The effect of protein kinase C and its alpha subtype on human vascular smooth muscle cell proliferation, migration and fibronectin production

BACKGROUND: Vascular smooth muscle cell (SMC) migration, proliferation and extracellular matrix protein production are key steps in the formation of intimal hyperplasia, a process that leads to failure of vascular reconstructions. Protein kinase C (PKC) may be involved in all 3 cellular events. PKC consists of a family of 11 isotypes, 8 of which we have identified in human vascular SMCs. In this study we evaluate the role of PKCalpha as a second messenger for proliferation, migration and fibronectin production induced by human saphenous vein SMCs. METHODS: DNA synthesis was evaluated by using (3)H-thymidine incorporation. Mitogen-activated protein kinase (MAP-K) activation was quantified by Western blotting with an antibody to its phosphorylated substrate, Elk-1. Chemotaxis was evaluated by using a microchemotaxis chamber. SMC fibronectin was measured by Western blotting. For all experiments, PKCalpha was blocked with a selective inhibitor, G?6976. RESULTS: G?6976, at concentrations that allow selective inhibition of PKCalpha, inhibited platelet-derived growth factor-stimulated SMC proliferation and MAP-K activation by 30% to 40% and 30% to 60%, respectively. SMC chemotaxis was stimulated approximately 2-fold by the PKCalpha inhibitor. Neither basal nor transforming growth factor-betaI induced fibronectin production was affected by G?6976. CONCLUSIONS: Our data suggest that PKCalpha is a positive mediator of SMC proliferation and MAP-K activity, a negative regulator of migration and has no effect on SMC fibronectin production. These data suggest that modulating activities of specific PKC isotypes might be useful in both the study and control of intimal hyperplasia.     

Midazolam stimulates vascular endothelial growth factor release in aortic smooth muscle cells: role of the mitogen-activated protein kinase superfamily

BACKGROUND: Intravenous anesthetics used during perioperative periods affect the vascular signaling molecules and the vascular reactivity. Vascular endothelial growth factor (VEGF), an angiogenesis factor produced in and secreted from aortic smooth muscle cells, is a specific mitogen for vascular endothelial cells. This study investigated the effects of various intravenous anesthetics on VEGF release, and the underlying mechanism, in a rat aortic smooth muscle cell line, A10 cells. METHODS: Intravenous anesthetics (midazolam and propofol) were continuously administered to rats by infusion. Cultured A10 cells were stimulated by intravenous anesthetics (midazolam, propofol, and ketamine). VEGF was evaluated by immunoassay. The phosphorylation of mitogen-activated protein (MAP) kinases was evaluated by Western blotting. RESULTS: Continuous infusion of midazolam, but not propofol, increased the VEGF concentration in rat plasma. In cultured cells, midazolam stimulated VEGF release, but propofol and ketamine did not. Midazolam induced phosphorylation of p44/p42 MAP kinase and stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK), without affecting p38 MAP kinase. PD98059 and U0126, specific inhibitors of MAP kinase kinase, significantly reduced the midazolam-stimulated release of VEGF. SP600125, a specific inhibitor of SAPK/JNK, significantly reduced midazolam-stimulated VEGF release. Applied together, PD98059 and SP600125 produced an additive reduction in midazolam-stimulated VEGF release. Moreover, a bolus injection of PD98059 truly inhibited the midazolam-increased VEGF concentration in rat plasma in vivo. CONCLUSIONS: Midazolam, but not propofol or ketamine, stimulates VEGF release in aortic smooth muscle cells. Its effect is mediated at least in part via activation of p44/p42 MAP kinase and SAPK/JNK.     

Characterization of glucose-induced in situ protein kinase C activity in cultured vascular smooth muscle cells.

The VSMC is an important target for the injurious effects of hyperglycemia in vivo. PKC plays a key role in the regulation of VSMC contraction and growth. This study examines whether elevated extracellular glucose concentrations (10-30 mM [180-540 mg/dl]) activate PKC in cultured rat VSMCs in vitro. A new, rapid, and highly specific assay was used to determine in situ PKC activity in digitonin-permeabilized VSMCs. PKC activity in VSMCs responded rapidly to variations in extracellular glucose concentrations. PKC was activated significantly within 10 min of exposure to D-glucose (20 mM) versus glucose (5 mM). Moreover, with continued exposure to D-glucose (20 mM), PKC activation was sustained for up to 48 h. Reducing D-glucose concentrations to 5 mM restored PKC activity to control values within 1 h. PKC activation was also glucose-concentration dependent. A threshold of only 15 mM (270 mg/dl) was required to significantly and maximally activate PKC in VSMC. PKC was not activated in the presence of osmotic control media that contained either elevated mannitol or L-glucose concentrations. In marked contrast to the sustained PKC activation induced by D-glucose in VSMCs, the normal physiological PKC response to the pressor hormones, AII and AVP, was short-lived and returned to base line within minutes. Sustained PKC activation in the presence of elevated D-glucose concentrations in vitro could disturb the normal physiological regulation of VSMC function and growth and thereby may contribute to the apparent vasotoxicity of hyperglycemia in vivo.     

Heparin inhibits thrombin-induced mitogen-activated protein kinase signaling in arterial smooth muscle cells

Purpose: Smooth muscle cell proliferation is a key event in the development of intimal hyperplasia after arterial injury. Heparin can suppress smooth muscle cell proliferation in vitro and prevents intimal hyperplasia after arterial injury, but the mechanisms of action are poorly understood. Recently, we observed that heparin inhibited serum-induced activation of mitogen-activated protein kinase in smooth muscle cells, but heparin did not inhibit signaling induced by platelet-derived growth factor BB and basic fibroblast growth factor, both ligands of tyrosine kinase receptors. Here, we examined the possibility that heparin inhibits signaling by thrombin and other activators of heterotrimeric G-proteins. Design of Study: Baboon aortic smooth muscle cells were stimulated with thrombin, angiotensin II, endothelin-1, and lysophosphatidic acid in the presence or absence of heparin. After stimulation, mitogen-activated protein kinase activity was measured with an in-gel phosphorylation assay, mitogen-activated protein kinase kinase-1 was immunoprecipitated from the same samples, and activity was measured with recombinant mitogen-activated protein kinase as a substrate. DNA synthesis was measured by ³H-thymidine labeling and scintillation counting. Results: Heparin inhibited sustained activity of mitogen-activated protein kinase kinase-1 and mitogen-activated protein kinase and prevented DNA synthesis induced by thrombin, angiotensin II, endothelin-1, and lysophosphatidic acid. Conclusions: Heparin inhibits growth of baboon smooth muscle cells by preventing prolonged mitogen-activated protein kinase activation elicited by ligands of seven transmembrane domain receptors and heterotrimeric G-proteins. The results indicate that heparin interferes with a specific pathway in smooth muscle cell growth, which could be a future target in attempts to inhibit lesion development after vascular surgery. (J Vasc Surg 1998;27:512-20.)     

The effect of growth factors, cytokines, and extracellular matrix proteins on fibronectin production in human vascular smooth muscle cells

PURPOSE: Although 60% to 80% of the mature intimal hyperplastic plaque is composed of extracellular matrix (ECM) proteins, little is known about the factors that stimulate smooth muscle cells (SMCs) to produce these proteins. A major component of the ECM protein is fibronectin. Thus we studied fibronectin production and its response to various growth factors, cytokines, and other ECM proteins that are released at the time of vascular injury. METHODS: Quiescent cultured human SMCs were stimulated for varying intervals with increasing concentrations of agonist. Fibronectin in the cell medium was assayed by immunoblotting with a fibronectin-specific antibody. RESULTS: After 72 hours of stimulation, transforming growth factor-beta (10 ng/mL) had the most profound effect on fibronectin production (9.6- +/- 2.1-fold; P <.05), followed by epidermal growth factor (100 ng/mL; 5.0- +/- 0.1-fold; P <.05, for both). Surprisingly, the platelet-derived growth factors (AA, AB, and BB) and fibroblast growth factor did not stimulate fibronectin production. Among the matrix proteins studied, only collagen type I (20 microg/mL) stimulated fibronectin production (1.9- +/- 0.1-fold; P <.05), whereas collagen type IV and laminin had no effect. The contractile protein angiotensin II (100 ng/mL) was a weak stimulant of fibronectin (1.6- +/- 0.2-fold; P <.05). Time course studies of fibronectin production up to 72 hours revealed kinetics that varied with each agonist. Transforming growth factor-beta stimulated significant early production of fibronectin, whereas fibronectin production in response to epidermal growth factor and collagen type I was initially modest but increased with time. The effect of angiotensin II did not become evident until 72 hours. CONCLUSION: Cytokines, growth factors, and matrix proteins have varying quantitative effects on ECM protein production by human vascular SMCs. Knowledge of the factors that influence ECM protein production may allow for the design of specific inhibitors that can prevent intimal hyperplasia.     

Protein kinase C isoforms in human aortic smooth muscle cells

Purpose: To identify the protein kinase C (PKC) isoforms in human arterial smooth muscle cells (SMC) and define their subcellular location in the resting state and in response to the PKC activator, 12-O-tetradecanoylphorbol 13-acetate (TPA). Methods: Arterial SMC cultures established from transplant donor aorta were treated with 100 nM TPA or control media, then mechanically lysed. PKC from the soluble and particulate fraction were separated by centrifugation, and protein normalized immunoblots were performed with antibodies to the PKC isoforms α, β_I, β_II, δ, ϵ, γ, and ζ. Bands were detected by enhanced chemiluminescence and analyzed densitometrically, with results expressed as the mean percentage of each fraction ± SEM. Translocation was defined as a significant (p < 0.05) change in the particulate fraction for each isoform. Immunofluorescent staining of cultured SMC visualized the resting location and stimulated translocation of each isoform. Results: Isoforms α and β_I were detected primarily in the soluble fraction, translocating to the particulate fraction with TPA stimulation (p < 0.0001). The isoforms β_II, δ, and ϵ were found primarily in the particulate fraction and did not translocate. Immunofluorescent staining confirmed these locations. Neither γ or ζ were detected in these SMC. Conclusions: The PKC isoforms expressed in human arterial SMC differ from those reported in animal models. Their specific locations and response to stimulation suggest unique functions in cellular regulation and provide the groundwork for further investigation into their role in the development of vascular disease and regulation of matrix metabolism. (J Vasc Surg 1998;27:919-27.)     

Fucoidan inhibits smooth muscle cell proliferation and reduces mitogen-activated protein kinase activity.

OBJECTIVES AND DESIGN: fucoidan has previously been shown to inhibit the proliferation of arterial smooth muscle cells both in animal models and in vitro. However, the mechanisms behind the anti-proliferative effects of this polysulfated polysaccharide are not known in detail. Here, the inhibitory effect of fucoidan on rat aortic smooth muscle cell proliferation was examined and compared with the effects of heparin after stimulation with fetal calf serum, platelet-derived growth factor BB, basic fibroblast growth factor, heparin-binding epidermal growth factor, and angiotensin II. MATERIALS AND METHODS: the cultures were analysed with respect to cell proliferation and DNA synthesis by cell counting and measurement of(3)H-thymidine incorporation. Phosphorylation of mitogen-activated protein kinase and nuclear translocation of phosphorylated mitogen-activated protein kinase were studied by immunoblotting and immunocytochemistry. RESULTS: fucoidan was shown to be a more potent inhibitor of smooth muscle cell proliferation than heparin. Fucoidan also reduced growth factor-induced activation of mitogen-activated protein kinase and prevented nuclear translocation of phosphorylated mitogen-activated protein kinase. CONCLUSION: fucoidan is a more potent anti-proliferative polysulphated polysaccharide than heparin and may mediate its effects through inhibition of the mitogen-activated protein kinase pathway in a similar manner as heparin.     

Isoflurane activates sarcolemmal adenosine triphosphate-sensitive potassium channels in vascular smooth muscle cells: a role for protein kinase A

BACKGROUND: Recent evidence indicates that vascular adenosine triphosphate-sensitive potassium (K(ATP)) channels in vascular smooth muscle cells are critical in the regulation of vascular tonus under both physiologic and pathophysiologic conditions. Studies of the interaction of volatile anesthetics with vascular K(ATP) channels have been limited. In the current study, the authors investigated the molecular mechanism of isoflurane's action on vascular K(ATP) channels. METHODS: Electrophysiologic experiments were performed using cell-attached and inside-out patch clamp techniques to monitor native vascular K(ATP) channels, and recombinant K(ATP) channels comprised of inwardly rectifying potassium channel subunits (Kir6.1) and the sulfonylurea receptor (SUR2B). Isometric tension experiments were performed in rat thoracic aortic rings without endothelium. RESULTS: Application of isoflurane (0.5 mM) to the bath solution during cell-attached recordings induced a significant increase in K(ATP) channel activity, which was greatly reduced by pretreatment with a selective inhibitor of protein kinase A (PKA), Rp-cAMPS (100 microM). In inside-out patches, isoflurane did not activate K(ATP) channels. Isoflurane significantly activated wild-type recombinant SUR2B/Kir6.1 in cell-attached patches. Isoflurane-induced activation of wild-type channels was diminished in the PKA-insensitive mutant SUR2B-T633A/Kir6.1, SUR2B-S1465A/Kir6.1, and SUR2B/Kir6.1-S385A. In addition, the authors demonstrated that isoflurane-induced PKA activation was associated with isoflurane-induced decreases in isometric tension in the rat aorta. CONCLUSION: These results indicate that isoflurane activates K(ATP) channels via PKA activation. PKA-dependent vasodilation induced by isoflurane also was observed in isometric tension experiments. Analysis of expressed vascular-type K(ATP) channels suggested that PKA-mediated phosphorylation of both Kir6.1 and SUR2B subunits plays a pivotal role in isoflurane-induced vascular K(ATP) channel activation.     

人类脑动脉瘤血管平滑肌细胞表型蛋白与雌激素受体表达

目的 探讨正常人体动脉血管和脑动脉瘤的血管平滑肌(VSMC)雌激素受体(ERs)和α-平滑肌肌动蛋白(α-SMA)和结蛋白(Desmin)表达变化的关系.方法 临床取材8例非动脉瘤患者手术区头部动脉血管和脑动脉瘤切除标本18例.采用免疫组织化学和形态学分析方法 检测正常动脉血管和脑动脉瘤ERs、α-SMA和Desmin的表达.结果 脑动脉瘤VSMC的ERα和ERβ表达水平均高于对照动脉血管表达水平,差异有统计学意义(P<0.01).脑动脉瘤VSMC的α-SMA和Desmin的表达水平均低于对照动脉血管表达水平,差异有统计学意义(P<0.01).脑动脉瘤VSMCERs的表达与α-SMA和Desmin的表达水平明显相关.结论 人类脑动脉瘤VSMC的ERs表达水平普遍升高,其VSMC表型蛋白表达水平普遍降低,两者之间呈负相关,这可能与脑动脉瘤的形成有关.     

Requirement of aldose reductase for the hyperglycemic activation of protein kinase C and formation of diacylglycerol in vascular smooth muscle cells

Activation of protein kinase C (PKC) has been linked to the development of secondary diabetes complications. However, the underlying molecular mechanisms remain unclear. We examined the contribution of aldose reductase, which catalyzes the first, and the rate-limiting, step of the polyol pathway of glucose metabolism, to PKC activation in vascular smooth muscle cells (VSMCs) isolated from rat aorta and exposed to high glucose in culture. Exposure of VSMCs to high glucose (25 mmol/l), but not iso-osmotic mannitol, led to an increase in total membrane-associated PKC activity, which was prevented by the aldose reductase inhibitors tolrestat or sorbinil or by the ablation of aldose reductase by small interfering RNA (siRNA). The VSMCs were found to express low levels of sorbitol dehydrogenase, and treatment with the sorbitol dehydrogenase inhibitor CP-166572 did not prevent high-glucose-induced PKC activation. Stimulation with high glucose caused membrane translocation of conventional (alpha, beta1, beta2, and gamma) and novel (delta and epsilon) isoforms of PKC. Inhibition of aldose reductase prevented membrane translocation of PKC-beta2 and -delta and delayed the activation of PKC-beta1 and -epsilon, whereas membrane translocation of PKC-alpha and -gamma was not affected. Treatment with tolrestat prevented phosphorylation of PKC-beta2 and -delta. High glucose increased the formation of diacylglycerol (DAG) and enhanced phosphorylation of phospholipase C-gamma1 (PLC-gamma1). Inhibition of aldose reductase prevented high glucose-induced DAG formation and phosphorylation of PLC-gamma1 and PLC-beta2 and -delta. Inhibition of phospholipid hydrolysis by D609, but not by the synthetic alkyl-1-lysophospholipid 1-O-octadecyl-2-O-methyl-rac-glycerophosphocholine, or edelfosine, prevented DAG formation. Treatment with sorbinil decreased the levels of reactive oxygen species in high-glucose-stimulated VSMCs. Hence, inhibition of aldose reductase, independent of sorbitol dehydrogenase, appears to be effective in diminishing oxidative stress and hyperglycemic changes in signaling events upstream to the activation of multiple PKC isoforms and PLC-gamma1 and may represent a useful approach for preventing the development of secondary vascular complications of diabetes.     

Mechanism of vascular smooth muscle cells activation by hydrogen peroxide: role of phospholipase C gamma.

BACKGROUND: Hydrogen peroxide (H2O2) formation is a critical factor in processes involving ischaemia/ reperfusion. However, the precise mechanism by which reactive oxygen species (ROS) induce vascular damage are insufficiently known. Specifically, activation of phospholipase C gamma (PLCgamma) is a probable candidate pathway involved in vascular smooth muscle cells (VSMC) activation by H2O2. METHODS: The activation of human venous VSMC was measured as cytosolic free calcium mobilization, shape change and protein phosphorylation, focusing on the role of tyrosine phosphorylation-activated PLCgamma. RESULTS: The exposure of VSMC to exogenous H2O2 caused a rapid increase in cytosolic free calcium concentration ([Ca2+]i), and induced a significant VSMC shape change. Both effects were dependent on a tyrosine kinase-mediated mechanism, as determined by the blockade of short-term treatment of VSMC with the protein tyrosine kinase inhibitor, genistein. Giving further support to the putative role of phospholipase C (PLC)-dependent pathways, the [Ca2+]i and VSMC shape change response were equally inhibited by the specific PLC blocker, 1-(6-((17-beta-methoxyestra-1,3,5(10)trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122). In addition, U73122 had a protective effect against the deleterious action (24 h) of H2O2 on non-confluent VSMC. As a further clarification of the specific pathway involved, the exposure to H2O2 significantly stimulated the tyrosine phosphorylation of PLCgamma with a concentration- and time-profile similar to that of [Ca(2+)](i) mobilization. CONCLUSIONS: The present study reveals that H(2)O(2) activates PLCgamma on VSMC through tyrosine phosphorylation and that this activation has a major role in rapid [Ca(2+)](i) mobilization, shape-changing actions and damage by H(2)O(2) in this type of cells. These findings have direct implications for understanding the mechanisms of the vascular actions of H(2)O(2) and may help to design pharmacologically protective strategies.     

Effects of tautomycetin on proliferation and fibronectin secretion in vascular smooth muscle cells and glomerular mesangial cells

Tautomycetin (TMC), a newly developed immunosuppressive agent, induces T-lymphocyte apoptosis through the inhibition of tyrosine kinase and protein phosphatase 1. We examined the effects of TMC on platelet-derived growth factor (PDGF)-induced proliferation and extracellular matrix synthesis in cultured vascular smooth muscle cells (VSMCs) and mesangial cells (MCs) of Sprague-Dawley rats, and investigated the molecular mechanisms involved. Different concentrations of TMC were administered 1 hour before the addition of 10 ng/mL PDGF into the growth-arrested and synchronized cells. Cell proliferation was assessed by methylthiazoletetrazolium (MTT) assay, fibronectin secretion, and the activation of Akt, ERK, and p38 MAPK by Western blot analysis. PDGF increased cell proliferation, fibronectin secretion, and the activation of Akt, ERK, and p38 MAPK in both VSMCs and MCs. In both cultured cells, TMC at >1 mug/mL significantly reduced basal MTT. TMC at 100 ng/mL significantly decreased the PDGF-induced VSMC and MC proliferation. However, fibronectin secretion and the activation of Akt, ERK, and p38 MAPK were not affected by this nontoxic concentration of TMC. The present data demonstrate that low-dose TMC reduced PDGF-induced VSMC and MC proliferation without affecting the fibronectin secretion and cellular kinase activation.     

Propofol increases pulmonary artery smooth muscle myofilament calcium sensitivity: role of protein kinase C

BACKGROUND: Vascular smooth muscle tone is regulated by changes in intracellular free Ca2+ concentration ([Ca2+]i) and myofilament Ca2+ sensitivity. These cellular mechanisms could serve as targets for anesthetic agents that alter vasomotor tone. This study tested the hypothesis that propofol increases myofilament Ca2+ sensitivity in pulmonary artery smooth muscle (PASM) via the protein kinase C (PKC) signaling pathway. METHODS: Canine PASM strips were denuded of endothelium, loaded with fura-2/AM, and suspended in modified Krebs-Ringer's buffer at 37 degrees C for simultaneous measurement of isometric tension and [Ca2+]i. RESULTS: The KCl (30 mm) induced monotonic increases in [Ca2+]i and tension. Verapamil, an L-type Ca2+ channel blocker, attenuated KCl-induced increases in [Ca2+]i and tension to an equal extent. In contrast, propofol attenuated KCl-induced increases in [Ca2+]i to a greater extent than concomitant changes in tension and caused an upward shift in the peak tension-[Ca2+]i relation. Increasing extracellular Ca2+ in the presence of 30 mM KCl resulted in similar increases in [Ca2+]i in control and propofol-pretreated strips, whereas concomitant increases in tension were greater during propofol administration. The Ca2+ ionophore, ionomycin (0.1 microm), increased [Ca2+]i to approximately 50% of the value induced by 60 mm KCl. Under these conditions, propofol (10, 100 microm) caused increases in tension equivalent to 11 +/- 2 and 28 +/- 3% of the increases in tension in response to 60 mM KCl, whereas [Ca2+]i was slightly decreased. Similar effects were observed in response to the PKC activator, phorbol 12-myristate 13-acetate (PMA, 1 microm). Specific inhibition of PKC with bisindolylmaleimide I before ionomycin administration decreased the propofol- and PMA-induced increases in tension and abolished the propofol- and PMA-induced decreases in [Ca2+]i. Selective inhibition of Ca2+ -dependent PKC isoforms with G? 6976 also attenuated propofol-induced increases in tension. CONCLUSION: These results suggest that propofol increases myofilament Ca2+ sensitivity in PASM, and this effect involves the PKC signaling pathway.     

p38 mitogen-activated protein kinase and alkaline phosphatase in human dental pulp cells.

OBJECTIVE: The objective of this study was to investigate the implication of p38 mitogen-activated protein kinase (MAPK) in mediating alkaline phosphatase (ALPase) activity in human dental pulp cells (HPCs). STUDY DESIGN: Nuclear translocation of p38 was observed by immunofluorescence in isolated HPCs treated with transforming growth factor beta1 (TGF-beta1). TGF-beta1 was used to examine the interaction between p38 MAPK and Smad pathway. Role of p38 kinase in mediating ALPase activity was determined with SB203580, a specific inhibitor for the p38 pathway. Lipopolysaccharide (LPS) or TGF-beta1 was added to inhibit or increase ALPase activity. Statistical analysis was performed by unpaired t test. RESULTS: TGF-beta1 induced nuclear translocalization of p38. Blockage of p38 pathway with SB203580 inhibited translocation of Smad2/3 to nuclei. ALPase activity decreased in cells treated with SB203580, in contrast to its vehicle (P < .05). Inhibition on enzyme activity by LPS was exacerbated by SB203580 (P < .05). Treatment with SB203580 before addition of TGF-beta1 also made a significant decrease in ALPase activity (P < .05). CONCLUSIONS: These results suggest that p38 MAPK is implicated in regulating ALPase activity in HPCs and may interact with Smad pathway.     

Mechanical stretch-induced fibronectin and transforming growth factor-beta1 production in human mesangial cells is p38 mitogen-activated protein kinase-dependent

Hemodynamic abnormalities are important in the pathogenesis of the excess mesangial matrix deposition of diabetic and other glomerulopathies. p38-Mitogen-activated protein (MAP) kinase, an important intracellular signaling molecule, is activated in the glomeruli of diabetic rats. We studied, in human mesangial cells, the effect of stretch on p38 MAP kinase activation and the role of p38 MAP kinase in stretch-induced fibronectin and transforming growth factor-beta1 (TGF-beta1) accumulation. p38 MAP kinase was activated by stretch in a rapid (11-fold increase at 30 min, P < 0.001) and sustained manner (3-fold increase at 33 h, P < 0.001); this activation was mediated by protein kinase C (PKC). Stretch-induced fibronectin and TGF-beta1 protein levels were completely abolished (100% inhibition, P < 0.001; and 92% inhibition, P < 0.01, respectively) by SB203580, a specific p38 MAP kinase inhibitor. At 33 h, TGF-beta1 blockade did not affect stretch-induced fibronectin production, but partially prevented stretch-induced p38 MAP kinase activation (59% inhibition, P < 0.05). TGF-beta1 induced fibronectin accumulation after 72 h of exposure via a p38 MAP kinase-dependent mechanism (30% increase over control, P < 0.01). In human mesangial cells, stretch activates, via a PKC-dependent mechanism, p38 MAP kinase, which independently induces TGF-beta1 and fibronectin. In turn, TGF-beta1 contributes to maintaining late p38 MAP kinase activation, which perpetuates fibronectin accumulation.     

Interleukin-17--induced interleukin-8 release in human airway smooth muscle cells: role for mitogen-activated kinases and nuclear factor-kappaB.

BACKGROUND: It has recently become clear that interleukin (IL)-8 plays a role in chronic neutrophilic inflammatory disorders, such as chronic rejection after lung transplantation. We have shown that IL-17--stimulated human airway smooth muscle cells (HASMC) are able to produce IL-8. The aim of this study was to determine whether p38 mitogen-activated protein kinase (MAPK), c-Jun amino-terminal kinase (JNK), p42/p44 extracellular signal-related kinase (ERK) and nuclear factor-kappaB (NF-kappaB) are involved in IL-17--induced IL-8 production in HASMC in vitro. METHODS: We used human airway smooth muscle cells in culture. Western blotting was done to obtain data regarding activation of MAPK. Furthermore, we used specific inhibitors of MAPK to investigate their involvement in IL-17--induced IL-8 release, which was measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Western blotting clearly demonstrated that p38 MAPK, JNK and p42/p44 ERK were activated by IL-17 in HASMC. Using SB203580, a specific inhibitor of p38 MAPK, we detected a concentration-dependent inhibition of IL-17--induced IL-8 production with a maximal decrease of 63 +/- 5% (n=8, p<0.01). Curcumin, a specific inhibitor of JNK, also concentration-dependently reduced IL-17--induced IL-8 production, with a maximal decrease of 82+/-4% (n=8, p<0.01). U0126, a specific inhibitor of p42/p44 ERK, induced a maximal decrease of 84+/-5% (n=8, p<0.001). Pyrrolydine dithiocarbamate (PDTC), an inhibitor of NF-kappaB, caused a 70+/-5% (n=8, p<0.01) decrease in IL-17--induced IL-8 production. CONCLUSIONS: We found that IL-17 induces activation of p38MAPK, JNK and p42/p44ERK in HASMC. We also found that p38MAPK, JNK, p42/p44 ERK and NF-kappaB play an important role in IL-17--induced IL-8 production in HASMC in vitro. This may open up new opportunities for further treatment of this disease.     

Fatty acids carried on albumin modulate proximal tubular cell fibronectin production: a role for protein kinase C.

BACKGROUND: Proteinuric renal disease is associated with accumulation of tubulointerstitial matrix proteins. Human proximal tubular cells (PTCs) produce fibronectin in response to serum proteins but not albumin alone. It has been suggested that renal toxicity of filtered albumin depends on its lipid moiety. We therefore investigated the functional consequences of different fatty acids (FAs) carried on human albumin after exposure to human PTCs in culture. METHODS: Confluent human PTCs were exposed to recombinant human serum albumin (rHSA) or palmitate (P)-, stearate (S)-, oleate (O)-, and linoleate (L)-complexed rHSA. In all experimental conditions, test media contained 1 mg/ml rHSA alone or carrying 100 mmol FAs. Mitogenic response was assessed by [(3)H]thymidine incorporation. Cell culture supernatants were assayed for fibronectin. Protein kinase C (PKC) activity was assessed in cell lysates. RESULTS: Apical exposure to rHSA alone or the O-rHSA complex stimulated a significant increase in [(3)H]thymidine incorporation, whereas the L-rHSA complex was markedly inhibitory to human PTC growth. The L-rHSA complex was associated with severe cytotoxicity as assessed by lactate dehydrogenase release. Among all conditions, O-rHSA was the only test media that significantly increased fibronectin levels over control conditions (150.1+/-10.6% over control, P<0.05, n=3). Pre-treatment of PTCs with PKC inhibitors before O-rHSA exposure resulted in a dose-dependent decrease in fibronectin secretion. O-rHSA activated PKC significantly compared with controls. CONCLUSIONS: We conclude that rHSA has a mitogenic effect on human PTCs, but fibronectin secretion was only induced by O-complexed rHSA and the O-rHSA effect was mediated via PKC activation. Involvement of PKC signal transduction pathway may be a novel therapeutic target for ameliorating proteinuria-induced tubular injury.