Wenhan Chang

EXPRESSION AND SIGNAL TRANSDUCTION OF CALCIUM-SENSING RECEPTORS IN CARTILAGE AND BONE

We previously showed that Ca 21 -sensing receptors (CaRs) are expressed in chondrogenic RCJ3.1C5.18 (C5.18) cells and that changes in extracellular [Ca 21 ] ([Ca 21 ]o) modulate nodule formation and chondrogenic gene expression. In the present study, we detected expression of CaRs in mouse, rat, and bovine cartilage and bone by in situ hybridization, immunocytochemistry, immunoblotting, and RT-PCR; and we tested the effects of CaR agonists on signal transduction in chondrogenic and osteogenic cell lines. In situ hybridization detected CaR transcripts in most articular chondrocytes and in the hypertrophic chondrocytes of the epiphyseal growth plate. Expression of CaR transcripts was weak or absent, however, in proliferating and maturing chondrocytes in the growth plate. In bone, CaR transcripts were present in osteoblasts, osteocytes, and bone marrow cells, but rarely in osteoclasts. A complementary DNA was amplified from mouse growth plate cartilage, which was highly homologous to the human parathyroid CaR sequence. Immunocytochemistry of cartilage and bone with CaR antisera confirmed these findings. Western blotting revealed specific bands (;140 ‐190 kDa) in membrane fractions isolated from growth plate cartilage, primary cultures of rat chondrocytes, and several osteogenic cell lines (SaOS-2, UMR-106, ROS 17/2.8, and MC3T3-E1). InsP responses to high [Ca 21 ]o were evident in C5.18 cells and all osteogenic cell lines tested except for SaOS-2 cells. In the latter, high [Ca 21 ]o reduced PTH-induced cAMP formation. Raising [Ca 21 ]o also increased intracellular free [Ca 21 ]i n SaOS-2 and C5.18 cells. These studies confirm expression of CaRs in cartilage and bone and support the concept that changes in [Ca 21 ]o may couple to signaling pathways important in skeletal metabolism. (Endocrinology 140: 5883‐5893, 1999)

The Extracellular Calcium-Sensing Receptor (CaSR) Is a Critical Modulator of Skeletal Development

The extracellular calcium-sensing receptor (CaSR) is essential for embryonic and postnatal skeletal development. Bone Censor The extracellular calcium-sensing receptor (CaSR) is a guanine nucleotide-binding protein (G protein)–coupled receptor (GPCR) that responds to changes in the concentration of extracellular calcium ([Ca2+]e) and modulates various functions of parathyroid cells (PTCs), chondrocytes (the cells that produce cartilage), osteoblasts, and renal tubular cells. Previous attempts to characterize the CaSR in Casr−/− mice have been hampered by the expression of an alternatively spliced form of the receptor that provides at least partial compensation for loss of the full-length receptor (see the Perspective by Brown and Lian). Chang et al. have now developed mice with cell type–specific knockout of Casr that do not express the alternative receptor. Mice with PTC- or osteoblast-specific deletion of Casr were viable but had postnatal skeletal defects. Unexpectedly, knockout of Casr in chondrocytes was lethal. Mice in which chondrocyte-specific knockout of Casr was induced late in embryonic life were viable but had delayed growth plate development. Together, these findings reveal a previously unappreciated role for the CaSR in embryonic and postnatal skeletal development. The extracellular Ca2+-sensing receptor (CaSR) plays a nonredundant role in the functions of the parathyroid gland (PTG) and the kidney. Severe hyperparathyroidism, premature death, and incomplete gene excision in Casr−/− mice have precluded the assessment of CaSR function in other tissues. We generated mice with tissue-specific deletion of Casr in the PTG, bone, or cartilage. Deletion of Casr in the PTG or bone resulted in profound bone defects, whereas deletion of Casr in chondrocytes (cartilage-producing cells) resulted in death before embryonic day 13 (E13). Mice in which chondrocyte-specific deletion of Casr was induced between E16 and E18 were viable but showed delayed growth plate development. Our data show a critical role for the CaSR in early embryogenesis and skeletal development.

The Calcium Sensing Receptor and Its Alternatively Spliced Form in Murine Epidermal Differentiation

We have recently reported that human keratinocytes express both the full-length calcium sensing receptor (CaR) and an alternatively spliced form lacking exon 5, which were suggested to be involved in calcium induced keratinocyte differentiation. To understand further the role of these CaRs, we analyzed the structure of mouse CaRs, and investigated their role using a mouse model in which only the full-length CaR was disrupted. Our results show that both the full-length and the alternatively spliced variant lacking exon 5 encoding 77 amino acids of the extracellular domain were expressed in mouse epidermis. The deletion of the full-length CaR increased the production of the alternatively spliced form of CaR in mutant mice. The keratinocytes derived from these mutant mice did not respond to extracellular calcium, suggesting that the full-length CaR is required to mediate calcium signaling in the keratinocytes. The loss of the full-length CaR altered the morphologic appearance of the epidermis and resulted in a reduction of the mRNA and protein levels of the keratinocyte differentiation marker, loricrin. These results indicate that CaR is important in epidermal differentiation, and that the alternatively spliced form does not fully compensate for loss of the full-length CaR.

Inactivation of the Calcium Sensing Receptor Inhibits E-cadherin-mediated Cell-Cell Adhesion and Calcium-induced Differentiation in Human Epidermal Keratinocytes

Extracellular Ca2+ (Ca2+o) is a critical regulator that promotes differentiation in epidermal keratinocytes. The calcium sensing receptor (CaR) is essential for mediating Ca2+ signaling during Ca2+o-induced differentiation. Inactivation of the endogenous CaR-encoding gene CASR by adenoviral expression of a CaR antisense cDNA inhibited the Ca2+o-induced increase in intracellular free calcium (Ca2+i) and expression of terminal differentiation genes, while promoting apoptosis. Ca2+o also instigates E-cadherin-mediated cell-cell adhesion, which plays a critical role in orchestrating cellular signals mediating cell survival and differentiation. Raising Ca2+o concentration ([Ca2+]o) from 0.03 to 2 mm rapidly induced the co-localization of α-, β-, and p120-catenin with E-cadherin in the intercellular adherens junctions (AJs). To assess whether CaR is required for the Ca2+o-induced activation of E-cadherin signaling, we examined the impact of CaR inactivation on AJ formation. Decreased CaR expression suppressed the Ca2+o-induced AJ formation, membrane translocation, and the complex formation of E-cadherin, catenins, and the phosphatidylinositol 3-kinase (PI3K), although the expression of these proteins was not affected. The assembly of the E-cadherin-catenin-PI3K complex was sensitive to the pharmacologic inhibition of Src family tyrosine kinases but was not affected by inhibition of Ca2+o-induced rise in Ca2+i. Inhibition of CaR expression blocked the Ca2+o-induced tyrosine phosphorylation of β-, γ-, and p120-catenin, PI3K, and the tyrosine kinase Fyn and the association of Fyn with E-cadherin and PI3K. Our results indicate that the CaR regulates cell survival and Ca2+o-induced differentiation in keratinocytes at least in part by activating the E-cadherin/PI3K pathway through a Src family tyrosine kinase-mediated signaling.

Coupling of calcium receptors to inositol phosphate and cyclic AMP generation in mammalian cells and Xenopus laevis oocytes and immunodetection of receptor protein by region-specific antipeptide antisera.

Ca2+ and other divalent cations modulate parathyroid hormone secretion by interacting with cell‐surface Ca2+‐sensing receptors (CaRs). We assessed the ability of these receptors to couple to Ca2+ mobilization, inositol phosphate (InsP) accumulation, and cyclic AMP production in different expression systems. In Xenopus laevis oocytes injected with bovine parathyroid CaR cRNA, the addition of extracellular cations to 1.5 mM Ca2+, 5.5 mM Mg2+, or 10 μM Gd3+ significantly increased45Ca efflux (p < 0.01). InsP accumulation also increased dramatically when adding these cations to human embryonic kidney (HEK) 293 cells stably transfected with wild‐type bovine parathyroid CaR cDNA. Raising the extracellular [Ca2+] ([Ca2+]o) from 0.1 to >1.4 mM in oocytes and to >1.0 mM in HEK 293 cells stimulated significant increments in45Ca efflux and InsP accumulation, respectively (p < 0.05). In contrast, Ca2+ and Mg2+ increased InsPs to a lesser extent in COS 7 cells transiently transfected with CaR cDNA. In HEK 293 cells stably expressing CaR cDNA, there were significant reductions in cAMP content when adding high Ca2+, Mg2+, Gd3+, or the CaR modulator NPS R‐467. Three region‐specific anti‐CaR peptide antisera immunoblotted bands of ∼140 and 155 kDa in membranes from CaR‐transfected HEK 293 cells and bovine parathyroid tissue. Immunocytochemistry demonstrated strong cell‐surface staining in CaR‐transfected HEK 293 cells and parathyroid tissue, which was absent when antisera were preabsorbed with CaR peptides. These results indicate that the activation of the recombinant CaR by extracellular Ca2+ can couple negatively to adenylate cyclase but positively to phospholipase C (PLC), the latter at physiological [Ca2+]o.

Epidermal expression of the full-length extracellular calcium-sensing receptor is required for normal keratinocyte differentiation

The importance of the extracellular calcium‐sensing receptor (CaR) in the stringent control of extracellular Ca2+ concentration is well established. However, the presence of CaR in tissues not directly involved in regulating mineral ion homeostasis such as the epidermis suggests a role for CaR in other cellular functions. Although extracellular Ca2+ regulates the differentiation of epidermal keratinocytes, the role of CaR in this process in the epidermis is not fully understood. In this study we showed using in situ hybridization and immunohistochemistry that CaR is expressed in suprabasal keratinocytes of the mammalian epidermis. We then evaluated the changes in epidermal keratinocyte morphology and differentiation in Casr−/− mice lacking the full‐length CaR. These mice show increased expression of an alternatively spliced form of CaR which lacks acute Ca2+‐signaling properties. The absence of the full‐length CaR in the epidermis resulted in ultrastructural changes (abnormal keratohyalin granule formation and precocious lamellar body secretion) in the terminally differentiated granular keratinocytes. Furthermore, the expression of both mRNA and protein for the calcium inducible keratinocyte differentiation markers, filaggrin and loricrin, were down‐regulated in the epidermis of Casr−/− mice, whereas the number of proliferating cells were increased even though the calcium gradient within the epidermis was enhanced. Our results demonstrate that the epidermal expression of the full‐length CaR is required for the normal terminal differentiation of keratinocytes.

IGF-1R signaling in chondrocytes modulates growth plate development by interacting with the PTHrP/Ihh pathway.

Systemic derangements and perinatal death of generalized insulin‐like growth factor 1 (IGF‐1) and IGF‐1 receptor (IGF‐1R) knockout mice preclude definitive assessment of IGF‐1R actions in growth‐plate (GP) chondrocytes. We generated cartilage‐specific Igf1r knockout (CartIgf1r−/−) mice to investigate local control of chondrocyte differentiation in the GP by this receptor. These mice died shortly after birth and showed disorganized chondrocyte columns, delayed ossification and vascular invasion, decreased cell proliferation, increased apoptosis, and increased expression of parathyroid hormone–related protein (Pthrp) RNA and protein in their GPs. The increased Pthrp expression in the knockout GPs likely was due to an increase in gene transcription, as determined by the increased activity of a LacZ reporter that was inserted downstream of the endogenous PTHrP promoter and bred into the knockout mice. To circumvent the early death of CartIgf1r−/− mice and investigate the role of IGF‐1R during postnatal growth, we made tamoxifen (Tam)–inducible, cartilage‐specific Igf1r knockout (TamCartIgf1r−/−) mice. At 2 weeks of age and 7 to 8 days after Tam injection, the TamCartIgf1r−/− mice showed growth retardation with a disorganized GP, reduced chondrocyte proliferation, decreased type 2 collagen and Indian Hedgehog (Ihh) expression, but increased expression of PTHrP. Consistent with in vivo observations, in vitro knockout of the Igf1r gene by adenoviral expression of Cre recombinase suppressed cell proliferation, promoted apoptosis, and increased Pthrp expression. Our data indicate that the IGF‐1R in chondrocytes controls cell growth, survival, and differentiation in embryonic and postnatal GPs in part by suppression of Pthrp expression.

Extracellular Ca2+-Sensing Receptors Modulate Matrix Production and Mineralization in Chondrogenic RCJ3.1C5.18 Cells

Previous studies in chondrogenic RCJ3.1C5.18 (C5.18) cells showed that growth of these cells at high extracellular Ca2+ concentrations ([Ca2+]o) reduced the expression of markers of early chondrocyte differentiation. These studies addressed whether raising [Ca2+]o accelerates C5.18 cell differentiation and whether Ca2+ receptors (CaRs) are involved in coupling changes in [Ca2+]o to cellular responses. We found that high [Ca2+]o increased expression of osteopontin (OP), osteonectin, and osteocalcin, all markers of terminal differentiation, in C5.18 cells and increased the production of matrix mineral. Overexpression of wild-type CaR cDNA in C5.18 cells suppressed proteoglycan synthesis and aggrecan RNA, two early differentiation markers, and increased OP expression. The sensitivity of these parameters to changes in [Ca2+]o was significantly increased, as indicated by left-shifted dose-responses. In contrast, stable expression of a signaling-defective CaR mutant (Phe707Trp CaR) in C5.18 cells, presumably th...

Complex Formation with the Type B γ-Aminobutyric Acid Receptor Affects the Expression and Signal Transduction of the Extracellular Calcium-sensing Receptor STUDIES WITH HEK-293 CELLS AND NEURONS

We co-immunoprecipitated the Ca2+-sensing receptor (CaR) and type B γ-aminobutyric acid receptor (GABA-B-R) from human embryonic kidney (HEK)-293 cells expressing these receptors and from brain lysates where both receptors are present. CaRs extensively co-localized with the two subunits of the GABA-B-R (R1 and R2) in HEK-293 cell membranes and intracellular organelles. Coexpressing CaRs and GABA-B-R1s in HEK-293 cells suppressed the total cellular and cell surface expression of CaRs and inhibited phospholipase C activation in response to high extracellular [Ca2+] ([Ca2+]e). In contrast, coexpressing CaRs and GABA-B-R2s enhanced CaR expression and signaling responses to raising [Ca2+]e. The latter effects of the GABA-B-R2 on the CaR were blunted by coexpressing the GABA-B-R1. Coexpressing the CaR with GABA-B-R1 or R2 enhanced the total cellular and cell surface expression of the GABA-B-R1 or R2, respectively. Studies with truncated CaRs indicated that the N-terminal extracellular domain of the CaR participated in the interaction of the CaR with the GABA-B-R1 and R2. In cultured mouse hippocampal neurons, CaRs co-localized with the GABA-B-R1 and R2. CaRs and GABA-B-R1s also co-immunoprecipitated from brain lysates. The expression of the CaR was increased in lysates from GABA-B-R1 knock-out mouse brains and in cultured hippocampal neurons with their GABA-B-R1 genes deleted in vitro. Thus, CaRs and GABA-B-R subunits can form heteromeric complexes in cells, and their interactions affect cell surface expression and signaling of CaR, which may contribute to extracellular Ca2+-dependent receptor activation in target tissues.

Osteoblast extracellular Ca2+-sensing receptor regulates bone development, mineralization, and turnover

The extracellular Ca2+‐sensing receptor (CaR), a G protein‐coupled receptor responsible for maintenance of calcium homeostasis, is implicated in regulation of skeletal metabolism. To discern the role of the osteoblast CaR in regulation of bone development and remodeling, we generated mice in which the CaR is excised in a broad population of osteoblasts expressing the 3.6‐kb a1(I) collagen promoter. Conditional knockouts had abnormal skeletal histology at birth and developed progressively reduced mineralization secondary to retarded osteoblast differentiation, evident by significantly reduced numbers of osteoblasts and decreased expression of collagen I, osteocalcin, and sclerostin mRNAs. Elevated expression of ankylosis protein, ectonucleotide pyrophosphatase/phosphodiesterase 1, and osteopontin mRNAs in the conditional knockout indicate altered regulation of genes important in mineralization. Knockout of the osteoblast CaR also resulted in increased expression of the receptor activator of NF‐κB ligand (RANKL), the major stimulator of osteoclast differentiation and function, consistent with elevated osteoclast numbers in vivo. Osteoblasts from the conditional knockouts exhibited delayed differentiation, reduced mineralizing capacity, altered expression of regulators of mineralization, and increased ability to promote osteoclastogenesis in coculture experiments. We conclude that CaR signaling in a broad population of osteoblasts is essential for bone development and remodeling and plays an important role in the regulation of differentiation and expression of regulators of bone resorption and mineralization.