Yokoo T, Kitamura M
Department of Medicine, University College London Medical School, Rayne Institute, England, United Kingdom.
Kidney Int. 1996 Sep;50(3):894-901. doi: 10.1038/ki.1996.389.
Glomerular mesangial cells express matrix metalloproteinase sromelysin in response to the proinflammatory cytokine IL-1 beta. The present study was conducted to identify intracellular machinery involved in this IL-1 action, especially focusing on the role of the TPA response element (TRE) located in the 5'-flanking region of the stromelysin gene. Using transient transfection with a pTRE-LacZ reporter plasmid, we detected no obvious up-regulation of TRE activity in rat mesangial cells following the IL-1 stimulation. However, the basal activity of TRE was found to be essential to the stromelysin induction, since (i) mesangial cells stably expressing a transdominant negative mutant of c-Jun, which effectively suppressed both basal and inducible TRE activity, exhibited the blunted expression of stromelysin in response to IL-1 beta, whereas (ii) transfection with a c-fos antisense gene, which suppressed only the inducible TRE activity, did not affect the stromelysin induction. To seek cooperative pathways required for the IL-1 action, we next focused on protein kinases, the potential regulators of the stromelysin gene. Stimulation of mesangial cells with a protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), induced the stromelysin transcript without affecting TRE activity. Depletion of intracellular PKC by high-dose PMA or inhibition of PKC activity with calphostin C suppressed the stromelysin induction by IL-1 beta, suggesting the crucial contribution of a PKC-mediated, but TRE-independent pathway. In contrast, either cAMP inducer forskolin or dibutyryl cAMP suppressed the IL-1-mediated stromelysin expression. An inhibitor of cAMP-dependent protein kinase A (PKA), HA1004, enhanced the IL-1 effect in a dose-dependent manner. Unexpectedly, the inhibitory action of PKA was not through cAMP response element (CRE) but through TRE, because (i) activation of CRE was not induced by IL-1 beta, and (ii) cAMP-mediated activation of PKA suppressed the basal TRE activity. These findings elucidated the unique, binary regulation of stromelysin by IL-1 beta; that is, IL-1 up-regulated the transcript via the PKC-dependent pathway under the cooperation with constitutively active TRE, and this stimulatory effect was in part counterbalanced by the IL-1-inducible PKA which down-regulated the basal TRE activity.
肾小球系膜细胞在促炎细胞因子白细胞介素-1β（IL-1β）的作用下表达基质金属蛋白酶（stromelysin）。本研究旨在确定参与这种IL-1作用的细胞内机制，特别关注位于stromelysin基因5'侧翼区域的佛波酯反应元件（TPA反应元件，TRE）的作用。使用pTRE-LacZ报告质粒进行瞬时转染，我们发现在IL-1刺激后大鼠系膜细胞中TRE活性没有明显上调。然而，发现TRE的基础活性对于stromelysin的诱导至关重要，因为（i）稳定表达c-Jun的反式显性负突变体的系膜细胞，该突变体有效抑制基础和诱导性TRE活性，对IL-1β的反应中stromelysin的表达减弱，而（ii）用c-fos反义基因转染，其仅抑制诱导性TRE活性，并不影响stromelysin的诱导。为了寻找IL-1作用所需的协同途径，我们接下来关注蛋白激酶，stromelysin基因的潜在调节因子。用蛋白激酶C（PKC）激活剂佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）刺激系膜细胞可诱导stromelysin转录本，而不影响TRE活性。高剂量PMA耗尽细胞内PKC或用钙泊三醇C抑制PKC活性可抑制IL-1β诱导的stromelysin表达，提示PKC介导但不依赖TRE的途径起关键作用。相反，cAMP诱导剂福斯可林或二丁酰cAMP均抑制IL-1介导的stromelysin表达。cAMP依赖性蛋白激酶A（PKA）的抑制剂HA1004以剂量依赖性方式增强IL-1的作用。出乎意料的是，PKA的抑制作用不是通过cAMP反应元件（CRE）而是通过TRE，因为（i）IL-1β未诱导CRE的激活，并且（ii）cAMP介导的PKA激活抑制基础TRE活性。这些发现阐明了IL-1β对stromelysin的独特二元调节；即，IL-1在与组成性活性TRE协同作用下通过PKC依赖性途径上调转录本，并且这种刺激作用部分被下调基础TRE活性的IL-1诱导的PKA所抵消。
