From the Pediatric Surgical Research Laboratory, Stanford University School of Medicine, Stanford, California 94305-5148
Received for publication, October 13, 2002 , and in revised form, February 27, 2003.
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ABSTRACT |
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INTRODUCTION |
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Cornification is a complex and ordered system of epidermal terminal differentiation. Proliferating basal keratinocytes lose contact with the basement membrane and differentiate stepwise into spinous cells, then granular cells, and lastly into anuclear, keratin-filled, corneocytes. These cells form the corneal layer of the stratified squamous epithelium, which is eventually shed from skin.
During epidermal terminal differentiation in stratified squamous epithelia, the CE forms on the intracellular side of the plasma membrane (3). The precursor proteins of CE are cross-linked via isopeptide bonds formed by the action of transglutaminases (10). A growing number of proteins are now known to be components of the epidermal CE, including cystatin , desmoplakin, elafin, loricrin, involucrin, plasminogen activator inhibitor-2, and the SPRs (4, 5, 11, 12). Interestingly, the components of the CE vary in different epithelia and body sites. Loricrin comprises the major component (70%) of the CE in interfollicular cutaneous keratinocytes (13, 14), whereas SPR1 is the major component of the CE in human oral keratinocytes (15).
Among the CE precursor proteins, loricrin is a glycine-rich protein (55% of total protein) with a molecular mass of about 29 kDa (16), whereas the SPRs have high content of proline (19%) and their molecular size is relative small, i.e. around 1425 kDa (7). The SPR family has four groups of proteins with a high degree of homology in their head and tail domains (8, 12). Sequence analysis indicates that SPR1 in different species has unique, conserved, repeated elements, which may cause a folded structure and form a rigid protein (17). SPRs function as cross-bridges in CE by joining with other proteins with multiple adjacent glutamines and lysines on only the amino and carboxyl termini. The amount of SPRs varies in the CE of different epithelia. For example, during development SPR1 is not detectable in E15 mice but is expressed on the periderm of E16 mice, and it is not detectable in adult mouse epidermis (18). It is absent from the interfollicular epidermis in newborn and adult mice but is expressed in epidermis of lip, foot pad, and hair follicles (17). Some investigators suggest (5) that SPR1 may modify tissue biomechanical properties by changing its relative amount in different tissues.
Other members of the CE proteins include repetin, filaggrin, trichohyalin, and hornerin. They exhibit both terminal differentiation-related and intermediate filament-associated protein characteristics and are thought to be cross-linked with other CE proteins (19). These proteins have high molecular weights and are insoluble under physiological conditions (2022). Furthermore, profilaggrin, and possibly hornerin, is cleaved with post-translational proteolytic processing during the process of terminal differentiation.
In the current study, we describe a new rat gene, keratinocyte proline-rich protein (KPRP), encoding a protein of 699-amino acids with a high (19%) proline content. KPRP is expressed exclusively in stratified squamous epithelial layers, beginning during fetal development. A human mRNA-derived hypothetical protein with 576 amino acids, and chromosome 1q21 [PDB] origin, shows high homology to the encoded rat KPRP protein. A mouse genomic DNA sequence located on chromosome 3 is 89% homologous to rat KPRP. In addition, the BLAST analysis of rat genomic DNA indicates KPRP is located on chromosome 2 near the location of rat CE proteins. KPRP antibody demonstrates cytoplasmic localization with accumulation adjacent to the nuclear and plasma membranes. We believe KPRP is a new marker for epidermal differentiation during skin development, and likely has function in the keratinocyte-cornified cell envelope protein layer.
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EXPERIMENTAL PROCEDURES |
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RNA CollectionTime-dated pregnant rats at E17, E18, E19, E20, and E21 day gestational ages were used for the experiments (Term = E22). Rats were anesthetized with ketamine (87 mg/kg) and xylazine (13 mg/kg) by subcutaneous injection. Hair on the abdomen was shaved and the skin was scrubbed with Betadine and 75% ethanol before a midline incision was made to expose the uterus. Fetal rats were removed and fetal skin was collected from dorsal thorax. Skin was pooled according to each gestational age, and at least 5 rat skins were pooled for each group. Total RNA was purified from skin of each gestational group by RNAzol B as described by the manufacturer.
cDNA Library ConstructionTotal RNA from skin on E17, E18, E20, and E21 rats was pooled and mRNA was purified by the FastTrack mRNA isolation kit. Our cDNA library was constructed by Creater SMART cDNA library construction kit according to the manufacturer's instructions. Briefly, 1 µg of poly(A)+ RNA was reverse-transcribed to synthesize first strand cDNA. Next, first strand cDNA was mixed with 5'-PCR primer and other PCR reagents at 72 °C for 10 min, 95 °C for 20 s, and 3 cycles of 95 °C for 5 s, 68 °C for 8 min to synthesize double strand DNA. After digestion by proteinase K, purification by phenol-chloroform, digestion by SfiI, and fractional purification, double-stranded DNA was ligated to pDNR-LIB vectors. The library plasmids were then transformed to ElectroMAX DH5-E cells by eletroporation at 1.6 kV, 200 ohms for 4 s. After titering the plasmid library, about 20,000 colony forming units were cultured on 150 mm plates for library screening.
Probe DetailscDNA probe (800 bp) was cloned by RT-PCR from rat skin total RNA using forward primer 5'-ACAACCCCAGTCCTCGTTCAC and reverse primer 5'-GCTGCCAACCATAACCTTGT, based on the cloned KPRP sequence. The probe fragment was purified and ligated into a dual-promoter vector and further amplified by bacteria transformation. The sequence of the probe was checked after the ligation to the dual-promoter vector. For Northern blot probe, vectors were cut first by EcoRI and purified probes were labeled by using a Ready-To-Go dCTP labeling beads, using 50 ng cDNA and 50 µCi [
-32P]dCTP at 37 °C for 30 min. The 32P-labeled probes were purified using a NICK column and boiled at 100 °C for 5 min. For in situ hybridization probes, sense and antisense RNA probes were transcribed from linearized plasmid by using T7 or SP6 RNA polymerases with digoxigenin-labeled UTP.
Northern BlottingAdult and E21 fetal rats were used and brain, heart, lung, esophagus, liver, intestine, kidney, and skin were collected for RNA purification. Total RNA from each organ was extracted by RNAzol B, and purified RNA was reconstituted in water, quantified and fractionated (20 µg/lane) by electrophoresis on a 2.1 M formadehyde, 1% agarose gel in 1x MOPS buffer. Separated RNA was then trans-blotted to a GeneScreen Plus nylon membrane and immobilized by UV irradiation. Blots were hybridized with Northern blot probe prepared as above, in a rolling incubator at 42 °C for 1620 h. Blots were washed with 2x SSC buffer with 0.1% SDS for 15 min, 3 times and 0.5x SSC with 0.1% SDS for 10 min. After washing, blots were exposed to BioMax film (Eastman Kodak). In addition, blots were analyzed using a phosphorimaging device.
In Situ HybridizationTen percent formalin-fixed E18, E19, and neonatal rat skin was dehydrated and blocked in paraffin. Seven-µm sections were mounted on slides. After de-paraffin, rehydration, treatment with proteinase K, and acetylation with 0.25% acetic anhydride, sections were pre-hybridized in buffer containing 50% formamide, 2x SSC, 5 mM EDTA, 10% dextran sulfate, 100 µg/ml yeast tRNA, 100 µg/ml sperm DNA, and 10 mM dithiothreitol for1hat52 °C. Denatured sense and antisense RNA probes were added and hybridization continued for 16 h at 52 °C. After hybridization, sections were washed with 50% formamide for 30 min and 2x SSC for 1 h, treated with RNase A (8 units/ml) for 20 min at 37 °C. After serial washing, sections were blocked for 1 h at room temperature in buffer containing 0.1 M Tris, pH 7.4, 150 mM NaCl, 2% horse serum, 2% goat serum, and 1.2 mg/ml lavamisole. Sections then were incubated in 1:250 dilution of sheep anti-digoxigenin conjugated with alkaline phosphatase for 1 h at room temperature. Color detection for 20 min was by Vector Red for alkaline phosphatase as instructed by the manufacturer.
RT-PCRTotal RNA from E17, E18, E19, E20, and E21 was purified by RNAzol B and followed by phenol-chloroform extraction. After treatment with DNase I for 30 min at 37 °C and purification, two µg of total RNA from each sample was reverse transcribed with superscript II at 42 °C for 1 h. KPRP fragments were next amplified by PCR using the first strand cDNA, which was from 0.5 µg of original total RNA and the upstream primer 5'-CTCCATGCCAATCTCAGGTC-3' and the downstream primer 5'-CTCAGGTCTGGGCAAGAGAC-3', forming a 900-bp product. -actin was used as the RNA positive control, with first strand cDNA from 0.1 µg original total RNA and the upstream primer 5'-AAGATTTGGCACCACACTTTCT-3' and the downstream primer 5'-TCTCTTTAATGTCACGCACGAT-3', which formed a
500-bp cDNA product.
AntibodiesPolyclonal antibodies were produced by Genemed Synthesis Inc (South San Francisco, CA). The peptide, PCPSPELRPRPRPEP, from the KPRP hypothetical protein sequence was synthesized and injected into rabbits for antibody generation.
Primary Culture of Rat KeratinocytesEmbryonic rat skin at E21 was collected and incubated in 0.3% dispase at 4 °C overnight. The epidermal layer was then separated from dermal layer with fine forceps, washed and minced with fine scissors, and digested with 0.05% trypsin and 0.02% EDTA at 37 °C for 15 min. The cell suspension was filtered through a 100-µm cell strainer, and keratinocytes were centrifuged at 800 rpm for 10 min. Keratinocytes were cultured on plastic in defined-keratinocyte SFM growth medium (Invitrogen) with daily media changes.
Extraction of Protein and ImmunoblottingProtein was prepared from cultured E21 rat keratinocytes by scraping cells in lysis buffer containing 0.25 M Tris-HCL (pH 7.5), 5% SDS, and 20% -mercaptoethanol. The negative control protein was harvested from cultured rat dermal fibroblasts. Fifty µg of protein from keratinocytes or fibroblasts was heated at 100 °C for 5 min, separated by 10% SDS-polyacrylamide gel, and blotted onto a polyvinylidene difluoride membrane (Millipore). The protein was detected by polyclonal anti-KPRP antiserum, and bands were visualized by blot exposure to x-ray film after 1 min reaction with ECL Western blotting detection reagents (Amersham Biosciences).
ImmunostainingParaffin sections from adult rat skin, tongue, and esophagus were de-waxed and re-hydrated, washed three times with phosphate-buffered saline, and treated with proteinase K (DAKO ready to use kit) for 10 min at 37 °C and followed by the DAKO LSAB 2 System peroxidase kit instruction. Briefly, sections were washed with Tris-buffered saline and treated with H2O2 for 5 min, blocked in Tris-buffered saline with 0.1% Tween 20 and 1% bovine serum albumin for 30 min, and followed by reaction with anti-KPRP antibody (1:100 dilution) for 1 h at room temperature. After wash and color detection, sections were counter-stained with hematoxylin and mounted. For immunofluorescent staining, keratinocytes cultured on matrigel-coated cover slides were fixed with 4% paraformadehyde in phosphate-buffered saline for 30 min, washed, and blocked with phosphate-buffered saline containing 0.1% saponin and 1% normal goat serum for 1 h. Sections were reacted with anti-KPRP polyclonal antibody diluted 1:500 in blocking buffer for 2 h at room temperature, washed with blocking buffer, and reacted with Alexa Fluor goat anti-rabbit IgG (Molecular Probes) for 1 h, and counter-stained with DAPI. Fluorescein isothiocyanate-labeled staining was visualized and pictured by an Axioplan 2 microscope (Zeiss). Sections without primary antibody were used as negative controls.
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RESULTS |
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For KPRP, an open reading frame protein with 699 amino acids was predicted and further searched in the GenBankTM protein data base. BLAST search results showed that KPRP protein has 56% similarity to a human putative protein encoded from an mRNA (accession number XM_060108) whose genomic DNA is located on chromosome 1q21 [PDB] (Fig. 2). The KPRP open reading frame protein has a predicted 76.4 kDa molecular mass and 8.4 IP. The proline content in KPRP is relatively high, about 19%, similar to the SPRs. However, unlike the SPRs, within the carboxyl-terminal half of KPRP, proline content reaches 28% (99 proline/350 amino acids).
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KPRP Is Expressed in Stratified Squamous EpitheliaTo examine which tissues express KPRP, Northern blot of RNAs isolated from different organs in adult and E21 rats was performed. Fig. 3A demonstrates KPRP mRNA expression by Northern blot in adult rats. In adult rat esophagus and skin, KPRP probe hybridized to a single 3.0 kb band. This band was also seen in the skin of E21 fetal rats (Fig. 3B). The size of the KPRP mRNA in the Northern blot corresponds to the full-length cDNA clone size. In situ hybridization on skin, tongue, and esophagus in adult rats localized KPRP expression in their respective stratified squamous epithelial layers. The sense riboprobe control did not show positive stain (Fig. 4). KPRP expression did not appear different between both follicular and interfollicular keratinocytes, nor within the stratified epithelial layers.
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Detection of KPRP ProteinImmunostaining with KPRP antibody showed the presence of KPRP protein in the stratified epithelial layers of skin, tongue, and esophagus in rats (Fig. 5). These data further support the in situ hybridization findings for the localization of KPRP. To determine the intracellular localization of KPRP protein, primary rat dermal keratinocytes in calcium-free SFM medium were selected for immunostaining. KPRP localized to the cytoplasm with signal enhancement adjacent to the nuclear and plasma membranes (Fig. 6). Immunoblotting of cell lysate protein from rat dermal fibroblasts and keratinocytes showed two KPRP bands with sizes of 76 and 55 kDa (Fig. 7). The 55-kDa band was denser than the 76-kDa band, suggesting possible post-translational modification of KPRP.
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Localization of KPRP to Keratinocytes in E19 SkinIn situ hybridization was performed on E18, E19, and 4-day neonatal rat skin. Fig. 8 shows Hematoxylin and Eosin (a, c, and e) stain and in situ hybridization (b, d, and f) on E18, E19, and 4-day neonatal rat skin, respectively. KPRP expression was not detectable in E18 skin but was detected in keratinocytes and hair-germ cells at E19.
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KPRP Expression During Skin DevelopmentFetal rat skin RNA was treated with DNase I before in vitro reverse transcription. No expression was detected in E17 and E18 skin. Weak expression was detected in E19 with strong expression in E20 and E21 (Fig. 9). This experiment was repeated two times with different RNA samples, and the results corresponded to the preliminary differential display-PCR data (data not shown). The -actin control showed equal amplification bands at all five gestational ages.
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DISCUSSION |
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Our data strongly suggest that the expression of KPRP is unique to stratified squamous epithelium and is initiated in fetal skin keratinocytes during development. KPRP expression is not likely related to the initiation of epidermis or epidermal appendage formation. Primitive forms of these structures are present in E18 fetal skin but KPRP expression is undetectable on E18 by both in situ hybridization and RT-PCR.
KPRP may be an epidermal terminal differentiation-related gene with expression starting during late gestation. The detection of KPRP in the epidermis began on E19, when the epidermis is two or three cell layers thick, and its expression extends through the entire epidermis by E21. This pattern correlates with other epidermal terminal differentiation proteins such as SPR1, whose expression starts on E16 mouse periderm (17).
Many of the CE proteins initiate expression during fetal development, with persistent postnatal expression during the terminal differentiation process. During human fetal development, involucrin is detected from 14 weeks of gestation, whereas loricrin and the SPR proteins are detected from 16 weeks gestational age (9). All are expressed postnatally during terminal differentiation and are components of the CE. KPRP shares these characteristics, and thus may belong to the group of terminal differentiation CE proteins.
Although KPRP has a high proline content similar to the SPRs, the amino acid sequences of the head and tail of the putative KPRP protein do not correspond to those of the SPRs. The molecular mass of KPRP is also much larger, at least 55 kDa, compared with the SPRs (1425 kDa). In addition, the conserved, repeated elements of the SPRs are not present in the predicted KPRP protein sequence. Thus, KPRP appears to belong to a class of CE proteins distinct from the SPRs.
KPRP expression did not change from the basal keratinocyte layer to the outer keratinocyte layers. Graded CE protein expression occurs from basal layer keratinocytes to outer layer cells for loricrin and the SPRs. However, involucrin and other CEs do not show graded expression differences. Similar to KPRP, involucrin expression starts in keratinocytes during fetal development and persists postnatally through the epidermal layers (24). Intracellular localization of KPRP indicates a cytoplasmic distribution pattern similar to hornerin and filaggrin, (22) which are high molecular weight, insoluble, intermediate filament-associated proteins. KPRP shares these characteristics, suggesting KPRP may be a terminal differentiation CE protein that is related to the intermediate filament-associated types. The immunoreactive granules we observed in keratinocytes that are detaching from their substrate (Fig. 6) have a striking similarity to those observed with filaggrin (20), further supporting a possible KPRP function with keratin-associated intermediate filaments.
KPRP shares more characteristics with the keratin-associated intermediate filament proteins of the CE. KPRP is insoluble under physiological conditions (data not shown) as are filaggrin and hornerin. The molecular mass of profilaggrin is >400 kda in humans, and it undergoes post-translational modification into 1012 filaggrin repeats flanked by N- and C-terminal domains (25). Hornerin also undergoes post-translational proteolytic cleavage (22). We observed two different sizes of molecular mass KPRP bands with immunoblotting, 76 and 55 kDa, suggesting the possibility of post-translational modification of KPRP. Alternatively, KPRP may have been partially degraded by the strong reducing reagents used in our experiment.
In summary, KPRP is a new keratinocyte-specific protein with many similarities to the CE proteins, some shared with the SPR family and some with the intermediate filament-associated family. Whether KPRP is a structural protein in the CE or another type of functional protein remains unknown. KPRP is expressed in interfollicular and follicular keratinocytes in stratified squamous epithelia. It begins expression in fetal squamous epithelium during late development and may represent a new family of proline-rich proteins.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant DE00463-03. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Stanford University School of Medicine, 257 Campus Dr., Stanford, CA 94305-5148. Tel.: 650-723-8315; Fax: 650-736-1705; E-mail: plorenz{at}stanford.edu.
1 The abbreviations used are: CE, cornified envelope; SPR, small proline-rich protein; KPRP, keratinocyte proline-rich protein; RT, reverse transcription; MOPS, 4-morpholinepropanesulfonic acid; DAPI, 4',6-diamidino-2-phenylindole.
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REFERENCES |
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