Focal adhesion proteins Pinch1 and Pinch2 regulate bone homeostasis in mice
Yishu Wang, Qinnan Yan, Yiran Zhao, Xin Liu, Simin Lin, Peijun Zhang, Liting Ma, Yumei Lai, Xiaochun Bai, Chuanju Liu, Chuanyue Wu, Jian Q. Feng, Di Chen, Huiling Cao, Guozhi Xiao
Yishu Wang, Qinnan Yan, Yiran Zhao, Xin Liu, Simin Lin, Peijun Zhang, Liting Ma, Yumei Lai, Xiaochun Bai, Chuanju Liu, Chuanyue Wu, Jian Q. Feng, Di Chen, Huiling Cao, Guozhi Xiao
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Research Article Bone biology

Focal adhesion proteins Pinch1 and Pinch2 regulate bone homeostasis in mice

Abstract

Mammalian focal adhesion proteins Pinch1 and Pinch2 regulate integrin activation and cell–extracellular matrix adhesion and migration. Here, we show that deleting Pinch1 in osteocytes and mature osteoblasts using the 10-kb mouse Dmp1-Cre and Pinch2 globally (double KO; dKO) results in severe osteopenia throughout life, while ablating either gene does not cause bone loss, suggesting a functional redundancy of both factors in bone. Pinch deletion in osteocytes and mature osteoblasts generates signals that inhibit osteoblast and bone formation. Pinch-deficient osteocytes and conditioned media from dKO bone slice cultures contain abundant sclerostin protein and potently suppress osteoblast differentiation in primary BM stromal cells (BMSC) and calvarial cultures. Pinch deletion increases adiposity in the BM cavity. Primary dKO BMSC cultures display decreased osteoblastic but enhanced adipogenic, differentiation capacity. Pinch loss decreases expression of integrin β3, integrin-linked kinase (ILK), and α-parvin and increases that of active caspase-3 and -8 in osteocytes. Pinch loss increases osteocyte apoptosis in vitro and in bone. Pinch loss upregulates expression of both Rankl and Opg in the cortical bone and does not increase osteoclast formation and bone resorption. Finally, Pinch ablation exacerbates hindlimb unloading–induced bone loss and impairs active ulna loading–stimulated bone formation. Thus, we establish a critical role of Pinch in control of bone homeostasis.

Authors

Yishu Wang, Qinnan Yan, Yiran Zhao, Xin Liu, Simin Lin, Peijun Zhang, Liting Ma, Yumei Lai, Xiaochun Bai, Chuanju Liu, Chuanyue Wu, Jian Q. Feng, Di Chen, Huiling Cao, Guozhi Xiao

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Figure 6

BMSCs from dKO mice display decreased osteoblastic, but increased adipogenic, differentiation capacity.

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BMSCs from dKO mice display decreased osteoblastic, but increased adipog...
(A) Colony forming unit-fibroblast (CFU-F) assays. n = 6 mice per group. (B) CFU-osteoblast (CFU-OB) assays. n = 6 mice per group. (C) BMSC proliferation assay. BMSCs were seeded in a 96-well plate at a density of 2,000 cells/well. The absorbance was measured at the time points of 12, 24, 48, and 72 hours. *P < 0.05, **P < 0.01 vs. controls. Unpaired Student’s t test. Results are expressed as mean ± SD. (D–F) In vitro osteoblastic differentiation. Primary BMSCs from 5-month-old female control and dKO mice were seeded in 6-well plates at a density of 4 × 105 cells/well in 2 mL α-MEM media (complete α-MEM containing 50 μg/mL L-ascorbic acid) for 7 days. Whole cell extracts were used for Western blot analysis with the indicated antibodies (D), for qPCR analysis (E), or for ALP staining (F). n = 3 mice for each group. *P < 0.05 vs. controls. Unpaired Student’s t test. Results are expressed as mean ± SD. (G–I) In vitro adipogenic differentiation. Primary BMSCs from 5-month-old female control and dKO mice were seeded 6-well plates at a density of 4 × 105 cells/well and cultured in adipogenic differentiation media (Stemcell Technology, catalog 05503) for 14 days, followed by Western blot analysis with the indicated antibodies (G), and qPCR analysis (H), or for 21 days, followed by Oil Red O staining (I). n = 3 mice for each group. *P < 0.05, **P < 0.01 vs. controls. Unpaired Student’s t test. Results are expressed as mean ± SD. Scale bar: 100 μm. (J) BM adiposity. Tibial sections of 6-month-old female control and dKO mice were subjected to H&E staining. (K and L) Western blotting. Protein extracts isolated from BMSCs of 6-month-old female mice of the 2 genotypes were subjected to Western blotting using the indicated antibodies. n = 5 mice for each group. *P < 0.05 vs. controls. Unpaired Student’s t test. Results are expressed as mean ± SD. (M) qPCR analysis. Total RNAs were isolated from BMSCs of 6-month-old female mice of the 2 genotypes and subjected to qPCR analysis. n = 3 mice for each group. *P < 0.05, **P < 0.01 vs. controls. Unpaired Student’s t test. Results are expressed as mean ± SD. Western blotting analyses in this figure were repeated at least 3 times.