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Protein kinase C enhances the transcriptional activity of the porcine P-450 side-chain cleavage insulin-like response element

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

	ABSTRACT

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IGF-I enhances steroidogenesis in granulosa cells by stimulating the expression of the rate-limiting steroidogenic enzyme, cytochrome P-450 side-chain cleavage (P-450_scc). This effect is mediated through an IGF response element (IGFRE) that binds polypyrimidine tract-binding protein (PTB)-associated splicing factor (PSF) and Sp1. Sp1 is essential for activation of the IGFRE, and PSF functions as a repressor. We investigated mechanisms of modulation of the IGFRE by the atypical protein kinase C (PKC) in a porcine stable granulosa cell line, JC-410. PKC was found in nuclear extracts, and levels were increased by IGF-I after 24 and 48 h of treatment. Immunoprecipitation experiments demonstrated that PSF and PKC associated with each other in nuclear extracts from JC-410 cells. Transient transfection with expression plasmids of kinase-active and kinase-deficient PKC isoforms enhanced transcriptional activity of the IGFRE regardless of kinase catalytic activity. Depletion of PKC protein by small interfering RNA suppressed basal IGFRE activity but did not prevent IGF-I stimulation of the IGFRE. We conclude that PKC enhances transcriptional activity of the porcine P-450_scc IGFRE independently of kinase activity by a mechanism involving protein-protein interaction with PSF.

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-450_scc) gene (32). P-450_scc 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-450_scc 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-450_scc (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-450_scc gene expression (9, 10), and in NWTb3 cells, a mouse fibroblast cell line that is responsive to IGF-I stimulation of P-450_scc 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-450_scc IGFRE, we studied the mechanism by which PKC enhances the transcriptional activity of the porcine P-450_scc IGFRE by protein-protein interaction with PSF in the stable porcine granulosa cell line JC-410.

	METHODS

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Materials

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-450_scc luciferase construct containing 2,320 bp of 5' porcine P-450_scc 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-450_scc, 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-450_scc 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 20–30% 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

 PCR amplified a 675-bp band on a Southern gel that was excised and TA cloned into the pCR2.1 vector (Invitrogen, Carlsbad, CA) for sequencing. Sequencing (DNA Recombinant Laboratory, University of Texas Medical Branch) identified the band as PKC (compared with the human sequence), and oligonucleotides were selected from the porcine sequence as potential siRNA target sites for the PKC gene. Beginning at the start codon, the length of the gene was scanned for poly(A) sequences, and the 3'-adjacent 19 nucleotides were scanned as potential siRNA target sites. Potential sites were compared with the appropriate database to eliminate any target sequences with significant homology to other genes. Ambion's "siRNA Template Design Tool for the Silencer siRNA Construction Kit" was used to design two 29-mer oligonucleotides for each siRNA being tested. The siRNAs were prepared using the Silencer siRNA Construction Kit by Ambion. Briefly described, the DNA oligonucleotides were hybridized to the T7 Promoter Oligonucleotide, and a double stranded DNA template was prepared using DNA polymerase. A separate transcription reaction was set up for each template and incubated for 2–4 h at 37°C. The two reactions corresponding to the opposite strands of each siRNA were then mixed and incubated overnight at 37°C. The transcription reactions were treated with DNAse and a single-strand RNase to eliminate DNA templates, siRNA leader sequences, and unhybridized RNA. The siRNAs were column purified. The 260-nm absorbance of the siRNA sample was used to assess the concentration of the siRNA preparation.

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

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.

	RESULTS

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PKC Expression in Granulosa Cells

To investigate whether PKC influenced the activity of the P-450_scc 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-450_scc IGFRE by IGF-I (32).

View larger version (31K): [in this window] [in a new window]   Fig. 1. Protein kinase C (PKC) expression with and without IGF-I treatment in primary cultures of porcine granulosa cells and JC-410 cells. Cultures of primary porcine granulosa cells and of JC-410 cells were treated with IGF-I (20 nM) for 24 and 48 h with control cultures. Cells were harvested for nuclear extracts, and Western analysis was conducted for each cell culture using a PKC antibody with 25 µg of nuclear protein extract per lane. Actin (data not shown) was used to ensure equal protein loading in each lane. Top: 2 representative blots, one from porcine granulosa cells and the other from JC-410 cells. PKC antibody identifies a 74-kDa band in Western blot. Bottom: band densities of 3 experiments in JC-410 cells after IGF-I treatment and Western blot analysis for PKC. Units expressed are arbitrary, having been normalized to actin band intensities. *P 0.05, as determined by ANOVA with Tukey multiple comparison test.

PKC and PSF Associate in Nuclear Extracts

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.

View larger version (36K): [in this window] [in a new window]   Fig. 2. Immunoprecipitation (IP) experiments showing PKC and polypyrimidine tract-binding protein-associated splicing factor (PSF) association from nuclear extracts of JC-410 cells. Nuclear extract (1.5 mg) from JC-410 cells was incubated overnight with anti-PSF antibody and nonspecific IgG (top) and anti-PKC antibody and nonspecific IgG (bottom). IP extracts were Western blotted and probed with the reciprocal antibody (blot). Recombinant PSF protein served as a control for PSF, whereas Start represents nuclear protein before IP (20 µg). PKC-PSF association could be shown in both IP experiments. This Western blot is representative of 3 IP experiments.

View larger version (20K): [in this window] [in a new window]   Fig. 3. IP of PSF from nuclear extracts of NWTb3 cells shows association with PKC. Nuclear proteins (1.0 mg) from NWTb3 cells were incubated overnight with anti-PSF antibody or nonspecific IgG. IP PSF and nonspecific IgG were Western blotted and probed with anti-PKC (Blot). PKC was associated with IP PSF and not with IgG. Start represents nuclear protein (20 µg) from NWTb3 cells before IP as a control for the PKC band. This Western blot is representative of 3 IP experiments.

Transfection Experiments with PKC Expression Plasmids in JC-410 Cells

To determine the physiological significance of the PSF and PKC association in relationship to the transcriptional activity of the P-450_scc 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-450_scc IGFRE, suggesting that PKC catalytic activity is not required to affect expression.

View larger version (9K): [in this window] [in a new window]   Fig. 4. Results from transient transfection experiments with expression vectors for PKC in JC-410 cells, stably transfected with the porcine cytochrome P-450 side-chain cleavage (P-450scc) IGF response element (IGFRE) in a luciferase construct. REP 4 represents the expression vector without construct. CaPKC expressed a constitutively active form of PKC, and KdPKC expressed a kinase-dead PKC isoform. Data are means ± SE of 6 experiments. Arbitrary units represent light units from luciferase activity corrected by protein concentration of treated cells. *P 0.05, as determined by ANOVA.

siRNA Depletion of PKC Protein in JC-410 Cells

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-450_scc 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.

View larger version (37K): [in this window] [in a new window]   Fig. 5. Porcine PKC small interfering (si)RNA specifically depletes PKC and reduces transcriptional activity of porcine P-450scc IGFRE in a dose-dependent manner in JC-410 cells. From the sequence of porcine PKC, an siRNA was synthesized and transfected into JC-410 cells cotransfected with a porcine P-450scc luciferase construct containing the IGFRE. Top: a Western blot of JC-410 cell nuclear extract protein (15 µg) hybridized with PKC antibody after transfection over a dose response of PKC siRNA (Control, 10 and 20 nM). Middle: a 2nd blot was hybridized with PKC antibody to demonstrate specific depletion of PKC but not related PKC isoforms. Graph shows measured light units (arbitrary units) corrected by protein concentrations from 6 experiments in JC-410 cells transfected with the indicated concentrations of PKC siRNA. Statistical significance as determined by ANOVA: *P < 0.05, Control vs. 10 nM siRNA. **P < 0.05, 10 to 20 nM siRNA.

View larger version (25K): [in this window] [in a new window]   Fig. 6. Depletion of PKC by siRNA modulates IGF-I stimulation of transcriptional activity of the porcine P-450scc IGFRE. Top: JC-410 cells were transfected with 20 nM PKC siRNA and treated with 20 nM IGF-I, and nuclear extracts were Western blotted with PKC antibody. Graph (bottom) shows transcriptional activity of the porcine P-450scc IGFRE (arbitrary light units corrected for protein concentration, means ± SE from 6 experiments) in JC-410 cells transfected with PKC siRNA and treated with IGF-I. Statistically significant increase (*P 0.05) from control to IGF-I treatment as determined by ANOVA using Tukey multiple comparison test; a statistically significant decrease (**P < 0.05) from control to treatment with siRNA.

	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-450_scc 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-450_scc IGFRE.

	GRANTS

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This work was supported by National Institute of Child Health and Human Development Grant HD-36092 (to R. J. Urban).

	FOOTNOTES

 

Address for reprint requests and other correspondence: R. J. Urban, Division of Endocrinology, 301 Univ. Blvd., The Univ. of Texas Medical Branch, Galveston, TX 77555-1060 (E-mail: rurban{at}utmb.edu).

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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This Article Abstract Full Text (PDF) All Versions of this Article: 286/6/E975    most recent 00520.2003v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via ISI Web of Science (1) Citing Articles via Google Scholar Google Scholar Articles by Urban, R. J. Articles by Chedrese, J. Search for Related Content PubMed PubMed Citation Articles by Urban, R. J. Articles by Chedrese, J.