Figure 5.

Periostin promotes integrin-β3 signaling in vivo and in vitro. (A) pFAK expression in WT/KO NTS mice is shown by Western blot. The increase in FAK phosphorylation observed in WT mice after NTS is blunted in KO mice. (B) Immunohistochemical staining of pAKT shows upregulation in glomeruli and vessels in WT mice after NTS, which is blunted in KO mice. (C) Double immunofluorescence for p-FAK and p-AKT (green) with CD44 or nestin (red) shows that both proteins are expressed by activated parietal epithelial cells and podocytes in the glomeruli of NTS mice. (D) E11 immortalized podocytes were incubated with recombinant mouse periostin (400 ng/ml) for the indicated time points and Western blot analysis was performed for integrin-β3, pFAK, pAKT, and GAPDH, with quantification graphs shown on the right. At 6 hours of periostin incubation integrin-β3 and its downstream effectors pFAK and pAKT were upregulated. (E) E11 cells were incubated for 6 hours with increasing doses of recombinant mouse periostin (400 and 1000 ng/ml), followed by Western blot analysis of integrin-β3, pFAK, FAK, pAKT, AKT, and GAPDH. Quantifications are shown on the right. pFAK and pAKT remain elevated after incubation with increasing doses of periostin. Scale bars, 100 μm (B and C). n=6–7 mice per group. Animals were euthanized after 14 days of NTS administration. For cell cultures, quantifications of three independent experiments performed in triplicates are shown. *P<0.05 versus control; **P<0.01 versus WT PBS or control.