Functional AP1 and CRE response elements in the human keratinocyte transglutaminase promoter mediating Whn suppression

Gene 254 (2000) 77–85 www.elsevier.com/locate/gene

Functional AP1 and CRE response elements in the human keratinocyte transglutaminase promoter mediating Whn suppression Bart A. Jessen, Qin Qin, Robert H. Rice * Department of Environmental Toxicology, University of California, One Shields Avenue, Davis, CA 95616-8588, USA Received 28 January 2000; received in revised form 16 June 2000; accepted 22 June 2000 Received by R. Di Lauro

Abstract Expression of keratinocyte transglutaminase (TGM1) is critical for maturation of mammalian epidermis and occurs during squamous metaplasia. Examination of the TGM1 5∞-flanking region in transient transfections of human epidermal cells revealed an AP1 site #1.5 kb upstream from the transcription start site and a CRE site #0.5 kb upstream that, combined, accounted for as much as 90% of the transcriptional activity. Upon incubation with nuclear extract, three electrophoretically separable protein complexes were formed by a CRE site oligonucleotide, one of which was competed by an AP1 response element. In super-shift analysis, c-Jun and JunD formed complexes with both the AP1 and CRE sequences. The AP1 and CRE sites were found to mediate the suppressive effects of the Whn transcription factor on the activity of the TGM1 promoter. Similarly, two previously described AP1 sites mediated Whn suppression of involucrin promoter activity. © 2000 Elsevier Science B.V. All rights reserved. Keywords: cJun; Involucrin; JunD

1. Introduction Transglutaminases catalyze the transamidation of the c-carboxamide group of available glutamine residues in proteins ( Folk and Chung, 1973). Among the members of this diverse enzyme family, keratinocyte transglutaminase (TGM1, previously called TGK ) is a membranebound isozyme expressed primarily in keratinocytes of the stratified squamous epithelia (Parenteau et al., 1986). TGM1 is responsible for formation of the cornified envelope in maturing cells of the epidermis by crosslinking a variety of structural proteins (Nemes and Steinert, 1999). Lack of envelope formation due to loss of TGM1 expression or function leads to defective lipid barrier function and a subtype of the rare skin disease lamellar ichthyosis (Huber et al., 1995; Russell et al., 1995). In preparation for envelope cross-linking in the Abbreviations: CRE, cAMP response element; GAPDH, glyceraldehyde phosphate dehydrogenase; hEp, normal human epidermal cells; rB, rat bladder cells; rEp, rat epidermal cells; rEs, rat esophageal cells; SIK, spontaneously immortalized keratinocytes; TGM1, keratinocyte transglutaminase. * Corresponding author. Tel.: +1-530-752-5176; fax: +1-530-752-3394. E-mail address: [email protected] (R.H. Rice)

granular layer of epidermis, expression of TGM1 is elevated in suprabasal cells in vivo and in culture, making it a valuable marker of keratinocyte differentiation ( Thacher and Rice, 1985). It also serves as a marker for squamous metaplasia in vivo (Jetten et al., 1987) and the similar reprogramming in culture of a number of rat epithelial cell types, including bladder (Parenteau et al., 1986). Keratinocytes recapitulate well their normal differentiation program under culture conditions that are optimal for growth (Green, 1979). The stratified colonies express the usual markers of keratinocyte differentiation, including high levels of keratins, TGM1, involucrin, and they readily form cornified envelopes. As shown in transgenic mice, the 5∞-flanking sequences of the TGM1 coding region required for appropriate tissue and cell type expression are contained within #2 kb of the transcription start site ( Yamada et al., 1997). Thus examination of this 2 kb promoter in culture has the potential to reveal physiologically relevant sites of transcriptional activity. In previous work, the TGM1 promoter was analyzed by transient transfection of the highly responsive rB rat bladder epithelial cell line (Mariniello et al., 1995). Initial findings pointed to an active region #500 bp

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upstream from the transcription start site. Improved transfection methods have now permitted analysis of the TGM1 5∞ flanking region in human epidermal cells and have led to the discovery of two response elements (AP1 and CRE) in the human promoter critical for expression. A preliminary account of this work has appeared (Jessen and Rice, 1998). The identification of functional promoter response elements is anticipated to help understand more complex physiological regulation. To this end, the influence of the Whn transcription factor has been investigated. Mutations in the Whn locus are associated with the nude phenotype in mice, rats, and humans (Segre et al., 1995; Frank et al., 1999) and characterized by congenital athymia and hairlessness. Whn is specifically expressed in the epithelial cells of the skin and thymus where it maintains a balance between growth and differentiation. Overexpression of Whn in mouse keratinocyte cultures transfected with reporter constructs containing TGM1 and involucrin 5∞-flanking regions showed a dramatic suppression of these promoter activities (Brissette et al., 1996). Here we present evidence that Whn suppression is mediated through the AP1 and CRE sites in the TGM1 promoter and AP1 sites in the involucrin promoter.

2. Methods and materials 2.1. Cell culture Cells employed were normal human epidermal (hEp), the SIK spontaneously immortalized human epidermal line (Rice et al., 1993) and continuous rat epithelial lines from bladder (rB), epidermis (rEp) and esophagus (rEs) (Heimann and Rice, 1983; Phillips and Rice, 1983). All keratinocytes were grown in a 3:1 mixture of Dulbecco–Vogt Eagle’s and Ham’s F12 media containing 5% fetal bovine serum, 5 mg/ml transferrin, 5 mg/ml insulin, 0.18 mM adenine, 20 pM triiodothyronine, antibiotics and 0.4 mg/ml hydrocortisone using a feeder layer of lethally irradiated mouse 3T3 cells (Allen-Hoffman and Rheinwald, 1984). SIK and hEp culture medium also contained 10 ng/ml cholera toxin and 10 ng/ml epidermal growth factor. 2.2. Constructs A l clone containing human TGM1 genomic DNA (Phillips et al., 1992) was used as a template for polymerase chain reaction to generate the largest promoter construct, designated 2.2 kb, and smaller fragments containing deletions of the 5∞ sequence and designated according to the length of the genomic sequence. All PCR fragments were subcloned into Promega pGL3 basic and pGL3 pro vectors, which contain the firefly luciferase gene. Mutagenesis of the CRE and AP1 sites

in the TGM1 promoter was performed by generating overlapping PCR fragments with mutations in the 5∞ sequence of the primers. The CRE (5∞-TGATGTCA-3∞) was mutated to an EcoR1 site (5∞-AGAATTCG-3∞) and the AP1 site (5∞-TGACTCA-3∞) was mutated to a Nhe1 site (5∞-GCTAGCA-3∞), permitting rejoining of the PCR fragments. The CRE–AP1 double mutant was constructed by generating point mutations (5∞-TGACTCA-3∞ was changed to 5∞-TGAGCCA-3∞) in the AP1 site of the CRE mutant construct using the Stratagene Pfu PCR mutagenesis kit. A pRLCMV plasmid (Promega) containing the cytomegalovirus promoter driving transcription of the renilla luciferase gene was used to normalize for transfection efficiency. Large-scale plasmid preparations were purified by Qiagen plasmid kits or CsCl gradients. The expression construct containing mouse Whn cDNA in pcDNA 3 (Invitrogen) was kindly provided by Dr Janice Brissette (Brissette et al., 1996). The 2.4 kb involucrin promoter construct, used previously ( Kachinskas et al., 1997), was obtained from Dr Howard Green. 2.3. Transfections Transfections of TGM1 promoter constructs were performed using calcium phosphate co-precipitation. In most experiments, 10 mg (#2.5 pmol ) of pGL3 were co-transfected with 0.1 mg of pRLCMV per 6 cm culture. To optimize precipitate formation, pGL3Basic was added to the transfections so that each contained 20 mg of DNA. Rat cells were transfected at 30% confluence, while human cells were transfected at 70% confluence. Cultures were harvested between 2 and 5 days after transfection, and firefly and renilla luciferase activities were assayed using a commercial dual luciferase assay kit (Promega). In co-transfections with the Whn expression vector, typically 3 mg of the latter were employed. At this level an equal amount of the empty pcDNA3 vector, added in the parallel comparison samples lacking Whn in each experiment, had little effect on measured transcription. The co-transfections also included pRLCMV for normalization. 2.4. DNase I protection In each reaction, 10 pmol of 32P end-labeled TGM1 restriction fragment DNA were incubated with 100 mg of nuclear extract (Chodosh, 1993) in binding buffer [12.5% glycerol, 12.25 mM Hepes (pH 7.9), 0.01% Nonidet P-40, 0.1 mM EDTA, 1 mM dithiothreitol ] and 5 mg poly(dI-dC ) for 1 h on ice. DNase I digestion was carried out (Leblanc and Moss, 1994) using 0.02–2 units of DNase I (Sigma Chemical Co.). Control reactions lacking nuclear extract used 0.002–0.005 units of DNase I and 1 mg of poly(dI-dC ). The aqueous fraction of the phenol/chloroform extracted samples was ethanol precipitated and subjected to electrophoresis through a 6%

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polyacrylamide gel containing 8.5 M urea. A 32P-end labeled 10 bp ladder (GibcoBRL) was used as a size marker. The dried gels were subjected to autoradiography. 2.5. Electrophoretic mobility shift assays For each reaction, 10 mg of keratinocyte nuclear extract was incubated for 10 min at room temperature in binding buffer containing 150±20 mM final concentration NaCl and 1 mg of poly(dI-dC ). A 100 fold molar excess of unlabeled double-stranded oligonucleotide was subsequently added as a competitor where appropriate, and incubated for an additional 10 min at room temperature. Subsequently 50 fmol of 32P-radiolabeled oligonucleotides were added and incubated for 10 min at room temperature. In electrophoretic mobility super-shift assays, 1 mg of the appropriate antibody [cJun (H-79), JunB (N-17-G), JunD (329-G), cFos (4-10G), Fra-1 (R-20), Fra-2 (L-15), ATF-1 ( F1-1), CREB-1 (C-21), and CREM-1 ( X-12), all from Santa Cruz Biotechnology] was added, and the reaction incubated on ice for an additional 1 h. Complexes were separated by electrophoresis through a 4% non-denaturing polyacrylamide gel in a buffer containing 50 mM Tris base, 380 mM glycine and 2 mM EDTA. The dried gels were then phosphorimaged. The sequences of the oligonucleotides used, except for the commercial consensus CRE (5∞-GAGCTCTCTGACGTCAGCCAATC-3∞), contain the sequences shown as protected in Fig. 2A, B and flanking promoter sequence. 2.6. Partial purification of nuclear proteins Nuclear extracts were diluted 1:4 with binding buffer so that the final KCl concentration was 0.1 M. Extracts were then added to a 5 ml column containing affi-gel heparin (BioRad ) that had been equilibrated with binding buffer. After washing the column with 10 ml of binding buffer containing 0.1 M NaCl, nuclear proteins were eluted by a step gradient of NaCl (0.2–1.2 M ) in binding buffer. Fractions (1 ml ) were collected and 2 ml of each was assayed by mobility shift for the presence of AP1 and CRE complexes. The NaCl concentration of each fraction was determined by conductivity.

3. Results 3.1. Functional mapping of the TGM1 promoter TGM1 promoter 5∞ deletions were tested for their ability to stimulate luciferase expression in transient transfection experiments in human epidermal cells (the spontaneously immortalized SIK line) to identify sequences involved in transcriptional regulation. As shown in Fig. 1, the most active construct contained

Fig. 1. Luciferase activities of constructs containing TGM1 5∞-flanking DNA transfected into SIK cultures. Shown are the activities generated by a series of constructs containing the designated lengths of flanking DNA (extending from −30) positioned upstream of the firefly luciferase reporter gene in the pGLpro vector and normalized to pRLCMV (renilla). The highest activity observed, given by the −1.6 kb construct, was normalized to 100. The means of three to eight transfections± standard deviations with each construct are shown.

1.6 kb of the 5∞ flanking DNA, and the greatest drop in activity occurred between −1.6 and −1.5 kb. In fact, constructs of 1.5 kb and smaller displayed little more activity than the empty vector. The present profile stands in considerable contrast to previous observations from this laboratory using the rB line of rat bladder epithelial cells, where the longest upstream region (2.2 kb) had the greatest activity, and promoter activity generally decreased with decreasing length of the 5∞ flanking region (Mariniello et al., 1995). 3.2. DNase I footprinting of active TGM1 promoter regions Regions endowing the promoter with marked transcriptional activity were considered likely to contain response elements for binding of regulatory proteins that could be identified by footprinting. Thus, footprinting of the TGM1 5∞ flanking region between −1.6 and −1.3 kb with SIK nuclear extract revealed a protected area spanning −1531 to −1504 bp. As illustrated in Fig. 2A, this area contained a consensus AP1 site (5∞-TGACTCA-3∞) between −1520 and −1514 bp. In the original survey of transcriptional activity in rB cells, a larger drop in activity was found between −540 and −390 bp than over any similar sized region of the promoter. DNase I protection of the region between −540 and −315 bp with rB nuclear extract (Fig. 2B) now revealed the binding of a protein between −482 and −463 bp. Analysis of the sequence of this region revealed a near consensus CRE between −462 and −453 bp. The sequence, 5∞-TGATGTCA-3∞, differs from

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Fig. 2. DNase I protection of TGM1 5∞-flanking regions and complex formation by nuclear proteins. DNase I protection assays were performed on transcriptionally active regions of the DNA in the presence (+) or absence (−) of 0.1 mg of nuclear proteins. (A) SIK nuclear extract (NE) was incubated with 32P end-labeled DNA spanning the region from −1.6 to −1.3 kb. The protected sequence, shown to the right, contains a consensus AP1 site (boxed ). (B) rB nuclear extract (NE ) was incubated with 32P end-labeled DNA spanning from −540 to − 315 bp in the promoter. The protected sequence contains a near consensus CRE (boxed ), from which it differs by a single base. 32P-labeled synthetic oligonucleotides corresponding to the protected CRE ( left) and AP1 (right) regions in the TGM1 promoter were incubated with nuclear extracts from SIK (C ) and rB (D) cultures in the presence of various unlabeled competitor oligonucleotides (100-fold molar excess). The competitors (CO) are none (−), the CRE sequence taken from the TGM1 promoter ( TC ), the AP1 sequence taken from the TGM1 promoter, and a commercial consensus CRE (CC ). To the left of the CRE lanes in (C, D) is an enlarged image showing three complexes (marked by arrows) formed with the CRE oligomer in the absence of competitor. Note that all three were competed with both CRE oligomers, but only one was competed by the AP1 oligomer. On the right edge of (C, D), an arrow marks the complex formed with the AP1 oligomer, which is competed by all three competitors.

the consensus 5∞-TGACGTCA-3∞ by a single base. The same sequence was also protected in DNase I assays using SIK nuclear extracts (not shown). 3.3. Electrophoretic mobility shift of AP1 and CRE sequences Oligomers of 30 bp containing either the TGM1 CRE or AP1 sites and flanking sequence from the promoter

were labeled with 32P and used as probes in mobility shift assays. Nuclear extract proteins (10 mg) from SIK or rB cultures were incubated with the labeled probes in the presence or absence of a 100-fold molar excess of the TGM1 CRE and AP1 site oligomers or a commercially available consensus CRE oligonucleotide. As shown in Fig. 2C, D, results from the mobility shift assays from the two nuclear extracts were similar. Three self-competing complexes were formed with the CRE

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oligomer. All three bands were similarly competed by both the TGM1 CRE sequence and the consensus CRE oligomer. The AP1 oligomer competed effectively for the middle band produced in the CRE shift assay. The labeled AP1 probe produced only one clear specific complex in the mobility shift assay. Formation of this complex was prevented to the greatest extent by selfcompetition, but both the TGM1 and consensus CRE oligomers competed well for AP1 binding. These results suggested that proteins binding the middle CRE complex were also capable of binding the AP1 site, while those in the upper and lower CRE complexes were not. 3.4. Chromatographic separation of CRE and AP1 binding complexes Heparin column chromatography of SIK and rB nuclear extracts was used to characterize further the CRE and AP1 binding complexes. After binding to the column, these complexes were eluted with a NaCl step gradient, and fractions were assayed for their ability to shift CRE and AP1 labeled oligomers. The elution profiles, illustrated in Fig. 3, showed that two AP1 binding activities were separable from SIK nuclear extracts (bottom panel ). The peak activities coincided with two peaks of CRE binding activity. In rB nuclear extracts, three peaks of AP1-binding activity were evident (top panel ). The material in the second and third

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of these peaks co-eluted with substantial CRE binding activity, but the material in the first peak contained little CRE binding activity. Despite the separations achieved, the three protein complexes seen in the mobility shift experiments with the CRE oligonucleotide were not resolved by the chromatography, nor were electrophoretic differences in the chromatographically separable AP1 complexes evident. 3.5. Electrophoretic mobility super-shift analysis To identify proteins involved in the CRE and AP1 binding complexes, super-shift assays were performed using antibodies to Jun and Fos family members. As seen in Fig. 4, antibodies to the Jun family of transcription factors super-shifted AP1 and CRE complexes from both SIK and rB nuclear extracts. c-Jun was clearly evident in AP1 and CRE complexes from SIK extracts, and JunD was prevalent in these complexes from both SIK and rB extracts. At most, only traces of c-Jun were present in the complexes from rB cells and of JunB in complexes from either cell extract. The only clearly super-shifted complex obtained with antibodies to the Fos family of transcription factors was that using Fra-1 antiserum with the AP1 oligonucleotide and rB extract. The presence of Fra-1 in AP1 complexes from rB but not SIK extracts could explain the additional rB AP1 activity found by the heparin fractionation. Antibodies to ATF-1, CREB-1, and CREM-1 did not give supershifted bands with the CRE oligonucleotide (data not shown). 3.6. Functional significance of AP1 and CRE sites in the TGM1 promoter

Fig. 3. Heparin chromatography of nuclear proteins isolated from rB and SIK cultures. Nuclear extract protein (1 mg) isolated from rB or SIK cultures was eluted by an NaCl step gradient from a heparin column. Fractions were assayed for ability to form complexes with 32P-labeled CRE (solid circles) and AP1 (open circles) oligomers.

Mutation of either the AP1 or CRE site or mutation of both sites resulted in substantially decreased promoter activity in transient transfection assays. For this purpose, the CRE sequence was changed from 5∞-TGATGTCA-3∞ to 5∞-AGAATTCG-3∞, while the AP1 site was changed from 5∞-TGACTCA-3∞ to 5∞-GCTAGCA-3∞. The mutated constructs were tested in several cell types, and the results were expressed as percent of wild-type fulllength TGM1 promoter activity (Fig. 5A). Two of the rat lines, rEp and rEs, and normal human epidermal cells showed #50% decrease in promoter activity due to mutation of either site. The human SIK line showed a similar decrease in activity from mutation of the CRE but a marginally greater effect (70% decrease) in response to the AP1 mutation. The rB line was only slightly affected (20% decrease) by the AP1 mutation. A critical finding, however, was that these cell lines all lost between 70 and 90% of their transcriptional activity in the double mutants, indicative of cooperation between the sites.

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Fig. 4. Identification of transcription factors binding to AP1 and CRE sites. Complexes between 32P-labeled oligomers (AP1 left and center, CRE right) and nuclear extracts from SIK (A) and rB (B) cultures were incubated with various antibodies. The antibodies used were none (−), c-Jun, JunB, JunD, c-Fos, Fra-1 and Fra-2. The complexes formed between the oligomers and nuclear proteins are indicated by brackets, while the supershifted complexes are marked by arrows.

3.7. AP1 and CRE mediation of transcriptional suppression by Whn Expression of the Whn transcription factor in mouse epidermal cultures suppressed the transcriptional activities of transiently transfected human TGM1 and involucrin promoters (Brissette et al., 1996). The present experiments investigated the importance of functional AP1 and CRE elements in mediating this phenomenon. As shown in Fig. 5B, transfection of a mouse Whn expression vector into SIK cultures greatly reduced the activity of the cotransfected TGM1 promoter. In constructs where either the AP1 or the CRE site was inactivated by mutation, Whn still produced suppression. The double mutant had much reduced activity compared with the native sequence, but the cotransfected Whn produced no further suppression. A similar phenomenon was observed using the involucrin promoter. Mutation of either the proximal or the distal AP1 sites known to be functional in the involucrin

promoter ( Welter et al., 1995; Kachinskas et al., 1997) did not prevent Whn from reducing the activity still further, but the activity of the double mutant was not significantly reduced. In transfected hEp, Whn also suppressed transcription from the promoters with the native sequence and with singly inactivated response elements essentially to the same extent as in SIK cultures, but again the double mutants were not affected (not shown). In all cases, activities of the double mutants were low but readily measurable, so that further reduction in reporter transcription would have been easily detected.

4. Discussion Studies in transgenic mice have shown that 2.5 kb of the human and 2.9 kb of the rabbit 5∞-flanking sequence are sufficient to give proper tissue-specific expression of the TGM1 coding region ( Yamada et al., 1997;

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Fig. 5. Effects of mutations in the CRE and AP1 sites and Whn expression on TGM1 promoter activity. (A) Constructs containing 2.2 kb of the TGM1 5∞-flanking DNA in the pGL vector and bearing mutations in either the AP1 or CRE sites or a combination of both were used to transfect various rat and human cells. Firefly luciferase activities were normalized to renilla luciferase activities, and shown as percent of wild-type (unmutated) activities measured in parallel. Each value represents the mean and standard deviation of three samples. (B) An expression vector containing mouse Whn cDNA (black bars) or the empty vector (clear bars) was co-transfected with either a TGM1 (top) or involucrin (bottom) reporter construct and pRLCMV for normalization. Constructs with the native promoter sequence or with mutations in AP1 or CRE sites (or both) are indicated. The involucrin AP1 sites are numbered as in ( Welter et al., 1995). Values represent the mean and standard error of at least three samples.

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Medvedev et al., 1999). This finding indicates that transient transfections using the upstream region have the potential to reveal important regulatory elements. In a previous study, the 2.2 kb human flanking sequence had little transcriptional activity in human keratinocytes, but was quite active in rat bladder epithelial cells (Mariniello et al., 1995). Initial study of the promoter in these rB cells permitted identification of a transcriptionally important region #0.5 kb upstream from the transcription start site. Although not testable at the time, this region was considered likely to be important in human epidermal cells because rB cultures adopt a keratinocyte-like phenotype (Phillips and Rice, 1983), but uncertain because rat bladder epithelium in vivo does not ordinarily express TGM1 (Parenteau et al., 1986). Results now demonstrate that human epidermal cells and rat lines derived from keratinizing epithelia (epidermal, esophageal ) exhibit transcriptional dependence on a CRE in the region identified. A careful study of the rabbit TGM1 promoter, which appeared as this work was being prepared for publication, has also shown this CRE to be important in rabbit tracheobronchial epithelial and human epidermal cells (Medvedev et al., 1999). Finding the extent to which this site mediates TGM1 expression during the reprogramming in culture of nonkeratinizing rat epithelial cell types (Parenteau et al., 1986) will be of considerable interest. Additionally, the present study has demonstrated that a distal AP1 element is critical for high-level expression of the TGM1 promoter in human keratinocytes. Mutagenesis of individual sequence elements revealed the AP1 site to be important in rEp and rEs cultures, but much less critical in rB cultures, where the response is dominated by the CRE. Dominance by the CRE may also occur in rabbit tracheobronchial cells, since the transcriptional response in these cells showed considerably less dependence upon sequences further upstream than in human epidermal cells (Medvedev et al., 1999). The distal promoter beyond −819 has also been stated to confer little responsiveness in a line of fetal rat keratinocytes ( Ueda et al., 1996), probably for the same reason. The dependence of expression on a distal AP1 and a more proximal CRE site in human epidermal cells resembles superficially the situation with the involucrin promoter, where a distal and a proximal AP1 site are both important ( Welter et al., 1995). Just as the distal promoter region, containing a functional AP1 element, is known to be important for proper regulation of involucrin expression in vivo (Crish et al., 1998), the distal promoter of TGM1 is likely to be important as well. One indication of that is the sensitivity of distal regions in both promoters to transcriptional suppression by Whn. Loss of Whn function, responsible for the hairless phenotype in nude mice (Segre et al., 1995), results in abnormal development (premature or excessive differentiation) of hair, skin and

thymus and dramatic sensitization of keratinocytes from nude mice to phorbol ester stimulation of differentiation. The sensitization could result in part from Whn suppression of transcriptional stimulation by AP1 sites, which present results demonstrate. Thus localization of Whn primarily in the first suprabasal layer of mouse skin (Lee et al., 1999) could account for involucrin and TGM1 being expressed only more superficially, where Whn is lacking. Paradoxically, overexpression of Whn in the suprabasal layers (driven by the involucrin promoter) does not suppress involucrin expression (Prowse et al., 1999). However, similar to findings in human skin treated with retinoic acid ( Rosenthal et al., 1992), where involucrin and transglutaminase appear elevated in contrast to their suppression by retinoids in culture, the resulting hyperplasia of the epidermis may dominate the outcome. The observation that AP1 elements function in expression of genes for several keratinocyte structural proteins has led to studies of transcription factors that bind to this element in keratinocytes. The present results, showing that JunD and to a lesser extent c-Jun are active in band shift assays using nuclear extracts, are consistent with immunohistochemical studies showing that these and other members of the Jun and Fos families are present in the upper spinous and granular layers of human epidermis ( Welter and Eckert, 1995) where TGM1 is expressed. Most striking is the present finding of an AP1-like factor that binds to the TGM1 CRE, evidence for cross-talk between the sites. However, making correlations between transcription factor protein levels and their functional activity and, particularly, reaching definite conclusions regarding which factors drive keratinocyte differentiation have proven difficult. In part this reflects the numerous combinations of bZIP proteins that are now known to heterodimerize and bind to AP1 or CRE elements. In addition to the Jun and Fos family members are ATF, Maf, CREB and CREM and their isoforms, some combinations of which are negative regulators ( Karin et al., 1997; Rutberg et al., 1999). Nevertheless, tracing the signal transduction pathway leading to transcriptional activation through these sites will be of great interest. Beyond super-shift assays, which provide limited information, physical identification of the transcription factors actually bound to the response elements will likely prove illuminating. This will lay the groundwork for understanding interactions of factors such as Whn in complex physiological regulation.

Acknowledgements We thank Dr M.A. Phillips for valuable advice and discussion. This research was supported by USPHS Grants AR27130, ES04699, ES05707 and ES07059.

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