The epidermis is a dynamic tissue in which keratinocytes proliferate in the basal layer and undergo a tightly controlled differentiation while moving into the suprabasal layers. The balance between keratinocyte proliferation, differentiation, and death is essential, and its perturbation can result in pathological changes. Some common skin diseases, such as psoriasis, are characterized by hyperproliferation accompanied by inflammatory reactions, suggesting that molecules with topical anti-inflammatory and ROS scavenging abilities may be useful for their treatment. Here we investigate the potential of the flavone Luteolin-7-glucoside (LUT-7G) as a treatment for psoriasis. We show that LUT-7G leads to a modification of the cell cycle and the induction of keratinocyte differentiation, with modification of energy, fatty acid, and redox metabolism. LUT-7G treatment also neutralizes the proliferative stimulus induced by the proinflammatory cytokines IL-22 and IL-6 in HEKn. Moreover, in the Imiquimod (IMQ) mouse model of psoriasis, topical administration of LUT-7G leads to a marked reduction of acanthosis and re-expression of epidermal differentiation markers. Dissection of the IL-22 signalling pathway, activated by IMQ treatment, demonstrates that LUT-7G impairs the nuclear translocation of phosphorylated (activated) STAT3, blocking the IL-22 signalling cascade. Thus LUT-7G appears to be a promising compound for the treatment of hyperproliferative and inflammatory skin diseases, such as psoriasis.

CCND1 Cl HO O O Cl N H YK-4-279 DHX9 pncCCND1_B EWS-FLI1 Sam68, DHX9, and pncCCND1_B form a multimolecular inhibitory complex on the CCND1 promoter, fostered by EWS-FLI1 dissociation and counteracted by mitogenic stimuli, such as IGF1 stimulation. Sam68 Most Ewing sarcomas are characterized by the inframe chromosomal translocation t(11;22) generating the EWS-FLI1 oncogene. EWS-FLI1 protein interacts with the RNA helicase DHX9 and affects transcription and processing of genes involved in neoplastic transformation, including CCND1 (the cyclin D1 gene), which contributes to cell-cycle dysregulation in cancer. In this study, we found that CCND1 expression is significantly higher in patients with Ewing sarcoma compared with other sarcomas and that the pncCCND1_B RNA, a previously uncharacterized CCND1 promoterassociated noncoding (pnc) transcript, is expressed in Ewing sarcoma cells. PncCCND1_B interacted with the RNA-binding protein Sam68 and repressed CCND1 expression. Notably, knockdown of Sam68 affected pncCCND1_B subcellular localization and cyclin D1 expression. Pharmacologic impairment of DHX9/EWS-FLI1 interaction promoted RNA-dependent association of Sam68 with DHX9 and recruitment of Sam68 to the CCND1 promoter, thus repressing it. Conversely, mitogenic stimulation of Ewing sarcoma cells with IGF1 impaired Sam68/DHX9 interaction and positively regulated CCND1 expression. These studies uncover a fine-tuned modulation of the proto-oncogene CCND1 in Ewing sarcoma cells via alternative complexes formed by DHX9 with either EWS-FLI1 or pncCCND1_B-Sam68. Significance: A pncRNA-based mechanism represses expression of CCND1 through the formation of a protein-RNA complex and provides new therapeutic opportunities for patients with Ewing sarcoma.

Flavonoids have been demonstrated to affect the activity of many mammalian enzyme systems. Their functional phenolic groups are able to mediate antioxidant effects by scavenging free radicals. Molecules of this class have been found able to modulate the activity of kinases, phospholipase A2, cyclooxygenases, lipoxygenase, glutathione S-transferase, and many others. Recently, it has been demonstrated that luteolin, in the form of Luteolin-7-O-β-d-glucoside (LUT-7G) is able to induce the keratinocyte differentiation process in vitro. This flavonoid is able to counteract the proliferative effects of IL-22/IL6 pathway by the inhibition of STAT3 activity also in vivo in a psoriatic mouse model. Observations on energy metabolism changes of differentiating cells led us to perform a complete metabolomics analysis using human primary keratinocytes treated with LUT-7G. Our results show that LUT-7G, is not only able to impair the nuclear translocation of STAT3, but it also blocks the energy metabolism pathway, depressing the glycolytic and Krebs pathway by the inhibition of hexokinase 2 activity. These data confirm that LUT-7G can be proposed as a potential candidate for the treatment of inflammatory and proliferative diseases, but its role as a hexokinase 2 (HEK2) inhibitor opens new perspectives in nutritional science, and especially in cancer therapy, in which the inhibition of the Warburg effect could be relevant.

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Lamellar Ichthyosis (LI) is a form of congenital ichthyosis that is caused by mutations in the TGM1 gene that encodes for the transglutaminase 1 (TG1) enzyme. Functional inactivation of TG1 could be due to mutations, deletion or insertions. In this study, we have screened 16 patients affected by LI and found six new mutations: two transition/transversion (R37G, V112A), two nonsense mutations and two putative splice site both leading to a premature stop codon. The mutations are localized in exons 2 (N-terminal domain), 5, 11 (central catalytic domain), and none is located in the two beta-barrel C-terminal domains. In conclusion, this study expands the current knowledge on TGM1 mutation spectrum, increasing the characterization of mutations would provide more accurate prenatal genetic counselling for parents at-risk individuals.

Alternative splicing is a combinatorial mechanism by which exons are joined to produce multiple mRNA variants, thus expanding the coding potential and plasticity of eukaryotic genomes. Defects in alternative splicing regulation are associated with several human diseases, including cancer. Ewing sarcoma is an aggressive tumor of bone and soft tissue, mainly affecting adolescents and young adults. DHX9 is a key player in Ewing sarcoma malignancy, and its expression correlates with worse prognosis in patients. In this study, by screening a library of siRNAs, we have identified splicing factors that regulate the alternative inclusion of a poison exon in DHX9 mRNA, leading to its downregulation. In particular, we found that hnRNPM and SRSF3 bind in vivo to this poison exon and suppress its inclusion. Notably, DHX9 expression correlates with that of SRSF3 and hnRNPM in Ewing sarcoma patients. Furthermore, downregulation of SRSF3 or hnRNPM inhibited DHX9 expression and Ewing sarcoma cell proliferation, while sensitizing cells to chemotherapeutic treatment. Hence, our study suggests that inhibition of hnRNPM and SRSF3 expression or activity could be exploited as a therapeutic tool to enhance the efficacy of chemotherapy in Ewing sarcoma.

OTX proteins, homologs of the Drosophila orthodenticle (Otd), are important for the morphogenesis of the neuroectoderm, and for the central nervous system formation. OTX1 and OTX2 are important for the cochlea and macula development, indeed when OTX1 is knocked down, these organs undergo developmental failure. Moreover OTX2 transfection revert this effect in OTX1−/− mice. The TA isoform of TP63, involved in Notch regulation pathway, has a critical function in the cochlear neuroepithelium differentiation. TAp63 positively regulates Hes5 and Atoh1 transcription. This pathway has been also demonstrated in p63−/− mice, and in patients p63 mutated, affected by Ectodermal Dysplasia (ED, OMIM 129810). These patients are affected by mild sensorineural deafness, most likely related to the mutation in p63 gene impairing the Notch pathway. We demonstrated the role of OTX2 on TAp63 regulation necessary for the correct formation of macular neuroepithelium and we confirmed the impairment of vestibular function caused by p63 mutations. Although the abnormalities found in our patient were still at a subclinical extent, aging could exacerbate this impairment and cause a decrease in quality of life.