EMBRYOGLYCAN ECTODOMAINS REGULATE BIOLOGICAL-ACTIVITY OF FGF-2 TO EMBRYONIC STEM-CELLS

Basic fibroblast growth factor (FGF-2) functions as a natural inducer of mesoderm, regulator of cell differentiation and autocrine modulator of cell growth and transformation. The FGF-2 signals are transduced t hrough receptors with intrinsic protein tyrosine kinase activity. Howe ver, receptor binding and activation is governed by extracellular matr ix, cell surface or soluble proteoglycans. This paper focuses on the r ole of proteoglycans synthesized by embryonic cells, embryoglycans, in FGF-2 signaling via FGF receptor-1 (FGFR-1). We found that embryoglyc an ectodomain Lewis X, analog of developmentally regulated embryonic c ell surface epitope TEC 1, promotes oligomerization of FGF-2 in the ce ll free chemical crosslinking, In vitro assays show that a large molar excess of extracellular Lewis X does not inhibit binding of FGF-2 to embryonic stem (ES) cells, but prevents the mitogenic effect of FGF-2. Western blot analysis of ES cells revealed the presence of abundant 5 2 kDa and trace amounts of 67 and 125 kDa isoforms of FGFR-1. However, none of these isoforms undergo any detectable changes in tyrosine pho sphorylation under the conditions that modulate the mitogenic effect o f FGF-2. Rather, a primary substrate of all receptor tyrosine kinases, phospholipase Cy (PLC gamma), is activated by both FGF-2 and Lewis X. The combination, FGF-2 plus Lewis X, leads to weak inhibition, when c ompared with the effects of FGF-2 and Lewis X, respectively. In accord ance, the level of phosphorylation of non-receptor tyrosine kinase c-S rc is reduced in a reversed pattern to PLC gamma Furthermore, in this particular cell type we show the presence of activated forms of extrac ellular signal-related kinase (ERK) in all nontreated and treated cell s. These findings demonstrate that embryoglycan ectodomains may act as negative regulators of FGF-2-induced ES cell proliferation, most like ly through the FGFR-1-independent signaling pathway.