Chang Cuifang, Zhao Weiming, Luo Yaru, Xi Lingling, Chen Shasha, Zhao Congcong, Wang Gaiping, Guo Jianlin, Xu Cunshuan
State Key Laboratory Cultivation Base for Cell Differentiation Regulation and Henan Engineering Laboratory for Bioengineering and Drug Development, College of Life Science, Henan Normal University, Xinxiang, China.
Cell Biochem Funct. 2017 Aug;35(6):339-348. doi: 10.1002/cbf.3288.
Serine peptidase inhibitor Kazal type I (SPINK1) has the similar spatial structure as epidermal growth factor (EGF); EGF can interact with epidermal growth factor receptor (EGFR) to promote proliferation in different cell types. However, whether SPINK1 can interact with EGFR and further regulate the proliferation of hepatocytes in liver regeneration remains largely unknown. In this study, we investigated the role of SPINK1 in a rat liver hepatocyte line of BRL-3A in vitro. The results showed the upregulation of endogenous Spink1 (gene addition) significantly increased not only the cell viability, cell numbers in S and G /M phase, but also upregulated the genes/proteins expression related to cell proliferation and anti-apoptosis in BRL-3A. In contrast, the cell number in G phase and the expression of pro-apoptosis-related genes/proteins were significantly decreased. The similar results were observed when the cells were treated with exogenous rat recombinant SPINK1. Immunoblotting suggested SPINK1 can interact with EGFR. By Ingenuity Pathway Analysis software, the SPINK1 signalling pathway was built; the predicted read outs were validated by qRT-PCR and western blot; and the results showed that p38, ERK, and JNK pathways-related genes/proteins were involved in the cell proliferation upon the treatment of endogenous Spink1 and exogenous SPINK1. Collectively, SPINK1 can associate with EGFR to promote the expression of cell proliferation-related and anti-apoptosis-related genes/proteins; inhibit the expression of pro-apoptosis-related genes/proteins via p38, ERK, and JNK pathways; and consequently promote the proliferation of BRL-3A cells. For the first time, we demonstrated that SPINK1 can associate with EGFR to promote the proliferation of BRL-3A cells via p38, ERK, and JNK pathways. This work has direct implications on the underlying mechanism of SPINK1 in regulating hepatocytes proliferation in vivo and liver regeneration after partial hepatectomy.
丝氨酸蛋白酶抑制剂Kazal I型（SPINK1）具有与表皮生长因子（EGF）相似的空间结构；EGF可与表皮生长因子受体（EGFR）相互作用，促进不同细胞类型的增殖。然而，SPINK1是否能与EGFR相互作用并进一步调节肝再生中肝细胞的增殖，目前仍知之甚少。在本研究中，我们在体外研究了SPINK1在大鼠肝脏BRL-3A肝细胞系中的作用。结果显示，内源性Spink1（基因添加）的上调不仅显著增加了细胞活力、S期和G/M期的细胞数量，还上调了BRL-3A中与细胞增殖和抗凋亡相关的基因/蛋白质表达。相反，G期细胞数量以及促凋亡相关基因/蛋白质的表达显著降低。当用外源性大鼠重组SPINK1处理细胞时，观察到了类似的结果。免疫印迹表明SPINK1可与EGFR相互作用。通过 Ingenuity Pathway Analysis软件构建了SPINK1信号通路；预测结果通过qRT-PCR和western blot进行验证；结果表明，内源性Spink1和外源性SPINK1处理后，p38、ERK和JNK通路相关基因/蛋白质参与了细胞增殖。总体而言，SPINK1可与EGFR结合，促进细胞增殖相关和抗凋亡相关基因/蛋白质的表达；通过p38、ERK和JNK通路抑制促凋亡相关基因/蛋白质的表达；从而促进BRL-3A细胞的增殖。我们首次证明，SPINK1可与EGFR结合，通过p38、ERK和JNK通路促进BRL-3A细胞的增殖。这项工作对SPINK1在体内调节肝细胞增殖和部分肝切除术后肝再生的潜在机制具有直接意义。
