Expression and function of chitinase-3 like protein 1 (CHI3L1) in brain glial tumors

摘要

Chitinase-3 like protein 1 (CHI3L1, HC gp-39 or YKL-40) is a human cartilage glycoprotein encoded by CHI3L.1 gene revealed by us previously among the highly upregulated genes in glioblastoma – the most aggressive type of human brain tumors. Increased levels of circulating CHI3L1 have been reported in both patients with breast and colorectal cancer and patients with liver cirrhoses. In connective tissue cells CHI3L1 initiates signaling cascade which leads to increased cell proliferation. The objective of this project is to find possible involvement of CHI3L1 into the main cellular signaling pathways, mainly into MAPK and P13K cascades in glial cells as well as to find possible CHI3L1 protein partners. Methods. cDNA CHI3L1 was cloned into pET--24a vector, expressed in E. coda cells, a recombinant protein was purified on Ni-NTA-agarose. U-87 MG and HEK-293 cells were grown about to the confluence in DMEM supplemented with 10 % FBS and 100 tg/m1 penicillin and 100 units/ml streptomycin in 6-well tissue-culture plates. Human embryonic kidney 293 (HEK-293) cells were serum-starved for 24 h, followed by exposure to MG-б3 cell medium enriched with CНI3L1 or recombinant CHI3L1 for lh. At the end of the incubation period, cell layers were washed twice with ice-cold PBS, lysed in SDS buffer, and the cell lysates were analysed by SDS/PAGE and Western blotting. The blots were exposed to the phosphorylation-specific antibodies at dilutions recommended by the manufacturer and reprobed with the pan-specific antibodies to determine total ERK1/2 protein. Results. The results obtained have shown that CHI3L1 plays a certain role in the mitogen-activated protein signaling cascade (MAPK) involved in the fibroblast mitogenic response. To test a possibility of CHI3L1 participation in the activation of extracellular signal-regulated protein kinases (ERK1/ERK2), we used the human embryonic kidney 293 (HEK-293) cell line, which like many other cell types, in monolayer culture grows in unsupplemented culture medium for no longer than one week loosing whereupon its viability. The results suggest that addition of CHI3L1 stimulates ERK1/ERK2 phosphorylation in these cells. No ERK1/ERK2 phosphorylation was observed either in the cells exposed to the medium without CHI3L1 or to the medium supplemented with BSA. In contrast to HEK-293, phosphorylation of ERK1 /ERK2 could be seen in U-87 MG cells exposed to the medium without CНI3L1 addition or in cells exposed to the medium supplemented with BSA. The reason for this difference may be explained by our previous finding that U-87 MG cells produce CHI3L1. We cloned CHI3L1 cDNA into expression vector pCMV-flag N-terminal to search for potential CHI3L1 protein partners. The expression of recombinant protein was confirmed by transfection in mammalian cells and further Western blot analysis of the lysates. The search for partners of CHI3L1 with co-immunoprecipitation is the next step of our research. Conclusions. In this work we have found that CHI3L1 is involved in the activation of mitogen-activated cellular signaling pathway (MAPK) through phosphorylation of ERK1/ERK2 in human embryonic kidney cells and human glial cells.