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Citrate accumulation and secretion are physiological functions of the prostate gland that are regulated by testosterone and prolactin. The metabolic pathway for citrate production in the prostate involves the activity of mitochondrial aspartate aminotransferase (mAAT). The expression of mAAT in the prostate is regulated by prolactin through a signal transduction pathway mediated by protein kinase C (PKC). In this report we determined which PKC isoforms are expressed in rat lateral prostate epithelial cells and their activation by prolactin. Eight PKC isoforms are expressed in the ventral and lateral prostate lobes. Although all eight isoforms are expressed, only PKCalpha and PKCvarepsilon were stimulated by prolactin and only in the lateral prostate lobe. Activator protein-1 (AP-1) appears to be the target of prolactin-PKC signaling because prolactin stimulated nuclear protein binding to an AP-1 consensus oligodeoxynucleotide. Moreover, the nuclear binding protein stimulated by prolactin also bound an mAAT oligodeoxynucleotide that contained an AP-1 consensus sequence and which competed for binding with the consensus AP-1 oligodeoxynucleotide. A PKCvarepsilon antisense oligodeoxynucleotide blocked expression of mAAT mRNA. Thus, we conclude that PKCvarepsilon is a specific PKC isoform that mediates via AP-1 the signal for prolactin regulation of mAAT gene expression in rat lateral prostate epithelial cells.

Citrate production is a major physiological function of the prostate that is regulated by testosterone and prolactin. Mitochondrial aspartate aminotransferase (mAAT) is a key enzyme in the metabolic pathway of prostate citrate production. In addition, prolactin stimulates expression of mAAT in the rat lateral prostate. In this report we establish the role of prolactin in the regulation of mAAT in two prostate cancer cell lines, LNCaP and PC-3. LNCaP cells respond to hormonal stimulation with increased secretion of prostate specific products. PC-3 cells, on the other hand, are testosterone independent and apparently do not respond to other growth factors either. Results showed that both LNCaP and PC-3 cells responded to prolactin with increased mAAT activity and an increased steady state level of mAAT mRNA. Prolactin also increased protein kinase C (PKC) activity in both these cell lines. Treatment of LNCaP and PC-3 cells with the phorbol ester 12-O-tetradecanoylphorbol (TPA) caused the same effect on mAAT activity and mRNA level as prolactin. The results suggest that the diacylglycerol-PKC signal transduction system mediates the prolactin effect on mAAT. In addition, these results also show that the prolactin effect on mAAT is independent of androgens since PC-3 cells reportedly lack androgen receptor expression. Thus, these results provide evidence that prolactin is a physiological regulator of prostate function in human as well as rat prostate. In addition, the results also show that though prostate cancer cells are androgen independent, they remain responsive to prolactin. This could have important implications for the treatment and management of prostate cancer.

Mitochondrial aspartate aminotransferase (mAAT) is one of two key enzymes in the pathway of citrate production in prostate. Expression of mAAT is modulated by testosterone and prolactin in prostate. We cloned the promoter and 5'-flanking region of the rat mAAT gene and sequenced 2.0 kilobases of the DNA. This fragment contains the 5'-regulatory promoter region that lacks a TATA and a CCAAT box but is G+C rich. The 5'-upstream flanking region contains sequences that have high homology with the consensus glucocorticoid response element/androgen response element (ARE) and a reported ARE sequence that is different from the consensus sequence. Functional transcription studies showed that a 481-base region containing the two ARE sequences was sufficient for androgen-regulated gene expression. There are multiple transcription start sites that are regulated by testosterone in prostate. In liver, on the other hand, castration did not affect transcription from any of the start sites. Therefore, these data provide evidence that transcriptional regulation of the rat pmAAT gene occurs through an ARE located in the 5'-region. In addition, not only is gene expression modulated by testosterone, but the effect of testosterone on transcription is cell specific.

The effect of testosterone on the precursor mitochondrial aspartate aminotransferase (pmAAT) gene and on pmAAT-mRNA was studied in rat ventral prostate (VP) and pig prostate epithelial cells. Castration significantly decreased the level of nuclear pmAAT transcripts in VP; whereas testosterone treatment of castrated animals restored the level of pmAAT transcripts. Correspondingly, castration resulted in a marked decrease in the transcription rate of the pmAAT gene; whereas testosterone treatment markedly increased the transcription rate. In vitro studies with isolated pig prostate epithelial cells demonstrated that testosterone directly and rapidly induced a transient increase in the transcription rate of the pmAAT gene. The increase in transcription was associated with an increase in the steady-state level of pmAAT-mRNA. Similar in vitro effects were observed with isolated VP epithelial cells. In addition to its stimulatory effect on transcription of the pmAAT gene, testosterone also increased the half-life of pmAAT-mRNA from 2 h in the absence of hormone to 16 h in its presence. Consequently, testosterone appears to stabilize the pmAAT-mRNA. The combination of its immediate effect on stimulating the transcription of the pmAAT gene and its stabilizing effect on pmAAT-mRNA would account for the increase in the steady-state level of pmAAT-mRNA by testosterone. These studies support our proposal that, through these effects, testosterone increases the biosynthesis of mAAT thereby increasing the transamination of aspartate to oxaloacetate and ultimately increasing the synthesis of citrate. This appears to provide at least one of the mechanisms by which testosterone regulates prostate citrate production.

Prolactin (PRL) has been reported to stimulate citrate production and the activity of mitochondrial aspartate aminotransferase (mAAT) and its precursor form pmAAT in prostate epithelial cells. The phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) caused the same result as PRL, which suggests that the PRL effect on mAAT activity might be mediated by protein kinase C (PKC) stimulation of pmAAT gene transcription. Both PRL and TPA increased the level of pmAAT mRNA by 2.5- to 3-fold in pig prostate cells. The PKC inhibitor gossypol completely inhibited the PRL and TPA induced increases. In addition, the effects of both PRL and TPA were inhibited by down-regulation of prostate PKC. Nuclear run-off assays indicated that PRL and TPA induction of pmAAT occurred primarily at the transcriptional level. The stimulation of pmAAT transcription by TPA suggests that the pmAAT gene contains a TPA response element. Thus, these results are consistent with our previous observation that PRL directly induces pmAAT and that the mechanism of this PRL effect might involve stimulation of PKC.

The effect of testosterone on precursor mitochondrial aspartate aminotransferase (pmAAT) mRNA was studied in rat ventral prostate and primary cell cultures of mini-pig prostate. Testosterone induced a 2-3-fold increase in pmAAT mRNA level in both rat ventral prostate and mini-pig prostate cultures. The pmAAT mRNA induction occurred 30 min after testosterone treatment and was maximal by 1.5 h. Prostatic mAAT activity was also induced by testosterone with a 1-2 h lag period. The time-course of induction of pmAAT mRNA, pmAAT activity and mAAT activity was consistent with stimulation of mRNA synthesis followed by increased synthesis and import of pmAAT into mitochondria. The effect of testosterone on pmAAT mRNA was specific because the increase in pmAAT mRNA was at least 2-fold greater than the increase in poly (A+) RNA. These results suggest that testosterone stimulated mAAT activity by induction of pmAAT mRNA. This continues to support our proposal that a major physiological effect of testosterone is increased pmAAT mRNA steady-state levels which result in increased pmAAT synthesis and increased mAAT activity. These changes ultimately result in increased citrate production by prostate epithelial cells.

Prolactin, in vitro, significantly increased citrate production, mAAT (mitochondrial aspartate aminotransferase) and pmAAT (precursor form of mAAT) activity of prostate epithelial cells derived from rat lateral prostate (LP) and pig prostate cultures. In contrast, prolactin had no effect on the cytosolic isozyme, cAAT. This prolactin effect appeared to be independent of testosterone. The phorbol ester TPA (12-O-tetradecanoyl-phorbol-13-acetate) induced the same effects as prolactin thereby indicating the involvement of protein kinase C. This report demonstrates that prolactin directly regulates citrate production of prostate epithelial cells and the availability of an in vitro model to elucidate the mechanism of action of prolactin.

Mitochondrial aspartate transamination was investigated as a major source of oxalacetate for citrate synthesis in rat ventral prostate. Citrate accumulation was measured in isolated mitochondria incubated with acetyl coenzyme A and various combinations of amino acids. Aspartate plus alpha ketoglutarate in the presence of acetyl coenzyme A resulted in significant citrate accumulation. Neither aspartate nor alpha ketoglutarate alone resulted in any significant citrate accumulation. Aspartate and alpha ketoglutarate use was comparable to glutamate and citrate production. The results indicated the presence of a mitochondrial aspartate aminotransferase. Castration (3 days) caused a significant decrease in citrate production from aspartate plus alpha ketoglutarate as well as a decrease in mitochondrial AAT activity in prostate although no effect on kidney activity occurred. A single injection of 1 mg. testosterone propionate to castrate rats significantly increased prostate mitochondrial AAT activity within 24 hours while MDH activity was unaltered. A double reciprocal plot indicated that testosterone might regulate the level of mitochondrial AAT in prostate. Ventral prostate also contain a uniquely high level of endogenous aspartate. These studies indicate that aspartate might be the major 4-carbon source of oxalacetate for citrate synthesis. Also testosterone possibly regulated prostate citrate production by its effect on the level of mitochondrial AAT activity.

Stimulation of mitochondrial aspartate aminotransferase (mAAT) activity by testosterone was determined in organ cultures of rat ventral prostate. The effect of testosterone on citrate accumulation in the culture medium was also determined. Testosterone stimulation of citrate accumulation and mAAT occurred in a dose dependent manner. Stimulation of mAAT activity occurred after a 1-3 h lag period and appeared to involve the synthesis of specific RNA since the response was inhibited by actinomycin D. Studies utilizing [3H]L-leucine indicated that unlike the total tissue, testosterone stimulated the incorporation of [3H]leucine into proteins of the mitochondrial fraction. The results suggested that mitochondrial proteins may be more sensitive to testosterone stimulation than cytosol proteins. The response was specific for mAAT since testosterone had no effect on mitochondrial malic dehydrogenase activity. The data suggested that testosterone may regulate prostate citrate content by the induction of mAAT in prostate mitochondria, which results in a source oxalacetic acid for citrate synthesis through transamination of aspartate by alpha ketoglutarate.

The net production of citrate from exogenous substrates by rat ventral prostate was investigated. The preparation of isolated prostate epithelial cells was described. These cells were capable of oxidizing pyruvate (5 mmol/l) as a source of acetyl coenzyme A. The addition of aspartate + alpha-ketoglutarate (5 mmol/l) in the presence of pyruvate resulted in significant net production of citrate and excess oxalacetate. In the presence of aspartate and glutamate, the cells were capable of producing citrate without excessive oxalacetate production. Neither glucose alone nor glucose plus pyruvate resulted in net citrate production. The results demonstrated that aspartate could serve as a 4-carbon source of oxalacetate for citrate synthesis. Furthermore, the results indicate the intramitochondrial operation of a glutamate-aspartate-citrate pathway involving mitochondrial aspartate aminotransferase and glutamic dehydrogenase activities in prostate epithelial cells.

The prostate gland is unusual in that it has a high content of citric acid compared with other soft tissues. The mechanism of citrate accumulation in prostate is unknown. One possibility is that aspartate aminotransferase (AAT) could supply an extra Krebs cycle source of oxaloacetate for citrate formation. Since testosterone regulates citrate production and secretion and strong transaminase activity exists in prostate, a relationship between AAT and citrate production could exist if testosterone also regulates AAT activity. To investigate this possibility, animals were castrated and given testosterone. After a total castration period of 72 hours, animals were sacrificed and mitochondrial AAT activity determined. Results revealed that castration decreased AAT activity and testosterone replacement stimulated activity. Neither castration nor testosterone affected malate dehydrogenase (MDH) activity. Both actinomycin D and cycloheximide blocked the testosterone stimulation of AAT activity. These studies indicate that testosterone may regulate AAT activity in rat ventral prostate. Furthermore, regulation may occur through testosterone stimulation of specific RNA and protein synthesis.

The effects of testosterone on mitochondrial aspartate aminotransferase (mAAT) synthesis in rat ventral prostate was investigated. Procedures for the isolation, purification and characterization of AAT isozymes were developed and described. Purified mAAT preparations contained no demonstrable contaminating proteins. Prostatic mAAT was characterized as a cationic protein with an estimated mol. wt of 120,000. Cytoplasmic AAT (cAAT) isozyme was identified as an anionic protein with an estimated mol. wt of 132,000. A cytosolic cationic isozyme, similar to mAAT, was also identified as pre-mAAT. Testosterone administration to castrated rats resulted in significant increases in leucine incorporation into mAAT, in the level of mAAT, and in mAAT activity. These effects of testosterone were observed within 2 h of administration. Conversely, testosterone administration had none of these effects on cAAT or on non-AAT protein pool. Testosterone treatment did appear to increase leucine incorporation into pre-mAAT. Testosterone treatment in organ cultures and in prostate epithelial cell cultures resulted in the same stimulatory effects on mAAT as observed in the in vivo studies. The hormone was effective at the physiological concentration of 2 X 10(-9) M. These results indicated that testosterone has a rapid and specific effect on the biosynthesis of mAAT. This continues to support our proposal that testosterone regulates prostate citrate production via a stimulatory effect on mAAT which results in increased mitochondrial synthesis of citrate from aspartate.