Analysis of the rat clusterin gene promoter and cyclic AMP-regulated mRNA stability in testicular cells.
Clusterin, also known as SGP-2 or TRPM-2, is expressed in the male reproductive tissues at different levels. The genomic structure of the rat clusterin gene was recently reported by our laboratory and others. In this study, we have determined the promoter responsible for the basal expression of the rat clusterin gene in testicular cells by analyzing the transient expression of the bacterial chloramphenicol acetyl transferase (CAT) reporter gene in MA-10 cells driven by different segments of the 5'-flanking region and the first intron of the clusterin gene. The region required for maximal basal expression was identified at -266 to +54. Addition of DNA fragments of the rat clusterin gene from -1298 to -266 bp, or from +54 to +1153 to (-266/+54)CAT resulted in a 87% decrease in CAT activity, suggesting the presence of inhibitory DNA elements in both the 5'-flanking region and the first intron. When DNA fragment in the first intron, +1153 to +2874, was included, CAT activity in the (-266/+2874)CAT construct increased to 70% of the clusterin promoter (-266/+54)CAT, indicating that stimulatory DNA elements may be present in this region of the first intron. Treatment of MA-10 cells with cyclic AMP (cAMP) neither decreased CAT activity driven by any of the clusterin/CAT chimeric plasmids examined in transient transfection studies, nor reduced the synthesis of nuclear clusterin RNA in nuclear run-on assays, indicating that the reduction of clusterin mRNA levels by cAMP previously reported in our laboratory is not exerted at the transcriptional level. Furthermore, addition of transcriptional or translational inhibitors (actinomycin D and cycloheximide respectively) abolished the cAMP effect observed in MA-10 cells. In summary, we have demonstrated that the basal transcription of the rat clusterin gene in testicular cells is under the control of both positive and negative regulatory sequences at the 5'-flanking region as well as in the first intron. The reduction of clusterin mRNA after exposure of MA-10 cells to cAMP is not due to a decrease in its transcriptional activity, but rather to an increase in the degradation of this mRNA through synthesis of a destabilizing protein(s) and its mRNA.
Rosemblit
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Population Council, New York, New York 10021, USA.
Feng
Z M
ZM
Chen
C L
CL
D000818:Animals; D015415:Biomarkers; D003994:Bucladesine / Q000494:pharmacology*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D003001:Cloning, Molecular; D051152:Clusterin; D000242:Cyclic AMP / Q000378:metabolism*; D003609:Dactinomycin / Q000494:pharmacology; D017930:Genes, Reporter; D006023:Glycoproteins / Q000096:biosynthesis* / Q000235:genetics*; D007438:Introns; D008297:Male; D018832:Molecular Chaperones; D011401:Promoter Regions, Genetic / Q000187:drug effects; D012333:RNA, Messenger / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis; D013737:Testis / Q000378:metabolism*; D014162:Transfection; D001616:beta-Galactosidase / Q000096:biosynthesis
D015415:Biomarkers; D051152:Clusterin; D006023:Glycoproteins; D018832:Molecular Chaperones; D012333:RNA, Messenger; D011994:Recombinant Proteins; D003609:Dactinomycin; D003994:Bucladesine; D000242:Cyclic AMP; D015500:Chloramphenicol O-Acetyltransferase; D001616:beta-Galactosidase