Nair D, Misra R P, Sallis J D, Cheung H S
Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
J Biol Chem. 1997 Jul 25;272(30):18920-5. doi: 10.1074/jbc.272.30.18920.
Calcium deposition diseases caused by calcium pyrophosphate dihydrate (CPPD) and basic calcium phosphate (BCP) crystals are a significant source of morbidity in the elderly. We have shown previously that both types of crystals can induce mitogenesis, as well as metalloproteinase synthesis and secretion by fibroblasts and chondrocytes. These responses may promote degradation of articular tissues. We have also shown previously that both CPPD and BCP crystals activate expression of the c-fos and c-jun proto-oncogenes. Phosphocitrate (PC) can specifically block mitogenesis and proto-oncogene expression induced by either BCP or CPPD crystals in 3T3 cells and human fibroblasts, suggesting that PC may be an effective therapy for calcium deposition diseases. To understand how PC inhibits BCP and CPPD-mediated cellular effects, we have investigated the mechanism by which BCP and CPPD transduce signals to the nucleus. Here we demonstrate that BCP and CPPD crystals activate a protein kinase signal transduction pathway involving p42 and p44 mitogen-activated protein (MAP) kinases (ERK 2 and ERK 1). BCP and CPPD also cause phosphorylation of a nuclear transcription factor, cyclic AMP response element-binding protein (CREB), on serine 133, a residue essential for CREB's ability to transactivate. Treatment of cells with PC at concentrations of 10(-3) to 10(-5) M blocked both the activation of p42/p44 MAP kinases, and CREB serine 133 phosphorylation, in a dose-dependent fashion. At 10(-3) M, a PC analogue, n-sulfo-2-aminotricarballylate and citrate also modulate this signal transduction pathway. Inhibition by PC is specific for BCP- and CPPD-mediated signaling, since all three compounds had no effect on serum-induced p42/P44 or interleukin-1beta induced p38 MAP kinase activities. Treatment of cells with an inhibitor of MEK1, an upstream activator of MAPKs, significantly inhibited crystal-induced cell proliferation, suggesting that the MAPK pathway is a significant mediator of crystal-induced signals.
由二水焦磷酸钙（CPPD）和碱性磷酸钙（BCP）晶体引起的钙沉积疾病是老年人发病的一个重要原因。我们之前已经表明，这两种类型的晶体都可以诱导有丝分裂，以及成纤维细胞和软骨细胞合成并分泌金属蛋白酶。这些反应可能会促进关节组织的降解。我们之前还表明，CPPD和BCP晶体都会激活原癌基因c-fos和c-jun的表达。磷酸柠檬酸（PC）可以特异性地阻断BCP或CPPD晶体在3T3细胞和人成纤维细胞中诱导的有丝分裂和原癌基因表达，这表明PC可能是治疗钙沉积疾病的有效疗法。为了了解PC如何抑制BCP和CPPD介导的细胞效应，我们研究了BCP和CPPD将信号转导至细胞核的机制。在此我们证明，BCP和CPPD晶体激活了一条涉及p42和p44丝裂原活化蛋白（MAP）激酶（ERK 2和ERK 1）的蛋白激酶信号转导途径。BCP和CPPD还会导致一种核转录因子——环磷酸腺苷反应元件结合蛋白（CREB）的丝氨酸133位点发生磷酸化，该位点是CREB具有反式激活能力所必需的。用浓度为10^(-3)至10^(-5) M的PC处理细胞，会以剂量依赖的方式阻断p42/p44 MAP激酶的激活以及CREB丝氨酸133位点的磷酸化。在10^(-3) M时，一种PC类似物——n-磺基-2-氨基三羧酸盐和柠檬酸盐也会调节这条信号转导途径。PC的抑制作用对BCP和CPPD介导的信号传导具有特异性，因为这三种化合物对血清诱导的p42/P44或白细胞介素-1β诱导的p38 MAP激酶活性均无影响。用MAPKs的上游激活剂MEK1的抑制剂处理细胞，可显著抑制晶体诱导的细胞增殖，这表明MAPK途径是晶体诱导信号的重要介导者。
