Mtgratton of vascular smooth muscle cells (VSMCs) 1s a crucial response to vascular mjury resulting m neomtima formatton and atherosclerosis Platelet-derived growth factor (PDGF-BB) functions as a potent chemoattractant for VSMCs and enhances these pathologies m the vasculature. However, little is known about the mtracellular pathways that mediate VSMC mtgratton In the present study, we mvestigated the role of mltogen-achvated protein kmase (MAPK) activatton m this function, since PDGF-BB as well as other growth factors activate this pathway Usmg an m-gel kmase assay, we observed that PD 98059, an mhlbttor of MEK that activates MAP kmase, inhibited PDGF-BB-induced activation of ERK-1 and ERK-2 m cultured rat aorttc smooth muscle cells m a concentration-dependent manner In contrast, PDGF-mediated acttvation of mtracellular calcmm release was not affected by PD 98059 The chemotacttc response of both rat aorttc smooth muscle cells (RASMCs) and human umbilical vem smooth muscle cells (HUSMCs) toward PDGF-BB (10 ng/mL) was stgmticantly reduced by PD 98059 (10 pmol/L) to 41 7?7 1% m RASMCs (P<.Ol) and to 47 2 (1) increased mtracellular calcmm and the activation of calcmm/calmodulm-dependent protein kmase II', (2) mcreased phosphattdylinosttol turnover linked to the acttvatton of phosphohpase Cy or phospholipase Cp3, (3) actrvatron of phosphatidylmosuol3-kmase4; and (4) activation of RAS and RAF 8 The mrtogenic stgnalmg pathway for PDGF involves the activation of RAS, which activates the serme threonme kmase RAF, which triggers a protein kmase cascade.9 The signal for prohferatton 1s transmitted into the nucleus though the actrvatton of MAPK, which phosphorylates transcription factors that induce expresston of c-fos and other early growth response genes. The involvement of the RAS-RAF-MAPK cascade m cell migration 1s controverstal In fibroblasts expressing a dominant negative RAS, directed migration toward PDGF was suppressed,* suggesting this pathway contributed to cell migration In contrast, IGF-I and PDGF-BB were shown to equally induce VSMC migration, but IGF-1 only weakly mduced MAPK m contrast to PDGF 3 The authors concluded that actrvatron of MAPK may not required for VSMC migration The avatlabrlity of the MEK mhtbrtor PD 98059, which is widely used as a pharmacologrcal mhibttor of the MAPK pathway, and antisense ODN against MAPK mRNA now provides cnttcal tools to determine the specific role of MAPK activation m cell functions such as migration

Angiotensin II (Ang II) and insulin are implicated in the mesangial cell hypertrophy and excessive accumulation of mesangial matrix seen in glomerulosclerosis. Therefore, the effects of Ang II with and without insulin on mRNA levels of several important extracellular matrix genes and transforming growth factor beta-1 (TGF-beta 1) were examined. Ang II alone (1 microM) added to quiescent, murine mesangial cells in serum-free, insulin-free media slightly but not significantly increased TGF-beta 1, fibronectin, collagen I, collagen IV and laminin message levels. The slight elevations in message expression were reversed by losartan, suggesting that these modest effects are mediated by the AT-1 receptor. Ang II alone also had no significant effects on TGF-beta 1 and extracellular matrix message levels in quiescent rat mesangial cells. In contrast, significant increases in mRNA for collagen 1 (6-fold), collagen IV (4-fold), fibronectin 1 (4-fold) and TGF-beta 1 (2-fold) were seen with insulin alone (10(-6)M) in rat mesangial cells, and a dose-response effect could be demonstrated for insulin (10(-9) to 10(-6)M). Ang II plus insulin further significantly increased collagen I (9-fold), collagen IV (9-fold), fibronectin 1 (5-fold) and TGF-beta 1 (3-fold) message expression. These effects were partially reversed in the presence of losartan. The Northern analyses were supported by measurements of active and total TGF-beta 1 activity (pg/ml/ 5 x 10(6) cells): 1145 +/- 76 and 1960 +/- 199, serum free control; 1121 +/- 92 and 1932 +/- 214, Ang II (10(-6)M); 4589 +/- 103 (P < 0.001 vs. control) and 11071 +/- 1952 (P < 0.01 vs. control), insulin (10(-6)M); and 6881 +/- 183 (P < 0.001 vs. control) and 16626 +/- 1435 (P < 0.01 vs. control), insulin plus Ang II. These results suggest that insulin, itself, significantly increases TGF-beta 1 and extracellular matrix gene expression in rat mesangial cells. Ang II alone has modest effects, while Ang II and insulin have additive effects. To explain the mechanism of these additive effects, we investigated the action of Ang II on insulin signaling and the effect of insulin on Ang II AT1 receptor mRNA expression. Ang II did not enhance insulin-induced insulin receptor substrate-1 (IRS-1) phosporylation or phosphatidylinositol3 (PI-3) kinase activity, but did enhance insulin-induced mitogen activated protein (MAP) kinase activity. Insulin increased message levels of AT1 receptor by twofold. These results suggest that enhancement of MAP kinase activity and AT1 receptor regulation by insulin may contribute to the additive effects of insulin and Ang II in mesangial cells.

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Troglitazone (TRO) is an oral insulin-sensitizer that has direct effects on the vasculature to inhibit cell growth and migration. In vascular smooth muscle cells (VSMCs), insulin transduces a mitogenic signal that is dependent on the ERK1/2 MAP kinases. We examined the effects of TRO on this pathway and found that it inhibits mitogenic signaling. In quiescent VSMCs, insulin (1 microM) induced a 3.2-fold increase in DNA synthesis. TRO (1-20 microM) inhibited insulin-stimulated DNA synthesis by 72.8% at the maximal concentration. TRO at I and 10 microM had no significant effect on insulin-stimulated ERK1/2 activity. At 20 microM, however, TRO modestly enhanced insulin-stimulated ERK1/2 activity by 1.5-fold. ERKs transduce a mitogenic signal by phosphorylating transcription factors such as Elk-1. which regulate critical growth-response genes. We used GAL-Elk-1 expression plasmids to detect ERK-dependent activation of Elk-1. TRO at 1-20 microM potently inhibited insulin-stimulated, ERK1/2-dependent Elk-1 transcription factor activity. Neither early steps in insulin signaling nor the phosphatidylinositol 3-kinase (PI3K) branch of this pathway were affected by TRO, because it had no effect on IRS-1 phosphorylation, PI3K/IRS-1 association, or Akt phosphorylation. Because TRO is a known ligand for the nuclear transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma), we tested two other ligands for this receptor, rosiglitazone (RSG) and 15-deoxy-delta12,14 prostaglandin J2 (15d-PGJ2). Both also inhibited insulin-induced DNA synthesis. In summary, these data show that TRO inhibits mitogenic signaling by insulin at a point distal of ERK1/2 activation, potentially by a PPARgamma-mediated inhibition of ERK-dependent phosphorylation and activation of nuclear transcription factors that regulate cell growth.

The aim of this study was to investigate the effect of cardiac troponin I-interacting kinase (TNNI3K) on sepsis-induced myocardial dysfunction (SIMD) and further explore the underlying molecular mechanisms. In this study, a lipopolysaccharide- (LPS-) induced myocardial injury model was used. qRT-PCR was performed to detect the mRNA expression of TNNI3K. Western blot was conducted to quantitatively detect the expression of TNNI3K and apoptosis-related proteins (Bcl-2, Bax, and caspase-3). ELISA was performed to detect the content of lactate dehydrogenase (LDH) and creatine kinase (CK). TUNEL assay was used to detect the apoptosis of H9C2 cells. In LPS-induced H9C2 cells, TNNI3K was up regulated. Besides, the CK activity, the content of LDH, and the apoptosis of H9C2 cells were significantly increased after treatment with LPS. Silencing TNNI3K decreased the LDH release activity and CK activity and inhibited apoptosis of H9C2 cell. Further research illustrated that si-TNNI3K promoted the protein expression of Bcl-2 and decreased the protein expression of Bax and cleaved caspase-3. The study concluded that TNNI3K was upregulated in LPS-induced H9C2 cells. Importantly, functional research findings indicated that silencing TNNI3K alleviated LPS-induced H9C2 cell injury by regulating apoptosis-related proteins.