Characterization of Retinoic Acid Receptor-deficient Keratinocytes
2000; Elsevier BV; Volume: 275; Issue: 22 Linguagem: Inglês
10.1074/jbc.m909382199
ISSN1083-351X
AutoresPhilippe Goyette, Chang Feng Chen, Wei Wang, F. Seguin, David Lohnes,
Tópico(s)NF-κB Signaling Pathways
ResumoRetinoids are essential for normal epidermal growth and differentiation and show potential for the prevention or treatment of various epithelial neoplasms. The retinoic acid receptors (RARα, -β, and -γ) are transducers of the retinoid signal. The epidermis expresses RARγ and RARα, both of which are potential mediators of the effects of retinoids in the epidermis. To further investigate the role(s) of these receptors, we derived transformed keratinocyte lines from wild-type, RARα, RARγ, and RARαγ null mice and investigated their response to retinoids, including growth inhibition, markers of growth and differentiation, and AP-1 activity. Our results indicate that RARγ is the principle receptor contributing to all-trans-retinoic acid (RA)-mediated growth arrest in this system. This effect partially correlated with inhibition of AP-1 activity. In the absence of RARs, the synthetic retinoid N-(4-hydroxyphenyl)-retinamide inhibited growth; this was not observed with RA, 9-cis RA, or the synthetic retinoid (E)-4-[2-(5, 5, 8, 8 tetramethyl-5,6,7,8-tetrahydro-2-naphthalenyl)-1-propenyl] benzoic acid. Finally, both RARα and RARγ differently affected the expression of some genes, suggesting both specific and overlapping roles for the RARs in keratinocytes. Retinoids are essential for normal epidermal growth and differentiation and show potential for the prevention or treatment of various epithelial neoplasms. The retinoic acid receptors (RARα, -β, and -γ) are transducers of the retinoid signal. The epidermis expresses RARγ and RARα, both of which are potential mediators of the effects of retinoids in the epidermis. To further investigate the role(s) of these receptors, we derived transformed keratinocyte lines from wild-type, RARα, RARγ, and RARαγ null mice and investigated their response to retinoids, including growth inhibition, markers of growth and differentiation, and AP-1 activity. Our results indicate that RARγ is the principle receptor contributing to all-trans-retinoic acid (RA)-mediated growth arrest in this system. This effect partially correlated with inhibition of AP-1 activity. In the absence of RARs, the synthetic retinoid N-(4-hydroxyphenyl)-retinamide inhibited growth; this was not observed with RA, 9-cis RA, or the synthetic retinoid (E)-4-[2-(5, 5, 8, 8 tetramethyl-5,6,7,8-tetrahydro-2-naphthalenyl)-1-propenyl] benzoic acid. Finally, both RARα and RARγ differently affected the expression of some genes, suggesting both specific and overlapping roles for the RARs in keratinocytes. alltrans retinoic acid retinoid X receptor retinoic acid receptor CREB-binding protein c-Jun N-terminal kinase N-(4-hydroxyphenyl)-retinamide (E)-4-[2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthalenyl)-1-propenyl] benzoic acid 4-morpholinepropanesulfonic acid keratin activator protein retinoic acid response element phosphorylated c-Jun Vitamin A derivatives (retinoids) play central roles in embryonic development and maintenance of various tissues in the adult (1.$$$$$$ ref data missingGoogle Scholar, 2.Kastner P. Mark M. Chambon P. Cell. 1996; 83: 859-869Abstract Full Text PDF Scopus (935) Google Scholar, 3.Morriss-Kay G. Ward S. Sokolova N. Arch. Toxicol. 1994; 16: 112-117Google Scholar). 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The retinoid signal is transduced by two families of nuclear receptors, the retinoic acid (RA)1receptors (RARα, -β, and -γ and their isoforms) and the retinoid x receptors (RXRα, -β, and -γ) (10.Leid M. Kastner P. Chambon P. Trends Biochem. Sci. 1992; 17: 427-433Abstract Full Text PDF PubMed Scopus (803) Google Scholar, 11.Mangelsdorf D.J. Borgmeyer U. Heyman R.A. Zhou J.Y. Ong E.S. Oro A.E. Kakizuka A. Evans R.M. Genes Dev. 1992; 6: 329-344Crossref PubMed Scopus (1061) Google Scholar, 12.Houle B. Rochette-Egly C. Bradley W.E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 985-989Crossref PubMed Scopus (283) Google Scholar, 13.Mangelsdorf D.J. Thummel C. Beato M. Herrlich P. Schultz G. Umesono K. Blumberg B. Kastner P. Evans R.M. Cell. 1996; 83: 835-839Abstract Full Text PDF Scopus (6039) Google Scholar). RARs function as ligand-inducible transcription regulators by binding, together with an RXR partner, to specific cis-acting response elements (RAREs). RARs can be activated by both RA and its stereoisomer, 9-cis RA, whereas RXRs are activated only by 9-cis RA (14.Heyman R.A. Mangelsdorf D.J. Dyck J.A. Stein R.B. Eichele G. Evans R.M. Thaller C. Cell. 1992; 68: 397-406Abstract Full Text PDF PubMed Scopus (1560) Google Scholar). RXRs are also essential heterodimeric partners for a number of other nuclear receptor signaling pathways, including thyroid hormone, vitamin D, and certain orphan receptors (15.Kliewer S.A. Umesono K. Mangelsdorf D.J. Evans R.M. Nature. 1992; 355: 446-449Crossref PubMed Scopus (1233) Google Scholar,16.Mangelsdorf D.J. Evans R.M. Cell. 1996; 83: 841-850Abstract Full Text PDF Scopus (2822) Google Scholar). Although 9-cis RA is not obligatory for transcriptional regulation via these pathways, some results suggest that RXR-specific ligands can elicit transcriptional activation in certain settings (e.g. Refs. 17.Shiohara M. Dawson M.I. Hobbs P.D. Sawai N. Higuchi T. Koike K. Komiyama A. Koeffler H.P. 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FEBS Lett. 1999; 448: 45-48Crossref PubMed Scopus (14) Google Scholar, 27.Chen Y.C. Freund R. Listerud M. Wang Z. Talmage D.A. Oncogene. 1999; 18: 139-148Crossref PubMed Scopus (19) Google Scholar, 28.Caelles C. Gonzalez-Sancho J.M. Munoz A. Genes Dev. 1997; 11: 3351-3364Crossref PubMed Scopus (289) Google Scholar, 29.Lee H.Y. Sueoka N. Hong W.K. Mangelsdorf D.J. Claret F.X. Kurie J.M. Mol. Cell. Biol. 1999; 19: 1973-1980Crossref PubMed Scopus (92) Google Scholar). Gene targeting of the various RARs has revealed essential and diverse roles for these receptors (2.Kastner P. Mark M. Chambon P. Cell. 1996; 83: 859-869Abstract Full Text PDF Scopus (935) Google Scholar, 30.Lohnes D. Mark M. Mendelsohn C. Dolle P. Decimo D. LeMeur M. Dierich A. Gorry P. Chambon P. J. Steroid Biochem. Mol. Biol. 1995; 53: 475-486Crossref PubMed Scopus (126) Google Scholar, 31.Chambon P. FASEB J. 1996; 10: 940-954Crossref PubMed Scopus (2589) Google Scholar). However, because of perinatal or embryonic lethality inherent to many of these RAR null backgrounds, there is a void in our knowledge of RAR function in a number of contexts, such as tumorigenesis. Exogenous retinoids can attenuate the effects of tumor promoters in the two stage skin carcinogenesis protocol (9.Lotan R. Semin. Cancer Biol. 1991; 2: 197-208PubMed Google Scholar, 32.De Luca L.M. FASEB J. 1991; 5: 2924-2933Crossref PubMed Scopus (813) Google Scholar). Among the retinoid receptors, normal epidermis expresses RARγ and RARαγ as well as RXRα and RXRβ, with RARγ and RXRα as the predominant heterodimer (33.Darwiche N. Celli G. Tennenbaum T. Glick A.B. Yuspa S.H. Deluca L.M. Cancer Res. 1995; 55: 2774-2782PubMed Google Scholar, 34.Fisher G.J. Voorhees J.J. FASEB J. 1996; 10: 1002-1013Crossref PubMed Scopus (349) Google Scholar). This pattern of expression prompted us to investigate the roles of RARα and RARγ in mediating the antitumorigenic effects of retinoids in epithelial keratinocytes. To this end, we established RARα, RARγ, and RARαγ null keratinocyte lines by transformation with a dominant-negative p53 expression vector and compared the properties of these various lines. Our results demonstrate that RARα and RARγ affect different aspects of retinoid response in these transformed cells, with RARγ being the primary mediator of RA-induced growth inhibition. However, other synthetic ligands affected proliferation independent of the RARs. RAR-dependent, but not -independent, growth inhibitory effects generally correlated with the attenuation of AP-1 transcriptional activity. Finally, the effects of RARα and RARγ on expression of certain keratinocyte markers suggests that each RAR may perform a subset of specific functions, which cannot be entirely fulfilled by other RARs in this cell type. The RAR null mice used in these studies have been described previously (35.Lufkin T. Lohnes D. Mark M. Dierich A. Gorry P. Gaub M.P. LeMeur M. Chambon P. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7225-7229Crossref PubMed Scopus (474) Google Scholar,36.Lohnes D. Kastner P. Dierich A. Mark M. LeMeur M. Chambon P. Cell. 1993; 73: 643-658Abstract Full Text PDF PubMed Scopus (527) Google Scholar). RARα, RARγ, and RARαγ mutants were generated from the appropriate matings, whereas wild-type offspring were obtained from RARγ+/− intercrosses. Fetuses were procured by caesarean section at 18.5 days post coitus, and genotype was determined by polymerase chain reaction as described (37.Iulianella A. Folberg A. Petkovich M. Lohnes D. Dev. Biol. 1999; 205: 33-48Crossref PubMed Scopus (80) Google Scholar). Primary keratinocyte cultures were established from the epidermis by standard means (38.Greenhalgh D.A. Welty D.J. Strickland J.E. Yuspa S.H. Mol. Carcinogen. 1989; 2: 199-207Crossref PubMed Scopus (42) Google Scholar) and cultured in S-minimal essential medium with 10% chelex-treated fetal calf serum (calcium concentration of 0.5 mm), insulin (5 μg/ml), hydrocortisone (0.5 μm), MgCl2 (1.5 mm), cholera toxin (1.2 × 10−11m), adenine (24 μg/ml), and gentimycin (10 μg/ml). The next day, the cells were fed with medium further supplemented with epidermal growth factor (10 ng/ml) and expanded for several days. Cultures were treated at 3–5 days post-plating with versene (0.5 mm EDTA in phosphate-buffered saline) to remove contaminating fibroblasts. The cells were subcultured at a 1:3 ratio at most 2 times prior to transformation. A single 10-cm plate of cells (∼2 × 106) of each genotype was harvested, and cells were resuspended in 800 μl of medium. The cells were then electroporated (250 mV, 960 microfarads in a 0.4-cm gap cuvette) with 25 μg of a linearized expression vector harboring a mutated p53 from the Friend erythroleukemia cell line CB7 (39.Johnson P. Chung S. Benchimol S. Mol. Cell. Biol. 1993; 13: 1456-1463Crossref PubMed Scopus (133) Google Scholar). Cells were plated and routinely subcultured until past crisis. All experiments were performed using cultures between passage 16 and 26. Transformed keratinocytes were seeded into 96-well plates at a cell density of 500 cells/well and were treated the following day with vehicle (Me2SO) or the appropriate retinoid (RA, 9-cis RA, 4-HPR, or TTNPB). Medium was replenished every second day. Growth was assessed either in response to varying concentrations of retinoid at eight days post-plating or over time in response to 10−6m ligand. DNA content was assessed as a measure of cell growth using crystal violet staining as described previously (40.Kueng W. Silber E. Eppenberger U. Anal. Biochem. 1989; 182: 16-19Crossref PubMed Scopus (592) Google Scholar). Relative dye binding was assessed by OD at 590 nm using a microplate reader. Results were expressed either asA 590 values or as growth relative to untreated controls and were derived from the mean (± S.D.) of four replicate wells. Transfections were performed using Lipofect ACE reagent (Life Technologies, Inc.). Briefly, cells were plated in 6-well cluster plates at 4 × 104 cells/well. Transfections consisted of 0.5 μg of AP-1 reporter or appropriate control (41.Angel P. Karin M. Matrix. 1992; 1 (suppl.): 156-164Google Scholar), either alone or with expression vectors encoding c-Fos, c-Jun, CBP, p300, or RARs. Total DNA (5 μg; normalized with KS+) was mixed with 10 μl of lipid and added to 100 μl of serum-free S-minimal essential medium. The lipid/DNA mixture was then added to the cells in 1 ml of complete medium and incubated at 37 °C overnight. Medium was changed daily, and luciferase activity was assessed 48 h post-transfection. Results were corrected for protein concentration and are expressed as the mean (± S.D.) from three independent transfections. All experiments were repeated at least three times with comparable results. Cells were cultured in 10-cm plates in the presence of RA (10−6m) or vehicle for 48 h prior to harvest. Nuclear proteins were isolated from each cell line, and protein concentration was determined using the DC protein assay kit (Bio-Rad). Electrophoretic mobility shift assays were performed essentially as before (42.Zechel C. Shen X.Q. Chen J.Y. Chen Z.P. Chambon P. Gronemeyer H. EMBO J. 1994; 13: 1425-1433Crossref PubMed Scopus (232) Google Scholar). Briefly, binding reactions containing ∼2 ng of probe (50,000 cpm) and 5 μg of nuclear protein were separated by electrophoresis through a 6% polyacrylamide gel containing 0.25 × Tris borate and EDTA. Specificity of binding was assessed by competition with a 10-fold excess of unlabeled RARE (5′-GGGTAGGGTTCACCGAAAGTTCACTCGCA) or AP-1 (5′-GATCCGATGAGTCAGCCA) double-stranded oligonucleotides. For Western blot analysis, 40 μg of nuclear protein from the various cell lines were size fractionated on a 10% SDS-polyacrylamide gel electrophoresis and electroblotted to Immobilon-P polyvinylidene difluoride membrane as recommended by the supplier (Millipore). Proteins of interest were detected by incubation with the desired antibodies and detection with an ECL kit (Amersham Pharmacia Biotech) as per the manufacturer's instructions. Antibodies were purchased from Santa Cruz Biotechnology. Fifteen micrograms of total RNA, isolated by Trizol reagent (Life Technologies, Inc.), were size fractionated on a 1% agarose-formaldehyde gel in MOPS buffer and transferred to a MAGNA nylon membrane (MSI). Fragments were isolated by restriction digestion of cDNAs followed by purification by Geneclean and used to generate probes by labeling with [α-32P]CTP by random priming with an oligo labeling kit (Amersham Pharmacia Biotech). Membranes were hybridized according to the manufacturer's directions. Primary cultures of wild-type and RAR null keratinocytes showed no major differences in morphology, growth, or immortalization with dominant-negative p53. All lines grew well for at least 40 passages, suggesting that they were immortalized. None of the lines formed colonies in soft agar or were tumorigenic in nude mice. 2P. Goyette, C. F. Chen, W. Wang, F. Seguin, and D. Lohnes, unpublished observation. Electrophoretic mobility shift assay revealed that, relative to wild-type extracts, disruption of RARα, and to a greater extent RARγ, decreased specific binding to an RARE, and association was completely abolished in extracts from RARαγ double null cultures (Fig. 1). Northern blot analysis confirmed the disruption of RARα and/or RARγ message in the appropriate cell line (data not shown). RARβ transcripts were undetectable in all lines by Northern blot or polymerase chain reaction approaches, consistent with previous studies indicating that this receptor type is not expressed in epidermal keratinocytes (33.Darwiche N. Celli G. Tennenbaum T. Glick A.B. Yuspa S.H. Deluca L.M. Cancer Res. 1995; 55: 2774-2782PubMed Google Scholar, 43.Chen L.C. Sly L. Deluca L.M. Carcinogenesis. 1994; 15: 2383-2386Crossref PubMed Scopus (26) Google Scholar). These data suggest that there is no compensatory up-regulation of the remaining receptors in response to disruption of a given RAR. All transformed cell lines exhibited similar morphology and growth characteristics in the absence of retinoid treatment (Fig. 2). However, wild-type and RARα−/− cultures were growth inhibited by 10−6m RA (Fig. 2). In marked contrast, RARγ−/− cells were highly resistant and RARαγ−/− cultures were completely resistant to these effects. The growth arrest observed in wild-type and RARα null cultures was likely because of the inhibition of proliferation as opposed to apoptosis, as judged by thymidine incorporation and programmed cell death assays (data not shown). Dose-response experiments were performed to determine the relative sensitivity of the various cell lines to growth arrest by RA, 9-cis RA, or the synthetic retinoids TTNPB or 4-HPR. As shown in Fig. 3, wild-type keratinocytes exhibited a significant reduction in proliferation at 10−9m RA, with the maximal affect at 10−7-10−6m RA. RARα−/− keratinocytes exhibited a similar profile, although their response to RA was slightly more pronounced than wild-type cultures. Consistent with time-course analysis, RARγ−/− keratinocytes were only marginally inhibited by the highest dose of RA examined (10−6m), and RARαγ−/− keratinocytes were not significantly affected by RA at any dose tested. 9-cis RA is a ligand for both RARs and RXRs, and RXR agonists have been shown to induce effects on growth or differentiation in several model systems. Proliferation of both wild-type and RARα−/− cultures was inhibited by 9-cis RA, although higher concentrations were required compared with RA (Fig. 3). Interestingly, 9-cis RA had no significant outcome on the growth of either RARγ−/− or RARαγ−/−cultures. This finding suggests that RXR activation does not lead to growth arrest in this model system, at least in the absence of RARs. Whether RXR-specific signaling has other biological consequences remains to be investigated. The RAR agonist TTNPB was a very potent inhibitor of growth in wild-type or RARα−/− cultures with an effect evident at 10−11-10−10m (Fig. 3). However, TTNPB affected RARγ−/− and RARαγ−/−cultures only at the highest dose tested (10−6m). Whether this is indicative of effects on other pathways or is because of nonspecific cytotoxicity is unknown. The synthetic retinoid 4-HPR has been shown to be a potent inducer of growth arrest and/or apoptosis in several model systems (44.Decensi A. Formelli F. Torrisi R. Depalo G. Costa A. Oncol. Rep. 1994; 1: 817-824PubMed Google Scholar, 45.Oridate N. Lotan D. Mitchell M.F. Hong W.K. Lotan R. J. Cell. Biochem. 1995; 23 (suppl.): 80-86Crossref Scopus (57) Google Scholar, 46.Wang T.T.Y. Phang J.M. Cancer Lett. 1996; 107: 65-71Crossref PubMed Scopus (41) Google Scholar). This compound was the least efficient of all those tested in inhibiting proliferation of wild-type and RARα−/− cultures (Fig.3). However, in marked contrast to the other retinoids, 4-HPR affected the growth of RARγ and RARαγ cultures at high doses, consistent with receptor-dependent and -independent mechanisms of action for this compound (47.Clifford J.L. Menter D.G. Wang M. Lotan R. Lippman S.M. Cancer Res. 1999; 59: 14-18PubMed Google Scholar, 48.Giandomenico V. Andreola F. de la Concepcion M.L.R. Collins S.J. De Luca L.M. Carcinogenesis. 1999; 20: 1133-1135Crossref PubMed Scopus (17) Google Scholar, 49.Sheikh M.S. Shao Z.M. Li X.S. Ordonez J.V. Conley B.A. Wu S.L. Dawson M.I. Han Q.X. Chao W.R. Quick T. Niles R.M. Fontana J.A. Carcinogenesis. 1995; 16: 2477-2486Crossref PubMed Scopus (134) Google Scholar, 50.Fanjul A.N. Delia D. Pierotti M.A. Rideout D. Qiu J. Pfahl M. J. Biol. Chem. 1996; 271: 22441-22446Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). RARs can repress AP-1 transcriptional activity, and this mechanism of action has been proposed to underlie at least some of the antitumorigenic effects of retinoids (8.Hong W.K. Lippman S.M. Itri L.M. Karp D.D. Lee J.S. Byers R.M. Schantz S.P. Kramer A.M. Lotan R. Peters L.J. Dimery I.W. Brown B.W. Goepfert H. N. Eng. J. Med. 1990; 323: 795-801Crossref PubMed Scopus (1193) Google Scholar, 9.Lotan R. Semin. Cancer Biol. 1991; 2: 197-208PubMed Google Scholar, 43.Chen L.C. Sly L. Deluca L.M. Carcinogenesis. 1994; 15: 2383-2386Crossref PubMed Scopus (26) Google Scholar, 51.Oridate N. Lotan D. Mitchell M.F. Hong W.K. Lotan R. Intl. J. Oncol. 1995; 7: 433-441PubMed Google Scholar). In transient transfection assays, we found that RA (10−6m) inhibited AP-1 activity 8–10-fold in wild-type and RARα−/− cultures (Fig.4 A). AP-1 activity in RARγ−/− cultures was more modestly affected, typically exhibiting 10–30% reduction, whereas activity in RARαγ−/− cultures was not affected. The latter line was capable of response following re-introduction of either RARα or RARγ by transient transfection (Fig. 4 B). Thus, attenuation of AP-1 activity requires the presence of at least one functional RAR, although there does not appear to be discrimination between receptor types for this outcome. Dose-response studies revealed a close parallel between AP-1 activity and growth inhibition mediated by all four compounds in wild-type cultures (Fig. 5). However, growth arrest induced by 4-HPR in RARγ and RARαγ mutant lines never correlated with a reduction of AP-1 activity (data not shown). This finding underscores a unique and unknown mechanism of action for this retinoid in affecting proliferation. We next determined the effect of RA on the expression of AP-1 members in wild-type and RAR null lines. Both the basal mRNA levels and RA response of several of the AP-1 members varied across the different RAR null lines. In untreated cells, c-fosexpression was comparable across all four lines, although it was slightly reduced in RARαγ cells (Fig.6). RA strongly inhibited c-fos in both wild-type and RARα−/− lines but had no effect in RARγ or RARαγ cultures. This pattern was also observed at the protein level (Fig.7). In contrast, treatment affected c-jun expression only in RARα null cultures, although basal mRNA levels varied across the lines. However, c-Jun protein did not reflect its cognate mRNA levels and was reduced by RA treatment in wild-type, RARα−/−, and RARγ−/− cultures. Phosphorylated c-Jun (P-Jun) levels paralleled those of c-Jun, suggesting that variations in phosphorylation were because of alterations in total c-Jun levels, rather than affects on JNK activity.Figure 7Effects of RAR ablation and RA treatment on c-Fos, c-Jun, and P-jun protein levels. Nuclear protein extracts (40 μg) from cells treated for 48 h with carrier (−) or 10−6m RA (+) were used to prepare a Western blot. Specific antibodies used to probe the blots are denoted to the right, and the various cell lines are noted at thetop. WT, wild type.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Fra-1 expression was barely detectable in wild-type, RARα−/−, and RARαγ−/− lines but was elevated in RARγ−/− cultures; Western blot analysis was inconclusive, as the signal was too weak to be distinguished (data not shown). junB and junD expression did not vary significantly with the exception that junB levels were slightly reduced in the RARαγ−/− line (Fig. 6 and data not shown). A number of mechanisms have been suggested to underlie retinoid repression of AP-1 activity. These include inhibition of JNK activity, which is unlikely given the observation that P-Jun levels appear to change as a function of c-Jun levels. Alternatively, the observed down-regulation of c-Fos and/or c-Jun proteins might play a role, especially if either of them are limiting. A third mechanism involves competition for limiting ancillary factors common to both RAR and AP-1 transcriptional complexes, such as p300/CBP (24.Kamei Y. Xu L. Heinzel T. Torchia J. Kurokawa R. Gloss B. Lin S.C. Heyman R.A. Rose D.W. Glass C.K. Rosenfeld M.G. Cell. 1996; 85: 403-414Abstract Full Text Full Text PDF PubMed Scopus (1917) Google Scholar). We addressed the latter two possibilities by assessing the ability of exogenous CBP, p300, c-Fos, or c-Jun to negate the effects of RA treatment on AP-1 activity. CBP or p300 transfection in wild-type cells resulted in a dose-dependent increase in AP-1 activity in the absence of RA (Fig. 8 A). Interestingly, p300 appeared to be more potent in affecting AP-1 activity, suggesting that it may be preferred over CBP in this context. Despite this increase in activity, expressing the data as fold inhibition indicated that both factors resulted in only a modest reversal of inhibition (Fig. 8 B). Overexpression of either c-Fos or c-Jun also resulted in an increase in basal AP-1 activity, again with only a marginal reduction in fold repression mediated by RA (Fig. 8). Although this rescue effect was more pronounced when both c-Jun and c-Fos were co-transfected, repression was not completely abolished (Fig. 8 B). These observations suggest that several mechanisms, including titration of limiting co-factors and inhibition of expression of AP-1 family members, act in concert in an RAR-dependent manner to attenuate AP-1 activity in these transformants. Northern blot analysis was performed to study the effect of receptor disruption on the expression levels of several genes implicated in keratinocyte growth and differentiation. The major integrin isoforms found in epidermis are integrin α2, α3, α6, β1, and β4. These are expressed in basal keratinocytes, and a decrease in integrin expression is generally correlated with differentiation and loss of proliferative potential (52.Fuchs E. Byrne C. Curr. Opin. Genet. Dev. 1994; 4: 725-736Crossref PubMed Scopus (222) Google Scholar, 53.Jones P.H. Harper S. Watt F. Cell. 1995; 80: 83-93Abstract Full Text PDF PubMed Scopus (719) Google Scholar). Northern blot analysis revealed that, with the exception of the α3 isoform, all integrins were down-regulated by RA treatment in wild-type, RARα−/−, and RARγ−/− cultures in a manner that correlated with the effects of treatment on proliferation (Fig.9). Moreover, although integrin expression was not affected by RA treatment in RARαγ cultures, basal expression of integrins α2, α3, and β4 was substantially reduced. The seemingly contradictory observation that both RA excess and RAR loss can reduce expression of several integrins is perhaps indicative of an altered differentiation state in the double mutant line. Interestingly, integrin β1 expression decreased in untreated RARα−/− cells and was up-regulated in untreated RARγ mutant cultures. Keratin expression patterns reflect the differentiation states of the various epithelial strata (52.Fuchs E. Byrne C. Curr. Opin. Genet. Dev. 1994; 4: 725-736Crossref PubMed Scopus (222) Google Scholar, 53.Jones P.H. Harper S. Watt F. Cell. 1995; 80: 83-93Abstract Full Text PDF PubMed Scopus (719) Google Scholar). K5/K14 are expressed in basal epidermal cells, whereas K1/K10 are associated with early differentiation steps and predominate in suprabasal cells. K6 and K19 are not expressed in normal epidermal keratinocytes but are often observed in situations of aberrant proliferation, such as psoriasis, wound healing, and propagation in tissue culture. With the exception of RARαγ null cultures, RA treatment repressed expression of K10 (Fig.9). This observation may be related to the fact that RA excess can inhibit keratinocyte differentiation (54.Stellmach V. Leask A. Fuchs E. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4582-4586Crossref PubMed Scopus (86) Google Scholar, 55.Kopan R. Traska G. Fuchs E. J. Cell Biol. 1987; 105: 427-440Crossref PubMed Scopus (269) Google Scholar). However, RAR disruption did not result in up-regulation of this differentiation marker, suggesting that K10 is not normally regulated by the RARs but responds to pharmacological levels of RA. RA suppressed K6 expression in wild-type, RARα−/−, and (to a lesser ex
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