Thrombospondin-1 mediates Rho-kinase inhibitor-induced increase in outflow-facility.

Rho-kinase (ROCK) inhibitors, a novel class of anti-glaucoma agents, act by increasing the aqueous humor outflow through the conventional trabecular meshwork pathway. However, the downstream signaling consequences of the ROCK inhibitor are not completely understood. Our data show that Y39983, a selective ROCK inhibitor, could induce filamentous actin remodeling, reduced cell motility (as measured by cell migration), and transepithelial resistance in primary human TM (hTM) cells. After 2 days Y39983 treatment of hTM cells, a proteomic study identified 20 proteins whose expression was significantly altered. Pathway analysis of those proteins revealed the involvement of the p53 pathway, integrin signaling pathway, and cytoskeletal pathway regulation by Rho GTPase. Thrombospondin-1 (TSP1), a matricellular protein that is increased in glaucoma patients, was downregulated fivefold following Y39983 treatment. More importantly, both TSP1 antagonist leucine-serine-lysine-leucine (LSKL) and small interfering RNA (siRNA) reduced TSP1 gene and protein expressions as well as hTM cell migration. In the presence of Y39983, no further inhibition of cell migration resulted after LSKL and TSP1 siRNA knockdown. Likewise, LSKL triggered a dose-dependent increase in outflow facility in ex vivo mouse eyes, to a similar extent as Y39983 (83.8% increase by Y39983 vs. 71.2% increase by LSKL at 50 µM). There were no additive effects with simultaneous treatment with LSKL and Y39983, supporting the notion that the effects of ROCK inhibition were mediated by TSP1.

PTPσ inhibitors promote hematopoietic stem cell regeneration.

Receptor type protein tyrosine phosphatase-sigma (PTPσ) is primarily expressed by adult neurons and regulates neural regeneration. We recently discovered that PTPσ is also expressed by hematopoietic stem cells (HSCs). Here, we describe small molecule inhibitors of PTPσ that promote HSC regeneration in vivo. Systemic administration of the PTPσ inhibitor, DJ001, or its analog, to irradiated mice promotes HSC regeneration, accelerates hematologic recovery, and improves survival. Similarly, DJ001 administration accelerates hematologic recovery in mice treated with 5-fluorouracil chemotherapy. DJ001 displays high specificity for PTPσ and antagonizes PTPσ via unique non-competitive, allosteric binding. Mechanistically, DJ001 suppresses radiation-induced HSC apoptosis via activation of the RhoGTPase, RAC1, and induction of BCL-XL. Furthermore, treatment of irradiated human HSCs with DJ001 promotes the regeneration of human HSCs capable of multilineage in vivo repopulation. These studies demonstrate the therapeutic potential of selective, small-molecule PTPσ inhibitors for human hematopoietic regeneration.

Recombinant Slit2 attenuates neuronal apoptosis via the Robo1-srGAP1 pathway in a rat model of neonatal HIE.

Apoptosis following hypoxic-ischemic injury to the brain plays a major role in neuronal cell death. The neonatal brain is more susceptible to injury as the cortical neurons are immature and there are lower levels of antioxidants. Slit2, an extracellular matrix protein, has been shown to be neuroprotective in various models of neurological diseases. However, there is no information about the role of Slit2 in neonatal hypoxia-ischemia. In this study, we evaluated the effect of Slit2 and its receptor Robo1 in a rat model with neonatal HIE. 10-day old rat pups were used to create the neonatal HIE model. The right common carotid artery was ligated followed by 2.5 h of hypoxia. Recombinant Slit2 was administered intranasally 1 h post HI, recombinant Robo1 was used as a decoy receptor and administered intranasally 1h before HI and srGAP1-siRNA was administered intracerebroventricularly 24 h before HI. Brain infarct area measurement, short-term and long-term neurological function tests, Western blot, immunofluorescence staining, Fluoro-Jade C staining, Nissl staining and TUNEL staining were the assessments done following drug administration. Recombinant Slit2 administration reduced neuronal apoptosis and neurological deficits after neonatal HIE which were reversed by co-administration of recombinant Robo1 and srGAP1-siRNA administration. Recombinant Slit2 showed improved outcomes possibly via the robo1-srGAP1 pathway which mediated the inhibition of RhoA. In this study, the results suggest that Slit2 may help in attenuation of apoptosis and could be a therapeutic agent for treatment of neonatal hypoxic ischemic encephalopathy.

Arsenic Trioxide Inhibits the Metastasis of Small Cell Lung Cancer by Blocking Calcineurin-Nuclear Factor of Activated T Cells (NFAT) Signaling.

BACKGROUND The inhibitory effect of arsenic trioxide (As2O3) on lung cancer has been reported in some preclinical studies. However, its effect on small cell lung cancer (SCLC) has been poorly explored. Calcineurin and its substrate, nuclear factor of activated T cells (NFAT), mediate the downstream signaling of VEGF, and is critical in the process endothelium activation and tumor metastasis. In this study, we aimed to evaluate whether As2O3 had inhibitory effects on endothelial cells activation and the metastasis of SCLC, and to explore the possible mechanisms. MATERIAL AND METHODS In vitro, human umbilical vein endothelial cells (HUVECs) were used. Cell Counting Kit-8 assay and cell migration assay were performed to determine the effect of As2O3 on HUVECs proliferation and migration. The level of calcineurin, NFAT, downstream factors for Down syndrome candidate region 1 (DSCR1), and the endogenous inhibitor of calcineurin, were evaluated by quantitative PCR and western blotting. In vivo, SCLC metastasis models were established by injecting NCI-H446 cells into tail veins of nude mice. Tumor-bearing mice were treated with As2O3 or calcineurin inhibitor for 10 days, after which tumor metastasis in target organs was evaluated. RESULTS As2O3 significantly inhibited the proliferation and migration of endothelial cells. Also, As2O3 inhibited the expression levels of calcineurin, NFAT, and the downstream target genes CXCR7 and RND1, while it upregulated the level of DSCR1. Both As2O3 and calcineurin inhibitor exhibited notable inhibitory effect on the metastasis of SCLC, without obvious side effects. CONCLUSIONS These findings suggested that As2O3 had remarkable inhibitory effects on the endothelial cell activation and SCLC metastasis, and the mechanism might be related to the blocking of calcineurin-NFAT signaling by upregulating DSCR1.

Rho GTPases as therapeutic targets in Alzheimer's disease.

The progress we have made in understanding Alzheimer's disease (AD) pathogenesis has led to the identification of several novel pathways and potential therapeutic targets. Rho GTPases have been implicated as critical components in AD pathogenesis, but their various functions and interactions make understanding their complex signaling challenging to study. Recent advancements in both the field of AD and Rho GTPase drug development provide novel tools for the elucidation of Rho GTPases as a viable target for AD. Herein, we summarize the fluctuating activity of Rho GTPases in various stages of AD pathogenesis and in several in vitro and in vivo AD models. We also review the current pharmacological tools such as NSAIDs, RhoA/ROCK, Rac1, and Cdc42 inhibitors used to target Rho GTPases and their use in AD-related studies. Finally, we summarize the behavioral modifications following Rho GTPase modulation in several AD mouse models. As key regulators of several AD-related signals, Rho GTPases have been studied as targets in AD. However, a consensus has yet to be reached regarding the stage at which targeting Rho GTPases would be the most beneficial. The studies discussed herein emphasize the critical role of Rho GTPases and the benefits of their modulation in AD.

High-Salt Intake Augments the Activity of the RhoA/ROCK Pathway and Reduces Intracellular Calcium in Arteries From Rats.

BACKGROUND: We investigated the influence of salt overconsumption on the functionality of the RhoA/Rho-associated kinase (ROCK) pathway and calcium regulation in arteries. METHODS: The aorta and small mesenteric arteries from rats fed a chow containing 2%, 4%, or 8% NaCl were evaluated in organ baths for the activity of the RhoA/ROCK pathway and intracellular calcium mobilization. Components of these pathways and intracellular calcium levels were also assessed in samples from 4% NaCl group. RESULTS: In arteries from animals fed regular chow, the ROCK inhibitor Y-27632 reduced the responses to phenylephrine, even when the smallest concentrations (1 and 3 µM) were tested. However, only higher concentrations of Y-27632 (10 and 50 µM) reduced phenylephrine-induced contraction in vessels from high-salt groups. Immunoblotting revealed augmented phosphorylation of the myosin phosphatase targeting subunit 1 and increased amounts of RhoA in the membrane fraction of aorta homogenates from the 4% NaCl group. Under calcium-free solution, vessels from NaCl groups presented reduced contractile responses to phenylephrine and caffeine, compared with the regular chow group. Moreover, decreased intracellular calcium at rest and after stimulation with ATP were found in aortic smooth muscle cells from 4% NaCl-fed rats, which also showed diminished levels of SERCA2 and SERCA3, but not of IP3 and ryanodine receptors, or STIM1 and Orai1 proteins. CONCLUSIONS: Arteries from rats subjected to high-salt intake are unable to properly regulate intracellular calcium levels and present augmented activity of the calcium sensitization pathway RhoA/ROCK. These changes may precede the development of vascular diseases induced by high-salt intake.

The anticancer phytochemical rocaglamide inhibits Rho GTPase activity and cancer cell migration.

Chemotherapy is one of the pillars of anti-cancer therapy. Although chemotherapeutics cause regression of the primary tumor, many chemotherapeutics are often shown to induce or accelerate metastasis formation. Moreover, metastatic tumors are largely resistant against chemotherapy. As more than 90% of cancer patients die due to metastases and not due to primary tumor formation, novel drugs are needed to overcome these shortcomings. In this study, we identified the anticancer phytochemical Rocaglamide (Roc-A) to be an inhibitor of cancer cell migration, a crucial event in metastasis formation. We show that Roc-A inhibits cellular migration and invasion independently of its anti-proliferative and cytotoxic effects in different types of human cancer cells. Mechanistically, Roc-A treatment induces F-actin-based morphological changes in membrane protrusions. Further investigation of the molecular mechanisms revealed that Roc-A inhibits the activities of the small GTPases RhoA, Rac1 and Cdc42, the master regulators of cellular migration. Taken together, our results provide evidence that Roc-A may be a lead candidate for a new class of anticancer drugs that inhibit metastasis formation.

Enriched environment reduces glioma growth through immune and non-immune mechanisms in mice.

Mice exposed to standard (SE) or enriched environment (EE) were transplanted with murine or human glioma cells and differences in tumour development were evaluated. We report that EE exposure affects: (i) tumour size, increasing mice survival; (ii) glioma establishment, proliferation and invasion; (iii) microglia/macrophage (M/Mφ) activation; (iv) natural killer (NK) cell infiltration and activation; and (v) cerebral levels of IL-15 and BDNF. Direct infusion of IL-15 or BDNF in the brain of mice transplanted with glioma significantly reduces tumour growth. We demonstrate that brain infusion of IL-15 increases the frequency of NK cell infiltrating the tumour and that NK cell depletion reduces the efficacy of EE and IL-15 on tumour size and of EE on mice survival. BDNF infusion reduces M/Mφ infiltration and CD68 immunoreactivity in tumour mass and reduces glioma migration inhibiting the small G protein RhoA through the truncated TrkB.T1 receptor. These results suggest alternative approaches for glioma treatment.

Diallyl disulfide induces G2/M arrest and promotes apoptosis through the p53/p21 and MEK-ERK pathways in human esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is an aggressive tumor with high incidence and mortality worldwide. Diallyl disulfide (DADS) is a natural organosulfur compound, isolated from garlic. In this study, MTT assay showed that DADS significantly reduced cell viability in a dose- and time-dependent manner in ESCC cells, with lower toxicity in normal liver cells. Cell cycle analysis revealed that DADS made G2/M phase arrest. Molecular analysis suggested that this cell cycle arrest was likely made by the decrease of cyclin B1, cdc2, p-cdc2, cdc25c in concomitance with activation of the p53/p21 pathway. Apoptosis was detected by Annexin V/PI staining. The molecule markers showed that DADS induced apoptosis through activating caspases, altering the Bax/Bcl-2 balance and suppressing the MEK-ERK pathway. Our data indicated that DADS has the potential to be an effective and safe anticancer agent for ESCC therapy in the near future.

Local translation of TC10 is required for membrane expansion during axon outgrowth.

The surface of developing axons expands in a process mediated by the exocyst complex. The spatio-temporal regulation of the exocyst is only partially understood. Here we report that stimulated membrane enlargement in dorsal root ganglion (DRG) axons is triggered by intra-axonal synthesis of TC10, a small GTPase required for exocyst function. Induced membrane expansion and axon outgrowth are inhibited after axon-specific knockdown of TC10 mRNA. To determine the relationship of intra-axonal TC10 synthesis with the previously described stimulus-induced translation of the cytoskeletal regulator Par3, we investigate the signalling pathways controlling their local translation in response to NGF. Phosphoinositide 3-kinase (PI3K)-dependent activation of the Rheb-mTOR pathway triggers the simultaneous local synthesis of TC10 and Par3. These results reveal the importance of local translation in the control of membrane dynamics and demonstrate that localized, mTOR-dependent protein synthesis triggers the simultaneous activation of parallel pathways.

Hinokitiol inhibits cell growth through induction of S-phase arrest and apoptosis in human colon cancer cells and suppresses tumor growth in a mouse xenograft experiment.

Hinokitiol (1), a tropolone-related natural compound, induces apoptosis and has anti-inflammatory, antioxidant, and antitumor activities. In this study, the inhibitory effects of 1 were investigated on human colon cancer cell growth and tumor formation of xenograft mice. HCT-116 and SW-620 cells derived from human colon cancers were found to be similarly susceptible to 1, with IC50 values of 4.5 and 4.4 µM, respectively. Compound 1 induced S-phase arrest in the cell cycle progression and decreased the expression levels of cyclin A, cyclin E, and Cdk2. Conversely, 1 increased the expression of p21, a Cdk inhibitor. Compound 1 decreased Bcl-2 expression and increased the expression of Bax, and cleaved caspase-9 and -3. The effect of 1 on tumor formation when administered orally was evaluated in male BALB/c-nude mice implanted intradermally separately with HCT-116 and SW-620 cells. Tumor volumes and tumor weights in the mice treated with 1 (100 mg/kg) were decreased in both cases. These results suggest that the suppression of tumor formation by compound 1 in human colon cancer may occur through cell cycle arrest and apoptosis.

Phenylboronic acid is a more potent inhibitor than boric acid of key signaling networks involved in cancer cell migration.

Previous studies from our lab have shown that both boric (BA) and phenylboronic- acid (PBA) inhibit the migration of prostate cancer cell lines, as well as non-tumorigenic prostate cells. Our results indicate that PBA is more potent than BA in targeting metastatic and proliferative properties of cancer cells. Here we focus on the impact of BA and PBA on Rho family of GTP-binding proteins and their downstream targets. Treatment with 1mM PBA and BA decreases activities of RhoA, Rac1, and Cdc42 in DU-145 metastatic prostate cancer cells, but not in normal RWPE-1 prostate cells. Furthermore, ROCKII activity and phosphorylation of myosin light chain kinase decrease as a result of either PBA or BA treatment in DU-145 cells, suggesting these compounds target actomyosin-based contractility.

Ibuprofen improves functional outcome after axotomy and immediate repair in the peripheral nervous system.

BACKGROUND: Activation of the Small GTP-binding protein Rho following the nerve injury contributes to the lack of regeneration in the peripheral nervous system. By elucidating the mechanisms leading to Rho activation, a nonsteroidal anti-inflammatory drug ibuprofen has been identified as a potent inhibitor of Rho activity. In this study we tested the hypothesis, that inhibiting Rho activity by ibuprofen will enhance posttraumatic regeneration after peripheral nerve injury. METHODS: In adult female Wistar rats we introduced an experimental injury by excising a 5 mm piece of the tibial nerve and returning it to the injury site as an interpositional graft. The animals then received ibuprofen or phosphate buffered saline through an osmotic pump for a period of 3 weeks. Following the injury we recorded tibial functional index (TFI) on a weekly basis. After 3 months we measured nerve conduction velocity and peak amplitude of action potential (PAAP). Also, the histomorphometric analysis was carried out in the zone distal to the injury site. RESULTS: We found that the animals receiving ibuprofen recovered the tibial nerve function more rapidly, with the TFI being significantly different 8, 9, 11 and 12 weeks after the injury. We also detected the values of the PAAP, the area of axons and the area of myelin to be significantly greater in the experimental group. CONCLUSIONS: Our results show that ibuprofen significantly enhanced regeneration after tibial nerve axotomy and repair in rats. This study is expected to set a stage for testing the ibuprofen in the human patients.

Bacterial protein toxins that modify host regulatory GTPases.

Many bacterial pathogens produce protein toxins to outmanoeuvre the immune system of the host. Some of these proteins target regulatory GTPases such as those belonging to the RHO family, which control the actin cytoskeleton of the host cell. In this Review, I discuss a diversity of mechanisms that are used by bacterial effectors and toxins to modulate the activity of host GTPases, with a focus on covalent modifications such as ADP-ribosylation, glucosylation, adenylylation, proteolysis, deamidation and transglutamination.

Isoflurane neurotoxicity is mediated by p75NTR-RhoA activation and actin depolymerization.

BACKGROUND: The mechanisms by which isoflurane injured the developing brain are not clear. Recent work has demonstrated that it is mediated in part by activation of p75 neurotrophin receptor. This receptor activates RhoA, a small guanosine triphosphatase that can depolymerize actin. It is therefore conceivable that inhibition of RhoA or prevention of cytoskeletal depolymerization might attenuate isoflurane neurotoxicity. This study was conducted to test these hypotheses using primary cultured neurons and hippocampal slice cultures from neonatal mouse pups. METHODS: Primary neuron cultures (days in vitro, 4-7) and hippocampal slice cultures from postnatal day 4-7 mice were exposed to 1.4% isoflurane (4 h). Neurons were pretreated with TAT-Pep5, an intracellular inhibitor of p75 neurotrophin receptor, the cytoskeletal stabilizer jasplakinolide, or their corresponding vehicles. Hippocampal slice cultures were pretreated with TAT-Pep5 before isoflurane exposure. RhoA activation was evaluated by immunoblot. Cytoskeletal depolymerization and apoptosis were evaluated with immunofluorescence microscopy using drebrin and cleaved caspase-3 staining, respectively. RESULTS: RhoA activation was increased after 30 and 120 min of isoflurane exposure in neurons; TAT-Pep5 (10 µm) decreased isoflurane-mediated RhoA activation at both time intervals. Isoflurane decreased drebrin immunofluorescence and enhanced cleaved caspase-3 in neurons, effects that were attenuated by pretreatment with either jasplakinolide (1 µm) or TAT-Pep5. TAT-Pep5 attenuated the isoflurane-mediated decrease in phalloidin immunofluorescence. TAT-Pep5 significantly attenuated isoflurane-mediated loss of drebrin immunofluorescence in hippocampal slices. CONCLUSIONS: Isoflurane results in RhoA activation, cytoskeletal depolymerization, and apoptosis. Inhibition of RhoA activation or prevention of downstream actin depolymerization significantly attenuated isoflurane-mediated neurotoxicity in developing neurons.

[HCV DF protein inhibits expression of p16 and p21 in HepG2 cells].

AIM: To investigate the effect of HCV DF (Double-shift F) protein on the expression p16 and p21 in HepG(2); cells. METHODS: DF gene was amplificated from the whole HCV 1b genome, and cloned into pCDNA3.0 vecter. The recombinant plasmid (pCDNA3.0/HCV-DF) and empty vector were transfected into HepG(2); cells. Screening was performed with G418. p16 and p21 mRNA were detected by semi-quantitative RT-PCR, and protein by Western blot. RESULTS: Stable expression of the recombinant plasmid was found in HCV DF protein. The expression of p16 and p21 in HepG();2 cells transfected with pCDNA3.0/HCV-DF were lower than those with blank plasmid. CONCLUSION: HCV DF protein inhibits expression of p16 and p21 in HepG(2); cells. This suggested that HCV DF protein may participate in the progress of hepatocellular carcinoma.

Cigarette smoke impairs clearance of apoptotic cells through oxidant-dependent activation of RhoA.

RATIONALE: Cigarette smoke (CS) is the primary cause of chronic obstructive pulmonary disease (COPD), an effect that is, in part, due to intense oxidant stress. Clearance of apoptotic cells (efferocytosis) is a critical regulator of lung homeostasis, which is defective in smokers and in patients with COPD, suggesting a role in disease pathogenesis. OBJECTIVES: We hypothesized that CS would impair efferocytosis through oxidant-dependent activation of RhoA, a known inhibitor of this process. METHODS: We investigated the effect of CS on efferocytosis in vivo and ex vivo, using acute, subacute, and long-term mouse exposure models. MEASUREMENTS AND MAIN RESULTS: Acute and subacute CS exposure suppressed efferocytosis by alveolar macrophages in a dose-dependent, reversible, and cell type-independent manner, whereas more intense CS exposure had an irreversible effect. In contrast, CS did not alter ingestion through the Fc gamma receptor. The inhibitory effect of CS on apoptotic cell clearance depended on oxidants, because the effect was blunted in oxidant-resistant ICR mice, and was prevented by either genetic or pharmacologic antioxidant strategies in vivo and ex vivo. CS inhibited efferocytosis through oxidant-dependent activation of the RhoA-Rho kinase pathway because (1) CS activated RhoA, (2) antioxidants prevented RhoA activation by CS, and (3) inhibitors of the RhoA-Rho kinase pathway reversed the suppressive effect of CS on apoptotic cell clearance in vivo and ex vivo. CONCLUSIONS: These findings advance the hypothesis that impaired efferocytosis may contribute to the pathogenesis of COPD and suggest the therapeutic potential of drugs targeting the RhoA-Rho kinase pathway.

Lipopolysaccharide-induced endothelial barrier breakdown is cyclic adenosine monophosphate dependent in vivo and in vitro.

OBJECTIVES: To determine whether cyclic adenosine monophosphate (cAMP) is critically involved in lipopolysaccharide (LPS)-induced breakdown of endothelial barrier functions in vivo and in vitro. DESIGN: Experimental laboratory research. SETTING: Research laboratory. SUBJECTS: Wistar rats and cultured human microvascular endothelial cells. INTERVENTION: Permeability measurements in single postcapillary venules in vivo and permeability measurements and cell biology techniques in vitro. MEASUREMENTS AND RESULTS: We demonstrate that within 120 minutes LPS increased endothelial permeability in rat mesenteric postcapillary venules in vivo and caused a barrier breakdown in human dermal microvascular endothelial cells in vitro. This was associated with the formation of large intercellular gaps and fragmentation of vascular endothelial cadherin immunostaining. Furthermore, claudin 5 immunostaining at cell borders was drastically reduced after LPS treatment. Interestingly, activity of the small GTPase Rho A, which has previously been suggested to mediate the LPS-induced endothelial barrier breakdown, was not increased after 2 hours. However, activity of Rac 1, which is known to be important for maintenance of endothelial barrier functions, was significantly reduced to 64 +/- 8% after 2 hours. All LPS-induced changes of endothelial cells were blocked by a forskolin-mediated or rolipram-mediated increase of cAMP. Consistently, enzyme-linked immunosorbent assay-based measurements demonstrated that LPS significantly decreased intracellular cAMP. CONCLUSION: In summary, our data demonstrate that LPS disrupts endothelial barrier properties by decreasing intracellular cAMP. This mechanism may involve inactivation of Rac 1 rather than activation of Rho A.

Negative impact of statins on oligodendrocytes and myelin formation in vitro and in vivo.

Statins are widely prescribed drugs in cardiovascular diseases. Recent studies also demonstrated anti-inflammatory and immunomodulatory properties of statins by modulating the activity of small GTPases. Statins are thus considered as potential therapeutic drug for the inflammatory demyelinating disease multiple sclerosis (MS). However, little is known about the effects of statins on myelin-forming oligodendrocytes. Here, we show that statins hamper process and myelin formation in vitro by interfering with Ras and Rho signaling in mature oligodendrocytes and provide evidence that statins impair ongoing remyelination in vivo. Our findings may have significant implications for the application of statins in MS patients and in other demyelinating diseases of the CNS.

Type I collagen receptor (alpha2beta1) signaling promotes prostate cancer invasion through RhoC GTPase.

The most frequent site of metastasis in human prostate cancer (PCa) is the bone. Preferential adhesion of PCa cells to bone-specific factors may facilitate the selective metastasis of the skeleton. The most abundant protein within the skeleton is type I collagen. We previously demonstrated that PCa cells selected in vitro for collagen I binding (LNCaP(col)) are highly motile and acquired the capacity to grow within the bone compared to nontumorigenic LNCaP parental cells. Treatment with alpha(2)beta(1)-neutralizing antibodies selectively blocked collagen-stimulated migration, suggesting that integrin signaling mediates PCa migration. To elucidate the mechanism of collagen-stimulated migration, we evaluated integrin-associated signaling pathways in non-collagen-binding LNCaP parental cells and in collagen-binding isogenic C4-2B and LNCaP(col) PCa cells. The expression and activity of RhoC guanosine triphosphatase was increased five- to eightfold in collagen-binding LNCaP(col) and C4-2B cells, respectively, compared to parental LNCaP cells. RhoC activation was selectively blocked with antibodies to alpha(2)beta(1) where treatment with a small hairpin RNA specific for RhoC suppressed collagen-mediated invasion without altering the PCa cells' affinity for collagen I. We conclude that the ligation of alpha(2)beta(1) by collagen I activates RhoC guanosine triphosphatase, which mediates PCa invasion, and suggests a mechanism for the preferential metastasis of PCa cells within the bone.