Duca Laurent, Lambert Elise, Debret Romain, Rothhut Bernard, Blanchevoye Charlotte, Delacoux Frédéric, Hornebeck William, Martiny Laurent, Debelle Laurent
Laboratoire de Biochimie, UMR CNRS 6198, IFR53 Biomolécules, Faculty of Sciences, Université de Reims Champagne Ardenne, Moulin de la Housse, BP 1039, 51687 Reims Cedex 2, France.
Mol Pharmacol. 2005 Apr;67(4):1315-24. doi: 10.1124/mol.104.002725. Epub 2005 Jan 13.
Elastin peptides (EPs) produced during cancer progression bind to the elastin binding protein (EBP) found at the surface of dermal fibroblasts, leading to the expression of collagenase-1 gene. The production of this enzyme involved in stromal reaction is caused by the sustained activation of the extracellular signal-regulated kinases 1/2 (ERK1/2) pathway via cAMP/protein kinase A (PKA) and phosphatidylinositol 3-kinase (PI3K). However, the mechanism of these signaling events remains unknown. We show that kappa-elastin (kappaE), a commonly used EP, induces maximum phosphorylation of mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK)1/2 and ERK1/2 after 30 min. The simultaneous inhibition of PKA and PI3K, by N-(2-(p-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide (H89) and 2-(4-morpholynil)-8-phenyl-4H-1-bemzopyran-4-one (LY294002), respectively, blocked MEK1/2 and ERK1/2 phosphorylation, as did lactose, an EBP antagonist. kappaE induced Raf-1 phosphorylation and activation in a PI3K-dependent manner. In our system, the PI3K p110gamma is expressed and activated by betagamma-derived subunits from a pertussis toxin-sensitive G protein after fibroblast stimulation. Pertussis toxin also blocks the Raf-1/MEK1/2/ERK1/2 phosphorylation cascade. In addition, we found that B-Raf is expressed in dermal fibroblasts and activated in a PKA-dependent manner after kappaE treatment, thereby integrating PKA signals to MEK1/2. It is noteworthy that Ras involvement was excluded because ERK1/2 activation by kappaE was not blocked in RasN17-transfected fibroblasts. Together, our results identify a novel Ras-independent ERK1/2 activation system in which p110gamma/Raf-1/MEK1/2 and PKA/B-Raf/MEK1/2 cooperate to activate ERK1/2. Thus, p110gamma and B-Raf seem to be important modulators of dermal fibroblasts physiology and should now qualify as therapeutic targets in strategies aiming at limiting elastin degradation contribution to cancer progression.
癌症进展过程中产生的弹性蛋白肽（EPs）与真皮成纤维细胞表面发现的弹性蛋白结合蛋白（EBP）结合，导致胶原酶-1基因的表达。这种参与基质反应的酶的产生是由细胞外信号调节激酶1/2（ERK1/2）途径通过cAMP/蛋白激酶A（PKA）和磷脂酰肌醇3-激酶（PI3K）的持续激活引起的。然而，这些信号事件的机制仍然未知。我们发现，常用的EPκ-弹性蛋白（κE）在30分钟后诱导丝裂原活化蛋白激酶/细胞外信号调节激酶（MEK）1/2和ERK1/2的最大磷酸化。分别用N-（2-（对溴肉桂氨基）乙基）-5-异喹啉磺酰胺（H89）和2-（4-吗啉基）-8-苯基-4H-1-苯并吡喃-4-酮（LY294002）同时抑制PKA和PI3K，以及用EBP拮抗剂乳糖，均可阻断MEK1/2和ERK1/2的磷酸化。κE以PI3K依赖的方式诱导Raf-1磷酸化和激活。在我们的系统中，PI3K p110γ在成纤维细胞刺激后由百日咳毒素敏感G蛋白的βγ衍生亚基表达并激活。百日咳毒素也阻断Raf-1/MEK1/2/ERK1/2磷酸化级联反应。此外，我们发现B-Raf在真皮成纤维细胞中表达，并在κE处理后以PKA依赖的方式激活，从而将PKA信号整合到MEK1/2。值得注意的是，由于在RasN17转染的成纤维细胞中κE对ERK1/2的激活未被阻断，因此排除了Ras的参与。总之，我们的结果确定了一种新的不依赖Ras的ERK1/2激活系统，其中p110γ/Raf-1/MEK1/2和PKA/B-Raf/MEK1/2协同激活ERK1/2。因此，p110γ和B-Raf似乎是真皮成纤维细胞生理学的重要调节因子，现在应该被视为旨在限制弹性蛋白降解对癌症进展影响的治疗策略中的治疗靶点。
