Roles of Rac1 GEFs, Tiam1 and Tiam2, in the ROCK1-regulated Rac1 and RhoA activity

To determine the roles of Rac1 GEFs, Tiam1 and Tiam2, in the ROCK1 feedback mechanism to regulate upstream Rac1 and RhoA, PC-3 cells were transfected with Tiam1 siRNA or Tiam2 siRNA (siTiam1/2) and subsequently treated with fasudil. Tiam1 siRNA diminished Tiam1 protein expression and decreased GTP-Rac1 (Fig. 4A). Tiam1 siRNA also abolished the ability of fasudil to increase GTP-Rac1 and decrease GTP-RhoA, whereas fasudil alone increased GTP-Rac1 and decreased GTP-RhoA in all control samples (Fig. 4A). The averages of RhoA and Rac1 activity results indicate that Tiam1 siRNA abolished the effects of direct ROCK1 inhibition upon RhoA (top panel) and Rac1 activity (bottom panel) (Fig. 4C).

Fig. 4.

Tiam1 is required for the ROCK1 regulation of Rac1 and RhoA activity. PC-3 cells with no transfection (NT), mock transfection (M), a non-targeting, negative control siRNA (siCtrl), and Tiam1 or Tiam2 siRNA (siTiam1/2). Then, cells were treated with vehicle or fasudil (25 µM) at 37°C for 5 min (designated as +F). (A) Examples of Western blots of GTP-RhoA, total RhoA, GTP-Rac1, total Rac1, Tiam1, and GAPDH from two independent experiments of Tiam1 siRNA. (B) Examples of Western blots of GTP-RhoA, total RhoA, GTP-Rac1, total Rac1, Tiam2, and GAPDH from two independent experiments of Tiam2 siRNA. (C) Average of RhoA activity (top) and Rac1 activity (bottom) from G-LISA™ analysis from 3 separate experiments of 4 samples for each treatment as performed in Panel A and B. The GTP-Rac1 and GTP-RhoA in siCtrl and siCtrl+F samples were not included in the Western blots; however, RhoA activity and Rac1 activity in these samples was measured by G-LISA. The RhoA activity and Rac1 activity was normalized to the untreated control cells. The results are mean ± s.e.m.; , not significantly different; *, significantly lower than control; #, significantly higher than control, p < 0.05.

Tiam2 siRNA transfection decreased Tiam2 protein expression and decreased GTP-Rac1. In contrast to the results from the Tiam1 siRNA, the inhibition of ROCK1 by fasudil still increased GTP-Rac1 and decreased GTP-RhoA in PC-3 cells transfected with Tiam2 siRNA (Fig. 4C). These results are similar to the effects of fasudil alone to increase GTP-Rac1 and decrease GTP-RhoA in all control samples (Fig. 4B). The averages of RhoA and Rac1 activity results (Fig. 4C) indicate that Tiam2 siRNA did not block the ability of fasudil to alter the RhoA and Rac1 activity.

Effects of direct inhibition of ROCK1 on p-Tiam1 (Tyr 384)

The effect of the direct inhibition of ROCK1 on the phosphorylation of Tiam1 (Tyr 384) was determined by treating PC-3 cells with fasudil at various time form 0, 1, 3 and 5 min. Then, p-Tiam1 was detected by Western blot analysis. Fasudil treatments did not change p-Tiam1 levels during these times of treatment (Fig. 5).

Fig. 5.

Effects of ROCK1 inhibition on phosphorylated Tiam1 (Tyr 384). (A) Examples of Western blots of p-Tiam1 (Tyr 384) and total Tiam1 in PC-3 cells treated with fasudil (25 µM) at 37°C for 0, 1, 3, 5 min. (B) Average of the ratios of p-Tiam1 to t-Tiam1 for each time point from 4 separate experiments. The results are mean ± s.e.m.

Discussion

As evidenced by these results, ROCK1 regulates Rac1 and RhoA activity in a dichotomous fashion. Our findings also suggest that ROCK1 suppresses the function of Rac1-GEF Tiam1 function, resulting in the reduction of Rac1 activity. Thus, the direct inhibition of ROCK1 likely increases the function of Tiam1, which enhances the activation of Rac1 (Fig. 5). Studies have demonstrated that the phosphorylation of Tiam1 and Tiam2 (STEF) by a number of kinases abolishes their ability to activate Rac1 [31–36]. Another study has demonstrated that ROCK1 phosphorylates Tiam2 (STEF) and Tiam1 [35]. Since association or complexation of Tiam1 or Tiam2 with other molecules is essential for the activation of Rac1, the phosphorylation of Tiam1 and Tiam2 likely disrupts these complexes [35,37,38] or enhances the degradation of Tiam1 and Tiam2 [34,39], resulting in diminished Rac1 activation. However, our results do not support this hypothesis because the direct inhibition of ROCK1 by fasudil did not significantly change the phosphorylation of Tiam1 (Tyr 384). Thus, an alternative mechanism likely explains the role of Tiam1 and Tiam2 in the ROCK1-induced regulation of Rac1 and RhoA activity.

Among the complexes associated with small GTPases, the Par complex is critical in the regulation of the small GTPase signaling [40–43]. The Par complex, comprised of protein partitioning defective-3 (Par3), partitioning defective-6 (Par6) and atypical Protein Kinase C (aPKC) [44–46]. The Rac1-GEFs, Tiam1 and Tiam2, directly interact with Par3 to induce the formation of the Par cell polarity complex [9], activate Rac1, and promote the formation of neurite outgrowths in N1E-115 cells [9,35,47]. These results explain our observations that ROCK1 inhibition with either fasudil or ROCK1 siRNA increased Rac1 activity, and that treatment of cells with fasudil induced cell membrane extension and protrusion. This is also supported by the results from NSC23766 treatment. NSC23766 disrupts Tiam1/Tiam2 interaction with Rac1, causing cell rounding and blocking the ability of fasudil to enhance cell protrusion.

Interestingly, ROCK1 has been demonstrated to phosphorylate Par3 at Thr833 and the phosphorylation disrupts its interaction with aPKC and Par6 of the Par complex [48]. Since Par3 directly interacts with Tiam1 [9,49,50], the phosphorylation of Par3 will diminish Rac1 activation by Tiam1 [9]. Importantly, the phosphorylation of Par3 by ROCK1 does not disrupt its interaction with Tiam2 [48]. Thus, there is a strong possibility that this mechanism may be responsible for our findings that Tiam1 siRNA decreased Rac1 activity and blocked the ability of fasudil to increase Rac1 activity while Tiam2 siRNA decreased Rac1 activity but failed to block the increase of Rac1 activity by fasudil (Fig. 4). The change in phosphorylated Par3 (p-Par3) caused by fasudil will be important information to support this hypothesis. Unfortunately, the primary antibody for p-Par3 is not available for application to this study. The results from Tiam1 and Tiam2 siRNA clearly indicate that Tiam1 has a significant role in the inhibition of ROCK1 by fasudil to increase Rac1 activity and decrease RhoA activity, but Tiam2 appears to have no major role in the ROCK1 feedback loop.

The results indicate that the inhibition of Rac1 by NSC23766 or Rac1 siRNA abolished the ability of fasudil, a ROCK1 inhibitor, to reduce RhoA activity. These results suggest that Rac1 is an intermediate in the loop of ROCK1 regulation of upstream RhoA (Fig. 6) and also illustrate the induced reciprocal activity between Rac1 and RhoA. This relationship between an increase of Rac1 activity and a decrease of RhoA activity has been demonstrated by others [10,51–55].

Fig. 6.

A proposed ROCK1 feedback loop for the regulation of Rac1 and RhoA activation. Under normal conditions, active (GTP-bound) RhoA activates ROCK1, which in turn inhibits Tiam1 function and subsequently suppresses the Rac1 activity. Direct ROCK1 inhibition by fasudil increases Tiam1 function to increase Rac1 activity and results in a diminished RhoA activity. The decreased levels of GTP-RhoA diminishes the activity of ROCK1, resulting in a cyclical feedback mechanism. Fasudil and Y27632 were used to inhibit ROCK1 function and ROCK1 siRNA was used to knockdown ROCK1 protein and diminish its function. NSC23766 was used to inhibit the interaction of Tiam1/2 with Rac1 and Rac1 activation, and Rac1 siRNA was used to knockdown Rac1 protein and diminish its function. Tiam1/2 siRNA were used to knockdown Tiam1/2 proteins and diminish their ability to activate Rac1.

Conclusions

This study provides clear evidence that ROCK1 regulates upstream RhoA activity. Inhibition of ROCK1 enhances the function of Rac1-GEF Tiam1, which catalyzes the activation of Rac1. The increase in Rac1 activity inhibits RhoA activation, thus completing a feedback loop. Recognition of these multiple effects of ROCK1 inhibition indicates the need to reinterpret the mechanism of action of ROCK1 inhibitors that possess broader physiological and pathological significance. The ROCK1 regulation of upstream Tiam1, Rac1, and RhoA will undoubtedly be of importance in understanding factors affecting cytoskeletal reorganization in various diseases. More importantly, this feedback loop adds to our understanding of the mechanisms by which the cell balances Rho GTPase activities and how this balance is crucial for proper cell behavior. The ROCK1 feedback loop is only the first example of a motif that will be recurrently observed in other signal transduction pathways as our understanding of small GTPase signaling becomes increasingly complete.

Highlights.

• The direct inhibition of ROCK1 negatively regulates the activity of the upstream RhoA.

• The inhibition of ROCK1 stimulates the activity of Rac1.

• Tiam1 is an intermediate player in the ROCK1-mediated Rac1 and RhoA activity.

• The direct inhibition of the downstream effector ROCK1 in RhoA/ROCK pathway induces a unique cell morphology.