Characterization of a ultraviolet B-induced corticotropin-releasing hormone-proopiomelanocortin system in human melanocytes

CRH, the main regulator of the systemic response to stress, is also expressed in the skin where it is incorporated into a local homolog of the hypothalamic-pituitary-adrenal axis. To investigate the mechanisms of the induction of the CRH-proopiomelanocortin (POMC) response in human melanocytes, we used UVB as an epidermal-specific stressor. Human normal melanocytes cultured in vitro were irradiated with graded doses of UVB, and the CRH-POMC responses were measured in cell extracts and/or supernatants. UVB stimulated the CRH promoter, the CRH mRNA expression, and peptide release. The UVB-induced stimulation of the CRH promoter was suppressed by pharmacological inhibitors of protein kinase A or by plasmid overexpressing a dominant mutant cAMP response element (CRE)-binding protein (CREB). UVB also stimulated phosphorylation of CREB, binding of phosphorylated CREB to CRE sites in the CRH promoter, and activity of the reporter gene construct driven by consensus CRE sites. Mutation in the CRE site in the CRH promoter rendered the corresponding reporter gene construct less responsive to UVB in both normal and malignant melanocytes. In addition to CRH effects, UVB activated the POMC promoter, POMC mRNA expression, and ACTH release, whereas an antagonist of the CRH receptor 1 abrogated the UVB-stimulated induction of POMC. In conclusion, UVB induces CRH production in human melanocytes through stimulation of the protein kinase A pathway, with sequential involvement of CRH-CRH receptor 1 in the stimulation of POMC expression.     

Corticotropin-releasing hormone induces keratinocyte differentiation in the adult human epidermis

Previously we documented that human epidermis exclusively expresses corticotropin releasing hormone receptor 1 (CRH-R1). To define the role of CRH in the epidermis, we investigated its effects on differentiation of normal human adult epidermal keratinocytes. Thus, CRH inhibited proliferation in a dose dependent fashion and significantly decreased Ki-67 antigen expression. This effect was independent of either the presence or the absence of growth factors in the medium. Flow cytometry analysis demonstrated that CRH inhibited the transition from G0/1 to S phase of the cell cycle, which was accompanied by an increased expression of cdk inhibitor p16 (Ink4a) protein. The antiproliferative effect was attenuated by protein kinase C inhibitor (GF109203X) but not by H89 (protein kinase A inhibitor), PD98059, or SB203580 (MAP kinase inhibitors). The cell cycle withdrawal was associated with the induction of keratinocyte differentiation. Thus, CRH stimulated the expression of cytokeratin 1 and involucrin, and inhibited cytokeratin 14 on both mRNA and protein levels. It also increased cell granularity and cell size. Furthermore, CRH induced signal transduction cascade that included stimulation of inositol 1,4,5-triphosphate, which was time and dose dependent. CRH also increased activator protein-1 DNA binding activity with JunD identified as the most important element. Thus, activation of CRH-R1 induces a non-random and sequential signal transduction cascade governing both keratinocyte differentiation and the inhibition of cell proliferation through G0/1 arrest. We propose that this program, triggered by CRH interaction with CRH-R1, includes induction of a transduction pathway involving the sequential activation of phospholipase C, protein kinase C, activator protein-1 (including Jun D), and p16.     

Tryptophan hydroxylase expression in human skin cells

We attempted to further characterize cutaneous serotoninergic and melatoninergic pathways evaluating the key biosynthetic enzyme tryptophan hydroxylase (TPH). There was wide expression of TPH mRNA in whole human skin, cultured melanocytes and melanoma cells, dermal fibroblasts, squamous cell carcinoma cells and keratinocytes. Gene expression was associated with detection of TPH immunoreactive species by Western blotting. Characterization of the TPH immunoreactive species performed with two different antibodies showed expression of the expected protein (55-60 kDa), and of forms with higher and lower molecular weights. This pattern of broad spectrum of TPH expression including presumed degradation products suggests rapid turnover of the enzyme, as previously reported in mastocytoma cells. RP-HPLC of skin extracts showed fluorescent species with the retention time of serotonin and N-acetylserotonin. Immunocytochemistry performed in skin biopsies localized TPH immunoreactivity to normal and malignant melanocytes. We conclude that while the TPH mRNA and protein are widely expressed in cultured normal and pathological epidermal and dermal skin cells, in vivo TPH expression is predominantly restricted to cells of melanocytic origin.     

A simple and rapid method for detection of the 106Gln mutation in Wilson-Konovalov disease

A simple and rapid method for detecting the 1069Gln mutation in gene ATP7B based on a PCR specific for this allele has been developed. The 1069Gln mutation is the main cause of Wilson disease (WD) in Russia and accounts for approximately 40% of all mutant alleles of gene ATP7B. Therefore, the method proposed makes the postnatal and prenatal diagnosis of Wilson disease in Russia considerably more rapid and less expensive.     

Splenic eumelanin differs from hair eumelanin in C57BL/6 mice

The presence of melanin in spleens of black C57BL/6 mice has been known for long. Although its origin and biological functions are still obscure, the relation of splenic melanin to the hair follicle and skin pigmentation was suggested. Here, we demonstrated using for the first time electron paramagnetic resonance spectroscopy that black-spotted C57BL/6 spleens contain eumelanin. Its presence here is a "yes or no" phenomenon, as even in the groups which revealed the highest percentage of spots single organs completely devoid of the pigment were found. Percentage of the spotted spleens decreased, however, with the progress of telogen after spontaneously-induced hair growth. The paramagnetic properties of the spleen eumelanin differed from the hair shaft or anagen VI skin melanin. The splenic melanin revealed narrower signal, and its microwave power saturability betrayed more heterogenous population of paramagnetic centres than in the skin or hair shaft pigment. Interestingly, the pigment of dry hair shafts and of the wet tissue of depilated anagen VI skin revealed almost identical properties. The properties of splenic melanin better resembled the synthetic dopa melanin (water suspension, and to a lesser degree - powder sample) than the skin/hair melanin. All these findings may indicate a limited degradation of splenic melanin as compared to the skin/hair pigment. The splenic eumelanin may at least in part originate from the skin melanin phagocyted in catagen by the Langerhans cells or macrophages and transported to the organ.     

Analysis of the glutathione S-transferase P1 gene Ile105Val polymorphism in the patients with sporadic motor neuron disease from Russia

Ile105Val polymorphism in exon 5 of glutathione S-transferase (GSTP1) gene was examined in a group of patients with motor neuron disease (MND) and control sample. No statistically significant differences in the allele and genotype frequency distributions between the samples examined were demonstrated. We conclude that Ile105Val polymorphism is not associated with the risk of the disease development in the patients from Russia with sporadic form of MND.     

Molecular genetic analysis of hereditary neurodegenerative diseases

The review summarizes the results of a decade of molecular genetic studies of several high-incidence hereditary neurodegenerative diseases, including primary parkinsonism, various forms of hereditary dystonia and ataxia, polyglutamine disorders, hepatolenticular degeneration, essential tremor, etc. Various relevant mutations were studied. The character and frequencies of particular mutations and the corresponding genetic disorders were established for the Russian population. Particular genotypes were associated with various clinical variants of the diseases. Genetic loci were identified for several unique hereditary diseases of the nervous system (X-linked cerebellar hypoplasia, an atypical form of autosomal recessive muscular dystrophy, etc.). Nosological positions of the relevant clinical forms were clarified on the basis of the molecular genetic data. Protocols were developed for direct or indirect DNA diagnostics of the diseases under study to improve medical genetic counseling and prevention of new disease cases in affected families.     

Molecular-genetic analysis of torsion dystonia in Russia

For the first time in Russia, analysis of the GCH-I and DYT1 genes was carried out for the purpose of direct DNA diagnostics in families with various forms of hereditary torsion dystonia (TD). Four new missense mutations (Met102Lys, Thr94Lys, Cys141Trp, and Ser176Thr) in the GCH-I gene were found in patients with dopa-responsive dystonia (DRD), testifying to a genetic heterogeneity of this clinical form of TD. The distribution of the major del GAG mutation in exon 5 of the DYT1 gene was studied in patients with non-dopa-responsive dystonia (NDRD). In total, the mutation was found in 68% of the patients. The frequency of this mutation in Ashkenazi Jews with NDRD was 100% (twice higher than in Slavonic families), suggesting the founder effect reported for NDRD in this ethnic group. Mutations of the GCH-I and DYT1 genes were also found in patients with atypical and questionable cases of TD, which are difficult to diagnose with methods other than DNA analysis. The data obtained made it possible to extend the spectrum of clinical signs of DRD and NDRD and to revise the views on true penetrance of the corresponding mutant genes, which is important for medical genetic counseling in affected families.