1Department of Internal Medicine, The University of Texas Medical Branch, Galveston, Texas 77555-1060; and 2Department of Obstetrics/Gynecology and Reproductive Sciences, University of Saskatchewan, Saskatoon, SK S7N 0W8, Canada
Submitted 14 November 2003 ; accepted in final form 25 January 2004
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ABSTRACT |
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protein kinase C iota; polypyrimidine tract-binding protein-associated splicing factor; insulin-like growth factor I
In this study, we investigate PKC modulation of the transcriptional activity of an insulin-like growth factor response element (IGFRE) in the upstream region of the porcine cytochrome P-450 side-chain cleavage (P-450scc) gene (32). P-450scc is the rate-limiting enzyme in the steroidogenic pathway and is responsible for cleavage of the C21-C22 bond that frees the C22-C27 side chain of cholesterol (14). The gene has been isolated and cloned from human (15, 16), rat (17), mouse (19), and bovine (1) genomic libraries. We isolated and cloned the porcine P-450scc gene from a genomic library (32) and identified an IGFRE in the upstream region of the gene (32). The IGFRE is a 30-bp GC-rich domain
100 bp upstream from the start site in the TATA-driven promoter of porcine P-450scc (32). The GC-rich domain binds Sp1 (28) and an additional transcription factor, polypyrimidine tract-binding protein (PTB)-associated splicing factor (PSF) (29).
PSF was isolated and cloned by Patton et al. (18) in 1993. It is a 76-kDa protein that migrates anomalously on SDS gels because it is highly basic. The protein associates with PTB to form spliceosomes for splicing of pre-mRNA. The PSF amino terminus is rich in proline and glutamine residues. Similar proline/glutamine-rich regions comprise the transactivation domains of Sp1 (5, 6). PSF is the product of only one gene; however, alternative splicing results in two isoforms that vary in length from their carboxyl terminus but retain the proline/glutamine-rich regions and two RNA-binding domains (18). The rapidly expanding body of knowledge regarding PSF and its many functions in the nucleus has recently been reviewed (24).
For our system, we determined that PSF binds to the IGFRE upstream of the GC box and functions as a repressor of IGFRE transcriptional activity (29). Moreover, we conducted studies in Y1 adrenal cells, a mouse steroidogenic cell line unresponsive to IGF-I for stimulating P-450scc gene expression (9, 10), and in NWTb3 cells, a mouse fibroblast cell line that is responsive to IGF-I stimulation of P-450scc gene expression (29). In these models, we observed increased PSF protein expression in Y1 adrenal cells compared with the NWTb3 cells (26). However, in Y1 cells, the repressive actions of PSF on the IGFRE could be overcome by overexpression of Sp1 (26), indicating that the cellular machinery for a functional IGFRE was present in Y1 cells and under the regulatory control of PSF.
To further investigate PSF regulation of the porcine P-450scc IGFRE, we studied the mechanism by which PKC enhances the transcriptional activity of the porcine P-450scc IGFRE by protein-protein interaction with PSF in the stable porcine granulosa cell line JC-410.
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METHODS |
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The recombinant PSF polyclonal antibody was produced by Bio-Molecular Technology (Frederick, MD). The PKC antibody was obtained from B D Biosciences (Boston, MA). The PKC
antibody was obtained from Dr. Alan Fields (Mayo Clinic, Jacksonville, FL). Nitrocellulose filters were obtained from Bio-Rad (Hercules, CA).
Plasmid Constructs
The PKC constitutively active and kinase-deficient REP4 expression plasmids were obtained from Dr. Alan Fields (Mayo Clinic). The porcine P-450scc luciferase construct containing 2,320 bp of 5' porcine P-450scc sequence, including the IGFRE, was previously described (32).
Cell Cultures
Porcine granulosa cells. Granulosa cells for primary culture were obtained from ovaries of immature pigs collected at a local slaughterhouse. Granulosa cells were cultured as previously described (30). Briefly, granulosa cells isolated from 1- to 5-mm follicles were cultured at 1.5 x 106 cells per 35-mm well on a 6-well plate with 2 ml of Eagle's MEM and 3% FBS.
JC-410 cells.
This cell line was developed from a primary culture of porcine granulosa cells (4). The levels of expression of key steroidogenic enzymes P-450scc, cytochrome P-450 aromatase, and 3-hydroxysteroid dehydrogenase are similar in amount to primary granulosa cells (4). The JC-410 cells lack expression of gonadotropin receptors but retain the ability to respond to forskolin and cholera toxin (4). The JC-410 cells were validated for studies in granulosa cell physiology in a number of previous investigations (3, 20, 25). JC-410 cells were grown to 85% confluence and harvested according to the experiments described.
NWTb3 cells.
This cell line was developed from a mouse fibroblast cell line (NIH 3T3) and is stably transfected with a plasmid overexpressing the IGF-I receptor (2). We have shown in previous studies that this cell line is responsive to IGF-I when transfected with the porcine P-450scc IGFRE (31). This cell line was used for immunoprecipitation experiments with PKC and PSF, so we could demonstrate that PKC
and PSF association occurs in more than one cell line.
Immunoprecipitation Experiments with PSF and PKC
Immunoprecipitation (IP) experiments with nuclear extract from JC-410 cells and NWTb3 cells were performed using standard protocols. Beads were washed 4 times with RIPA buffer (PBS, 1% Nonidet, 0.5% sodium deoxycholate, and 0.1% SDS).
Transient Transfection in JC-410 Cells and Luciferase Assay
JC-410 cells were cultured at 1.5 x 106 cells per 35-mm well on a 6-well plate with 2 ml of Eagle's MEM and 3% FBS per well. After overnight culture, cells were transfected with the appropriate plasmid constructs with LipofectAMINE 2000 (GIBCO-BRL, Rockville, MD). DNA-LipofectAMINE reagent complexes were left on cells for 6 h before treatment medium was added directly to wells containing complexes, and then were incubated at 37°C for 48 h. Cells were rinsed with PBS and harvested for luciferase activity with Promega's (Madison, WI) luciferase assay system. Light production was measured with a Turner TD-20e luminometer. Bio-Rad Protein DC Assay Reagent was used to measure protein concentrations of the lysates to normalize the experiments, as previously described (32). We found 2030% transfection efficiency in this system when a plasmid expressing green fluorescent protein was transfected in JC-410 cells.
Western Gel and Immunoblotting
Samples of cellular protein were fractionated by discontinuous 10% SDS-PAGE under reducing conditions. The gel was then electroblotted onto nitrocellulose (TransBlot, Bio-Rad) with electrophoretic transfer buffer for 1 h. The blot was then blocked for 2 h in 10% milk and Tris-buffered saline (TBS) and was incubated overnight with primary antibody in 5% milk-TBS. The secondary (0.5 µg) antibody, anti-rabbit (or appropriate species) IgG-horseradish peroxidase in 5% milk-TBS, was added to the blot and incubated for 1 h. Bands were detected using enhanced chemiluminescence Western blotting reagent (Amersham, Piscataway, NJ) and autoradiography.
Small Interfering RNA
We cloned porcine PKC cDNA so that species-specific small interfering (si)RNA oligonucleotides could be designed. We used human PKC
sequence to design oligonucleotides for isolation of porcine PKC
cDNA by RT-PCR from total RNA isolated from porcine granulosa cells. The oligonucleotides used were as follows.
RT-PCR primers (5' to 3') for porcine PKC
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Porcine JC-410 cells were transfected with siRNAs between 0.1 and 20 nM per ml of media by use of Lipofectamine 2000 (Invitrogen, Carlsbad, CA) and were incubated between 36 and 48 h. PKC protein levels were evaluated posttransfection by Western immunoblotting with PKC
antibody (Upstate). From the two siRNAs designed, in initial dose-response studies, one was more effective than the other in depletion of PKC
protein. The siRNA sequence selected is located 116 bp downstream from the ATG start site. An 8-bp sequence complementary to the T7 Promoter Primer was added to the 3' end of both oligonucleotides for the siRNA. The siRNA sequence used in these experiments is given below.
siRNA oligonucleotides (5' to 3') for porcine PKC iota
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Statistical Analysis
Statistical analysis was done using ANOVA with the Tukey multiple comparison test. Statistical significance was designated as P 0.05. All data are expressed as means ± SE.
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RESULTS |
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To investigate whether PKC influenced the activity of the P-450scc IGFRE, we first demonstrated that PKC
was present in nuclear extracts from primary cultures of both granulosa cells and JC-410 cells (Fig. 1). We found that IGF-I treatment of JC-410 cells significantly increased protein levels of PKC
in nuclear extracts (Fig. 1). This increase in PKC
levels by IGF-I follows the same time course of activation of the P-450scc IGFRE by IGF-I (32).
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Having established that PKC was present in the nucleus of granulosa cells, we next performed co-IP experiments to determine whether PKC
and PSF associate with each other. As shown in Fig. 2, we immunoprecipitated with PKC
(top) or PSF (bottom) and then Western blotted with the reciprocal antibody. We found (Fig. 2) that PSF and PKC
associate in nuclear extracts from JC-410 cells with both co-IP experiments. To demonstrate that this association generalized to other cell lines and was not specific to JC-410 cells, we performed a similar experiment on nuclear extracts from NWTb3 cells, using PSF for the IP and PKC
for the Western blot (Fig. 3). Again, an association of PSF and PKC
was found in the nuclear extracts. Performing the IP with IgG and showing the absence of PKC
by Western blotting confirmed the specificity of these interactions. All IP experiments were reproduced three times.
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To determine the physiological significance of the PSF and PKC association in relationship to the transcriptional activity of the P-450scc IGFRE, we tested the hypothesis that the transcriptional effects of PKC
on IGFRE were independent of the phosphorylation activity of the kinase. We performed transient transfection experiments with plasmids expressing an active PKC
(CaPKC
) or a kinase-deficient isoform (KdPKC
). As shown in Fig. 4, both plasmids increased the transcriptional activity of the porcine P-450scc IGFRE, suggesting that PKC
catalytic activity is not required to affect expression.
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We next tested the hypothesis that depletion of PKC protein suppressed IGFRE activity. Transfection of JC-410 cells with PKC
siRNA reduced PKC
protein below basal levels and inhibited the transcriptional activity of the IGFRE in a dose-dependent manner (Fig. 5). Moreover, we found that depletion of PKC
with siRNA did not prevent IGF-I stimulation of transcriptional activity of the porcine P-450scc IGFRE, but it did modulate the IGF-I effect (Fig. 6). These results are consistent with PKC
modulating IGF-I stimulation of the IGFRE but not directly regulating the stimulatory mechanism.
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DISCUSSION |
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Our observation that PKC associates with PSF and functions in a kinase-independent manner through direct protein-protein interactions is consistent with previous studies demonstrating that several PKCs function by a similar mechanism. In Neuro-2a neuroblastoma cells, overlay assays and glutathione S-transferase (GST) pull-down assays with GST-PKC
showed that PSF associated with PKC
(21) while showing only a weak phosphorylation of PSF by PKC
(21). Additional studies found that PKC
binds to v-src tyrosine kinase (22), whereas PKC
binds to actin, nonmuscle actin (11), and GAP-43 (8). Furthermore, PKC
binds to receptors for activated C kinase (RACK)1 (12) and RACK2 (7).
There is an accumulating body of evidence that PSF represses transcription in addition to many other cellular functions (13, 24). From our previous studies, we know that PSF binds to the IGFRE upstream of the GC box (29). Using COOH terminus-truncated proteins, we have shown that the amino acid residues responsible for binding to the IGFRE were located in the NH2 terminus of PSF (27). Moreover, we demonstrated that PSF must bind to the IGFRE to function as a repressor (27). The mechanism whereby association of PSF and PKC prevents PSF repression of the IGFRE in cells cannot be determined from these studies and will be the focus of future studies.
In conclusion, we determined that PKC modulates the transcriptional activity of the porcine P-450scc IGFRE through a mechanism independent of kinase catalytic activity. Association of PKC
with PSF to prevent repression of the IGFRE is an apparent mechanism whereby PKC
enhances the activity of the porcine P-450scc IGFRE.
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GRANTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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