Role of RHEB in Regulating Differentiation Fate of Mesenchymal Stem Cells for Cartilage and Bone Regeneration

Abstract

Advances in mesenchymal stem cells (MSCs) and cell replacement therapies are promising approaches to treat cartilage and bone defects since substantial differentiation capacities of MSCs match the demands of tissue regeneration. Our understanding of the dynamic process requiring indispensable differentiation of MSCs remains limited. Herein, we describe the role of RHEB (Ras homolog enriched in brain) regulating gene signature for differentiation of human adipose derived mesenchymal stem cells (ASCs) into chondrogenic, osteogenic, and adipogenic lineages. RHEB-overexpression increases the proliferation of the ASCs. RHEB enhances the chondrogenic differentiation of ASCs in 3D culture via upregulation of SOX9 with concomitant increase in glycosaminoglycans (GAGs), and type II collagen (COL2). RHEB increases the osteogenesis via upregulation of runt related transcription factor 2 (RUNX2) with an increase in the calcium and phosphate contents. RHEB also increases the expression of osteogenic markers, osteonectin and osteopontin. RHEB knockdown ASCs were incapable of expressing sufficient SRY (Sex determining region Y)-box 9 (SOX9) and RUNX2, and therefore had decreased chondrogenic and osteogenic differentiation. RHEB-overexpression impaired ASCs differentiation into adipogenic lineage, through downregulation of CCAAT/enhancer binding protein beta (C/EBPβ). Conversely, RHEB knockdown abolished the negative regulation of adipogenesis. We demonstrate that RHEB is a novel regulator, with a critical role in ASCs lineage determination, and RHEB-modulated ASCs may be useful as a cell therapy for cartilage and bone defect treatments.

Keywords: Ras homolog enriched in brain (RHEB); adipogenesis; chondrogenesis; differentiation; mesenchymal stem cells; osteogenesis.

Figure 1

The differential role of Ras homolog enriched in brain (RHEB) in adipose derived stem cells (ASCs). (A) Left panel: ASCs cells were transfected with Mock and RHEB vectors that harbors sequence for GFP. Transfected cells showed green fluorescent protein (GFP) expression as visualized under fluorescent microscope; right panel: Analysis of transfection efficiency by detecting GFP expression thorough flow cytometry; (B,C) cell proliferation analysis after RHEB overexpression and knockdown, respectively, using CCK-8. During CCK-8 assay, an orange formazan product is produced by cellular dehydrogenases. The amount of formazan produced is directly proportional to the number of living cells. Absorbance of orange formazan was measured at 450 nm; (D) left panel: protein expression analysis by Western blotting after RHEB overexpression; right panel: quantification of the Western blots using ImageJ (1.48v, Wayne Rasband, National Institute of Health, Bethesda, MD, USA) and normalizing with GAPDH; (E) left panel: protein expression analysis by Western blotting after RHEB knockdown; right panel: quantification of the Western blots using ImageJ and normalizing with GAPDH. (Data are shown for a representative experiment as averages of triplicates with standard deviation. The statistical significance of differences was calculated using the Student’s t-test. * p < 0.05; ** p < 0.01; *** p < 0.001; Scale bar: 100 μm).

Figure 2

Effect of RHEB overexpression and knockdown on chondrogenic differentiation of ASCs in differentiation media. (A) Alcian blue staining in monolayer culture of ASCs after RHEB overexpression for evaluating glycosaminoglycan (GAG) matrix; (B) quantification of the Alcian blue staining extraction from monolayer cultured cells; (C) the mRNA analysis by quantitative real-time PCR from monolayer cultured ASCs; (D) pellet culture; Alcian blue staining, immunohistochemistry (IHC) was performed for determination of RHEB, COL2 and SOX9 expression; (E) the mRNA expression analysis by quantitative real-time PCR in pellet culture; (F) Alcian blue staining in monolayer culture ASCs after RHEB knockdown; (G) quantification of the Alcian blue staining extraction in RHEB knockdown ASCs; (H) the mRNA expression analysis in monolayer cells after RHEB knockdown. (Data are shown for a representative experiment as averages of triplicates with standard deviation. The statistical significance of differences was calculated using the Student’s t-test. * p < 0.05; Scale bar: 100 μm).

Figure 3

Effect of RHEB overexpression and knockdown on osteogenic differentiation of ASCs in differentiation media. (A) von Kossa staining showed higher phosphate and calcium mineralization in RHEB-overexpressed ASCs; (B) estimation of calcium concentration in the cells after RHEB overexpression; (C) the mRNA expression analysis by quantitative real-time PCR after RHEB overexpression; (D) von Kossa staining after RHEB knockdown in ASCs; (E) estimation of calcium concentration in the cells after RHEB knockdown; (F) the mRNA expression analysis by quantitative real-time PCR after RHEB knockdown. (Data are shown for a representative experiment as averages of triplicates with standard deviation. The statistical significance of differences was calculated using the Student’s t-test. * p < 0.05; Scale bar: 100 μm).

Figure 4

Effect of RHEB overexpression and knockdown on adipogenic differentiation of the ASCs. (A) Oil Red O staining as seen by higher levels of red colored fat droplets in mock-differentiated cells as compared to RHEB-differentiated cells; (B) quantification of extracted Oil Red O staining; (C) the mRNA expression analysis by quantitative real-time PCR after RHEB overexpression; (D) Oil Red O staining after RHEB knockdown; (E) quantification of extracted Oil Red O staining; (F) the mRNA expression analysis by quantitative real-time PCR after RHEB knockdwon. (Datas are shown for a representative experiment as averages of triplicates with standard deviation. The statistical significance of differences was calculated using the Student’s t-test. * p < 0.05; Scale bar: 100 μm).