Christina N. Bennett

TSC2 Integrates Wnt and Energy Signals via a Coordinated Phosphorylation by AMPK and GSK3 to Regulate Cell Growth

Mutation in the TSC2 tumor suppressor causes tuberous sclerosis complex, a disease characterized by hamartoma formation in multiple tissues. TSC2 inhibits cell growth by acting as a GTPase-activating protein toward Rheb, thereby inhibiting mTOR, a central controller of cell growth. Here, we show that Wnt activates mTOR via inhibiting GSK3 without involving beta-catenin-dependent transcription. GSK3 inhibits the mTOR pathway by phosphorylating TSC2 in a manner dependent on AMPK-priming phosphorylation. Inhibition of mTOR by rapamycin blocks Wnt-induced cell growth and tumor development, suggesting a potential therapeutic value of rapamycin for cancers with activated Wnt signaling. Our results show that, in addition to transcriptional activation, Wnt stimulates translation and cell growth by activating the TSC-mTOR pathway. Furthermore, the sequential phosphorylation of TSC2 by AMPK and GSK3 reveals a molecular mechanism of signal integration in cell growth regulation.

Wnt signaling stimulates osteoblastogenesis of mesenchymal precursors by suppressing CCAAT/enhancer-binding protein α and peroxisome proliferator-activated receptor γ

Mesenchymal precursor cells have the potential to differentiate into several cell types, including adipocytes and osteoblasts. Activation of Wnt/β-catenin signaling shifts mesenchymal cell fate toward osteoblastogenesis at the expense of adipogenesis; however, molecular mechanisms by which Wnt signaling alters mesenchymal cell fate have not been fully investigated. Our prior work indicates that multipotent precursors express adipogenic and osteoblastogenic transcription factors at physiological levels and that ectopic expression of Wnt10b in bipotential ST2 cells suppresses expression of CCAAT/enhancer-binding protein α (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ) and increases expression of Runx2, Dlx5, and osterix. Here, we demonstrate that transient activation of Wnt/β-catenin signaling rapidly suppresses C/EBPα and PPARγ, followed by activation of osteoblastogenic transcription factors. Enforced expression of C/EBPα or PPARγ partially rescues lipid accumulation and decreases mineralization in ST2 cells expressing Wnt10b, suggesting that suppression of C/EBPα and PPARγ is required for Wnt/β-catenin to alter cell fate. Furthermore, knocking down expression of C/EBPα, PPARγ, or both greatly reduces adipogenic potential and causes spontaneous osteoblastogenesis in ST2 cells and mouse embryonic fibroblasts, suggesting that Wnt signaling alters the fate of mesenchymal precursor cells primarily by suppressing C/EBPα and PPARγ.

Role of Wnt10b and C/EBPα in spontaneous adipogenesis of 243 cells☆☆☆

Abstract This report examines the balance of positive and negative adipogenic factors in a line of immortalized 243 embryonic fibroblasts that undergo spontaneous preadipocyte differentiation. Control of adipogenesis reflects the interplay of factors that promote or inhibit expression of C/EBPα and PPARγ. The 243 cells express C/EBPα early and at elevated levels compared to 3T3-F442A preadipocytes or adipocytes. Cell clones were derived from the heterogeneous 243 population for ability or inability to differentiate into adipocytes. Wnt10b, a secreted protein that inhibits adipogenesis, is expressed at high levels in cells with low adipogenic potential and is undetectable in preadipocytes that spontaneously differentiate. In contrast, C/EBPα is expressed at reduced levels in cells with low adipogenic potential, and is expressed at high levels in preadipocytes that spontaneously differentiate. These data are consistent with a model in which decreased Wnt10b, coupled with increased C/EBPα, results in induction of PPARγ and spontaneous adipogenesis of 243 cells.

Disruption of cell-matrix interactions by heparin enhances mesenchymal progenitor adipocyte differentiation

Differentiation of marrow-derived mesenchymal progenitors to either the osteoblast or adipocyte lineage is reciprocally regulated. Factors that promote osteoblastogenesis inhibit adipogenesis, while adipogenic factors are inhibitory to osteoblast differentiation. Heparin, a soluble glycosaminoglycan, inhibits bone formation in vivo and osteoblast cell differentiation and function in vitro, and has been shown to promote adipocyte differentiation. To elucidate the role that heparin plays in the adipogenic induction of murine mesenchymal progenitors, we studied immortalized marrow stromal cells (IM-MSC), the MSC cell line, ST2, and 3T3L1 pre-adipocytes. Heparin alone was not sufficient to induce adipogenesis, but enhanced the induction under a variety of adipogenic cocktails. This effect was both dose- and time-dependent. Heparin showed a positive effect at concentrations > 0.1 microg/ml when applied before day 3 during the induction course. Heparins effect on adipogenesis was independent of cell proliferation, cell density, and extracellular lipid. This effect is likely related to the unique structure of heparin because another polyanionic glycosaminoglycan, dextran sulfate, did not promote adipogenic differentiation. Heparin treatment altered morphology and adhesion characteristics of progenitor cells, resulting in cell rounding and aggregation. As well, heparin counteracted the known inhibitory effect of fibronectin on adipogenesis and decreased basal focal adhesion kinase and paxillin phosphorylation. We conclude that heparin-mediated disruption of cell-matrix adhesion enhances adipogenic potential.