Extracellular Matrix of the Skin: 50 Years of Progress

The extracellular connective tissue matrix of the skin is a complex aggregate of distinct collagenous and non-collagenous components. Optimal quantities and delicate interactions of these components are necessary to maintain normal physiologic properties of skin. This overview summarizes the progress made in understanding the normal biology and biochemistry of the extracellular matrix, and will highlight cutaneous diseases with underlying molecular defects in the structure and expression of extracellular matrix components

Suppression of Ornithine Decarboxylase Gene Expression by Retinoids in Cultured Human Keratinocytes

Modulation of ornithine decarboxylase (ODC) gene expression by retinoids was analyzed in human keratinocyte cultures maintained in serum-free medium containing 0.15mM Ca++. Cells were incubated with all-trans-retinoic acid, 13-cis-retinoic acid or arotinoid Ro15-0778 (10−10 to 10−5 M), total RNA was isolated, and mRNA transcripts for ODC were analyzed by Northern and slot blot hybridizations with a human ODC cDNA. Treatment of cells for 24h resulted in a dose-dependent decrease in ODC mRNA levels, with an estimated IC50 of ∼1 × 10−8 M for all-trans- and 13-cis-retinoic acid, while Ro15-0778 was somewhat less effective (IC50 ∼1-5 × 10−7 M). The suppression of ODC mRNA levels by retinoids was detectable at ∼3h of incubation, with essentially a maximal inhibition at 12h. Reduced ODC mRNA levels noted after 24h of incubation with 5 × 10−7 M all-trans-retinoic acid were accompanied by a reduction in ODC enzyme activity. To determine if all-trans-retinoic acid was regulating ODC gene expression directly, or if protein synthesis was required, ODC expression was analyzed in cultures treated with protein synthesis inhibitors. In the presence of cycloheximide or puromycin, all-trans-retinoic acid did not suppress ODC mRNA levels. These findings suggest that suppression of ODC gene expression is not a direct effect of all-trans-retinoic acid, but depends on ongoing protein synthesis

Fluorescent protein markers to tag collagenous proteins: The paradigm of procollagen VII

Fluorescent proteins are powerful markers allowing tracking expression, intracellular localization, and translocation of tagged proteins but their effects on the structure and assembly of complex extracellular matrix proteins has not been investigated. Here, we analyzed the utility of fluorescent proteins as markers for procollagen VII, a triple-helical protein critical for the integrity of dermal-epidermal junction. DNA constructs encoding a red fluorescent protein-tagged wild type mini-procollagen VII α chain and green fluorescent protein-tagged α chains harboring selected mutations were genetically engineered. These DNA constructs were co-expressed in HEK-293 cells and the assembly of heterogeneous triple-helical mini-procollagen VII molecules was analyzed. Immunoprecipitation and fluorescence resonance energy transfer assays demonstrated that the presence of different fluorescent protein markers at the C-termini of individual α chains neither altered formation of triple-helical molecules nor affected their secretion to the extracellular space. Our study provides a basis for employing fluorescent proteins as tags for complex structural proteins of extracellular matrix

Cytotoxic activity of Treponema denticola

published_or_final_versio

Changes in dermal fibroblasts from Abcc6-/- mice are present before and after the onset of ectopic tissue mineralization

Pseudoxanthoma elasticum (PXE), a rare genetic disease caused by mutations in the ABCC6 gene, is characterized by progressive calcification of elastic fibers in the skin, eyes, and the cardiovascular system. The pathomechanism of the mineralization is still obscure. Several hypotheses have been proposed, one of them suggesting a role for fibroblasts in controlling the amount and the quality of the calcified extracellular matrix. This hypothesis raises the question whether changes in mesenchymal cells are the cause and/or the consequences of the calcification process. In this study, fibroblasts were isolated and cultured from Abcc6+/+ and Abcc6-/- mice of different ages to investigate parameters known to be associated with the phenotype of fibroblasts from PXE patients. Results demonstrate that a few changes (Ank and Opn downregulation) are already present before the occurrence of calcification.By contrast, a modification of other parameters (intracellularO2- content, Tnap activity, and Bmp2 upregulation) can be observed inAbcc6-/- mice after the onset of tissue mineralization.These data suggest that in the Abcc6 -/- genotype, dermal fibroblasts actively contribute to changes that promote matrix calcification and that these cells can be further modulated with time by the calcified environment, thus contributing to the age-dependent progression of the disease

Mouse models for pseudoxanthoma elasticum: Genetic and dietary modulation of the ectopic mineralization phenotypes

Pseudoxanthoma elasticum (PXE), a heritable ectopic mineralization disorder, is caused by mutations in the ABCC6 gene. Null mice ( Abcc6 -/-) recapitulate the genetic, histopathologic and ultrastructural features of PXE, and they demonstrate early and progressive mineralization of vibrissae dermal sheath, which serves as a biomarker of the overall mineralization process. Recently, as part of a mouse aging study at The Jackson Laboratory, 31 inbred mouse strains were necropsied, and two of them, KK/HlJ and 129S1/SvImJ, were noted to have vibrissae dermal mineralization similar to Abcc6-/- mice. These two strains were shown to harbor a single nucleotide polymorphism (rs32756904) in the Abcc6 gene, which resulted in out-of-frame splicing and marked reduction in ABCC6 protein expression in the liver of these mice. The same polymorphism is present in two additional mouse strains, DBA/2J and C3H/HeJ, with similar reduction in Abcc6 protein levels, yet these mice did not demonstrate tissue mineralization when kept on standard rodent diet. However, all four mouse strains, when placed on experimental diet enriched in phosphate and low in magnesium, developed extensive ectopic mineralization. These results indicate that the genetic background of mice and the mineral composition of their diet can profoundly modulate the ectopic mineralization process predicated on mutations in the Abcc6 gene. These mice provide novel model systems to study the pathomechanisms and the reasons for strain background on phenotypic variability of PXE. © 2014 Li et al

Assessment of the risk and characterization of non-melanoma skin cancer in Kindler syndrome: study of a series of 91 patients.

BACKGROUND: Kindler Syndrome (KS) is a rare genodermatosis characterized by skin fragility, skin atrophy, premature aging and poikiloderma. It is caused by mutations in the FERMT1 gene, which encodes kindlin-1, a protein involved in integrin signalling and the formation of focal adhesions. Several reports have shown the presence of non-melanoma skin cancers in KS patients but a systematic study evaluating the risk of these tumors at different ages and their potential outcome has not yet been published. We have here addressed this condition in a retrospective study of 91 adult KS patients, characterizing frequency, metastatic potential and body distribution of squamous cell carcinoma (SCC) in these patients. SCC developed in 13 of the 91 patients. RESULTS: The youngest case arose in a 29-year-old patient; however, the cumulative risk of SCC increased to 66.7% in patients over 60 years of age. The highly aggressive nature of SCCs in KS was confirmed showing that 53.8% of the patients bearing SCCs develop metastatic disease. Our data also showed there are no specific mutations that correlate directly with the development of SCC; however, the mutational distribution along the gene appears to be different in patients bearing SCC from SCC-free patients. The body distribution of the tumor appearance was also unique and different from other bullous diseases, being concentrated in the hands and around the oral cavity, which are areas of high inflammation in this disease. CONCLUSIONS: This study characterizes SCCs in the largest series of KS patients reported so far, showing the high frequency and aggressiveness of these tumors. It also describes their particular body distribution and their relationship with mutations in the FERMT-1 gene. These data reinforce the need for close monitoring of premalignant or malignant lesions in KS patients