117 EXPRESSlON OF A HIGH Km 25.HYDROXYVITAMIN D-la- HYDROXYLASE IN A HUMAN HEPATOMA CELL LINE HEP G2. &W. Hollis. D.3. D minick and L.R. Braina. Depts. Pediatrics, Biochemistlv and &zcular Biolonv. Medical University of South Carolina, Charleston, SC 29425 Ufi

The enzymatic conversion of 2S(OH)D to l,SS(OH),D is a critical step in the metabolic activation of iitamin D. This enzyme, originally thoueht to be confined to renal tissue, has subsequently been identified in several extrarenal sites including liver tissue. -To f&her investigate the role of hepatic tissue in the metabolism of 2S(OH)D we carried out experiments using a human hepatoma-derived cell line, Hep G2. Cells were grown in T75 llasks containing Earle’s MEM supplemented with L-glutamine, penicillin and 10% FCS. For experiments, cells were plated on Falcon multiwell plates and grown to late log phase. 24 hrs prior to incubation with 2S(OH)D, FCS was removed from cells. Monolayers were washed 3 times with MEM prior to incubation with 2S(OH)D,. Incubations took place from O-6 hn at 37°C and each well contained 2 ml media. Experiments were performed to determine time course generation of l;ZS(OH),D, apparent q and V, of the hepatic I- hydroxylase, substrate specificity of the enzyme and effect of inhibitors f,n enzyme activity, Cells and media were removed from culture wells and analyzed in- total for 1,25(0H),D, content using solidephase extraction. silicn ore-ourification. HPLC semtration and RRA quantitrtion with b∈ thymus l,&(OH),D r&eptor. :Time e~urse studies demonstrated a linear production of 1,25(OH),D, for up to6 hrsin the presence of 20 pM 25(OH)DS. Kineticanulysisofthe I- hydroxylation reaction in Hep G2 cells wtih increasing concentrations of substrate (O-25 PM) demonstrated rm apparent k of 4.6 pM and V, of 10%) pgl1.6 x 10* cells/6 hr. The I-hydroxylation was 62% inhibited in the presence of I 1M ketoconazole and 48% increased in the presence of 1 PM 1,2=dianilinoethane. This enzyme appeared to l- hydroxyha Zs(OH)D, and ZS(OH)D, equally but would not l- hydroxylateD,orD,at up to50 pM.m:HepG2 cellsexpress a high K. I=hydroxylase, probably of the cytochrome Puo class. This cell line should prove to be a useful tool in which to study the control and function of the hepatic 2Sahydrortyvitamin D-I-hydrovlase.

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11 c~LcEnIc HORNONES AND CHR~MO~~RANXN A (CGA) PEPTIDES REGULATE EACH OTHER'S PRODUCTION. L.J. Deftos, D.W. Brandt, E.M. Nolan, and Medicine, University of California,

Department of Sin Diego, and San

Diego VA Medical Center, La Jolla, California. We have demonstrated reoulatorv interactions amona

and between the calcemic hijrmones-and CgA-derived * peptides. These interactions affect the aene exDression ind secretlon of the calcemic oeotides and renreient new1

r -appreciated paracrine anh iutocrine paihw&s of

regu ation. To further delineate these regulator pathways, we studied the effect of calcitonin (CT J and other peptides on the transcription. translation. and secrctloi! of the parathyroid hbrmoni-like protein (PLP) In human cell lines. Secretion sttldies were performed by radioimmunoassay of cell culture medium, messenger RNA (mRNA) studies by Northern anaiysjs and by polymerase chain reaction (PCR) amplification of cDNA, and transcription studies by the analysis of cells transfected with calcemic-hormone promoters cloned into a growth hormone(GH)-reporter plasmid. Our studies demnstrated that CT stimulated and CgAi-40 inhibited the secretion of PLP. PCR studies demonstrated a dose- dependent stimulation by CT of the PLP mRNA species that are representative of alternative promotor and 3' coding splice pathway usage. CT treatment of mammalian cells transfected with PLP promoter-GM reporter demonstrated direct regulation of PLP gene

plasmids

transcription. These studies are one example of the regulatory

interactions that exists among the calcemic hormones, They demonstrate that CT stimulates the gene expression and secretion of PLP. demonstrated regulatory

In corresponding studies, we also interactions among other members

of this endocrine family of hormones. These pathways, along with other interregulatory pathways involving the calcemic-hormone and CgA-peptides, indicate the importance of hormonal interactions as endocrine, paracrine, autocrine, and intracrine regulators of calcium and skeletal metabolism.

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w V/VQ POLAR METABOLITES OF PS-HYDROXY- DIHYDROTACHYSTEROL!I IN THE RAT ARE 16. HYDROXYLATED. Schroeder. N.J.. m. F.. v Tlafford. Department of Chemical Pathology. The London Hospital Medical College, London El 2AD, UK, Department of Biochemistry, Queen’s University, Ontario K7L 3N6 Canada. and Chemical Research. Leo Pharmaceutical Producls. DK-2750 Balierup, Denmark.

Dihydrotachysteroig (DHT3). after hepatic 2Shydroxyiation, undergoes extensive side chain modification &&Q along the same metabolic pathways already described for 2bhydroxyvitamin D3 (25- OHD3). It is also further melaboiised at an as yet unidentilied non- renal site producing a mixture of la- and lg-hydroxylated metabolites - lf1,25(OH)2DHTs being found in 10x higher concentration than la,25(OH)zDHT3. Further metaboiites of DHTs, more polar lhan 1,25(OH)2DHTs, are found in rat plasma. We have investigated the identity of these polar metaboiites after extraction and purilication by HPLC (using straight-phase, reverse-phase and cyano- systems). Using ‘chekical modification and ‘gas chromatography-mask spectmmetry, we have identified these polar compounds as metaboiites of 1,25(OH)sDHTs (namely 24-0~0, 24- and 26.hydroxy-, and 23,26-laclone derivatives). incubation of synthetic la,25(OH)2DHT3 in a rat osteosarcoma cell line (UMR 106), previously demonstrated to metaboise 25.OHDHTsinthe same way is the perfused rat kidney. Pave rise 10 a number if polar oroduck Some bf these had sim'iia? chromatogrephic and '&ss'~speclral characteristics to polar compounds isolated from rat Diasma. However close comoarkon showed that thev were‘ not identical. 1~,25(0H)@HT3, obtained in small amour& by incubating synthetic lp-OHDHTs in a human hepaloma ceil line (Hep 38). was also incubated in the osteosarcoma ceil line. lp,24,2&6H)@HT3, which was the only compound in sufficient amounts to allow further study, was compared to the 24.hydroxy metabolite isolated from rat plasma. Bolh metaboiites had identical chromatographic and mass spectral properties. We conclude that the polar metabolites seen in rat plasma after administration of DHTs are target ceil metaboiites of 1,25(OH)2DHTs and are lp-hydroxyiated. It is not clear why lp- hydroxy metabolites are formed in such large amounts nor do we know where or how these hitherto unrecognised lp-hydroxy compounds are formed.

I20 REGULATION OF THE EXPRESSION OF THE GENE FOR THE PARATHYROID HORMONE-LIKE PROTEIW. P.W. Brandt and L.J. Oeftos, Department of Medicine, University of %lifornia, San Diego, and San Diego VA Medical Center, La Jolla, California.

We stidied the expression of the gene for parathyroid hormone-like protein (PLP) in human cells by evaluating its transcription with transfection analysis, its mRNA splicing products by the polymerase chain reaction (PCR), and its polypeptide products by applying region- specific antibodies to several immunochemical formats, including laieh performance size exclusion chromatography,‘Western analysis, and immunoassay.

For PCR studies, we desioned a series of exon- specific oligonucleotide pr%ners that hybridize to DNA sequences adjacent to exon-intron splice junctions throuohout the PLP aene. These Drimers were used to evaluate cDNA prepa;ed from mRNA'of PLP-producing cell lines during different experimental maniuulations. Our analysis wiih promoter-spkcific primers &d transfected cells indicated differential activity from all three putative promoters. Our analysis with C-terminal coding specific primers indicated the utilization of splicing pathways that produce all three mature PLP polypeptides, PLPl-141, l-139, and I-173. Treatment of the various cell lines with apentc that act throuah orotein Kinase-C (e.g., phorbol) aiid protein Kinase-A {e.b., CT) stimulated different patterns of splicing of these alternative promoter and coding mRNA pathways. Northern analyses generally correlated with PCR findings, but they did not have the sensitivity or resolving power of PCR. Corresponding immunochemical studies demonstrated the presence and correlated requlation of PLP forms representing all three of the PLP species predicted by its gene.

PCR in combination with other procedures allows the elucidation of the complex regulatory patterns that exist for the expression of the various PLP mRNA species and their several promoters and encoded products.

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