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BOR - Papers in Press, published online ahead of print April 19, 2006.
Biol Reprod 2006, 10.1095/biolreprod.105.049486
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BIOLOGY OF REPRODUCTION 75, 279–288 (2006)
DOI: 10.1095/biolreprod.105.049486
© 2006 by the Society for the Study of Reproduction, Inc.

Research Article

Transcriptional Regulation of Cyclin D2 by the PKA Pathway and Inducible cAMP Early Repressor in Granulosa Cells1

Luis C. Muñiz 3, Ghassan Yehia 4, Elisabeth Mémin 4, Pillarisetty V.A.L. Ratnakar 4, and Carlos A. Molina 2 34 

Department of Biochemistry and Molecular Biology and Graduate School of Biomedical Sciences3 and Department of Obstetrics, Gynecology and Women's Health,4 University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey 07103

ABSTRACT

Cyclin D2 (Ccnd2) is an essential gene for folliculogenesis, as null mutation in mice impairs granulosa cell proliferation in response to FSH. Ccnd2 mRNA is induced during the estrus cycle by FSH and is rapidly inhibited by LH. Yet, the responsive elements and transcription factors accounting for the gene expression of cyclin D2 in the ovary have not been fully characterized. Using primary cultures of rat granulosa cells and immortalized mouse granulosa cells, we demonstrate a mechanism for the regulation of cyclin D2 at the level of transcription via a PKA-dependent signaling mechanism. The promoter activity of cyclin D2 was shown to be induced by FSH and the catalytic alpha subunit of PKA (PRKACA), and this activity was repressible by inducible cAMP early repressor (ICER), a cAMP response element (CRE) modulator isoform. In silico analysis of the mouse, rat, and human cyclin D2 promoters identified two CRE-binding protein sites, a conserved proximal element and a less conserved distal element relative to the translation start site. The mutation on the proximal element drastically decreases the effects of PRKACA and ICER on the promoter activity, whereas the mutation on the distal element did not contribute to the decrease in the promoter activity. Electrophoretic mobility shift assays and deoxyribonuclease footprint analysis confirmed ICER binding to the proximal element, and chromatin immunoprecipitation analysis demonstrated the occurrence of this binding in vivo. These results showed a CRE within the upstream region of Ccnd2 that is (at least partly) implicated in the stimulation and repression of cyclin D2 transcription. Finally, our data suggest that ICER involvement in the regulation of granulosa cell proliferation as overexpression of ICER results in the inhibition of PRKACA-induced DNA synthesis.

cAMP, Ccnd2, cyclic adenosine monophosphate, cyclin D2, follicle-stimulating hormone, folliculogenesis, gene regulation, granulosa, granulosa cells, ICER, mechanisms of hormone action, ovarian

INTRODUCTION

The D-type cyclins (D1, D2, and D3) are a family of cell cycle regulators involved in the G1/S phase transition of the cell cycle [1–7]. On binding to cyclin-dependent kinases CDK4 or CDK6 and activation by a CDK-activating kinase complex, the cyclin-CDK complex activates numerous genes involved in DNA synthesis and cell cycle progression [1–6]. D-type cyclins have been shown to be differentially expressed in a number of isolated cell types and cell lines [7–10].

In the ovaries, cyclin D2 expression is localized to the granulosa cells of the follicles, and its expression has been shown to be crucial for their proper growth [9]. Female mice carrying a null mutation on the cyclin D2 gene (Ccnd2) are infertile because of impairment in granulosa cell proliferation in response to FSH, resulting in small follicles with trapped oocytes [9]. Furthermore, hypophysectomized female rats display low levels of cyclin D2 in the ovaries and are unable to sustain follicular growth or to stimulate granulosa cell proliferation [11]. Conversely, human granulosa cell tumors display high levels of cyclin D2 mRNA compared with wild-type ovaries [9]. Therefore, cyclin D2 is an important factor in the regulation of granulosa cell proliferation during normal folliculogenesis, and the deregulation of Ccnd2 may contribute to the development of granulosa cell tumors. Hence, the mechanisms regulating cyclin D2 gene expression must be essential for normal ovarian function.

Hormonal regulation of the cell cycle machinery in granulosa cells is critical for folliculogenesis, ovulation, and luteinization. The anterior pituitary glycoprotein FSH is essential for folliculogenesis. Hypophysectomized rats lack sustained follicular growth because of the lack of gonadotropins. However, treatment with FSH promotes the formation of large antral follicles [12]. Another major effect of FSH on granulosa cells is to induce the expression of LH receptors and to acquire LH responsiveness [13]. FSH has been shown to stimulate cyclin D2 mRNA via a cAMP/PKA pathway in granulosa cells [9, 11]. However, a luteinizing dose of LH in hormonally primed hypophysectomized female rats results in a rapid decrease in cyclin D2 mRNA and protein levels [11]. It is unclear how cAMP mediates the actions of FSH and LH in producing their contrasting effects on granulosa cell growth and cyclin D2 expression [14–18]. One possibility might be related to differential expression of the transcription factors, mediating the cAMP pathway.

The nuclear response to the cAMP pathway is mediated by a large family of transcription factors [19, 20]. The best characterized of these factors are the cAMP response element (CRE)-binding protein (CREB), the CRE modulator (CREM), and the shorter isoforms of CREM, the inducible cAMP early repressors (ICERs) [21–23]. Creb and Crem genes encode several nuclear factors that can act as transcriptional activators or repressors of cAMP-responsive genes [21, 23–29]. These transcription factors exert their effects on binding to CREs within the promoters of cAMP-responsive genes. ICER is unique among the other CREBs in that its expression is induced by cAMP from an internal promoter within Crem [23]. This CREM isoform shares the DNA binding and dimerization domains with the other CREM isoforms but lacks the kinase and transactivation domains. Therefore, ICER functions as a dominant negative transcriptional repressor by binding as a homodimer or heterodimer with other CRE-binding family members [23].

This unique feature endows ICER with a key role in mediating the repression of cAMP-dependent transcription. In the ovaries of adult cycling rats, Crem mRNA levels of ICER isoforms have been shown to be selectively induced in the granulosa cells of preovulatory follicles in response to the ovulatory surge of LH [30]. Similarly, ICER expression was found to be induced in granulosa cells of eCG-primed immature rats injected with hCG, whereas eCG alone did not induce ICER expression [30]. This induction of ICER in response to LH/hCG has been proposed to mediate the suppression of FSH-inducible genes, such as inhibin {alpha} (Inha) [30] and cytochrome P450 (Cyp19a1) [31].

In this study, we investigated the regulation of cyclin D2 promoter via PRKACA and the effects of ICER on the promoter activity. Transfection experiments in granulosa cells using the human cyclin D2 promoter linked to a luciferase reporter gene showed the promoter to be inducible by PRKACA, and subsequent expression of ICER was able to repress this inducibility. This stimulation and repression was mapped to a newly discovered CRE located at position –294 with respect to the translation initiation site of the human cyclin D2 promoter. This CRE was found to be conserved in humans, mice, and rats. Finally, we show that overexpression of ICER was able to block PRKACA-induced DNA synthesis in cultured primary granulosa cells. These results suggest that the CRE within the cyclin D2 promoter is (at least partly) responsible for the stimulation and repression of cyclin D2 transcription. Our data further support the role of ICER in mediating the granulosa to luteal cell transition by moderating the sequential hormone-dependent gene expression necessary for the normal maturation of the ovarian follicle.

MATERIALS AND METHODS

Animals and Hormone Treatments

Immature (<25 days old) female Sprague-Dawley rats (Taconic Farms, Germantown, NY) were used for primary granulosa cell culture studies. At age 21 days, rats were injected subcutaneously with 1.5 mg of 17ß-estradiol (Sigma, St. Louis, MO) in 0.2 ml of propylene glycol once daily for 3 days to stimulate the development of large preantral follicles, as described previously [32]. On the fourth day, the animals were humanely killed, ovaries were removed, and granulosa cells were expressed from the ovaries according to the methods of Campbell [32]. All protocols were approved by the University of Medicine and Dentistry of New Jersey Institutional Animal Care and Use Committee.

Cell Culture Conditions

Primary granulosa cells were cultured in Dulbecco modified Eagle medium (DMEM): Ham F12 nutrient mixture (1:1 DMEM:F12), supplemented with 20 mM Hepes (pH 7.4), 400 mM glutamine, 100 IU of penicillin, and 100 µg/ml of streptomycin without fetal bovine serum (FBS) (Hyclone, Logan, UT), as described previously [32]. Before plating the cells, the dishes were coated with 5% fetal calf serum for 30 min at 37°C and washed twice with DMEM-F12. Cells were then cultured for up to 72 h in a humidified incubator at 37°C and 5% CO2 and were transfected with plasmids using the calcium phosphate coprecipitation technique, as described previously [33].

GRMO2 cells [34] (N.V. Innogenetics, Ghent, Belgium) were cultured in DMEM-F12, supplemented with insulin-transferrin-sodium selenite media (Sigma) and 2% FBS in a humidified incubator at 37°C and 5% CO2. GRMO2 cells were transiently transfected using LipofectAMINE 2000 reagent (Invitrogen, Carlsbad, CA) [30].

Anti-ICER Polyclonal Antibody

This antibody was raised against bacterially purified ICER-II{gamma} and has been previously characterized [35]. It has been shown to cross-react with other CREM isoforms and ubiquitinated forms of ICER and does not cross-react with CREB [36].

Plasmids

The pSV ICER-II{gamma} plasmid contains ICER cDNA under the control of the SV40 promoter [23, 27]. The pCEV-neo PKA plasmid contains the catalytic {alpha} subunit of PKA (PRKACA) and is under the control of mouse Mt-1 metallothionein-1 promoter [37]. Luciferase reporter plasmids D2 –1632, D2 {Delta}–767, and D2 {Delta}–345 have been described previously [38]. Renilla luciferase (pRL-SV40) (Promega, Madison, WI) cDNA is under the control of the SV40 promoter. D2 {Delta}–289 was generated by PCR using D2 {Delta}–345 as a template and cyclin D2 promoter primers corresponding to bases –289/–275 of the forward primer 5'-CCGCTCGAGTCACCGCTTCAGAGC-3' and –12/–3 for the reverse primer 5'-CATGCCATGGCCAGCCCGGC-3'. The underscoring denotes flanking restriction cut sites for XhoI and NcoI. The PCR product was subcloned into pBluescript II KS linearized with EcoRV containing a single T overhang at the 3' end. The plasmid was then digested with XhoI to release the 330-base pair (bp) fragment. This fragment was then cloned into pGL2 plasmid (Promega).

Site-Directed Mutagenesis

Mutations on the distal or proximal elements were generated with the QuikChange Site-Directed Mutagenesis Kit (Stratagene, LaJolla, CA) according the manufacturer's instructions using the following primers: mutProximal: 5'-CCGCTCTGAAGCGGTAACACTAGCTGGCTGGGCAG-3'; mutProximal: 5'-CTGCCCAGCCAGCTAGTGTTACCGCTTCAGAGCGG-3'; mutDistal: 5'-CTAAATTGTCTAGATCTCCCCATCTTC-3'; and mutDistal: 5'-GAAGATGGGGAGATCTAGACAATTTAG-3'. D2 {Delta}–769 plasmid was used to generate D2 –769 prox-mut, D2 –769 dist-mut, and D2 –769 prox/dist-mut. D2 {Delta}–345 plasmid was used to generate D2 –345 prox-mut. All mutations were confirmed by sequencing.

Luciferase Assay

Luciferase activity assay was performed as previously described [30]. Where indicated, cells were subjected to 8 h of hormone treatment with 100 ng/ml of FSH (Sigma) 24 h after transfection. Briefly, cells were washed with PBS and lysed using Promega's passive lysis buffer. Luciferase activity was measured using a commercial luciferase assay kit (Promega) and was read in a luminometer, essentially as previously described [39].

In Silico Analysis

Sequences pertaining to the cyclin D2 promoter region were retrieved from GenBank: human GI:1276975, rat GI:4205091, and mouse GI:2555094. The promoter region was aligned using the SeqWeb's multiple alignment program (Accelrys, Inc., San Diego, CA). Putative transcription factor binding sites were determined using TFSEARCH: Searching Transcription Factor Binding Sites (version 1.3) (http://www.cbrc.jp/research/db/TFSEARCH.html) [40].

Electrophoretic Mobility Shift Assays

Gel mobility shift analysis was performed as previously described [30]. GRMO2 cells were cultured in 10-cm plates transiently transfected with 10 to 20 µg of pSV CREM{tau} (CREM{tau}), pSV ICER-II{gamma} (ICER), or CMV2-FLAG ICER-II{gamma} (FLAG-ICER) plasmid DNA. Two double-stranded oligonucleotides spanning the distal or proximal putative CRE (underscored) (oligo-294: 5'-GCCAGCTTGCGTCACCGCTT-3' and oligo-712: 5'-ATTGTCTGAGGTCACCCC-3') of the human cyclin D2 promoter [38] were labeled with [{gamma}32P] ATP (Amersham Biosciences, Piscataway, NJ) and were used as probes in the binding reactions. Whole-cell protein extracts were prepared as previously described [30] 24 h after transfection. For competition experiments, unlabeled oligo-294 was used at concentrations of 75 ng, 150 ng, or 300 ng, whereas the unlabeled mutated version (mutant oligo-294: 5'-CCAGCCAGCTAGATCTCCGCTTCAGAG-3') was used at a concentration of 300 ng. Binding reactions were performed for 20 min with 20 µg of whole-cell protein extracts using 10 000 cpm of 32P-labeled oligonucleotide probe. For antibody supershift analysis, protein extracts were incubated for an additional 20 min with 1 µl of rabbit anti-ICER polyclonal antibody [35] or 1 µl of anti-mouse FLAG M2 monoclonal antibody (Sigma).

Chromatin Immunoprecipitation

GRMO2 cells were cultured on a 60-mm dish and were subjected to 8 h of 0.5 mM 8-Br cAMP treatment or were transfected with plasmids expressing FLAG-ICER (CMV2-FLAG ICER-II{gamma}) or FLAG (CMV2-FLAG). The chromatin immunoprecipitation (ChIP) assay was performed according to the manufacturer's instructions (Upstate Cell Signaling Solutions, Charlottesville, VA). Briefly, cells were grown to a confluency of 1 x 106 cells and were cross-linked with 1% formaldehyde for 15 min at room temperature. Glycine was added to a final concentration of 0.125 M to stop cross-linking. Cells were then rinsed twice with ice-cold PBS containing protease inhibitors (1:1000 of Sigma protease inhibitor cocktail and 1.0 mM PMSF), scrapped, and collected by centrifugation. The cell pellets were resuspended in 0.3 ml of lysis buffer (1% SDS, 5 mM EDTA, 50 mM Tris-HCl [pH 8.0]) and were incubated on ice for 10 min. The samples were sonicated on ice using a Misonix Ultrasonics sonicator (Misonix, Inc., Farmingdale, NY) with a microtip at a setting of 5 for three 10-sec pulses to yield a mean length of 1000 bp and were then centrifuged. The chromatin solution was precleared with the addition of protein A Sepharose (Sigma) (50% slurry in TE buffer blocked previously with 0.5 mg/ml of BSA and 200 µg/ml of salmon sperm DNA) at 4°C for 2 h. Precleared chromatin was incubated with 1 µl of rabbit anti-mouse ICER polyclonal antibody [35], 1 µl of anti-mouse FLAG M2 monoclonal antibody (Sigma), 1 µl of preimmune control rabbit IgG, or no antibody mock (2 µg/ml of BSA) and was rotated at 4°C overnight. Immunoprecipitation, washing, elution, and purification were carried out as described in the ChIP assay kit protocol (Upstate Cell Signaling Solutions), with the following exceptions: proteinase K treatment was performed for 3 h at 55°C after the overnight reverse cross-linking, and the chromatin sample was purified using the Qiaquick PCR purification kit (Qiagen), according to the manufacturer's protocol. Immunoprecipitated samples were analyzed by PCR using primers for the mouse cyclin D2 promoter (forward primer: 5'-TTGAAGTTTGGTCAGGCCAGCTGC-3' and reverse primer: 5'-AGTGCTTCCCTTACCTCCTTCC-3') and the internal Crem promoter (forward primer: 5'-AGGGCTTTGCTTTCAGTGAGCTGCAC-3' and reverse primer: 5'-TCTCCAGTTACAGCCATGTTGGGC-3'). The PCR products were analyzed by electrophoresis on 1.2% agarose gels.

DNA Sequencing

The cyclin D2 luciferase reporter plasmid containing the 1.6-kilobase (kb) upstream sequence (D2 –1632) was sequenced by PCR with the dideoxy chain termination method [41] using reverse primers from the internal sequences that were 5' end labeled with T4 polynucleotide kinase and [{gamma}-32P] ATP (Amersham Biosciences). The relative positions of these primers are numbered relative to the translation start site: oligo-664: *5'-TTAAGATCCAGGAATGTAGGG-3'; oligo-426: *5'-TTATTAAGGAGAACAGCAGCTGGC-3'; and oligo-223: *5'-CACCGGTCCTCCCCTTAAAACTGG-3'.

Deoxyribonuclease Footprint Analysis

PCR was used to generate labeled probes from the upstream portion of Ccnd2 spanning from –884 to –664, –664 to –426, and –443 to –223 relative to the translational start site using the 32P 5' end-labeled primer generated for the DNA sequencing reaction and its corresponding forward primer: oligo-664/oligo-884: 5'-CAGAAGGGACGTTGTTCTGGTCC-3'; oligo-426/oligo-664: 5'-AATACAAGGGCAGGAGGATTAGG-3'; and oligo-223/oligo-443: 5'-TGCTGTTCTCCTTAATAACGAGAGGGG-3'. All PCR reactions used D2 –1632 as a template. An additional PCR reaction was performed with oligo-223 and oligo-443 using D2 –767 prox/dist mut as a template. The PCR-amplified fragment was then purified using Qiaquick PCR purification kit. Footprinting was performed with or without 200 ng of purified N-terminal 6His ICER II{gamma} using the Core Footprinting System according to the supplier's recommendations (Promega).

Ribonuclease Protection Assay

RNA and protein were extracted from primary granulosa cells using TRIZOL Reagent (Invitrogen) according to the manufacturer's instructions. Aliquots of 5 µg of total RNA were subjected to ribonuclease protection analysis, essentially as previously described [23, 33, 39, 42]. The rat cyclin D2 riboprobe was generated by RT-PCR from RNA of primary culture of granulosa cells treated with 8-Br cAMP to induce the levels of cyclin D2 mRNA. The oligonucleotides used to amplify the partial region of the rat cyclin D2 cDNA were 5'-CGCGAATTCCATGGAGCTGCTGTGCTG-3' and 5'- GCGCGGATCCTTCTGCACGCACTTGAA-3'. A BamHI-EcoRI 146-bp fragment that included the first exon of cyclin D2 ATG was subcloned into pBluescript II SK(–). The antisense riboprobe was generated using T3 RNA polymerase after digestion with HindIII to linearize the plasmid. For loading control, pTRI-GAPDH-Rat antisense control template (Ambion, Austin, TX) was used as directed by the supplier to generate a riboprobe and resulted in a protected fragment of 316 nucleotides.

DNA [3H]-Thymidine ([3H]TdR) Incorporation Assays

Primary granulosa cells were harvested and seeded in 6 wells plated at a density of 5 x 104 cells per well. Cells were transfected using the calcium phosphate coprecipitation technique with plasmids expressing PRKACA, ICER, or both, as described previously [33]. After DNA transfection, the cells were washed, and [3H]TdR (5 µCi/ml) was added for 16 h overnight to assess the rate of DNA synthesis. The cells were then fixed with trichloroacetic acid (TCA), and TCA-insoluble radioactivity was determined by scintillation counting.

Statistical Analysis

Differences between treatments were analyzed for significance using Student t-test.

RESULTS

PKA-Dependent Activation of the Cyclin D2 Promoter

The cyclin D2 mRNA levels have been shown to be strongly induced by FSH via a cAMP/PKA pathway in granulosa cells [9, 11]. Yet, luteinizing doses of LH in hormonally primed hypophysectomized female rats result in the rapid decrease of cyclin D2 mRNA [11]. Therefore, we sought to characterize the regulatory elements within the promoter region of the human cyclin D2 gene that account for the FSH inducibility. We used a luciferase reporter construct containing a 1.6-kb portion of the human cyclin D2 promoter (D2 –1632) [38] to assess for FSH responsiveness by primary culture of rat granulosa cells. In cotransfection cell assays, this reporter construct was used in conjunction with a plasmid expressing PRKACA or in the presence of FSH. PRKACA and FSH induced the activity of the –1632 construct by 3-fold compared with basal levels, with a slightly better induction from the FSH treatment (Fig. 1A). Similar experiments were conducted using established mouse granulosa cell line GRMO2 cells [34]. As seen with the primary culture of rat granulosa cells, PRKACA induced promoter activity 3-fold, but FSH treatment only resulted in a 0.5-fold increase in promoter activity (Fig. 1B).


Figure 1
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  		FIG. 1. PKA-dependent activation of the cyclin D2 promoter. The effects of FSH or PRKACA on the promoter activity of cyclin D2. The figure shows the activity of the 1.6-kb portion of the human cyclin D2 promoter and its truncated variants linked to firefly luciferase cDNA. Luciferase activity was measured in primary culture of rat granulosa cells (A) or in GRMO2 cells (B and C) transiently transfected as described in the Materials and Methods section with plasmids expressing PRKACA or in cells treated with 100 ng/ml of FSH. C) On the left, in a diagrammatic representation of the truncated variants of the cyclin D2 promoter, numbers indicate the 5' ends of the promoter DNA inserts relative to the translational start site (+1). Renilla luciferase vector was used to normalize for transfection efficiency, and relative luciferase activity is expressed as a ratio of firefly:renilla. Data are presented as the mean ± SEM of 3 independent experiments. Bars with (*) differ significantly from no treatment (A and B) or control (C) (P < 0.05)

We next sought to determine the responsive element involved in the PRKACA-dependent activation of the cyclin D2 promoter. Deletion analysis of the human cyclin D2 promoter was used to assess the subsequent effects of PRKACA on the promoter activity in GRMO2 cells. Four luciferase reporter constructs containing different segments (–1632, –769, –345, and –290) on the human cyclin D2 promoter were used to study PKA-dependent regulation of cyclin D2. These constructs were used in cotransfection experiments with PRKACA (Fig. 1C). PRKACA induced the activity of the –1632 construct by 6-fold compared with the basal level. Similarly, PRKACA induced the activity of the –769 and –345 truncated constructs by 6-fold and 5-fold, respectively. However, further truncation of the –345 promoter to –290 bp drastically reduced basal levels by 6-fold and PRKACA inducibility by 9-fold. Results suggest that in the region between –345 and –290 of the cyclin D2 promoter resides a responsive element accounting for cyclin D2 inducibility via PRKACA.

Comparison of the 5' Region of the Cyclin D2 Gene in Mouse, Rat, and Human

The previous results suggested the presence of CRE within the cyclin D2 promoter. In silico analysis of the human cyclin D2 promoter using TFSEARCH [40] predicted two motifs as binding sites for CREB. These two putative binding sites share sequence similarity to the canonical somatostatin (Sst) CRE [43]. These elements are located on positions –712 (TGAGGTCA) and –294 (TTGCGTCA), herein referred to as the distal and proximal elements relative to the translation start site. This proximal CRE resides within the region shown in the previously cited figures to be responsive to PRKACA. Sequence alignments among human, mouse, and rat cyclin D2 promoters (Fig. 2A) located the two putative elements at similar positions. Furthermore, in contrast to the putative distal CRE, the putative proximal CRE is conserved among the three species.


Figure 2
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  		FIG. 2. Nucleotide sequence comparison among human, rat, and mouse upstream regions of the cyclin D2 gene. Two potential cis elements within the 5' region of the cyclin D2 gene sharing sequence similarity to the CRE of the somatostatin promoter. A) Sequence alignment among the human, rat, and mouse 5' regions of the cyclin D2 gene revealed the location, herein referred to as the distal and proximal elements. Also shown are the nucleotide sequences of the proximal or distal element, numbered relative to the translational start site. B and C) Mutational analysis of the human cyclin D2 promoter and the subsequent effects of ICER and PRKACA on promoter activity in granulosa cells. On the left is a diagrammatic representation of the cyclin D2 promoter and its truncated variants with or without mutations on the distal, proximal, or both elements simultaneously linked to a firefly luciferase cDNA. The numbers shown are relative to the translational start site (+1). Luciferase activity was measured in GRMO2 cells (B) or primary culture of rat granulosa cells (C) transiently transfected with plasmid expressing ICER or PRKACA as indicated in the figure. A renilla luciferase vector was used to normalize for transfection efficiency; relative luciferase activity is expressed as a ratio of firefly:renilla. Data are presented as the mean ± SEM of 3 independent experiments. Bars with (*) differ significantly from control; bars with (**) differ significantly from PRKACA treatment (P < 0.05)

The presence of putative CREs within the cyclin D2 promoter would suggest potential binding sites for transcription factors that mediate the nuclear response to the cAMP pathway. To confirm the involvement of the distal or proximal putative CREs in the PRKACA-induced cyclin D2 promoter activity, constructs bearing point mutations on either element were tested by luciferase assay in GRMO2 cells and in primary culture of rat granulosa cells (Fig. 2, B and C). A 769-bp cyclin D2 promoter harboring a mutation on the distal element maintained PRKACA-induced activity by 6.5-fold relative to basal levels. Furthermore, a 2-fold enhancement in the overall basal and PRKACA-induced promoter activity was observed compared with the wild type. A mutation on the proximal element of the –769-bp construct decreased basal levels by 6-fold, and PRKACA induced activity by 7-fold relative to a later construct bearing a mutation only on the distal element. Comparable results were obtained with the –769-bp construct harboring mutations on both putative CREs (Fig. 2B). Moreover, a mutation on the putative proximal CRE of the truncated –345-bp construct resulted in a further decrease in the basal and PRKACA-induced activity. However, in luciferase experiments using primary culture of granulosa cells (Fig. 2C), the basal levels remained unchanged between the construct with a mutation on the proximal element compared with the wild type. The 1.6-kb construct displayed a 4.5-fold induction on PRKACA transfection relative to basal levels. The –345-bp construct also displayed PRKACA-induced activity, but this activity was reduced by 2.3-fold in the –345-bp construct harboring the proximal mutation. These data suggest that the proximal element mediates PRKACA-induced activity and functions as a potential CRE. Hence, we next sought to determine if ICER modulates this activity.

ICER has been shown in the ovaries of adult cycling rats to be selectively induced in the granulosa cells of preovulatory follicles in response to the LH surge at the transcriptional level [30]. This is in contrast to cyclin D2 transcript levels, which have been shown to be strongly induced by FSH [9, 11] and rapidly decreased within 4 h in response to luteinizing doses of LH [11]. Therefore, we hypothesized that ICER may be involved in the cAMP-mediated regulation of cyclin D2 [38]. To demonstrate the potential role of ICER, cotransfection experiments were conducted using constructs containing a wild-type or mutated proximal element and ICER-expressing plasmids. On cotransfection with ICER, constructs containing a wild-type proximal element displayed a 4.5-fold repression in PRKACA-induced promoter activity in GRMO2 cells (Fig. 2B) and a complete repression of activity in primary culture of rat granulosa cells (Fig. 2C). ICER did not affect the promoter activity in constructs containing a mutated proximal element or those lacking the proximal element (Fig. 2, B and C). This suggests that ICER mediates the repression of PRKACA-induced activity through the proximal element. These data implicate the involvement of the putative proximal CRE on the regulation of the cyclin D2 promoter activity by PRKACA and ICER.

CREM and ICER Preferentially Bind to the Proximal Element of the Cyclin D2 Promoter

Electrophoretic mobility shift assays (EMSAs) were performed to establish whether the CREM isoforms (ICER and CREM{tau}) could directly interact with the putative proximal CRE of the human cyclin D2 promoter. Protein extracts from GRMO2 cells transiently transfected with plasmid expression of CREM{tau} or ICER were incubated with radiolabeled double-stranded oligonucleotide probes containing the putative distal (oligo-712) or proximal (oligo-294) CRE of the human cyclin D2 promoter. Figure 3A shows that ICER and CREM{tau} strongly bind to the proximal element (lanes 1–3); however, the binding to the distal element was much weaker (lanes 4–6). To assess the specificity of ICER binding to the proximal element, protein extracts from GRMO2 cells transfected with FLAG-tagged ICER were used in EMSA (Fig. 3B). The binding of FLAG-tagged ICER to the proximal element (lane 2) was shown to be competed out with excess unlabeled oligo-294 (lanes 3–5). Inversely, the excess of unlabeled oligonucleotides containing a mutation on oligo-294 was unable to compete with the binding of ICER protein to the labeled wild-type oligo-294 (lane 6). Moreover, incubation with antibodies against FLAG or ICER proteins resulted in the disappearance of the shifted band and the appearance of supershifted complexes of a lesser mobility, indicating that ICER was a component of the protein complex formed on the putative proximal cyclin D2 CRE (lanes 7 and 8). These data strongly suggest that the putative proximal CRE is a target for CRE-binding factors required to modulate the cyclin D2 promoter activity via the cAMP pathway.


Figure 3
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  		FIG. 3. ICER preferentially binds to the proximal element of the cyclin D2 promoter. A) EMSA was performed using whole-cell protein extracts from GRMO2 cells transfected with the indicated amount of DNA construct expressing CREM{tau} (lanes 1 and 4) or ICER (lanes 2, 3, 5, and 6). The lysates were incubated with 32P-labeled double-stranded oligonucleotides containing the proximal element (oligo-294) or the distal element (oligo-712). B) EMSA using whole-cell protein extracts from GRMO2 cells transfected with CMV-FLAG-ICERII{gamma} plasmid. Lysates were incubated with 32P-labeled oligo-294 (lane 2). The binding reaction was incubated with increasing concentration of excess nonradioactive oligo-294 (lanes 3, 4, and 5), mutant oligo-294 (lane 6), anti-FLAG M2 antibody (lane7), or anti-ICER antibody (lanes 8). Lane 1 is a control without cell lysate. The specific protein-DNA complex, the antibody supershifted complex, and the free probe are indicated by arrows

The Proximal Element is the Binding Site for ICER

Deoxyribonuclease (DNase) I footprint analysis was used as an unbiased approach to determine ICER binding sites on the cyclin D2 promoter and to determine if the proximal element is the sole CRE present in the promoter. Three regions spanning 662 bp of the human cyclin D2 promoter were used for analysis (Fig. 4A). These regions were generated by PCR using D2 –1632 as a template and 32P end-labeled reverse primers, with the PCR products referred to as fragments I, II, and III. D2 –769 prox-mut was used as a template to generate the PCR product (III-mut) containing the mutated proximal element as a negative control. PCR sequencing reactions were also performed for the corresponding fragments (I, II, and III) using the same end-labeled primer. Figure 4, B through D, shows the autoradiograms of the PCR sequencing reactions and DNase I digestion for the PCR fragments spanning the three regions. Incubation of ICER with PCR fragment I failed to produce a protected pattern along the region corresponding to the distal element when subjected to increasing treatment duration with DNase I (Fig. 4, B and D). Similar results were obtained with PCR fragment II, which displayed no protected pattern along the entire length of the PCR fragment corresponding to the region between the distal and proximal putative CREs (Fig. 4D). However, DNase I treatment of PCR fragment III displayed a protected pattern along the region corresponding to the proximal element as shown by the PCR sequencing reaction when incubated with ICER (Fig. 4, C and D). Conversely, the same PCR fragment harboring a mutation within the proximal element failed to produce the same protected pattern (Fig. 4, C and D). These data suggest that the proximal element is a potential site for ICER binding and authenticate this element as a CRE.


Figure 4
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  		FIG. 4. The proximal element is the binding site for ICER. DNase I footprint analysis of the 5' region of the human cyclin D2 gene. A) Diagrammatic representation of the cyclin D2 promoter regions used for DNase I footprint assay. Three regions spanning 662 bp of the promoter were used for analysis. PCR fragment I from –824 to –663 contains the distal element, and fragment III from –444 to –225 contains the proximal element. PCR fragment III-mut contains a mutated proximal element, the sequence of which is shown. Fragment II from –664 to –426 overlaps a region between fragments I and III. Representative autoradiograms of DNase I reactions with PCR fragments I (B), III, and III-mut (C) along with their corresponding sequencing reaction are shown. The boxed region pertains to the location of the distal and proximal elements. DNase I reactions include 3 incubation times with DNase I (30 sec, 1 min, and 2 min) with probe DNA using 200 ng ICER II{gamma} or in the absence of ICER protein (control). D) Representative autoradiogram subjected to longer exposure spanning the entire length of the gel, including DNase I reaction with PCR fragment II

CREM Binds to the Cyclin D2 Promoter In Vivo

The data presented thus far suggest a probable role of ICER in regulating cyclin D2 promoter activity through its ability to bind the proximal CRE of the cyclin D2 promoter in vitro. To study the interactions of ICER with cyclin D2 promoter in vivo, we performed a ChIP assay. The chromatin from GRMO2 cells treated with 8-Br cAMP for 8 h was used in the following ChIP experiments, because ICER has been shown to be strongly induced between 5 and 12 h after 8-Br cAMP treatment in GRMO2 cells (see supplemental Fig. 1 available online at http://www.biolreprod.org). After the chromatin was fixed and sheared, the sample was divided into samples immunoprecipitated with an antibody recognizing ICER or was subjected to preimmune rabbit IgG or mock immunoprecipitation using BSA as negative controls. The chromatin was then analyzed by PCR using primers flanking the proximal CRE-284 of the mouse cyclin D2 promoter or primers flanking the four CREs within the Crem internal promoter [23] as a positive control for ICER binding (Fig. 5A). PCR analysis on the chromatin immunoprecipitated against ICER resulted in the amplification of the targeted cyclin D2 promoter region producing the expected 366-bp PCR product (Fig. 5B). Likewise, the 181-bp fragment of the targeted internal Crem promoter was amplified. As expected, the use of IgG or BSA did not produce any significant amplification of cyclin D2 or the internal Crem promoter regions. These data demonstrate in vivo binding of CREM family members to the proximal CRE of the cyclin D2 promoter.


Figure 5
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  		FIG. 5. CREM binds to the cyclin D2 promoter in vivo. A) Diagrammatic representation of the mouse promoter regions of ICER and Ccnd2. CRE sites are indicated by rectangular boxes. The positions of the PCR primers used for the ChIP assay are indicated (tick mark) and are numbered relative to the translational start site; in addition, the sizes of the expected PCR products are shown in parentheses. B) Representative ChIP analyses using GRMO2 cells are shown. Chromatin was prepared from GRMO2 cells treated with 0.5 mM 8-Br-cAMP for 8 h. Immunoprecipitation was performed using anti-ICER, preimmune control rabbit IgG, or no antibody (BSA) mock. Following DNA purification, samples were subjected to PCR amplification using a pair of primers that cover the regions of cyclin D2 and ICER gene promoter as indicated in panel A. The sizes of the PCR products are indicated on the left. These data are representative of 3 independent experiments with similar results

ICER Binds to the Cyclin D2 Promoter In Vivo

Because ICER antibody used is known to cross-react with other CREM family members, the data presented thus far demonstrate in vivo binding of CREM family members to the proximal CRE of the cyclin D2 promoter. As an alternative approach, GRMO2 cells were transiently transfected using a plasmid expressing FLAG-tagged ICER to specifically immunoprecipitate ICER using anti-FLAG antibody. Figure 6A shows Western blot analysis against anti-FLAG and anti-ICER showing that FLAG-ICER was efficiently expressed in GRMO2 cells. In addition, FLAG-ICER was shown (Fig. 3B) to function in EMSA-binding studies.


Figure 6
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  		FIG. 6. ICER binds to the cyclin D2 promoter in vivo. A) Western blot analysis of whole-cell protein extracts prepared from GRMO2 cells transfected with CMV2-FLAG or CMV2-FLAG-ICERII{gamma} expressing plasmid using an anti-ICER antibody or anti-FLAG antibody. The relative migration of protein molecular mass markers is indicated (kDa). B) Representative ChIP analyses using GRMO2 cells are shown. Chromatin was prepared from GRMO2 cells transfected with CMV2-FLAG or CMV2-FLAG-ICERII{gamma} plasmids. Immunoprecipitation was performed using anti-FLAG antibody or preimmune control rabbit IgG. Following DNA purification, samples were subjected to PCR amplification using a pair of primers that cover the region of Ccnd2 gene promoter. The size of the PCR product is indicated on the left. These data are representative of 3 independent experiments with similar results

PCR analysis of chromatin immunoprecipitated against FLAG resulted in the amplification of the targeted cyclin D2 promoter region producing the expected 366-bp PCR product (Fig. 6B). However, PCR analysis from the IgG immunoprecipitation did not detect any significant amplification of the cyclin D2 promoter region. Furthermore, chromatin immunoprecipitated with anti-FLAG antibody from GRMO2 cells transfected with FLAG vector did not produce detectable amplification of the cyclin D2 promoter region (Fig. 6B). These data demonstrate in vivo binding of ICER to the proximal CRE of the cyclin D2 promoter.

ICER Decreases the PRKACA-Induced Cyclin D2 mRNA Level

Next, we sought to determine the role of ICER binding to the CRE and the consequent effect on the endogenous levels of cyclin D2 mRNA. For this purpose, we used a previously characterized antisense approach to block the endogenous levels of ICER [33, 35, 39] and to assess the consequent effect of the lack of ICER on cyclin D2 expression. Primary cultures of rat granulosa cells were transfected with PRKACA alone or were cotransfected with a plasmid containing antisense ICER. Transfection with an empty vector served as a control for basal cyclin D2 transcript levels. Following transfection, RNA was isolated for further analysis. The levels of endogenous cyclin D2 mRNA were measured by ribonuclease protection assay using a riboprobe against rat cyclin D2 mRNA. Figure 7 shows representative data of the ribonuclease protection assay normalized against rat Gapdh. Cells transfected with PRKACA or ICER antisense alone did not result in a significant change in cyclin D2 mRNA levels compared with transfection with empty vector. However, we saw a significant increase in cyclin D2 mRNA when cotransfected with PRKACA and ICER antisense compared with cells transfected with PKKACA alone (P < 0.05).


Figure 7
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  		FIG. 7. Antisense ICER enhances PRKACA-induced cyclin D2 mRNA levels. The levels of cyclin D2 in primary rat granulosa cells were measured by ribonuclease assay. A) Representative autoradiogram using 5 µg of RNA for hybridization with probes specific for rat cyclin D2 and Gapdh. Primary cultures of rat granulosa cells were transiently transfected with plasmids expressing PRKACA and antisense ICER (ICER[AS]) independently or together. Transfection with empty vector served as a control. B) Autoradiograms were scanned, and densitometry was used to quantify the relative changes in cyclin D2 mRNA expression with different transfection treatments. Densitometric analysis of cyclin D2 mRNA levels was normalized against Gapdh. Data are presented as the mean ± SEM of 2 independent experiments. Bars with (*) differ significantly from cells transfected with PRKACA alone (P < 0.05)

ICER Inhibits DNA Synthesis in Granulosa Cells

The physiological consequences of ICER repressing the promoter activity of cyclin D2 in granulosa cells would imply a halt in cell cycle progression, as cyclin D2 has been shown to be essential in granulosa cell proliferation [9, 11]. Although FSH and LH act predominantly via the cAMP pathway, only LH/hCG induces ICER expression in granulosa cells [30]. To determine whether ICER might be involved in LH-mediated granulosa cell growth inhibition, the rate of DNA synthesis was measured by [3H]-thymidine ([3H]TdR) incorporation into primary cultures of rat granulosa cells. The data shown in Figure 8 demonstrate that the rate of DNA synthesis in granulosa cells transfected with PRKACA increased 24-fold. However, in granulosa cells cotransfected with ICER, a complete abrogation of PRKACA-induced DNA synthesis was observed. These data suggest a potential physiological role of ICER in mediating granulosa cell proliferation, which is essential for normal folliculogenesis.


Figure 8
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  		FIG. 8. ICER inhibits DNA synthesis in primary rat granulosa cells. The rate of [3H]TdR incorporation in primary rat granulosa cells was measured. Cells were transfected with an empty vector alone (control) or with plasmids expressing PRKACA, ICER, or both. Data are the fold induction of [3H]TdR incorporation into DNA relative to the control. Data are presented as the mean ± SEM of 4 independent experiments. Bars with (*) differ significantly from PRKACA treatment (P < 0.05)

DISCUSSION

An inherent aspect of proper luteal development involves the transition of actively dividing granulosa cells into nonproliferating luteal cells, which constitutes the luteal compartment. Previous studies have demonstrated that granulosa cell proliferation requires normal expression of the cell cycle regulator cyclin D2 [9, 11]. Cyclin D2 is necessary for the G1/S transition of the cell cycle [1–6], and its expression is induced by FSH in granulosa cells [9, 11]. Granulosa cells from mice lacking cyclin D2 fail to proliferate in response to FSH [9, 11]. Furthermore, the absence of Cdkn1b (previously known as p27Kip), an inhibitor of G1 cyclin-dependent kinases, in female mice results in normal differentiation but impaired growth arrest of luteal cells [44]. Collectively, these findings unveil the existence of a concerted effort to maintain tight regulation of cyclin D2 by controlling its functional activity and expression levels, consistent with the pivotal role of cyclin D2 in granulosa cell proliferation. Although cyclin D2 transcript levels have been shown to be regulated by FSH and LH, the molecular basis for this transcriptional regulation is not fully understood [9, 11]. In this study, we identified a CRE within the 5' region of Ccnd2, important for FSH-mediated induction, and demonstrated that the CREM isoform ICER can negatively regulate this activity. Hence, we presented yet another level of regulation by which ICER contributes to the maintenance of the tight control of cyclin D2, potentially inhibiting granulosa cell growth during folliculogenesis.

From our data and other findings, a more comprehensive model of the hormonal regulation of granulosa cell growth and differentiation is emerging. It has been postulated that the cAMP/PKA pathway mediates the high levels of cyclin D2 in response to FSH; however, the regulatory elements involved in cyclin D2 cAMP responsiveness were largely unknown [9, 11]. Luciferase analysis using a 1.6-kb portion of the 5' region of the human cyclin D2 gene displayed FSH and PRKACA responsiveness. Differences in the relative inducibility with FSH treatment between primary culture of rat granulosa cells and GRMO2 cells are likely due to the fact that GRMO2 cells have been characterized as more closely resembling an early luteinized cell type [34]. However, the fact that PKA is downstream of FSH may explain the similar response from these cell types on PRKACA transfection.

The region accounting for the FSH/PKKACA-induced promoter activity was mapped within positions –345 and –290 relative to the translation start site. This area was originally considered to be a potential binding site for AP2, Myb, and C/EBP [38]. However, in silico analysis of the 1.6-kb promoter region of the human cyclin D2 gene using the TFSEARCH program predicted the previous C/EBP binding site as a CREB binding site. We used comparative genomics to determine whether this binding site is likely to have functional relevance. Sequence alignment of the human, mouse, and rat cyclin D2 promoter revealed conservation in the sequence and in the relative location of this binding site. This putative binding site, TTGCGTCA, shares sequence homology with the somatostatin CRE (TGACGTCA). Because it is presumed that the functionally important regions are evolutionally conserved, the use of the human promoter in mice and rats would be relevant. Based on the sequence analysis, we speculated that regulation through this element by CRE-binding proteins such as ICER would occur similarly in human, mice, and rat granulosa cells. However, because the distal element is less conserved, the regulation through this element may differ among species.

Mutational analysis of this CRE resulted in a decrease of the PRKACA-induced promoter activity, suggesting that FSH mediated cyclin D2 induction through this element. However, the actions of FSH and LH are mediated mainly through the cAMP pathway [15, 18]. Therefore, we hypothesized that different members of the nuclear factors that control the transcriptional response to cAMP would mediate the disparate effect on cyclin D2 expression in granulosa cells. Indeed, we found that ICER, a member of the CREM family, may contribute to the transcriptional repression of cyclin D2.

The results of our study demonstrate that ICER can negatively regulate the PRKACA-induced activity of the human cyclin D2 promoter. However ICER repression on PRKACA-induced cyclin D2 promoter activity was more efficient in primary culture of rat granulosa cells than in GRMO2 cells. The fact that immortalized cells are less responsive to a gene that is potentially involved in regulating cell growth is not surprising. It has been shown that ICER has a shorter half-life in immortalized cells [36], which may explain the weaker response to ICER in GRMO2 cells.

In light of these data, our findings suggest a more comprehensive model of transcriptional regulation of cyclin D2 during folliculogenesis. FSH in conjunction with activin elevates the pre-FSH repression of FOXO1 on cyclin D2 transcription, an essential step for the exponential surge in granulosa cell proliferation required for follicular maturation [45]. With the preovulatory surge of LH, ICER expression is induced in the granulosa cells [30]. The cyclin inhibitors are also upregulated [46]. Collectively, this allows for the coupled regulation of cyclin D2 activity and transcription, which causes the granulosa cells of the ruptured follicle to cease proliferation and to differentiate into luteal cells. The data complement a model for regulation of cyclin D2 expression.

The fact that all of our cyclin D2 promoter mutants were stimulated with PRKACA using GRMO2 cell extract comparatively to rat primary granulosa cells suggests that more than one element regulates inducibility by PRKACA, whereas ICER may have more of an effect on the proximal CRE. In addition, GRMO2 cells are transformed tumorigenic cells, such that several signal transducing pathways could be converging to activate cyclin D2 expression. Collectively, this may account for differences observed in ICER repression of PRKACA-induced wild-type promoter activity between primary culture of rat granulosa cells and GRMO2 cells.

Moreover, the data herein demonstrate that ICER binds in vitro and in vivo to the cyclin D2 promoter. In determining the effect of ICER on the endogenous levels of cyclin D2 mRNA, we used a previously characterized antisense approach to block the endogenous levels of ICER [33, 39]. An RNA interference approach was not feasible because of the lack of ICER-specific small interfering RNA, as well as without affecting other CREM isoforms. However, the antisense approach has been shown to be effective in inhibiting ICER expression without affecting the expression of the other CREM isoforms [33, 39]. It is only when cells were cotransfected with ICER antisense that a moderate yet significant increase in PRKACA-induced cyclin D2 mRNA was observed, suggesting a role for ICER in the transcriptional repression of cyclin D2. Overexpression of ICER inhibits PRKACA-induced DNA synthesis in primary culture of rat granulosa cells. Hence, our data suggest that the antiproliferative activity associated with LH could be in part mediated by ICER through yet another level of control by inhibiting cyclin D2 transcription.

Previous findings [30], in addition to the results of the present study, have suggested a role of ICER in the transition from granulosa to luteal cell in the mammalian ovary. Mukherjee et al. [30] showed that LH/hCG induces the expression of ICER in granulosa cells. In contrast to ICER, cyclin D2 expression has been shown to be reduced by LH, and its expression is low in the corpus luteum [9, 11]. ICER has been implicated in the regulation of cAMP-responsive genes in the ovary. For example, ICER was shown to inhibit the promoter activity of the Inha and Cyp19a1 genes in rat granulosa cells [30, 31]. Similar to cyclin D2 expression patterns, FSH induces the expression of Inha and Cyp19a1 gene products in the ovaries by virtue of a CRE-like sequence and is downregulated by LH after ovulation.

An ovarian-specific ICER-null mice model is not available to fully validate the role of ICER during folliculogenesis. Crem-mutant mice have been generated, resulting in male sterility; however, the female mice were reported to be fertile [13, 47]. Judging by the apparent pleiotropic function of CREM, such a specific phenotype is surprising. Moreover, the homozygous mutant mice lacked activators and repressors such as CREM and ICER. Therefore, we suspect that the balance mechanism controlling CREM-mediated gene expression in Crem-null mice might be affected in two opposite directions, which would result in mutual cancellation without other observed apparent phenotypic differences. In the case of cyclin D2 regulation, we postulate that the balance mechanisms controlling its expression and repression will be equally counterbalanced in the Crem-null mice model. Because ICER is unique among the CRE-binding factors, the generation of an ICER-null mice and ovarian-specific ICER transgenic animals will aid in answering these questions by disrupting this balance. In accord with this postulate, we recently observed that ovarian-specific ICER transgenic female mice are hypersensitive to the ovulatory effects of FSH and LH (L.C.M., unpublished data).

Collectively, our data suggest that ICER is an essential player in the regulation of genes necessary in the granulosa to luteal cell transition. Herein, we present for the first time (to our knowledge) evidence suggesting that the observed downregulation of cyclin D2 during the LH surge may be in part caused by the observed upregulation of ICER. In conclusion, these control mechanisms may assist LH in conditioning granulosa cells to enter a nonproliferative differentiated state that is characteristic of luteal cells.

ACKNOWLEDGMENTS

We thank Dov Shiffman (CV Therapeutics, Inc., Palo Alto, CA) for the human cyclin D2 promoter constructs, N.V. Innogentics for providing us with the GRMO2 cell line, and the Molecular Resource Facility of the University of Medicine and Dentistry of New Jersey for providing us with oligonucleotides. We also thank Nuris E. Portuondo and Lorraine Ampaw for their assistance in the preparation of the manuscript.

FOOTNOTES

1 Supported by National Institute of Child Health and Human Development grant R03HD045503 and fellowship F31HD43691 to L.C.M. Back

2 Correspondence: Carlos A. Molina, Department of Obstetrics, Gynecology and Women's Health, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, 185 South Orange Avenue, MSB E-510, Newark, NJ 07103-2714. FAX: 973 972 4574; e-mail: molinaca@umdnj.edu Back

Received: 21 November 2005.

First decision: 15 December 2005.

Accepted: 18 April 2006.

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