The Role of Melanotransferrin (CD228) in the regulation of the differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells (hBM-MSC)

Abstract

Melanotransferrin (CD228), firstly reported as a melanoma-associated antigen, is a membrane-bound glycoprotein of an iron-binding transferrin homolog. CD228 was found to be expressed significantly higher in human bone marrow-derived mesenchymal stem cells (hBM-MSC) than in human embryonic fibroblasts (FB) by RT-PCR, western blotting and flow cytometry. The expression of CD228 declined in aged hBM-MSC as osteogenesis-related genes did. We examined a possible role for CD228 in the regulation of osteogenesis and adipogenesis of hBM-MSC. Surprisingly, siRNA-mediated CD228 knockdown increased the expression of the transcription factor DLX5 and enhanced osteogenesis of hBM-MSC evidenced by an increased expression of the runt-related transcription factor 2 (RUNX2), osterix (Osx), and osteocalcin (OC), as well as higher alkaline phosphatase (ALP) activity and extracellular calcium deposition. Interestingly, hBM-MSC transfected with CD228 siRNA also showed an increase in intracellular lipid level during adipogenesis, indicated by oil red O staining of differentiated adipocytes. Overall, our study unveils CD228 as a cell surface molecule expressed by young hBM-MSC, but not by FB. It also provides evidence to suggest a role for CD228 as a negative regulator of osteogenesis and of lipid accumulation during adipogenesis in hBM-MSC in vitro.

Keywords: Melanotransferrin; adipogenesis; cell surface markers; differentiation; mesenchymal stem cells; osteogenesis.

Figure 1

Analysis of CD228 expression in hBM-MSC and FB. Comparison of the expression of CD228 between hBM-MSC and human FB by (A) RT-PCR analysis, (B) western blot analysis, and (C) FACS analysis. Established MSC markers were also detected by FACS analysis in hBM-MSC and FB. β-Actin was used as an internal control for RT-PCR analysis and western blot analysis. Representative histograms show the expression of CD228 as indicated. Bar graphs show the percentage of positive events presented as the mean ± SD of three independent experiments. Red curves: positive samples, white curves: negative isotype control. p-values were calculated using the Student's t-test, ns: not significant.

Figure 2

Analysis of differentiation-related markers and CD228 between young and old hBM-MSC. The expression of CD228 was analyzed in young and old hBM-MSC by (A) RT-PCR analysis, (B) western blot analysis, and (C) FACS analysis. Established MSC markers were also detected by FACS analysis in young and old hBM-MSC. β-Actin was used as an internal control. Representative histograms show the expression of CD228 as indicated. Bar graphs show the percentage of positive events presented as the mean ± SD of three independent experiments. Red curves: positive samples, white curves: negative isotype control. p-values were calculated using the Student's t-test, ns: not significant. (D) RT-PCR analysis for osteogenic or adipogenic differentiation-related genes in young and old hBM-MSC. β-Actin was used as an internal control.

Figure 3

Expression of osteogenesis or adipogenesis-related markers after CD228 knockdown in hBM-MSC. hBM-MSC were transfected with scrambled siRNA (Scr siRNA) or siRNA against CD228 (CD228 siRNA) for 48 h. (A) Knockdown of CD228 by siRNA transfection was evaluated by RT-PCR (left), western blot analysis (middle), and FACS analysis (right). Red curves: positive samples, white curves: negative isotype control. β-Actin was used as an internal control. (B) RT-PCR analysis and (C) western blot analysis for the indicated osteogenesis- or adipogenesis-related markers in hBM-MSC transfected with Scr siRNA or CD228 siRNA for 48 h. β-Actin was used as an internal control. Bar graphs show the fold change in the expression of each gene for CD228 siRNA-transfected cells compared to Scr siRNA control ones. Data is presented as the mean ± SD of three to four independent experiments. p-values were calculated using the Student's t-test; ns: not significant.

Figure 4

CD228 knockdown enhances osteogenic differentiation of hBM-MSC. (A) hBM-MSC were transfected with scrambled siRNA (Scr siRNA) or siRNA against CD228 (CD228 siRNA) for 48 h, collected by trypsinization, re-seeded, and kept for up to 13 days under osteogenic differentiation conditions. Western blot analysis for CD228 was performed at the indicated time points. β-Actin was used as an internal control. (B) Alkaline phosphatase activity was determined in Scr siRNA or CD228 siRNA-transfected hBM-MSC after the induction of osteogenic differentiation for 6 days. Alkaline phosphatase activity was normalized using MTT values for each group. Data is presented as the mean ± SD fold change of treated cells (ODM) compared to control (CON) cell of four independent experiments (n=5 for each). p-values were calculated using ANOVA or Student's t-test. (C) Phase contrast micrographs of Scr siRNA or CD228 siRNA-transfected hBM-MSC after the induction of osteogenic differentiation for 13 days. CON: control medium; ODM: osteogenic differentiation medium. Magnification: x 100. (D) Alizarin red S staining was performed to evaluate the mineralization of Scr siRNA- or CD228 siRNA-transfected hBM-MSC after 13 days of osteogenic differentiation induction. Alizarin red S staining intensity was normalized using MTT values for each group and expressed as the mean ± SD fold change of treated cells (ODM) compared to control (CON) cells for three independent experiments (n=5 for each). p-values were calculated using ANOVA or Student's t-test.

Figure 5

CD228 knockdown enhances the adipogenic differentiation of hBM-MSC. hBM-MSC were transfected with scrambled siRNA (Scr siRNA) or siRNA against CD228 (CD228 siRNA) for 48 h. Then, cells were collected, re-seeded, and kept for up to 16 days under adipogenic differentiation conditions. (A) Phase contrast micrographs of Scr siRNA or CD228 siRNA-transfected hBM-MSC after the induction of adipogenic differentiation for 16 days. CON: control media; ADM: adipogenic differentiation media. Magnification: x 100. (B) Differentiated adipocytes were stained with oil red O after 16 days to evaluate lipid accumulation. Oil red O staining intensity was normalized using MTT values for each group and expressed as the mean ± SD fold change of treated cells (ADM) compared to control cells (CON) for three independent experiments (n=5 for each). p-values were calculated using ANOVA or Student's t-test.