Cyclic adenosine 3',5'-monosphosphate mediate prolactin regulation of mitochondrial aconitase in human prostate carcinoma cells

Mitochondrial aconitase (mACON) is regarded as the key enzyme for citrate oxidation in human prostatic epithelial cells. The results of RT-PCR and immunoblot assays indicated that human prostatic carcinoma cells (PC-3 cells) express the long-form of the prolactin receptor. In vitro studies determined that prolactin upregulates mACON enzymatic activity and cell proliferation of PC-3 cells. Immunoblot assay revealed that prolactin treatments increase the gene expression of mACON. Transient gene expression assay indicated that the regulation by prolactin of mACON gene expression depends on the presence of the cyclic adenosine 3',5'-monosphosphate (cAMP) response element on the promoter of the mACON gene. Both prolactin and dibutyryl-cAMP doubled the promoter activity of the mACON gene; however, adding H-89, a specific protein kinase A inhibitor, suppressed the prolactin response. The intracellular cAMP levels, but not the cGMP levels, increased after treatment with prolactin. This study showed that prolactin regulates the expression of the mACON gene via the cAMP signal pathway in human prostatic carcinoma cells.     

A placenta-specific receptor for the fusogenic, endogenous retrovirus-derived, human syncytin-2

Syncytin-2 is an envelope gene from the human endogenous retrovirus FRD (HERV-FRD) co-opted by an ancestral primate host, conserved in evolution over >40 Myr, specifically expressed in the placenta, and with a cell–cell fusogenic activity likely contributing to placenta morphogenesis. Here, using the GeneBridge4 human/Chinese hamster radiation hybrid panel, we mapped and identified the human receptor for syncytin-2. This receptor—namely Major Facilitator Superfamily Domain Containing 2 (MFSD2)—belongs to a large family of presumptive carbohydrate transporters with 10–12 membrane-spanning domains, is located at chromosomal position 1p34.2, and is conserved in evolution. An expression vector for MFSD2 confers fusogenicity to otherwise insusceptible cells upon trans-fection of syncytin-2. It also confers infectivity to syncytin-2 pseudotypes, consistent with this protein being the receptor for the ancestrally acquired HERV-FRD family of endogenous retroviruses. At variance with the human gene, neither mouse nor rat MFSD2 can mediate membrane fusion, which is consistent with the fact that the envelope-derived syncytin genes co-opted by rodents during evolution are not orthologous to the human syncytin genes. Remarkably, a real-time quantitative RT-PCR analysis of MFSD2 in various human tissues demonstrates specific expression in the placenta, as well as in the human BeWo choriocarcinoma cell line, which discloses enhancement of receptor expression upon induction by forskolin of cell–cell fusion and syncytium formation. In situ hybridization of human placental tissue using an MFSD2-specific probe further unambiguously demonstrates receptor expression at the level of the syncytiotrophoblast, again consistent with a role in placenta morphogenesis.     

Dopamine inhibits prolactin release when cyclic adenosine 3',5'-monophosphate levels are elevated

We purified lactotrophs from pituitary tumors induced by estrogen in ovariectomized female Fischer 344 rats from 80% of the population before to more than 90% after purification through a continuous Percoll density gradient. The percentage of lactotrophs was evaluated by immunofluorescence. The patterns of PRL release stimulated by 100 nM TRH, 20 microM A23187 (a Ca++ ionophore), 50 nM 12-O-tetradecanoyl-phorbol-13-acetate (a C-kinase activator), or combinations of these agents, or inhibited by 10 microM dopamine were similar in perifused primary cultures of tumor lactotrophs to patterns in cultures of anterior pituitary cells from female retired breeders used previously. In particular, dopamine completely inhibited the release stimulated by forskolin. Intracellular cAMP concentrations and PRL accumulation in the medium were measured in monolayer cultures of purified tumor lactotrophs. In 9 separate experiments, forskolin (10 microM) increased intracellular cAMP concentrations more than 60-fold above control after 30 min of incubation. Preincubation (30 min) with dopamine (10 microM) reduced the cAMP accumulation caused by forskolin, but levels were still at least 20-fold above basal levels in most experiments. PRL release was stimulated 2-fold with forskolin alone, but there was no stimulation of PRL release by forskolin in the presence of dopamine even though cAMP levels were elevated above basal. Therefore, a decrease in cAMP levels is not necessary to inhibit PRL release, and dopamine must have a mechanism for inhibiting PRL release in addition to inhibiting adenylate cyclase.     

Adenosine 3',5'-monophosphate-dependent phosphoproteins in human placenta

cAMP modulates estrogen, hCG, and lactate syntheses by the human placenta. The major effects of cAMP are presumably mediated through the phosphorylation of specific regulatory phosphoproteins after cAMP activation of cAMP-dependent protein kinase. cAMP-dependent phosphoproteins have not been identified in the placenta. Homogenates and cytosol from term human placenta were subjected to an endogenous protein phosphorylation assay using [gamma-32P]ATP in the presence and absence of 1.0 microM cAMP. Protein phosphorylation was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. cAMP stimulated 32P incorporation into proteins with mol wt of 25,000, 27,000, 39,000, 45,000, 52,000, 58,000, and 73,000 (P less than 0.02). Half-maximal 32P incorporation was observed with 1.0 X 10(-7) M cAMP, which was similar to the concentration required for half-maximal histone kinase activity (8.5 +/- 2.9 X 10(-8) M). cGMP induced 32P incorporation into the same placental proteins as cAMP, but 2 orders of magnitude greater cGMP concentrations were required to achieve phosphorylation levels similar to those caused by cAMP. cAMP-dependent protein kinase inhibitor completely blocked cGMP-induced phosphorylation, even when histone protein was added. Therefore, no evidence of a cGMP-dependent protein kinase or specific cGMP-dependent phosphoproteins were detected. CaCl2 (10(-8) - 10(-4) M) had no effect on cAMP-induced 32P incorporation into the seven cAMP-dependent phosphoproteins. However calcium induced 32P incorporation into four other proteins (mol wt, 97,000, 90,000, 20,000, and 19,000). Regulation of placental metabolism by catecholamines and other hormones known to mediate intracellular cAMP or calcium levels may be accomplished by phosphorylation of these phosphoproteins.     

Alternative splicing to exon 11 of human prolactin receptor gene results in multiple isoforms including a secreted prolactin-binding protein

Endocrine and autocrine prolactin (PRL) exerts effects on normal breast and breast cancer cells, and high serum PRL is a poor prognostic factor for colorectal cancer. Here we tested the hypothesis that short isoforms of the PRL receptor (PRLR) in human tissue regulate the actions of PRL in cancer. Using 3' RACE we isolated five splice variants of the human PRLR (hPRLR), three of which encode the complete extracellular binding domain. Two of these isoforms, short form 1a (SF1a) and short form 1b (SF1b), possess unique intracellular domains encoded by splicing to exon 11 from exons 10 and 9 respectively. A third novel isoform (delta7/11) reflects alternative splicing from exon 7 to exon 11 and encodes a secreted soluble PRL-binding protein. Additional splice variants of SF1b and delta7/11 that lacked exon 4 (delta4-SF1b and delta4-delta7/11) were also identified. Functional analyses indicated that hPRLR-SF1b is a strong dominant-negative to the differentiative function of the PRLR long form while hPRLR-SF1a is a weaker dominant-negative. Differential abundance of SF1a, SF1b and delta7/11 expression was detected in normal breast, colon, placenta, kidney, liver, ovary and pancreas, and breast and colon tumors. Taken together, these data indicate the presence of multiple isoforms of the hPRLR that may function to modulate the endocrine and autocrine effects of PRL in normal human tissue and cancer.     

Glycosylated human prolactin

A glycosylated form of human PRL (G-hPRL) was isolated from pituitary glands. The glycoprotein was separated from the major form of PRL on columns of lentil lectin-Sepharose 4B. The major form of PRL did not bind to the lentil lectin, whereas the glycosylated modification did and could be eluted with methyl-alpha-D-mannopyranoside. By gel electrophoresis in sodium dodecyl sulfate, a mol wt of 25,000 was estimated for the glycosylated PRL. The mol wt of hPRL is 23,000. In a RIA for hPRL, the glycosylated hormone was about one third as reactive as the principal form. Since there is only one Asn-X-Ser(Thr) sequence in hPRL, the asparagine at position 31 is the likely point of N-linked glycosylation.     

The role of adenosine 3',5'-monophosphate in dopaminergic inhibition of prolactin release in anterior pituitary cells

The relationship between cAMP stimulation and dopaminergic inhibition of PRL release was studied in primary cultures of rat anterior pituitary cells. Bromocriptine, a dopaminergic agonist, and cholera enterotoxin, isobutylmethylxanthine, theophylline, and 8-bromo-cAMP, agents which mimic cAMP action or cause increases in cAMP, were used. Short term PRL release (that which occurs within 1 or 2 h) was stimulated 1.5- to 2-fold by all of the cAMP agents. Bromocriptine (5 nM) decreased basal release and completely abolished any short term stimulation above basal caused by cholera enterotoxin and 8-bromo-cAMP. Isobutylmethylxanthine and theophylline did cause some stimulation of PRL release above basal, but the magnitude of the increase was half that in the absence of bromocriptine. The short term release stimulated by 8-bromo-cAMP was dependent on extracellular calcium. PRL accumulation in the medium for 1-3 days was increased by all of the cAMP agents. A long term increase caused by these agents was also observed in the presence of bromocriptine. The magnitude of the increase in release above basal was the same with and without bromocriptine, but the total PRL in the medium was always less in the presence of bromocriptine, since basal release was reduced. These results show that the short term inhibition of PRL release by bromocriptine cannot be completely reversed by agents which increase cAMP.