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• W2068454411 abstract "Retinoids inhibit the expression of migration inhibitory factor-related protein-8 (MRP-8), a marker of hyperproliferative or abnormal keratinocyte differentiation, in a retinoic acid receptor (RAR)-dependent manner in various cell culture systems. MRP-8 expression is also down-regulated in vivo in psoriatic lesions after topical application of an anti-psoriatic RARβ/γ-selective synthetic retinoid, tazarotene. We demonstrate that an MRP-8 promoter linked to a chloramphenicol acetyltransferase reporter (MRP8CAT) faithfully replicates the differentiation-specific regulation of the endogenous keratinocyte MRP-8 gene. Further, interferon γ and serum-induced expression of MRP8CAT is inhibited by retinoid receptors in a ligand-dependent manner. We also show that NF-IL6 acts as a transcriptional enhancer of MRP-8, and that RARs inhibit MRP8CAT by inhibiting the enhancer action of nuclear factor-interleukin-6 (NF-IL6). The NF-IL6 antagonism function of RAR is a complex of the core of the DNA binding domain and the hydrophobic zipper region. This manuscript identifies NF-IL6 as another transcription factor, in addition to AP1, whose activity is inhibited by RAR in a ligand-dependent manner. The interdiction of NF-IL6-dependent signal transduction pathway by RARs may explain some of the therapeutic effects of retinoids in inflammatory and proliferative diseases. Retinoids inhibit the expression of migration inhibitory factor-related protein-8 (MRP-8), a marker of hyperproliferative or abnormal keratinocyte differentiation, in a retinoic acid receptor (RAR)-dependent manner in various cell culture systems. MRP-8 expression is also down-regulated in vivo in psoriatic lesions after topical application of an anti-psoriatic RARβ/γ-selective synthetic retinoid, tazarotene. We demonstrate that an MRP-8 promoter linked to a chloramphenicol acetyltransferase reporter (MRP8CAT) faithfully replicates the differentiation-specific regulation of the endogenous keratinocyte MRP-8 gene. Further, interferon γ and serum-induced expression of MRP8CAT is inhibited by retinoid receptors in a ligand-dependent manner. We also show that NF-IL6 acts as a transcriptional enhancer of MRP-8, and that RARs inhibit MRP8CAT by inhibiting the enhancer action of nuclear factor-interleukin-6 (NF-IL6). The NF-IL6 antagonism function of RAR is a complex of the core of the DNA binding domain and the hydrophobic zipper region. This manuscript identifies NF-IL6 as another transcription factor, in addition to AP1, whose activity is inhibited by RAR in a ligand-dependent manner. The interdiction of NF-IL6-dependent signal transduction pathway by RARs may explain some of the therapeutic effects of retinoids in inflammatory and proliferative diseases. All-trans-retinoic acid (RA) 1The abbreviations used are: RA, retinoic acid; RAR, retinoic acid receptor; RXR, retinoid X receptor; RARE, retinoic acid response element; MRP-8, migration inhibitory factor-related protein 8; NF-IL6, nuclear factor-interleukin 6; DBD, DNA binding domain; CAT, chloramphenicol acetyltransferase; IFN-γ, interferon γ; PCR, polymerase chain reaction; bp, base pair(s); kb, kilobase(s); HSV, herpes simplex virus; tk, thymidine kinase; TPA, 12-O-tetradecanoylphorbol-13acetate. 1The abbreviations used are: RA, retinoic acid; RAR, retinoic acid receptor; RXR, retinoid X receptor; RARE, retinoic acid response element; MRP-8, migration inhibitory factor-related protein 8; NF-IL6, nuclear factor-interleukin 6; DBD, DNA binding domain; CAT, chloramphenicol acetyltransferase; IFN-γ, interferon γ; PCR, polymerase chain reaction; bp, base pair(s); kb, kilobase(s); HSV, herpes simplex virus; tk, thymidine kinase; TPA, 12-O-tetradecanoylphorbol-13acetate. and its synthetic analogs not only modulate cell growth and differentiation in vitro, but are also therapeutically effective in the treatment of a variety of diseases involving cell proliferation, abnormal differentiation, and inflammation (1Nagpal S. Chandraratna R.A.S. Curr. Pharm. Design. 1996; 2: 295-316Google Scholar, 2Boehm M.F. Heyman R.A. Patel S. Stein R.B. Nagpal S. Exp. Opin. Invest. Drugs. 1995; 4: 593-612Crossref Scopus (54) Google Scholar). The pleiotropic effects of retinoids are mediated through two families of nuclear retinoid receptors, retinoic acid receptors (RARα, -β, and -γ) and retinoid X receptors (RXRα, -β, and -γ). RARs and RXRs are modular proteins containing domains responsible for sequence-specific DNA binding (C-region), ligand independent transactivation (AF-1, A/B region), and ligand-dependent transactivation (AF-2, E-region) (1Nagpal S. Chandraratna R.A.S. Curr. Pharm. Design. 1996; 2: 295-316Google Scholar, 3Chambon P. Semin. Cell Biol. 1994; 5: 115-125Crossref PubMed Scopus (499) Google Scholar). Retinoid receptors transactivate the expression of genes in a ligand-dependent manner by sequence-specific binding to the retinoic acid-responsive elements (RAREs) present in the promoter regions of responsive genes. In contrast to the steroid receptors, which function as homodimers, retinoid receptors function inside the cells as RAR-RXR heterodimers (4Nagpal S. Friant S. Nakshatri H. Chambon P. EMBO J. 1993; 12: 2349-2360Crossref PubMed Scopus (272) Google Scholar). Although the retinoid-dependent transactivation of genes can thus be explained by the binding of RAR-RXR to the RAREs, recruitment of co-activators, and subsequent activation of the transcriptional machinery, this paradigm does not hold true for the genes whose expression is repressed by retinoids. While RARs have been shown to inhibit the expression of certain genes by antagonizing the enhancer actions of the transcription factor AP1 (5Fanjul A. Dawson M.I. Hobbs P.D. Jong L. Cameron J.F. Harlev E. Graupner G. Lu X.P. Pfahl M. Nature. 1994; 372: 107-111Crossref PubMed Scopus (316) Google Scholar, 6Nagpal S. Athanikar J. Chandraratna R.A.S. J. Biol. Chem. 1995; 270: 923-927Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar), the mechanism of retinoid-mediated inhibition of other non-AP1-dependent genes remains unclear. Since various inflammatory and hyperproliferative conditions appear to be maintained or perpetuated by the products of genes which are under the control of an AP1 motif, the antagonism of AP1-dependent gene expression appears to be an important underlying mechanism of the therapeutic action of RA.Tazarotene is an RARβ/γ-selective synthetic retinoid which is therapeutically effective in the treatment of psoriasis (6Nagpal S. Athanikar J. Chandraratna R.A.S. J. Biol. Chem. 1995; 270: 923-927Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar), a highly prevalent skin disease characterized by hyperproliferation and inflammation (7Krueger G.G. Duvic M. J. Invest. Dermatol. 1994; 102: 14S-18SCrossref PubMed Scopus (115) Google Scholar). To understand the mechanism of tazarotene action in psoriasis, we have identified several tazarotene-responsive genes in an in vitro skin system by subtractive hybridization (8Nagpal S. Patel S. Asano A.T. Johnson A.T. Duvic M. Chandraratna R.A.S. J. Invest. Dermatol. 1996; 106: 269-274Abstract Full Text PDF PubMed Scopus (109) Google Scholar, 9Nagpal S. Thacher S.M. Patel S. Friant S. Malhotra M. Shafer J. Krasinski G. Asano A.T. Teng M. Duvic M. Chandraratna R.A.S. Cell Growth Differ. 1996; 7: 1783-1791PubMed Google Scholar). The expression of one of the genes, migration inhibitory factor-related protein-8 (MRP-8), was inhibited in an RAR-dependent manner in vitro in cultured keratinocytes and in skin raft organ cultures, as well as in vivo in psoriatic lesions (9Nagpal S. Thacher S.M. Patel S. Friant S. Malhotra M. Shafer J. Krasinski G. Asano A.T. Teng M. Duvic M. Chandraratna R.A.S. Cell Growth Differ. 1996; 7: 1783-1791PubMed Google Scholar). MRP-8 is highly expressed in psoriatic epidermis, while its expression is absent in normal epidermis (10Wilkinson M.M. Busuttil A. Hayward C. Brock D.J. Dorin J.R. Van Heyningen V. J. Cell Sci. 1988; 91: 221-230Crossref PubMed Google Scholar). MRP-8 is also expressed in other chronic inflammatory diseases such as chronic polyarthritis and cystic fibrosis and displays a 73% amino acid homology to a murine cytokine CP-10 (11Odink K. Cerletti N. Bruggen J. Clerc R.G. Tarcsay L. Zwadlo G. Gerhards G. Schlegel R. Sorg C. Nature. 1987; 330: 80-82Crossref PubMed Scopus (553) Google Scholar, 12Madsen P. Rasmussen H.H. Leffers H. Honore B. Celis J.E. J. Invest. Dermatol. 1992; 99: 299-305Abstract Full Text PDF PubMed Scopus (220) Google Scholar, 13Barthe C. Figarella C. Carrere J. Guy-Crotte O. Biochim. Biophys. Acta. 1991; 1096: 175-177Crossref PubMed Scopus (40) Google Scholar, 14Lackmann M. Rajasekariah P. Iismaa S.E. Jones G. Cornish C.J. Hu S. Simpson R.J. Moritz R.L. Geczy C.L. J. Immunol. 1993; 150: 2981-2991PubMed Google Scholar).In this manuscript we demonstrate that retinoids inhibit the expression of MRP-8 by directly acting at the promoter level. We also show that the MRP-8 promoter is activated by NF-IL6 and retinoids inhibit MRP-8 expression by antagonizing the enhancer action of NF-IL6. We further demonstrate that the integrity of the core of the DNA binding domain (DBD) and the hydrophobic zipper region of RAR is mandatory for the inhibition of the MRP-8 promoter, thus indicating that the NF-IL6 antagonism domain of RAR is a complex of the DBD and the hydrophobic zipper region.DISCUSSIONWe have recently demonstrated that MRP-8 expression is inhibited by retinoids in vitro in skin systems such as cultured keratinocytes and skin rafts. Further, we have demonstrated that the topical treatment of psoriatic plaques with the anti-psoriatic retinoid tazarotene inhibits the expression of MRP-8 in psoriatic lesions during the course of clinical improvement of the disease (9Nagpal S. Thacher S.M. Patel S. Friant S. Malhotra M. Shafer J. Krasinski G. Asano A.T. Teng M. Duvic M. Chandraratna R.A.S. Cell Growth Differ. 1996; 7: 1783-1791PubMed Google Scholar). To delineate the mechanism of negative regulation of MRP-8 by retinoids, the promoter region of MRP-8 was cloned into a reporter plasmid and analyzed by transient transfection. The expression of MRP8CAT was induced in primary keratinocytes, a keratinocyte cell line (HaCaT), and HeLa cells, thus indicating that the elements involved in the serum and IFN-γ induction of the endogenous MRP-8 gene are present in the cloned upstream sequences. Further, both serum- and IFN-γ-induced expression of MRP8CAT was inhibited by retinoids in keratinocytes and in HeLa cells (Fig. 2). These results demonstrate that the 1.5 kb of cloned MRP-8 promoter also harbors the retinoid regulatory sequences responsible for the repression of the gene. A motif search of the MRP-8 promoter did not turn up any obvious RAREs. An AP1-like motif (5′-TGACTAA-3′) present at −465 bp from the TATA box, however, was inactive since the MRP8CAT construct was non-responsive to TPA induction in both cultured keratinocytes and HeLa cells (Fig.2 c and data not shown). A search of the MRP-8 promoter revealed the presence of three putative NF-IL6 binding motifs. Co-transfection experiments with MRP8CAT and an NF-IL6 expression vector showed that NF-IL6 induced the MRP8CAT reporter (Fig.3 a). NF-IL6, like c-Jun/c-Fos, is a bZip transcription factor and belongs to the family of C/EBPs (20Brasier A.R. Kumar A. J. Biol. Chem. 1994; 269: 10341-10351Abstract Full Text PDF PubMed Google Scholar). RAR inhibited the NF-IL6-induced expression of MRP8CAT in a ligand-dependent manner (Fig. 3 b). NF-IL6 is the second transcription factor identified so far whose enhancer action, like that of AP1, is antagonized by retinoids. Using various mutants of RARs, we demonstrate that the integrity of the DBD as well as the hydrophobic zipper region is obligatory for inhibition of MRP-8 gene expression (Fig. 4). The dispensability of the A/B regions, together with the requirement of the core of the DBD and the intact hydrophobic zipper suggested that the NF-IL6 antagonism function of RAR is a complex of two distinct domains (Fig. 4). Note that tazarotene, which is an RARβ/γ-selective retinoid in transactivation assays, inhibited the expression of the MRP-8 promoter through RARα (Fig. 2). Therefore, like the anti-AP1 property of RARα (6Nagpal S. Athanikar J. Chandraratna R.A.S. J. Biol. Chem. 1995; 270: 923-927Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar), the anti-NF-IL6 activity of RARα appears to be separable from its transactivation function.Retinoids are widely accepted as therapeutic modalities for the treatment of dermatological diseases including psoriasis and have been shown to be effective in the treatment of various cancers (e.g. acute promyelocytic leukemia, squamous cell carcinoma, Kaposi's sarcoma, etc.). Further, retinoids inhibit the proliferation of a number of other cancer cell lines (e.g. breast, ovarian, colon etc.) in vitro (1Nagpal S. Chandraratna R.A.S. Curr. Pharm. Design. 1996; 2: 295-316Google Scholar). Although activation of genes has been thought to be the major mode of action of RARs, so far only one retinoid-induced gene has been identified which can transduce the anti-proliferative and anti-inflammatory signals of retinoids. Insulin-like growth factor-binding protein 3 is induced by retinoids in transformed cervical cells and inhibits the growth-promoting activities of insulin-like growth factor 1 (22Andreatta-Van Leyen S. Hembree J.R. Eckert R.L. J. Cell Physiol. 1994; 160: 265-274Crossref PubMed Scopus (35) Google Scholar). In contrast, a number of retinoid-repressed genes are associated with therapeutic effects of retinoids (6Nagpal S. Athanikar J. Chandraratna R.A.S. J. Biol. Chem. 1995; 270: 923-927Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). The antagonism of AP1-dependent gene expression can explain some of the therapeutic effects of retinoids in various proliferative and inflammatory diseases. Since NF-IL6 is another pro-inflammatory and proliferative transcription factor, the inhibition of its activity delineates another pathway for the therapeutic effects of retinoids. NF-IL6 regulates the expression of IL-6 (23Ray A. LaForge K.S. Sehgal P.B. Mol. Cell. Biol. 1990; 10: 5736-5746Crossref PubMed Scopus (237) Google Scholar), and as shown here, MRP-8, both of which are highly expressed in psoriasis (10Wilkinson M.M. Busuttil A. Hayward C. Brock D.J. Dorin J.R. Van Heyningen V. J. Cell Sci. 1988; 91: 221-230Crossref PubMed Google Scholar, 24Grossman R.M. Krueger J. Yourish D. Granelli-Piperno A. Murphy D.P. May L.T. Kupper T.S. Sehgal P.B. Gottlieb A.B. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6367-6371Crossref PubMed Scopus (732) Google Scholar) and involved in the pathophysiology of the disease. Therefore, by antagonizing NF-IL6 action, retinoids would inhibit both IL-6 and MRP-8 in psoriatic lesions, resulting in disease amelioration. Retinoids, in fact, inhibit the expression of IL-6 in cultured keratinocytes and skin rafts. 3S. Thacher and R. A. S. Chandraratna, unpublished observations. Further, RAR-NF-IL6 antagonism can also account for the normalization of abnormal keratinocyte differentiation, since MRP-8 is associated with the abnormal differentiation of keratinocytes. In summary, we describe a novel pathway for the anti-inflammatory and anti-proliferative effects of retinoids. This novel mode of retinoid action involves the interdiction of NF-IL6 signal transduction pathway by RARs in a ligand-dependent manner. This pathway can be therapeutically exploited by the systematic chemical synthesis of more potent anti-NF-IL6 retinoids. Such retinoids may exhibit increased therapeutic:toxic ratios for the treatment of certain retinoid-responsive diseases involving NF-IL6 as one of the pathophysiological signals. All-trans-retinoic acid (RA) 1The abbreviations used are: RA, retinoic acid; RAR, retinoic acid receptor; RXR, retinoid X receptor; RARE, retinoic acid response element; MRP-8, migration inhibitory factor-related protein 8; NF-IL6, nuclear factor-interleukin 6; DBD, DNA binding domain; CAT, chloramphenicol acetyltransferase; IFN-γ, interferon γ; PCR, polymerase chain reaction; bp, base pair(s); kb, kilobase(s); HSV, herpes simplex virus; tk, thymidine kinase; TPA, 12-O-tetradecanoylphorbol-13acetate. 1The abbreviations used are: RA, retinoic acid; RAR, retinoic acid receptor; RXR, retinoid X receptor; RARE, retinoic acid response element; MRP-8, migration inhibitory factor-related protein 8; NF-IL6, nuclear factor-interleukin 6; DBD, DNA binding domain; CAT, chloramphenicol acetyltransferase; IFN-γ, interferon γ; PCR, polymerase chain reaction; bp, base pair(s); kb, kilobase(s); HSV, herpes simplex virus; tk, thymidine kinase; TPA, 12-O-tetradecanoylphorbol-13acetate. and its synthetic analogs not only modulate cell growth and differentiation in vitro, but are also therapeutically effective in the treatment of a variety of diseases involving cell proliferation, abnormal differentiation, and inflammation (1Nagpal S. Chandraratna R.A.S. Curr. Pharm. Design. 1996; 2: 295-316Google Scholar, 2Boehm M.F. Heyman R.A. Patel S. Stein R.B. Nagpal S. Exp. Opin. Invest. Drugs. 1995; 4: 593-612Crossref Scopus (54) Google Scholar). The pleiotropic effects of retinoids are mediated through two families of nuclear retinoid receptors, retinoic acid receptors (RARα, -β, and -γ) and retinoid X receptors (RXRα, -β, and -γ). RARs and RXRs are modular proteins containing domains responsible for sequence-specific DNA binding (C-region), ligand independent transactivation (AF-1, A/B region), and ligand-dependent transactivation (AF-2, E-region) (1Nagpal S. Chandraratna R.A.S. Curr. Pharm. Design. 1996; 2: 295-316Google Scholar, 3Chambon P. Semin. Cell Biol. 1994; 5: 115-125Crossref PubMed Scopus (499) Google Scholar). Retinoid receptors transactivate the expression of genes in a ligand-dependent manner by sequence-specific binding to the retinoic acid-responsive elements (RAREs) present in the promoter regions of responsive genes. In contrast to the steroid receptors, which function as homodimers, retinoid receptors function inside the cells as RAR-RXR heterodimers (4Nagpal S. Friant S. Nakshatri H. Chambon P. EMBO J. 1993; 12: 2349-2360Crossref PubMed Scopus (272) Google Scholar). Although the retinoid-dependent transactivation of genes can thus be explained by the binding of RAR-RXR to the RAREs, recruitment of co-activators, and subsequent activation of the transcriptional machinery, this paradigm does not hold true for the genes whose expression is repressed by retinoids. While RARs have been shown to inhibit the expression of certain genes by antagonizing the enhancer actions of the transcription factor AP1 (5Fanjul A. Dawson M.I. Hobbs P.D. Jong L. Cameron J.F. Harlev E. Graupner G. Lu X.P. Pfahl M. Nature. 1994; 372: 107-111Crossref PubMed Scopus (316) Google Scholar, 6Nagpal S. Athanikar J. Chandraratna R.A.S. J. Biol. Chem. 1995; 270: 923-927Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar), the mechanism of retinoid-mediated inhibition of other non-AP1-dependent genes remains unclear. Since various inflammatory and hyperproliferative conditions appear to be maintained or perpetuated by the products of genes which are under the control of an AP1 motif, the antagonism of AP1-dependent gene expression appears to be an important underlying mechanism of the therapeutic action of RA. Tazarotene is an RARβ/γ-selective synthetic retinoid which is therapeutically effective in the treatment of psoriasis (6Nagpal S. Athanikar J. Chandraratna R.A.S. J. Biol. Chem. 1995; 270: 923-927Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar), a highly prevalent skin disease characterized by hyperproliferation and inflammation (7Krueger G.G. Duvic M. J. Invest. Dermatol. 1994; 102: 14S-18SCrossref PubMed Scopus (115) Google Scholar). To understand the mechanism of tazarotene action in psoriasis, we have identified several tazarotene-responsive genes in an in vitro skin system by subtractive hybridization (8Nagpal S. Patel S. Asano A.T. Johnson A.T. Duvic M. Chandraratna R.A.S. J. Invest. Dermatol. 1996; 106: 269-274Abstract Full Text PDF PubMed Scopus (109) Google Scholar, 9Nagpal S. Thacher S.M. Patel S. Friant S. Malhotra M. Shafer J. Krasinski G. Asano A.T. Teng M. Duvic M. Chandraratna R.A.S. Cell Growth Differ. 1996; 7: 1783-1791PubMed Google Scholar). The expression of one of the genes, migration inhibitory factor-related protein-8 (MRP-8), was inhibited in an RAR-dependent manner in vitro in cultured keratinocytes and in skin raft organ cultures, as well as in vivo in psoriatic lesions (9Nagpal S. Thacher S.M. Patel S. Friant S. Malhotra M. Shafer J. Krasinski G. Asano A.T. Teng M. Duvic M. Chandraratna R.A.S. Cell Growth Differ. 1996; 7: 1783-1791PubMed Google Scholar). MRP-8 is highly expressed in psoriatic epidermis, while its expression is absent in normal epidermis (10Wilkinson M.M. Busuttil A. Hayward C. Brock D.J. Dorin J.R. Van Heyningen V. J. Cell Sci. 1988; 91: 221-230Crossref PubMed Google Scholar). MRP-8 is also expressed in other chronic inflammatory diseases such as chronic polyarthritis and cystic fibrosis and displays a 73% amino acid homology to a murine cytokine CP-10 (11Odink K. Cerletti N. Bruggen J. Clerc R.G. Tarcsay L. Zwadlo G. Gerhards G. Schlegel R. Sorg C. Nature. 1987; 330: 80-82Crossref PubMed Scopus (553) Google Scholar, 12Madsen P. Rasmussen H.H. Leffers H. Honore B. Celis J.E. J. Invest. Dermatol. 1992; 99: 299-305Abstract Full Text PDF PubMed Scopus (220) Google Scholar, 13Barthe C. Figarella C. Carrere J. Guy-Crotte O. Biochim. Biophys. Acta. 1991; 1096: 175-177Crossref PubMed Scopus (40) Google Scholar, 14Lackmann M. Rajasekariah P. Iismaa S.E. Jones G. Cornish C.J. Hu S. Simpson R.J. Moritz R.L. Geczy C.L. J. Immunol. 1993; 150: 2981-2991PubMed Google Scholar). In this manuscript we demonstrate that retinoids inhibit the expression of MRP-8 by directly acting at the promoter level. We also show that the MRP-8 promoter is activated by NF-IL6 and retinoids inhibit MRP-8 expression by antagonizing the enhancer action of NF-IL6. We further demonstrate that the integrity of the core of the DNA binding domain (DBD) and the hydrophobic zipper region of RAR is mandatory for the inhibition of the MRP-8 promoter, thus indicating that the NF-IL6 antagonism domain of RAR is a complex of the DBD and the hydrophobic zipper region. DISCUSSIONWe have recently demonstrated that MRP-8 expression is inhibited by retinoids in vitro in skin systems such as cultured keratinocytes and skin rafts. Further, we have demonstrated that the topical treatment of psoriatic plaques with the anti-psoriatic retinoid tazarotene inhibits the expression of MRP-8 in psoriatic lesions during the course of clinical improvement of the disease (9Nagpal S. Thacher S.M. Patel S. Friant S. Malhotra M. Shafer J. Krasinski G. Asano A.T. Teng M. Duvic M. Chandraratna R.A.S. Cell Growth Differ. 1996; 7: 1783-1791PubMed Google Scholar). To delineate the mechanism of negative regulation of MRP-8 by retinoids, the promoter region of MRP-8 was cloned into a reporter plasmid and analyzed by transient transfection. The expression of MRP8CAT was induced in primary keratinocytes, a keratinocyte cell line (HaCaT), and HeLa cells, thus indicating that the elements involved in the serum and IFN-γ induction of the endogenous MRP-8 gene are present in the cloned upstream sequences. Further, both serum- and IFN-γ-induced expression of MRP8CAT was inhibited by retinoids in keratinocytes and in HeLa cells (Fig. 2). These results demonstrate that the 1.5 kb of cloned MRP-8 promoter also harbors the retinoid regulatory sequences responsible for the repression of the gene. A motif search of the MRP-8 promoter did not turn up any obvious RAREs. An AP1-like motif (5′-TGACTAA-3′) present at −465 bp from the TATA box, however, was inactive since the MRP8CAT construct was non-responsive to TPA induction in both cultured keratinocytes and HeLa cells (Fig.2 c and data not shown). A search of the MRP-8 promoter revealed the presence of three putative NF-IL6 binding motifs. Co-transfection experiments with MRP8CAT and an NF-IL6 expression vector showed that NF-IL6 induced the MRP8CAT reporter (Fig.3 a). NF-IL6, like c-Jun/c-Fos, is a bZip transcription factor and belongs to the family of C/EBPs (20Brasier A.R. Kumar A. J. Biol. Chem. 1994; 269: 10341-10351Abstract Full Text PDF PubMed Google Scholar). RAR inhibited the NF-IL6-induced expression of MRP8CAT in a ligand-dependent manner (Fig. 3 b). NF-IL6 is the second transcription factor identified so far whose enhancer action, like that of AP1, is antagonized by retinoids. Using various mutants of RARs, we demonstrate that the integrity of the DBD as well as the hydrophobic zipper region is obligatory for inhibition of MRP-8 gene expression (Fig. 4). The dispensability of the A/B regions, together with the requirement of the core of the DBD and the intact hydrophobic zipper suggested that the NF-IL6 antagonism function of RAR is a complex of two distinct domains (Fig. 4). Note that tazarotene, which is an RARβ/γ-selective retinoid in transactivation assays, inhibited the expression of the MRP-8 promoter through RARα (Fig. 2). Therefore, like the anti-AP1 property of RARα (6Nagpal S. Athanikar J. Chandraratna R.A.S. J. Biol. Chem. 1995; 270: 923-927Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar), the anti-NF-IL6 activity of RARα appears to be separable from its transactivation function.Retinoids are widely accepted as therapeutic modalities for the treatment of dermatological diseases including psoriasis and have been shown to be effective in the treatment of various cancers (e.g. acute promyelocytic leukemia, squamous cell carcinoma, Kaposi's sarcoma, etc.). Further, retinoids inhibit the proliferation of a number of other cancer cell lines (e.g. breast, ovarian, colon etc.) in vitro (1Nagpal S. Chandraratna R.A.S. Curr. Pharm. Design. 1996; 2: 295-316Google Scholar). Although activation of genes has been thought to be the major mode of action of RARs, so far only one retinoid-induced gene has been identified which can transduce the anti-proliferative and anti-inflammatory signals of retinoids. Insulin-like growth factor-binding protein 3 is induced by retinoids in transformed cervical cells and inhibits the growth-promoting activities of insulin-like growth factor 1 (22Andreatta-Van Leyen S. Hembree J.R. Eckert R.L. J. Cell Physiol. 1994; 160: 265-274Crossref PubMed Scopus (35) Google Scholar). In contrast, a number of retinoid-repressed genes are associated with therapeutic effects of retinoids (6Nagpal S. Athanikar J. Chandraratna R.A.S. J. Biol. Chem. 1995; 270: 923-927Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). The antagonism of AP1-dependent gene expression can explain some of the therapeutic effects of retinoids in various proliferative and inflammatory diseases. Since NF-IL6 is another pro-inflammatory and proliferative transcription factor, the inhibition of its activity delineates another pathway for the therapeutic effects of retinoids. NF-IL6 regulates the expression of IL-6 (23Ray A. LaForge K.S. Sehgal P.B. Mol. Cell. Biol. 1990; 10: 5736-5746Crossref PubMed Scopus (237) Google Scholar), and as shown here, MRP-8, both of which are highly expressed in psoriasis (10Wilkinson M.M. Busuttil A. Hayward C. Brock D.J. Dorin J.R. Van Heyningen V. J. Cell Sci. 1988; 91: 221-230Crossref PubMed Google Scholar, 24Grossman R.M. Krueger J. Yourish D. Granelli-Piperno A. Murphy D.P. May L.T. Kupper T.S. Sehgal P.B. Gottlieb A.B. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6367-6371Crossref PubMed Scopus (732) Google Scholar) and involved in the pathophysiology of the disease. Therefore, by antagonizing NF-IL6 action, retinoids would inhibit both IL-6 and MRP-8 in psoriatic lesions, resulting in disease amelioration. Retinoids, in fact, inhibit the expression of IL-6 in cultured keratinocytes and skin rafts. 3S. Thacher and R. A. S. Chandraratna, unpublished observations. Further, RAR-NF-IL6 antagonism can also account for the normalization of abnormal keratinocyte differentiation, since MRP-8 is associated with the abnormal differentiation of keratinocytes. In summary, we describe a novel pathway for the anti-inflammatory and anti-proliferative effects of retinoids. This novel mode of retinoid action involves the interdiction of NF-IL6 signal transduction pathway by RARs in a ligand-dependent manner. This pathway can be therapeutically exploited by the systematic chemical synthesis of more potent anti-NF-IL6 retinoids. Such retinoids may exhibit increased therapeutic:toxic ratios for the treatment of certain retinoid-responsive diseases involving NF-IL6 as one of the pathophysiological signals. We have recently demonstrated that MRP-8 expression is inhibited by retinoids in vitro in skin systems such as cultured keratinocytes and skin rafts. Further, we have demonstrated that the topical treatment of psoriatic plaques with the anti-psoriatic retinoid tazarotene inhibits the expression of MRP-8 in psoriatic lesions during the course of clinical improvement of the disease (9Nagpal S. Thacher S.M. Patel S. Friant S. Malhotra M. Shafer J. Krasinski G. Asano A.T. Teng M. Duvic M. Chandraratna R.A.S. Cell Growth Differ. 1996; 7: 1783-1791PubMed Google Scholar). To delineate the mechanism of negative regulation of MRP-8 by retinoids, the promoter region of MRP-8 was cloned into a reporter plasmid and analyzed by transient transfection. The expression of MRP8CAT was induced in primary keratinocytes, a keratinocyte cell line (HaCaT), and HeLa cells, thus indicating that the elements involved in the serum and IFN-γ induction of the endogenous MRP-8 gene are present in the cloned upstream sequences. Further, both serum- and IFN-γ-induced expression of MRP8CAT was inhibited by retinoids in keratinocytes and in HeLa cells (Fig. 2). These results demonstrate that the 1.5 kb of cloned MRP-8 promoter also harbors the retinoid regulatory sequences responsible for the repression of the gene. A motif search of the MRP-8 promoter did not turn up any obvious RAREs. An AP1-like motif (5′-TGACTAA-3′) present at −465 bp from the TATA box, however, was inactive since the MRP8CAT construct was non-responsive to TPA induction in both cultured keratinocytes and HeLa cells (Fig.2 c and data not shown). A search of the MRP-8 promoter revealed the presence of three putative NF-IL6 binding motifs. Co-transfection experiments with MRP8CAT and an NF-IL6 expression vector showed that NF-IL6 induced the MRP8CAT reporter (Fig.3 a). NF-IL6, like c-Jun/c-Fos, is a bZip transcription factor and belongs to the family of C/EBPs (20Brasier A.R. Kumar A. J. Biol. Chem. 1994; 269: 10341-10351Abstract Full Text PDF PubMed Google Scholar). RAR inhibited the NF-IL6-induced expression of MRP8CAT in a ligand-dependent manner (Fig. 3 b). NF-IL6 is the second transcription factor identified so far whose enhancer action, like that of AP1, is antagonized by retinoids. Using various mutants of RARs, we demonstrate that the integrity of the DBD as well as the hydrophobic zipper region is obligatory for inhibition of MRP-8 gene expression (Fig. 4). The dispensability of the A/B regions, together with the requirement of the core of the DBD and the intact hydrophobic zipper suggested that the NF-IL6 antagonism function of RAR is a complex of two distinct domains (Fig. 4). Note that tazarotene, which is an RARβ/γ-selective retinoid in transactivation assays, inhibited the expression of the MRP-8 promoter through RARα (Fig. 2). Therefore, like the anti-AP1 property of RARα (6Nagpal S. Athanikar J. Chandraratna R.A.S. J. Biol. Chem. 1995; 270: 923-927Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar), the anti-NF-IL6 activity of RARα appears to be separable from its transactivation function. Retinoids are widely accepted as therapeutic modalities for the treatment of dermatological diseases including psoriasis and have been shown to be effective in the treatment of various cancers (e.g. acute promyelocytic leukemia, squamous cell carcinoma, Kaposi's sarcoma, etc.). Further, retinoids inhibit the proliferation of a number of other cancer cell lines (e.g. breast, ovarian, colon etc.) in vitro (1Nagpal S. Chandraratna R.A.S. Curr. Pharm. Design. 1996; 2: 295-316Google Scholar). Although activation of genes has been thought to be the major mode of action of RARs, so far only one retinoid-induced gene has been identified which can transduce the anti-proliferative and anti-inflammatory signals of retinoids. Insulin-like growth factor-binding protein 3 is induced by retinoids in transformed cervical cells and inhibits the growth-promoting activities of insulin-like growth factor 1 (22Andreatta-Van Leyen S. Hembree J.R. Eckert R.L. J. Cell Physiol. 1994; 160: 265-274Crossref PubMed Scopus (35) Google Scholar). In contrast, a number of retinoid-repressed genes are associated with therapeutic effects of retinoids (6Nagpal S. Athanikar J. Chandraratna R.A.S. J. Biol. Chem. 1995; 270: 923-927Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). The antagonism of AP1-dependent gene expression can explain some of the therapeutic effects of retinoids in various proliferative and inflammatory diseases. Since NF-IL6 is another pro-inflammatory and proliferative transcription factor, the inhibition of its activity delineates another pathway for the therapeutic effects of retinoids. NF-IL6 regulates the expression of IL-6 (23Ray A. LaForge K.S. Sehgal P.B. Mol. Cell. Biol. 1990; 10: 5736-5746Crossref PubMed Scopus (237) Google Scholar), and as shown here, MRP-8, both of which are highly expressed in psoriasis (10Wilkinson M.M. Busuttil A. Hayward C. Brock D.J. Dorin J.R. Van Heyningen V. J. Cell Sci. 1988; 91: 221-230Crossref PubMed Google Scholar, 24Grossman R.M. Krueger J. Yourish D. Granelli-Piperno A. Murphy D.P. May L.T. Kupper T.S. Sehgal P.B. Gottlieb A.B. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6367-6371Crossref PubMed Scopus (732) Google Scholar) and involved in the pathophysiology of the disease. Therefore, by antagonizing NF-IL6 action, retinoids would inhibit both IL-6 and MRP-8 in psoriatic lesions, resulting in disease amelioration. Retinoids, in fact, inhibit the expression of IL-6 in cultured keratinocytes and skin rafts. 3S. Thacher and R. A. S. Chandraratna, unpublished observations. Further, RAR-NF-IL6 antagonism can also account for the normalization of abnormal keratinocyte differentiation, since MRP-8 is associated with the abnormal differentiation of keratinocytes. In summary, we describe a novel pathway for the anti-inflammatory and anti-proliferative effects of retinoids. This novel mode of retinoid action involves the interdiction of NF-IL6 signal transduction pathway by RARs in a ligand-dependent manner. This pathway can be therapeutically exploited by the systematic chemical synthesis of more potent anti-NF-IL6 retinoids. Such retinoids may exhibit increased therapeutic:toxic ratios for the treatment of certain retinoid-responsive diseases involving NF-IL6 as one of the pathophysiological signals. We thank Drs. P. Chambon and P. Gill for providing some of the plasmids used in these studies and we thank Dr. S. Thacher for kindly providing the HaCaT cell line. We also thank Drs. L. Wheeler, M. Rosenthal, S. Thacher, and C. Ghosn for reviewing the manuscript. During the review of this manuscript, two supporting papers were published describing the functional significance of RAR-NF-IL6 antagonism in the inhibition of adipogenesis and Kaposi's sarcoma cell proliferation (25Schwarz E.J. Reginato M.J. Shao D. Krakow S.L. Lazar M. Mol. Cell. Biol. 1997; 17: 1552-1561Crossref PubMed Google Scholar, 26Nagpal S. Cai J. Zheng T. Patel S. Masood R. Lin G.Y. Friant S. Johnson A. Smith D.L. Chandraratna R.A.S. Gill P. Mol. Cell. Biol. 1997; 17: 4159-4168Crossref PubMed Scopus (61) Google Scholar)." @default.
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