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. 2020 Oct 23;11(1):450.
doi: 10.1186/s13287-020-01965-4.

HSPB7 regulates osteogenic differentiation of human adipose derived stem cells via ERK signaling pathway

Chanyuan Jin  1 , Ting Shuai  1 , Zhihui Tang  2
Affiliations

HSPB7 regulates osteogenic differentiation of human adipose derived stem cells via ERK signaling pathway

Chanyuan Jin et al. Stem Cell Res Ther. .

Abstract

Background: Heat shock protein B7 (HSPB7), which belongs to small heat shock protein family, has been reported to be involved in diverse biological processes and diseases. However, whether HSPB7 regulates osteogenic differentiation of human adipose derived stem cells (hASCs) remains unexplored. VSports手机版.

Methods: The expression level of HSPB7 during the osteogenesis of hASCs was examined by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot analysis. Lentivirus transfection was used to knock down or overexpress HSPB7, which enabled us to investigate the effect of HSPB7 on osteogenic differentiation of hASCs. U0126 and extracellular signal-regulated protein kinase 1/2 (ERK1/2) siRNA were used to identify the mechanism of the HSPB7/ERK1/2 axis in regulating osteogenic differentiation of hASCs. Moreover, ectopic bone formation in nude mice and osteoporosis mice model was used to investigate the effect of HSPB7 on osteogenesis in vivo V体育安卓版. .

Results: In this study, we found the expression of HSPB7 was significantly downregulated during the osteogenic differentiation of hASCs. HSPB7 knockdown remarkably promoted osteogenic differentiation of hASCs, while HSPB7 overexpression suppressed osteogenic differentiation of hASCs both in vitro and in vivo. Moreover, we discovered that the enhancing effect of HSPB7 knockdown on osteogenic differentiation was related to the activation of extracellular signal-regulated protein kinase (ERK) signaling pathway. Inhibition of ERK signaling pathway with U0126 or silencing ERK1/2 effectively blocked the stimulation of osteogenic differentiation induced by HSPB7 knockdown. Additionally, we found that HSPB7 expression was markedly increased in mouse bone marrow mesenchymal stem cells (mBMSCs) from the osteoporotic mice which suggested that HSPB7 might be utilized as a potential target in the development of effective therapeutic strategies to treat osteoporosis and other bone diseases V体育ios版. .

Conclusion: Taken together, these findings uncover a previously unrecognized function of HSPB7 in regulating osteogenic differentiation of hASCs, partly via the ERK signaling pathway. VSports最新版本.

Keywords: Bone formation; Heat shock protein; Osteogenesis; cvHSP. V体育平台登录.

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VSports手机版 - Conflict of interest statement

The authors indicate no potential conflicts of interest.

Figures

Fig. 1
Fig. 1
HSPB7 was downregulated during the osteogenic differentiation of hASCs. a Relative mRNA expression of HSPB7 was measured by qRT-PCR at days 0, 1, 4, 7, 10, and 14 during the osteogenic differentiation of hASCs. b–d Relative mRNA expression of RUNX2, ALP, and OCN during the osteogenic differentiation of hASCs. e After osteogenic induction, the protein expression of HSPB7 was significantly reduced, accompanied by the increased expression of RUNX2. f The quantitative results of e by Image J software. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a control. Data are presented as the mean ± SD (*P < 0.05, **P < 0.01, compared with day 0)
Fig. 2
Fig. 2
HSPB7 overexpression inhibited the osteogenic differentiation of hASCs in vitro. a ALP staining in the HSPB7 overexpression (HSPB7) and control (NC) groups at day 7 of osteogenic differentiation. b Quantification of ALP activity in HSPB7 overexpression and control hASCs at day 7 of osteogenic differentiation. c Alizarin Red S staining was performed at day 14 of osteogenic differentiation. d Quantification of Alizarin Red S (ARS) staining. e–g Relative mRNA expression of osteogenic genes RUNX2, ALP, and OCN was assessed by qRT-PCR at day 14 of osteogenic differentiation. h, i HSPB7 overexpression downregulated the protein expression of OCN, as determined by Western blot. j Confocal microscopy confirmed the downregulation of OCN protein expression in HSPB7 overexpression hASCs at day 14 of osteogenic differentiation. Scale bar = 50 μm. Data are presented as the mean ± SD (*P < 0.05, **P < 0.01, compared with NC)
Fig. 3
Fig. 3
HSPB7 knockdown promoted the osteogenic differentiation of hASCs in vitro. a ALP staining in the HSPB7 knockdown (shHSPB7-1, shHSPB7-2) and control (shNC) groups at day 7 of osteogenic differentiation. b Histogram shows the 7-day quantification of ALP activity. c ARS staining at day 14 of osteogenic differentiation. d Relative quantitative analysis of ARS staining. e–g Relative mRNA expression of RUNX2, ALP, and OCN at day 14 of osteogenic differentiation. h, i HSPB7 knockdown increased the protein expression of OCN. j Confocal microscopy confirmed the upregulation of OCN protein expression in HSPB7 knockdown hASCs at day 14 of osteogenic differentiation. Scale bar = 50 μm. (*P < 0.05, **P < 0.01, compared with shNC)
Fig. 4
Fig. 4
HSPB7 knockdown activated ERK signaling pathway. a qRT-PCR showed HSPB7 knockdown increased the expression of FGFR1. b HSPB7 knockdown increased the level of phosphorylated ERK1/2 in hASCs. c The quantitative results of b by Image J software. Data are presented as the mean ± SD (**P < 0.01, compared with shNC)
Fig. 5
Fig. 5
Inhibiting ERK signaling pathway with U0126 reversed the enhancing effect of HSPB7 knockdown on osteogenesis of hASCs. a U0126 reduced the level of phosphorylated ERK1/2 in shNC and shHSPB7 hASCs. b The quantitative results of a by Image J software. c ALP staining at day 7 of osteogenic differentiation. U0126 (10 μM) or DMSO was incubated for 7 days. d Histogram shows the 7-day quantification of ALP activity. e ARS staining in shNC, shHSPB7 groups treated with U0126 (10 μM) or DMSO (control) for 14 days. f Relative quantitative analysis of ARS staining. g–i Relative mRNA expression of RUNX2, ALP, and OCN at day 14 of osteogenic differentiation in the presence or absence of U0126 (10 μM). DMSO was used as a control. Data are presented as the mean ± SD (*P < 0.05, **P < 0.01, compared with shNC)
Fig. 6
Fig. 6
HSPB7 knockdown promoted bone formation of hASCs in vivo. a HSPB7 knockdown promoted bone formation capacity of hASCs in vivo, as determined by HE staining (HE) and Masson’s trichrome staining (Masson). b HSPB7 overexpression inhibited bone formation capacity of hASCs. c Quantitative measurements of bone-like tissue showed the area of bone-like tissue was significantly increased in HSPB7 knockdown hASCs but decreased in HSPB7 overexpression hASCs. Black arrows indicate new bone-like tissue. Scale bar = 50 μm. Data are presented as the mean ± SD (**P < 0.01, compared with shNC or NC)
Fig. 7
Fig. 7
Schematic diagram of the regulation of osteogenesis by HSPB7. HSPB7 regulated the osteogenic differentiation of hASCs via ERK1/2 signaling
See this image and copyright information in PMC

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