Biomedical subjects

B C Berk

Publications and source records attributed to B C Berk.

At least 19 recordsLinked to original sources

PubMed1997

PKC-epsilon is required for mechano-sensitive activation of ERK1/2 in endothelial cells.

Mechano-sensitive regulation of endothelial cells (EC) function by shear stress is critical for flow-induced vasodilation and gene expression. Previous studies by our laboratory demonstrated that shear stress activates the 44- and 42-kDa extracellular signal-regulated kinases (ERK1/2) in EC in a time- and force-dependent manner. ERK1/2 activation was inhibited by protein kinase C (PKC) down-regulation with phorbol 12,13-dibutyrate (1 microM for 24 h) but not by calcium chelation with BAPTA-AM (acetoxymethyl ester of BAPTA) (75 microM for 30 min), suggesting that a novel PKC isoform (delta, epsilon, eta, theta) mediates shear stress-induced ERK1/2 activation. Western blotting with PKC isoform-specific antibodies demonstrated expression of PKC-alpha, -epsilon, and -zeta isoforms in EC. PKC-epsilon was specifically inhibited by transfection with antisense PKC-epsilon phosphorothioate oligonucleotides (1,000 nM for 6 h). Antisense treatment decreased PKC-epsilon protein levels by 80 +/- 13% after 72 h and completely inhibited shear stress-stimulated ERK1/2 activation. Scrambled PKC-epsilon oligonucleotides and antisense PKC-alpha and PKC-zeta oligonucleotides had no effect on ERK1/2 activity. PKC-epsilon appeared specific for mechano-sensitive ERK1/2 activation, as antisense PKC-epsilon oligonucleotides did not inhibit ERK1/2 activation by EGF or bradykinin but did inhibit ERK1/2 activation upon EC adhesion to fibronectin. These results define a pathway for shear stress-mediated ERK1/2 activation and establish a new function for PKC-epsilon as part of a mechano-sensitive signal transduction pathway in EC.

Animals↗

PubMed1997

Vitamins C and E inhibit O2- production in the pig coronary artery.

BACKGROUND: We previously found in a pig coronary balloon injury model that vitamins C and E as well as probucol had beneficial effects on the vessel response to injury measured by morphometry These effects correlated with an inhibition in the ability to oxidize LDLs ex vivo, suggesting that the morphological response was due to the antioxidant effect of the treatments. METHODS AND RESULTS: In the present study, the production of O2- by vessels 14 days after balloon injury was determined and correlated with circulating and tissue levels of vitamins C and E. Twenty-five domestic pigs were divided into four groups: control (n=7), vitamin C (500 mg/d, group C, n=6), vitamin E (1000 IU/d, group E, n=6), and vitamins C and E (500 mg/d and 1000 IU/d, group C+E, n=6). Vitamins were administered 7 days before oversized balloon injury of the left anterior descending coronary artery (LAD) and continued for 14 days after injury. Vitamin C and E concentrations were determined in plasma and lymphocytes as an index for tissue levels. Vessels were harvested after animals were killed, and O2- production was measured by lucigenin chemiluminescence. O2- production by the injured LAD was 2.5-fold greater than O2- production by the uninjured LAD or right coronary artery (RCA). The increase in O2- was caused primarily by cells present in the media and neointima. All vitamin-treated groups showed significantly decreased O2- production in both the RCA and LAD (approximately 45% inhibition) relative to vessels in the control, untreated group. There was a significant correlation between LAD O2- production and lymphocyte vitamin E levels. CONCLUSIONS: The present study is the first to show increased O2- production in injured vessels and to demonstrate that antioxidant vitamins reduce O2- production. These results suggest that beneficial effects of antioxidant vitamins in coronary artery disease are related, in part, to alterations in vessel redox state.

Animals↗

PubMed1997

c-Src is required for oxidative stress-mediated activation of big mitogen-activated protein kinase 1.

Big mitogen-activated kinase 1 (BMK1) or extracellular signal-regulated kinase-5 (ERK5) has recently been identified as a new member of the mitogen-activated protein kinase family. We have shown that BMK1 is activated to a greater extent by H2O2 than growth factors, suggesting that in comparison with other mitogen-activated protein kinase family members, BMK1 is a redox-sensitive kinase. Previous investigations indicate that the tyrosine kinase c-Src mediates signal transduction by reactive oxygen species, including H2O2. Therefore, the role of Src kinase family members (c-Src and Fyn) in activation of the BMK1 by H2O2 in mouse fibroblasts was studied. An essential role for c-Src was suggested by four experiments. First, H2O2 stimulated c-Src activity rapidly in fibroblasts (peak at 5 min), which preceded peak activity of BMK1 (20 min). Second, specific Src family tyrosine kinase inhibitors (herbimycin A and CP-118,556) blocked BMK1 activation by H2O2 in a concentration-dependent manner. Third, BMK1 activation in the response to H2O2 was completely inhibited in cells derived from mice deficient in c-Src, but not Fyn. Finally, BMK1 activity was much greater in v-Src-transformed NIH-3T3 cells than wild type cells. These results demonstrate an essential role for c-Src in H2O2-mediated activation of BMK1 and suggest that redox-sensitive regulation of BMK1 is a new function for c-Src.

Animals↗

PubMed1997

A mathematical model of the cytosolic-free calcium response in endothelial cells to fluid shear stress.

Important among the responses of endothelial cells to flow stimuli are cytosolic-free calcium transients. These transients are mediated by several factors, including blood-borne agonists, extracellular calcium, and fluid-imposed shear forces. A mathematical model has been developed describing the recognition and transduction of shear stress to the second messenger cytosolic calcium. Shear stress modulates the calcium response via at least two modalities. First, mass transfer of agonist to the cell surface is enhanced by perfusion and is thus related to shear stress. Second, the permeability of the cell membrane to extracellular calcium increases upon exposure to shear stress. A mass balance for agonist in the perfusate is coupled to a previously published calcium dynamics model. Computations indicate a flow region where the transient moves from transport limited to kinetically limited. Parametric studies indicate distinct contributions to the time course by each step in the process. These steps include the time to develop the concentration boundary layer of agonist, receptor activation, and the mobilization of calcium from intracellular stores. Exogenous calcium is presumed to enter the cell via shear stress-gated ion channels. The model predicts a sigmoidal dependence of calcium influx upon shear stress. The peak value of the transient is determined largely by the agonist pathway, whereas the plateau level is governed by calcium influx. The model predicts the modulation of the calcium transient in the physiologically relevant range of flow and the associated shear stress. This implies that hemodynamics is important in regulating endothelial biology.

Animals↗

PubMed1997

Fluid shear stress-mediated signal transduction: how do endothelial cells transduce mechanical force into biological responses?

We propose a model for signaling events induced by fluid shear stress that incorporates many of the features discussed in this paper (FIG. 4). First, heterotrimeric G-proteins, as well as a small G-proteins, are activated by flow. Indeed, a G protein appears to be required for ERK1/2 activation by flow because ERK1/2 activation is completely inhibited by GDP-beta S. Then, flow activates phospholipase C and generates IP3 and diacylglycerol (DG). IP3 releases Ca2+ from internal Ca2+ stores via IP3 receptor and DG activates PKC. Nollert and colleagues have shown that flow activates PLC and increases IP3. It is possible that several different PKC isozymes are activated by flow including both Ca(2+)-dependent and Ca(2+)-independent isozymes. These different isozymes may have specific downstream substrates. For example, PKC-epsilon may be involved in activation of ERK1/2, while the PKC isozyme responsible for activation of JNK remains unknown. It is also possible that these PKC isozymes may be important in gene transcription events. For example, PKC-zeta has been suggested to be involved in NF-kappa B-mediated gene transcription. Longer term changes in endothelial cell morphology and structure are likely to involve separate kinases. Important candidates for these changes include members of the c-Src and FAK families. c-Src is now considered to be a component of the focal adhesion complex and regulate focal adhesion formation and/or cytoskeletal rearrangement. Recently, stretch, another mechanostress, has been shown to activate c-Src in fetal rat lung cells. It has been clarified that ERK1/2 and JNK are regulated by the small G-proteins, Ras and Rac/Cdc42H, respectively, and their effectors in parallel with each other. Rac and Rho are also thought to be involved in membrane ruffling and/or cytoskeletal rearrangement. Fluid shear stress causes stress fiber formation and focal adhesion rearrangement. Recent study by Malek and Izumo suggested the importance of microtubules in shear stress-induced morphological change and actin stress fiber formation. It is clear that the focal adhesion complex plays an important role in shear stress-induced signal and it is interesting to speculate that shear stress-induced signaling has cross-talk with signaling induced by integrins. As a general model we propose that the integration between the rapid events stimulated by shear stress and the longer term events is mediated by tyrosine kinases that serve to regulate these multiple signal transduction pathways.

Animals↗

PubMed1997

Protein kinase C-zeta mediates angiotensin II activation of ERK1/2 in vascular smooth muscle cells.

Activation of 44 and 42 kDa extracellular signal-regulated kinases (ERK)1/2 by angiotensin II (angII) plays an important role in vascular smooth muscle cell (VSMC) function. The dual specificity mitogen-actived protein (MAP) kinase/ERK kinase (MEK) activates ERK1/2 in response to angII, but the MEK activating kinases remain undefined. Raf is a candidate MEK kinase. However, a kinase other than Raf appears responsible for angII-mediated signal transduction because we showed previously that treatment with 1 microM phorbol 12, 13-dibutyrate (PDBU) for 24 h completely blocked Raf-Ras association in VSMC but did not inhibit activation of MEK and ERK1/2 by angII. We hypothesized that an atypical protein kinase C (PKC) isoform, which lacks a phorbol ester binding domain, mediated ERK1/2 activation by angII. Western blot analysis of rat aortic VSMC with PKC isoform-specific antibodies showed PKC-alpha, -beta1, -delta, -epsilon, and -zeta in relative abundance. All isoforms except PKC-zeta were down-regulated by 1 microM PDBU for 24 h suggesting that PKC-zeta was responsible for angII-mediated ERK1/2 activation. In response to angII, PKC-zeta associated with Ras as shown by co-precipitation of PKC-zeta with anti-H-Ras antibody. To characterize further the role of PKC-zeta, PKC-zeta protein was depleted specifically by transfection with antisense PKC-zeta oligonucleotides. Antisense PKC-zeta oligonucleotide treatment significantly decreased PKC-zeta protein expression (without effect on other PKC isoforms) and angII-mediated ERK1/2 activation in a concentration-dependent manner. In contrast, ERK1/2 activation by platelet-derived growth factor and phorbol ester was not significantly inhibited. These results demonstrate an important difference in signal transduction by angII compared with PDGF and phorbol ester in VSMC, and suggest a critical role for PKC-zeta and Ras in angII stimulation of ERK1/2.

Angiotensin II↗

PubMed1997

Oxidized LDL stimulates mitogen-activated protein kinases in smooth muscle cells and macrophages.

It has been proposed that oxidized LDL is more atherogenic than native LDL. However, the mechanisms by which native LDL and oxidized LDL alter function of cells in the vessel wall remain undefined. A signal transduction pathway that mediates many changes in cell function is the mitogen-activated protein (MAP) kinase cascade. We therefore examined the effect of native LDL and oxidized LDL on MAP kinase activity in cultured vascular smooth muscle cells (VSMC), endothelial cells, and macrophages by using an in-gel-kinase assay and anti-phosphotyrosine MAP kinase antibodies. Native LDL and LDL oxidized by the addition of Cu2+ (Cu(2+)-oxidized LDL) stimulated MAP kinase in a time- and dose-dependent manner in baboon and rat VSMC but not in bovine endothelial cells. Cu(2+)-oxidized LDL stimulated MAP kinase in human monocyte-derived macrophages, but the effect was much greater in cells cultured for 7 days compared with 1 day, suggesting dynamic regulation of the cellular response to oxidized LDL. In rat VSMC, the maximal MAP kinase response to Cu(2+)-oxidized LDL was significantly greater than the response to native LDL. Cu(2+)-oxidized LDL was more potent, with half-maximal activation at 15 micrograms/mL versus 30 micrograms/mL for native LDL. Stimulation of MAP kinase appeared to involve protein kinase C, since phorbol ester pretreatment for 24 hours blocked MAP kinase activation. Oxidation of LDL by other methods showed that activation of MAP kinase was not well correlated with lipid peroxides or aldehydes, suggesting that other components present in oxidized LDL were responsible. The active moiety appeared to be lipid based on extraction of oxidized LDL with organic solvents. These data indicate that LDL stimulates MAP kinase in VSMC, oxidation of LDL potentiates the effect, a lipid moiety is involved, and Cu(2+)-oxidized LDL activation of MAP kinase is cell-type specific. These findings suggest a role for MAP kinase in the pathways by which oxidized LDL contributes to altered cellular function associated with atherogenesis.

Animals↗

PubMed1997

A 90-kD Na(+)-H+ exchanger kinase has increased activity in spontaneously hypertensive rat vascular smooth muscle cells.

Increased activity of the Na(+)-H+ exchanger (NHE-1 isoform) has been observed in cells and tissues from hypertensive humans and animals, including the spontaneously hypertensive rat (SHR). No mutation in NHE-1 DNA sequence or alteration in NHE-1 mRNA and protein expression has been demonstrated in hypertension, indicating that alterations in proteins that regulate NHE-1 activity are responsible for increased activity. The recent finding that NHE-1 phosphorylation in SHR vascular smooth muscle cells (VSMCs) was greater than in Wistar-Kyoto rat (WKY) VSMCs suggested that NHE-1 kinases may represent an abnormal regulatory pathway present in hypertension. To define NHE-1 kinases altered in the hypertensive phenotype. We measured NHE-1 kinase activity by an in-gel-kinase assay using a recombinant glutathione S-transferase NHE-1 fusion protein as a substrate. At least 7 NHE-1 kinases (42 to 90 kD) were present in VSMCs. We studied a 90-kD kinase because it was the major NHE-1 kinase and exhibited differences between SHR and WKY. Comparison of 90-kD kinase activity revealed that SHR VSMCs had increased activity in growth-arrested cells and in cells stimulated by angiotensin II (100 nmol/L for 5 minutes). Activation of the 90-kD kinase by angiotensin II was Ca2+ dependent, PKC independent, and partially dependent on the mitogen-activated protein kinase pathway. These findings indicate that increased activity of a 90-kD NHE-1 kinase is a characteristic of SHR VSMCs in culture and suggest that alterations in the 90-kD NHE-1 kinase and/or proteins that regulate its activity may be a pathogenic component in hypertension in the SHR.

Animals↗

PubMed1997

Angiotensin II signal transduction in vascular smooth muscle: role of tyrosine kinases.

In this review, the role of tyrosine kinases in angiotensin II-mediated signal transduction pathways in vascular smooth muscle is discussed. Angiotensin II was isolated by virtue of its vasoconstrictor abilities and has long been thought to play a critical role in hypertension. However, recent studies indicate important roles for angiotensin II in inflammation, atherosclerosis, and congestive heart failure. The expanding role of angiotensin II indicates that multiple signal transduction pathways are likely to be activated in a tissue-specific manner. Exciting recent data show that angiotensin II directly stimulates tyrosine kinases, including pp60(c-src) kinase (c-Src), focal adhesion kinase (FAK), and Janus kinases (JAK2 and TYK2). Angiotensin II may activate receptor tyrosine kinases, such as Axl and platelet-derived growth factor, by as-yet-undefined autocrine mechanisms. Finally, unknown tyrosine kinases may mediate tyrosine phosphorylation of Shc, Raf, and phospholipase C-gamma after angiotensin II stimulation. These angiotensin II-regulated tyrosine kinases appear to be required for angiotensin II effects, such as vasoconstriction, proto-oncogene expression, and protein synthesis, on the basis of studies with tyrosine kinase inhibitors. Thus, understanding angiotensin II-stimulated signaling events, especially those related to tyrosine kinase activity, may form the basis for the development of new therapies for cardiovascular diseases.

Angiotensin II↗

PubMed1997

Angiotensin II stimulates p90rsk in vascular smooth muscle cells. A potential Na(+)-H+ exchanger kinase.

Angiotensin II is a multifunctional agonist for vascular smooth muscle cells (VSMCs), stimulating increases in signal events, cell growth, and ion flux. We previously defined protein kinase C (PKC)-dependent and -independent mechanisms by which angiotensin II stimulated activity of the Na(+)-H+ exchanger isoform-1 (NHE-1) and identified a 90-kD kinase that exhibited increased activity in VSMCs isolated from genetically hypertensive rats. To determine whether this 90-kD kinase was p90rsk (RSK), VSMCs were stimulated with 100 nmol/L angiotensin II, and NHE-1 kinase activity was measured by phosphorylation of recombinant NHE-1 (a glutathione S-transferase fusion protein containing amino acids 516 to 815 of the cytoplasmic carboxyl tail) in vitro. NHE-1 kinase (90 kD) activity was markedly decreased by immunodepletion of RSK. Characterization of RSK activation by angiotensin II revealed many similarities to the 90-kD NHE-1 kinase, including time course and NHE-1 domain phosphorylation, as well as regulation by extracellular signal-regulated kinases (ERK1/2), intracellular Ca2+, and PKC. Specifically, angiotensin II stimulated a rapid and transient (peak, 5 minutes) increase in RSK activity. Analysis of several NHE-1 fusion proteins revealed that only proteins containing amino acids 670 to 714 were phosphorylated by RSK. Inhibiting ERK1/2 (30 mumol/L PD098059 for 30 minutes) or chelating intracellular Ca2+ prevented RSK activation. In contrast, downregulating PKC (1 mumol/L phorbol dibutyrate for 24 hours) had little effect. These findings establish RSK as a putative NHE-1 kinase and potential mediator of increased Na(+)-H+ exchange in hypertension.

Amino Acid Sequence↗

PubMed1997

Flow-induced vascular remodeling in the rat carotid artery diminishes with age.

Vascular remodeling is regulated by a combination of hemodynamic, environmental, and genetic factors and may be influenced by age. To evaluate age-dependent remodeling in rats, we developed and used a quantitative highly reproducible model of carotid flow alteration. Fourteen juvenile (99+/-3 g) and 9 adult (199+/-5 g) male inbred Fischer rats underwent ligation of the left internal and external carotid arteries under anesthesia. Left common carotid blood flow immediately decreased by approximately 93%, whereas flow in the contralateral carotid increased by approximately 46%. After 4 weeks, the left carotid outer diameter (OD) significantly decreased in both juvenile and adult rats (as measured in vivo and by histological morphometry) compared with sham-operated rats. Changes in shear stress acutely mirrored the changes in blood flow. OD increased and shear stress returned to initial values after chronic exposure to increased flow in juvenile but not adult rats. To develop a simple quantitative index of remodeling that would not require killing the animals, we measured the OD in vivo and compared the ratio of right to left OD (OD ratio [ODR]) between groups. The initial ODR for all groups was approximately 1.0. After 4 weeks of altered flow, the ODR was significantly greater in juvenile than in adult rats (1.48+/-0.05 versus 1.29+/-0.04, respectively; P=.030), indicating that juvenile rats experienced more extensive remodeling than did the adult rats. We also found that unilateral carotid ligation caused a left versus right difference in endothelial NO synthase protein levels after 4 weeks that was not present in the sham-operated animals. Thus, the model described here shows that flow-induced vascular remodeling is dependent on age and supports the hypothesis that the driving force for remodeling involves shear stress and possibly NO. Because the model is quantitative, it allows dissection of the genetic factors that regulate remodeling in inbred rat strains.

Aging↗

PubMed1997

Platelet-derived growth factor ligand and receptor expression in response to altered blood flow in vivo.

Blood flow and the tractive force shear stress are important determinants of artery caliber, and reduced shear predisposes arteries to intimal thickening and atherosclerosis. The molecular basis for shear-induced changes in artery wall structure is poorly defined. A number of factors associated with normal and pathological artery wall remodeling are induced by shear stress in endothelial cell cultures. These include platelet-derived growth factor (PDGF), a potent mitogen, chemoattractant, and vasoconstrictor. To determine whether similar changes occur in vivo, we examined the effects of reduced blood flow on endothelial cell PDGF expression and proliferation in the rat carotid artery. Branches of the right internal and external carotid arteries were ligated, reducing common carotid artery blood flow from 8.0+/-0.6 to 0.5+/-0.1 mL/min while increasing flow in the left carotid from 7.1+/-0.6 to 10.8+/-0.7 mL/min. Shear stress following the procedure was 1.4+/-0.2 and 33.4+/-1.1 dyne/cm2 in carotids with reduced blood flow (RF) and increased blood flow (IF), respectively. Arteries were harvested 6, 24, 48, or 72 hours after ligation, perfusion-fixed, and opened longitudinally. Endothelial cell proliferation (bromodeoxyuridine [BrdU] labeling) was assessed en face at 24, 48, and 72 hours; expression of mRNA for PDGF-A and -B chains and PDGF alpha- and beta-receptors (in situ hybridization) was determined at 6, 48, and 72 hours after unilateral flow reduction. RF induced endothelial cell proliferation, which peaked at 48 hours (RF BrdU labeling: 24 hours, 0.4+/-0.2%; 48 hours, 7.2+/-2.0%; and 72 hours, 4.1+/-0.6%; n=5). PDGF-B expression increased in RF compared with IF endothelium within 48 hours and persisted at 72 hours (percent labeling [RF/IFx100]: 6 hours, 76+/-20%; 48 hours, 395+/-179%; and 72 hours, 208+/-44%; n=3). PDGF-A expression was similarly increased in RF endothelium. In contrast, expression of PDGF alpha- and beta-receptors was undetectable in RF and IF endothelium at all times. We conclude that endothelial cell PDGF ligand expression is induced by reduced shear stress in vivo and may play an important role in flow-mediated remodeling and atherogenesis.

Animals↗

PubMed1997

Angiotensin II stimulates tyrosine phosphorylation of phospholipase C-gamma-associated proteins. Characterization of a c-Src-dependent 97-kD protein in vascular smooth muscle cells.

Stimulation of phospholipase C-gamma (PLC-gamma) is a critical event in angiotensin II (Ang II) signal transduction. We have previously shown that in rat aortic smooth muscle (RASM) cells Ang II stimulates tyrosine phosphorylation of PLC-gamma via activation of c-Src. Because we failed to demonstrate a direct association between c-Src and PLC-gamma, we hypothesized that a linker protein mediates the interaction between these molecules. To identify PLC-gamma-associated proteins, RASM cells were labeled with [32P]orthophosphate and stimulated with 100 nmol/L Ang II for 5 minutes. PLC-gamma was immunoprecipitated, and associated proteins were characterized by autoradiography and Western blotting with anti-phosphotyrosine antibodies. Ang II stimulated the phosphorylation of 47-, 60-, 84-, and 97-kD PLC-gamma-associated proteins. Because Ang II increased tyrosine phosphorylation of only the 97-kD protein, we characterized p97 further. An important role for Src in tyrosine phosphorylation of p97 was suggested by findings that p97 phosphorylation was inhibited by the selective Src-family kinase inhibitor CP-118,556, diminished in mouse aortic smooth muscle (MASM) cells from c-Src knockout mice compared with wild-type MASM cells, and increased in v-Src-transformed NIH-3T3 cells compared with wild-type NIH-3T3 cells. These studies are the first to define a PLC-gamma-associated protein that may be required for Ang II-mediated signal transduction.

Angiotensin II↗

PubMed1997

Angiotensin II signal transduction in vascular smooth muscle cells: role of tyrosine kinases.

Originally known to be a vasoconstrictor and thought to play a critical role in hypertension, angiotensin II has recently emerged to be important in inflammation, atherosclerosis and congestive heart failure. The expanding role of angiotensin II implies that multiple signal transduction pathways are likely to be activated in a tissue-specific manner. Recent data show that angiotensin II stimulates not only cytoplasmic tyrosine kinases including c-Src, focal adhesion kinase (FAK), and Janus kinases (JAK2 and TYK2), but also may transactivate receptor tyrosine kinases such as Axl and PDGF by as yet undefined autocrine/paracrine mechanisms. Finally, tyrosine kinases, which mediate tyrosine phosphorylation of key signal mediators such as Shc, Raf, and phospholipase C-gamma following angiotensin II stimulation, remain to be defined. These tyrosine kinases, activated by angiotensin II, appear to be required for angiotensin II effects such as vasoconstriction, proto-oncogene expression, protein synthesis, and cell proliferation. Thus, it is important to understand angiotensin II-mediated signaling events, especially those related to tyrosine kinase activity, to develop new therapies for cardiovascular diseases.

Angiotensin II↗

PubMed1996

Mitogen-activated protein kinase (ERK1/2) activation by shear stress and adhesion in endothelial cells. Essential role for a herbimycin-sensitive kinase.

Fluid shear stress modulates vascular function and structure by stimulating mechanosensitive endothelial cell signal events. Cell adhesion, mediated by integrin-matrix interactions, also regulates intracellular signaling by mechanosensitive events. To gain insight into the role of integrin-matrix interactions, we compared tyrosine phosphorylation and extracellular signal-regulated kinase (ERK1/2) activation in adhesion- and shear stress-stimulated human umbilical vein endothelial cells (HUVEC). Adhesion of HUVEC to fibronectin, but not to poly-L-lysine, rapidly activated ERK1/2. Fluid shear stress (12 dyn/cm2) enhanced ERK1/2 activation stimulated by adhesion, suggesting the presence of a separate pathway. Two differences in signal transduction were identified: focal adhesion kinase phosphorylation was increased rapidly by adhesion but not by shear stress; and ERK1/2 activation in response to adhesion was inhibited to a significantly greater extent when actin filaments were disrupted by cytochalasin D. Two similarities in activation of ERK1/2 were observed: protein kinase C (PKC) activity was necessary as shown by complete inhibition when PKC was downregulated; and an herbimycin-sensitive (genistein- and tyrphostin-insensitive) tyrosine kinase was required. c-Src was identified as a candidate tyrosine kinase as it was activated by both shear stress and adhesion. These findings suggest that adhesion and shear stress activate ERK1/2 via a shared pathway that involves an herbimycin-sensitive tyrosine kinase and PKC. In addition, shear stress activates ERK1/2 through another pathway that is partially independent of cytoskeletal integrity.

Antigen-Antibody Complex↗

PubMed1996

Big mitogen-activated protein kinase 1 (BMK1) is a redox-sensitive kinase.

Mitogen-activated protein (MAP) kinases are a multigene family activated by many extracellular stimuli. There are three groups of MAP kinases based on their dual phosphorylation motifs, TEY, TPY, and TGY, which are termed extracellular signal-regulated protein kinases (ERK1/2), c-Jun N-terminal kinases, and p38, respectively. A new MAP kinase family member termed Big MAP kinase 1 (BMK1) or ERK5 was recently cloned. BMK1 has a TEY sequence similar to ERK1/2 but has unique COOH-terminal and loop-12 domains. To define BMK1 regulation, its activation in cultured rat vascular smooth muscle cells was characterized. Angiotensin II, phorbol ester, platelet-derived growth factor, and tumor necrosis factor-alpha were the strongest stimuli for ERK1/2 but were weak activators of BMK1. In contrast, H2O2 caused concentration-dependent activation of BMK1 but not ERK1/2. Sorbitol activated both BMK1 and ERK1/2. BMK1 activation by H2O2 was calcium-dependent and appeared ubiquitous as shown by stimulation in human skin fibroblasts, human vascular smooth muscle cells, and human umbilical vein endothelial cells. These findings demonstrate that activation of BMK1 is different from ERK1/2 and suggest an important role for BMK1 as a redox-sensitive kinase.

Amino Acid Sequence↗

PubMed1996

A mathematical model of cytosolic calcium dynamics in human umbilical vein endothelial cells.

Important among the responses of endothelial cells are cytosolic free calcium transients. These transients are mediated by several factors, including blood-borne agonists, extracellular calcium, and fluid-imposed shear forces. The transients are characterized by a rapid rise followed by a plateau phase. A base mathematical model is presented that reasonably reproduces the measured calcium transient in cultured human umbilical vein endothelial cells responding to thrombin. Kinetic equations for receptor activation and calcium mobilization comprise the model. A graded response of intracellular free calcium to increasing concentrations of agonist is predicted. Also predicted is the elevation of the peak value and the plateau level by steady nonspecific leak of calcium across the plasma membrane. The influences of capacitative calcium entry, calcium-induced calcium release, and buffering by cytosolic proteins are investigated parametrically. The model predicts significant depletion of cellular calcium in response to agonist stimulation.

Buffers↗

PubMed1996

Ca(2+)-dependent mitogen-activated protein kinase activation in spontaneously hypertensive rat vascular smooth muscle defines a hypertensive signal transduction phenotype.

The mechanisms responsible for altered vascular smooth muscle cell (VSMC) function in hypertension remain unknown. In the spontaneously hypertensive rat (SHR) model of genetic hypertension, there are multiple abnormalities in VSMC function, including increased growth, Na(+)-H+ exchange, and increased signal transduction by protein kinase C. The family of kinases termed mitogen-activated protein (MAP) kinases has recently been shown to be essential mediators of growth factor signal transduction. In the present study, alterations in MAP kinase function in the hypertensive phenotype were investigated using early-passage SHR and Wistar-Kyoto (WKY) VSMCs stimulated with angiotensin II (Ang II, 100 nmol/L) or platelet-derived growth factor-BB (PDGF-BB, 10 ng/mL). MAP kinase activity was measured by in-gel kinase assays and Western blot analysis. Two differences between SHR and WKY rats were observed for Ang II-mediated MAP kinase activation: (1) Inactivation after Ang II stimulation was more rapid in SHR than WKY VSMCs. (2) Activity in SHR VSMCs showed a greater dependence on Ca2+ mobilization, since chelation of intracellular Ca2+ with BAPTA inhibited maximal activity by 95% in SHR VSMCs but by only 50% in WKY VSMCs. In contrast to the results with Ang II, no differences in PDGF-stimulated MAP kinase activity were observed. These findings establish activation of MAP kinase by Ang II as a feature that distinguishes SHR VSMCs from WKY VSMCs and suggest that differences in regulation of MAP kinase signaling may alter cellular events that are increased in the SHR genetic model of hypertension.

Angiotensin II↗