©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Cloning and Regulation of Cornifin , a New Member of the Cornifin/spr Family
SUPPRESSION BY RETINOIC ACID RECEPTOR-SELECTIVE RETINOIDS (*)

(Received for publication, August 2, 1995; and in revised form, November 17, 1995)

Stephen J. Austin Wataru Fujimoto (§) Keith W. Marvin Thomas M. Vollberg (¶) Laslo Lorand (**) Anton M. Jetten (§§)

From the Cell Biology Section, Laboratory of Pulmonary Pathobiology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

In this study, we describe the isolation and characterization of a cDNA clone C12 that encodes a new member of the cornifin/small proline-rich protein (spr) family, which we have named cornifin beta. C12 encodes a 1.1-kilobase pair mRNA and a 24.3-kDa cytosolic protein with a high proline content (19%). Its total amino acid sequence exhibits a 37-66% identity while the first 30 amino acids at the amino terminus are 87% identical to that of members of the cornifin family. At its carboxyl terminus, cornifin beta contains 21 tandem repeats of an octapeptide. Cornifin beta expression is restricted to several squamous epithelia. It is highly expressed in esophagus, tongue, and oral mucosa but, in contrast to cornifin alpha, is not detectable in the epidermis. Both retinoic acid and a retinoid selective for the nuclear retinoic acid receptors were very potent suppressors of cornifin beta expression while an analog selective for the nuclear retinoid X receptors was much less effective, suggesting that a specific retinoid signaling pathway is involved in this suppression. Cornifin beta can function through some of its Gln residues as an amine acceptor in transglutaminase-catalyzed cross-linking reactions. These results indicate that cornifin beta functions as a cross-linked envelope precursor.


INTRODUCTION

Squamous differentiation is a multistage process that is accompanied by irreversible growth arrest and expression of squamous cell-specific genes(1, 2, 3, 4) . The formation of the cross-linked envelope is a characteristic feature of squamous differentiation in many tissues (5, 6) . This structure consists of a layer of covalently cross-linked protein deposited just beneath the plasma membrane(6, 7, 8, 9) . These linkages are catalyzed by transglutaminases, enzymes that carry out the formation of -(-glutamyl)lysine bonds between precursor proteins (7, 8, 9, 10, 11, 12) . The formation of the cross-linked envelope is believed to occur in several stages and to involve multiple membrane-associated and cytosolic precursor proteins, including involucrin, cornifin/small proline-rich protein (spr), (^1)and loricrin (11) . The first described envelope precursor, involucrin, is a glutamine-rich protein induced early during squamous differentiation (9, 13, 14) . Loricrin, a glycine-rich precursor protein, is induced at later stages of differentiation than involucrin and cornifin and appears to be the major constituent of the mature cross-linked envelope (15, 16) . Cornifins and sprs are a family of related envelope precursor proteins(17, 18, 19, 20, 21, 22) which contain a highly repeated octapeptide (nonapeptide for spr2) at their carboxyl terminus and a high percentage of proline. Cornifin alpha has been reported to be an excellent substrate for epidermal (type I) transglutaminase and has also been shown to be assembled into the cross-linked envelope(20) .

In this study, we describe the characterization of C12, a cDNA clone isolated from a library prepared from poly(A) RNA of squamous-differentiated rabbit tracheal epithelial (RbTE) cells. This clone represents an mRNA that is present at high abundance in squamous-differentiated cells but not in undifferentiated RbTE cells. Based on its amino acid sequence homology with the previously described cornifin/sprs, C12 is a new member of the cornifin/spr family(17, 18, 19, 20, 21, 22) . We have named the previously described cornifin (SQ37)(20) , cornifin alpha, and the protein encoded by C12, cornifin beta. We show that cornifin beta functions as a substrate for transglutaminase type I indicating that it can also function as a cross-linked envelope precursor. The fact that the sequence of the tandem repeats and pattern of tissue-specific expression are different between the two cornifins may suggest distinct roles for specific cornifins, perhaps in determining the physical properties of the cross-linked envelope.


EXPERIMENTAL PROCEDURES

Cell Culture and Materials

RbTE cells were isolated and cultured as described previously(11, 23) . Retinoic acid (RA) and the calcium ionophore Ro 2-2985 were obtained from Hoffmann-La Roche. The RAR-selective retinoid SRI-6751-84, RXR-selective retinoid SRI11217, and the analog SR11302, which does not activate transactivation by RARs or RXRs but exhibits anti-AP1 activity, were provided by Dr. Marcia Dawson, SRI International, Menlo Park, CA(24, 25) .

Differential Screening of cDNA Library

A cDNA library was constructed in the vector Zap using poly(A) RNA from squamous-differentiated RbTE cells(12) . The library was screened using P-labeled cDNAs synthesized from poly(A) RNA isolated from undifferentiated and differentiated RbTE cells as reported previously(17) . One of the differentially expressed clones, C12, was used for further analysis. The clone C12-3 was isolated after screening the cDNA library with the labeled insert of C12.

Sequencing

Sequencing of C12 was carried out in both directions by the dideoxynucleotide chain termination method with a Sequenase kit (U. S. Biochemical Corp.)(26) . The DNA and deduced protein sequences were analyzed by the GCG sequence analysis software package(27) .

Generation of Antiserum and Immunoblot Analysis

The peptide PQPGYTKVPESGCTSVPGSGYSVI (C12-PEPB) was cross-linked to bovine serum albumin with maleimide using a cross-linking kit from Pierce. Immunization of New Zealand White rabbits was carried out as described elsewhere(28) . Immunoblot analysis was performed as described using an ECL kit (Amersham Corp.)(21) .

Immunohistochemical Staining

Sections from rabbit tissue specimens were prepared and analyzed with C12-PEPB or SQ37B antisera as described previously using biotinylated goat-anti-rabbit IgG (Jackson Laboratories) and streptavidin-horseradish peroxidase(20, 21) . Involucrin was detected using the involucrin immuno-kit from Biomedical Technologies (Stoughton, MA)

Transglutaminase Catalyzed Cross-linking

Total cell extracts were prepared in 50 mM Tris-HCl (pH 8.0) containing 2 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and leupeptin and aprotinin (1 µg/ml each) and incubated at 37 °C with and without 1 mM dansylcadaverine(20, 29) . The transglutaminase catalyzed cross-linking was initiated by the addition of CaCl(2) (10 mM final concentration). The reaction was stopped by the addition of 10 mM EDTA. Dansylated proteins were analyzed by Western blotting using a rabbit anti-dansyl polyclonal antiserum E7(29) .

Northern Blot Analysis

Samples of total RNA (30 µg) were separated on a 1.2% agarose-formaldehyde gel, and transferred to Nytran maximum strength membrane (Schleicher & Schuell) (20) . Northern blots were probed either with the EcoRI-excised, 0.9-kb insert of C12, HindII/EcoRI-excised, 0.71-kb insert of SQ37 encoding cornifin alpha, or with a probe (pGAD28) encoding chicken glyceraldehyde-3-phosphate dehydrogenase(20, 21) . Probes were labeled with [alpha-P]dCTP (3000 Ci/mmol; Amersham Corp.) using a random priming kit (Stratagene). Following hybridization (1-4 h at 68 °C) in Quikhyb (Stratagene), blots were washed (65 °C and 0.5 times SSC for 30 min) as described previously (20) .

In Situ Hybridization

Biopsy specimens of rabbit lip, tongue, and esophagus were fixed with 4% paraformaldehyde, dehydrated, and then embedded in paraffin. Ribonucleotide probes were synthesized with alpha-S-UTP (Amersham Corp.) from the full-length coding region of C12. In situ hybridization was performed using sense and antisense C12 probes as previously described(30) .


RESULTS

Differential screening of a cDNA library prepared from poly(A) RNA isolated from squamous-differentiated RbTE cells yielded several cDNA clones encoding mRNAs that were abundantly expressed in squamous differentiated RbTE cells but not in undifferentiated cells(17) . In this study, we describe the characterization of one of these cDNA clones named C12 and its derivative C12-3. These cDNAs contain inserts encoding overlapping 3`- and 5`-fragments of a novel squamous cell-specific mRNA. These inserts were sequenced in both directions; the cDNA sequence is shown in Fig. 1. A putative initiation codon was present 27 bases from the 5`-end of the cDNA. The open reading frame (ORF) terminates with a stop codon at nucleotide 720. A polyadenylation signal (AATAAA) was found 308 nucleotides further from the stop codon. Based on the deduced amino acid sequence, the mRNA encodes a hydrophilic 24.3-kDa protein with an estimated pI of 8 (Fig. 1).


Figure 1: Nucleotide and deduced amino acid sequence of C12 (cornifin beta). The putative initiation codon (ATG), termination codon (TAG), and the polyadenylation signal (AATAAA) are indicated in boldface type. The amino acid sequence is shown in the single-letter code. The underlined amino acid sequence represents the peptide C12-PEPB used to raise antibodies.



Data base searching (GCG FastA on the combined nucleic acid data base) with the ORF of C12 revealed substantial similarity to sprs and cornifin alpha(18, 19, 20) . The DNA coding sequence of C12 exhibited a 51% identity with that of cornifin alpha(20) . The amino acid sequence of C12 was 49, 57, 37, and 66% identical to cornifin alpha, spr1, 2, and 3, respectively(18, 19, 20) . The first 30 amino acids at the amino terminus were the most highly conserved (87%) between C12, cornifin alpha and the spr's (Fig. 2A). Like cornifin alpha/spr1 and spr3, C12 has a high proline content (19%) and contains a highly repeated octapeptide at the carboxyl terminus. However, these repeats deviate substantially from the consensus repeat sequences of cornifin alpha/spr1 and spr3 (Fig. 2B). Moreover, the octapeptide repeats in C12 were not as highly conserved between one another as those in cornifin alpha or the sprs. Four subclasses of octapeptides could be identified in C12 exhibiting a 40-60% identity between one another and a 25-50% and 50-87% identity with sequences in cornifin alpha and spr3, respectively (Fig. 2B). These octapeptides each were duplicated two to six times. These results indicate that C12 encodes a protein that is distinct but closely related to cornifin alpha and sprs. This protein was named cornifin beta.


Figure 2: Comparison of the amino acid sequence of cornifins and sprs. A, comparison of the first 30 amino acids at the amino terminus. The amino acids shown in bold vary from the amino acid sequence of cornifin beta (C12). SPR1, 2, and 3 are the sequences of the human small proline-rich proteins(18, 19) . B, comparison of the different octapeptides present in the tandem repeats of cornifins alpha and beta, and spr3. The arrows line up the repeats that are identical or exhibit a high degree of homology. Two (A and B) such repeats can be identified in cornifin alpha, four (C, D, E, and F) in cornifin beta and two in spr3 (G and H).



We next examined by Northern blot analysis and in situ hybridization the tissue-specific expression of cornifin beta mRNA and its regulation during squamous differentiation. Northern blot analysis showed that C12 represents a 1.1-kb mRNA that is induced when RbTE cells undergo squamous cell differentiation (Fig. 3A). Squamous differentiated RbTE cells express more than 50-fold higher levels of cornifin beta mRNA than undifferentiated cells. Furthermore, Northern blot analysis of RNA from different rabbit tissues showed that cornifin beta mRNA was highly expressed in esophagus and tongue and present at low levels in lip, but was not detectable in kidney, liver, brain, or testis (Fig. 3B). Although expression of cornifin beta is highly restricted to several squamous differentiating tissues, it was, in contrast to cornifin alpha, undetectable in rabbit skin. Similar observations were obtained with human tissues. Cornifin beta was highly expressed in human esophagus but was undetectable in epidermis and squamous-differentiated human epidermal keratinocytes in culture (not shown). Thus, cornifin beta, rather then being a general marker for squamous differentiation, is expressed only in certain squamous tissues.


Figure 3: Expression of C12 mRNA in squamous differentiating cells. Total RNA (30 µg) prepared from RbTE cells and various rabbit tissues were fractionated, blotted to Nytran, and hybridized to P-labeled probes for C12 or glyceraldehyde 3-phosphate dehydrogenase (GPDH). A, RNA from undifferentiated and squamous differentiated RbTE cells. B, RNA from various rabbit tissues.



The conclusion that expression of cornifin beta is induced during squamous cell differentiation was further confirmed in studies examining the localization of cornifin beta transcripts by in situ hybridization. Expression of cornifin beta mRNA was restricted to the suprabasal layers of the rabbit esophageal epithelium (Fig. 4) and other squamous epithelia such as the tongue and the oral mucosa (not shown). This pattern of expression is very similar to those reported previously for cornifin alpha and transglutaminase type I (21, 31) and confirms that cornifin beta expression is associated with squamous differentiation. However, once again cornifin beta mRNA was not detectable in the epidermis (not shown).


Figure 4: Localization of C12 mRNA in rabbit esophagus by in situ hybridization. In situ hybridization on sections of rabbit esophagus was carried out as described under ``Experimental Procedures'' using S-labeled sense (A and C) and antisense (B and D) C12 probes. A and B, bright field; C and D, dark field exposure.



To analyze the expression of cornifin beta at the protein level, an antiserum was raised against the synthetic peptide C12-PEPB (Fig. 1A). The antiserum recognized a major protein in total protein extracts of squamous differentiated RbTE cells that migrated at an apparent molecular mass of about 32 kDa (Fig. 5A). The latter is higher than the predicted molecular mass, as has also been observed for cornifin alpha(20) . In several experiments the antiserum reacted weakly with another protein migrating at a slightly lower molecular mass (28 kDa). This smaller immunoreactive protein may have been derived from cornifin beta by proteolytic digestion. The specificity of the immunoreactivity was shown by competitive blocking of the protein-antiserum interaction with the homologous peptide but not with a heterologous peptide (Fig. 5A). In addition, preimmune serum did not react with any protein in extracts from squamous differentiated RbTE cells (not shown). Fig. 5, B and C, shows the induction of cornifin beta in relation to the onset of squamous differentiation in RbTE cells, which is induced when cultures reach confluence (at day 8 and 9)(1, 11) . Cornifin beta was detectable only in confluent, squamous-differentiated cultures but not in logarithmic cultures containing undifferentiated cells. Cornifin beta was increased more than 50-fold when cultures of RbTE cells reached confluence.


Figure 5: Immunoblot analysis of cornifin beta protein expression. Proteins from undifferentiated and squamous-differentiated RbTE cells were examined by immunoblot analysis using C12-PEPB-Ab antiserum. A, immunoblot analysis of total cellular protein from squamous-differentiated cells (lane 1), in the presence of the homologous peptide (lane 2), or heterologous peptide (lane 3). B and C, induction of cornifin beta protein during differentiation of RbTE cells. RbTE cells were plated at 5 times 10^4 cells/60-mm dish and at the times indicated cells were collected for the determination of cell number (B) and cornifin beta protein (C) by immunoblot analysis. The molecular mass of protein markers (kDa) is indicated on the right.



In agreement with the results obtained by Northern blotting and in situ hybridization, immunoblot and immunohistochemical analysis indicated that cornifin beta expression is associated with squamous differentiation in several, but not all, squamous tissues. Cornifin beta was detectable in extracts from rabbit tongue, esophagus, and oral mucosa but undetectable in skin, trachea. muscle, and liver (Fig. 6). The localization of cornifin beta was analyzed by immunohistochemical staining and compared with that of cornifin alpha and involucrin. In sections of esophageal epithelium, immunoreactivity with the C12-PEPB antiserum is restricted to the suprabasal layers (Fig. 7A). Although the staining pattern for cornifin beta is very similar to that for involucrin, it appears that the induction of involucrin occurs somewhat earlier (Fig. 7C). The immunoreactivity for cornifin alpha occurs in layers that are closer to the lumen than those stained for involucrin and cornifin beta. These results suggest that both cornifin beta and involucrin are induced at an earlier stage of differentiation than cornifin alpha.


Figure 6: Expression of cornifin beta in different rabbit tissues. Protein extracts from the epithelium of the rabbit tongue (1), esophagus (2), oral mucosa (3), skin (4), trachea (5), muscle (6), and liver (7) were examined by immunoblot analysis using anti-C12-PEPB antiserum. The molecular mass (kDa) of protein markers is indicated on the left.




Figure 7: Comparison of the expression of cornifin alpha and beta and involucrin in the esophagus by immunohistochemistry. Sections of human esophagus were analyzed by immunohistochemistry using rabbit antisera against A, cornifin beta; B, cornifin alpha; C, involucrin; and D, rabbit preimmune serum.



Fractionation of the cellular lysates showed that cornifin beta was associated predominantly with the soluble fraction suggesting that it is a cytosolic protein (Fig. 8A). Since cornifin beta is related to the cross-linked envelope precursor cornifin alpha(20) , we determined whether it can also serve as a substrate for transglutaminase type I which catalyzes the formation of (-glutamyl)lysine isopeptide bonds between cross-linked envelope precursors(7) . We first examined whether cornifin beta becomes cross-linked when cells are treated with calcium ionophore. Such an exposure increases the intracellular Ca level which activates transglutaminase type I leading to subsequent cross-linking of envelope precursors(10, 20) . Fig. 8A shows that the reactivity of cornifin beta with the antibody against C12-PEPB is abrogated after the cells are treated with the calcium ionophore Ro 2-2985 in agreement with the concept that it becomes cross-linked into high molecular weight aggregates. Varying degrees of epitope masking have been observed previously during cross-linking of envelope precursors (20) and is probably responsible for the loss of immunoreactivity of the cross-linked C12 protein as well.


Figure 8: Transglutaminase-induced cross-linking of cornifin beta protein. A, total cellular protein (T), the soluble protein fraction (S) and the particulate protein fraction (P) from untreated and Ca-ionophore-treated squamous differentiated RbTE cells were examined by immunoblot analysis with C12-PEPB-Ab. B, identification of proteins in differentiated RbTE cells that are covalently cross-linked with dansylcadaverine. Total cellular extracts were incubated in the presence of dansylcadaverine for 0, 2, 5, and 10 min (lanes 1, 2, 3, and 4). Samples were then examined by immunoblot analysis using E7 monoclonal antibody (DANS) (29) or antisera against C12-PEPB or SQ37A-Ab (20) for the presence of dansylated proteins, cornifins beta and cornifin alpha, respectively.



In order to confirm cross-linking of cornifin beta, dansylcadaverine was supplied as an amine donor in in vitro cross-linking reactions(10, 20) . As shown in Fig. 8B, two major proteins of 21 and 32 kDa in crude extracts prepared from differentiated RbTE cells were covalently linked to dansylcadaverine in a time-dependent manner. The smaller protein comigrated with cornifin alpha(20) , the larger one with cornifin beta.

The expression of many squamous cell-specific genes have been reported to be down-regulated by retinoids(1, 2) . Therefore, we examined the effect of several retinoids on the expression of cornifin beta. As shown in Fig. 9A, 10M retinoic acid totally suppressed the induction of cornifin beta mRNA. To obtain more insight in the signaling pathway involved in this retinoid action, the effect of two retinoid receptor selective retinoids was determined. Nanomolar concentrations the RAR-selective retinoid SRI 6751-84 were able to suppress the expression of both cornifin beta and alpha (SQ37) very effectively, whereas the RXR-selective retinoid SRI-11217 was much less potent. Cornifin beta may be slightly less sensitive to retinoids than cornifin alpha. The repression of cornifin beta by retinoids was also observed at the level of the protein (Fig. 10). The retinoid SR11302, which does not induce RAR- or RXR-dependent transactivation but inhibits AP1-dependent transactivation(24) , had no effect on the expression of cornifin beta (not shown).


Figure 9: Effect of RAR- and RXR-selective retinoids on cornifin alpha and beta mRNA expression. RbTE cells were grown to confluence and then treated with RA, RAR-selective (RAR) or RXR-selective (RXR) retinoids at the concentrations indicated. Five days later cells were collected and total RNA isolated. RNA (30 µg) was analyzed by Northern blot analysis using P-labeled probes for C12 (A), cornifin alpha (SQ37) (B), and GPDH.




Figure 10: Suppression of cornifin beta protein by the RAR-selective retinoid SRI 6751-84. Subconfluent cultures of RbTE cells were treated with the RAR-selective retinoid SRI 6751-84 (RAR) at the indicated concentrations and four days later cells were collected and examined by immunoblot analysis using anti-C12-PEPB antiserum.




DISCUSSION

In this study, we describe the isolation and characterization of a novel cross-linked envelope precursor which is a new member of the cornifin/spr family. This protein was named cornifin beta. The lines of evidence supporting this classification include the presence of a highly conserved amino-terminal region characteristic of cross-linked envelope precursors and of a characteristic, highly repeated octapeptide. In addition, the strong association of its expression with squamous differentiation and its ability to serve as a substrate in transglutaminase-catalyzed cross-linking reactions. The predicted amino acid sequence of cornifin beta is 49% identical to that of the previously reported cornifin, referred to now as cornifin alpha(20) , and exhibits a 57, 37, and 66% identity to that of spr1, 2, and 3, respectively(18, 19) . The sequence of the 30 amino acids at the amino-terminal region are remarkably well conserved, 87% between rabbit cornifin alpha and cornifin beta. It is also well conserved across species (Fig. 2). Interestingly, this sequence also shows considerable homology to the amino-terminal region of two other cross-linked envelope precursors, involucrin and loricrin(13, 15, 16) . Although one could expect this highly conserved region to be derived by the duplication of a single exon, no intron was found at the borders of this region(19) . (^2)

As for cornifins and sprs, cornifin beta is rich in amino acids that can disrupt protein secondary structure. Cornifin beta has a proline content of 19% compared to 31% for cornifin alpha and 22% for spr3(19, 20) and a glycine content of 8% versus 0 and 9% for the other two, respectively. The percentage of glutamine, lysine, and cysteine in cornifin beta (8.6, 6.9, and 3.0%, respectively) is much lower than that in cornifin alpha (20, 13, and 11%, respectively).

Cornifin beta contains a highly repeated octapeptide at its carboxyl terminus as do cornifin alpha, spr1 and 3. Cornifin alpha contains 12 repetitions of the highly conserved consensus sequence EPCQPKVP, whereas cornifin beta contains 21 octapeptide repeats. These sequences are not as highly conserved as those in cornifin alpha and spr3. The cornifin beta repeat sequences fall into four subclasses: ESGCTSVP, QP(G/S)YTKVP, GPGYPTVP, and GSGYSV(V/I)P which are repeated, respectively, five, five, two, and three times (Fig. 2B). These octapeptides can be viewed as being organized in groups of three as repeats of a 24-amino acid sequence. During evolution, the cornifin beta sequence may have arisen from duplications of the octapeptide followed first by mutations leading to a diversion in amino acid composition between the octapeptides and subsequent duplications of the 24-amino acid sequence. The sequence and organization of the repeats in cornifin beta deviate substantially from those found in cornifin alpha and spr3, yet still one-third of the amino acids in this region tend to disrupt conventional secondary structure. The repeat sequences exhibit a 25-50% and 50-87% identity with sequences in cornifin alpha and spr3. No similarity in the sequence of these repeats were observed with those found in other squamous cell marker genes such as involucrin, loricrin, and filaggrin(13, 15, 16, 31) .

As cornifin alpha, cornifin beta can function as a substrate for transglutaminase type I(20) . This was indicated by treatment of RbTE cells with calcium ionophore Ro 2-2985 which results in the activation of transglutaminase type I and the disappearance of immunoreactive cornifin beta when it becomes cross-linked and associated with the cross-linked envelope. In addition, labeling of proteins with dansylcadaverine revealed two major labeled proteins of 21 and 32 kDa. Previous studies identified the 21-kDa band as cornifin alpha and showed that the larger 32-kDa protein did not immunoprecipitate with anti-cornifin alpha antibodies(20) . This protein comigrates at the same position as cornifin beta, suggesting that this dansylated protein is cornifin beta.

The expression of cornifin beta is associated with squamous differentiation. This is demonstrated by Northern blot, in situ hybridization, and immunohistochemical analyses showing that the presence of cornifin beta mRNA and protein was restricted to squamous epithelia and limited to the suprabasal layers of the squamous epithelium. The tissue-specific expression of cornifin beta appears to be more restricted than that of cornifin alpha. In contrast to cornifin alpha, cornifin beta was expressed in neither rabbit nor human skin nor in cultured NHEK cells; however, cornifin alpha and beta each were abundantly expressed in both rabbit and human oral mucosa, esophagus, and tongue ( Fig. 4and Fig. 6)(20, 21) . (^3)The differential expression of cornifin alpha and beta may through alterations in the composition of the cross-linked envelope determine different physical properties of that structure as may be required in different tissues. Immunohistochemical analyses indicate that cornifin alpha and beta, involucrin and loricrin are induced at different points during squamous differentiation(14, 15, 21) . This sequential induction supports the hypothesis that the formation of the cross-linked envelope is a multistep process(20) . As proposed previously, involucrin and cornifin may form a scaffold upon which loricrin and perhaps other cross-linked envelope precursors are assembled.

The induction of cornifin beta protein and mRNA is suppressed by retinoic acid. Retinoids have been shown to mediate their action on gene expression through specific nuclear retinoid receptors, RARs and RXRs (reviewed in Giguere(32) ). To examine what retinoic acid signaling pathway is involved in this suppression, the action of SRI 6751-84 and SRI-11217, an RAR- and an RXR-selective retinoid, respectively, and of SR11302, a retinoid that exhibits anti-AP-1 activity but that is unable to induce transactivation through the retinoid response elements, RARE or RXRE(25) , were studied. In contrast to the RXR-selective retinoid, the RAR-selective retinoid was very effective in suppressing the expression of cornifin beta mRNA while SR11302 had no effect. These observations suggest that the suppression of C12 is mediated through activation of RARs rather than RXRs and appears not to require the anti-AP1 activity of retinoids. Characterization of the DNA elements involved in the up-regulation and the retinoid-mediated repression of this gene has to await the isolation of the promoter region of cornifin beta.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U40631[GenBank].

§
Present address: Department of Dermatology, Okayama University Medical School, Okayama, Japan.

Present address: Department of Medical Sciences, Creighton University School of Medicine, Omaha, NE 68178.

**
Present address: Department of Cell, Molecular, and Structural Biology, Northwestern University Medical School, Chicago, IL 60611.

§§
To whom correspondence should be addressed. Tel.: 919-541-2768; Fax.: 919-541-4133; :jetten{at}niehs.nih.gov.

(^1)
The abbreviations used are: spr, small proline-rich protein; RbTE, rabbit tracheal epithelial; RAR, retinoic acid receptor; RXR, retinoid X receptor; RA, retinoic acid; ORF, open reading frame; kb, kilobase pair(s).

(^2)
K. Marvin and A. M. Jetten, unpublished observations.

(^3)
W. Fujimoto and A. M. Jetten, unpublished observations.


REFERENCES

  1. Jetten, A. M., Nervi, C., and Vollberg, T. M. (1992) J. Natl. Cancer Inst. Monogr. 13, 93-100 [Medline] [Order article via Infotrieve]
  2. Eckert, R. L., and Rorke, E. A. (1989) Environ. Health Perspect. 80, 109-116 [Medline] [Order article via Infotrieve]
  3. Watt, F. M. (1989) Curr. Opin. Cell Biol. 1, 1107-1115 [Medline] [Order article via Infotrieve]
  4. Fuchs, E. (1990) J. Cell Biol. 111, 2807-2814 [Medline] [Order article via Infotrieve]
  5. Hohl, D. (1990) Dermatologica 180, 201-211 [Medline] [Order article via Infotrieve]
  6. Sun, T.-T., and Green, H. (1976) Cell 9, 511-522 [Medline] [Order article via Infotrieve]
  7. Rice, R. H., and Green, H. (1979) Cell 18, 681-694 [Medline] [Order article via Infotrieve]
  8. Abernethy, J. L., Hill, R. L., and Goldsmith, L. A. (1977) J. Biol. Chem. 252, 1837- 1839 [Abstract]
  9. Simon, M., and Green, H. (1985) Cell 40, 677-683 [Medline] [Order article via Infotrieve]
  10. Thacher, S. M., and Rice, R. H. (1985) Cell 40, 685-695 [Medline] [Order article via Infotrieve]
  11. Jetten, A. M., and Shirley, J. E. (1986) J. Biol. Chem. 261, 15097-15101 [Abstract/Free Full Text]
  12. Floyd, E. E., and Jetten, A. M. (1989) Mol. Cell . Biol. 9, 4846-4851 [Medline] [Order article via Infotrieve]
  13. Eckert, R. L., and Green, H. (1986) Cell 46, 583-589 [Medline] [Order article via Infotrieve]
  14. Eckert, R. L., Yaffe, M. B., Crish, J. F., Murthy, S., Rorke, E. A., and Welter, J. F. (1993) J. Invest. Dermatol. 100, 613-617 [Abstract]
  15. Mehrel, T., Hohl, D., Rothnagel, J. A., Longley, M. A., Bundman, D., Cheng, C., Lichti, U., Bischer, M. E., Steven, A. C., Steinert, P. M., Yuspa, S. H., and Roop, D. R. (1990) Cell 61, 1103-1112 [Medline] [Order article via Infotrieve]
  16. Hohl, D., Mehrel, T., Lichti, U., Turner, M. L., Roop, D. R., and Steinert, P. M. (1991) J. Biol. Chem. 266, 6626-6636 [Abstract/Free Full Text]
  17. Smits, H. L., Floyd, E., and Jetten, A. M. (1987) Mol. Cell. Biol. 7, 4017-4023 [Medline] [Order article via Infotrieve]
  18. Kartasova, T., and van de Putte, P. (1988) Mol. Cell. Biol. 8, 2195-2203 [Medline] [Order article via Infotrieve]
  19. Gibbs, S., Lohman, F., Teubel, W., van de Putte, P., and Backendorf, C. (1990) Nucleic Acids Res. 18, 4401-4407 [Abstract]
  20. Marvin, K. W., George, M. D., Fujimoto, W., Saunders, N. A., Bernacki, S. H., and Jetten, A. M. (1992) Proc. Natl. Acad. Sci. U. S. A. 87, 9333-9337 [Abstract]
  21. Fujimoto, W., Marvin, K. W., George, M. D., Celli, G., Darwiche, N., De Luca, L. M., and Jetten, A. M. (1993) J. Invest. Dermatol. 101, 268-274 [Abstract]
  22. An, G., Tesfaigzi, J., Chuu, Y.-J., and Wu, R. (1993) J. Biol. Chem. 268, 10977-10982 [Abstract/Free Full Text]
  23. Rearick, J. I., Albro, P. W., and Jetten, A. M. (1987) J. Biol. Chem. 262, 13069-13074 [Abstract/Free Full Text]
  24. Lehmann, J. M., Jong, L., Fanjul, A., Cameron, J. F., Ping, L., Haefner, P., Dawson, M. I., and Pfahl, M. (1992) Science 258, 1944-1946 [Medline] [Order article via Infotrieve]
  25. Fanjul, A., Dawson, M. I., Hobbs, P. D., Jong, L., Cameron, J. F., Harlev, E., Graupner, G., Lu, X., and Pfahl, M. (1994) Nature 372, 107-111 [CrossRef][Medline] [Order article via Infotrieve]
  26. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5463-5467 [Abstract]
  27. Devereux, J., Haeberli, P., and Smithies, O. (1984) Nucleic Acids Res. 12, 387-395 [Abstract]
  28. Jetten, A. M., Bernacki, S. H., Floyd, E. E., Saunders, N. A., Pieniazek, J., and Lotan, R. (1992) Cell Growth Differ. 3, 549-556 [Abstract]
  29. Murthy, S. N., Wilson, J., Zhang, Y., and Lorand, L. (1994) J. Biol. Chem. 269, 22907-22911 [Abstract/Free Full Text]
  30. Noji, S., Takahashi, N., Nohno, T., Koyama, E., Yamaai, T., Muramatsu, M., and Taniguchi, S. (1990) Acta Histochem. Cytochem. 23, 353-366
  31. Presland, R. B., Haydock, P. V., Fleckman, P., Nirunsuksiri, W., and Dale, B. A. (1994) J. Biol. Chem. 267, 23772-23781 [Abstract/Free Full Text]
  32. Giguere, V. (1994) Endocr. Rev. 15, 61-79 [Medline] [Order article via Infotrieve]

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