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SPRR4, a novel cornified envelope precursor: UV-dependent epidermal expression and selective incorporation into fragile envelopes

¹ Department of Molecular Genetics, Leiden Institute of Chemistry, Leiden University, PO Box 9502, 2300 RA Leiden, The Netherlands
² Department of Dermatology, Leiden University Medical Centre, The Netherlands

View larger version (43K): [in a new window]   Fig. 1. Molecular characterization of the SPRR4 gene and protein. (A) Physical map of the SPRR4 gene in the SPRR locus. Three overlapping cosmid clones (South et al., 1999) are represented. Black boxes indicate either the genomic sequences of individual genes or the SPRR2 cluster as a whole. EcoRI restriction sites (E) are indicated. (B) Nucleotide and deduced amino acid sequence of the SPRR4 gene. The sequence used as a probe is underlined, and the position of the single intron is indicated by an asterisk. Grey boxes indicate the four internal octapeptide repeats. Putative polyadenylation sites are double underlined. GenBank accession number AF335109. (C) Comparison of internal repeat consensi from the two groups (I/II) and four classes of human SPRR proteins (Cabral et al., 2001). The number of repetitive units for each class is represented between brackets. Amino acids conserved in three out of four classes are in red. (D) Amino acid sequence of N- and C-terminal SPRR domains. Differences between various classes are in bold. Amino acids involved in the transglutaminase mediated crosslinking reaction in SPRR1, -2 or -3, and conserved in SPRR4, are in red.

View larger version (45K): [in a new window]   Fig. 2. SPRR4 is a CE precursor protein. (A) Subcellular location of SPRR4-GFP and GFP in living cells. Monolayers of normal human keratinocytes were transfected with either SPRR4-GFP (1,2) or GFP (3,4), as a control. Cultures were either maintained in medium without calcium (1,3) or induced to differentiate for 40 hours in 1.8 mM Ca2+ (2,4). Cells were analyzed with a laser scanning confocal microscope. (B) Isolation of cornified envelopes from keratinocytes transfected with SPRR4-GFP. Transfected cells were incubated for 48 hours with A23187 ionophore, treated with 2% SDS and 10 mM DTT and examined using a fluorescence microscope (1, fluorescence; 2, phase-contrast). The middle image in panels 1 and 2 shows a rigid envelope and the flanking images show fragile envelopes. Bar, 15 µm.

View larger version (80K): [in a new window]   Fig. 3. In vivo screening for SPRR4 expression. (A) RT-PCR analysis of SPRR4 expression. Epidermal RNA (lanes 1-7) and RNA isolated from primary cultured keratinocytes (lanes 8-11) were analyzed with SPRR4, involucrin and GAPDH-specific primers. (B) In situ hybridization performed on different skin samples. Sense (a,c,e) and antisense (b,d,f) probes from either SPRR4 (a-d) and SPRR2 (e,f) were analyzed. Melanin caps (c, filled arrows) and SPRR2-positive staining (f, open arrows) are indicated. Bar, 30 µm.

View larger version (81K): [in a new window]   Fig. 4. Influence of calcium-induced differentiation and UV irradiation on SPRR expression in vitro. Normal human keratinocytes (NHK) were cultured in complete medium to confluency and subsequently incubated with calcium-free medium to remove differentiated cells. Cells were either irradiated with 500 J/m2 UV-A, 300 J/m2 UV-B or 30 J/m2 UV-C, or mock irradiated, and maintained for 24 or 48 hours in medium containing 1.8 mM Ca2+. Total RNA was isolated from the different cultures and hybridized with either SPRR class specific, involucrin, or 28S ribosomal RNA probes.

View larger version (118K): [in a new window]   Fig. 5. In vivo SPRR4 expression after chronic UV exposure. In situ hybridization was performed with SPRR4 sense (a,c,e,g) and antisense (b,d,f,h) probes. Results for chronic exposure from phototype II (a-d) and phototype III (e-h) volunteers, before (a,b,e,f) and after (c,d,g,h) the UV treatment are represented. Bar, 30 µm.