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Steroid hormone regulation of the human osteocalcin gene promoter
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SESSION 2 - MOLECULAR AND CELLULAR BONE BIOLOGY 2 STEROID IiGRMO8E REGULATION OF THE HUMAN OSTEOCALClN GENE PROMOTER Ni@ MonilO” and John Ebrnan Galvan lnniwr OfMediC0l &search. sr. “i”cc”ll, “O~~i,Ol, Sydney.NSW 2010. *u.wo,;o Ommkin isn gammwarboxyktcd pm,cin pmduccd by or,eoblas,s during the synthesisof new bone where i, is ,hO”gk,,O p,ay a rnki” minemlisaionofbonc mnrix. or,eaca,cin liberated imolhc remmis BSsaytias acunica, indicamr of bone fonnuioe. Tkltlserum level of,bis prain is increarcd by treamen, wilh 1.25-dihydmxytimmin Ds (I.25 D?) and decMscd by glwxcnicoids. with similar effecu in cell cullwe syslems. 7% osteocakin gem pmmoar was fused 10 ule baclcrial chlommphenicol aatyl tmnsfemse (CAT) gene ,o provide an assayable n@cr. Bo,h lransklu and pcrmpnem ,rsnSCc,ion expcdmcw demoUrtm,ed a dose &pan&n, induction of ,hc.as,ewakin promae, by I,2S 4 consis,en, wilh ,he Kd of Ihe vilsmin D receplor. fnd.~i~~
in ra o~f~owcoma cells was S-fold fw R0SI’IR.g
and 20.fold for UMRlDd
cells while human
o~~eowcoma ceUs (SAOS-2) mached 40 fold The vitamin D responsiveclement (VDRE) was identified by deklion and reconsuwlion ewabnenls. The VDRE conlaim an unusual double Dalindmmc tTfGGTGAmCACCGGGTGAA) which is &ntid for function. Induction by 1.25 D) and ipression by~glucoconicoids appcand to bc co,,,n,lkd by ,wo “plrslc regions of the pmmcw. A symhedc “DRE inset,& upswam of a haemlogovs promoter (pTRCAT) functions in other ec,, Iines. pmviag the genem,i,y of this sequence in mediating vitamin D nsponss. Abhough rhe VDRR bearssome similarity 10 mher hormonerespon8ivcelcmcms. il is dislinn from prrviously describedektwnls and is unresponsive10 tier prum,er w in Ihe hetemlogous scning.
steroid hommncs citber in i,, native
Tix palent synthetic glucomtimid. dexamthawm. reptessedbolh the contitutivc and Ihc 1.25 D, induced kvcl of gene cxpnssion a, conccn,m,ions of deramabamnc ccmpuabk lo ,hox achieved clinically. O(her synthtdc and nmulsl glucwonicoidr mprcsscdCAT activily 10 a degreemn*r,en, ti their known afSni,ies for ,be glucocwdcoid ~ccp,or. The anti-pmgcs,erow anti-g,ucosa(icoid ccmpovnd RU4Bb wsli abk u) reverse glucoconicoid mediaed reptession of 1.25 DI indwcd expression. Howwer, RU486 alom had some agonist activity, significandy z%presring,bc basal level ofCATanivi,y a, low conccn,mdonsand 1.25 D, induced aclivi,y a, high eoncc~tradans. Tlxse daa dcmonm’a,c ,ha, the omcakin gene is regulated by physiological vimmin D, meUbo,i,cp and glucaconicaids. The fact ,hDI the VDRE was unresponsive10 ahcr homvma of Ihc stcmidhhymid family. rhicb all Bn lhmugh hormone rqansive ebmelUS. nflec,s the unique role Ihc viumti Da mctabolilw play in bone and calcium metabolism. In addilisn. the osleocalcin gene-CAT CO~S,IIIE,~pmvide a powerful mans ofanalyring ,be efficacy of syndwic and naruml vilamin D rela,ed com~unds a, UK gene Icvcl.
ADRNYLATE CYCLASE BLOCKERS DISSOCIATE PARATHYROID HORMONE.STIMULATED BONE RESORPTION FROM CAMP PRODUCTION IN NEONATAL MOUSE CALVARIA Jillian Corn&b. Carolyn Law. D. Harley Gray. Stephen 1.M. Skinner. Ian R. Reid Deparrmcnlr ofhfedicins and surgery. Auc&u!d. New zeo,mld I1 is uncenain whether CAMP or ,be inosilol-calcium pubray mediQLeS ,he rlimulltion of bone ,wxp,ion by pam,hymid homwnc (PTH). Incubation of bone organ cukuns with CAMP analogws and forskolin has nn refolved Uris question becauseof the allular inhomogencily of born and Ihc mnscquen, prwcncc of adenylate cyclase-linked neepors fw bmb PTH and calciwnin hamanes with opposite cffecw on bDnc rc~orption. We have used IWO KW inhibilors of adenylau cyclare. 9.f,euahydro-2.furyl)adenine (SQ 22S36) and Z’J’dideoxyadamrim (DDA). to diwdy rursus Ihc role of CAMP in PTH-nimulatcd oneolysis. SQ 22% (0.01-1.0 mM) and DDA (0.01-1.0 mM) completely blwked PM sdmulation of CAMP produnion measuredin ,hc absenceof B phospbodicsterascblcckcr. In ,bc prcwncc of I mM isobu,ybne,hy,xamhine. inhibition of PTH-induced EAMP produclio” cccured wkb 0.2 mM SQ and 0.1 mM DDA. nrpcnively. These concentrpllor.~of SQ and DDA had no effect on FIX-slimulakd “Ca nlcase from calwia. lhough kab agems inhibilcd bone rwxplion when preoa a, concawmions of L-2 mM. A, these levcI8. SQ and DDA caused equivalent inbibilica of% nlcasc s,imula,ed by 1,25dihydmxyviumin Da but did IIM affea Lwal ‘%a lclcasc nor [3HI-pbenyfalanim Inwpontion. I1 is concluded lha, substaatiti block&. of Pl’H%,duccd cAMP pmducdon dots no, affcc, this hormons’r stimulalionof bow wxp,io(I. which is thereforelikely robe mediaed byanaherin,~l,ubrmcsscngasyslan. possibly calcium. In millimolw concentrations SQ and DDA appmr 10 be nonspecific blockers of os,ecc,as,ic bone nsoqxion.
SESSION 2 - MOLECULAR AND CELLULAR BONE BIOLOGY 2 STEROID IiGRMO8E REGULATION OF THE HUMAN OSTEOCALClN GENE PROMOTER Ni@ MonilO” and John Ebrnan Galvan lnniwr OfMediC0l &search. sr. “i”cc”ll, “O~~i,Ol, Sydney.NSW 2010. *u.wo,;o Ommkin isn gammwarboxyktcd pm,cin pmduccd by or,eoblas,s during the synthesisof new bone where i, is ,hO”gk,,O p,ay a rnki” minemlisaionofbonc mnrix. or,eaca,cin liberated imolhc remmis BSsaytias acunica, indicamr of bone fonnuioe. Tkltlserum level of,bis prain is increarcd by treamen, wilh 1.25-dihydmxytimmin Ds (I.25 D?) and decMscd by glwxcnicoids. with similar effecu in cell cullwe syslems. 7% osteocakin gem pmmoar was fused 10 ule baclcrial chlommphenicol aatyl tmnsfemse (CAT) gene ,o provide an assayable n@cr. Bo,h lransklu and pcrmpnem ,rsnSCc,ion expcdmcw demoUrtm,ed a dose &pan&n, induction of ,hc.as,ewakin promae, by I,2S 4 consis,en, wilh ,he Kd of Ihe vilsmin D receplor. fnd.~i~~
in ra o~f~owcoma cells was S-fold fw R0SI’IR.g
and 20.fold for UMRlDd
cells while human
o~~eowcoma ceUs (SAOS-2) mached 40 fold The vitamin D responsiveclement (VDRE) was identified by deklion and reconsuwlion ewabnenls. The VDRE conlaim an unusual double Dalindmmc tTfGGTGAmCACCGGGTGAA) which is &ntid for function. Induction by 1.25 D) and ipression by~glucoconicoids appcand to bc co,,,n,lkd by ,wo “plrslc regions of the pmmcw. A symhedc “DRE inset,& upswam of a haemlogovs promoter (pTRCAT) functions in other ec,, Iines. pmviag the genem,i,y of this sequence in mediating vitamin D nsponss. Abhough rhe VDRR bearssome similarity 10 mher hormonerespon8ivcelcmcms. il is dislinn from prrviously describedektwnls and is unresponsive10 tier prum,er w in Ihe hetemlogous scning.
steroid hommncs citber in i,, native
Tix palent synthetic glucomtimid. dexamthawm. reptessedbolh the contitutivc and Ihc 1.25 D, induced kvcl of gene cxpnssion a, conccn,m,ions of deramabamnc ccmpuabk lo ,hox achieved clinically. O(her synthtdc and nmulsl glucwonicoidr mprcsscdCAT activily 10 a degreemn*r,en, ti their known afSni,ies for ,be glucocwdcoid ~ccp,or. The anti-pmgcs,erow anti-g,ucosa(icoid ccmpovnd RU4Bb wsli abk u) reverse glucoconicoid mediaed reptession of 1.25 DI indwcd expression. Howwer, RU486 alom had some agonist activity, significandy z%presring,bc basal level ofCATanivi,y a, low conccn,mdonsand 1.25 D, induced aclivi,y a, high eoncc~tradans. Tlxse daa dcmonm’a,c ,ha, the omcakin gene is regulated by physiological vimmin D, meUbo,i,cp and glucaconicaids. The fact ,hDI the VDRE was unresponsive10 ahcr homvma of Ihc stcmidhhymid family. rhicb all Bn lhmugh hormone rqansive ebmelUS. nflec,s the unique role Ihc viumti Da mctabolilw play in bone and calcium metabolism. In addilisn. the osleocalcin gene-CAT CO~S,IIIE,~pmvide a powerful mans ofanalyring ,be efficacy of syndwic and naruml vilamin D rela,ed com~unds a, UK gene Icvcl.
ADRNYLATE CYCLASE BLOCKERS DISSOCIATE PARATHYROID HORMONE.STIMULATED BONE RESORPTION FROM CAMP PRODUCTION IN NEONATAL MOUSE CALVARIA Jillian Corn&b. Carolyn Law. D. Harley Gray. Stephen 1.M. Skinner. Ian R. Reid Deparrmcnlr ofhfedicins and surgery. Auc&u!d. New zeo,mld I1 is uncenain whether CAMP or ,be inosilol-calcium pubray mediQLeS ,he rlimulltion of bone ,wxp,ion by pam,hymid homwnc (PTH). Incubation of bone organ cukuns with CAMP analogws and forskolin has nn refolved Uris question becauseof the allular inhomogencily of born and Ihc mnscquen, prwcncc of adenylate cyclase-linked neepors fw bmb PTH and calciwnin hamanes with opposite cffecw on bDnc rc~orption. We have used IWO KW inhibilors of adenylau cyclare. 9.f,euahydro-2.furyl)adenine (SQ 22S36) and Z’J’dideoxyadamrim (DDA). to diwdy rursus Ihc role of CAMP in PTH-nimulatcd oneolysis. SQ 22% (0.01-1.0 mM) and DDA (0.01-1.0 mM) completely blwked PM sdmulation of CAMP produnion measuredin ,hc absenceof B phospbodicsterascblcckcr. In ,bc prcwncc of I mM isobu,ybne,hy,xamhine. inhibition of PTH-induced EAMP produclio” cccured wkb 0.2 mM SQ and 0.1 mM DDA. nrpcnively. These concentrpllor.~of SQ and DDA had no effect on FIX-slimulakd “Ca nlcase from calwia. lhough kab agems inhibilcd bone rwxplion when preoa a, concawmions of L-2 mM. A, these levcI8. SQ and DDA caused equivalent inbibilica of% nlcasc s,imula,ed by 1,25dihydmxyviumin Da but did IIM affea Lwal ‘%a lclcasc nor [3HI-pbenyfalanim Inwpontion. I1 is concluded lha, substaatiti block&. of Pl’H%,duccd cAMP pmducdon dots no, affcc, this hormons’r stimulalionof bow wxp,io(I. which is thereforelikely robe mediaed byanaherin,~l,ubrmcsscngasyslan. possibly calcium. In millimolw concentrations SQ and DDA appmr 10 be nonspecific blockers of os,ecc,as,ic bone nsoqxion.