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Focal adhesion proteins Pinch1 and Pinch2 regulate bone homeostasis in mice
Yishu Wang, … , Huiling Cao, Guozhi Xiao
Yishu Wang, … , Huiling Cao, Guozhi Xiao
Published November 14, 2019
Citation Information: JCI Insight. 2019;4(22):e131692. https://doi.org/10.1172/jci.insight.131692.
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Research Article Bone biology

Focal adhesion proteins Pinch1 and Pinch2 regulate bone homeostasis in mice

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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 1

Pinch1Dmp1; Pinch2–/– (dKO), but not Pinch1Dmp1 or Pinch2–/–, mice display a low bone mass.

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Pinch1Dmp1; Pinch2–/– (dKO), but not Pinch1Dmp1 or Pinch2–/–, mice disp...
(A) Three-dimensional (3-D) reconstruction from micro-computerized tomography (μCT) scans of distal femurs from 3-month-old female control (Dmp1-Cre), Pinch1Dmp1, Pinch2–/–, and dKO mice. Scale bar: 100 μm. (B–D) Quantitative analyses of bone mineral density (BMD) (B), bone volume/tissue volume (BV/TV) (C), and cortical thickness (Ct.Th) (D) of distal femurs from A. n = 6 mice per group. ***P < 0.001 vs. controls, 2-way ANOVA. Results are expressed as mean ± SD. (E) H&E staining of tibial sections of 6-month-old female control and dKO mice. (F) qPCR analyses. Total RNAs isolated from 3-month-old female middiaphyseal femoral shafts (with their BM flushed) and other indicated tissues were used for qPCR analysis for expression of Pinch1 gene, which was normalized to Gapdh mRNA. n = 3 mice per group. **P < 0.01 vs. controls, unpaired Student’s t test. Results are expressed as mean ± SD. (G) Western blot analysis. Protein extracts were isolated from osteocyte-enriched middiaphyseal femoral shafts (with their BM flushed) and other indicated tissues of 6-month-old female mice of control and dKO mice and subjected to Western blotting using an antibody against Pinch1. Western blotting was repeated 3 times. (H) IHC staining. Longitudinal tibial sections of 6-month-old female control and dKO mice were stained with an antibody against Pinch1. (I) Quantification of H. n = 6 mice per group. ***P < 0.001 vs. control. Unpaired Student’s t test. Results are expressed as mean ± SD. (J) Growth curve. n = 6 mice per group. Results are expressed as mean ± SD.

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