ARTICLE |
Correspondence to: Nam-ho Huh, Dept. of Cell Biology, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8558, Japan. E-mail: namu@md.okayama-u.ac.jp
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Summary |
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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.
(J Histochem Cytochem 51:485492, 2003)
Key Words: hornerin, profilaggrin, keratohyalin granule, keratinocyte, differentiation
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Introduction |
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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 (
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 (
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Materials and Methods |
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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 (
Antibodies
Antibodies against hornerin were prepared as previously reported (
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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 x 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 (70100 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.
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Results |
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Immunostaining of Mouse Tissues
We raised antibodies against two different regions of hornerin protein (Fig 1, upper panel), and the staining patterns with both antibodies were very similar, as shown in Fig 1C and Fig 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 (Fig 1B1D). Hornerin transcript was detected in the granular layer (Fig 1B), and positive staining for hornerin protein was observed in the granular and horny layers (Fig 1C and Fig 1D). Preimmune serum used as a first antibody showed no staining (Fig 1E). The antibody against filaggrin gave a localization of staining similar to that for hornerin (Fig 1F).
Stratified squamous epithelia other than that of the skin (i.e., those of the tongue, esophagus, and forestomach) were stained under similar conditions (Fig 2). In all three tissues, clear staining of hornerin was observed in the upper parts of the epithelia (Fig 2B, Fig 2F, and Fig 2J) with a distribution similar to that of filaggrin (Fig 2D, Fig 2H, and Fig 2L). The use of preimmune serum consistently resulted in negative staining (Fig 2C, Fig 2G, and Fig 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 (Fig 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 (Fig 3C, Fig 3F, Fig 3I, and Fig 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 (
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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 (Fig 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.
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Immunoelectron Microscopy
In accordance with the observation by immunofluorescent staining (Fig 3), gold particles for hornerin and profilaggrin were detected in keratohyalin granules of the epidermal granular cells of newborn mice (Fig 5A and Fig 5B). In the horny layer, gold particles for hornerin were observed in the region corresponding to cornified envelopes (Fig 5C), whereas those for profilaggrin were distributed more diffusely in the matrixfilament complex (Fig 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 (Fig 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 (Fig 6A). The numbers of particles in three concentric oval regions of keratohyalin granules were counted (Fig 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.
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Discussion |
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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 (
Profilaggrin appeared to be distributed equally within the granules (Fig 6C), a finding that is in accordance with previously reported findings (
In our previous study (
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 (
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Acknowledgments |
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Supported by a grant-in-aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
We sincerely thank Dr Motomu Manabe, Akita University, for critical comments on the results of our electron microscopy.
Received for publication June 27, 2002; accepted October 2, 2002.
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