Loricrin Expression in Cultured Human Keratinocytes Is Controlledby a Complex Interplay between Transcription Factors of the Sp1,CREB, AP1, and AP2 Families*

Received for publication, June 5, 2002, and in revised form, August 21, 2002Published, JBC Papers in Press, August 27, 2002, DOI 10.1074/jbc.M205593200

Shyh-Ing Jang‡ and Peter M. Steinert

From the Laboratory of Skin Biology, NIAMS, National Institutes of Health, Bethesda, Maryland 20892-8023

The major protein component of the cornified cell en-velope barrier structure of the epidermis is loricrin, andit is expressed late during terminal differentiation inepidermal keratinocytes. We have previously shownthat an AP1 site located in the proximal promoter region(position �55) is essential for human loricrin promoteractivity (Rossi, A., Jang, S-I., Ceci, R., Steinert, P. M., andMarkova, N. G. (1998) J. Invest. Dermatol. 110, 34–40). Inthis study we show that its regulation requires complexcooperative and competitive interactions between mul-tiple transcription factors in keratinocytes located indifferent compartments of the epidermis. We show thatas few as 154 base pairs of 5�-upstream sequences fromthe cap site can direct the keratinocyte-specific expres-sion in cultured keratinocytes. Mutation and DNA-pro-tein analyses show that Sp1, c-Jun, an unidentified reg-ulator, and the co-activator p300/CREB-binding proteinup-regulate whereas Sp3, CREB-1/CREM�/ATF-1, Jun B,and an AP2-like protein (termed the keratinocyte-spe-cific repressor-1 (KSR-1)) suppress loricrin promoter ac-tivity. We show that CREB protein can compete withc-Jun for the AP1 site and repress loricrin promoteractivity. We show here that the protein kinase A path-way can activate loricrin expression by manipulation ofthe Sp1, Sp3, and KSR-1 levels in the nucleus. Thus, inundifferentiated cells, loricrin expression is suppressedby Jun B, Sp3, and KSR-1 proteins. But in advanceddifferentiated cells, levels of Sp3, KSR-1, and CREB pro-teins are lower; the unidentified regulator protein canbind; Sp1 and c-Jun are increased; and then p300/CBP isrecruited. Together, these events allow loricrin tran-scription to proceed. Indeed, the synergistic effects ofthe Sp1, c-Jun, and p300 factors indicate that p300/CBPmight act as bridge to form an active transcriptioncomplex.

The epidermis provides an essential protective barrieragainst the environment. As a stratified squamous epithelium,some inner basal cells become committed to terminal differen-tiation and move outward to form spinous, granular, and even-tually fully differentiated, dead cornified layers. During thismigration and differentiation program, unique sets of biochem-ical markers including specific structural proteins, such askeratins, their associated proteins, and processing enzymes,

are synthesized (1–3). Another characteristic feature is theformation of the cornified cell envelope (CE),1 which is crucialfor barrier function of the epidermis (4). The CE is a highlyinsoluble structure and contains a complex mixture of specificproteins such as involucrin, loricrin, small proline-rich pro-teins, XP-5 family members, cystatin A, elafin, S100 familymembers, and lipids that are covalently cross-linked by trans-glutaminases (5–10). The quantitatively major CE protein innormal epidermis is loricrin (6, 10). However, the loricrin-deficient mouse model shows only minor defects in barrierfunction (11), probably because other CE proteins such as smallproline-rich proteins, XP-5 proteins, and repetin are up-regu-lated late during fetal development and apparently compensatefor the loss of loricrin (12).

The expression of loricrin is strictly linked to keratinocyteterminal differentiation both in vivo and in vitro (13, 14). Theloricrin transcripts are localized in the granular layers of theepidermis and some related orthokeratinizing epithelia (15,16). Also, it has been demonstrated that loricrin transcriptioncan be up-regulated when cultured keratinocytes are inducedto differentiate by calcium (14). Thus, the tissue and differen-tiation specificity of loricrin expression make it an excellentmodel to study the sequences and transcription factors in-volved in the control of keratinocyte- and differentiation-spe-cific gene expression. It has been reported that 6.5 kbp ofsequences upstream of the cap site are required for the epithe-lium-specific expression of the mouse loricrin gene (17). Fur-ther study showed that a total of 14-kbp sequences are requiredto direct the differentiation-specific expression of mouse lori-crin in transgenic mice (18).

Recent studies have shown that the regulation of epidermalgene expression appears to be controlled primarily at the tran-scription level. Several ubiquitously expressed transcriptionfactors such as Sp1, NF-�B, AP2, AP1, Ets, and POU domainproteins, together with some unidentified factors (for exampleAP2-like factors), are involved in the specificity of epidermalgene expression. It is thought that the control of specificity ofgene expression can be achieved through various arrangementsof binding sites and the complex combinatory interplay be-tween these regulators (19–24).

The transcriptional regulation of the human loricrin geneexpression is poorly understood. In our previous study, wereported that a functional AP1 motif (located 55 bp upstream ofthe cap site) is indispensable for human loricrin promoter ac-

* The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked“advertisement” in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.

‡ To whom all correspondence should be addressed: Bldg. 50, Rm.1529, NIAMS, National Institutes of Health, Bethesda, MD 20892-8023. Tel.: 301-451-8231; Fax: 301-402-3417; E-mail: jangs@mail.nih.gov.

1 The abbreviations used are: CE, cell envelope; CRE, cAMP-respon-sive element; PKA, protein kinase A; CREB, cAMP-responsive element-binding protein; CBP, CREB-binding protein; CAT, chloramphenicolacetyltransferase; NHEK, normal human epidermal keratinocytes;NHF, normal human fibroblasts; KSSE, keratinocyte-specific silencingelement; KSR-1, keratinocyte-specific repressor 1; HA, hemagglutinin;CMV, cytomegalovirus; TBS, Tris-buffered saline.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 44, Issue of November 1, pp. 42268–42279, 2002Printed in U.S.A.

This paper is available on line at http://www.jbc.org42268

by guest on April 1, 2018

http://ww

w.jbc.org/

Dow

nloaded from

tivity, and c-Jun/c-Fos heterodimers activate whereas Jun Bsuppresses promoter activity (25). The aim of the present workis to further characterize the regulatory elements and thetranscriptional control of the human loricrin promoter in cul-tured NHEK cells. Our study shows that, unlike the mouseloricrin gene, as few as 154 bp upstream of the transcriptioninitiation site are sufficient for keratinocyte-specific expres-sion. However, the calcium-responsiveness elements are notlocated within the first 1.3 kbp of the promoter region. Our datademonstrate that the regulation of loricrin gene expressionrequires interactions among multiple transcription factors, afine balance of the availability of these proteins, and recruit-ment of the co-activator p300/CBP. Together, these allow max-imal loricrin promoter activity in NHEK cells.

MATERIALS AND METHODS

Construction of Plasmids—To construct the pCAT(�1370/�9) plas-mid, the following primers were used (PstI and XbaI restriction sitesused for cloning are shown in lowercase): forward, 5�-AActgcagCTGT-CATTTCAAAACATAACTGGGC-3�; reverse, 5�-GctctagaAGATGCTG-GCAATGTGAGGAC-3�. These were used for amplification by PCR us-ing a human loricrin genomic clone (26). The PCR product was digestedwith PstI and XbaI and cloned into the pCAT-Basic vector, whichcontains the chloramphenicol acetyltransferase (CAT) gene but does nothave any eukaryotic regulatory elements (Promega, Madison, WI). Toobtain a series of deletion constructs, the pCAT(�1370/�9) constructwas subjected to the following digestion: HindIII digestion, which elim-inated a 415-bp fragment and generated the pCAT (�955/�9) con-struct. This construct was further digested with HindIII/BglII, HindIII/BspHI, or HindIII/SacI to generate the CAT constructs containingfragments spanning the region of �370/�9, �154/�9, and �45/�9,respectively. The pCAT(�112/�9) construct was obtained by PCR using5�-AActgcagACAGCCACGGGTCACGT-3� paired with the same reverseprimer used above. For the pCAT (�67/�9) construct, the pCAT (�154/�9) was digested with both EcoRII and HindIII, blunt-ended, andcloned into the pCAT-Basic vector. All constructs were purified by 2�CsCl2 banding and confirmed by DNA sequencing. All experimentswere performed using at least two different batches of plasmidpreparation.

Site-directed Mutagenesis—Mutations of functional elements in theloricrin promoter were made in the pCAT (�150/�9) construct usingthe QuikChange method (Stratagene, La Jolla, CA). Mutations of de-sired sequences (underlined) are as follows: Sp1 site (GGGATA-AGGGGC), AP2-like site (GCCGACATTTACG), and AP1 site (ATTT-GTCAG). To construct the pCAT (�150/�9)-Gal4 plasmid, thesequences between �130 and �110 were replaced with a Gal4 bindingsite, 5�-GCGGAGTACTGTCCTCCGA-3� (27). For this substitution, theQuikChange protocol was modified as follows. The first two PCR cyclesof synthesis were performed in two separate tubes containing either theplus or minus strand of an oligonucleotide carrying Gal4 binding se-quences, together with the template and Pfu polymerase. Both reac-tions were then mixed together, and PCR was continued for another 25cycles before DpnI digestion as suggested by manufacturer. All of themutations were confirmed by DNA sequencing.

Cell Cultures, Transfections, and Enzyme Assays—CryopreservedNHEK cells and primary normal human fibroblasts (NHF) were pur-chased from Clonetics (San Diego, CA). NHEK cells were seeded in calfskin collagen (Sigma)-coated dishes, and the cultures were maintainedin serum-free keratinocyte growth medium (KGM-2; Clonetics) with0.05 mM calcium. NHF cells were grown and maintained in FGM-2medium (Clonetics) supplemented with 2% fetal bovine serum. Thethird passage of NHEK and NHF cultures were used for transfectionexperiments and preparation of cellular and nuclear extracts. HEK-293, A431, COS-7, HeLa, and NIH-3T3 cells were purchased fromAmerican Tissue Culture Collection (Manassas, VA) and were grownand maintained using the recommended procedures. HaCaT cells werea gift from Dr. Norbert E. Fusening and were grown in Dulbecco’smodified Eagle’s medium supplemented with 4.5 g/liter glucose, 10%fetal bovine serum, and nonessential amino acids (Invitrogen).

Cells were seeded on six-well plates 24 h prior to transfection. Tran-sient transfections were performed in duplicate using FuGENE 6(Roche Molecular Biochemicals) for NHEK, NHF, HaCaT, HeLa, A-431,cells, or LipofectAMINE Plus (Invitrogen) for HEK-293, COS-7, andNIH-3T3 cells as recommended by the manufacturers. Typically, trans-fections were done when cultures reached 60–70% confluence. For

NHEK and NHF transfections, cell cultures were washed once at 37 °Cwith warm phosphate-buffered saline and then were preincubated at37 °C for 30 min with either Keratinocyte-SFM (Invitrogen) for NHEK,HaCaT, and NHF cells or Opti-MEM I medium (Invitrogen) for A-431,HeLa, HEK-293, COS-7, and NIH-3T3 cells. For each well, 1.0 �g ofreporter plasmids and 0.5 �g of tk-�-gal plasmid were mixed with 6 �gof either FuGENE 6 or LipofectAMINE Plus in Keratinocyte-SFM andincubated for 20 min at 23 °C. The lipid/DNA mixture was then addedinto each well and incubated for 16–18 h in the case of the NHEK, NHF,or HaCaT cells or 4–6 h for other cell types. At the end of the trans-fection period, the medium was replaced with the fresh medium inwhich the cells normally grow. For NHEK cells, unless indicated, allcultures were maintained in KGM-2 medium containing 1.2 mM cal-cium. Forskolin (10 �M; Calbiochem) was dissolved in Me2SO and addedinto cultures after the transfection was terminated. The control sam-ples were treated with Me2SO (�0.1%, v/v) alone. Cells were harvested48 h post-transfection. Cellular extracts were prepared as described byPothier et al. (28). CAT assay, �-galactosidase assay, and total proteinmeasurements were performed as previously described (23). The valuesfor CAT were normalized by protein content and the �-galactosidaseactivity. Unless indicated, all of the data are the averages of at leastthree independent experiments with duplicated samples.

For co-transfection experiments, typically 1 �g of loricrin CAT con-structs were co-transfected with 0.5 �g of expression vector as indi-cated. The corresponding parent vectors were used to ensure the sametotal amount of plasmid in each sample. All expression vectors em-ployed in this study include pRSV-c-Jun (from Dr. M. Karin) and c-JunTAM under the control of cytomegalovirus promoter (a gift from Dr. M.Birrer). The Gal4/Sp1 expression vectors used were generously pro-vided by Dr. R. Tjian (University of California, Berkeley, CA) and arepSG424 (parent vector), pSG-Sp1Wt (containing Sp1 amino acid resi-dues 83–778, which include domains A–D), pSG-Sp1N (containing do-mains A–C, amino acid residues 83–621), pSG-Sp1A&B (containingdomains A and B, amino acid residues 83–542), and pSG-Sp1B (con-taining domain B, amino acid residues 263–542). All of these vectorscontain cDNA encoding the Gal4 binding domain, amino acids 1–147 ofthe Gal4 protein, which were driven by an SV40 promoter (29).

The pCMV-Sp3 and pCMV-Sp3/DBD, in which the 168 C-terminalcodons of Sp3 were removed, were gifts generously provided by Dr. G.Suske. The pCMV�p300-CHA was kindly provided by Dr. S. Grossmanand contains a p300 cDNA insert from nucleotides 1134–8329 with aC-terminal HA tag cloned into the pCI vector. The expression plasmidsof pRc/RSV-CBP.HA.RK, pRc/RSV-mCREB-341, pRc/RSV-CREM�, andpRc/RSV-KCREB were kindly provided by Dr. R. Goodman. The pSKG4plasmid was a gift from Dr. S. K. Hanks and contains the cDNA of thehuman protein kinase A (PKA) type � catalytic subunit driven by theCMV promoter. The pECE/ATF-1 expression plasmids under control ofthe SV40 promoter and enhancer were obtained from Dr. M. Green. Theexpression vector pRSV-AP2� was kindly provided by Dr. R. Tjian. ThepRSV-AP2� and pRSV-AP2� were gifts from Dr. H. C. Hurst.

Gel Mobility Shift Assays—Nuclear extracts from NHEK, NHF, andHeLa cells were prepared as previously described (23). The cytosolicfractions were obtained after the cells were homogenized with a tightlyfitting glass homogenizer and centrifuged at 3000 � g in a refrigeratedmicrocentrifuge for 10 min. Supernatants were used for gel shift assays.Forskolin-treated NHEK cells were grown in high calcium KGM-2medium for 4 days and treated with forskolin (50 �M) for 6 h beforeharvesting. All buffers also contained phosphatase inhibitors: sodiumvanadate (200 �M), sodium fluoride (10 mM), and okadaic acid (2 nM).The double-stranded oligonucleotide probes were end-labeled with T4polynucleotide kinase in the presence of [�-32P]ATP and then purifiedon 6% TBE gels (Invitrogen). Mobility shift experiments were per-formed as previously described (24). For supershift experiments, thenuclear extracts were first incubated with 2 �g of the correspondingcommercially available antibodies (Santa Cruz Biotechnology, Inc.,Santa Cruz, CA) for 2 h at 4 °C and then treated with the probe foranother 30 min. The complexes were resolved on nondenaturing 6%polyacrylamide gels in 0.5� TBE for 1 h at 14 V/cm. The gels were driedand exposed to x-ray film (Bio-Max MR, Eastman Kodak Co.) with anintensifying screen at �80 °C overnight.

Western Blot Analysis—For Western immunoblot analysis on theexpression of pCMV-Sp3, 10 �g of nuclear extracts from the transfectedNHEK were fractionated on 4–12% gradient Tris-glycine gels andtransferred to a polyvinylidene difluoride membrane (Invitrogen). Aftertransfer, the membranes were washed once in 1� Tris-buffered saline(TBS), pH 7.5, and then incubated in blocking buffer (1� TBS, 0.1%Tween 20, 5% nonfat dry milk) for 1 h at 23 °C. The membranes werewashed three times for 5 min each with 1� TBS/T (1� TBS, 0.1%

Complexity of Human Loricrin Transcription 42269

by guest on April 1, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Tween 20) and then incubated with a 1:1000 dilution of either c-Jun orSp3 polyclonal antibody (Santa Cruz Biotechnology) in blocking bufferfor 1 h at 23 °C. Membranes were then washed three times with 1�TBS/T and incubated with a 1:2000 dilution of the horseradish peroxi-dase-conjugated secondary antibody (Bio-Rad) in blocking buffer for 1 hat 23 °C. Following three washes with 1� TBS/T, signals were detectedby using the SuperSignal West Pico Luminol/Enhancer kit (Pierce) andthe manufacturer’s protocol prior to exposure to x-ray film.

RESULTS

Human Loricrin Promoter Activity in Cultured Cells—In aprevious study, we reported that an AP1 motif located 55 bpupstream of the cap site (position �55) is essential for theactivity of the loricrin promoter in NHEK cells (25). Usingcultured keratinocytes (18) and a transgenic mouse approach,DiSepio et al. (17) showed that an AP1 site situated in theproximal promoter region is likewise critical for the expressionof the mouse loricrin gene. However, it is highly unlikely thatthe tight control of the loricrin transcription is determinedsolely by interactions over the AP1 motif. Thus, the aim of thepresent study is to further elucidate the recognition sequencesand their cognate binding factors that modulate the AP1-de-pendent activity of the human loricrin promoter. A series offragments spanning increasing lengths of the 5� regulatoryregion were cloned into the pCAT-Basic vector and their activ-ities assessed after transfection into NHEK cells (Fig. 1). Thehighest activity was detected with construct �67/�9, whichencompasses the loricrin TATA box and the AP1 site. In ac-cordance with our previous results (25), deletion of the AP1 sitecaused a 10-fold reduction of expression. The addition of 45 bpof upstream sequences (�112/�9) reduced the CAT expressionby 40%, 87 bp (construct �154/�9) reduced by 70%, and moresequences out to �450 reduced the expression to the level of thebasal promoter (construct �45/�9, which encompasses only theTATA box). Such reductions of promoter activity could not beovercome by extension of the regulatory region to �1370. Theloricrin constructs tested in this study gave similar resultswhen NHEK cells were maintained under high calcium condi-tions (data not shown). Thus, this region does not encompasspresumed calcium-responsive regulatory elements.

To assess whether these sequences functioned in a cell type-specific mode, the constructs were transfected into a variety ofcells (Table I). The promoter activity of construct �45/�9 wascomparable in all cell types (data not shown) and was about15–20% of the level of the pCAT-Promoter construct containing

the SV-40 early promoter (23). Inclusion of the AP1 site (con-struct �67/�9) resulted in a 9-fold enhancement in NHEK andHaCaT cells and about 5-fold enhancement in HeLa cells,which, although transformed, have preserved some features oftheir squamous cell origin (30). In contrast, this constructshowed only basal promoter activity in simple epithelial andnonepithelial cells, consistent with the notion that the AP1 siteis involved in cell type specificity of loricrin expression. Inter-estingly, mutation of the AP2-like site (keratinocyte-specificsilencer element (KSSE); see below) showed a 3-fold increaseover that of construct �154/�9 for both NHEK and HaCaTcells but not in HeLa or other cell types. This suggests that theKSSE site is involved in keratinocyte-specific repression of theloricrin promoter. These results also indicate that as few as 67bp upstream of the transcription initiation site may be suffi-cient to restrict the activity of the human loricrin promoter tostratified squamous epithelial cells. Elements residing between�67 and �450 participate in the regulation of loricrin promoteractivity and are further studied in detail here.

Identification of Transcription Factors That Bind on theProximal Promoter Region of Human Loricrin—Initially, wefocused our studies on the region between �67 and �154,which was responsible for a 2-fold repression of the loricrinproximal promoter. Comparison of these sequences with tran-scription factor databases (31) revealed an Sp1 site (position�125), an AP2-like site (�115), and a CRE-like motif (�100).Involvement of Sp1, AP2, and cyclic AMP response elements intranscriptional regulation in keratinocytes have been previ-ously reported (32–34). Therefore, we first explored the abilityof these motifs to interact with nuclear proteins using gel shiftexperiments. The specificity of the interaction was verified incompetition experiments with unlabeled oligonucleotides car-rying wild type or mutated sequences from this region or withdI/dC oligomers.

A double-stranded oligonucleotide (�132/�90) encompassingthe potential Sp1, AP2-like, and CRE-like sites (Fig. 2A) waslabeled and incubated with nuclear extracts from NHEK cells.Four specific DNA-protein complexes (Fig. 2B, lane 1) wereobserved. Specific competition with unlabeled probe abolishedcomplexes A, B, and D and reduced the intensity of complex C(Fig. 2B, lane 2). The origin of complexes A–D was determinedin competition experiments with an array of oligomers in whichthree bp mutations had been created within the putative Sp1,AP2-like, and CRE-like recognition motifs. Mutant B1 com-peted out complexes A and B (lane 3). Mutants B2, B3, and B4,which affected the Sp1 motif, failed to interfere with the for-mation of complexes A and B (lanes 4–6). Mutants of theputative AP2-like motif (B4–B7) did not interfere with complexC (lanes 6–9). The competition for complex D was prevented bythe mutants B1, B2, B4, B5, B6, and B11, where the CRE-likesite was located (lanes 3, 4, 6, 7, 8, and 13). Taken together,these results demonstrate that complexes A and B originatefrom interactions with the Sp1 site that and complex C wasinvolved with the AP2-like site. Complex D appeared to beinvolved with all three recognition motifs.

To identify the proteins that participated in the above inter-actions, the binding reactions were performed in the presenceof oligomers containing the corresponding recognition motifs orwith corresponding specific antibodies. The formation of com-plexes A and B was abolished in the presence of an excess ofunlabeled Sp1 consensus oligonucleotide (Fig. 2C, lane 3), con-firming that both complexes resulted from the interactionswith Sp1 proteins at the Sp1 site. Accordingly, the bindingreactions were performed in the presence of antibodies againstSp1, Sp2, Sp3, and Sp4 transcription factors, or against tran-scription factor GKLF, which served as an unrelated antibody

FIG. 1. Transient expression of the CAT enzyme under thecontrol of loricrin 5�-upstream sequences in NHEK cells. Theloricrin CAT constructs are shown with the AP1 site (�55) indicated.These constructs were transfected into NHEK cells as described under“Materials and Methods.” The CAT activity was determined and nor-malized by �-galactosidase activity from the ptk-�-gal plasmid. Dataare presented as CAT expression in -fold change over that from �45/�9construct.

Complexity of Human Loricrin Transcription42270

by guest on April 1, 2018

http://ww

w.jbc.org/

Dow

nloaded from

control. None of these antibodies affected the mobility or inten-sity of complexes C and D. The antibody against Sp1 super-shifted complex A (arrowhead b, lane 4), whereas complex Bwas supershifted by the antibody against Sp3 (arrowhead a,lane 6). Since the antibodies against Sp2 (lane 5) and Sp4 (lane7) did not result in a supershift, it is unlikely that these familymembers are present in either complex. Together, these resultsconfirmed that interactions of Sp1 and Sp3 transcription fac-tors over the sequence AGGTGGGGC were responsible for theformation of complexes A and B, respectively.

Likewise, the mutation scanning experiments determinedthat the potential AP2 recognition sequence GGCCGACAGCCis involved in the interactions resulting in the formation of bothcomplexes C and D. Interestingly, when a shorter oligonucleo-tide (�117/�105), which encompasses only the AP2-like site,was used for competition (Fig. 2D, lane 3), both complexes Cand D were competed out, suggesting that the AP2-like motif isresponsible for the formation of both complexes. We could notdesign experiments to determine whether these two complexeswere formed by two different proteins or whether the fastermigrating band resulted from the degradation of bindingproteins.

The identity of the proteins in complexes C and D wereexplored in interference experiments with antibodies againstthe known AP2 family members �, �, and �. The intensity ofcomplex C was reduced but not competed out in the presence ofantibodies against AP2-� and AP2-� but not AP2-� (Fig. 2D,lanes 4–6, respectively). None of the other three complexes (A,B, and D) was affected by these AP2 antibodies.

Since the antibodies against Sp1 and AP2 proteins were notable to interfere with complex D formation (Fig. 2, C and D) andCRE-like site mutant oligonucleotides failed to compete forcomplex D (Fig. 2B, lanes 11 and 12), we suspected that com-plex D might involve members of the CREB family of transcrip-tion factors. Accordingly, we performed the binding reactions ofoligonucleotide �132/�90 in the presence of antibodies againsta number of CREB proteins but could not detect any interfer-ence. We reasoned that the strong interactions between theSp1 site and the high level of endogenous Sp1 proteins mightmask protein binding over the CRE element. Indeed, when thebinding reactions were performed with an oligonucleotide thatencompassed only the AP2- and CRE-like motifs (�120/�90),in addition to the AP2-containing complex C, two relativelyweak complexes were observed (Fig. 2E, lane 1) that weresuccessfully competed by an excess of unlabeled probe (lane 2).Antibodies against CREB-1, CREM-1, and ATF-1 reduced theintensity of the upper band of the doublet (lanes 3, 5, and 6),and the supershifted bands were observed from both CREB-1

FIG. 2. Gel shift analyses on the loricrin promoter region. A,the sequences of probes used for gel shift assays (�132/�90 for A–D;�120/�90 for E). The oligonucleotides (�117/�105, flanked by HindIIIand XbaI linkers) and mutant variants (B1–B11) were used for compe-tition assays (see “Materials and Methods”). The Sp1, AP2-like, andCRE-like sites are indicated. B, the binding profile of oligonucleotides(�132/�90, lane 1) with NHEK cell nuclear extracts (NE) and poly(dI/dC). Competition assays were carried with a 100-fold molar excess ofunlabeled probe and mutation B1 to B11 (lanes 2–13) oligonucleotides.The arrows denote the specific complexes formed. C, competition withconsensus Sp1 oligomer (lane 3, TGAATTCATCGGGGCGGGGCGAG-C). Supershift assays are shown for complexes A and B with antibodiesspecific for Sp1 (lane 4), Sp2 (lane 5), Sp3 (lane 6), Sp4 (lane 7), andcontrol GKLF (lane 8). The arrowheads denote the supershift caused bySp1 (b) and Sp3 (a) antibodies. D, complexes C and D were competed outby the �117/�105 oligonucleotide (lane 3). E, the effect of gel shift bythe antibody against CREB-1 (lane 3), CREB-2 (lane 4), CREM-1 (lane5), ATF-1 (lane 6), ATF-2 (lane 7), and ATF-3 (lane 8). The two arrowsdenote the specific complexes; the arrowhead represents the super-shifted bands.

TABLE IExpression of the loricrin constructs in different cell types

The relative CAT expression of the five loricrin constructs were normalized with the activity of tk-�-gal construct and are expressed as -foldchange over the basal promoter activity of the loricrin construct �45/�9, which was comparable in all cell types studied.

Cell typeLoricrin constructs

�450/�9 �154/�9 �154/�9(KSSE) �67/�9

Keratinocyte cellsNHEKa 1.2 � 0.1 4.2 � 0.4 14.5 � 1.2 8.8 � 0.4HaCaT 1.5 � 0.2 4.0 � 0.3 13.2 � 0.8 9.0 � 0.5

Stratified squamous epithelial cellsHeLa 1.1 � 0.1 2.2 � 0.3 2.0 � 0.3 5.0 � 0.7A431 1.2 � 0.2 2.0 � 0.4 2.2 � 0.2 1.8 � 0.2

Epithelial cellsHEK-293 0.8 � 0.1 2.0 � 0.3 3.1 � 0.2 1.1 � 0.3COS-7 1.5 � 0.1 2.1 � 0.2 2.5 � 0.3 2.0 � 0.4

Nonepithelial cellsNHF 1.1 � 0.1 1.2 � 0.1 2.8 � 0.4 1.2 � 0.2NIH 3T3 1.2 � 0.2 2.1 � 0.1 3.0 � 0.2 1.2 � 0.1

a Data from NHEK cells under high calcium condition (1.2 mM) are shown.

Complexity of Human Loricrin Transcription 42271

by guest on April 1, 2018

http://ww

w.jbc.org/

Dow

nloaded from

and ATF-1 antibodies (Fig. 2E, arrowhead). The identity of theprotein that forms the second weak band was not clear. Wehave tried the antibody against the phosphorylated CREB pro-tein, but no change in binding profile was observed (data notshown). One possibility may be the presence of an unknownmember of the CREB family in keratinocytes, but this has notbeen investigated further. Together, these data demonstratedthat the CRE-like site is capable of interacting with the mem-bers of the CREB family present in keratinocytes.

Functional Analyses of the Human Loricrin Promoter—Wenext examined the role of Sp1, AP2-like, and AP1 motifs in thecontrol of the loricrin proximal promoter. A series of mutatedloricrin CAT constructs were prepared and transfected intoNHEK and HeLa cells.

As shown in Fig. 3, mutation of the Sp1 site (construct A)showed about 75 and 55% decrease of the CAT activity inNHEK and HeLa cells, respectively, suggesting that interac-tions over this motif positively regulate the loricrin promoter inboth cell types. However, this motif alone is not sufficient forthe keratinocyte-specific expression of loricrin. When the AP2-like site was mutated (construct B), the expression level of CATincreased more than 2.5-fold compared with wild type inNHEK cells, but no effect was observed in HeLa cells. We alsoobserved similar activation in HaCaT cells when the AP-2 likesite was mutated (data not shown). This suggests that theAP2-like site plays a key role in the keratinocyte-specific re-pression on the loricrin promoter activity. Hence, we termedthis AP2-like element the keratinocyte-specific silencer ele-ment (KSSE) and the protein that binds to it the keratinocyte-specific repressor-1 (KSR-1). The mutation of the CRE-like site(construct C) only has little effect in both cell types. The effectof mutation of the AP1 site (construct D) was similar to that ofthe Sp1 site in both HeLa and NHEK cells. However, doublemutations of the Sp1 and KSSE sites (construct E), KSSE andCRE-like sites (construct F), or KSSE and AP1 sites (constructG) reduced the promoter activity by 85, 40, and 80%, respec-tively, compared with the activity from construct B in NHEKcells. For HeLa cells, there is only a slight change (about5–10%) in CAT activity for construct E, F, and G when com-pared with construct B. Taken together, the data show thatSp1, CRE-like, and AP1 elements play a positive role, whereasthe KSSE motif is involved in a keratinocyte-specific repressionon loricrin promoter activity. Furthermore, the CRE-like siteonly showed its positive effect when the KSSE site was mu-tated, suggesting that the presence of KSR-1 binding on theKSSE site might interfere with the binding of the regulator onCRE-like site.

Sp1 Activates and Sp3 Represses Human Loricrin PromoterActivity in NHEK Cells—The role of Sp1 as a transcriptionalactivator and Sp3 as a transcriptional repressor have been welldocumented (35, 36). To further confirm their involvement inthe control of human loricrin promoter activity, the pRSV-Sp1and pCMV-Sp3 expression vectors were co-transfected withwild type and mutant �154/�9 constructs. As shown in Fig.4A, in the presence of pRSV-Sp1 plasmid, the level of CATactivity of the �154/�9 construct increased 50% comparedwith the control. A similar increase was observed when theKSSE site was mutated (construct �154/�9(KSSE)). This mod-est increase can be expected because of the high level of endog-enous Sp1 in keratinocytes (37). However, no regulatory effectfrom forced expression of Sp1 was observed when the Sp1 sitewas mutated. To explore the role of Sp3 in the control of loricrinexpression, loricrin CAT constructs were co-transfected withthe pCMV-Sp3 expression vector. Forced expression of Sp3repressed CAT expression of the �154/�9 construct more than50% compared with the control (Fig. 4A, blank). Forced expres-sion of Sp3 also suppressed CAT expression even when theKSSE site was mutated, indicating that Sp3 alone is sufficientto exert the repression effect on the loricrin promoter. Whenthe loricrin constructs were co-transfected with the fingerlessSp3 expression vector (pCMV-Sp3/DBD), which lacked theDNA binding domain, CAT activity levels were similar to thecontrol (Fig. 4A, hatched bar). This suggests that Sp3 requiresits DNA binding domain to exert suppression on the loricrinpromoter. No effect from forced expression of Sp1 or Sp3 wasobserved when the Sp1 site of the loricrin promoter was mu-tated. Furthermore, when equal amounts of Sp1 and Sp3 ex-pression vectors were introduced together into NHEK cellswith the loricrin constructs, Sp3 did not inhibit the CAT activ-ity induced by Sp1 on �154/�9 and �154/�9(KSSE) constructs(Fig. 4B). Since NHEK cells have relatively low levels of Sp3compared with Sp1 (Fig. 2B, lane 1), in an attempt to modulatethe Sp1/Sp3 ratio, we transfected various amounts (0–6 �g) ofpCMV-Sp3 into NHEK cells and evaluated the effects on lori-crin promoter activities. Western blot analysis showed increas-ing level of Sp3 (Fig. 4C). The c-Jun protein was detected inparallel to serve as a loading control. Co-transfection of loricrinCAT construct with various amounts of pCMV-Sp3 cDNAshowed a dose-dependent decrease in loricrin promoter activity(Fig. 4D). Together, these results show that both Sp1 and Sp3are capable of binding and most likely compete with each otherfor the Sp1 site on the loricrin promoter. Furthermore, theseresults also confirm that Sp1/Sp3 ratios are involved in thecontrol of loricrin promoter activity.

To further identify which transcriptional activation domainsof Sp1 were involved in the transactivation of the loricrinpromoter, we tested several expression vectors encoding differ-ent Sp1 domains fused with Gal4 protein. To exclude the effectfrom the endogenous Sp1 on the loricrin promoter activity, boththe Sp1 and KSSE sites were replaced with a Gal 4 bindingmotif. The KSSE site was also eliminated to release its repres-sion effect on the loricrin promoter and to maintain the properlength of the proximal promoter region, since both sites arevery close and overlap each other (see Fig. 2A). As shown in Fig.4E, the activation domain B of Sp1 (pSG-Sp1/B) showed a2-fold increase of reporter activity of construct �154/�9(Gal4)over that of the same construct co-transfected with pSG424parent vector alone. The combined glutamine-rich domains Aand B (pSG-Sp1/A�B) increased the promoter activity morethan 6-fold. The addition of activation domain C (pSG-Sp1/N)enhanced the promoter activity by 8.5-fold. The addition ofdomain D (pSG-Sp1/Wt) showed only a slight further increaseof transactivation activity when compared with that from pSG-

FIG. 3. The effects of mutations on Sp1, AP2-like, CR-like, andAP1 sites on loricrin promoter activity. The indicated Sp1, AP2-like, and AP1 motifs were subjected to site-directed mutagenesis asdescribed under “Materials and Methods.” The constructs were trans-fected into HeLa cells (open) and NHEK cells (hatched) with the me-dium maintained in high calcium after transfection. The relative CATvalues are expressed as -fold change over the activity of wild type CATconstruct �154/�9 from NHEK.

Complexity of Human Loricrin Transcription42272

by guest on April 1, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Sp1/N expression plasmid. Together, these data demonstratethat the transactivation domains A, B, and C of Sp1 play amajor role in the up-regulation of the human loricrin promoterin keratinocytes.

Forskolin Up-regulates Loricrin Promoter Activity—Fig. 3showed that the CRE-like site plays a positive role in thecontrol of loricrin promoter activity. Since CREB family pro-teins were involved in the formation of the specific complexes atthe CRE-like site (Fig. 2E), we next assessed whether activa-tion of the PKA signal pathway can lead to activation of humanloricrin promoter. Various loricrin CAT constructs were trans-fected into NHEK cells, and the CAT activity was measured inthe absence or presence of forskolin, an activator of the PKApathway (38). As shown in Fig. 5, compared with the activity ofuntreated cells, the CAT activities of the �154/�9 constructshowed 50% increase in cells treated with forskolin under highcalcium medium. Little effect of forskolin was observed whenthe CRE-like site was either mutated (constructs �154/�9(CRE), �154/�9(AP2/CRE)) or deleted (construct �67/�9).These findings suggest that the up-regulation of loricrin pro-

moter activity by forskolin is exerted through the CRE-like siteof the loricrin promoter.

The Effect of PKA, p300, CBP, and CREB Family Proteins onthe Control of Loricrin Promoter Activity—Phosphorylation ofCREB protein at Ser-133 is necessary for increasing its tran-scription activity (39), and this can be achieved by severalkinases like protein kinase C, PKA, and calmodulin-dependentkinases (40–42). It has been reported that CREB exists as aphosphoprotein in differentiated keratinocytes (43). We nextexamined the role of CREB proteins in the control of loricrinexpression and whether the effect was through the CRE-likesite on the loricrin promoter. Expression plasmids of severalmembers of the CREB family were used in co-transfectionexperiments. To our surprise, the expression of mouse mCREB-341 cDNA reduced the level of CAT expression of the �154/�9loricrin construct by 70% (Fig. 6A). On the other hand, co-transfection of the dominant negative CREB expression vector(pRSV-KCREB) increased CAT expression by 80% above thecontrol. Since the mutation carried by KCREB abolishes itsbinding to DNA, the KCREB/CREB heterodimers should be

FIG. 4. Sp1 activates and Sp3 suppresses loricrin promoter activity in NHEK cells. A, loricrin CAT constructs were co-transfected witheither pRSV-Sp1 (solid) or pCMV-Sp3 (open) or pCMV-Sp3/DBD (hatched). Relative CAT activity is presented as -fold change over the �154/�9co-transfected with each respective parent vector, pRSV or pCMV plasmid alone. B, loricrin CAT constructs were co-transfected with pRSV andpCMV parent vector (solid), pRSV-Sp1 (dotted), or pCMV-Sp3 (open) or with pCMV-Sp3/DBD (hatched) or with both pRSV-Sp1 and pCMV-Sp3/DBD (shaded). The pRSV and pCMV vectors were added to ensure that equal amounts of plasmids were used for transfection. C, the indicatedamount of pCMV-Sp3 plasmid was transfected into NHEK cells, and the nuclear extracts were isolated and subjected to Western blot analysis.c-Jun protein was used as loading control. D, loricrin �154/�9 CAT construct was co-transfected with indicated amount of pCMV-Sp3 in NHEKcells. E, the loricrin CAT construct of �154/�9(Gal4) was co-transfected with Gal-424 or Gal424/Sp1 expression vectors: pSG-Sp1(Wt), pSG-Sp1(N), pSG-Sp1(A�B), and pSG-Sp1(B) (see “Materials and Methods”). Relative CAT activity of the various chimeric vectors was compared withthe pSG424 control vector containing the Gal4 binding domain alone.

Complexity of Human Loricrin Transcription 42273

by guest on April 1, 2018

http://ww

w.jbc.org/

Dow

nloaded from

transcriptionally inactive. Thus, our data suggest thatmCREB-341 acts as a repressor for loricrin promoter activity.Further, the KCREB protein apparently can dimerize withendogenous CREB protein and release the repression. Also,both mCREM� and ATF-1 expression vectors reduced the CATactivity to the same level as mCREB-341, suggesting that allthree are negative regulators for the loricrin promoter (Fig.6A). Since all CREB-1, ATF-1, and mCREM� antibodies causedsupershifted bands at the CRE-like site (Fig. 2E), and co-transfection of all three expression vectors showed similar de-grees of repression on loricrin, we have chosen to report onlythe findings observed with mCREB-341.

It is well known that phosphorylation of CREB allows forrecruitment of the coactivators p300 and CBP (44–46). Sincetreatment with forskolin increased, whereas mCREB-341 re-pressed the loricrin promoter activity, we next explored the roleof the catalytic subunit of the PKA, p300, and CBP to modulatethe expression of the �154/�9 loricrin construct. In the pres-ence of the PKA � subunit, the CAT activity increased morethan 2-fold (Fig. 6A). This confirmed the observation from theforskolin experiments. The presence of CPB and p300 alsoshowed an increase of CAT expression by 3- and 5-fold, re-spectively. Interestingly, after co-transfection of expressionplasmids of either CBP and mCREB-341 or p300 andmCREB-341 together with the loricrin construct, the CATactivities were reduced to a similar level as for the sameloricrin construct co-transfected with mCREB-341 alone.These results suggest that the presence of mCREB-341 com-promised the activation of the loricrin �154/�9 construct byCBP and p300.

CREB Represses Loricrin Promoter Activity through the AP1Site—The above experiments showed that forced expression ofPKA � subunit, CBP, and p300 up-regulated but that mCREB-341 down-regulated loricrin promoter activity. Additional ex-periments were done to clarify these apparently contradictoryobservations. Deleted loricrin CAT constructs were used toexamine where mCREB-341 displayed its repression effect onthe loricrin promoter in NHEK cells. As shown in Fig. 6B, withthe exception of the �45/�9 construct, mCREB-1 reduced thelevel of CAT activity to less than 50% in all loricrin CAT con-structs compared with each respective control. The presence of

KCREB showed opposite effects compared with mCREB-341 onconstructs �154/�9, �112/�9, and �67/�9. No effect by KCREBwas observed on constructs �154/�9(CRE) and �45/�9.

The negative effect by mCREB-341 on loricrin promoter ac-tivity is contradictory to the observation from both mutation(Fig. 3) and forskolin (Fig. 5) experiments where the CRE-likesite appears to serve as a positive element for loricrin promoteractivity. Furthermore, the repression by mCREB-341 and theenhancement by KCREB on the �67/�9 construct (Fig. 6B),which contained only the AP1 site and TATA box without anycryptic CRE site, prompted us to investigate whether mCREB-341 exerts its repression by interfering with the binding of AP1proteins at the AP1 site. The ability of CREB proteins to formheterodimers with AP1 factors has been reported (47–49). Also,it has been documented that CREB can bind at an AP1 site andrepress the AP1 reporter activity (43). First, to see if endoge-nous CREB dimerized with AP1 factors, the antibody againstCREB protein was used in supershift analyses, but we did notsee any interference of AP1 complex formation (data notshown). Second, we examined whether mCREB-341 competeswith AP1 transcription factors for the AP1 site on the loricrinpromoter. As shown in Fig. 6C, co-transfection of the pRSV-c-Jun expression vector increased the CAT activity of the �67/�9construct more than 4-fold over the control. However, suchup-regulation by c-Jun was abolished in the presence of themCREB-341 expression plasmid. On the other hand, in thepresence of mCREB-341, the increased amounts of pRSV-c-Junfailed to overcome the repression. Thus, these results demon-strate that mCREB-341 exerted its suppression through theAP1 site and prevented the binding of c-Jun. These resultsfurther support the observation that forced expression ofKCREB increased loricrin promoter activity, most likelythrough the dimerization with endogenous CREB protein, andthereby releases suppression (Fig. 6B).

As shown in the supershift analyses, the CREB proteins bindon the CRE-like site (Fig. 2E). We next examined whether theCREB protein binds on the CRE-like site and exerted its effecton the loricrin promoter. To prevent the binding of CREB onthe AP1 site, the AP1 site was mutated in the �154/�9 con-struct. The forced expression of mCREB-341 protein did notenhance but slightly reduced the CAT activity of �154/�9(AP1) construct (Fig. 6D, white bar). One possibility is thatthe binding of KSR-1 on the KSSE site interferes with thebinding of mCREB-341 on the CRE-like site. However, whenthe KSSE site was mutated, mCREB-341 still showed a repres-sion effect. After double mutations either on the KSSE andCRE-like sites or on the KSSE and AP1 sites, mCREB-341reduced more than 50% of CAT activity compared with thecontrol (black bar). The mCREB-341 did not suppress onlywhen both CRE-like and AP1 sites were mutated. Further-more, it is known that CREB acts as a repressor in the hy-pophosphorylated state (50). Thus, when we tested the co-transfection of mCREB-341 with loricrin constructs in thepresence of forskolin, we still observed the repression on theloricrin promoter by mCREB-341 (data not shown). On theother hand, the presence of pKCREB expression vector (Fig.6D, hatched bar) increased the CAT expression compared withthe control in all loricrin constructs except when CRE and AP1sites were both mutated. Taken together, these data indicatethe presence of other unidentified CREB family members thatcan up-regulate loricrin promoter in keratinocytes. Clearly,however, these results demonstrate that mCREB-341 acts as arepressor on loricrin promoter through two mechanisms; it caneither compete with c-Jun for the binding on AP1 site, or it candimerize with the unidentified endogenous regulator and in-terfere the CRE site-mediated transcription regulation.

FIG. 5. Forskolin up-regulates loricrin promoter activity inNHEK cells. Wild type and mutated loricrin CAT constructs weretransfected into NHEK cells. After termination of transfection, thecultures were maintained either in high calcium (1.2 mM, solid), ortreated with forskolin (10 �M, hatched) under high calcium condition for48 h before harvest. Data are presented as relative CAT activity in -foldchange as described in the legend to Fig. 1.

Complexity of Human Loricrin Transcription42274

by guest on April 1, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Gel Shift Analysis Displays the Translocation of Sp1, Sp3,and KSR-1 Proteins by Forskolin—In an attempt to understandthe possible mechanism for the up-regulation by forskolin ofloricrin promoter activity, both cytosolic fractions and nuclearextracts from keratinocyte cultures treated with or withoutforskolin were prepared for gel shift analysis. As shown in Fig.7A, whereas the intensity of the Sp1 complex was comparablebetween forskolin-treated and untreated samples, the intensi-ties of the Sp3 and KSR-1 complexes were both less in theforskolin-treated samples than in the untreated control (com-pare lanes 1 and 6). Also, the complex D band was diminishedin the forskolin-treated sample. The treatment of forskolinmight either affect the DNA binding capability of Sp3 andKSR-1, or cause the translocation of proteins from the nucleusto cytoplasmic compartments. The latter indeed proved to bethe case. When the same probe was incubated with the cyto-plasmic fraction from forskolin-treated and untreated samples(Fig. 7B), two binding complexes (KSR-1 and D), which werecompeted out by �117/�105 oligonucleotides (lane 5), were

found in untreated samples. In contrast, the complexes of Sp1,Sp3, and KSR-1 were found in the cytosol fractions of forskolin-treated samples. In addition, the �117/�105 oligomer reducedthe intensity of the KSR-1 complex and competed out the com-plex D (lane 10). These results suggest that activation of thePKA signal pathway by forskolin can lead to the translocationof Sp1, Sp3, and KSR-1 proteins (for complexes C and D) fromthe nucleus to the cytoplasm. It therefore seems likely that thisdeparture of especially Sp3 and KSR-1 results in the release ofthe suppression of loricrin expression, leading to the activationof the loricrin promoter. Furthermore, an increase of calciumconcentration may not be involved in this process, since similarresults were observed for forskolin-treated NHEK cells main-tained either in low or high calcium medium (data not shown).

The Interplay of Sp1, c-Jun, and p300 on Loricrin PromoterActivity—The role of transcriptional cofactor p300 involved inthe activation of Jun proteins (51) and the collaboration withSp1 in the activation of the p21 promoter in HeLa cells (52) hasbeen documented. Accordingly, we next explored the role of

FIG. 6. The effects of CREB, ATF-1,CBP, p300, and PKA � on the loricrinpromoter in NHEK cells. A, the loricrin�154/�9 CAT construct was co-trans-fected with the expression vectors as in-dicated into NHEK cells. The relativeCAT activity is in -fold change over thesame loricrin construct co-transfectedwith the parent vector alone. The respec-tive parent vectors included pRC/RSV (formCREB-341, KCREB, and CBP), pECE(for ATF-1), and pCMV (for p300 and PKA�). B, indicated loricrin CAT constructswere co-transfected with pRC/RSV-mCREB-341 (solid) or pRC/RSV-KCREB(hatched). The data are presented as de-scribed for A. C, the loricrin �67/�9 CATconstruct was co-transfected with the in-dicated amounts of pRSV, mCREB-341,c-Jun, and c-Jun/TAM expression vectors.The pRSV plasmid was used to ensureequal amounts of DNA were used in eachsample. The data are shown as relativeCAT activity in -fold change over �67/�9construct co-transfected with the pRSVplasmid alone. D, indicated loricrin CATconstructs were co-transfected with pRSV(black), mCREB-341 (open), and KCREB(hatched) expression vectors. The dataare shown as relative CAT activity in -foldchange over that from �154/�9 constructco-transfected with pRSV plasmid alone.

Complexity of Human Loricrin Transcription 42275

by guest on April 1, 2018

http://ww

w.jbc.org/

Dow

nloaded from

p300 in modulating the activity of Sp1 and c-Jun on the loricrinpromoter in NHEK cells. Loricrin constructs were co-trans-fected with vectors expressing either Sp1, p300, c-Jun, or c-Jun/TAM (a dominant negative mutant of c-Jun with a trun-cated transactivation domain). Co-transfection of Sp1 had amodest effect on the activity of �154/�9 construct (�50% high-er; Fig. 8A, solid), suggesting that Sp1 alone is incapable ofovercoming the repression effect on the loricrin promoter.Forced expressed of Sp1 did not show any effect when theKSSR site was mutated (Fig. 8A, shaded bar), suggesting thehigh level of endogenous Sp1 is sufficient to exert its maximaleffect on loricrin promoter activity. In the presence of c-Jun orp300, the CAT expression of construct �154/�9 increased4-fold over the control. Since the level of increase by c-Jun or

p300 on the wild type construct is 80% higher than that ofconstruct �154/�9 (KSSE) co-transfected with parent vectors(shaded bar); thus, forced expression of c-Jun or p300 is able toovercome the silencing effect and activate the loricrin pro-moter. Simultaneous co-transfection of c-Jun and Sp1 or c-Junand p300 expression vectors synergistically increased the levelof CAT activity 8-fold higher than the control. However, Sp1and p300 together did not show a synergistic effect on the wildtype construct. In fact, the level of expression in the presence ofSp1 and p300 was similar to that by p300 alone. Interestingly,when the KSSE site was mutated, co-transfection of Sp1 andp300 exerted a synergistic effect and activated the CAT expres-sion to the same level as by c-Jun and p300 together (shadedbar). In the presence of c-Jun, Sp1, and p300 expression vec-tors, the CAT activity of the wild type construct increased more

FIG. 7. Sp1, Sp3, and KSR-1 are translocated into the cyto-plasm in forskolin-treated NHEK cells. A, binding profile of thenuclear extracts (NE) isolated from NHEK cultures either grown underhigh calcium (1.2 mM) for 2 days (lane 1) or under the same conditionbut treated with 50 �M forskolin for 8 h before harvesting (NE/F, lane6). The �132/�90 oligonucleotide was used as the probe. Equivalentamount of proteins were loaded in each lane. The binding reactionswere incubated with a 100-fold molar excess of unlabeled probe (lanes 2and 7), Sp1 antibody (lanes 3 and 8), Sp3 antibody (lanes 4 and 9), or�117/�105 oligonucleotide (lanes 5 and 10). The arrows indicate thebinding complex with Sp1, Sp3, KSR-1, and complex D. B, bindingprofile of cytosolic extracts isolated from forskolin-treated (CE/F) oruntreated (CE) NHEK cultures as described above. The arrows indicatethe binding complexes from Sp1, Sp3, KSR-1, and complex D.

FIG. 8. Sp1, c-Jun, and p300 up-regulate the loricrin promoterin NHEK cells. A, the loricrin �154/�9 (solid) and loricrin with KSSEmutation (shaded) CAT constructs were co-transfected with pRSV-Sp1,pRSV-c-Jun, pRSV-c-Jun/TAM, and pCMV-p300. The parent vectors ofpRSV and pCMV were added to achieve equal amounts of DNA in eachsample. The results are from one representative experiment. Similarresults were observed from other experiments. B, the loricrin �154/�9construct (1.0 �g of each sample) was co-transfected with the indicatedSp1, c-Jun, and p300 expression vectors into NHEK cells. The asteriskindicates the control transfected with pRSV and pCMV at 0.5 �g each.The plus sign represents 0.25 �g of expression vector used in eachsample. The increased amounts of expression vectors represent 0.1, 0.5,and 1.0 �g for each expression vector.

Complexity of Human Loricrin Transcription42276

by guest on April 1, 2018

http://ww

w.jbc.org/

Dow

nloaded from

than 16-fold, indicating that Sp1 can potentiate the synergisticeffect caused by both p300 and c-Jun on the loricrin promoter.The transactivation effect of c-Jun was abolished in all caseswhen the transactivation domain of c-Jun was mutated (c-Jun/TAM). Also, the transactivation activity by c-Jun, Sp1, andp300 were either significantly reduced or abolished by muta-tions on either the Sp1 or AP1 sites, suggesting that both sitesare indispensable for loricrin promoter activity (data notshown). Furthermore, Sp1, c-Jun, and p300 showed a dose-de-pendent activation on the loricrin promoter. Both c-Jun andp300 showed a higher dose responsiveness than that of Sp1(Fig. 8B). These results support the observation from theco-transfection experiments that both c-Jun and p300 play acritical role in the control of loricrin expression. Taken to-gether, these data show that the effect of p300 on loricrinexpression could be exerted through the modulation of thec-Jun transactivation activity. Sp1 can exert its positiveeffect in the presence of c-Jun, suggesting that the two mightinteract directly or indirectly on the control of loricrin expres-sion. The possible interplay between Sp1 and p300 can beachieved only when the KSSE site is mutated. Furthermore,these results also demonstrate that the high level of loricrinpromoter activity can be achieved when Sp1, c-Jun, and p300factors are present in NHEK cells.

DISCUSSION

In the present study, we have characterized the regulatoryelements that are involved in cell type-specific expression of thehuman loricrin gene. Our study demonstrates that the tran-scription of this gene depends upon both proximal promoterand distal regions. The regulatory elements that are necessaryto restrict the loricrin expression during the late epidermaldifferentiation lie beyond the first 1.3 kbp 5�-upstream from thetranscription start site. However, we describe in detail here thepresence and function of keratinocyte-specific elements withinthe first 154 bp upstream from the transcription initiation site.

The AP1 Motif—A comparison of the similar proximal pro-moter region of the human and mouse loricrin genes reveals acommon AP1 motif. However, different family members of AP1proteins are involved in the two species. We have previouslyreported that c-Jun/c-Fos and Jun D activate and Jun B down-regulates human loricrin promoter activity (25). On the otherhand, c-Fos/Jun B heterodimers activate, whereas c-Jun andJun D show no effect on mouse loricrin promoter activity (18).Rutberg et al. (53) also reported that c-Fos/Jun B heterodimersinduce AP1 transcription activity in mouse keratinocytes. Onepossible explanation as to why there are differences betweenhumans and mice is that the expression pattern of AP1 familyproteins is different in the epidermis of humans and mice.Indeed, Jun B, Jun D and Fra-2 are predominantly expressedin basal and suprabasal layers, whereas c-Jun, c-Fos, and Fra-1are expressed in spinous and/or granular layer of human epi-dermis (54). However, in mouse epidermis, c-Jun is expressedin the basal layer, Jun D and Fra-1 are found in basal andspinous layers, and Jun B and Fra-2 are present in basal andgranular layers (55). Thus, depending upon the availability ofAP1 protein factors in the granular layer of the epidermis, thedimerization among different members of the AP1 family mightlead to different regulatory effects on loricrin expression inhuman or mouse epidermis.

Furthermore, it is likely that the interactions between AP1protein factors and other regulatory proteins in the proximalpromoter region contribute to the control of cell type specificityof loricrin expression. For example, we have found here that inaddition to the AP1 site, functional Sp1, KSSE, and CRE-likemotifs are also present in the proximal human loricrin pro-moter, whereas only functional AP1 and Sp1 sites were re-

ported in the mouse loricrin proximal promoter region (18, 56).Further, we found that the AP1 site alone is not sufficient toconfer keratinocyte-specific expression to the human loricrinpromoter, which is unlike another late differentiation markerprofilaggrin in which the AP1 motif is enough to restrict theexpression to epidermal cells (23). Thus, our data have shownthat at least these four elements together contribute both ac-tivation and repression in a keratinocyte-specific manner toloricrin expression in cultured NHEK cells.

Competitive Interrelationship between Sp1 and Sp3 Pro-teins—The involvement of Sp1 and Sp3 proteins has been dem-onstrated in the regulation of several epidermally expressedkeratin genes (20, 34, 57). This study on the human loricrinpromoter is the first report that both Sp1 and Sp3 regulate alate epidermal differentiation marker. In general, Sp1 acts asan activator and Sp3 can act as either an activator or a repres-sor for transcriptional regulation. Since both recognize a simi-lar GC-box motif, their relative availability during keratinocytedifferentiation becomes critical (i.e. the balance between thelevels of Sp1 and Sp3 may be responsible, at least in part, forthe activation or repression on their target genes in the epider-mis). Indeed, changes in the ratios of Sp1 and Sp3 have beenreported to modulate promoter activity in other cell types (58).Further, one study reported that the level of Sp1 is up-regu-lated and the level of Sp3 is down-regulated when humankeratinocytes are induced to differentiate by raising the extra-cellular calcium (59). Significantly, the present study confirmsthat the ratio of Sp1/Sp3 affects the loricrin promoter activityin NHEK cells (Fig. 4, B–D). On the other hand, when the PKAsignal pathway was activated by forskolin, we found that bothSp1 and Sp3 were translocated from the nucleus to the cytosol(Fig. 7B), which will lead to changes in the relative ratios of Sp1and Sp3 within nucleus. Because there is a relatively higherlevel of Sp1 compared with Sp3 in nuclear extracts of NHEKcells (Fig. 2B), it is therefore likely that Sp1 competitivelyreplaces Sp3 binding, and this in turn activates the loricrinpromoter. However, further work is needed to explore the na-ture of the nuclear-cytosolic translocation and to addresswhether phosphorylation might be involved.

Since Sp1 is a ubiquitously expressed transcription factor, itis possible that Sp1 interacts with other regulators to conferkeratinocyte-specific gene expression. The involvement of Sp1in the keratinocyte-specific expression of the human KRT14gene has been demonstrated (24). We found that forced expres-sion of Sp1 alone showed only modest effects on human loricrinpromoter activity. However, when the Sp1 site was mutated,the promoter activity was reduced to the basal level (Fig. 3),indicating its critical role in the transcriptional control of hu-man loricrin gene. By using the Gal4-Sp1 expression plasmids,we identified that the transactivation domains A, B, and C ofSp1 protein contribute the transactivation. Furthermore, ourdata showed that Sp1 and c-Jun act synergistically to increaseloricrin promoter activity. A similar observation was reportedin the study of the involucrin promoter in which the Sp1 sitecould enhance the activation by AP1 (60). Further study alsoindicated that Sp1 and AP1 motifs together are essential forinvolucrin expression during late keratinocyte differentiation(61). Besides the AP1 regulator, it has been reported that p300is required for the induction of p21 expression in keratinocytedifferentiation and that Sp1 and p300 might interact indirectlyfor the activation of the p21 promoter (52). In this study on thehuman loricrin promoter, we found a synergistic effect betweenSp1 and p300 only when the KSSE site was eliminated, indi-cating that the putative KSR-1 protein might interfere with theinteraction between Sp1 and p300. These data indicate a pos-sible mutually exclusive binding between Sp1 and KSR-1.

Complexity of Human Loricrin Transcription 42277

by guest on April 1, 2018

http://ww

w.jbc.org/

Dow

nloaded from

The AP2-like (KSSE) Motifs—The involvement of the AP2transcription factor in the regulation of tissue-specific expres-sion of K1, K5, and K14 has been reported (19, 62, 64). Inaddition, several functional AP2-like response elements werefound in the promoters of keratinocyte-specific genes such asbullous pemphigoid antigen (65), transglutaminase 1 (66), andinvolucrin (67). These AP2-like elements serve as enhancersand are believed to be involved in tissue-specific and differen-tiation-dependent expression. We have identified a KSSE sitein the human loricrin promoter that has a similar recognitionsequence (GCCGACAGCC) as the generic AP2 motif but in-stead is critical for keratinocyte-specific repression (Table I). Inaddition, we identified the presence of the putative KSR-1protein, but its identity remains unknown. KSR-1 may notbelong to the AP2 transcription factor family, since 1) forcedexpression of known AP2 family proteins activated the loricrinpromoter (data not shown) and 2) KSR-1 was found to be asilencer not an activator of loricrin promoter activity. KSR-1 islocated in both the cytoplasm and nucleus in keratinocytes(Fig. 7). Activation of PKA signal pathway leads to the reloca-tion of KSR-1 through an unknown mechanism. It is not clearwhether KSR-1 protein interacts with Sp3 directly or indirectlyand together contributes the silencing effect on loricrin expres-sion. However, forced expression of Sp3 reduced the promoteractivity to a similar degree regardless of the presence of KSSEsite (Fig. 4A). This suggests that Sp3 works independently forrepression. Further work is needed to determine whether theKSR-1 protein is related to other proteins such as KTF-1 (65)and KDF-1 (67) that bind to AP2-like response elements foundin the promoter region of keratinocyte-specific genes.

The CRE-like Motif—One intriguing finding in this study isthe involvement of CREB family proteins in the regulation ofloricrin transcription in keratinocytes. We found that CREB-1,CREM�, and ATF-1 proteins act as inhibitors of the loricrinpromoter. It has been documented that CREB and AP1 pro-teins can form heterodimers and bind to AP1 sites (48, 68, 69).The involvement of AP1 family members in the transcriptionregulation of epidermal genes has been well documented (23,63), but there are very few studies on the role of CREB pro-teins. In the human small proline-rich protein 1 promoterregion, a CRE-like site (�588) was identified, but no detailedstudy was conducted on its role in the regulation of smallproline-rich protein 1 expression (32). Jessen et al. (33) re-ported that the functional AP1 and CRE motifs were involvedin the regulation of human transglutaminase 1 gene expres-sion, but the possible interaction between the two was notstudied. Rutberg et al. (43) demonstrated that CREB familymembers are capable of binding to AP1 sites and repress thetranscription activity of a AP1 reporter in differentiated mousekeratinocytes in vitro.

Our new work on the human loricrin promoter is the firstepidermal gene in which there is a clear interaction betweenCREB and AP1 proteins at the AP1 site. Our data demon-strated that CREB proteins suppress the AP1-mediated acti-vation of the loricrin promoter by binding on the AP1 site (Fig.6C). Although we did not detect any change by CREB-1 anti-body on the AP1 binding profile in the supershift assays, thepossible dimerization between CREB and AP1 factors in kera-tinocytes cannot be ruled out. Since forced expressed c-Junalone can overcome the repression of �154/�9 construct (Fig.8A), it is possible that the high levels of introduced c-Jun cancompete with the endogenous CREB protein for the binding onAP1 site and activate loricrin promoter.

Interestingly, a CRE-like site is located just 40 bp down-stream from the AP1 site and 10 bp upstream of the KSSE site.This CRE-like site serves as a positive element only when the

KSSE motif is mutated, suggesting the likelihood of mutuallyexclusive binding between the proteins for these two sites. Thepossible existence of the unidentified positive regulator thatcan bind on the CRE-like site is based on two lines of evidence:1) there is a faster moving band of specific doublet complex, asshown in Fig. 2E, and 2) when the AP1 and KSSE sites weremutated, mCREB-341 still repressed the promoter activity, yetin the presence of KCREB, the promoter activity increased overthe control. The effects from both mCREB-341 and KCREBdiminished only when both CRE and AP1 sites were mutated(Fig. 6D). One likely possibility is that the CREB proteindimerizes with the unidentified positive regulator, which bindson this CRE-like site. This should result in suppression of theloricrin promoter activity in NHEK cells. Thus, the CREBprotein can either bind on the AP1 site or dimerize with AP1proteins and/or with an unidentified regulator, leading to thesuppression of loricrin promoter activity. The distribution pat-tern of the members of CREB family in the skin of newbornmice has been reported (43). The CREB, CREM�, and ATF-1are abundant in nuclei in both basal and spinous layers but notin granular layers. Accordingly, it is likely that the CREBprotein represses loricrin promoter activity, whereas the kera-tinocytes are in basal and spinous layers but not in the gran-ular layer, where c-Jun levels are high. Thus loricrin expres-sion can be initiated.

Model for the Control of the Human Loricrin Promoter—Wepropose the following model to account for our new data (Fig.9). When NHEK cells are in the basal and spinous layers, theloricrin promoter activity is suppressed through 1) the interac-tions of Jun B, Sp3, and KSR-1 proteins by binding to their

FIG. 9. A model for the regulation of human loricrin expres-sion in NHEK cells. This model accounts for the interactions betweenthe basal transcription machinery and the AP1, Sp1, Sp3, KSR-1,CREB-1, and p300/CBP proteins over the loricrin promoter in the basaland spinous (A and B) and terminally differentiating granular (C)layers of the epidermis described in this paper. The Sp1, KSSE, CRE-like, and AP1 motifs are indicated. Y represents the unknown activatorthat binds on the CRE-like site and, together with Sp1 and AP1 pro-teins, recruits the p300/CBP co-activator, resulting in the stabilizationof basal transcription machinery for activation of loricrin transcription.

Complexity of Human Loricrin Transcription42278

by guest on April 1, 2018

http://ww

w.jbc.org/

Dow

nloaded from

respective motifs, which prevents the binding of Sp1 and c-Jun(Fig. 9A); or 2) the CREB proteins bind on the AP1 site and/ordimerize with other positive regulators (Fig. 9B). When kera-tinocytes move to the granular layer a number of changesoccur: the Sp1/Sp3 ratio changes due to the departure of Sp3and KSR-1 proteins from the nucleus; the levels of CREBproteins become lower; relative levels of Sp1 and c-Jun becomehigher; and p300/CBP is recruited. Thus, a cooperative inter-action among these factors results in the assembly of basaltranscription machinery over the initiation site and leads to theactivation of human loricrin transcription (Fig. 9C). Thus, theprecise coordination in the combinatory interactions providesan on-off mechanism for optimal loricrin expression in thegranular layer of human epidermis.

Acknowledgments—We thank Antonello Rossi, Maria Morasso, andNedialka Markova for constructive comments during the course of thiswork.

REFERENCES

1. Watt, F. M. (1989) Curr. Opin. Cell Biol. 1, 1107–11152. Fuchs, E., and Byrne, C. (1994) Curr. Opin. Genet. Dev. 4, 725–7363. Fuchs, E. (1995) Annu. Rev. Cell Dev. Biol. 11, 123–1534. Roop, D. R. (1995) Science 267, 474–4755. Rice, R. H., and Green, H. (1979) Cell 18, 681–6946. Steinert, P. M., and Marekov, L. N. (1995) J. Biol. Chem. 270, 17702–177117. Marekov, L. N., and Steinert, P. M., (1998) J. Biol. Chem. 273, 17763–177708. Steinert, P. M., Kartasova, T., and Marekov, L. N. (1998) J. Biol. Chem. 273,

11758–117699. Steinert, P. M., Candi, E., Kartasova, T., and Marekov, L. N. (1998) J. Struct.

Biol. 122, 76–8510. Steven, A. C., and Steinert, P. M. (1994) J. Cell Sci. 107, 693–70011. Koch, P., de Viragh, P. A., Scharer, E., Bundman, D., Longley, M. A.,

Bickenbach, J., Kawachi, Y., Suga, Y., Zhou, Z., Huber, M., Hohl, D.,Kartasova, T., Jarnik, M., Steven, A. C., and Roop, D. R. (2000) J. Cell Biol.151, 389–400

12. Marshall, D., Hardman, M. J., Nield, K. M., and Byrne, C. (2001) Proc. Natl.Acad. Sci. U. S. A. 98, 3031–13036

13. Hohl, D. (1993) Am. J. Dermatophthol. 15, 20–2714. Hohl, D., Lichti, U., Bretkreutz, D., Steinert, P. M., and Roop, D. R. (1991)

J. Invest. Dermatol. 96, 414–41815. Mehrel, T., Hohl, D., Rothnagel, J. A., Longley, M. A., Bundman, D., Cheng, C.,

Lichti, U., Bisher, M. E., Steven, A. C., Steinert, P. M., Yuspa, S. H., andRoop, D. R. (1990) Cell 61, 1103–1112

16. Hohl, D., Mehrel, T., Lichti, U., Turner, M. L., Roop, D. R., and Steinert, P. M.(1991) J. Biol. Chem. 266, 6626–6636

17. DiSepio, D., Jones, A., Longley, M. A., Bundman, D., Rothnagel, J. A., andRoop, D. R. (1995) J. Biol. Chem. 270, 10792–10799

18. DiSepio, D., Bickenbach, J., Longley, M. A., Bundman, D., Rothnagel, J. A.,and Roop, D. R. (1995) Differentiation 64, 225–235

19. Ohtsuki, M., Flanagan, S., Freedberg, I., and Blumenberg, M. (1993) GeneExpr. 3, 201–213

20. Byrne, C., and Fuchs, E. (1993) Mol. Cell. Biol. 13, 3176–319021. Wu, R.-L., Cheng, T.-T., and Sun, T.-T. (1994) J. Biol. Chem. 269,

28450–2845922. Welter, J. F., Crish, J. F., Agarwal, C., and Echert, R. L. (1995) J. Biol. Chem.

270, 12614–1262223. Jang, S.-I., Steinert, P. M., and Markova, N. G. (1996)) J. Biol. Chem. 271,

24105–2411424. Lee, J.-H., Jang, S.-I., Yang, J.-M., Markova, N. G., and Steinert, P. M. (1996)

J. Biol. Chem. 271, 4561–456825. Rossi, A., Jang, S-I., Ceci, R., Steinert, P. M., and Markova, N. G. (1998)

J. Invest. Dermatol. 110, 34–4026. Yoneda, K., Hohl, D., McBride, O. W., Wang, M., Cehrs, K. U., Idler, W. W.,

and Steinert, P. M. (1992), J. Biol. Chem. 267, 18060–1806627. Sadovsky, Y., Webb, P., Lopez, G., Baxter, J. D., Fitzpatrick, P. M., Gizang-

Ginsburg, E., Cavilles, V., Parker, M. G., and Kushner, P. J. (1995) Mol.Cell. Biol. 15, 1554–1563

28. Pothier, F., Ouellet, M., Julien, J.-P., and Guerin, S. (1992) DNA Cell Biol. 11,83–90

29. Gill, G., Pascal, E., Tseng, Z. H., and Tjian, R. (1994) Proc. Natl. Acad. Sci.U. S. A. 91, 192–196

30. Blumenberg, M., Connolly, D. M., and Freedberg, I. M. (1992) J. Invest.Dermatol. 98, S42–S49

31. Heinemeyer, T., Wingender, E., Reuter, I., Hermjakob, H., Kel, A. E., Kel,O. V., Ignatieva, E. V., Ananko, E. A., Podkolodnaya, O. A., Kolpakov, F. A.,Podkolodny N. L., and Kolchanov, N. A. (1998) Nucleic Acids Res. 26,364–370

32. Reddy, S. P. M., Chuu, Y-J., Lao, P. N., Donn, J., Ann, D. K., and Wu, R. (1995)J. Biol. Chem. 270, 26451–26459

33. Jessen, B. A., Qin, Q., and Rice, R. H. (2000) Gene (Amst.) 254, 77–8534. Sinha, S., and Fuchs, E. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 2455–246035. Courey, A., and Tjian, R. (1988) Cell 55, 887–89836. Hagen, G., Muller, S., Beato, M., and Suske, G. (1994) EMBO J. 13, 3843–385137. Saffer, J. D., Jackson, S. P., and Annarella, M. B. (1991) Mol. Cell. Biol. 11,

2189–219938. Seamon, K. B., Padgett, W., Daly, J. W. (1981) Proc. Natl. Acad. Sci. U. S. A.

78, 3363–336739. Gonzalez, G. A., Yamamoto, K. K., Fischer, W. H., Karr, D., Menzel, P., Biggs,

W., III, Vale, W. W., and Montminy, M. R. (1989) Nature 337, 749–75240. Ginty D. D., Bonni, A., and Greenberg, M. E. (1994) Cell 77, 713–72541. Xing, J., Ginty, D. D., and Greenburg, M. E. (1996) Science 273, 959–96342. Monaco, L., and Sassone-Corsi, P. (1997) Oncogene 15, 2493–250043. Rutberg, S. E., Adams, T. L., Olive, M., Alexander, N., Vinson, C., and Yuspa,

S. H. (1999) Oncogene 18, 1569–157944. Chrivia, J. C., Kwok, R. P. S., Lamb, N., Hagiwara, M., Montminy, M., and

Goodman, R. H. (1993) Nature 65, 855–85945. Kwok, R. P. S., Lundblad, J. R., Chrivia, J. C., Richard, J. P., Bachinger, H. P.,

Brennan, R. G., Roberts, S. G. E., Green, M. R., and Goodman, R. H. (1994)Nature 370, 223–226

46. Lee, J. S., Zhang, X., and Shi, Y. (1996) J. Biol. Chem. 271, 17666–1767447. Hai T. W., Liu, F, Coukos, W. J., Green, M. R. (1989) Genes Dev. 3, 2083–209048. Hai, T. W., and Curran, T. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 3720–372449. Ryseck, R. P., and Bravo, R. (1991) Oncogene 6, 533–54250. Hagiwara, M., Alberts, A., Brindle, P., Meinkoth, J., Feramisco, J., Deng, T.,

Karin, M., Shenolikar, S., and Montminy, M. (1992) Cell 70, 105–11351. Lee, J.-S., See, R. H., Deng, T., and Shi, Y. (1996) Mol. Cell. Biol. 16,

4312–432652. Xiao, H., Hasegawa, T., and Isobe, K. (2000) J. Biol. Chem. 275, 1371–137653. Rutberg, S. E, Saez, E., Sam, L., Jang, S-I., Markova, N., Spiegelman, B. M.,

and Yuspa, S. H. (1997) Oncogene 15, 1337–134654. Welter, J. F., and Echert, R. L. (1995) Oncongene, 11, 2681–268755. Rutberg, S. E, Saez E., Glick, A., Dlugosz, A. A., Spiegelman, B. M., and Yuspa,

S. H. (1996)) Oncogene 13, 167–17656. Kawachi, Y., and Roop, D. R. (2001) J. Invest. Dermatol. 119, 376 (part 2)57. Brembeck, F. H., and Rustgi, A. K. (2000) J. Biol. Chem. 275, 28230–2823958. Hata, Y., Duh, E., Zhang, K., Robinson, G. S., and Aiello, L. P. (1998) J. Biol.

Chem. 273, 19249–1930359. Apt, D., Watts, R. M., Suske, G., and Bernard, H-U. (1996) Virology 224,

281–29160. Banks, E. B., Crish, J. F., Welter, J. F., and Eckert, R. L. (1998) Biochem. J.

331, 61–6861. Crish, J. F., Bone, F., Banks, E. B., and Eckert, R. L. (2002) Oncogene 21,

738–74762. Leask, A., Byrne, C., and Fuchs, E. (1991) Proc. Natl. Acad. Sci. U. S. A. 88,

7948–795263. Eckert, R. L., Crish, J. F., Banks, E. B., and Welter, J. F. (1997) J. Invest.

Dermatol. 109, 501–50964. Mariniello, L., Qin, Q., Jessen, B. A., Rice, R. H. (1995) J. Biol. Chem. 270,

31358–3136365. Tamai, K., Li, K., and Uitto, J. (1994) J. Biol. Chem. 269, 493–50266. Magnaldo, T., Bernerd, F., Freedberg, I. M., Ohtsuki, M., and Blumenberg, M.

(1993) DNA Cell Biol. 12, 911–92367. LaPres, J. J., and Hudson, L. G. (1996) J. Biol. Chem. 271, 23154–2316068. Hadman, M., Loo, M., and Bos, T. J. (1993) Oncongene, 8, 1895–190369. Chatton, B., Bocco, J. L., Goetz, J., Gaire, M., and Jorcano, J. L. (1995)

Oncongene 9, 375–385

Complexity of Human Loricrin Transcription 42279

by guest on April 1, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Shyh-Ing Jang and Peter M. SteinertInterplay between Transcription Factors of the Sp1, CREB, AP1, and AP2 FamiliesLoricrin Expression in Cultured Human Keratinocytes Is Controlled by a Complex

doi: 10.1074/jbc.M205593200 originally published online August 27, 20022002, 277:42268-42279.J. Biol. Chem. 

  10.1074/jbc.M205593200Access the most updated version of this article at doi:

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/277/44/42268.full.html#ref-list-1

This article cites 69 references, 32 of which can be accessed free at

by guest on April 1, 2018

http://ww

w.jbc.org/

Dow

nloaded from