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Gene regulation in osteoblastic cells by carboxylterminal PTH peptides: Effects of hPTH (53–84) in human osteoblastic SaOS-2 cells
24 CHARACTERIZATION OF A RATGENE ENCODING THERECEPTOROF PARATHYRDID HORtlONEAND PARATNYROID HORMONE-RELATED PEPTIDa. C. Kona. 9. Arena. E. Schipani. H. JiiDDner. H. Kconenberg, G. Senre, A.B. Abou-Samra. Endocrine Unit, Hassachusetts General Hospital, Harvard Medical School, Boston, HA! USA. Genomic DNA encoding the rat PTWPTHrPreceptor was cloned by hybridization screening of a lambda phage library vith rat bone receptor cDNA (ASBMR meeting, By comparing the sequences of the cDNA with 1991). those of the genomic DNA (a 12 Kb fragment), we can cDNA-based primers were define intron-exon borders. used for polymerase chain reaction and DNA sequence Unlike the genes encoding the adrenergic and analysis. the PTH/PTHrP receptor gene receptors, muscarinic Thus far, ve contains multiple intervening sequences. have identified 8 introns interrupting :he region Therefore, the rat encoding amino acids 25-322. PT.H/PTHrP receptor gene contains at least 9 exons. An intrrveninp sequence separates the coding region(s) of the signal peptfdc from tb? nwns encoding the amfnoThe amino-terminal tsrmlnal rxtracsllular domain, sxrretxllulet domain is encoded by k exons, one ol which encodes a region (40 amino acids) containing all 4 petantiel glycesyletion siks. Each of the remaining 3 axons thus far characterized encodes a potential trnnsmembrane domain (I, II, and III); intervening scqusncas interrupt DNA encoding the extracellular or cy~oplasmls loops, Thus the available datn Indicate that the rat PTH/PTHrP receptor gene contains several exons; each The encodes a functional domain of the receptor. multiale intervening sequences in the rat PTWPTHrP racepiar gene rais& the possibility that receptor variants may be generated through alternative splicing Complete characterization of the receptor of the gene. gene and the search for PTWPTHrP receptor variants are under current investigation in our laboratory.
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GENEREGULATION IN OSTEOBLASTIC CELLS BY CARBOXYLTERMINALPTU PEPTIDES:EFFECTS OF hPTH (53.84) IN HUUAN Se. Nichael’s Hoopitzsl and University of Toronto, Torooto, Canada, end Dapartmont of Biochemistry, UWrersity of Arizona, Tucson, AZ, USA. Previous work in our laboratory has provided evidence for bind@ sites specific for the carboxylt.srminal (G-terminal) fragment of PTH comprising rrsiduesl 53 to 84 in kidney and bone. In ROS 17/2.8 cells, 80% of added intact bPTH (l-84) binds via Cterminal residues within the (53-84) realon llZndoct=ineloa, 11?:1832, 1983). In addition, wg have recently shown that hPTH (53-84) can stimulate alkaline ohosphatase (ALP) nativity in rat osteoblasttc ROS lj/2.8 cells, which mntrasts with the suppressive effects of hPTH (l-34) and hPTH (l-84) on the enzyme (Calcif. Tiss. Int. 49:120, 1991). We have now studied the effects of hPTH W-84) on gene regulation in the human osteoblastic SaOS-2 cell line with a panel of cDNA probes, using Northern and slot-blot analyses on cell extracts of total RNA. The C-terminal (53-84) pepcide increases steady-state mRNA levels for ALP, osteocalcin (OC), and receptors for 1,25(OH),D.. estroaen. and T.. but not type I~procollagrn (cOLif.*. Incre&e; in mR& levels are %+en at doses of hPTit (53-81) as low as 10 pk and the effects differ from thase of hPTH (l-34) and (l-84); these latter peptidas increased mRNA levels fop GGLl but not ALP or OG. The increase in mRNA levrk fer ALP and OC are dose-related with maximumeffects (1.5.fold, p< .OS) at 10 nN hPTH (53-84). These findings indicate a specific role for the C-terminal portion of the PTH molecule in skeletal metabolism. Since the residues in hPTH (53-84) differ from the homologous PTHrP sequence, it is possible that this region confers qualitative differences between PTH and
FTHrP in biological
25 INTRACELLULAR CALCIUM MOBILIZATION: MOLECULAR CLONING OF THE RAT LACTOTROPX THYROTROPINRELEASING HORMONE RECEPTOR AND THE ACTIONS OF CYCLIC ADP-RIBOSE IN MAMMALIAN CELLS. ,4.H. Tashiian. Jr.. D. Zhao. J. Ya n. K.F Jo es. C. Gerald, P. Hogan. W.W. Chin and H. Koshi:a. Dep: Mol. & Cell.
Toxicol., Harvard Sch. Pub. Hlth.; Depts. of Biol. Chem. & Mol. Pharmacol.. Med., and Neurobiol., Harvard Med. Sch., Boston, MA, USA. The plasma membrane receptor for thyrotropin-releasing hormone (TRH-R) is prototypic of d protein-linked receptors that mobilize intracellular Caz’ as a mediator r,f hormone action. We have cloned a cDNA for tire TRH-R from prolactin-producing rat GHJ!, cells using the PCR technique and pools of cloned cDNAs prepared from a GH,C, libnry. Two cBNA clones were identified from which in vitro transcribed mRNA encoded a functional TRH-R as detected by a characteristic conductance response to TRH in microinjected oocytes. DNA sequence analysis revealed a molecule of 412 amino acid residues containing 7 putative transmembrane domains. Becaure TRH-induced mobilization of intracellular Ca*+ in GH& cells occurs more rapidly than can be explained by generation of Ins(1,4,5)P,, we have investigated other mechanisms for Ca*’ mobiliz&ion and have discovered that GHJCl cells can synthesize a novel mediator called cyclic ADP-ribose (cADPR), which was first identified by H.C. Lee and his colleagues (Dept. Physiol., Univ. Minnesota, Minneapolis, MN, USA) as a regulator of Ca*’ release in sea urchin eggs. cADPR acts in permeabilized OH&!, cellsto release sequestered Ca*’ by a mechanism that differs from that used by Ins(1,4,5)P,. These findings broaden conventional views on the mechanisms by which Cazt regulating hormones act on target cells.
27 VITWIN DAM) ADAPTATION To MINERAL(Ca,P) DEFICIEEFI&XETS
INCREASE THE NUMBER OF PNl’ESTINAL PLASMA MnulBRANE Cd PW UNITS. R.H. Wassernran,C.A. Smith, M.E. Brindak. N. de Talam3ni. C.S. FMlmer, Q. Cai, J.T. Penniston and R. Kw, Cornell University, Ithaca, NY 14853 and Mayo m and Foundation, Rochester, MN55905, USA. The terminal step in the active transport of Ca2+
ac~ss intestinal ey.ithelia is the uphill extrusion of Ca across the basolateral mbrqne by an ATP-dependent Vitamin D increases Ca absorption by affecting Ca pq. steps in the transryrt path prior to the extrusion step, as well as the actctrvity of the Ca punp as determined :dith isolated basolateral &rane vesicles (BIMV) by several groups. A mDnpflona1 antibody produced against the hmn erythrocyte Ca punp was now shown to react with a Ca pq epitope of chick intestine. Using this antibody, the Ca purtp was localized almost exclusivnly on the basolateral membraneby indirect imnunofluorescent staining of cryosectioned chick intestinal segments (duodenm, jejunun, ilecnr, colon). Western blotting of electrophoretically separated mucosal proteins further showed that vitamin D (500 i.u., 72 hr.) given to D-deficient chicks increased the density of the Ca punp epitope bands by a factor of 23. Maptation of vitamin D-replete chicks to either a low Ca (0.06% Ca, 0.86% P) or a low P (1.2% Ca, 0.32% P) diet Similarly increased the density of the Cd pwcip band as canpared to the normal group (1.2% Ca, 0.869 P). These results show for the first tinrz a net increase in Ca pq units by vitamin D, and also by adaptation of animals to mineral deficient diets. These data further indiqate that the increase in the activity of ATP-dependent Cd‘* uptake by isolated BCMVby vitamin D is due, at least in part. to the net synthesis of BLMCa pw units. Preliminary data show a vitamin D-dependent increase in Ca planpmRNA, suggestive of stimulation of Ca planp gene transcription. (Supported by NIH Grants DK-04652 (Cornell), and DK-25409 and a-28835 (Mayo Clinic).
regulatfon.
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GENEREGULATION IN OSTEOBLASTIC CELLS BY CARBOXYLTERMINALPTU PEPTIDES:EFFECTS OF hPTH (53.84) IN HUUAN Se. Nichael’s Hoopitzsl and University of Toronto, Torooto, Canada, end Dapartmont of Biochemistry, UWrersity of Arizona, Tucson, AZ, USA. Previous work in our laboratory has provided evidence for bind@ sites specific for the carboxylt.srminal (G-terminal) fragment of PTH comprising rrsiduesl 53 to 84 in kidney and bone. In ROS 17/2.8 cells, 80% of added intact bPTH (l-84) binds via Cterminal residues within the (53-84) realon llZndoct=ineloa, 11?:1832, 1983). In addition, wg have recently shown that hPTH (53-84) can stimulate alkaline ohosphatase (ALP) nativity in rat osteoblasttc ROS lj/2.8 cells, which mntrasts with the suppressive effects of hPTH (l-34) and hPTH (l-84) on the enzyme (Calcif. Tiss. Int. 49:120, 1991). We have now studied the effects of hPTH W-84) on gene regulation in the human osteoblastic SaOS-2 cell line with a panel of cDNA probes, using Northern and slot-blot analyses on cell extracts of total RNA. The C-terminal (53-84) pepcide increases steady-state mRNA levels for ALP, osteocalcin (OC), and receptors for 1,25(OH),D.. estroaen. and T.. but not type I~procollagrn (cOLif.*. Incre&e; in mR& levels are %+en at doses of hPTit (53-81) as low as 10 pk and the effects differ from thase of hPTH (l-34) and (l-84); these latter peptidas increased mRNA levels fop GGLl but not ALP or OG. The increase in mRNA levrk fer ALP and OC are dose-related with maximumeffects (1.5.fold, p< .OS) at 10 nN hPTH (53-84). These findings indicate a specific role for the C-terminal portion of the PTH molecule in skeletal metabolism. Since the residues in hPTH (53-84) differ from the homologous PTHrP sequence, it is possible that this region confers qualitative differences between PTH and
FTHrP in biological
25 INTRACELLULAR CALCIUM MOBILIZATION: MOLECULAR CLONING OF THE RAT LACTOTROPX THYROTROPINRELEASING HORMONE RECEPTOR AND THE ACTIONS OF CYCLIC ADP-RIBOSE IN MAMMALIAN CELLS. ,4.H. Tashiian. Jr.. D. Zhao. J. Ya n. K.F Jo es. C. Gerald, P. Hogan. W.W. Chin and H. Koshi:a. Dep: Mol. & Cell.
Toxicol., Harvard Sch. Pub. Hlth.; Depts. of Biol. Chem. & Mol. Pharmacol.. Med., and Neurobiol., Harvard Med. Sch., Boston, MA, USA. The plasma membrane receptor for thyrotropin-releasing hormone (TRH-R) is prototypic of d protein-linked receptors that mobilize intracellular Caz’ as a mediator r,f hormone action. We have cloned a cDNA for tire TRH-R from prolactin-producing rat GHJ!, cells using the PCR technique and pools of cloned cDNAs prepared from a GH,C, libnry. Two cBNA clones were identified from which in vitro transcribed mRNA encoded a functional TRH-R as detected by a characteristic conductance response to TRH in microinjected oocytes. DNA sequence analysis revealed a molecule of 412 amino acid residues containing 7 putative transmembrane domains. Becaure TRH-induced mobilization of intracellular Ca*+ in GH& cells occurs more rapidly than can be explained by generation of Ins(1,4,5)P,, we have investigated other mechanisms for Ca*’ mobiliz&ion and have discovered that GHJCl cells can synthesize a novel mediator called cyclic ADP-ribose (cADPR), which was first identified by H.C. Lee and his colleagues (Dept. Physiol., Univ. Minnesota, Minneapolis, MN, USA) as a regulator of Ca*’ release in sea urchin eggs. cADPR acts in permeabilized OH&!, cellsto release sequestered Ca*’ by a mechanism that differs from that used by Ins(1,4,5)P,. These findings broaden conventional views on the mechanisms by which Cazt regulating hormones act on target cells.
27 VITWIN DAM) ADAPTATION To MINERAL(Ca,P) DEFICIEEFI&XETS
INCREASE THE NUMBER OF PNl’ESTINAL PLASMA MnulBRANE Cd PW UNITS. R.H. Wassernran,C.A. Smith, M.E. Brindak. N. de Talam3ni. C.S. FMlmer, Q. Cai, J.T. Penniston and R. Kw, Cornell University, Ithaca, NY 14853 and Mayo m and Foundation, Rochester, MN55905, USA. The terminal step in the active transport of Ca2+
ac~ss intestinal ey.ithelia is the uphill extrusion of Ca across the basolateral mbrqne by an ATP-dependent Vitamin D increases Ca absorption by affecting Ca pq. steps in the transryrt path prior to the extrusion step, as well as the actctrvity of the Ca punp as determined :dith isolated basolateral &rane vesicles (BIMV) by several groups. A mDnpflona1 antibody produced against the hmn erythrocyte Ca punp was now shown to react with a Ca pq epitope of chick intestine. Using this antibody, the Ca purtp was localized almost exclusivnly on the basolateral membraneby indirect imnunofluorescent staining of cryosectioned chick intestinal segments (duodenm, jejunun, ilecnr, colon). Western blotting of electrophoretically separated mucosal proteins further showed that vitamin D (500 i.u., 72 hr.) given to D-deficient chicks increased the density of the Ca punp epitope bands by a factor of 23. Maptation of vitamin D-replete chicks to either a low Ca (0.06% Ca, 0.86% P) or a low P (1.2% Ca, 0.32% P) diet Similarly increased the density of the Cd pwcip band as canpared to the normal group (1.2% Ca, 0.869 P). These results show for the first tinrz a net increase in Ca pq units by vitamin D, and also by adaptation of animals to mineral deficient diets. These data further indiqate that the increase in the activity of ATP-dependent Cd‘* uptake by isolated BCMVby vitamin D is due, at least in part. to the net synthesis of BLMCa pw units. Preliminary data show a vitamin D-dependent increase in Ca planpmRNA, suggestive of stimulation of Ca planp gene transcription. (Supported by NIH Grants DK-04652 (Cornell), and DK-25409 and a-28835 (Mayo Clinic).
regulatfon.
76