Figure 4. Integrin α11 is required for osteogenic differentiation and Wnt pathway activation in response to Osteolectin but exogenous collagen does not activate Wnt pathway signaling.
(A) Osteogenic differentiation in culture of parental or Itga11 deficient MC3T3-E1 cells, hBMSC#1 cells, and hBMSC#2 cells with PBS or recombinant mouse Osteolectin (n = 3 independent experiments). (B) qRT-PCR analysis of Dmp1 transcript levels in MC3T3-E1 cells, hBMSC#1 cells, and hBMSC#2 cells after 14 to 21 days of osteogenic differentiation (n = 5 independent experiments). (C) Parental or Itga11 deficient MC3T3-E1 cells, hBMSC#1 cells, and hBMSC#2 cells were transferred into osteogenic differentiation medium with or without recombinant mouse Osteolectin then lysed 24 hr later and immunoblotted for phospho-GSK3, β-catenin, and Actin (this blot is representative of blots from three independent experiments). (D) qRT-PCR analysis of Wnt target gene transcript levels in parental or Itga11 deficient MC3T3-E1 cells, hBMSC#1 cells, and hBMSC#2 cells 24 hr after transfer into osteogenic differentiation medium with PBS or Osteolectin (n = 5 independent experiments). (E) MC3T3-E1 cells, hBMSC#1 cells, and hBMSC#2 cells were transferred into osteogenic differentiation medium with PBS or 30 ng/ml Osteolectin, or 30 ng/ml recombinant Pro-Collagen 1α, then lysed 24 hr later and immunoblotted for phospho-GSK3, β-catenin, and Actin (this blot is representative of blots from three independent experiments). (F) qRT-PCR analysis of Wnt target gene transcript levels in MC3T3-E1 cells, hBMSC#1 cells, and hBMSC#2 cells 24 hr after transfer into osteogenic differentiation medium with PBS or 30 ng/ml Osteolectin, or 30 ng/ml Pro-Collagen 1α (n = 5 independent experiments). (G) Primary mouse bone marrow stromal cells were adherently cultured in osteogenic differentiation medium. PBS (control), recombinant human Pro-Collagen 1α, or recombinant human Osteolectin was added, then the cells were lysed 1 hr later and lysates were immunoblotted for phospho-FAK, phospho-GSK3, total FAK, and Actin (this blot is representative of blots from three independent experiments). (H) Primary mouse bone marrow stromal cells adherently cultured in osteogenic differentiation medium were treated with PBS (control), recombinant human Pro-Collagen 1α, or recombinant human Osteolectin then lysed 24 hr later and lysates were immunoblotted for phospho-FAK, phospho-GSK3, β-catenin, total FAK, and Actin (this blot is representative of blots from three independent experiments). (I) Primary mouse bone marrow stromal cells adherently cultured in osteogenic differentiation medium were treated with 30 ng/ml recombinant human Osteolectin or PBS (control) then lysed 2, 4, 6, 12, or 24 hr later. Nuclear and cytosolic/membrane-associated fractions were isolated from lysates by centrifugation then immunoblotted for β-catenin. As loading controls, Histone H3 was blotted in the nuclear fraction and Actin was blotted in the cytosolic/membrane-associated fraction (this blot is representative of blots from three independent experiments). (J) qRT-PCR analysis of Ctnnb1 transcript levels in cells from the experiment in panel (I) (n = 3 independent experiments). (K) Primary mouse bone marrow stromal cells from Lepr-Cre; Itga11fl/fl or littermate control mice were adherently cultured in osteogenic differentiation medium. PBS (control), recombinant human Pro-Collagen 1α, or recombinant human Osteolectin was added then the cells were lysed 1 hr later and lysates were immunoblotted for phospho-FAK and FAK (this blot is representative of blots from two independent experiments). All numerical data reflect mean ±standard deviation. Statistical significance was determined with two-way ANOVAs with Tukey’s multiple comparisons tests (A, B and D), Dunnett’s multiple comparisons tests (F), or Sidak’s multiple comparisons tests (J).