Relaxin inhibits early steps in vascular inflammation

Increased expression of endothelial adhesion molecules, high levels of the monocyte chemoattractant protein-1 (MCP-1) and enhanced VLA4 integrin/VCAM-1 and CCR-2/MCP-1 interactions are initial steps in vascular inflammation. We sought to determine whether relaxin, a potent vasodilatory and anti-fibrotic agent, mitigates these early events compromising endothelial integrity. The effect of relaxin coincubation on the TNF-α-stimulated expression of the adhesion molecules VCAM-1, ICAM-1 and E-selectin; the MCP-1 expression by human umbilical vein endothelial cells (HUVEC) and human aortic smooth muscle cells (HAoSMC); as well as on direct monocyte–endothelium cell adhesion was quantified by ELISA or adhesion assay. CCR-2 and PECAM expression on HUVEC and THP-1 monocytes was investigated by FACS analysis. Relaxin treatment suppressed significantly TNF-α-induced upregulation of VCAM-1 and PECAM, CCR-2, and MCP-1 levels and direct monocyte adhesion to HUVEC. Our findings identify relaxin as a promising inhibitory factor in early vascular inflammation. By attenuating the upregulation of VCAM-1, key adhesion molecule in early vascular inflammation, and of MCP-1, a chemokine pivotal to monocyte recruitment, relaxin decreased initial monocyte–endothelium contact. This may be of relevance for the prevention and treatment of atherosclerosis and of other pro-inflammatory states.     

Distinct signaling pathways for MCP-1-dependent integrin activation and chemotaxis

Transmigration of monocytes to the subendothelial space is the initial step of atherosclerotic plaque formation and inflammation. Integrin activation and chemotaxis are two important functions involved in monocyte transmigration. To delineate the signaling cascades leading to integrin activation and chemotaxis by monocyte chemoattractant protein-1 (MCP-1), we have investigated the roles of MAPK and Rho GTPases in THP-1 cells, a monocytic cell line. MCP-1 stimulated beta1 integrin-dependent, but not beta2 integrin-dependent cell adhesion in a time-dependent manner. MCP-1-mediated cell adhesion was inhibited by a MEK inhibitor but not by a p38-MAPK inhibitor. In contrast, MCP-1-mediated chemotaxis was inhibited by the p38-MAPK inhibitor but not by the MEK inhibitor. The inhibitor of Rho GTPase, C3 exoenzyme, and a Rho kinase inhibitor abrogated MCP-1-dependent chemotaxis but not integrin-dependent cell adhesion. Further, C3 exoenzyme and the Rho kinase inhibitor blocked MCP-1-dependent p38-MAPK activation. These data indicate that ERK is responsible for integrin activation, that p38-MAPK and Rho are responsible for chemotaxis mediated by MCP-1, and that Rho and the Rho kinase are upstream of p38-MAPK in MCP-1-mediated signaling. This study demonstrates that two distinct MAPKs regulate two dependent signaling cascades leading to integrin activation and chemotaxis induced by MCP-1 in THP-1 cells.     

Thy-1-Induced Migration Inhibition in Vascular Endothelial Cells through Reducing the RhoA Activity

Our previous study indicated that Thy-1, which is expressed on blood vessel endothelium in settings of pathological and a specific of physiological, but not during embryonic, angiogenesis, may be used as a marker for angiogenesis. However, the function of Thy-1 during angiogenesis is still not clear. Here, we demonstrate that knock-down of the endogenous Thy-1 expression by Thy-1 siRNA transfection promoted the migration of human umbilical vein endothelial cells (HUVEC). In contrast, treatment with interleukin-1β (IL-1β) or phorbol-12-myristate-13-acetate (PMA) increased the level of Thy-1 protein and reduced the migration of HUVEC. These effects were abolished by pre-transfection of HUVEC with Thy-1 siRNA to knock-down the expression of Thy-1. Moreover, over-expression of Thy-1 by transfection of HUVEC with Thy-1 pcDNA3.1 decreased the activity of RhoA and Rac-1 and inhibited the adhesion, migration and capillary-like tube formation of these cells. These effects were prevented by co-transfection of the cell with constitutively active RhoA construct (RhoA V14). On the other hand, pre-treatment with a ROCK (a kinase associated with RhoA for transducing RhoA signaling) inhibitor, Y27632, abolished the RhoA V14-induced prevention effect on the Thy-1-induced inhibition of endothelial cell migration and tube formation. Taken together, these results indicate that suppression of the RhoA-mediated pathway might participate in the Thy-1-induced migration inhibition in HUVEC. In the present study, we uncover a completely novel role of Thy-1 in endothelial cell behaviors.     

Inhibition of protein geranylgeranylation and RhoA/RhoA kinase pathway induces apoptosis in human endothelial cells

Geranylgeranylation of RhoA small G-protein is essential for its localization to cell membranes and for its biological functions. Many RhoA effects are mediated by its downstream effector RhoA kinase. The role of protein geranylgeranylation and the RhoA pathway in the regulation of endothelial cell survival has not been elucidated. The hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitor lovastatin depletes cellular pools of geranylgeranyl pyrophosphate and farnesol pyrophosphate and thereby inhibits both geranylgeranylation and farnesylation. Human umbilical vein endothelial cells (HUVECs) were exposed to lovastatin (3 microm-30 microm) for 48 h, and cell death was quantitatively determined by cytoplasmic histone-associated DNA fragments as well as caspase-3 activity. The assays showed that lovastatin caused a dose-dependent endothelial cell death. The addition of geranylgeraniol, which restores geranylgeranylation, rescued HUVEC from apoptosis. The geranylgeranyltransferase inhibitor GGTI-298, but not the farnesyltransferase inhibitor FTI-277, induced apoptosis in HUVEC. Cell death was also induced by a blockade of RhoA function by exoenzyme C3. In addition, treatment of HUVEC with the RhoA kinase inhibitors Y-27632 and HA-1077 caused dose-dependent cell death. Y-27632 did not inhibit other well known survival pathways, such as NF-kappa B, ERK, and phosphatidylinositol 3-kinase/Akt. However, there was an increase in p53 protein level concomitant with Y-27632-induced cell death. Unlike the apoptosis induced by TNF-alpha, which occurs only with inhibition of new protein synthesis, apoptosis induced by inhibitors of HMG-CoA reductase, geranylgeranyltransferase, or RhoA kinase was blocked by cycloheximide. Our data indicate that inhibition of protein geranylgeranylation and RhoA pathways induce apoptosis in HUVEC and that induction of p53 or other proapoptotic proteins is required for this process.     

Cardiotrophin-1 stimulates intercellular adhesion molecule-1 and monocyte chemoattractant protein-1 in human aortic endothelial cells

Intercellular adhesion molecule-1 (ICAM-1) and monocyte chemoattractant protein-1 (MCP-1) play critical roles in mediating monocyte adhesion to the vascular endothelium and monocyte migration into the subendothelial regions of the vessels. Inasmuch as cardiotrophin-1 (CT-1), an IL-6-type cytokine, was expressed in human atherosclerotic plaque, we examined whether CT-1 induces monocyte adhesion and migration by stimulating gene and protein expressions of ICAM-1 and MCP-1 in human aortic endothelial cells (HAECs). Immunocytochemistry revealed that CT-1 increased intensity of ICAM-1 and MCP-1 immunoreactivity in HAECs. Adhesion assay and chemotaxis assay revealed that CT-1 increased human monocytic THP-1 cell adhesion to HAECs and promoted chemotaxis in THP-1 cells, which were attenuated by anti-ICAM-1 and anti-MCP-1 antibody, respectively. Western blot analysis showed that CT-1 increased phosphorylation of ERK1/2 MAP kinase, p38 MAP kinase, and Akt and that their inhibitors, PD-98059, SB-203580, and LY-294002, respectively, inhibited phosphorylation. RNase protection assay and ELISA demonstrated that CT-1 increased gene and protein expressions of ICAM-1 and MCP-1. EMSA revealed that CT-1 enhanced NF-kappaB DNA-binding activity. CT-1-mediated upregulation of ICAM-1 and MCP-1 was suppressed by PD-98059, SB-203580, LY-294002, and parthenolide. The present study demonstrates that CT-1 promotes monocyte adhesion and migration by stimulating ICAM-1 and MCP-1 through mechanisms that involve ERK1/2 MAP kinase, p38 MAP kinase, phosphatidylinositol 3-kinase, and NF-kappaB pathways and suggests that CT-1 plays an important role in the pathophysiology of vascular inflammation and atherosclerosis.     

Role of phosphoinositide 3-kinase in monocyte recruitment under flow conditions

Chemokines such as the monocyte chemol attractant protein-1 (MCP-1) convert monocyte rolling to firm arrest under physiological flow conditions via integrin activation and simultaneously activate phosphoinositide 3-kinase (PI3K). Here we used adenoviral gene transfer and biochemical inhibitors to manipulate PI3K-dependent pathways in human monocytes. In in vitro lipid kinase assays from purified human monocytes, we showed that MCP-1 activates the "classical" PI3Kalpha pathway and not PI3Kgamma, a PI3K isoform thought to be activated only by the betagamma complex of heterotrimeric G proteins. The activity of PI3Kalpha in purified human monocytes was evident within 30 s. MCP-1-induced monocyte arrest was significantly inhibited both by wortmannin (n = 4; p < 0.01) and LY294002 (n = 4; p < 0.01) with restoration of the rolling phenotype (p < 0.05 for both inhibitors, compared with rolling of control monocytes after MCP-1 treatment). To test the hypothesis that activation of PI3K is sufficient to induce monocyte adhesion, we transduced the monocytic THP-1 cell line with a recombinant adenovirus (Ad) carrying a constitutively active mutant of PI3K (Ad.BD110). We examined the ability of these cells to adhere to human vascular endothelium (HUVEC) transduced with adenoviruses carrying E-selectin, intercellular adhesion molecule-1 (ICAM-1), and VCAM-1. Under flow conditions, ICAM-1- and VCAM-1-dependent firm adhesion of Ad.BD110-transduced THP-1 cells was enhanced compared with THP-1 cells infected with control Ad (n = 4; p < 0.01 for both). Adhesion augmented by constitutive PI3K activation was entirely abrogated by pretreatment with wortmannin (n = 3; p < 0.01). In contrast, a constitutively active Akt construct had no effect on THP-1 adhesion (n = 3; p = NS). We conclude that PI3K activation is necessary and sufficient to enhance monocytic adhesion under physiological flow conditions. BD110-expressing THP-1 cells should provide a useful tool for identifying the signaling pathways downstream of PI3K that are necessary for monocyte recruitment relevant to a variety of human vascular pathologies.     

Sphingosine 1-phosphate (S1P) inhibits monocyte-endothelial cell interaction by regulating of RhoA activity

Recent studies suggest that sphingosine 1-phosphate (S1P) protects against atherosclerosis. We assessed the effects of S1P on monocyte-endothelial interaction in the presence of inflammatory mediators. Pretreatment of THP-1 cells with S1P abolished Phorbol 12 myristate 13-acetate (PMA)-induced THP-1 cell adhesion to human umbilical vein endothelial cells (HUVECs). S1P inhibited PMA-induced activation of RhoA, but not PKCs. S1P activated p190Rho GTPase activation protein (GAP) only in the presence of PMA, suggesting an inhibitory effect of S1P and PMA to suppress RhoA. In conclusion, S1P inhibited monocyte-endothelial interactions by inhibiting RhoA activity which may explain its anti-atherogenic effects.     

Cilostazol reduces MCP-1-induced chemotaxis and adhesion of THP-1 monocytes by inhibiting CCR2 gene expression

The chemotaxis and adhesion of monocytes to the injured endothelium in the early atherosclerosis is important. Cilostazol, a specific phosphodiesterase type III inhibitor, is known to exhibit anti-atherosclerotic effects mediated by different mechanisms. This study aimed to investigate the modulating effect of cilostazol on the MCP-1-induced chemotaxis and adhesion of monocytes. The gene expression of CCR2, the major receptor of MCP-1 in THP-1 monocytes, was also analyzed. The chemotaxis of monocytes toward MCP-1 was investigated using the transwell filter assay. Cilostazol dose-dependently inhibited the MCP-1-induced chemotaxis of monocytes which was shown to be cAMP-dependent. Using western blot analysis and flow cytometry method, we demonstrated the decrease of CCR2 protein at the cell membrane of monocytes by cilostazol treatment. Results from RT/real-time PCR confirmed the decrease of CCR2 mRNA expression by cilostazol which was also mediated by cAMP. Similar inhibition was also noted in human peripheral monocytes. The post-CCR2 signaling pathways including p44/42 and p38 MAPK were examined by western blot analysis. Result confirmed the inhibitory effect of cilostazol on the phosphorylation of p44/42 and p38 MAPK after MCP-1 stimulation. The activation of monocytes after MCP-1 treatment exhibited enhanced adhesion to vascular endothelial cells which was dose-dependently suppressed by cilostazol. Together, cilostazol was demonstrated, for the first time, to inhibit the CCR2 gene expression and MCP-1-induced chemotaxis and adhesion of monocytes which might therefore reduce the infiltration of monocytes during the early atherosclerosis. The present study provides an additional molecular mechanism underlying the anti-atherosclerotic effects of cilostazol.     

Modulation of COX-2 expression by statins in human aortic smooth muscle cells. Involvement of geranylgeranylated proteins

Cyclooxygenase (COX)-2 and COX-1 play an important role in prostacyclin production in vessels and participate in maintaining vascular homeostasis. Statins are inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, which is crucial in cholesterol biosynthesis. Recently, cholesterol-independent effects of statins have been described. In this study, we evaluated the effect of two inhibitors of HMG CoA reductase, mevastatin and lovastatin, on the production of prostacyclin and the expression of COX in human aortic smooth muscle cells. Treatment of cells with 25 microm mevastatin or lovastatin resulted in the induction of COX-2 and increase in prostacyclin production. Mevalonate, the direct metabolite of HMG CoA reductase, and geranylgeranyl-pyrophosphate reversed this effect. GGTI-286, a selective inhibitor of geranylgeranyltransferases, increased COX-2 expression and prostacyclin formation, thus indicating the involvement of geranylgeranylated proteins in the down-regulation of COX-2. Furthermore, Clostridium difficile toxin B, an inhibitor of the Rho GTP-binding protein family, the Rho selective inhibitor C3 transferase, and Y-27632, a selective inhibitor of the Rho-associated kinases, targets of Rho A, increased COX-2 expression whereas the activator of the Rho GTPase, the cytotoxic necrotizing factor 1, blocked interlukin-1alpha-dependent COX-2 induction. These results demonstrate that statins up-regulate COX-2 expression and subsequent prostacyclin formation in human aortic smooth muscle cells in part through inhibition of Rho.     

Salusin-β accelerates inflammatory responses in vascular endothelial cells via NF-κB signaling in LDL receptor-deficient mice in vivo and HUVECs in vitro

The bioactive peptide salusin-β is highly expressed in human atheromas; additionally, infusion of antiserum against salusin-β suppresses the development of atherosclerosis in atherogenic mice. This study examined the roles of salusin-β in vascular inflammation during atherogenesis. Infusion of antiserum against salusin-β attenuated the induction of VCAM-1, monocyte chemoattractant protein (MCP)-1, and IL-1β and as well as nuclear translocation of NF-κB in aortic endothelial cells (ECs) of LDL receptor-deficient mice, which led to the prevention of monocyte adhesion to aortic ECs. In vitro experiments indicated that salusin-β directly enhances the expression levels of proinflammatory molecules, including VCAM-1, MCP-1, IL-1β, and NADPH oxidase 2, as well as THP-1 monocyte adhesion to cultured human umbilical vein ECs (HUVECs). Both salusin-β-induced VCAM-1 induction and monocyte/HUVEC adhesion were suppressed by pharmacological inhibitors of NF-κB, e.g., Bay 11-7682 and curcumin. Furthermore, the VCAM-1 induction was significantly prevented by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY-294002, whereas it was accelerated by the ERK inhibitor, U-0126. Treatment of HUVECs with salusin-β, but not with salusin-α, accelerated oxidative stress and nuclear translocation of NF-κB as well as phosphorylation and degradation of IκB-α, an endogenous inhibitor of NF-κB. Thus, salusin-β enhanced monocyte adhesion to vascular ECs through NF-κB-mediated inflammatory responses in ECs, which can be modified by PI3K or ERK signals. These findings are suggestive of a novel role of salusin-β in atherogenesis.     

Ginsenoside metabolite compound K differentially antagonizing tumor necrosis factor-α-induced monocyte-endothelial trafficking

Human leukocyte endothelial adhesion and transmigration occur in the early stage of the pathogenesis of atherosclerosis. Vascular endothelial cells are targeted by pro-inflammatory cytokines modulating many gene proteins responsible for cell adhesion, thrombosis and inflammatory responses. This study examined the potential of compound K to inhibit the pro-inflammatory cytokine TNF-α induction of monocyte adhesion onto TNF-α-activated human umbilical vein endothelial cells (HUVEC). HUVEC were cultured with 10 ng/ml TNF-α with individual ginsenosides of Rb1, Rc, Re, Rh1 and compound K (CK). Ginsenosides at doses of ?50 μM did not show any cytotoxicity. TNF-α induced THP-1 monocyte adhesion to HUVEC, and such induction was attenuated by Rh1 and CK. Consistently, CK suppressed TNF-α-induced expression of HUVEC adhesion molecules of VCAM-1, ICAM-1 and E-selectin, and also Rh1 showed a substantial inhibition. Rh1 and CK dampened induction of counter-receptors, α4/β1 integrin VLA-4 and αL/β2 integrin LFA-1 in TNF-α-treated THP-1 cells. Additionally, CK diminished THP-1 secretion of MMP-9 required during transmigration, inhibiting transendothelial migration of THP-1 cells. CK blunted TNF-α-promoted IL-8 secretion of HUVEC and CXCR1 expression of THP-1 monocytes. Furthermore, TNF-α-activated endothelial IκB phosphorylation and NF-κB nuclear translocation were disturbed by CK, and TNF-α induction of α4/β1 integrin was abrogated by the NF-κB inhibitor SN50. These results demonstrate that CK exerts anti-atherogenic activity with blocking leukocyte endothelial interaction and transmigration through negatively mediating NF-κB signaling.     

Involvement of Arp2/3 complex in MCP-1-induced chemotaxis

The migrating monocyte shows dynamic actin polymerization in response to MCP-1. We investigated the involvement of the actin-related protein 2 and 3 complex (Arp2/3 complex) during chemotaxis of a human monocyte cell line (THP-1). To clarify whether the Arp2/3 complex directly polymerizes actin in response to MCP-1 stimulation, THP-1 cells were transfected with complementary DNA constructs encoding ScarWA. In ScarWA-transfected cells, neither recruitment of Arp2/3 complex at the leading edge nor actin polymerization was detected. Indeed, migration induced by MCP-1 was almost completely blocked. At the same time, transfection also interfered with the recruitment of integrin beta-1 at the leading edge and reduced affinity binding to fibronectin. Immunoprecipitation with an anti-Arp2 antibody showed that integrin beta-1 and WASP were co-precipitated under the condition of MCP-1 stimulation. These results indicate that interaction between the Arp2/3 complex and WASP stimulates actin polymerization and integrin beta-1-mediated adhesion during MCP-1-induced chemotaxis of THP-1 cells.     

Involvement of Rac GTPase activation in phosphatidylcholine hydroperoxide-induced THP-1 cell adhesion to ICAM-1

Increasing evidence indicates that phospholipid oxidation plays important roles in atherosclerosis. Here, we investigated the involvement of Rho-family GTPases inphosphatidylcholine hydroperoxide (PCOOH)-induced THP-1 cell adhesion to ICAM-1. Isoprenoid depletion by fluvastatin and geranylgeranyltransferase inhibition by GGTI-286 suppressed PCOOH-induced cell adhesion to ICAM-1 and F-actin-rich membrane protrusion formation. Pull-down assays demonstrated the activation of Rac1 and Rac2 in PCOOH-treated cells. Pan-Rho-family GTPase inhibitor Clostridium difficile toxin B, Rac-specific inhibitor NSC23776, and RNA interference of the Rac isoforms suppressed the cell adhesion. These findings indicate the involvement of Rac GTPase activation in PCOOH-induced cell adhesion to ICAM-1 via actin reorganization.     

Pharmacological targeting of HSP90 with 17-AAG induces apoptosis of myogenic cells through activation of the intrinsic pathway

Endothelial inflammation and monocyte plays an essential role in the initiation and progression of atherosclerosis. Ghrelin is beneficial for atherosclerosis progression. However, the detailed and precise molecular mechanisms of how ghrelin regulates endothelial inflammation are not clear. In this study, we investigated the regulation mechanism of ghrelin on TNF-α-activated endothelial inflammation and monocyte adhesion. It was found that TNF-α-induced monocyte adhesion on HUVEC was significantly attenuated by ghrelin. Furthermore, we found that ghrelin effectively suppressed TNF-α-induced inflammatory factors’ (including ICAM-1, VCAM-1, MCP-1, and IL-1β) expression through inhibiting AMPK phosphorylation and p65 expression both in HUVEC and THP-1. This phenomenon was further demonstrated by using AMPK agonist AICAR and inhibitor compound C, respectively. Our findings suggest that ghrelin may mediate TNF-α-induced endothelial inflammation and monocyte adhesion, in part via AMPK/NF-κB signaling pathway. These novel anti-inflammatory and immunoregulatory actions of ghrelin may play a certain role in understanding the formation and development of atherosclerosis.     

CX3C-chemokine, fractalkine-enhanced adhesion of THP-1 cells to endothelial cells through integrin-dependent and -independent mechanisms

Leukocyte adhesion and trafficking at the endothelium requires both cellular adhesion molecules and chemotactic factors. A newly identified CX3C chemokine, fractalkine, expressed on activated endothelial cells, plays an important role in leukocyte adhesion and migration. We examined the functional effects of fractalkine on beta1 and beta2 integrin-mediated adhesion using a macrophage-like cell line, THP-1 cells. In this study, we report that THP-1 cells express mRNA encoding a receptor for fractalkine, CX3CR1, determined by Northern blotting. Scatchard analysis using fractalkine-SEAP (secreted form of placental alkaline phosphatase) chimeric proteins revealed that THP-1 cells express a single class of CX3CR1 with a dissociation constant of 30 pM and a mean expression of 440 sites per cell. THP-1 cells efficiently adhered, in a fractalkine-dependent manner, to full-length of fractalkine immobilized onto plastic and to the membrane-bound form of fractalkine expressed on ECV304 cells or TNF-alpha-activated HUVECs. Moreover, soluble-fractalkine enhanced adhesion of THP-1 cells to fibronectin and ICAM-1 in a dose-dependent manner. Pertussis toxin, an inhibitor of Gi, inhibited the fractalkine-mediated enhancement of THP-1 cell adhesion to fibronectin and ICAM-1. Finally, we found that soluble-fractalkine also enhanced adhesion of freshly separated monocytes to fibronectin and ICAM-1. These results indicate that fractalkine may induce firm adhesion between monocytes and endothelial cells not only through an intrinsic adhesion function itself, but also through activation of integrin avidity for their ligands.     

Effects of 25-hydroxycholesterol, cholesterol, and isoprenoid derivatives on the G1 progression in Swiss 3T3 cells

The effect of inhibition of 3-Hydroxy-3-methylglutaryl Coenzyme A reductase (HMG CoA reductase) on cell cycle progression in proliferating 3T3 cells was studied. It was found that short transient exposures to the HMG CoA reductase inhibitor 25-hydroxycholesterol temporarily blocked the cell cycle traverse in the postmitotic half of G1 (G1pm), whereas cells in the subsequent cell cycle phases were unaffected. The kinetics of the cell cycle delay, induced by 25-hydroxycholesterol, resembled the kinetics of the delay induced by serum depletion, which also inhibited the activity of HMG CoA reductase. In contrast to the case of serum depletion, platelet derived growth factor (PDGF), which efficiently prevented the decrease of HMG CoA reductase in serum-free medium, was not capable of preventing the growth inhibitory effect following treatment by 25-hydroxycholesterol. However, cholesterol and two isoprenoids, dolichol and coenzyme Q, were effective in this respect. In addition, dolichol counteracted the cell cycle delay following short periods of serum starvation.     

MCP-1-stimulated chemotaxis of monocytic and endothelial cells is dependent on activation of different signaling cascades

Monocyte chemoattractant protein-1 (MCP-1) is important in attracting monocytes to sites of inflammation. Besides induction of monocyte recruitment, MCP-1 can also affect chemotactic response of endothelial cells. The molecular mechanisms involved in MCP-1-induced cell migration are poorly understood. In the current investigation, we demonstrate activation of p42/44(ERK1/2) and p38 mitogen-activated protein kinases (MAPKs), phosphatydilinositol-3-kinase (PI3K) and Src-kinases in both monocytes and endothelial cells stimulated with MCP-1 in vitro. The response was rapid and time-dependent, detectable within 3 min of MCP-1 stimulation. MCP-1-induced phosphorylation of p42/44(ERK1/2) MAPKs was partially blocked by inhibitor of PI3K LY294002, while phosphorylation of p38 MAPK was diminished to a greater extent in presence of Src-kinase inhibitor PP2. There was a substantial inhibition of monocyte migration upon treatment with inhibitors of p38 MAPK, at the same time inhibition of p42/44(ERK1/2) MAPK activation had no effect. On the contrary, the MCP-1-stimulated chemotaxis of endothelial cells was completely abolished by inhibitors of PI3K and p42/44(ERK1/2), but not by p38 MAPK inhibitors. These results suggest that parallel signal transduction pathways are activated by MCP-1, and that depending on the cell type these pathways differentially contribute to cell chemotactic activity.     

Analysis of the coordinate expression of 3-hydroxy-3-methylglutaryl coenzyme A synthase and reductase activities in Chinese hamster ovary fibroblasts

Decreased activities of both 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) synthase and HMG CoA reductase are observed in the presence of sterol in the Chinese hamster ovary (CHO) fibroblast. In three different genotypes of CHO cell mutants resistant to 25-hydroxycholesterol both enzyme activities exhibit a decreased response to 25-hydroxycholesterol compared to wild-type cells. Permanently repressed levels of both HMG CoA synthase and HMG CoA reductase activities are observed in another CHO mutant, phenotypically a mevalonate auxotroph. Mevinolin, a competitive inhibitor of HMG CoA reductase, has no effect on HMG CoA synthase activity measured in vitro. Incubation of CHO cells with sublethal concentrations of mevinolin produces an inhibition of the conversion of [14C]acetate to cholesterol and results in elevated levels of both HMG CoA synthase and HMG CoA reductase activities. Studies of CHO cells in sterol-free medium supplemented with cycloheximide indicate that continuous protein synthesis is not required for the maximal expression of HMG CoA synthase activity and provide an explanation for the lack of temporal similarity between HMG CoA synthase and reductase activities after derepression. These results support the hypothesis of a common mode of regulation for HMG CoA synthase and HMG CoA reductase activities in CHO fibroblasts.