Enhanced expression of the osteoblastic phenotype on substrates that modulate fibronectin conformation and integrin receptor binding.
Integrins represent the primary mechanism of cell-extracellular matrix interactions and control cell morphology, proliferation, and differentiation. We have previously shown that substrate-dependent modulation of adsorbed fibronectin (Fn) conformation alters alpha5beta1 integrin binding to Fn and directs C2C12 myoblast proliferation and differentiation (Mol. Biol. Cell 10 (1999) 785). The model substrates used in these experiments were bacteriological (untreated) polystyrene (B), tissue culture polystyrene (T), and type-I collagen-coated T (C). In the present study, we examined MC3T3-EI osteoblast-like cell differentiation on Fn-coated B, T, and C substrates. Immunofluorescence staining revealed substrate-dependent differences in integrin alpha5beta1 binding and clustering into focal adhesions (C > T > B), consistent with our previous integrin binding analysis. Alkaline phosphatase activity and matrix mineralization showed substrate-dependent differences (C > T > B, p < 0.05). Similar trends were observed for alkaline phosphatase, osteocalcin, and bone sialoprotein gene expression. Blocking experiments with antibodies directed against Fn completely inhibited matrix mineralization on Fn-coated C, indicating that Fn is critical to expression of the osteoblastic phenotype on this extracellular matrix component. These substrate-dependent differences in osteoblast differentiation correlated with differences in alpha5beta1 binding, suggesting that these differences arise from substrate modulation of integrin-matrix interactions. Substrate-dependent modulation of cell function may provide a versatile mechanism to control cell responses in numerous biomedical applications.
Stephansson
Sean N
SN
GTEC Undergraduate Research Scholars Program, Atlanta, GA 30332-0363, USA.
Byers
Benjamin A
BA
García
Andrés J
AJ
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D000906:Antibodies / Q000378:metabolism; D002113:Calcification, Physiologic / Q000502:physiology; D002417:Cattle; D002460:Cell Line; D005353:Fibronectins / Q000737:chemistry / Q000378:metabolism*; D022001:Focal Adhesions / Q000378:metabolism; D006801:Humans; D039081:Integrin alpha5beta1 / Q000378:metabolism*; D051379:Mice; D010006:Osteoblasts / Q000166:cytology / Q000502:physiology*; D010641:Phenotype; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D017434:Protein Structure, Tertiary
D000906:Antibodies; D005353:Fibronectins; D039081:Integrin alpha5beta1; D000469:Alkaline Phosphatase