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Identification of sequences in the rat calbindin-D9k gene which confer basal activation and estrogen. butyrate, and cAMP inducible responses
81 TRANSMEMBRANE SlCNAL PATHWAYS INVOLVED EN CALCIUM MEDIATED PARATHYROID SECRETION. A _ Bourdeau. J.C! Souberbielle. M. Liebcrheq. CNRS URA 553 and Depanment of bhysiology, Hopitrl Necker - Enfams Maladcs, Paris, France.
The mechanism by which high extracellularcalcium (Caz+ec) signals the suppression of ptuathyroid hormone {PTH) release are unknown. We examined if the effects of high Ca*+econ .P?‘Hrelease are mediated through membrane phospholipid hydrolysis, phospholipase A2 (PL-AZ) activation and arachidonic acid (AA) metal@ism in porcine parathyroid cell suspension. Two types of expertmental studies were performed : 1) determination of labeled products from cells (in 0.5 mM Ca2+ medium) labeled with either [3HJmyo-inosito!. [*4C]AA or [3H] AA, exposed to 2 mM Ca2+ for different time penods ; 2) measurement of PTH release from cells (in 0.5 mM Ca2+ medium) treated with exogenous PL-A2 (l-500 mU/ml). AA (5-40 PM), and from cells (in 2 mM Ca*+ medium) exposed to inhibitors of PL-A2 activity and of AA metabolism. Blockers used were : mepacrine (50 PM) and indomethacin (30 &I) for PL-A:! activity inhibition, baicalein (0.1 PM), phenidone (1 PM), NDGA (10 PM) for lipoxygenase (LO) pathway inhibition and ibuprofen (40 pM) for cyclooxygenase (CO) pathway inhibition. Resu!#s show that : 1) high Ca*+ec provokes within 120.5 a biphasic and dose-dependent increase in inosito! erisshosphate and diacylglycerol formation and AA release ; 2) exogenous PL-AZ and AA inhibit PTll secretion in a dose dependent manner ; 3) PL-A2 inhibitors, LO (12.LO but not 5-LO) blockers and not CO blockers, restore PTH secretion. In conclusion : Ca*+eceffect on PTH secretion appears to act via a receptor-mediated process linked to phospholipase C and phospholipase AZ,and involves AA metabolites through the LO-pathway.
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lDENTlFlCATlON OF 8EQUENCES 1N THE RAT CALBINDIN-Dgk GENE WHICH CONFER BASAL ACTIVATION AND ESTROGEN, BUTYRATE. AND CAMP INDUCIBLE RESPONSES. Hui Ll and Sylvia Christakos. Department of Biochemistry, UMDNJ-New Jersey Medicai School, Newark, NJ 07103. Wk have lsolared the 5’ flanklng region of the calblndin-D9k gene from a nat genomic Agtll library and llnked this region to a chloramuhenicol acetvltransfernse (CAT) reporter Rene. n fragment ‘of the crldlndin-D9k gene wlth coordlnaees -1009/+106 was capable of ptomoclny! the expression of CAT in a bovine kidney cell line, EXDBK (which contains calbindin-D9k endogenously) and in Tq7D breast cancer cells but nor in ROS 17/2.8 cells. Deletion mutant analvsis of the uromoter usinn bo;h MDBK and T47D cells indicatkd that the. basal level of CAT expresslon increased up to 4 fold with deletion of 893 basepairs of upi.tream sequence, suggesting the presence of The 5’ flanking reelon of the upstream repressor sequences. gene was found to mediate dose dependenr stimulation by sodium butyrate (up to 7 fold) in MDBK cells but nor in T47D cells, suggesting that buryrate may activate cell specific enhancer bindfne ototeins. This reeion was also found to mediate dose dependent stimulation by &bromo-cAMIP, forskolln and isobutyl methvl xanthine (UD to 6.3 fold) in both MDBK and T47D cells. The ;AMP and -dutyrate response regions were napped to 61, suggesting that this fragment sequences between -il6/+ Purther contains target reeions for regulation of the gene. analysis revealed sequence homology to the esriogen response element (ERE) at two positions (-832/-820 and +50/+65). Only the construct which cdntained the downstream ERE was able to confer estrogen responsiveness in T47D but not in MDBK Transfeceion of estrogen receptor expression vectors cells. enhnnced this responsiveness. -No char& tn CAT actlvi~y was observed in the presence of 1,25(Otl)2D3 - . or the phorbol ester PMA, even afte; cotransfectlon with vitamin D receptor or c-Jun and c-fos expression vectors. In summary, these findings, which reoresent the first functional analysis of the calbindln-Dgk promote;, Indicate cell s,l?ciflc tr.rnscrlptlonal regulation by butyrate and estrogen which may reflect cell spcclfic inducible regulatory factors as well as regulation by phosphorylation of the transcriptional activatio;. of the calbindin D-9k gene.
SALMON CALCITONIN-LIKE PEPTIDE IS PRESENT IN EXTRACTS OF RAT BRAIN DIENCEPHALON. Patrick M. Saxron and Jnanne M. Hilton. St. Vincent’s Institute of Medical Research, Melhournr, Australia. Cidcitonin (CT), ;I 32 amino acid peplidehormone is a major peripheral calciotrqir: hormone. High densities of CT receptorsarc present in bone, kidney and brain. Central injection of CT induces potent effects including aniIlgt?sia and deureasctl appetite. with the actions of Ihe p+ide correlating with the Iucatiun of CT rcreptrrs in the central nervuus system (CNS). However. I htle of the circularing form of the peptide is present in the CNS. ruising questions on the identity of the edogenous ligand for crntral rrcqmxs. This study idrntilies a s,ilnum CT @CT)-like pepride in extracts of rat brain. BrSn exirash are prepared as follows: fresh or frozen tissue was homcjgrnisrd in 20 vol~~~~wsof IM HCI, bnilcd fur 3 min, then centrifuged a( 48.000 g flir 30 min. Supernsrams were concentrated by application to a Cl8 Sep-Pak column. bulk elated with 75% CH&N/25% 0.05% TFA and dried down in e Sav;mt overnight. Samples were reconsWltrd in O.OlM acetic arid. Aliquuts of rsrtrnsthuted m;lterial were assayed f~)r sCT-like ilnmllrlclr~;lclivity using ;I r;ltlioinllllrlnr,;lssay (RIA) bXW.l on a guinr:i pig ;mti-*CT antihodv (dctcction limir 0. I ng). The ass:i was less than 0. I % crohs-rcictive wirh human CT. ptrrcine CT. rai CGRP-I and human CGRP-I. Extracts of tlirncephalon revd;lldd sCT-like activity (- 100 rig/g wet weight). while extracts frlim cortex, crrrhullum and nxdull;~ did nut clmGl rle~eut;ihlr :Icfivi(y. Reverse ph;cse HPLC analysis demonsrntrd corlu~i~ui with symh& sCT. Further, the peak mstcrial was active in 3 Cl hio;lssay sy.\tem\: (I J stimulatiun of cAMP production in UMR 106-06 IXIIS. (2) inhihitiun of “&CT hintling in UMR IO&O6 cells and (3) inhihililm nf “51-sCT binding in sheep brain Inumbranus. Thr hioxtivhy of matrri;ll in theseassayswas similar tn that esGmated U~roughthe RIA. Th;: resuh; identify. fijr the first time, presence of sCT-like m;ltarial in the r;lt hr;iin ml constitutes Ihe first dcmunstraticm of biologiselly active sCT-like material frum brain extrilcts. Salmon CT-like peptitle mily rrprcsL’nt rhr! endugmuus lig;mtl for cmrr;il CT rrcep!ptlirs,
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The mechanism by which high extracellularcalcium (Caz+ec) signals the suppression of ptuathyroid hormone {PTH) release are unknown. We examined if the effects of high Ca*+econ .P?‘Hrelease are mediated through membrane phospholipid hydrolysis, phospholipase A2 (PL-AZ) activation and arachidonic acid (AA) metal@ism in porcine parathyroid cell suspension. Two types of expertmental studies were performed : 1) determination of labeled products from cells (in 0.5 mM Ca2+ medium) labeled with either [3HJmyo-inosito!. [*4C]AA or [3H] AA, exposed to 2 mM Ca2+ for different time penods ; 2) measurement of PTH release from cells (in 0.5 mM Ca2+ medium) treated with exogenous PL-A2 (l-500 mU/ml). AA (5-40 PM), and from cells (in 2 mM Ca*+ medium) exposed to inhibitors of PL-A2 activity and of AA metabolism. Blockers used were : mepacrine (50 PM) and indomethacin (30 &I) for PL-A:! activity inhibition, baicalein (0.1 PM), phenidone (1 PM), NDGA (10 PM) for lipoxygenase (LO) pathway inhibition and ibuprofen (40 pM) for cyclooxygenase (CO) pathway inhibition. Resu!#s show that : 1) high Ca*+ec provokes within 120.5 a biphasic and dose-dependent increase in inosito! erisshosphate and diacylglycerol formation and AA release ; 2) exogenous PL-AZ and AA inhibit PTll secretion in a dose dependent manner ; 3) PL-A2 inhibitors, LO (12.LO but not 5-LO) blockers and not CO blockers, restore PTH secretion. In conclusion : Ca*+eceffect on PTH secretion appears to act via a receptor-mediated process linked to phospholipase C and phospholipase AZ,and involves AA metabolites through the LO-pathway.
83
82
lDENTlFlCATlON OF 8EQUENCES 1N THE RAT CALBINDIN-Dgk GENE WHICH CONFER BASAL ACTIVATION AND ESTROGEN, BUTYRATE. AND CAMP INDUCIBLE RESPONSES. Hui Ll and Sylvia Christakos. Department of Biochemistry, UMDNJ-New Jersey Medicai School, Newark, NJ 07103. Wk have lsolared the 5’ flanklng region of the calblndin-D9k gene from a nat genomic Agtll library and llnked this region to a chloramuhenicol acetvltransfernse (CAT) reporter Rene. n fragment ‘of the crldlndin-D9k gene wlth coordlnaees -1009/+106 was capable of ptomoclny! the expression of CAT in a bovine kidney cell line, EXDBK (which contains calbindin-D9k endogenously) and in Tq7D breast cancer cells but nor in ROS 17/2.8 cells. Deletion mutant analvsis of the uromoter usinn bo;h MDBK and T47D cells indicatkd that the. basal level of CAT expresslon increased up to 4 fold with deletion of 893 basepairs of upi.tream sequence, suggesting the presence of The 5’ flanking reelon of the upstream repressor sequences. gene was found to mediate dose dependenr stimulation by sodium butyrate (up to 7 fold) in MDBK cells but nor in T47D cells, suggesting that buryrate may activate cell specific enhancer bindfne ototeins. This reeion was also found to mediate dose dependent stimulation by &bromo-cAMIP, forskolln and isobutyl methvl xanthine (UD to 6.3 fold) in both MDBK and T47D cells. The ;AMP and -dutyrate response regions were napped to 61, suggesting that this fragment sequences between -il6/+ Purther contains target reeions for regulation of the gene. analysis revealed sequence homology to the esriogen response element (ERE) at two positions (-832/-820 and +50/+65). Only the construct which cdntained the downstream ERE was able to confer estrogen responsiveness in T47D but not in MDBK Transfeceion of estrogen receptor expression vectors cells. enhnnced this responsiveness. -No char& tn CAT actlvi~y was observed in the presence of 1,25(Otl)2D3 - . or the phorbol ester PMA, even afte; cotransfectlon with vitamin D receptor or c-Jun and c-fos expression vectors. In summary, these findings, which reoresent the first functional analysis of the calbindln-Dgk promote;, Indicate cell s,l?ciflc tr.rnscrlptlonal regulation by butyrate and estrogen which may reflect cell spcclfic inducible regulatory factors as well as regulation by phosphorylation of the transcriptional activatio;. of the calbindin D-9k gene.
SALMON CALCITONIN-LIKE PEPTIDE IS PRESENT IN EXTRACTS OF RAT BRAIN DIENCEPHALON. Patrick M. Saxron and Jnanne M. Hilton. St. Vincent’s Institute of Medical Research, Melhournr, Australia. Cidcitonin (CT), ;I 32 amino acid peplidehormone is a major peripheral calciotrqir: hormone. High densities of CT receptorsarc present in bone, kidney and brain. Central injection of CT induces potent effects including aniIlgt?sia and deureasctl appetite. with the actions of Ihe p+ide correlating with the Iucatiun of CT rcreptrrs in the central nervuus system (CNS). However. I htle of the circularing form of the peptide is present in the CNS. ruising questions on the identity of the edogenous ligand for crntral rrcqmxs. This study idrntilies a s,ilnum CT @CT)-like pepride in extracts of rat brain. BrSn exirash are prepared as follows: fresh or frozen tissue was homcjgrnisrd in 20 vol~~~~wsof IM HCI, bnilcd fur 3 min, then centrifuged a( 48.000 g flir 30 min. Supernsrams were concentrated by application to a Cl8 Sep-Pak column. bulk elated with 75% CH&N/25% 0.05% TFA and dried down in e Sav;mt overnight. Samples were reconsWltrd in O.OlM acetic arid. Aliquuts of rsrtrnsthuted m;lterial were assayed f~)r sCT-like ilnmllrlclr~;lclivity using ;I r;ltlioinllllrlnr,;lssay (RIA) bXW.l on a guinr:i pig ;mti-*CT antihodv (dctcction limir 0. I ng). The ass:i was less than 0. I % crohs-rcictive wirh human CT. ptrrcine CT. rai CGRP-I and human CGRP-I. Extracts of tlirncephalon revd;lldd sCT-like activity (- 100 rig/g wet weight). while extracts frlim cortex, crrrhullum and nxdull;~ did nut clmGl rle~eut;ihlr :Icfivi(y. Reverse ph;cse HPLC analysis demonsrntrd corlu~i~ui with symh& sCT. Further, the peak mstcrial was active in 3 Cl hio;lssay sy.\tem\: (I J stimulatiun of cAMP production in UMR 106-06 IXIIS. (2) inhihitiun of “&CT hintling in UMR IO&O6 cells and (3) inhihililm nf “51-sCT binding in sheep brain Inumbranus. Thr hioxtivhy of matrri;ll in theseassayswas similar tn that esGmated U~roughthe RIA. Th;: resuh; identify. fijr the first time, presence of sCT-like m;ltarial in the r;lt hr;iin ml constitutes Ihe first dcmunstraticm of biologiselly active sCT-like material frum brain extrilcts. Salmon CT-like peptitle mily rrprcsL’nt rhr! endugmuus lig;mtl for cmrr;il CT rrcep!ptlirs,
Y
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