Proc. Natl. Acad. Sci. USA Vol. 88, pp. 7605-7609, September 1991 Biochemistry

Repression of the interleukin 6 gene promoter by p53 and the retinoblastoma susceptibility gene product (wild-type murine and human p53 and their ansforming mutants/wild-type human RB product/interleukin 6 promoter induction/c-fos, ,B-actin, and major histocompatibility class I gene promoters)

UMA SANTHANAM, ANURADHA RAY, AND PRAVINKUMAR B. SEHGAL* The Rockefeller University, 1230 York Avenue, New York, NY 10021

Communicated by Igor Tamm, May 1, 1991 (received for review April 1, 1991)

ABSTRACT The aberrant overexpression of interleukin 6 (IL-6) is implicated as an autocrine mechanism in the enhanced proliferation of the neoplastic cell elements in various B- and T-cell malignancies and in some carcinomas and sarcomas; many of these neoplasms have been shown to be associated with a mutated p53 gene. The possibility that wild-type (wt) p53, a nuclear tumor-suppressor protein, but not its transforming mutants might serve to repress IL-6 gene expression was investigated in HeLa cells. We transiently cotransfected these cells with constitutive cytomegalovirus (CMV) enhancer/promoter expression plasmids overproducing wt or mutant human or murine p53 and with appropriate chloramphenicol acetyltransferase (CAT) reporter plasmids containing the promoter elements of human IL-6, c-fos, or T-actin genes or of porcine major histocompatibility complex (MHC) class I gene in pN-38 to evaluate the effect of the various p53 species on these promoters. Murine and human wt p53 derived from pCMVNc9 and pC53-SN3, respectively, strongly repressed the IL-6 (promoter position -225 to +13), c-fos (-711 to +42), fi-actin (-3400 to +912), and MHC (-528 to -38) promoters in serum-induced HeLa cells; additionally, IL-6 promoter/CAT transcription unit constructs induced by IL-1, phorbol ester, or pseudorabies virus were also repressed by wt human and murine p53. The murine transforming mutant p53 (pCMVc5) was less active in repressing the IL-6, c-fos, /3-actin, and MHC promoter constructs. The human p53 mutant derived from pC53-SCX3 was also less active than the wt protein in repressing the IL-6, c-fos, /3-actin, and MIIC promoters, except that serum-induced IL-6/CAT expression was equally repressed by both human wt and mutant p53. In similar transient transfection experiments in HeLa cells, overexpression of the wt human retinoblastoma susceptibility gene product, RB, was found to repress the serum-induced IL-6 (-225 to +13), c-fos (-711 to +42), and /8-actin (-3400 to +912) promoters but not the PRV-induced IL-6 (-110 to +13) or the serum-induced MHC (-528 to -38) promoters. These observations identify transcriptional repression as a property of p53 and suggest that p53 and RB may be involved as transcriptional repressors in modulating IL-6 gene expression during cellular differentiation and oncogenesis. The aberrant production of interleukin 6 (IL-6) by neoplastic cell elements has been implicated as a strong contributory factor to the growth of multiple myeloma and other B-cell dyscrasias, T-cell lymphoma, renal and ovarian cell carcinomas, and Kaposi sarcoma (reviewed in refs. 1-4). Increased local IL-6 production by keratinocytes also appears to contribute to the development of hyperproliferative plaques in psoriasis (5, 6). Strong-to-moderate IL-6 immunoreactivity is observed in the neoplastic cell elements present in primary

squamous cell carcinomas; in adenocarcinomas of mammary, colonic, ovarian, and endometrial origin; in various adenocarcinomas metastatic to lymph nodes; and in soft tissue tumors such as leiomyosarcoma and neurofibrosarcoma (7). In cell culture experiments, IL-6 enhances the

proliferation of normal human keratinocytes, mesangial cells, some renal and ovarian carcinoma and Kaposi sarcomaderived cell lines, and of Epstein-Barr virus-transformed or multiple myeloma-derived B-cell lines (1-8). Although the inducible expression of the IL-6 gene by serum, cytokines, second messengers, and viruses in a variety of cell types and its down-modulation by glucocorticoids has been studied in considerable molecular detail (9-13), there is little information about the molecular basis for the apparently "constitutive" production of IL-6 by neoplastic cells. The p53 protein and the retinoblastoma susceptibility gene product RB are considered to be "tumor suppressor" proteins that are frequently mutated in a variety of neoplasms (reviewed in refs. 14-16). Stimulated by the observation that the same neoplastic tissues that exhibit mutations in p53 and RB also show increased IL-6 immunostaining (5, 14-16), we have investigated the possibility that wild-type (wt) p53 and RB might repress the promoter of the IL-6 gene (IL-6 promoter) and that transforming mutations in these proteins might relieve the repression. It has been reported (17) that wt RB but not its mutants can repress c-fos expression and activator protein AP-1 transcriptional activity in both serum-induced and cycling murine NIH 3T3 cells. This repression is mediated through a cis-acting element in the c-fos promoter called the retinoblastoma control element (RCE). We have noted (12) that a region highly analogous to the RCE is also present in the IL-6 promoter between positions -126 and -101. In the present study we show that human and murine wt p53 derived from appropriate constitutive expression plasmids transfected into HeLa cells strongly repress various cotransfected constructs incorporating the promoter element of the IL-6 gene and the chloramphenicol acetyltransferase (CAT) transcription unit. Human and murine wt p53 proteins also repress the serum-responsive c-fos promoter (-711 to +42)/CAT gene, f8-actin gene promoter (-3400 to +912)/ CAT gene, and major histocompatibility complex (MHC) class I gene promoter (-528 to -38)/CAT gene constructs. Overall, the transforming mutants of murine and human p53 display a decreased ability to repress transcription of the IL-6, c-fos, f3-actin, and MHC genes. Furthermore, wt RB also represses the cotransfected IL-6 (-225 to +13), c-fos, and f3-actin promoters in serum-induced HeLa cells but not Abbreviations: CAT, chloramphenicol acetyltransferase; CMV, cytomegalovirus; IL, interleukin; MHC promoter, major histocompatibility class I porcine gene promoter in pN-38; PRV, pseudorabies virus; RB, product of human retinoblastoma susceptibility gene, RB; RCE, RB control element; RSV, Rous sarcoma virus; wt, wild-type. *To whom reprint requests should be addressed.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Biochemistry: Santhanam et al.

the pseudorabies virus (PRV)-induced IL-6 (-110 to +13) or the serum-induced MHC promoters. These data define transcriptional repression as a novel property of p53 and suggest the hypothesis that p53 and RB might be involved as transcriptional repressors in the regulation of IL-6 gene expression.

MATERIALS AND METHODS Cell Culture and DNA Transfection Assays. HeLa cells were cultured in Dulbecco's modified Eagle's medium as described (9). Procedures for DNA transfection into cells were essentially as described (11, 12) except that either the plasmid pCH110 (2.5 ,ug), a constitutive early simian virus 40 promoter-driven expression vector for /-galactosidase, or the plasmid pRSVf8gal (5 pZg), a constitutive Rous sarcoma virus (RSV) long terminal repeat promoter-driven expression vector for B-galactosidase, was included in the DNA mixture added to cells in every 100-mm Petri dish as a marker for the overall effectiveness of the transfection experiment. All cell extracts were first assayed for 3-galactosidase activity, and CAT assays were carried out as described (11-13) with aliquots of each extract that contained a defined amount of 3-galactosidase activity. CAT activity was calculated as described earlier (9). DNA Plasmids. The human IL-6 (-225 to +13)/CAT (pIC225), IL-6 (- 10 to +13)/CAT (pICl10), the human c-fos (-711 to +42)/CAT (pFC700), and pCH110 constructs used have been described (9, 11, 12). pRSVj8gal was constructed by inserting the BamHI (filled in)-HindIII fragment of pCH110 (Pharmacia) into the EcoRV-HindIII sites of the plasmid pRcRSV (Invitrogen). pHBAC is a construct containing the 4.3-kilobase (kb) EcoRI-Alu I fragment from the human /3-actin gene isolate p14TB-17 (18) linked to the CAT transcription unit. All of the p53 constitutive expression constructs were in a cytomegalovirus (CMV) promoter/ enhancer expression vector. The murine wt p53 expression plasmid pCMVNc9 and the transforming mutant pCMVc5 (mutations Glu-168 -* Gly and Met-234--+ Ile) (19, 20) and the pdrcine MHC class I promoter (-528 to -38) linked to the CAT transcription unit, pN-38 (21), were obtained from Moshe Oren (The Weizmann Institute for Science, Rehovot, Israel). The htiman wt p53 expression plasmid pC53-SN3 and the transfdrming mutant pC53-SCX3 (mutation Val-143 Ala) were obtained from Bert Vogelstein (The Johns Hopkins University School of Medicine, Baltimore, MD; ref. 22). The human wt RB expression vector pJ3QHRbC was obtained from Robert Weinberg (The Whitehead Institute, Cambridge, MA); it has been described in ref. 17 also. Antibodies and Immunoprecipitation Analyses. Three monoclonal antibodies (mAbs) to p53, Ab-1, Ab-2, and Ab-3, were purchased from Oncogene Science (Manhasset, NY). According to the manufacturer's specifications, p53 Ab-1, which is a derivative of clone PAb421 (23), reacts with p53 from a broad range of mammalian species. Ab-2 derived from clone P1801 (24) reacts preferentially with human p53. Ab-3 made by clone PAb420 (25) recognizes a common conformational epitope on mutant forms of p53 from different species. For immunoprecipitation analyses, HeLa cell cultures in 100-mm Petri dishes transfected 40 hr earlier with 20 ,g of the appropriate p53 expression vectors were labeled with [35S]methionine (0.1 mCi per ml, DuPont/NEN; 1 Ci = 37 GBq) for 4 hr in methionine-free medium, the labeled cell extracts were immunoprecipitated with the appropriate p53 antibodies, and the labeling of p53 was evaluated by polyacrylamide gel electrophoresis and autoradiography.

Proc. Natl. Acad. Sci. USA 88 (1991)

RESULTS Expression of p53 in Transfected HeLa Cells. Labeling of transfected HeLa cells with [35S]methionine followed by immunoprecipitation analysis of the cellular proteins with three different anti-p53 mAbs is illustrated in Fig. 1. Although wt p53 has been reported to possess a short half-life (26), wt murine and human p53 are clearly synthesized in transfected HeLa cells and could be immunoprecipated by Ab-1 (Fig. 1 Left, lanes 1 and 3). Synthesis of mutant murine and human p53 could be detected by immunoprecipitation with Ab-3, a monoclonal antibody that specifically recognizes mutant forms of p53 (Fig. 1 Right, lanes 2 and 4). In addition, wt and mutant human p53 were also visualized by immunoprecipitation with Ab-2; a monoclonal antibody specific for human p53 (Fig. 1 Center, lanes 3 and 4). Repression of the IL-6 Promoter by wt p53 and RB. The effect of overexpression ofwt or mutant murine or human p53 or wt human RB was evaluated in transient transfection experiments in which the human IL-6 (-225 to +13)/CAT (pIC225) reporter construct was cotransfected into HeLa cells, and IL-6 transcription was induced with serum 40 hr later by using a protocol described earlier (9, 11). In this and all subsequent experiments, ,3-galactosidase activity (derived either from the control plasmid pCH110 or from the plasmid pRSV-,8gal) was used to monitor and normalize for transfection efficiency. It can be seen from Fig. 2A and Table 1 that the induction by serum of pIC225 was markedly inhibited by both murine and human wt p53 and by wt RB. The extent of inhibition was less with the transforming mutant of murine p53 (pCMVc5), whereas the human mutant p53 (pC53-SCX3) used in this study was as effective as the wt p53 in repressing the induction of pIC225 by serum. Fig. 2B and Table 1 show that the induction of pIC225 or of pIC110 by PRV was also repressed by wt murine and human p53; the respective transforming mutants were less active. The wt RB was less active in repressing PRV-induced expression of the IL-6/ CAT constructs pIC225 or pIC110 compared with seruminduced expression of pIC225 (Fig. 2 and Table 1). The wt murine and human p53 were also able to repress induction of pIC225 in response to the inducers IL-la and phorbol ester (Fig. 3). Thus, p53 can inhibit the IL-6 promoter irrespective of the inducer used. Repression of the c-fos Promoter by wt p53 and RB. Given the functional similarity between the IL-6 and c-fos promoters (9-11), we examined the effect of p53 on the c-fos promoter (-711 to +42)/CAT (pFC700) construct in transfected HeLa cells. As with the pIC225 construct, pFC700 was also strongly repressed by wt human and murine p53 (Fig. 4 1 2 3 4 5

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FIG. 2. Repression of the serum-induced (A) or PRV-induced (B) IL-6 promoter by p53 and RB. (A) HeLa cells were transfected with 10 ,g of pIC225 and 2.5 pg of pCH11O alone or together with 5 or 0.5 ,ug of pCMVNc9 (wt murine p53), pCMVcS (mutant murine p53), pC53-SN3 (wt human pS3), pC53-SCX3 (mutant human p53), or pJ3HRbC (pJ3QlHRbC, wt human RB). Cells were transferred to serum-free medium 16-20 hr after transfection; after 24 hr, cells were induced with 20o fetal bovine serum for 4 hr. CAT activity in the cytoplasmic extracts normalized for f-galactosidase expression is shown. (B) HeLa cells were transfected with 10 Mg of pIC225 or pIC110 and 2.5 ,g of pCH110 together with 4 Mg of the p53 or RB constructs. The experimental protocol was as above except that after 6 hr in serum-free medium, cells (in all the lanes) were induced with PRV (at a multiplicity of infection of 1) for 18 hr.

and Table 1). The two transforming mutants tested have a reduced ability to repress c-fos transcription compared with the respective wt p53 (Fig. 4 and Table 1). Furthermore, the

FIG. 3. Repression of the IL-1-induced (A) or phorbol 12myristate 13-acetate (PMA)-induced (B) IL-6 promoter by p53. HeLa cells were transfected with 10 Mug of pIC225 along with 4 ug of the various p53 expression plasmids. Via the protocol described in Fig. 2, the cells were induced with IL-la (50 ng/ml) or PMA (100 ng/ml) for 16 hr. pCH110 (2.5,ug per plate) was used as the control plasmid.

right-most lane in Fig. 4 verifies the previously reported (17) ability of human RB to inhibit the c-fos promoter in HeLa cells. Mixed cotransfection ofthe c-fos promoter with RB and p53 expression vectors resulted in an additive inhibition of transcription (data not shown). Results similar to those in Fig. 4 were also obtained in NIH 3T3 cells (data not shown). Repression of the fi-Actin and MHC Promoters by wt p53. The transcription modulatory phenotype of the p53 species tested and of wt RB could be different for strongly inducible promoters compared with those for housekeeping genes or for weakly inducible promoters. We examined the effect of p53 and RB on transcription from the f3-actin and the MHC promoters (Fig. 5 A and B, respectively, and Table 1). wt murine and human p53 markedly inhibited both promoters, while the mutants were less active. wt RB had no discernible effect on the MHC promoter as has been reported (17), but it clearly inhibited the 83-actin promoter.

DISCUSSION IL-6 gene expression is up-regulated in the neoplastic cell elements in a variety of B- and T-cell malignancies and in

Table 1. Repression of various promoters by p53 and RB CAT activity,*

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Ile) (19, 20) was used in the present study. This mutant p53 transformed rat fibroblasts in association with Ha-ras while the wt p53 did not (19, 20). Furthermore, in transfection studies, the wt p53 expression plasmid inhibited the transforming ability of this mutant p53 plus ras (20). wt but not the mutant murine p53 plasmid inhibited the induction of transformed foci by combinations of other oncogenes (myc plus ras or E1A plus ras) (20). In the present study, we found that this transforming mutant p53 was less active than wt p53 in repressing transcription from the promoters studied. The human wt and mutant p53 expression plasmids we have used in our experiments have also been characterized for their effects on cell growth (22). The p53 mutant (Val-143 -- Ala) was cloned from a human colorectal carcinoma (22). Expression of the wt but not the mutant p53 gene dramatically inhibited the growth of human colorectal carcinoma lines (22). In the present study, the human wt p53 was more effective than the mutant in inhibiting IL-6 transcription induced by PRV, IL-1, or phorbol 12-myristate 13-acetate. A difference between the functional effects of the human wt and mutant p53 proteins was also observed on the c-fos, 83-actin, and MHC promoter constructs, with the wt protein being more effective in exerting transcriptional repression. However, we could not discriminate between the ability of the wt human p53 and the mutant human p53 to repress seruminduced transcription of the IL-6 promoter (both were equally effective).

Although p53 is a nuclear protein, the biochemical mechanisms by which it exerts its transcriptional and growthmodulatory effects are unclear. The wt and various trans-

Biochemistry: Santhanam et al. forming mutants of p53 have different abilities to complex with cellular proteins (29), bind to RNA (30), or bind to DNA (31). The NH2-terminal fragment (residues 1-73) or the entire wt p53 protein fused to the DNA binding domain of yeast GAL4 has been shown to possess a transcription-activating function (32-34), whereas the transforming mutants tested had lost this ability (33). Nevertheless, this transcriptional activation function of p53 fusion proteins has only been observed by using a p53-GAL4 chimeric fusion protein acting on an artificial promoter containing multiple copies of the GALA-binding DNA motif (32-34). Our data, as well as those of Oren and colleagues (personal communication), indicate that wt p53 by itself is a strong transcriptional repressor of many but not all RNA polymerase II promoters in different mammalian cells. Although p53 has been reported to bind to DNA (31), we have thus far been unable to obtain evidence for direct binding between IL-6 promoter DNA and p53 using a sequential DNA binding-immunoprecipitation assay. The observation that p53 inhibits IL-6 gene expression irrespective of the inducer used (serum, PRV, IL-1, or phorbol ester) suggests that p53 may interact with and inhibit the function of a wide range of cellular transcription factors. It has previously been suggested that RB may not directly bind to its functionally repressible target DNA (RCE) in the c-fos promoter (17). The detection of a DNA motif in the IL-6 promoter (from -126 to -101) that bears a strong resemblance to the RCE element in the c-fos promoter (12) indicates that the mechanism of RB repression of the c-fos and IL-6 promoters may involve interactions with the same transcription factors. The present data identify wt p53 and RB proteins as candidate repressor proteins that may serve to limit IL-6 expression in nonneoplastic cells. We propose that one consequence of the various transforming mutations in p53 and RB is a relief of this repression leading to the enhanced or dysregulated production of IL-6 in neoplastic tissues. We thank Dr. Igor Tamm for his enthusiastic support, Dr. Moshe Oren for the plasmids pCMVNc9, pCMVc5, and pN-38 and for sharing his unpublished data with us, Dr. Bert Vogelstein for the plasmids pC53-SN3 and pC53-SCX3, Dr. Robert Weinberg for the plasmid pJ3flHRbC, and Mr. K. Steven LaForge and Ms. Barbara Klock for technical assistance. This work was supported by National Institutes of Health Research Grant AI-16262 and a contract from the National Foundation for Cancer Research. 1. Sehgal, P. B., Grieninger, G. & Tosato, G. (1989) Ann. N. Y. Acad. Sci. 557, 1-583. 2. Kishimoto, T. (1989) Blood 74, 1-10. 3. Sehgal, P. B. (1990) Proc. Soc. Exp. Biol. Med. 195, 183-191. 4. Kishimoto, T. (1990) Immunol. Today 11, 443-449. 5. Grossman, R. M., Kreuger, J., Yourish, D., Granelli-Piperno, A., Murphy, D. P., May, L. T., Kupper, T. S., Sehgal, P. B. & Gottleib, A. (1989) Proc. Nail. Acad. Sci. USA 86, 63676371. 6. Krueger, J., Ray, A., Tamm, I. & Sehgal, P. B. (1991) J. Cell. Biochem. 45, 327-334.

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Repression of the interleukin 6 gene promoter by p53 and the retinoblastoma susceptibility gene product.

The aberrant overexpression of interleukin 6 (IL-6) is implicated as an autocrine mechanism in the enhanced proliferation of the neoplastic cell eleme...
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