Study of the role of protein kinase C-phosphoipase D signaling pathway in pigment cells.

众所周知，磷脂酶D （PLD）在多种细胞的信号转导中起着至关重要的作用，当细胞受到刺激时，它被蛋白激酶Cα (PKCα)激活。为了阐明PLD在黑色素瘤中的作用，我们利用免疫组织学技术研究了PLD1和PKCα在肢端色素性黑色素瘤（ALM）和浅表性扩散黑色素瘤（SSM）原发和转移灶中的表达。此外，利用腺病毒介导的基因转移技术，在人黑色素瘤细胞系HM3KO中检测了PKCα调控PLD1的机制。PLD1和DKCα在SSM原发和转移性病变中均有强烈表达。相反，在ALM病变中，这两种蛋白的表达随着肿瘤的进展而显著增加；PLD1和PKCα的表达在原发性ALM病变的径向生长期几乎为阴性，在晚期ALM病变（包括垂直生长）中以进展相关的方式同步增加。免疫沉淀研究表明，PLD1和PKCα在静息黑色素瘤细胞中存在生理关联。腺病毒介导的同时过表达PLD1和PKCα，或PLD1和PKCα激酶阴性突变体的HM3KO细胞进一步免疫沉淀研究显示，在缺乏外部信号的情况下，黑色素瘤细胞中PKCα和PKCα激酶阴性突变体都与PLD1相关。腺病毒载体在黑色素瘤细胞中过表达PKCα或PKCα激酶阴性突变体，以病毒剂量依赖的方式增强基础PLD活性。此外，基础PLD活性的增强增加了HM3KO细胞的体外侵袭潜能。这些结果表明，PLD1和PKCα的上调在ALM从径向生长期向垂直生长期的发展过程中起着重要作用。目前的研究结果还表明，PKCα与PLD1相关，并在黑色素瘤细胞中以蛋白磷酸化不依赖的方式增强基础PLD活性，这有助于细胞的高侵袭潜力。少

It is well known that phospholipase D (PLD) plays a crucial role in the signal transduction of many types of cells, and is activated by protein kinase C α (PKCα) when cells are stimulated. To elucidate the role of PLD in melanoma, the expression of PLD1 and PKCα in primary and metastatic lesions of acral lentiginous melanoma (ALM) and superficial spreading melanoma (SSM) was investigated using immunohistological techniques. In addition, the mechanism of regulation of PLD1 by PKCα was examined in a human melanoma cell line HM3KO using an adenovirus-mediated gene transfer technique. Both PLD1 and DKCα were strongly expressed in primary and metastatic lesions of SSM. Conversely, in ALM lesions, the expression of these two proteins increased dramatically with tumor progression ; the expression of both PLD1 and PKCα was almost negative in the radial growth phase of primary ALM lesions, and increased synchronously in a progression-related manner in advanced ALM esions, including vertical gro … More wth phase and metastatic lesions. Immunoprecipitation study showed that PLD1 and PKCα are associated physiologically in resting melanoma cells. Further immunoprecipitation study using HM3KO cells after adenovirus-mediated simultaneous overexpression of PLD1 and PKCα, or PLD1 and the kinase-negative mutant of PKCα revealed that both PKCα and the kinase-negative mutant of PKCα are associated with PLD1 in melanoma cells in the absence of an extemal signal. Overexpression of PKCα or the kinase-negative mutant of PKCα in melanoma cells by the adenovirus vectors resulted in the enhancement of basal PLD activity in a viral dose-dependent manner. Furthermore, enhanced basal PLD activity increased the in vitro invasive potential of HM3KO cells. These results suggest that upregulation of PLD1 and PKCα plays a rote in the progression of ALM from the radial growth phase to the vertical growth phase. The present results also suggest that PKCα associates with PLD1 and enhances basal PLD activity in a protein phosphorylation-independent manner in melanoma cells, which contributes to the cell's high invasive potential. Less