help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2007-0154
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Li, D.
Right arrow Articles by Liu, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Li, D.
Right arrow Articles by Liu, M.
Endocrinology Vol. 148, No. 10 4821-4828
Copyright © 2007 by The Endocrine Society

Estrogen Regulates KiSS1 Gene Expression through Estrogen Receptor {alpha} and SP Protein Complexes

Dali Li, Dianne Mitchell, Jian Luo, Zhengfang Yi, Sung-Gook Cho, Jingjing Guo, Xiaoying Li, Guang Ning, Xiushan Wu and Mingyao Liu

Institute of Biosciences and Technology (D.L., D.M., J.L., Z.Y., S.-G.C., J.G., M.L.), and Department of Molecular and Cellular Medicine, Texas A&M University System Health Science Center, Houston, Texas 77030; Endocrine and Metabolic Division (X.L., G.N., M.L.), E-Institutes of Shanghai Universities, Shanghai Clinical Center for Endocrine and Metabolic Diseases, Shanghai Jiaotong University Medical School, Shanghai 200025, China; and College of Life Sciences (D.L., J.L., X.W., M.L.), Hunan Normal University, Changsha, Hunan 410081, China

Address all correspondence and requests for reprints to: Mingyao Liu, Institute of Biosciences and Technology, Texas A&M Health Science Center, 2121 West Holcombe Boulevard, Houston, Texas 77030. E-mail: mliu{at}ibt.tamhsc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Kisspeptins are natural ligands of G protein-coupled receptor-54. Activation of KiSS1/G protein-coupled receptor-54 signaling pathways results in potent activation of the hypothalamus-pituitary-gonadal axis and initiates puberty. Recent data have shown that in female mice, KiSS1 is positively regulated by estradiol (E2) in the anteroventral periventricular nucleus, an important reproductive neuroendocrine brain region, but negatively regulated in the arcuate nucleus. However, little is known about the molecular mechanisms governing E2-modulated KiSS1 expression. Here, we demonstrate that the expression level of the KiSS1 gene was up-regulated with the administration of E2 in estrogen receptor {alpha} (ER{alpha})-positive hypothalamic GT1–7 cells. Using transient transfection of human KiSS1 gene promoter coupled to a luciferase reporter, E2 increases promoter activity in the presence of ER{alpha}. Deletion analysis of KiSS1 promoter indicates that the E2-regulated increase in promoter activity depends on the Sp1 sites of the proximal promoter region. Using both EMSAs and chromatin immunoprecipitation analysis, we determined that both Sp1 and Sp3 proteins constitutively associate with the four putative Sp1 sites in vitro, whereas the association of ER{alpha} with the KiSS1 promoter is dependent on E2 exposure. Sp1 and ER{alpha} form a complex in vivo to mediate the E2-induced activation of KiSS1 promoter. Interestingly, Sp1 transactivates KiSS1 promoter activity, whereas Sp3 functions as a transcriptional repressor. Together, these results demonstrate that E2-dependent transcriptional activation of KiSS1 gene is mediated by ER{alpha} through the interaction of Sp1/Sp3 proteins with the GC-rich motifs of KiSS1 promoter, providing a molecular mechanism of how steroid hormone feedback regulates KiSS1 expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PUBERTY IS DEFINED as the short period between childhood and adulthood during which reproductive ability is attained by the awakened hypothalamic-pituitary-gonadal (HPG) axis. The hypothalamic decapeptide GnRH has been considered the central player that triggers the onset of puberty (1). In late 2003, independent reports by de Roux (2) and Seminara (3) et al. linked the inactivating mutations of G protein-coupled receptor-54 (GPR54) to idiopathic hypogonadotropic hypogonadism, both in human and mouse. Further studies revealed that the GnRH secreting neurons are colocalized with KiSS1 expressing neurons in sheep (4) and activated by the KiSS1 peptides, the natural ligand of GPR54 (5, 6, 7). Moreover, central and peripheral administration of KiSS1 increases plasma concentrations of pituitary hormones, LH, and FSH, which are the direct downstream target hormones of GnRH in the HPG axis (6, 8, 9, 10). Since then, more and more data indicate that KiSS1 and GPR54 are the new gatekeepers of reproduction (11, 12, 13). Understanding how the expression of pubertal KiSS1 is regulated has become important to our understanding of the onset of puberty.

Estradiol (E2) is the dominant sex steroid in females, and is stimulated by the pituitary hormones, LH, and FSH. Through negative feedback, estrogen down-regulates the expression of many hypothalamic and pituitary hormones, such as GnRH, LH, and FSH (14, 15, 16). Although KiSS1 has been identified as the hormone that triggers the onset of puberty via its regulation of LH and FSH, the molecular mechanism of KiSS1 regulation by estrogens still remains unclear. Recent data demonstrated that KiSS1 expression was differentially affected by estrogens both in the hypothalamus and in the pituitary of different mammalian species (17, 18, 19). Furthermore, KiSS1 expression was not altered in response to E2 treatment in ovariectomy (OVX) mice, in which functional estrogen receptor {alpha} (ER{alpha}) was ablated, whereas in ERß null OVX mice, KiSS1 responded the same as wild-type female mice, suggesting that ER{alpha} directly mediates the regulation of E2 (17).

ER{alpha}, an estrogen-inducible transcription factor, can directly bind to a specific DNA sequence, defined as an estrogen response element (ERE), through its conserved DNA-binding domain. Alternatively, ER{alpha} can interact with AP1 or Sp proteins, thus indirectly associating with target DNA elements and inducing transcriptional modification (20, 21, 22, 23). The members of the Sp protein family are ubiquitously expressed transcription factors that bind and act through GC-rich elements to regulate gene expression in mammalian cells (23, 24, 25). Sp proteins are commonly detected in hypothalamic, pituitary, and mammary gland tissues and cell lines. Sp1 and Sp3 are structurally similar SP protein family members, usually recruited by nuclear receptors as well as other nuclear receptor cofactors to activate or repress the expression of target genes (25, 26, 27, 28). These two proteins function together through dimerization to regulate the expression of target genes depending on the ratio of the two proteins (29, 30, 31).

KiSS1 is regulated by E2 in the hypothalamus and pituitary through ER{alpha} in vivo, but the molecular mechanism is still unclear. To examine how KiSS1 is regulated at the transcriptional level in the hypothalamus and pituitary, the ER{alpha} positive immortalized hypothalamic cell line GT1–7 was used as a cell model, and the expression of KiSS1 mRNA was monitored upon E2 stimulation. The promoter region of the human KiSS1 gene was then cloned and analyzed for functional cis-elements mediating effects of E2. Deletion analysis of the promoter indicates that the region between –193 and –45 is required for both basal activity and hormone-induced activation, and mutations of the two Sp1 binding sites within this region result in the loss of transactivation. Further results from EMSA and chromatin immunoprecipitation (ChIP) assay showed that Sp1 and Sp3 directly bind to the GC-rich motif, and ER{alpha} binds to Sp proteins upon induction by E2. Our study suggests that the regulation of KiSS1 expression by E2 in the hypothalamus is due to the association of ER{alpha} and Sp proteins through GC-rich motifs on the KiSS1 promoter.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines, chemicals, biochemicals, and constructs
MCF-7, 293T (American Type Culture Collection, Manassas, VA) and GT1–7 cells were grown in DMEM (Hyclone Laboratories, Inc., Logan, UT) supplemented with 10% fetal bovine serum (FBS) (Hyclone), 4.5 mg/ml glucose, and penicillin/streptomycin, and maintained at 37 C in 5% CO2, as described by Mellon and colleagues (32). [{gamma}-32P]ATP (300 Ci/mmol) was obtained from PerkinElmer Life Sciences (Wellesley, MA). Poly (dI-dC) and T4 polynucleotide kinase were purchased from Roche Molecular Biochemicals (Indianapolis, IN). Antibodies for Sp1, Sp3, and ER{alpha} were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Luciferase reagent and lysis buffer were obtained from Promega Corp. (Madison, WI). The human KiSS1 promoter was cloned from BAC clone RP11-203F10 using primers consisting of XhoI and KpnI sites for ligation into the pGL3-basic vector, as previously described (33). All other human KiSS1 promoter truncated fragments were likewise cloned into XhoI and KpnI sites in the pGL3-basic vector, and the primers for subcloning were: hKiSS1 + 1 R, 5'-CCGCTCGAGGTTCTCCCCAGCTCCCT-3'; hKiSS1–2K F, 5'-GGGGTACCCTGAGGAGCCCAG-3'; hKiSS1–1K F, 5'-GGGGTACCTGTATGTGCCCAGCAATGG-3'; hKiSS1–534 F, 5'-GGGGTACCTCAAAACCCTGCGCTGAGG-3'; and hKiSS1–190 F, 5'-ATGGTACCAGTCCCGCCTCGGAGGG-3'. The 45-bp pGL3-KiSS1 promoter was generated by digestion of the 2-kb promoter construct with KpnI and SmaI followed by blunt-end ligation. Expression vectors for Sp1 were cloned as previously described (33). The pCMV-SP3 and pCMV-DNSP3 (G-313) that encode the dominant-negative form of SP3 in pCDNA3.1/His C were generously provided by Dr. Miles F. Wilkinson (The University of Texas, M.D. Anderson Cancer Center, Houston, TX). The ER{alpha} and ER{alpha} deletion constructs, HE11C and HE19C, were kindly provided by Dr. Stephen Safe (Texas A&M University, College Station, TX).

Transfection and luciferase assay
Cells were cultured in six- or 24-well plates in DMEM supplemented with 10% FBS. After 18–20 h when cells were approximately 60% confluent, reporter gene constructs were transfected with indicated expression vectors using Lipofectamine reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. After 6 h, medium was replaced with phenol red-free DMEM-F12 medium supplemented with 5% dextran-coated charcoal-stripped FBS. Cells were then treated with Me2SO or other treatments. The final concentration of E2 used in the experiments was 100 nM if not further described. Forty-eight hours after transfection, cells were harvested, and the luciferase activity in the various treatment groups was determined using the luciferase assay system (Promega) normalized to ß-galactosidase enzyme activity, as previously described (33).

Site-directed mutagenesis
Site-directed mutagenesis of Sp1 binding sites within the human KiSS1 promoter reporter constructs were performed with the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) using mutagenic primers listed (the mutations are underlined and substituted bases are indicated in bold): –188 Sp1 mut, 5'-GAAGGAGAAAGTCCTACCTCGGAGGGCTCGGG-3'; –175 Sp1 mut, 5'-GGAGGGCTCGGATGGGATAGGAGGAGAGGC-3'; –87 Sp1 mut, 5'-GTCTGAGGAGGAGTTAGGGGCGGCAGAGTGG-3'; and –80 Sp1 mut, 5'-GGAGGAGGGAGTGGAGGCAGAGTGGGCCTG-3'. All the mutants were identified by DNA sequencing.

Semiquantitative RT-PCR analysis
Total RNA was harvested using TRIzol (Invitrogen). First-strand complementary DNA synthesis was performed using Moloney murine leukemia virus reverse transcriptase and oligo-dT (Promega) according to the manufacturer’s protocol. Primer sequences used for detection of KiSS1 transcripts were 5'-AGCCTCGAGCCCAGAATGATCTCAATGGCTT-3' and 5'-CCCAAGCTTGGCCTCTACAATCCACCTGCAG-3'. Amplified products were about 430 bases in length. In addition, primers for ß-actin were 5'-GGCTCCGGCATGTGCAAGGC-3' and 5'-AGATTTTCTCCATGTCGTCC-3', which resulted in PCR products of about 200 bases. Optimal PCR cycles required for linear amplification were determined for each (ß-actin required 21–23, KiSS1 required 28–30 cycles). PCR products were separated on 2% agarose gels and quantitated using {alpha}-Imager software (Alpha Innotech, San Leandro, CA).

EMSA
MCF-7 and GT1–7 cells were cultured in six-well plates in DMEM containing 10% FBS. The following day, cells were harvested by manual scraping and resuspended in 25 mM HEPES, 1.5 mM EDTA, and 1 mM dithiothreitol (pH 7.6), and homogenized with 10% glycerol. Cell lysates were then centrifuged at 800 x g/10 min at 4 C, and protein concentration of nuclear extracts was then determined using BCA assay (Pierce, Rockford, IL). Aliquots of nuclear protein were then frozen and stored at –80 C until use. KiSS1 promoter derived Sp1 binding site oligonucleotides were synthesized and annealed, and 5pmole were 5' end-labeled using T4 polynucleotide kinase and [{gamma}32-P]ATP. A 30-µl EMSA reaction containing approximately 100 mM KCl, 5 µg crude nuclear extract, 1 µg poly (dI-dC), and 1 µl of 1 mM ZnCl2 with or without unlabeled competitor oligonucleotide, and 10 fmol labeled probe was incubated on ice for 20 min. Sp1- and Sp3-specific antibodies were then incubated in appropriate reactions for 20 min on ice. DNA-protein complexes were then resolved on 5% PAGE gels at approximately 120 V at 4 C for 2.5 h. The gels were dried, and protein DNA complexes were visualized by autoradiography.

Coimmunoprecipitation analysis
Binding of ER{alpha} and Sp1 in transfected 293T cells was examined by immunoprecipitation (IP) and by Western blot analysis. Briefly, cells were transfected with pcDNA-Sp1 and pcDNA-ER{alpha} or empty vector. After 6 h, medium was changed with phenol red-free DMEM-F12 medium supplemented with 5% dextran-coated charcoal-stripped FBS. Thirty-six hours later, cells were treated with 100 nM E2 for 3 h, then lysed with radioimmunoprecipitation assay buffer containing 0.1% sodium dodecyl sulfate, 0.5% sodium deoxycholate, 1% Triton X-100, 10 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 20 mM NaF, 10 mg/ml phenylmethylsulfonyl fluoride, 1 mg/ml aprotinin, and 1 mg/ml leupeptin, and immunoprecipitated with the indicated antibodies. Anti-Sp1 immunocomplexes were recovered using protein A beads (Santa Cruz Biotechnology). All immunoprecipitates were washed four times with lysis buffer, separated by SDS-PAGE, and then transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA). After incubation in Tris-buffered saline with Tween 20 [20 mM Tris-HCl (pH 7.5), 500 mM NaCl, and 0.02% Tween 20] containing 5% dry milk powder for 2 h, the membranes were probed with the indicated antibodies and visualized with the SuperSignal West Pico detection system (Pierce).

ChIP assay
ChIP assays were performed as previously described (33). Briefly, MCF-7 cells were grown in 100-mm tissue culture plates and treated with 20 nM E2 for various lengths of time. Formaldehyde was then added to the medium to a final concentration of 1%, and the reaction was incubated at room temperature with shaking for 10 min, after which glycine (0.125 M) was added, and the reaction was incubated for another 10 min. The media were then removed, and cells were washed twice with cold PBS and 1 mM phenylmethylsulfonyl fluoride, scraped, collected by centrifugation, and lysed in sodium dodecyl sulfate lysis buffer containing 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, and 1 µg/ml pepstatin A. DNA was sheared to fragments of 500–1000 bp by eight 10-sec sonications. The chromatin was precleared with salmon sperm DNA/protein A-agarose slurry (Upstate Biotechnology, Waltham, MA) for 1 h at 4 C with gentle agitation. The agarose beads were pelleted, and the precleared supernatant was incubated with anti-Sp1, anti-Sp3, anti-ER{alpha} antibodies or nonspecific IgG overnight at 4 C, respectively. The reactions were subsequently washed with low-salt wash buffer [0.1% sodium dodecyl sulfate, 1% Triton X-100, 2 mM EDTA, 20 mM Tris (pH 8.1), and 150 mM NaCl], high-salt wash buffer [0.1% sodium dodecyl sulfate, 1% Triton X-100, 2 mM EDTA, 20 mM Tris (pH 8.1), and 500 mM NaCl], LiCl wash buffer [0.25 M LiCl, 1% IGEPAL CA-630, 1% deoxycholate, 1 mM EDTA, 10 mM Tris (pH 8.1)], and twice in TE buffer [10 mM Tris-HCl (pH 8.0) and 1 mM EDTA]. After reversing the protein/DNA cross-links, DNA was recovered by phenol/chloroform extraction and ethanol precipitation. The region between –230 and +1 of the human KiSS1 promoter was amplified from the immunoprecipitated chromatin using the following primers: sense, 5'-TGGAGGATGGAAAGAGCCGG-3'; and antisense, 5'-GTTCTCCCCAGCTCCCT-3'. After PCR, the products were resolved on a 2.0% agarose gel and stained with ethidium bromide. Samples were visualized under UV light. PCR products were purified and sequenced. MCF-7 cells were chosen for the ChIP assays because the GT1–7 hypothalamic cell line is derived from mouse, and the promoter we analyzed is based on the human KiSS1 gene. In addition, the MCF-7 cell line is a human KiSS1 positive cell line from our previous study (34) and is well established to investigate the E2-induced transactivation through ER{alpha} and Sp protein complex (28, 35, 36).

Statistical analysis
Experiments were repeated two or more times, and data are expressed as the mean ± SD for at least three replicates for each treatment group. Statistical differences between treatment groups were determined by Scheffé’s test. Treatments were considered significantly different from controls if P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
E2 stimulates KiSS1 mRNA expression and KiSS1 promoter activity through ER{alpha}
The GT1–7 cell line was established by Mellon et al. (37) as an immortalized hypothalamic cell model to study GnRH secretion and hormone regulation. Although KiSS1 positive neurons were found to colocalize partially with GnRH neurons in sheep but not in rodents (4), we found that the immortalized mouse hypothalamic GT1–7 cell line expresses KiSS1 gene using 20 different pairs of RT-PCR primers(data not shown). GT1–7 cells were maintained in phenol red-free DMEM-F12 medium supplemented with 5% charcoal-stripped FBS before treatment using different concentrations (from 0–1 µM) of E2 for 6 h. Then KiSS1 mRNA levels were determined by RT-PCR. As shown in Fig. 1AGo, E2 treatment significantly increased KiSS1 mRNA expression at 10 nM, but higher concentrations (more than 100 nM) of E2 did not show any further stimulation. To examine further the transcriptional regulation of KiSS1 in hypothalamus by E2, the 2-kb full-length human KiSS1 promoter linked to a luciferase reporter was transiently transfected into GT1–7 cells that were treated with E2 varying from 0–1 µM. The promoter activity was significantly increased and exhibited a responsive pattern that correlated with an increased mRNA level of KiSS1 (Fig. 1BGo). The concentrations of E2 lower than 1 nM did not significantly affect the promoter activity. These data suggest that E2 up-regulates KiSS1 gene expression through the 2-kb region proximal to the KiSS1 start site.


Figure 1
View larger version (25K):
[in this window]
[in a new window]

  		FIG. 1. Hormone-induced activation of KiSS1 gene expression. A, Regulation of KiSS1 mRNA expression levels by different concentrations of E2. GT1–7 cells were treated with E2 for 6 h in different concentrations (from 0–1 µM), and KiSS1 mRNA levels were determined by RT-PCR analysis as described under Materials and Methods. A concentration of 10 nM E2 strongly up-regulated the expression level of the KiSS1 gene. The intensity values were quantified using {alpha}-Imager software and were normalized to the values of ß-actin mRNA. Significant induction (P < 0.05) is indicated by an asterisk. B, Transient activation of KiSS1 promoter reporter by E2. GT1–7 cells were transfected with full-length human KiSS1 promoter reporter (pGL3–2k), then treated with E2 in different concentrations. Luciferase and ß-galactosidase activities were determined as described in Materials and Methods. Significant induction (P < 0.05) is indicated by an asterisk.

 
To determine whether the transcriptional regulation of KiSS1 is through ER{alpha}-dependent pathways, the ER{alpha} positive cell line GT1–7 and the ER{alpha} negative cell line 293T were transiently transfected with pGL3-hKiSS1 2-kb promoter and pGL3-basic control plasmid with or without ER{alpha} cotransfection, respectively. In 293T cells, E2 treatment increased KiSS1 promoter activity approximately 3-fold in the presence of ER{alpha}, whereas no hormone-induced effect was observed in the cells without ER{alpha}. In the pGL3-basic control group, E2 had no stimulatory effect, despite the presence of ER{alpha} (Fig. 2AGo). In the ER{alpha} positive hypothalamic GT1–7 cell line, E2 responsive effects of the KiSS1 promoter were observed without exogenous ER{alpha}, as well as with the pGL3–3xERE positive control, but not with the pGL3-basic negative control. When exogenous ER{alpha} was cotransfected, KiSS1 promoter activity increased about 3-fold in the presence of E2 (Fig. 2BGo). These data suggest that ER{alpha} plays a pivotal role in hormone-induced KiSS1 promoter.


Figure 2
View larger version (17K):
[in this window]
[in a new window]

  		FIG. 2. ER{alpha} is necessary for hormone responsiveness of KiSS1 promoter. ER{alpha}-negative cell line 293T (A) and positive cell line GT1–7 (B) were transfected with full-length KiSS1 promoter and pCDNA-ER{alpha} or pcDNA plasmids and treated with E2 (E) or without E2 [dimethyl sulfoxide (DMSO)]. pGL3-Basic and pGL3–3xERE plasmids were used as negative and positive control of hormone inductions. Luciferase and ß-galactosidase activities were determined as described under Materials and Methods. Results are expressed as means ± SD for at least three replicate determinations for each treatment group, and significant induction (P < 0.01) is indicated with an asterisk.

 
Analysis of human KiSS1 promoter and identification of E2 responsive region.
The full-length human KiSS1 promoter sequence was submitted to the Transcription Element Search Software (http://www.cbil.upenn.edu/cgi-bin/tess/tess) and analyzed. The analysis of E2 response-related motifs demonstrated the presence of three half-palindromic EREs in positions –1587, –468, and –198, and two AP1 binding sites in positions –1272 and –418 (Fig. 3Go). In addition, the human KiSS1 promoter has a high GC-content, and multiple GC-rich Sp1 binding sites were predicted as shown in Fig. 3Go. At –190 bp to the transcriptional initiation site, there are four potential Sp1 binding sites, one of which was identified as a functional binding site responsible for vitamin D receptor interacting protein-130 (DRIP-130) mediated transcriptional activation (33).


Figure 3
View larger version (13K):
[in this window]
[in a new window]

  		FIG. 3. Deletion analysis of human KiSS1 promoter for hormone (E2) regulation. 293T cells were transiently transfected with various human KiSS1 promoter deletion constructs together with 500 ng ER{alpha} plasmid, and treated with the ethanol vehicle or 10 nM E2. The –190 region of the KiSS1 promoter contains the binding sites for hormone (E2) regulation. KiSS1-luciferase activity was determined as described in Materials and Methods. Results are expressed as means ± SD for at least three replicate determinations for each treatment group, and significant (P < 0.01) induction is indicated with an asterisk. DMSO, Dimethyl sulfoxide.

 
To investigate which region of the human KiSS1 promoter was responsible for the stimulatory effect of E2 mediated by ER{alpha}, truncated deletions of the KiSS1 promoter were performed. The deletion plasmids were transfected into 293T cells together with pcDNA-ER{alpha}. The deletion of the region from –2000 to –1000 did not affect either the basal or hormone-induced promoter activity by E2 (Fig. 3Go). Although the basal promoter activity dramatically decreased after –1000 to –500 bp was ablated, the deletion did not significantly affect hormone stimulation (P < 0.05). Similar results were observed when the –500 to –190-bp region was deleted. Strikingly, the activity of the proximal –45-bp promoter lost almost all the basal promoter activity as well as the hormone-induced activity (Fig. 3Go). These results suggest that the proximal –190 to –45-bp region of the human KiSS1 promoter is responsible for both basal promoter activity and E2-induced KiSS1 up-regulation. Because the putative half-ERE and Ap1 sites are not in this region, the data suggest that one or more of the proximal Sp1 binding sites may be very important in response to E2/ER{alpha}-induced KiSS1 transcriptional regulation.

SP proteins modulate KiSS1 promoter activity and mediate hormone responsiveness.
To analyze the contribution of Sp1 to the activity of the KiSS1 promoter, 293T cells were cotransfected with truncated promoter reporters and different amounts of pcDNA-Sp1 construct. Overexpression of Sp1 dramatically increased the activity of pGL3–190 promoter, which contains three Sp1 binding sites, however, no significant changes were observed in the group cotransfected with the Sp1 binding site-free promoter pGL3–45 and pcDNA-Sp1 (Fig. 4AGo). The induction of KiSS1 promoter activation was dependent on the amount of transfected Sp1 construct. Even at a very low concentration of pcDNA-Sp1 (0.01 µg) (Fig. 4AGo), the promoter activity was stimulated 3-fold compared with the control group.


Figure 4
View larger version (27K):
[in this window]
[in a new window]

  		FIG. 4. Roles of the Sp1/Sp3 sites in the proximal region of human KiSS1 promoter in estrogen-mediated KiSS1 regulation. A, Sp1 protein activates KiSS1 transcriptional activity through the Sp1 sites located between –190 and –45 bp of KiSS1 promoter. The 293T cells were transfected with 190- and 45-bp human KiSS1 promoter constructs, together with increasing amounts of pcDNA-Sp1 plasmids, respectively. Deletion of the Sp1 sites between –190 and –45 bp completely abolished the Sp1-regulated activity. Luciferase activity was determined as described in Materials and Methods. B, Sp3 attenuated Sp1 induced KiSS1 promoter activity. 293T cells were transfected with 190-bp KiSS1 promoter with Sp1 and Sp3 or a dominant-negative Sp3 constructs. The amount of each plasmid DNA was listed. Luciferase activity was determined as described in Materials and Methods. C, Sp1 sites at –87 to –80 are essential for hormone regulation of KiSS1 promoter by mutation (mut) analysis. 293T cells were cotransfected with ER{alpha} and mutated 190-bp human KiSS1 promoter constructs. Hormone-induced activity was determined as described in Materials and Methods, and significant (P < 0.01) induction is indicated by an asterisk. DMSO, Dimethyl sulfoxide; WT, wild type.

 
Sp1 and Sp3 are the ubiquitously expressed members in the SP protein family, and usually function together through dimerization either synergistically or antagonistically by regulating target gene expression (29, 30, 31, 38). In some cases the ratio of Sp1 to Sp3 is the key mechanism to regulate downstream gene expression (29, 39, 40). Our data showed that increasing the Sp1 to Sp3 ratio by overexpression Sp1 in 293T cells resulted in transactivation of KiSS1 promoter activity (Fig. 4Go, A and B), whereas the promoter activity was repressed when decreasing the Sp1 to Sp3 ratio by increasing the exogenous amount of Sp3 but not dominant-negative Sp3 constructs (Fig. 4BGo). When both the full-length (data not shown) and 190-bp reporter plasmids were cotransfected with a fixed amount of Sp1 expression vector and increasing amounts of Sp3 expression plasmid, Sp1 induction of KiSS1 transcription was significantly repressed by Sp3 (Fig. 4AGo).

To identify further the specific Sp1 binding site that contributes to the hormonal induction, site-directed mutagenesis was performed to disturb the individual Sp1 binding sites. Wild-type or mutant pGL3–190 promoter reporters were cotransfected with pcDNA-ER{alpha} into 293T cells and left unstimulated or treated with E2. Individual mutations of all Sp1 sites reduced the promoter activity in unstimulated cells by about 40% compared with wild-type promoter, whereas double mutations of the Sp1 sites (–87 and –80) resulted in 70% loss of promoter activity compared with the wild-type promoter (Fig. 4CGo). Hormone-induced promoter activity did not change in the constructs containing the Sp1 site mutations at –188 or –175 Sp1 sites. However, the hormonal responsiveness of KiSS1 promoters with individual Sp1 mutations at –87 or –80 Sp1 sites showed significant reduction, and double mutations of these two Sp1 sites resulted in ablation of hormonal responsiveness (Fig. 4CGo). These data suggest that all the Sp1 binding sites contribute to the basal promoter activity of KiSS1, and the –87 and –80 Sp1 sites function together to be responsible for E2 stimulation.

Sp proteins directly bind to KiSS1 promoter on multiple sites.
Sp1 stimulates activation of the KiSS1 promoter and mediates hormone-induced KiSS1 expression, however, whether this occurs directly through direct DNA binding of the KiSS1 promoter or indirectly remains unclear. To answer this question, EMSAs were performed using individual Sp1 binding site DNA sequences found in the KiSS1 promoter. As shown in Fig. 5Go, nuclear extracts from ER{alpha}-positive MCF-7 and GT1–7 cell lines bound to a [{gamma}32-P]-labeled probe and showed several bands of which the intensity was decreased by incubation of a 100-fold excessive unlabeled probe. A major low mobility Sp1-[{gamma}32P]Sp1 complex (indicated with an arrow) was observed, and this band was unaffected by nonspecific IgG antibody (Fig. 5Go, A and B, lane 4). However, the complex was super-shifted with addition of specific Sp1 antibody (Fig. 5Go, A and B, lane 5). In addition, by using a Sp3-specific antibody, a weak super-shift band was observed, and the intensity of the major complex was decreased, indicating competitive binding of the probe to the Sp3 protein complex (Fig. 5Go, A and B, lane 6). When both Sp1 and Sp3 antibodies were used in the reaction, the intensity of the major complex was dramatically decreased, and the lower Sp3-[32P]Sp1 complex disappeared (Fig. 5Go, A and B, lane 7). Together, these results suggest that Sp1 and Sp3 proteins directly bind to the Sp1 binding sites located on the proximal region of human KiSS1 promoter.


Figure 5
View larger version (75K):
[in this window]
[in a new window]

  		FIG. 5. Identification of Sp1 and Sp3 binding to the GC-rich Sp1 sites of KiSS1 promoter by EMSAs. Nuclear extracts from MCF-7 cells (A, lanes 1–7) and GT1–7 cells (B, lanes 8–14) were incubated with [32P]radiolabeled –87 Sp1 oligonucleotides (lanes 2 and 9) or competed with 100x unlabeled cold competitor (lanes 3 and 10), nonspecific IgG (lanes 4 and 11), Sp1 antibody (lanes 5 and 12), Sp3 antibody (lanes 6 and 13), or both Sp1 and Sp3 antibodies (A and B, lane 7), as described in Materials and Methods. Oligo sequence used for EMSA is GTCTGAGGAGGAGGGAGGGGCGGCAGAGTGG. Results in these assays are from triplicate experiments. F.P., Free probe; ND, not determined.

 
ER{alpha} complexes with Sp proteins to interact with KiSS1 promoter.
To determine which domain of ER{alpha} is essential for estrogen-induced KiSS1 expression, we cotransfected pGL3–190 luciferase reporter with ER{alpha} and its mutant constructs containing deletions either at the DNA-binding domain (HE11) or at the activation function 1 domain (HE19) into the cells. As shown in Fig. 6AGo, induction of pGL3–190 reporter gene activity by E2 was observed in cells cotransfected with ER{alpha} or HE11, but not in cells cotransfected with a deletion mutant of the activation domain of ER{alpha}. These data suggest that ER{alpha}-induced KiSS1 expression is not mediated through the DNA-binding domain but through the activation function domain of ER{alpha}. Previous publications have demonstrated that Sp1 and Sp3 interact with ER{alpha} through the zinc finger domain (41, 42). Thus, we hypothesize that E2-activated ER{alpha} binds to Sp proteins to form a complex and to stimulate KiSS1 promoter activity. To confirm this hypothesis, we examined the direct interaction of ER{alpha} with Sp1 in the cells using immunoprecipitation (Fig. 6BGo). As shown in Fig. 6BGo, protein complexes immunoprecipitated by Sp1 contain ER{alpha} (Fig. 6BGo, bottom). To confirm further the complex formation of ER{alpha} and Sp proteins and their association with the human KiSS1 promoter, we performed ChIP assays (43). Cells were treated with E2 at different time points and then cross-linked with formaldehyde. Nuclear extracts were sonicated and incubated with Sp1, Sp3, or ER{alpha} antibody, respectively. DNA-protein complexes were precipitated, and DNA was purified. The interactions of proteins with the human KiSS1 promoter were determined by PCR directed to the –230 to +1 region of the promoter. As shown in Fig. 6CGo, Sp1 and Sp3 proteins form complexes with the KiSS1 promoter in MCF-7 cells in the presence or absence of E2. On the other hand, the complex formation of ER{alpha} on the KiSS1 promoter is dependent on the stimulation of the cells by E2, and ER{alpha} did not bind to KiSS1 promoter in untreated cells (Fig. 6CGo, time 0). However, in the presence of E2, the interaction of ER{alpha} with the KiSS1 promoter was increased over time.


Figure 6
View larger version (47K):
[in this window]
[in a new window]

  		FIG. 6. Analysis of ER{alpha}/Sp1 interaction and the association of the ER/Sp protein complex with KiSS1 promoter. A, Role of wild-type and deletion mutants of ER{alpha} on the activation of KiSS1 promoter. 293T cells were cotransfected with the 190-bp hKiSS1 promoter and wild-type (WT) or mutant ER{alpha} constructs. Hormone-induced activity was determined as described in Materials and Methods, and significant (P < 0.01) induction is indicated by an asterisk. B, Interaction of ER{alpha} with Sp1 protein in vivo using IP assays. The 293T cells were cotransfected with Sp1 and ER{alpha} or control vector. Cells were harvested after E2 treatment. Protein complex was immunoprecipitated by specific antibody against Sp1 antibody and followed by Western blot with ER{alpha}-specific antibody, as described in Materials and Methods. C, ChIP analysis of ER{alpha}-Sp protein complexes with KiSS1 promoter region. MCF-7 cells were treated with 100 nM E2 for different time points, harvested, and processed. After IP of the cross-linked complexes, the chromatin was analyzed by PCR. Primers amplified a 230-bp region ranging from –230 to +1, as described in Materials and Methods, and comparable results were observed in at least two replicate experiments. Single PCR products were obtained for all antibodies, and the identity of these bands was confirmed by sequencing (data not shown). DMSO, Dimethyl sulfoxide; WB, Western blot.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It has long been believed that the onset of puberty in mammals is coupled to changes in hormone levels as mediated by the HPG axis, which is first triggered by a pulsatile release of GnRH. Recent studies on GPR54 mutations in humans and the targeted deletion in mice link this G protein-coupled receptor to idiopathic hypogonadotropic hypogonadism, which is caused by a deficiency of GnRH (2, 3). Administration of KiSS1 induced LH and FSH secretion through GnRH-dependent pathway (5, 8, 10, 44). Like other hypothalamic and pituitary hormones, the expression of KiSS1 has recently been regulated by sex steroids such as estrogen and androgen through unknown feedback regulation (18, 45). In female mice, OVX results in a significant increase of total hypothalamic content of KiSS1 mRNA, which was reversed by E2 replacement (46). However, recent studies suggest that the regulation by E2 is different in various regions of the forebrain because KiSS1 mRNA was stimulated by E2/ER{alpha} in the anteroventral periventricular nucleus (AVPV) and the preoptic periventricular nucleus (PeN) but repressed in the arcuate nucleus (Arc). In this study we found that E2 elevated KiSS1 mRNA expression in ER-positive hypothalamic cell lines and stimulated luciferase-conjugated KiSS1 promoter activity. EMSAs found that the conserved half-ERE site is not responsive to E2 induction, whereas the four proximal GC-rich Sp1 binding sites at –188 to –87 of the KiSS1 promoter are essential for both basal promoter activity and E2-mediated hormone responsiveness. The binding of Sp1 and ER{alpha} to the KiSS1 promoter was confirmed by EMSA and ChIP assay. Sp1 and Sp3 are ubiquitously expressed transcription factors recognizing the same GC-rich motif. Sp proteins function either as transcriptional activators or repressors to regulate downstream gene expression. In some cases Sp1 and Sp3 function antagonistically in the complex (47). Decreasing Sp1 to Sp3 ratios negatively regulates KiSS1 promoter activity. The expression ratio of Sp1 and Sp3 proteins may vary from different tissue or cell types in the forebrain, which may be one of the reasons why the distribution of KiSS1 is not equal in different regions.

GT1–7 cells are GnRH and KiSS1 positive cells. Because KiSS1 activator GnRH secretion through an unknown mechanism, many efforts have been made to determine how KiSS1 regulates GnRH expression in GT1–7 cells and in hypothalamic explants (48). Recent data showed KiSS1 stimulated GnRH secretion in the explants, but not in the GT1–7 cell line (48, 49). Our unpublished data also showed that kisspeptin-10 failed to activate GnRH promoter reporter activity in GT1–7 cells, suggesting that the immortalized GT1–7 cell line is not a good cell model for studying the control of KiSS1 on GnRH releasing in vivo. These data may elucidate why GnRH in GT1–7 cells was negatively regulated by E2, whereas KiSS1 was positively regulated because GnRH may not be the direct target of KiSS1 in these cells.

Estrogen receptors are hormone-induced transcriptional factors that regulate gene transcription through direct DNA binding or through recruitment of other transcription factors such as Sp1 and Sp3 (22). Our data demonstrate that E2 up-regulated KiSS1 expression through an ER{alpha}/Sp protein complex at the molecular level and confirmed the in vivo data by other groups that E2 increased KiSS1 transcription in AVPV (17). However, a discrepancy remains regarding how estrogen regulates the expression of KiSS1 in different regions of the brain, such as in Arc and PeN. There are multiple possibilities for the observed differences. First, ER{alpha} may associate with a coactivator in AVPV and PeN, resulting in up-regulation of KiSS1 mRNA; on the other hand, ER{alpha} could also associate with a co-repressor in Arc, leading to negative regulation of KiSS1 expression (Fig. 7Go). In addition, the differential regulation of KiSS1 expression by E2 in different brain regions can be achieved by the different ratio of Sp1 to Sp3 in the protein complexes in different nuclei of the forebrain. In AVPV and PeN, higher ratios of Sp1 in the complex may mediate positive hormone responsiveness, whereas in Arc higher ratios of Sp3 in the complex may repress hormone-induced transcription of KiSS1 expression. The exact molecular mechanism will be tested in our future studies.


Figure 7
View larger version (21K):
[in this window]
[in a new window]

  		FIG. 7. Proposed model of regulation for KiSS1 gene expression by hormone (E2) via ER{alpha}/Sp protein complexes. Without hormone stimulation, Sp1 and Sp3 proteins form dimers that bind to the GC-rich motif of the promoter and stably stimulate KiSS1 expression. With the induction by E2, ER{alpha} is activated after binding with the ligand and associates with coactivators or co-repressors in different regions in the hypothalamus. The ligand-receptor-cofactor complex is then recruited to the most proximal Sp1/Sp3 binding sites, where it either activates or represses KiSS1 expression, depending on the types of interacting cofactors.

 
KiSS1 has been identified as a novel hormone that regulates puberty onset and as a tumor metastasis suppressor (8, 9, 10, 11, 14, 50, 51, 52). We recently have shown that KiSS1 can be regulated at the transcriptional level by AP-2, Sp1, and its cofactors in different tumor cells (33, 34). Our current studies on transcriptional regulation of KiSS1 are very important in understanding how puberty is triggered and how steroid hormone feedback works within the HPG axis. To our knowledge, the present work is the first study to identify molecular mechanisms that regulate the expression of KiSS1 at the transcriptional level in the HPG axis. More studies will be needed to determine which cofactors are recruited in the ER{alpha}/Sp complex, and/or what is the critical ratio of Sp1 and Sp3 that mediates the positive/negative hormone responsiveness of KiSS1 expression.


   Acknowledgments
 
We thank Dr. Stephen Safe at the Institute of Biosciences and Technology, Texas A&M Health Science Center for Sp1, estrogen receptor {alpha}-constructs, and related reagents, and Dr. Pam L. Mellon (University of California San Diego, La Jolla, CA) for providing the GT1–7 cells.


   Footnotes
 
This work was supported partially by Grants 1R01CA106479 and 5R01HL064792 (to M.L.) from the National Institutes of Health, a collaborative grant from Shanghai Education Commission, a predoctoral fellowship from Department of Defense Breast Cancer Research Program (W81XWH-05-1-0353; to S.-G.C.), and a grant from the National Basic Research Program of China (2005 CB522505; to X.W.).

Disclosure Summary: The authors have nothing to declare.

First Published Online July 26, 2007

Abbreviations: Arc, Arcuate nucleus; AVPV, anteroventral periventricular nucleus; ChIP, chromatin immunoprecipitation; E2, estradiol; ER{alpha}, estrogen receptor {alpha}; ERE, estrogen response element; FBS, fetal bovine serum; GPR54, G protein-coupled receptor-54; HPG, hypothalamic-pituitary-gonadal; IP, immunoprecipitation; OVX, ovariectomy; PeN, preoptic periventricular nucleus.

Received February 2, 2007.

Accepted for publication July 13, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Terasawa E, Fernandez DL 2001 Neurobiological mechanisms of the onset of puberty in primates. Endocr Rev 22:111–151[Abstract/Free Full Text]
  2. de Roux N, Genin E, Carel JC, Matsuda F, Chaussain JL, Milgrom E 2003 Hypogonadotropic hypogonadism due to loss of function of the KiSS1-derived peptide receptor GPR54. Proc Natl Acad Sci USA 100:10972–10976[Abstract/Free Full Text]
  3. Seminara SB, Messager S, Chatzidaki EE, Thresher RR, Acierno Jr JS, Shagoury JK, Bo-Abbas Y, Kuohung W, Schwinof KM, Hendrick AG, Zahn D, Dixon J, Kaiser UB, Slaugenhaupt SA, Gusella JF, O’Rahilly S, Carlton MB, Crowley Jr WF, Aparicio SA, Colledge WH 2003 The GPR54 gene as a regulator of puberty. N Engl J Med 349:1614–1627[Abstract/Free Full Text]
  4. Pompolo S, Pereira A, Estrada KM, Clarke IJ 2006 Colocalization of kisspeptin and gonadotropin-releasing hormone in the ovine brain. Endocrinology 147:804–810[Abstract/Free Full Text]
  5. Irwig MS, Fraley GS, Smith JT, Acohido BV, Popa SM, Cunningham MJ, Gottsch ML, Clifton DK, Steiner RA 2004 Kisspeptin activation of gonadotropin releasing hormone neurons and regulation of KiSS-1 mRNA in the male rat. Neuroendocrinology 80:264–272[CrossRef][Medline]
  6. Matsui H, Takatsu Y, Kumano S, Matsumoto H, Ohtaki T 2004 Peripheral administration of metastin induces marked gonadotropin release and ovulation in the rat. Biochem Biophys Res Commun 320:383–388[CrossRef][Medline]
  7. Muir AI, Chamberlain L, Elshourbagy NA, Michalovich D, Moore DJ, Calamari A, Szekeres PG, Sarau HM, Chambers JK, Murdock P, Steplewski K, Shabon U, Miller JE, Middleton SE, Darker JG, Larminie CG, Wilson S, Bergsma DJ, Emson P, Faull R, Philpott KL, Harrison DC 2001 AXOR12, a novel human G protein-coupled receptor, activated by the peptide KiSS-1. J Biol Chem 276:28969–28975[Abstract/Free Full Text]
  8. Gottsch ML, Cunningham MJ, Smith JT, Popa SM, Acohido BV, Crowley WF, Seminara S, Clifton DK, Steiner RA 2004 A role for kisspeptins in the regulation of gonadotropin secretion in the mouse. Endocrinology 145:4073–4077[Abstract/Free Full Text]
  9. Navarro VM, Castellano JM, Fernandez-Fernandez R, Tovar S, Roa J, Mayen A, Nogueiras R, Vazquez MJ, Barreiro ML, Magni P, Aguilar E, Dieguez C, Pinilla L, Tena-Sempere M 2005 Characterization of the potent luteinizing hormone-releasing activity of KiSS-1 peptide, the natural ligand of GPR54. Endocrinology 146:156–163[Abstract/Free Full Text]
  10. Shahab M, Mastronardi C, Seminara SB, Crowley WF, Ojeda SR, Plant TM 2005 Increased hypothalamic GPR54 signaling: a potential mechanism for initiation of puberty in primates. Proc Natl Acad Sci USA 102:2129–2134[Abstract/Free Full Text]
  11. Seminara SB, Kaiser UB 2005 New gatekeepers of reproduction: GPR54 and its cognate ligand, KiSS-1. Endocrinology 146:1686–1688[Free Full Text]
  12. Lapatto R, Pallais JC, Zhang D, Chan YM, Mahan A, Cerrato F, Wei Le W, Hoffman GE, Seminara SB 2007 Kiss1–/– mice exhibit more variable hypogonadism than Gpr54–/– mice. 26 June 2007 Endocrinology 10.1210/en.2007-0078
  13. d’Anglemont de Tassigny X, Fagg LA, Dixon JP, Day K, Leitch HG, Hendrick AG, Zahn D, Franceschini I, Caraty A, Carlton MB, Aparicio SA, Colledge WH 2007 Hypogonadotropic hypogonadism in mice lacking a functional Kiss1 gene. Proc Natl Acad Sci USA 104:10714–10719[Abstract/Free Full Text]
  14. Stafford LJ, Xia C, Ma W, Cai Y, Liu M 2002 Identification and characterization of mouse metastasis-suppressor KiSS1 and its G-protein-coupled receptor. Cancer Res 62:5399–5404[Abstract/Free Full Text]
  15. Terasawa E 1985 Developmental changes in the positive feedback effect of estrogen on luteinizing hormone release in ovariectomized female rhesus monkeys. Endocrinology 117:2490–2497[Abstract/Free Full Text]
  16. Lania A, Gangi E, Romoli R, Losa M, Travaglini P, Meringolo D, Ambrosi B, Faglia G, Beck-Peccoz P, Spada A 2002 Impaired estrogen-induced negative feedback on gonadotropin secretion in patients with gonadotropin-secreting and nonfunctioning pituitary adenomas. Eur J Clin Invest 32:335–340[CrossRef][Medline]
  17. Smith JT, Cunningham MJ, Rissman EF, Clifton DK, Steiner RA 2005 Regulation of Kiss1 gene expression in the brain of the female mouse. Endocrinology 146:3686–3692[Abstract/Free Full Text]
  18. Estrada KM, Clay CM, Pompolo S, Smith JT, Clarke IJ 2006 Elevated KiSS-1 expression in the arcuate nucleus prior to the cyclic preovulatory gonadotrophin-releasing hormone/lutenising hormone surge in the ewe suggests a stimulatory role for kisspeptin in oestrogen-positive feedback. J Neuroendocrinol 18:806–809[CrossRef][Medline]
  19. Kuohung W, Kaiser UB 2006 GPR54 and KiSS-1: role in the regulation of puberty and reproduction. Rev Endocr Metab Disord 7:257–263[CrossRef][Medline]
  20. Lonard DM, O’Malley BW 2006 The expanding cosmos of nuclear receptor coactivators. Cell 125:411–414[CrossRef][Medline]
  21. Fleming JG, Spencer TE, Safe SH, Bazer FW 2006 Estrogen regulates transcription of the ovine oxytocin receptor gene through GC-rich SP1 promoter elements. Endocrinology 147:899–911[CrossRef][Medline]
  22. Safe S, Kim K 2004 Nuclear receptor-mediated transactivation through interaction with Sp proteins. Prog Nucleic Acid Res Mol Biol 77:1–36[Medline]
  23. Khan S, Abdelrahim M, Samudio I, Safe S 2003 Estrogen receptor/Sp1 complexes are required for induction of cad gene expression by 17ß-estradiol in breast cancer cells. Endocrinology 144:2325–2335[Abstract/Free Full Text]
  24. Suske G 1999 The Sp-family of transcription factors. Gene 238:291–300[CrossRef][Medline]
  25. Cawley S, Bekiranov S, Ng HH, Kapranov P, Sekinger EA, Kampa D, Piccolboni A, Sementchenko V, Cheng J, Williams AJ, Wheeler R, Wong B, Drenkow J, Yamanaka M, Patel S, Brubaker S, Tammana H, Helt G, Struhl K, Gingeras TR 2004 Unbiased mapping of transcription factor binding sites along human chromosomes 21 and 22 points to widespread regulation of noncoding RNAs. Cell 116:499–509[CrossRef][Medline]
  26. Wang W, Dong L, Saville B, Safe S 1999 Transcriptional activation of E2F1 gene expression by 17ß-estradiol in MCF-7 cells is regulated by NF-Y-Sp1/estrogen receptor interactions. Mol Endocrinol 13:1373–1387[Abstract/Free Full Text]
  27. Wang F, Wang W, Safe S 1999 Regulation of constitutive gene expression through interactions of Sp1 protein with the nuclear aryl hydrocarbon receptor complex. Biochemistry 38:11490–11500[CrossRef][Medline]
  28. Krishnan V, Wang X, Safe S 1994 Estrogen receptor-Sp1 complexes mediate estrogen-induced cathepsin D gene expression in MCF-7 human breast cancer cells. J Biol Chem 269:15912–15917[Abstract/Free Full Text]
  29. Pang RT, Lee LT, Ng SS, Yung WH, Chow BK 2004 CpG methylation and transcription factors Sp1 and Sp3 regulate the expression of the human secretin receptor gene. Mol Endocrinol 18:471–483[Abstract/Free Full Text]
  30. Magee C, Nurminskaya M, Faverman L, Galera P, Linsenmayer TF 2005 SP3/SP1 transcription activity regulates specific expression of collagen type X in hypertrophic chondrocytes. J Biol Chem 280:25331–25338[Abstract/Free Full Text]
  31. Wooten LG, Ogretmen B 2005 Sp1/Sp3-dependent regulation of human telomerase reverse transcriptase promoter activity by the bioactive sphingolipid ceramide. J Biol Chem 280:28867–28876[Abstract/Free Full Text]
  32. Nelson SB, Lawson MA, Kelley CG, Mellon PL 2000 Neuron-specific expression of the rat gonadotropin-releasing hormone gene is conferred by interactions of a defined promoter element with the enhancer in GT1–7 cells. Mol Endocrinol 14:1509–1522[Abstract/Free Full Text]
  33. Mitchell DC, Stafford LJ, Li D, Bar-Eli M, Liu M 2006 Transcriptional regulation of KiSS-1 gene expression in metastatic melanoma by specificity protein-1 and its coactivator DRIP-130. Oncogene 26:1739–1747[CrossRef][Medline]
  34. Mitchell DC, Abdelrahim M, Weng J, Stafford LJ, Safe S, Bar-Eli M, Liu M 2006 Regulation of KiSS-1 metastasis suppressor gene expression in breast cancer cells by direct interaction of transcription factors activator protein-2alpha and specificity protein-1. J Biol Chem 281:51–58[Abstract/Free Full Text]
  35. Lee J, Safe S 2007 Coactivation of estrogen receptor alpha (ER alpha)/Sp1 by vitamin D receptor interacting protein 150 (DRIP150). Arch Biochem Biophys 461:200–210[CrossRef][Medline]
  36. Porter W, Wang F, Wang W, Duan R, Safe S 1996 Role of estrogen receptor/Sp1 complexes in estrogen-induced heat shock protein 27 gene expression. Mol Endocrinol 10:1371–1378[Abstract/Free Full Text]
  37. Mellon PL, Windle JJ, Goldsmith PC, Padula CA, Roberts JL, Weiner RI 1990 Immortalization of hypothalamic GnRH neurons by genetically targeted tumorigenesis. Neuron 5:1–10[CrossRef][Medline]
  38. Osawa H, Yamada K, Onuma H, Murakami A, Ochi M, Kawata H, Nishimiya T, Niiya T, Shimizu I, Nishida W, Hashiramoto M, Kanatsuka A, Fujii Y, Ohashi J, Makino H 2004 The G/G genotype of a resistin single-nucleotide polymorphism at –420 increases type 2 diabetes mellitus susceptibility by inducing promoter activity through specific binding of Sp1/3. Am J Hum Genet 75:678–686[CrossRef][Medline]
  39. Chadjichristos C, Ghayor C, Kypriotou M, Martin G, Renard E, Ala-Kokko L, Suske G, de Crombrugghe B, Pujol JP, Galera P 2003 Sp1 and Sp3 transcription factors mediate interleukin-1 beta down-regulation of human type II collagen gene expression in articular chondrocytes. J Biol Chem 278:39762–39772[Abstract/Free Full Text]
  40. Ghayor C, Chadjichristos C, Herrouin JF, Ala-Kokko L, Suske G, Pujol JP, Galera P 2001 Sp3 represses the Sp1-mediated transactivation of the human COL2A1 gene in primary and de-differentiated chondrocytes. J Biol Chem 276:36881–36895[Abstract/Free Full Text]
  41. Porter W, Saville B, Hoivik D, Safe S 1997 Functional synergy between the transcription factor Sp1 and the estrogen receptor. Mol Endocrinol 11:1569–1580[Abstract/Free Full Text]
  42. Stoner M, Wang F, Wormke M, Nguyen T, Samudio I, Vyhlidal C, Marme D, Finkenzeller G, Safe S 2000 Inhibition of vascular endothelial growth factor expression in HEC1A endometrial cancer cells through interactions of estrogen receptor alpha and Sp3 proteins. J Biol Chem 275:22769–22779[Abstract/Free Full Text]
  43. Weinmann AS, Bartley SM, Zhang T, Zhang MQ, Farnham PJ 2001 Use of chromatin immunoprecipitation to clone novel E2F target promoters. Mol Cell Biol 21:6820–6832[Abstract/Free Full Text]
  44. Plant TM, Ramaswamy S, Dipietro MJ 2006 Repetitive activation of hypothalamic G protein-coupled receptor 54 with intravenous pulses of kisspeptin in the juvenile monkey (Macaca mulatta) elicits a sustained train of gonadotropin-releasing hormone discharges. Endocrinology 147:1007–1013[Abstract/Free Full Text]
  45. Smith JT, Dungan HM, Stoll EA, Gottsch ML, Braun RE, Eacker SM, Clifton DK, Steiner RA 2005 Differential regulation of KiSS-1 mRNA expression by sex steroids in the brain of the male mouse. Endocrinology 146:2976–2984[Abstract/Free Full Text]
  46. Blondet A, Gout J, Durand P, Begeot M, Naville D 2005 Expression of the human melanocortin-4 receptor gene is controlled by several members of the Sp transcription factor family. J Mol Endocrinol 34:317–329[Abstract/Free Full Text]
  47. Lee LT, Tan-Un KC, Pang RT, Lam DT, Chow BK 2004 Regulation of the human secretin gene is controlled by the combined effects of CpG methylation, Sp1/Sp3 ratio, and the E-box element. Mol Endocrinol 18:1740–1755[Abstract/Free Full Text]
  48. Nazian SJ 2006 Role of metastin in the release of gonadotropin-releasing hormone from the hypothalamus of the male rat. J Androl 27:444–449[Abstract/Free Full Text]
  49. Castellano JM, Navarro VM, Fernandez-Fernandez R, Castano JP, Malagon MM, Aguilar E, Dieguez C, Magni P, Pinilla L, Tena-Sempere M 2006 Ontogeny and mechanisms of action for the stimulatory effect of kisspeptin on gonadotropin-releasing hormone system of the rat. Mol Cell Endocrinol 257–258:75–83
  50. Lee JH, Miele ME, Hicks DJ, Phillips KK, Trent JM, Weissman BE, Welch DR 1996 KiSS-1, a novel human malignant melanoma metastasis-suppressor gene. J Natl Cancer Inst 88:1731–1737[Abstract/Free Full Text]
  51. Bilban M, Ghaffari-Tabrizi N, Hintermann E, Bauer S, Molzer S, Zoratti C, Malli R, Sharabi A, Hiden U, Graier W, Knofler M, Andreae F, Wagner O, Quaranta V, Desoye G 2004 Kisspeptin-10, a KiSS-1/metastin-derived decapeptide, is a physiological invasion inhibitor of primary human trophoblasts. J Cell Sci 117:1319–1328[Abstract/Free Full Text]
  52. Ohtaki T, Shintani Y, Honda S, Matsumoto H, Hori A, Kanehashi K, Terao Y, Kumano S, Takatsu Y, Masuda Y, Ishibashi Y, Watanabe T, Asada M, Yamada T, Suenaga M, Kitada C, Usuki S, Kurokawa T, Onda H, Nishimura O, Fujino M 2001 Metastasis suppressor gene KiSS-1 encodes peptide ligand of a G-protein-coupled receptor. Nature 411:613–617[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Nucleic Acids ResHome page
D. Li, Z. Niu, W. Yu, Y. Qian, Q. Wang, Q. Li, Z. Yi, J. Luo, X. Wu, Y. Wang, et al.
SMYD1, the myogenic activator, is a direct target of serum response factor and myogenin
Nucleic Acids Res., September 25, 2009; (2009) gkp773v1.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
M. L. Gottsch, V. M. Navarro, Z. Zhao, C. Glidewell-Kenney, J. Weiss, J. L. Jameson, D. K Clifton, J. E. Levine, and R. A. Steiner
Regulation of Kiss1 and Dynorphin Gene Expression in the Murine Brain by Classical and Nonclassical Estrogen Receptor Pathways
J. Neurosci., July 22, 2009; 29(29): 9390 - 9395.
[Abstract] [Full Text] [PDF]

Home page
 J Mol EndocrinolHome page
S. Safe and K. Kim
Non-classical genomic estrogen receptor (ER)/specificity protein and ER/activating protein-1 signaling pathways
J. Mol. Endocrinol., November 1, 2008; 41(5): 263 - 275.
[Abstract] [Full Text] [PDF]

Home page
 Endocr Relat CancerHome page
D Bonofiglio, H Qi, S Gabriele, S Catalano, S Aquila, M Belmonte, and S Ando
Peroxisome proliferator-activated receptor {gamma} inhibits follicular and anaplastic thyroid carcinoma cells growth by upregulating p21Cip1/WAF1 gene in a Sp1-dependent manner
Endocr. Relat. Cancer, June 1, 2008; 15(2): 545 - 557.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Li, D.
Right arrow Articles by Liu, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Li, D.
Right arrow Articles by Liu, M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals