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Am J Physiol Endocrinol Metab. Author manuscript; available in PMC Sep 21, 2009.
Published in final edited form as:
PMCID: PMC2747322
NIHMSID: NIHMS139660

Expression and Regulation of Kruppel-like factors 4, 9 and 13 and the steroidogenic genes LDLR, StAR and CYP11A in ovarian granulosa cells

Abstract

Kruppel-like factors (KLF's) are important Sp1-like eukaryotic transcriptional proteins, which mediate cell growth, differentiation and apoptosis. The LDLR, StAR and CYP11A genes exhibit GC-rich Sp1-like sites, which have the potential to bind KLF's in multiprotein complexes. We now report that KLF4, KLF9 and KLF13 transcripts and cognate proteins are expressed in and regulate ovarian cells. KLF4 and 13, but not KLF9, mRNA expression was induced and then repressed over time (P < 0.001). Combined LH and IGF-I stimulation increased KLF4 mRNA at 2 h (P < 0.01), whereas LH decreased KLF13 mRNA at 6 h (P < 0.05) and IGF-I did the so at 24 h (P < 0.01) compared with untreated controls. KLF9 was not regulated by either hormone. Transient transfection of KLF4, KLF9 and KLF13 suppressed LDLR/luc, StAR/luc and CYP11A/luc by 80 to 90% (P < 0.001). Histone-deacetylase (HDAC) inhibitors stimulated LDLR/luc by 5-6 fold and StAR/luc and CYP11A/luc activity by 2-fold (P < 0.001), and partially reversed suppression by all 3 KLF's (P < 0.001). Deletion of the zinc-finger domain of KLF13 abrogated repression of LDLR/luc. Lentiviral overexpression of the KLF13 gene suppressed LDLR mRNA (P < 0.001) and CYP11A mRNA (P = 0.003), but increased StAR mRNA (P = 0.007). Collectively, these data suggest that KLF's may recruit inhibitory complexes containing HDAC corepressors, thereby repressing LDLR and CYP11A transcription. Conversely, KLF13 may recruit unknown coactivators or stabilize StAR mRNA, thereby explaining enhancement of in situ StAR gene expression. These data introduce a new cohort of potent gonadal regulator of genes encoding proteins that mediate sterol uptake and steroid biosynthesis.

Keywords: KLF4, KLF9, KLF13, LDLR, StAR, CYP11A, sterol, gonad, luteal

Introduction

Ovarian steroidogenesis is critical for the physiological regulation of both reproductive and nonreproductive tissues, such as the uterus, breast, vascular endothelium, adipose tissue, skeleton and brain (18). De novo synthesis of steroids is directly dependent on the mitochondrial availability of free cholesterol. The primary source of intracellular sterol substrate for steroidogenesis in the cow, pig, monkey and human is blood-borne low-density lipoprotein (LDL) cholesterol, which is taken up by cognate membrane receptors (2; 15; 41; 51; 52). Steroidogenic acute regulatory protein (StAR) transports cytoplasmic cholesterol from the outer to the inner mitochondrial membrane, where the cytochrome P450 cholesterol side-chain cleavage (CYP11A) complex resides. StAR and CYP11A are rate limiting in absolute terms and enzymatically, respectively. These genes contain several response element, including steroidogenic factor-1 (SF-1), Sp1, CAAT/enhancer-binding protein-β (C/EBPβ), cAMP response-element binding protein (CRE), sterol regulatory element-binding protein-1 (SREBP-1) and GATA-4 sites (23; 26). Regulation of LDL receptor (LDLR) transcription in mammalian cells is also complex, requiring interactions among cholesterol-sensitive SREBP-1 and sequence-specific but generic coregulatory factors like Sp1, CBP, C/EBPβ, YY1 and NF-Y (14). In addition, a recent study disclosed that the Sp1-like protein, Krüppel-like factor 13 (KLF13), can repress the proximal LDLR promoter in a sterol-independent manner (27).

Triple Cys2/His2 (C2H2) zinc-finger sequences characteristic of the Sp1/KLF superfamily constitute the most common DNA-binding domain in human's making up approximately 2% of the genome (7; 43). At least 24 such nuclear proteins have been identified in mammals, which are involved in a variety of cellular activities such as cell growth, development, differentiation and apoptosis (22; 42). Sp1/KLF13′s bind conservatively to GC or GT-rich sites containing either a CGCCC or CT/ACC core sequence (7). DNA microarray technology and other exploratory analyses have identified several KLF genes (KLF2, KLF4, KLF5, KLF6, KLF9, KLF13, KLF15 and KLF17) in the mammalian ovary, but their functions in this organ are not known (8; 9; 13; 20; 53). In addition, KLF4 is highly expressed in the testis and acutely regulated by FSH (34), but its role in gonadal cells has not been delineated (42). In relation to reproduction, ablation of the klf9 gene in female mice results in uterine hypoplasia, reduced litter size, and increased neonatal death (38). In endometrial cells, KLF9 binds progesterone receptor isoforms, PR-A and PR-B, and functionally activates PR-B in the presence of progesterone (55).

The present study identifies genes that encode Krüppel-like factors 4, 9 and 13 in porcine granulosa-luteal cells. KLF4 and KLF13 expression is regulated by LH and/or insulin/IGF-I. Conversely, all 3 of KLF4, KLF9 and KLF13 repress the proximal promoters of LDLR, StAR and CYP11A. Transcriptional repression is antagonized by HDAC inhibitors, trichostatin A (TSA) and sodium butyrate (SB). These data suggest that KLF4, KLF9 and KLF13 may participate in the hormonally regulated transcription of steroidogenic genes in gonadal cells.

Materials and Methods

Reagents

Ovine follicle-stimulating hormone (NIDDK oFSH-18; potency 65 × NIH-oFSH-S1), OLH-26 and human IGF-I were obtained from the National Hormone and Pituitary Program, NIH (Bethesda MD); porcine insulin, trichostatin A and estradiol-17 beta from Sigma Chemical Co. (St. Louis, MO); Eagle's Minimum Essential Medium (MEM), penicillin/streptomycin, gentamicin, fetal bovine serum, trypsin-EDTA and lipofectamine reagent from Life Technologies (Grand Island, NY); rabbit anti-GKLF (H-180; KLF4), goat antibody to KLF9 and KLF13 (human C-terminus), from Santa Cruz Biotechnology, Inc (Santa Cruz, CA); sodium butyrate from Upstate Cell Signaling Solutions (Lake Placid, NY) and the Luciferase Reporter Assay System from Promega (Madison, WI). Oligonucleotides were synthesized by IDT (Integrated DNA Technologies, Inc., Coraville, IA) or by the Advanced Genomics Technology Center, Mayo Clinic, Rochester, MN.

Ovarian granulosa-cell culture

Ovaries from prepubertal (60-70 kg) swine were collected at an abattoir and transported to the laboratory in iced saline. Granulosa cells were obtained from small and medium-sized (1-5 mm) antral Graafian follicles by fine-needle aspiration under sterile conditions, and washed three times by low-speed centrifugation (3000 rpm) in MEM. Approximately 5 × 106 viable cells were plated in 12-well culture dishes (Corning, NY) containing bicarbonate-buffered MEM and 3% fetal bovine serum (FBS) cells so cultured for 48 h were termed granulosa cells, and used in Western-blot studies. In all other experiments, cells were maintained additionally with insulin (1 μg/ml), estradiol (0.5 μg/ml) and FSH (5 ng/ml) to permit partial luteinization (53) and attachment for 48 h at 37 C in 5% CO2. The resultant cells were termed granulosa-luteal cells. The human granulosa cell line, SVOG-4o was produced as described previously from cells otherwise discarded at in vitro fertilization, and immortalized by SV40 Tag (19).

Transient transfection

Transient transfection analyses utilized porcine LDLR/luc (-1076 to +11 bp), StAR/luc (-1423 to + 130 bp) and CYP11A (-2320 to + 23 bp) proximal 5′-upstream regulatory sequences driving cytoplasmically targeted firefly luciferase (16; 29; 36). Granulosa-luteal cell monolayers were incubated in serum-free MEM without antibiotics for 20-30 min. Transfection medium (0.5 mL/well) comprised serum-free MEM without antibiotics containing 1 μg total plasmid DNA and 6 μL lipofectamine. Based on optimizing time-course experiments, 5% serum-containing medium was replaced after 6 h of transfection. After an additional 24 h of recovery to allow vector expression, cells were exposed to serum-free MEM containing antibiotics and the indicated effectors or vehicle for 24 h. Where indicated, cells were cotransfected with 0.3 μg/well of pcDNA3.1/His-C-KLF13 or pcDNA3.1/His-C-KLF9 or pcDNA 3.1/His-C-KLF4 and/or empty control pCMV/His-C vector. Full-length KLF13 expression vector, a C-terminal zinc finger-deleted KLF13 construct (1-172 a.a) or a C-terminal deleted KLF13 construct retaining only N-terminal a.a 1-35 were generated from a pCMV/His-C vector by PCR. Expression vectors containing just the zinc-finger motif or the zinc-finger combined with any 1 of 3 different N-terminal repression domains (1-24 a.a or 55-74 a.a or 75-114 a.a) were reported previously (12). To quantify reporter expression, cultures were rinsed once at room temperature with Dulbecco's phosphate-buffered saline (PBS), lysed in 100 μL of 1 × lysis buffer (Luciferase Assay System, Promega Corp., Madison, WI), and stored at -70 C until later assay. Luciferase activity was measured using 100 μL firefly luciferin substrate (Promega Corp., Madison, WI) and 20 μL cellular lysate in a Turner TD-20/20 DLR luminometer (Turner Designs, Sunnyvale, CA). Data were normalized as relative light units/100 μg of protein and expressed as the mean ± SEM. Total protein was used to normalize luciferase activity rather than coexpression of SV40_or RSV/Renilla or β-galactosidase, which are induced in this and some other systems_(27; 48). All experiments were performed at least three times each with triplicate incubations.

Cloning of KLF13 cDNA into pSIN-CSGW-UnEm plasmid and viral transduction

KLF13 cDNA was amplified by PCR from total RNA from pig ovarian granulosa cells using gene-specific primers containing BamHI and NotI, as described previously (27). PCR products were subcloned into a pSIN-CSGW-UNEm (6) (provided by Dr. Y Ikeda Mayo Clinic, Rochester, MN) to create KLF13/pSIN-E or empty control vector pSIN-E. Purified KLF13 expression Lentivirus or empty vectors (1.5 μg) were transfected with 1 μg of pMD.G and 1 μg of p8.9-QV helper plasmids (provided by Dr. Y. Ikeda.Y) using 15 μL of Fugene 6 (Roche Applied science, Indianapolis, IN) into 293FT cells using the ViraPower Lentiviral Expression System (Invitrogen, Carlsbad, CA). Medium containing DNA-Fugene 6 complexes was removed the next day and replaced with complete medium for 48 h. Viral supernatants were harvested 72 h after transfection and centrifuged at 3000 rpm for 15 min at 4C to remove cell debris. To determine viral titers, granulosa cells were transduced with serial dilutions of the lentiviral stocks, and 72 h later GFP expression was assessed by flow cytometry (6). Granulosa-luteal cells were then virally transduced with the indicated multiplicity-of-infection units, and incubated overnight in growth medium containing 6 μg/mL polybrene followed by complete medium for 48 h to allow gene and protein expression.

Real-time quantitative polymerase-chain reaction (PCR)

Total RNA was isolated using Trizol Reagent and reverse transcribed using the SuperScript III Reverse transcriptase kit (Invitrogen, Carlsbad, CA) with 2.5 μM of oligo d(T)15, 0.2 μM 18S-reverse template and 1 μg RNA. The cDNA was amplified in 25 μL PCR buffer with IQ SYBR Green Master Mix (Bio-Rad) in the presence of specified primers for KLF4, 9 and 13 as well as for LDLR, StAR and CYP11A coding sequences (Table 1). The PCR conditions comprised a hot-start by 95°C for 10 min, followed by 40 cycles of 95°C for 30 s, 60°C for 30 s and 72°C for 30 s. Samples were run in duplicate on the MyiQ™ Single color real-time PCR detection system (Bio-Rad, Hercules, CA) to determine the threshold cycle (Ct) (5). Expression levels were normalized to 18S by the ΔΔCt method (30).

Table 1
Real-time PCR primer sequences

Western blot

Granulosa-luteal and granulosa-cell extracts (50 μg) were resolved by 4-20% SDS-PAGE (Bio-Rad, Hercules, CA) and electroblotted to nitrocellulose membranes. Basal KLF peptide levels were not detectable consistently in granulosa-luteal cells. Thus, granulosa cells were used in Western studies. For KLF13 protein expression studies, 600 μg total protein per sample were subjected to immunoprecipitaion using ExactaCruz™ IP/Western blot kits (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Western-blot analyses were carried out using goat anti-human KLF9 or KLF13 (1:700 dilution) or rabbit anti-human KLF4 [(1: 1000, Santa Cruz Biotechnology, Inc, Santa Cruz, CA)]. Signals were visualized by chemiluminescence using ECL Western blotting substrate from Pierce (Pierce Biotechnology, Inc., Rockford, IL).

Statistical methods

Observations were based on 3 or more independent experiments conducted using separate batches of 150-200 ovaries. Data from separate experiments were subjected to one- or two-way ANOVA in a repeated-measures design (21). Ratio values (observed to control) were log-transformed to limit the dispersion of residual variance. Means were compared by the post hoc Tukey multiple-comparison test at P < 0.05. For mRNA expression data (Figure 1), a one-sided F-ratio test was performed against the null hypothesis of no difference from time-matched control.

Figure 1
Time course of LH and/or insulin/IGF-I-stimulated expression of KLF4, KLF9 and KLF13 gene transcripts

Results

Kruppel-like factor mRNA and protein expression in granulosa-luteal cells

To understand the expression and regulation of ovarian KLF's, gene transcripts were assessed by real-time PCR. Granulosa-luteal cells were exposed to control, solvent, LH and/or IGF-I for 2, 6 and 24 h (N = 4 to 14 experiments): Figure 1. In unstimulated cultures, relative abundancies of KLF4, 9 and 13 mRNA were 48, 1.0, and 25, respectively. Based on ANOVA, KLF4 and KLF13, but not KLF9, mRNA expression differed significantly over time (P < 0.001). Incubation with LH and IGF-I together (but not alone) for 2 h increased KLF4 mRNA expression by 4-6 fold compared with untreated control (P < 0.01). KLF9 mRNA expression was unaffected by different experimental stimuli. KLF13 mRNA expression was reduced by LH at 6 h (P < 0.05) and by IGF-I at 24 h (P < 0.01) compared with respective untreated controls obtained at the same time points. Thus, all three KLF genes are expressed in pig granulosa-luteal cells, and regulated (KLF13 and KLF4) or not regulated (KLF9) by hormone(s) over time.

Granulosa-cell expression of KLF proteins was evaluated basally and after stimulation with gonadotropins or insulin in three experiments. Immature cells were used since KLF proteins were not consistently detectable in granulosa-luteal cells. Western analysis revealed a single specific band for KLF13 (~ 37 kDa), which was not significantly altered by exposure to either forskolin or insulin for 24 h. The HDAC inhibitor, SB (1.0 mM for 24 h), increased KLF13 protein expression by 2-fold: Figure 2C. Immunoprecipitation of total cellular or nuclear protein did not yield bands specific to KLF9, suggesting very low expression or the need for a species-specific antibody. Analysis of total cellular protein using 4-20% SDS-PAGE showed three bands of KLF4 immunoreactivity. All bands were evaluated together by densitometry. Other studies have also reported multiple variants of KLF4 protein/mRNA in myeloid cells (31) and testis (37). Stimulation with FSH or forskolin for 2 and 24 h decreased total KLF4 protein expression by 40 to 50% compared with untreated control (P < 0.01). Insulin/IGF-I or the HDAC inhibitor, SB (1 mM), did not alter KLF4 expression significantly. KLF4 mRNA expression was downregulated by treatment of IGF-I (Figure 1), whereas protein levels were not altered. This discrepancy might be due to a longer half-life of KLF protein or greater sensitivity of PCR than Western blot.

Figure 2
KLF4 and KLF13 protein expression in less mature granulosa cells

KLF's suppress gene expression in ovarian cells

To asses regulation of ovarian steroidogenic genes, porcine granulosa-luteal cells were transiently transfected with the promoter-reporter constructs, LDLR/luc (-1076 to + 11bp), StAR/luc (-1423 to + 130bp) and CYP11A/luc (-2320 to + 23 bp) along with cDNA's for full-length coding sequences of KLF4, KLF9 and KLF13. Statistical analyses (N=8 experiments) showed that exogenous KLF13, KLF9 and KLF4 decreased LDLR/luc activity by 92%, 78% and 85%, respectively (P < 0.001) Figure 3. Exposure to histone deacetylase (HDAC) inhibitors, TSA (10 ng/ml) or SB (1.0 mM), for 24 h after transfection increased basal LDLR/luc reporter expression by 4-5 fold (P < 0.001), and significantly attenuated repression of LDLR/luc activity by each of the 3 KLF's (P < 0.001).

Figure 3
Repression of LDLR/luc, StAR/luc and CYP11A/luc by each of KLF4, 9 and 13 in porcine granulosa-luteal cells

In steroidogenic tissues, cholesterol is transported from the outer to the inner mitochondrial membrane by StAR protein and converted into pregnenolone by the CYP11A enzyme. To understand how KLF proteins influence these 2 steroidogenic genes, studies were extended to include the corresponding reporters (Figure 3). Cotransfection of KLF13, KLF9 and KLF4 with StAR or CYP11A promoter fragments repressed luciferase activity by 80-90% compared with basal (in each case P < 0.001). In addition, StAR/luc and CYP11A/luc were upregulated by TSA or SB treatment by ~2 fold over basal (P < 0.01). Thus, each of the 3 KLF's can repress promoter-reporter constructs of all 3 of LDLR, StAR and CYP11A. HDAC inhibitors relieve such repression, suggesting that histone deacetylases contribute to promoter silencing.

To evaluate whether transcriptional repression is consistent, SVOG-4o cells derived from a human granulosa-cell line (19) were studied also. Transfection of KLF4, 9 or 13 reduced the expression of LDLR/luc (by > 95%) and StAR/luc (by 50%) (P < 0.001): Figure 4. Exposure to SB partially reversed inhibition of LDLR/luc but not StAR/luc. CYP11A/luc did not express well in the immortalized cells.

Figure 4
Repression of LDLR/luc and StAR/luc by KLF's in human granulosa-cell line, SVOG-4o

To assess the structural requirements for gene repression by KLF13, we created a series of deletional pcDNA3.1/His C constructs containing or excluding the N-terminal regulatory or C-terminal triple zinc-finger DNA-binding domains. As a positive control, transfection of CMV-driven full-length KLF13 suppressed LDLR/luc activity by 80-90%. As a negative control, empty His C vector did not alter basal promoter expression. Figure 5 shows that vectors lacking the C-terminal zinc-finger motif (1-172 a.a) or C-terminal-deleted KLF13 construct retaining only N-terminal sequence (1-35 a.a) did not repress LDLR/luc. Conversely, expression vectors containing just the zinc-finger motif or the latter along with any of 3 different N-terminal repressor domains (1-24 a.a or 55-74 a.a or 75 to 114 a.a) suppressed luciferase activity comparably to wild-type KLF13 (P < 0.001, N = 5 experiments). These observations suggest the DNA-interacting zinc finger domain is essential for the repressive effects of KLF13 on the LDLR promoter.

Figure 5
Functional characterization of KLF13 protein

Viral transfer of the KLF13 minigene

A lentivirus expression vector, pSIN-CSGW-UNEm (Methods), was used to overexpress the full-length coding sequence of porcine KLF13. Granulosa-luteal cells were transduced with various concentrations or multiplicities of infection (5 to 50 MOI) of KLF13 lentivirus or empty lentivirus and cultured for 3 days to allow gene expression. Overexpression of KLF13 message was confirmed by real-time RT-PCR and of KLF13 protein by Western blot. Transcripts of the putatively targeted genes, LDLR, StAR and CYP11A, were quantified by real-time PCR in a total of 13-19 experiments: Figure 6. Regression analyses showed that increased expression of KLF13 message predicted decreased expression of LDLR mRNA (P < 0.001). A similarly strong negative correlation was found between KLF13 mRNA and CYP11A mRNA (P < 0.003). In contrast, KLF13 and StAR mRNA were positively correlated in granulosa-luteal cells (P < 0.007). The last finding distinguishes regulation of the subcloned 1.4 kb StAR 5′-upstream fragment contained in StAR/luc from that of the native gene studied in situ.

Figure 6
Lentiviral-mediated transfer of KLF13 minigene regulates expression of endogenous LDLR, StAR and CYP11A genes in porcine granulosa-luteal cells

Discussion

An earlier study showed that KLF13 suppresses LDLR promoter expression (27). The present work establishes the following new observations: 1) KLF 4 and KLF13 mRNA expression are regulated by LH and/or IGF-I; 2) all three KLF's suppress not only the LDLR promoter but also that of StAR and CYP11A; and 3) using lentiviral overexpression of KLF13 in granulosa-luteal cells, endogenous LDLR and CYP11A mRNA expression are downregulated, whereas StAR mRNA is unregulated.

Earlier gel mobility-shift analyses reported that pig ovarian cells contain immunoactive Sp1 and Sp3 and an unknown protein-DNA complex. Each nuclear protein bound all 3 Sp1-like 5′-TCCTCC-3′ sequences contained in the porcine proximal LDLR promoter (27; 30). Recent rat ovarian gene-expression databases identified KLF4, KLF5, KLF9, KLF13 and KLF15, which were of unknown function (9). The current study delineates expression of KLF4, KLF9 and KLF13 gene transcripts and KLF13 and KLF4 proteins in granulosa-luteal or granulosa cells. Transient transfection experiments established that each of the 3 KLF's represses a putative proximal promoter fragment of the LDLR, StAR and CYP11A genes. Viral overexpression studies documented that KLF13 is a strong repressor of both LDLR and CYP11A gene expression in situ in granulosa-luteal cells, but unexpectedly a potent inducer of StAR. Mechanistic analyses demonstrated that HDAC inhibitors significantly overcome repression by each of KLF4, 9 and 13 of promoter-reporter constructs for LDLR, StAR and CYP11A. Moreover, the C-terminal triple zinc-finger motif of KLF13 rather than its N-terminus provides a critical repressive signal for LDLR expression. The collective data introduce evidence for a novel nuclear transcriptional system that may participate in steroidogenic-gene regulation in the ovary.

Activation and repression of selected genes by distinct transcriptional factors play important roles in gonadal function, including in cellular replication, apoptosis, differentiation and steroid biosynthesis (33). Among mammalian transcription factors, the zinc-finger motif is the most abundant DNA-binding sequence recognized (43). The C2H2 zinc-finger motif is generally present in tandem arrays containing the sequence Y/F-X-C-X2-4-C-X3-F-X5-L-X2-H-X3-5-H, where X represents variable amino-acid residues. Examples of proteins in this family include the gonadotropin-inducible transcription factors, GIOT1 and GIOT2, which are transiently regulated by FSH (25). Other zinc-finger proteins, such as GATA4 and GATA6, are also expressed in the ovary, and are positively regulated by FSH in granulosa cells (17). Recently, a novel nuclear zinc-finger protein called GATA-like protein-1 (GLP-1), a critical nuclear repressor, was discovered in somatic cells of developing gonads, including in Leydig and granulosa cells (18). Yan et al., reported a zinc-finger protein ZFP393 expressed exclusively in the testis and ovary (53), which was later shown to be KLF17 (49). Furthermore, analyses of a rat gene database and DNA microarrays disclosed expression of KLF4, KLF5, KLF9, KLF10, KLF13 and KLF15 in gonadal tissues (9; 13; 20; 44). These studies did not elucidate any functional roles of KLF's. However, clinical investigations point to dysregulation of KLF2 and KLF4 in the polycyctic ovary syndrome (8) and of KLF2 (50) and KLF6 (5) in ovarian tumors.

The accompanying data document regulated expression of KLF4 and KLF13 gene transcripts in ovarian granulosa-luteal cells. KLF9 gene expression did not vary over time, and was not responsive to LH or IGF-I stimulation. However, exposure to LH increased the abundance of KLF4 transcripts by ~ 5-fold. By real-time PCR, KLF4 mRNA concentrations peaked at 2 h and returned to control after 6 and 24 h. FSH analogously induced KLF4 gene expression in the murine testis within 4 h with recovery to baseline before 24 h (34). In addition, in a fibroblastic cell line (NIH 3T3), KLF4 transcripts were elevated in growth-arrested compared with proliferating cells (37). Northern blot and RT-PCR analyses showed ubiquitous expression of the KLF13 gene in mouse and human tissues and also in several cell lines (24; 35; 39). During mouse embryonic development, KLF13 is highly expressed in the heart and cephalic mesenchyme (24). Hormonal regulation of gonadal KLF13 mRNA was inferred from DNA-microarray data, in which immature rats were treated with human chorionic gonadotropin. The latter increased KLF13 mRNA by 2-fold at 6 h with a return to basal after 12 h (9). In contrast, we observed that IGF-I (but not LH) induces KLF13 gene transcripts by 2-3-fold within 2 h in granulosa-luteal cells. Further studies will be required to understand multi-hormonal regulation of the KLF13 gene in gonadal cells. Hormone-specific and time-dependent control of ovarian KLF4 and KLF13 gene expression would suggest distinct roles for these transcriptional factors in gonadal function. Although species-specific antibody or more sensitive protein-detention methods are needed to quantify the regulation of KLF peptides in granulosa-luteal cells, an HDAC inhibitor and FSH respectively increased KLF13 and decreased KLF4 protein in less mature granulosa cells.

A previous investigation identified TCCTCC-rich sequences in the LDLR gene that bind immunoreactive Sp1, Sp3 and an unidentified third protein. Electrophoretic gel-mobility analysis suggested that KLF13 is an important candidate protein (27; 30). The present data reveal further that KLF13 represses not only an LDLR-promoter construct, but also the native LDLR gene in granulosa-luteal cells. Moreover KLF13 inhibits expression of CYP11A, while stimulating that of StAR. The latter data suggest that the StAR promoter fragment does not include all KLF13-responsive sequences, and highlight that in situ gene regulation may differ from that inferred using promoter-reporter constructs. The mechanism of gene repression appears to involve deacetylation of histones, because two HDAC inhibitors significantly opposed reporter repression by KLF13 as well as by KLF9 and KLF4.

Several transcriptional-factor binding sequences have been localized 5′-upstream of the transcriptional start site of the StAR gene (3). Recently, Clem and Clark identified a critical CG-rich motif located at -180/-150 bp that binds Sp3 and mediates repression of murine StAR (4). Based on analyses using the transcription-element search system, the pig StAR promoter contains multiple Sp1-like binding sites within the region -170 to -145 bp (5′-CTGCCTCCCC CCCCCATCCC CCGCC-3′). These elements may participate in StAR regulation (10), given that the segment -219 to -31 bp is hormonally responsive (16; 28). Our data do not define the locus of KLF's effects on the StAR promoter, which will important to dissect further. However, in MA-10 cells, addition of the HDAC inhibitor, TSA, increased wild-type mouse StAR promoter activity, whereas mutations within the CAGA/Sp3 repressor region (-174 5′-CAGAGTCTGGTCCTCCC-3′ -157 bp) diminished reporter activity. HDAC inhibitors also stimulated basal expression of LH-receptor gene, which contains two Sp1-like binding sites between -180 to +1 bp (32). Thus, Sp1-like sites might mediate gene repression or induction. IGF-I increases CYP11A reporter activity through IGF-I-responsive elements (IGFRE, -130 to -100 bp), which represent GC-rich domains that can bind both Sp1 (45; 47; 48) and polypyrimidine tract-binding protein (PTB) (46). In this context, the porcine CYP11A promoter exhibits two CCCCTCC-rich sequences located -211 to-190 bp and -130 to-110 bp upstream of the transcriptional start site. However, whether KLF4, 9 or 13 can act via these or other cis elements is not yet known. What is clear is that all 3 KLF's are expressed in the ovary, and each can repress a proximal promoter sequence, and KLF13 can inhibit expression of the native LDL-R and CYP11A genes.

DNA-binding studies have shown that many Sp1-like and KLF proteins have similar but not identical affinities for GC-rich motifs (11; 42). Both the GC-rich sequence and gene context appear to determine binding and recruitment of KLF and coactivators such as CBP/p300/pCAF (1; 40), or corepressors such as HDAC/mSin3A (4; 27; 54). Preliminary mechanistic analysis presented here indicate that, at least in the case of KLF13, the C-terminal triple zinc-finger domain is needed to mediate transrepression of an LDLR-promoter fragment in granulosa-luteal cells. Whether the same requirement is true for KLF4 and KLF9 or is relevant to other sterol-regulated or steroidogenic genes targeted by KLF's has not been defined.

In summary, potent Sp1-like transcriptional regulators, KLF4, KLF9 and KLF13, are expressed in ovarian granulosa cells. Expression of the KLF4 and 13 transcripts is hormonally regulated, and all 3 KLF's can repress LDLR, StAR and CYP11A hybrid promoter-reporter constructs. In addition, KLF13 decreases native gene transcripts for LDLR and CYP11A, while increasing transcripts for StAR. Repression is antagonized by HDAC inhibitors, suggesting a role for histone deacetylation in gene silencing by these KLF's. Further studies will be required to elucidate the precise mechanisms by which any given KLF controls the transcription of a particular steroidogenic gene in gonadal cells.

Acknowledgments

This work was supported by National Institute of Child Health and Human Development Grant R01 HD-16393.

Bibliography

1. Ahn YT, Huang B, McPherson L, Clayberger C, Krensky AM. Dynamic interplay of transcriptional machinery and chromatin regulates “late” expression of the Chemokine RANTES in T lymphocytes. Mol Cell Biol. 2007;27:253–266. [PMC free article] [PubMed]
2. Chaffin CL, Dissen GA, Stouffer RL. Hormonal regulation of steroidogenic enzyme expression in granulosa cells during the peri-ovulatory interval in monkeys. Mol Hum Reprod. 2000;6:11–18. [PubMed]
3. Christenson LK, Osborne TF, McAllister JM, Strauss JF., III Conditional response of the human steroidogenic acute regulatory protein gene promoter to sterol regulatory element binding protein-1a. Endocrinology. 2001;142:28–36. [PubMed]
4. Clem BF, Clark BJ. Association of the mSin3A-histone deacetylase 1/2 corepressor complex with the mouse steroidogenic acute regulatory protein gene. Mol Endocrinol. 2006;20:100–113. [PubMed]
5. DiFeo A, Narla G, Hirshfeld J, Camacho-Vanegas O, Narla J, Rose SL, Kalir T, Yao S, Levine A, Birrer MJ, Bonome T, Friedman SL, Buller RE, Martignetti JA. Roles of KLF6 and KLF6-SV1 in ovarian cancer progression and intraperitoneal dissemination. Clin Cancer Res. 2006;12:3730–3739. [PubMed]
6. Ikeda Y, Takeuchi Y, Martin F, Cosset FL, Mitrophanous K, Collins M. Continuous high-titer HIV-1 vector production. Nat Biotechnol. 2003;21:569–572. [PubMed]
7. Iuchi S. Three classes of C2H2 zinc finger proteins. Cell Mol Life Sci. 2001;58:625–635. [PubMed]
8. Jansen E, Laven JS, Dommerholt HB, Polman J, van Rijt C, van den HC, Westland J, Mosselman S, Fauser BC. Abnormal gene expression profiles in human ovaries from polycystic ovary syndrome patients. Mol Endocrinol. 2004;18:3050–3063. [PubMed]
9. Jo M, Gieske MC, Payne CE, Wheeler-Price SE, Gieske JB, Ignatius IV, Curry TE, Jr, Ko C. Development and application of a rat ovarian gene expression database. Endocrinology. 2004;145:5384–5396. [PubMed]
10. Jonathan S. Using TESS to predict transcription factor binding sites in DNA sequence. Hoboken, NJ: John Wiley & Sons, Inc; 2003. pp. 1–15.
11. Kaczynski J, Cook T, Urrutia R. Sp1- and Kruppel-like transcription factors. Genome Biol. 2003;4:206. [PMC free article] [PubMed]
12. Kaczynski J, Zhang JS, Ellenrieder V, Conley A, Duenes T, Kester H, van der BB, Urrutia R. The Sp1-like protein BTEB3 inhibits transcription via the basic transcription element box by interacting with mSin3A and HDAC-1 co-repressors and competing with Sp1. J Biol Chem. 2001;276:36749–36756. [PubMed]
13. Kezele PR, Ague JM, Nilsson E, Skinner MK. Alterations in the ovarian transcriptome during primordial follicle assembly and development. Biol Reprod. 2005;72:241–255. [PubMed]
14. Kong WJ, Liu J, Jiang JD. Human low-density lipoprotein receptor gene and its regulation. J Mol Med. 2006;84:29–36. [PubMed]
15. LaVoie HA, Benoit AM, Garmey JC, Dailey RA, Wright DJ, Veldhuis JD. Coordinate developmental expression of genes regulating sterol economy and cholesterol side-chain cleavage in the porcine ovary. Bio Reprod. 1997;57:402–407. [PubMed]
16. LaVoie HA, Garmey JC, Veldhuis JD. Mechanisms of insulin-like growth factor I augmentation of follicle-stimulating hormone-stimulated porcine steroidogenic acute regulatory protein gene promoter activity in granulosa cells. Endocrinology. 1999;140:146–153. [PubMed]
17. LaVoie HA, McCoy GL, Blake CA. Expression of the GATA-4 and GATA-6 transcription factors in the fetal rat gonad and in the ovary during postnatal development and pregnancy. Mol Cell Endocrinol. 2004;227:31–40. [PubMed]
18. Li S, Lu MM, Zhou D, Hammes SR, Morrisey EE. GLP-1: A novel zinc finger protein required in somatic cells of the gonad for germ cell development. Dev Biol. 2006;301:106–116. [PMC free article] [PubMed]
19. Lie BL, Leung E, Leung PC, Auersperg N. Long-term growth and steroidogenic potential of human granulosa-lutein cells immortalized with SV40 large T antigen. Mol Cell Endocrinol. 1996;120:169–176. [PubMed]
20. Liu HC, He Z, Rosenwaks Z. Application of complementary DNA microarray (DNA chip) technology in the study of gene expression profiles during folliculogenesis. Fertil Steril. 2001;75:947–955. [PubMed]
21. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25:402–408. [PubMed]
22. Lomberk G, Urrutia R. The family feud: turning off Sp1 by Sp1-like KLF proteins. Biochem J. 2005;392:1–11. [PMC free article] [PubMed]
23. Manna PR, Wang XJ, Stocco DM. Involvement of multiple transcription factors in the regulation of steroidogenic acute regulatory protein gene expression. Steroids. 2003;68:1125–1134. [PubMed]
24. Martin KM, Metcalfe JC, Kemp PR. Expression of Klf9 and Klf13 in mouse development. Mech Dev. 2001;103:149–151. [PubMed]
25. Mizutani T, Yamada K, Yazawa T, Okada T, Minegishi T, Miyamoto K. Cloning and characterization of gonadotropin-inducible ovarian transcription factors (GIOT1 and -2) that are novel members of the (Cys)(2)-(His)(2)-type zinc finger protein family. Mol Endocrinol. 2001;15:1693–1705. [PubMed]
26. Monte D, DeWitte F, Hum DW. Regulation of the human P450scc gene by steroidogenic factor 1 is mediated by CBP/p300. J Biol Chem. 1998;273:4585–4591. [PubMed]
27. Natesampillai S, Fernandez-Zapico ME, Urrutia R, Veldhuis JD. A novel functional interaction between the Sp1-like protein KLF13 and SREBP/Sp1 activation complex underlies regulation of low density lipoprotein-receptor promoter function. J Biol Chem. 2006;281:3040–3047. [PubMed]
28. Natesampillai S, LaVoie HA, Veldhuis JD. Concerted regulation of steroidogenic acute regulatory gene expression by luteinizing hormone and insulin (or insulin-like growth factor I) in primary cultures of porcine granulosa-luteal cells. Endocrinology. 2000;141:3983–3992. [PubMed]
29. Natesampillai S, Veldhuis JD. Concerted transcriptional activation of the low density lipoprotein (LDL) receptor gene by insulin and luteinizing hormone in cultured porcine granulosa-luteal cells: possible convergence of protein kinase A, phosphatidylinositol 3-kinase and mitogen activated protein-kinase signaling pathways. Endocrinology. 2001;142:2921–2928. [PubMed]
30. Natesampillai S, Veldhuis JD. Involvement of Sp1 and SREBP-1a in transcriptional activation of the low density lipoprotein-receptor gene by insulin and luteinizing hormone in cultured porcine granulosa-luteal cells. Am J Physiol Endocrinol Metab. 2004;287:E128–E135. [PubMed]
31. Noti JD, Johnson AK, Dillon JD. The leukocyte integrin gene CD11d is repressed by gut-enriched Kruppel-like factor 4 in myeloid cells. J Biol Chem. 2005;280:3449–3457. [PubMed]
32. Phillips RJ, Tyson-Capper Nee Pollard AJ, Bailey J, Robson SC, Europe-Finner GN. Regulation of expression of the chorionic gonadotropin/luteinizing hormone receptor gene in the human myometrium: involvement of specificity protein-1 (Sp1), Sp3, Sp4, Sp-like proteins, and histone deacetylases. J Clin Endocrinol Metab. 2005;90:3479–3490. [PubMed]
33. Roy A, Matzuk MM. Deconstructing mammalian reproduction: using knockouts to define fertility pathways. Reproduction. 2006;131:207–219. [PubMed]
34. Sadate-Ngatchou PI, Pouchnik DJ, Griswold MD. Follicle-stimulating hormone induced changes in gene expression of murine testis. Mol Endocrinol. 2004;18:2805–2816. [PubMed]
35. Scohy S, Gabant P, Van Reeth T, Hertveldt V, Dreze PL, Van Vooren P, Riviere M, Szpirer J, Szpirer C. Identification of KLF13 and KLF14 (SP6), novel members of the SP/XKLF transcription factor family. Genomics. 2000;70:93–101. [PubMed]
36. Seals RC, Urban RJ, Sekar N, Veldhuis JD. Upregulation of basal transcriptional activity of the cytochrome P450 cholesterol side-chain cleavage (CYP11A) gene by isoform-specific calcium calmodulin-dependent protein kinase in primary cultures of ovarian granulosa cells. Endocrinology. 2004;145:5616–5622. [PubMed]
37. Shields JM, Christy RJ, Yang VW. Identification and characterization of a gene encoding a gut-enriched Kruppel-like factor expressed during growth arrest. J Biol Chem. 1996;271:20009–20017. [PMC free article] [PubMed]
38. Simmen RC, Eason RR, McQuown JR, Linz AL, Kang TJ, Chatman L, Jr, Till SR, Fujii-Kuriyama Y, Simmen FA, Oh SP. Subfertility, uterine hypoplasia, and partial progesterone resistance in mice lacking the Kruppel-like factor 9/basic transcription element-binding protein-1 (Bteb1) gene. J Biol Chem. 2004;279:29286–29294. [PubMed]
39. Song A, Chen YF, Thamatrakoln K, Storm TA, Krensky AM. RFLAT-1: a new zinc finger transcription factor that activates RANTES gene expression in T lymphocytes. Immunity. 1999;10:93–103. [PubMed]
40. Song CZ, Keller K, Murata K, Asano H, Stamatoyannopoulos G. Functional interaction between coactivators CBP/p300, PCAF, and transcription factor FKLF2. J Biol Chem. 2002;277:7029–7036. [PMC free article] [PubMed]
41. Soumano K, Price CA. Ovarian follicular steroidogenic acute regulatory protein, low-density lipoprotein receptor, and cytochrome P450 side-chain cleavage messenger ribonucleic acids in cattle undergoing superovulation. Bio Reprod. 1997;56:516–522. [PubMed]
42. Suske G, Bruford E, Philipsen S. Mammalian SP/KLF transcription factors: bring in the family. Genomics. 2005;85:551–556. [PubMed]
43. Tupler R, Perini G, Green MR. Expressing the human genome. Nature. 2001;409:832–833. [PubMed]
44. Uchida S, Tanaka Y, Ito H, Saitoh-Ohara F, Inazawa J, Yokoyama KK, Sasaki S, Marumo F. Transcriptional regulation of the CLC-K1 promoter by myc-associated zinc finger protein and kidney-enriched Kruppel-like factor, a novel zinc finger repressor. Mol Cell Biol. 2000;20:7319–7331. [PMC free article] [PubMed]
45. Urban RJ, Bodenburg Y. Transcriptional activation of the porcine P450 11A insulin-like growth factor response element in MCF-7 breat cancer cells. J Biol Chem. 1996;271:31695–31698. [PubMed]
46. Urban RJ, Bodenburg Y, Kurosky A, Wood TG, Gasic S. Polypyrimidine tract-binding protein-associated splicing factor is a negative regulator of transcriptional activity of the porcine p450scc insulin-like growth factor response element. Mol Endocrinol. 2000;14:774–782. [PubMed]
47. Urban RJ, Nagamani M, Bodenburg Y. Tumor necrosis factor alpha inhibits transcriptional activity of the porcine P-45011A insulin-like growth factor response element. J Biol Chem. 1996;271:31699–31703. [PubMed]
48. Urban RJ, Shupnik MA, Bodenburg YH. Insulin-like growth factor-1 increases expression of the porcine P-450 cholesterol side chain cleavage gene through a GC-rich domain. J Biol Chem. 1994;269:25761–25769. [PubMed]
49. van VJ, Crofts LA, Quinlan KG, Czolij R, Perkins AC, Crossley M. Human KLF17 is a new member of the Sp/KLF family of transcription factors. Genomics. 2006;87:474–482. [PubMed]
50. Wang F, Zhu Y, Huang Y, McAvoy S, Johnson WB, Cheung TH, Chung TK, Lo KW, Yim SF, Yu MM, Ngan HY, Wong YF, Smith DI. Transcriptional repression of WEE1 by Kruppel-like factor 2 is involved in DNA damage-induced apoptosis. Oncogene. 2005;24:3875–3885. [PubMed]
51. Yadav VK, Muraly P, Medhamurthy R. Identification of novel genes regulated by LH in the primate corpus luteum: insight into their regulation during the late luteal phase. Mol Hum Reprod. 2004;10:629–639. [PubMed]
52. Yamada S, Fujiwara H, Kataoka N, Honda T, Nakayama T, Higuchi T, Mori T, Maeda M. Stage-specific uptake of apolipoprotein-B in ovarian follicles and corpora lutea of the menstrual cycle and early pregnancy. Hum Reprod. 1998;13:944–952. [PubMed]
53. Yan W, Burns KH, Ma L, Matzuk MM. Identification of Zfp393, a germ cell-specific gene encoding a novel zinc finger protein. Mech Dev. 2002;118:233–239. [PubMed]
54. Zhang JS, Moncrieffe MC, Kaczynski J, Ellenrieder V, Prendergast FG, Urrutia R. A conserved alpha-helical motif mediates the interaction of Sp1-like transcriptional repressors with the corepressor mSin3A. Mol Cell Biol. 2001;21:5041–5049. [PMC free article] [PubMed]
55. Zhang XL, Zhang D, Michel FJ, Blum JL, Simmen FA, Simmen RC. Selective interactions of Kruppel-like factor 9/basic transcription element-binding protein with progesterone receptor isoforms A and B determine transcriptional activity of progesterone-responsive genes in endometrial epithelial cells. J Biol Chem. 2003;278:21474–21482. [PubMed]
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