Fluvastatin promotes chondrogenic differentiation of adipose-derived mesenchymal stem cells by inducing bone morphogenetic protein 2

Abstract

Background: Adipose-derived mesenchymal stem cells (ADMSCs) are a promising source of material source for medical regeneration of cartilage. Growth factors, including transforming growth factor-β (TGFβ) subfamily members and bone morphogenetic proteins (BMPs), play important roles in inducing and promoting chondrogenic differentiation of MSCs. However, these exogenous growth factors have some drawbacks related to their cost, biological half-life, and safety for clinical application. Several studies have reported that statins, the competitive inhibitors of 3-hydroxy-2-methylglutaryl coenzyme A (HMG-CoA) reductase, induce the expression of BMP2 in multiple cell types as the pleotropic effects. The objective of this study was to investigate the effects of fluvastatin during chondrogenic differentiation of human ADMSCs (hADMSCs).

Methods: The effects of fluvastatin were analyzed during chondrogenic differentiation of hADMSCs in the pellet culture without exogenous growth factors by qRT-PCR and histology. For functional studies, Noggin, an antagonist of BMPs, mevalonic acid (MVA) and geranylgeranyl pyrophosphate (GGPP), metabolites of the mevalonate pathway, ROCK inhibitor (Y27632), or RAC1 inhibitor (NSC23766) were applied to cells during chondrogenic differentiation. Furthermore, RhoA activity was measured by RhoA pulldown assay during chondrogenic differentiation with or without fluvastatin. Statistically significant differences between groups were determined by Student's t-test or the Tukey-Kramer test.

Results: Fluvastatin-treated cells expressed higher levels of BMP2, SOX9, ACAN, and COL2A1 than control cells, and accumulated higher levels of glycosaminoglycans (GAGs). Noggin significantly inhibited the fluvastatin-mediated upregulation of ACAN and COL2A1. Both MVA and GGPP suppressed the effects of fluvastatin on the expressions of BMP2, SOX9, ACAN, and COL2A1. Furthermore, fluvastatin suppressed the RhoA activity, and inhibition of RhoA-ROCK signaling by Y27632 increased the expressions of BMP2, SOX9, ACAN, and COL2A1, as well as fluvastatin.

Conclusions: Our results suggest that fluvastatin promotes chondrogenic differentiation of hADMSCs by inducing endogenous BMP2, and that one of the mechanisms underlying the effects is inhibition of RhoA-ROCK signaling via suppression of GGPP. Fluvastatin is a safe and low-cost compound that holds promise for use in transplantation of hADMSCs for cartilage regeneration.

Keywords: ADMSCs; BMP2; Chondrogenic differentiation; Fluvastatin; GGPP; Mevalonate pathway; RhoA–ROCK signaling.

Fig. 1

Effects of fluvastatin during chondrogenic differentiation of hADMSCs. A Effects of fluvastatin on cell viability of hADMSCs. hADMSCs were treated with fluvastatin at 0, 0.01, 0.1 or 1 μM for 2 weeks. Cell viability is shown relative to the level on treatment with 0 μM fluvastatin. Error bars denote the means ± standard deviation (n = 5). ^**p < 0.01, NS: not significant by Tukey–Kramer test. B Relative SOX9 mRNA levels on day 4 during chondrogenic differentiation of hADMSCs assessed by qRT-PCR; control, fluvastatin, and TGFβ1 groups are shown. Gene expression is given relative to the level in the control group. Error bars denote means ± standard deviation (n = 4). ^*p < 0.05, ^**p < 0.01 by Tukey–Kramer test. C Effects of fluvastatin on gene expression during chondrogenic differentiation of hADMSCs, as determined by qRT-PCR. Relative mRNA levels of SOX9, ACAN, COL2A1, and COL10A1 in chondrogenic differentiation of hADMSCs on days 0, 7, and 14 among control, fluvastatin, and TGFβ1 groups. Gene expression at each stage is shown relative to the level on day 0. Error bars denote the means ± standard deviation (n = 5). ^*p < 0.05, ^**p < 0.01 by Tukey–Kramer test

Fig. 2

Histological appearance of pellets of hADMSCs cultured in chondrogenic medium for 14 days. A Histological appearance of pellets of hADMSCs stained with hematoxylin and eosin (H&E) and Alcian blue; control, fluvastatin, and TGFβ1 groups are shown. Bars represent 100 μm. B Percentages of Alcian blue–positive areas in the control, fluvastatin, and TGFβ1 groups. Error bars denote means ± standard deviation (n = 10). ^**p < 0.01 by Tukey–Kramer test

Fig. 3

Fluvastatin-induced BMP2 expression and its effect on chondrogenic differentiation of hADMSCs. A Relative BMP2 mRNA levels on day 4 during chondrogenic differentiation of hADMSCs assessed by qRT-PCR; control, fluvastatin, and TGFβ1 groups are shown. Gene expression is given relative to the level in the control group. Error bars denote means ± standard deviation (n = 4). ^*p < 0.05 by Tukey–Kramer test. B Effects of Noggin on gene expression promoted by fluvastatin in chondrogenic differentiation of hADMSCs assessed by qRT-PCR. Relative ACAN and COL2A1 mRNA levels on day 14 in chondrogenic differentiation of hADMSCs cultured with or without fluvastatin or Noggin. Gene expression is expressed relative to the level in cells cultured without fluvastatin or Noggin. Error bars denote means ± standard deviation (n = 4). ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 by Tukey–Kramer test

Fig. 4

Effects of MVA or GGPP on fluvastatin-induced gene expression by during chondrogenic differentiation of hADMSCs. A Relative BMP2 and SOX9 mRNA levels on day 4 and B relative ACAN and COL2A1 mRNA levels on day 7 in chondrogenic differentiation of hADMSCs cultured with or without fluvastatin, MVA, or GGPP, as determined by qRT-PCR. Gene expression is expressed relative to the level in cells cultured without fluvastatin, MVA, or GGPP. Error bars denote means ± standard deviation (n = 4). ^*p < 0.05, ^**p < 0.01 by Tukey–Kramer test

Fig. 5

Effects of fluvastatin on the RhoA activity in chondrogenic differentiation of hADMSCs. A Levels of GTP-RhoA, total RhoA, and GAPDH on day 1 in chondrogenic differentiation of hADMSCs cultured with or without fluvastatin, as determined by western blotting. GAPDH was used as a loading control. These blot images were cropped. Error bars denote means ± standard deviation (n = 3). ^*p < 0.05 by Student’s t-test. B Relative BMP2 and SOX9 mRNA levels on day 4 and (C) relative ACAN and COL2A1 mRNA levels on day 7 in chondrogenic differentiation of hADMSCs cultured with Y27632 or NSC23766, as determined by qRT-PCR. Gene expression is expressed relative to the level in cells cultured without Y27632 or NSC23766. Error bars denote means ± standard deviation (BMP2 and SOX9, n = 4; ACAN and COL2A1, n = 5). ^*p < 0.05 by Tukey–Kramer test