Cooperation Between TGF-β 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-␤ and Wnt signaling pathways during stimulation of chondrogenesis and inhibition of adipogenesis. Combining these signals may be useful in marrow stromal cell therapies.Introduction: 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)-␤ promote chondrocyte differentiation at the expense of adipocyte differentiation. In other processes, TGF-␤ 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. Materials and Methods: 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-␤1 on Wnt signaling-associated genes, chondrocyte differentiation genes, and TGF␤/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-␤ 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. Results: As indicated by macroarray, RT-PCR, and Western blot, TGF-␤ activated genes in the TGF-␤/Smad pathway, upregulated Wnt2, Wnt4, Wnt5a, Wnt7a, Wnt10a, and Wnt co-receptor LRP5, and increased nuclear accumulation and stability of ␤-catenin in KM101 cells. TGF-␤ 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-␤1 to express aggrecan and collagen type II mRNA, whereas expression of lipoprotein lipase was inhibited. Conclusions: In summary, TGF-␤ stimulated chondrocyte differentiation and inhibited adipocyte differentiation of hMSCs in vitro. The activation of both TGF-␤ and Wnt signal pathways by TGF-␤, and synergy between TGF-␤ and Wnt signals, supports the view that Wnt-mediated signaling is one of the mechanisms of TGF-␤'s effects on chondrocyte and adipocyte differentiation of hMSCs.

TWIST Family of Basic Helix-Loop-Helix Transcription Factors Mediate Human Mesenchymal Stem Cell Growth and Commitment

“…These findings support a recent findings demonstrating that Twist-1 interferes with BMP-2 induced expression of collagen type II and aggrecan [50]. Interestingly, the present study found that adipose accumulation and adipogenic related gene expression were enhanced in human MSC cultures following enforced expression of either Twist-1 or Dermo-1, consistent with studies showing that suppression of osteo/chondrogenesis via BMP/ TGFb signaling is associated with increased adipogenesis [50,[63][64][65][66][67]. Therefore, it is likely that a reciprocal regulation of the Twist transcription factors occurs with different BMP/TGFb and Wnt signaling components that collectively act to regulate the lineage commitment of MSC toward adipogenic or ostechondrogenic development [49,50,62,65,68].…”

Novel Wnt Regulator NEL-Like Molecule-1 Antagonizes Adipogenesis and Augments Osteogenesis Induced by Bone Morphogenetic Protein 2

“…64,65 NELL-1 reduction of PPARg expression was blocked by canonical Wnt inhibitors, indicating that NELL-1 down-regulation of adipogenesis was dependent on intact Wnt signaling. It was recently reported that BMP2 represses Wnt signaling during bone repair in a 1-mm in vivo monocortical trephine defect model.…”

Wnt pathway regulation by demineralized bone is approximated by both BMP‐2 and TGF‐β1 signaling

“…A study with hMSCs showed that TGF-b and Wnt cooperatively stimulate their proliferation and inhibit their differentiation to osteoblasts. 30,33 We reported that crosstalk between TGF-b and Wnt signaling regulates chondrocyte 20 and adipocyte 20,34 differentiation, and that TGF-b1 activates b-catenin signaling pathway and modulates osteoblastogenesis in hMSCs. 31 Crosstalk between BMP and Wnt can be synergistic or antagonistic depending on the context.…”

“…Western immunoblotting was performed as described. 20 The primary antibody anti-b-catenin (E5), anti-b-actin, and the second antibody anti-mouse IgG-HRP were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The antibody-associated protein bands were revealed with the ECL-plus Western immunoblotting system (Amersham Biosciences, Little Chalfont, UK).…”