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Background: Prostate epithelial cells accumulate a high level of aspartate that is utilized as a substrate for their unique function of production and secretion of enormously high levels of citrate. In most mammalian cells aspartate is synthesized; and, therefore is a non-essential amino acid. In contrast, in citrate-producing prostate cells, aspartate is an essential amino acid that must be derived from circulation. The prostate intracellular/extracellular conditions present a 40:1 concentration gradient. Therefore, these cells must possess a plasma membrane-associated aspartate uptake transport process to achieve their functional activity. In earlier kinetic studies we identified the existence of a unique Na+-dependent high-affinity L-aspartate transport process in rat prostate secretory epithelial cells. The present report is concerned with the identification of this putative L-aspartate transporter in rat and human prostate cells., Results: The studies show for the first time that EAAC1 is expressed in normal rat prostate epithelial cells, in normal and hyperplastic human prostate glands, and in human malignant prostate cell lines. EAAC1 expression and high-affinity L-aspartate transport are correspondingly down-regulated by EAAC1 siRNA knock down. Exposure of prostate cells to physiological levels of prolactin or testosterone results in an up-regulation of EAAC1 expression and a corresponding increase in the high-affinity transport of L-aspartate into the cells., Conclusion: This study shows that EAAC1 functions as the high-affinity L-aspartate transporter that is responsible for the uptake and accumulation of aspartate in prostate cells. In other cells (predominantly excitable tissue cells), EAAC1 has been reported to function as a glutamate transporter rather than as an aspartate transporter. The regulation of EAAC1 expression and L-aspartate transport by testosterone and prolactin is consistent with their regulation of citrate production in prostate cells. The identification of EAAC1 as the high-affinity L-aspartate transporter now permits studies to elucidate the mechanism of hormonal regulation of EAAC1 gene expression, and to investigate the mechanism by which the cellular environment effects the functioning of EAAC1 as an aspartate transporter or as a glutamate transporter.

The control and alteration of key regulatory enzymes is a determinant of the reactions and pathways of intermediary metabolism in mammalian cells. An important mechanism in the metabolic control is the hormonal regulation of the genes associated with the transcription and the biosynthesis of these key enzymes. The secretory epithelial cells of the prostate gland of humans and other animals possess a unique citrate-related metabolic pathway regulated by testosterone and prolactin. This specialized hormone-regulated metabolic activity is responsible for the major prostate function of the production and secretion of extraordinarily high levels of citrate. The key regulatory enzymes directly associated with citrate production in the prostate cells are mitochondrial aspartate aminotransferase, pyruvate dehydrogenase, and mitochondrial aconitase. Testosterone and prolactin are involved in the regulation of the corresponding genes associated with these enzymes (which we refer to as "metabolic genes"). The regulatory regions of these genes contain the necessary response elements that confer the ability of both hormones to control gene transcription. In this report, we describe the role of protein kinase c (PKC) as the signaling pathway for the prolactin regulation of the metabolic genes in prostate cells. Testosterone and prolactin regulation of these metabolic genes (which are constitutively expressed in all mammalian cells) is specific for these citrate-producing cells. We hope that this review will provide a strong basis for future studies regarding the hormonal regulation of citrate-related intermediary metabolism. Most importantly, altered citrate metabolism is a persistent distinguishing characteristic (decreased citrate production) of prostate cancer (PCa) and also (increased citrate production) of benign prostatic hyperplasia (BPH). An understanding of the role of hormonal regulation of metabolism is essential to understanding the pathogenesis of prostate disease. The relationships described for the regulation of prostate cell metabolism provides insight into the mechanisms of hormonal regulation of mammalian cells in general.

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.

Background: m-aconitase catalyzes the first step leading to the oxidation of citrate via the Krebs cycle. It is a constituitive enzyme in virtually all mammalian cells, found in excess, and is considered to be a regulatory or regulated enzyme. In contrast to these general relationships, prostate secretory epithelial cells possess a uniquely limiting mitochondrial (m-) aconitase which minimizes the oxidation of citrate. This permits the unique prostate function of accumulating and secreting extraordinarily high levels of citrate. Previous animal studies demonstrated that testosterone and prolactin regulate the level of m-aconitase specifically in citrate-producing prostate cells. The present studies were conducted to determine if testosterone and prolactin regulated the expression of the m-aconitase gene in prostate cells, and to determine the effect of the hormones on human prostate cells., Methods: The studies were conducted with freshly prepared rat ventral, rat lateral, and pig prostate epithelial cells, and with the human malignant cell lines LNCaP and PC-3. The effects of 1 nM testosterone and 3 nM prolactin on the level of m-aconitase mRNA and on the transcription rate of m-aconitase were determined., Results: The studies revealed that both prolactin and testosterone increase the levels of m-aconitase mRNA and the transcription rates of m-aconitase in rat ventral prostate cells, pig prostate cells, and human malignant prostate cells (LNCaP and PC-3). In contrast, both hormones decreased the level of m-aconitase mRNA and repressed m-aconitase gene transcription in rat lateral prostate cells. The hormonal regulation of m-aconitase corresponded with the levels of m-aconitase enzyme, m-aconitase activity, and citrate oxidation., Conclusions: In addition to the constitutive expression of m-aconitase, the m-aconitase gene is testosterone- and prolactin-regulated in specifically targeted prostate cells. The hormonal regulation of m-aconitase gene expression and biosynthesis of m-aconitase provide a regulatory mechanism for the oxidation of citrate, and consequently, the level of net citrate production by prostate. The hormonally increased expression and biosynthesis of m-aconitase in human malignant cells might be involved in the increased citrate oxidation associated with the development of true malignant cells in prostate cancer., (Copyright 2000 Wiley-Liss, Inc.)

The prostate gland of humans and other animals has the unique function of accumulating and secreting extraordinarily high levels of citrate. The prostate secretory epithelial cells synthesize citrate which, due to a limiting mitochondrial (m-) aconitase, accumulates rather than being oxidized. Thus citrate is essentially an end product of metabolism in prostate. For continued net citrate production, a continual source of oxaloacetate (OAA) and acetyl CoA is required. Glucose via pyruvate oxidation provides the source of Acetyl CoA. In prostate cells, citrate production is regulated by testosterone and/or by prolactin. Both hormones selectively regulate the level and activity of pyruvate dehydrogenase E1 alpha (E1a) in animal prostate cells; thereby regulating the availability of acetyl CoA for citrate synthesis. Studies were conducted to determine if testosterone and prolactin might regulate the expression of the E1a gene in prostate epithelial cells. Prolactin treatment of rat ventral and lateral prostate cells and human PC3 cells increased the levels of E1a mRNA and the rates of transcription of the E1a gene. Testosterone also increased the mRNA level and transcription of E1a in rat ventral prostate cells, and in PC3 cells transfected with androgen receptor. However, testosterone treatment resulted in a repression of E1a gene expression in lateral prostate cells. Evidence is presented which supports the view that prolactin regulation of E1a is mediated via PKC. The rapidity of the effects of both hormones is representative of an immediate-early gene response. To our knowledge this represents the first report in any mammalian cells that, in addition to its constitutive expression in all mammalian cells, the E1a gene is a hormonally-regulated gene in specifically targeted prostate epithelial cells.

The glandular epithelial cells of the human prostate gland have the unique capability and function of accumulating the highest zinc levels of any soft tissue in the body. Zinc accumulation in the prostate is regulated by prolactin and testosterone; however, little information is available concerning the mechanisms associated with zinc accumulation and its regulation in prostate epithelial cells. In the present studies the uptake and accumulation of zinc were determined in the human malignant prostate cell lines LNCaP and PC-3. The results demonstrate that LNCaP cells and PC-3 cells possess the unique capability of accumulating high levels of zinc. Zinc accumulation in both cell types is stimulated by physiological concentrations of prolactin and testosterone. The studies reveal that these cells contain a rapid zinc uptake process indicative of a plasma membrane zinc transporter. Initial kinetic studies demonstrate that the rapid uptake of zinc is effective under physiological conditions that reflect the total and mobile zinc levels in circulation. Correspondingly, genetic studies demonstrate the expression of a ZIP family zinc uptake transporter in both LNCaP and PC-3 cells. The rapid zinc uptake transport process is stimulated by treatment of cells with physiological levels of prolactin and testosterone, which possibly is the result of the regulation of the ZIP-type zinc transporter gene. These zinc-accumulating characteristics are specific for prostate cells. The studies support the concept that these prostate cells express a unique hormone-responsive, plasma membrane-associated, rapid zinc uptake transporter gene associated with their unique ability to accumulate high zinc levels.

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.

The prostate gland of many animals accumulates extremely high levels of zinc and citrate. Evidence currently exists in support of a concept that zinc might be an important regulator of m-aconitase and citrate oxidation of prostate epithelial cells. No information concerning the mitochondrial levels of zinc has been available. The roles of testosterone and prolactin in the regulation of zinc have not been established. In this report, we determined the levels of tissue and mitochondrial zinc of rat lateral prostate (LP), ventral prostate (VP), dorsal prostate (DP), liver, and kidney. The results demonstrate that the mitochondrial zinc levels of the prostate cells were higher than levels of nonprostatic cells. The LP contained severalfold higher zinc levels than DP and VP. The effects of testosterone and prolactin in vivo and in vitro on the zinc levels were also determined. Both hormones significantly increased cellular and mitochondrial zinc levels of LP cells; decreased the zinc levels of VP cells; and had no effect on the zinc levels of DP, liver, or kidney cells. These effects are direct and physiological effects of the hormones on the targeted prostate epithelial cells. The hormonal effects on mitochondrial zinc of LP and VP epithelial cells correlate perfectly with their effects on citrate oxidation. The results support the concept that mitochondrial zinc is an inhibitor of m-aconitase and citrate oxidation; and that prolactin and testosterone regulation of mitochondrial zinc provides a mechanism for their regulation of citrate oxidation in citrate-producing prostate epithelial cells.

Objectives: The function of the prostate gland in many animals, including humans, is to accumulate and secrete large quantities of citrate. This function derives from the metabolic characteristics of the prostate secretory epithelial cells. These cells possess a uniquely limiting mitochondrial aconitase (m-aconitase) that minimizes citrate oxidation and thus permits citrate to accumulate. Unfortunately, the characteristics of prostate m-aconitase and its manner of regulation have not been established. The hormones testosterone and prolactin, however, are significantly involved in regulating prostate citrate production. Thus it is reasonable to hypothesize that these hormones may be involved in the regulation of both m-aconitase and citrate oxidation., Methods: Using freshly prepared pig prostate epithelial cells, we attempted to determine the effects of testosterone and prolactin treatment on the level of m-aconitase enzyme, on the level of m-aconitase activity, and on citrate utilization. The epithelial cells were incubated for 3 hours with either testosterone (10(-9) M), prolactin (1 microgram/mL), or vehicle (control)., Results: Both hormone applications caused a marked increase in the level of m-aconitase. In contrast, neither hormone had any effect on the m-aconitase level of pig seminal vesicle cells, which are also citrate-producing cells. Moreover, neither hormone had any effect on pyruvate dehydrogenase E1a. These findings suggest that testosterone and prolactin regulation of prostate m-aconitase is a highly specific effect. Along with the increase in the level of m-aconitase enzyme, both hormones also increased m-aconitase activity and prostate-cell utilization of citrate., Conclusions: These studies demonstrate that testosterone and prolactin can regulate m-aconitase and sub-sequent citrate oxidation of specific prostate epithelial cells. This unique aconitase relationship is not observed in other mammalian cells.

The prostate gland of many animals, including humans, produces and secretes extremely high levels of citrate. To achieve this function, prostate secretory epithelial cells possess unique metabolic properties that permit accumulation and ultimate secretion (net citrate production) of citrate. Mounting evidence continues to support the concept that prostate epithelial cells possess a limiting mitochondrial (m)-aconitase activity that minimizes citrate oxidation and results in the accumulation of citrate synthesized by the cells. Recent studies have revealed that prolactin (PRL) stimulates net citrate production of rat lateral prostate (RLP). The mechanism of this PRL effect has not been established. The current studies were concerned with the possibility that PRL might be involved in the regulation of citrate oxidation and m-aconitase of prostate cells. Studies were conducted with RLP, RVP (rat ventral prostate), RDP (rat dorsal prostate), and kidney cells. The results showed that PRL in vitro and in vivo decreased citrate utilization and the level of m-aconitase in RLP cells, and conversely increased citrate utilization and m-aconitase in RVP cells. Furthermore, PRL had no effect on either RDP or kidney cells. The effects of PRL on both citrate utilization and m-aconitase of RLP and RVP were abolished by cycloheximide and actinomycin. Mitochondrial studies revealed that PRL decreased citrate oxidation of RLP and increased citrate oxidation of RVP, but had no effect on isocitrate oxidation. In conclusion, these studies establish that PRL has a physiological role in the regulation of citrate oxidation in prostate, and that this action is associated with PRL regulation of the biosynthesis of m-aconitase. Furthermore, the effects of PRL are cell-specific and targeted at m-aconitase.

Prolactin is an important regulator of prostate citrate production. In rats this regulatory effect of prolactin is specific for lateral prostate, and has no effect on either ventral or dorsal prostate. The mechanisms by which prolactin regulates prostate citrate production have not been elucidated. Two key regulatory enzymes involved in citrate synthesis by prostate epithelial cells are mitochondrial aspartate aminotransferase (mAAT) which provides oxalacetate, and PDH E1 alpha (pyruvate dehydrogenase) which provides acetyl CoA for citrate synthesis. Our previous studies demonstrated that prolactin regulates mAAT. However, an increase in citrate synthesis would require an increase in both oxalacetate and acetyl CoA. Therefore, we investigated the possibility that prolactin might also regulate PDH E1 alpha in LP epithelial cells. The present studies demonstrate that prolactin administration (1 mg/rat) to rats resulted in an increased level of E1 alpha in LP epithelial cells within 6 hr, but had no effect on the E1 alpha level of VP epithelial cells. In vitro studies demonstrated that exposure of freshly prepared LP epithelial cells to prolactin (0.1-1.0 microgram/ml) resulted in increased levels of E1 alpha. Prolactin had no effect on either VP or DP epithelial cells. The stimulatory effect of prolactin on E1 alpha was inhibited by actinomycin and cycloheximide, thereby indicating that prolactin stimulated the biosynthesis of E1 alpha. The studies reveal that prolactin specifically stimulates E1 alpha levels of LP epithelial cells, whereas testosterone specifically stimulates E1 alpha levels of VP epithelial cells. At this time, we propose that the effects of prolactin and testosterone involve increased expression of the E1 alpha gene of LP and VP epithelial cells, respectively.

We propose that: 1. Prolactin is a trophic hormone required for normal development and growth of prostate as well as other tissues. 2. Citrate production, the major function of prostate, is directly regulated by prolactin. 3. The mechanisms of prolactin regulation of citrate production by prostate epithelial cells include a. Regulation of the pmAAT gene, which results in an increase in the biosynthesis of mAAT and, ultimately, an increase in citrate synthesis (the prolactin regulation of gene transcription is mediated via PKC). b. Inhibition of citrate oxidation, possibly via zinc inhibition of m-aconitase. c. Stimulation of aspartate transport, possibly via regulation of the biosynthesis of the high-affinity aspartate transporter. 4. These relationships apply to normal human prostate, and might have important implications in the pathogenesis of prostate neoplasms. 5. The regulation of prostate citrate production is the reproductive function of prolactin in males.

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.

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.