Prostate androgen-regulated mucin-like protein 1: a novel regulator of progesterone metabolism.

The LH surge reprograms preovulatory follicular cells to become terminally differentiated luteal cells which produce high levels of progesterone and become resistant to apoptosis. PARM1 (prostate androgen regulated mucin-like protein 1) has been implicated in cell differentiation and cell survival in nonovarian cells, but little is known about PARM1 in the ovary. This study demonstrated that the LH surge induced a dramatic increase in Parm1 expression in periovulatory follicles and newly forming CL in both cycling and immature rat models. We further demonstrated that hCG increases Parm1 expression in granulosa cell cultures. The in vitro up-regulation of Parm1 expression was mediated by hCG-activated multiple signaling pathways and transcriptional activation of this gene. Parm1 knockdown increased the viability of cultured granulosa cells but resulted in a decrease in progesterone levels. The inhibitory effect of Parm1 silencing on progesterone was reversed by adenoviral mediated add-back expression of Parm1. Parm1 silencing had little effect on the expression of genes involved in progesterone biosynthesis and metabolism such as Scarb1, Ldlr, Vldlr, Scp2, Star, Cyp11a1, Hsd3b, and Srd5a1, while decreasing the expression of Akr1c3. Analyses of culture media steroid levels revealed that Parm1 knockdown had no effect on pregnenolone levels, while resulting in time-dependent decreases in progesterone and 20α-dihydroprogesterone and accelerated accumulation of 5α-pregnanediol. This study revealed that the up-regulation of Parm1 expression promotes progesterone and 20α-dihydroprogesterone accumulation in luteinizing granulosa cells by inhibiting progesterone catabolism to 5α-pregnanediol. PARM1 contributes to ovulation and/or luteal function by acting as a novel regulator of progesterone metabolism.

PARM-1 promotes cardiomyogenic differentiation through regulating the BMP/Smad signaling pathway.

PARM-1, prostatic androgen repressed message-1, is an endoplasmic reticulum (ER) molecule that is involved in ER stress-induced apoptosis in cardiomyocytes. In this study, we assessed whether PARM-1 plays a role in the differentiation of stem cells into cardiomyocytes. While PARM-1 was not expressed in undifferentiated P19CL6 embryonic carcinoma cells, PARM-1 expression was induced during cardiomyogenic differentiation. This expression followed expression of mesodermal markers, and preceded expression of cardiac transcription factors. PARM-1 overexpression did not alter the expression of undifferentiated markers and the proliferative property in undifferentiated P19CL6 cells. Expression of cardiac transcription factors during cardiomyogenesis was markedly enhanced by overexpression of PARM-1, while expression of mesodermal markers was not altered, suggesting that PARM-1 is involved in the differentiation from the mesodermal lineage to cardiomyocytes. Furthermore, overexpression of PARM-1 induced BMP2 mRNA expression in undifferentiated P19CL6 cells and enhanced both BMP2 and BMP4 mRNA expression in the early phase of cardiomyogenesis. PARM-1 overexpression also enhanced phosphorylation of Smads1/5/8. Thus, PARM-1 plays an important role in the cardiomyogenic differentiation of P19CL6 cells through regulating BMP/Smad signaling pathways, demonstrating a novel role of PARM-1 in the cardiomyogenic differentiation of stem cells.

Human PARM-1 is a novel mucin-like, androgen-regulated gene exhibiting proliferative effects in prostate cancer cells.

In this paper we characterize hPARM-1, the human ortholog of rat PARM-1 (prostatic androgen-repressed message-1) and demonstrate its role in prostate cancer. Immunofluorescence microscopy and ultrastructural analysis revealed the localization of hPARM-1 to Golgi, plasma membrane and the early endocytic pathway but not in lysosomes. Biochemical and deglycosylation studies showed hPARM-1 as a highly glycosylated, mucin-like type I transmembrane protein. Analysis of expression of hPARM-1 in various human tissues revealed its presence in most human tissues with especially high expression in heart, kidney and placenta. Androgen controls the expression of the gene as a marked 7-fold increase is seen in the androgen-dependent prostate cancer cell line, LNCaP on androgen stimulation. This is further supported by its decrease in expression in CWR22 xenograft upon castration. Moreover, ectopic expression of hPARM-1 in PC3 prostate cancer cells increased colony formation, suggesting a probable role in cell proliferation. These results suggest that hPARM-1 may have a role in normal biology of the prostate cell and in prostate cancer.

Identification of putative androgen receptor interaction protein modules: cytoskeleton and endosomes modulate androgen receptor signaling in prostate cancer cells.

We have developed a novel androgen receptor (AR) expression system in the 293 human embryonic kidney cell line that recapitulates AR biochemical activity as a steroid hormone receptor in prostate cancer cells. We used this system to identify putative AR-binding proteins in the cytosolic and nuclear compartments of mammalian cells using a large scale co-immunoprecipitation strategy coupled to quantitative mass spectrometry. For example, the heat shock 70 and 90 chaperones, which are known regulators of steroid hormone receptor, were identified as AR-binding proteins. AR purification enriched for proteins involved in RNA processing, protein transport, and cytoskeletal organization, suggesting a functional link between AR and these protein modules in mammalian cells. For example, AR purification in the nuclear compartment led to the specific enrichment of alpha-actinin-4, clathrin heavy chain, and serine-threonine protein kinase C delta. Short interfering RNA knockdown studies and co-transcriptional reporter assays revealed that clathrin heavy chain possessed co-activator activity during AR-mediated transcription, whereas alpha-actinin-4 and protein kinase C delta displayed both co-activator and co-repressor activity during AR-mediated transcription that was dependent upon their relative expression levels. Lastly immunohistochemical staining of prostate tissue showed that alpha-actinin-4 levels decreased in the nucleus of high grade cancerous prostate samples, suggesting its possible deregulation in advanced prostate cancers as previously observed in late stage metastatic breast cancers. Taken together, these findings suggest AR binds to specific protein modules in mammalian cells and that these protein modules may provide a molecular framework for interrogating AR function in normal and cancerous prostate epithelial cells.

Bull testicular haploid germ cells express a messenger encoding for a truncated form of the protein tyrosine kinase HCK.

Protein tyrosine phosphorylation is a process that has been studied worldwide during sperm capacitation and acrosomal exocytosis events. Although few capacitation-induced phosphotyrosine-containing proteins have been identified, little is known about the tyrosine kinases directly involved in this post-translational modification. Different studies from our and other groups using tyrosine kinase inhibitors suggest the involvement of members of the family of src-related tyrosine kinases in the sperm capacitation associated increase in protein tyrosine phosphorylation. Using a molecular biology approach, we report for the first time messengers encoding for members from the src-related tyrosine kinase family in bovine spermatogenic cells. Degenerated primers were designed within a highly homologous region specific to the family of src tyrosine kinases, and RNAs coding for c-src, c-yes, lyn, lck, and hck were identified in bull testis and haploid germ cells by RT-PCR. We also report the presence of a messenger in haploid bull germ cells that could encode for a truncated isoform of the hck tyrosine kinase. This messenger was detected by screening of a haploid germ cells cDNA library using the RT-PCR product homologous to hck as a probe. The presence of this transcript in haploid germ cell RNA preparations was validated by RT-PCR, 3'RACE, 5'RACE as well as Northern blot. Such a truncated protein could function as an adaptor protein or as a competitive inhibitor in spermiogenesis or mature sperm functions.

A role for RKIP in cell motility.

The target of locostatin, a small-molecule inhibitor of cell movement, has been identified as RKIP, a Raf-1 kinase modulator [1]. In addition to advancing our understanding of cell locomotion, this work represents a major landmark in the development of chemical genetics.

A chemical inhibitor reveals the role of Raf kinase inhibitor protein in cell migration.

Raf kinase inhibitor protein (RKIP) is a modulator of cell signaling that functions as an endogenous inhibitor of multiple kinases. We demonstrate here a positive role for RKIP in the regulation of cell locomotion. We discovered that RKIP is the relevant cellular target of locostatin, a cell migration inhibitor. Locostatin abrogates RKIP's ability to bind and inhibit Raf-1 kinase, and it acts by disrupting a protein-protein interaction, an uncommon mode of action for a small molecule. Small interfering RNA-mediated silencing of RKIP expression also reduces cell migration rate. Overexpression of RKIP converts epithelial cells to a highly migratory fibroblast-like phenotype, with dramatic reduction in the sensitivity of cells to locostatin. RKIP is therefore the compound's valid target and a key regulator of cell motility.

Raf kinase inhibitory protein inhibits beta-cell proliferation.

BACKGROUND: Raf-1 kinase inhibitory protein (RKIP) was recently identified as a physiologic endogenous inhibitor of the extracellular signal-regulated kinase (ERK) pathway. The expression and role of RKIP within the pancreas are unknown. METHODS: RKIP expression in normal pancreas and human insulinomas was examined by using paraffin-embedded sections. Co-localization of RKIP within islet cell subtypes was performed by using double immunofluorescence staining with antibodies directed toward RKIP and endocrine markers. To examine the role of RKIP in beta-cell proliferation, stable expression of sense (ss) and antisense (as) RKIP was established in HIT-T15 beta cells. The effect of RKIP on the ERK-signaling pathway in beta cells was determined by Western blotting with the use of phospho-specific antibodies directed against mitogen-activated protein kinase kinase (MEK) and ERK. The role of RKIP in beta-cell proliferation was assessed by using MTS assay and FACS analysis. RESULTS: RKIP was expressed only within pancreatic islet cells. Immunofluorescent double staining revealed that RKIP was expressed in most beta cells and a subset of pancreatic polypeptide-expressing cells. Based on the known function of RKIP, we hypothesized that RKIP expression would be downregulated in insulinomas: 8 of 9 human insulinomas demonstrated no RKIP staining, with decreased expression in 1 of 9 insulinomas. Studies using asRKIP and ssRKIP demonstrated that RKIP blocked activation of MEK and ERK by Raf-1 in beta cells. We also showed that RKIP inhibited beta-cell proliferation by altering cell cycle distribution, rather than by promoting apoptosis. CONCLUSIONS: RKIP is important in beta-cell proliferation, and its downregulation may play a role in islet neoplasia.

Raf-1 kinase inhibitor protein: structure, function, regulation of cell signaling, and pivotal role in apoptosis.

The acquisition of resistance to conventional therapies such as radiation and chemotherapeutic drugs remains the major obstacle in the successful treatment of cancer patients. Tumor cells acquire resistance to apoptotic stimuli and it has been demonstrated that conventional therapies exert their cytotoxic activities primarily by inducing apoptosis in the cells. Resistance to radiation and chemotherapeutic drugs has led to the development of immunotherapy and gene therapy approaches with the intent of overcoming resistance to drugs and radiation as well as enhancing the specificity to eliminate tumor cells. However, cytotoxic lymphocytes primarily kill by apoptosis and, therefore, drug-resistant tumor cells may also be cross-resistant to immunotherapy. To evade apoptosis, tumor cells have adopted various mechanisms that interfere with the apoptotic signaling pathways and promote constitutive activation of cellular proliferation and survival pathways. Thus, modifications of the antiapoptotic genes in cancer cells are warranted for the effectiveness of conventional therapies as well as novel immunotherapeutic approaches. Such modifications will avert the resistant phenotype of the tumor cells and will render them susceptible to apoptosis. Current studies, both in vitro and preclinically in vivo, have been aimed at the modification and regulation of expression of apoptosis-related gene products and their activities. A novel protein designated Raf-1 kinase inhibitor protein (RKIP) has been partially characterized. RKIP is a member of the phosphatidylethanolamine-binding protein family. RKIP has been shown to disrupt the Raf-1-MEK1/2 [mitogen-activated protein kinase-ERK (extracellular signal-regulated kinase) kinase-1/2]-ERK1/2 and NF-kappaB signaling pathways, via physical interaction with Raf-1-MEK1/2 and NF-kappaB-inducing kinase or transforming growth factor beta-activated kinase-1, respectively, thereby abrogating the survival and antiapoptotic properties of these signaling pathways. In addition, RKIP has been shown to act as a signal modifier that enhances receptor signaling by inhibiting G protein-coupled receptor kinase-2. By regulating cell signaling, growth, and survival through its expression and activity, RKIP is considered to play a pivotal role in cancer, regulating apoptosis induced by drugs or immune-mediated stimuli. Overexpression of RKIP sensitizes tumor cells to chemotherapeutic drug-induced apoptosis. Also, induction of RKIP by drugs or anti-receptor antibodies sensitizes cancer cells to drug-induced apoptosis. In this review, we discuss the discovery, structure, function, and significance of RKIP in cancer.

RKIP sensitizes prostate and breast cancer cells to drug-induced apoptosis.

Cancer cells are more susceptible to chemotherapeutic agent-induced apoptosis than their normal counterparts. Although it has been demonstrated that the increased sensitivity results from deregulation of oncoproteins during cancer development (Evan, G. I., and Vousden, K. H. (2001) Nature 411, 342-348; Green, D. R., and Evan, G. I. (2002) Cancer Cell 1, 19-30), little is known about the signaling pathways leading to changes in the apoptotic threshold in cancer cells. Here we show that low RKIP expression levels in tumorigenic human prostate and breast cancer cells are rapidly induced upon chemotherapeutic drug treatment, sensitizing the cells to apoptosis. We show that the maximal RKIP expression correlates perfectly with the onset of apoptosis. In cancer cells resistant to DNA-damaging agents, treatment with the drugs does not up-regulate RKIP expression. However, ectopic expression of RKIP resensitizes DNA-damaging agent-resistant cells to undergo apoptosis. This sensitization can be reversed by up-regulation of survival pathways. Down-regulation of endogenous RKIP by expression of antisense and small interfering RNA (siRNA) confers resistance on sensitive cancer cells to anticancer drug-induced apoptosis. Our studies suggest that RKIP may represent a novel effector of signal transduction pathways leading to apoptosis and a prognostic marker of the pathogenesis of human cancer cells and tumors after treatment with clinically relevant chemotherapeutic drugs.

Dynamics of testicular germ cell apoptosis in normal mice and transgenic mice overexpressing rat androgen-binding protein.

The number and type of testicular germ cells undergoing apoptosis in different age groups of mice (from 7 to 360 days of age) was determined and compared in age-matched wild type (WT) control and in a transgenic (TG) mice homozygous to rat androgen binding protein (ABP) using flow cytometry. Flow cytometric quantification revealed that the total number of germ cells undergoing apoptosis did not differ significantly in WT and TG mice up to Day 14. From Day 21 to Day 60, the number of germ cells undergoing apoptosis was consistently higher in TG than in WT mice. Starting from Day 90, the number of germ cells undergoing apoptosis in TG mice was lower than controls until Day 360. In 21-60 days old TG mice, spermatogonia, S-Phase cells, and primary spermatocytes are the cell types undergoing apoptosis at significantly greater numbers than those in WT mice. However, starting from day 60, the total number of spermatids undergoing apoptosis was significantly lower in TG mice than in age-matched WT controls. TdT-mediated dUTP-biotin nick end labeling (TUNEL) in testicular sections from TG mice of 21 and 30 days of age confirmed the presence of increased numbers of apoptotic germ cells compared to their age matched controls. These data indicate that the continuous presence of greater than physiological concentrations of ABP in the mouse testis has a biphasic effect on the frequency of apoptosis in germ cells. The initial pre-pubertal increase in testicular germ cell apoptosis may result from direct or indirect actions of ABP and is likely to determine the subsequent life-death balance of germ cell populations in TG mice, whereas the subsequent reduction may result from maturation depletion. A wave of apoptosis during the pre-pubertal period is required for normal spermatogenesis to develop, and our data indicate that this apoptotic wave may be regulated by ABP and/or androgens.

Age-dependent histopathological findings in the prostate of probasin/SV40 T antigen transgenic rats: lack of influence of carcinogen or testosterone treatment.

Sequential changes in the phenotype of prostatic lesions and the impact of additional carcinogen treatment or castration on development and progression of prostate cancers were examined in probasin/simian virus 40 (SV40) T antigen transgenic (TG) rats. Non-invasive prostate adenocarcinomas were evident in all lobes at 15 weeks of age. Invasive tumors were limited to the anterior lobe at this time point and were found in all lobes in an age-dependent manner thereafter. No metastasis was apparent at any age. Additional carcinogen treatment or castration did not enhance progression or generate selective growth of hormone-independent prostate cancer cells. These results suggest that our TG rats are suitable for clarification of mechanisms in early stages of prostate carcinogenesis, that is, from prostatic intraepithelial neoplasia (PIN) to non-invasive and then invasive lesions.

Effect of prostatein, the major protein produced by the rat ventral prostate, on phagocytic cell functions.

PROBLEM: To determine whether prostatein, the major protein produced and secreted into the seminal fluid by the rat ventral prostate has any effect on the phagocytic cell functions in vitro. METHOD OF STUDY: Analysis was done by determining if purified prostatein added to cells obtained from the peritoneal cavity has any effect on their phagocytic and intracellular killing capacity. Also, we analyzed the effect of prostatein on the production of oxygen and nitrogen intermediates, measuring these metabolites by Nitroblue tetrazolium assay and by the Griess reaction respectively. RESULTS: Prostatein possess the ability to inhibit in vitro the phagocytic and killing properties of peritoneal rat leukocytes in a dose-dependent manner. The addition of a polyclonal antiserum against prostatein specifically blocks this inhibitory effect. Moreover, prostatein inhibits the production of oxygen and nitrogen intermediates by these cells. CONCLUSION: Regulation of the production of reactive oxygen species in the reproductive tract is extremely necessary to avoid their deleterious effects on the sperm motility and the fertilization process. We propose that prostatein, a protein supplied by an accessory gland like prostate, can inhibit the macrophage function, showing an important antioxidant effect.

The roles of androgen receptors and androgen-binding proteins in nongenomic androgen actions.

The biological activity of testosterone and dihydrotestosterone is thought to occur predominantly through binding to the androgen receptor (AR), a member of the nuclear receptor superfamily that functions as a ligand-activated transcription factor. However, androgens have also been reported to induce the rapid activation of kinase-signaling cascades and modulate intracellular calcium levels. These effects are considered to be nongenomic because they occur in cell types that lack a functional AR, in the presence of inhibitors of transcription and translation, or are observed to occur too rapidly to involve changes in gene transcription. Such nongenomic effects of androgens may occur through AR functioning in the cytoplasm to induce the MAPK signal cascade. In addition, androgens may function through the sex hormone binding globulin receptor and possibly a distinct G protein-coupled receptor to activate second messenger signaling mechanisms. The physiological effect of nongenomic androgen action has yet to be determined. However, it may ultimately contribute to regulation of transcription factor activity, including mediation of the transcriptional activity of AR.

Female preference for male saliva: implications for sexual isolation of Mus musculus subspecies.

We studied the effects of a single genetic change on a complex mammalian behavior using animals congenic for two variants of Abpa, the gene for the alpha subunit of mouse salivary androgen-binding protein (ABP), in two-way preference tests. Females exhibited a preference for investigating salivas of males of their own genetic type of ABP but not for urines of either type of male. This preference behavior is consistent for samples of mice from geographically diverse populations of Mus musculus domesticus and M. m. musculus. These findings provide an explanation for the observation that this gene is evolving under strong selection.

Differential expression of c-erb B2/neu, epidermal growth factor receptor, cytokeratin 8, and the prostatic steroid-binding protein gene in rat ventral prostate during postnatal development.

BACKGROUND: The development and progression of prostate neoplasia may recapitulate the early developmental pattern of expression of genes in the prostate. The study of prostate development may, therefore, provide insights into the molecular mechanisms important in prostate neoplasia and reveal new markers. METHODS: We compared postnatal expression of four genes: neu and epidermal growth factor receptor genes (EGFR), androgen-upregulated in the ventral prostate of adult rats (C-3), and androgen-repressed (CK8) in Sprague-Dawley rats. In situ hybridization was performed on prostate frozen sections collected on postnatal days 1, 5, 10, 15, 20, 30, and 60 from five rats per day. Staining intensities for antisense probes specific for each gene were determined relative to day 1 intensity. RESULTS: Growth factor receptors including neu and EGFR may be coordinately regulated in the basal-cell population during prostate development. CK8 and C-3 show evidence of similar androgen regulation during development. CONCLUSIONS: CK8 and C-3 have distinct patterns of expression in the postnatal period of development and these genes may be good markers of differentiation. Both neu and EGFR may be involved in androgen-independent growth of basal cell population in prostate. Prostate 47:164-171, 2001.

Rat probasin: structure and function of an outlier lipocalin.

Probasin (PB) occurs both as a secreted and a nuclear protein that is abundantly expressed in the epithelial cells of the rat prostate. A genomic clone of 17.5 kb gene was isolated from a rat liver genomic library, determining that the probasin gene was comprised of seven exons where the splice donor/acceptor sites conformed to the GT/AG consensus sequence. The exon number and size are remarkably similar to those of aphrodisin, rat alpha(2)-urinary globulin and major urinary protein, outlier members of the lipocalin superfamily. In addition, alignment of the deduced amino acids determined that the probasin gene also contains the glycine-X-tryptophan (G-X-W) motif similar to that of human retinol serum binding protein which binds retinol, and the C-X-X-X-C motif also found in insect lipocalins that bind pheromones. The cysteine residues in exons 3 and 6 are conserved, predicting a secondary structure of eight beta-sheets and the alpha-helix commonly seen in the lipocalin superfamily. Unique PB characteristics include a large genomic fragment (17.5 kb compared to the 3-5 kb seen in other lipocalin genes) and an isoelectric point (pI) of 11.5 which is very basic compared to that of the other more acidic lipocalins. Functionally, PB gene expression is regulated by androgens and zinc in the epithelial cells of the rodent prostate. The 5'-flanking region of probasin contains two androgen receptor binding sites that allow androgen-specific gene expression as well as prostate-specific elements that target and maintain high levels of transgene expression in several PB transgenic mouse models.

Sequence and functional relationships between androgen-binding protein/sex hormone-binding globulin and its homologs protein S, Gas6, laminin, and agrin.

Androgen-binding protein/sex hormone-binding globulin (ABP/SHBG) is an extracellular binding protein that regulates the bioavailability of sex steroids. ABP/SHBG is closely related to the globular (G) domain of vitamin K-dependent protein S family of proteins and more distantly related to the G domains of several extracellular matrix proteins. ABP/SHBG appears to have evolved from the fusion of two ancestral G domains. Expanding evidence suggests that ABP/SHBG has other functions that are mediated through membrane binding, including signal transduction; however, the types of binding proteins (receptors) have not been identified. Sequence comparisons of ABP/SHBG with G domains of its homologs protein S, Gas6, laminin, and agrin have identified regions of ABP/SHBG that may bind receptors related to homolog receptors. These membrane receptors include beta-integrins, alpha-dystroglycan, and receptor tyrosine kinases. The G domains of laminin and related proteins have clearly evolved from a common ancestor to interact with specific receptors and binding proteins. It remains to be determined if ABP/SHBG followed this evolutionary pathway.

Estructura del gen del receptor de andrógeno y función de la proteína: defectos estructurales del gen que determinan resistencia a andrógenos en el hombre / Androgen receptor gene structure and protein function: structural gene defects that promote androgen resistance in man

The human androgen receptor is a member of the superfamily of steroid hormone receptors and contains three functional domains: an amino-terminal region involved in the expression of androgen regulated genes, a central cystein-rich DNA binding region and a carboxy-terminal hormone binding region. Proper functioning of this protein is a prerequisite for normal male sexual differentiation and development. Androgen action is currently studied in vitro, using fibroblasts culture from genital skin and complementary DNA of the androgen receptor gene has been recently cloned and sequenced. During recent years a substantial progress has been made elucidating the structure-function relationship of the androgen receptor and the characterization of the molecular defects associated with androgen insensitivity syndromes. There appears to be a broad correlation between the degree of receptor dysfunction caused by the mutation and the patient phenotype