Characterization of Mouse Pro®laggrin: Evidence for NuclearEngulfment and Translocation of the Pro®laggrin B-Domainduring Epidermal Differentiation

Dan Zhang,1 Seetha Karunaratne, Monica Kessler, Donna Mahony, and Joseph A. RothnagelDepartment of Biochemistry and Molecular Biology and the Center for Functional and Applied Genomics, University of Queensland, Brisbane,

Australia

Filaggrin is a keratin ®lament associated protein thatis expressed in granular layer keratinocytes andderived by sequential proteolysis from a polyproteinprecursor termed pro®laggrin. Depending on thespecies, each pro®laggrin molecule contains between10 and 20 ®laggrin subunits organized as tandemrepeats with a calcium-binding domain at the N-terminal end. We now report the characterization ofthe complete mouse gene. The structural organ-ization of the mouse gene is identical to the humanpro®laggrin gene and consists of three exons with a4 kb intron within the 5¢ noncoding region and a1.7 kb intron separating the sequences encoding thecalcium-binding EF-hand motifs. A processedpseudogene was found embedded within the secondintron. The third and largest exon encodes thesecond EF-hand, a basic domain (designated the B-domain) followed by 12 ®laggrin repeats and aunique C-terminal tail domain. A polyclonal anti-

body raised against the conceptually translatedsequence of the B-domain speci®cally stained kerato-hyalin granules and colocalized with a ®laggrin anti-body in granular layer cells. In upper granular layercells, B-domain containing keratohyalin granuleswere in close apposition to the nucleus and, in somecells, appeared to be completely engulfed by thenucleus. In transition layer cells, B-domain stainingwas evident in the nucleus whereas ®laggrin stainingremained cytoplasmic. Nuclear staining of the B-domain was also observed in primary mousekeratinocytes induced to differentiate. This study hasalso revealed signi®cant sequence homology betweenthe mouse and human promoter sequences and inthe calcium-binding domain but the remainder ofthe protein-coding region shows substantial diver-gence. Key words: expression/®laggrin/gene structure/protein domains. J Invest Dermatol 119:905±912, 2002

Filaggrin is a principal component of the mammalianepidermis that is expressed late in the differentiationprogram of keratinocytes (reviewed in Presland and Dale,2000). The major gene products of epidermal suprabasalcells are the differentiation-speci®c keratins, the keratin

associated proteins, and the proteins that are assembled into thecorni®ed cell envelope. The most predominant of the nonkeratinproteins are loricrin and ®laggrin, which initially accumulate asnonmembrane bound cytoplasmic aggregates known as kerato-hyalin granules that are characteristic of granular layer cells (Stevenet al, 1990; Manabe et al, 1991). It is thought that these granulesserve to sequest these proteins away from other cellular constituentsuntil they are required, and indeed their dispersal from thesegranules appears to be highly regulated (Presland and Dale, 2000;Kalinin et al, 2001).

Filaggrin is generated by the proteolytic cleavage of a short-lived,very high molecular weight (> 300 kDa), phosphorylated poly-protein precursor termed pro®laggrin (Scott and Harding, 1981;

Ramsden et al, 1983; Lonsdale-Eccles et al, 1984). Gene mappingand sequencing studies have determined that pro®laggrin iscomposed of multiple repeating units of ®laggrin, up to 20 ormore in rodents (Rothnagel and Steinert, 1990) and 10±12 copiesin humans (Presland et al, 1992; Markova et al, 1993). Theliberation of individual ®laggrin subunits from pro®laggrin and thecoincident dissolution of keratohyalin occurs only after the removalof phosphate groups from the precursor. This processing is carriedout by protein phosphatase PP2A and at least three differentproteases, including pro®laggrin proteinase 1, calpain, and furin,resulting in ®laggrin peptides of 27 kDa in mice and 35 kDa inhuman (Presland and Dale, 2000). Early biochemical studiesestablished that ®laggrin (and not the phosphorylated precursor)could speci®cally interact with keratin ®laments to form highlyordered structures known as macro®brils (Steinert et al, 1981;Lonsdale-Eccles et al, 1982; Harding and Scott, 1983). Someauthors have questioned the relevance of this interaction in vivo, asin diseases such as ichthyosis vulgaris, where there is little or no®laggrin present, keratin ®laments appear to condense normally(Sybert et al, 1985; Weidenthaler et al, 1993). Others havesuggested that there is enough residual ®laggrin left in theepidermis of these patients to account for the apparently normalappearance of their keratin ®lament bundles (Manabe et al, 1991),but another possibility is that this function is shared with relatedproteins such as hornerin (Makino et al, 2001). Remarkably,

0022-202X/02/$15.00 ´ Copyright # 2002 by The Society for Investigative Dermatology, Inc.

905

Manuscript received May 23, 2002; accepted for publication May 30,2002

Reprint requests to: Dr. Joseph Rothnagel, Department of Biochemistryand Molecular Biology, University of Queensland, Brisbane, QLD 4072,Australia; Email: j.rothnagel@uq.edu.au

1Present address: Lions Eye Institute af®liated with the University ofWestern Australia, Nedlands, WA 6009, Australia.

®laggrin is subjected to further modi®cations in the fully differen-tiated cells of the stratum corneum, where it is proteolyzed into freeamino acids that are thought to provide the high osmolaritynecessary for the retention of water and maintenance of tissue¯exibility (Scott et al, 1982; Horii et al, 1983).

Chromosomal mapping studies have determined that the humanpro®laggrin gene maps to chromosome 1q21 (McKinley-Grant et al,1989) and the mouse gene to the syntenic region on chromosome 3(Rothnagel et al, 1994). The pro®laggrin gene resides within a genecluster that makes up the epidermal differentiation complex, whichis a growing family of genes expressed by differentiatingkeratinocytes. The epidermal differentiation complex genes mostlikely have a common evolutionary history given their similargenomic organization and the characteristic repetitive motifs oftheir open reading frames. Three classes of epidermal differentiationcomplex genes have been recognized within a 1.9 Mb region of1q21: the S100 family of small calcium-binding proteins, thecorni®ed envelope precursor proteins such as the corni®ns(SPRRs), involucrin, loricrin, and the late envelope proteins(LEPs), and the S100-fused proteins of pro®laggrin, repetin, andtrichohyalin (Marenholz et al, 2001; Marshall et al, 2001). Sequenceanalysis of the human gene revealed that pro®laggrin contains twocalcium-binding motifs at the N-terminal with high homology tothe S100 family of calcium-binding proteins (Presland et al, 1992;1995; Markova et al, 1993). It has been postulated that thepro®laggrin calcium-binding domain (denoted the A-domain) maybe involved in calcium signal transduction in differentiatingkeratinocytes (Markova et al, 1993) or, alternatively, in regulatingcalcium availability to calcium-dependent enzymes such aspeptidylarginine deiminase and transglutaminase (Presland et al,1992; Markova et al, 1993). Sandwiched between the calcium-binding domain and the ®laggrin repeats lies a region of about 200amino acids (denoted the B-domain) that contains a highproportion of charged residues (Presland et al, 1992; Markovaet al, 1993). The function of this region is not known but hasrecently been postulated to be involved in triggering nuclear eventsassociated with the ®nal stages of epidermal differentiation (Ishida-Yamamoto et al, 1998; Presland and Dale, 2000). The C-terminalof pro®laggrin contains a distinct hydrophobic region of about 30amino acids that is somewhat conserved between species and maybe involved in the formation of pro®laggrin keratohyalin granules(Presland et al, 1992).

We now report the complete sequence of the mouse pro®laggringene.2 The exon/intron organization of the mouse gene is identicalto human pro®laggrin and the other multifunctional S100 genes,trichohyalin and repetin (Presland et al, 1992; Fietz et al, 1993; Leeet al, 1993; Markova et al, 1993; Krieg et al, 1997). The ®rst intronis located within the 5¢ noncoding region and the second intron liesbetween the two EF-hands of the calcium-binding domain. Thereare no introns in the ®laggrin repeat portion of the gene, whichcomprises the third and largest exon. As was observed for humanpro®laggrin (Ishida-Yamamoto et al, 1998), the mouse B-domain istranslocated to the nucleus after separation from the ®laggrinsubunits in the ®nal stages of keratinocyte differentiation. Inaddition, we found that nuclear translocation of the B-domain ispreceded by nuclear engulfment of keratohyalin granules.

MATERIALS AND METHODS

Preparation of P1 plasmid DNA A mouse (129/SVJ) P1 genomiclibrary (Genome Systems, St. Louis, MO) was screened by polymerasechain reaction (PCR) using primers to the 3¢ ¯anking region of themouse pro®laggrin gene (Rothnagel and Steinert, 1990), and onepositive clone (P1/2900) was isolated. Bacterial colonies transfected withthe P1/2900 clone were inoculated into 3 ml of LB medium containing25 mg per ml kanamycin and grown overnight at 37°C. The overnightculture was diluted 1:30 with LB medium containing the antibiotic andincubated for 1.5 h. Ampli®cation of P1 copy number was carried out

by the addition of isopropyl-b-D-thiogalactoside to a ®nal concentrationof 1 mM and incubation for a further 5 h. The P1 plasmid DNA wasisolated by alkaline lysis using the High Pure plasmid isolation kit(Boehringer Mannheim, Sydney, Australia).

Restriction mapping and southern blot analysis Mouse genomicand P1/2900 plasmid DNA was digested with BamH1, Rsa1, and EcoR1(New England Biolabs, Beverly, MA) and electrophoresed on 0.6%±0.8%agarose/TAE gels. The DNA fragments were immobilized on Hybond-N or NX nylon membranes (Amersham, Sydney, Australia) according tothe manufacturer's instructions. A 750 bp ®laggrin repeat probe (type B;Rothnagel and Steinert, 1990) and a 180 bp 3¢ noncoding probe(Rothnagel et al, 1987) were labeled with [a32P]dCTP using theRadPrime DNA labeling system (Gibco BRL, Gaithersburg, MD).Hybridization and washing were carried out using established protocols(Sambrook et al, 1989).

DNA subcloning and sequence analysis Restriction fragments fromthe P1/2900 clone were puri®ed using the QIAEX II gel extraction kit(Qiagen, Melbourne, Australia) and cloned into pGEMz vectors(Promega, Maddison, WI). Automatic DNA sequencing was performedusing a PCR-based cycle sequencing system (Amersham). In a fewinstances, manual sequencing was carried out using the SequenaseVersion 2.0 (Amersham). Larger DNA fragments were sequenced inboth orientations using internal primers. The sequence data wereanalyzed using the programs available through ANGIS (Sydney,Australia; http://angis.org.au/), NCBI (Bethesda, MD; http://www.ncbi.nlm.nih.gov), and Celara (Rockville, MD; http://www.celera.com/).

Rapid ampli®cation of cDNA ends (RACE) Total skin RNA wasisolated from newborn pups (Bickenbach et al, 1995). One microgram ofunfractionated RNA was reverse transcribed using 200 units ofSuperscript II reverse transcriptase (Gibco BRL) according to themanufacturer's protocol. A gene-speci®c primer (5¢-CTGGTCAGC-CCTGACTGG-3¢) corresponding to bp 682±699 of exon 3 was usedfor ®rst strand synthesis. After removal of the RNA template by RNaseH (Gibco BRL) digestion, the single-stranded cDNA was puri®ed usingQIAEX II (QIAGEN) and C-tailed with 200 mM dCTP and 15 units ofterminal deoxynucleotidyl transferase (Gibco BRL). The tailed cDNAreaction mixture was diluted 1:50 for PCR ampli®cation. The 5¢RACE reaction (Frohman et al, 1988) contained the anchor primer(5¢-AAGCGATCGCAATCTCTCGGGIIGGGIIGGGIIG-3¢) and anadapter primer (5¢-AAGCGATCGCAATCTCTC-3¢) in 1:8 ratio, theexon 3 speci®c primer used for cDNA synthesis, and 5 ml of the tailedcDNA. The reaction was heated to 95°C for 5 min prior to the additionof 2.5 units of Taq polymerase (Amersham). The PCR conditionsconsisted of 30 cycles of 94°C for 1 min, 48°C for 1 min, 72°C for1 min, followed by a ®nal extension at 72°C for 10 min. The sameconditions were used for a second round of ampli®cation using theadapter primer with a nested exon 3 primer (5¢-GGCTTC-TCGTTCTGTCAC-3¢) corresponding to bp 266±283 of exon 3, and1 ml of puri®ed ®rst round product. The ®nal product of about 500 bpwas cloned into pGEMT (Promega) and sequenced.

Antibodies Two synthetic peptides corresponding to residues 202±217(NYDEIYDNGKYNEDWE; B-domain peptide) and 328±343 (DSQ-VHSGVQVEGRRGQ; ®laggrin repeat peptide) of the pro®laggrin openreading frame were conjugated to diphtheria toxoid (Chiron,Melbourne, Australia) and used to elicit antibodies in rabbits and inguinea pigs. The speci®c antibodies were af®nity-puri®ed using apeptide-coupled sepharose column. All other antibodies used wereobtained commercially and include a monoclonal lamin B antibody,antirabbit IgG conjugated to Cy5 and biotin (Zymed, San Francisco,CA), antiguinea pig IgG conjugated to biotin (Sigma, St Louis, MO),antirabbit IgG conjugated to Alexa 488 and Alexa 594 ¯uorophores(Molecular Probes, Eugene, OR), and antirabbit IgG conjugated to5 nm or 10 nm gold particles (BBInternational, U.K.).

Immunohistochemistry Back skin from 5-d-old mouse was dissectedand embedded in Tissue Tek II OCT (Miles, Elkhart, ID). Sections 5±8 mm thick were cut at ±20°C and mounted on poly L-lysine coatedslides and dried at room temperature. Excess OCT was removed prior tostaining by washing in phosphate-buffered saline (PBS). For in vitrostudies, neonatal mouse primary keratinocytes were cultured in lowcalcium (0.05 mM) medium for 24 h and then switched to high calcium(0.12±0.16 mM) medium following the method described by Yuspa et al(1989). After harvesting, cells were ®xed in cold methanol and stored at±20°C. Skin sections and ®xed primary keratinocytes were blocked in5% fetal bovine serum, 1% bovine serum albumin (BSA), 0.05% Tween20 in TM buffer (10 mM Tris±HCl pH 7.5, 100 mM MgCl2) for 1 h at

2These sequences have been deposited in GenBank under the accessionnumbers AY094988and AF500171.

906 ZHANG ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

room temperature. Sections were incubated overnight with either rabbitanti-B-domain or rabbit anti®laggrin diluted at 1:100 in blockingsolution. The keratinocytes were usually incubated in primary antibodiesfor 1±2 h at room temperature. Where double labeling was required, amixture of guinea pig anti-B-domain and rabbit anti®laggrin were usedat appropriate dilutions. Nuclear membrane staining was detected byincubation with a monoclonal lamin B antibody (1:1000) for 1±2 h atroom temperature. Sections/keratinocytes were then washed three timesfor 5 min in Tris-buffered saline solution containing 0.05% Tween 20,incubated in speci®c secondary antibodies at predetermined optimaldilutions for 1 h at room temperature, washed as before, and mounted in¯uorescent mounting medium (Dako, Carpinteria, CA). Fluorescenceimages were captured as a Z series at 0.2 mm increments, using a Bio-Rad MRC600 (Hercules, CA) confocal laser scanning system attached toa Zeiss microscope with appropriate ®lters. NIH Image and AdobePhotoshop software programs were used for pseudo-coloring andmerging of the optical sections.

Immunoelectron microscopy Freshly harvested skin samples fromnewborn mice were diced into 1±2 mm square blocks and ®xed in 4%paraformaldehyde±PBS for 2 h at room temperature, prior todehydration and embedding in Lowicryl as described by Steven et al(1990). These samples were cut into 50 nm thin sections and mountedon carbon-coated formvar ®lms supported by copper grids. The gridswere ¯oated section-side down on droplets of the blocking solution(10% fetal bovine serum in PBS) for about 15 min and then incubatedon droplets of primary antibodies diluted in 1% BSA in PBS at roomtemperature for 2 h. Sections were then washed by ¯oating the grids ondroplets of 0.1% BSA in PBS at room temperature. Subsequentincubation with gold-conjugated IgG (BBInternational) was performedfor 1 h following the manufacturer's instructions. This was followed bythree washes as before and a 1 h wash in ultra pure water. Sections werefaintly stained with 5% uranyl acetate and Reynolds lead citrate (1±2 minin each) and examined using a JEOL 1010 transmission electronmicroscope at 80 kV.

RESULTS

Structure of the mouse pro®laggrin gene Restrictionmapping and southern blot analysis was used to identify ®laggrinsequences within the P1/2900 clone isolated from the mousegenomic library (data not shown). A coding probe generated fromthe 750 bp ®laggrin repeat (Rothnagel and Steinert, 1990) detecteda single high molecular weight fragment (> 30 kb) in an EcoR1digest of the P1/2900 clone, which was of similar size to thatobserved in a genomic DNA digest (Rothnagel et al, 1987). Thesame band was also identi®ed using a 3¢ noncoding probe,indicating that this fragment most probably contained the entiregene. The coding probe also detected three BamHI digestedfragments (0.8, 1.8, and 11.0 kb) and three RsaI digested fragments(2.3, 3.6, and 6.0 kb), which were all subcloned for further

characterization. Restriction mapping and sequence analysisshowed that the 11.0 kb BamHI fragment contained part of exon3, the ®rst and second introns, the promoter region, and 4.0 kb of5¢ ¯anking information. Similarly the 6.0 kb RsaI fragmentcontained 0.5 kb of intron 2 and 5.5 kb of exon 3 sequences.Restriction mapping of the 6.0, 3.6, and 2.3 kb RsaI clonesrevealed that they encode 5.5, 3.5, and 3 ®laggrin repeats,respectively. Further analysis revealed that the mouse pro®laggringene in the P1/2900 clone encodes an 11.5 kb mRNA consistingof 12 full ®laggrin repeats (three type A and nine type B) and twopartial repeats (Fig 1).

Determination of the transcription initiation site and intronboundaries We used 5¢ RACE to identify sequencescorresponding to the 5¢ end of the mRNA. The largest fragmentisolated after ampli®cation contained the sequences for the 5¢untranslated region, the calcium-binding domain, and most of theB-domain in exon 3. By comparing this sequence with thegenomic sequence ascertained from the P1 clone, we determinedthat the transcription start site occurs 24 bp downstream of theputative TATA box motif. In addition, we were able to identify thesplice junctions for both introns (Fig 2). This analysis revealed thatintron 1 is 4.0 kb in length and intron 2 is 1.67 kb long, whichcompares to 9.6 kb and 0.5 kb for the respective introns of thehuman homolog. The introns are located in the same relativepositions in both mouse and human pro®laggrin genes.

Comparison of mouse and human N-terminal domainsConceptual translation of the 5¢ RACE product revealed acalcium-binding domain and a basic domain of 202 residues. Thecalcium-binding domain of 81 residues has signi®cant sequencehomology to human pro®laggrin and other EF-hand proteins of theS100 family (Fig 3). The mouse B-domain has much lesshomology with the human sequence but nevertheless has asimilar pI and a similar distribution of charged residues (Fig 4).Of interest is the presence of four overlapping nuclear localizationsignals of the bipartite type (Dingwall and Laskey, 1991) in themouse sequence compared with only one in human B-domain(Ishida-Yamamoto et al, 1998). In addition, the mouse sequencecontains the conserved furin protease recognition sequence, whichhas been shown to be the cleavage site that removes the B-domainfrom the ®laggrin repeat portion of human pro®laggrin (Presland etal, 1997).

Immuno¯uorescent analysis of mouse pro®laggrin expres-sion We produced an antibody against a synthetic peptidededuced from the N-terminal B-domain sequence in order tofurther characterize pro®laggrin expression. The peptide

Figure 1. Schematic showing the organization of the pro®laggrin gene and the protein coding domains. The domains are denoted after theterminology of Presland et al (1997) as follows: (a) calcium-binding domain; (b) B-domain; (c) partial ®laggrin peptides; (d) ®laggrin repeats, and (e) C-terminal hydrophobic domain. Two forms of ®laggrin repeat sequences have been identi®ed based on their length of 255 and 250 residues (765/750 bp), which are denoted as types A and B, respectively (see Rothnagel and Steinert, 1990). The gene contains nine type B repeats and three type Arepeats.

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NYDEIYDNGKYNEDWE is located 201 residues downstream ofthe initiation codon within the unique B-domain and was chosenin preference to sequences within the calcium-binding domain tominimize cross-reaction with S100 proteins. Western blot analysisof whole skin and epidermal extracts showed that the anti-B-domain antibody detected pro®laggrin and some lower molecularweight peptides with a molecular weight of 40±50 kDa but not®laggrin itself (data not shown). This is consistent with the earlierstudies on human pro®laggrin, which found that the B-domain iscleaved from the ®laggrin repeat portion of pro®laggrin duringprocessing (Markova et al, 1993; Presland et al, 1995; 1997).Double-label immuno¯uorescence analysis on frozen sections of

neonatal back skin showed that the B-domain antibody speci®callystained keratohyalin granules and colocalized with ®laggrin ingranular layer keratinocytes (Fig 5A). Colocalization of the twoantibodies was less evident in upper granular and corni®ed layercells, however. Double-label immuno¯uorescence with antilaminB revealed the presence of the B-domain, but not ®laggrin, in thenuclei of some upper granular and transition layer cells (Fig 5B±E).The B-domain was mainly localized to the perinuclear region orinside the nucleus whereas ®laggrin antibody staining was neverobserved in the nucleus. A similar expression pattern was also seenin cultured mouse primary keratinocytes that had been induced todifferentiate by the addition of calcium (0.12±0.16 mM) into thegrowth medium for 24±48 h. Both the B-domain and ®laggrinantibodies showed ®ne granular staining in primary mousekeratinocytes grown in high calcium for 24 h that wasexclusively cytoplasmic (Fig 5F, G, respectively). After 48 h inhigh calcium medium, however, this expression pattern becamemore punctate and resembled keratohyalin granules with some B-domain staining now evident inside the nucleus (Fig 5H), whereas®laggrin staining remained cytoplasmic (Fig 5I). This wascon®rmed by examining the full series of optical sections througha labeled cell. Interestingly, some granules were so closely associatedwith the nucleus that they distorted the nuclear membrane,whereas others were completely invaginated, giving the appearanceof being physically inside the nucleus.

Immunoelectron microscopy analysis of mouse pro®laggrinexpression Immunoelectron microscopy using neonatal mouseskin labeled with the ®laggrin and the B-domain antibodies, eithersingly or in combination, con®rmed the immuno¯uorescence®ndings. Attempts to gold label the nuclear membrane with thelamin B antibody were not successful but nuclear membranes could

Figure 3. Comparison of the N-terminal calcium-bindingdomain of mouse pro®laggrin with human pro®laggrin andrelated proteins. The conceptual translated N-terminal residues ofmouse pro®laggrin (mFlg) are compared with human pro®laggrin (FLG)(Markova et al, 1992; Presland et al, 1992), human trichohyalin (THH)(Lee et al, 1993), mouse hornerin (mHorn) (Makino et al, 2001), andmouse repetin (mRptn) (Krieg et al, 1997). The conserved helix-turn-helix motifs of the EF-hands (Lewit-Bentley and Rety, 2000) areindicated. The sequences encoding the two EF-hands are separated by anintron whose position is conserved (indicated by an arrowhead). Thesequences were aligned using CLUSTALW.

Figure 4. Sequence comparison of mouse and human pro-®laggrin B-domains. The conceptually translated B-domain sequenceswere aligned using BLAST2. Identical residues are indicated with darkshading and conserved nonidentical residues with light shading. Thenuclear localization signals were identi®ed using PSORTII and areindicated by an asterisk. The furin protease recognition sequence thatdemarcates the C-terminal end of the B-domain (Presland et al, 1997) isunderlined.

Figure 2. Sequence of the 5¢ end of the mouse pro®laggrin gene.The sequence shows the 5¢ end of the transcription unit, the 5¢noncoding region (exon 1), two introns, and the ®rst 554 amino acids.The TATA box, OctB, Sp1, ets/jen, AP1, NF-kB, Dlx3/OctA, andintron splice sites are indicated. The transcription initiation site isindicated by a bent arrow. The sequences of exons 1, 2, and 3 are inuppercase. The promoter region and introns are in lowercase. Theconceptual translation of the open reading frame is shown below thenucleotide sequence. The furin recognition sequence demarcating thejunction between the N-terminal leader sequence and the partial®laggrin repeat is indicated by shaded lettering (residues 280±283). Thehydrophobic residues of the ®rst ®laggrin linker are highlighted (residues455±462). The B-domain (residues 202±217) and ®laggrin repeat(residues 328±343) peptide sequences used to produce antibodies areunderlined.

908 ZHANG ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

be clearly seen after uranyl acetate staining. Gold labeling with theB-domain and ®laggrin antibodies revealed colocalization of®laggrin subunits and the B-domain in keratohyalin granules ofearly granular layer cells (data not shown) but not in the outerepidermal layers (Fig 6A, B). Notably, the nuclei in uppergranular/transition layer cells were labeled with the B-domainantibody and in some cells this could be attributed to engulfment ofa dissolving keratohyalin granule by a crenulated nucleus (Fig 6C±F). This is clearly seen in sections through the mid-axis of akeratohyalin granule that is being invaginated by the nucleus(Fig 6D, E). The nuclear membrane in this cell is still intact and a

thin layer of nucleoplasm surrounds the keratohyalin granuleundergoing dissolution. In more advanced stages of differentiation,where nuclear breakdown is well under way, keratohyalin granulesare no longer evident but B-domain staining is associated withnuclear remnants (Fig 6A, B).

DISCUSSION

Features of the mouse pro®laggrin gene The organization ofthe mouse pro®laggrin gene is identical to human pro®laggrin andthat of the trichohyalin and repetin genes (Presland et al, 1992; Fietzet al, 1993; Lee et al, 1993; Markova et al, 1993; Krieg et al, 1997).All these genes contain a large intron within their 5¢ untranslatedregion and a second smaller intron separating the two EF-hands ofthe calcium-binding domain. The ®rst intron of the mouse gene is4.0 kb long compared to 9.6 kb in the human pro®laggrin genewith little apparent homology apart from sequences specifying thesplice junctions (Fig 2). The second intron of the mousepro®laggrin gene is 1.67 kb long, which compares to 0.5 kb forthe human gene. The additional 1.2 kb can be wholly attributed tothe presence of a processed pseudogene for the mouse acidicribosomal phosphatase (MARP) gene, which is not found in thehuman gene. Comparison with the published MARP sequence(Krowczynska et al, 1989) revealed that the intron II sequencecontains a complete MARP transcript including a short poly A tailbut the open reading frame has several changes that would beincompatible with producing a functional product. A comparisonof the mouse and human ®laggrin proximal promoter regionsrevealed very high homology (75%) for sequences between±300 bp and +25 bp relative to the transcription start site, and anumber of regulatory motifs previously identi®ed in the humangene (Presland et al, 1992; Markova et al, 1993; Jang et al, 1996;2000; Morasso et al, 1996) are conserved in the mouse sequenceincluding OctB, Sp1, ets/jen, AP1, and NF-kB sites (Fig 2). Thecis-element for Dlx3, a homeodomain protein related to DrosophilaDistal-less and found to be an important regulator of epidermaldifferentiation (Morasso et al, 1996), is also present in the mousegene.

Comparison of the mouse pro®laggrin gene in the P1/2900clone with the published sequence for mouse ®laggrin and the 3¢end of the pro®laggrin gene (Rothnagel et al, 1987; Rothnagel andSteinert, 1990) showed only minor variation between thesesequences. The mapping analysis did show differences in thenumber of ®laggrin repeats, however (12 in the P1/2900 cloneversus 20 in the cosmid clone of the earlier study), and in theirsequential order (see Fig 1 and Rothnagel and Steinert, 1990). Asthe P1/2900 DNA is from the 129/SVJ strain and the cosmid clonefrom NIH3T3 cells, it suggests that the repeat number variesmarkedly between different mouse strains. In a preliminary analysiswe digested genomic DNA from several strains with BstZ17I,which cuts only in a sequence found in type A repeats (GTATAC),and found large differences in the number and size of ®laggrincontaining fragments (data not shown). This suggests that both thenumber of ®laggrin repeats and the organization of the type A/typeB sequences differ between the different mouse strains.

Expression analysis of the mouse pro®laggrin gene Earlierin situ mRNA hybridization and immunolocalization studiesdetermined that pro®laggrin is expressed in the granular layer(Rothnagel et al, 1987; Steven et al, 1990; Manabe et al, 1991). In

Figure 5. Double-label immuno¯uorescence analysis of B-domain and ®laggrin expression in 5-d-old mouse skin and inmouse primary keratinocytes. (A) Granular layer and corni®ed layercells are double labeled with ®laggrin repeat (red) and the B-domain(green) antibodies. Colocalization is observed as yellow. (B) A view ofupper granular layer and transition cells double labeled with the B-domain (green) and lamin B (red). (C) A view of upper granular layer andtransition cells double labeled with the ®laggrin repeat (green) and laminB (red) antibodies. (D) Enlarged view of the boxed region in (B)showing B-domain staining within a nucleus of an upper granular layer/transition cell. (E) Enlarged view of the boxed region in (C) showing®laggrin repeat staining in the cytoplasm of an upper granular layer/transition cell. (F) A differentiating keratinocyte double labeled with B-domain (green) and nuclear lamin B (red) antibodies. (G) A differentiatingkeratinocyte double labeled with ®laggrin repeat (green) and lamin B (red)antibodies. Note the ®ne granular appearance of the B-domain and®laggrin staining pattern in (F) and (G). (H) A late-differentiatingkeratinocyte labeled for the B-domain (green) and lamin B (red). (I) Alate-differentiating keratinocyte double labeled for the ®laggrin repeat(green) and lamin B (red) antibodies. Note the larger aggregates of B-domain and ®laggrin staining in (H) and (I) and the B-domain staining inand around the nucleus and the cytoplasmic localization of the ®laggrinrepeat. CL, corni®ed layer; GL, granular layer; TL, transition layer. Scalebar: 10 mm.

Figure 6. Immunoelectron microscopy analysis of B-domain and ®laggrin expression. Ultrathin sections of a 5-d-old mouse backskinepidermis showing the expression of ®laggrin repeat (10 nm gold, pseudocolored red) and the B-domain (5 nm gold, pseudocolored green). (A) Asection showing a transition cell (TC) and a neighboring corni®ed layer cell (CL) above it and a granular layer cell (GC) below it. (B) The boxed areain (A) enlarged to show B-domain labeling associated with nuclear remnants and ®laggrin repeat labeling over ®laments. (C), (D) Two different uppergranular layer cells labeled for the B-domain. Note the keratohyalin granule (KG) being engulfed by the nucleus in (D). (E) A close up of thekeratohyalin granule in (D) showing B-domain labeling of the granule, which is surrounded by an apparently intact nuclear membrane. (F) A sectionstained with uranyl acetate and lead citrate showing a granular layer cell with a deeply indented nucleus. CL, corni®ed layer; GC, granular layer cell;KG, keratohyalin granule; NUC, nucleus; NM, nuclear membrane; NR, nuclear remnants; PM, plasma membrane; TC, transition cell. Scale bar:100 nm.

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this study, the B-domain and the ®laggrin repeats were ®rstobserved in the ®ne keratohyalin granules of the lower granularlayer. In more mature granular layer cells, both antibodies localizedto larger keratohyalin granules of which many were in a perinuclearposition. In the upper granular layer the antibodies began to showdifferential localization consistent with the enzymatic release of the®laggrin subunits from the pro-protein and the dissolution of thekeratohyalin granule. As has been shown in human skin (Ishida-Yamamoto et al, 1998), the ®laggrin repeats were distributed alongkeratin ®laments whereas the B-domain tended to localize in andaround the nucleus (Figs 5, 6).

The B-domain of human pro®laggrin has been shown to containa nuclear targeting sequence, which is believed to be responsible formediating the translocation of the B-domain in the transition layernucleus (Ishida-Yamamoto et al, 1998). Similarly, four nucleartargeting sequences of the bipartite type are present in the mouseB-domain (Fig 4), which suggests a conserved role for this domain.We observed discrete gold particles in the nuclei of upper granularand transition layer cells in ultrathin sections of mouse skin labeledwith the anti-B-domain antibody (Fig 6), con®rming the obser-vations on human B-domain. We also observed keratohyalingranules, however, that had been partially or completely engulfedby the nucleus in a subset of these cells. Similar keratohyalin-likenuclear inclusions that are associated with nuclear invaginations ofupper granular layer cells have been reported previously (Wier et al,1971; Karasek et al, 1972; Karasek, 1988). According to one report,the occurrence of keratohyalin-like granules in the nuclear regionof differentiating granular cells has been attributed to the degrad-ation of the nucleus (Lavker and Matoltsy, 1970). In this model, it issuggested that the breakdown of the nucleus allows the migrationof keratohyalin granules into the space previously occupied by thenucleus. Our observations, however, suggest that keratohyalingranules are actively engulfed by the nucleus prior to nucleardegradation. One outcome of this process is that the surface area ofthe nuclear membrane that is in contact with the keratohyalingranule is markedly increased, which may facilitate the trans-location of the B-domain into the nucleus. We favor a two-stepmodel that is consistent with past morphologic observations and theantibody localization data of this study. In this model, pro®laggrin-containing keratohyalin granules preferentially localize to peri-nuclear sites where some are partially internalized by upper granularlayer nuclei before nuclear degradation begins. The second stepinvolves the dissolution of the granule and the translocation of theB-domain into the nucleus. It remains to be determined whetherthe B-domain, perhaps with the calcium-binding domain in tow,signals the ®nal nuclear events of terminal differentiation.

JAR was supported by a Wellcome Trust Senior Research Fellowship in Medical

Research (Australia). This work was supported by an NHMRC grant to JAR. We

thank Dr. Pierre de Viragh for his input into the initial stages of this project.

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