Intracellular targets of RGDS peptide in melanoma cells

<p>Abstract</p> <p>Background</p> <p>RGD-motif acts as a specific integrins-ligand and regulates a variety of cell-functions via extracellular action affecting cell-adhesion properties. However, increasing evidence identifies additional RGDS-functions at intracellular level. Previous reports show RGDS-internalization in endothelial cells, cardiomyocytes and lymphocytes, indicating intracellular targets such as caspase-8 and caspase-9, and suggest RGDS specific activity at cytoplasmic level. Given the role RGDS-peptides play in controlling proliferation and apoptosis in several cell types, investigating intracellular targets of RGDS in melanoma cells may un-reveal novel molecular targets and key pathways, potentially useful for a more effective approach to melanoma treatment.</p> <p>Results</p> <p>In the present study we show for the first time that RGDS-peptide is internalized in melanoma cells in a time-dependent way and exerts strong anti-proliferative and pro-apoptotic effects independently from its extracellular anti-adhesive action. RGES control-peptide did not show biological effects, as expected; nevertheless it is internalized, although with slower kinetics. Survivin, a known cell-cycle and survival-regulator is highly expressed in melanoma cells. Co-immunoprecipitation assays in cell lysates and overlay assays with the purified proteins showed that RGDS interacts with survivin, as well as with procaspase-3, -8 and -9. RGDS-peptide binding to survivin was found to be specific, at high affinity (Kd 27.5 μM) and located at the survivin C-terminus. RGDS-survivin interaction appeared to play a key role, since RGDS lost its anti-mitogenic effect in survivin-deprived cells with a specific siRNA.</p> <p>Conclusions</p> <p>RGDS inhibits melanoma growth with an adhesion-independent mechanism; it is internalized in melanoma cells and specifically interacts with survivin. The present data may indicate a novel role of RGDS-containing peptides physiologically released from the extracellular matrix and may suggest a possible novel anti-proliferation strategy in melanoma.</p

Ichthyosis Linearis Circumflexa as the Only Clinical Manifestation of Netherton Syndrome.

Ichthyosis linearis circumflexa (ILC) presents as serpiginous and migratory erythematous patches with double-edged scales. ILC is rarely an isolated skin manifestation, but most commonly a part of Netherton syndrome (NS). NS is caused by SPINK5 mutations, which lead to absent or sometimes reduced expression of the serine protease inhibitor LEKTI. NS is characterised by congenital ichthyosiform erytroderma, trichorrhexis invaginata (TI) and atopy. We report 2 children who presented since the first months of life cheek erythema followed by the appearance of sparse ILC lesions on the face, trunk and proximal extremities. Erythroderma at birth, TI and atopy were absent. LEKTI immunoreactivity was reduced in patient epidermis, and serine protease activity was modestly increased, while desmoglein-1 expression remained unaffected. SPINK5 mutation and expression analysis in patient keratinocytes revealed compound heterozygous splicing variants, which allowed residual LEKTI secretion. Our results show that ILC can be the only clinical manifestation of NS

Early immunopathological diagnosis of ichthyosis with confetti in two sporadic cases with new mutations in keratin 10.

Ichthyosis with confetti (IC) is a severe non-syndromic ichthyosis due to heterozygous mutations in the KRT10 gene. The disease manifests at birth with erythroderma and scaling and is characterised by the gradual development of numerous confetti-like spots of normal skin. Diagnosis of IC is frequently delayed until adolescence or even adulthood. We report 2 young children who were first diagnosed as having congenital ichthyosiform erythroderma. However, the development of thick, confluent hyperkeratotic plaques together with the histopathological finding of keratinocyte vacuolisation in the suprabasal epidermis evoked IC. Immunofluorescence analysis showed a highly reduced keratin 10 expression within the cytoplasm of suprabasal keratinocytes and its characteristic mislocalisation to the nuclei. The diagnosis was confirmed by the identification of 2 previously unreported mutations in intron 6 and exon 7 of KRT10. Careful clinical examination then showed the presence of the first spots of normal skin in both patients at the age of 2.5 and 5 years, respectively. These cases point to the usefulness of immunofluorescence analysis of keratin 10 expression for an early diagnosis of IC

Chemerin expression marks early psoriatic skin lesions and correlates with plasmacytoid dendritic cell recruitment

Psoriasis is a type I interferon-driven T cell–mediated disease characterized by the recruitment of plasmacytoid dendritic cells (pDC) into the skin. The molecules involved in pDC accumulation in psoriasis lesions are unknown. Chemerin is the only inflammatory chemotactic factor that is directly active on human blood pDC in vitro. The aim of this study was to evaluate the role of the chemerin/ChemR23 axis in the recruitment of pDC in psoriasis skin. Prepsoriatic skin adjacent to active lesions and early lesions were characterized by a strong expression of chemerin in the dermis and by the presence of CD15+ neutrophils and CD123+/BDCA-2+/ChemR23+ pDC. Conversely, skin from chronic plaques showed low chemerin expression, segregation of neutrophils to epidermal microabscesses, and few pDC in the dermis. Chemerin expression was localized mainly in fibroblasts, mast cells, and endothelial cells. Fibroblasts cultured from skin of psoriatic lesions expressed higher levels of chemerin messenger RNA and protein than fibroblasts from uninvolved psoriatic skin or healthy donors and promoted pDC migration in vitro in a chemerin-dependent manner. Therefore, chemerin expression specifically marks the early phases of evolving skin psoriatic lesions and is temporally strictly associated with pDC. These results support a role for the chemerin/ChemR23 axis in the early phases of psoriasis development

De novo variants of CSNK2B cause a new intellectual disability-craniodigital syndrome by disrupting the canonical Wnt signaling pathway

CSNK2B encodes for casein kinase II subunit beta (CK2b), the regulatory subunit of casein kinase II (CK2), which is known to mediate diverse cellular pathways. Variants in this gene have been recently identified as a cause of Poirier-Bienvenu neurodevelopmental syndrome (POBINDS), but functional evidence is sparse. Here, we report five unrelated individuals: two of them manifesting POBINDS, while three are identified to segregate a new intellectual disability-craniodigital syndrome (IDCS), distinct from POBINDS. The three IDCS individuals carried two different de novo missense variants affecting the same codon of CSNK2B. Both variants, NP_001311.3; p.Asp32His and NP_001311.3; p.Asp32Asn, lead to an upregulation of CSNK2B expression at transcript and protein level, along with global dysregulation of canonical Wnt signaling. We found impaired interaction of the two key players DVL3 and b-catenin with mutated CK2b. The variants compromise the kinase activity of CK2 as evident by a marked reduction of phosphorylated b-catenin and consequent absence of active b-catenin inside nuclei of the patient-derived lymphoblastoid cell lines (LCLs). In line with these findings, whole-transcriptome profiling of patient-derived LCLs harboring the NP_001311.3; p.Asp32His variant confirmed a marked difference in expression of genes involved in the Wnt signaling pathway. In addition, whole-phosphoproteome analysis of the LCLs of the same subject showed absence of phosphorylation for 313 putative CK2 substrates, enriched in the regulation of nuclear b-catenin and transcription of the target genes. Our findings suggest that discrete variants in CSNK2B cause dominant-negative perturbation of the canonical Wnt signaling pathway, leading to a new craniodigital syndrome distinguishable from POBINDS

TFIIH-dependent MMP-1 overexpression in trichothiodystrophy leads to extracellular matrix alterations in patient skin

Mutations in the XPD subunit of the DNA repair/transcription factor TFIIH result in distinct clinical entities, including the cancer-prone xeroderma pigmentosum (XP) and the multisystem disorder trichothiodystrophy (TTD), which share only cutaneous photosensitivity. Gene-expression profiles of primary dermal fibroblasts revealed overexpression of matrix metalloproteinase 1 (MMP-1), the gene encoding the metalloproteinase that degrades the interstitial collagens of the extracellular matrix (ECM), in TTD patients mutated in XPD compared with their healthy parents. The defect is observed in TTD and not in XP and is specific for fibroblasts, which are the main producers of dermal ECM. MMP-1 transcriptional up-regulation in TTD is caused by an erroneous signaling mediated by retinoic acid receptors on the MMP-1 promoter and leads to hypersecretion of active MMP-1 enzyme and degradation of collagen type I in the ECM of cell/tissue systems and TTD patient skin. In agreement with the well-known role of ECM in eliciting signaling events controlling cell behavior and tissue homeostasis, ECM alterations in TTD were shown to impact on the migration and wound-healing properties of patient dermal fibroblasts. The presence of a specific inhibitor of MMP activity was sufficient to restore normal cell migration, thus providing a potential approach for therapeutic strategies. This study highlights the relevance of ECM anomalies in TTD pathogenesis and in the phenotypic differences between TTD and XP

Multiple Skin Squamous Cell Carcinomas in Junctional Epidermolysis Bullosa Due to Altered Laminin-332 Function

Variably reduced expression of the basement membrane component laminin-332 (&alpha;3a&beta;3&gamma;2) causes junctional epidermolysis bullosa generalized intermediate (JEB-GI), a skin fragility disorder with an increased susceptibility to squamous cell carcinoma (SCC) development in adulthood. Laminin-332 is highly expressed in several types of epithelial tumors and is central to signaling pathways that promote SCC tumorigenesis. However, laminin-332 mutations and expression in individuals affected by JEB-GI and suffering from recurrent SCCs have been poorly characterized. We studied a JEB-GI patient who developed over a hundred primary cutaneous SCCs. Molecular analysis combined with gene expression studies in patient skin and primary keratinocytes revealed that the patient is a functional hemizygous for the p.Cys1171* mutant allele which is transcribed in a stable mRNA encoding for a &beta;3 chain shortened of the last two C-terminal amino acids (Cys1171-Lys1172). The lack of the Cys1171 residue involved in the C-terminal disulphide bond to &gamma;2 chain did not prevent assembly, secretion, and proteolytic processing of the heterotrimeric molecule. Immunohistochemistry of SCC specimens revealed accumulation of mutant laminin-332 at the epithelial-stromal interface of invasive front. We conclude that the C-terminal disulphide bond is a structural element crucial for laminin-332 adhesion function in-vivo. By saving laminin-332 amount, processing, and signaling role the p.Cys1171* mutation may allow intrinsic pro-tumorigenic properties of the protein to be conveyed, thus contributing to invasiveness and recurrence of SCCs in this patient