We previously identified transcripts encoding a G protein-coupled, extracellular calcium/polyvalent cation-sensing receptor, RaKCaR, in rat kidney (D. Riccardi, J. Park, W.-S. Lee, G. Gamba, E. M. Brown, and S. C. Hebert. Proc. Natl. Acad. Sci. USA 92: 131–135, 1994), which was proposed to provide the mechanism for modulating a variety of renal functions in response to changes in extracellular Ca2+ (E. M. Brown. In: Handbook of Physiology. Bethesda, MD: Am. Physiol. Soc., 1992, sect. 8, vol. 2, chapt. 39, p. 1841–1916; and S. C. Hebert. Kidney Int. 50: 2129–2139, 1996). Here, we examine the cellular and regional distribution of receptor protein by immunofluorescence microscopy using a polyclonal antibody raised against a 22 amino acid region of the NH2 terminus of the receptor. The most intense fluorescence was seen at the basolateral border of cortical thick ascending limb cells. Basolateral staining for the receptor was also detected in medullary thick ascending limbs, in macula densa cells identified by costaining with antibody to brain nitric oxide synthase, NOS-B1, and in distal convoluted tubule cells distinguished by costaining for the apical thiazide-sensitive Na+-Cl−cotransporter. Apical anti-RaKCaR staining was detected at the base of the brush border of proximal tubules with decreasing intensity from S1 to S3 segments. In cortical collecting ducts, anti-RaKCaR staining was detected in some, but not all, type A intercalated cells identified by costaining with anti-H+-ATPase and anti-AE1 Cl−/[Formula: see text]exchanger antibodies. The present study demonstrates that RaKCaR protein is expressed in many different nephron segments and that the polarity of receptor expression varies with cell type along the nephron. These results suggest potential roles for the extracellular Ca2+/polyvalent cation-sensing receptor in responding to both circulating and urinary concentrations of divalent minerals and potentially other polyvalent cations (e.g., aminoglycoside antibiotics) to modulate nephron function.

Th17 cells produce IL-17, and the latter promotes bone loss in collagen-induced arthritis in mice. Blocking IL-17 action in mouse model of rheumatoid arthritis reduces disease symptoms. These observations suggest that Th17 cells may be involved in the pathogenesis of bone loss. However, the role of Th17 cell in estrogen (E2) deficiency-induced bone loss is still not very clear. We investigated the effect of E2 on Th17 differentiation in vivo and IL-17 mediated regulation of osteoclast and osteoblast differentiation. Additionally, effect of IL-17 functional block under E2 deficiency-induced bone loss was studied. In murine bone marrow cells, E2 suppressed IL-17 mediated osteoclast differentiation. IL-17 inhibited formation of mineralized nodules in osteoblasts and this effect was suppressed by E2. E2 treatment to mouse calvarial osteoblasts inhibited the IL-17-induced production of osteoclastogenic cytokines and NF-kB translocation. In ovariectomized mice, there was increase in the number of Th17 cells, transcription factors promoting Th17 cell differentiation and circulating IL-17 levels. These effects were reversed by E2 supplementation. Treatment of neutralizing IL-17 monoclonal antibody to Ovx mice mitigated the E2 deficiency-induced trabecular bone loss and reversed the decreased osteoprotegerin-to-receptor activator of nuclear factor kappa B ligand (RANKL) transcript levels in long bones, increased osteoclast differentiation from the bone marrow precursor cells and decreased osteoblast differentiation from the bone marrow stromal cells. Our findings indicate that E2 deficiency leads to increased differentiation of Th17 cells with attendant up regulation of STAT3, ROR-γt and ROR-α and downregulation of Foxp3 which antagonizes Th17 cell differentiation. Increased IL-17 production in turn induces bone loss by increasing pro-osteoclastogenic cytokines including TNF-α, IL-6 and RANKL from osteoblasts and functional block of IL-17 prevents bone loss. IL-17 thus plays a critical causal role in Ovx-induced bone loss and may be considered a potential therapeutic target in pathogenesis of post menopausal osteoporosis.

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The extracellular calcium ([Formula: see text])-sensing receptor (CaR) plays vital roles in [Formula: see text] homeostasis, but no data are available on its expression in small and large intestine. Polymerase chain reaction products amplified from reverse-transcribed duodenal RNA using CaR-specific primers showed >99% homology with the rat kidney CaR. Northern analysis with a CaR-specific cRNA probe demonstrated 4.1- and 7.5-kb transcripts in all intestinal segments. Immunohistochemistry with CaR-specific antisera showed clear basal staining of epithelial cells of small intestinal villi and crypts and modest apical staining of the former, whereas there was both basal and apical staining of colonic crypt epithelial cells. In situ hybridization and immunohistochemistry also demonstrated CaR expression in Auerbach’s myenteric plexus of small and large intestines and in the submucosa in the region of Meissner’s plexus. Our results reveal CaR expression in several cell types of small and large intestine, in which it may modulate absorptive and/or secretomotor functions.

The recent cloning of a [Ca2+]o-sensing receptor from several different tissues in several species directly demonstrates that a variety of cells can directly recognize and respond to small changes in their ambient level of [Ca2+]o through a G protein-coupled, cell surface receptor. This finding directly documents that [Ca2+]o can act as an extracellular, first messenger in addition to subserving its better known role as an intracellular second messenger. Several of the tissues expressing the CaR are important elements in the calcium homeostatic system that have long been known to be capable of sensing [Ca2+]o, such as parathyroid and thyroidal C cells. The presence of the receptor in the kidney, however, provides strong evidence that several of the long-recognized but poorly understood direct actions of [Ca2+]o on renal function could be mediated by the CaR. These actions include the up-regulation of urinary calcium and magnesium excretion in the setting of hypercalcemia, which complements the indirect inhibition of renal tubular reabsorption of calcium that results from high [Ca2+]o-mediated inhibition for PTH secretion. The impaired renal concentrating capacity in hypercalcemia is likely a manifestation of a homeostatically important interaction between the regulation of renal calcium and water handling that reduces the risk of pathological deposition of calcium in the kidney when there is a need to dispose of excess, calcium in the urine. In this regard, the availability of human syndromes of [Ca2+]o "resistance" or "overresponsiveness" due to loss-of-function or gain-of-function mutations in the CaR, respectively, have provided useful experiments in nature that have clarified the importance of the receptor in both abnormal and normal physiology. Much remains to be learned, however, about the role of the CaR in locations, such as the brain, where it likely responds to local rather than systemic levels of [Ca2+]o. In such sites, it may represent an important modulator of neuronal function, responding to [Ca2+]o as a neuromodulator or even neurotransmitter. The development of therapeutics that either activate or inhibit the function of the CaR may be useful for treating a variety of conditions in which the receptor is either under- or overactive. Finally, it would not be surprising to discover additional receptors for [Ca2+]o or for other ions (the CaR may, in fact, be an important [Mg2+]o-sensor) that could function abnormally in certain disease states and be amenable to pharmacological manipulation with ion receptor-based therapeutics.

The G protein-coupled, extracellular calcium-sensing receptor (CaR) regulates parathyroid hormone secretion and parathyroid cellular proliferation as well as the functions of diverse other cell types. The CaR resides in caveolae-plasma membrane microdomains containing receptors and associated signaling molecules that are thought to serve as cellular "message centers." An additional mechanism for coordinating cellular signaling is the presence of scaffold proteins that bind and organize components of signal transduction cascades. With the use of the yeast two-hybrid system, we identified filamin-A (an actin-cross-linking, putative scaffold protein that binds mitogen-activated protein kinase (MAPK) components activated by the CaR) as an intracellular binding partner of the CaR's carboxyl (COOH)-terminal tail. A direct interaction of the two proteins was confirmed by an in vitro binding assay. Moreover, confocal microscopy combined with two color immunofluorescence showed co-localization of the CaR and filamin-A within parathyroid cells as well as HEK-293 cells stably transfected with the CaR. Deletion mapping localized the sites of interaction between the two proteins to a stretch of 60 amino acid residues within the distal portion of the CaR's COOH-terminal tail and domains 14 and 15 in filamin-A, respectively. Finally, introducing the portion of filamin-A interacting with the CaR into CaR-transfected HEK-293 cells using protein transduction with a His-tagged, Tat-filamin-A fusion protein nearly abolished CaR-mediated activation of ERK1/2 MAPK but had no effect on ERK1/2 activity stimulated by ADP. Therefore, the binding of the CaR's COOH-terminal tail to filamin-A may contribute to its localization in caveolae, link it to the actin-based cytoskeleton, and participate in CaR-mediated activation of MAPK.

Intracellular transduction pathways that are dependent on activation of the CaR by Ca(o)2+ have been studied extensively in parathyroid and other cell types, and include cytosolic calcium, phospholipases C, A2, and D, protein kinase C isoforms and the cAMP/protein kinase A system. In this study, using bone marrow cells isolated from CaR-/- mice as well as DN-CaR-transfected RAW 264.7 cells, we provide evidence that expression of the CaR plays an important role in osteoclast differentiation. We also establish that activation of the CaR and resultant stimulation of PLC are involved in high Ca(o)2+-induced apoptosis of mature rabbit osteoclasts. Similar to RANKL, Ca(o)2+ (20 mM) appeared to trigger rapid and significant nuclear translocation of NF-kappaB in a CaR- and PLC-dependent manner. In summary, our data suggest that stimulation of the CaR may play a pivotal role in the control of both osteoclast differentiation and apoptosis in the systems studied here through a signaling pathway involving activation of the CaR, phospholipase C, and NF-kappaB.

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