Cartilage, a highly specialized connective tissue, contains an extensive extracellular matrix and provides mechanical strength to resist compression in joints. In development, cartilage serves as the template for the growth and development of most bones. Our focus has been on protein factors that regulate differentiation of chondrocytes and osteoblasts to understand the molecular mechanisms of these factors in tissue formation and diseases. In tissue development and homeostasis, cells perceive micro-environmental cues from soluble mediators, the extracellular matrix (ECM), and neighboring cells. Adhesion complexes, such as adherens junctions, tight junctions, and gap junctions, mediate cell-cell and cell-ECM interactions and links between the cell and its environment, which integrate signaling cascades for cell proliferation and differentiation. Gap junction proteins consist of two families, connexins (Cxs) and pannexins (Panxs). The Cx family has more than 20 members. Mutations of Cxs cause human diseases, such as cancer, hypertension, atherosclerosis, and developmental abnormalities. The Panx family consists of three members, Panx1, 2, and 3. 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 functions to promote differentiation of chondrocytes and osteoblasts. Osteoblasts differentiate from mesenchyme stem cells and form bone through endochondral and intramembranous ossification. BMP2 induces the master osteogenic transcription factors Runx2 and osterix. This leads to the activation of osteogenic marker genes and subsequently to terminal differentiation of osteoblasts and mineralization. Ca2+ is a universal intracellular signaling molecule that regulates cell proliferation, differentiation, morphology, and function. Intracellular Ca2+ concentrations can rise more than five-fold via Ca2+ influx from the extracellular space and/or release from the endoplasmic reticulum (ER), an intracellular Ca2+ storage organelle. This occurs when cells are activated by extracellular stimuli such as ATP. We previously showed that Panx3 functions as a unique Ca2+ channel in the endoplasmic reticulum (ER), which was activated by ATP receptor/PI3K/Akt signaling for differentiation. Panx3 also forms hemichannels that allow the release of ATP into the extracellular space. ATP in the extracellular space activates ATP receptors via autocrine and non-autocrine mechanism following the activation of PI3K/Akt signaling. In addition, Panx3 forms gap junctions and propagates Ca2+ waves between adjacent cells. Blocking the Panx3 Ca2+ channel and gap junction activities inhibits osteoblast differentiation. These findings reveal that Panx3 promotes osteoblast differentiation by functioning as an ER Ca2+ channel, a hemichannel, and by forming gap junctions. The Panx3 hemichannel triggers these Panx3 signaling pathways. Because Panx3 is induced in the transitional stage from proliferation to differentiation during osteogenesis, we hypothesized that Panx3 may also play a role in the inhibition of osteoprogenitor cell proliferation. Canonical Wnt/beta-catenin signaling and BMP promote the proliferation and differentiation of osteoprogenitors, respectively. However, the regulatory mechanism involved in the transition from proliferation to differentiation is unclear. We show that Panx3 plays a key role in this transition by inhibiting proliferation and promoting cell cycle exit. Using cell cultures and calvaria explants, we found that Panx3 overexpression inhibited cell growth, whereas the inhibition of endogenous Panx3 expression increased it. We found 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. In addition, the Panx3 ER Ca2+ channel induced transcription and phosphorylation of p21 through the calmodulin/Smad pathway, resulting in cell cycle exit. Thus, Panx3 is a novel regulator that promotes the switch from proliferation to differentiation of osteoprogenitors. The apical ectodermal ridge (AER) at the distal edge of the developing limb bud is a major signaling center for limb development. Epiprofin (Epfn)/Sp6 and Buttonhead/Sp8 are two members of the Sp zinc-finger transcription family that are expressed in the limb bud ectoderm. Sp6 and Sp8 function downstream of Wnt/beta-catenin signaling for Fgf 8 induction. Sp6 knockout (KO) mice show a mild syndactyly phenotype, while Sp8 KO mice exhibit severe limb truncations. In collaboration with Dr. Marian Ros and her group, 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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