ROCK Inhibitor H-1152 Research Articles

Tankyrase inhibition interferes with junction remodeling, induces leakiness, and disturbs YAP1/TAZ signaling in the endothelium

Tankyrase inhibitors are increasingly considered for therapeutic use in malignancies that are characterized by high intrinsic β-catenin activity. However, how tankyrase inhibition affects the endothelium after systemic application remains poorly understood. In this study, we aimed to investigate how the tankyrase inhibitor XAV939 affects endothelial cell function and the underlying mechanism involved. Endothelial cell function was analyzed using sprouting angiogenesis, endothelial cell migration, junctional dynamics, and permeability using human umbilical vein endothelial cells (HUVEC) and explanted mouse retina. Underlying signaling was studied using western blot, immunofluorescence, and qPCR in HUVEC in addition to luciferase reporter gene assays in human embryonic kidney cells. XAV939 treatment leads to altered junctional dynamics and permeability as well as impaired endothelial migration. Mechanistically, XAV939 increased stability of the angiomotin-like proteins 1 and 2, which impedes the nuclear translocation of YAP1/TAZ and consequently suppresses TEAD-mediated transcription. Intriguingly, XAV939 disrupts adherens junctions by inducing RhoA-Rho dependent kinase (ROCK)-mediated F-actin bundling, whereas disruption of F-actin bundling through the ROCK inhibitor H1152 restores endothelial cell function. Unexpectedly, this was accompanied by an increase in nuclear TAZ and TEAD-mediated transcription, suggesting differential regulation of YAP1 and TAZ by the actin cytoskeleton in endothelial cells. In conclusion, our findings elucidate the complex relationship between the actin cytoskeleton, YAP1/TAZ signaling, and endothelial cell function and how tankyrase inhibition disturbs this well-balanced signaling.

Mechanism of α1-Adrenergic Receptor-Induced Increased Contraction of Rat Mesenteric Artery in Aging Hypertension Rats

Introduction: Vasoconstriction is triggered by an increase in intracellular-free calcium concentration. Growing evidence indicates that contraction is also regulated by calcium-independent mechanisms involving RhoA-Rho kinase (ROCK), protein kinase C (PKC), and so on. In this study, we studied the changes of vascular reactivity as well as the underlying signaling pathways in aging spontaneously hypertensive rats (SHRs). Methods: The artery tension induced by α1-adrenergic receptor activator (α1-AR) phenylephrine (PE) was measured in the absence or presence of myosin light chain kinase (MLCK), PKC, and ROCK inhibitors. The α1-AR, PKC, ROCK, phosphorylation of myosin light chain (MLC), and PKC-potentiated phosphatase inhibitors of 17 kDa (CPI-17) of rat mesenteric arteries were analyzed at the mRNA level or protein level. Results: The vascular tension measurements showed that there was a significant increase in the mesenteric artery contraction induced by PE in old SHR. MLCK inhibitor ML-7 can similarly inhibit PE-induced vasoconstriction. PKC inhibitor GF109203X has the weakest inhibitory effect on PE-induced contraction in old SHR. At the presence of ROCK inhibitor H1152, PE-induced contraction was significantly reduced in young Wistar-Kyoto (WKY) rats, but this phenomenon disappeared in other rats. Furthermore, in old SHR the protein expression of α1-AR decreased and phosphorylation of MLC and CPI-17 were upregulated and MLC phosphatase (MLCP) activity was significantly lower. The expressions of PKC were upregulated in SHR and old rats. In addition, the expression of ROCK-1 was decreased and ROCK-2 was significantly upregulated with age in SHR. Conclusion: In aging hypertension, the expression/activity of PKC or ROCK-2/CPI-17 excessively increased, MLCP activity decreased and MLC phosphorylation enhanced, leading to increased α1-AR-induced vasoconstriction.

Mechanopharmacology and Synergistic Relaxation of Airway Smooth Muscle.

Asthmatic airways are stiffer than normal. We have shown that the cytoskeletal passive stiffness of airway smooth muscle (ASM) can be regulated by intracellular signaling pathways, especially those associated with Rho kinase (ROCK). We have also shown that an oscillatory strain reduces the passive stiffness of ASM and its ability to generate force. Here, we investigated the combined effect of inhibiting the ASM contraction with β2 agonist and decreasing the ASM cytoskeletal stiffness with ROCK inhibitor and/or force oscillation (FO) on the relaxation of contracted ASM. We hypothesize that the ASM relaxation can be synergistically enhanced by the combination of these interventions, because drug-induced softening of the cytoskeleton enhances the FO-induced relaxation and vice versa. Sheep tracheal strips were isotonically contracted to acetylcholine (3 × 10−5 M). At the plateau of shortening, β2 agonist salbutamol (10−7 M), ROCK inhibitor H1152 (10−7 M), and FO (square wave, 1 Hz, amplitude 6% maximal active force) were applied either alone or in combination. After adjusting for nonspecific time-dependent variation, relengthening by individual interventions with low-dose salbutamol or H1152, or small amplitude FO was not significantly different from zero. However, significant relengthening was observed in all combination treatments. The relengthening was greater than the mathematical sum of relengthening caused by individual treatments thereby demonstrating synergistic relaxation. The ASM stiffness did not change with salbutamol or H1152 treatments, but was lower with FO in combination with H1152. The results suggest that the mechanopharmacological treatment can be an effective therapy for asthma.

Constriction of Retinal Venules to Endothelin-1: Obligatory Roles of ETA Receptors, Extracellular Calcium Entry, and Rho Kinase.

PurposeEndothelin-1 (ET-1) is a potent vasoconstrictor peptide implicated in retinal venous pathologies such as diabetic retinopathy and retinal vein occlusion. However, underlying mechanisms contributing to venular constriction remain unknown. Thus, we examined the roles of ET-1 receptors, extracellular calcium (Ca2+), L-type voltage-operated calcium channels (L-VOCCs), Rho kinase (ROCK), and protein kinase C (PKC) in ET-1-induced constriction of retinal venules.MethodsPorcine retinal venules were isolated and pressurized for vasoreactivity study using videomicroscopic techniques. Protein and mRNA were analyzed using molecular tools.ResultsRetinal venules developed basal tone and constricted concentration-dependently to ET-1. The ETA receptor (ETAR) antagonist BQ123 abolished venular constriction to ET-1, but ETB receptor (ETBR) antagonist BQ788 had no effect on vasoconstriction. The ETBR agonist sarafotoxin S6c did not elicit vasomotor activity. In the absence of extracellular Ca2+, venules lost basal tone and ET-1–induced constriction was nearly abolished. Although L-VOCC inhibitor nifedipine also reduced basal tone and blocked vasoconstriction to L-VOCC activator Bay K8644, constriction of venules to ET-1 remained. The ROCK inhibitor H-1152 but not PKC inhibitor Gö 6983 prevented ET-1-induced vasoconstriction. Protein and mRNA expressions of ETARs and ETBRs, along with ROCK1 and ROCK2 isoforms, were detected in retinal venules.ConclusionsExtracellular Ca2+ entry via L-VOCCs is essential for developing and maintaining basal tone of porcine retinal venules. ET-1 causes significant constriction of retinal venules by activating ETARs and extracellular Ca2+ entry independent of L-VOCCs. Activation of ROCK signaling, without involvement of PKC, appears to mediate venular constriction to ET-1 in the porcine retina.

In vivo Effects in Melanoma of ROCK Inhibition-Induced FasL Overexpression.

Ectopic Fas-ligand (FasL) expression in tumor cells is responsible for both tumor escape through tumor counterattack of Fas-positive infiltrating lymphocytes and tumor rejection though inflammatory and immune responses. We have previously shown that RhoA GTPase and its effector ROCK negatively control FasL membrane expression in murine melanoma B16F10 cells. In this study, we found that B16F10 treatment with the ROCK inhibitor H1152 reduced melanoma development in vivo through FasL membrane overexpression. Although H1152 treatment did not reduce tumor growth in vitro, pretreatment of tumor cells with this inhibitor delayed tumor appearance, and slowed tumor growth in C57BL/6 immunocompetent mice. Thanks to the use of mice-bearing mutated Fas receptors (B6/lpr), we found that reduced tumor growth, observed in immunocompetent mice, was linked to FasL overexpression induced by H1152 treatment. Tumor growth analysis in immunosuppressed NUDE and IFN-γ-KO mice highlighted major roles for T lymphocytes and IFN-γ in the H1152-induced tumor growth reduction. Histological analyses of subcutaneous tumors, obtained from untreated versus H1152-treated B16F10 cells, showed that H1152 pretreatment induced a strong intratumoral infiltration of leukocytes. Cytofluorometric analysis showed that among these leukocytes, the number of activated CD8 lymphocytes was increased. Moreover, their antibody-induced depletion highlighted their main responsibility in tumor growth reduction. Subcutaneous tumor growth was also reduced by repeated intravenous injections of a clinical ROCK inhibitor, Fasudil. Finally, H1152-induced ROCK inhibition also reduced pulmonary metastasis implantation independently of T cell-mediated immune response. Altogether, our data suggest that ROCK inhibitors could become interesting pharmacological molecules for melanoma immunotherapy.

RhoA/ROCK Signaling Triggers Innate Immune Responses in Ischemia-Stressed Liver Through Activation of c-Jun N-Terminal Kinase Pathway.

Background: The RhoA/Rho kinase (ROCK) signaling plays an important role in innate immune cell activation and the development of inflammatory diseases. However, little is known on the functional role of RhoA/ROCK signaling in the activation of innate immune responses during organ ischemia-reperfusion injury (IRI). This study was designed to explore potential molecular mechanisms of Rho/ROCK signaling in innate immune responses triggered by IR stress in a mouse liver. Methods: Using a partial hepatic warm ischemia model (90 min), C57BL/6 wide-type (WT) mice (n=6/gr) were pretreated with ROCK inhibitor H1152 (3 mg/kg, i.p.) or vehicle at 1h prior to ischemia, and then sacrificed at 6 h of reperfusion. In parallel in vitro study, bone marrow-derived WT macrophages (BMMs) were transfected with JNK1 siRNA/nonspecific siRNA (100 nM) or treated with H1152 (10μM), and then incubated with LPS (100 ng/ml). Results: Hepatic IR increased ROCK1 and c-Jun N-terminal kinase (JNK) expression and enhanced PTEN activity, whereas anti-ROCK (H1152) treatment decreased JNK and PTEN/TLR4 activation yet increased PI3K/Akt expression. Unlike in vehicle-treated WT controls, H1152 preconditioning improved hepatic function, reduced hepatocellular damage, and depressed macrophage/neutrophil trafficking, pro-inflammatory cytokine programs, and hepatocellular necrosis/apoptosis, while increasing anti-apoptotic functions in ischemic liver lobes. LPS-stimulated BMMs in vitro revealed increased expression of ROCK1, JNK, PTEN, and TLR4. However, knockdown of JNK with siRNA readily suppressed PTEN/TLR4, leading to diminished NF-κB activation and decreased expression of proinflammatory mediators. Consistent with our in vivo findings, pretreatment of LPS-stimulated BMMs with H1152 decreased JNK expression and upregulated Akt phosphorylation. Conclusion: This study demonstrates that IR-triggered RhoA/ROCK signaling induced JNK activation, leading to enhanced PTEN/TLR4 signaling. Inhibition of RhoA/ROCK protected livers from IR-mediated inflammation. Knockdown of JNK depressed PTEN yet promoted PI3K/Akt, which in turn negatively regulated TLR4 activation. By documenting the critical role of RhoA/ROCK-JNK signaling cascade in the mechanism of innate immune-driven local inflammation responses, our findings provide the rationale for a novel therapeutic strategy against IRI in organ transplant recipients.

Role of Endothelium in Vasomotor Responses to Endothelin System and Protein Kinase C Activation in Porcine Retinal Arterioles

Endothelial cells synthesize vasodilator nitric oxide (NO) and vasoconstrictor endothelin-1 (ET-1) from NO synthase (eNOS) and endothelin-converting enzyme-1 (ECE-1), respectively. Protein kinase C (PKC) and Rho kinase (ROCK) are major signaling molecules mediating vasoconstriction. Although endothelial cells express eNOS, ECE-1, endothelin B (ET(B)) receptors, PKC, and ROCK, their influences on ET-1-induced vasoconstriction remain elusive. We studied whether these endothelial signaling molecules modulate retinal arteriolar constriction to ET-1. Porcine retinal arterioles were isolated and pressurized for vasomotor study, under conditions with intact or denuded endothelium, using videomicroscopic techniques. Retinal arterioles developed similar resting tone (≈45% of maximum diameter) with or without endothelium. Endothelial denudation attenuated vasoconstriction to ET-1 precursor, big ET-1, by almost equal to 50%, but did not affect vasoconstrictions to ET-1, ET(B) agonist sarafotoxin S6c, or PKC activator phorbol-12, 13-dibutyrate (PDBu). The ROCK inhibitor H-1152 caused vasodilation, and abolished vasoconstrictions to ET-1 and PDBu independent of endothelium. With L-type voltage-operated calcium channel (L-VOCC) blocker nifedipine, PDBu-induced vasoconstriction was abolished and converted to NO-mediated vasodilation in the presence of endothelium. The ET-1-induced vasoconstriction was unaffected by NO released from endothelium during flow elevation. Endothelial and smooth muscle ECE-1 contribute equally to synthesis of vasoactive ET-1 in retinal arterioles, with nominal role of endothelial ETB receptors in vasoconstriction to ET-1. The PKC activation leads to endothelium-dependent NO-mediated vasodilation when smooth muscle contraction is ablated by L-VOCC blockade. Endothelial cells and NO appear to have modest roles in modulating ROCK-dependent vasoconstriction, and are insufficient to counteract smooth muscle contractions to ET-1 and PKC activation.

Role of Endothelium in Vasomotor Responses to Endothelin System and Protein Kinase C Activation in Porcine Retinal Arterioles

Purpose.: Endothelial cells synthesize vasodilator nitric oxide (NO) and vasoconstrictor endothelin-1 (ET-1) from NO synthase (eNOS) and endothelin-converting enzyme-1 (ECE-1), respectively. Protein kinase C (PKC) and Rho kinase (ROCK) are major signaling molecules mediating vasoconstriction. Although endothelial cells express eNOS, ECE-1, endothelin B (ETB) receptors, PKC, and ROCK, their influences on ET-1–induced vasoconstriction remain elusive. We studied whether these endothelial signaling molecules modulate retinal arteriolar constriction to ET-1. Methods.: Porcine retinal arterioles were isolated and pressurized for vasomotor study, under conditions with intact or denuded endothelium, using videomicroscopic techniques. Results.: Retinal arterioles developed similar resting tone (≈45% of maximum diameter) with or without endothelium. Endothelial denudation attenuated vasoconstriction to ET-1 precursor, big ET-1, by almost equal to 50%, but did not affect vasoconstrictions to ET-1, ETB agonist sarafotoxin S6c, or PKC activator phorbol-12, 13-dibutyrate (PDBu). The ROCK inhibitor H-1152 caused vasodilation, and abolished vasoconstrictions to ET-1 and PDBu independent of endothelium. With L-type voltage-operated calcium channel (L-VOCC) blocker nifedipine, PDBu-induced vasoconstriction was abolished and converted to NO-mediated vasodilation in the presence of endothelium. The ET-1–induced vasoconstriction was unaffected by NO released from endothelium during flow elevation. Conclusions.: Endothelial and smooth muscle ECE-1 contribute equally to synthesis of vasoactive ET-1 in retinal arterioles, with nominal role of endothelial ETB receptors in vasoconstriction to ET-1. The PKC activation leads to endothelium-dependent NO-mediated vasodilation when smooth muscle contraction is ablated by L-VOCC blockade. Endothelial cells and NO appear to have modest roles in modulating ROCK-dependent vasoconstriction, and are insufficient to counteract smooth muscle contractions to ET-1 and PKC activation.

Atorvastatin inhibited Rho-associated kinase 1 (ROCK1) and focal adhesion kinase (FAK) mediated adhesion and differentiation of CD133+CD44+ prostate cancer stem cells

Prostate cancer has become a global health concern and is one of the leading causes of cancer death of men after lung and gastric cancers. It has been suggested that the 3-hydroxy-3-methyl-glutarylcoenzyme-CoA (HMG-CoA) reductase inhibitor atorvastatin shows anticancer activity in prostate cancer cell lines. To this end, we analyzed the influence of atorvastatin on the cell adhesion and differentiation of CD133+CD44+ cells derived from prostate cancer biopsies and peripheral blood. CD133+CD44+ cells were treated with atorvastatin (16–64μM) for different time periods. Cell adhesion to endothelial cell monolayers and differentiation into prostate cancer cells were evaluated. α1, β1 and α2β1 integrins adhesion receptors and the downstream target of atorvastatin Rho-dependent kinase (ROCK) and focal adhesion kinase (FAK) were analyzed by Western blot. Further blocking studies with the ROCK inhibitor H1152, anti-FAK antibody and anti-integrin α1 and β1 antibodies were carried out. Atorvastatin treatment inhibited dose-dependently cell attachment to endothelium and differentiation. The inhibitory effect of atorvastatin on cell adhesion was associated with decreased expression of integrins α1 and β1 and phosphorylated MYPT1 and FAK. Furthermore, atorvastatin strongly reduced ROCK1 and FAK mediated differentiation of CD133+CD44+ cells, which was confirmed by antibody treatment. Atorvastatin modified the expression of cell adhesion molecules and differentiation markers. These beneficial effects of atorvastatin may be mediated by ROCK and FAK signaling pathway. The data presented may point to novel treatment options for prostate cancer.

S127 Influenza A and Poly(I:C) Induce α Vβ6-Integrin-Mediated TGFβ Activity in Human Epithelial Cells Via Stimulation of TLR3

People with chronic lung disease are more susceptible to influenza infection which may lead to exacerbation of pre-existing conditions such as fibrosis. Transforming growth factor-β (TGFβ) is a profibrotic cytokine,...

Constriction of Retinal Arterioles to Endothelin-1: Requisite Role of Rho Kinase Independent of Protein Kinase C and L-Type Calcium Channels

Although endothelin-1 (ET-1) is a potent vasoconstrictor peptide implicated in several retinal pathologies, the underlying mechanism of vasoconstriction is understood incompletely. We addressed this issue by assessing the contributions of extracellular calcium (Ca²⁺), L-type voltage-operated calcium channels (L-VOCCs), Rho kinase (ROCK), and protein kinase C (PKC) to ET-1-induced constriction of porcine retinal arterioles, all of which have been implicated commonly in vascular smooth muscle contraction. Porcine retinal arterioles (~50-100 μm) were isolated for vasomotor study and molecular assessment of ROCK isoforms. Isolated arterioles developed stable basal tone at 55 cmH₂O luminal pressure and constricted to ET-1 (0.1 nM) with a 40 ± 6% reduction in resting diameter in 20 minutes. In the absence of extraluminal Ca²⁺, arterioles lost basal tone and failed to constrict to ET-1. Although L-VOCC inhibitor nifedipine reduced basal tone and blocked vasoconstriction to PKC activator PDBu, vasoconstriction to ET-1 was unaffected. The broad-spectrum PKC inhibitor Gö-6983 abolished vasoconstriction to PDBu, but did not alter ET-1-induced vasoconstriction or basal tone. Incubation of arterioles with ROCK inhibitor H-1152 abolished basal tone and vasoconstrictions to ET-1 and PDBu. Both ROCK1 and ROCK2 isoforms were expressed in the retinal arteriolar wall. Extracellular Ca²⁺ entry via L-VOCCs and basal ROCK activity play important roles in the maintenance of basal tones of porcine retinal arterioles. ET-1-induced constriction is mediated by extracellular Ca²⁺ entry independent of L-VOCCs and by ROCK activation without the involvement of PKC. However, direct PKC activation can cause vasoconstriction via L-VOCC and ROCK signaling.

Endothelin‐1 Signaling in Coronary Arteriolar Constriction

Endothelin‐1 (ET‐1) is a potent vasoconstrictor implicated in coronary heart diseases, but its signaling for vasoconstriction remains elusive. Herein, we probed the role of voltage‐gated Ca2+ channels (VGCCs), phospholipase C (PLC), myosin light chain kinase (MLCK), protein kinase C (PKC) and Rho kinase (ROCK) in the porcine coronary arteriolar response to a pathologic dose of ET‐1 (0.1 nM). Isolated coronary arterioles (~70 μm) constricted to ET‐1 (from 52±3% to 32±3% max. diameter) in a manner sensitive to ETA receptor blocker BQ123, zero‐extraluminal Ca2+, and PLC inhibitor U73122. Inhibiting VGCCs by nifedipine or MLCK by ML‐9 abolished basal tone but did not affect ET‐1 response. ROCK inhibitor H1152 blocked vasoconstrictions to ET‐1 but not PKC activator PDBu. Broad‐spectrum PKC inhibitor Gö6983 blocked vasoconstriction to PDBu but not ET‐1. The PKC‐activated CPI‐17 protein was undetectable in arterioles. Moreover, ET‐1 elicited phosphorylation of myosin‐binding subunit (MBS) in H1152‐sensitive manner. ROCK2, but not ROCK1, siRNA inhibited vasoconstriction to ET‐1. We conclude that VGCCs and MLCK contribute to the generation of basal tone rather than the constriction to ET‐1. The sequential activation of ETA receptors, PLC and the VGCC‐independent Ca2+ entry leads to vasoconstriction to ET‐1 via a pathway involved in ROCK2‐associated MBS phosphorylation independent of PKC signaling.

Phorbol 12,13-Dibutyrate-Induced, Protein Kinase C-Mediated Contraction of Rabbit Bladder Smooth Muscle

Contraction of bladder smooth muscle is predominantly initiated by M3 muscarinic receptor-mediated activation of the Gq/11-phospholipase C β-protein kinase C (PKC) and the G12/13-RhoGEF-Rho kinase (ROCK) pathways. However, these pathways and their downstream effectors are not well understood in bladder smooth muscle. We used phorbol 12,13-dibutyrate (PDBu), and 1,2-dioctanoyl-sn-glycerol (DOG), activators of PKC, in this investigation. We were interested in dissecting the role(s) of PKC and to clarify the signaling pathways in bladder smooth muscle contraction, especially the potential cross-talk with ROCK and their downstream effectors in regulating myosin light chain phosphatase activity and force. To achieve this goal, the study was performed in the presence or absence of the PKC inhibitor bisindolylmaleimide-1 (Bis) or the ROCK inhibitor H-1152. Phosphorylation levels of Thr38-CPI-17 and Thr696/Thr850 myosin phosphatase target subunit (MYPT1) were measured during PDBu or DOG stimulation using site specific antibodies. PDBu-induced contraction in bladder smooth muscle involved both activation of PKC and PKC-dependent activation of ROCK. CPI-17 as a major downstream effector, is phosphorylated by PKC and ROCK during PDBu and DOG stimulation. Our results suggest that Thr696 and Thr850-MYPT1 phosphorylation are not involved in the regulation of a PDBu-induced contraction. The results also demonstrate that bladder smooth muscle contains a constitutively active isoform of ROCK that may play an important role in the regulation of bladder smooth muscle basal tone. Together with the results from our previous study, we developed a working model to describe the complex signaling pathways that regulate contraction of bladder smooth muscle.

Rho-kinase inhibition attenuates calcium-induced contraction in β-escin but not Triton X-100 permeabilized rabbit femoral artery

K+-depolarization (KCl) of smooth muscle has long been known to cause Ca2+-dependent contraction, but only recently has this G protein-coupled receptor (GPCR)-independent stimulus been associated with rhoA kinase (ROCK)-dependent myosin light chain (MLC) phosphatase inhibition and Ca2+ sensitization. This study examined effects of ROCK inhibition on the concentration-response curves (CRCs) generated in femoral artery by incrementally adding increasing concentrations of KCl to intact tissues, and Ca2+ to tissues permeabilized with Triton X-100, β-escin and α-toxin. For a comparison, tissue responses were assessed also in the presence of protein kinase C (PKC) and MLC kinase inhibition. The ROCK inhibitor H-1152 induced a strong concentration-dependent inhibition of a KCl CRC. A relatively low GF-109203X concentration (1 μM) sufficient to inhibit conventional PKC isotypes also inhibited the KCl CRC but did not affect the maximum tension. ROCK inhibitors had no effect on the Ca2+ CRC induced in Triton X-100 or α-toxin permeabilized tissues, but depressed the maximum contraction induced in β-escin permeabilized tissue. GF-109203X at 1 μM depressed the maximum Ca2+-dependent contraction induced in α-toxin permeabilized tissue and had no effect on the Ca2+ CRC induced in Triton X-100 permeabilized tissue. The MLC kinase inhibitor wortmannin (1 μM) strongly depression the Ca2+ CRCs in tissues permeabilized with Triton X-100, α-toxin and β-escin. H-1152 inhibited contractions induced by a single exposure to a submaximum [Ca2+] (pCa 6) in both rabbit and mouse femoral arteries. These data indicate that β-escin permeabilized muscle preserves GPCR-independent, Ca2+- and ROCK-dependent, Ca2+ sensitization.

Simvastatin modulates the adhesion and growth of hepatocellular carcinoma cells via decrease of integrin expression and ROCK

Hepatocellular carcinoma (HCC) has become a global health concern and is one of the leading causes of cancer death after lung and gastric cancers. It has been suggested that the 3-hydroxy-3-methyl-glutarylcoenzyme-CoA (HMG-CoA) reductase inhibitor simvastatin exhibits anticancer properties. To this end, we analyzed the influence of simvastatin on the cell growth and adhesion of HCC and evaluated the yet poorly characterized mechanism of action of simvastatin in HCC. HepG2 and Huh7 cells were treated with simvastatin (16-64 μM) for different time periods. Cell proliferation using the MTT assay and tumor cell adhesion to endothelial cell monolayers were evaluated. ß1, ß3 and α2 integrin adhesion receptors and the downstream target of simvastatin Rho-dependent kinase (ROCK) were analyzed by Western blot. Further blocking studies with the ROCK-inhibitor H1152 and anti-integrin ß1 and ß3 antibodies were carried out. Simvastatin treatment inhibited dose-dependently tumor cell growth and attachment to endothelium. The inhibitory effect of simvastatin on cell adhesion was associated with decreased expression of ß1, ß3 and α2 integrins. Furthermore, simvastatin strongly reduced the expression of ROCK-I and activated MYPT, an indicator of ROCK activity. Also, the ROCK-inhibitor H1152 reduced the adhesive capacity of the tumor cells. Anti-adhesive effects of simvastatin were prevented by exogenous mevalonate, a downstream product of HMG-CoA. Tumor cell adhesion to endothelium was significantly impaired following incubation with functional anti-ß1 antibody. Simvastatin modifies the expression of cell adhesion molecules leading to reduced tumor cell growth and invasion. These beneficial effects of simvastatin may be mediated by ROCK. The data presented may point to novel treatment options for HCC.

Carbachol-induced rabbit bladder smooth muscle contraction: roles of protein kinase C and Rho kinase

Smooth muscle contraction is regulated by phosphorylation of the myosin light chain (MLC) catalyzed by MLC kinase and dephosphorylation catalyzed by MLC phosphatase. Agonist stimulation of smooth muscle results in the inhibition of MLC phosphatase activity and a net increase in MLC phosphorylation and therefore force. The two pathways believed to be primarily important for inhibition of MLC phosphatase activity are protein kinase C (PKC)-catalyzed CPI-17 phosphorylation and Rho kinase (ROCK)-catalyzed myosin phosphatase-targeting subunit (MYPT1) phosphorylation. The goal of this study was to determine the roles of PKC and ROCK and their downstream effectors in regulating MLC phosphorylation levels and force during the phasic and sustained phases of carbachol-stimulated contraction in intact bladder smooth muscle. These studies were performed in the presence and absence of the PKC inhibitor bisindolylmaleimide-1 (Bis) or the ROCK inhibitor H-1152. Phosphorylation levels of Thr(38)-CPI-17 and Thr(696)/Thr(850)-MYPT1 were measured at different times during carbachol stimulation using site-specific antibodies. Thr(38)-CPI-17 phosphorylation increased concurrently with carbachol-stimulated force generation. This increase was reduced by inhibition of PKC during the entire contraction but was only reduced by ROCK inhibition during the sustained phase of contraction. MYPT1 showed high basal phosphorylation levels at both sites; however, only Thr(850) phosphorylation increased with carbachol stimulation; the increase was abolished by the inhibition of either ROCK or PKC. Our results suggest that during agonist stimulation, PKC regulates MLC phosphatase activity through phosphorylation of CPI-17. In contrast, ROCK phosphorylates both Thr(850)-MYPT1 and CPI-17, possibly through cross talk with a PKC pathway, but is only significant during the sustained phase of contraction. Last, our results demonstrate that there is a constitutively activate pool of ROCK that phosphorylates MYPT1 in the basal state, which may account for the high resting levels of MLC phosphorylation measured in rabbit bladder smooth muscle.

Calcium-independent phospholipase A2 participates in KCl-induced calcium sensitization of vascular smooth muscle

In vascular smooth muscle, KCl not only elevates intracellular free Ca2+ ([Ca2+]i), myosin light chain kinase activity and tension (T), but also can inhibit myosin light chain phosphatase activity by activation of rhoA kinase (ROCK), resulting in Ca2+ sensitization (increased T/[Ca2+]i ratio). Precisely how KCl causes ROCK-dependent Ca2+ sensitization remains to be determined. Using Fura-2-loaded isometric rings of rabbit artery, we found that the Ca2+-independent phospholipase A2 (iPLA2) inhibitor, bromoenol lactone (BEL), reduced the KCl-induced tonic but not early phasic phase of T and potentiated [Ca2+]i, reducing Ca2+ sensitization. The PKC inhibitor, GF-109203X (≥3μM) and the pseudo-substrate inhibitor of PKCζ produced a response similar to BEL. BEL reduced basal and KCl-stimulated myosin phosphatase phosphorylation. Whereas BEL and H-1152 produced strong inhibition of KCl-induced tonic T (∼50%), H-1152 did not induce additional inhibition of tissues already inhibited by BEL, suggesting that iPLA2 links KCl stimulation with ROCK activation. The cPLA2 inhibitor, pyrrolidine-1, inhibited KCl-induced tonic increases in [Ca2+]i but not T, whereas the inhibitor of 20-HETE production, HET0016, acted like the ROCK inhibitor H-1152 by causing Ca2+ desensitization. These data support a model in which iPLA2 activity regulates Ca2+ sensitivity.