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Biomedical subjects

J Uitto

Publications and source records attributed to J Uitto.

At least 109 records · Page 6Linked to original sources
PubMed1999

Cytokine modulation of type XV collagen gene expression in human dermal fibroblast cultures.

The expression of type XV collagen was studied in cultured human dermal fibroblasts exposed to transforming growth factor-beta (TGF-beta), tumor necrosis factor-alpha (TNF-alpha) or interleukin-1beta (IL-1beta), cytokines which have been shown previously to alter the expression of several extracellular matrix genes. TGF-beta enhanced the expression of the type XV collagen gene (COL15A1) in a time-dependent manner, up to 4.3-fold after 24 h of incubation, whereas TNF-alpha and IL-1beta reduced the mRNA steady-state levels by 32 and 80%, respectively. When TGF-beta and TNF-alpha were added together to the cultures, the stimulatory effect of TGF-beta on type XV collagen gene expression was abrogated, indicating antagonistic modulation by these 2 cytokines. These data suggest that the cytokines tested in this study may contribute to the regulation of type XV collagen synthesis in a variety of tissues which have recently been shown to express this particular collagen gene.

Cells, Cultured↗
PubMed1999

Targeted inactivation of the type VII collagen gene (Col7a1) in mice results in severe blistering phenotype: a model for recessive dystrophic epidermolysis bullosa.

Dystrophic forms of epidermolysis bullosa (DEB) are associated with mutations in the type VII collagen gene (Col7a1) which encodes the major component of anchoring fibrils. To develop a DEB animal model, type VII collagen deficient mice were generated by targeted homologous recombination. The targeting vector replaced exons 46-69 of Col7a1 with the neomycin-resistance gene, in reverse transcriptional orientation, resulting in elimination of most of the collagenous domain 1. Col7a1 heterozygous (+/-) mice were phenotypically normal. Mating of Col7a1 +/- mice revealed that Col7a1 null (-/-) mice, which were born with extensive cutaneous blistering, died during the first two weeks of life probably due to complications arising from the blistering. Transmission electron microscopy revealed subepidermal blistering below the lamina densa and absence of anchoring fibrils. Immunohistochemical staining with anti-human type VII collagen antibody stained the dermal-epidermal junction in control mice, but did not stain the skin of Col7a1 null mice. Collectively, the DEB mice recapitulate the clinical, genetic, immunohistochemical and ultrastructural characteristics of recessive DEB in humans. These mice provide an animal model to study the pathomechanisms of DEB and serve as a system to test therapeutic approaches, including gene replacement, towards the cure of this devastating skin disease.

Animals↗
PubMed1999

Expression of the elastin promoter in novel tissue sites in transgenic mouse embryos.

We have previously shown in a transgenic mouse line, in which 5.2 kb of the elastin promoter was linked to the reporter enzyme chloramphenicol acetyltransferase (CAT), that the highest levels of expression were found in embryonic lungs and aorta, while lower levels were detected in other elastin-containing tissues. Furthermore, in general, expression of the transgene showed developmental regulation similar to that of the endogenous gene. However, the precise location of cellular expression could not be determined in this model. To overcome this limitation, we have developed a similar model, but replaced CAT with the reporter enzyme beta-galactosidase. Enzyme activity was readily detected in the transgenic mouse embryos in expected regions of tissue forming elastic fibers, including the dermis and elastic cartilage. Of considerable interest, however, was the novel finding of expression in specific areas of neuroepithelium of the brain and in the perichondrium surrounding areas destined to form hyaline cartilage in endochondral bone formation. These latter areas included all the bones of the limbs, the spine and rib cage. It appeared that these segments of elastin expression demarcated the border between the developing cartilage and the surrounding mesenchymal tissue. Elastin promoter expression was also found in developing somites, in the mesenchymal layer of the forming cornea of the eye, in the genital tubercle and in the epithelium destined to form the olfactory epithelium. These findings indicate that the elastin promoter is activated during embryonic development in a variety of tissues, suggesting that elastin gene expression may play a role in organizing cutaneous, skeletal and neural structures.

Animals↗
PubMed1999

Cloning of multiple keratin 16 genes facilitates prenatal diagnosis of pachyonychia congenita type 1.

Pachyonychia congenita type 1 (PC-1) is an autosomal dominant ectodermal dysplasia characterized by severe nail dystrophy, focal non-epidermolytic palmoplantar keratoderma (FNEPPK) and oral lesions. We have previously shown that mutations in keratin K16 cause fragility of specific epithelia resulting in phenotypes of PC-1 or FNEPPK alone. These earlier analyses employed an RT-PCR approach to avoid amplification of K16-like pseudogenes. Here, we have cloned the K16 gene (KRT16A) and two homologous pseudogenes (psiKRT16B and psiKRT16C), allowing development of a genomic mutation detection strategy based on a long-range PCR, which is specific for the functional K16 gene. We report a novel heterozygous 3 bp deletion mutation (388del3) in K16 in a sporadic case of PC-1. The mutation was detected in genomic DNA and confirmed at the mRNA level by RT-PCR, showing that our genomic PCR system is reliable for K16 mutation detection. Using this system, we carried out the first prenatal diagnosis for PC-1 using CVS material, correctly predicting a normal fetus. This work will greatly improve K16 mutation analysis and allow predictive testing for PC-1 and the related phenotype of FNEPPK.

Abnormalities, Multiple↗
PubMed1998

SMAD3/4-dependent transcriptional activation of the human type VII collagen gene (COL7A1) promoter by transforming growth factor beta.

The human type VII collagen gene (COL7A1) recently has been identified as an immediate-early response gene for transforming growth factor beta (TGF-beta)/SMAD signaling pathway. In this study, by using MDA-MB-468 SMAD4-/- breast carcinoma cells, we demonstrate that expression of SMAD4 is an absolute requirement for SMAD-mediated promoter activity. We also demonstrate that the SMAD binding sequence (SBS) representing the TGF-beta response element in the region -496/-444 of the COL7A1 promoter functions as an enhancer in the context of a heterologous promoter. Electrophoretic mobility-shift assays with nuclear extracts from COS-1 cells transfected with expression vectors for SMADs 1-5 indicate that SMAD3 forms a complex with a migration similar to that of the endogenous TGF-beta-specific complex observed in fibroblast extracts. Electrophoretic mobility-shift assays using recombinant glutathione S-transferase-SMAD fusion proteins indicate that both SMAD4 and C-terminally truncated SMAD3, but not SMAD2, can bind the COL7A1 SBS. Coexpression of SMAD3 and SMAD4 in COS-1 cells leads to the formation of two complexes: a DNA/protein complex containing SMAD3 alone and another slower-migrating complex containing both SMAD3 and SMAD4, the latter complex not being detected in fibroblasts. Maximal transactivation of COL7A1 SBS-driven promoters in either MDA-MB-468 carcinoma cells or fibroblasts requires concomitant overexpression of SMAD3 and SMAD4. These data may represent the first identification of a functional homomeric SMAD3 complex regulating a human gene.

Animals↗
PubMed1998

Smad-dependent transcriptional activation of human type VII collagen gene (COL7A1) promoter by transforming growth factor-beta.

We have previously shown that transforming growth factor-beta (TGF-beta) increases type VII collagen gene (COL7A1) expression in human dermal fibroblasts in culture (Mauviel, A., Lapière, J.-C., Halcin, C., Evans, C. H., and Uitto, J. (1994) J. Biol. Chem. 269, 25-28). To gain insight into the molecular mechanisms underlying the up-regulation of COL7A1 by this growth factor, we performed transient cell transfections with a series of 5'-deletion promoter/chloramphenicol acetyltransferase reporter gene constructs. We identified a 68-base pair region between nucleotides -524 and -456, relative to the transcription start site, as critical for TGF-beta response. Using electrophoresis mobility shift assays (EMSAs) with an oligonucleotide spanning the region from -524 to -444, we discovered that a TGF-beta-specific protein-DNA complex was formed as early as 11 min after TGF-beta stimulation and persisted for 1 h after addition of the growth factor. Deletion analysis of the TGF-betaresponsive region of the COL7A1 promoter by EMSA identified segment -496/-444 as the minimal fragment capable of binding the TGF-beta-induced complex. Furthermore, two distinct segments, -496/-490 and -453/-444, appeared to be necessary for TGF-beta-induced DNA binding activity, suggesting a bipartite element. Supershift experiments with a pan-Smad antibody unambiguously identified the TGF-beta-induced complex as containing a Smad member. This is the first direct identification of binding of endogenous Smad proteins to regulatory sequences of a human gene.

Base Sequence↗
PubMed1998

cDNA cloning, mRNA expression, and chromosomal mapping of human and mouse periplakin genes.

A portion of the intracellular domain of Type XVII collagen, used as a bait in a yeast two-hybrid screen of an epidermal keratinocyte cDNA library, identified overlapping cDNA clones that showed a high degree of homology to envoplakin and other members of the plakin family of intermediate filament connector molecules. Subsequent cloning allowed identification of contiguous cDNA sequences with an open reading frame of 5268 bp encoding a putative polypeptide of 1756 amino acids with a computed molecular mass of 204.7 kDa. Northern analysis using these cDNA clones revealed a prominent band of approximately 6.5 kb in keratinocytes, which was barely detectable in fibroblasts. Multiple tissue RNA analysis showed that this protein is highly expressed in tissues with a prominent component of epithelial cells. This novel member of the plakin family was designated periplakin. The human gene (PPL) was mapped to the interval between D16S510 and D16S509 by radiation hybrid mapping, corresponding to chromosomal band 16p13. Murine ESTs having 97.2% amino acid identity to the human sequence were identified. Interspecific backcross mapping was used to place the murine periplakin gene (Ppl) 0.53 cM distal to marker D16mit32 on the proximal part of murine chromosome 16, close to the locus of mahoganoid (md), a mouse hair mutant. Mapping of this gene in human and mouse will allow evaluation of periplakin as a candidate locus for disorders of epithelial fragility, with or without other phenotypes.

Amino Acid Sequence↗
PubMed1998

Direct interaction between the intracellular domains of bullous pemphigoid antigen 2 (BP180) and beta 4 integrin, hemidesmosomal components of basal keratinocytes.

Bullous pemphigoid antigen 2 (BPAG2/BP180, also known as type XVII collagen) and alpha 6 beta 4 integrin are both transmembrane proteins and hemidesmosomal components of basal keratinocytes. In this study, using yeast two-hybrid system, we demonstrate direct protein-protein interaction between the intracellular domains of BP180 and the beta 4 integrin subunit. Detailed analysis revealed that a bait construct spanning amino acids 13-89 of BP180 contained sufficient information for the protein protein interaction, but further deletion of 13 amino-terminal amino acids, which eliminates a predicted beta-sheet, abolished the interaction. The intracellular domain of the beta 4 integrin subunit contains two pairs of fibronectin type III (FNIII) repeats separated by a connecting segment. Series of expression constructs, sequentially deleting each domain, revealed that the connecting segment, the second pair of FNIII repeats and the tail region of the beta 4 integrin subunit were necessary for the interaction with BP180 in yeast two-hybrid system.

Alternative Splicing↗
PubMed1998

Different ocular abnormalities in individuals of a three-generation family caused by a new nonsense mutation in the PST domain of the PAX6 gene. Mutations in brief no. 189. Online.

PAX6 is a candidate gene for familial aniridia. We have carried out a mutational analysis of the PAX6 gene in a three-generation family from Germany, containing 5 individuals affected with ocular abnormalities. In all affected individuals, a heterozygous mutation was detected in the PAX6 gene, exchanging tyrosine 369 by a stop codon. The mutation is located in the 3' moiety of the PST domain, at the C terminus of the PAX6 protein. In the affected family members, the same heterozygous mutation leads to distinct phenotypes of varying severity. Most notably, no aniridia was observed in one of the family members carrying the mutation, although other ocular abnormalities (underdeveloped iris and cataracts) were present. We discuss the possibility that small C terminal truncations of the PAX6 protein might lead to less severe or more divergent phenotypes than trancations at internal positions.

Aniridia↗
PubMed1998

A proximal element within the human alpha 2(I) collagen (COL1A2) promoter, distinct from the tumor necrosis factor-alpha response element, mediates transcriptional repression by interferon-gamma.

Previous studies have shown that interferon-gamma (IFN-gamma) inhibits type I collagen gene expression through both transcriptional and post-transcriptional mechanisms (Kahäri et al., 1990). In the present study, using transient cell transfections of human dermal fibroblast cultures with a series of 5' deletion promoter/CAT reporter gene constructs, we have identified the IFN-gamma-response element of the human alpha 2(I) collagen gene (COL1A2) promoter. Specifically, we have identified a segment of the proximal promoter region, located between nucleotides -161 and -125 relative to the transcription start site, as critical for down-regulation of COL1A2 promoter activity by IFN-gamma. This IFN-gamma response element (IgRE) is clearly distinct from the previously described tumor necrosis factor-alpha response element (TaRE) located between nucleotides -265 and -241 of the COL1A2 promoter, a difference which is likely to explain the additive inhibitory effect of these two cytokines. The inhibitory effect of IFN-gamma was dose-dependent and rapidly induced, requiring less than 5 min exposure of fibroblast cultures. Gel mobility shift assays indicated that a highly specific nuclear protein complex bound to this 37-base pair region of promoter. Competition experiments with oligonucleotides spanning discrete segments of this promoter region mapped the binding element within a distinctive pyrimidine-rich sequence. Point mutations within the latter revealed that this element plays a crucial role not only in the IFN-gamma response, but also in the basal activity of the proximal promoter. Substitution mutations within the IgRE of the -161/CAT construct attenuated the promoter response to IFN-gamma, as measured in transient cell transfections, and eliminated specific DNA/protein complex formation, as measured by gel mobility shift assay. UV-crosslinking experiments indicated that two DNA/protein complexes were formed with the IgRE, with molecular weights around 55 kDa and 30 kDa, corresponding to proteins of approximately 30 kDa and approximately 6 kDa, respectively. Our results further clarify the molecular mechanisms involved in the regulation of type I collagen gene expression by IFN-gamma.

Binding, Competitive↗
PubMed1998

Two-hybrid analysis reveals multiple direct interactions for thrombospondin 1.

The yeast two-hybrid system was used to reveal the interactions between proteins residing within the cutaneous basement membrane zone and other gene products expressed in cultured human keratinocytes. The proteins of interest included type VII collagen, the predominant component of anchoring fibrils, and laminin 5, a component of anchoring filaments. Although the two-hybrid system was not able to verify a direct interaction between the type VII collagen NC1 domain and the short arm of Lam(beta)3, the type VII collagen NC1 domain (tVII/NC1) and the laminin 5 beta3 chain globular domain VI (lam5/beta3) cDNAs, when used as baits, detected four overlapping cDNA clones encoding thrombospondin 1 (TSP1). The overlapping region of these cDNAs encodes amino acids 400-459, a segment included within a 70 kDa chymotryptic fragment known to bind type V collagen, laminin-1 and other matrix components. The type VII collagen NC1/TSP1 interaction was confirmed by exchanging the vectors, and the interacting domain was mapped by testing a set of both 5' and 3' deletion constructs. The central region of TSP1, when used as a bait in two-hybrid system, showed strong binding to the fibronectin (FN) type III-like repeats 4-7 of type VII collagen NC1 domain. The TSP1 bait also interacted with laminin 5 beta3 chain domain V/III, and the TSP1/laminin 5 beta3 chain interaction was verified by a GST-fusion protein interaction assay. The transcripts encoding TSP1, TSP2, Lam(beta)3 and type VII collagen were abundant in cultured foreskin keratinocytes, and the expression of TSP1 and TSP2 in a wide variety of adult and fetal tissues was confirmed by PCR analysis of multiple tissue cDNA panels. Furthermore, TSP1 type I repeats showed self interaction, and recognized a clone for extracellular matrix protein fibrillin-2. In addition, clones encoding angiogenesis related protein Jagged1 and a platelet enzyme phospholipase scramblase were identified. Thus, the results indicate several previously undetected interactions of TSP1, which is known to be highly expressed during embryonic development, tissue remodeling and wound healing.

Adult↗
PubMed1998

Keratin 17 mutations cause either steatocystoma multiplex or pachyonychia congenita type 2.

Pachyonychia congenita type 2 (PC-2; Jackson-Lawler syndrome) is an autosomal dominant disorder characterized by hypertrophic nail dystrophy, mild focal keratoderma, multiple pilosebaceous cysts and other features of ectodermal dysplasia. Keratin 17 (K17) is a differentiation-specific keratin expressed in the nail bed, hair follicle, sebaceous gland and other epidermal appendages. Previously, we have demonstrated that PC-2 is caused by mutations in K17 and that similar mutations in this gene can present as steatocystoma multiplex with little or no nail dystrophy. Here, we describe three unrelated kindreds carrying K17 mutations. Two of these families have identical missense mutations (R94C) in the 1A domain of K17. However, while affected members of one kindred have the classical features of PC-2, affected persons in the other family have the steatocystoma multiplex phenotype. In a third family with PC-2, mutation N92S was detected, bringing the total number of distinct mutations reported in K17 thus far to 11. These results demonstrate that K17 mutations commonly underlie both PC-2 and steatocystoma multiplex and that the alternate phenotypes which arise from these genetic lesions in K17 are independent of the specific mutation involved.

Adolescent↗
PubMed1998

Pyloric atresia-junctional epidermolysis bullosa syndrome: mutations in the integrin beta4 gene (ITGB4) in two unrelated patients with mild disease.

Junctional epidermolysis bullosa associated with pyloric atresia (EB-PA; OMIM 226730) is a rare autosomal recessively inherited disease in which mucocutaneous fragility is associated with gastrointestinal atresia. This disease is usually fatal within the first few weeks or months of life even following surgical correction of the intestinal obstruction. Recently, mutations in the genes encoding the epithelial integrin alpha6beta4 (ITGA6 and ITGB4) have been identified in several patients with EB-PA. We report two unrelated patients with this disease who have survived into early childhood with mild cutaneous involvement, in whom we have identified pathogenetic mutations in ITGB4. The first patient was a compound heterozygote for a splice site mutation in exon 30 (3793 + 1G-to-A) and a non-sense mutation in exon 36 (W1478X), and the second was a compound heterozygote for a missense mutation in exon 3 (C38R) and a 1 bp deletion in exon 36 (4776delG). Although the non-sense and deletion mutations are predicted to result in markedly reduced beta4 integrin mRNA levels, the presence of the missense or splice site mutation on the second allele may enable the synthesis of some functional, albeit perturbed, beta4 polypeptide. Determination of the molecular mechanisms in these two cases increases our understanding of EB-PA and may enable correlation between genotype and phenotype.

Antigens, CD↗
PubMed1998

Cycloheximide facilitates the identification of aberrant transcripts resulting from a novel splice-site mutation in COL17A1 in a patient with generalized atrophic benign epidermolysis bullosa.

Patients with generalized atrophic benign epidermolysis bullosa often show decreased expression of type XVII collagen, a transmembrane hemidesmosomal protein encoded by COL17A1. This report documents a novel splice-site mutation in COL17A1 in a patient with generalized atrophic benign epidermolysis bullosa, and applies a new methodology to define and characterize the resulting mRNA splice variants. Mutational analysis of COL17A1 identified a maternally inherited G-to-T transversion at the -1 position of exon 32. This acceptor splice-site mutation led to the formation of aberrant transcripts present at extremely low levels. Based on our recent finding that cycloheximide stabilized mutant COL17A1 transcripts in keratinocytes homozygous for a frameshift mutation, the effects of the splice-site mutation on splicing of COL17A1 transcripts were determined using reverse transcriptase polymerase chain reaction of total RNA from keratinocytes incubated for 2.5 h in the presence or absence of 10 microg cycloheximide per ml. Using this approach, an abnormally spliced transcript was identified that contains an extra 264 bases upstream from exon 32, resulting in a premature termination codon 27 bp downstream from the cryptic splice site. Three other splice variants, including one derived from the skipping of exon 32, were also identified. These results indicate the usefulness of cycloheximide treatment in evaluating the abnormal processing of mRNA due to splice-site mutations, because: (i) aberrant splicing often generates a premature termination codon, (ii) transcripts with premature termination codons can occur at low or undetectable levels due to nonsense-mediated mRNA decay, and (iii) the levels of these transcripts can be increased by cycloheximide.

Adult↗
PubMed1998

Novel mutations in the LAMB3 gene shared by two Japanese unrelated families with Herlitz junctional epidermolysis bullosa, and their application for prenatal testing.

The LAMB3 gene encoding the beta3 chain of laminin 5 is a candidate gene for mutations in the autosomal recessive blistering skin disorder, junctional epidermolysis bullosa. In this study, we performed genetic analyses in two unrelated Japanese families with Herlitz junctional epidermolysis bullosa and identified two novel nonsense mutations in the LAMB3 gene. One of them, Q166X (CAG --> TAG), was found in the maternal allele of family 1 and the paternal allele of family 2. Conversely, the other mutation, W610X (TGG --> TGA), was found in the paternal allele of family 1 and the maternal allele of family 2. Thus, probands of both families were compound heterozygotes for these nonsense mutations. Haplotype analyses with intragenic LAMB3 polymorphisms suggested that both mutations had arisen independently in these two families. Both mutations create a premature translation termination codon predicting truncated beta3 chains that lead to absent expression of laminin 5 in the epidermal basement membrane zone. Based on these results, DNA-based prenatal diagnosis was performed by chorionic villus sampling for subsequent pregnancies in both families. Both fetuses were found to be heterozygous carriers of the W610X mutation together with a normal LAMB3 allele, indicating that they were phenotypically unaffected. These findings expand the repertoire of LAMB3 mutations in junctional epidermolysis bullosa, and emphasize the notion that premature termination codons in both alleles of the laminin 5 genes result in Herlitz junctional epidermolysis bullosa.

Base Sequence↗
PubMed1998

A novel in vivo model for evaluating agents that protect against ultraviolet A-induced photoaging.

Increasing evidence demonstrates that ultraviolet A radiation (UVA) contributes to photoaging, which results in the accumulation of massive amounts of abnormal elastic material in the dermis of photoaged skin. To study UVA-induced photoaging in an in vivo system, we utilized a line of transgenic mice containing the human elastin promoter linked to a chloramphenicol acetyl transferase reporter gene. Our prior work demonstrates promoter activation in response to ultraviolet B radiation (UVB), UVA, and psoralen plus ultraviolet A radiation in the skin of these mice. The addition of psoralen (8-MOP) prior to administration of UVA results in substantial increases in promoter activation, as compared with UVA alone. To demonstrate the utility of these mice as a model of UVA-induced photodamage, we administered four lotions to the skin of our transgenic mice that included: a sunscreen containing octyl methoxycinnamate and benzophenone-3 with a sun protection factor (SPF) of 15, the UVA filter butyl methoxydibenzoylmethane, the SPF 15 sunscreen and the UVA filter together, and the lotion vehicle alone. Following sunscreen administration, mice received a single psoralen plus ultraviolet A radiation treatment. All sunscreens decreased chloramphenicol acetyl transferase activity with the SPF 15 sunscreen, the UVA filter, and the combination SPF 15 sunscreen and UVA filter, resulting in increasing degrees of protection against psoralen plus ultraviolet A radiation. These results demonstrate that this model functions as a rapid and sensitive model of UVA photodamage for the identification and comparison of compounds that protect against UVA-induced photoaging.

Animals↗
PubMed1998

Maternal uniparental meroisodisomy in the LAMB3 region of chromosome 1 results in lethal junctional epidermolysis bullosa.

Herlitz junctional epidermolysis bullosa (OMIM#226700) is a lethal, autosomal recessive blistering disorder caused by mutations in one of the three genes LAMA3, LAMB3, or LAMC2, encoding the constitutive polypeptide subunits of laminin 5. In this study, we describe a patient homozygous for a novel nonsense mutation Q936X in exon 19 of LAMB3, which has been mapped to chromosome 1q32. The patient was born with extensive blistering and demonstrated negative immunofluorescence staining for laminin 5, and transmission electron microscopy revealed tissue separation within lamina lucida of the dermal-epidermal junction, diagnostic of Herlitz junctional epidermolysis bullosa. The mother of the proband was found to be a heterozygous carrier for this mutation, whereas the father demonstrated the wild-type LAMB3 allele only. Nonpaternity was excluded by 13 microsatellite markers in six different chromosomes. Genotype analysis using 28 microsatellite markers spanning chromosome 1 revealed that the patient had maternal primary heterodisomy, as well as meroisodisomy within two regions of chromosome 1, one on 1p and the other one on 1q, the latter region containing the maternal LAMB3 mutation. These results suggest that Herlitz junctional epidermolysis bullosa in this patient developed as a result of reduction to homozygosity of the maternal LAMB3 mutation on chromosome 1q32.

Cell Adhesion Molecules↗