Serine peptidase inhibitor Kazal type I (SPINK1) promotes BRL-3A cell proliferation via p38, ERK, and JNK pathways

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(2)/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(1) 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.