Integrin alphaVbeta3 contains a cell surface receptor site for thyroid hormone that is linked to activation of mitogen-activated protein kinase and induction of angiogenesis

Integrin alpha(V)beta(3) is a heterodimeric plasma membrane protein whose several extracellular matrix protein ligands contain an RGD recognition sequence. This study identifies integrin alpha(V)beta(3) as a cell surface receptor for thyroid hormone [L-T(4) (T(4))] and as the initiation site for T(4)-induced activation of intracellular signaling cascades. Integrin alpha(V)beta(3) dissociably binds radiolabeled T(4) with high affinity, and this binding is displaced by tetraiodothyroacetic acid, alpha(V)beta(3) antibodies, and an integrin RGD recognition site peptide. CV-1 cells lack nuclear thyroid hormone receptor, but express plasma membrane alpha(V)beta(3); treatment of these cells with physiological concentrations of T(4) activates the MAPK pathway, an effect inhibited by tetraiodothyroacetic acid, RGD peptide, and alpha(V)beta(3) antibodies. Inhibitors of T(4) binding to the integrin also block the MAPK-mediated proangiogenic action of T(4). T(4)-induced phosphorylation of MAPK is inhibited by small interfering RNA knockdown of alpha(V) and beta(3). These findings suggest that T(4) binds to alpha(V)beta(3) near the RGD recognition site and show that hormone-binding to alpha(V)beta(3) has physiological consequences.     

MEK1/2-ERK1/2 mediates alpha1-adrenergic receptor-stimulated hypertrophy in adult rat ventricular myocytes

We examined the relative roles of the mitogen-activated protein kinases (MAPK) in mediating the alpha1-adrenergic receptor (alpha1-AR) stimulated hypertrophic phenotype in adult rat ventricular myocytes (ARVM). Norepinephrine (NE; 1 microM) in the presence of the beta -AR antagonist propranolol (Pro; 2 microM) caused activation of Ras (>six-fold), MAPK/ERK kinase 1 and 2 (MEK1/2, >10-fold) and extracellular signal-regulated kinases 1 and 2 (ERK1/2, approximately 30-fold) within 5 min, as determined by kinase activity assays and Western blots using phospho-specific antibodies. Conversely, p38 and c-Jun amino-terminal kinases (JNK) were not activated by NE/Pro. Activated MEK1/2 signals remained detectable at 2 h, and activated ERK1/2 remained detectable at 48 h. The alpha1-AR selective inhibitor prazosin (100 nM) completely inhibited the NE/Pro-stimulated activation of Ras, MEK1/2 and ERK1/2. The MEK inhibitor PD98059 caused a concentration-dependent inhibition of NE/Pro-stimulated protein synthesis (as assessed by [3H]leucine incorporation and cellular protein accumulation) and ERK1/2 activation, with approximately 50% inhibition at a concentration between 10 and 50 microM, which is consistent with the known IC50 values of PD98059 for MEK1 (4 microM) and MEK2 (50 microM). Thus, these data show that alpha1-AR stimulated hypertrophy in ARVM is dependent on the MEK1/2-ERK1/2 signaling pathway.     

Integrin-mediated mechanotransduction in vascular smooth muscle cells: frequency and force response characteristics

Blood vessels are continuously exposed to mechanical forces that lead to adaptive remodeling and atherosclerosis. Although there have been many studies characterizing the responses of vascular cells to mechanical stimuli, the precise mechanical characteristics of the forces applied to cells to elicit these responses are not clear. We designed a magnetic exposure system capable of producing a defined normal force on ferromagnetic beads that are specifically bound to cultured cells coated with extracellular matrix proteins or integrin-specific antibodies. Rat aortic smooth muscle cells were incubated with engineered fibronectin-coated ferromagnetic beads and then exposed to a magnetic field. With activation of extracellular signal-regulated mitogen-activated protein kinase 1/2 (ERK 1/2(MAPK)) used as a prototypical marker for cell responsiveness to mechanical forces, Western blot analysis demonstrated an increase in phosphorylated ERK 1/2(MAPK) expression reaching a maximal response of a 3.5-fold increase at a total force of approximately 2.5 pN per cell. The peak response occurred after 5 minutes of exposure and slowly decreased to baseline after 30 minutes. A cyclic, rather than static, force was required for this activation, and the frequency-response curve increased approximately 2-fold between 0.5 and 2.0 HZ: Vitronectin- and beta(3) antibody-coated beads showed a response nearly identical to those coated with engineered fibronectin, whereas forces applied to beads coated with alpha(2) and beta(1) antibodies did not significantly activate ERK 1/2(MAPK). Mechanical activation of the ERK 1/2(MAPK) system in rat aortic smooth muscle cells occurs through specific integrin receptors and requires a cyclic force with a magnitude estimated to be in the piconewton range.     

Tri-iodothyronine induces proliferation in cultured bovine thyroid cells: evidence for the involvement of epidermal growth factor-associated tyrosine kinase activity

The effects of the tri-iodothyronine (T(3)) secreted by thyroid cells on the growth of the thyrocyte are poorly known. In this study we analyzed the effects of T(3) on the proliferation of bovine thyroid follicles in primary culture previously depleted of endogenous T(3). Cellular deoxiribonucleic acid (DNA) synthesis, determined by [(3)H]thymidine incorporation, was stimulated by T(3) (0.1-5.0 nM) for 24 h in a concentration-dependent fashion with a maximal effect at 1.0 nM T(3) (P<0.01). This T(3) action was time-dependent when assayed from 12 to 72 h. The induction of mitogenic activity was corroborated by the increase in proliferating cell nuclear antigen (PCNA) measured by Western blot analysis. PCNA increased after treatment with T(3) (0.1-5.0 nM) in a concentration-dependent manner. Since T(3) modifies the activity of growth factors whose actions are mainly mediated by tyrosine kinase (TK) activation in diverse cellular types, we assayed the effects of genistein, a general TK inhibitor, and tyrphostin A25, a specific epidermal growth factor (EGF)-receptor (EGFR)-dependent TK activity inhibitor, on the proliferative effects of T(3). The T(3)-induced [(3)H]thymidine incorporation was inhibited by both agents in a concentration-dependent manner. A significant increase in the total TK activity measured in cellular protein extracts was induced by 0.5 and 1.0 nM T(3) (P<0.001). Tyrosine phosphorylation of the EGFR was also stimulated by T(3) (P<0.001) with no change in the EGFR expression as determined by Western blot analysis. Both, the T(3)-stimulated [(3)H]thymidine incorporation and the TK activity were inhibited by a anti-mouse EGF antibody. These results lead us to propose that T(3) could operate as a proliferative agent in bovine thyroid cells through a mechanism involving an autocrine/paracrine EGF/EGFR-dependent regulation.     

Extracellular HIV-1 Tat protein differentially activates the JNK and ERK/MAPK pathways in CD4 T cells.

OBJECTIVE: To investigate the intracellular signals elicited by extracellular HIV-1 Tat protein in lymphoid CD4 T cells. METHODS: CD4 Jurkat T cells were treated with a series of glutathione S-transferase (GST)-Tat fusion proteins: full-length two-exon GST-Tat (GST-Tat2E); one-exon Tat, in which the second exon of Tat was deleted (GST-Tat1E); two-exon Tat, in which the seven arginine residues have been changed to alanine residues (GST-TatArg(mut)), GST-TatdeltaN, which shows a deletion of the N-terminal 21 amino acids. The cells were either treated with soluble GST-Tat proteins or seeded on plates coated with GST-Tat proteins immobilized on plastic. At various time points, Jurkat cells were lysed and examined for c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) activity. RESULTS: Soluble and immobilized GST-Tat2E, but not GST-Tat1E, GST-TatArg(mut) and GST-TatdeltaN, activated JNK in a dose-dependent manner, induced a rapid phosphorylation of c-Jun on Ser63 and promoted the de novo synthesis of c-Jun protein. Moreover, both GST-Tat2E and GST-Tat1E also stimulated ERK/MAPK. However, the activation of JNK was maximal at concentrations of 100 nM of GST-Tat2E and was blocked by the S6-kinase inhibitor rapamycin, whereas the activation of ERK/MAPK was already maximal at 1 nM of GST-Tat2E and was enhanced by rapamycin. CONCLUSIONS: Tat-mediated activation of JNK requires the second exon of Tat, which is dispensable for the activation of ERK/MAPK. The ability to stimulate JNK and ERK/MAPK does not require Tat internalization.     

Two distinct roles of mitogen-activated protein kinases in platelets and a novel Rac1-MAPK-dependent integrin outside-in retractile signaling pathway

Mitogen-activated protein kinases (MAPK), p38, and extracellular stimuli-responsive kinase (ERK), are acutely but transiently activated in platelets by platelet agonists, and the agonist-induced platelet MAPK activation is inhibited by ligand binding to the integrin alpha(IIb)beta(3). Here we show that, although the activation of MAPK, as indicated by MAPK phosphorylation, is initially inhibited after ligand binding to integrin alpha(IIb)beta(3), integrin outside-insignaling results in a late but sustained activation of MAPKs in platelets. Furthermore, we show that the early agonist-induced MAPK activation and the late integrin-mediated MAPK activation play distinct roles in different stages of platelet activation. Agonist-induced MAPK activation primarily plays an important role in stimulating secretion of platelet granules, while integrin-mediated MAPK activation is important in facilitating clot retraction. The stimulatory role of MAPK in clot retraction is mediated by stimulating myosin light chain (MLC) phosphorylation. Importantly, integrin-dependent MAPK activation, MAPK-dependent MLC phosphorylation, and clot retraction are inhibited by a Rac1 inhibitor and in Rac1 knockout platelets, indicating that integrin-induced activation of MAPK and MLC and subsequent clot retraction is Rac1-dependent. Thus, our results reveal 2 different activation mechanisms of MAPKs that are involved in distinct aspects of platelet function and a novel Rac1-MAPK-dependent cell retractile signaling pathway.     

Ligation of CD31 (PECAM-1) on endothelial cells increases adhesive function of alphavbeta3 integrin and enhances beta1 integrin-mediated adhesion of eosinophils to endothelial cells.

We determined the role of the heterophilic interaction of alphavbeta3 integrin on endothelial cells with CD31 on leukocytes in mediating leukocyte-endothelial cell interactions. Preincubation of interleukin-4 (IL-4)-stimulated human umbilical vein endothelial cells (HUVECs) with anti-CD31 monoclonal antibodies (MoAbs) enhanced eosinophil adhesion to the IL-4-stimulated HUVECs, and the endothelial CD31-induced enhancement of eosinophil adhesion to IL-4-stimulated HUVECs was prevented by anti-vascular cell adhesion molecule-1 (VCAM-1) MoAb and anti-very late activation antigen-4 (VLA-4) MoAb, but not by anti-intercellular adhesion molecule-1 (ICAM-1) MoAb, anti-lymphocyte function-associated antigen-1 (LFA-1) MoAb, anti-P-selectin MoAb, or anti-E-selectin MoAb. CD31 stimulation of HUVECs increased the adhesive function of alphavbeta3 integrin to its ligand RGD peptide, the binding of which reached a maximum at 10 minutes after the stimulation, and the CD31-induced alphavbeta3 integrin activation on HUVECs was inhibited by inhibitors of protein kinase C and phosphatidylinositol 3 kinase (PI3-kinase). Furthermore, anti-alphavbeta3 integrin MoAb and RGD peptide as well as soluble CD31 inhibited endothelial CD31-induced enhancement of eosinophil adhesion to IL-4-stimulated HUVECs. However, anti-alphavbeta3 integrin MoAb had no significant inhibitory effect on the eosinophil adhesion to IL-4-stimulated or unstimulated HUVECs without CD31 stimulation of HUVECs. Finally, CD31 stimulation of eosinophils increased the adhesive function of alpha4beta1 integrin (VLA-4) to its ligand fibronectin and their adhesion to IL-4-stimulated HUVECs in a VLA-4-dependent manner. These results indicate that CD31-mediated inside-out signaling activates alphavbeta3 integrin on endothelial cells, that the heterophilic alphavbeta3 integrin/CD31 interaction induces beta1 integrin-mediated adhesion of eosinophils to endothelial cells, and that the heterophilic interaction itself is not significantly involved in firm adhesion of eosinophils to endothelial cells.     

Glucagon receptor-mediated extracellular signal-regulated kinase 1/2 phosphorylation in rat mesangial cells: role of protein kinase A and phospholipase C

Glucagon, a major insulin counterregulatory hormone, binds to specific Gs protein-coupled receptors to activate glycogenolytic and gluconeogenic pathways, causing blood glucose levels to increase. Inappropriate increases in serum glucagon play a critical role in the development of insulin resistance and target organ damage in type 2 diabetes. We tested the hypotheses that: (1) glucagon induces proliferation of rat glomerular mesangial cells through glucagon receptor-activated phosphorylation of mitogen-activated protein kinase extracellular signal-regulated kinase 1/2 (p-ERK 1/2); and (2) this phosphorylation involves activation of cAMP-dependent protein kinase A (PKA) and phospholipase C (PLC)/[Ca2+]i signaling pathways. In rat mesangial cells, glucagon (1 nM) stimulated [3H]-thymidine incorporation by 96% (P<0.01). This proliferative effect was blocked by the specific glucagon receptor antagonist [Des-His1-Glu9] glucagon (1 micromol/L; P<0.01), a mitogen-activated protein kinase/ERK kinase inhibitor PD98059 (10 micromol/L; P<0.01), a PLC inhibitor U73122 (1 micromol/L; P<0.01), or a PKA inhibitor H-89 (1 micromol/L; P<0.01). The proliferation was associated with a 2-fold increase in p-ERK 1/2 that peaked 5 minutes after glucagon stimulation (P<0.01) and also was blocked by [Des-His1-Glu9] glucagon. Total ERK 1/2 was not affected by glucagon. Pretreating of mesangial cells with U73122 or H89 significantly attenuated ERK 1/2 phosphorylation induced by glucagon. We believe that these are the first data showing that glucagon activates specific receptors to induce ERK 1/2 phosphorylation and thereby increase mesangial cell proliferation and that this effect of glucagon involves both PLC/[Ca2+]i- and cAMP-dependent PKA-activated signaling cascades.     

Inhibitory guanine nucleotide regulatory protein activation of mitogen-activated protein kinase in experimental hepatocellular carcinoma in vitro.

OBJECTIVE: Hepatocellular carcinoma (HCC) is associated with altered expression and function of inhibitory guanine nucleotide regulatory proteins (Gi-proteins). This study addresses the interaction between Gi-proteins and the extracellular regulated kinase (ERK) component of the mitogen activated protein kinase (MAPK) cascade in regulating mitogenesis in an experimental model of HCC. DESIGN: Pharmacological agents which selectively interact with specific target proteins involved in signal transduction through a Gi-MAPK pathway have recently become available. These agents in combination with scientific assays allow us to address the role of individual components of this cascade in the regulation of mitogenesis in HCC. METHODS: These studies were performed using rat hepatic tumorigenic cells (H4IIE) and isolated cultured hepatocytes in vitro in conjunction with pharmacological agents which interact with Gi-protein or MAPK components of intracellular signalling. RESULTS: Direct activation of Gi-proteins with mastoparan M7 (M7) significantly increased nuclear thymidine incorporation in hepatic tumorigenic H4IIE cells in a dose-dependent manner (10-1000 nM, n = 4, P < 0.05), an effect that was abolished by treatment with either pertussis toxin (PTx) or the selective mitogen-activated ERK-regulated kinase (MEK) inhibitor PD098059. In contrast, M7 inhibited nuclear thymidine incorporation in serum-stimulated isolated hepatocytes. ERK2 activity was then determined as the ability of immunoprecipitated ERK2 proteins to phosphorylate the ERK substrate myelin basic protein. These studies demonstrated a time- and dose-dependent increase in ERK2 activity in H4IIE cells following Gi-protein activation with M7, a maximal response being attained at 20 min. In contrast, M7 failed to significantly alter ERK2 activity in isolated cultured hepatocytes at any of the doses or time points assayed (10-5000 nM, 10-120 min). Gi-stimulated ERK activation was completely blocked in tumorigenic cells following treatment with PTx. CONCLUSIONS: These data demonstrate for the first time a Gi-linked MAPK cascade in experimental HCC, activation of which stimulates cellular mitogenesis.     

TIMP-2 disrupts FGF-2-induced downstream signaling pathways

We have previously reported that tissue inhibitor of metalloproteinases-2 (TIMP-2), an endogenous inhibitor of matrix metalloproteinase, modulates angiogenic responses through the MMP inhibition-independent activity. In this study, we investigate the molecular mechanisms of TIMP-2-mediated growth inhibition in response to fibroblast growth factor-2 (FGF-2). Pre-treatment with a protein tyrosine phosphatase inhibitor orthovanadate or expression of a dominant negative Shp-1 mutant fails to induce TIMP-2 inactivation of FGF-2 signaling pathways in human microvascular endothelial cells. We also show that TIMP-2 inhibition of FGF-2-induced p42/44(MAPK) activation and cell proliferation is associated with TIMP-2 binding to integrin alpha3beta1 on endothelial cell surfaces, as demonstrated by use of anti-integrin alpha3 or beta1 blocking antibodies, or disruption of integrin alpha3 expression by siRNA. Collectively, our results indicate that TIMP-2 inhibits FGF-2 signaling pathways through association with integrin alpha3beta1 and Shp-1-dependent inhibition of p42/44(MAPK) signaling, which in turn, results in suppression of FGF-2-stimulated endothelial cell mitogenesis.     

Regulation of c-jun-NH2 terminal kinase and extracellular-signal regulated kinase in human platelets

Platelets are an interesting model for studying the relationship betwen adhesion and mitogen-activated protein (MAP) kinase activation. We have recently shown that in platelets, ERK2 was activated by thrombin and downregulated by alpha(IIb)beta(3) integrin engagement. Here we focused our attention on the c-Jun NH2-terminal kinases (JNKs) and their activation in conditions of platelet aggregation. We found that JNK1 was present in human platelets and was activated after thrombin induction. JNK1 phosphorylation was detected with low concentrations of thrombin (0. 02 U/mL) and after 1 minute of thrombin-induced platelet aggregation. JNK1 activation was increased (fivefold) when fibrinogen binding to alpha(IIb)beta(3) integrin was inhibited by the Arg-Gly-Asp-Ser (RGDS) peptide or (Fab')(2) fragments of a monoclonal antibody specific for alpha(IIb)beta(3), demonstrating that, like ERK2, alpha(IIb)beta(3) integrin engagement negatively regulates JNK1 activation. Comparison of JNK1 activation by thrombin in stirred and unstirred platelets in the presence of RGDS peptide showed a positive regulation by stirring itself, independently of alpha(IIb)beta(3) integrin engagement, which was confirmed in a thrombasthenic patient lacking platelet alpha(IIb)beta(3). The same positive regulation by stirring was found for ERK2. These results suggest that MAP kinases (JNK1 and ERK2) are activated positively by thrombin and stirring. In conclusion, we found that JNK1 is present in platelets and can be activated after thrombin induction. Moreover, this is the first report showing that two different MAP kinases (ERK2 and JNK1) are regulated negatively by alpha(IIb)beta(3) engagement and positively by mechanical forces in platelets.     

Effects of estriol on the proliferation and differentiation of human osteoblastic MG-63 cells

Estriol has been showed to prevent bone loss in osteoporotic rats and postmenopausal women, but the mechanisms remain unclear. In the present study, we evaluated the effect of estriol on osteoblastic MG-63 cells in vitro, and compared its action with 17beta-estradiol (E2). Cell proliferation was determined by measuring total cell numbers and [3H]thymidine incorporation. Cell function was studied by measuring alkaline phosphatase (ALP) activity and secreted osteocalcin. Our data showed that estriol stimulated MG-63 cells proliferation in a dose-dependent manner, but had no influence on ALP activity in MG-63 cells and osteocalcin production. Compared with estriol treatment, E2 showed a stronger proliferation. Estrogen receptor (ER) alpha and beta expression in MG-63 cells can be detected by Western immunoblot analysis, and the proliferative response to E2 and estriol can be all abrogated by ER antagonist ICI 182,780. In conclusion, estriol stimulates osteoblastic MG-63 cells proliferation, but has no effects on differentiation. The proliferative response to estriol is mediated by the ER. These results suggest that estriol has an effect on osteoblastic proliferation, and this may contribute to its actions on prevention of bone loss.     

H-Ras is involved in the inside-out signaling pathway of interleukin-3-induced integrin activation

The proto-oncogene product, p21(ras), has been implicated in the cellular mechanism of adhesion, although its precise role has been controversial. Numerous cytokines and growth-factors activate Ras, which is an important component of their growth-promoting signaling pathways. On the other hand, the role of Ras in cytokine-induced adhesion has not been elucidated. We therefore investigated the function of H-Ras in the inside-out signaling pathway of interleukin-3 (IL-3)-induced integrin activation in the murine Baf3 cell line after transfection of cells with either constitutively active, dominant-negative, or wild-type H-Ras cDNAs. Adhesion of Baf3 cells to fibronectin was induced by IL-3 in a dose-dependent manner via very late antigen-4 (VLA-4; alpha4beta1 integrins) and VLA-5 (alpha5beta1 integrins) activation. On the other hand, IL-4 did not induce the adhesion of Baf3 cells to fibronectin, although IL-4 did stimulate the cell proliferation of Baf3 cells. Constitutively active H-Ras-transfected Baf3 cells adhered to fibronectin without IL-3 stimulation through VLA-4 and VLA-5, whereas dominant-negative H-Ras-transfected Baf3 cells showed significantly less adhesion induced by IL-3 compared with wild-type and constitutively active H-Ras-transfected Baf3 cells. Anti-beta1 integrin antibody (clone; 9EG7), which is known to change integrin conformation and activate integrins, induced the adhesion of dominant-negative H-Ras-transfected Baf3 cells as much as the other types of H-Ras-transfected Baf3 cells. 8-Br-cAMP, Dibutyryl-cAMP, Ras-Raf-1 pathway inhibitors, and PD98059, a MAPK kinase inhibitor, suppressed proliferation and phosphorylation of MAPK detected by Western blotting with anti-phospho-MAPK antibody, but not adhesion of any type of H-Ras-transfected Baf3 cells, whereas U-73122, a phospholipase C (PLC) inhibitor, suppressed adhesion of these cells completely. These data indicate that H-Ras and PLC, but not Raf-1, MAPK kinase, or the MAPK pathway, are involved in the inside-out signaling pathway of IL-3-induced VLA-4 and VLA-5 activation in Baf3 cells.     

Transforming growth factor-beta-activated protein kinase 1-binding protein (TAB)-1alpha, but not TAB1beta, mediates cytokine-induced p38 mitogen-activated protein kinase phosphorylation and cell death in insulin-producing cells

Previous studies have indicated that the p38 MAPK participates in signaling events that lead to the death of the insulin-producing beta-cell. The aim of the present study was to elucidate the role of the TGF-beta-activated protein kinase 1-binding protein 1 (TAB1) in the cytokine-induced activation of p38. Levels of TAB1 mRNA and protein were analyzed by real-time PCR and immunoblotting, and TAB1 expression in mouse and human islet cells was down-regulated using lipofection of diced-small interfering RNA. TAB1 overexpression in beta-TC6 cells was achieved by transient transfections followed by fluorescence activated cell sorting. Phosphorylation of p38, c-Jun N-terminal kinase, and ERK was assessed by immunoblotting, and viability was determined using vital staining with bisbenzimide and propidium iodide. We observed that TAB1 is expressed in insulin-producing cells. Cytokine (IL-1beta + interferon-gamma)-stimulated p38 phosphorylation was significantly increased by TAB1alpha overexpression, but not TAB1beta overexpression, in beta-TC6 cells. The TAB1alpha-augmented p38 phosphorylation was paralleled by an increased cell death rate. Treatment of islet cells with diced-small interfering RNA specific for TAB1, but not for TGF-beta-activated kinase 1, resulted in lowered cytokine-induced p38 phosphorylation and protection against cell death. The cytokine-induced phosphorylation of c-Jun N-terminal kinase and ERK was not affected by changes in TAB1 levels. Finally, TAB1 phosphorylation was decreased by the p38 inhibitor SB203580. We conclude that TAB1alpha, but not TAB1beta, plays an important role in the activation of p38 in insulin-producing cells and therefore also in cytokine-induced beta-cell death.     

Involvement of Rho-kinase in prostaglandin F2alpha-stimulated interleukin-6 synthesis via p38 mitogen-activated protein kinase in osteoblasts

We have previously reported that prostaglandin F(2alpha) (PGF(2alpha)) stimulates interleukin-6 (IL-6), a potent bone resorptive agent, through p44/p42 mitogen-activated protein (MAP) kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether Rho-kinase is implicated in the PGF(2alpha)-stimulated IL-6 synthesis in MC3T3-E1 cells. PGF(2alpha) time-dependently induced the phosphorylation of myosin phosphatase targeting subunit (MYPT-1), a Rho-kinase substrate. Y27632, a specific Rho-kinase inhibitor, significantly reduced the PGF(2alpha)-stimulated IL-6 synthesis as well as the MYPT-1 phosphorylation. Fasudil, another inhibitor of Rho-kinase, suppressed the PGF(2alpha)-stimulated IL-6 synthesis. Y27632 and fasudil failed to affect the PGF(2alpha)-induced phosphorylation of p44/p42 MAP kinase. SB203580 and BIRB0796, potent inhibitors of p38 MAP kinase, suppressed the IL-6 synthesis induced by PGF(2alpha). While SP600125, an inhibitor of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), failed to reduce the synthesis. Y27632 as well as fasudil attenuated the PGF(2alpha)-induced phosphorylation of p38 MAP kinase. These results strongly suggest that Rho-kinase regulates PGF(2alpha)-stimulated IL-6 synthesis via p38 MAP kinase activation in osteoblasts.     

Role of endocytosis in the activation of the extracellular signal-regulated kinase cascade by sequestering and nonsequestering G protein-coupled receptors

Acting through a number of distinct pathways, many G protein-coupled receptors (GPCRs) activate the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) cascade. Recently, it has been shown that in some cases, clathrin-mediated endocytosis is required for GPCR activation of the ERK/MAPK cascade, whereas in others it is not. Accordingly, we compared ERK activation mediated by a GPCR that does not undergo agonist-stimulated endocytosis, the alpha(2A) adrenergic receptor (alpha(2A) AR), with ERK activation mediated by the beta(2) adrenergic receptor (beta(2) AR), which is endocytosed. Surprisingly, we found that in COS-7 cells, ERK activation by the alpha(2A) AR, like that mediated by both the beta(2) AR and the epidermal growth factor receptor (EGFR), is sensitive to mechanistically distinct inhibitors of clathrin-mediated endocytosis, including monodansylcadaverine, a mutant dynamin I, and a mutant beta-arrestin 1. Moreover, we determined that, as has been shown for many other GPCRs, both alpha(2A) and beta(2) AR-mediated ERK activation involves transactivation of the EGFR. Using confocal immunofluorescence microscopy, we found that stimulation of the beta(2) AR, the alpha(2A) AR, or the EGFR each results in internalization of a green fluorescent protein-tagged EGFR. Although beta(2) AR stimulation leads to redistribution of both the beta(2) AR and EGFR, activation of the alpha(2A) AR leads to redistribution of the EGFR but the alpha(2A) AR remains on the plasma membrane. These findings separate GPCR endocytosis from the requirement for clathrin-mediated endocytosis in EGFR transactivation-mediated ERK activation and suggest that it is the receptor tyrosine kinase or another downstream effector that must engage the endocytic machinery.     

Insulin-like growth factor-binding protein 1 stimulates human trophoblast migration by signaling through alpha 5 beta 1 integrin via mitogen-activated protein Kinase pathway

A highly migratory subpopulation of the human placental trophoblast, known as the extravillous trophoblast (EVT), invades the uterus and its vasculature, to establish adequate exchange of key molecules between the maternal and fetal circulations. During their formation, EVT cells selectively acquire alpha 5 beta 1 integrin. We had shown that alpha 5 beta 1 is required for their migratory function, and that EVT cell migration is stimulated by insulin-like growth factor-binding protein (IGFBP)-1 produced by the uterine decidua. The present study examined whether this stimulation is dependent on binding of the Arg-Gly-Asp (RGD) domain of IGFBP-1 to an RGD binding site on the alpha 5 beta 1 integrin, followed by activation of focal adhesion kinase (FAK) and stimulation of the mitogen-activated protein kinase (MAPK) pathway. IGFBP-1 treatment increased migration of EVT cells, whereas an anti-alpha 5 beta 1 integrin antibody blocked migration regardless of IGFBP-1 treatment. Migration stimulation by IGFBP-1 was abrogated by pretreatment with a Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP), but not a Gly-Arg-Gly-Glu-Ser-Pro (GRGESP) hexapeptide, and by mutation of the RGD domain of IGFBP-1 to Trp-Gly-Asp (WGD). IGFBP-1 treatment caused a rapid localization of immunoreactive FAK to cellular lamellipodia, a rapid increase in phosphorylation of FAK and extracellular-signal regulated kinases 1 and 2. Preincubation of EVT cells with Herbimycin A, a tyrosine kinase inhibitor, abrogated IGFBP-1 effects; whereas an MAPK kinase inhibitor, PD 98059, reduced migration regardless of IGFBP-1 treatment. These results indicate that IGFBP-1 stimulation of EVT cell migration occurs by binding of its RGD domain to the alpha 5 beta 1 integrin, leading to activation of FAK and stimulation of MAPK pathway.