Collagen I Regulates the Self-Renewal of Mouse Embryonic Stem Cells Through α2β1 Integrin- and DDR1-Dependent Bmi-1

Adhesion of cells to extracellular matrix (ECM) influences vital aspects of anchorage-dependent cell behavior including survival, proliferation, and differentiation. However, the role of collagen I in mouse embryonic stem cells (mESCs) is not well-known. Therefore, in the present study we examined the effect of collagen I on mESC self-renewal and related signal pathways. Collagen I (10mg/ml) maintained mESCs in an undifferentiated state (Nanog, OCT4, and SSEA-1) and did not affect differentiation (GATA4, Tbx5, Fgf5, and Cdx2) in the presence of leukemia inhibitory factor (LIF). Treatment with collagen I bound both alpha 2 beta 1 integrin and discoidin domain receptor 1 (DDR1), and stimulated intracellular signaling pathways. Collagen I-bound alpha 2 beta 1 integrin increased integrin-linked kinase (ILK) phosphorylation, cleaved Notch protein expression in the nuclear fraction, and Gli-1 mRNA expression. In addition, collagen I-bound DDR1 increased GTP-bound Ras, phosphoinositide 3-kinase (PI3K) p85a catalytic subunit protein expression, and Akt and ERK phosphorylation. Importantly, collagen I increased Bmi-1 protein expression in the nucleus which was blocked by small interfering RNA (siRNA) specific for Gli-1 and ERK, showing that parallel pathways of integrins and DDR1 merge at Bmi-1. Furthermore, collagen I-induced p16 decrease and p-Rb increase were reversed by Bmi-1-specific siRNA. Moreover, Bmi-1 silencing abolished the collagen I-induced increase of proliferation indices and undifferentiation markers. These results indicate that collagen I stimulates the self-renewal of mESCs mediated by Bmi-1 through alpha 2 beta 1 integrin-dependent ILK, Notch, Gli-1, and DDR1-dependent Ras, PI3K/Akt, and ERK. J. Cell. Physiol. 226: 3422-3432, 2011. (C) 2011 Wiley Periodicals, Inc.