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1.
J Cell Biochem ; 74(4): 587-95, 1999 Sep 15.
Article in English | MEDLINE | ID: mdl-10440928

ABSTRACT

Commitment of members of the monocyte/macrophage family to the bone resorptive phenotype, in vitro, requires contact, of these osteoclast precursors, with osteoblasts or related stromal cells. The osteoclast-inductive properties of these stromal cells are typically expressed, however, only in the presence of steroid hormones such as 1,25 dihydroxyvitamin D (1,25D3) and dexamethasone (DEX). To gain insight into the means by which steroid treated accessory cells induce osteoclast differentiation we asked, using differential RNA display (DRD), if gene expression by this stromal cell population differs from that of their untreated, non-osteoclastogenic counterpart. We identified four known genes specifically expressed by 1,25D3/DEX-treated ST2 stromal cells: 1) a family of rat organic anion transporters, 2) Na/K ATPase ss-subunit, 3) tazarotene-induced gene 2 (TIG2), and 4) prostaglandin G/H synthase I, or cyclooxygenase 1 (Cox-1). The regulation of these genes in 1,25D3/DEX-treated ST2 cells was demonstrated by Northern blot analysis of treated (osteoclast-supporting) and untreated (non-osteoclast-supporting) ST2 cells; the genes have a limited and specific tissue mRNA expression pattern. Northern blot analysis of treated and untreated ST2 cell total RNA using either a DRD-derived Cox-1 cDNA or a Cox-1 specific oligonucleotide confirmed the steroid regulation of Cox-1 mRNA. Surprisingly, there is no detectable expression by untreated or steroid exposed ST2 cells, of Cox-2, the classical regulated cyclooxygenase isoform. In contrast to 1, 25D3/DEX, serum treatment rapidly induces Cox-2 mRNA, substantiating the capacity of ST2 cells to express the gene. These data establish that steroid induction of the osteoclastogenic properties of stromal cells is attended by Cox gene expression, a phenomenon consistent with the capacity of eicosinoids to impact the resorptive process. The response of osteoclast-supporting ST2 cells to 1,25D3/DEX treatment may be one prostaglandin-mediated event which specifically involves Cox-1 regulation.


Subject(s)
Calcitriol/pharmacology , Dexamethasone/pharmacology , Isoenzymes/genetics , Osteoclasts/cytology , Prostaglandin-Endoperoxide Synthases/genetics , Stromal Cells/drug effects , Stromal Cells/enzymology , Animals , Bone Remodeling/drug effects , Bone Remodeling/genetics , Cell Line , Cyclooxygenase 1 , Cyclooxygenase 2 , Gene Expression Regulation, Enzymologic/drug effects , Membrane Proteins , Mice , Osteoblasts/cytology , Osteoblasts/metabolism , Osteoclasts/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Sodium-Potassium-Exchanging ATPase/genetics , Stromal Cells/cytology
2.
Endocrinology ; 137(7): 2748-57, 1996 Jul.
Article in English | MEDLINE | ID: mdl-8770894

ABSTRACT

We have generated a series of stably transfected HEK-293 cell lines expressing the newly identified alternate human PTH receptor (hPTH2 receptor). This receptor subtype is selectively activated by N-terminal PTH-(1-34) and not the corresponding N-terminal (1-34) region of the functionally and structurally related hormone, PTH-related protein (PTHrP). A total of 20 distinct clones displaying different levels of PTH-responsive cAMP production were analyzed. None responded to PTHrP-(1-34). One of these clones (BP-16), displaying maximal PTH responsiveness, was chosen for more detailed evaluation. The BP-16 clone (and the parental HEK-293 cell line lacking both the hPTH/PTHrP receptor and the hPTH2 receptor) were examined for PTH binding, PTH-stimulated cAMP accumulation, PTH-stimulated changes in intracellular calcium ([Ca2+]i) levels, and hPTH2 receptor messenger RNA expression. In addition, we studied the photomediated cross-linking of a potent PTH agonist, namely [Nle8,18,Lys13 (epsilon-pBz2), 2-L-Nal23,Tyr34]bPTH(1-34)NH2 (K13), to the hPTH2 receptor on BP-16 cells. Photoaffinity cross-linking identified an approximately 90-kDa cell membrane component that was specifically competed by PTH-(1-34) and other receptor-interacting ligands. PTH-(1-34) and K13 are potent stimulators of both cAMP accumulation and increases in (Ca2+]i levels, and both bind to the hPTH2 receptor with high affinity (apparent Kd, 2.8 +/- 0.9 x 10(-8) and 8.5 +/- 1.7 x 10(-8) M, respectively). There was no apparent binding, cAMP-stimulating activity, or [Ca 2+]i signaling observed, nor was specific competition vs. binding of a PTH-(1-34) radioligand ([125I]PTH) with PTHrP-(1-34)NH2 found. PTHrP-(1-34) failed to inhibit cross-linking of the hPTH2 receptor by radiolabeled K13 ([125I]K13). However, effective competition vs. [125I]PTH and [125I]K13 binding and [125I]K13 cross-linking were observed with the potent PTH/PTHrP receptor antagonists, PTHrP-(7-34)NH2 and PTH-(7-34)NH2. PTHrP-(7-34)NH2 was shown to be a partial agonist that weakly stimulates both cAMP accumulation and increases in [Ca 2+]i levels in BP-16 cells. These data suggest that the hPTH2 receptor is distinct from the hPTH/PTHrP receptor in the structural features it requires for ligand binding in the family of PTH and PTHrP peptides.


Subject(s)
Parathyroid Hormone/pharmacology , Peptide Fragments/pharmacology , Proteins/pharmacology , Receptors, Parathyroid Hormone/physiology , Signal Transduction , Calcium/metabolism , Cell Line , Cross-Linking Reagents , Cyclic AMP/metabolism , Egtazic Acid/pharmacology , Humans , Ionomycin/pharmacology , Kidney , Kinetics , Parathyroid Hormone/metabolism , Parathyroid Hormone-Related Protein , Peptide Fragments/metabolism , Proteins/metabolism , Radioligand Assay , Receptor, Parathyroid Hormone, Type 2 , Receptors, Parathyroid Hormone/biosynthesis , Receptors, Parathyroid Hormone/isolation & purification , Receptors, Parathyroid Hormone/metabolism , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Teriparatide , Transfection
4.
Endocrinology ; 135(4): 1713-6, 1994 Oct.
Article in English | MEDLINE | ID: mdl-7925136

ABSTRACT

Parathyroid hormone (PTH) exerts its biological action by binding to membrane-bound, G-protein coupled receptors expressed predominantly in bone and kidney. In this study, we describe the production and characterization of a panel of cell lines, derived from a human embryonic kidney cell line (HEK-293), each of which stably express different amounts of the recombinant human PTH/parathyroid hormone-related protein (PTHrP) receptor (Rc). A total of 52 distinct clones displaying different levels of PTH-responsive cAMP production were analyzed; three clones were chosen for more detailed evaluation. These clones (and the receptor-lacking parental cell line) were examined for PTH binding, PTH-stimulated cyclic AMP accumulation and PTH/PTHrP Rc mRNA expression. Receptor-positive clones display a spectrum of PTH-responsiveness that correlates with receptor number/cell and level of receptor mRNA present. The interaction of a C-terminal hPTH-(52-84) peptide with the stably expressed human receptor was examined in cells expressing the highest amount of Rc (> 400,000 Rc/cell). There was no direct binding of hPTH-(52-84) or specific competition versus radiolabeled PTH-(1-34). However, competition versus radiolabeled PTH-(1-34) was observed with bPTH-(1-34), hPTH-(1-84) and hPTHrP-(1-34). These data suggest that hPTH-(52-84) does not interact with the only known form of the human PTH/PTHrP Rc. Therefore, the reported effects of PTH-(52-84) in other systems must be via an alternate (as yet unidentified) mechanism(s). The expression of various amounts of the human PTH/PTHrP Rc in a human target cell background should facilitate characterization of the ligand-binding properties and physiological signal transduction mechanism of the Rc.


Subject(s)
Kidney/chemistry , Kidney/cytology , Receptors, Parathyroid Hormone/analysis , Cell Line , Cyclic AMP/metabolism , DNA/analysis , DNA/genetics , Humans , Kidney/metabolism , Parathyroid Hormone/analysis , Parathyroid Hormone/genetics , Parathyroid Hormone/metabolism , Peptide Fragments/analysis , Peptide Fragments/genetics , Peptide Fragments/metabolism , RNA, Messenger/analysis , RNA, Messenger/genetics , Receptor, Parathyroid Hormone, Type 1 , Receptors, Parathyroid Hormone/genetics , Receptors, Parathyroid Hormone/metabolism , Recombinant Proteins/analysis , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Signal Transduction/physiology , Teriparatide
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