Int J Stem Cells.  2024 Nov;17(4):418-426. 10.15283/ijsc23165.

IGF-1 Induces Osteogenic Differentiation of Rat Bone Marrow Mesenchymal Stem Cells by Promoting SOX4 via the MAPK/ERK Pathway

• Zeng J
• Deng J
• He C
• Xiong Qa
• Li X
• Wang Z

• KMID: 2562361
• DOI: http://doi.org/10.15283/ijsc23165

Abstract

Tissue engineering envisions functional substitute creation for damaged tissues. Insulin-like growth factor-1 (IGF-1) plays roles in bone marrow mesenchymal stem cell (BMSC) osteogenic differentiation (OD), and we investigated its specific mechanism. BMSCs were cultured and OD was induced. Surface antigens (CD105, CD90, CD44, CD45, CD34) were identified by flow cytometry. Adipogenic, chondrogenic, and osteogenic differentiation abilities of BMSCs were observed. BMSCs were cultured in osteogenic medium containing 80 ng/mL IGF-1 for 3 weeks. Alkaline phosphatase activity, calcification level, osteogenic factor (runt related protein 2 [RUNX2], osteocalcin [OCN], osterix [OSX]), total (t-) ERK1/2 and phosphorylated-(p-) ERK1/2 levels, and SRY-related high-mobility-group box 4 (SOX4) levels were assessed by alkaline phosphatase staining and Alizarin Red staining, Western blot, and reverse transcription-quantitative polymerase chain reaction. The mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway inhibitor (PD98059) was used to inhibit the MAPK/ERK pathway in IGF-1-treated BMSCs. Small interfering-SOX4 was transfected into BMSCs to down-regulate SOX4. IGF-1 increased alkaline phosphatase activity, cell calcification, and osteogenic factor (RUNX2, OCN, OSX) levels in BMSCs, indicating that IGF-1 induced rat BMSC OD. SOX4, and p-ERK1/2 and t-ERK1/2 levels were elevated in IGF-1-induced BMSCs, which were annulled by PD98059. PD98059 partly averted IGF-1-induced rat BMSC OD. SOX4 levels, alkaline phosphatase activity, cell calcification, and osteogenic factor (RUNX2, OCN, OSX) levels were reduced after SOX4 down-regulation, showing that downregulation of SOX4 averted the effect of IGF-1 on inducing rat BMSC OD. IGF-1 induced rat BMSC OD by stimulating SOX4 via the MAPK/ERK pathway.

Keyword

Insulin-like growth factor I; Bone marrow mesenchymal stem cells; MAPK/ERK; SRY-related high-mobility- group box 4; Osteogenic differentiation

Figure

• 

  Fig. 1 Insulin-like growth factor-1 (IGF-1) induced rat bone marrow mesenchymal stem cell (BMSC) osteogenic differentiation. (A) Alkaline phosphatase (ALP) activity of cells was assessed by ALP staining. (B) Calcification level of cells was determined by Alizarin Red staining. (C) Runt related protein 2 (RUNX2), osteocalcin (OCN), and osterix (OSX) protein levels in BMSCs were measured by Western blot. The cellular experiments were independently repeated three times. Data were expressed as mean±SD. Comparisons between the two groups were performed by independent sample t-test. GAPDH: glyceraldehyde-3-phosphate dehydrogenase. *p<0.05, **p<0.01.

• 

  Fig. 2 Insulin-like growth factor-1 (IGF-1) up-regulated SRY-related high-mobility-group box 4 (SOX4) expression by activating the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway. (A) Total- (t-) ERK1/2 and phosphorylated- (p-) ERK1/2 levels in bone marrow mesenchymal stem cells (BMSCs) were assessed by Western blot. (B) SOX4 mRNA expression in BMSCs was measured by reverse transcription-quantitative polymerase chain reaction. (C) SOX4 protein expression in BMSCs was determined by Western blot. The cellular experiments were independently repeated three times. Data were expressed as mean±SD. One-way ANOVA was applied for comparisons among groups, followed by Tukey’s test. GAPDH: glyceraldehyde-3-phosphate dehydrogenase, DMSO: dimethyl sulfoxide. *p<0.05, **p<0.01, ***p<0.001.

• 

  Fig. 3 The mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway inhibition partially nullified the induction of insulin-like growth factor-1 (IGF-1) on rat bone marrow mesenchymal stem cell osteogenic differentiation. (A) Alkaline phosphatase (ALP) activity of cells was detected by ALP staining. (B) Calcification level of cells was determined by Alizarin Red staining. (C) Runt related protein 2 (RUNX2), osteocalcin (OCN), and osterix (OSX) levels were measured by Western blot. The cellular experiments were independently repeated three times. Data were expressed as the mean±SD. One-way ANOVA was adopted for comparisons among groups, followed by Tukey’s test. DMSO: dimethyl sulfoxide, GAPDH: glyceraldehyde-3-phosphate dehydrogenase. *p<0.05, **p<0.01.

• 

  Fig. 4 Knockdown of SRY-related high-mobility-group box 4 (SOX4) partially reversed the effect of insulin-like growth factor-1 (IGF-1) on inducing rat bone marrow mesenchymal stem cell (BMSC) osteogenic differentiation. (A) SOX4 mRNA expression in BMSCs was assessed by reverse transcription-quantitative polymerase chain reaction. (B) SOX4 protein level in BMSCs was determined by Western blot. (C) Alkaline phosphatase (ALP) activity of cells was measured by ALP staining. (D) Calcification level of cells was measured by Alizarin Red staining. (E) Runt related protein 2 (RUNX2), osteocalcin (OCN), and osterix (OSX) protein levels in BMSCs were assessed by Western blot. The cellular experiments were independently repeated three times. Data were expressed as the mean±SD. One-way ANOVA was adopted for comparisons among groups, followed by Tukey’s test. GAPDH: glyceraldehyde-3-phosphate dehydrogenase, si: small interfering. *p<0.05, ***p<0.001.

Reference

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