Role of PP2A in the regulation of p38 MAPK activation in bovine aortic endothelial cells exposed to cyclic strain

We have previously reported that cyclic strain results in rapid phosphorylation of p38 mitogen activated protein kinase (MAPKs). The aim of this study was to examine the role of protein phosphatase type 2A (PP2A) in regulating p38 MAPK activation in bovine aortic endothelial cells exposed to cyclic strain. In this study, we demonstrate that the catalytic subunit of PP2A is tyrosine phosphorylated by cyclic strain, resulting in inhibition of phosphatase activity. Okadaic acid, an inhibitor of PP2A at lower concentrations increased phosphorylation of p-38. Phospho-p38 MAPK physically associated with the catalytic subunit, PP2Ac. Phospho-p38 MAPK was dephosphorylated by purified PP2Ac in cell lysates, but if pretreated with okadaic acid, phospho-p38 MAPK was maintained. Taken together, our result suggests that PP2A plays a regulatory role in p38 MAPK activation in endothelial cells exposed to cyclic strain.     

Strain activation of bovine aortic smooth muscle cell proliferation and alignment: Study of strain dependency and the role of protein kinase A and C signaling pathways

Smooth muscle cell (SMC) phenotype can be altered by physical forces as demonstrated by cyclic strain-induced changes in proliferation, orientation, and secretion of macromolecules. However, the magnitude of strain required and the intracellular coupling pathways remain ill defined. To examine the strain requirements for SMC proliferation, we selectively seeded bovine aortic SMC either on the center or periphery of silastic membranes which were deformed with 150 mm Hg vacuum (0–7% center; 7–24% periphery). SMC located in either the center or peripheral regions showed enhanced proliferation compared to cells grown under the absence of cyclic strain. Moreover, SMC located in the center region demonstrated significantly (P < 0.005) greater proliferation as compared to those in the periphery. In contrast, SMC exposed to high strain (7–24%) demonstrated alignment perpendicular to the strain gradient, whereas SMC in the center (0–7%) remained aligned randomly. To determine the mechanisms of these phenomena, we examined the effect of cyclic strain on bovine aortic SMC signaling pathways. We observed strain-induced stimulation of the cyclic AMP pathway including adenylate cyclase activity and cyclic AMP accumulation. In addition, exposure of SMC to cyclic strain caused a significant increase in protein kinase C (PKC) activity and enzyme translocation from the cytosol to a particulate fraction. Further study was conducted to examine the effect of strain magnitude on signaling, particularly protein kinase A (PKA) activity as well as cAMP response element (CRE) binding protein levels. We observed significantly (P < 0.05) greater PKA activity and CRE binding protein levels in SMC located in the center as compared to the peripheral region. However, inhibition of PKA (with 10 μM Rp-cAMP) or PKC (with 5–20 ng/ml staurosporine) failed to alter either the strain-induced increase in SMC proliferation or alignment. These data characterize the strain determinants for activation of SMC proliferation and alignment. Although strain activated both the AC/cAMP/PKA and the PKC pathways in SMC, singular inhibition of PKA and PKC failed to prevent strain-induced alignment and proliferation, suggesting either their lack of involvement or the multifactorial nature of these responses. J. Cell. Physiol. 170:228–234, 1997. © 1997 Wiley-Liss, Inc.     

Protein kinase C activators suppress stimulation of capillary endothelial cell growth by angiogenic endothelial mitogens

The intracellular events regulating endothelial cell proliferation and organization into formalized capillaries are not known. We report that the protein kinase C activator beta-phorbol 12,13-dibutyrate (PDBu) suppresses bovine capillary endothelial (BCE) cell proliferation (K50 = 6 +/- 4 nM) and DNA synthesis in response to human hepatoma-derived growth factor, an angiogenic endothelial mitogen. In contrast, PDBu has no effect on the proliferation of bovine aortic endothelial cells and is mitogenic for bovine aortic smooth muscle and BALB/c 3T3 cells. Several observations indicate that the inhibition of human hepatoma-derived growth factor-stimulated BCE cell growth by PDBu is mediated through protein kinase C. Different phorbol compounds inhibit BCE cell growth according to their potencies as protein kinase C activators (12-O-tetradecanoylphorbol 13-acetate greater than PDBu much greater than beta-phorbol 12,13-diacetate much much greater than beta-phorbol; alpha-phorbol 12,13-dibutyrate; alpha-phorbol 12,13-didecanoate). PDBu binds to a single class of specific, saturable sites on the BCE cell with an apparent Kd of 8 nM, in agreement with reported affinities of PDBu for protein kinase C in other systems. Specific binding of PDBu to BCE cells is displaced by sn-1,2-dioctanoylglycerol, a protein kinase C activator and an analog of the putative second messenger activating this kinase in vivo. The weak protein kinase C activator, sn-1,2-dibutyrylglycerol, does not affect PDBu binding. A cytosolic extract from BCE cells contains a calcium/phosphatidylserine-dependent protein kinase that is activated by sn-1,2-dioctanoylglycerol and PDBu, but not by beta-phorbol. These findings indicate that protein kinase C activation can cause capillary endothelial cells to become desensitized to angiogenic endothelial mitogens. This intracellular regulatory mechanism might be invoked during certain phases of angiogenesis, for example when proliferating endothelial cells become differentiated to organize into nongrowing tubes.     

Identification of protein phosphatase 2A as the major tyrosine hydroxylase phosphatase in adrenal medulla and corpus striatum: evidence from the effects of okadaic acid

(i) The major sites on bovine adrenal tyrosine hydroxylase (TH) phosphorylated by calmodulin-dependent multiprotein kinase (CaM-MPK) and cyclic AMP-dependent protein kinase were shown to be Ser-19 and Ser-40, respectively, while Ser-40 was also phosphorylated slowly by CaM-MPK. (ii) Type 2A and type 2C phosphatases accounted for approximately 90% and approximately 10% of TH phosphatase activity, respectively, in extracts of adrenal medulla and corpus striatum assayed at near physiological free Mg2+ (1 mM), while type 1 and type 2B phosphatases had negligible activity towards TH. (iii) Incubation of adrenal chromaffin cells with okadaic acid increased TH phosphorylation by 206% and activity by 77%, establishing that type 2A phosphatases play a major role in regulating TH in vivo.     

Changes associated with tyrosine phosphorylation during short-term hypoxia in retinal microvascular endothelial cells in vitro

The occlusion of capillary vessels results in low oxygen tension in adjacent tissues which triggers a signaling cascade that culminates in neovascularization. Using bovine retinal capillary endothelial cells (BRCEC), we investigated the effects of short-term hypoxia on DNA synthesis, phosphotyrosine induction, changes in the expression of basic fibroblast growth factor receptor (bFGFR), protein kinase C (PKCα), heat shock protein 70 (HSP70), and SH2-containing protein (SHC). The effect of protein tyrosine kinase (PTK) and phosphatase inhibitors on hypoxia-induced phosphotyrosine was also studied. Capillary endothelial cells cultured in standard normoxic (pO₂ = 20%) conditions were quiesced in low serum containing medium and then exposed to low oxygen tension or hypoxia (pO₂ = 3%) in humidified, 5% CO₂, 37°C, tissue culture chambers, on a time-course of up to 24 h. DNA synthesis was potentiated by hypoxia in a time-dependent manner. This response positively correlated with the cumulative induction of phosphotyrosine and the downregulation of bFGFR (M_r ～ 85 kDa). Protein tyrosine kinase inhibitors, herbimycin-A, and methyl 2,5-dihydroxycinnamate, unlike genistein, markedly blocked hypoxia-induced phosphotyrosine. Prolonged exposure of cells to phosphatase inhibitor, sodium orthovanadate, also blocked hypoxia-induced phosphotyrosine. The expression of HSP70, PKCα, and SHC were not markedly altered by hypoxia. Taken together, these data suggest that short-term hypoxia activates endothelial cell proliferation in part via tyrosine phosphorylation of cellular proteins and changes in the expression of the FGF receptor. Thus, endothelial cell mitogenesis and neovascularization associated with low oxygen tension may be controlled by abrogating signaling pathways mediated by protein tyrosine kinase and phosphatases. © 1995 Wiley-Liss, Inc.     

Induction of KDR expression in bovine arterial endothelial cells by thrombin: involvement of nitric oxide.

Thrombin, a multifunctional serine protease, is generated at the site with vascular injuries. It not only participates in the coagulation cascade, but also can induce a lot of events related to cell mitogenesis and migration. In this study, we investigated the effect of thrombin on endothelial cell proliferation induced by vascular endothelial growth factor (VEGF). Thrombin promoted proliferation of cultured bovine carotid endothelial cells in a time- and dose-dependent manner. Moreover, it drastically enhanced the cell growth stimulated by VEGF. This stimulatory effect was reduced by inhibitors of either protein kinase C (PKC) or mitogen-activated protein kinase kinase (MAPKK). Thrombin induced a significant increase in the level of mRNA of the kinase domain-containing receptor (KDR), but not tms-like tyrosine kinase (Flt-1), in a time-dependent manner, which reached the maximum after 24 h of stimulation. This increase coincides well with the KDR protein expression. The luciferase assay showed that thrombin induced an about 7.5-fold increase in the KDR promoter activity compared with the control. This enhanced KDR promoter activity was also abolished by inhibitors of either PKC or MAPKK. The deletion analyses indicated that the region between -115 and -97 (containing Sp1 binding region) within the KDR promoter gene was required for the enhanced KDR expression induced by thrombin and VEGF. Moreover, the nitric oxide synthase (NOS) inhibitor abolished both the accelerated cell proliferation and the increased KDR expression induced by thrombin and VEGF. This inhibition was abrogated by DETA NONOate, a NO donor with long half-life. These findings suggest that thrombin might potentiate the VEGF-induced angiogenic activity through increasing the level of the VEGF receptor KDR, in which production of NO is involved.     

Involvement of protein kinase C in the mitogenic and chemotaxis effects of basic fibroblast growth factor on bovine cerebral cortex capillary endothelial cells

Basic fibroblast growth factor is increasingly implicated in cellular growth, differentiation, angiogenesis and oncogenesis. In culture, basic fibroblast growth factor greatly improved the growth rate of bovine brain cortex capillary endothelial cells. Down-regulation of protein kinase C by prolonged treatment with phorbol esters prevented the mitogenic effect of basic fibroblast growth factor on capillary endothelial cells. Furthermore, staurosporine, a potent protein kinase inhibitor, showed strong antiproliferative activity against basic fibroblast growth factor-induced endothelial cell growth. Similarly, the chemotaxis effect of basic fibroblast growth factor on capillary endothelial cells was abolished by down-regulation of protein kinase C or by staurosporine treatment. Therefore, it is suggested that protein kinase C could account for part of the angiogenic effect of basic fibroblast growth factor.     

Mechanical stimulation and mitogen-activated protein kinase signaling independently regulate osteogenic differentiation and mineralization by calcifying vascular cells

Ectopic calcification of vascular tissue is associated with several cardiovascular pathologies and likely involves active regulation by vascular smooth muscle cells and osteoblast-like vascular cells. This process often occurs in sites with altered mechanical environments, suggesting a role for mechanical stimuli in calcification. In this study, we investigated the effect of mechanical stimulation on the proliferation, osteogenic differentiation, calcification, and mitogen-activated protein kinase (MAPK) signaling in calcifying vascular cells (CVCs), a subpopulation of aortic smooth muscle cells putatively involved in vascular calcification. Application of equibiaxial cyclic strain (7%, 0.25 Hz) to CVCs had no effect on cell proliferation, but accelerated alkaline phosphatase expression and significantly increased mineralization by 3.1-fold over unstrained cells. Fluid motion in the absence of strain also enhanced mineralization, but to a lesser degree. Because MAPK pathways mediate mechanically regulated osteoblast differentiation, we tested whether similar signaling was involved in mineralization by CVCs. In static cultures, pharmacological inhibition of the extracellular signal-regulated kinase (ERK1/2), p38 MAPK, and c-Jun N-terminal kinase pathways significantly attenuated mineral production by as much as -94%, compared with uninhibited CVCs. Strikingly, although mechanical stimulation activated each of the MAPK pathways, inhibition of these pathways had no effect on the mechanically induced enhancement of alkaline phosphatase activity or mineralization. These novel data indicate that mechanical signals regulate calcification by CVCs, and although MAPK signaling is critical to CVC osteogenic differentiation and mineralization, it is not involved directly in transduction of mechanical signals to regulate these processes under the conditions utilized in this study.     

Dephosphorylation of Microtubule Proteins by Brain Protein Phosphatases 1 and 2A, and Its Effect on Microtubule Assembly

Protein phosphatase C was purified 140-fold from bovine brain with 8% yield using histone H1 phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase (cyclic AMP-kinase). Brain protein phosphatase C was considered to consist of 10 and 90%, respectively, of the catalytic subunits of protein phosphatases 1 and 2A on the basis of the effects of ATP and inhibitor-2. Protein phosphatase C dephosphorylated microtubule-associated protein 2 (MAP2), tau factor, and tubulin phosphorylated by a multifunctional Ca2+/calmodulin-dependent protein kinase (calmodulin-kinase) and the catalytic subunit of cyclic AMP-kinase. The properties of dephosphorylation of MAP2, tau factor, and tubulin were compared. The Km values were in the ranges of 1.6-2.7 microM for MAP2 and tau factor. The Km value for tubulin decreased from 25 to 10-12.5 microM in the presence of 1.0 mM Mn2+. No difference in kinetic properties of dephosphorylation was observed between the substrates phosphorylated by the two kinases. Protein phosphatase C did not dephosphorylate the native tubulin, but universally dephosphorylated tubulin phosphorylated by the two kinases. The holoenzyme of protein phosphatase 2A from porcine brain could also dephosphorylate MAP2, tau factor, and tubulin phosphorylated by the two kinases. The phosphorylation of MAP2 and tau factor by calmodulin-kinase separately induced the inhibition of microtubule assembly, and the dephosphorylation by protein phosphatase C removed its inhibitory effect. These data suggest that brain protein phosphatases 1 and 2A are involved in the switch-off mechanism of both Ca2+/calmodulin-dependent and cyclic AMP-dependent regulation of microtubule formation.     

cAMP-dependent protein kinase inhibits the mitogenic action of vascular endothelial growth factor and fibroblast growth factor in capillary endothelial cells by blocking Raf activation

Proliferation of endothelial cells is regulated by angiogenic and antiangiogenic factors whose actions are mediated by complex interactions of multiple signaling pathways. Both vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) stimulate cell proliferation and activate the mitogen-activated protein kinase (MAPK) cascade in bovine brain capillary endothelial (BBE) cells. We have extended these findings to show that both mitogens activate MAPK via stimulation of Raf-1. Activation of Raf/MAPK is inhibited by increasing intracellular cAMP levels pharmacologically or via stimulation of endogenously expressed β-adrenergic receptors. Both VEGF- and bFGF-induced Raf-1 activity are blocked in the presence of forskolin or 8-bromo-cAMP by 80%. The actions of increased cAMP appear to be mediated by cAMP-dependent protein kinase (PKA), since treatment with H-89, a the specific inhibitor of PKA, reversed the inhibitory effect of elevated cAMP levels on mitogen-induced cell proliferation and Raf/MAPK activation. Moreover, elevations in cAMP/PKA activity inhibit mitogen-induced cell proliferation. These findings demonstrate, in cultured endothelial cells, that the cAMP/PKA signaling pathway is potentially an important physiological inhibitor of mitogen activation of the MAPK cascade and cell proliferation. J. Cell. Biochem. 67:353–366, 1997. © 1997 Wiley-Liss, Inc.     

Protein phosphorylation in cultured endothelial cells

We have investigated the protein phosphorylation systems present in cultured bovine aortic and pulmonary artery endothelial cells. The cells contain cyclic AMP-dependent protein kinase, three calcium/calmodulin-dependent protein kinases, protein kinase C, and at least one tyrosine kinase. No cyclic GMP-dependent protein kinase activity was found. The cells also contained numerous substrates for cyclic AMP-dependent protein kinase and protein kinase C. Fewer substrates were found for the calcium/calmodulin-dependent protein kinases. There was little difference between either protein kinase activities or substrates when pulmonary artery endothelium was compared to aortic endothelium grown under similar culture conditions. It is likely that these various protein kinases and their respective substrate proteins are involved in mediating several of the actions of the hormones and drugs which affect the vascular endothelium.     

Possible role of protein phosphorylation in the mitogenic effect of high density lipoproteins on cultured vascular endothelial cells

The implication of protein phosphorylation in the mitogenic action of high density lipoproteins (HDL) on bovine vascular endothelial cells was investigated by incubating endothelial cell cultures in the presence of 32P-labeled phosphoric acid. The incorporation of 32P into proteins was measured after fractionation by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and autoradiography of the gel. In endothelial cells seeded at low density and made quiescent by serum starvation, HDL markedly and consistently enhanced the degree of phosphorylation of a Mr 27,000 protein in a time- and dose-dependent manner. Using 500 micrograms/ml HDL, 32P labeling of the 27-kDa protein was already measurable after 10 min of incubation and reached a maximum at 20-30 min. Minimal effective dose of HDL during a 30-min incubation period was in the range of 5-10 micrograms/ml. While the apolipoprotein moiety of HDL was able to mimic the effect of total HDL, the lipid part of HDL was not. Furthermore, fibroblast growth factor appeared to potentiate the effect of HDL on 27-kDa protein phosphorylation, in agreement with the synergism observed between fibroblast growth factor and HDL on endothelial cell proliferation. Two activators of protein kinase C, 12-O-tetradecanoylphorbol 13-acetate and 1-oleoyl-2-acetylglycerol also induced the phosphorylation of the 27-kDa protein. These results suggest that the 27-kDa protein may be a physiological substrate for protein kinase C and that HDL could exert their mitogenic effect on endothelial cells through activation of protein kinase C and subsequent protein phosphorylation.     

Translocation of PKC isoforms in bovine aortic smooth muscle cells exposed to strain

Our laboratory has previously reported that the exposure of smooth muscle cells (SMC) to the cyclic strain results in significant stimulation of protein kinase C (PKC) activity by translocating the enzyme from the cytosol to the particulate fraction. We now sought to examine the strain-induced translocation of individual PKC isoforms in SMC. Confluent bovine aortic SMC grown on collagen type I-coated plates were exposed to cyclic strain for up to 100 s at average 10% strain with 60 cycles/min. Immunoblotting analysis demonstrates that SMC express PKC-alpha, -beta and -zeta in both cytosolic and particulate fractions. Especially, PKC-alpha and -zeta were predominantly expressed in the cytosolic fraction. However, cyclic strain significantly (P < 0.05) increased PKC-alpha and -zeta in the particulate fraction and decreased in the cytosolic fraction. Thus, the cyclic strain-mediated stimulation of PKC activity in SMC may be due to the translocation of PKC-alpha and -zeta from the cytosolic to the particulate fraction. These results demonstrate that mechanical deformation causes rapid translocation of PKC isoforms, which may initiate a cascade of proliferation responses of SMC since NF-kappaB, which is involved in the cellular proliferation has been known to be activated by these PKC isoforms.     

Defining the role of protein kinase c in calcium-ionophore-(A23187)-mediated activation of phospholipase A2 in pulmonary endothelium.

We sought to investigate the mechanisms by which the calcium ionophore A23187 triggers arachidonic acid release in bovine pulmonary endothelial cells and to test the hypothesis that protein kinase C is involved in this process. Our results indicate that the mechanism by which A23187 increases phospholipase A2 activity and arachidonic acid release in bovine pulmonary arterial endothelial cells depends upon the concentration studied. At concentrations of 1 microM and 2.5 microM, A23187 increases phospholipase A2 activity and arachidonic acid release without stimulating protein kinase C. At concentrations of 5-12.5 microM, A23187 increases arachidonic acid release and phospholipase A2 activity in conjunction with a dose-dependent activation of membrane-bound protein kinase C. To test the hypothesis that these doses of A23187 increase phospholipase A2 activity by stimulating protein kinase C, we studied the effect of prior treatment with the protein kinase C inhibitor sphingosine. Sphingosine inhibits the increase in phospholipase A2 activity and arachidonic acid release caused by A23187 over the range 5-12.5 microM. To investigate further the potential role of protein kinase C, we studied the effects of the inactive phorbol ester 4 alpha-phorbol 12 beta-myristate 13 alpha-acetate (4 alpha-PMA) and an active phorbol ester 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (4 beta PMA). Neither 4 alpha-PMA nor 4 beta-PMA affected basal arachidonic acid release. 4 alpha-PMA also did not augment the effects of A23187. In contrast, 4 beta-PMA significantly augments the increase in phospholipase A2 activity and arachidonic acid release caused by lower doses of A23187. Under these conditions, sphingosine completely inhibits the stimulatory effects of 4 beta-PMA on protein kinase C translocation, phospholipase A2 and arachidonic acid release. Thus, at low doses (1 microM and 2.5 microM) A23187 increases phospholipase A2 activity and arachidonic acid release by a mechanism that does not involve protein kinase C. At these A23187 doses, activating membrane-bound protein kinase C with 4 beta-PMA causes a synergistic increase in phospholipase A2 activity and arachidonic acid release. At higher doses (5-12.5 microM), A23187 acts in large part by stimulating protein kinase C translocation. Overall, our results indicate that activating membrane-bound protein kinase C by itself is an insufficient stimulus to increase phospholipase A2 activity and arachidonic acid release in pulmonary endothelial cells, but activating protein kinase C can substantially augment the increase in phospholipase A2 activity and arachidonic acid caused by a small increase in intracellular calcium.     

Protein phosphatase 3 differentially modulates vascular endothelial growth factor- and fibroblast growth factor 2-stimulated cell proliferation and signaling in ovine fetoplacental artery endothelial cells

A critical process for vascular endothelial growth factor (VEGF)- and fibroblast growth factor 2 (FGF2)-regulated cellular function is reversible protein phosphorylation, which is tightly controlled by a balance of protein kinases and phosphatases. We have reported that in ovine fetoplacental artery endothelial (OFPAE) cells, VEGF and FGF2 stimulate cell proliferation in part via activation of mitogen-activated protein kinase kinase 1/2 (MAP2K1/2)/mitogen-activated protein kinase 3/1 (MAPK3/1) and phosphoinositide 3-kinase (PI3K)/v-akt murine thymoma viral oncogene homolog 1 (AKT1) pathways. In the present study, we examined if protein phosphatase 3 (PPP3) mediated VEGF- and FGF2-stimulated OFPAE cell proliferation via modulating activation of MAPK3/1 and AKT1. Small interfering RNA (siRNA) targeting human PPP3 catalytic subunit alpha (PPP3CA) was used to suppress PPP3CA protein expression in OFPAE cells. Compared with the scrambled siRNA, PPP3CA siRNA decreased PPP3CA protein levels by approximately 97% without altering protein levels of protein phosphatase 2 catalytic subunit alpha, total MAPK3/1, total AKT1, or glyceraldehyde-3-phosphate dehydrogenase. Knockdown of PPP3CA protein expression enhanced VEGF-stimulated, but not FGF2-stimulated, cell proliferation. Knockdown of PPP3CA protein expression did not significantly affect VEGF-induced MAPK3/1 and AKT1 phosphorylation but attenuated FGF2-induced MAPK3/1 and AKT1 phosphorylation. Thus, to our knowledge, the present study is the first to demonstrate successful knockdown of PPP3CA protein expression in any cell model using a single pair of double-strained siRNA. Moreover, specific knockdown of PPP3CA protein expression enhances VEGF-stimulated, but not FGF2-stimulated, OFPAE cell proliferation and attenuates FGF2-induced, but not VEGF-induced, MAPK3/1 and AKT1 activation. Thus, PPP3CA differentially modulates the VEGF- and FGF2-stimulated cell proliferation and signaling cascades in OFPAE cells. These data also suggest that signaling molecules other than MAPK3/1 and AKT1 play an important role in VEGF- and FGF2-stimulated cell proliferation after knockdown of PPP3CA in OFPAE cells.     

Tumor necrosis factor employs a protein-tyrosine phosphatase to inhibit activation of KDR and vascular endothelial cell growth factor-induced endothelial cell proliferation

Vascular endothelial cell growth factor (VEGF) binds to and promotes the activation of one of its receptors, KDR. Once activated, KDR induces the tyrosine phosphorylation of cytoplasmic signaling proteins that are important to endothelial cell proliferation. In human umbilical vein endothelial cells (HUVECs), tumor necrosis factor (TNF) inhibits the phosphorylation and activation of KDR. The ability of TNF to diminish VEGF-stimulated KDR activity was impaired by sodium orthovanadate, suggesting that the inhibitory activity of TNF was mediated by a protein-tyrosine phosphatase. KDR-initiated responses specifically associated with endothelial cell proliferation, mitogen-activated protein kinase activation and DNA synthesis, were also inhibited by TNF, and this was reversed by sodium orthovanadate. Stimulation of HUVECs with TNF induced association of the SHP-1 protein-tyrosine phosphatase with KDR, identifying this phosphatase as a candidate negative regulator of VEGF signal transduction. Heterologous receptor inactivation mediated by a protein-tyrosine phosphatase provides insight into how TNF may inhibit endothelial cell proliferative responses and modulate angiogenesis in pathological settings.     

Cyclic strain stimulates isoform-specific PKC activation and translocation in cultured human keratinocytes

Previous studies have demonstrated that cyclic strain induces keratinocyte proliferative and morphological changes. Since protein kinase C (PKC) is known to play an important role in the regulation of keratinocyte growth and differentiation, the objective of this study was to determine the role of the PKC signaling pathway as a mediator of strain modulation of the keratinocyte phenotype. In particular, we tested the following specific hypotheses: (1) cyclic strain stimulates PKC activity and translocation, (2) cyclic strain activates PKC in an isoform-specific manner, and (3) PKC mediates the strain activated proliferative and morphological response in cultured human keratinocytes. To test these hypotheses, keratinocytes were subjected to vacuum-generated cyclic strain (10% average strain), followed by measurement of PKC activity, PKC isoform distribution by Western blot analysis and confocal microscopy, and examination of the effect of PKC inhibitors (calphostin C and staurosporine) on strain induced proliferative and morphological changes. We observed stimulation of PKC activity (62.3 ± 5.1% increase) coupled with translocation of PKC from the cytosolic to the membrane fraction in keratinocytes subjected to acute cyclic strain. Cyclic strain also caused translocation of PKC α and δ, but not ζ isoforms, from the cytosolic to the membrane fraction as demonstrated by both Western blot analysis and confocal microscopy. PKC β was not detected in these cells. PKC inhibitors, calphostin C (10 nM), and staurosporine (5 nM), inhibited strain-induced PKC activation and keratinocyte proliferation, but did not block the effects of strain on cellular morphology or alignment. We conclude that these data support our hypothesis that cyclic strain stimulates PKC activity and translocation in an isoform-specific manner in cultured human keratinocytes. Moreover, our studies with PKC inhibitors support the hypothesis that strain-induced changes in the keratinocyte phenotype may be selectively modulated by PKC. J. Cell. Biochem. 67:327–337, 1997. © 1997 Wiley-Liss, Inc.