cAMP-response element-binding protein mediates acid-induced NADPH oxidase NOX5-S expression in Barrett esophageal adenocarcinoma cells

Gastroesophageal reflux disease complicated by Barrett esophagus (BE) is a major risk factor for esophageal adenocarcinoma (EA). The mechanisms whereby acid reflux may accelerate the progression from BE to EA are not known. We found that NOX1 and NOX5-S were the major isoforms of NADPH oxidase in SEG1-EA cells. The expression of NOX5-S mRNA was significantly higher in these cells than in esophageal squamous epithelial cells. NOX5 mRNA was also significantly higher in Barrett tissues with high grade dysplasia than without dysplasia. Pulsed acid treatment significantly increased H(2)O(2) production in both SEG1-EA cells and BE mucosa, which was blocked by the NADPH oxidase inhibitor apocynin. In SEG1 cells, acid treatment increased mRNA expression of NOX5-S, but not NOX1, and knockdown of NOX5 by NOX5 small interfering RNA abolished acid-induced H(2)O(2) production. In addition, acid treatment increased intracellular Ca(2+) and phosphorylation of cAMP-response element-binding protein (CREB). Acid-induced NOX5-S expression and H(2)O(2) production were significantly inhibited by removal of extracellular Ca(2+) and by knockdown of CREB using CREB small interfering RNA. Two novel CREB-binding elements TGACGAGA and TGACGCTG were identified in the NOX5-S gene promoter. Overexpression of CREB significantly increased NOX5-S promoter activity. Knockdown of NOX5 significantly decreased [(3)H]thymidine incorporation, which was restored by 10(-13) M H(2)O(2). Knockdown of NOX5 also significantly decreased retinoblastoma protein phosphorylation and increased cell apoptosis and caspase-9 expression. In conclusion, in SEG1 EA cells NOX5-S is overexpressed and mediates acid-induced H(2)O(2) production. Acid-induced NOX5-S expression depends on an increase in intracellular Ca(2+) and activation of CREB. NOX5-S contributes to increased cell proliferation and decreased apoptosis.     

STAT5 mediates PAF-induced NADPH oxidase NOX5-S expression in Barrett's esophageal adenocarcinoma cells

We have shown that NADPH oxidase NOX5-S is overexpressed in Barrett's esophageal adenocarcinoma (EA) cells and may contribute to the progression from Barrett's esophagus (BE) to EA presumably by increasing cell proliferation and decreasing apoptosis (Fu X, Beer DG, Behar J, Wands J, Lambeth D, Cao W. J Biol Chem 281: 20368-20382, 2006). The mechanism(s) of NOX5-S overexpression in EA, however, is not fully understood. In SEG1 EA cells we found that acid treatment significantly increased platelet-activating factor (PAF) production, which in turn markedly increased NOX5-S expression and hydrogen peroxide (H(2)O(2)) production. Knockdown of NOX5-S by NOX5-S small interfering RNA (siRNA) blocked PAF-dependent H(2)O(2) production. PAF-dependent induction of NOX5-S expression and H(2)O(2) production were significantly decreased by the MAPK kinase 1 inhibitor PD-98059, by the cytosolic phospholipase A(2) (cPLA(2)) inhibitor AACOCF3, and by STAT5 downregulation with STAT5 siRNA. PAF significantly increased the phosphorylation of ERK1/2 MAPK, cPLA(2), and STAT5. Using inhibitors, we demonstrated that PAF-induced STAT5 phosphorylation depends on activation of ERK1/2 MAPK and cPLA(2), whereas PAF-induced cPLA(2) phosphorylation was associated with activation of ERK1/2 MAPK. Given that STAT5 bound to the c-sis-inducible element (TTCTGGTAA) of the NOX5-S promoter, overexpression of STAT5 significantly increased NOX5-S promoter activity. We conclude that acid-induced NOX5-S expression and H(2)O(2) production is mediated in part by production of PAF in SEG1 EA cells, and that PAF-induced increase in NOX5-S expression depends on sequential activation of ERK MAP kinases, cPLA(2), and STAT5 in these cells.     

NADPH oxidase NOX5-S mediates acid-induced cyclooxygenase-2 expression via activation of NF-kappaB in Barrett's esophageal adenocarcinoma cells

We have shown that the NADPH oxidase NOX5-S may play an important role in the progression from Barrett's esophagus to esophageal adenocarcinoma (EA) by increasing cell proliferation and decreasing apoptosis. However, the mechanism of the acid-induced NOX5-S-mediated increase in cell proliferation is not known. We found that, in SEG1 EA cells, the acid-induced increase in prostaglandin E2 (PGE2) production was mediated by activation of cyclooxygenase-2 (COX2) but not by COX1. Acid treatment increased intracellular Ca2+, and a blockade of intracellular Ca2+ increase inhibited the acid-induced increase in COX2 expression and PGE2 production. Knockdown of NOX5-S or NF-kappaB1 p50 by their small interfering RNA significantly inhibited acid-induced COX2 expression and PGE2 production in SEG1 cells. Acid treatment significantly decreased IkappaBalpha and increased luciferase activity when SEG1 cells were transfected with an NF-kappaB in vivo activation reporter plasmid, pNF-kappaB-Luc. In a novel Barrett's cell line overexpressing NOX5-S, IkappaBalpha was significantly reduced, and luciferase activity increased when these Barrett's cells were transfected with pNF-kappaB-Luc. Overexpression of NOX5-S in Barrett's cells significantly increased H2O2 production, COX2 expression, PGE2 production, and thymidine incorporation. The increase in thymidine incorporation occurring in NOX5-S-overexpressing Barrett's cells or induced by acid treatment in SEG1 EA cells was significantly decreased by COX2 inhibitors or small interfering RNA. We conclude that acid-induced COX2 expression and PGE2 production depend on an increase in cytosolic Ca2+ and sequential activation of NOX5-S and NF-kappaB in SEG1 cells. COX2-derived PGE2 production may contribute to NOX5-S-mediated cell proliferation in SEG1 cells.     

Role of Rac1 in regulation of NOX5-S function in Barrett's esophageal adenocarcinoma cells

We have shown that a novel NADPH oxidase isoform, NOX5-S, is the major isoform of NADPH oxidases in an esophageal adenocarcinoma (EA) cell line, FLO, and is overexpressed in Barrett's mucosa with high-grade dysplasia. NOX5-S is responsible for acid-induced reactive oxygen species production. In this study, we found that mRNA levels of NOX5-S were significantly higher in FLO EA cells than in the normal human esophageal squamous cell line HET-1A or in a Barrett cell line, BAR-T. The mRNA levels of NOX5-S were also significantly increased in EA tissues. The data suggest that NOX5-S may be important in the development of EA. Mechanisms of functional regulation of NOX5-S are not fully understood. We show that small G protein Rac1 was present in HET-1A cells, BAR-T cells, and EA cell lines FLO and OE33. Rac1 protein levels were significantly higher in FLO and OE33 cells than in HET-1A or BAR-T cells. Knockdown of Rac1 with Rac1 small interfering RNA significantly decreased acid-induced increase in H(2)O(2) production in FLO EA cells. Overexpression of constitutively active Rac1 significantly increased H(2)O(2) production, an increase that was blocked by knockdown of NOX5-S. By immunofluorescence staining and immunoprecipitation, we found that NOX5-S was present in the cytosol of FLO EA cells and colocalized with Rac1 and SERCA1/2 Ca(2+)-ATPase which is located in the endoplasmic reticulum membrane. We conclude that Rac1 may be important in activation of NOX5-S in FLO EA cells.     

NOX4 Mediates BMP4-Induced Upregulation of TRPC1 and 6 Protein Expressions in Distal Pulmonary Arterial Smooth Muscle Cells

Rationale

Our previous studies demonstrated that bone morphogenetic protein 4 (BMP4) mediated, elevated expression of canonical transient receptor potential (TRPC) largely accounts for the enhanced proliferation in pulmonary arterial smooth muscle cells (PASMCs). In the present study, we sought to determine the signaling pathway through which BMP4 up-regulates TRPC expression.

Methods

We employed recombinant human BMP4 (rhBMP4) to determine the effects of BMP4 on NADPH oxidase 4 (NOX4) and reactive oxygen species (ROS) production in rat distal PASMCs. We also designed small interfering RNA targeting NOX4 (siNOX4) and detected whether NOX4 knockdown affects rhBMP4-induced ROS, TRPC1 and 6 expression, cell proliferation and intracellular Ca²⁺ determination in PASMCs.

Results

In rhBMP4 treated rat distal PASMCs, NOX4 expression was (226.73±11.13) %, and the mean ROS level was (123.65±1.62) % of that in untreated control cell. siNOX4 transfection significantly reduced rhBMP4-induced elevation of the mean ROS level in PASMCs. Moreover, siNOX4 transfection markedly reduced rhBMP4-induced elevation of TRPC1 and 6 proteins, basal [Ca²⁺]_i and SOCE. Furthermore, compared with control group (0.21±0.001), the proliferation of rhBMP4 treated cells was significantly enhanced (0.41±0.001) (P<0.01). However, such increase was attenuated by knockdown of NOX4. Moreover, external ROS (H₂O₂ 100 µM, 24 h) rescued the effects of NOX4 knockdown, which included the declining of TRPC1 and 6 expression, basal intracellular calcium concentration ([Ca²⁺]_i) and store-operated calcium entry (SOCE), suggesting that NOX4 plays as an important mediator in BMP4-induced proliferation and intracellular calcium homeostasis.

Conclusion

These results suggest that BMP4 may increase ROS level, enhance TRPC1 and 6 expression and proliferation by up-regulating NOX4 expression in PASMCs.     

Differential regulation of adhesion complex turnover by ROCK1 and ROCK2

Background

ROCK1 and ROCK2 are serine/threonine kinases that function downstream of the small GTP-binding protein RhoA. Rho signalling via ROCK regulates a number of cellular functions including organisation of the actin cytoskeleton, cell adhesion and cell migration.

Methodology/Principal Findings

In this study we use RNAi to specifically knockdown ROCK1 and ROCK2 and analyse their role in assembly of adhesion complexes in human epidermal keratinocytes. We observe that loss of ROCK1 inhibits signalling via focal adhesion kinase resulting in a failure of immature adhesion complexes to form mature stable focal adhesions. In contrast, loss of ROCK2 expression results in a significant reduction in adhesion complex turnover leading to formation of large, stable focal adhesions. Interestingly, loss of either ROCK1 or ROCK2 expression significantly impairs cell migration indicating both ROCK isoforms are required for normal keratinocyte migration.

Conclusions

ROCK1 and ROCK2 have distinct and separate roles in adhesion complex assembly and turnover in human epidermal keratinocytes.     

Activation of RhoA and FAK induces ERK-mediated osteopontin expression in mechanical force-subjected periodontal ligament fibroblasts

The precise mechanism by which Rho kinase translates the mechanical signals into OPN up-regulation in force-exposed fibroblasts has not been elucidated. Human periodontal ligament fibroblasts (hPLFs) were exposed to mechanical force by centrifuging the culture plates at a magnitude of 50 g/cm² for 60 min. At various times of the force application, they were processed for analyzing cell viability, trypan blue exclusion, and OPN expression at protein and RNA levels. Cellular mechanism(s) of the force-induced OPN up-regulation was also examined using various kinase inhibitors or antisense oligonucleotides specific to mechanosensitive factors. Centrifugal force up-regulated OPN expression and induced a rapid and transient increase in the phosphorylation of focal adhesion kinase (FAK), extracellular signal-regulated kinase (ERK), and Elk1. Pharmacological blockade of RhoA/Rho-associated coiled coil-containing kinase (ROCK) signaling markedly reduced force-induced FAK and ERK1/2 phosphorylation. Transfecting hPLFs with FAK antisense oligonucleotide diminished ERK1/2 activation and force-induced OPN expression. Further, ERK inhibitor inhibited significantly OPN expression, Elk1 phosphorylation, and activator protein-1 (AP-1)-DNA binding activation, but not FAK phosphorylation, in the force-applied cells. These results demonstrate that FAK signaling plays critical roles in force-induced OPN expression in hPLFs through interaction with Rho/ROCK as upstream effectors and ERK-Elk1/ERK-c-Fos as downstream effectors.     

The Lbc Rho Guanine Nucleotide Exchange Factor/α-Catulin Axis Functions in Serotonin-induced Vascular Smooth Muscle Cell Mitogenesis and RhoA/ROCK Activation

Serotonin (5-hydroxytryptamine, 5-HT) is mitogenic for several cell types including pulmonary arterial smooth muscle cells (PASMC), and is associated with the abnormal vascular smooth muscle remodeling that occurs in pulmonary arterial hypertension. RhoA/Rho kinase (ROCK) function is required for 5-HT-induced PASMC mitogenesis, and 5-HT activates RhoA; however, the signaling steps are poorly defined. Rho guanine nucleotide exchange factors (Rho GEFs) transduce extracellular signals to Rho, and we found that 5-HT treatment of PASMC led to increased membrane-associated Lbc Rho GEF, suggesting modulation by 5-HT. Lbc knockdown by siRNA attenuated 5-HT-induced thymidine uptake in PASMC, indicating a role in PASMC mitogenesis. 5-HT triggered Rho-dependent serum response factor-mediated reporter activation in PASMC, and this was reduced by Lbc depletion. Lbc knockdown reduced 5-HT-induced RhoA/ROCK activation, but not p42/44 ERK MAP kinase activation, suggesting that Lbc is an intermediary between 5-HT and RhoA/ROCK, but not ERK. 5-HT stimulation of PASMC led to increased association between Lbc, RhoA, and the α-catulin scaffold. Furthermore, α-catulin knockdown attenuated 5-HT-induced PASMC thymidine uptake. 5-HT-induced PASMC mitogenesis was reduced by dominant-negative G_q protein, suggesting cooperation with Lbc/α-catulin. These results for the first time define a Rho GEF involved in vascular smooth muscle cell growth and serotonin signaling, and suggest that Lbc Rho GEF family members play distinct roles. Thus, the Lbc/α-catulin axis participates in 5-HT-induced PASMC mitogenesis and RhoA/ROCK signaling, and may be an interventional target in diseases involving vascular smooth muscle remodeling.     

Role of Rho kinase in sphingosine 1-phosphate-mediated endothelial and smooth muscle cell migration and differentiation

The role of sphingosine 1-phosphate (S1P)-induced Rho kinase (ROCK) activation in the angiogenic responses of pulmonary artery-derived endothelial cells (PAEC) and smooth muscle cells (PASMC) was examined. S1P, a biologically active phospholipid that regulates angiogenesis, promoted PAEC chemotaxis and capillary morphogenesis; furthermore, this activity was unaltered by pretreatment with the pharmacological inhibitor of ROCK, H1152. In contrast, S1P (500 nM) significantly inhibited spontaneous PASMC chemotaxis and differentiation; however, this inhibition was eradicated upon H1152 pretreatment. Similarly, PASMCs transfected with ROCK II siRNA diminished S1P-induced inhibition of the development of multi-cellular structures. Analysis by RT-PCR identified the presence of S1P₁ and S1P₃ receptors on both PAECs and PASMCs, while S1P₂ receptor expression was confined to only PASMCs. Consistent with this observation, the S1P₁ and S1P₃ receptor antagonist, VPC23019, virtually abolished the S1P-initiated PAEC differentiation but did not impede the S1P-induced inhibition of PASMC differentiation. However, the S1P₂ receptor antagonist, JTE013, had no effect on S1P-mediated differentiation of PAECs but abolished the S1P-induced inhibition of PASMC function. Co-cultured endothelial and smooth muscle cells differentiated into “neovascular-like” networks, which were significantly inhibited by S1P. The inhibition of co-culture differentiation in both PAECs and PASMCs was negated by H1152 pretreatment. However, when smooth muscle cells were added to S1P-initiated endothelial cell networks, additional S1P treatment did not inhibit the cellular networks generated by these cells. In conclusion, S1P-induced PAEC angiogenic responses are regulated by S1P₁ and/or S1P₃ receptors independent of Rho kinase activation, whereas S1P₂ receptor-mediated curtailment of PASMC function by S1P.     

Hypoxia-induced ARHGAP26 deficiency inhibits the proliferation and migration of human ductus arteriosus smooth muscle cell through activating RhoA-ROCK-PTEN pathway

The Rho family plays crucial roles in O₂-induced vasoconstriction, cell proliferation, and migration. Rho GTPase-activating protein 26 (ARHGAP26) is a GTPase-activating protein for the small GTPases of the Rho family. Our previous studies have demonstrated that ARHGAP26 expression was significantly downregulated in patent human ductus arteriosus (DA) tissue. However, its role underlying the maintenance of DA patency is unclear. In this study, patent (fetal) and constricted (newborn) mouse DA tissues were harvested to confirm the differences in the levels of expression of ARHGAP26. Human DA smooth muscle cells (DASMCs) were isolated and cultured in vitro and used to test the function of ARHGAP26. The expression of ARHGAP26 was significantly lower in patent (fetal) than constricted (newborn) mouse DA. ARHGAP26-knocked-down human DASMCs showed reduced proliferation and migration, which are both crucial to anatomic closure of DA. Moreover, after culturing under hypoxic conditions, the expression of ARHGAP26 in human DASMCs was significantly lower and hypoxia-induced ARHGAP26 deficiency activated the phosphorylation level of phosphatase and tensin homolog (PTEN) in DASMCs by mediating the activity of RhoA and RhoA-associated kinase 1 (ROCK1). Use of Y27632, an inhibitor of ROCK which further reduces the phospholipid activity of PTEN can reverse the inhibitory effect of PTEN on the proliferation and migration of human DASMCs. This provides insight into the molecular regulation of the RhoA-ROCK-PTEN pathway in DA smooth muscle cells, which may be a suitable therapeutic target or diagnostic biomarker for perinatal DA tone management.     

G12 signaling through c-Jun NH2-terminal kinase promotes breast cancer cell invasion

Signaling through the heterotrimeric G protein, G12, via Rho induces a striking increase in breast cancer cell invasion. In this study, evidence is provided that the c-Jun NH(2)-terminal kinase (JNK) is a key downstream effector of G12 on this pathway. Expression of constitutively-active Gα12 or activation of G12 signaling by thrombin leads to increased JNK and c-Jun phosphorylation. Pharmacologic inhibition of JNK or knockdown of JNK expression by siRNA significantly decreases G12-induced JNK activation as well as the ability of breast cancer cells to invade a reconstituted basement membrane. Furthermore, expression of dominant-negative Rho or treatment of cells with an inhibitor of the Rho kinase, ROCK, reduces G12-induced JNK and c-Jun activation, and ROCK inhibitor treatment also inhibits G12-induced cellular invasion. JNK knockdown or ROCK inhibitor treatment has no effect on activation of Rho by G12. Taken together, our data indicate that JNK activation is required for G12-induced invasion of breast cancer cells and that JNK is downstream of Rho and ROCK on this pathway. This study implicates a G12-stimulated mitogen-activated protein kinase cascade in cancer cell invasion, and supports a role for JNK in cancer progression.     

The functional role of rho and rho-associated coiled-coil forming protein kinase in eotaxin signaling of eosinophils

The CC chemokine eotaxin plays a pivotal role in local accumulation of eosinophils. Very little is known about the eotaxin signaling in eosinophils except the activation of the mitogen-activated protein (MAP) kinase family. The p21 G protein Rho and its substrate Rho-associated coiled-coil forming protein kinase (ROCK) regulate the formation of stress fibers and focal adhesions. In the present study, we studied the functional relevance of Rho and ROCK in eosinophils using the ROCK inhibitor (Y-27632) and exoenzyme C3, a specific Rho inhibitor. Eotaxin stimulates activation of Rho A and ROCK II in eosinophils. Exoenzyme C3 almost completely inhibited the ROCK activity, indicating that ROCK is downstream of Rho. We then examined the role of Rho and ROCK in eosinophil chemotaxis. The eotaxin-induced eosinophil chemotaxis was significantly inhibited by exoenzyme C3 or Y-27632. Because extracellular signal-regulated kinase (ERK)1/2 and p38 MAP kinases are activated by eotaxin and are critical for eosinophil chemotaxis, we investigated whether Rho and ROCK are upstream of these MAP kinases. C3 partially inhibited eotaxin-induced phosphorylation of ERK1/2 but not p38. In contrast, neither ERK1/2 nor p38 phosphorylation was abrogated by Y-27632. Both C3 and Y-27632 reduced reactive oxygen species production from eosinophils. We conclude that both Rho and ROCK are important for eosinophil chemotaxis and reactive oxygen species production. There is a dichotomy of downstream signaling pathways of Rho, namely, Rho-ROCK and Rho-ERK pathways. Taken together, eosinophil chemotaxis is regulated by multiple signaling pathways that involve at least ROCK, ERK, and p38 MAP kinase.     

RhoA/Rho Kinase Mediates Neuronal Death Through Regulating cPLA<Subscript>2</Subscript> Activation

Activation of RhoA/Rho kinase leads to growth cone collapse and neurite retraction. Although RhoA/Rho kinase inhibition has been shown to improve axon regeneration, remyelination and functional recovery, its role in neuronal cell death remains unclear. To determine whether RhoA/Rho kinase played a role in neuronal death after injury, we investigated the relationship between RhoA/Rho kinase and cytosolic phospholipase A₂ (cPLA₂), a lipase that mediates inflammation and cell death, using an in vitro neuronal death model and an in vivo contusive spinal cord injury model performed at the 10th thoracic (T10) vertebral level. We found that co-administration of TNF-α and glutamate induced spinal neuron death, and activation of RhoA, Rho kinase and cPLA₂. Inhibition of RhoA, Rho kinase and cPLA₂ significantly reduced TNF-α/glutamate-induced cell death by 33, 52 and 43 %, respectively (p < 0.001). Inhibition of RhoA and Rho kinase also significantly downregulated cPLA₂ activation by 66 and 60 %, respectively (p < 0.01). Furthermore, inhibition of RhoA and Rho kinase reduced the release of arachidonic acid, a downstream substrate of cPLA₂. The immunofluorescence staining showed that ROCK₁ or ROCK₂, two isoforms of Rho kinase, was co-localized with cPLA₂ in neuronal cytoplasm. Interestingly, co-immunoprecipitation (Co-IP) assay showed that ROCK₁ or ROCK₂ bonded directly with cPLA₂ and phospho-cPLA₂. When the Rho kinase inhibitor Y27632 was applied in mice with T10 contusion injury, it significantly decreased cPLA₂ activation and expression and reduced injury-induced apoptosis at and close to the lesion site. Taken together, our results reveal a novel mechanism of RhoA/Rho kinase-mediated neuronal death through regulating cPLA₂ activation.     

The development of benzimidazoles as selective rho kinase inhibitors

Rho Kinase (ROCK) is a serine/threonine kinase whose inhibition could prove beneficial in numerous therapeutic areas. We have developed a promising class of ATP-competitive inhibitors based upon a benzimidazole scaffold, which show excellent potency toward ROCK (IC₅₀ <10 nM). This report details the optimization of selectivity for ROCK over other related kinases such as Protein kinase A (PKA).     

Novel ROCK inhibitors for the treatment of pulmonary arterial hypertension

A novel class of selective inhibitors of ROCK1 and ROCK2 has been identified by structural based drug design. PK/PD experiments using a set of highly selective Rho kinase inhibitors suggest that systemic Rho kinase inhibition is linked to a reversible reduction in lymphocyte counts. These results led to the consideration of topical delivery of these molecules, and to the identification of a lead molecule 7 which shows promising PK and PD in a murine model of pulmonary hypertension after intra-tracheal dosing.     

Selective glucocorticoid control of Rho kinase isoforms regulate cell-cell interactions

The two Rho kinase isoforms ROCK1 and ROCK2 are downstream effectors of the small GTPase RhoA, although relatively little is known about potential isoform specific functions or the selective control of their cellular activities. Using Con8 rat mammary epithelial cells, we show that the synthetic glucocorticoid dexamethasone strongly stimulates the level of ROCK2 protein, which accounts for the increase in total cellular ROCK2 activity, whereas, steroid treatment down-regulated ROCK1 specific kinase activity without altering ROCK1 protein levels. In Con8 cells, the glucocorticoid induced formation of tight junctions requires the steroid-mediated down-regulation RhoA and function of the RhoA antagonist Rnd3. Treatment with the ROCK inhibitor Y-27632 ablated both the glucocorticoid-induced and Rnd3-mediated stimulation in tight junction sealing. Taken together, our results demonstrate that the expression and activity of ROCK1 and ROCK2 can be uncoupled in a signal-dependent manner, and further implicate a new function for ROCK2 in the steroid control of tight junction dynamics.     

Rho kinase regulation of vasopressin-induced calcium entry in vascular smooth muscle cell: Comparison between rat isolated aorta and cultured aortic cells

In addition to its role in artery contraction, Rho kinase (ROCK) is reported to be involved in the Ca²⁺ response to vasoconstrictor agonist in rat aorta. However the signaling pathway mediated by ROCK had not been investigated so far and it was not known whether ROCK also contributed to Ca²⁺ signaling in cultured vascular smooth muscle cells (VSMC), which undergo profound phenotypic changes. Our results showed that in VSMC, ROCK inhibition by Y-27632 or H-1152 had no effect on the Ca²⁺ response to vasopressin, while in aorta the vasopressin-induced Ca²⁺ entry was significantly decreased. The inhibition of myosin light chain kinase (MLCK) by ML-7 depressed the vasopressin-induced Ca²⁺ signal in aorta but not in VSMC. The difference in ROCK sensitivity of vasopressin-induced Ca²⁺ entry between aorta and VSMC was not related to an alteration of the RhoA/ROCK pathway. However, MLCK expression and activity were depressed in cultured cells compared to aorta. We concluded that the regulation of vasopressin-induced Ca²⁺ entry by ROCK in aorta could involve the myosin cytoskeleton and could be prevented by the downregulation of MLCK in VSMC. These results underline the important differences in Ca²⁺ regulation between whole tissue and cultured cells.     

Regulation of Rho/ROCK signaling in airway smooth muscle by membrane potential and [Ca2+]i

Recently, we have shown that Rho and Rho-activated kinase (ROCK) may become activated by high-millimolar KCl, which had previously been widely assumed to act solely through opening of voltage-dependent Ca(2+) channels. In this study, we explored in more detail the relationship between membrane depolarization, Ca(2+) currents, and activation of Rho/ROCK in bovine tracheal smooth muscle. Ca(2+) currents began to activate at membrane voltages more positive than -40 mV and were maximally activated above 0 mV; at the same time, these underwent time- and voltage-dependent inactivation. Depolarizing intact tissues by KCl challenge evoked contractions that were blocked equally, and in a nonadditive fashion, by nifedipine or by the ROCK inhibitor Y-27632. Other agents that elevate intracellular calcium concentration ([Ca(2+)](i)) by pathways independent of G protein-coupled receptors, namely the SERCA-pump inhibitor cyclopiazonic acid and the Ca(2+) ionophore A-23187, evoked contractions that were also largely reduced by Y-27632. KCl directly increased Rho and ROCK activities in a concentration-dependent fashion that paralleled closely the effect of KCl on tone and [Ca(2+)](i), as well as the voltage-dependent Ca(2+) currents that were measured over the voltage ranges that are evoked by 0-120 mM KCl. Through the use of various pharmacological inhibitors, we ruled out roles for Ca(2+)/calmodulin-dependent CaM kinase II, protein kinase C, and protein kinase A in mediating the KCl-stimulated changes in tone and Rho/ROCK activities. In conclusion, Rho is activated by elevation of [Ca(2+)](i) (although the signal transduction pathway underlying this Ca(2+) dependence is still unclear) and possibly also by membrane depolarization per se.     

Rho and Rho-associated kinase modulate the tyrosine kinase PYK2 in T-cells through regulation of the activity of the integrin LFA-1

We have examined the role of the small GTPase Rho and its downstream effector, the Rho-associated kinase (ROCK), in the control of the adhesive and signaling function of the lymphocyte function-associated antigen-1 (LFA-1) integrin in human T-lymphocytes. Inhibition of Rho (either by treatment with C3-exoenzyme or transfection with a dominant-negative form of Rho (N19Rho)) or ROCK (by treatment with Y-27632) results in the following: (a) partial disorganization and aggregation of cortical filamentous actin (F-actin); (b) induction of LFA-1-mediated cellular adhesion to the LFA-1 ligand intercellular adhesion molecule-1 (ICAM-1) through a mechanism involving clustering of LFA-1 molecules, rather than alterations in the level of expression or in the affinity state of this integrin; and (c) induction of cellular polarization and activation of the tyrosine kinase PYK2. Transfection of T-cells with a constitutively active form of Rho (V14Rho) blocks the clustering of LFA-1 on the membrane and the LFA-1-mediated activation of PYK2. Importantly, the activation of PYK2 caused by inhibition of Rho or ROCK takes place only when the T-cells are plated onto ICAM-1 but not when they are either prevented from interacting with ICAM-1 with anti-LFA-1 blocking antibodies or when they are plated on the nonspecific poly-l-lysine substrate. These results indicate that the small GTPase Rho regulates the tyrosine kinase PYK2 in T-cells through the F-actin-mediated control of the activity of the integrin LFA-1. These findings represent a novel paradigm for the regulation of the activity of a cytoplasmic tyrosine kinase by the small GTPase Rho.