Retinoids exert their biologic effects through two families of nuclear receptors, retinoic acid receptors (RARs) and retinoid X receptors (RXRs), which belong to the superfamily of steroid/thyroid hormone nuclear receptors. By using a subtraction hybridization approach, we have identified a cDNA sequence TIG2 (Tazarotene-induced gene 2), whose expression is up-regulated by the treatment of skin raft cultures by an RAR beta/gamma-selective anti-psoriatic synthetic retinoid tazarotene [AGN 190168/ethyl 6-[2-(4,4-dimethylthiochroman-6-yl)-ethynyl] nicotinate]. The retinoid-mediated up-regulation in the expression of TIG2 was confirmed by Northern blot analysis. Upon sequencing, TIG2 was found to be a cDNA whose complete sequence was not in the GenBank and EMBL data bases. The TIG2 cDNA is 830 bp long and encodes a putative protein product of 164 amino acids. TIG2 is neither expressed nor induced by tazarotene in primary keratinocyte and fibroblast cultures. Thus, TIG2 is expressed and induced by tazarotene only when keratinocytes and fibroblasts form a tissue-like 3-dimensional structure. We further demonstrate that RAR-specific retinoids increase TIG2 mRNA levels. In contrast, neither RXR-specific retinoids nor 1,25-dihydroxyvitamin D3 increased TIG2 levels. Finally, we demonstrate that TIG2 is expressed at high levels in nonlesional psoriatic skin but at lower levels in the psoriatic lesion and that its expression is up-regulated in psoriatic lesions after topical application of tazarotene.
Retinoids, synthetic and natural analogs of retinoic acid, exhibit potent growth inhibitory and cell differentiation activities that account for their beneficial effects in treating hyperproliferative diseases such as psoriasis, actinic keratosis, and certain neoplasias. Tazarotene is a synthetic retinoid that is used in the clinic for the treatment of psoriasis. To better understand the mechanism of retinoid action in the treatment of hyperproliferative diseases, we used a long-range differential display-PCR to isolate retinoid-responsive genes from primary human keratinocytes. We have identified a cDNA, tazarotene-induced gene 3 (TIG3; Retinoic Acid Receptor Responder 3) showing significant homology to the class II tumor suppressor gene, H-rev 107. Tazarotene treatment increases TIG3 expression in primary human keratinocytes and in vivo in psoriatic lesions. Increased TIG3 expression is correlated with decreased proliferation. TIG3 is expressed in a number of tissues, and expression is reduced in cancer cell lines and some primary tumors. In breast cancer cell lines, retinoid-dependent TIG3 induction is observed in lines that are growth suppressed by retinoids but not in nonresponsive lines. Transient over-expression of TIG3 in T47D or Chinese hamster ovary cells inhibits colony expansion. Finally, studies in 293 cells expressing TIG3 linked to an inducible promoter demonstrated decreased proliferation with increased TIG3 levels. These studies suggest that TIG3 may be a growth regulator that mediates some of the growth suppressive effects of retinoids.Retinoic acid (RA) and its synthetic analogs are therapeutically effective in the treatment of hyperproliferative dermatological diseases, such as psoriasis and cutaneous neoplasias, and in certain cancers (1-6). The biological effects of retinoids are mediated through two families of nuclear receptors, retinoic acid receptor (RAR␣, -, and -␥) and retinoid X receptor (receptors ␣, , and ␥) (6, 7). RARs and retinoid X receptors are ligand-dependent transcription factors that, as heterodimers, function by changing the expression of RAresponsive genes (5-8). Tazarotene is an RAR͞␥-selective synthetic retinoid that is used clinically for the treatment of psoriasis, a skin disorder characterized by epidermal hyperproliferation and inflammation (9). Despite the therapeutic efficacy of retinoids in various hyperproliferative dermatological diseases, the molecular basis of their action in skin is largely unknown. Various genes whose expression is induced in cell culture systems by retinoids have been described. Of these, cellular retinoic acid binding protein II and tazaroteneinduced genes (TIG) 1 and 2 are the only markers whose expression is induced in a retinoid-dependent manner in skin (10-12). The increased levels of cellular retinoic acid binding protein II in psoriatic plaques indicate that it may not be a useful efficacy marker of retinoid action in psoriatic lesions. In addition, several lines of evidence suggests that cellular retinoic acid binding p...
Loricrin gene expression is limited to terminally differentiating keratinocytes of stratified squamous epithelia. To define the regulatory elements that mediate the expression of the loricrin gene, we replaced the loricrin coding sequences from a 6.5-kilobase genomic fragment with the chloramphenicol acetyltransferase gene and transfected this construct into cultured mouse keratinocytes. High expression levels were observed in both undifferentiated as well as differentiating cells. Transgenic mice bearing a similar construct, but with beta-galactosidase as the reporter gene, corroborated these in vitro findings and showed tissue- and cell type-specific, but not differentiation-specific expression. Deletion analysis of the promoter region determined that sequences up to -60 base pairs from the start of transcription could be removed without significant loss of promoter activity. Within these proximal 60 base pairs is an evolutionarily conserved AP-1 element that is recognized by both purified c-Jun and AP-1 factors from keratinocytes in vitro. Mutation of this AP-1 site abolished the activity of the loricrin promoter. These studies show that elements directing expression of the loricrin gene to the stratified squamous epithelia are contained within a 6.5-kilobase genomic fragment, and those elements required to restrict expression to differentiated keratinocytes lie outside this region.
Targeted recruitment of histone acetyltransferase (HAT) activities by sequence-specific transcription factors, including the retinoic acid receptors (RARs) and retinoid X receptors (RXRs), has been proposed to lead to destabilization of nucleosomal cores by acetylation of core histones. However, biochemical evidence indicates that destabilization and depletion of linker H1 histones must also occur at the promoter regions of actively transcribing genes. Mechanisms by which nuclear receptors and other transcription factors affect the removal of histone H1 from transcriptionally silent chromatin have not been previously described. In this report, we show that RARs interact in a ligand-dependent manner with HMG-I, which is known to displace histone H1 from chromatin. We further show that HMG-I and a novel related protein, HMG-R, also interact with other transcription factors. Using sense and antisense constructs of HMG-I/R in transient transfection assays with a retinoid responsive reporter, we also demonstrate that HMG-I/R is important for retinoid dependent transcriptional activity of RAR. These findings suggest a step wise mechanism by which RARs and other transcription factors can cause a targeted unfolding of compact chromatin as a first step in transcriptional activation, which would then be followed by recruitment of HAT activity and subsequent events.Retinoic acid receptors and retinoid X receptors (RAR and RXR ␣, , and ␥) are sequence-specific, ligand-dependent transcription factors belonging to the superfamily of steroid/thyroid/vitamin D 3 nuclear receptors (1). RAR-RXR 1 heterodimers induce gene expression in a ligand dependent manner through RA responsive elements (RAREs) present in the promoter regions of responsive genes (2). Recently, CBP/p300, Sug1/Trip1, TIF1, SRC-1/N-CoA1, TIF2/GRIP1, and ACTR have been identified as co-factors, which interact with RARs and other nuclear receptors in a ligand-dependent manner (3, 4). Biochemical evidence supports models involving depletion of the nucleosomal core as well as H1 histones at the promoter regions of actively transcribing genes. CBP/p300, its associated protein p/CAF, SRC-1, and ACTR have intrinsic histone acetyltransferase (HAT) activity (5-7). It has been proposed that recruitment of HAT activity by sequence-specific transcription factors leads to acetylation of core histones and a destabilization of the nucleosomal core, thereby facilitating transcriptional activation (8 -10). However, an earlier obligatory step in transcriptional activation involves an unfolding of the compact, 30-nm chromatin fiber, which results only from a displacement of the potent transcriptional repressor, histone H1, from chromatin (11, 12). Mechanisms by which nuclear receptors and other transcription factors affect this obligatory removal of histone H1 from transcriptionally silent chromatin have not been described previously. In this report, we provide evidence that RARs interact in a ligand-dependent manner with HMG-I, which is known to displace histone H1 from chromati...
ABSTRACTsplice-junction mutation, an inner set of primers, TS12F and TS12R, was used for secondary PCR. Thermocycling conditions were similar to those used in the secondary PCR for exon 11, except that primer annealing occurred at 60TC. Four microliters of a solution containing 5x buffer (500 mM NaCl/250 mM Tris.HCl/50 mM MgCl2/5 mM dithiothreitol, pH 7.9) and 20-25 units ofDde I restriction endonuclease was added to 16 p1 of amplified 176-bp product, with digestion at 370C for 90 min and electrophoresis in a 10%o polyacrylamide minigel (Bio-Rad) ZFX and ZFY (11), using 2 p1 of PEP product for the primary PCRs. The CFTR AF508 locus was analyzed by Abbreviation: PEP, primer extension preamplification.
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