RCAS1 increases cell morphological changes in murine fibroblasts by reducing p38 phosphorylation

Abstract

Receptor‑binding cancer antigen expressed on SiSo cells (RCAS1) is a tumor‑associated antigen that is expressed in a number of human malignancies. RCAS1 acts as a ligand for a putative RCAS1 receptor that is present on various human cells including T and B lymphocytes and natural killer cells, in which it induces cell growth inhibition and apoptosis. It has been suggested that RCAS1 might serve an important role in tumor cell evasion from the host immune system. In fact, RCAS1 expression is related to malignant characteristics including tumor size, invasion depth, clinical stage and poor overall survival. The authors previously established doxycycline‑induced RCAS1 overexpression murine fibroblast L cells to analyze the biological functions of RCAS1 and reported that its expression inhibited cell cycle progression via the downregulation of cyclin D3, which subsequently induced apoptosis. Additionally, it was found that RCAS1 expression induced cell morphological changes prior to caspase‑mediated apoptosis. Thus, the present study examined signaling pathways associated with changes in cell morphology that were induced by RCAS1 expression. The data showed that increased RCAS1 expression caused a reduction in actin stress fibers and decreased cofilin phosphorylation. Recent studies have shown that p38 signaling regulates actin polymerization. The data the present study showed that increased RCAS1 expression significantly decreased p38 phosphorylation.

Keywords: actin dynamics; cell morphological change; p38 MAP kinase; receptor‑binding cancer antigen expressed on SiSo cells.

Figure 1.

The effect of soluble-form RCAS1 on wild-type L cells. (A) Detection of soluble-form RCAS1 in concentrated SiSo culture supernatant was measured by ELISA. SiSo Sup/RCAS1⁺: serum-free concentrated SiSo culture supernatant; SiSo Sup/RCAS1⁻: fraction of SiSo Sup/RCAS1⁺ with RCAS1 immunodepletion. (B) Observation of morphological changes in cells stimulated with SiSo Sup/RCAS1⁺ or SiSo Sup/RCAS1⁻; scale bar, 40 µm. RCAS1, Receptor-binding cancer antigen expressed on SiSo cells.

Figure 2.

Effect of RCAS1 expression on the morphology of L/ind RCAS1 cells. (A) Western blot analysis of RCAS1 expression in L/ind RCAS1 cells after Dox induction. Results are presented as the mean ± standard deviation of three independent experiments; **P<0.01 vs. Dox-0 h. (B) Observations of cell morphologies induced by RCAS1; scale bar, 40 µm. (C) Effect of RCAS1 expression on actin stress fibers (F-actin) in L/ind RCAS1 cells. RCAS1 (green) was detected with an anti-RCAS1 antibody and FITC-conjugated goat-anti mouse IgG. F-actin (red) was stained with rhodamine phalloidin; scale bar, 25 µm. (D) Cell index measurement by RTCA system. RCAS1, Receptor-binding cancer antigen expressed on SiSo cells; Dox, doxycycline; RTCA, real-time cell-monitoring analysis.

Figure 3.

Effect of RCAS1 expression on actin polymerization in L/ind RCAS1 cells. (A) F- and G-actin from L/ind RCAS1 cells were separated by ultracentrifugation and determined by western blotting. (B) Analysis of cofilin phosphorylation by western blotting in L/ind RCAS1 cells after Dox induction. Results are presented as the mean ± standard deviation of three independent experiments; *P<0.05 vs. Dox −0 h. RCAS1, Receptor-binding cancer antigen expressed on SiSo cells; G, G-actin; F, F-actin; Dox, doxycycline; p-, phosphorylated.

Figure 4.

Effect of RCAS1 expression on MAPKs in L/ind RCAS1 cells. The amounts and phosphorylation status of (A) p38 and (B) ERK1/2 were analyzed by western blotting. (C) Time-dependent change of intracellular molecules following RCAS1 expression in L/ind RCAS1. The results are presented as the mean ± standard deviation of three independent experiments; *P<0.05 vs. Dox −0 h; **P<0.01 vs. Dox −0 h. RCAS1, Receptor-binding cancer antigen expressed on SiSo cells; Dox, doxycycline; G, G-actin; F, F-actin.