FIBRONECTIN GENE-EXPRESSION, SYNTHESIS, AND ACCUMULATION DURING IN-VITRO DIFFERENTIATION OF CHICKEN OSTEOBLASTS

A well-defined chicken osteoblast culture system((18)) has been used t o examine fibronectin (FN) mRNA levels, synthesis, and accumulation du ring in vitro differentiation and matrix mineralization, Immunofluores cent staining of cells after 6 or 18 days in culture revealed that FN was initially associated with the cell surface and in partial coalignm ent with cytoskeletal elements while at the latter time most FN was as sociated with the extracellular matrix as a ubiquitous fibrillar netwo rk Western blot analysis of total cell-associated proteins also detect ed FN at all culture times, However, when results were normalized to c ellular DNA, FN levels increased until days 12-16 and remained relativ ely constant thereafter, Similarly, FN synthesis as measured by [S-35] -methionine labeling, and immunoprecipitation was greatest in early cu ltures (culture day 3) and then declined such that synthesis decreased 60% at day 18 and 94% after 24-31 days. FN mRNA levels as measured by Northern blot analysis were well correlated with FN synthesis. These results clearly show that FN is made by primary osteoblasts during the ir in vitro maturation, In contrast to other osteoblast markers such a s alkaline phosphatase, osteocalcin, and osteopontin, whose expression increases as cells differentiate, FN accumulates in the matrix during periods of early cell growth and attachment and then remains proporti onal to cell number, Results with FN differ from those obtained,vith c ollagen which continues to accumulate in the extracellular matrix duri ng osteoblast maturation, These results are consistent with FN being i mportant for the initial attachment of early osteoblasts or osteoblast precursors to the pericellular matrix.