Cartilage, a highly specialized connective tissue, contains an extensive extracellular matrix and provides mechanical strength to resist compression in joints. Bones are formed through two mechanisms: endochondral ossification and intramembranous ossification. Endochondral ossification, observed in most of the long bones, involves the formation of cartilage as a template initiated by mesenchymal cell condensation. Mesenchymal cells in the condensed area differentiate into chondrocytes, which proliferate and then further differentiate into mature hypertrophic chondrocytes to be later replaced by bone cells. Mesenchymal cells at the periphery of the condensation give rise to the perichondrium, which differentiates into osteoblasts and forms a bone collar. Bones that are not formed through the endochondral process, such as the skull, are formed by intramembranous ossification. Through this process, mesenchymal cells directly differentiate into osteoblasts and deposit bone matrix proteins. Our focus has been on the mechanisms that regulate proliferation and differentiation of chondrocytes ad osteoblasts in development and diseases of cartilage and bones. Panx3 regulates the transition from proliferation to differentiation of osteoprogenitors: Cell-cell and cell-matrix communication regulates the activation of signaling pathways involved in cell functioning, proliferation, differentiation, and cell death. Gap junction proteins play important roles in such cellular communication. Gap junction proteins consist of two families, connexins (Cxs) and pannexins (Panxs). Although Panx3 is expressed in certain soft tissues, we found high levels of Panx3 expression in developing hard tissues, including cartilage, bone, and teeth. We previously showed that Panx3 promotes osteoblast differentiation by functioning as an endoplasmic reticulum (ER) Ca2+ channel and a hemichannel, and by forming gap junctions. Panx 3 is induced in the transition stage from proliferation to differentiation of osteoprogenitor cells. Osteoprogenitor proliferation and differentiation are coordinately regulated during osteogenesis. Canonical Wnt/beta-catenin signaling and BMP promote the proliferation and differentiation, respectively, of osteoprogenitors. However, the regulatory mechanism involved in the transition from proliferation to differentiation was unclear. We show that Panx3 plays a key role in this transition by inhibiting proliferation and promoting cell cycle exit. Using C2C12 cells, primary calvarial cells, and calvaria explants, we found that Panx3 overexpression inhibited cell growth, whereas the inhibition of endogenous Panx3 expression increased it. Mechanistically, we showed that the Panx3 hemichannel inhibited cell growth by promoting beta-catenin degradation through GSK3beta which was activated by reduced cAMP/PKA signaling. The Panx3 hemichannel also reduced CREB signaling, which inhibited cyclin D1 transcription and Rb phosphorylation. Furthermore, the Panx3 ER Ca2+ channel induced transcription and phosphorylation of p21 through the calmodulin/Smad pathway, resulting in cell cycle exit. Our results revealed that Panx3 is a novel regulator that promotes the switch from proliferation to differentiation of osteoprogenitors. Cooperative roles of Epfn (Sp6) and Sp8 in limb development: The formation and maintenance of the apical ectodermal ridge (AER) is critical for the outgrowth and patterning of the limb. Epiprofin (Epfn/Sp6) is expressed in the limb ectoderm and the AER. We previously showed that Sp6 KO mice have a defective autopod that shows mesoaxial syndactyly in the forelimb, synostosis (bony fusion) in the hindlimb, and partial bidorsal digital tips. Sp6 KO mice also show a defect in the maturation of the AER, which appears flat and broad, with a double ridge phenotype. Buttonhead/Sp8, another member of the Sp zinc-finger transcription family, is also expressed in the limb bud ectoderm and AER. The Sp8 KO mice show severe limb truncations. In collaboration with Dr. Marian Ros, we studied the role of Sp6 and Sp8 in limb development by creating double KO mice for Sp6 and Sp8. We found that Sp6 and Sp8 worked together as indispensable mediators of Wnt/beta-catenin and Bmp signaling in the limb ectoderm. Our results suggest that Sp6 and Sp8 are required in a dose-dependent manner for Fgf8 and En1 induction and function as an important link between the induction of the AER and the establishment of dorso-ventral patterning during limb development.
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