NH2 terminus of PTB-associated splicing factor binds to the porcine P450scc IGF-I response element

Randall J. Urban1, Yvonne H. Bodenburg1, and Thomas G. Wood2

1 Division of Endocrinology, Department of Internal Medicine, and 2 Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, Texas 77555


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

An insulin-like growth factor (IGF) I response element (IGFRE) in the porcine P-450 cholesterol side-chain cleavage gene (P450scc) regulates transcription through the binding of two proteins, Sp1 and polypyrimidine tract-binding protein-associated splicing factor (PSF). PSF is a component of spliceosomes and contains RNA-binding domains. In this study, we localized the NH2-terminal amino acid residues necessary for binding of PSF to the IGFRE. Three COOH-terminal truncated proteins (aa 304, 214, and 134) of PSF were designed to empirically partition the NH2-terminal region while excluding the RNA-binding domains. Southwestern analysis showed that only the largest expressed truncated protein, P3, strongly bound the porcine P450scc IGFRE. Truncated PSF protein expression in Y1 adrenal cells showed that P3 repressed transcriptional activity of the IGFRE similar to full-length PSF, whereas P2 (minimal binding to the IGFRE) had no effect. In conclusion, the NH2-terminal region of PSF contains the amino acid residues necessary for binding to the porcine P450scc IGFRE and repressing the transcriptional activity of the element.

polypyrimidine tract-binding protein; P-450 cholesterol side-chain cleavage; steroidogenesis; insulin-like growth factor-I; Sp1; transcription


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

INSULIN-LIKE GROWTH FACTOR I (IGF-I) is a growth factor that stimulates steroidogenesis in the ovary (1, 2). We determined that IGF-I increases steroidogenesis in porcine granulosa cells by increasing the expression of P-450 cholesterol side-chain cleavage (P450scc) enzyme (23). IGF-I stimulates P450scc gene expression through a 30-bp GC-rich domain (IGF-I response element, IGFRE) located ~100 bp from a classical TATA box in the P450scc gene (25). Moreover, we determined that the IGFRE binds Sp1 (21) and polypyrimidine tract-binding protein (PTB)-associated splicing factor (PSF) (22). Sp1 binds to the GC box of the IGFRE, whereas PSF binds to a nonoverlapping site upstream of the GC box, the palindrome CTGAGTC (22). Transient transfection experiments in porcine granulosa cells with an Sp1 expression plasmid stimulated IGFRE-mediated transcriptional activity of the P450scc gene, whereas PSF expression repressed transcriptional activity even during concomitant expression of Sp1 (22).

PSF was isolated and cloned by Patton in 1993 (19). 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 the splicing of pre-mRNA. It is an intriguing protein in that the amino terminus is rich in proline and glutamine residues (see Fig. 1). Similar proline/glutamine-rich regions comprise the transactivation domains of Sp1 (7, 8). 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 (19). A protein with 70% homology to PSF, nuclear RNA-binding protein (54 kDa) shows DNA binding in the NH2 terminus of the protein (4). The NH2-terminal region of PSF was shown to bind response sequences for thyroid hormone and retinoid X receptors (16). Therefore, this study focuses on determining whether the NH2 terminus of PSF contains the amino acid residues that bind the porcine P450scc IGFRE and how this affects the function of the response element.


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Fig. 1.   Western blot of expressed truncated proteins of polypyrimidine tract-binding protein-associated splicing factor (PSF). Top: the PSF protein. R1 and R2 indicate the RNA-binding domains; P and P,Q indicate regions rich in proline and glutamine. The truncated proteins are indicated by P1-P3, and their sizes in amino acids (aa) are shown by the lines below. Bottom: Western blot shows their expression with the use of a polyclonal antibody to PSF. The protein shown is a crude bacterial extract resulting in nonspecific bands in addition to the expressed truncated proteins. Molecular mass markers are in the margins (kDa). Isopropyl-beta -D-thiogalactopyranoside (IPTG) was used to induce protein expression. PSF represents full-length PSF that had been previously purified from bacterial extract.

Studies show that the 30-bp IGFRE in P450scc serves a much broader role in controlling P450scc gene expression than just as an IGF-I response element. Stimulation of steroidogenesis by luteinizing hormone and follicle-stimulating hormone (FSH) occurs by increasing cAMP and activating protein kinase A, which can translocate to the nucleus and phosphorylate transcription factors (12). The 30-bp IGFRE of P450scc mediates a threefold stimulation of P450scc gene expression in transient transfection experiments in porcine granulosa cells treated with FSH or forskolin (25). This same region is also responsive to cAMP in bovine, rat, and human P450scc genes (3, 18). Moreover, GC-rich cAMP response regions that bind Sp1 occur in other steroidogenic enzyme genes (14) and in other cell types (27). The impact of tumor necrosis factor-alpha (TNF-alpha ) on steroidogenesis is also controlled by the IGFRE. In porcine granulosa cells, TNF-alpha inhibits expression of P450scc mRNA concentrations stimulated by insulin or IGF-I (24, 26). The inhibition of steroidogenesis by TNF-alpha was mediated through the IGFRE (24). Finally, there is evidence that the protein kinase C pathway can also regulate steroidogenesis by effects on FSH-stimulated steroidogenesis, indicating that this important cellular pathway may influence P450scc expression through the IGFRE (10, 15). Therefore, understanding the mechanisms and interactions of PSF and Sp1 on this important 30-bp response element can result in an increased understanding of P450scc gene expression by multiple cellular pathways.

In the present study, we focused on determining the PSF amino acid residues necessary for binding to the IGFRE. We used COOH-terminal truncated PSF proteins to show that the amino acid residues important to PSF binding/function were located in the NH2 terminus and did not require the RNA binding domains to repress transcriptional activity of the element.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. The antibody to PSF was made from recombinant PSF by Bio-Molecular Technology (Frederick, MD). Nitrocellulose filters were obtained from Bio-Rad (Hercules, CA).

Plasmid constructs. The PSF cDNA clone was obtained from Dr. James Patton (Vanderbilt University) in a pET-15b expression vector (19). PSF truncation mutants P1-P3 were created using a PCR-based strategy. Primers introduced unique cloning sites 5' (NdeI) and 3' (XhoI) to facilitate construction of the expression plasmids by use of a pET-15b vector (Novagen) for bacterial expression. PCR-based strategy was also used for cloning PSF and the PSF truncation mutants P2 and P3 into the pcDNA3.1/V5-His expression vector for mammalian expression (Invitrogen). Both strategies used PCR-introduced unique cloning sites that maintained the normal reading frame for PSF and the truncation mutants. The anti-V5 antibody (Invitrogen) was used to verify protein expression of the truncation mutants in Y1 adrenal cells. The anti-V5 antibody is directed against the epitope found in the P and V proteins of the paramyxovirus, SV5, resulting in a marker of expression that is highly sensitive with low background. The plasmid pSVPLUC is a modified pGEM3 plasmid containing the luciferase gene and an enhancerless SV40 early region promoter (5). The complete sequenced upstream region of porcine P450scc (approximately -2,000 bp and including the IGFRE) was used in a reporter gene construct with the pSVPLUC plasmid. The control plasmid in transfection experiments is pSV2Apap containing the SV40 early promoter enhancer region and the human placental alkaline phosphatase gene (13).

Transient transfection in Y1 adrenal cells. Y1 adrenal cells were cultured as previously described (25). Transient transfection was carried out by lipofection (Tfx-50 Reagent, Promega). Transfection experiments were done on 60-mm plates following the Promega protocol for Tfx-50 Reagent. A 3:1 ratio (1 µg of DNA/2.5 µl of Tfx-50) was used for each transfection. Cells were harvested, and luminescence was measured 48 h after cotransfection using Promega's luciferase assay system.

Southwestern analysis. Protein samples were taken from the bacterial protein expression solutions described in Fig. 1 and were denatured for 5 min at 95°C and then loaded onto a 10% SDS-PAGE gel. Proteins were electroblotted onto nitrocellulose using 25 mM Tris, 192 mM glycine, and 20% (vol/vol) methanol for 1 h. The filter was then rinsed with TNE-50 (10 mM Tris · HCl, pH 7.5, 50 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol) briefly and then blocked in Blotto (5% milk-TNE-50) for 2 h at room temperature with gentle shaking. The blots were rinsed two times in TNE-50 before being placed in hybridization buffer. (TNE-50 buffer with 1 µg/µl poly(dI-dC) and 1.5 × 106 cpm/ml labeled probe). The probe is the 30-bp porcine P450scc IGFRE end-labeled with 32P. The blot was hybridized for 2-4 h at room temperature, rinsed in TNE-50 three times for 5 min at room temperature, and exposed to an autoradiogram.

Western gel and immunoblotting. Nuclear extract protein samples were obtained from Y1 adrenal and NWTb3 cells by use of a standard protocol to isolate nuclear protein. Samples of cell nuclear extract protein were fractionated by discontinuous 10% SDS-PAGE gel under reducing conditions. The gel was then electroblotted onto nitrocellulose (TransBlot, Bio-Rad) by use of electrophoretic transfer buffer for 1 h. The blot was then blocked for 2 h in 5% milk-TBS and then incubated overnight with primary antibody, in 1% milk-TBS. The secondary 0.5-µg antibody was added to the blot and incubated for 1 h. The blot was exposed to film and developed. This standard method was used for Western analysis with antibody to PSF and V5.

Truncated PSF protein expression in bacteria. The truncated proteins were expressed using competent BL21DE3 plysS cells (Novagen) that were transformed with each clone. Detailed protein expression methods have been previously described (22).

Statistical analysis. Statistical analysis on the transient transfection experiments was done either by ANOVA on ranks with Student-Newman-Keuls multiple comparison or by paired t-test. Data are presented as means ± SE. Statistical significance is reached at P <=  0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Localization of DNA binding of PSF. Having shown previously that PSF bound to the palindrome CTGAGTC, located 5' of the GC box of the porcine P450scc IGFRE (22), we wanted to determine the amino acid residues of PSF necessary for binding to the porcine P450scc IGFRE. Therefore, we made three COOH-terminal truncated proteins of PSF (Fig. 1). We partitioned the NH2 terminus of the PSF protein into three regions (aa 304, 214, 134), intentionally excluding the two RNA-binding domains (Fig. 1). The NH2-terminal region is rich in prolines and glutamines, as indicated in Fig. 1. We used a PCR-based strategy to create the three truncated PSF domains, cloned the three cDNAs into the pET15b expression vector, and expressed all three truncated proteins (Fig. 1). PSF is a unique protein in that it is 76 kDa in size but runs on SDS-PAGE at 100 kDa because of the basic nature of the protein (19).

We used Southwestern analysis with the wild-type porcine P450scc IGFRE to determine whether any of the three truncated proteins would bind the IGFRE. As shown in Fig. 2, the largest truncated protein, P3, strongly bound to the IGFRE. P2 shows faint binding to the IGFRE, and no binding is seen for P1 (Fig. 2). Therefore, the NH2-terminal region contains amino acid residues that are necessary for binding of PSF to the porcine P450scc IGFRE. This occurs primarily between amino acids 214 and 304. 


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Fig. 2.   Southwestern analysis of truncated PSF proteins. The proteins described in Fig. 1 were analyzed in a standard Southwestern analysis with the porcine cholesterol P-450 side-chain cleavage (P450scc) insulin-like growth factor-I response element (IGFRE). P3 was the only truncated protein that strongly bound the IGFRE. Purified expressed full-length PSF is included as a reference as are molecular mass markers (kDa).

Truncated PSF expression in Y1 adrenal cells. We hypothesized that the truncated protein P3 could have two possible functions. It could compete with endogenous PSF and prevent repression of the transcriptional activity of the IGFRE; however, it could also function like endogenous PSF and repress the response element. We selected mouse Y1 adrenal cells to express P3 because we had previously found that this steroidogenic cell line was not responsive to IGF-I in transient transfection experiments using the P450scc IGFRE (25). We reasoned that this cell line expressed abundant amounts of PSF that would enable competition with expressed P3. Figure 3 shows the verification that PSF is expressed in Y1 adrenal cells by Western analysis with a PSF-derived antibody. Bacterially expressed PSF was used as the positive control for the Western.


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Fig. 3.   Western analysis of PSF expression in Y1 adrenal cells. Shown is a Western blot analyzed with an antibody to PSF derived from recombinant PSF. Recombinant PSF (0.5 µl) was run in the first lane (rP). Y1 (20 µg) nuclear extract protein was run in the next lane. The size of PSF is also shown in the figure.

PSF, P2, and P3 constructs were ligated in the pcDNA3.1/V5-His expression vector for transfection experiments (see METHODS). Figure 4 shows that PSF, P2, and P3 were expressed in Y1 cells after transient transfection (V5 epitope tag). Both P2 and P3 expressed as doublet bands on Western analysis at similar intensities. PSF expressed at a lower level than the two truncated proteins but as only one band. Also shown in Fig. 4 are the results of cotransfection experiments that show that P3 (strong IGFRE binding) expression represses the P450scc IGFRE similarly to full-length PSF, whereas P2 (faint IGFRE binding) expression does not influence the transcriptional activity of the IGFRE. Therefore, the RNA-binding domains and COOH-terminal region of PSF are not necessary for the inhibitory actions of the protein on the IGFRE.


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Fig. 4.   Expression of COOH-terminal truncated proteins of PSF in Y1 cells. Y1 adrenal cells were transfected with either the empty pcDNA3.1/V5-His plasmid (V5) or the V5 constructs containing PSF (100 kDa), P2 (49 kDa), and P3 (65 kDa). Top: Western blot analysis showing expression of the truncated proteins by epitope labeling with an antibody to V5 (epitope found in the P and V proteins of the paramyxovirus, SV5). The arrows indicate the protein expression bands for PSF, P2, and P3. Both truncated proteins showed doublet expression, while full-length PSF expressed only 1 band. An SV40 luciferase construct of the porcine P450scc IGFRE was co-transfected with a control plasmid, pSV2Apap. Bottom: graph of transfection results expressed as means ± SE from 6 replicates. Arbitrary units are luminescence of the lysate after treatment divided by absorbance (alkaline phosphatase). *Statistical significance, P <=  0.05. The V5 vector was the negative control.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we have found that the NH2 terminus of PSF contains the amino acid sequences necessary for binding to the porcine P450scc IGFRE. The truncated PSF protein alone, without the RNA-binding domains, acts on the proximal 2 kb of porcine P450scc to repress transcriptional activity of the IGFRE.

The findings in this study increase our understanding of PSF. The NH2 terminus of this protein contains many proline and glutamine residues (Fig. 1), and these basic amino acids result in a slowed migration in SDS-PAGE gels (19). The 304-amino acid truncated protein P3 contained the sequences necessary for IGFRE binding of PSF (Fig. 2) as well as maintaining repression of the transcriptional activity of the IGFRE (Fig. 4). Therefore, from our studies, PSF acts much like two distinct proteins. The NH2 terminus binds to DNA and inhibits transcription, whereas the RNA-binding domains and COOH terminus function for RNA splicing and preparation for protein translation. The physiological relevance of such a multitasking protein for ovarian steroidogenesis is yet to be determined. P450scc is the rate-limiting enzyme in the steroidogenic pathway, and the porcine P450scc IGFRE controls many cellular pathways (17). As shown previously, TNF-alpha suppresses the activity of the IGFRE, implying that this effect could be mediated through the repressive action of PSF (24). Regulation could derive from increased PSF expression or from changes in PSF or Sp1 phosphorylation states. Abnormalities in cellular pathways that regulate PSF binding to the porcine P450scc IGFRE could reduce repression of the promoter by PSF and make P450scc expression more sensitive to insulin/IGF-I stimulation in conditions such as polycystic ovarian disease that are genetically linked to P450scc (6, 11).

Our study also adds further confirmation to the developing literature on PSF serving as a repressor of transcription. Mathur et al. (16) identified PSF as a corepressor of the type II nuclear hormone receptors thyroid hormone receptor and retinoid X receptor. PSF bound to the DNA-binding domain of these receptors and associated with Sin3A, a protein known to mediate transcriptional repression by recruitment of class I histone deacetylases (16). The repressor activity of PSF in their system required amino acid residues containing one of the RNA-binding domains (aa 361-464), whereas our studies showed repression and binding to the IGFRE with residues closer to the NH2 terminus and excluding the RNA-binding domains (aa 214-304). Subnuclear localization studies using green fluorescent protein found that loss of the RNA recognition motif 2 in PSF resulted in diffuse accumulation of PSF in the nucleus and loss of localized speckle accumulation (9). The truncated PSF protein P3 lacked both RNA motifs but was found in nuclear extract protein and repressed P450scc transcriptional activity (Fig. 4). Whether P3 lost localized nuclear speckle accumulation while retaining functional activity will need to be determined in future studies. To add another level of complexity to PSF, a recent study found that, during apoptosis in bone marrow cells, PSF is hyperphosphorylated, dissociates from PTB, associates with new protein partners, and becomes insensitive to proteolysis (20). Therefore, PSF is a highly complex protein, showing that it may be an important component of transcriptional repression for many different genes by many different mechanisms.

In conclusion, the NH2 terminus of PSF contains the sequences necessary to bind the porcine P450scc IGFRE. The COOH-terminal truncated PSF protein that binds the IGFRE represses its transcriptional activity without the RNA-binding domains. The physiological relevance of gene-specific repression of the rate-limiting enzyme in steroidogenesis, P450scc, could be important in syndromes of abnormal ovarian steroidogenesis such as polycystic ovary syndrome.


    ACKNOWLEDGEMENTS

This study was supported by National Institutes of Health Grant HD-36092 (to R. J. Urban).


    FOOTNOTES

Address for reprint requests and other correspondence: R. J. Urban, 8.138 MRB, 1060, Div. of Endocrinology, 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.

April 23, 2002;10.1152/ajpendo.00057.2002

Received 11 February 2002; accepted in final form 20 April 2002.


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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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