Marrow adipocytes inhibit the differentiation of mesenchymal stem cells into osteoblasts via suppressing BMP-signaling

Abstract

Background: Reduced bone formation is associated with increased bone marrow fat in many bone-loss related diseases including aging, post-menopause, and anorexia nervosa. Several lines of evidence suggested the regulation of osteogenesis and adipogenesis of the bone marrow-derived mesenchymal (skeletal) stem cells (BMSCs) by paracrine mediators. This study aimed to investigate the impact of adipocytes-secreted factors on the cell proliferation and osteoblast differentiation of BMSCs.

Methods: Serum free conditioned medium (CM-Adipo) was collected from stromal ST2 cells-derived adipocytes. Cell viability, quantitative alkaline phosphatase (ALP) activity assay, Alizarin red staining for matrix mineralization and osteogenic gene array expression were performed to determine the effect of CM-Adipo on cell proliferation and osteoblast differentiation of primary murine BMSCs (mBMSCs). Regulation of BMPs and NF-κB signaling pathways by CM-Adipo were detected by Western blot analysis and gene reporter assay.

Results: CM-Adipo showed no effect on cell viability/proliferation of primary mBMSCs as compared to CM-control. On the other hand, CM-Adipo significantly inhibited the commitment of mBMSCs into osteoblastic cell lineage in dose-dependent manner. CM-Adipo was found to dramatically inhibit the BMP2-induced osteoblast differentiation and to activate the inflammatory NF-κB signaling in mBMSCs. Interestingly, treatment of mBMSCs with the selective inhibitor of NF-κB pathway, BAY11-770682, showed to retrieve the inhibitory effect of CM-Adipo on BMP2-induced osteoblast differentiation in mBMSCs.

Conclusions: Our data demonstrated that the marrow adipocytes exert paracrine inhibitory effect on the osteoblast differentiation of mBMSCs by blocking BMPs signaling in a mechanism mediated by adipokines-induced NF-κB pathway activation.

Keywords: Adipocyte; BMSCs; Mesenchymal stem cells; Osteoblast; Paracrine factors; osteoblast differentiation.

Figure 1

Adipocytes-derived CM has no effect on the cell viability or the cell proliferation of mBMSCs. a Efficient differentiation of stromal mouse ST2 cell line into adipocytes as measured by Oil red O staining and its quantification. b Quantitative real time RT-PCR (qPCR) analysis of the adipogenic markers mRNA expression at day 12 of the adipocyte differentiation of ST2 cells. Each target gene was normalized to reference genes and represented as fold change over non-induced control cells. c Effect of CM-Adipo (100%) versus CM-Control (100%) on cell viability and (d) cell proliferation of cultured mBMSCs. Values are mean ± SD of three independent experiments, (*p < 0.05, **p < 0.005)

Figure 2

CM-Adipo exerts paracrine inhibitory effect on osteoblast differentiation of mBMSCs. a Dose dependent inhibitory effect of the CM-Adipo on the osteoblast differentiation of mBMSCs as measured by quantitative alkaline phosphatase activity (ALP) after 6 days of osteogenic induction and (b) Alizarin red staining for matrix mineralization after 12 days of induction. Representative images of the ALP activity and ALZ red staining were shown at 100% concentration of the CM. c Downregulated osteogenic genes in mBMSCs when cultured in CM-Adipo versus CM-Control after 6 days of osteogenic induction. Gene expression was measured by qPCR using osteogenic RT2 profiler array as described in M&M. Values are mean ± SD of three independent experiments, (*p < 0.05, **p < 0.005)

Figure 3

CM-Adipo inhibits BMP2-induced osteoblast differentiation of mBMSCs. a Studying the effect of CM-Adipo versus CM-Control on different osteogenic signaling pathways. Cultured mBMSCs were induced for osteogenesis without (control) or with regular osteogenic induction medium (induced), PDGF-BB (100 ng/ml), Wnt3a (10 ng/ml), BMP2 (100 ng/ml) and insulin (10ug/ml) in 100% of either CM-Adipo or CM-Control. ALP activity was quantified after 6 days of induction and represented as fold change over control non-induced cells. b Dose-dependent inhibitory effect of CM-Adipo on BMP2-induced matrix mineralization in m BMSCs. Alizarine Red staining and its quantification were performed after 12 days of induction. c qPCR analysis of osteoblastic gene expression in mBMSCs induced to osteoblast differentiation by BMP2 in either CM-Adipo or CM-Control for 6 days. d Western blot analysis of Smad1/5/8 phosphorylation in BMP2 treated mBMSCs in either CM-Adipo or CM-Control for 5-20 min. Values are mean ± SD of three independent experiments, (*p < 0.05, **p < 0.005)

Figure 4

The inhibitory effect of CM-Adipo on BMP2-induced osteogenesis is mediated by activating NF-κB signaling pathway. b Western blot analysis of NF-κB subunit p-65 phosphorylation in mBMSCs cultured in CM-Adipo versus CM-Control. Cells were incubated with the CM for 30 min and cell laystes were subjected to western blot analysis. b CM-Adipo stimulates NF-κB signaling activity. HEK 293 cells were transfected with Cignal NF-κB Reporter negative control, or positive control. Cells were incubated with 50% and 100% CM-Control or CM-Adipo for 24 h. Dual-luciferase assays were performed, and reporter activity was represented as arbitrary units after normalization to the internal Renilla reporter. c Effect of NF-κb inhibitor, BAY 11-7082 on retrieving the inhibition of BMP2-induced osteoblast differentiation, as measured by ALP activity quantification and (d) Alizarin Red staining of matrix mineralization. Cells were treated with different concentrations of the inhibitor, 1 h prior the treatment with BMP2. ALP activity was measured after 6 days and represented as fold change after normalization to the cell viability. Alizarin Red was measured after 10 days. Values are mean ± SD of three independent experiments, (*p < 0.05, **p < 0.005)