• Studies on prolactin regulation of mitochondrial aspartate aminotransferase gene expression and protein kinase C activity and expression in prostate cells

      Gorski, Ewa; Franklin, Renty B. (1996)
      Citrate accumulation and secretion are physiological functions of the normal prostate gland in most species. Prolactin (PRL) stimulates citrate accumulation in the rat lateral prostate (LP) and pig prostate by increasing the expression of mitochondrial aspartate aminotransferase (mAAT), the enzyme that catalyzes aspartate transamination. In the present studies, we established the role of PRL and the phorbol ester 12-O-tetra-decanoylphorbol 13-acetate (TPA) in the regulation of mAAT in LNCaP and PC-3 cells. We isolated total RNA from the cells and hybridize it with a mAAT cDNA. Next, we assayed protein kinase C (PKC) activity and expression in PRL and TPA treated prostate cells. We, then determined the effect of PRL and TPA on gene transcription using a mAAT-chloramphenicol acetyltransferase (CAT) reporter gene construct, transiently transfected into PC-3 cells. A fragment of the 5{dollar}\sp\prime{dollar} gene region containing putative TPA response elements (TRE) was cloned into the pCAT-Promoter vector from Promega. The reporter vector was introduced into the cells by the calcium phosphate-DNA co-precipitation method. The cells were then incubated for 48 h with or without PRL or TPA.;The results indicated that PRL and TPA increased the mAAT mRNA level 2-4 fold. Upon PRL and TPA treatment the level of PKC activity in rat LP, and LNCaP and PC-3 cells increased 20-60% and 40-207%, respectively. In addition, long term PRL and TPA treatment resulted in increased PKC expression (10-60% and 10-240%, respectively) in the LP cytosol fraction. Furthermore, the results showed that PRL at a concentration of {dollar}1\times10\sp{lcub}-9{rcub}{dollar} M and TPA (0.1 {dollar}\mu{dollar}g/ml) treatment increased CAT activity in PC-3 cells. These results suggest that PRL regulates mAAT at the transcriptional level. Moreover, these effects of PRL on mAAT mRNA and PKC activity were eliminated by PKC down-regulation. Since both PRL and TPA induced PKC activity and since the effects of PRL and TPA were eliminated by PKC down-regulation, we postulate that the PRL effect on mAAT is mediated via the diacylglycerol (DAG)-PKC signal transduction pathway in rat LP and prostate cancer (CA) cells.
    • Testosterone regulates mitochondrial aspartate aminotransferase gene expression in rat ventral prostate

      Qian, Kaifeng; Franklin, Renty B.; Costello, Leslie (1991)
      One of the physiological functions of the rat ventral prostate, like human prostate, is to secrete an extraordinarily high quantity of citrate. This unique characteristic is under the influence of testicular androgen. The continuous secretion of citrate from prostate results in loss of a 6-carbon compound from the metabolic pool which must be replenished. Citrate is synthesized from the concentration of acetyl-CoA and oxaloacetate. Acetyl-CoA may come from various sources. However, recent studies indicated that the possible available source for oxaloacetate is the transamination of aspartate. Mitochondrial aspartate amino-transferase (mAAT) is an important enzyme which regulates citrate production in rat ventral prostate. Testosterone, as a major testicular androgen, stimulates citrate production and mAAT activity in a similar pattern. The hypothesis in this dissertation is that the ability of testosterone to increase citrate production and mAAT activity is the result of stimulation of mAAT gene expression. To verify this hypothesis, numerous in vivo and in vitro studies were utilized to analyze the steady-state level of mAAT mRNA in response to testosterone depletion and repletion. The rats were castrated, followed by testosterone or oil vehicle injection. Twenty-four (or forty-eight) hours later all animals were killed, prostates removed, RNA and nuclei isolated. In vitro studies were performed using primary cultured pig prostate cells. The quantity of mAAT mRNA was measured using northern hybridization. The rates of transcription and degradation of mAAT mRNA were determined by in vitro transcription assay and pulse chase labeling assay, respectively. Results show that castration caused a significant decrease in the content of mAAT mRNA and the transcription rate of the mAAT gene. However, testosterone administration reversed these hormonal depletion effects. Moreover, testosterone administration also prolonged the half life of mAAT mRNA. These results support the hypothesis that one of the major physiological functions of testosterone is to stimulate mAAT mRNA gene expression, which in turn enhances citrate production in rat ventral prostate. Furthermore, these results confirm that the increase in the steady-state level of mAAT mRNA in response to testosterone administration results from stimulation of mAAT gene transcription and inhibition of mAAT mRNA degradation.