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  • Author: C.-L. C. Chen x
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Regulators for the rat clusterin gene: DNA methylation and cis-acting regulatory elements

N Rosemblit and C-L C Chen

ABSTRACT

Clusterin, also known as sulphated glycoprotein-2 or testosterone-repressed prostate message-2, is a ubiquitous protein found in a variety of tissues and species. In the reproductive tract of the male rat, clusterin is regulated in a complex age-dependent and cell-specific manner. It is expressed at high levels in the epididymis and testis and at very low levels in the prostate under basal conditions. The expression of this gene in the prostate and seminal vesicles is associated with androgen withdrawal, while in the testis clusterin mRNA is repressed by cyclic AMP (cAMP). To understand the mechanisms that control the expression of the clusterin gene better, we isolated and characterized the gene encoding rat clusterin, and analysed its cytosine methylation pattern in various tissues. Several putative regulatory DNA elements were identified, including a consensus AP-1 site in the 5′ flanking region. Two AP-1 sites and two transforming growth factor-β inhibitory elements, one AP-2 site and eight half-sites for glucocorticoid/androgen response elements were found within the first intron, and one cAMP response element was found in the first exon. The cytosine methylation pattern indicated that testicular or epididymal DNA in the rat is hypomethylated in the region between positions −534 and −99 of the clusterin gene, when compared with tissues with lower levels of expression such as prostate as well as liver, lung, kidney and spleen.

DOI:
https://doi.org/10.1677/jme.0.0130069
Volume 13: Issue 1,
Pages:
69–76 (8 total)
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Negative control of the rat inhibin α subunit promoter in MA-10 Leydig tumour cells

Z-M Feng and C-L C Chen

ABSTRACT

The promoter/regulatory sequences responsible for the transcription of the rat inhibin α subunit gene in the testis were identified by the transient expression in an MA-10 Leydig tumour cell line of a bacterial reporter gene, chloramphenicol acetyltransferase (CAT), which was driven by different regions of the 5′ flanking sequence of the inhibin α subunit gene. The CAT activity was elevated when the 2·0 kb 5′ flanking α subunit gene fragment was progressively shortened from its 5′ end, and a maximal increase was reached when the CAT gene was driven by an α subunit gene promoter extending to −163 bp. This construct was termed AαBstCAT. Furthermore, when either the −2·0 to −1·6 kb or the −2·0 to −1·0 kb α subunit DNA fragment was fused to AαBstCAT, the CAT activity was markedly suppressed, indicating the presence of negative regulatory DNA elements (NREs) in the upstream region of the gene. The cyclic AMP (cAMP) responsiveness of the α subunit gene, which was dependent upon the putative cAMP response element within the 67 bp α subunit promoter, was not affected by the upstream NREs. The inhibitory effect was also demonstrated when the −2·0 to −1·0 kb fragment was placed in either orientation with respect to the α subunit promoter or to a thymidine kinase promoter, suggesting that the NRE(s) can act as a silencer. Based on our observations we conclude that the basal expression of the rat inhibin α subunit gene in testicular MA-10 cells may, at least in part, be controlled by the upstream silencer(s) and NRE(s).

DOI:
https://doi.org/10.1677/jme.0.0130039
Volume 13: Issue 1,
Pages:
39–47 (9 total)
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Influence of chronic restraint stress on proopiomelanocortin mRNA and β-endorphin in the rat hypothalamus

A. López-Calderón, C. Ariznavarreta, and C.-L. C. Chen

ABSTRACT

It has been postulated that some endocrine responses to stressful stimuli are mediated through the activation of hypothalamic pro-opiomelanocortin (POMC)-derived peptides. The aim of the present study was to analyse the effect of chronic stress on expression of the POMC gene in the medial basal hypothalamus and pituitary, and on serum concentrations of LH, β-endorphin and corticosterone. Adult male rats were killed after being subjected to restraint stress for 6 h/day over 2, 3 or 4 days. Chronic restraint induced an increase in serum concentrations of β-endorphin and corticosterone and a decrease in serum LH levels. To determine whether chronic stress induced any change in POMC synthesis, a dot-blot method was used to measure POMC mRNA levels. No significant changes were detected either in the β-endorphin content or in POMC mRNA levels in the medial basal hypothalamus after 2, 3 or 4 days of chronic restraint. This observation contrasts with the stimulation of POMC mRNA levels in both lobes of the pituitary. The data suggest that although chronic restraint induces an increase in POMC synthesis and secretion in the pituitary and a decrease in LH secretion, it has no effect on hypothalamic POMC neurones.

DOI:
https://doi.org/10.1677/jme.0.0070197
Volume 7: Issue 3,
Pages:
197–204 (8 total)
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Expression of the clusterin gene in the tissues of Booroola sheep which were homozygotes or non-carriers of the fecundity gene FecB

J. S. Fleming, P. J. Greenwood, and C.-L. C. Chen

ABSTRACT

Clusterin or sulphated glycoprotein-2 is a major component of the rete testis fluid, synthesized by the rete testis epithelial cells and Sertoli cells. Differences in the two-dimensional polyacrylamide gel electrophoresis pattern of clusterin-like proteins have been reported in rete testis fluid from Booroola rams carrying the fecundity gene FecB, when compared with that from non-carrier rams. In order to determine whether the FecB gene influences the expression of the clusterin gene, we used a rat clusterin cRNA probe to investigate mRNA species in the tissues of homozygous (BB) or non-carrier (+ +) Booroola sheep. Northern blots of polyadenylated RNA showed hybridization to the cRNA probe in the testis, ovarian follicles, corpora lutea and stroma, pituitary and liver. A major mRNA transcript was observed at 2·3 kb and a minor transcript in some tissues at 0·8 kb. Densitometry of the autoradiographs revealed no FecB-specific differences in the densities of the hybridization signals from + + and BB testis or ovarian follicle, corpora lutea or stromal RNA. We conclude that the gene for ovine clusterin is expressed widely in the tissues of sheep and that its expression is not affected by the presence of the FecB gene.

DOI:
https://doi.org/10.1677/jme.0.0090207
Volume 9: Issue 3,
Pages:
207–211 (5 total)
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Analysis of the rat clusterin gene promoter and cyclic AMP-regulated mRNA stability in testicular cells

N Rosemblit, Z-M Feng, and C-L C Chen

ABSTRACT

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.

DOI:
https://doi.org/10.1677/jme.0.0160287
Volume 16: Issue 3,
Pages:
287–296 (10 total)
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Insulin-like growth factor-I mRNA expression in the interstitial cells of the rat testis

A Moore, C-L C Chen, J R E Davis, and I D Morris

ABSTRACT

IGF-I mRNA has been demonstrated in testicular tissue and, more recently, localized specifically to Leydig cells. This study investigated the expression of IGF-I and side-chain cleavage enzyme (SCC) mRNA in two preparations of rat interstitial testicular cells which were separated by buoyant density into Leydig cell-enriched and -depleted fractions. RNA was prepared from interstitial cells obtained from the testes of untreated adult and immature rats and adult rats treated with human chorionic gonadotrophin (hCG) or ethane dimethanesulphonate (EDS; to destroy Leydig cells). IGF-I mRNA was detected in all samples, with five major transcripts ranging from 7·5 to 0·6 kb. Leydig cells (3β-hydroxysteroid dehydrogenase-positive and sensitive to EDS) expressed abundant IGF-I and SCC mRNAs, and levels of both were increased following hCG treatment. However, in addition, IGF-I mRNA which was derived from non-Leydig interstitial cells was detected, in the complete absence of SCC message, either in the more buoyant interstitial cells or in both interstitial cell fractions following the destruction of Leydig cells by EDS treatment. IGF-I expression in the Leydig cell-depleted cell fraction was also increased by hCG treatment, and it is therefore suggested that at least part of this non-Leydig interstitial cell IGF-I mRNA originates in Leydig cell precursors. In conclusion, Leydig cells are not the sole origin of IGF-I mRNA in the testis, and the non-Leydig cell expression may be an important component of testicular IGF-I production.

DOI:
https://doi.org/10.1677/jme.0.0110319
Volume 11: Issue 3,
Pages:
319–324 (6 total)