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. 2019 Nov;28(11):1420-1431.
doi: 10.1177/0963689719871000. Epub 2019 Aug 19.

αCGRP Affects BMSCs' Migration and Osteogenesis via the Hippo-YAP Pathway

Bin Wang  1   2   3 , Jie Lin  2   3 , Qin Zhang  1   2 , Xinyuan Zhang  1   2 , Hui Yu  1 , Ping Gong  1   2 , Lin Xiang  1   2
Affiliations

αCGRP Affects BMSCs' Migration and Osteogenesis via the Hippo-YAP Pathway

Bin Wang et al. Cell Transplant. 2019 Nov.

Abstract

Alpha-calcitonin gene-related peptide (αCGRP) plays a significant pathophysiological role in the regulation of bone metabolism. Our previous research indicated that αCGRP might have a potential application in enhancing osseointegration in vivo. To further uncover the intrinsic mechanism of its networks in bone regeneration, here we investigate the impact of αCGRP on osteogenic differentiation in bone marrow-derived mesenchymal stem cells (BMSCs) from both wild-type and αCGRP-/- mice. Considering the half-life of αCGRP in plasma is only 10 min, we applied αCGRP lentivirus and stably transfected it into BMSCs, followed by transfection identification and cell cycle assay. We further conducted a series of in vitro tests, and the results revealed that biological functions including migratory ability and osteogenicity exhibited positive correlation with BMSCs' αCGRP expression. Meanwhile, this phenomenon was associated with an enhanced expression of YAP (Yes-associated protein), the key downstream effector of the Hippo pathway. To sum up, our data together with previous in vivo observations is likely to elucidate the intrinsic mechanism of αCGRP in bone remodeling, and αCGRP would appear to be a novel treatment to promote bone wound healing.

Keywords: Hippo-YAP pathway; alpha-calcitonin gene-related peptide; bone marrow-derived mesenchymal stem cells; bone remodeling; osteogenesis.

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Conflict of interest statement

Declaration of Conflicting Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Figures

Figure 1.
Figure 1.
Western blot analysis of αCGRP expression via different MOI levels. (A) Wild-type BMSCs. (B) αCGRP-/- BMSCs.
Figure 2.
Figure 2.
Results of cell cycle assay. (A and D) The SPF parameter in each group. (B and E) The PI value in each group. (C and F) Proliferation activity was reflected by the cell cycle distribution in the logarithmic growth phase.
Figure 3.
Figure 3.
Results of Transwell assays. (A) Western blot analysis. (B) The migratory response of BMSCs at 12 h and 24 h in each group. (C) Quantitation of the migratory response of BMSCs. Scale Bar=50 μm. *p<0.05.
Figure 4.
Figure 4.
Immunofluorescent staining of ALP. Scale Bar=50 μm.
Figure 5.
Figure 5.
Immunofluorescent staining of COL-1 in each group. Scale Bar=50 μm.
Figure 6.
Figure 6.
Alizarin red staining of BMSCs. (A–D) Microscopy observation of alizarin red-positive mineral nodules in the αCGRP-/- control group (A), αCGRP-/- recovery group (B), αCGRP+/+ control group (C), and αCGRP+/+ overexpression group (D), respectively. (E) Quantitation of the density of alizarin red-positive nodules. Scale Bar=500 μm. *p<0.05.
Figure 7.
Figure 7.
Osteoblast-associated phenotype expressions. Levels of (A) ALP, (B) COL-1,(C) OCN, and (D) OPN. (E) Western blot analysis. All data are expressed as mean±SD from three independent experiments. *p<0.05.
See this image and copyright information in PMC

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