J L Sanders

Brigham and Women's Hospital , Boston, MA, United States

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Publications (8)28.42 Total impact

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    ABSTRACT: Prostate cancer metastasizes frequently to bone. Elevated extracellular calcium concentrations ([Ca(2+)](o)) stimulate parathyroid hormone-related protein (PTHrP) secretion from normal and malignant cells, potentially acting via the [Ca(2+)](o)-sensing receptor (CaR). Because prostate cancers produce PTHrP, if high [Ca(2+)](o) stimulates PTHrP secretion via the CaR, this could initiate a mechanism whereby osteolysis caused by bony metastases of prostate cancer promotes further bone resorption. We investigated whether the prostate cancer cell lines LnCaP and PC-3 express the CaR and whether polycationic CaR agonists stimulate PTHrP release. Both PC-3 and LnCaP prostate cancer cell lines expressed bona fide CaR transcripts by Northern analysis and RT-PCR and CaR protein by immunocytochemistry and Western analysis. The polycationic CaR agonists [Ca(2+)](o), neomycin, and spermine each concentration dependently stimulated PTHrP secretion from PC-3 cells, as measured by immunoradiometric assay, with maximal, 3.2-, 3.6-, and 4.2-fold increases, respectively. In addition, adenovirus-mediated infection of PC-3 cells with a dominant negative CaR construct attenuated high [Ca(2+)](o)-evoked PTHrP secretion, further supporting the CaR's mediatory role in this process. Finally, pretreating PC-3 cells with transforming growth factor (TGF)-beta(1) augmented both basal and high [Ca(2+)](o)-stimulated PTHrP secretion. Thus, in PTHrP-secreting prostate cancers metastatic to bone, the CaR could initiate a vicious cycle, whereby PTHrP-induced bone resorption releases [Ca(2+)](o) and TGF-beta stored within bone, further increasing PTHrP release and osteolysis.
    AJP Endocrinology and Metabolism 01/2002; 281(6):E1267-74. · 4.51 Impact Factor
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    ABSTRACT: We have previously shown the expression of the extracellular calcium (Ca2+o)-sensing receptor (CaR) in osteoblast-like cell lines, and others have documented its expression in sections of murine, bovine, and rat bone. The existence of the CaR in osteoblasts remains controversial, however, since some studies have failed to document its expression in the same osteoblast-like cell lines. The goals of the present study were twofold. 1) We sought to determine whether the CaR is expressed in the human osteoblast-like cell line, MG-63, which has recently been reported by others not to express this receptor. 2) We investigated whether the CaR, if present in MG-63 cells, is functionally active, since most previous studies have not proven the role of the CaR in mediating known actions of Ca2+o on osteoblast-like cells. We used immunocytochemistry and Western blotting with the specific, affinity-purified anti-CaR antiserum 4637 as well as Northern blot analysis and RT-PCR using a riboprobe and PCR primers specific for the human CaR, respectively, to show readily detectable CaR protein and mRNA expression in MG-63 cells. Finally, we employed the patch-clamp technique to show that an elevation in Ca2+o as well as the specific, allosteric CaR activator NPS R-467 (0.5 microM), but not its less active stereoisomer NPS S-467 (0.5 microM), activate an outward K+ channel in MG-63 cells, strongly suggesting that the CaR in MG-63 cells is not only expressed but is functionally active.
    AJP Cell Physiology 03/2001; 280(2):C382-93. · 3.71 Impact Factor
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    ABSTRACT: Metastasis of breast cancer to bone occurs with advanced disease and produces substantial morbidity. Secretion of PTH-related peptide (PTHrP) from breast cancer cells is thought to play a key role in osteolytic metastases and is increased by transforming growth factor-beta (TGFbeta), which is released from resorbed bone. Elevated extracellular calcium (Ca2+(o)) also stimulates PTHrP secretion from various normal and malignant cells, an action that could potentially be mediated by the Ca2+(o)-sensing receptor (CaR) originally cloned from the parathyroid gland. Indeed, we previously showed that both normal breast ductal epithelial cells and primary breast cancers express the CaR. In this study we investigated whether the MCF-7 and MDA-MB-231 human breast cancer cell lines express the CaR and whether CaR agonists modulate PTHrP secretion. Northern blot analysis and RT-PCR revealed bona fide CaR transcripts, and immunocytochemistry and Western analysis with a specific anti-CaR antiserum demonstrated CaR protein expression in both breast cancer cell lines. Furthermore, elevated Ca2+(o) and the polycationic CaR agonists, neomycin and spermine, stimulated PTHrP secretion dose dependently, with maximal, 2.1- to 2.3-fold stimulation. In addition, pretreatment of MDA-MB-231 cells overnight with TGFbeta1 (0.2, 1, or 5 ng/ml) augmented both basal and high Ca2+-stimulated PTHrP secretion. Thus, in PTHrP-secreting breast cancers metastatic to bone, the CaR could potentially participate in a vicious cycle in which PTHrP-induced bone resorption raises the levels of Ca2+(o) and TGFbeta within the bony microenvironment, which then act in concert to evoke further PTHrP release and worsening osteolysis.
    Endocrinology 01/2001; 141(12):4357-64. · 4.72 Impact Factor
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    ABSTRACT: Recently, substantial evidence has accumulated that the G-protein-coupled, extracellular calcium (Ca2+o)-sensing receptor (CaR) is expressed in bone marrow-derived cells, including osteoblasts, stromal cells, monocytes–macrophages, and osteoclast precursor cells. Our previous studies have shown that the mouse osteoblastic MC3T3-E1 cell line also expresses the CaR and exhibits mitogenic responses when exposed to various CaR agonists. In this study, in order to understand the signaling pathway(s) mediating this response, we studied the effects of CaR agonists on the phosphorylation of p42/44 mitogen-activated protein kinase (MAPK) (Erk1/2), p38 MAPK, and c-Jun N-terminal kinase (JNK) in MC3T3-E1 cells. Raising the level of Ca2+o (4.5 mM) or addition of the polycationic CaR agonists, gadolinium (Gd3+) (25 μM), neomycin (300 μM) or spermine (1 mM), each stimulated phosphorylation of both p42/44 and p38 MAPKs, but not JNK, as assessed using phospho-specific antibodies to the respective MAPKs. Furthermore, phosphorylation of p42/44 and p38 MAPK were markedly inhibited by their selective and potent inhibitors, PD98059 (50 μM) and SB203580 (10 μM), respectively. Finally, the two inhibitors suppressed [3H]thymidine incorporation into DNA in MC3T3-E1 cells at a normal level of Ca2+o (1.8 mM) as well as when stimulated by high (4.5 mM) Ca2+o, Gd3+, or neomycin. Thus, in mouse osteoblastic MC3T3-E1 cells, both the p42/44 and p38 MAPK cascades play pivotal roles in CaR-stimulated mitogenic responses.
    Biochemical and Biophysical Research Communications 01/2001; · 2.28 Impact Factor
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    ABSTRACT: Parathyroid hormone-related protein (PTHrP) is the major mediator of the humoral hypercalcemia of malignancy and of malignant osteolysis associated with skeletal metastases of common epithelial cancers. PTHrP secretion is regulated by the extracellular calcium concentration ([Ca(2+)](o)) in several types of normal and malignant cells. Because the [Ca(2+)](o)-sensing receptor (CaR) is a key mediator of [Ca(2+)](o)-regulated hormone secretion [e.g., of parathyroid hormone (PTH) by parathyroid chief cells], we investigated the expression of the CaR and PTHrP in normal and neoplastic glial cells and studied the effects of [Ca(2+)](o) on PTHrP secretion. Our results show that primary embryonic human astrocytes (HPA) express CaR mRNA and protein as detected by RT-PCR and Western analysis, respectively. Furthermore, astrocytomas and meningiomas also express the CaR at similar levels as assessed by RT-PCR and Northern and Western blot analyses. HPA and astrocytomas express transcripts encoding all three known isoforms of PTHrP [PTHrP(139), PTHrP(141), and PTHrP(173), comprising 139, 141, and 173 predicted amino acid residues, respectively] as assessed by RT-PCR, whereas meningiomas express only the first two of these. Finally, elevated levels of [Ca(2+)](o) and other polycationic CaR agonists dose dependently stimulate PTHrP secretion from HPA, astrocytomas, and meningiomas, although both basal and high [Ca(2+)](o)-stimulated rates of PTHrP secretion are approximately 2. 5-fold higher in HPA than in the glial tumors studied here. Therefore, our results show that HPA, astrocytomas, and meningiomas express both the CaR and PTHrP and that CaR agonists stimulate PTHrP secretion.
    AJP Cell Physiology 10/2000; 279(3):C691-9. · 3.71 Impact Factor
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    ABSTRACT: The existence in osteoblasts of the G-protein-coupled extracellular calcium (Ca(o)(2+))-sensing receptor (CaR) that was originally cloned from parathyroid and kidney remains controversial. In our recent studies, we utilized multiple detection methods to demonstrate the expression of CaR transcripts and protein in several osteoblastic cell lines, including murine MC3T3-E1 cells. Although we and others have shown that high Ca(o)(2+) and other polycationic CaR agonists modulate the function of MC3T3-E1 cells, none of these actions has been unequivocally shown to be mediated by the CaR. Previous investigations using neurons and lens epithelial cells have shown that activation of the CaR stimulates Ca(2+)-activated K(+) channels. Because osteoblastic cells express a similar type of channel, we have examined the effects of specific "calcimimetic" CaR activators on the activity of a Ca(2+)-activated K(+) channel in MC3T3-E1 cells as a way of showing that the CaR is not only expressed in those cells but is functionally active. Patch-clamp analysis in the cell-attached mode showed that raising Ca(o)(2+) from 0.75 to 2.75 mmol/L elicited about a fourfold increase in the open state probability (P(o)) of an outward K(+) channel with a conductance of approximately 92 pS. The selective calcimimetic CaR activator, NPS R-467 (0.5 micromol/L), evoked a similar activation of the channel, while its less active stereoisomer, NPSS-467 (0.5 micromol/L), did not. Thus, the CaR is not only expressed in MC3T3-E1 cells, but is also functionally coupled to the activity of a Ca(2+)-activated K(+) channel. This receptor, therefore, could transduce local or systemic changes in Ca(o)(2+) into changes in the activity of this ion channel and related physiological processes in these and perhaps other osteoblastic cells.
    Bone 08/2000; 27(1):21-7. · 4.46 Impact Factor
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    ABSTRACT: Humoral hypercalcemia of malignancy (HHM) occurs when secretion of parathyroid hormone-related peptide (PTHrP) by cancer cells causes hypercalcemia in the absence of skeletal metastases. High extracellular calcium (Ca2+o) increases secretion of PTH-like bioactivity by rat H-500 leydig cells, a transplantable model of HHM, an action potentially mediated by the Ca2+o-sensing receptor (CaR). In this study we investigated whether H-500 cells express the CaR and, if so, whether CaR agonists modulate PTHrP secretion. Northern blot analysis and RT-PCR revealed bona fide CaR transcript(s), and immunocytochemistry and Western analysis with a specific anti-CaR antiserum demonstrated CaR protein expression in H-500 cells. Furthermore, high Ca2+o and neomycin stimulated PTHrP secretion dose-dependently with maximal 2.7- and 3.3-fold increases at 5 mM Ca2+o and 300 μM neomycin, respectively. Thus in HHM caused by H-500 cells, the CaR could participate in a vicious cycle whereby PTHrP-induced increases in Ca2+o further stimulate PTHrP release and exacerbate hypercalcemia.
    Biochemical and Biophysical Research Communications 04/2000; · 2.28 Impact Factor
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    ABSTRACT: . Human promyelocytic leukemia cells (HL-60) have been used widely as a model for studying the differentiation of hematopoietic progenitor cells in vitro. After treatment with phorbol-12-myristate-13-acetate (PMA) or 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], HL-60 cells differentiate into cells with the phenotype of monocytes/macrophages. We previously showed that peripheral blood monocytes and the murine J774 monocytic cell line express the CaR, and myeloid progenitors in the bone marrow and myeloid cells in peripheral blood other than monocytes express lower levels of the CaR. Therefore, we investigated whether undifferentiated HL-60 cells express a functional G protein-coupled, extracellular calcium (Ca2+ o)-sensing receptor (CaR) and if the expression of the CaR increases as these cells differentiate along the monocytic lineage. The use of reverse transcription-polymerase chain reaction (RT-PCR) with CaR-specific primers, followed by sequencing of the amplified products, identified an authentic CaR transcript in undifferentiated HL-60 cells. Both immunocytochemistry and Western blot analysis using a CaR-specific antiserum detected low levels of CaR protein expression in undifferentiated HL-60 cells. The levels of CaR protein increased considerably following treatment of the cells with PMA (50 nM) or 1,25(OH)2D3 (100 nM) for 5 days. Northern analysis using a CaR-specific riboprobe identified CaR transcripts in undifferentiated HL-60 cells, but CaR mRNA levels did not change appreciably after treatment with either agent, suggesting that upregulation of CaR protein occurs at a translational level. PMA-treated HL-60 cells expressed a nonselective cation channel (NCC), and the calcimimetic CaR activator, NPS R-467, but not its less active stereoisomer, NPS S-467, as well as the polycationic CaR agonist, neomycin, activated this NCC, demonstrating that the CaR expressed in these cells is functionally active. Therefore, HL-60 cells exhibit an increase in CaR protein expression, occurring at a translational level during their differentiation into cells with a monocyte/macrophage phenotype in response to treatment with PMA or 1,25(OH)2D3, which is functionally linked to activation of a nonselective cation channel.
    Calcified Tissue International 01/2000; 66(5):375-382. · 2.75 Impact Factor