Kaiura T L, Itoh H, Kent K C
Division of Vascular Surgery, New York Hospital/Cornell University Medical Center, New York, New York, 10021, USA.
J Surg Res. 1999 Jun 15;84(2):212-7. doi: 10.1006/jsre.1999.5646.
After evaluating various growth factors, cytokines, and extracellular matrix (ECM) proteins, we found that the most potent agonists of smooth muscle cell (SMC) fibronectin (Fn) production were transforming growth factor-beta (TGF-beta) and epidermal growth factor (EGF). To determine the possible signaling pathways involved in the production of this matrix protein, we investigated the role of the intracellular proteins, protein kinase C (PKC) and mitogen-activated protein kinase (MAP-K), in TGF-beta- and EGF-induced human vascular SMC Fn production.
After stimulation of human SMCs with TGF-beta (10 ng/ml) and EGF (100 ng/ml), Fn in the cell medium was assayed by immunoblotting using a specific antibody. PKC was activated by brief stimulation of SMC with phorbol 12,13-dibutyrate (PDBu) and inhibited by downregulation with PDBu or the inhibitor, GF109203X. MAP-K was inhibited with PD098059.
PKC activation increased basal and synergistically enhanced TGF-beta- and EGF-induced Fn production. However, inhibition of PKC by downregulation and GF109203X did not diminish Fn production by TGF-beta and EGF. Surprisingly, these two methods of inhibition slightly increased basal and agonist-induced Fn production. The MAP-K kinase inhibitor, PD098059, produced an almost complete inhibition of EGF and a partial inhibition of TGF-beta-induced Fn production.
Activation of PKC stimulates Fn production; however, neither TGF-beta nor EGF produce Fn through a PKC-dependent pathway. EGF and TGF-beta both stimulate Fn production at least in part through the intracellular signaling protein MAP-K. Understanding the signaling pathways involved in extracellular matrix protein production will allow the design of specific inhibitors of intimal hyperplasia.
在评估了多种生长因子、细胞因子和细胞外基质（ECM）蛋白后，我们发现平滑肌细胞（SMC）纤连蛋白（Fn）产生的最有效激动剂是转化生长因子-β（TGF-β）和表皮生长因子（EGF）。为了确定参与这种基质蛋白产生的可能信号通路，我们研究了细胞内蛋白蛋白激酶C（PKC）和丝裂原活化蛋白激酶（MAP-K）在TGF-β和EGF诱导的人血管SMC Fn产生中的作用。
用TGF-β（10 ng/ml）和EGF（100 ng/ml）刺激人SMC后，使用特异性抗体通过免疫印迹法检测细胞培养基中的Fn。通过用佛波醇12,13-二丁酸酯（PDBu）短暂刺激SMC激活PKC，并用PDBu或抑制剂GF109203X下调来抑制PKC。用PD098059抑制MAP-K。
PKC激活增加了基础Fn产生，并协同增强了TGF-β和EGF诱导的Fn产生。然而，通过下调和GF109203X抑制PKC并没有减少TGF-β和EGF诱导的Fn产生。令人惊讶的是，这两种抑制方法略微增加了基础和激动剂诱导的Fn产生。MAP-K激酶抑制剂PD09805产生了几乎完全抑制EGF和部分抑制TGF-β诱导的Fn产生。
PKC激活刺激Fn产生；然而，TGF-β和EGF均不通过PKC依赖性途径产生Fn。EGF和TGF-β均至少部分通过细胞内信号蛋白MAP-K刺激Fn产生。了解参与细胞外基质蛋白产生的信号通路将有助于设计内膜增生的特异性抑制剂。
