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TGF Beta and BMP Signaling Pathways Influence Regenerative Capacity of Calvarial Bones via Cross-Talk and Modulation of Apoptosis: The Potential Therapeutic Role of Small Molecule Inhibitors of TGF Beta Signaling.
Kshemendra Senarath-Yapa, MA (Cantab) MBBChir MRCS, Nathaniel Meyer, BS, Shuli Li, MD PhD, Michael T. Longaker, MD MBA, Natalina Quarto, PhD.
The Hagey Laboratory for Pediatric Regenerative Medicine, Stanford University, Stanford, CA, USA.
Craniofacial skeletal defects pose a significant clinical burden that is insufficiently met by current reconstructive approaches. A better understanding of calvarial osteoblast biology is essential in order to promote endogenous skeletal regeneration. Studies on transgenic mice with a Wnt1-Cre construct and a reporter R26R established that frontal bones arise from neural crest and parietal bones from paraxial mesoderm. Frontal neural crest-derived osteoblasts (FOb) possess greater osteogenic potential relative to parietal bone osteoblasts (POb) and reduced apoptotic activity. This is partly due to increased TGF-β1 signaling in parietal bones, which is known to promote apoptosis in different cell types. Determining a signaling network that modulates apoptotic activity is therefore worthwhile. We investigated the potential for enhancing calvarial regeneration by inhibiting TGF-β1 signaling with a specific small molecule inhibitor, SB431542 and the importance of apoptosis in calvarial healing with a specific caspase-3 inhibitor. Furthermore, we investigate the effect of TGF-β1 on apoptotic activity in calvarial osteoblasts and dural cells.
Non-critical calvarial defects were made in parietal bones of CD-1 mice. A collagen sponge was used to deliver TGF-β1 (400ng) or SB431542 (26mM), a small molecule inhibitor of TGF-β1 signaling. Micro-CT evaluation of bone regeneration was done for 6 weeks and osseous healing calculated using GE Microview. The same in vivo model was use to test the effect of Ac-DEVD-CHO (1μM) a specific Caspase 3 inhibitor on calvarial healing. IHC was performed on sections through calvarial defects for Phospho-Smad 2 and 3, downstream effectors for TGFβ signaling, to assess whether SB431542 (SB) was effectively inhibiting this pathway in vivo. A Caspase-3 fluorometric protease assay was used to assess apoptotic activity in dural cells and POb during osteogenic differentiation. POb were collected at different time points of osteogenic differentiation and immunoblotting was performed for down stream effectors of the TGF-β1 pathway (P-Smad2) and BMP pathway (P-Smad5) on cell lysates during osteogenic differentiation.
Delivery of TGF-β1 significantly reduced the percentage healing relative to non-treated defects (*p<0.05). Inhibition of TGF-β1 signaling using SB resulted in significantly enhanced bone regeneration (*p<0.05). Direct inhibition of apoptosis with Ac-DEVD-CHO significantly improved calvarial defect healing (P<0.05). IHC for downstream effectors of TGF-β1 signaling, Phospho-Smad 2 and 3, established effective TGF-β1 inhibition in-vivo. In vitro apoptosis assays demonstrated a significant reduction in apoptotic activity in POb and dura with TGF-β1 inhibition. Immunoblotting analysis for P-Smad2 showed effective inhibition of TGF-β1 signaling by SB. Intriguingly, SB treatment also led to increased P-Smad 5, a downstream effector of BMP signaling, suggesting cross-talk between these two pathways.
We demonstrate that increased TGF-β1 signaling impairs calvarial healing whereas inhibition promotes regeneration. TGF-β1 inhibition using a specific small molecule inhibitor significantly reduces apoptotic activity in POb and dural cells undergoing osteogenic differentiation. We establish the presence of cross-talk between TGF-β and BMP signaling pathways during osteogenic differentiation and the importance of apoptosis during calvarial healing. The study also provides an insight into the use of small molecule inhibitors of TGF-β signaling as a novel therapeutic approach for treatment of craniofacial skeletal defects.
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