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Loss Of TGF-beta Activated Kinase (TAK1) Activity Induces Cellular Proliferation And Diminishes Differentiation During Bone Healing
Hsiao Hsieh, Hsung, DDS, Shawn Loder, BS, David Cholok, BS, Michael Chung, MD, Kavitha Ranganathan, MD, Shailesh Agarwal, MD, John Li, MD, Christopher Breuler, BS, Caitlin Priest, BS, Joseph Habbouche, BS, Arminder Kaura, BS, John Butts, BS, Shuli Li, PhD, Yuji Mishina, PhD, Benjamin Levi, MD.
University of Michigan, Ann Arbor, MI, USA.

PURPOSE: The ability to modulate signaling pathways at the site of injury represents a novel paradigm in wound healing with implications for regenerative medicine and tissue engineering. Transforming growth factor-beta (TGF-β) activating kinase 1 (TAK1) is a key regulator in the TGF-β and bone morphogenetic protein (BMP) signaling pathways with central roles in proliferation, differentiation, survival, and apoptosis of myriad tissues during development and in a range of pathologic processes. Here we examine the effect of TAK1 inhibition both in vitro and in the in vivo wound environment. We introduce a novel dual inducible Cre/loxP and Flp/FRT recombinase system to precisely control the expression of TAK1 during wound healing and identify a role for TAK1 as a molecular switch between mesenchymal cell proliferation and differentiation.
METHODS: We developed a novel dual recombinase system (Cre/loxP; Flp/FRT) allowing TAK1 to be specifically inactivated (Ad.Cre) and sequentially re-activated afterwards (Ad.Flp). We first evaluated the effect of this system in vitro mesenchymal cells harvested from sites of musculoskeletal injury. Osteogenic differentiation was assayed via alizarin red and alkaline phosphatase staining. SMAD protein signaling was analyzed directly via immunocytochemistry and western blot analysis. Proliferation was assayed directly via BrdU and cell counting. In vivo, critical sized-calvarial defects (4mm) were performed and mice received either: 1. Ad.LacZ (control) or 2. Ad.Cre (inactivation) or 3. Ten days Ad.Cre (inactivation) followed by Ad.FLP (reactivation) to modulate Tak1 expression. Calvarial tissue was harvested to assay for gene expression and cellular proliferation and differentiation were quantified histologically.
RESULTS: In vitro analysis of mesenchymal cells carrying the Ad.Flp/Ad.Cre construct demonstrated a significant loss in osteogenic potential (p<0.05) and pSMAD1/5 signaling in the absence of Tak1 (Fig. 1A,B). Reactivation of Tak1 was sufficient to restore pSMAD1/5 signaling and osteogenic differentiation (Fig. 1A,B). Tak1 knockout demonstrated an opposite effect on cell proliferation with immediate and significant (p<0.05) increases in cell growth upon knockout and normalization of cell proliferation on gene reactivation (Fig. 1C,D). Loss of Tak1 in the calvarial wound environment resulted in increased presence of mesenchymal cells (Fig. 1C) and increased expression of proliferative genes including Ccnd1, E2f1, and Ki67, an effect reversed by Ad.Flp reactivation of Tak1. Consistent with our in vitro data, loss of Tak1 in calvarial tissue led to diminished osteogenic differentiation genes including Bmp2, Tgfβ1, Col1, Ocn, and Runx2. Again this effect was reversed by Ad.Flp reactivation of Tak1.
CONCLUSION: We demonstrate that precise control of Tak1 can be used to modulate a switch between proliferation and osteogenic differentiation in mesenchymal cells. These findings are possible due to a novel dual-recombinase system with applications in other animal models studying TGF-B signaling and TAK1. Our in vivo data suggests that therapeutic modulation of Tak1 may provide a target to control the proliferation/differentiation switch required during tissue regeneration.


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