Neuropeptide-stimulated cell migration in prostate cancer cells is mediated by RhoA kinase signaling and inhibited by neutral endopeptidase.

The neuropeptides bombesin and endothelin-1 stimulate prostate cancer (PC) cell migration and invasion (J Clin Invest, 2000; 106: 1399-1407). The intracellular signaling pathways that direct this cell movement are not well delineated. The monomeric GTPase RhoA is required for migration in several cell types including neutrophils, monocytes and fibroblasts. We demonstrate that bombesin-stimulated PC cell migration occurs via the heterotrimeric G-protein-coupled receptors (G-protein) G alpha 13 subunit leading to activation of RhoA, and Rho-associated coiled-coil forming protein kinase (ROCK). Using siRNA to suppress expression of the three known G-protein alpha-subunit-associated RhoA guanine nucleotide exchange factors (GEFs), we also show that two of these RhoA GEFs, PDZ-RhoGEF and leukemia-associated RhoGEF (LARG), link bombesin receptors to RhoA in a non-redundant manner in PC cells. We next show that focal adhesion kinase, which activates PDZ-RhoGEF and LARG, is required for bombesin-stimulated RhoA activation. Neutral endopeptidase (NEP) is expressed on normal prostate epithelium whereas loss of NEP expression contributes to PC progression. We also demonstrate that NEP inhibits neuropeptide activation of RhoA. Together, these results establish a contiguous signaling pathway from the bombesin receptor to ROCK in PC cells, and they implicate NEP as a major regulator of neuropeptide-stimulated RhoA in these cells. This work also identifies members of this signaling pathway as potential targets for rational pharmacologic manipulation of neuropeptide-stimulated migration of PC cells.

Rho activation in excitatory agonist-stimulated vascular smooth muscle.

Small GTPase Rho and its downstream effector, Rho kinase, have been implicated in agonist-stimulated Ca(2+) sensitization of 20-kDa myosin light chain (MLC(20)) phosphorylation and contraction in smooth muscle. In the present study we demonstrated for the first time that excitatory receptor agonists induce increases in amounts of an active GTP-bound form of RhoA, GTP-RhoA, in rabbit aortic smooth muscle. Using a pull-down assay with a recombinant RhoA-binding protein, Rhotekin, we found that a thromboxane A(2) mimetic, U-46619, which induced a sustained contractile response, induced a sustained rise in the amount of GTP-RhoA in a dose-dependent manner with an EC(50) value similar to that for the contractile response. U-46619-induced RhoA activation was thromboxane A(2) receptor-mediated and reversible. Other agonists including norepinephrine, serotonin, histamine, and endothelin-1 (ET-1) also stimulated RhoA, albeit to lesser extents than U-46619. In contrast, ANG II and phorbol 12,13-dibutyrate failed to increase GTP-RhoA. The tyrosine kinase inhibitor genistein substantially inhibited RhoA activation by these agonists, except for ET-1. Thus excitatory agonists induce Rho activation in an agonist-specific manner, which is thought to contribute to stimulation of MLC(20) phosphorylation Ca(2+) sensitivity.

Involvement of phospholipase D in sphingosine 1-phosphate-induced activation of phosphatidylinositol 3-kinase and Akt in Chinese hamster ovary cells overexpressing EDG3.

Phospholipase D (PLD), phosphatidylinositol 3-kinase (PI3K), and Akt are known to be involved in cellular signaling related to proliferation and cell survival. In this report, we provide evidence that PLD links sphingosine 1-phosphate (S1P)-induced activation of the G protein-coupled EDG3 receptor to stimulation of PI3K and its downstream effector Akt in Chinese hamster ovary (CHO) cells. S1P stimulation of EDG3-overexpressing CHO cells but not vector-transfected cells induced activation of PLD, PI3K, and Akt in a time- and dose-dependent manner. Akt phosphorylation was prevented by the PI3K inhibitors wortmannin and LY294002 (2-(4-monrpholinyl)-8-phenyl-4H-1-benzopyran-4-one), indicating that Akt activation was dependent on PI3K. S1P-induced activation of PI3K and Akt was abrogated by 1-butanol, which inhibited S1P-induced accumulation of phosphatidic acid by serving as a phosphatidyl group acceptor in the transphosphatidylation reaction catalyzed by PLD, whereas both PI3K and Akt activation were not inhibited by 2-butanol without such reaction. Co-expression of wild-type PLD2 with myc-Akt resulted in increased Akt activation in response to S1P. In contrast, co-expression of a catalytically inactive mutant of PLD2 eliminated the S1P-induced Akt activation. The treatment of EDG3-expressing CHO cells with exogenous Streptomyces chromofuscus PLD, which caused an accumulation of phosphatidic acid, resulted in increases in PI3K activity and the phosphorylation of Akt, the latter of which was completely abolished by LY294002. Furthermore, S1P-induced membrane ruffling, which was dependent on PI3K and Rac, was inhibited by 1-butanol, but not by 2-butanol. These results demonstrate that PLD participates in the activation of PI3K and Akt stimulation of EDG3 receptor.

Subtype-specific, differential activities of the EDG family receptors for sphingosine-1-phosphate, a novel lysophospholipid mediator.

The lysosphingolipid sphingosine-1-phosphate (S1P) and the structurally related lipid lysophosphatidic acid (LPA) elicit a wide spectrum of biological responses in a variety of cell types, including mitogenesis, cell-shape changes, migration and contraction. Recent studies have unveiled the existence of the G protein-coupled heptahelical receptor subfamily for the biologically active lysophospholipids, which consists of the two receptor subgroups specific for S1P and LPA, respectively. The S1P receptor subgroup comprises four members, i.e. EDG-1, EDG-3, EDG-5/AGR16 and EDG-6, with considerable amino acid similarity among them. The S1P receptor subtypes are coupled to different heterotrimeric G proteins, leading to the activation of a unique set of multiple intracellular signaling pathways. The expression of transcripts of the S1P receptor subtypes is wide-spread, except for EDG-6 which exhibits lymphoid tissue-specific expression. Plasma contains substantial concentrations of S1P as well as LPA. Activated platelets appear to be a major source of S1P and LPA in blood. In addition, accumulating evidence demonstrates that S1P and LPA are released from a variety of cell types in response to various extracellular stimuli. These observations demonstrate the existence of the novel signaling system comprising the lysosphingolipids and their cognate receptors, suggesting physiological and pathological roles.

Regulation of cell cycle molecules by the Ras effector system.

Eukaryotic cell cycle progression is driven by an ordered array of phosphorylation events that are specifically catalyzed by members of CDK (cyclin-dependent kinase) family serine/threonine protein kinases, each consisting of a catalytic subunit CDK and a positive regulatory subunit cyclin. In mammalian somatic cells extracellular cues act mainly during the G1 phase to regulate the activity of D type cyclin-dependent CDKs, which, in turn, serve as key regulators of G1--S phase progression by phosphorylating and functionally inactivating the tumor suppressor retinoblastoma (Rb) protein. The small molecular weight G protein Ras has been implicated as a crucial molecule that transduces extracellular growth stimuli into intracellular signals. Recent studies, including our own, have demonstrated that maintained cellular Ras activity is required until late in the G1 phase for inactivation of the Rb protein and the G1/S transition and mediates both upregulation of cyclin D1 and downregulation of p27kip1 CDK inhibitor.

The MEK1-ERK map kinase pathway and the PI 3-kinase-Akt pathway independently mediate anti-apoptotic signals in HepG2 liver cancer cells.

Primary liver cancers, which are generally hypervascular in nature, depend highly on blood supply. So far there are few reports on apoptosis of liver cancer cells upon deprivation of serum-derived survival factors. The aim of our study is to clarify molecular mechanisms by which liver cancer cells survive with the aid of serum. In HepG2 liver cancer cells, serum deprivation induced time-dependent increase in the number of apoptotic cells, which was detected by fragmentation of genomic DNA and fluorescent nuclear staining. The activity of extracellular signal-regulated kinase (ERK) did not decrease considerably after serum deprivation, although it increased after serum stimulation. However, we found that the MEK1 inhibitor PD98059, but not the p38 kinase inhibitor SB203580, potently induced apoptosis of the liver cancer cells in the presence of serum, indicating that the MEK-ERK signaling pathway is required for serum-dependent survival of HepG2 cells. In agreement with this notion, transient expression of active MEK1 prevented apoptosis in serum-deprived condition. We also found that the protective effect of serum against apoptosis was totally abrogated by LY294002 or wortmannin, which are the inhibitors of phosphatidylinositol (PI) 3-kinase. The activity of Akt, the target of PI 3-kinase, decreased gradually after deprivation of serum, whereas it was rapidly reactivated upon serum stimulation. These data indicate that survival of HepG2 liver cancer cells depends upon serum and that both the MEK1-ERK- and the PI 3-kinase-Akt- pathways are required for survival signaling to the nucleus.

Ambient temperatures preferred by humans acclimated to heat given at a fixed daily time.

We investigated preferred ambient temperatures (T(pref)) of heat-acclimated humans to assess their behavioral thermoregulation. Seven male volunteers were exposed to an ambient temperature (T(a)) of 42 degrees C and relative humidity (RH) of 40% for 4 h (14:00-18:00 h)/day for 9-10 consecutive days. Rectal temperature (T(re)) was measured, and T(pref) was determined at two distinct times of day, 09:00-11:00 h (AM test) and 14:00-16:00 h (PM test), in both heat- and nonheat-acclimated (control) conditions. Heat acclimation significantly decreased T(re) only in the PM test. There was no difference in the T(pref) between the two tests in the control condition. However, T(pref) in the PM test was significantly lower than that of the AM test in the heat-acclimated condition. The findings suggest that repeated heat exposure in humans for 4 h at a fixed time daily alters the core temperature level and behavioral thermoregulatory function, particularly during the period when the subjects had previously been exposed to heat.

Mechanisms of angiotensin II-induced platelet-derived growth factor gene expression.

Angiotensin II induces the expression of platelet-derived growth factor A-chain and B-chain in cultured vascular smooth muscle cells at the level of transcription. The renin-angiotensin system has also been implicated in the increased expression of platelet-derived growth factor in the mechanically-injured artery wall. This review is concerned with recent developments in our understanding of the signaling and transcriptional mechanisms mediating the inducible expression of one vasoconstrictor by another.

Inhibitory regulation of Rac activation, membrane ruffling, and cell migration by the G protein-coupled sphingosine-1-phosphate receptor EDG5 but not EDG1 or EDG3.

Sphingosine-1-phosphate (S1P) is a bioactive lysophospholipid that induces a variety of biological responses in diverse cell types. Many, if not all, of these responses are mediated by members of the EDG (endothelial differentiation gene) family G protein-coupled receptors EDG1, EDG3, and EDG5 (AGR16). Among prominent activities of S1P is the regulation of cell motility; S1P stimulates or inhibits cell motility depending on cell types. In the present study, we provide evidence for EDG subtype-specific, contrasting regulation of cell motility and cellular Rac activity. In CHO cells expressing EDG1 or EDG3 (EDG1 cells or EDG3 cells, respectively) S1P as well as insulin-like growth factor I (IGF I) induced chemotaxis and membrane ruffling in phosphoinositide (PI) 3-kinase- and Rac-dependent manners. Both S1P and IGF I induced a biphasic increase in the amount of the GTP-bound active form of Rac. In CHO cells expressing EDG5 (EDG5 cells), IGF I similarly stimulated cell migration; however, in contrast to what was found for EDG1 and EDG3 cells, S1P did not stimulate migration but totally abolished IGF I-directed chemotaxis and membrane ruffling, in a manner dependent on a concentration gradient of S1P. In EDG5 cells, S1P stimulated PI 3-kinase activity as it did in EDG1 cells but inhibited the basal Rac activity and totally abolished IGF I-induced Rac activation, which involved stimulation of Rac-GTPase-activating protein activity rather than inhibition of Rac-guanine nucleotide exchange activity. S1P induced comparable increases in the amounts of GTP-RhoA in EDG3 and EDG5 cells. Neither S1P nor IGF I increased the amount of GTP-bound Cdc42. However, expression of N(17)-Cdc42, but not N(19)-RhoA, suppressed S1P- and IGF I-directed chemotaxis, suggesting a requirement for basal Cdc42 activity for chemotaxis. Taken together, the present results demonstrate that EDG5 is the first example of a hitherto-unrecognized type of receptors that negatively regulate Rac activity, thereby inhibiting cell migration and membrane ruffling.

Biological activities of novel lipid mediator sphingosine 1-phosphate in rat hepatic stellate cells.

Sphingosine 1-phosphate (S-1-P), a lipid mediator shown to be a ligand for aortic G protein-coupled receptor [corrected] (AGRs), endothelial differentiation gene (EDG)1, EDG3, and AGR16/EDG5, is stored in platelets and released on their activation. Platelet consumption occurs in acute liver injury. Hepatic stellate cells (HSCs) play an important role in wound healing. Effects of S-1-P on HSCs were investigated. S-1-P enhanced proliferation of culture-activated HSCs. The mitogenic effect was pertussis toxin sensitive, mitogen-activated protein kinase dependent, and more prominent at lower cell density. S-1-P increased contraction of collagen lattices containing HSCs, irrespective of activation state, in a C3 exotoxin-sensitive manner. mRNAs of EDG1 and AGR16, but not of EDG3, were detected in HSCs. In HSC activation, EDG1 mRNA levels were downregulated, whereas AGR16 mRNA levels were unchanged. Considering that HSCs are capable of production of extracellular matrices and modulation of blood flow in sinusoids, our results suggest that S-1-P may play a role in wound healing process in the liver.

Rho kinase inhibitor HA-1077 prevents Rho-mediated myosin phosphatase inhibition in smooth muscle cells.

In smooth muscle, a Rho-regulated system of myosin phosphatase exists; however, it has yet to be established whether Rho kinase, one of the downstream effectors of Rho, mediates the regulation of myosin phosphatase activity in vivo. In the present study, we demonstrate in permeabilized vascular smooth muscle cells (SMCs) that the vasodilator 1-(5-isoquinolinesulfonyl)-homopiperazine (HA-1077), which we show to be a potent inhibitor of Rho kinase, dose dependently inhibits Rho-mediated enhancement of Ca(2+)-induced 20-kDa myosin light chain (MLC(20)) phosphorylation due to abrogating Rho-mediated inhibition of MLC(20) dephosphorylation. By an immune complex phosphatase assay, we found that guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) stimulation of permeabilized SMCs caused a decrease in myosin phosphatase activity with an increase in the extent of phosphorylation of the 130-kDa myosin-binding regulatory subunit (MBS) of myosin phosphatase in a Rho-dependent manner. HA-1077 abolished both of the Rho-mediated events. Moreover, we observed that the pleckstrin homology/cystein-rich domain protein of Rho kinase, a dominant negative inhibitor of Rho kinase, inhibited GTPgammaS-induced phosphorylation of MBS. These results provide direct in vivo evidence that Rho kinase mediates inhibition of myosin phosphatase activity with resultant enhancement of MLC(20) phosphorylation in smooth muscle and reveal the usefulness of HA-1077 as a Rho kinase inhibitor.

Angiotensin II stimulates platelet-derived growth factor-B chain expression in newborn rat vascular smooth muscle cells and neointimal cells through Ras, extracellular signal-regulated protein kinase, and c-Jun N-terminal protein kinase mechanisms.

Platelet-derived growth factors (PDGFs) have been implicated in the pathogenesis of vascular proliferative disorders. Vascular smooth muscle cells (VSMCs) are one of the cell types that produce PDGF-B chain in proliferative lesions, although the mechanism of regulation of PDGF-B chain production in these cells is not well understood. In the present study, we demonstrate that angiotensin II (Ang II), which is also implicated in vascular stenosis after angioplasty and atherosclerosis, markedly stimulates PDGF-B chain mRNA expression in cultured newborn rat medial VSMCs and neointimal VSMCs via an AT(1), but not in adult rat VSMCs. In newborn rat VSMCs, Ang II activates extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal protein kinase (JNK), and p38 mitogen-activated protein kinase. The mitogen-activated protein/ERK (MEK) inhibitor PD98059, but not the p38 inhibitor SB203580, abrogates Ang II-induced PDGF-B mRNA expression. Transient transfection analysis using a PDGF-B promoter-luciferase gene reporter construct reveals that Ang II induces transcriptional activation of PDGF-B chain gene, which is abolished by the expression of a dominant negative form of either ERK or JNK, but not of p38. The expression of a dominant negative form of Ras abolishes the stimulatory effects of Ang II on ERK activity and PDGF-B mRNA expression. In adult rat VSMCs, Ang II activates ERK and JNK, but weakly induces Egr-1, a transcription factor implicated in PDGF-B chain gene expression, compared with newborn VSMCs. These data indicate that Ang II activates PDGF-B chain gene expression in VSMCs through mechanisms involving Ras-ERK and JNK.

Targeting endogenous platelet-derived growth factor B-chain by adenovirus-mediated gene transfer potently inhibits in vivo smooth muscle proliferation after arterial injury.

Platelet-derived growth factor (PDGF), especially its B chain, has been implicated in the pathogenesis of vascular proliferative disorders such as atherosclerosis and restenosis after angioplasty. We constructed a replication-deficient recombinant adenovirus containing the gene encoding the extracellular region of PDGF beta-receptor (PDGFXR) that binds PDGF-B chain and acts as its antagonist. The administration into balloon-injured rat carotid arteries of an adenovirus containing the Escherichia coli lacZ gene as a marker gene at 5 days after injury markedly facilitated efficacy of gene transfer, as compared with its administration immediately after injury. Adenovirus-mediated gene transfer of PDGFXR into injured arteries performed at 5 days resulted in a more than 50% reduction in the neointimal area of injured arteries at 14 days. In contrast, the administration of control adenoviruses containing lacZ gene or containing no foreign gene was without suppressive effects on neointima formation. The inhibition of neointima formation by the expression of PDGFXR was accompanied by a reduction in bromodeoxyuridine-labeled cells and nearly complete inhibition of tyrosine phosphorylation of both alpha- and beta-receptors for PDGF, but not of epidermal growth factor receptor, in injured arteries. This is the first report to indicate the usefulness of targeting a growth factor by expressing an extracellular binding region of a receptor using an adenovirus for the treatment of vascular proliferative disorders, and provide direct evidence that PDGF-B chain plays an essential role in neointimal formation.

EDG3 is a functional receptor specific for sphingosine 1-phosphate and sphingosylphosphorylcholine with signaling characteristics distinct from EDG1 and AGR16.

AGR16/H218/EDG5 and EDG1 are functional receptors for lysosphingolipids, whereas EDG2 and EGD4 are receptors for lysophosphatidic acid (LPA). The present study demonstrates that EDG3, the yet poorly defined member of the EDG family G protein-coupled receptors, shows identical agonist specificity, but distinct signaling characteristics, compared to AGR16 and EDG1. Overexpression of EDG3 conferred a specific [32P]S1P binding, which was displaced by S1P and sphingosylphosphorylcholine (SPC), but not by LPA or other related lipids. In cells overexpressing EDG3, S1P induced inositol phosphate production and [Ca2+]i increase in a manner only partially sensitive to pertussis toxin (PTX), which was similar to the case of AGR16, but quite different from the case of EDG1, in which the S1P-induced responses were totally abolished by PTX. EDG3 also mediated activation of mitogen-activated protein kinase (MAPK) in PTX-sensitive and Ras-dependent manners, as in the cases of EDG1 and AGR16, although EDG3 and EDG1 were more effectively coupled to activation of MAPK, compared to AGR16. Additionally, EDG3 mediated a decrease in cellular cyclic AMP content, like EDG1, but contrasting with AGR16 which mediated an increase in cyclic AMP. These and previous results establish that EDG1, AGR16 and EDG3 comprise the lysosphingolipid receptor subfamily, each showing distinct signaling characteristics.

Cyclin D1 expression mediated by phosphatidylinositol 3-kinase through mTOR-p70(S6K)-independent signaling in growth factor-stimulated NIH 3T3 fibroblasts.

Phosphatidylinositol (PI) 3-kinase is required for G1 to S phase cell cycle progression stimulated by a variety of growth factors and is implicated in the activation of several downstream effectors, including p70(S6K). However, the molecular mechanisms by which PI 3-kinase is engaged in activation of the cell cycle machinery are not well understood. Here we report that the expression of a dominant negative (DN) form of either the p110alpha catalytic or the p85 regulatory subunit of heterodimeric PI 3-kinase strongly inhibited epidermal growth factor (EGF)-induced upregulation of cyclin D1 protein in NIH 3T3(M17) fibroblasts. The PI 3-kinase inhibitors LY294002 and wortmannin completely abrogated increases in both mRNA and protein levels of cyclin D1 and phosphorylation of pRb, inducing G1 arrest in EGF-stimulated cells. By contrast, rapamycin, which potently suppressed p70(S6K) activity throughout the G1 phase, had little inhibitory effect, if any, on either of these events. PI 3-kinase, but not rapamycin-sensitive pathways, was also indispensable for upregulation of cyclin D1 mRNA and protein by other mitogens in NIH 3T3 (M17) cells and in wild-type NIH 3T3 cells as well. We also found that an enforced expression of wild-type p110 was sufficient to induce cyclin D1 protein expression in growth factor-deprived NIH 3T3(M17) cells. The p110 induction of cyclin D1 in quiescent cells was strongly inhibited by coexpression of either of the PI 3-kinase DN forms, and by LY294002, but was independent of the Ras-MEK-ERK pathway. Unlike mitogen stimulation, the p110 induction of cyclin D1 was sensitive to rapamycin. These results indicate that the catalytic activity of PI 3-kinase is necessary, and could also be sufficient, for upregulation of cyclin D1, with mTOR signaling being differentially required depending upon cellular conditions.

The novel sphingosine 1-phosphate receptor AGR16 is coupled via pertussis toxin-sensitive and -insensitive G-proteins to multiple signalling pathways.

In the present study, we determined the agonist specificity and the signalling mechanisms of a putative sphingosine 1-phosphate (S1P) receptor, AGR16. In CHO cells transiently transfected with an AGR16 expression vector, but not in cells transfected with an empty vector, the addition of a low concentration of S1P (1 nM) caused an increase in the intracellular free Ca2+ concentration ([Ca2+]i) by mobilization of Ca2+ from both intra- and extra-cellular pools. To determine the spectrum of agonists for AGR16, we employed K562 cells, which in the naive state do not respond at all to either S1P or structurally related lipids with an increase in [Ca2+]i. In K562 cells stably expressing AGR16, S1P and sphingosylphosphorylcholine (SPC) dose-dependently increased [Ca2+]i with half-maximal values of 3 nM and 100 nM respectively. In CHO cells stably expressing AGR16 (CHO-AGR16), but not in parental CHO cells, we observed specific binding of [32P]S1P, which was displaced by unlabelled S1P and SPC. In CHO-AGR16 cells, but not in parental CHO cells, S1P stimulated the production of inositol phosphates and Ca2+ mobilization which was only 30% inhibited by pertussis toxin (PTX), different from the case of the recently identified S1P receptor EDG1. Also in CHO-AGR16 cells, but not in CHO cells, S1P at higher concentrations activated mitogen-activated protein kinase (MAPK) in a PTX-sensitive and Ras-dependent manner. S1P also induced the activation of two stress-activated MAPKs, c-Jun N-terminal kinase and p38, in a manner that was totally insensitive to PTX. In CHO-AGR16 cells, S1P induced stress-fibre formation, with an increase in myosin light chain phosphorylation, in a PTX-insensitive and Rho-dependent manner. S1P also induced an increase in the cellular cAMP content in CHO-AGR16 cells, which contrasts sharply with the case of EDG1. These results establish that the S1P receptor AGR16 is coupled via both PTX-sensitive and -insensitive G-proteins to multiple effector pathways.

Inhibitory effects of trapidil on PDGF signaling in balloon-injured rat carotid artery.

Trapidil, which was originally developed as an anti-platelet agent, is among the few agents thus far proven to be clinically effective in preventing restenosis after percutaneous coronary interventions. Trapidil was previously shown to inhibit platelet-derived growth factor (PDGF)-induced cellular responses in vitro in cultured cells. However, its mechanism of action is poorly understood. In this study, we investigated by using a rat carotid balloon-injury model whether and how trapidil inhibited the in vivo action of PDGF, which is regarded as a most important growth factor implicated in proliferation and migration of vascular smooth muscle cells. The combination of both oral and topical administration of trapidil reduced the intimal lesion size by more than 70% and nearly completely suppressed injury-induced increases in phosphotyrosine content of PDGF alpha- and beta- receptors of carotid artery. Moreover, trapidil was found to decrease mRNA levels of PDGF alpha- and beta- receptors strongly and of PDGF A- and B- chains moderately in injured arteries. These results indicate that trapidil potently suppresses the action of PDGF with inhibition of neointima formation in injured artery, which is mediated at least in part through decreasing the expression of both PDGF ligands and their receptors.

[The molecular mechanism of vasospasm and the attenuation by fasudil].

Increase in cytosolic Ca2+ concentration and phosphorylation of myosin light chain (MLC) are key steps of normal and abnormal contractility in smooth muscle. In pathological condition like vasospasm, artery exhibits a hyperreactive contraction associated with an enhanced and sustained MLC phosphorylation, particularly diphosphorylation (Thr18/Ser19). Phosphorylation levelis regulated by a balance between phosphorylation and dephosphorylation. Recent studies propose that myosin phosphatase activity is regulated through the phosphorylation of 130K-myosin binding subunit (MBS130K) of the myosin phosphatase in smooth muscle. It is known that in normar artery, receptor agonists like PGF2 alpha induce certain contraction of smooth muscle associated with MLC diphosphorylation. We recently confirm that this PGF2 alpha stimulation also evokes the phosphorylation of MBS130K and fasudil, Rho-kinase inhibitor, inhibits this phosphorylation in parallel with MLC phosphorylation. Furthermore, we reveal that in vasospastic artery, Rho-kinase activity is augmented to induce high level phosphorylation of MBS130K. In this study, we also confirm that phosphorylation of MBS130K is Rho-kinase-dependent and its site is Thr654 residue. Now we speculate that fasudil may shift the phosphorylation-dephosphorylation cycle to dephosphorylation side through the inhibition of Thr654phosphorylation in MBS130K. These results also suggest that augmented activation of Rho-kinase may be involved in the pathophysiology of vasospasm associated with MLC diphosphorylation.

Successive occupancy by immediate early transcriptional factors of the tyrosine hydroxylase gene TRE and CRE sites in PACAP-stimulated PC12 pheochromocytoma cells.

We previously demonstrated that pituitary adenylate cyclase-activating polypeptide (PACAP) coordinately upregulates the expression of the tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH) genes by activating the cyclic AMP (cAMP) and protein kinase C (PKC) signaling pathways. In this study, we examined the effects of PACAP on the expression of fos and jun immediate early gene (IEG) families, expression of which can be up-regulated by both PKC and cAMP signaling pathways, in rat pheochromocytoma cell line PC12 cells. PACAP potently stimulated the expression of c-fos, fosB junB and junD, but not c-jun mRNAs, at doses of 0.1-10 nM, as revealed by Northern blot analysis. The effects of PACAP on the expression of these mRNAs in PC12 cells was rapid (30-60 min) and dose-dependent. PACAP administration induced maximum expression of c-fos, fosB and junB mRNA after 60 min, and of junD mRNA after 8 h. Gel mobility shift assays using synthetic DNA oligonucleotides corresponding to the TH 5'-flanking region and nuclear extracts from PC12 cells demonstrated that PACAP enhanced formation of the specific protein complexes which bind to the TPA-responsive element (TRE) and cAMP-responsive element (CRE), respectively. Gel shift and supershift analyses showed that the TRE-binding factors and CRE-binding factors comprised fosB, c-fos, junB, and junD, and CRE-binding protein (CREB) and junD, respectively. JunB was dominant in the TRE-binding complexes at 4 h after addition of PACAP, whereas both JunD and JunB were dominant at 12 h. These results suggest that agonist occupancy of PACAP receptors activates transcriptional factors (Fos/Jun families and CREB) that interact with the TRE and CRE sites of the TH 5'-flanking region, contributing to transcriptional activation of TH gene.

EDG1 is a functional sphingosine-1-phosphate receptor that is linked via a Gi/o to multiple signaling pathways, including phospholipase C activation, Ca2+ mobilization, Ras-mitogen-activated protein kinase activation, and adenylate cyclase inhibition.

In Chinese hamster ovary (CHO) cells transiently transfected with an expression vector for EDG1, but not an empty vector, sphingosine-1-phosphate (SP) at a concentration as low as 10(-10) M caused an increase in the intracellular free Ca2+ concentration ([Ca2+]i) as a result of mobilization of Ca2+ from both intracellular and extracellular pools. In a CHO clone stably expressing EDG1 receptor (CHO-EDG1 cells), SP induced increases in the production of inositol phosphates and the [Ca2+]i and inhibited forskolin-induced increase in the cellular cAMP content, all in a manner sensitive to pertussis toxin. SP also activated mitogen-activated protein kinase in CHO-EDG1 cells in pertussis toxin-sensitive and Ras-dependent manners. To evaluate the spectrum of agonists for EDG1, we used human erythroleukemia (HEL) cells, which at naive state do not respond to SP or structurally related lipids with an increase in the [Ca2+]i. In HEL cells stably expressing EDG1 receptor (HEL-EDG1 cells), SP dose-dependently increased the [Ca2+]i with half-maximal and maximal concentration values of 10(-9) and 3 x 10(-7) M, respectively; sphingosylphosphorylcholine at exclusively high concentrations, but not sphingosine at all, also increased the [Ca2+]i. HEL-EDG1 cells bound 32P-labeled SP, which was displaced dose dependently by unlabeled SP. These results indicate that EDG1, a member of the EDG family G protein-coupled receptors, is a specific, high-affinity SP receptor.