Two closely related human members of chitinase-like family, CHI3L1 and CHI3L2, activate ERK1/2 in 293 and U373 cells but have the different influence on cell proliferation

Abstract

The activation of extracellular signal-regulated kinases (ERK1/2) has been associated with specific outcomes. Sustained activation of ERK1/2 by nerve growth factor (NGF) is associated with translocation of ERKs to the nucleus of PC12 cells and precedes their differentiation into sympathetic-like neurons whereas transient activation by epidermal growth factor (EGF) leads to cell proliferation. It was demonstrated that different growth factors initiating the same cellular signaling pathways may lead to the different cell destiny, either to proliferation or to the inhibition of mitogenesis and apoptosis. Thus, further investigation on kinetic differences in activation of certain signal cascades in different cell types by biologically different agents are necessary for understanding the mechanisms as to how cells make a choice between proliferation and differentiation.It was reported that chitinase 3-like 1 (CHI3L1) protein promotes the growth of human synovial cells as well as skin and fetal lung fibroblasts similarly to insulin-like growth factor 1 (IGF1). Both are involved in mediating the mitogenic response through the signal-regulated kinases ERK1/2. In addition, CHI3L1 which is highly expressed in different tumors including glioblastomas possesses oncogenic properties. As we found earlier, chitinase 3-like 2 (CHI3L2) most closely related to human CHI3L1 also showed increased expression in glial tumors at both the RNA and protein levels and stimulated the activation of the MAPK pathway through phosphorylation of ERK1/2 in 293 and U87 MG cells. The work described here demonstrates the influence of CHI3L2 and CHI3L1 on the duration of MAPK cellular signaling and phosphorylated ERK1/2 translocation to the nucleus. In contrast to the activation of ERK1/2 phosphorylation by CHI3L1 that leads to a proliferative signal (similar to the EGF effect in PC12 cells), activation of ERK1/2 phosphorylation by CHI3L2 (similar to NGF) inhibits cell mitogenesis and proliferation.

Keywords: ERK1/2; HC gp-39; MAP kinase).; YKL-39) protein; YKL-40) protein; chitinase 3-like 1 (CHI3L1; chitinase 3-like 2 (CHI3L2.

Figure 1

Effect of CHI3L1 and CHI3L2 on 293 cells and U373 cells growth. Dose-dependent effect of CHI3L1 and CHI3L2 application on [³H]thymidine incorporation in 293 cells (A) and U373 cells (B). Dose-dependent effect of CHI3L1 and CHI3L2 on 293 cells (C) and U373 cells (D) proliferation. *p < 0.05, **p < 0.01 and ***p < 0.001 vs control group. Values were means±SD (n=6).

Figure 2

CHI3L2-inhibition of CHI3L1 proliferative effect and CHI3L1-inhibition of CHI3L2-inducible apoptosis in 293 cells and U373 cells. Cultured 293 cells (A, C) or U373 cells (B, D) were untreated (0 ng/ml), treated for 24 hours with CHI3L1, CHI3L2, or IGF1 (concentrations indicated) and treated with a mixture of these proteins. (A, B) DNA synthesis rate analyzed by [3H]thymidine incorporation evaluation. (C, D) Caspase 3/7 activity determined by a caspase 3/7-Glo assay. *p < 0.05, **p < 0.01 and ***p < 0.001 vs control group. Values were means±SD (n=6).

Figure 3

Dose-dependent phosphorylation of ERK1/2 induced by CHI3L1 and CHI3L2. (A) Western-blot analysis of U373 cells with anti-phospho-ERK and pan-anti-ERK antibodies after CHI3L1 or CHI3L2 cell treatment (60 min). (B) Western-blot analysis of U373 cell, 293 cell, and MG-63 cell media or purified CHI3L1 and CHI3L2 proteins with anti-CHI3L1, -CHI3L2 or -ACTB antibodies.

Figure 4

Influence of U0126 inhibitor on activation of ERK1/2-mediated MAPK signaling pathway. (A) 293 cells and U373 cells were serum-starved and exposed or not exposed to the ERK1/2 inhibitor U0126 (20 µM) for 30 min prior to addition of CHI3L1 or CHI3L2 (100 ng/ml). Cell lysates were prepared after 60 min of stimulation and levels of phosphorylated and total ERK1/2 were analyzed as described for Figure 3 A. (B) Cell proliferation rate of 293 cells and U373 cells exposed or not exposed to the U0126 inhibitor after CHI3L1 or CHI3L2 (200 ng/ml) stimulation (24 hrs). *p < 0.05, **p < 0.01. Values were means±SD (n=6).

Figure 5

Time course of CHI3L1- or CHI3L2-induced ERK1/2 phosphorylation and nuclear accumulation in 293 cells and U373 cells. (A,E), Time course of ERK1/2 phosphorylation by CHI3L1 or CHI3L2 (100 ng/ml) treatment in 293 cells(A) or in U373 cells (E) . (B, F) Densitometric analysis of Figure A (B) or Figure E (F). Results are expressed as fold of phosphorylation of ERK1/2 compared with total ERK1/2. (C, G) Phosphorylated ERK1/2 cellular localization invoked by CHI3L1 in 293 cells (C) or in U373 cells (G). (D,H) Phosphorylated ERK1/2 cellular localization invoked by CHI3L2 in 293 cells (D) or in U373 cells (H).

Figure 6

Key functional motifs of human CHI3L1 and CHI3L2. Pairwise sequence alignment was produced with ESPript 2.2. Elements of secondary structure of CHI3L1 are shown. B - basic amino acid, X - any, but proline.