Expression of Hornerin in Stratified Squamous Epithelium in the Mouse: A Comparative Analysis with Profilaggrin

Abstract

We have recently identified a novel protein named hornerin, the structural features of which are most similar to those of profilaggrin, an essential protein for keratinization of epidermal tissues. In this study we examined the expression of hornerin compared with that of profilaggrin in various mouse tissues. Hornerin was expressed in the upper epidermis of newborn mouse skin, as was profilaggrin. In addition, both hornerin and profilaggrin were expressed in the tongue, esophagus, and forestomach. In all four tissues, immunostaining for hornerin and profilaggrin showed a granular pattern, and most of the signals for the two proteins were co-localized on keratohyalin granules. This was confirmed by double immunoelectron microscopy. Within keratohyalin granules, hornerin was detected more frequently in the periphery, whereas profilaggrin was equally distributed. A quantitative RT-PCR revealed that both genes were expressed at highest levels in the forestomach and at the next highest levels in skin. Profilaggrin mRNA was most abundant in the forestomach. In skin, the amount of hornerin mRNA was more than fourfold greater than the amount of profilaggrin mRNA. These results form the basis for a better understanding of possible overlapping and/or differential functions of hornerin and profilaggrin.

Stratification and cornification of the squamous epithelium are essential for resistance to various insults from the external environment. Filaggrin, among other proteins, plays unique pleiotropic roles in that process (Markova et al. 1993). Synthesized as a high molecular weight precursor phosphoprotein, profilaggrin (Dale 1977; Lonsdale-Eccles et al. 1984), filaggrin promotes the aggregation of keratin intermediate filaments, resulting in the formation of disulfide bonds among them (Dale et al. 1993,1994). In accordance with such functions of filaggrin, a subset of ichthyosis vulgaris patients who lack the epidermal granular layer and exhibit an abnormality in cornification (termed IV AGL) express reduced amounts of profilaggrin mRNA and protein in keratinocytes (Sybert et al. 1985; Nirunsuksiri et al. 1995). A mouse mutant flaky tail (ft/ft), showing skin abnormalities similar to those of human ichthyosis vulgaris AGL, completely lacked the mature filaggrin protein (Presland et al. 2000).

We have recently identified a novel protein named hornerin, which comprises EF-hand domains at the N-terminus, followed by a spacer sequence and a large repetitive domain (Makino et al. 2001). The structural features of hornerin indicate that it is a novel member of the “fused gene”-type cornified envelope precursor protein family (Presland et al. 1992; Lee et al. 1993), to which profilaggrin also belongs. The hornerin protein was detected in keratohyalin granules of epidermal granular cells together with profilaggrin. Furthermore, the hornerin protein appeared to undergo post-translational proteolytic processing, as is observed in profilaggrin. Considering the essential role of filaggrin and the similarity of hornerin to profilaggrin, it is important to determine possible overlapping and/or differential functions of hornerin and profilaggrin. In this study we therefore examined the expression of hornerin compared with that of profilaggrin in various mouse tissues as a step towards this goal.

Materials and Methods

Tissue Samples

ICR mice (Nippon SLC; Hamamatsu, Japan) were used throughout the experiments. The skin of the back was isolated from mice 3 days after birth. The tongue, esophagus, and forestomach were resected from adult mice. All procedures were performed according to conventional principles for animal care and the guidelines of Okayama University.

In Situ Hybridization

In situ hybridization (ISH) was performed using an ∼500-bp 3′-untranslated region of hornerin cDNA as a probe under conditions described previously (Takaishi and Huh 1999). Skin tissues of newborn mice were fixed in 4% paraformaldehyde/PBS and embedded in paraffin for making sections. The probe was labeled with digoxigenin (Roche Diagnostics; Basel, Switzerland).

Antibodies

Antibodies against hornerin were prepared as previously reported (Makino et al. 2001). Briefly, cDNA fragments covering either the EF-hand region plus a part of the spacer or a repeat A3 (Figure 1, upper panel) were amplified by RT-PCR and subcloned into pGEX-6P-1 (glutathione S-transferase gene fusion vector; Amersham Pharmacia Biotech, Poole, UK). The recombinant proteins were purified using glutathione-Sepharose 4B (Amersham Pharmacia Biotech) and injected into rabbits with an adjuvant (TiterMax Gold, CytRx). The resulting antibodies were affinity-purified using a Hitrap NHS-activated column (Amersham Pharmacia Biotech) conjugated with the purified immunogens without a GST moiety.

Figure 1

Immunogens for the preparation of antibodies against hornerin and expression of hornerin in mouse skin. (Upper panel) Two regions of hornerin protein, the EF-hand and repeat A3 regions, were used for immunizing rabbits. The repeats were classified into two groups, i.e., As and Bs (see Makino et al. 2001). (A) hematoxylin-eosin staining; (B) in situ hybridization; (C) immunohistochemistry with anti-repA antibody; (D) immunohistochemistry with anti-EF antibody; (E) immunohistochemistry with preimmune serum; (F) immunohistochemistry with anti-filaggrin (anti-Filg) antibody. Bar = 20 μm.

A polyclonal antibody against mouse filaggrin (anti-Filg) and FITC-labeled anti-Filg were purchased from Berkeley Antibody (Richmond, CA).

Immunostaining

Paraffin sections were made from tissues that had been fixed in 4% paraformaldehyde/PBS for 3 hr to overnight and had then been treated with 1 μg/ml proteinase K at 37C for 15 min. After blocking with 1% goat serum, 0.1% bovine serum albumin, and 0.02% sodium azide in buffer, the antibody against the EF-hand region (anti-EF) or the repetitive region (anti-repA) of hornerin was applied. The signals were detected with Envision+ (DAKO; Carpinteria, CA), followed by staining using a Diaminobenzidine Reagent Set (Kirkegaard Perry Laboratories; Gaithersburg, MD). For immunofluorescent observation, AlexaFluor 488 goat anti-rabbit IgG (H+L) or Alexa Fluor 546 goat anti-rabbit IgG (H+L) (Molecular Probes: Leiden, The Netherlands) was used as the second antibody. For double staining, FITC-labeled anti-Filg (Berkley Antibody) was applied after thorough washing. The tissue sections were observed under a confocal laser microscope, LSM510 (Carl Zeiss; Oberkochen, Germany).

Immunoelectron Microscopy

Skin tissues from newborn mice were cut into 2 × 2-mm pieces and fixed in 4% paraformaldehyde/PBS at 4C overnight. After dehydration, the tissues were embedded in LR White Resin (HARD GRADE Acrylic Resin; London Resin, London, UK) and the resin was polymerized with an accelerating agent at 45C overnight. Thin sections (∼70–100 nm in thickness) were blocked with 1% bovine serum albumin (BSA)/PBS and then incubated at 4C overnight with the first antibodies, anti-repA, anti-Filg, or 1% BSA as a negative control. After washing with 1% BSA/PBS, the sections were incubated with anti-rabbit IgG antibody labeled with 20-nm gold particles (British Biocell; Cardiff, UK) at room temperature for 2 hr. The samples were finally stained with uranium acetate and lead citrate under conventional conditions.

For double immunostaining, the skin sections were successively treated with anti-repA, anti-rabbit IgG antibody labeled with 20-nm gold particles (British Biocell), anti-Filg labeled with FITC (Berkley Antibody), and anti-FITC antibody labeled with 10-nm gold particles (British Biocell), with thorough washings between each incubation. The final staining with uranium acetate and lead citrate was the same as described above. To determine the relative distributions of hornerin and profilaggrin in keratohyalin granules, we counted the number of gold particles in three concentric oval regions of the granules with the same length of radii on electron microscopic pictures. The results are expressed as arbitrary units of density.

Quantitative RT-PCR

Quantitative RT-PCR was performed using a QuantTect SYBR RT-PCR kit according to the protocol of the manufacturer (Qiagen; Bothell, WA). Primers used and the expected sizes of amplified fragments are shown in Table 1. RNA samples pretreated with DNase I were reverse-transcribed by Superscript II (Invitrogen; Carlsbad, CA), and the resulting cDNAs were amplified with SYBR Green under thermocycling conditions at 50C for 2 min and 95C for 15 min, followed by 40 cycles at 95C for 30 sec, 55C for 30 sec, and 72C for 30 sec. Numbers of copies of hornerin and profilaggrin mRNA were estimated by comparison with standard curves and expressed as copy ratios to GAPDH.

Table 1

Primers used for quantitative RT-PCR

Primer/probe	Sequence	Product length
Hornerin		158 bp
Sense	5′-ATGAGTATGTCCAAGAGCAGAG-3′
Antisense	5′-ATGGATGAGAGCAGATATGTCC-3′
Profilaggrin		137 bp
Sense	5′-GCAAGTGGTCAGGGAGGATAT-3′
Antisense	5′-GGAACGATATACCTGGAGATGC-3′
GAPDH		236 bp
Sense	5′-CTTCACCACCATGGAGAAGGC-3′
Antisense	5′-GGCATGGACTGTGGTCATGAG-3′

Results

Immunostaining of Mouse Tissues

We raised antibodies against two different regions of hornerin protein (Figure 1, upper panel), and the staining patterns with both antibodies were very similar, as shown in Figures 1C and 1D. Therefore, we show only the results of immunostaining using anti-repA antibody (i.e., the antibody to the repeat region A3) hereafter.

Hornerin was expressed in the upper epidermis of mice at 3 days after birth, which we used because the epidermis was thickest (Figures 1B–1D). Hornerin transcript was detected in the granular layer (Figure 1B), and positive staining for hornerin protein was observed in the granular and horny layers (Figures 1C and 1D). Preimmune serum used as a first antibody showed no staining (Figure 1E). The antibody against filaggrin gave a localization of staining similar to that for hornerin (Figure 1F).

Stratified squamous epithelia other than that of the skin (i.e., those of the tongue, esophagus, and forestomach) were stained under similar conditions (Figure 2). In all three tissues, clear staining of hornerin was observed in the upper parts of the epithelia (Figures 2B, 2F, and 2J) with a distribution similar to that of filaggrin (Figures 2D, 2H, and 2L). The use of preimmune serum consistently resulted in negative staining (Figures 2C, 2G, and 2K). In the granular layer, both hornerin and profilaggrin showed a granular pattern. To observe this staining pattern more clearly, we stained hornerin and profilaggrin with fluorescence-labeled antibodies (marked red and green, respectively) and observed the tissue sections using a confocal laser microscope (Figure 3). In all four tissues, hornerin and profilaggrin showed a granular pattern, and most of the granules were yellow when the corresponding two pictures were merged (Figures 3C, 3F, 3I, and 3L). In the tongue, hornerin was detected in the anterior portion of the filiform papillae in a distribution similar to that reported for profilaggrin and trichohyalin (Manabe and O'Guin 1994). This indicates that hornerin and profilaggrin are co-localized in the granules, most likely in keratohyalin granules.

Figure 2

Expressions of hornerin and profilaggrin in mouse stratified squamous epithelia. (A-D) Tongue; (E-H), esophagus; (I-L) forestomach. (A,E,I) Hematoxylin-eosin staining; (B,F,J) immunohistochemistry with anti-repA antibody; (C,G,K) immunohistochemistry with preimmune serum; (D,H,L) immunohistochemistry with anti-Filg antibody. Staining with preimmune serum consistently gave negative results. Bar = 20 μm.

Figure 3

Double staining for hornerin and profilaggrin in various mouse tissues. (A-C) skin; (D-F) tongue; (G-I) esophagus; (J-L) forestomach. (A,D,G,J) Immunostaining with anti-repA antibody; (B,E,H,K) immunostaining with anti-Filg antibody; (C,F,I,L) merged pictures.

We also analyzed skin tissues from the auricle, the region surrounding the lip, and the foot pad, and we found that hornerin was present in the granular and horny layers in a granular pattern similar to that observed in the back skin (data not shown).

Quantitative RT-PCR

Relative expressions of hornerin and profilaggrin in the skin, tongue, esophagus, and forestomach were assessed by quantitative RT-PCR (Figure 4). The expression levels of both genes were highest in the forestomach and next highest in the skin. The tongue and esophagus expressed the genes at lower levels. The amount of profilaggrin mRNA was most abundant in the forestomach, about fivefold greater than that of hornerin. In skin, the amount of hornerin mRNA was fourfold greater than that of profilaggrin mRNA.

Figure 4

Quantitative RT-PCR of hornerin and profilaggrin transcripts. The results are shown as ratios of the estimated copy number of each mRNA to that of GAPDH. Sk, skin; Ton, tongue; Eso, esophagus; FS, forestomach.

Immunoelectron Microscopy

In accordance with the observation by immunofluorescent staining (Figure 3), gold particles for hornerin and profilaggrin were detected in keratohyalin granules of the epidermal granular cells of newborn mice (Figures 5A and 5B). In the horny layer, gold particles for hornerin were observed in the region corresponding to cornified envelopes (Figure 5C), whereas those for profilaggrin were distributed more diffusely in the matrix-filament complex (Figure 5D). Although the distribution pattern was not conclusive because of the limited labeling efficiency, this tendency was also visible when the tissues were doubly stained with gold particles of different sizes (Figure 5E; hornerin, particles 20 nm in diameter; filaggrin, particles 10 nm in diameter). In keratohyalin granules, the signals for hornerin were observed more frequently in the periphery than in the inner region (Figure 6A). The numbers of particles in three concentric oval regions of keratohyalin granules were counted (Figure 6B). The density of hornerin signals was about eightfold higher in the outer region than in the inner and intermediate regions, whereas profilaggrin was distributed equally among the three regions. No granules were labeled solely with either 20-nm or 10-nm gold particles, indicating that hornerin and profilaggrin compositely form the deposits and are not segregated.

Figure 5

Immunoelectron microscopy for hornerin and profilaggrin in the mouse epidermis. (A,C) With anti-repA antibody; (B,D) with anti-Filg antibody. (A,B) A keratohyalin granule; (C,D) stratum corneum. (E) Stratum corneum doubly stained for hornerin (20 nm) and profilaggrin (10 nm). Negative control using bovine serum albumin showed no particles. Bars = 1 μm.

Figure 6

Relative distributions of hornerin and profilaggrin in keratohyalin granules. (A) A keratohyalin granule doubly stained for hornerin (20 nm) and profilaggrin (10 nm). Bar = 1 μm. (B) Relative distributions of hornerin and profilaggrin within keratohyalin granules. The granules were divided into three concentric oval regions with equal length of radii. Int, internal region; Med, intermediate region; Out, outer region. The results are expressed as densities in arbitrary units.

Discussion

In this study we demonstrated that hornerin was expressed in the four stratified squamous epithelia examined and that hornerin protein was co-localized with profilaggrin in keratohyalin granules. Keratohyalin granules are the most distinctive cytoplasmic organelle of granular cells in the stratified squamous epithelium. They are not membrane-bound and are rather insoluble aggregates of proteins. In rodents there are two distinguishable granules, PF-granules and L-granules, the major components of which are profilaggrin and loricrin, respectively (Dale et al. 1994). Because the keratohyalin granules were consistently labeled with both 20-nm (for hornerin) and 10-nm gold particles (for profilaggrin) in double immunoelectron microscopy (Figure 6), hornerin is believed to be present with profilaggrin in PF-granules. Trichohyalin, another member of the “fused gene”-type cornified envelope precursor protein family (Presland et al. 1992; Lee et al. 1993), was not completely co-localized with profilaggrin even when expressed in the same cells (Ishida-Yamamoto et al. 1997).

Profilaggrin appeared to be distributed equally within the granules (Figure 6C), a finding that is in accordance with previously reported findings (Steven et al. 1990). On the other hand, hornerin was present more frequently in the periphery of the granules (Figure 6C). The biological or biochemical relevance of this distribution is not clear at present. The order of protein synthesis in the process of differentiation is an unlikely cause for this phenomenon because hornerin was expressed at a slightly earlier stage of embryonic skin development than was profilaggrin (Makino et al. 2001). In the mouse embryo, the epidermis differentiates stepwise from a simple epithelial cell layer to a fully differentiated cornified epithelium during the period from 13.5 dpc to 17.5 dpc, and the developmental stages thus reflect distinct differentiation steps.

In our previous study (Makino et al. 2001), we compared the expressions of hornerin and profilaggrin in different tissues by Northern blotting analysis. The forestomach appeared to show the highest expression levels of both genes, but the results were not clear because the large sizes of both transcripts (>10 kb) prevented precise assessment. Quantitation of the proteins has been almost impossible because of the proteolytic processing (Kubilus et al. 1985; Presland et al. 1997; Makino et al. 2001). We therefore performed quantitative RT-PCR and found that hornerin and profilaggrin were expressed at the highest levels in the forestomach and at the next highest levels in the skin. The tongue and esophagus expressed the genes at lower levels. However, it should be noted that the apparent expression levels depend on relative amounts of granular cells expressing hornerin and profilaggrin in the whole tissues.

Keratinization is crucial for protection of the epithelium. Because profilaggrin plays unique roles in the keratinizing process, partial or complete lack of profilaggrin results in abnormality of skin functions (Sybert et al. 1985; Nirunsuksiri et al. 1995; Presland et al. 2000). Hornerin is structurally and functionally very similar to profilaggrin. Further studies are needed to understand the physiological and pathological relevance of both genes. Examination of the expression of hornerin in tissues with a reduced amount of or no profilaggrin and characterization of human hornerin are now under way in our laboratory.