Estrogen inhibits mechanical strain-induced mitogenesis in human vascular smooth muscle cells via down-regulation of Sp-1.

OBJECTIVE: The cellular basis of the cardioprotective effects of estrogen are largely unknown. An inhibitory effect on vascular smooth muscle (VSM) growth has been proposed. We examined the effect of 17beta-estradiol (E2) on mechanical strain-induced mitogenesis in human fetal VSM cells. METHODS AND RESULTS: Cells were grown on fibronectin-coated plates with silicone-elastomer bottoms, and exposed to cyclic mechanical strain (60 cycles/min), with and without E2 (1 nmol/l), for 48 h. [3H]-Thymidine incorporation was measured during the last 6 h. Strain induced 1.5-2 fold increases in DNA synthesis that were attenuated by antibodies to platelet-derived growth factor (PDGF) AA and BB. Strain also induced increases both in mRNA and protein levels of Sp-1, a transcription factor that binds to the PDGF-A gene promoter site. E2 attenuated strain-induced mitogenesis, and also increases in mRNA and protein levels of Sp-1. The estrogen receptor (ER) antagonist ICI 182,780 (100 nmol/l) reversed the inhibitory effect of E2 on strain-induced increases in DNA synthesis and Sp-1 protein. RT-PCR analysis showed presence of both ER-alpha and -beta in these cells. CONCLUSIONS: Estrogen inhibits strain-induced mitogenesis in human VSM cells via an ER mediated process involving down-regulation of the transcription factor Sp-1.

Tesio catheter: radiologically guided placement, mechanical performance, and adequacy of delivered dialysis.

PURPOSE: Tunneled catheters are an alternative means of vascular access for patients in need of hemodialysis who cannot undergo dialysis through a surgical shunt. This study was undertaken to evaluate the performance of the Tesio dialysis catheter. MATERIALS AND METHODS: A prospective study of the Tesio catheter was performed. Follow-up data regarding catheter function and adequacy of dialysis were obtained from nine hemodialysis facilities. RESULTS: Seventy-nine Tesio catheters were placed in 71 patients. Immediate technical success was 99% (78 of 79 catheters). The procedure complication rate was 9% (seven catheters). Only two complications required intervention: one fatal air embolism and one chest wall hematoma. Sixty-seven catheters in 60 patients were followed up for a total of 4,367 catheter days. Overall, catheter-related infection occurred in 9% (six of 67 catheters). Primary catheter patency was 87% at 1 week, 82% at 1 month, 72% at 3 months, and 66% at 6 months. Mean blood flow was 286 mL/min immediately after insertion, 301 mL/min at 3 months, and 306 mL/min at 6 months. Adequate dialysis dose as reflected by a urea reduction ratio of 60 or more or a urea kinetic modeling, or Kt/V, value of 1.2 or more was observed on at least one occasion for 74% and 76% of catheters, respectively. CONCLUSION: The Tesio catheter is a reasonable means of vascular access for patients who undergo dialysis but are not candidates for surgical shunt placement.

Matrix-dependent gene expression of egr-1 and PDGF A regulate angiotensin II-induced proliferation in human vascular smooth muscle cells.

We have previously shown, in a neonatal rat cell line, that angiotensin II (Ang II)-induced proliferation in vascular smooth muscle cells is extracellular matrix (ECM) dependent. We hypothesized that such an effect might be mediated via differences in Ang II-induced increases in the transcriptional factor early growth response-1 (Egr-1) gene and, consequently, in platelet-derived growth factor (PDGF). Cultured human newborn aortic smooth muscle cells were studied on 4 different surfaces: (1) plastic, (2) laminin, (3) collagen, and (4) fibronectin. Ang II-induced increases in DNA synthesis were significantly greater on collagen (2.0+/-0.3-fold) and fibronectin (1.9+/-0.3-fold) than on laminin (1.0+/-0.2-fold) or plastic (1.4+/-0.2-fold). As with DNA synthesis, at 48 and 72 hours, Ang II-induced increases in cell numbers occurred only in cells grown on collagen and fibronectin culture plates and were blocked by an antagonist to the angiotensin type 1 (losartan, 10 micromol/L) but not the angiotensin type 2 (PD 123319, 10 micromol/L) receptor. Anti-PDGF AA antibody (6 microg/mL) blocked the increase in DNA synthesis by 60% to 64% in cells on collagen or fibronectin cultures but not on plastic cultures. When PDGF-AA (10 ng/mL) and Ang II were added together, DNA synthesis increased 2-fold and did not differ on the various ECM proteins. Increases in PDGF A-chain mRNA were observed only in cells grown on collagen (3.21+/-0.65-fold) and fibronectin (2.86+/-0.49-fold) plates 2 to 8 hours after the addition of Ang II and were blocked by losartan but not PD 123319. Expression of Egr-1, an early growth response gene, increased at 15 minutes, peaked at 30 minutes, and returned to normal after 2 hours with Ang II treatment. Ang II-induced increases in Egr-1 mRNA were greater on collagen (4. 82+/-0.66-fold at maximum) and fibronectin (4.01+/-0.56-fold) than on laminin (2.74+/-0.45-fold) or plastic (2.53+/-0.40-fold) and were blocked by losartan but not PD 123319. Thus, in human vascular smooth muscle cells in culture, Ang II-induced proliferation is mediated via the angiotensin type 1 receptor, dependent on ECM proteins, and regulated by differential gene expression of Egr-1 and PDGF-1.

Mechanical strain increases PDGF-B and PDGF beta receptor expression in vascular smooth muscle cells.

Cyclic mechanical strain causes proliferation of vascular smooth muscle cells, mediated in part by platelet-derived growth factor (PDGF). We examined the effect of cyclic strain on expression of PDGF-B and the PDGF beta receptor. Neonatal rat vascular smooth muscle cells were exposed to 1 hertz cyclic strain on silicone elastomer plates. PDGF-B mRNA increased after 6 h of strain. In cells transfected with a PDGF-B promoter chloramphenicol acetyl transferase construct (psisCAT 6A), activity increased by 12-fold following 12 h of strain. Two neutralizing antibodies to the PDGF beta receptor both reduced strain-induced [(3)H]thymidine incorporation by 50%. Expression of the PDGF beta receptor protein increased 1.8-fold following 24 h of strain. During strain, PDGF beta receptor expression was not significantly altered by neutralizing antibodies to PDGF-B. Thus, both PDGF-B and the PDGF beta receptor are induced by cyclic mechanical strain and both contribute to cell proliferation in response to strain.

Rapid induction and translocation of Egr-1 in response to mechanical strain in vascular smooth muscle cells.

The effect of mechanical strain on transcription and expression of the immediate-early genes, early growth response gene-1 (Egr-1), c-jun, and c-fos, was investigated in neonatal rat aortic vascular smooth muscle (VSM) cells. Cells grown on silicone elastomer plates were subjected to cyclic mechanical strain (1 Hz) at various durations and magnitudes. Egr-1 mRNA increased rapidly in response to cyclic strain, reached a maximum of 10-fold after 30 minutes, and returned to baseline after 4 hours. c-jun exhibited a similar pattern, whereas c-fos mRNA expression was unaffected by strain. Cycloheximide prolonged the increase in Egr-1 and c-jun mRNA and caused superinduction of both. The threshold level of continuous cyclic strain needed to induce expression was 5% for Egr-1 and c-jun. Even a single cycle of mechanical strain that lasted 1 second was sufficient to induce Egr-1 and c-jun mRNA. Strain also increased expression of a transiently transfected Egr-1 promoter-reporter construct. The effect of varying extracellular matrices on strain-induced Egr-1 and c-jun mRNA was examined. In contrast to collagen type 1- and pronectin-coated plates, strain did not significantly alter expression of Egr-1 and c-jun was less induced on laminin-coated plates. On collagen type 1, strain increased Egr-1 protein levels by 2.1-fold at 60 minutes. Immunofluorescence microscopy revealed translocation of Egr-1 to the nucleus in response to strain. These observations indicate that Egr-1 expression and translocation are sensitive to mechanical perturbation of the cell. c-jun is also induced by strain, but c-fos is not. The signal for this induction may involve specific cell-matrix interactions.

IP3 receptor blockade fails to prevent intracellular Ca2+ release by ET-1 and alpha-thrombin.

The effect of inositol 1,4,5-trisphosphate (IP3) receptor blockade on platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), endothelin-1 (ET-1), or alpha-thrombin receptor-mediated intracellular Ca2+ (Ca2+i) release was examined using fura 2 microspectrofluorometry in single Chinese hamster ovary cells and myoblasts. Blockade of the IP3 receptor was achieved by microinjection of heparin or monoclonal antibody (MAb) 18A10 into the IP3 type 1 receptor. Heparin completely inhibited Ca2+i release after flash photolysis with caged IP3 and after exposure to PDGF and FGF. In contrast, heparin failed to block Ca2+i release after alpha-thrombin and ET-1. After application of ligand, IP3 levels were five- to sevenfold higher for alpha-thrombin than for ET-1 or PDGF. IP3 levels after PDGF and ET-1 were comparable. Similar to heparin, MAb 18A10 blocked Ca2+i release after PDGF but failed to block Ca2+i release after ET-1 or alpha-thrombin. These data suggest that the mechanisms of Ca2+i release by tyrosine kinase and certain 7-transmembrane receptors may differ. Although both receptor types use the IP3-signaling system, the ET-1 and alpha-thrombin receptors may have a second, alternative mechanism for activating CA2+i release.

Strain-responsive regions in the platelet-derived growth factor-A gene promoter.

Proliferation of cultured neonatal vascular smooth muscle (VSM) cells is enhanced by exposure to cyclic mechanical strain, in part through autocrine action of secreted platelet-derived growth factor (PDGF). We examined transcription factors and DNA response elements that may participate in the induction of PDGF-A gene transcription by mechanical strain. PDGF-A mRNA increased gradually over 4 to 24 hours exposure to cyclic (1 Hz) strain. This was due, at least in part, to increased transcription since a full length (890 bp) PDGF-A promoter reporter construct was induced 3.5-fold in transfected VSM cells exposed to strain for 24 hours. A series of PDGF-A promoter truncation reporter constructs was used to identify potential regions of the promoter involved in regulation by strain. Strain-responsive regions were found between -262 bp and -92 bp and between -92 bp and -41 bp of the promoter. Since these regions are GC-rich and contain response elements for Egr-1 and Sp-1, we examined expression of these transcription factors in response to strain. mRNA for both factors increased over 0.5 to 4 hours of strain, while protein expression for both increased gradually over a 24 hours period. Gel shift assays with a probe specific for Egr-1 demonstrated at least 1 prominent new shifted band after 4 to 12 hours exposure to strain. An Sp-1 probe demonstrated constitutive shifted bands that did not change in response to strain. Thus, GC-rich regions in the proximal 92 bp of the PDGF-A promoter contain mechanical strain-responsive elements that bind Egr-1 and possibly Sp-1.

Non-capacitative calcium entry in Chinese hamster ovary cells expressing the platelet-derived growth factor receptor.

Platelet-derived growth factor (PDGF) is believed to produce intracellular calcium (Ca2+i) transients by inositol trisphosphate (InsP3)-mediated release of intracellular Ca2+ stores followed by "capacitative" Ca2+ entry due to emptying of these stores. We examined the roles for the phospholipase Cgamma-InsP3 pathway and the emptying of InsP3-dependent intracellular Ca2+ stores in PDGF-mediated Ca2+ entry. Intracellular Ca2+ release and Ca2+ entry were measured with fluorometric methods in Chinese hamster ovary cells expressing wild type or mutant PDGF receptors. Activation of the wild type PDGF receptor caused both intracellular "Ca2+ release, " measured in nominally 0 Ca2+ extracellular medium, and "Ca2+ entry, " measured upon addition of 2 mM Ca2+ medium. Both phases were absent in Chinese hamster ovary cells expressing a PDGF receptor mutant (Y977F,Y989F) that fails to bind phospholipase Cgamma. Blockade of the InsP3 receptor, by microinjection of single cells with low molecular weight heparin (5-50 mg/ml), blocked only Ca2+i release (following PDGF or flash photolysis of caged InsP3) and had no effect on PDGF-induced Ca2+ entry. In whole cell patch-clamp experiments, intracellular heparin also failed to block PDGF-evoked ion currents. Release of InsP3-dependent intracellular Ca2+ stores, by flash photolysis of caged InsP3, was apparently not sufficient to maximally activate Ca2+ entry. Intracellular InsP3 caused significantly less Ca2+ entry than PDGF alone. These data suggest that InsP3 alone is not sufficient to maximally activate Ca2+ entry by the capacitative pathway and that products of phosphatidylinositol 4,5-bisphosphate breakdown other than InsP3 probably play a role in PDGF-mediated Ca2+ entry.

Activation of JNK/SAPK and ERK by mechanical strain in vascular smooth muscle cells depends on extracellular matrix composition.

Application of cyclic mechanical strain to vascular smooth muscle (VSM) cells elicits distinct cellular responses depending on extracellular matrix composition. We now examine activation of p42/p44 MAP kinase (ERK) and c-jun amino terminal kinase (JNK/SAPK) by cyclic (1 Hz) mechanical strain in neonatal rat VSM cells cultured on pronectin or laminin. In cells grown on pronectin, mechanical strain activated both ERKs (peak 10-30 min) and JNK/SAPK (peak 15-30 min). On laminin, mechanical strain induced a comparable activation of JNK/SAPK to that seen on pronectin, but no significant activation of ERKs. In contrast, application of strain to adult VSM cells activated both enzymes independently of extracellular matrix composition. In neonatal VSM cells, cyclic strain induced SM-1 smooth muscle myosin in cells cultured on laminin, but not on pronectin.. Thus in neonatal VSM cells, activation of ERKs and induction of SM-1 myosin by mechanical strain depend on extracellular matrix composition.

Mechanical strain increases smooth muscle and decreases nonmuscle myosin expression in rat vascular smooth muscle cells.

The effect of cyclic (1-Hz) mechanical strain on expression of myosin heavy chain isoforms was examined in neonatal rat vascular smooth muscle cells cultured on silicone elastomer plates. Myosin heavy chain isoforms were identified by immunoblot using antibodies recognizing (1) smooth muscle myosin heavy chain isoforms SM-1 and SM-2, (2) SM-1 exclusively, and (3) nonmuscle myosin heavy chains A and B. In response to 36 to 72 hours of strain, SM-1 and SM-2 increased by fourfold to sixfold, whereas nonmuscle myosin A decreased to 30% of control. Nonmuscle myosin B was unaffected by strain. SM-1 mRNA increased by twofold to threefold after 12 hours of strain but decreased toward control levels thereafter. SM-2 mRNA was only barely detectable. Nonmuscle myosin A mRNA decreased to 50% of control after 3 hours of strain and then returned to the control level. Since these cells secrete platelet-derived growth factor (PDGF) in response to strain, we assessed the effects of PDGF on myosin isoform expression. Exogenous PDGF (10 ng/mL) decreased SM-1 expression by 35% and increased nonmuscle myosin expression twofold, opposite the effect of strain. In cells exposed to strain with neutralizing antibodies to PDGF-AB, the strain-induced increase in SM-1 was enhanced 10-fold, and nonmuscle myosin A was reduced to 40% of control. Finally, the effect of extracellular matrix on transduction of the strain signal was studied. Forty-eight hours of cyclic strain increased SM-1 by twofold in cells cultured on collagen type 1 and threefold in cells cultured on laminin. In fibronectin-cultured cells, strain elicited no increase in SM-1. Thus, mechanical strain, sensed through specific interactions with the matrix, can alter myosin isoform expression toward that found in a more differentiated state.

Mechanical strain of rat vascular smooth muscle cells is sensed by specific extracellular matrix/integrin interactions.

Cyclic mechanical strain (1 Hz) causes a mitogenic response in neonatal rat vascular smooth muscle cells due to production and secretion of PDGF. In this study, the mechanism for sensing mechanical strain was investigated. Silicone elastomer strain plates were coated at varying densities with elastin, laminin, type I collagen, fibronectin, or vitronectin. Strain was applied by cyclic application of a vacuum under the dishes. Cells adhered, spread, and proliferated on each matrix protein, but the mitogenic response to strain was matrix dependent. Strain increased DNA synthesis in cells on collagen, fibronectin, or vitronectin, but not in cells on elastin or laminin. When strain was applied on matrices containing both laminin and vitronectin, the mitogenic response to strain depended upon the vitronectin content of the matrix. Fibronectin, in soluble form (0-50 micrograms/ml), and the integrin binding peptide GRGDTP (100 micrograms/ml) both blocked the mitogenic response to mechanical strain in cells grown on immobilized collagen. Neither soluble laminin nor the inactive peptide GRGESP blocked the response to strain. GRGDTP did not alter the mitogenic response to exogenous PDGF or alpha-thrombin but did prevent the secretion of PDGF in response to strain. Furthermore, GRGDTP, but not GRGESP, prevented strain-induced expression of a PDGF-A chain promoter 890 bp-chloramphenicol acetyltransferase construct that was transiently transfected into vascular smooth muscle cells. Finally, the response to strain was abrogated by antibodies to both beta 3 and alpha v beta 5 integrins but not by an antibody to beta 1 integrins. Thus interaction between integrins and specific matrix proteins is responsible for sensing mechanical strain in vascular smooth muscle cells.

Osmotic activation of a Na(+)-dependent Cl-/HCO3- exchanger.

In many systems, osmotically induced cell shrinkage activates the Na+/H+ exchanger. To assess the role of H(+)-extruding transporters in the response to osmotic shrinkage in vascular smooth muscle (VSM) and Chinese hamster ovary (CHO) cells, intracellular pH (pHi) was measured with 2',7'-bis(carboxy-ethyl)-5(6)- carboxyfluorescein-acetoxymethyl ester (BCECF-AM) after exposing cells to hypertonic medium. In nominally HCO(3-)-free medium, addition of 200 mM sucrose caused pHi to increase 0.33 pH unit on average in VSM cells but only 0.13 pH unit in CHO cells. Permeant solutes failed to increase pHi significantly. Cytochalasin B (1-20 microM), colchicine (1-10 microM), Ca2+ removal, and downregulation of protein kinase C activity did not affect osmotic activation of H+ extrusion in either cell type. Additional work was carried out to determine why osmotic activation of H+ extrusion was less in CHO than in VSM cells. In CHO cells, the osmotically induced delta pHi was only weakly sensitive to amiloride, suggesting that osmotic forces may activate an H+ transport system other than Na+/H+ exchange. In the presence of 10 mM HCO3-, osmotically induced delta pHi decreased by 60% in VSM cells but increased by 50% in CHO cells compared with the delta pHi in HCO(3-)-free medium. Lastly, removal of extracellular Cl- did not affect osmotically induced delta pHi in VSM cells but completely abolished the response in CHO cells. We conclude that in VSM cells osmotically induced changes in pHi are mediated by Na+/H+ exchange, whereas in CHO cells they are most likely mediated by a Na(+)-dependent Cl-/HCO3- exchanger.

Activation of Na+/H+ exchange by platelet-derived growth factor involves phosphatidylinositol 3'-kinase and phospholipase C gamma.

The effect of site-specific mutations in the mouse platelet-derived growth factor (PDGF) beta-receptor on activation of the Na+/H+ exchanger was examined in normal murine mammary gland epithelial (NMuMG) and Chinese hamster ovary (CHO) cells. These cells, which do not normally express PDGF receptors, were stably transfected with PDGF beta-receptor cDNA. Intracellular pH and Ca2+ were monitored using fluorescent probes. In both NMuMG and CHO cells expressing wild-type PDGF beta-receptors, PDGF B/B activated the amiloride-sensitive Na+/H+ exchanger. In both cell types, cell alkalinization was reduced by approximately 50% with a receptor mutant Y708F,Y719F which cannot bind phosphatidylinositol (PI) 3'-kinase. An inhibitor of PI 3'-kinase, LY294002, also inhibited alkalinization by 43% in cells with wild-type, but not Y708F,Y719F receptors. PDGF-induced intracellular Ca2+ release was not affected by this mutation. Both alkalinization and Ca2+ release were reduced by nearly 100% with the mutant Y977F,Y989F, which cannot bind phospholipase C gamma (PLC gamma). Y739F, a mutant that fails to bind the GTPase-activating protein did not affect PDGF-induced alkalinization. In protein kinase C (PKC) down-regulated NMuMG cells (wild-type receptor), PDGF no longer activated the Na+/H+ exchanger. In contrast, in PKC down-regulated CHO cells (wild-type receptor), PDGF-induced alkalinization was attenuated by only 37%. This residual activity was unaffected by the Y708F,Y719F mutation, but was completely eliminated by removal of medium Ca2+. These findings indicate that phospholipase C gamma is essential for activation of Na+/H+ exchange. PI 3'-kinase participates in PKC-dependent activation of Na+/H+ exchange by PDGF. In CHO cells, there is a second, Ca(2+)-dependent mechanism for activation of the exchanger.

Phospholipase C gamma activation, phosphotidylinositol hydrolysis, and calcium mobilization are not required for FGF receptor-mediated chemotaxis.

Basic fibroblast growth factor (FGF) is a potent angiogenic factor that stimulates several cell types to migrate along a chemotactic gradient. Most chemoattractant receptors appear to share a common mechanism that involves activation of phospholipase C (PLC), hydrolysis of phosphotidylinositol, and mobilization of intracellular calcium. We transfected two different cell lines with either human FGF receptor-1 cDNA or chimeric FGF receptor cDNA. Ligand stimulation induced chemotaxis, activation of PLC gamma, phosphotidylinositol hydrolysis, and calcium mobilization in both wild-type receptor cell lines. No such response was elicited in control cells. Mutation of the two fibroblast growth factor receptors at residue 766, replacing tyrosine with phenylalanine, made the receptors incapable of associating with and activating PLC gamma following ligand stimulation. These mutant receptors also failed to mediate phosphotidylinositol hydrolysis and calcium mobilization. However, cells transfected with the mutant fibroblast growth factor receptors were as chemotactically responsive to the appropriate ligand as were cells transfected with the wild-type receptors. These findings demonstrate that the ability of the fibroblast growth factor receptor to promote chemotaxis is not dependent on increased activation of PLC gamma, increased hydrolysis of phosphotidylinositol, or increased global mobilization of calcium.

Mechanical strain and collagen potentiate mitogenic activity of angiotensin II in rat vascular smooth muscle cells.

The effects of extracellular matrix proteins and mechanical strain on the mitogenic activity of angiotensins I and II (AI and AII) were examined in cultured rat vascular smooth muscle (VSM) cells. VSM cells on various extracellular matrices were exposed to AII (1 microM) for 48 h. On plastic, AII induced only a 1.6-fold increase in [3H]thymidine incorporation, but on fibronectin- or type I collagen-coated plastic, the response to AII was enhanced from two- to fourfold. On a type I collagen-coated silicone elastomer, to which mechanical strain was applied, [3H]thymidine incorporation dramatically increased to a maximum of 53-fold. Dup 753 (10(-5) M) blocked the AII-induced increase in DNA synthesis. AI also increased DNA synthesis in VSM cells, and this response was also enhanced by mechanical strain. Mitogenic activity of AI was blocked by ramiprilat (10(-5) M), indicating that its mitogenic activity was via conversion to AII. The synergy between AII and strain was completely eliminated by neutralizing antibodies to PDGF AB (3 micrograms/ml). Furthermore, the mitogenic effect of AII in unstrained cells was also synergistic with submaximal concentrations of PDGF AB (1 ng/ml). Thus, the synergy between AII and mechanical strain probably results from synergism between AII and PDGF secreted in response to strain.

Mechanical strain induces growth of vascular smooth muscle cells via autocrine action of PDGF.

The effect of cyclic mechanical strain on growth of neonatal rat vascular smooth muscle (VSM) cells were examined. Cells were grown on silicone elastomer plates subjected to cyclic strain (60 cycle/min) by application of a vacuum under the plates. A 48 h exposure to mechanical strain increased the basal rate of thymidine incorporation by threefold and increased cell number by 40% compared with cells grown on stationary rubber plates. Strain also increased the rate of thymidine incorporation in response to alpha-thrombin (from 15- to 33-fold), but not to PDGF. As determined by thymidine autoradiography, strain alone induced a fourfold increase in labeled nuclei at the periphery of dishes, where strain is maximal, and a 2-3-fold increase at the center of dishes. Strain appeared to induce the production of an autocrine growth factor(s), since conditioned medium from cells subjected to strain induced a fourfold increase in DNA synthesis in control cells. Western blots of medium conditioned on the cells subjected to strain indicate that the cells secrete both AA and BB forms of PDGF in response to strain. Northern blots of total cell RNA from cells exposed to strain for 24 h show increased steady-state level of mRNA for PDGF-A. Lastly, polyclonal antibodies to the AA form of PDGF reduced by 75% the mitogenic effect of strain and polyclonal antibodies to AB-PDGF reduced mitogenicity by 50%. Antibodies to bFGF did not significantly reduce the strain-induced thymidine incorporation. Thus, the mechanism of strain-induced growth appears to involve the intermediary action of secreted PDGF.

Differential modulation of fos and jun gene expression by 1,25-dihydroxyvitamin D3.

1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) modulates the mitogenic response to alpha-thrombin either positively or negatively in vascular smooth muscle (VSM) cells depending upon the time at which cells are exposed to 1,25-(OH)2D3. We now examine the impact of 1,25-(OH)2D3 on the induction by alpha-thrombin of c-jun and c-fos mRNA. When 1,25-(OH)2D3 and alpha-thrombin were added simultaneously to rat VSM cells, c-jun expression was enhanced by five-fold compared to cells exposed to thrombin alone. However, when cells were exposed to 1,25-(OH)2D3 for 48h prior to thrombin, c-jun expression after alpha-thrombin was reduced to less than 25% of the level in cells exposed to thrombin alone. c-fos expression after alpha-thrombin was unaffected by 1,25-(OH)2D3. Thus 1,25-(OH)2D3 dramatically alters the ratio of jun/fos mRNA produced during the mitogenic response and may thus modulate the response to mitogens.

Role of cytoplasmic domain of the Na+/H+ exchanger in hormonal activation.

The growth factor-activated Na+/H+ exchanger is regulated by numerous stimuli, including polypeptide hormones, phorbol esters, cell acidity, and cell shrinkage. To determine whether this regulation occurs at a common site on the cytoplasmic domain of the Na+/H+ exchanger, we microinjected polyclonal antibodies (RP1-c28) to the C-terminal 157 amino acids of the molecule and measured cell pH changes after application of a variety of stimuli known to activate the Na+/H+ exchanger. Microinjection of approximately 10 fg of RP1-c28 antibody, but not control IgG, into single cultured fibroblasts blocked subsequent activation of the exchanger by both endothelin and alpha-thrombin. In contrast, microinjected RP1-c28 did not prevent activation of Na+/H+ exchange by phorbol esters, consistent with the observation that both endothelin-1 and alpha-thrombin retained the ability to activate exchange activity in protein kinase C-depleted cells. Finally, activation of Na+/H+ exchange by both cell acidity and osmotic shrinkage was also unaffected by microinjected RP1-c28 antibody. These data indicate that activation of Na+/H+ exchange by endothelin-1 and alpha-thrombin is mechanistically distinct both from activation by protein kinase C and activation by physical factors and probably occurs at a separate site on the exchanger molecule.

Point mutation of an FGF receptor abolishes phosphatidylinositol turnover and Ca2+ flux but not mitogenesis.

Stimulation of certain receptor tyrosine kinases results in the tyrosine phosphorylation and activation of phospholipase C gamma (PLC gamma), an enzyme that catalyses the hydrolysis of phosphatidylinositol (PtdIns). This hydrolysis generates diacylglycerol and free inositol phosphate, which in turn activate protein kinase C and increase intracellular Ca2+, respectively. PLC gamma physically associates with activated receptor tyrosine kinases, suggesting that it is a substrate for direct phosphorylation by these kinases. Here we report that a fibroblast growth factor (FGF) receptor with a single point mutation at residue 766 replacing tyrosine with phenylalanine fails to associate with PLC gamma in response to FGF. This mutant receptor also failed to mediate PtdIns hydrolysis and Ca2+ mobilization after FGF stimulation. However, the mutant receptor phosphorylated itself and several other cellular proteins, and it mediated mitogenesis in response to FGF. These findings show that a point mutation in the FGF receptor selectively eliminates activation of PLC gamma and that neither Ca2+ mobilization nor PtdIns hydrolysis are required for FGF-induced mitogenesis.