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J Uitto

Publications and source records attributed to J Uitto.

At least 253 records · Page 14Linked to original sources
PubMed1994

Interleukin 10 up-regulates elastin gene expression in vivo and in vitro at the transcriptional level.

In immune cells, such as T cells and monocytes, interleukin 10 (IL-10) has regulatory functions on a number of cytokines, including IL-1, IL-2, IL-8 and tumour necrosis factor-alpha expression. However, the effects of IL-10 have not previously been studied in detail in connective-tissue cells. In the present study, we show that recombinant human IL-10 at physiological concentrations has direct effects on the expression of the human elastin gene both in vivo and in vitro. Transgenic mice expressing a human elastin promoter/chloramphenicol acetyltransferase (CAT) reporter gene construct were injected subcutaneously with IL-10 (1-100 ng) and the site of injection was biopsied after 24 h. CAT assay revealed an increase of up to 3.5-fold in the promoter activity with 10 ng of IL-10. Transforming growth factor-beta 2 (TGF-beta 2) is known to up-regulate elastin gene expression in cultured fibroblasts. When IL-10 was added to such cultures, the effects of TGF-beta 2 on elastin mRNA levels were synergistically potentiated. These results suggest that IL-10 has an up-regulatory effect on elastin gene expression.

Animals↗
PubMed1994

Transforming growth factor-beta up-regulates human elastin promoter activity in transgenic mice.

We have recently developed transgenic mice which express approximately 5.2 kb of the human elastin promoter linked to the chloramphenicol acetyl transferase (CAT) reporter gene (J. Biol. Chem. 269:18072-18075, 1994). Previously, transforming growth factor-beta (TGF-beta) has been shown to enhance elastin gene expression, as determined at the mRNA and protein levels. To examine whether this enhancement could be explained by upregulation of the elastin promoter, TGF-beta 1 (100 ng) was injected subcutaneously into the transgenic animals. CAT activity in the skin of treated animals was elevated in a time-dependent manner up to approximately 10-fold after a single injection. These results suggest that the 5.2-kb up-stream segment of the human elastin gene contains cis-elements responsive to TGF-beta 1 in vivo.

Animals↗
PubMed1994

Cloning of human type VII collagen. Complete primary sequence of the alpha 1(VII) chain and identification of intragenic polymorphisms.

Type VII collagen is the major, if not the exclusive, component of the anchoring fibrils, attachment structures at the dermal-epidermal basement membrane zone. In this study, we have isolated overlapping cDNA clones which correspond to full-length human type VII collagen mRNA of approximately 9.2 kilobases. The full-length cDNA sequence contains an 8,833 nucleotide open reading frame encoding 2,944 amino acids. The deduced amino acid sequence revealed the presence of a central collagenous domain flanked by a large NH2-terminal non-collagenous (NC-1) domain which consists of submodules with homology to known adhesive proteins, including nine fibronectin type III-like segments (FNIII), and a smaller COOH-terminal non-collagenous (NC-2) domain. In addition, we report six intragenic polymorphisms in the type VII collagen gene (COL7A1) which can be detected by restriction enzyme digestion of polymerase chain reaction-amplified segments. The complete cDNA sequence and polymorphisms in COL7A1 will facilitate mutational analysis and prenatal diagnosis for patients with the dystrophic forms of epidermolysis bullosa, in which mutations in COL7A1 have been demonstrated.

Amino Acid Sequence↗
PubMed1994

Tissue-specific and developmentally regulated expression of human elastin promoter activity in transgenic mice.

We have recently cloned the entire human elastin gene, including approximately 5.2 kilobases of the 5'-flanking sequences. To examine tissue-specific expression of the elastin gene, we have developed a transgenic mouse line that expresses the human elastin promoter linked to the chloramphenicol acetyltransferase (CAT) reporter gene. Assay of CAT activity in different tissues revealed the highest expression in the lungs and aorta, while lower levels were detected in the kidneys, heart, brain, and skin; this distribution parallels the accumulation of elastin in developing animals. Comparison of CAT activity in the lungs of fetal (15-day gestation) and newborn (5-day postnatal) animals revealed significantly (approximately 4-fold) higher activity in the fetal tissue. The relatively high activity in the lungs progressively declined during the postnatal period up to 6 months. The promoter activity in the aorta remained constant from 5 days to 3 months and then gradually declined, while in the skin, the activity peaked at 3 months, returning thereafter to the control (5-day) level. Thus, there is evidence for developmentally regulated, tissue-specific expression of the elastin promoter in vivo as tested in these transgenic mice.

Aging↗
PubMed1994

Premature termination codons in the type VII collagen gene (COL7A1) underlie severe, mutilating recessive dystrophic epidermolysis bullosa.

Epidermolysis bullosa (EB) is a group of heritable mechano-bullous skin diseases classified into three major categories on the basis of the level of tissue separation within the dermal-epidermal basement membrane zone. The most severe, dystrophic (scarring) forms of EB demonstrate blister formation below the cutaneous basement membrane at the level of the anchoring fibrils. Ultrastructural observations of altered anchoring fibrils and genetic linkage to the gene encoding type VII collagen (COL7A1), the major component of anchoring fibrils, have implicated COL7A1 as the candidate gene in the dystrophic forms of EB. We have recently cloned the entire cDNA and gene for human COL7A1, which has been mapped to 3p21. In this study, we describe mutations in four COL7A1 alleles in three patients with severe, mutilating recessive dystrophic EB (Hallopeau-Siemens type, HS-RDEB). Each of these mutations resulted in a premature termination codon (PTC) in the amino-terminal portion of COL7A1. One of the patients was a compound heterozygote for two different mutations. The heterozygous carriers showed an approximately 50% reduction in anchoring fibrils, yet were clinically unaffected. Premature termination codons in both alleles of COL7A1 may thus be a major underlying cause of the severe, recessive dystrophic forms of EB.

Adult↗
PubMed1994

Structural organization of the human type VII collagen gene (COL7A1), composed of more exons than any previously characterized gene.

The human type VII collagen (COL7A1) gene is the locus for mutations in at least some cases of dystrophic epidermolysis bullosa. Here we describe the entire intron/exon organization of COL7A1, which is shown to have 118 exons, more than any previously described gene. Despite this complexity, COL7A1 is compact. Consisting of 31,132 bp from transcription start site to polyadenylation site, it is only about three times the size of type VII collagen mRNA. Thus, COL7A1 introns are small. A 71-nucleotide COL7A1 intron is the smallest intron yet reported in a collagen gene, and only one COL7A1 intron is greater than 1 kb in length. All exons in the COL7A1 triple helix coding region that do not begin with sequences corresponding to imperfections of the triple helix begin with intact codons for Gly residues of Gly-X-Y repeats. This is reminiscent of the structure of fibrillar rather than other nonfibrillar collagen genes. In addition, the COL7A1 triple helix coding region contains many exons of recurring sizes (e.g., 25 exons are 36 bp, 12 exons are 45 bp, 8 exons are 63 bp), suggesting an evolutionary origin distinct from those of other nonfibrillar collagen genes. Sequences from the 5' portion of COL7A1 are presented along with the 3766-bp intergenic sequence, which separates COL7A1 from the upstream gene encoding the core I protein of the cytochrome bc1 complex. The COL7A1 promoter region is found to lack extensive homologies with promoter regions of other genes expressed primarily in skin.

Amino Acid Sequence↗
PubMed1994

Dominant dystrophic epidermolysis bullosa: identification of a Gly-->Ser substitution in the triple-helical domain of type VII collagen.

Epidermolysis bullosa (EB) represents a group of genodermatoses characterized by fragility and easy blistering of the skin. In the dystrophic forms of EB, blisters occur below the basement membrane of the skin, at the level of the anchoring fibrils. We have recently demonstrated tight genetic linkage between the type VII collagen gene (COL7A1) and both the dominant and recessive forms of dystrophic EB. We searched for mutations in dominant dystrophic EB by PCR amplification of genomic segments of COL7A1, followed by heteroduplex analysis. Examination of the PCR fragment corresponding to exon 73 of COL7A1 revealed a marked shift in the electrophoretic pattern in patients from a large Finnish dominant dystrophic EB family with genetic linkage to the COL7A1 locus (Z = 5.37, theta = 0). Sequence analysis revealed a G-->A transition at nucleotide 6118 in the triple helical domain of COL7A1, which converted a glycine residue to a serine (GGT-->AGT). This mutation occurs between interruptions 11 and 12 of the triple helix, in the seventh of a series of 24 uninterrupted Gly-Xaa-Yaa repeats. Pathogenetic glycine substitutions that disrupt the triple helix have been shown to exert a deleterious effect on the protein in several other disorders involving collagen genes. The clinical phenotype in this family probably arises due to a dominant negative mutation in type VII collagen, resulting in the formation of structurally abnormal anchoring fibrils.

Amino Acid Sequence↗
PubMed1994

Differential cytokine regulation of type I and type VII collagen gene expression in cultured human dermal fibroblasts.

Type VII collagen is the major component of anchoring fibrils in the cutaneous basement membrane zone. In this study, we have examined the effects of various cytokines on the expression of types I and VII collagen genes in dermal fibroblasts in culture. The pro-inflammatory cytokines, interleukin-1 alpha (IL-1 alpha), interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), and leukoregulin (LR) strongly elevated (approximately 5-9-fold) type VII collagen mRNA levels, as measured by Northern blot hybridizations. These effects were also observed at the protein level by indirect immunofluorescence using a monoclonal antibody specific for type VII collagen. By contrast, IL-1 beta had only a slight stimulatory effect (approximately 2-fold) on type I collagen gene expression, while TNF-alpha and LR markedly reduced type I collagen mRNA steady-state levels. Interestingly, IL-1, TNF-alpha and LR had additive effects with transforming growth factor-beta (TGF-beta) on type VII collagen gene expression, whereas they counteracted the up-regulatory effect of TGF-beta on type I collagen gene expression. Thus, our data indicate that the modulation of type I and type VII collagen gene expression by cytokines involves different regulatory pathways.

Adult↗
PubMed1994

Identification of a DNA-binding protein (keratinocyte transcriptional protein-1) recognizing a keratinocyte-specific regulatory element in the 230-kDa bullous pemphigoid antigen gene.

We have recently cloned and characterized the entire human 230-kDa bullous pemphigoid antigen gene, which is expressed at a relatively high level in the basal keratinocytes. A putative AP2 binding sequence (KRE2), identified in the position -1786 to -1778, was cloned in front of a heterologous thymidine kinase chloramphenicol acetyltransferase construct, and transient transfections of normal human keratinocytes indicated a marked enhancement of the promoter activity. Normal human keratinocyte nuclear extracts contained a protein, designated as keratinocyte transcriptional protein-1 (KTP-1), which complexed with the KRE2 oligomer when examined by gel mobility shift assays. This protein was not detected in human skin fibroblast or HeLa cell nuclear extracts that did, however, contain AP2. UV cross-linking studies and Southwestern analyses suggested that KTP-1 binds to DNA as a single polypeptide of approximately 110 kDa. These data suggest that KTP-1 is a DNA-binding protein clearly distinct from AP2, and this protein may be responsible for the basal keratinocyte-specific expression of the bullous pemphigoid antigen gene.

Adult↗
PubMed1994

Uncoordinate regulation of collagenase, stromelysin, and tissue inhibitor of metalloproteinases genes by prostaglandin E2: selective enhancement of collagenase gene expression in human dermal fibroblasts in culture.

The degradative effects of interleukin-1 (IL-1) on the extracellular matrix of connective tissue are mediated primarily by metalloproteinases and prostaglandins. Clinical observations suggest that these effects can be prevented, to some extent, by the use of non-steroidal anti-inflammatory drugs. We have examined the role of prostaglandin E2 (PGE2) in IL-1-induced gene expression by human skin fibroblasts in culture. Incubation of confluent fibroblast cultures with varying concentrations (0.01-1.0 microgram/ml) of PGE2 led to a dose-dependent elevation of collagenase mRNA steady-state levels, the promoter activity, and the secretion of the protein, whereas relatively little effect was observed on stromelysin and TIMP gene expression. Exogenous PGE2 had no additive or synergistic effect with IL-1 on collagenase gene expression. Furthermore, commonly used non-steroidal anti-inflammatory drugs (indomethacin, acetyl salicylic acid and ibuprofen), at doses which block prostaglandin synthesis in cultured fibroblasts, failed to counteract IL-1-induced collagenase and stromelysin gene expression, nor did they affect TIMP expression. Although the effects of PGE2 did not potentiate those of IL-1 on collagenase gene expression in vitro, one could speculate that massive production of PGE2 by connective tissue cells in vivo in response to inflammatory mediators such as IL-1 or tumor necrosis factor-alpha, could lead to sustained expression of collagenase in connective tissue cells after clearance of the growth factors.

Cells, Cultured↗
PubMed1994

Molecular basis for the dystrophic forms of epidermolysis bullosa: mutations in the type VII collagen gene.

Significant progress has recently been made in understanding the molecular basis of heritable skin diseases, such as epidermolysis bullosa, a group of mechano-bullous genodermatoses. In particular, the dystrophic forms of epidermolysis bullosa have been shown to result from distinct mutations in the gene encoding type VII collagen, the major, if not the exclusive, component of the anchoring fibrils. These mutations result in deficient synthesis and/or altered assembly of the anchoring fibrils, thus compromising the integrity of the cutaneous basement membrane zone. The mutations in the type VII collagen gene have implications for understanding the structure-function relationships of the type VII collagen molecule, and also provide the basis for prenatal DNA-based diagnosis in families at risk for recurrence of the disease. Finally, understanding the genetic basis of dystrophic forms of EB sets the stage for gene therapy approaches for the treatment of these devastating skin diseases.

Collagen↗
PubMed1994

Expression of laminin, type IV procollagen and 230 kDa bullous pemphigoid antigen genes by keratinocytes and fibroblasts in culture: application of the polymerase chain reaction for detection of small amounts of messenger RNA.

In order to clarify whether keratinocytes and/or fibroblasts express genes encoding basement membrane zone macromolecules, we examined laminin, type IV collagen and 230 kDa bullous pemphigoid antigen (BPAG1) gene expression in keratinocytes and fibroblasts in culture. Northern transfer analysis revealed the presence of specific mRNA transcripts for alpha 1(IV) and alpha 2(IV) chains of type IV collagen as well as B1 and B2 chains of laminin in both fibroblast and keratinocyte RNA. Laminin A mRNA, however, was detected in fibroblasts but not in keratinocytes. In contrast, BPAG1 mRNA was detected in keratinocytes but not in fibroblasts using the same RNA preparations. A polymerase chain reaction (PCR) using laminin A and BPAG1-specific primers produced amplified DNAs with the predicted sizes in reverse-transcripted cDNA derived from keratinocyte and fibroblast RNA, respectively. These results provide evidence that normal human skin keratinocytes and fibroblasts express genes encoding laminin A, B1, B2, alpha 1(IV), and BPAG1 at a steady-state level. Moreover, the PCR for detecting small amounts of mRNA suggested that both keratinocytes and fibroblasts can be utilized for the analysis of DNA mutations in inherited skin diseases affecting the basement membrane zone, such as epidermolysis bullosa.

Autoantigens↗
PubMed1994

Type VII collagen DNA linkage analysis in a Japanese family with dominant dystrophic epidermolysis bullosa.

Type VII collagen, a major component of anchoring fibrils in the basement membrane zone, is now considered to be a primary genetic factor in the pathogenesis of dominant dystrophic epidermolysis bullosa (DDEB). In this study, we performed genetic linkage analysis in a Japanese family with DDEB using a PvuII polymorphism in the type VII collagen gene. The pedigree consisted of 10 affected and 13 unaffected living individuals and was diagnosed as having Cockayne-Touraine type of DDEB. Electron microscopic examination of the skin demonstrated a diminished number and rudimentary structure of anchoring fibrils. PCR-based detection of PvuII polymorphism resulted in 3 genotypes and co-segregated with DDEB phenotype in this pedigree. The maximum lod score was 2.10 at recombination fraction (theta) of 0. The absence of recombination between DDEB and type VII collagen gene locus, as well as the observation of altered anchoring fibrils, suggested that type VII collagen is a candidate gene for the Japanese family with DDEB, although the lod score was statistically not significant.

Base Sequence↗
PubMed1994

Tufted angioma of the thigh.

Tufted angioma or angioblastoma of Nakagawa is a rare vascular tumor that usually appears in early childhood. It frequently is seen as an erythematous to red-brown, frequently indurated plaque that usually occurs on the trunk or neck. Typically tufted angiomas enlarge for a few years and then cease growing and remain stable. Histopathologic findings are pathognomonic. We describe a patient with a tufted angioma of the thigh who sought treatment for paroxysmal episodes of pain. Treatment with topical clobetasol propionate resulted in a decreased frequency of painful episodes.

Adolescent↗
PubMed1994

Mutations in the gamma 2 chain gene (LAMC2) of kalinin/laminin 5 in the junctional forms of epidermolysis bullosa.

Junctional epidermolysis bullosa (JEB) is an autosomal recessive disorder characterized by blister formation within the dermal-epidermal basement membrane. Genes for the lamina lucida protein, kalinin/laminin 5, have been proposed as candidates for some forms of JEB, based on immunofluorescence analysis recognizing kalinin epitopes. We studied the cDNA of laminin gamma 2 chain for mutations in JEB using heteroduplex analysis. One patient showed a homozygous splice site mutation while another was heterozygous for a deletion-insertion, resulting in a premature termination codon in one allele. Our data implicate mutations in the laminin gamma 2 chain gene (LAMC2) in some forms of JEB.

Base Sequence↗
PubMed1994

Herlitz's junctional epidermolysis bullosa is linked to mutations in the gene (LAMC2) for the gamma 2 subunit of nicein/kalinin (LAMININ-5).

We have linked Herlitz's junctional epidermolysis bullosa (H-JEB) to the gene (LAMC2) encoding the gamma 2 subunit of nicein/kalinin, an isolaminin (laminin-5) expressed by basal keratinocytes. In four H-JEB kindreds, a maximum two-point lod score of 5.33 at theta = 0 was observed between a microsatellite near LAMC2 at 1q25-31 and the disease. In one family, a homozygous point mutation leading to a premature stop codon (CGA to TGA) was identified in exon 3 of the gene. The segregation of the mutated allele implicates the mutation in the pathology of the disorder and corroborates the linkage results.

Base Sequence↗