Naibedya Chattopadhyay

Localization of the extracellular Ca2+/polyvalent cation-sensing protein in rat kidney

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.

Mouse Osteoblastic Cell Line (MC3T3-E1) Expresses Extracellular Calcium (Ca2+o)–Sensing Receptor and Its Agonists Stimulate Chemotaxis and Proliferation of MC3T3-E1 Cells

The calcium‐sensing receptor (CaR) is a G protein‐coupled receptor that plays key roles in extracellular calcium ion (Ca2+o) homeostasis in parathyroid gland and kidney. Osteoblasts appear at sites of osteoclastic bone resorption during bone remodeling in the “reversal” phase following osteoclastic resorption and preceding bone formation. Bone resorption produces substantial local increases in Ca2+o that could provide a signal for osteoblasts in the vicinity, leading us to determine whether such osteoblasts express the CaR. In this study, we used the mouse osteoblastic, clonal cell line MC3T3‐E1. Both immunocytochemistry and Western blot analysis, using an antiserum specific for the CaR, detected CaR protein in MC3T3‐E1 cells. We also identified CaR transcripts in MC3T3‐E1 cells by Northern analysis using a CaR‐specific riboprobe and by reverse transcription‐polymerase chain reaction with CaR‐specific primers, followed by nucleotide sequencing of the amplified products. Exposure of MC3T3‐E1 cells to high Ca2+o (up to 4.8 mM) or the polycationic CaR agonists, neomycin and gadolinium (Gd3+), stimulated both chemotaxis and DNA synthesis in MC3T3‐E1 cells. Therefore, taken together, our data strongly suggest that the osteoblastic cell line MC3T3‐E1 possesses both CaR protein and mRNA very similar, if not identical, to those in parathyroid and kidney. Furthermore, the CaR in these osteoblasts could play a key role in regulating bone turnover by stimulating the proliferation and migration of such cells to sites of bone resorption as a result of local release of Ca2+o.

Identification and localization of extracellular Ca2+-sensing receptor in rat intestine

The extracellular calcium (Ca2+o)-sensing receptor (CaR) plays vital roles in Ca2+o 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 Auerbachs myenteric plexus of small and large intestines and in the submucosa in the region of Meissners 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 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 Auerbachs myenteric plexus of small and large intestines and in the submucosa in the region of Meissners 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.

Expression of an Extracellular Calcium‐Sensing Receptor in Human and Mouse Bone Marrow Cells

The cloning of a G protein–coupled, extracellular calcium (Ca2+e)‐sensing receptor (CaR) from bovine parathyroid provided direct evidence that Ca2+e‐sensing can occur through receptor‐mediated activation of G proteins and their associated downstream regulators of cellular function. CaR transcripts and protein are present in various tissues of humans and other mammals that are involved in Ca2+e homeostasis, including parathyroid, kidney, and thyroidal C‐cells. The present study was performed to determine whether bone marrow cells express the CaR, since cells within the marrow space could be exposed to substantial changes in Ca2+e related to bone turnover. Using DNA and RNA probes from the human parathyroid CaR cDNA, we identified CaR transcripts of 5.2 and ∼4.0 kilobases by Northern analysis of poly(A+) RNA from low‐density mononuclear cells isolated from whole human bone marrow that are putatively enriched in marrow progenitor cells, including bone cell precursors. In situ hybridization also identified CaR transcripts in the same cell preparations. Reverse transcription‐polymerase chain reaction demonstrated >99% nucleotide identity between transcripts from human bone marrow cells and the corresponding regions of the human CaR cDNA. Antisera specific for several different regions within the extracellular domain of the CaR were reactive with low‐density human marrow cells that were either adherent or nonadherent to plastic. About one‐third of the adherent, CaR‐immunoreactive cells were also positive for alkaline phosphatase, a nonspecific marker of preosteoblasts, osteoblasts, and assorted cells of the colony‐forming unit‐fibroblast lineage. In addition, a substantial fraction (∼60%) of low density murine marrow cells cultured for 1 week at 4.8 mM Ca2+e expressed both CaR immunoreactivity and nonspecific esterase, an enzyme expressed by monocyte/macrophages and fibroblasts. Finally, erythroid precursors and megakaryocytes from murine marrow as well as blood platelets expressed abundant CaR immunoreactivity, while peripheral blood erythrocytes and most polymorphonuclear leukocytes did not. These studies indicate that the CaR is present in low‐density mononuclear bone marrow cells as well as in cells of several hematopoietic lineages and could potentially play a role in controlling the function of various cell types within the marrow space.

The calcium sensing receptor is directly involved in both osteoclast differentiation and apoptosis

Intracellular transduction pathways that are dependent on activation of the CaR by Cao2+ 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 Cao2+‐induced apoptosis of mature rabbit osteoclasts. Similar to RANKL, Cao2+ (20 mM) appeared to trigger rapid and significant nuclear translocation of NF‐κB 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‐κB.— Mentaverri, R., Yano, S., Chattopadhyay, N., Petit, L., Kifor, O., Kamel, S., Terwilliger, E. F., Brazier, M., Brown, E. M. The calcium sensing receptor is directly involved in both osteoclast differentiation and apoptosis. FASEB J. 20, E1945‐E1954 (2006)

Estrogen Deficiency Induces the Differentiation of IL-17 Secreting Th17 Cells: A New Candidate in the Pathogenesis of Osteoporosis

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.

Expression of an extracellular calcium-sensing receptor in rat stomach

BACKGROUND & AIMS Circulating levels of Ca2+ can influence secretory functions and myoelectrical properties of the stomach. A Ca2+-sensing receptor (CaR) has recently been identified in tissues that regulate systemic Ca2+ homeostasis. The aim of this study was to evaluate expression of CaR in the stomach of the rat. METHODS In forestomach and glandular stomach, reverse-transcription polymerase chain reaction was used to amplify a 380-base pair product, which is 99% homologous with transcripts obtained in parathyroid and kidney. RESULTS Northern analysis of gastric mucosal polyA+ RNA revealed 7. 5- and 4.1-kilobase transcripts, similar to those obtained in rat parathyroid and kidney. Immunohistochemistry revealed CaR expression in regions of the submucosal plexus and myenteric neurons. In sections of intact tissue, preparations of primary culture surface cells and surgically dissected gastric glands, staining was observed consistently in epithelial cells of the gastric glands and in gastric surface cells. In parietal cells in isolated gastric glands, intracellular levels of Ca2+ responded to conditions that are known to activate CaR. CONCLUSIONS These are the first reported observations that CaR is expressed in different epithelial cells of mammalian gastric mucosa and its enteric nerve regions. The effects of extracellular Ca2+ on gastric function may be attributable to activation of CaR.

Kaempferol has osteogenic effect in ovariectomized adult Sprague–Dawley rats

Kaempferol (K), a flavonol, is known to have anti-osteoclastogenic effect. We here show that K, from 0.2 to 5.0 microM, increased mineralized nodules in rat primary osteoblasts. K also significantly attenuated adipocyte formation from bone marrow cells (BMCs). A single oral dose of 1 mg/kg body weight of K in Sprague-Dawley (180-200 g) rats resulted in a peak serum level of 2.04+/-0.8 nM in 30 min (Tmax), suggesting its rapid absorption. The Cmax of K in bone marrow was 0.684 nM after 90 min. Rats were ovariectomized (OVx) along with sham-operated rats and left for 4 weeks. Daily oral administration of K (5 mg/kg body weight) was then started to one group of OVx rats, and continued for 10 weeks. K levels were found to be 0.311 and 0.838 nM at the end of 4 and 10 weeks, respectively. K exhibited no estrogenicity at the uterine level. The K-treated group exhibited significantly higher bone mineral density (BMD) in the trabecular regions (femur neck, proximal tibia and vertebrae) and lower serum ALP (bone turnover marker) compared with the OVx rats. The compressive energy of the vertebrae was significantly higher in the OVx+K-treated group compared with the OVx group. K treatment of OVx rats resulted in the increase in osteoprogenitor cells as well as inhibition of adipocyte differentiation from BMCs compared with the OVx group. Together we show that K is non-estrogenic in vivo and exerts bone anabolic activity with attendant inhibition of bone marrow adipogenesis.

Expression of peroxisome proliferator-activated receptors (PPARS) in human astrocytic cells: PPAR? agonists as inducers of apoptosis

We report the isolation by RT‐PCR of partial cDNAs encoding the human peroxisome proliferator‐activated receptor (PPAR) isoforms PPARβ and ‐γ in human primary astrocytes (HPA) as well as in the human malignant astrocytoma cell line T98G. In contrast, we failed to detect PPARα mRNA in either of these two cell types. Because PPARβ is ubiquitously expressed but has, as yet, no known function, we pursued our functional studies of these cells with regard to PPARγ. To that end, we showed that PPARγ protein is abundantly expressed in both cell types, having a molecular weight of approximately 50 kDa. Immunocytochemistry revealed a predominantly nuclear localization of this receptor. Moreover, incubation of the two cell types with 1–12 μM 15‐deoxy PGJ2 or 1–12 μM ciglitazone, both of which are agonists of PPARγ, induced loss of cellular viability as assessed by the MTT assay after a 4 hr incubation. Reduced cellular viability as a consequence of exposure to PGJ2 or ciglitazone resulted from induction of apoptosis, as assessed by DNA fragmentation and Hoechst staining, and involves activation of the CPP32 (caspase‐3) protease. These data show that modulation of the process of apoptosis is one function of PPARγ in cells derived from the human astrocytic lineage. J. Neurosci. Res. 61:67–74, 2000.

Extracellular calcium (Ca2+(o))-sensing receptor in a murine bone marrow-derived stromal cell line (ST2): potential mediator of the actions of Ca2+(o) on the function of ST2 cells

The calcium-sensing receptor (CaR) is a G protein-coupled receptor that plays key roles in extracellular calcium ion (Ca2+(o)) homeostasis by mediating the actions of Ca2+(o) on parathyroid gland and kidney. Bone marrow stromal cells support the formation of osteoclasts from their progenitors as well as the growth of hematopoietic stem cells by secreting humoral factors and through cell to cell contact. Stromal cells also have the capacity to differentiate into bone-forming osteoblasts. Bone resorption by osteoclasts probably produces substantial local increases in Ca2+(o) that could provide a signal for stromal cells in the immediate vicinity, leading us to determine whether such stromal cells express the CaR. In this study, we used the murine bone marrow-derived, stromal cell line, ST2. Both immunocytochemistry and Western blot analysis, using an antiserum specific for the CaR, detected CaR protein in ST2 cells. We also identified CaR transcripts in ST2 cells by Northern analysis using a CaR-specific probe and by RT-PCR with CaR-specific primers, followed by nucleotide sequencing of the amplified products. Exposure of ST2 cells to high Ca2+(o) (4.8 mM) or to the polycationic CaR agonists, neomycin (300 microM) or gadolinium (100 microM), stimulated both chemotaxis and DNA synthesis in ST2 cells. Therefore, taken together, our data strongly suggest that the bone marrow-derived stromal cell line, ST2, possesses both CaR protein and messenger RNA that are very similar if not identical to those in parathyroid and kidney. Furthermore, as ST2 cells have the potential to differentiate into osteoblasts, the CaR in stromal cells could participate in bone turnover by stimulating the proliferation and migration of such cells to sites of bone resorption as a result of local, osteoclast-mediated release of Ca2+(o) and, thereafter, initiating bone formation after their differentiation into osteoblasts.