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Molecular Endocrinology, doi:10.1210/me.2008-0035
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Molecular Endocrinology 22 (6): 1449-1463
Copyright © 2008 by The Endocrine Society

Protein Kinase C{alpha}-Induced Derepression of the Human Luteinizing Hormone Receptor Gene Transcription through ERK-Mediated Release of HDAC1/Sin3A Repressor Complex from Sp1 Sites

Mingjuan Liao, Ying Zhang and Maria L. Dufau

Section on Molecular Endocrinology, Endocrinology and Reproduction Research Branch, Program in Developmental Endocrinology and Genetics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-4510

Address all correspondence and requests for reprints to: Maria L. Dufau, Building 49, Room 6A-36, 49 Convent Drive, MSC 4510, National Institutes of Health, Bethesda, Maryland 20892-4510. E-mail: dufaum{at}mail.nih.gov.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
LH receptor (LHR) gene transcription is subject to repression/derepression through various modes and multiple effectors. Epigenetic silencing and activation of the LHR is achieved through coordinated regulation at both histone and DNA levels. The LHR gene is subject to repression by deacetylation and methylation at its promoter region, where a HDAC/mSin3A repressor complex is anchored at Sp1 sites. The present studies revealed that protein kinase C (PKC) {alpha}/ERK signaling is important for the activation of LHR promoter activity, and the increase of endogenous transcripts induced by phorbol-12-myristate-13-acetate (PMA) in HeLa cells. Whereas these effects were attributable to PKC{alpha} activity, the ERK pathway was the downstream effector in LHR activation. PMA caused a significant enhancement of Sp1 phosphorylation at serine residue (s), which was blocked by PKC{alpha} or ERK inhibition. The interaction of activated phosphorylated ERK with Sp1 and ERK’s association with the LHR promoter points to Sp1 as a direct target of ERK. After Sp1 phosphorylation, the HDAC1/mSin3A repressor complex dissociated from Sp1 sites, histone 3 was acetylated, and transcription factor II B and RNA polymerase II were recruited. In addition, overexpression of a constitutively active PKC{alpha} (PKC{alpha} CA) strongly activated LHR transcription in MCF-7 cells (devoid of PKC{alpha}), induced Sp1 phosphorylation at serine residue (s) and caused derecruitment of HDAC1/mSin3A complex from the promoter. These effects were negated by cotransfection of a dominant-negative PKC{alpha}. In conclusion, these studies have revealed a novel regulatory signaling mechanism of transcriptional control in which the LHR is derepressed through PKC{alpha}/ERK-mediated Sp1 phosphorylation, causing the release of HDAC1/mSin3A complex from the promoter.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE LH RECEPTOR (LHR), a member of the G protein-coupled receptor family, has an essential role in sexual development and reproduction. The LHR gene is primarily expressed in gonads but is also found at several nongonadal sites including placenta and breast cancer tissues and cell lines (1, 2).

Characterization of the mechanisms controlling LHR gene transcription has revealed a complex regulatory network of multiple effectors. The basal promoter activity of the TATA-less LHR gene is governed by two activating Sp1/Sp3 binding domains, and an inhibitory nuclear orphan receptor-binding motif (3, 4, 5, 6, 7, 8). Transcriptional activators Sp1 and Sp3 are the anchor site of the HDAC1/2/mSin3A repressor complex within the LHR gene promoter. The LHR gene transcription is also subject to an epigenetic mechanism, whereby the coordinated changes of histone modification status and cytosine-phosphate-guanine dinucleotide island methylation state within its gene promoter region are required for gradations of silencing and derepression of this gene in human choriocarcinoma JAR and MCF-7 breast cancer cells (9, 10). The proximal Sp1/Sp3 binding site is required to mediate the histone deacetylase (HDAC) inhibitor trichostatin A (TSA)-induced activation of the LHR gene. Sp1 but not Sp3 was identified as the key participant and Sp1 phosphorylation triggered the LHR gene induction by TSA (11). The phosphatidylinositol 3-kinase and protein kinase C{zeta} (PI3K/PKC{zeta}) cascade has been shown to catalyze Sp1 phosphorylation at serine 641 upon TSA treatment, which in turn evokes the release of corepressor protein p107 from the LHR gene promoter and consequently gene activation (11).

Phosphorylation of transcription factors by various kinases in response to exogenous stimuli and environmental cues has been recognized as an efficient mechanism for fine tuning expression of diverse target genes (12, 13, 14). Among the kinases studied, the PKC family containing at least 12 isoforms with different physiological characteristics exhibits significant importance for various biological processes (15, 16, 17). In ovarian tissues, high level activity of the conventional and novel PKC isoforms ({alpha}, β1, β2, {gamma}, and {delta}), whose activation depends on calcium, phospholipid or diaceylglycerol (DAG), has been observed (18, 19, 20). Inhibition of the PKC activity in rat granulosa cells was found to contribute to the suppression of gonadotropin-induced ovulation (21). Dynamic changes of the PKC protein levels were also correlated to the ovarian follicular differentiation into corpora lutea (22). Furthermore, increasing body of evidence has indicated the dependence of PKCs in mediating signal transduction of GnRH, the first key hormone of the reproductive axis in mammals (23, 24, 25, 26, 27). PKCs are also the important regulator for GnRH receptor gene transcription (28, 29, 30). However, the molecular mechanism(s) participating in the transcriptional control of the LHR by conventional/novel PKCs has not previously addressed.

By the use of phorbol-12-myristate-13-acetate (PMA) to mimic DAG’s endogenous activating effect on the conventional/novel PKCs, we have identified the LH receptor gene as a target regulated by PKC{alpha} in HeLa cells. These findings have revealed a novel molecular mechanism in the regulation of the LHR gene, whereby its activation is achieved through dissociation of the HDAC1/mSin3A inhibitory complex in a PKC{alpha}/ERK-induced Sp1 phosphorylation-dependent manner.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Treatment of PMA Increases Transcription of the human LHR (hLHR) Gene through PKC{alpha}
To investigate whether hLHR gene activation/expression is regulated by PKC, PMA was used to induce the activities of PKCs in HeLa cells transfected with hLHR promoter/reporter gene construct. This cell type was selected because it has endogenous LHR and is responsive to PMA activation. Our previous study has demonstrated that the hLHR expressed in HeLa and JAR cells lack the first exon that is vital for the hormone binding; however, these systems are highly suitable for studies of the LHR transcriptional regulation. The two cell types (MCF-7 and JAR) in which we based on our previous studies on epigenetic and genetic aspects of LHR transcription (6, 8, 9, 10, 11), were unresponsive to PMA and also devoid of PKC{alpha}. In this investigation, we also used MCF-7 cells to address the function of transfected PKC{alpha}. PMA treatment of HeLa cells caused activation of the hLHR promoter in a time-dependent manner, with a significant increase of 2.8-fold over control at 3 h and subsequent increases at 6 and 9 h (5.4- and 6.3-fold, respectively; Fig. 1AGo). In parallel studies, we determined whether the endogenous expression of hLHR gene governed by its natural promoter could be induced by PMA. The hLHR mRNA level increased significantly (1.8-fold) after 1 h PMA treatment, and the maximal increases observed at 3 h (3.5-fold) were maintained for up to 6 h (Fig. 1BGo). These results demonstrated that the hLHR gene is transcriptionally up-regulated exogenously and endogenously by PMA-induced PKC activation in HeLa cells.


Figure 1
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  		Fig. 1. Effect of PKC Activation by PMA on Transcription of the hLHR Gene

A, Reporter gene analyses of hLHR gene promoter activity in response to PMA in HeLa cells. At 36 h after transfection with the wild-type hLHR promoter/reporter gene construct, cells were incubated with 100 nM PMA for 0–9 h. Relative luciferase activity was expressed as fold-induction (n-fold) over the activity in the absence of PMA (1-fold). Results shown in this and subsequent figures are the mean ± SE of three independent experiments in triplicate (*, P < 0.05). B, Real-time PCR analyses of hLHR expression in HeLa cells treated with 100 nM PMA or vehicle for 0–6 h. The relative mRNA levels are shown as n-fold induction over the levels in the absence of treatment (1-fold) (*, P < 0.05). C, Western blot analyses of endogenous expression of PKC isoforms. D, Reporter gene analyses in HeLa cells pretreated with or without PKC inhibitors (Gö6983, Gö6976) for 1 h before incubation with PMA (100 nM) or vehicle for additional 9 h. Relative luciferase activity was expressed as fold-induction over the activity in the absence of treatment (*, P < 0.05, compared with the PMA-induced activity in the absence of inhibitor).

 
PMA is known to activate the Ca2+-dependent PKC isoforms including conventional PKCs ({alpha}, β, {gamma}) and novel PKCs ({delta}, {epsilon}, {theta}, µ, {eta}), whereas PKC isoforms in atypical PKC family ({zeta}, {iota}) are insensitive to PMA stimulation. Western blot analyses of conventional and typical PKC isoforms from HeLa cell lysates revealed predominant expression of PKC{alpha} and PKC{delta} isoforms, relatively less but still significant expression of PKCβ1, whereas minimal expression of PKCβ2, PKC{theta} and PKCµ was observed. Expression of PKC{gamma}, PKC{epsilon}, and PKC{eta} was not detected (Fig. 1CGo). To identify the PKC isoform(s) responsible for the increased activity of hLHR gene, we examined the PMA effect on hLHR promoter gene in the presence of various PKC inhibitors (Fig. 1DGo). The activation of hLHR promoter activity by PMA was completely blocked by Gö6983, a broad inhibitor for both conventional and novel PKC isoforms (Fig. 1DGo). In addition, the PMA effect was also blocked by the selective inhibition of conventional PKCs with Gö6976 (Fig. 1DGo). These results indicated participation of conventional PKCs rather than novel PKCs in the PMA activation of hLHR gene transcription/expression. Because PKC{alpha} is the most abundant isoform in the conventional PKC family of proteins in HeLa cells, it was selected as the most likely candidate to mediate PMA-induced hLHR promoter activity. Knock-down of PKC{alpha} expression in HeLa cells with PKC{alpha} small interference RNA (siRNA) showed that its expression level was largely repressed compared with cells transfected with scramble siRNA (Fig. 2AGo, right). Repression of PKC{alpha} markedly reduced the basal activity of hLHR promoter by approximately 50% in the absence of PMA (Fig. 2AGo, left). Moreover, the PMA-induced hLHR activation was abolished by the silencing of PKC{alpha} (Fig. 2AGo, left). However, the knock-down of PKCβ or PKC{delta} by siRNAs did not change the basal and PMA-induced hLHR activity. These findings indicated that PKC{alpha} has an important role in maintaining the basal hLHR promoter activity in HeLa cells. They also demonstrated that PMA-induced activation of the hLHR promoter and gene expression is dependent on specific activation of PKC{alpha}.


Figure 2
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  		Fig. 2. PKC{alpha} Is Required for PMA Activation of the hLHR Gene

A, Reporter gene analysis of LHR gene promoter activity in HeLa cells cotransfected with PKC{alpha}, PKCβ, PKC{delta} siRNA, or negative control siRNA (NTC) incubated with and without PMA (100 nM) for 9 h. Relative luciferase activity was expressed as fold-induction over the promoter activity with vehicle treatment. Expression levels of PKC{alpha}, PKCβ1/2, and PKC{delta} in HeLa cells transfected with siRNA of PKC{alpha}, PKCβ, PKC{delta}, and NTC were analyzed by Western blot analyses (right) (*, P < 0.05, compared with NTC without PMA treatment. **, P < 0.05, compared with NTC with PMA treatment). B, Real-time PCR analyses of hLHR gene expression (left) and reporter gene analyses of its promoter activity (right) in MCF-7 cells that were cotransfected with increasing amount of PKC{alpha} CA (*, P < 0.05). C, Reporter gene analyses of hLHR promoter activity in MCF-7 cells that were transfected with CA or DN PKC constructs alone or both. HA-tag expression of transfected PKC{alpha} or PKC{delta} constructs was also shown. V, Empty vector; Ab, antibody; HA, hemagglutinin. ({alpha}CA, PKC{alpha}CA; {delta}CA, PKC{delta}CA; {alpha}DN, PKC{alpha}DN; {delta}DN, PKC{delta} DN) (*, P < 0.05).

 
To further confirm the specific role of PKC{alpha} in the regulation of hLHR gene, we analyzed the effect of a constitutively active form of PKC{alpha} (PKC{alpha} CA) construct that is devoid of the N-terminal regulatory domain in MCF-7 cells. Overexpression of PKC{alpha} CA dose-dependently increased both hLHR promoter activity and its expression levels (Fig. 2BGo). This activation was attributable to the kinase activity of PKC{alpha} because it was prevented by cotransfection of a dominant-negative mutant of PKC{alpha} (PKC{alpha} DN) (Fig. 2CGo). In contrast, transfection of CA or DN of PKC{delta} construct had no impact on the activity of hLHR promoter (Fig. 2CGo). Overexpression of PKC{alpha} or PKC{delta} constructs in these cells were verified by Western blot analyses (Fig. 2CGo).

PMA-Induced hLHR Activation Requires Two Sp1/Sp3 Binding Sites on the Promoter and Is Dependent on Sp1 But Not Sp3
The hLHR core promoter contains two Sp1/Sp3 binding sites (positive activation sites), Sp1(I) and Sp1(II), that bind Sp1 and Sp3, and are of central importance for basal promoter activity (31). To evaluate their roles in PMA/PKC{alpha}-induced hLHR promoter activation, reporter gene analyses were performed in HeLa cells transfected with the wild-type hLHR promoter construct or with binding-inactivating mutations on either Sp1/Sp3 sites or both (Fig. 3AGo, left). As shown above, PMA significantly enhanced the activity of the wild-type hLHR promoter in HeLa cells. This induction was reduced by approximately 50% with the disruption of either Sp1(I) or Sp1(II) on hLHR promoter, and was almost abolished by simultaneous mutation of Sp1(I) and Sp1(II) sites. Similar results were observed in MCF-7 cells where the PKC{alpha} CA overexpression-mediated effect was partially reduced by single mutation on either Sp1/Sp3 binding sites and further disrupted by mutation on both sites (Fig. 3AGo, right). Therefore, both Sp1(I) and Sp1(II) sites are required for PMA/PKC{alpha}-induced activation of hLHR promoter.


Figure 3
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  		Fig. 3. Requirement of Both Sp1/Sp3 Binding Sites and Involvement of Sp1 But Not Sp3 in PMA-Mediated hLHR Promoter Activation

A, Participation of Sp1/Sp3 binding sites Sp1(I) and Sp1(II) on the activation of hLHR promoter activity by PMA in HeLa cells (left) and by PKC{alpha} CA in MCF-7 cells (right). Reporter gene analyses of HeLa cells transfected with the wild-type hLHR promoter construct (WT) or with mutations on either Sp1(I) site (Sp1X) or Sp1(II) site (Sp2X) or on both sites (Sp1–2X), treated with vehicle or 100 nM PMA for 9 h. In MCF-7 cells, the hLHR promoter WT or mutated constructs were cotransfected with empty pcDNA 3.1 vector or PKC{alpha} CA. Luciferase activity was determined 36 h after transfection. Data were shown as the fold-induction of luciferase activity by PMA treatment (HeLa cells) or PKC{alpha} CA overexpression (MCF-7 cells) with the different promoter constructs over control in the absence of PMA (HeLa) or empty vector (MCF-7) (*, P < 0.05). B and C, Reporter gene analyses of hLHR promoter activity (left) and real-time PCR analyses of its expression (right) in HeLa and MCF-7 cells cotransfected with Sp1, Sp3 siRNA or NTC. HeLa cells were treated with 100 nM PMA or vehicle for 9 h. MCF-7 cells were cotransfected with pcDNA3.1 empty vector or PKC{alpha} CA. Relative luciferase activity was expressed as fold-induction over the promoter activity in the absence of PMA in HeLa cells or without PKC{alpha} CA in MCF-7 cells. Expression of Sp1 and Sp3 proteins in HeLa cells (B, top) and MCF-7 cells (C, top) transfected with Sp1, Sp3 or NTC siRNA was analyzed by Western blot analyses. The expression levels of β-actin served as loading control. (*, P < 0.05). D, Synergistic effect of Sp1 on PKC{alpha} CA-induced hLHR activation. Quantitative real-time PCR analyses of hLHR mRNA from MCF-7 cells transfected with empty vector only, Sp1 or pKC{alpha} CA or both. The relative amount of hLHR mRNA was expressed as fold-increase over the mRNA amount with transfection of pcDNA 3.1 empty vector (*, P < 0.05).

 
The requirement of both Sp1/Sp3 binding sites in the PMA-mediated hLHR promoter activity prompted us to study the involvement of Sp1 and Sp3 transcription factors in this signaling pathway. HeLa cells or MCF-7 cells were transfected with siRNA of Sp1 or Sp3 to reduce the expression of respective genes. In the negative control, scramble siRNA was transfected. The expression of Sp1 and Sp3 was specifically knocked down by transfection of their correspondent siRNA in both HeLa (Fig. 3BGo, top) and MCF-7 cells (Fig. 3CGo, top). PMA or PKC{alpha} CA overexpression dramatically increased hLHR promoter activity and its expression levels in cells transfected with scrambled siRNA, and these responses were markedly impaired by knockdown of Sp1 (Sp1 siRNA) in both cell lines (Fig. 3Go, B and C, bottom). In contrast, blockade of Sp3 expression (Sp3 siRNA) did not affect the induction of hLHR by PMA or PKC{alpha} CA. These results demonstrated that PMA/PKC{alpha} CA exerted their effects on hLHR gene through Sp1 but not Sp3. The dependence of Sp1 in the induction of hLHR by PKC{alpha} was also confirmed by quantifying the level of hLHR mRNA mediated by PKC{alpha} CA in the presence or absence of Sp1 protein. As expected, overexpression of Sp1 significantly increased transcription of endogenous hLHR gene, and coexpression of this factor with PKC{alpha} CA synergistically enhanced PKC{alpha} CA-induced accumulation of hLHR mRNA (Fig. 3DGo).

PMA Causes Increased Sp1 Phosphorylation in PKC{alpha}-Dependent Manner
We next examined whether Sp1-dependent transcription of the hLHR gene is associated with a change in Sp1 expression or its DNA binding activity with its binding sites. Western blot analyses of cytoplasmic and nuclear protein from MCF-7 cells transfected with empty vector or PKC{alpha} CA revealed that the Sp1 protein was predominantly present in the nuclear fraction and only minimally in the cytoplasm, and its level was not influenced by overexpression of PKC{alpha} CA (Fig. 4AGo). Consistent with previous studies (9, 31), EMSA showed that incubation of Sp1(I) sites with nuclear protein from MCF-7 cells formed Sp1 and Sp3 complexes, and that Sp1 and Sp3 binding was supershifted by Sp1 and Sp3 antibodies (Fig. 4BGo). Furthermore, the DNA binding activity of Sp1 and Sp3 was not affected by PKC{alpha} CA overexpression because a similar binding pattern and intensity was observed in the presence or absence of PKC{alpha} CA (Fig. 4BGo). Similarly, neither Sp1 protein level nor its DNA binding was influenced by PMA treatment in HeLa cells (data not shown).


Figure 4
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  		Fig. 4. PMA Treatement Enhances Sp1 Phosphorylation in a PKC{alpha}-Dependent Manner

A, Western blot analyses of cytosolic and nuclear extracts from MCF-7 cells overexpressing PKC{alpha} CA or vector using Sp1, Sp3, and β-actin antibodies. Expression of HDAC1 and tubulin, as nuclear and cytoplasmic protein markers, respectively, are also shown. B, EMSA of Sp1/Sp3 binding to the Sp1(I) site of the hLHR promoter. 32P-labeled double-strand DNA oligomer containing the Sp1(I) site of the hLHR promoter was incubated with nuclear extracts prepared from MCF-7 cells transfected with empty pcDNA 3.1 (vector) only, or pKC{alpha} CA. The reaction was carried out in the absence or presence of antibodies to Sp1, Sp3 or both. C, Effects of PMA on the phosphorylation of Sp1 in HeLa cells. Left, Nuclear extracts from PMA-treated (100 nM, 3 h) (+) or untreated (–) HeLa cells were immunoprecipitated (IP) with Sp1 antibody. Samples (immunoprecipitates) were analyzed by Western blots (IB) using p-Ser, p-Thr, and Sp1 antibodies. Right, Nuclear extracts from HeLa cells stimulated with 100 nM PMA (+) or vehicle (–) for 3 h in the presence or absence of Gö6976 were immunoprecipitated with Sp1 antibody. The immunocomplex was analyzed by Western blots for detection of Sp1 and p-Sp1. The relative levels of p-Sp1 were quantified by densitometry (*, P < 0.05). D, Effects of PKC{alpha} CA overexpression in MCF-7 cells on the phosphorylation of Sp1. Left, Nuclear extracts from MCF-7 cells that were transfected with empty vector (V) or PKC{alpha} CA were immunoprecipitated with Sp1 antibody, followed by Western blots using p-Ser, p-Thr, and Sp1 antibodies. Right, Nuclear extracts from MCF-7 cells overexpressing PKC{alpha} CA in the presence or absence of PKC{alpha} DN or vector only were immunoprecipitated by Sp1 antibody or normal IgG. The immunocomplexes were analyzed by Western blots using Sp1 and p-Ser antibodies.

 
Because neither Sp1 abundance nor its DNA binding activity was changed by PMA or PKC{alpha} CA, it was important to determine whether Sp1 modification, such as phosphorylation, was involved in this regulation. Immunoprecipitation of nuclear extracts from control and PMA-treated HeLa cells was performed, followed by Western blot analyses with phosphorylated (p-) Thr and p-Ser antibodies. Significant Sp1 phosphorylation at serine residue(s) was detected basally in HeLa cells (in the absence of PMA), whereas no phosphorylation was observed at threonine residue(s) (Fig. 4CGo, left). This result is consistent with the finding that Sp1 is endogenously phosphorylated at serine(s) in HeLa cells (32). PMA treatment resulted in a marked increase of Sp1 phosphorylation at serine residue(s), whereas phosphorylation at threonine was not detected (Fig. 4CGo, left). Furthermore, PMA-induced Sp1 phosphorylation was dependent on the activity of PKC{alpha} because treatment with Gö6976 significantly reduced such phosphorylation (Fig. 4CGo, right). The role of PKC{alpha} in Sp1 phosphorylation was further confirmed in MCF-7 cells, where overexpressing PKC{alpha} CA caused a profound increase in Sp1 phosphorylation at serine residue (s), whereas no induction at threonine residue(s) was observed (Fig. 4DGo, left). Moreover, cotransfection of PKC{alpha} DN diminished such Sp1 phosphorylation (Fig. 4DGo, right). Taken together, these results have demonstrated that PKC{alpha} causes induction of hLHR gene activation through modulation of Sp1 phosphorylation.

The ERK Cascade Participates in PMA-Induced hLHR Activation through Direct Phosphorylation of Sp1
The possibility of Sp1 as a direct nuclear target of PKC{alpha} was excluded by the absence of nuclear translocation of PKC{alpha} in HeLa cells in response to PMA during hLHR activation (data not shown). Therefore, a downstream effector may directly associate with and phosphorylate Sp1 in the induction of hLHR transcription/expression by PKC{alpha} activation.

The ERK/MAPK (ERK) has been reported to be activated by PKC{alpha} (33, 34), and it has been implicated in control of gene expression through direct modulation of transcriptional factors in the nucleus (13, 35, 36). Therefore, the possible involvement of ERK was examined using reporter gene analyses in PMA-treated and untreated HeLa cells in the presence of inhibitors for the three MAPKs, ERK, p38, and c-Jun N-terminal kinase (JNK) (Fig. 5AGo). Consistent with our previous findings (Figs. 1–3GoGoGo), PMA treatment led to a marked activation of hLHR promoter. This response was largely blocked by inhibition of the ERK cascade with U0126, a specific inhibitor of MEK that is directly upstream in the ERK signaling pathway. In contrast, neither an inhibitor of p38 (SB20290) nor that of JNK (SB600125) exhibited any effect. This result demonstrated that MEK/ERK, rather than p38 or JNK pathways, participates in the PMA response. The involvement of MEK/ERK was further confirmed by an siRNA study in which the increase of the hLHR expression levels and its promoter activity induced by PMA treatment in HeLa cells was prevented by suppression of ERK through knock-down of MEK1/2 (Fig. 5BGo). In accordance with the participation of ERK in the PMA effect on hLHR gene expression, PMA treatment for 15 min enhanced the level of activated p-ERK in HeLa cells, and this was inhibited by pretreatment with the MEK/ERK inhibitor, U0126 (Fig. 5CGo). ERK activity induced by PMA was also abrogated by Gö6976 (Fig. 5CGo), indicative of the role of the ERK cascade in downstream signaling of PKC{alpha}. Moreover, immunoprecipitation analyses showed that inhibition of ERK kinase with U0126 largely blocked Sp1 phosphorylation upon PMA treatment (Fig. 5DGo).


Figure 5
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  		Fig. 5. ERK/MAPK Pathway Participates in the PMA-Induced hLHR Activation

A, Reporter gene analyses of HeLa cells preincubated with or without 10 µM of inhibitors to ERK (U0126), p38 (SB20290), and JNK (SB600125) for 1 h before simulation with 100 nM PMA for 9 h (*, P < 0.05). B, Quantitative real-time PCR analyses of hLHR gene expression (left) and reporter gene analyses of its promoter activity (middle) in HeLa cells transfected NTC or MAPK kinase (MEK) 1/2 siRNA. Cells were treated with 100 nM PMA or vehicle for 9 h. Expression of MEK1/2 in HeLa cells transfected with siRNA of MEK1/2 or NTC analyzed by Western blot (right). (*, P < 0.05, compared with NCT treated with PMA). C, HeLa cells were pretreated with or without Gö6976 or U0126 for 1 h, followed by incubation with 100 nM PMA or vehicle for 15 min. Cell lysates were analyzed by Western blot using antibodies against p-ERK1/2, total ERK, and β-actin. D, Immunoprecipitation (IP) of Sp1 in nuclear extracts from HeLa cells treated with 100 nM PMA (+) or vehicle (–) for 3 h in the presence or absence of U0126. The phosphorylation level of Sp1 in the immunocomplex was determined by Western blot (IB) analyses with anti-p-Ser antibody. The total Sp1 immunoprecipitated is also shown. The relative level of p-Sp1 was quantified by densitometry (*, P < 0.05).

 
The dependence of Sp1 phosphorylation on the ERK cascade led us to determine whether Sp1 is the direct target of activated ERK in hLHR-induced transcription during PMA stimulation. In coimmunoprecipitation analyses of nuclear extracts from HeLa cells treated with vehicle alone, no p-ERK was present in the Sp1 precipitated complex (Fig. 6AGo). However, p-ERK was coimmunoprecipitated by Sp1 antibody when cells were stimulated by PMA for 15 min. No detectable p-ERK signal was observed in the precipitate of IgG controls (Fig. 6AGo). These results demonstrated that PMA treatment of cells induces an interaction between Sp1 and p-ERK, and that the latter directly targets Sp1 during PMA-activated hLHR expression. To further determine the association of p-ERK with Sp1 on the hLHR promoter, chromatin immunoprecipitation (ChIP) was performed in HeLa cells treated with or without PMA. The binding of p-ERK with the core promoter region of hLHR gene (–176 bp relative to translational starting site) that contain two Sp1 sites (–71 and –118 bp, respectively) was analyzed. A significant enrichment of p-ERK was observed in PMA-treated cells (Fig. 6BGo). This enrichment was dependent on activation of ERK because it was abrogated by inhibition of MEK/ERK with U0126. In addition, knock-down of Sp1 expression abolished the association of p-ERK with the hLHR promoter. These results provide further evidence that Sp1 is the substrate for p-ERK in PMA-induced hLHR activation. The presence of ERK and Sp1 at the same protein complex on the promoter during activation of LHR transcription in HeLa cells by PMA was verified using sequential ChIP (Re-ChIP) assay. As shown in Fig. 6CGo, 60% of the protein complex at the hLHR promoter precipitated by the Sp1 antibody (ChIP) was recovered by the p-ERK antibody (Re-ChIP). In contrast, only a trace amount was present in the supernatant from the second precipitation. Taken together, these results have demonstrated that the ERK cascade acted as the downstream target of PKC{alpha} that participates in PMA/PKC{alpha}-induced hLHR transcriptional activation through direct phosphorylation of Sp1.


Figure 6
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  		Fig. 6. ERK Interacts with Sp1 and Is Recruited to the hLHR Promoter in the Presence of PMA

A, Interaction of p-ERK and Sp1 determined by immunoprecipitation (IP). Whole cell lysates prepared from HeLa cells treated with PMA (+) or vehicle (–) for 15 min were immunoprecipitated with normal IgG or Sp1 antibody. The immunocomplex was analyzed by Western blot (IB) analyses with p-ERK antibody. The expression of p-ERK in the cells stimulated with PMA for 15 min is shown as input. B, Association of p-ERK with hLHR promoter by ChIP analyses. HeLa cells were treated with 100 nM PMA or vehicle for 15 min in the presence and absence of MEK inhibitor U0126 (10 µM), or Sp1 siRNA, and subjected to chromatin immunoprecipitation analyses with p-ERK antibody or normal rabbit IgG. The precipitated DNA was analyzed quantitatively by real-time PCR with primers amplifying the core promoter sequence of hLHR gene. The relative binding to promoter was expressed as the percentage amount over input (%). Data were presented as the mean ± SE from three independent experiments in triplicate (*, P < 0.05). C, Association of p-ERK to Sp1 bound to the LHR promoter determined Re-ChIP analyses. Sequential ChIP analyses of HeLa cells treated by PMA for 15 min using Sp1 as the first antibody followed by the second immunoprecipitation with p-ERK antibody. The DNA precipitates and supernatants after the second immunoprecipitation were examined by quantitative PCR, respectively. Co-occupancy of Sp1 set to 100% is also shown.

 
Dissociation of HDAC1/mSin3A from hLHR Promoter upon PKC{alpha} Activation
A possible mechanism by which PKC{alpha}/p-ERK/p-Sp1 cascade induces activation of the hLHR receptor gene transcription is by triggering the release of Sp1-associated inhibitory factors that contribute to the repression of the hLHR. Our previous studies have demonstrated that HDAC1 and HDAC2, which directly interact with Sp1, are potent repressors of hLHR transcription. They form repressive complexes with mSin3A corepressor to suppress hLHR gene expression (9). Therefore, the binding of HDAC1, HDAC2 and mSin3A was examined in HeLa cells control or treated with PMA by quantitative ChIP analyses (Fig. 7AGo). Consistent with previous findings (9), the presence of HDAC1, HDAC2/mSin3A complex on the hLHR promoter in basal state, suggested that their recruitment at the hLHR promoter provides a mechanism for repression of this gene. The association of HDAC1 with the hLHR promoter observed in untreated HeLa cells was markedly reduced after PMA treatment, and this was also the case for mSin3A. However, the binding of HDAC2 was not influenced by PMA. Consistent with our in vitro Sp1 binding experiments (see Fig. 4Go), the recruitment of Sp1 to hLHR promoter remained unchanged when cells were treated with PMA. Moreover, the dissociation of HDAC1 and mSin3A induced by PMA was impaired by inhibition of PKC{alpha} activity with Gö6976 and blockade of ERK with U0126. Similarly, overexpression of PKC{alpha} CA in MCF-7 cells induced release of both HDAC1 and mSin3A from the hLHR promoter, whereas no significant changes were observed in Sp1 and HDAC2 (Fig. 7BGo). The dissociation of HDAC1 and mSin3A induced by PKC{alpha} CA was prevented by the cotransfection of PKC{alpha} DN. Because p107 has been also identified as a potent corepressor of hLHR gene, the effect of PMA/PKC{alpha} CA on the association of p107 was investigated. We found that p107 associated with the hLHR gene in both HeLa cells and MCF-7 cells. This association was not altered by either PMA-induced activation of PKC or expression of PKC{alpha}CA (Fig. 7CGo), which is consistent with the notion that the derecruitment of p107 was dependent on the PI3K/PKC{zeta} cascade (11).


Figure 7
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  		Fig. 7. PMA Treatment Leads to PKC{alpha}/ERK-Dependent Release of HDAC1/mSin3A Complex from the hLHR Promoter

A and B, Quantitative ChIP analyses in HeLa cells (A) and MCF-7 cells (B). HeLa cells were treated with 100 nM PMA or vehicle for 3 h in the presence or absence of Gö6976 or U0126. MCF-7 cells were transfected with pcDNA3.1 vector (V) or PKC{alpha} CA (CA) alone or cotransfected with PKC{alpha} DN (CA/DN). Cells were subjected to ChIP assay with various antibodies (Sp1, HDAC1, HDAC2, and mSin3A). The precipitated DNA was analyzed by real-time PCR with primers for hLHR gene promoter. The relative binding of these factors with the promoter was presented as the percentage amount over input (%) (*, P < 0.05). C, Quantitative ChIP analyses of p107 association to hLHR promoter in HeLa cells treated with vehicle or PMA (100 nM, 3 h) (left) and MCF-7 cells transfected with pcDNA 3.1 vector (V) or PKC{alpha} CA (CA) (right). D, Nuclear extracts were prepared from HeLa cells treated with vehicle (–) or 100 nM PMA (+) for 3 h, and immunoprecipitated with Sp1 antibody. The immunocomplex was analyzed by Western blot analyses with HDAC1, HDAC2 or Sp1 antibodies. E, HeLa cells were pretreated with or without Gö6976 for 1 h before exposure to vehicle (–) or 100 nM PMA (+) for 3 h. Nuclear extracts were immmunoprecipitated with Sp1 antibody or IgG followed by Western blot analyses with HDAC1 antibody. Expression level of HDAC1 in these cells was shown as input. F, Nuclear extracts from MCF-7 cells transfected with vector or MEK1CA were immunoprecipitated with Sp1 antibody followed by Western blot analyses with HDAC1 antibody. Total immunoprecipitated Sp1 level was also shown.

 
To determine whether PMA affected the interaction between Sp1 and HDAC1 leading to release of mSin3A from the complex, nuclear extracts from control or PMA-treated HeLa cells were coimmunoprecipitated with Sp1 antibody followed by detection of HDAC1 and HDAC2. As expected, in controls both HDAC1 and HDAC2 interacted with Sp1 (Fig. 7DGo). Interaction between Sp1 and HDAC1 was significantly reduced in extracts from PMA-treated cells, whereas no significant change was observed in the interaction between Sp1 and HDAC2, indicating the differential role of HDAC1 and HDAC2 in the activation of hLHR mediated by PMA. These changes were not related to the Sp1 and HDAC1 protein levels because cellular expression of both proteins were not affected by PMA treatment (Fig. 7DGo). Furthermore, the reduction of HDAC1/Sp1 interaction induced by PMA was dependent on the activity of PKC{alpha} because it was prevented by inhibition of PKC{alpha} with Gö6976 (Fig. 7EGo). Similarly, coimmunoprecipitation studies revealed that overexpression of PKC{alpha} CA caused reduced interaction between Sp1 with HDAC1, but not HDAC2 and that the reduction of Sp1/HDAC1 interaction was blocked by cotransfection of PKC{alpha} DN (data not shown). We next tested whether the interaction of HDAC1 and Sp1 is regulated by ERK. Coimmunoprecipitation with nuclear extracts from MCF-7 cells that were overexpressed with MEK1 CA was performed (Fig. 7FGo). The interaction between Sp1 and HDAC1 was greatly reduced by MEK1 CA. Immunoprecipitation studies using HDAC1 antibody followed by Western blot analysis with specific antibodies to p-Ser, -Thr, -Tyr showed no basal or PMA-induced phosphorylation of HDAC1. This excluded the phosphorylation of HDAC1 as a participant in the dissociation of the corepressor (data not shown). Thus, our findings indicated that dissociation of HDAC1/mSin3A from promoter has a critical role in the transcription of hLHR regulated by PMA/PKC{alpha} CA, and this dissociation was accomplished through modulation of interaction between Sp1 and HDAC1.

Histone H3 Acetylation, and Recruitment of RNA Polymerase II (Pol II), and Transcription Factor II B (TFIIB) to the Promoter Are Dependent on PKC{alpha}/ERK
The effect of activated PKC{alpha} on the acetylation of histone 3 and recruitment of members of the transcriptional machinery on the hLHR promoter was subsequently evaluated (Fig. 8Go). ChIP assays with anti-acetyl-histone H3 on control HeLa cells revealed basal level of acetylated histone H3 at hLHR promoter, whereas a significant enrichment was observed after LHR activation with PMA. In addition, the association of Pol II and TFIIB with the hLHR promoter was significantly increased in PMA-stimulated cells. Similar findings were observed in MCF-7 cells transfected with PKC{alpha} CA (data not shown). These changes at the hLHR sites were dependent on PKC{alpha}/ERK kinase activities because they were largely blocked by Gö6976 or U0126 inhibitors. No changes on the methylation status of the hLHR promoter cytosine-phosphate-guanine dinucleotide island (70% methylated) was observed as a result of the activation of PKC{alpha}/ERK signal transduction pathway that leads to derepresion of hLHR in HeLa cells during incubation with PMA (not shown).


Figure 8
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  		Fig. 8. PMA Treatment Increases Acetylation of Histone H3, and Recruitment of Pol II and TFIIB to the LHR Promoter

HeLa cells were treated with vehicle or 100 nM PMA for 3 h in the presence or absence of Gö6976 (2 µM) or U0126 (10 µM), and subjected to quantitative ChIP analyses of AceH3, Pol II, and TFIIB at the hLHR promoter. The relative binding of indicated transcriptional factors was expressed as the percentage amount over input (%). Data were presented as the mean ± SE from three independent experiments in triplicate (*, P < 0.05).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
These studies have demonstrated that PMA treatment causes the induction of both hLHR promoter activity and its endogenous mRNA expression in HeLa cells, and that PKC{alpha} is responsible for this activation. Both Sp1 sites, which are of central importance for basal promoter activity, are required for the PMA/PKC{alpha}-mediated activation of hLHR, and Sp1 but not Sp3 is the relevant mediator of this activation. Furthermore, ERK signaling, as a downstream effector of PKC{alpha}, was required for the PMA-induced of hLHR expression, where it mediated Sp1 phosphorylation. This causes changes in the protein-protein interaction between Sp1 and HDAC1, and the release of HDAC1/mSin3A from the hLHR promoter, leading to enhanced transcription.

As a key component of signaling cascades in response to various stimuli, signaling from several PKC isoforms has been implicated in the regulation of numerous genes involved in diverse biological processes including proliferation, differentiation and apoptosis (37, 38, 39, 40, 41). This study has provided evidence that the hLHR gene is a regulatory target of PKC{alpha}. In HeLa cells, PMA treatment caused a significant increase of both hLHR promoter activity and its endogenous transcripts. Among the PKC isoforms expressed in HeLa cells, PKC{alpha} was shown to be responsible for the PMA effect, as revealed by studies using a specific PKC inhibitor (Gö6976) and siRNA specific to PKC{alpha}. Additional evidence for the role of PKC{alpha} in the regulation of hLHR gene transcription was provided by the demonstration in MCF-7 cells that overexpression of PKC{alpha} CA activated the hLHR gene transcription and endogenous mRNA expression. Also, cotransfection of PKC{alpha} DN antagonized the ability of PKC{alpha} CA to induce hLHR promoter activity. Identification of hLHR gene as a target of the PKC{alpha} signaling cascade implied its importance in physiological regulation of the hLHR, which undergoes endocrine changes during the growth and differentiation of gonads.

Mutational analyses of the hLHR gene promoter have shown that the activation of hLHR induced by PKC activation, or PKC{alpha}CA, is mediated by both Sp1/Sp3 binding sites. This is in contrast to the involvement of only one of those sites (Sp1-I) in the derepression of hLHR induced by TSA (9). These results suggested that although both Sp1 sites are of central importance for basal hLHR gene activity, they can be differentially involved in specific promoter activation in response to different stimuli. These findings have common features with the regulation of the Cdk (cyclin-dependent kinase) inhibitor gene, p21 WAF/Cip, where various Sp1-sites or combination of Sp1 sites were targeted by several stimuli such as PMA, TGF-β, tamoxifen, and HDAC inhibitors (42, 43, 44, 45). Although Sp1 and Sp3 exert similar binding activities to the Sp1 sites and are effective transcriptional activators of the hLHR basal promoter activity (31), Sp1 but not Sp3 was found to participate in PMA/PKC{alpha} and PKC{alpha} CA-induced hLHR activation. Furthermore, our previous studies showed that Sp1 is the critical mediator in TSA-induced hLHR activation in both JAR and MCF-7 cells, whereas Sp3 is not contributory (11). Similarly, Sp1 but not Sp3 was reported to be responsible for TGFβ-mediated activation of the p21 gene (44). In contrast, Sp3 but not Sp1 mediates the TSA response of the p21 gene in the MG63 human osterosarcoma cell line (46). The differential involvement of Sp1 and Sp3 in promoter regulation may relate to the cell system employed, or the mechanism involved in the activation.

The finding that activation of the LHR induced by PMA is inhibited by blockade of ERK activity by the U0126 inhibitor, or through knockdown of MEK, indicated the essential role of ERK in the transcriptional activation of the hLHR gene by PMA. This was further supported by the activation of ERK observed in PMA-treated cells and the suppression of its enzymatic activity caused by inhibition of PKC{alpha}. The ability of PKC{alpha}/ERK signaling to promote p15INK4b/p16INK4a gene expression has been observed in HepG2 cells treated with PMA or Saikosaponin {alpha} (47, 48). However, the mechanism by which the PKC{alpha}/ERK mediated the activation was not further defined in that case. This study has indicated that Sp1 phosphorylation is the molecular link between PKC{alpha}/ERK signaling and activation of the hLHR gene. In HeLa and MCF-7 cells, respectively, Sp1 phosphorylation on serine residue(s) was enhanced by PMA and overexpression of PKC{alpha} CA. Inhibition of PKC{alpha} or cotransfection of PKC{alpha} DN reduced Sp1 phosphorylation and abolished the ability of PMA and PKC{alpha} CA to activate the hLHR gene. Although there is evidence that PKC{alpha} can translocate into the nucleus in response to extracellular signal, and that PMA causes phosphorylation of transcriptional factor(s) for the regulation of gene expression (49, 50), we did not detect translocation of PKC{alpha} in HeLa cells after PMA treatment (data not shown). Consistent with this finding, no physical interaction between Sp1 and PKC{alpha} CA was observed in MCF-7 cells (data not shown). For this reason, we ruled out the possibility that PKC{alpha} could exert its action on the hLHR through direct phosphorylation on Sp1 in the nucleus. The fact that inhibition of ERK significantly reduced PMA-induced Sp1 phosphorylation indicated that ERK is responsible for the PKC{alpha} effect on Sp1 phosphorylation at serine residues, which consequently leads to the increase in LHR gene expression. This was confirmed by the finding that p-ERK interacts with Sp1 and coexisted in the same complex on the promoter during PMA-induced activation of hLHR gene in HeLa cells. In this regard, ERK-dependent Sp1 phosphorylation on serine was shown to be involved in regulation of the matrix metalloproteinase-2 gene by nonsteroidal antiinflammatory drugs in lung cancer cells (51). Sp1 is known to be phosphorylated by DNA-dependent protein kinase in unstimulated HeLa cells (52). However, it is not involved in PMA-induced Sp1 phosphorylation and/or activation of hLHR gene, because its blockade in these cells did not affect the PMA-induced of Sp1 phosphorylation and hLHR promoter activation (data not shown).

The unchanged binding pattern of Sp1 to its cognate site in the presence and absence of PMA or PKC{alpha} CA suggested that the ERK-Sp1 phosphorylation, associated with hLHR activation is not related to the changes in the binding activity of Sp1. Several studies have shown interactions of Sp1 with a variety of proteins including the TATA-box-binding protein TBP and associated factors, p107, YY1, E2F, HDAC1/2, p300, FBI-1, SMRT, NCoR, and BCoR (9, 53, 54, 55, 56, 57, 58, 59). Also, modulation of the interactions between Sp1 and these partner proteins has been shown to be crucial in the regulation of Sp1-dependent gene expression (11, 12, 60, 61). In addition, the phosphorylation/dephosphorylation of Sp1 has been suggested to be an important mechanism underlying regulation of its interaction with other factors (11, 12, 62). Our study provides the evidence that phosphorylation induced by PKC{alpha}/ERK signaling disrupts the interaction between Sp1 and HDAC1, and that this resulted in release of the HDAC1/mSin3A complex to activate transcription of the LHR gene. The simultaneous dissociation of HDAC1 and mSin3A, but not HDAC2 from the LHR gene promoter upon its activation by PMA, was consistent with our previous findings showing that mSin3A functions as a corepressor for HDAC1 but not HDAC2 in the regulation of LHR gene expression (9). MEK, the upstream regulator of ERK, has been reported to modulate the transcriptional activity of Sp1 through a molecular interaction between the zinc finger DNA binding domain (Sp1ZFDBD) (amino acids 622–720) and the inhibitory domain (amino acids 1–82) with corepressor SMRT, NCoR, and BCoR in HeLa cells (61). This Sp1ZFDBD domain is also involved in the interaction between Sp1 and HDAC1 (59). Therefore, it is possible that ERK-mediated Sp1 phosphorylation favors dissociation of HDAC1 from Sp1 by targeting the Sp1ZFDBD. In this regard, there is evidence showing that the EGF-Ras-MEK1-ERK2 signaling pathway abrogated mSin3A binding to the Sp1-like repressor protein TIEG2, through phosphorylation of four serine/threonine sites adjacent to the Sin3-interacting domain (63). HDAC1 phosphorylation has been shown to contribute to disruption of its interaction with corepressor mSin3A and YY1 (64). However, we have demonstrated that HDAC1 was not phosphorylated by PKC{alpha}/ERK and thus excluded its participation in the release of the corepressor.

Previous findings from our laboratory have shown that recruitment of HDAC1/2/mSin3A complex via the promoter of LHR gene induces promoter-localized chromatin condensation through histone hypoacetylation leading to repression of transcription (9). Release of the repressive HDAC/mSin3A complex from the promoter during TSA-derepression of hLHR gene was found to be dependent on TSA-induced changes of histone acetylation and a demethylated promoter (10). In this study, we identified a novel mechanism in which PKC{alpha}/ERK signaling and Sp1 phosphorylation-mediated release of HDAC/mSin3A leads to activation of the hLHR gene. However, PKC{alpha}/ERK-induced release of the repressor complex does not depend on the methylation status of the LHR promoter because it was not changed by PMA treatment of the cells. Release of the HDAC1/mSin3A complex from Sp1 facilitated histone acetylation, and recruitment of components of the basal transcriptional machinery (TFIIB and Pol II) to the hLHR promoter. These findings highlight the critical role of recruitment of HDAC/mSin3A on repression of the hLHR gene. Distinct mechanisms of dissociation of the HDAC complex from the hLHR promoter suggests that regulation of release of the HDAC/mSin3A complex represents an important mechanism for the control of repression/derepression of the hLHR gene.

Our previous studies have demonstrated a participation of PI3K/PKC{zeta} signaling in TSA-induced derepression of hLHR gene (11). In this study, we identified PKC{alpha}/ERK cascade as an essential pathway for hLHR gene activation. Sp1 phosphorylation has been shown to play critical roles in both PI3K/PKC{zeta}- (11) and PKC{alpha}/ERK- (this study) mediated activation of the hLHR gene; however, the mechanisms underlying these two processes are different. The phosphorylation site for PKC{zeta} resides at serine 641 in the vicinity of the Cys2 His2 DNA binding domain, and this change caused release of repressor p107. In contrast, this site is not phosphorylated by ERK. Presently the Ser site or sites (combinatorial) in Sp1 phosphorylated by ERK is/are under investigation, and such a change should be responsible for HDAC1 dissociation and hence the release of corepressor mSin3A.

Our previous and current studies on the transcriptional regulation of the LHR expression using nongonadal cell culture under well-established experimental conditions should provide solid basis for exploration into epigenetic/genetic/signal transduction regulation, and the understanding of molecular mechanism governing LHR transcription regulation during ovarian development and Leydig cell maturation. The LH surge or human chorionic gonadotropin treatment was found to induce through its cognate receptor the transcription and maturation of several EGF-like factors (amphiregulin, epiregulin, and betacellulin) and metalloproteases in granulosa cells via the cAMP/PKA pathway (65, 66). In addition, these growth factors subsequently activate epidermal growth factor receptor and ERK/MAPK signaling whereby this activation exerts paracrine (cumulus cells) and presumably autocrine LH effects (granulosa cells). Furthermore, other studies have demonstrated CA LHR coupling to Gq/phospholipase C β/phosphatidylinositol 4,5-bisphosphate 2/DAG/PKC pathway in addition to Gs{alpha}/adenylate cyclase/cAMP pathway (67). Both routes could lead to activation of downstream MAPK/ERK signaling, exerting an impact on the LHR gene transcription.

Taken together, the present studies have demonstrated that PKC{alpha} signaling transcriptionally activates the hLHR gene through ERK-dependent Sp1 phosphorylation and modulation of release of HDAC1/mSin3A complex in HeLa and MCF-7 cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Reagents and Antibodies
Phorbol-12-myristate-13-acetate (PMA), Gö6976, Gö6983, and U0126 were purchased from Calbiochem (La Jolla, CA). The antibodies against various PKC isoforms ({alpha}, β1, β2, {gamma}, {delta}, {epsilon}, {theta}, µ, {eta}), Sp1, TFIIB, RNAPol II, HDAC1, HDAC2, mSin3A, β-tubulin, and actin were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The efficiency of various PKC isoforms antibodies was verified as described previously (11). The Sp1 antibody for immunoprecipitation, and p-Ser antibody were purchased from Sigma-Aldrich (St. Louis, MO). ERK1/2, p-ERK1/2, MEK1/2, and p-Thr antibodies were provided by Cell Signaling Technology (Danvers, MA). HA.11 monoclonal antibody and anti-acetyl-histone H3 antibody were purchased from Covance (Berkeley, CA) and Upstate Biotechnology (Lake Placid, NY), respectively.

Reporter Gene Constructs and Expression Vector
The reporter gene constructs for wild type hLHR promoter or the promoters harboring the mutant Sp1-I, Sp1-II binding site, or both have been described previously (31). The PKC{alpha} CA and PKC{alpha} DN were kindly provided by Dr. Kevin Catt (Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health). The pCMV-Sp1 expression construct was purchased from OriGene (Rockville, MD). The MEK1 CA construct (MEK1CA) was obtained from Biomyx Technology (San Diego, CA).

Cell Culture, Transfection and Reporter Gene Assay
HeLa cells obtained from American Type Culture Collection (ATCC) (Manassas, VA) were maintained in Eagle’s MEM (ATCC). MCF-7A2 cells (MCF-7) were kindly provided by Dr. Erica Berleth (C. Roswell Park Cancer Institute, Buffalo, NY) (68), and cultured in RPMI 1640 medium (Invitrogen, Carlsbad, CA). All culture media were supplemented with 10% fetal bovine serum and 1% antibiotic/antimycotic solution (Invitrogen). Transfections were performed using Lipofectamine and Plus Reagent according to the manufacturer’s instruction. The DNA amount in each well was equalized with empty vector DNA. For HeLa cells treated with PMA, cells were incubated with 100 nM PMA or vehicle (dimethylsulfoxide) for the indicated time before termination. For HeLa cells treated with PMA in the presence of various inhibitors, cells were preincubated with inhibitors for 1 h before the PMA treatment. The luciferase activity was normalized to light units per microgram protein. All experiments were performed for at least three times in triplicate, and results were expressed as the mean ± SE. Statistical significance was evaluated by variance test analysis with computer programs Statview (Abacus Concepts, Berkeley, CA) and Superanova (Abacus Concept).

RNA Isolation and Real-Time RT-PCR
Total RNA from treated or untreated HeLa cells, or from MCF-7 cells transfected with indicated plasmids, was extracted with RNeasy Mini Kit (QIAGEN, Valencia, CA) followed by treatment with deoxyribonuclease I (Invitrogen) for 15 min at room temperature. For quantitative analyses of LHR gene mRNA expression level, 4 µg of total RNA were reversed transcribed with random primer for synthesis of the first-strand cDNA using the High Capacity cDNA Kit (Applied Biosystems, Foster City, CA). Real-time PCR was then carried out with SYBR-Green Master Mix in an ABI 7500 sequence detection system (Applied Biosystems). The PCR conditions used for all reactions included an initial cycle of 95 C for 10 min followed by 40 cycles of 95 C for 15 sec and 60 C for 1 min. The specificity of PCR products was verified by melting curve analyses. Relative LHR mRNA levels were calculated by the comparative CT method with human β-actin mRNA as an internal control. All samples from three individual experiments were performed in triplicate. The primers used are 5'-TCTACACCCTCACCGTCATCACTC-3' (LHR forward), 5'-AGCCATCCTCCAAGCATAATCA-3' (LHR reverse); 5'-GAGCGGGAAATCGTGCGTGACA-3' (β-actin forward), 5'-AGGAAGGAAGGCTGGAAG AGTGC-3' (β-actin reverse).

Preparation of Whole-Cell Lysate and Nuclear Protein, and Western Blot Analyses
HeLa and MCF-7 whole-cell lysates were extracted with the M-PER Mammalian Protein Extraction reagents (Pierce, Rockford, IL). The cytosolic and nuclear proteins from PMA- or vehicle-treated HeLa cells, and from MCF-7 cells transfected with indicated plasmids, were isolated with NE-PER Nuclear and Cytoplasmic Extraction reagents (Pierce). All of the preparations were performed in the presence of 1x protease inhibitor and 1x phosphatase inhibitor cocktails (Calbiochem). Protein concentration was determined by the colorimetric method of Bradford using Bio-Rad Protein Assay reagents (Bio-Rad, Philadelphia, PA).

For Western blots analyses, whole-cell lysates (50 µg) or nuclear proteins (15 µg) were used followed by detection with antibodies of indicated.

Immunoprecipitation (IP)
The 250-µg nuclear proteins from HeLa cells or MCF-7 cells were precleared with 25 µl Protein A/G plus agarose beads (Santa Cruz) in a total volume of 500 µl RIPA buffer in the presence of 1x protease inhibitor and 1x phosphatase inhibitor cocktail for 30 min at 4 C with gentle agitation. The supernatant were recovered by brief centrifugation and incubated with 1 µg rabbit Sp1 antibody or normal rabbit IgG overnight at 4 C. This was followed by addition of 36 µl Protein A/G plus beads and incubation for additional 1 h. The immunocomplexes precipitated were collected by brief centrifugation and washed three times with RIPA buffer for 5 min each time. The beads were resuspended in 36 µl of 2x sodium dodecyl sulfate protein sample buffer containing 2.5% of β-mercaptoethanol and boiled for 5 min before Western blot analyses.

siRNA Analyses
Validated siRNAs designed to knock down the endogenous expression of PKC{alpha}, PKCβ, PKC{delta}, Sp1, Sp3, or MEK1/2, and siRNA for the negative control (NTC) were purchased from Ambion (Austin, TX). Transfections of siRNA to HeLa or MCF-7 cells were carried out with siPORTNeoFX reagent (Ambion) as previously described (11). Briefly, diluted siPORTNeoFX were incubated with indicated siRNA (30 nM) in Opti-MEM I Reduced-Serum Medium (Invitrogen) at room temperature for 10 min. The transfection complexes formed were then dispensed into 24-well plates and overlaid with cells at a density of 1 x 105 cells/ml. At 24 h after transfection, cells were replaced with normal growth medium and cultured for additional 40 h before harvest. For reporter gene assays, the LHR gene promoter/reporter gene in the presence or absence of cotransfected PKC{alpha} constructs was introduced by Lipofectamine and Plus Reagent at 24 h after transfection of siRNA, and the luciferase activity was determined 40 h later. For cells treated with PMA, 100 nM PMA or vehicle was added to cells 9 h before termination. For each siRNA experiment, two different NTC siRNAs were used to exclude any nonspecific effect on gene expression. A representative example of these experiments was shown in all figures where siRNA was used.

EMSA
EMSA was performed as previously prescribed (6). Briefly, 20 µg nuclear extracts were incubated with {gamma}32P-labeled oligonucleotide probe harboring the Sp1-I site of hLHR promoter. For supershift assay, 2 µl of anti-Sp1, or -Sp3 antibody, or both was preincubated with the nuclear extracts for 20 min before the addition of probe. Protein-DNA complexes were resolved on 5% native polyacrylamide gels and visualized by autoradiography.

ChIP
ChIP assays were performed with the ChIP assay kit from Upstate Biotechnology (Lake Placid, NY). Briefly, 2 x 107 treated or untreated HeLa cells, or MCF-7 cells cotransfected with indicated expression plasmids, were cross-linked with 1% formaldehyde for 10 min at 37 C. The soluble chromatin prepared was immunoprecipitated overnight with 2 µg of rabbit antibodies against Sp1, p-ERK, HDAC1, HDAC2, mSin3A, RNAPol II, TFIIB, acetylated histone H3, or normal rabbit IgG. This is followed by incubation with 60 µl Protein A Agarose/Salmon Sperm DNA for additional 1 h. The immunoprecipitated complexes were sequentially washed with low salt, high salt, LiCl, and Tris-EDTA buffer followed by extraction twice with freshly prepared elution buffer (1% sodium dodecyl sulfate, 0.1 M NaHCO3.) The cross-link between DNA and protein was reversed, and DNA was purified by ethanol precipitation in the presence of Pellet Paint Co-Precipitatant (Calbiochem). The relative binding of transcriptional factors of interest to the hLHR promoter was quantitatively analyzed by real-time PCR assay of the precipitated DNA and input DNA using SYBER Green Master Mix in an ABI 7500 sequence detection system. The primers used for amplification of the hLHR gene promoter region were 5'-ACTGGGCACTGTCGCAGGTC 3' (forward) and 5' CATGGC CGGCGAACTGGGCT 3' (reverse).

For Re-ChIP analyses, complexes obtained from the primary immunoprecipitation were eluted from protein A agarose beads by incubation of samples with 5 mM dithiothreitol at 37 C for 20 min. Samples were mixed well by gentle vortex at every 5-min interval. The supernatant containing eluted protein complexes was recovered by brief centrifugation then was 1:20 diluted in ChIP dilution buffer (1% Trion X-100, 2 mM EDTA, 150 mM NaCl, and 20 mM Tris-HCl at pH 8.1). The second round of immunoprecipitation was performed with a relevant antibody that is different from one used in the first round. The protocol applied in subsequent steps of the re-ChIP assay was the same as that used in the single ChIP assay.


   FOOTNOTES
 
This work was supported by the Intramural Research Program of the National Institute of Child Health and Human Development, National Institutes of Health.

Disclosure Statement: The authors have nothing to disclose.

First Published Online March 27, 2008

Abbreviations: CA, Constitutively active; ChIP, chromatin immunoprecipitation; DAG, diaceylglycerol; DN, dominant negative; hLHR, human LHR; JNK, c-Jun N-terminal kinase; LHR, LH receptor; NTC, siRNA for the negative control; p-, phosphorylated; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; PMA, phorbol-12-myristate-13-acetate; Pol II, RNA polymerase II; Re-ChiP, sequential ChIP; siRNA, small interference RNA; TFIIB, transcription factor II B; TSA, trichostatin A.

Received for publication January 28, 2008. Accepted for publication March 20, 2008.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Dufau ML, Tsai-Morris C-H 2007 The Leydig cell in health and disease. In: Payne AH, Hardy MP, eds. Contemporary endocrinology. Totowa, NJ: Humana Press Inc.; 227–252
  2. Dufau ML 1998 The luteinizing hormone receptor. Annu Rev Physiol 60:461–496[CrossRef][Medline]
  3. Tsai-Morris C, Xie X, Wang W, Buczko E, Dufau M 1993 Promoter and regulatory regions of the rat luteinizing hormone receptor gene. J Biol Chem 268:4447–4452[Abstract/Free Full Text]
  4. Tsai-Morris C, Geng Y, Xie X, Buczko E, Dufau M 1994 Transcriptional protein binding domains governing basal expression of the rat luteinizing hormone receptor gene. J Biol Chem 269:15868–15875[Abstract/Free Full Text]
  5. Tsai-Morris CH, Geng Y, Buczko E, Dufau ML 1998 A novel human luteinizing hormone receptor gene. J Clin Endocrinol Metab 83:288–293[Abstract/Free Full Text]
  6. Zhang Y, Dufau ML 2000 Nuclear orphan receptors regulate transcription of the gene for the human luteinizing hormone receptor. J Biol Chem 275:2763–2770[Abstract/Free Full Text]
  7. Zhang Y, Dufau ML 2001 EAR2 and EAR3/COUP-TFI regulate transcription of the rat LH receptor. Mol Endocrinol 15:1891–1905[Abstract/Free Full Text]
  8. Zhang Y, Dufau ML 2003 Repression of the luteinizing hormone receptor gene promoter by cross talk among EAR3/COUP-TFI, Sp1/Sp3, and TFIIB. Mol Cell Biol 23:6958–6972[Abstract/Free Full Text]
  9. Zhang Y, Dufau ML 2002 Silencing of transcription of the human luteinizing hormone receptor gene by histone deacetylase-mSin3A complex. J Biol Chem 277:33431–33438[Abstract/Free Full Text]
  10. Zhang Y, Fatima N, Dufau ML 2005 Coordinated changes in DNA methylation and histone modifications regulate silencing/derepression of luteinizing hormone receptor gene transcription. Mol Cell Biol 25:7929–7939[Abstract/Free Full Text]
  11. Zhang Y, Liao M, Dufau ML 2006 Phosphatidylinositol 3-kinase/protein kinase C{zeta}-induced phosphorylation of Sp1 and p107 repressor release have a critical role in histone deacetylase inhibitor-mediated depression of transcription of the luteinizing hormone receptor gene. Mol Cell Biol 26:6748–6761[Abstract/Free Full Text]
  12. Chu S, Ferro TJ 2005 Sp1: Regulation of gene expression by phosphorylation. Gene 348:1–11[CrossRef][Medline]
  13. Milanini-Mongiat J, Pouyssegur J, Pages G 2002 Identification of two Sp1 phosphorylation sites for p42/p44 mitogen-activated protein kinases: their implication in vascular endothelial growth factor gene transcription. J Biol Chem 277:20631–20639[Abstract/Free Full Text]
  14. Fojas de Borja P, Collins NK, Du P, Azizkhan-Clifford J, Mudryj M 2001 Cyclin A-CDK phosphorylates Sp1 and enhances Sp1-mediated transcription. EMBO J 20:5737–5747[CrossRef][Medline]
  15. Parker PJ, Murray-Rust J 2004 PKC at a glance. J Cell Sci 117:131–132[Free Full Text]
  16. Newton AC 1995 Protein kinase C: structure, function, and regulation. J Biol Chem 270:28495–28498[Free Full Text]
  17. Teicher BA 2006 Protein kinase C as a therapeutic target. Clin Cancer Res 12:5336–5345[Free Full Text]
  18. Noland Jr T, Dimino M 1986 Characterization and distribution of protein kinase C in ovarian tissue. Biol Reprod 35:863–872[Abstract]
  19. Clark MR, Davis JS, Lemaire WJ 1983 Calcium- and lipid-dependent protein phosphorylation in the human ovary. J Clin Endocrinol Metab 57:872–874[Abstract/Free Full Text]
  20. Davis JS, Clark MR 1983 Activation of protein kinase in the bovine corpus luteum by phospholipid and Ca2+. Biochem J 214:569–574[Medline]
  21. Shimamoto T, Yamoto M, Nakano R 1993 Possible involvement of protein kinase C in gonadotropin-induced ovulation in the rat ovary. Endocrinology 133:2127–2132[Abstract/Free Full Text]
  22. Cutler Jr RE, Maizels ET, Brooks EJ, Mizuno K, Ohno S, Hunzicker-Dunn M 1993 Regulation of {delta} protein kinase C during rat ovarian differentiation. Biochim Biophys Acta 1179:260–270[Medline]
  23. Braden TD, Bervig T, Conn PM 1991 Protein kinase-C activation stimulates synthesis of gonadotropin-releasing hormone (GnRH) receptors, but does not mediate GnRH-stimulated receptor synthesis. Endocrinology 129:2486–2490[Abstract/Free Full Text]
  24. Anderson L 1996 Intracellular mechanisms triggering gonadotrophin secretion. Rev Reprod 1:193–202[Abstract]
  25. Vasilyev VV, Lawson MA, Dipaolo D, Webster NJ, Mellon PL 2002 Different signaling pathways control acute induction versus long-term repression of LHβ transcription by GnRH. Endocrinology 143:3414–3426[Abstract/Free Full Text]
  26. Leung P, Steele G 1992 Intracellular signaling in the gonads. Endocr Rev 13:476–498[Abstract/Free Full Text]
  27. Pati D, Habibi HR 2002 Involvement of protein kinase C and arachidonic acid pathways in the gonadotropin-releasing hormone regulation of oocyte meiosis and follicular steroidogenesis in the goldfish ovary. Biol Reprod 66:813–822[Abstract/Free Full Text]
  28. Cheng KW, Ngan ESW, Kang SK, Chow BKC, Leung PCK 2000 Transcriptional down-regulation of human gonadotropin-releasing hormone (GnRH) receptor gene by GnRH: role of protein kinase C and activating protein 1. Endocrinology 141:3611–3622[Abstract/Free Full Text]
  29. Yeung C-M, An B-S, Cheng CK, Chow BKC, Leung PCK 2005 Expression and transcriptional regulation of the GnRH receptor gene in human neuronal cells. Mol Hum Reprod 11:837–842[Abstract/Free Full Text]
  30. Lin X, Conn PM 1999 Transcriptional activation of gonadotropin-releasing hormone (GnRH) receptor gene by GnRH: involvement of multiple signal transduction pathways. Endocrinology 140:358–364[Abstract/Free Full Text]
  31. Geng Y, Tsai-Morris CH, Zhang Y, Dufau ML 1999 The human luteinizing hormone receptor gene promoter: activation by Sp1 and Sp3 and inhibitory regulation. Biochem Biophys Res Commun 263:366–371[CrossRef][Medline]
  32. Chun RF, Semmes OJ, Neuveut C, Jeang K-T 1998 Modulation of Sp1 phosphorylation by human immunodeficiency virus type 1 Tat. J Virol 72:2615–2629[Abstract/Free Full Text]
  33. Schonwasser DC, Marais RM, Marshall CJ, Parker PJ 1998 Activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway by conventional, novel, and atypical protein kinase C isotypes. Mol Cell Biol 18:790–798[Abstract/Free Full Text]
  34. Nakashima S 2002 Protein kinase C{alpha}: regulation and biological function. J Biochem (Tokyo) 132:669–675[Abstract/Free Full Text]
  35. Marais R, Wynne J, Treisman R 1993 The SRF accessory protein Elk-1 contains a growth factor-regulated transcriptional activation domain. Cell 73:381–393[CrossRef][Medline]
  36. Gille H, Sharrocks AD, Shaw PE 1992 Phosphorylation of transcription factor p62TCF by MAP kinase stimulates ternary complex formation at c-fos promoter. Nature 358:414–417[CrossRef][Medline]
  37. Soh J-W, Lee EH, Prywes R, Weinstein IB 1999 Novel roles of specific isoforms of protein kinase C in activation of the c-fos serum response element. Mol Cell Biol 19:1313–1324[Abstract/Free Full Text]
  38. Kang-Park S, Lee JH, Shin JH, Lee YI 2001 Activation of the IGF-II gene by HBV-X protein requires PKC and p44/p42 map kinase signalings. Biochem Biophys Res Commun 283:303–307[CrossRef][Medline]
  39. Pintus G, Tadolini B, Posadino AM, Sanna B, Debidda M, Carru C, Deiana L, Ventura C 2003 PKC/Raf/MEK/ERK signaling pathway modulates native-LDL-induced E2F-1 gene expression and endothelial cell proliferation. Cardiovasc Res 59:934–944[Abstract/Free Full Text]
  40. Lindemann RK, Braig M, Hauser CA, Nordheim A, Dittmer J 2003 Ets2 and protein kinase C {epsilon} are important regulators of parathyroid hormone-related protein expression in MCF-7 breast cancer cells. Biochem J 372:787–797[CrossRef][Medline]
  41. Soh JW, Lee YS, Weinstein IB 2003 Effects of regulatory domains of specific isoforms of protein kinase C on growth control and apoptosis in MCF-7 breast cancer cells. J Exp Ther Oncol 3:115–126[CrossRef][Medline]
  42. Biggs JR, Kudlow JE, Kraft AS 1996 The role of the transcription factor Sp1 in regulating the expression of the WAF1/CIP1 gene in U937 leukemic cells. J Biol Chem 271:901–906[Abstract/Free Full Text]
  43. Han JW, Ahn SH, Kim YK, Bae GU, Yoon JW, Hong S, Lee HY, Lee YW, Lee HW 2001 Activation of p21(WAF1/Cip1) transcription through Sp1 sites by histone deacetylase inhibitor apicidin: involvement of protein kinase C. J Biol Chem 276:42084–42090[Abstract/Free Full Text]
  44. Hu PP-c, Shen X, Huang D, Liu Y, Counter C, Wang X-F 1999 The MEK pathway is required for stimulation of p21WAF1/CIP1 by transforming growth factor-β. J Biol Chem 274:35381–35387[Abstract/Free Full Text]
  45. Huang L, Sowa Y, Sakai T, Pardee AB 2000 Activation of the p21WAF1/CIP1 promoter independent of p53 by the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) through the Sp1 sites. Oncogene 19:5712–5719[CrossRef][Medline]
  46. Sowa Y, Orita T, Minamikawa-Hiranabe S, Mizuno T, Nomura H, Sakai T 1999 Sp3, but not Sp1, mediates the transcriptional activation of the p21/WAF1/Cip1 gene promoter by histone deacetylase inhibitor. Cancer Res 59:4266–4270[Abstract/Free Full Text]
  47. Wen-Sheng W 2003 ERK signaling pathway is involved in p15INK4b/p16INK4a expression and HepG2 growth inhibition triggered by TPA and Saikosaponin a. Oncogene 22:955–963[CrossRef][Medline]
  48. Wen-Sheng W, Jun-Ming H 2005 Activation of protein kinase C {alpha} is required for TPA-triggered ERK (MAPK) signaling and growth inhibition of human hepatoma cell HepG2. J Biomed Sci 12:289–296[CrossRef][Medline]
  49. Gruber JR, Desai S, Blusztajn JK, Niles RM 1995 Retinoic acid specifically increases nuclear PKC {alpha} and stimulates AP-1 transcriptional activity in B16 mouse melanoma cells. Exp Cell Res 221:377–384[CrossRef][Medline]
  50. Martelli AM, Evangelisti C, Nyakern M, Manzoli FA 2006 Nuclear protein kinase C. Biochim Biophys Acta 1761:542–551[Medline]
  51. Pan M-R, Hung W-C 2002 Nonsteroidal anti-inflammatory drugs inhibit matrix metalloproteinase-2 via suppression of the ERK/Sp1-mediated transcription. J Biol Chem 277:32775–32780[Abstract/Free Full Text]
  52. Jackson SP, MacDonald JJ, Lees-Miller S, Tjian R 1990 GC box binding induces phosphorylation of Sp1 by a DNA-dependent protein kinase. Cell 63:155–165[CrossRef][Medline]
  53. Suzuki T, Kimura A, Nagai R, Horikoshi M 2000 Regulation of interaction of the acetyltransferase region of p300 and the DNA-binding domain of Sp1 on and through DNA binding. Genes Cells 5:29–41[Abstract]
  54. Emili A, Greenblatt J, Ingles CJ 1994 Species-specific interaction of the glutamine-rich activation domains of Sp1 with the TATA box-binding protein. Mol Cell Biol 14:1582–1593[Abstract/Free Full Text]
  55. Datta P, Raychaudhuri P, Bagchi S 1995 Association of p107 with Sp1: genetically separable regions of p107 are involved in regulation of E2F- and Sp1-dependent transcription. Mol Cell Biol 15:5444–5452[Abstract]
  56. Seto E, Lewist B, Shenk T 1993 Interaction between transcription factors Spl and YY1. Nature 365:462–464[CrossRef][Medline]
  57. Lin S, Black A, Kostic D, Pajovic S, Hoover C, Azizkhan J 1996 Cell cycle-regulated association of E2F1 and Sp1 is related to their functional interaction. Mol Cell Biol 16:1668–1675[Abstract]
  58. Lee D-K, Suh D, Edenberg HJ, Hur M-W 2002 POZ domain transcription factor, FBI-1, represses transcription of ADH5/FDH by interacting with the zinc finger and interfering with DNA binding activity of Sp1. J Biol Chem 277:26761–26768[Abstract/Free Full Text]
  59. Doetzlhofer A, Rotheneder H, Lagger G, Koranda M, Kurtev V, Brosch G, Wintersberger E, Seiser C 1999 Histone deacetylase 1 can repress transcription by binding to Sp1. Mol Cell Biol 19:5504–5511[Abstract/Free Full Text]
  60. Kaczynski J, Cook T, Urrutia R 2003 Sp1- and Kruppel-like transcription factors. Genome Biol 4:206[CrossRef][Medline]
  61. Lee J-A, Suh D-C, Kang J-E, Kim M-H, Park H, Lee M-N, Kim J-M, Jeon B-N, Roh H-E, Yu M-Y, Choi K-Y, Kim KY, Hur M-W 2005 Transcriptional activity of Sp1 is regulated by molecular interactions between the zinc finger DNA binding domain and the inhibitory domain with corepressors, and this interaction is modulated by MEK. J Biol Chem 280:28061–28071[Abstract/Free Full Text]
  62. Choi HS, Lee JH, Park JG, Lee YI 2002 Trichostatin A, a histone deacetylase inhibitor, activates the IGFBP-3 promoter by upregulating Sp1 activity in hepatoma cells: alteration of the Sp1/Sp3/HDAC1 multiprotein complex. Biochem Biophys Res Commun 296:1005–1012[CrossRef][Medline]
  63. Ellenrieder V, Zhang JS, Kaczynski J, Urrutia R 2002 Signaling disrupts mSin3A binding to the Mad1-like Sin3-interacting domain of TIEG2, an Sp1-like repressor. EMBO J 21:2451–2460[CrossRef][Medline]
  64. Galasinski SC, Resing KA, Goodrich JA, Ahn NG 2002 Phosphatase inhibition leads to histone deacetylases 1 and 2 phosphorylation and disruption of corepressor interactions. J Biol Chem 277:19618–19626[Abstract/Free Full Text]
  65. Hsieh M, Conti M 2005 G-protein-coupled receptor signaling and the EGF network in endocrine systems. Trends Endocrinol Metab 16:320–326[CrossRef][Medline]
  66. Park JY, Su YQ, Ariga M, Law E, Jin SL, Conti M 2004 EGF-like growth factors as mediators of LH action in the ovulatory follicle. Science 303:682–684[Abstract/Free Full Text]
  67. Liu G, Duranteau L, Carel JC, Monroe J, Doyle DA, Shenker A 1999 Leydig-cell tumors caused by an activating mutation of the gene encoding the luteinizing hormone receptor. N Engl J Med 341:1731–1736[Free Full Text]
  68. Ujhazy P, Klobusicka M, Babusikova O, Strausbauch P, Mihich E, Ehrke MJ 1994 Ecto-5'-nucleotidase (CD73) in multidrug-resistant cell lines generated by doxorubicin. Int J Cancer 59:83–93[Medline]

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S.-N. Kim, N. H. Kim, W. Lee, D.-W. Seo, and Y. K. Kim
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