Androgen receptor exon 1 mutation causes androgen insensitivity by creating phosphorylation site and inhibiting melanoma antigen-A11 activation of NH2- and carboxyl-terminal interaction-dependent transactivation.

Naturally occurring germ line mutations in the X-linked human androgen receptor (AR) gene cause incomplete masculinization of the external genitalia by disrupting AR function in males with androgen insensitivity syndrome. Almost all AR missense mutations that cause androgen insensitivity syndrome are located in the highly structured DNA and ligand binding domains. In this report we investigate the functional defect associated with an AR exon 1 missense mutation, R405S, that caused partial androgen insensitivity. The 46,XX heterozygous maternal carrier had a wild-type Arg-405 CGC allele but transmitted an AGC mutant allele coding for Ser-405. At birth, the 46,XY proband had a bifid scrotum, hypospadias, and micropenis consistent with clinical stage 3 partial androgen insensitivity. Androgen-dependent transcriptional activity of AR-R405S expressed in CV1 cells was less than wild-type AR and refractory in androgen-dependent AR NH(2)- and carboxyl interaction transcription assays that depend on the coregulator effects of melanoma antigen-A11. This mutation created a Ser-405 phosphorylation site evident by the gel migration of an AR-R405S NH(2)-terminal fragment as a double band that converted to the wild-type single band after treatment with λ-phosphatase. Detrimental effects of the R405S mutation were related to the proximity of the AR WXXLF motif (433)WHTLF(437) required for melanoma antigen-A11 and p300 to stimulate transcriptional activity associated with the AR NH(2)- and carboxyl-terminal interaction. We conclude that the coregulator effects of melanoma antigen-A11 on the AR NH(2)- and carboxyl-terminal interaction amplify the androgen-dependent transcriptional response to p300 required for normal human male sex development in utero.

Cloning and primary characterizations of rLcn9, a new member of epididymal lipocalins in rat.

Lipocalins are a structurally conserved and diversely functional family of proteins that are of potential importance in epididymis functions. The rat Lcn9 gene was cloned by in silico methods and genome walking based on homology to the rhesus monkey epididymal ESC513 and its polyclonal antisera were prepared. The rat Lcn9 gene is located on chromosome 3p13 spanning 7 exons, contains 2.3 kb and encodes 179 amino acids with a 17-amino acid signal peptide. Northern blot, western blot, and immunohistochemical staining analysis revealed that rat Lcn9 was a novel epididymis-specific gene, expressed selectively in the proximal caput region, influenced by luminal fluid testicular factors. Moreover, Lcn9 protein was modified by N-glycosylation and bound on the postacrosomal domain of caput sperm. In conclusion, the rat Lcn9 exhibited tissue-, region-, and temporal-specific expression patterns and its expression was regulated by luminal testicular factors. Its potential roles in sperm maturation are discussed.

Novel partners of SPAG11B isoform D in the human male reproductive tract.

Human sperm-associated antigen 11 (SPAG11) is closely related to beta-defensins in structure, expression, and function. Like the beta-defensins, SPAG11 proteins are predominantly expressed in the male reproductive tract, where their best-known major roles are in innate host defense and reproduction. Although several hypotheses have emerged to describe the evolution of beta-defensin and SPAG11 multifunctionality, few describe these multiple functions in terms of defensin interactions with specific proteins. To gain insight into the protein interaction potentials of SPAG11 and the signaling pathways that SPAG11 may influence, we used a yeast two-hybrid screening of a human testis-epididymis library. The results reveal human SPAG11B isoform D (SPAG11B/D) interactions with tryptase alpha/beta 1 (TPSAB1), tetraspanin 7 (TSPAN7), and attractin (ATRN). These interactions were confirmed by coimmunoprecipitation and glutathione S-transferase affinity matrix binding. SPAG11B/D and the three interacting proteins are expressed in the proximal epididymis, and all function in immunity and fertility pathways. We analyzed the functional consequences of SPAG11B/D interaction with TPSAB1 and showed that SPAG11B/D is both a substrate and a potent inhibitor of TPSAB1 activity. Furthermore, we show that (like SPAG11B/D) TSPAN7 and ATRN are associated with spermatozoa.

Hormone control and expression of androgen receptor coregulator MAGE-11 in human endometrium during the window of receptivity to embryo implantation.

The androgen receptor (AR) is a ligand-activated transcription factor of the male and female reproductive tracts whose activity is modulated by coregulator binding. We recently identified melanoma antigen gene protein-11 (MAGE-11) of the MAGEA gene family that functions as an AR coregulator by binding the AR N-terminal FXXLF motif. Here we report that MAGE-11 is expressed in a temporal fashion in endometrium of normally cycling women. Highest levels of MAGE-11 mRNA and protein occur in the mid-secretory stage, coincident with the window of uterine receptivity to embryo implantation. Studies in human endometrial cell lines together with the hormone profile of the menstrual cycle and pattern of estrogen receptor-alpha expression in cycling endometrium suggest the rise in MAGE-11 mRNA results from down-regulation by estradiol during the proliferative phase and up-regulation by cyclic AMP signaling in the early and mid-secretory stage. In agreement with its coregulatory function, MAGE-11 localizes with AR in glandular epithelial cell nuclei in the mid-secretory stage. The increase in AR protein in the mid-secretory endometrium without an increase in AR mRNA suggests MAGE-11 stabilizes AR in glandular epithelial cell nuclei. This was supported by expression studies at low androgen levels indicating AR stabilization by MAGE-11 dependent on the AR N-terminal transactivation domain. The results suggest that MAGE-11 functions as a coregulator that increases AR transcriptional activity during the establishment of uterine receptivity in the human female.

Comparative genomic analysis of a mammalian beta-defensin gene cluster.

Comparative genomic analyses have yielded valuable insights into conserved and divergent aspects of gene function, regulation, and evolution. Herein, we describe the characterization of a mouse beta-defensin gene cluster locus on chromosome 2F6. In addition, we present the evolutionary analysis of this cluster and its human, rhesus, and rat orthologs. Expression analysis in mouse revealed the occurrence of defensin cluster transcripts in multiple tissues, with the highest abundance in the urogenital tract. Molecular evolutionary analysis suggests that this cluster originated by a series of duplication events, and by positive selection occurring even after the rodent-primate split. In addition, the constraints analysis showed higher positive selection in rodents than in primates, especially distal to the six-cysteine array. Positive selection in the evolution of these defensins may relate not only to the evolving enhancement of ancestral host defense but also to functional innovations in reproduction. The multiplicity of defensins and their preferential overexpression in the urogenital tract indicate that defensins function in the protection and maintenance of fertility.

Characterization and functions of beta defensins in the epididymis.

The epididymal beta-defensins have evolved by repeated gene duplication and divergence to encode a family of proteins that provide direct protection against pathogens and also support the male reproductive tract in its primary function. Male tract defensins also facilitate recovery from pathogen attack. The beta-defensins possess ancient conserved sequence and structural features widespread in multi-cellular organisms, suggesting fundamental roles in species survival. Primate SPAG11, the functional fusion of two ancestrally independent beta-defensin genes, produces a large family of alternatively spliced transcripts that are expressed according to tissue-specific and species-specific constraints. The complexity of SPAG11 varies in different branches of mammalian evolution. Interactions of human SPAG11D with host proteins indicate involvement in multiple signaling pathways.

Genome-wide profiling of segmental-regulated transcriptomes in human epididymis using oligo microarray.

Sperm maturation during passage through the epididymis depends on regionalized gene expression which maintains the progressively changing environment within the epididymal tubule. Towards defining the genes that drive the sequential maturation of spermatozoa, we profiled regionally regulated gene expression pattern in the epididymis of a fertile young male donor using Affymetrix human genome U133 plus 2.0 microarray representing approximately the whole human genome. Over 15000 transcripts, almost one-third of the total on the array were identified in whole epididymis. Among them, 65% were detected in all three regions of the epididymis, 410 or 2.6% were present only in one region and the remaining 32.4% were distributed in two regions. Region-specific transcripts observed in caput (264), corpus (61) and cauda (81) epididymides were further classified as empirically determined reported genes or ESTs. This study revealed for the first time, the expression in human epididymis of a number of region-specific genes. The original data will be made publicly available on the Shanghai Science and Technology Database (http://www.scbit.org/human_epididymis_transcriptomes).

Identification, cloning and functional characterization of novel sperm associated antigen 11 (SPAG11) isoforms in the rat.

BACKGROUND: Sperm binding proteins and their C-terminal peptides of the Sperm Associated Antigen 11 (SPAG11) family were found to play an important role in epididymal innate immunity in addition to their role in sperm maturation. However, the expression of Spag11 transcripts in rodents is not well documented. METHODS: Computational analysis was employed to identify novel Spag11 isoforms in the rat. RT-PCR analyses were carried out on RNAs isolated from the male reproductive tract tissues of rat using gene specific primers for Spag11c and Spag11t. The identities of PCR products were confirmed by sequencing. Tissue distribution, developmental expression and androgen regulation of Spag11t and Spag11c were studied using RT-PCR. The antimicrobial activities of recombinant Spag11t and Spag11c were tested against E coli in a colony forming unit assay. RESULTS: In this study, we identified two novel Spag11 transcripts, namely, Spag11t and Spag11c derived from the long arm of chromosome 16 in the rat (Rattus norvegicus), using both in silico and molecular biology approaches. Spag11c is expressed in all three regions of the epididymis, in testis and in ovary but is absent from the seminal vesicle. Spag11t expression is confined to the caput and it is not expressed in the testis, seminal vesicle or ovary. Age dependent expression of Spag11t and Spag11c was observed in the epididymides of rats (10-60 day old). Their expression was found to be most abundant in the adult rat (60 day) suggesting roles in mature reproductive function. Further, both Spag11t and Spag11c expression was down regulated in castrated rat epididymides and the expression was maintained in the testosterone replaced castrated rats. SPAG11C is a potent antibacterial agent. SPAG11T also displayed bactericidal capacity although weaker than SPAG11C and SPAG11E. CONCLUSION: The abundant expression of Spag11t and Spag11c in the male reproductive tract suggests an important role in male reproductive tract immunity. Their expression is developmentally regulated and androgen dependent. Characterization of novel SPAG11 isoforms will contribute to our understanding of the role of epididymal proteins in sperm maturation and innate immunity.

Identification, cloning and functional characterization of novel beta-defensins in the rat (Rattus norvegicus).

BACKGROUND: Beta-defensins are small cationic peptides that exhibit broad spectrum antimicrobial properties. The majority of beta-defensins identified in humans are predominantly expressed in the male reproductive tract and have roles in non-immunological processes such as sperm maturation and capacitation. Characterization of novel defensins in the male reproductive tract can lead to increased understanding of their dual roles in immunity and sperm maturation. METHODS: In silico rat genomic analyses were used to identify novel beta-defensins related to human defensins 118-123. RNAs isolated from male reproductive tract tissues of rat were reverse transcribed and PCR amplified using gene specific primers for defensins. PCR products were sequenced to confirm their identity. RT-PCR analysis was performed to analyze the tissue distribution, developmental expression and androgen regulation of these defensins. Recombinant defensins were tested against E. coli in a colony forming unit assay to analyze their antimicrobial activities. RESULTS: Novel beta-defensins, Defb21, Defb24, Defb27, Defb30 and Defb36 were identified in the rat male reproductive tract. Defb30 and Defb36 were the most restricted in expression, whereas the others were expressed in a variety of tissues including the female reproductive tract. Early onset of defensin expression was observed in the epididymides of 10-60 day old rats. Defb21-Defb36 expression in castrated rats was down regulated and maintained at normal levels in testosterone supplemented animals. DEFB24 and DEFB30 proteins showed potent dose and time dependent antibacterial activity. CONCLUSION: Rat Defb21, Defb24, Defb27, Defb30 and Defb36 are abundantly expressed in the male reproductive tract where they most likely protect against microbial invasion. They are developmentally regulated and androgen is required for full expression in the adult epididymis.

Heregulin-induced activation of HER2 and HER3 increases androgen receptor transactivation and CWR-R1 human recurrent prostate cancer cell growth.

PURPOSE: The androgen receptor (AR) is a ligand-dependent transcription factor that mediates gene expression and growth of normal and malignant prostate cells. In prostate tumors that recur after androgen withdrawal, the AR is highly expressed and transcriptionally active in the absence of testicular androgens. In these "androgen-independent" tumors, alternative means of AR activation have been invoked, including regulation by growth factors and their receptors in prostate cancer recurrence. EXPERIMENTAL DESIGN AND RESULTS: In this report, we show that HER receptor tyrosine kinases 1 through 4 are expressed in the CWR-R1 recurrent prostate cancer cell line; their stimulation by epidermal growth factor (EGF) and heregulin activates downstream signaling, including mitogen-activated protein kinase and phosphatidylinositol-3 kinase and Akt pathways. We show that heregulin activates HER2 and HER3 and increases androgen-dependent AR transactivation of reporter genes in CWR-R1 cells. Tyrosine phosphorylation of HER2 and HER3, AR transactivation, and cell proliferation induced by heregulin were more potently inhibited by the EGFR/HER2 dual tyrosine kinase inhibitor GW572016 (lapatinib) than the EGFR-specific inhibitor ZD1839 (gefitinib). Basal proliferation in the absence of growth factors was also inhibited by GW572016 to a greater extent than ZD1839, suggesting that low level HER2/HER3 activation perhaps by an autocrine pathway contributes to the proliferation signal. CONCLUSIONS: These data indicate that heregulin signaling through HER2 and HER3 increases AR transactivation and alters growth in a recurrent prostate cancer cell line. Therefore, inhibition of low-level HER2 signaling may be a potential novel therapeutic strategy in prostate cancer.

The androgen axis in recurrent prostate cancer.

PURPOSE: Prostate cancer that recurs during androgen deprivation therapy is referred to as androgen-independent. High levels of expression of androgen receptor and androgen receptor-regulated genes in recurrent prostate cancer suggest a role for androgen receptor and its ligands in prostate cancer recurrence. EXPERIMENTAL DESIGN: Recurrent prostate cancer specimens from 22 men whose prostate cancer recurred locally during androgen deprivation therapy and benign prostate specimens from 48 men who had received no prior treatment were studied. Androgen receptor expression was measured using monoclonal antibody and automated digital video image analysis. Tissue androgens were measured using radioimmunoassay. RESULTS: Epithelial nuclei androgen receptor immunostaining in recurrent prostate cancer (mean optical density, 0.284 +/- SD 0.115 and percentage positive nuclei, 83.7 +/- 11.6) was similar to benign prostate (mean optical density, 0.315 +/- 0.044 and percentage positive nuclei, 77.3 +/- 13.0). Tissue levels of testosterone were similar in recurrent prostate cancer (2.78 +/- 2.34 pmol/g tissue) and benign prostate (3.26 +/- 2.66 pmol/g tissue). Tissue levels of dihydrotestosterone, dehydroepiandrosterone, and androstenedione were lower (Wilcoxon, P = 0.0000068, 0.00093, and 0.0089, respectively) in recurrent prostate cancer than in benign prostate, and mean dihydrotestosterone levels, although reduced, remained 1.45 nM. Androgen receptor activation in recurrent prostate cancer was suggested by the androgen-regulated gene product, prostate-specific antigen, at 8.80 +/- 10.80 nmol/g tissue. CONCLUSIONS: Testosterone and dihydrotestosterone occur in recurrent prostate cancer tissue at levels sufficient to activate androgen receptor. Novel therapies for recurrent prostate cancer should target androgen receptor directly and prevent the formation of androgens within prostate cancer tissue.

Domain interactions between coregulator ARA(70) and the androgen receptor (AR).

The coregulator function of AR-associated protein 70 (ARA(70)) was investigated to further characterize its interaction with the AR. Using a yeast two-hybrid assay, androgen-dependent binding of ARA(70) deletion mutants to the AR ligand-binding domain (LBD) was strongest with ARA(70) amino acids 321-441 of the 614 amino acid ARA(70) protein. Mutations adjacent to or within an FxxLF motif in this 120-amino acid region abolished androgen-dependent binding to the AR-LBD both in yeast and in glutathione-S-transferase affinity matrix assays. Yeast one-hybrid assays revealed an intrinsic ARA(70) transcriptional activation domain within amino acids 296-441. In yeast assays the ARA(70) domains for transcriptional activation and for binding to the AR-LBD were inhibited by the C-terminal region of ARA(70). Full-length ARA(70) increased androgen-dependent AR transactivation in transient cotransfection assays using a mouse mammary tumor virus-luciferase reporter in CV1 cells. ARA(70) also increased constitutive transcriptional activity of an AR NH(2)-terminal-DNA binding domain fragment and bound this region in glutathione-S-transferase affinity matrix assays. Binding was independent of the ARA(70) FxxLF motif. The results identify an ARA(70) motif required for androgen-dependent interaction with the AR-LBD and demonstrate that ARA(70) can interact with the NH(2)-terminal and carboxyl-terminal regions of AR.

The androgen-regulated epididymal sperm-binding protein, human beta-defensin 118 (DEFB118) (formerly ESC42), is an antimicrobial beta-defensin.

Spermatozoa bind a variety of proteins as they pass through the proximal regions of the epididymis, where they acquire forward motility and fertilizing ability. Recent evidence indicates that certain epididymis-specific secretory proteins that bind sperm have antibacterial activity and may function as part of the innate immune system. We reported earlier that ESC42, now designated human beta-defensin 118 (DEFB118), is a sperm-binding protein. In this study, we demonstrate that DEFB118 has potent antibacterial activity that is dose, time, and structure dependent. Incubation of Escherichia coli for 60 min with 10 microg/ml DEFB118 reduced bacterial survival to 20% of the control, and 25 microg/ml reduced survival to 5% of the control. DEFB118 concentrations of 50 and 100 microg/ml further reduced survival to less than 2 and 1%, respectively. A biphasic effect of salt concentration on the antibacterial activity of DEFB118 was observed. Reduction of disulfide bonds and alkylation of cysteines resulted in the complete loss of antibacterial activity. DEFB118 caused rapid permeabilization of both outer and inner membranes of E. coli and striking morphological alterations in the bacterial surfaces visible by scanning electron microscopy consistent with a membrane-disruptive mechanism of bacterial killing. In contrast, eukaryotic cell membranes were not permeabilized by DEFB118, as indicated by the rat erythrocyte hemolytic assay. Studies on DEFB118 inhibition of macromolecular synthesis and membrane permeability in E. coli were consistent with a primary effect at the cell membrane level. DEFB118 may contribute to epididymal innate immunity and protect the sperm against attack by microorganisms in the male and female reproductive tracts.

Cystatin 11: a new member of the cystatin type 2 family.

Cystatin (CST)11, a novel member of the CST type 2 family of cysteine protease inhibitors, was identified in Macaca mulatta epididymis by subtractive hybridization cloning. The human CST11 gene on chromosome 20p11.2 is located near three other CST genes expressed predominantly in the male reproductive tract. The CST11 gene spans three exons, a structure similar to that of other CST family 2 genes. An exon 2-deleted alternative transcript (CST11Delta2) was also identified. CST11 mRNA is expressed only in the epididymis as judged by Northern blot hybridization and is androgen regulated. The protein is most abundant in the initial segment, but is detected throughout the epididymis and on ejaculated human sperm. The calculated tertiary structure of CST11 reveals that the three regions corresponding to the protease inhibitory wedge of CST3 are similarly juxtaposed in CST11, consistent with protease inhibitor function. Intact and exon 2-deleted CST11 recombinant proteins were tested for antibacterial activity. After a 2-h incubation of Escherichia coli with 50 microg/ml recombinant CST11 or CST11Delta2, bacterial colony-forming units were reduced to 30% of control, indicating that both forms have antimicrobial activity.

Characterization of mouse Eppin and a gene cluster of similar protease inhibitors on mouse chromosome 2.

We have recently described a novel gene on human chromosome 20q 12-13.2 called Eppin (Epididymal protease inhibitor) that expresses three mRNAs encoding two isoforms of a cysteine-rich protein containing both Kunitz-type and WAP-type (four disulfide core) consensus sequences (Richardson et al., 2001). To further our studies on Eppin, we have cloned, sequenced and characterized mouse Eppin and report that it lies within a 200 Kb cluster of putative Eppin-like genes on mouse chromosome 2. Analysis of the homologies between the genes in the human and mouse Eppin clusters indicates that the first part of the cluster immediately surrounding Eppin represents a conserved linkage because the order of homologous genes is conserved. Sequencing of reverse transcription polymerase chain reaction (RT-PCR) products confirmed the expression of five of these novel Eppin-like genes in the mouse, which include the mouse homologue of HE-4. These genes are characterized by having either one or both of the Kunitz-type and WAP-type consensus sequences. Additional RT-PCR experiments revealed that expression of some of the Eppin-like genes is restricted to epididymis and testis while others are expressed in several somatic tissues. Northern blot analysis of 22 different mouse tissues identified Eppin transcripts only in the epididymis and testis. Immunostaining of Eppin with anti-recombinant mouse Eppin demonstrated Eppin predominantly on the postacrosomal region of mouse spermatozoa, in Sertoli cells, Leydig cells, and round spermatids in the testis, and in the principal cells of the cauda epididymidis epithelium. Eppin is first expressed by Sertoli cells of 12-day-old mice and subsequently in round spermatids, which is consistent with androgen regulation. Our results demonstrate that mouse chromosome 2 contains a conserved linkage of Eppin-like protease inhibitor genes that are expressed in the epididymis.

Partial androgen insensitivity with phenotypic variation caused by androgen receptor mutations that disrupt activation function 2 and the NH(2)- and carboxyl-terminal interaction.

Partial androgen insensitivity with sex phenotype variation in two unrelated families was associated with missense mutations in the androgen receptor (AR) gene that disrupted the AR NH(2)-terminal/carboxy terminal interaction. Each mutation caused a single amino acid change within the region of the ligand-binding domain that forms activation function 2 (AF2). In one family, the mutation I737T was in alpha helix 4 and in the other F725L was between helices 3 and 4. Neither mutation altered androgen binding as determined by assays of mutant AR in the patient's cultured genital skin fibroblasts or of recombinant mutant receptors transfected into COS cells. In transient cotransfection assays in CV1 cells, transactivation with the AR mutants at low concentrations of DHT was reduced several fold compared with wild-type AR but increased at higher concentrations. Defects in NH(2)-terminal/carboxy terminal interactions were identified in mammalian two hybrid assays. In similar assays, there was reduced binding of the p160 coactivators TIF2/SRC2 and SRC1 to the mutant AR ligand binding domains (LBD). In the family with AR I737T, sex phenotype varied from severely defective masculinization in the proband to a maternal great uncle whose only manifestation of AIS was severe gynecomastia. He was fertile and passed the mutation to two daughters. The proband of the F725L family was also incompletely masculinized but was raised as a male while his half-sibling by a different father was affected more severely and reared as a female. These studies indicate that the function of an AR AF2 mutant in male development can vary greatly depending on the genetic background.

Antimicrobial actions of the human epididymis 2 (HE2) protein isoforms, HE2alpha, HE2beta1 and HE2beta2.

BACKGROUND: The HE2 gene encodes a group of isoforms with similarities to the antimicrobial beta-defensins. We demonstrated earlier that the antimicrobial activity of HE2 proteins and peptides is salt resistant and structure dependent and involves permeabilization of bacterial membranes. In this study, we further characterize the antimicrobial properties of HE2 peptides in terms of the structural changes induced in E. coli and the inhibition of macromolecular synthesis. METHODS: E. coli treated with 50 microg/ml of HE2alpha, HE2beta1 or HE2beta2 peptides for 30 and 60 min were visualized using transmission and scanning electron microscopy to investigate the impact of these peptides on bacterial internal and external structure. The effects of HE2alpha, HE2beta1 and HE2beta2 on E. coli macromolecular synthesis was assayed by incubating the bacteria with 2, 10 and 25 microg/ml of the individual peptides for 0-60 min and measuring the incorporation of the radioactive precursors [methyl-3H]thymidine, [5-3H]uridine and L-[4,5-3H(N)]leucine into DNA, RNA and protein. Statistical analyses using Student's t-test were performed using Sigma Plot software. Values shown are Mean +/- S.D. RESULTS: E. coli treated with HE2alpha, HE2beta1 and HE2beta2 peptides as visualized by transmission electron microscopy showed extensive damage characterized by membrane blebbing, thickening of the membrane, highly granulated cytoplasm and appearance of vacuoles in contrast to the smooth and continuous membrane structure of the untreated bacteria. Similarly, bacteria observed by scanning electron microscopy after treating with HE2alpha, HE2beta1 or HE2beta2 peptides exhibited membrane blebbing and wrinkling, leakage of cellular contents, especially at the dividing septa, and external accumulation of fibrous materials. In addition, HE2alpha, HE2beta1 and HE2beta2 peptides inhibited E. coli DNA, RNA and protein synthesis. CONCLUSIONS: The morphological changes observed in E. coli treated with epididymal HE2 peptides provide further evidence for their membrane dependent mechanism of antibacterial action. HE2 C-terminal peptides can inhibit E. coli macromolecular synthesis, suggesting an additional mechanism of bacterial killing supplementary to membrane permeabilization.

LCN6, a novel human epididymal lipocalin.

BACKGROUND: The lipocalin (LCN) family of structurally conserved hydrophobic ligand binding proteins is represented in all major taxonomic groups from prokaryotes to primates. The importance of lipocalins in reproduction and the similarity to known epididymal lipocalins prompted us to characterize the novel human epididymal LCN6. METHODS AND RESULTS: LCN6 cDNA was identified by database analysis in a comprehensive human library sequencing program. Macaca mulatta (rhesus monkey) cDNA was obtained from an epididymis cDNA library and is 93% homologous to the human. The gene is located on chromosome 9q34 adjacent LCN8 and LCN5. LCN6 amino acid sequence is most closely related to LCN5, but the LCN6 beta-barrel structure is best modeled on mouse major urinary protein 1, a pheromone binding protein. Northern blot analysis of RNAs isolated from 25 human tissues revealed predominant expression of a 1.0 kb mRNA in the epididymis. No other transcript was detected except for weak expression of a larger hybridizing mRNA in urinary bladder. Northern hybridization analysis of LCN6 mRNA expression in sham-operated, castrated and testosterone replaced rhesus monkeys suggests mRNA levels are little affected 6 days after castration. Immunohistochemical staining revealed that LCN6 protein is abundant in the caput epithelium and lumen. Immunofluorescent staining of human spermatozoa shows LCN6 located on the head and tail of spermatozoa with the highest concentration of LCN6 on the post-acrosomal region of the head, where it appeared aggregated into large patches. CONCLUSIONS: LCN6 is a novel lipocalin closely related to Lcn5 and Lcn8 and these three genes are likely products of gene duplication events that predate rodent-primate divergence. Predominant expression in the epididymis and location on sperm surface are consistent with a role for LCN6 in male fertility.

Mechanism of androgen receptor corepression by CKßBP2/CRIF1, a multifunctional transcription factor coregulator expressed in prostate cancer.

The transcription factor coregulator Casein kinase IIß-binding protein 2 or CR6-interacting factor 1 (CKßBP2/CRIF1) binds the androgen receptor (AR) in prostate cancer cells and in response to dihydrotestosterone localizes with AR on the prostate-specific antigen gene enhancer, but does not bind DNA suggesting CKßBP2/CRIF1 localization in chromatin is determined by AR. In this study we show also that CKßBP2/CRIF1 inhibits wild-type AR and AR N-terminal transcriptional activity, binds to the AR C-terminal region, inhibits interaction of the AR N- and C-terminal domains (N/C interaction) and competes with p160 coactivator binding to the AR C-terminal domain, suggesting CKßBP2/CRIF1 interferes with AR activation functions 1 and 2. CKßBP2/CRIF1 is expressed mainly in stromal cells of benign prostatic hyperplasia and in stroma and epithelium of prostate cancer. CKßBP2/CRIF1 protein is increased in epithelium of androgen-dependent prostate cancer compared to benign prostatic hyperplasia and decreased slightly in castration recurrent epithelium compared to androgen-dependent prostate cancer. The multifunctional CKßBP2/CRIF1 is a STAT3 interacting protein and reported to be a coactivator of STAT3. CKßBP2/CRIF1 is expressed with STAT3 in prostate cancer where STAT3 may help to offset the AR repressor effect of CKßBP2/CRIF1 and allow AR regulation of prostate cancer growth.

Research resource: Genome-wide mapping of in vivo androgen receptor binding sites in mouse epididymis.

Epididymal function depends on androgen signaling through the androgen receptor (AR), although most of the direct AR target genes in epididymis remain unknown. Here we globally mapped the AR binding regions in mouse caput epididymis in which AR is highly expressed. Chromatin immunoprecipitation sequencing indicated that AR bound selectively to 19,377 DNA regions, the majority of which were intergenic and intronic. Motif analysis showed that 94% of the AR binding regions harbored consensus androgen response elements enriched with multiple binding motifs that included nuclear factor 1 and activator protein 2 sites consistent with combinatorial regulation. Unexpectedly, AR binding regions showed limited conservation across species, regardless of whether the metric for conservation was based on local sequence similarity or the presence of consensus androgen response elements. Further analysis suggested the AR target genes are involved in diverse biological themes that include lipid metabolism and sperm maturation. Potential novel mechanisms of AR regulation were revealed at individual genes such as cysteine-rich secretory protein 1. The composite studies provide new insights into AR regulation under physiological conditions and a global resource of AR binding sites in a normal androgen-responsive tissue.