Publications by authors named "Gail Grossman"

24 Publications

  • Page 1 of 1

Up-Regulation of Follistatin-Like 1 By the Androgen Receptor and Melanoma Antigen-A11 in Prostate Cancer.

Prostate 2017 04 14;77(5):505-516. Epub 2016 Dec 14.

Laboratories for Reproductive Biology, Department of Pediatrics, University of North Carolina, Chapel Hill, North Carolina.

Background: High affinity androgen binding to the androgen receptor (AR) activates genes required for male sex differentiation and promotes the development and progression of prostate cancer. Human AR transcriptional activity involves interactions with coregulatory proteins that include primate-specific melanoma antigen-A11 (MAGE-A11), a coactivator that increases AR transcriptional activity during prostate cancer progression to castration-resistant/recurrent prostate cancer (CRPC).

Methods: Microarray analysis and quantitative RT-PCR were performed to identify androgen-regulated MAGE-A11-dependent genes in LAPC-4 prostate cancer cells after lentivirus shRNA knockdown of MAGE-A11. Chromatin immunoprecipitation was used to assess androgen-dependent AR recruitment, and immunocytochemistry to localize an androgen-dependent protein in prostate cancer cells and tissue and in the CWR22 human prostate cancer xenograft.

Results: Microarray analysis of androgen-treated LAPC-4 prostate cancer cells indicated follistatin-like 1 (FSTL1) is up-regulated by MAGE-A11. Androgen-dependent up-regulation of FSTL1 was inhibited in LAPC-4 cells by lentivirus shRNA knockdown of AR or MAGE-A11. Chromatin immunoprecipitation demonstrated AR recruitment to intron 10 of the FSTL1 gene that contains a classical consensus androgen response element. Increased levels of FSTL1 protein in LAPC-4 cells correlated with higher levels of MAGE-A11 relative to other prostate cancer cells. FSTL1 mRNA levels increased in CRPC and castration-recurrent CWR22 xenografts in association with predominantly nuclear FSTL1. Increased nuclear localization of FSTL1 in prostate cancer was suggested by predominantly cytoplasmic FSTL1 in benign prostate epithelial cells and predominantly nuclear FSTL1 in epithelial cells in CRPC tissue and the castration-recurrent CWR22 xenograft. AR expression studies showed nuclear colocalization of AR and endogenous FSTL1 in response to androgen.

Conclusion: AR and MAGE-A11 cooperate in the up-regulation of FSTL1 to promote growth and progression of CRPC. Prostate 77:505-516, 2017. © 2016 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/pros.23288DOI Listing
April 2017

Melanoma antigen-A11 regulates substrate-specificity of Skp2-mediated protein degradation.

Mol Cell Endocrinol 2017 01 6;439:1-9. Epub 2016 Oct 6.

Laboratories for Reproductive Biology, Department of Pediatrics, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599, USA; Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27599, USA. Electronic address:

Melanoma antigen-A11 (MAGE-A11) is a proto-oncogene involved in androgen receptor signaling and androgen-dependent cell growth. In this report we provide evidence that MAGE-A11 interacts with Skp2 (S phase kinase-associated protein), the substrate recognition protein of the Skp1-Cullin1-F-box E3 ubiquitin ligase, and with Skp2 binding protein, cyclin A. A similar cyclin A binding motif in MAGE-A11 and Skp2 was consistent with a competitive relationship between MAGE-A11 and Skp2 in binding cyclin A. Skp2 inhibited MAGE-A11 interaction with cyclin A. Differential effects of MAGE-A11 on Skp2-mediated protein degradation were also revealed. MAGE-A11 increased Skp2-mediated degradation of cyclin A and retinoblastoma-related protein p130. In contrast, MAGE-A11 decreased Skp2-mediated degradation of E2F1 and Skp2 self-ubiquitination. Stabilization of E2F1 by MAGE-A11 was associated with sequestration and inactivation of Skp2 through the formation of an E2F1-MAGE-A11-Skp2 complex. We conclude that direct interactions of MAGE-A11 with Skp2 and cyclin A regulate the substrate-specificity of Skp2-mediated protein degradation.
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http://dx.doi.org/10.1016/j.mce.2016.10.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5123923PMC
January 2017

Post-translational Down-regulation of Melanoma Antigen-A11 (MAGE-A11) by Human p14-ARF Tumor Suppressor.

J Biol Chem 2015 Oct 1;290(41):25174-87. Epub 2015 Sep 1.

From the Laboratories for Reproductive Biology, Department of Pediatrics, Lineberger Comprehensive Cancer Center, Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599

X-linked primate-specific melanoma antigen-A11 (MAGE-A11) is a human androgen receptor (AR) coactivator and proto-oncogene expressed at low levels in normal human reproductive tract tissues and at higher levels in castration-resistant prostate cancer where it is required for androgen-dependent cell growth. In this report, we show that MAGE-A11 is targeted for degradation by human p14-ARF, a tumor suppressor expressed from an alternative reading frame of the p16 cyclin-dependent kinase inhibitor INK4a/ARF gene. MAGE-A11 degradation by the proteasome was mediated by an interaction with p14-ARF and was independent of lysine ubiquitination. A dose-dependent inverse relationship between MAGE-A11 and p14-ARF correlated with p14-ARF inhibition of the MAGE-A11-induced increase in androgen-dependent AR transcriptional activity and constitutive activity of a splice variant-like AR. Reciprocal stabilization between MAGE-A11 and AR did not protect against degradation promoted by p14-ARF. p14-ARF prevented MAGE-A11 interaction with the E2F1 oncoprotein and inhibited the MAGE-A11-induced increase in E2F1 transcriptional activity. Post-translational down-regulation of MAGE-A11 promoted by p14-ARF was independent of HDM2, the human homologue of mouse double minute 2, an E3 ubiquitin ligase inhibited by p14-ARF. However, MAGE-A11 had a stabilizing effect on HDM2 in the absence or presence of p14-ARF and cooperated with HDM2 to increase E2F1 transcriptional activity in the absence of p14-ARF. We conclude that degradation of MAGE-A11 promoted by the human p14-ARF tumor suppressor contributes to low levels of MAGE-A11 in nontransformed cells and that higher levels of MAGE-A11 associated with low p14-ARF increase AR and E2F1 transcriptional activity and promote the development of castration-resistant prostate cancer.
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http://dx.doi.org/10.1074/jbc.M115.663641DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4599020PMC
October 2015

Mechanism of androgen receptor corepression by CKβBP2/CRIF1, a multifunctional transcription factor coregulator expressed in prostate cancer.

Mol Cell Endocrinol 2014 Jan 5;382(1):302-313. Epub 2013 Oct 5.

Laboratories for Reproductive Biology, Department of Pediatrics, University of North Carolina, School of Medicine, Chapel Hill, NC, United States; Lineberger Comprehensive Cancer Center, University of North Carolina, School of Medicine, Chapel Hill, NC, United States. Electronic address:

The transcription factor coregulator Casein kinase IIβ-binding protein 2 or CR6-interacting factor 1 (CKβBP2/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 CKβBP2/CRIF1 localization in chromatin is determined by AR. In this study we show also that CKβBP2/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 CKβBP2/CRIF1 interferes with AR activation functions 1 and 2. CKβBP2/CRIF1 is expressed mainly in stromal cells of benign prostatic hyperplasia and in stroma and epithelium of prostate cancer. CKβBP2/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 CKβBP2/CRIF1 is a STAT3 interacting protein and reported to be a coactivator of STAT3. CKβBP2/CRIF1 is expressed with STAT3 in prostate cancer where STAT3 may help to offset the AR repressor effect of CKβBP2/CRIF1 and allow AR regulation of prostate cancer growth.
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http://dx.doi.org/10.1016/j.mce.2013.09.036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3880566PMC
January 2014

Proto-oncogene activity of melanoma antigen-A11 (MAGE-A11) regulates retinoblastoma-related p107 and E2F1 proteins.

J Biol Chem 2013 Aug 12;288(34):24809-24. Epub 2013 Jul 12.

Laboratories for Reproductive Biology, Department of Pediatrics, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599, USA.

Melanoma antigen-A11 (MAGE-A11) is a low-abundance, primate-specific steroid receptor coregulator in normal tissues of the human reproductive tract that is expressed at higher levels in prostate cancer. Increased expression of MAGE-A11 enhances androgen receptor transcriptional activity and promotes prostate cancer cell growth. Further investigation into the mechanisms of MAGE-A11 function in prostate cancer demonstrated interactions with the retinoblastoma-related protein p107 and Rb tumor suppressor but no interaction with p130 of the Rb family. MAGE-A11 interaction with p107 was associated with transcriptional repression in cells with low MAGE-A11 and transcriptional activation in cells with higher MAGE-A11. Selective interaction of MAGE-A11 with retinoblastoma family members suggested the regulation of E2F transcription factors. MAGE-A11 stabilized p107 by inhibition of ubiquitination and linked p107 to hypophosphorylated E2F1 in association with the stabilization and activation of E2F1. The androgen receptor and MAGE-A11 modulated endogenous expression of the E2F1-regulated cyclin-dependent kinase inhibitor p27(Kip1). The ability of MAGE-A11 to increase E2F1 transcriptional activity was similar to the activity of adenovirus early oncoprotein E1A and depended on MAGE-A11 interactions with p107 and p300. The immunoreactivity of p107 and MAGE-A11 was greater in advanced prostate cancer than in benign prostate, and knockdown with small inhibitory RNA showed that p107 is a transcriptional activator in prostate cancer cells. These results suggest that MAGE-A11 is a proto-oncogene whose increased expression in prostate cancer reverses retinoblastoma-related protein p107 from a transcriptional repressor to a transcriptional activator of the androgen receptor and E2F1.
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http://dx.doi.org/10.1074/jbc.M113.468579DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3750176PMC
August 2013

Excess androgen during perinatal life alters steroid receptor expression, apoptosis, and cell proliferation in the uteri of the offspring.

Reprod Toxicol 2013 Sep 10;40:1-7. Epub 2013 May 10.

Graduate Program in Cell and Structural Biology, Institute of Biology, State University of Campinas-UNICAMP, Campinas, SP, Brazil.

Exposure to environmental chemicals may contribute to reproductive disorders, especially when it occurs in critical periods of development. The female reproductive system can be a target for androgens derived from environmental contaminants or pathological conditions. The purpose of this study was to assess the long-term effects of androgens on uterine tissue after maternal exposure limited to the time of gestation and lactation. Pregnant Wistar rats were treated with testosterone propionate (TP) at 0.05 mg/kg, 0.1 mg/kg, 0.2 mg/kg or corn oil (vehicle), s.c., from gestational day 12 until the end of lactation. The results show changes in the pattern of expression of receptors for estrogen, progesterone, and androgen at all doses tested, and decreases in both apoptosis and cell proliferation indices at 0.1 and 0.2 mg/kg. We conclude that early TP exposure, under these experimental conditions, causes changes in cellular and molecular parameters that are essential for normal uterine function in the adult.
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http://dx.doi.org/10.1016/j.reprotox.2013.05.001DOI Listing
September 2013

Melanoma antigen-A11 (MAGE-A11) enhances transcriptional activity by linking androgen receptor dimers.

J Biol Chem 2013 Jan 21;288(3):1939-52. Epub 2012 Nov 21.

Laboratories for Reproductive Biology, Department of Pediatrics, University of North Carolina, Chapel Hill, NC 27599-7500, USA.

Prostate cancer growth and progression depend on androgen receptor (AR) signaling through transcriptional mechanisms that require interactions with coregulatory proteins, one of which is the primate-specific steroid receptor coregulator melanoma antigen-A11 (MAGE-A11). In this report, we provide evidence how increased expression of MAGE-A11 during prostate cancer progression enhances AR signaling and prostate cancer growth. MAGE-A11 protein levels were highest in castration-recurrent prostate cancer. The cyclic AMP-induced increase in androgen-dependent and androgen-independent AR transcriptional activity correlated with an increase in MAGE-A11 and was inhibited by silencing MAGE-A11 expression. MAGE-A11 mediated synergistic AR transcriptional activity in LAPC-4 prostate cancer cells. The ability of MAGE-A11 to rescue transcriptional activity of complementary inactive AR mutants and promote coimmunoprecipitation between unlike forms of AR suggests that MAGE-A11 links transcriptionally active AR dimers. A model for the AR·MAGE-A11 multidimeric complex is proposed in which one AR FXXLF motif of the AR dimer engages in the androgen-dependent AR NH(2)- and carboxyl-terminal interaction, whereas the second FXXLF motif region of the AR dimer interacts with dimeric MAGE-A11. The AR·MAGE-A11 multidimeric complex accounts for the dual functions of the AR FXXLF motif in the androgen-dependent AR NH(2)- and carboxyl-terminal interaction and binding MAGE-A11 and for synergy between reported AR splice variants and full-length AR. We conclude that the increased expression of MAGE-A11 in castration-recurrent prostate cancer, which is enhanced by cyclic AMP signaling, increases AR-dependent growth of prostate cancer by MAGE-A11 forming a molecular bridge between transcriptionally active AR dimers.
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http://dx.doi.org/10.1074/jbc.M112.428409DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3548502PMC
January 2013

Primate-specific melanoma antigen-A11 regulates isoform-specific human progesterone receptor-B transactivation.

J Biol Chem 2012 Oct 13;287(41):34809-24. Epub 2012 Aug 13.

Laboratories for Reproductive Biology, University of North Carolina, Chapel Hill, North Carolina 27599, USA.

Progesterone acting through the progesterone receptor (PR) and its coregulators prepares the human endometrium for receptivity to embryo implantation and maintains pregnancy. The menstrual cycle-dependent expression of melanoma antigen-A11 (MAGE-11) in the mid-secretory human endometrium suggested a novel function in human PR signaling. Here we show that MAGE-11 is an isoform-specific coregulator responsible for the greater transcriptional activity of human PR-B relative to PR-A. PR was recruited to progesterone response regions of progesterone-regulated FK506-binding protein 5 (FKBP5) immunophilin and small Ras family G protein cell growth inhibitor RASD1 genes. Expression of MAGE-11 lentivirus shRNA in human endometrial Ishikawa cells expressing PR-B showed that MAGE-11 is required for isoform-specific PR-B up-regulation of FKBP5. In contrast, MAGE-11 was not required for progesterone up-regulation of RASD1 in endometrial cells expressing the PR-A/B heterodimer. Target gene specificity of PR-B depended on the synergistic actions of MAGE-11 and p300 mediated by the unique PR-B NH(2)-terminal (110)LLXXVLXXLL(119) motif that interacts with the MAGE-11 F-box region in a phosphorylation- and ubiquitinylation-dependent manner. A progesterone-dependent mechanism is proposed in which MAGE-11 and p300 increase PR-B up-regulation of the FKBP5 gene. MAGE-11 down-regulates PR-B, similar to the effects of progesterone, and interacts with FKBP5 to stabilize a complex with PR-B. We conclude that the coregulator function of MAGE-11 extends to isoform-specific regulation of PR-B during the cyclic development of the human endometrium.
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http://dx.doi.org/10.1074/jbc.M112.372797DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3464583PMC
October 2012

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

Biol Reprod 2009 Oct 17;81(4):647-56. Epub 2009 Jun 17.

Departments of Pediatrics and Cell and Developmental Biology, Laboratories for Reproductive Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

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.
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http://dx.doi.org/10.1095/biolreprod.109.077545DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2754882PMC
October 2009

Immunolocalization of alpha(1A)-adrenoceptors in rat and human epididymis.

Cell Tissue Res 2008 Jun 20;332(3):509-22. Epub 2008 Mar 20.

Section of Experimental Endocrinology, Department of Pharmacology, Universidade Federal de São Paulo Escola Paulista de Medicina, INFAR, Vila Clementino, São Paulo, Brazil.

Immunohistochemistry was conducted to analyze the cellular localization of alpha(1A)-adrenoceptors along rat and human epididymis. ADR-A, a polyclonal antibody that recognizes the specific C-terminal region of alpha(1A)-adrenoceptors, immunostained this adrenoceptor subtype in smooth muscle cells surrounding the epididymal tubules and interstitial blood vessels and in subpopulations of epithelial cells from adult rat and human caput and cauda epididymidis. The same cell types from rat epididymidis were immunostained by ADR-1, a polyclonal antibody that recognizes a common region of the three alpha(1)-adrenoceptor subtypes, alpha(1A), alpha(1B), and alpha(1D). Immunostaining with both antibodies was also conducted in adult rat and human vas deferens and seminal vesicle used as positive controls because of the abundance of alpha(1A)-adrenoceptors in these tissues. ADR-A- and ADR-1-positive immunostaining was differentially distributed depending on the antibody, method of tissue fixation (Bouin-fixed and fresh frozen tissues), species (rat and human), tissue (caput and cauda epididymidis), and age (immature and adult rats) analyzed. This is the first report immunolocalizing alpha(1A)-adrenoceptor along rat and human epididymis. The presence of this adrenoceptor subtype in epididymal smooth muscle and epithelial cells indicates their contribution to smooth muscle contractile responses and a possible role in the absorptive and/or secretory activities of the epithelium lining the epididymal duct. Taken together, our results should contribute to a better understanding of the physiological role of alpha(1)-adrenoceptors in the epididymidis and the importance of the sympathetic nervous system for male (in)fertility.
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http://dx.doi.org/10.1007/s00441-008-0576-xDOI Listing
June 2008

Cloning, expression and immunolocalization of alpha1-adrenoceptor in different tissues from rhesus monkey and human male reproductive tract.

Mol Hum Reprod 2008 Feb 19;14(2):85-96. Epub 2008 Jan 19.

Section of Experimental Endocrinology, Department of Pharmacology, Universidade Federal de São Paulo, Escola Paulista de Medicina, Rua 3 de Maio 100, INFAR, Vila Clementino, São Paulo 04044-020, Brazil.

This study reports the genomic organization of the rhesus alpha(1A)-adrenoceptor gene (ADRA1A). Full-length cloning of rhesus ADRA1A splice variants was achieved by combining PCR screening of a seminal vesicle cDNA library and 5'-RACE assays with total RNA from seminal vesicle. The classical ADRA1A mRNA (ADRA1A_v1) and six full-length ADRA1A splice variants were identified representing transcripts that code for functional (ADRA1A_v1, ADRA1A_v2a, ADRA1A_v3a, ADRA1A_v3d, ADRA1A_v3e) and truncated (ADRA1A_v2c and ADRA1A_v3c) receptor isoforms. Comparative analysis of the deduced amino acid sequence indicated that rhesus ADRA1A_i1 isoform (corresponding to the ADRA1A_v1 transcript) shares high identity to the amino acid sequence present in the classical alpha(1A)-adrenoceptor from human and other mammalian species. Partial nucleotide sequences for rhesus alpha(1B)-(ADRA1B) and alpha(1D)-adrenoceptor (ADRA1D) transcripts were also characterized. RT-PCR studies indicated differential distribution of all ADRA1A-related splice variants as well as ADRA1B and ADRA1D mRNAs, in tissues from rhesus and human male reproductive tract. Immunohistochemistry revealed alpha(1A)-adrenoceptor (ADRA1A_i1) immunostaining in smooth muscle cells and epithelial cells of rhesus efferent ductules, epididymis and seminal vesicle. Taken together the present results demonstrate that the complexity of the splicing mechanisms involved in the regulation of the ADRA1A gene is not restricted to human and is a common characteristic among Old World monkeys.
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http://dx.doi.org/10.1093/molehr/gam084DOI Listing
February 2008

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

Mol Hum Reprod 2008 Feb 29;14(2):107-16. Epub 2007 Nov 29.

Laboratories for Reproductive Biology, University of North Carolina, Chapel Hill, NC 27599, USA.

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.
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http://dx.doi.org/10.1093/molehr/gam080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2701302PMC
February 2008

Novel aspects of the sperm-associated antigen 11 (SPAG11) gene organization and expression in cattle (Bos taurus).

Biol Reprod 2007 Jun 7;76(6):1103-16. Epub 2007 Mar 7.

Section of Experimental Endocrinology, Department of Pharmacology, Universidade Federal de São Paulo-Escola Paulista de Medicina, Rua 03 de Maio 100, Vila Clementino, São Paulo (SP) 04044-020, Brazil.

Beta-defensins are small cationic peptides exhibiting broad spectrum antimicrobial properties. In humans, many beta-defensin genes are located within a cluster on chromosome 8p23. The sperm associated antigen 11 (SPAG11) gene is contained in this cluster and is unusual among the human beta-defensins due to its complex genomic structure and mRNA splicing pattern. Here we report the genomic organization of the Bos taurus SPAG11 gene located on chromosome 27q1.2, within a cluster of beta-defensin genes. The exon structures of the fused bovine SPAG11 gene and of the mosaic transcripts initiated at both A and B promoters were established, including identification of novel exons and transcripts not previously found in primate or rodent. Evolutionary analysis against primate, rodent, canine, and porcine orthologs was performed. In adult bulls SPAG11C, SPAG11E, and SPAG11U mRNAs were detected predominantly in the male reproductive tract, while SPAG11D transcript was detected in reproductive and nonreproductive tissues and SPAG11V and SPAG11W mRNAs were confined to testis. Differential expression of all six transcripts was observed in tissues from fetal and adult bulls, suggesting that similar mRNA splicing mechanisms govern SPAG11 gene expression during pre- and postnatal development. Immunolocalization of SPAG11C and SPAG11D/E was demonstrated in the epithelium of the epididymis and testis, and SPAG11D in association with epididymal spermatozoa. Recombinant full-length SPAG11D protein was strongly antibacterial, while the SPAG11E C-terminal peptide that contains the beta-defensin motif in its structure was somewhat less potent. Taken together, the results suggest that SPAG11 isoforms perform both immune and reproductive functions in cattle.
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http://dx.doi.org/10.1095/biolreprod.106.059626DOI Listing
June 2007

Nuclear autoantigenic sperm protein (NASP), a linker histone chaperone that is required for cell proliferation.

J Biol Chem 2006 Jul 25;281(30):21526-34. Epub 2006 May 25.

Department of Cell and Developmental Biology, University of North Carolina, Chapel Hill, 27599, USA.

A multichaperone nucleosome-remodeling complex that contains the H1 linker histone chaperone nuclear autoantigenic sperm protein (NASP) has recently been described. Linker histones (H1) are required for the proper completion of normal development, and NASP transports H1 histones into nuclei and exchanges H1 histones with DNA. Consequently, we investigated whether NASP is required for normal cell cycle progression and development. We now report that without sufficient NASP, HeLa cells and U2OS cells are unable to replicate their DNA and progress through the cell cycle and that the NASP(-/-) null mutation causes embryonic lethality. Although the null mutation NASP(-/-) caused embryonic lethality, null embryos survive until the blastocyst stage, which may be explained by the presence of stored NASP protein in the cytoplasm of oocytes. We conclude from this study that NASP and therefore the linker histones are key players in the assembly of chromatin after DNA replication.
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http://dx.doi.org/10.1074/jbc.M603816200DOI Listing
July 2006

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

Reprod Biol Endocrinol 2006 Apr 28;4:23. Epub 2006 Apr 28.

Laboratories for Reproductive Biology, Department of Pediatrics, University of North Carolina, Chapel Hill, NC 27599-7500, USA.

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.
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http://dx.doi.org/10.1186/1477-7827-4-23DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1524968PMC
April 2006

Cells positive for microtubule-associated protein 1B (MAP 1B) are present along rat and human efferent ductules and epididymis.

Cell Tissue Res 2006 Jul 16;325(1):125-33. Epub 2006 Mar 16.

Section of Experimental Endocrinology, Department of Pharmacology, Universidade Federal de São Paulo-Escola Paulista de Medicina, Rua 3 de maio 100, INFAR, Vila Clementino, 04044-020 São Paulo, Brazil.

Microtubule-associated protein 1B (MAP 1B) is a neuronal cytoskeleton marker with predominant expression in the developing nervous system. The present study provides evidence for the expression of this cytoskeleton protein in non-neuronal and neuronal cells along rat and human efferent ductules and epididymis (initial segment, caput, and cauda). Reverse transcription/polymerase chain reaction and Western blot analysis were used to confirm the presence of MAP 1B (mRNA and protein) in rat tissues. Immunohistochemical studies revealed MAP-1B-positive staining in columnar ciliated cells present in efferent ductules and in narrow cells located in the initial segment, in both rat and human. MAP-1B-positive basal cells, located underneath the columnar cells, were only identified in the initial segment and caput epididymidis of the rat. Qualitative analysis of tissues from 40-day-old and 120-day-old rats indicated that the number of MAP-1B-positive ciliated, narrow, and basal cells per tubule increased with sexual maturation. These immunoreactive cells did not stain for dopamine beta-hydroxylase or acetylcholinesterase, indicating that they were not adrenergic or cholinergic in nature. Immunohistochemical studies also revealed the presence of MAP-1B-positive staining in interstitial nerve fibers in caput and cauda epididymidis from both rat and human. Thus, the expression of MAP 1B is not confined to a specific cell type in rat and human efferent ductules and epididymis. The functional significance of this cytoskeleton protein in tissues from the male reproductive tract requires further investigation.
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http://dx.doi.org/10.1007/s00441-005-0108-xDOI Listing
July 2006

Microarray analysis of androgen-regulated gene expression in testis: the use of the androgen-binding protein (ABP)-transgenic mouse as a model.

Reprod Biol Endocrinol 2005 Dec 9;3:70. Epub 2005 Dec 9.

Department of Cell and Developmental Biology and Laboratories for Reproductive Biology, The University of North Carolina at Chapel Hill, School of Medicine, Chapel Hill, NC 27599, USA.

Background: Spermatogenesis is an androgen-dependent process, yet the molecular mechanisms of androgens' actions in testis are poorly understood. Transgenic mice overexpressing rat androgen-binding protein (ABP) in their testes have reduced levels of intratesticular androgens and, as a result, show a progressive impairment of spermatogenesis. We used this model to characterize changes in global gene expression in testis in response to reduced bioavailability of androgens.

Methods: Total RNA was extracted from testes of 30-day old transgenic and wild-type control mice, converted to cRNA, labeled with biotin, and hybridized to oligonucleotide microarrays. Microarray results were confirmed by real-time reverse transcription polymerase chain reaction.

Results: Three-hundred-eighty-one genes (3.05% of all transcripts represented on the chips) were up-regulated and 198 genes (1.59%) were down-regulated by at least a factor of 2 in the androgen-deficient animals compared to controls. Genes encoding membrane proteins, intracellular signaling molecules, enzymes, proteins participating in the immune response, and those involved in cytoskeleton organization were significantly overrepresented in the up-regulated group. Among the down-regulated transcripts, those coding for extracellular proteins were overrepresented most dramatically, followed by those related to proteolysis, cell adhesion, immune response, and growth factor, cytokine, and ion channel activities. Transcripts with the greatest potential impact on cellular activities included several transcription factors, intracellular signal transducers, secreted signaling molecules and enzymes, and various cell surface molecules. Major nodes in the up-regulated network were IL-6, AGT, MYC, and A2M, those in the down-regulated network were IL-2, -4, and -10, MAPK8, SOCS1, and CREB1.

Conclusion: Microarray analysis followed by gene ontology profiling and connectivity analysis identified several functional groups of genes and individual genes responding to sustained reduction of androgen levels in the mouse testis. These include genes whose products function as transcription factors, cell surface molecules including ion channels, extra- and intracellular signaling molecules, and secreted enzymes with the potential of regulating cell-to-cell attachment. The transcription factors CREB1 (down-regulated) and MYC (up-regulated) may mediate the most important initial phases of the testicular response to reduced levels of androgens. These results suggest specific avenues for further research that will lead to a better understanding of how androgens regulate spermatogenesis.
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http://dx.doi.org/10.1186/1477-7827-3-70DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1327675PMC
December 2005

Altered bioavailability of testosterone in androgen-binding protein-transgenic mice.

Steroids 2005 Sep;70(10):704-14

Department of Cell and Developmental Biology and Laboratories for Reproductive Biology, University of North Carolina, Chapel Hill, NC 27599, USA.

Serum and intra-testicular total and free testosterone levels in different age groups of mice (7-360-day-old) were analyzed by radioimmunoassay (RIA) in age-matched wild type (WT)-control and in transgenic mice homozygous to rat androgen-binding protein (ABP-TG), in order to identify possible causes of increased pre-pubertal germ cell apoptosis, spermatogenetic defect and reduced fertility seen in ABP-TG mice. Total intra-testicular testosterone levels in the pre-pubertal ABP-TG (7, 14, 21 and 30-day-old) mice were significantly lower than those in age-matched WT-controls. After puberty (60 days and older) the total intra-testicular testosterone levels were higher than those in age-matched WT-controls and increased gradually, peaking on day 180. Serum total testosterone levels in ABP-TG mice did not differ from those in WT-control until day 30. However, a significant increase in the level of serum total testosterone was observed from day 60. Serum and intra-testicular free testosterone levels were significantly lower in 30, 120, 180 and 360-day-old ABP-TG mice than in age-matched WT-controls. Immunohistochemistry for the cholesterol side-chain cleavage (cytochrome P450) enzyme and quantitative real-time RT-PCR analysis of mRNAs for androgen receptor and for enzymes related to steroidogenesis did not show any changes in 30-day-old ABP-TG mice, indicating that the rates of steroidogenesis and utilization were not altered. Human chorionic gonadotrophin (hCG) administration to adult ABP-TG mice increased the intra-testicular total and free testosterone as well as total germ cell counts. We conclude that the presence of greater than physiological concentration of ABP in the mouse testis alters the ratio of free/bound testosterone, and thereby decreases the availability of free testosterone. As a result, a heightened wave of germ cell apoptosis during the pre-pubertal period followed by a reduction in germ cell numbers and reduced fertility is seen in these mice.
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http://dx.doi.org/10.1016/j.steroids.2005.03.015DOI Listing
September 2005

A three-generational study of transmission of risk for sexual abuse.

J Clin Child Adolesc Psychol 2004 Dec;33(4):662-72

Illinois School of Professional Psychology, Chicago, 60603, USA.

This intergenerational study investigates histories of both attachment relationships and abusive experiences and domains of current functioning that distinguish families of sexually abused children from families of nonabused children. The participants included (a) 199 nonoffending African American mothers of whom approximately half had children with documented sexual abuse histories and half had children with no documented abuse histories and (b) 106 maternal grandmothers of these children; approximately half had sexually abused grandchildren and half had grandchildren with no documented abuse. The children were 4 to 12 years old. Histories of abuse and attachment experiences and current functioning of the grandmother and mother were evaluated. Logistic regression analyses revealed that sexual abuse in a child was best predicted by 3 factors: maternal problems in adult functioning, a currently negative relationship between the grandmother and mother, and a disrupted pattern of caregiving during the mother's childhood. The findings underscore that troubled intergenerational attachment relationships in families can significantly heighten the risk of a child being sexually abused.
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http://dx.doi.org/10.1207/s15374424jccp3304_2DOI Listing
December 2004

Differential expression and antibacterial activity of epididymis protein 2 isoforms in the male reproductive tract of human and rhesus monkey (Macaca mulatta).

Biol Reprod 2004 Nov 30;71(5):1453-60. Epub 2004 Jun 30.

Section of Experimental Endocrinology, Department of Pharmacology, Universidade Federal de São Paulo--Escola Paulista de Medicina, Rua 03 de maio 100, INFAR, Vila Clementino, São Paulo, SP 04044-020, Brazil.

The epididymis protein 2 (EP2) gene, the fusion of two ancestral beta-defensin genes, is highly expressed in the epididymis and subject to species-specific regulation at the levels of promoter selection, transcription, and mRNA splicing. EP2 mRNA expression is also androgen dependent, and at least two of the secreted proteins bind spermatozoa. Alternative splicing produces more than 17 different EP2 mRNA variants. In this article, the expression of EP2 variants was profiled in different tissues from the human and rhesus monkey (Macaca mulatta) male reproductive tract using reverse transcriptase-polymerase chain reaction. Different EP2 mRNA variants were identified not only in human and rhesus testis and epididymis but also in the novel sites, seminal vesicle and prostate. Immunolocalization of EP2 protein in epithelial cells from rhesus and human seminal vesicle demonstrated that EP2 transcripts are translated in these tissues. In addition, two novel splicing variants, named EP2R and EP2S, were discovered. EP2C was the only splice variant expressed in all tissues tested from rhesus monkey. However, expression was not detected in human testis or seminal vesicle. For the first time, bactericidal function was demonstrated for EP2C, EP2K, and EP2L. Taken together, the results indicate that EP2 expression is more widespread in the male reproductive tract than realized previously. Whereas the activity of every EP2 variant tested thus far is antibacterial, further investigation may reveal additional physiological roles for EP2 peptides in the primate male reproductive tract.
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http://dx.doi.org/10.1095/biolreprod.104.031740DOI Listing
November 2004

LCN6, a novel human epididymal lipocalin.

Reprod Biol Endocrinol 2003 Nov 14;1:112. Epub 2003 Nov 14.

Department of Pediatrics, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599, USA.

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.
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http://dx.doi.org/10.1186/1477-7827-1-112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC293424PMC
November 2003

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

Reprod Biol Endocrinol 2003 Jun 12;1:48. Epub 2003 Jun 12.

Department of Cell and Developmental Biology and Laboratories for Reproductive Biology, University of North Carolina, Chapel Hill, NC 27599, USA.

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.
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http://dx.doi.org/10.1186/1477-7827-1-48DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC165588PMC
June 2003

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

Endocrinology 2002 Jul;143(7):2787-96

Department of Pediatrics, University of North Carolina School of Medicine, Chapel Hill 27599, USA.

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.
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http://dx.doi.org/10.1210/endo.143.7.8925DOI Listing
July 2002

Protein inhibitors of activated STAT resemble scaffold attachment factors and function as interacting nuclear receptor coregulators.

J Biol Chem 2002 May 4;277(19):16993-7001. Epub 2002 Mar 4.

Laboratories for Reproductive Biology, Department of Pediatrics, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599-7500, USA.

Protein inhibitor of activated STAT1 (PIAS1) functions as a nuclear receptor coregulator and is expressed in several cell types of human testis. However, the mechanism of PIAS1 coregulation is unknown. We report here that PIAS1 has characteristics of a scaffold attachment protein. PIAS1 localized in nuclei in a speckled pattern and bound A-T-rich double-stranded DNA, a function of scaffold attachment proteins in chromatin regions of active transcription. DNA binding was dependent on a 35-amino acid sequence conserved among members of the PIAS family and in scaffold attachment proteins. The PIAS family also bound the androgen receptor DNA binding domain, and binding required the second zinc finger of this domain. PIAS1 contained an intrinsic activation domain but had bi-directional effects on androgen receptor transactivation; lower expression levels inhibited and higher levels increased transactivation in CV1 cells. Other PIAS family members also had dose-dependent effects on transactivation, but they were in a direction opposite to those of PIAS1. When coexpressed with PIAS1, other PIAS family members counteracted PIAS1 coregulation of androgen receptor transactivation. The interaction of PIAS1 with other members of the PIAS family suggests a transcription coregulatory mechanism involving a multicomponent PIAS nuclear scaffold.
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http://dx.doi.org/10.1074/jbc.M109217200DOI Listing
May 2002