Calcium pantothenate modulates gene expression in proliferating human dermal fibroblasts.

Topical application of pantothenate is widely used in clinical practice for wound healing. Previous studies identified a positive effect of pantothenate on migration and proliferation of cultured fibroblasts. However, these studies were mainly descriptive with no molecular data supporting a possible model of its action. In this study, we first established conditions for an in vitro model of pantothenate wound healing and then analysed the molecular effects of pantothenate. To test the functional effect of pantothenate on dermal fibroblasts, cells were cultured and in vitro proliferation tests were performed using a standardized scratch test procedure. For all three donors analysed, a strong stimulatory effect of pantothenate at a concentration of 20 microg/ml on the proliferation of cultivated dermal fibroblasts was observed. To study the molecular mechanisms resulting in the proliferative effect of pantothenate, gene expression was analysed in dermal fibroblasts cultivated with 20 microg/ml of pantothenate compared with untreated cells using the GeneChip Human Exon 1.0 ST Array. A number of significantly regulated genes were identified including genes coding for interleukin (IL)-6, IL-8, Id1, HMOX-1, HspB7, CYP1B1 and MARCH-II. Regulation of these genes was subsequently verified by quantitative real-time polymerase chain reaction analysis. Induction of HMOX-1 expression by pantothenol and pantothenic acid in dermal cells was confirmed on the protein level using immunoblots. Functional studies revealed the enhanced suppression of free radical formation in skin fibroblasts cultured with panthenol. In conclusion, these studies provided new insight in the molecular mechanisms linked to the stimulatory effect of pantothenate and panthenol on the proliferation of dermal fibroblasts.

Genetic variations of the chemokine scavenger receptor D6 are associated with liver inflammation in chronic hepatitis C.

Chronic hepatitis C (HCV) represents one of the most common chronic infections worldwide and is a major indication for liver transplantation. Liver inflammation is the main predictor of advanced fibrosis in HCV. Inflammatory cells are recruited to the liver by chemokines. Recently, a novel class of chemokine receptors has been characterized that lack signaling functions and are termed scavenger receptors. We determine here whether genetic variations of the scavenger receptor D6 contribute to the grade of liver inflammation in HCV. Four haplotype tagging single nucleotide polymorphisms (SNPs) were identified from HapMap that cover the genetic information of D6 (CCBP2). Among these SNPs, rs4683336 was associated with liver inflammation in qualitative (p = 0.003) and quantitative (p = 0.0086) genotype analysis. This association was confirmed in an independent cohort of HCV-infected patients (p = 0.006 for qualitative and p = 0.0046 for quantitative analysis, respectively). Furthermore, the haplotype that is tagged by marker rs4683336 was significantly correlated with liver inflammation when compared with the most common D6 haplotype (p = 0.014). The importance of genetic variations in D6 was supported through the demonstration of an association of D6 mRNA expression with histologic inflammation in liver biopsies and a considerable range of D6 mRNA expression in isolated human hepatocytes. In conclusion, we demonstrate that variations in a chemokine scavenging receptor are significantly correlated with clinical inflammatory phenotypes such as HCV infection.

The fractalkine receptor CX3CR1 is involved in liver fibrosis due to chronic hepatitis C infection.

BACKGROUND/AIMS: The chemokine receptor CX3CR1 and its specific ligand fractalkine (CX3CL1) are known to modulate inflammatory and fibroproliferative diseases. Here we investigate the role of CX3CR1/fractalkine in HCV-induced liver fibrosis. METHODS: A genotype analysis of CX3CR1 variants was performed in 211 HCV-infected patients. Hepatic expression of CX3CR1 was studied in HCV-infected livers and isolated liver cell populations by RT-PCR and immunohistochemistry. The effects of fractalkine on mRNA expression of profibrogenic genes were determined in isolated hepatic stellate cells (HSC) and CX3CR1 genotypes were related to intrahepatic TIMP-1 mRNA levels. RESULTS: The intrahepatic mRNA expression of CX3CR1 correlates with the stage of HCV-induced liver fibrosis (P=0.03). The CX3CR1 coding variant V249I is associated with advanced liver fibrosis, independent of the T280M variant (P=0.009). CX3CR1 is present on primary HSC and fractalkine leads to a suppression of tissue inhibitor of metalloproteinase (TIMP)-1 mRNA in HSC (P=0.03). Furthermore, CX3CR1 genotypes are associated with TIMP-1 mRNA expression in HCV-infected liver (P=0.03). CONCLUSIONS: The results identify the fractalkine receptor CX3CR1 as susceptibility a gene for hepatic fibrosis in HCV infection. The modulation of TIMP-1 expression by fractalkine and CX3CR1 genotypes provides functional support for the observed genotype-phenotype association.

Dual porphyrias revisited.

The porphyrias are clinically and genetically heterogeneous metabolic diseases, which predominantly result from a hereditary dysfunction in the pathway of haeme biosynthesis. Currently, at least eight different forms of porphyrias can be differentiated, all of them characterized by a specific enzyme deficiency that is either inherited in an autosomal-dominant fashion, autosomal recessively or, in the case of porphyria cutanea tarda, might also be acquired. All genes encoding these enzymes have been cloned and several mutations underlying the different types of porphyrias have been reported. Traditionally, the diagnosis of porphyria is made on the basis of clinical symptoms, characteristic biochemical findings and enzyme assays. In some porphyria patients and families, however, these diagnostic tools can reveal simultaneous findings compatible with two different forms of porphyria, a phenomenon referred to as dual porphyria. Here, we give an overview on what is currently known about these peculiar variants of porphyria and suggest that, whenever feasible, molecular genetic analysis should complement the analytical techniques used to characterize patients and families in which a double enzymatic deficiency within the haeme biosynthetic pathway is assumed.

Skin retinoid concentrations are modulated by CYP26AI expression restricted to basal keratinocytes in normal human skin and differentiated 3D skin models.

Cellular levels of all-trans retinoic acid (RA) are meticulously regulated utilizing an array of systems to balance uptake, biosynthesis, catabolism, and efflux transport. Metabolic transformation of all-trans RA to 4-hydroxylated RA appears to be primarily catalyzed by the cytochrome P450 (CYP) 26AI. Analysis of monolayer cultures of normal human epidermal keratinocytes (NHEKs) and dermal fibroblasts by quantitative real-time PCR and reverse transcription-PCR revealed no basal levels of CYP26AI mRNA expression, whereas specific transcripts were detectable following addition of 10(-6) M all-trans RA. Immunofluorescence and Western blot analysis showed a weak expression of CYP26AI in NHEK, which was increased by stimulation with all-trans RA. Using a newly developed peptide antibody, we further examined the localization of CYP26AI expression in normal skin and three-dimensional (3D) skin models. In contrast to cell culture monolayers where CYP26AI was only weakly detectable, strong constitutive expression of CYP26AI in vivo and in organotypic culture was found to be restricted to basal epidermal keratinocytes, as well as eccrine sweat glands and sebaceous glands. These studies verify the capacity of human skin to metabolize RA, although substantial differences exist in CYP expression between normal skin and 3D skin models compared to monolayer cultures. Complex metabolic processes that maintain retinoid homeostasis may therefore be better studied in model systems more closely resembling in vivo skin. In light of our prior studies documenting the functional activity of RA metabolites, expression of CYP26 in the sebaceous gland epithelium supports the suggestion that altered RA metabolism may be involved in the pathogenesis of acne.

Allelic loss underlies type 2 segmental Hailey-Hailey disease, providing molecular confirmation of a novel genetic concept.

Hailey-Hailey disease (HHD) is an autosomal dominant trait characterized by erythematous and oozing skin lesions preponderantly involving the body folds. In the present unusual case, however, unilateral segmental areas along the lines of Blaschko showing a rather severe involvement were superimposed on the ordinary symmetrical phenotype. Based on this observation and similar forms of mosaicism as reported in other autosomal dominant skin disorders, we postulated that in such cases, 2 different types of segmental involvement can be distinguished. Accordingly, the linear lesions as noted in the present case would exemplify type 2 segmental HHD. In the heterozygous embryo, loss of heterozygosity occurring at an early developmental stage would have given rise to pronounced linear lesions reflecting homozygosity or hemizygosity for the mutation. By analyzing DNA and RNA derived from blood and skin samples as well as keratinocytes of the index patient with various molecular techniques including RT-PCR, real-time PCR, and microsatellite analysis, we found a consistent loss of the paternal wild-type allele in more severely affected segmental skin regions, confirming this hypothesis for the first time, to our knowledge, at the molecular and cellular level.

The molecular basis of porphyria cutanea tarda in Chile: identification and functional characterization of mutations in the uroporphyrinogen decarboxylase gene.

The porphyrias are heterogeneous disorders arising from predominantly inherited catalytic deficiencies of specific enzymes in heme biosynthesis. Porphyria cutanea tarda (PCT) results from a decreased activity of uroporphyrinogen decarboxylase, the fifth enzyme in heme biosynthesis. The disorder represents the only porphyria that is not exclusively inherited monogenetically. In PCT, at least two different types can be distinguished: acquired/sporadic (type I) PCT, in which the enzymatic deficiency is limited to the liver and inherited/familial (type II) PCT, which is inherited as an autosomal dominant trait with a decrease of enzymatic activity in all tissues. In an effort to characterize the molecular basis of PCT in Chile, we identified eight mutations in 18 previously unclassified PCT families by polymerase chain reaction, heteroduplex analysis, and automated sequencing. To study the role of these mutations in disease causality, in vitro expression of all novel missense mutations was studied. Our results indicate that the frequency of familial PCT in Chile is approximately 50%, thus, to our knowledge, representing the highest incidence of familial PCT reported to date. The data further emphasize the molecular heterogeneity in type II PCT and demonstrate the advantages of molecular genetic techniques as a diagnostic tool and in the detection of clinically asymptomatic mutation carriers.