ANALYSIS OF THE RAT CLUSTERIN GENE PROMOTER AND CYCLIC AMP-REGULATED MESSENGER-RNA STABILITY IN TESTICULAR CELLS

Clusterin, also known as SGP-2 or TRPM-2, is expressed in the male rep roductive tissues at different levels. The genomic structure of the ra t clusterin gene was recently reported by our laboratory and others. I n this study, we have determined the promoter responsible for the basa l expression of the rat clusterin gene in testicular cells by analyzin g the transient expression of the bacterial chloramphenicol acetyl tra nsferase (CAT) reporter gene in MA-10 cells driven by different segmen ts of the 5'-flanking region and the first intron of the clusterin gen e. 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 DN A elements in both the 5'-flanking region and the first intron. When D NA fragment in the first intron, +1153 to +2874, was included, CAT act ivity in the (-266/+2874)CAT construct increased to 70% of the cluster in promoter (-266/+54)CAT, indicating that stimulatory DNA elements ma y be present in this region of the first intron. Treatment of MA-10 ce lls with cyclic (cAMP) neither decreased CAT activity by any of the cl usterin/CAT chimeric plasmids examined in transient transfection studi es, nor reduced the synthesis of nuclear clusterin RNA in nuclear run- on assays, indicating that the reduction of clusterin mRNA levels by c AMP previously reported in our laboratory is not exerted at the transc riptional level. Furthermore, addition of transcriptional or translati onal inhibitors (actinomycin D and cycloheximide respectively) abolish ed the cAMP effect observed in MA-10 cells. In summary, we have demons trated that the basal transcription of the rat clusterin gene in testi cular cells is under the control of both positive and negative regulat ory 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 cAM P 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.