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 and osteoblasts in development and diseases of cartilage and bones. Panx3 inhibits cell proliferation and promotes differentiation of osteoblasts, chondrocytes, and odontoblasts: 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 such as skin and hair follicles, we found high levels of Panx3 expression in developing mineralized tissues, such as cartilage, bone, and teeth. We previously showed that Panx3 regulates the transition stage from proliferation to differentiation of osteoblasts, chondrocytes, and odontoblasts. We 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 showed that Panx3 plays a key role in this transition by inhibiting proliferation and promoting cell cycle exit by beta-catenin degradation through GSK3beta, which was activated by reduced cAMP/PKA signaling. Panx3 modulates skeletal development through distinct expression patterns and functions from Cx43: We have created Panx3 knockout (KO) mice to identify the in vivo functions of Panx3. Panx3 KO mice were small and had reduced cartilage and bone. Bone density was also reduced, and craniofacial development was abnormal. We found that the expression of VEGF and osteocalcin, markers of terminal differentiated hypertrophic chondrocytes, was reduced and that vascular invasion into the chondro-osseous boundary region was inhibited. The perichondrial layer, which contains osteoblast progenitors of the growth plate cartilage, was thin, and the formation of bone collars and the trabecular bone was reduced. In Panx3 KO mice, tooth eruption was delayed, and there were defects in dentin structures and enamel. The expression of the osteoblast markers osterix (Osx), alkaline phosphatase (ALP), and osteocalcin (Ocn) is reduced in the Panx3-/- tibias and calvaria. However, the expression levels of Runx2, a master transcription factor for osteoblast differentiation, were similar between Panx3-/- and WT mice. In Panx3-/- tibias and calvaria, proliferation of osteoprogenitor cells increased due to sustained beta-catenin activity. BMP2/Runx2 induced Panx3 expression. Cx43 is another abundantly expressed gap junction protein in osteoblastic cells. Cx43 mutations in humans cause enamel hypoplasia, syndactyly, and oculodentodigital dysplasia (ODDD). We explored whether Panx3 and Cx43 play any distinct roles during bone formation by comparing skeletal abnormalities and gene expression using Panx3-/-, Cx43-/-, and Panx3-/-; Cx43-/- mice as well as studies on primary calvarial cells from these mice. In Cx43-/- bones, Runx2, Osx, and ALP are expressed while the expression of Ocn, a marker of mature osteoblasts, is reduced. Cx43 expression is reduced in Panx3-/- bones, whereas Panx3 is normally expressed in Cx43-/- bones. Thus, Panx3 is upstream of Cx43 and regulates Cx43 expression in part through promoting Osx expression. Panx3 can replace Cx43 functions, while Cx43 cannot replace Panx3 functions using primary calvarial cell cultures. During differentiation of primary calvarial cells, Panx3 is induced at the early differentiation stage and its expression is reduced concomitant with an increase in Cx43 expression. Our data suggest that Panx3 is required for differentiation, and that at the maturation stage Cx43 plays a role. Our results show that Panx3 is a novel regulator essential for bone formation by regulating Wnt/beta-catenin signaling, Osx and Cx43 expression. In addition, Panx3 is required for differentiation of mature chondrocytes and the vascular invasion in cartilage required for endochondral ossification. Our findings reveal that Panx3 and Cx43 have distinct functions in skeletal formation. Deficiency of the ER cation channel TRIC-B protein disrupts intracellular calcium homeostasis that dysregulates collagen biosynthesis resulting in recessive osteogenesis imperfect: Osteogenesis imperfecta (OI) is characterized by growth deficiency and susceptibility to fractures. Autosomal dominant mutations in the type I collagen genes cause the majority of OI cases. Recently mutations in TMEM38B, encoding the ER membrane K+ channel TRIC-B, have been identified in the OI phenotype. However, the molecular mechanism causing OI by these mutations was not known. In collaboration with Dr. Joan Marini and her colleagues at the NICHD, using fibroblasts from three independent probands, we showed that deficiency of TRIC-B results in impaired ER Ca2+ flux and store-operated calcium entry (SOCE). These abnormalities disrupt intracellular calcium dynamics that cause alteration of the expression and activity of multiple collagen- interacting chaperones and - modifying enzymes within the ER. Thus TRIC-B deficiency causes OI by dysregulation of collagen synthesis, likley through the impairment of calcium-dependent gene expression and protein-protein interactions within the ER.
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