Transforming Growth Factor Associated Kinase-1 Regulates Chondrogenesis That Precedes Traumatic Heterotopic Ossification

Transforming growth factor associated kinase-1 regulates chondrogenesis that precedes traumatic heterotopic ossification 

Aberrant wound healing is often caused by the dysregulated proliferation and differentiation of progenitor cells. Heterotopic ossification (HO) is one instance of such aberrant wound healing during which extra-skeletal bone is formed in muscle and joint spaces through an endochondral process. TGF-β-activated kinase 1 (TAK1) is a MAP3K that mediates non-canonical TGF-β and BMP signaling during postnatal development of the growth plates and articular cartilage. We hypothesize that TAK1 signaling is necessary for traumatic HO and that genetic or pharmacologic inhibition of TAK1 will prevent HO formation.

We utilized two transgenic models to functionally knockout Tak1 expression, the first a Ub.cre-recombinase (Ub.cre/Tak1^fl/fl), tamoxifen inducible systemic knockout, and the second, a selective knockout in Prx lineage cells (Prx-cre/Tak1^fl/fl), a reliable marker of mesenchymal lineage. Mice underwent a dorsal burn injury with hindlimb Achilles’ tendon transection at 6-8 weeks of age. Separately, a cohort of mice were treated daily with NG-25, a known inhibitor of the TAK1 pathway, or vehicle control for six weeks post injury (n=6 per group). Injured limbs were assessed for HO using µCT analysis at 9 weeks. Injured limbs from Prx-cre/Tak1^fl/flmice were harvested at 3 weeks post-injury for histological analysis using H&E and Movat’s pentachrome staining. mRNA and proteins levels of TAK1 and downstream components were determined by qPCR and Western blotting, respectively. Proliferation was measured through Brdu assay.

Systemic knockout of Tak1 expression resulted in reduced HO volume at transection site (2.170 v. 3.135 mm³ p=0.426). Similarly drug treatment with NG-25, resulted in less HO at the same time points (6.803 v. 9641mm³ p=0.347). Histologically, the transection site from Prx-cre/Tak1^fl/fl yielded no evidence of chondro or osteogenesis, consistent with radiographic evidence.  TAK1 knockout cells and In vitro inhibition with TAK1 inhibitor, NG25, show significant decreased of differentiation (p=0.02/p=0.038, respectively). Brdu assay shows increase of proliferation rate in TAK1 knock out cells (p=0.001).The relative protein expression levels for TAK1 (p=0.009/0,003), p38MAPK (p=0.004/0.006), and pSmad1/5 (p=0.029/0.003) were significantly reduced in both groups, respectively. 

Loss of TAK1 signaling mitigates HO formation in both genetic and therapeutic models. These findings suggest the inhibition of TAK1 pathway (NG25) could be a possible therapeutic approach for treatment of HO.

 REFERENCES

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2. Gunnel L. et al. TAK1 regulates cartilage and joint development via the MAPK and BMP signaling pathway. JBMR 2010; 25(8): 1784-1797