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GONADALRECEPTORS AND ACTIONS OF PEPTIDES,
HORMONES, AND REGULATORY PROTEINS IN
STEROIDOGENESIS AND SPERMATOGENESIS
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Maria L. Dufau, MD,
PhD, Head, Section on
Molecular Endocrinology Chon-Hwa Tsai-Morris,
PhD, Staff Scientist Ying Zhang, PhD, Research Fellow Juying
Dong, PhD, Postdoctoral Fellow Aamer Qazi, PhD, Postdoctoral
Fellow Naheed Fatima, PhD, Adjunct Investigator
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We investigate the molecular basis of peptide
hormone control of gonadal function, with particular emphasis on the
structure and regulation of genes encoding the luteinizing hormone (LH) and prolactin
receptor (PRLR), and the regulatory mechanism(s) involved in the control of
steroidogenesis and spermatogenesis. Our studies focus on the regulation of
human LH receptor gene transcription (nuclear orphan receptors, epigenetic
regulation, DNA methylation) as well as on the multiple promoter control of hPRLR gene transcription. We are interested in
elucidating the function of two inhibitory short forms of PRL receptors and
their relevance to physiological regulation and breast cancer. We also investigate
hormone-regulated membrane coupling and intracellular events involved in the
modulation of steroid biosynthesis in the testis as well as novel
gonadotropin-regulated genes related to testicular and ovarian function and
other reproductive processes. We analyze the function of
gonadotropin/androgen–regulated RNA testicular helicase (GRTH/Ddx25),
define the mechanism of its regulation, and investigate its involvement in
the transport, storage, and translation of relevant mRNAs
at critical stages of spermatogenesis. We also investigate the proteins that
are regulated by GRTH and that contribute to the apoptosis and spermatogenic
arrest observed in the GRTH null mice developed in our laboratory. Regulation of DNA
methylation and histone modification by human LH receptor gene transcription Zhang, Fatima,
Dufau The LH receptor (LHR), a member of the G
protein–coupled, seven-transmembrane receptor family, is essential for
normal sexual development and reproductive function. LHR is expressed
primarily in the gonads but is also found in nongonadal
and cancer tissues. LH acts through LHR in gonadal cells (Leydig,
granulosa/thecal cells) to trigger intracellular responses that participate
in gonadal cell maturation and function as well as in the regulation of steroidogenic
enzymes and gametogenosis. We have previously
demonstrated two independent silencing mechanisms of regulation of
transcription of the LHR gene within its promoter domain by nuclear orphan
receptors (EAR3/COUP-TFI and EAR2) and histone deacetylase complexes. The
most proximal Sp1 site of the hLHR promoter serves
as a docking site for the silencing histone deacetylase (HDAC)/mSin3 A
complex, which plays a role in the control of human LHR gene expression (Zhang Y, Dufau ML, 2002). The
ability of histone acetylation–mediated local chromatin changes at the hLHR gene promoter region to cause derepression of hLHR gene transcription revealed that epigenetic
regulation was critical for hLHR gene expression. In recent studies, we initially characterized
the methylation status of the hLHR gene promoter in
human placenta choriocarcinoma JAR cells and subsequently explored its
functional connection with various histone modifications in the regulation of
hLHR gene transcription. The human LHR gene
promoter is highly methylated in JAR cells cultured under basal conditions
and TSA (histone deacetylase inhibitor), and 5´-AzaC (a DNA demethylating
agent) evoked marked functional synergism in the activation of hLHR gene expression. This effect was localized to the 176
bp hLHR promoter region, implying that the DNA
methylation and chromatin status of this promoter control hLHR
gene expression. Consistent with these observations, maximal derepression of hLHR gene promoter activity by the combined effect of TSA
and AzaC resulted from complete demethylation of a CpG island overlapping the promoter and adjacent 3´
coding domains and from significant changes of histone modification at the
promoter region. Both histone acetylation and methylation contribute to the
alteration of chromatin structure. More specifically, methylation of histone
H3 at Lys 4 was caused by TSA or AzaC, and
demethylation and acetylation at Zhang Y, Dufau ML. Dual mechanisms
of regulation of transcription of luteinizing hormone receptor gene by
nuclear orphan receptors and histone deacetylase complexes. J Steroid Bioch Mol Biol 2003;85:401-414. Zhang Y, Dufau ML. Gene silencing by nuclear orphan receptors. Vitam
Horm 2004;68:1-48. Zhang Y, Dufau ML. Repression of the luteinizing hormone receptor
gene promoter by cross talk among EAR3/COUP-TFI, Sp1/Sp3, and TFIIB. Mol
Cell Biol 2003;23:6958-6972. Zhang Y, Dufau ML. Silencing of transcription of the human
luteinizing hormone receptor gene by histone deacetylase-mSin3A complex. J
Biol Chem 2002;277:33431-33438. Requirement of gonadotropin-regulated testicular
RNA helicase GRTH/Ddx25 for completion of spermatogenesis Tsai-Morris,
Sheng, Dufau Spermatogenesis is a complex process that
depends on the integrated expression of an array of genes that must operate
in a precise temporal sequence to produce normal mature spermatozoa. Gene
expression in haploid spermatids requires temporal uncoupling of
transcription and translation. Translation of stored mRNAs
associated with protein (mRNP) in the cytoplasm of
spermatids at specific times is essential for the completion of
spermatogenesis. Modulation of RNA structure by members of the DEAD-box
family of RNA helicases is a crucial step in many fundament biological
processes. However, knowledge regarding the functional involvement of
DEAD-box proteins in testicular germ cells is limited in mammals to studies
on the mouse Vasa homolog, which is a specific RNA helicase of germ cells
whose deletion resulted in premeiotic arrest at the zygotene stage. Our laboratory previously discovered
GRTH/Ddx25, a novel gonadotropin-regulated testicular RNA helicase (Tang et
al., J Biol Chem 1999;274:37932). We cloned
the helicase, which is a member of the DEAD-box protein family, from human,
rat, and mouse testis libraries. GRTH displays ATPase and RNA helicase
activities and increases the in vitro translation of RNA templates.
The helicase is a male-specific protein expressed in the rat, mouse, and
human testis. It is present in Leydig cells and germ cells (meiotic
spermatocytes and round spermatids), is developmentally regulated, and is upregulated by gonadotropin/androgen at the
transcriptional level. In addition, cell-specific and hormone-dependent
regulation of GRTH translation occurs in the testis as a result of
alternative usage of AUG codons in GRTH mRNA. To gain insights into the
regulatory actions of GRTH in spermatogenesis, we examined the localization
of GRTH and its association with mRNA and subsequently generated GRTH-null
mice to determine the helicase’s functional
role in reproduction and testicular function. We observed that GRTH is
present in the nucleus, cytoplasm, and chromatoid body of germ cells and is
an integral com-ponent of messenger ribonuclear
protein particles. Male mice with a null mutation in the GRTH
gene displayed normal gonadotropin and androgen pro-files; however, they were
sterile, with azoospermia caused by a complete arrest of spermiogenesis at
step 8 of round spermatids and failure to elongate. Electron microscopy
studies in round spermatids of the null mice have shown marked diminution in
the size (by 90 percent) of chromatoid bodies, cytoplasmic organelles, viewed
as scaffolds of storage of mRNP (Figure 4.2). The
transcription of relevant messages remained altered, but the messages’
translation was abro-gated in a selective manner.
Protein expression of transition protein 1 and 2 and angiotensin-converting
enzyme was completely absent, whereas that of the transcriptional activator cAMP-responsive modulator (CREM) was intact. Thus, GRTH
protein may serve as a master translational regulator of a selective panel or
cascade of genes that are crucial for spermiogenesis. Although significant
apoptosis was present at the metaphase of meiosis in the GRTH-null mice,
spermatogenesis proceeded to step 8 of spermiogenesis, when complete arrest
occurred. This progression may relate to compensatory gene functions and/or
the observed upregulation of DNA repair proteins
Rad51 and Dmc1. From these studies, we deduce that GRTH
protein functions as a component of mRNP and/or may
be required for the formation of chromatoid bodies. GRTH is important in the
translation of crucial genes at specific times during spermatogenesis. GRTH
could also affect transport of poly(A)+ mRNA to the cytoplasm for
storage in chromatoid bodies of spermatids for later release for translation
in a time-specific manner during spermiogenesis. Furthermore, GRTH associated
with polyribosomes could influence the translation of genes. In summary, our
studies have demonstrated that GRTH is essential for spermatid development
(elongation) and completion of spermatogenesis. They have provided insights
into intrinsic requirements for spermiogenesis and established a model for
studies of male infertility and contraception. Sheng Y, Tsai-Morris CH, Dufau ML. Cell-specific
and hormonally regulated expression of gonadotropin-regulated testicular RNA
helicase gene (GRTH/Ddx25) resulting from alternative utilization of
translation initiation codons in the rat testis. J Biol
Chem 2003;278:27796-27803. Tsai-Morris CH, Lei CH, Jiang
Q, Sheng Y, Dufau ML.
Genomic organization and transcriptional analysis of gonadotropin-regulated
testicular RNA helicase. Gene 2004;331:83-94. Tsai-Morris CH,
Sheng Y, Lee E, Lei KJ, Dufau ML. Gonadotropin-regulated
testicular RNA helicase (GRTH/Ddx25) is essential for spermatid development
and completion of spermatogenesis. Proc Natl Acad
Sci USA 2004;101:6373-6378.
Prolactin receptors in human breast cancer Meng, Tsai-Morris, Qazi, Dong, Dufau The primary actions of prolactin (PRL) are to promote
growth and differentiation of the mammary gland during pregnancy and initiate
and maintain lactation. PRL also exerts diverse functions in its several
target tissues through specific membrane receptors (PRLRs)
and acts through the long form of the receptor (LF) to cause differentiation
of mammary epithelial cells through activation of the Jak2/Stat5 pathway and
subsequent transcriptional events. Our laboratory identified two novel short
forms (SF) with an abbreviated cytoplasmic domain (S1a, S1b) that are
products of alternative splicing and inhibit the activation induced by PRL
through the LF. PRL plays an essential role in the development of rodent
mammary tumors and is a potent mitogen in human normal and cancerous breast
tissues/cells. Local PRL production occurs in mammary epithelial cells, with
a correlation between serum PRL and the incidence and progression of breast
tumors. The available evidence strongly suggests that PRL has a role in the
development of human breast tumors. The role of PRLR variants in the actions
of PRL in breast cancer is unknown. During the year, we completed the first
phase of an evaluation of the expression of PRL receptors in breast tumors
and adjacent normal tissue, including the long receptor form (LF;
stimulatory) and two SFs (S1a and S1B; inhibitory).
Southern analysis of breast cancer profiling arrays revealed that 29 patients
(group I) expressed elevated LF, ten patients (group II) showed decreased LF,
and eight patients (group III) had no changes relative to the adjacent normal
tissue. The respective SF expression was increased in 21 patients of group I
and generally decreased in groups II and III. However, the ratio of SF to LF
decreased in 76 percent of the breast tumors and was distributed evenly among
the groups. Quantification of hPRLR variants by
real-time PCR in 15 pairs of human normal and breast tumor matched tissues
revealed a significant decrease in the ratio of SF to LF in the tumor tissue.
There is no specific correlation in the change of hPRLR
variant levels or SF/LF ratio with the type of breast tumor. Further evidence
linking the low SF/LF to breast tumor is provided by their relative
expression in normal versus mammary cancer cell lines from ductal, medullary,
lobular, and adenocarcinoma cancer cell lines. Lower SF/LF was found in eight
of ten breast cancer cell lines compared with normal mammary Hs578Bst and
MCF10A cells. These findings in cells support the SF/LF findings in breast
tumor tissues and provide useful information to select specific cells lines
for PRLR variants–related projects. In summary, we have observed a
general pattern of low ratio of the individual short forms to long form (SFs/LF) associated with both breast tumor tissues and
cancerous cell lines when compared with normal samples. These observations
provide an additional index for evaluation of human breast cancer. The
decreased ratio of SF to LF in tumors suggests that a loss of the inhibitory
regulation of SF to LF may accelerate abnormal cell proliferation and
differentiation. Meng J,
Tsai-Morris CH, Dufau ML. Human prolactin receptor
variants in breast cancer: low ratio of short forms to the long-form human
prolactin receptor associated with mammary carcinoma. Cancer Res 2004;64:5677-5682. COLLABORATORS Eric J.M. Lee, DVM, Laboratory
of Mammalian Genes and Development, NICHD, Ke-jian
G. Lei, PhD, Oral Infection and Immunity Branch, NIDCR, For further information, contact dufaum@mail.nih.gov |
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