Cooperation between TGF-beta and Wnt pathways during chondrocyte and adipocyte differentiation of human marrow stromal cells.

Abstract

Human marrow stromal cells have the potential to differentiate to chondrocytes or adipocytes. We show interactions between TGF-beta and Wnt signaling pathways during stimulation of chondrogenesis and inhibition of adipogenesis. Combining these signals may be useful in marrow stromal cell therapies. Human bone marrow stromal cells (hMSCs) have the potential to differentiate to lineages of mesenchymal tissues, including cartilage, fat, bone, tendon, and muscle. Agents like transforming growth factor (TGF)-beta promote chondrocyte differentiation at the expense of adipocyte differentiation. In other processes, TGF-beta and Wnt/wingless signaling pathways play major roles in controling certain developmental events and activation of specific target genes. We tested whether these pathways interact during differentiation of chondrocytes and adipocytes in human marrow stromal cells. Both a line of human marrow stromal cells (KM101) and freshly isolated hMSCs were studied. Reverse transcriptase-polymerase chain reaction (RT-PCR), Western blot, and macroarrays were used for analysis of the modulation of TGF-beta1 on Wnt signaling-associated genes, chondrocyte differentiation genes, and TGFbeta/bone morphogenetic protein (BMP) signaling-associated genes in KM101 cells. Early passage hMSCs obtained from 42- and 58-year-old women were used for the effects of TGF-beta and/or Wnt (mimicked by LiCl) signals on chondrocyte and adipocyte differentiation in two-dimensional (2-D) cultures, 3-D pellet cultures, and collagen sponges. As indicated by macroarray, RT-PCR, and Western blot, TGF-beta activated genes in the TGF-beta/Smad pathway, upregulated Wnt2, Wnt4, Wnt5a, Wnt7a, Wnt10a, and Wnt co-receptor LRP5, and increased nuclear accumulation and stability of beta-catenin in KM101 cells. TGF-beta upregulated chondrocyte gene expression in KM101 cells and also stimulated chondrocyte differentiation and inhibited adipocyte differentiation in hMSCs, synergistically with Wnt signal. Finally, hMSCs cultured in 3-D collagen sponges were stimulated by TGF-beta1 to express aggrecan and collagen type II mRNA, whereas expression of lipoprotein lipase was inhibited. In summary, TGF-beta stimulated chondrocyte differentiation and inhibited adipocyte differentiation of hMSCs in vitro. The activation of both TGF-beta and Wnt signal pathways by TGF-beta, and synergy between TGF-beta and Wnt signals, supports the view that Wnt-mediated signaling is one of the mechanisms of TGF-beta's effects on chondrocyte and adipocyte differentiation of hMSCs.