Expression profile of cornified envelope structural proteins and keratinocyte differentiation-regulating proteins during skin barrier repair.

BACKGROUND: Recent studies have emphasized the importance of heritable and acquired skin barrier abnormalities in common inflammatory diseases such as psoriasis and atopic dermatitis (AD). To date, no comprehensive studies on the effect of experimental barrier disruption on cornified envelope protein expression have been performed. OBJECTIVES: To analyse the effect of experimental skin barrier disruption on the expression of cornified envelope structural proteins and keratinocyte differentiation-regulating proteins. METHODS: We examined mRNA (day 1, 3 and 7) and protein (day 1, 2, 4 and 9) expression levels of structural proteins and regulatory molecules after sodium dodecyl sulphate (SDS) application on normal skin, and tape stripping of uninvolved epidermis of patients with psoriasis and AD and healthy controls. RESULTS: Upon tape stripping, several structural molecules were significantly downregulated (at the mRNA level as well as the protein level), including LCE5A, LCE2B, FLG, FLG2 and LOR, whereas others were upregulated: IVL, SPRR1, SPRR2, HRNR and most notably LCE3A. The epidermal crosslinking enzymes TGM1, TGM3 and TGM5 were all upregulated, whereas proteases involved in the desquamation process (CTSV, KLK5 and KLK7) were downregulated or unaffected. Most results were similar in SDS-instigated irritant contact dermatitis. There was no significant difference in response between normal epidermis and nonlesional skin of patients with psoriasis and AD. CONCLUSIONS: Skin barrier disruption induces a temporary barrier repair response composed of increased expression of several cornification-related proteins, and decreased expression of some structural and desquamation-related proteins.

The cystatin M/E-controlled pathway of skin barrier formation: expression of its key components in psoriasis and atopic dermatitis.

BACKGROUND: The antiprotease activity of cystatin M/E regulates skin barrier formation, as it inhibits the activity of cathepsin V, cathepsin L and legumain, thereby controlling the processing of transglutaminase 3. Misregulation of this pathway by unrestrained protease activity, as seen in cystatin M/E-deficient mice, leads to abnormal stratum corneum and hair follicle formation, and severe disturbance of skin barrier function. OBJECTIVES: Our major aim was to make a quantitative analysis of the expression of all players of this pathway in the epidermis of patients with inflammatory skin diseases. A second aim was to determine if reconstructed human skin could be used as an in vitro model system to investigate this pathway. METHODS: Autopsy material from normal human tissues, biopsies from normal skin of healthy volunteers, and lesional skin from patients with atopic dermatitis and psoriasis were used to study the expression of the above-mentioned molecules at the mRNA level by quantitative real-time polymerase chain reaction. Localization of the protein was performed by immunofluorescence microscopy, and expression was quantitated by image analysis. RESULTS: In skin, cystatin M/E is expressed at relatively higher levels than its target proteases, when compared with other tissues, which emphasizes its prominent role in cutaneous biology. We found decreased expression of cystatin M/E and cathepsin V in lesional atopic dermatitis and psoriasis epidermis at the mRNA level as well as the protein level. Cathepsin L and transglutaminase 3 were increased at the transcriptional level; however, this was not reflected by higher protein levels. Interestingly, the expression of all these molecules in reconstructed skin was qualitatively and quantitatively similar to the in vivo situation. CONCLUSIONS: Disturbance of the cystatin M/E-cathepsin pathway could contribute to the dysregulated skin barrier function observed in inflammatory dermatoses. Human reconstructed skin appears to be a valuable model to study this novel biochemical pathway in vitro.

Bacterial cryptochrome and photolyase: characterization of two photolyase-like genes of Synechocystis sp. PCC6803.

Photolyase is a DNA repair enzyme that reverses UV-induced photoproducts in DNA in a light-dependent manner. Recently, photolyase homologs were identified in higher eukaryotes. These homologs, termed crypto-chromes, function as blue light photoreceptors or regulators of circadian rhythm. In contrast, most bacteria have only a single photolyase or photolyase-like gene. Unlike other microbes, the chromosome of the cyanobacterium SYNECHOCYSTIS: sp. PCC6803 contains two ORFs (slr0854 and sll1629) with high similarities to photolyases. We have characterized both genes. The slr0854 gene product exhibited specific, light-dependent repair activity for a cyclo-butane pyrimidine dimer (CPD), whereas the sll1629 gene product lacks measurable affinity for DNA in vitro. Disruption of either slr0854 or sll1629 had little or no effect on the growth rate of the cyanobacterium. A mutant lacking the slr0854 gene showed severe UV sensitivity, in contrast to a mutant lacking sll1629. Phylogenetic analysis showed that sll1629 is more closely related to the cryptochromes than photolyases. We conclude that sll1629 is a bacterial cryptochrome. To our knowledge, this is the first description of a bacterial cryptochrome.

Respective roles of cyclobutane pyrimidine dimers, (6-4)photoproducts, and minor photoproducts in ultraviolet mutagenesis of repair-deficient xeroderma pigmentosum A cells.

The role of UV light-induced photoproducts in initiating base substitution mutation in human cells was examined by determining the frequency and spectrum of mutation in a supF tRNA gene in a shuttle vector plasmid transfected into DNA repair deficient cells (xeroderma pigmentosum complementation group A). To compare the role of two major UV-induced photoproducts, cis-syn cyclobutane-type pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs), each photoproduct was removed from UV-irradiated plasmid by photoreactivation before transfection. Removal of either CPDs or 6-4PPs by in vitro photoreactivation reduced the mutation frequency while keeping the mutation distribution and the predominance of G:C-A:T transitions as UV-irradiated plasmid without photoreactivation, indicating that both cytosine-containing CPDs and 6-4PPs were premutagenic lesions for G:C-A:T transitions. On the other hand, A:T-G:C transitions were not recovered from plasmids after the removal of 6-4PPs, whereas this type of mutation occurred at a significant level (11%) after the removal of CPDs. Thus, the premutagenic lesions for the A:T-G:C transition are 6-4PPs. Removal of both CPDs and 6-4PPs resulted in the disappearance of mutational hot spots and random distribution of mutation as observed in unirradiated control plasmids. However, the mutational spectrum of photoreactivated plasmids differed significantly from that of unirradiated plasmids. A characteristic feature is a high portion of A:T-T:A transversions (11%) in the photoreactivated plasmid. This mutation is due to nondipyrimidinic "minor" photoproducts, and the mutation spectrum suggests that TA*, the major photoproduct of thymidylyl-(3'-5')-deoxyadenosine, is the premutagenic lesion for this mutation. This is the first report revealing the distinct mutagenic roles of the major UV photoproducts and "minor" photoproducts by the use of (6-4)photolyase.

Transglutaminase 2 has opposing roles in the regulation of cellular functions as well as cell growth and death.

Transglutaminase 2 (TG2) is primarily known as the most ubiquitously expressed member of the transglutaminase family with Ca(2+)-dependent protein crosslinking activity; however, this enzyme exhibits multiple additional functions through GTPase, cell adhesion, protein disulfide isomerase, kinase, and scaffold activities and is associated with cell growth, differentiation, and apoptosis. TG2 is found in the extracellular matrix, plasma membrane, cytosol, mitochondria, recycling endosomes, and nucleus, and its subcellular localization is an important determinant of its function. Depending upon the cell type and stimuli, TG2 changes its subcellular localization and biological activities, playing both anti- and pro-apoptotic roles. Increasing evidence indicates that the GTP-bound form of the enzyme (in its closed form) protects cells from apoptosis but that the transamidation activity of TG2 (in its open form) participates in both facilitating and inhibiting apoptosis. A difficulty in the study and understanding of this enigmatic protein is that opposing effects have been reported regarding its roles in the same physiological and/or pathological systems. These include neuroprotective or neurodegenerative effects, hepatic cell growth-promoting or hepatic cell death-inducing effects, exacerbating or having no effect on liver fibrosis, and anti- and pro-apoptotic effects on cancer cells. The reasons for these discrepancies have been ascribed to TG2's multifunctional activities, genetic variants, conformational changes induced by the immediate environment, and differences in the genetic background of the mice used in each of the experiments. In this article, we first report that TG2 has opposing roles like the protagonist in the novel Dr. Jekyll and Mr. Hyde, followed by a summary of the controversies reported, and finally discuss the possible reasons for these discrepancies.

cDNA sequence for rkST1, a novel member of the sodium ion-dependent glucose cotransporter family.

A full length cDNA for rkST1, a novel member of the Na+/glucose cotransporter family, was cloned from rabbit kidney and sequenced. The coding sequence comprised 2022 base pairs and 674 amino acids. rkST1 beared 50-60% amino acid identity to the other cotransporters and was characteristic in respect of its expression in brain in addition to kidney among the cotransporters.

Molecular mechanism by which acyclic retinoid induces nuclear localization of transglutaminase 2 in human hepatocellular carcinoma cells.

Nuclear accumulation of transglutaminase 2 (TG2) is an important step in TG2-dependent cell death. However, the underlying molecular mechanisms for nuclear translocation of TG2 are still poorly understood. In this study, we demonstrated that acyclic retinoid (ACR) induced nuclear accumulation of TG2 in JHH-7 cells, a hepatocellular carcinoma (HCC) leading to their apoptosis. We further demonstrated molecular mechanism in nuclear-cytoplasmic trafficking of TG2 and an effect of ACR on it. We identified a novel 14-amino acid nuclear localization signal (NLS) (466)AEKEETGMAMRIRV(479) in the 'C' domain and a leucine-rich nuclear export signal (NES) (657)LHMGLHKL(664) in the 'D' domain that allowed TG2 to shuttle between the nuclear and cytosolic milieu. Increased nuclear import of GAPDH myc-HIS fused with the identified NLS was observed, confirming its nuclear import ability. Leptomycin B, an inhibitor of exportin-1 as well as point mutation of all leucine residues to glutamine residues in the NES of TG2 demolished its nuclear export. TG2 formed a trimeric complex with importin-α and importin-ß independently from transamidase activity which strongly suggested the involvement of a NLS-based translocation of TG2 to the nucleus. ACR accelerated the formation of the trimeric complex and that may be at least in part responsible for enhanced nuclear localization of TG2 in HCC cells treated with ACR.

Ascorbate-dependent expression of ubiquitin genes in guinea pigs.

In an attempt to characterize a gene(s), of which the expression is ascorbate-dependent, a cDNA fragment encoding ubiquitin was isolated from a subtracted cDNA library constructed from spleen RNAs of ascorbate-deficient or -replete guinea pigs. On Northern blot analysis, three transcripts (1.8 kb ubiX, 1.3 kb ubiY and 0.7 kb ubiZ) were detected. The ubiY encodes four direct repeats of the 76 amino acid ubiquitin sequence with seven additional amino acids, V-Y-A-S-P-I-F, at the C-terminus. The transcript ubiX appears to comprise more than five repeats of the ubiquitin-encoding unit. The ubiZ encodes a ubiquitin monomer fused to an 80 amino acid extension exhibiting 100% amino acid sequence identity to the human homolog, HUMUBA80R. The ubiX gene was expressed animal-dependently. The ubiY mRNA levels decreased under ascorbate-deficient conditions, and increased under ascorbate-replete conditions, whereas ubiZ mRNA remained unaltered at low levels under the feeding conditions used here.

Analysis of epidermal-type transglutaminase (TGase 3) expression in mouse tissues and cell lines.

In the formation of the cornified cell envelope in the epidermis, epidermal-type transglutaminase (TGase 3) cross-links a variety of structural proteins. However, its expression in other tissue has not been investigated. Furthermore, no cell line expressing TGase 3 has been found. The tissue distribution of TGase 3 in mice was investigated using reverse-transcription polymerase chain reaction (RT-PCR) and Western blotting analyses. TGase 3 mRNA was expressed in the brain, stomach, spleen, small intestine, testis, skeletal muscle and skin. The stomach and testis expressed TGase 3 protein in size similar to that observed in the epidermis. Screening various cell lines, a gastric human cancer cell line, MKN-1 and mouse neuroblast cell line, neuro2a, were found to express TGase 3.

Isolation and characterization of cDNA encoding rabbit reticulocyte 2,3-bisphosphoglycerate synthase.

We have isolated and sequenced a cDNA clone containing the entire coding region of rabbit reticulocyte 2,3-bisphosphoglycerate (DPG) synthase. The cDNA was verified by translation of the hybridization-selected RNA and by demonstrating identity of the deduced amino acid (aa) sequence to the sequences of CNBr peptides of the purified enzyme. The aa sequence of the enzyme was homologous to the reported sequence of the human enzyme [Haggarty et al., EMBO J. 2 (1983) 1213-1220], especially in the N-terminal half (aa 1-142). Northern blot analysis of rabbit reticulocyte poly(A)+ RNA revealed a single species of mRNA with about 1700 nucleotides.

Ascorbate-dependent elevation of mRNA levels for cytochrome P450s induced by polychlorinated biphenyls.

Trichlorobiphenyl induced only CYP1A2 mRNA, while pentachlorobiphenyl induced both CYP1A2 and CYP2B1 mRNAs in rat liver. The mRNA levels for these P450s were elevated when ascorbic acid-deficient ODS rats (mutant rats with a hereditary osteogenic disorder) were fed a diet supplemented with ascorbic acid. The amount of CYP2B1 mRNA increased rapidly and reached a maximum level of approximately double within 24 hr of injection of pentachlorobiphenyl. Thereafter, the amount of its mRNA decreased to a steady level. This pattern was roughly paralleled by changes in the amount of CYP1A2 mRNA.

Four types of calpastatin isoforms with distinct amino-terminal sequences are specified by alternative first exons and differentially expressed in mouse tissues.

Calpastatin, a specific inhibitor of calpain, consists of a unique N-terminal domain (domain L) and four repetitive protease-inhibitor domains (domains 1-4). The isolated cDNAs from various mammalian species have conspicuous differences in the regions encoding the N-terminal sequences and can be classified into four types. Mouse and bovine calpastatins (Type I and Type II, respectively), which also differ from each other in the uttermost N-terminal regions, possess longer domain L sequences than those of rabbit, pig, and human inhibitors (Type III). A sequence of a shorter isoform, registered in a DNA data bank, starts from a part of domain 2 with a different N-terminal sequence (Type IV). To clarify the source of this molecular diversity, we investigated the entire exon-intron organization of the mouse calpastatin gene. The previously obtained mouse calpastatin cDNA is encoded by as many as 31 exons including the first exon, designated 1xa. Three additional exons specifying the N-terminal sequences of the other types (designated exons 1xb, 1u, and 14t, respectively) were identified in the mouse genomic DNA sequence. While the mRNAs for Types I and III were expressed at high levels in liver, the Type II mRNA was abundant in heart and skeletal muscle and expressed at lower levels in liver, brain and testis. The Type IV mRNA was specifically expressed in testis among the tissues examined. These results suggest that the calpastatin isoforms possessing different N-terminal sequences are generated by alternative transcription initiation from their own promoters and skipping of the mutually exclusive exons.

Characterization of recombinant mouse epidermal-type transglutaminase (TGase 3): regulation of its activity by proteolysis and guanine nucleotides.

Epidermal-type TGase (TGase 3) is involved in the formation of the cornified cell envelope by cross-linking a variety of structural proteins in the epidermis. Unknown proteases activate this enzyme from the zymogen form by limited proteolysis during epidermal differentiation. It has been difficult to isolate sufficient quantities of native enzymes from tissues for biochemical studies of the properties of TGase 3. In this paper, we circumvented these problems by expressing recombinant full-length mouse TGase 3 in a baculovirus system, and purifying it to homogeneity by successive chromatography and HPLC. Treatment of the purified recombinant protein with dispase, a bacterial protease known to activate zymogens, produced activated TGase 3. The migration of TGase 3 zymogen in SDS-polyacrylamide gel electrophoresis was anomalous when the proTGase 3 was pre-incubated with calcium ion. GTP inhibited the enzymatic activity of recombinant TGase 3. Calpain, a calcium-dependent neutral protease, was a candidate protease, but had no effect on the activation of TGase 3 zymogen.

Calcium-induced exposure of a hydrophobic surface of mouse ALG-2, which is a member of the penta-EF-hand protein family.

ALG-2 is a 22 kDa EF-hand type Ca2+-binding protein associated with lymphocyte apoptosis. Comparison of the primary structure of ALG-2 with those of EF-hand type proteins revealed that it belongs to the penta-EF-hand (PEF) protein family including the small subunit of calpain. We established a convenient method for the purification of the recombinant mouse ALG-2 expressed in Escherichia coli. The recombinant protein was first pelleted from a lysate in the absence of a Ca2+-chelator, and then extracted with buffer containing EDTA/EGTA followed by purification by conventional column chromatographies. Estimation of the molecular mass by gel filtration suggested that the recombinant ALG-2 occurred as a monomeric form. Ca2+-dependent precipitation was blocked by inclusion of non-ionic detergent Triton X-100, suggesting hydrophobic self-aggregation at high concentrations of the protein. The N-terminal deletion mutant lacking the hydrophobic non-PEF region was found to be more soluble than the wild type in the presence of Ca2+. Analysis using a fluorescent hydrophobicity probe indicated that ALG-2 exposed a hydrophobic surface in a Ca2+-concentration dependent manner, the half-maximal effect occurring at approximately 6 microM. Mg2+ was not effective for the conformational change. On Western blotting, ALG-2 was detected in particulate fractions from cultured mammalian cells, suggesting the association of the protein with macromolecules in the cells.

Identification and characterization of two penta-EF-hand Ca(2+)-binding proteins in Dictyostelium discoideum.

Penta-EF-hand (PEF) proteins such as ALG-2 (apoptosis-linked gene 2 product) and the calpain small subunit are a newly classified family of Ca(2+)-binding proteins that possess five EF-hand-like motifs. We identified two mutually homologous PEF proteins, designated DdPEF-1 and DdPEF-2 (64% amino acid residue identities), in the cellular slime mold Dictyostelium discoideum. Both PEF proteins showed a higher similarity to mammalian ALG-2 and peflin (Group I PEF proteins) than to calpain and sorcin subfamily (Group II PEF proteins) in the first EF-hand (EF-1) regions. Northern blot analyses revealed that DdPEF-1 and DdPEF-2 were constitutively expressed throughout development of Dictyostelium, but their levels of expression were developmentally regulated. In situ hybridization analyses demonstrated that DdPEF-1 was expressed in both the anterior prestalk and the posterior prespore regions of the tipped aggregate, slugs and early culminants. On the other hand, DdPEF-2 was dominantly expressed in the anterior tip region of these multicellular structures. Both PEF proteins were detected as 22-23-kDa proteins in soluble fractions in the presence of EGTA but in particulate fractions in the presence of Ca(2+) by Western blotting using specific monoclonal antibodies. Together with the finding of PEF-like sequences in DNA databases of plants, fungi and protists, our results strongly suggest that Group I PEF proteins are ubiquitously present in all eukaryotes and play important roles in basic cellular functions.

Enhancement effect of O6-fluorobenzylguanines on chloroethylnitrosourea cytotoxicity in tumor cells.

O6-Alkylguanine derivatives sensitize tumor cells to chloroethylnitrosourea (CENU) chemotherapy by inactivation of O6-methylguanine-DNA methyltransferase (MGMT), which repairs CENU-induced O6-alkylguanines in DNA by accepting the alkyl group at a cysteine moiety. To test the biological significance of synthesized O6-fluorobenzylguanine derivatives, we measured their ability of inactivation of MGMT activity and their effects on the cytotoxicity of 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU) in comparison with the effects of O6-benzylguanine and O6-phenylguanine. The O6-(4- and 3-fluorobenzyl)guanines considerably reduced the MGMT activity of HeLa S3 cell-free extract as did O6-benzylguanine. In contrast, O6-(2-fluorobenzyl)guanine and O6-phenylguanine had less of an effect on the activity. Two-hour pretreatment of O6-(4- and 3-fluorobenzyl) guanines potentiated ACNU cytotoxicity in HeLa S3 cells to a greater extent than did O6-(2-fluorobenzyl)guanine and O6-phenylguanine. The enhancement effects were consistent with the depletion of MGMT activity after the pretreatment of O6-fluorobenzylguanine derivatives. O6-Fluorobenzylguanines with a fluoro-substitution at the 4- or 3-position of the benzyl group were comparable to O6-benzylguanine and were powerful MGMT inactivators. The chemical features of the O6-benzyl group are a biologically important determinant in the reaction evolution with MGMT.

Characterization of a human homolog of (6-4) photolyase.

(6-4)Photolyase catalyzes light-dependent repair of UV-induced pyrimidine (6-4) pyrimidone photoproducts. A human cDNA clone which has high sequence homology to the (6-4)photolyase gene (H64PRH gene) was identified. In this paper we also isolated a genomic clone corresponding to the H64PRH cDNA and mapped it to chromosome 12q24.1 by fluorescence in situ hybridization (FISH). Northern-blot analysis revealed transcription of this gene in all human tissues examined. The H64PRH protein was overproduced in E. coli, partially purified and characterized. Like (6-4)photolyase, the enzyme contains two chromophores, one of which is FAD. However, the enzyme does not show any detectable photolyase activity.

Human kidney glomeruli, with special reference to those in the aged person: scanning electron microscopic study of microvascular corrosion casts.

Blood vascular beds of fetal, adult and aged human kidneys were reproduced with methyl methacrylate and observed with a scanning electron microscope. The kidney glomeruli, including those from the fetal kidneys, had anastomosing capillaries. The glomeruli in the kidneys of an aged person contained many more capillaries which were much more tortuous than those of the adult and fetal kidneys. Furthermore, it was observed that the glomeruli in the kidneys of the aged person usually received tortuous afferent vessels and frequently emitted multiple efferent arterioles. The glomeruli in the juxtamedullary layer of the kidneys of the aged person were rather small in size and contained degenerative capillary networks. This observation suggests that the medulla of the kidneys of the aged is poorly supplied with blood.