JAK/STAT Inhibition Normalizes Lipid Composition in 3D Human Epidermal Equivalents Challenged with Th2 Cytokines.

Derangement of the epidermal barrier lipids and dysregulated immune responses are key pathogenic features of atopic dermatitis (AD). The Th2-type cytokines interleukin IL-4 and IL-13 play a prominent role in AD by activating the Janus Kinase/Signal Transduction and Activator of Transcription (JAK/STAT) intracellular signaling axis. This study aimed to investigate the role of JAK/STAT in the lipid perturbations induced by Th2 signaling in 3D epidermal equivalents. Tofacitinib, a low-molecular-mass JAK inhibitor, was used to screen for JAK/STAT-mediated deregulation of lipid metabolism. Th2 cytokines decreased the expression of elongases 1, 3, and 4 and serine-palmitoyl-transferase and increased that of sphingolipid delta(4)-desaturase and carbonic anhydrase 2. Th2 cytokines inhibited the synthesis of palmitoleic acid and caused depletion of triglycerides, in association with altered phosphatidylcholine profiles and fatty acid (FA) metabolism. Overall, the ceramide profiles were minimally affected. Except for most sphingolipids and very-long-chain FAs, the effects of Th2 on lipid pathways were reversed by co-treatment with tofacitinib. An increase in the mRNA levels of CPT1A and ACAT1, reduced by tofacitinib, suggests that Th2 cytokines promote FA beta-oxidation. In conclusion, pharmacological inhibition of JAK/STAT activation prevents the lipid disruption caused by the halted homeostasis of FA metabolism.

Targeting Fatty Acid Amide Hydrolase Counteracts the Epithelial-to-Mesenchymal Transition in Keratinocyte-Derived Tumors.

The endocannabinoid system regulates physiological processes, and the modulation of endogenous endocannabinoid (eCB) levels is an attractive tool to contrast the development of pathological skin conditions including cancers. Inhibiting FAAH (fatty acid amide hydrolase), the degradation enzyme of the endocannabinoid anandamide (AEA) leads to the increase in AEA levels, thus enhancing its biological effects. Here, we evaluated the anticancer property of the FAAH inhibitor URB597, investigating its potential to counteract epithelial-to-mesenchymal transition (EMT), a process crucially involved in tumor progression. The effects of the compound were determined in primary human keratinocytes, ex vivo skin explants, and the squamous carcinoma cell line A431. Our results demonstrate that URB597 is able to hinder the EMT process by downregulating mesenchymal markers and reducing migratory potential. These effects are associated with the dampening of the AKT/STAT3 signal pathways and reduced release of pro-inflammatory cytokines and tumorigenic lipid species. The ability of URB597 to contrast the EMT process provides insight into effective approaches that may also include the use of FAAH inhibitors for the treatment of skin cancers.

The Activation of PPARγ by (2Z,4E,6E)-2-methoxyocta-2,4,6-trienoic Acid Counteracts the Epithelial-Mesenchymal Transition Process in Skin Carcinogenesis.

Cutaneous squamous cell carcinoma (cSCC) is the most common UV-induced keratinocyte-derived cancer, and its progression is characterized by the epithelial-mesenchymal transition (EMT) process. We previously demonstrated that PPARγ activation by 2,4,6-octatrienoic acid (Octa) prevents cutaneous UV damage. We investigated the possible role of the PPARγ activators Octa and the new compound (2Z,4E,6E)-2-methoxyocta-2,4,6-trienoic acid (A02) in targeting keratinocyte-derived skin cancer. Like Octa, A02 exerted a protective effect against UVB-induced oxidative stress and DNA damage in NHKs. In the squamous cell carcinoma A431 cells, A02 inhibited cell proliferation and increased differentiation markers' expression. Moreover, Octa and even more A02 counteracted the TGF-ß1-dependent increase in mesenchymal markers, intracellular ROS, the activation of EMT-related signal transduction pathways, and cells' migratory capacity. Both compounds, especially A02, counterbalanced the TGF-ß1-induced cell membrane lipid remodeling and the release of bioactive lipids involved in EMT. In vivo experiments on a murine model useful to study cell proliferation in adult animals showed the reduction of areas characterized by active cell proliferation in response to A02 topical treatment. In conclusion, targeting PPARγ may be useful for the prevention and treatment of keratinocyte-derived skin cancer.

Bcl-2-like protein-10 increases aggressive features of melanoma cells.

Aim: B-cell lymphoma-2 (Bcl-2)-like protein-10 (Bcl2L10) is the less studied member of Bcl-2 family proteins, with the controversial role in different cancer histotypes. Very recently, Bcl2L10 expression in melanoma tumor specimens and its role in melanoma response to therapy have been demonstrated. Here, the involvement of Bcl2L10 on the in vitro and in vivo properties associated with melanoma aggressive features has been investigated. Methods: Endogenous Bcl2L10 protein expression was detected by western blotting analysis in a panel of patient-derived and commercially available human melanoma cells. In vitro assays to evaluate clonogenicity, cell proliferation, cell migration, cell invasion, and in vitro capillary-like structure formation [vasculogenic mimicry (VM)] have been performed by using human melanoma cells stably overexpressing Bcl2L10 or transiently transfected for loss/gain function of Bcl2L10, grown under two- or three-dimensional (3D) conditions Xenograft melanoma model was employed to evaluate in vivo tumor growth and angiogenesis. Results: Results demonstrated that Bcl2L10 acts as an inducer of in vitro cell migration, invasion, and VM, while in vitro cell proliferation, in vivo tumor growth, as well as colony formation properties were not affected. Dissecting different signaling pathways, it was found that Bcl2L10 positively affects the phosphorylation of extracellular-signal-regulated kinase (ERK) and the expression of markers of cell invasion, such as urokinase plasminogen activator receptor (uPAR) and matrix metalloproteinases (MMPs). Of note, Bcl2L10-dependent in vitro migration, invasion, and VM are linked to uPAR. Bcl2L10 also negatively regulates the intracellular calcium level. Finally, reduced invasion capability in 3D spheroid invasion assay of melanoma cells transiently overexpressing Bcl2L10 was observed after treatment with inhibitors of MMPs and uPAR. Conclusions: Overall, data reported in this paper provide evidence supporting a positive role of Bcl2L10 in melanoma aggressive features.

Altered epidermal proliferation, differentiation, and lipid composition: Novel key elements in the vitiligo puzzle.

Vitiligo is an acquired skin depigmentation disease involving multiple pathogenetic mechanisms, which ultimately direct cytotoxic CD8+ cells to destroy melanocytes. Abnormalities have been described in several cells even in pigmented skin as an expression of a functional inherited defect. Keratinocytes regulate skin homeostasis by the assembly of a proper skin barrier and releasing and responding to cytokines and growth factors. Alterations in epidermal proliferation, differentiation, and lipid composition as triggers for immune response activation in vitiligo have not yet been investigated. By applying cellular and lipidomic approaches, we revealed a deregulated keratinocyte differentiation with altered lipid composition, associated with impaired energy metabolism and increased glycolytic enzyme expression. Vitiligo keratinocytes secreted inflammatory mediators, which further increased following mild mechanical stress, thus evidencing immune activation. These findings identify intrinsic alterations of the nonlesional epidermis, which can be the prime instigator of the local inflammatory milieu that stimulates immune responses targeting melanocytes.

Sebocytes contribute to melasma onset.

Melasma is a hyperpigmentary disorder with photoaging features, whose manifestations appear on specific face areas, rich in sebaceous glands (SGs). To explore the SGs possible contribution to the onset, the expression of pro-melanogenic and inflammatory factors from the SZ95 SG cell line exposed to single or repetitive ultraviolet (UVA) radiation was evaluated. UVA up-modulated the long-lasting production of α-MSH, EDN1, b-FGF, SCF, inflammatory cytokines and mediators. Irradiated SZ95 sebocyte conditioned media increased pigmentation in melanocytes and the expression of senescence markers, pro-inflammatory cytokines, and growth factors regulating melanogenesis in fibroblasts cultures. Cocultures experiments with skin explants confirmed the role of sebocytes on melanogenesis promotion. The analysis on sebum collected from melasma patients demonstrated that in vivo sebocytes from lesional areas express the UVA-activated pathways markers observed in vitro. Our results indicate sebocytes as one of the actors in melasma pathogenesis, inducing prolonged skin cell stimulation, contributing to localized dermal aging and hyperpigmentation.

Anti-Inflammatory and Pro-Differentiating Properties of the Aryl Hydrocarbon Receptor Ligands NPD-0614-13 and NPD-0614-24: Potential Therapeutic Benefits in Psoriasis.

The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor expressed in all skin cell types, plays a key role in physiological and pathological processes. Several studies have shown that this receptor is involved in the prevention of inflammatory skin diseases, e.g., psoriasis, atopic dermatitis, representing a potential therapeutic target. We tested the safety profile and the biological activity of NPD-0614-13 and NPD-0614-24, two new synthetic AhR ligands structurally related to the natural agonist FICZ, known to be effective in psoriasis. NPD-0614-13 and NPD-0614-24 did not alter per se the physiological functions of the different skin cell populations involved in the pathogenesis of inflammatory skin diseases. In human primary keratinocytes stimulated with tumor necrosis factor-α or lipopolysaccharide the compounds were able to counteract the altered proliferation and to dampen inflammatory signaling by reducing the activation of p38MAPK, c-Jun, NF-kBp65, and the release of cytokines. Furthermore, the molecules were tested for their beneficial effects in human epidermal and full-thickness reconstituted skin models of psoriasis. NPD-0614-13 and NPD-0614-24 recovered the psoriasis skin phenotype exerting pro-differentiating activity and reducing the expression of pro-inflammatory cytokines and antimicrobial peptides. These data provide a rationale for considering NPD-0614-13 and NPD-0614-24 in the management of psoriasis.

Beta HPV Type 15 Can Interfere With NF-κB Activity and Apoptosis in Human Keratinocytes.

E7 protein from cutaneous as well as mucosal HPV types can alter NF-κB activity. Conflicting literature data show a HPV-induced up- or down-regulation of the NF-κB pathway in different cell lines. In a previous study we detected the expression of E7 gene of HPV15 in a subungual tumor of a patient affected by incontinentia pigmenti (IP). IP is a rare X-linked genodermatosis in which the IKKγ gene is altered. From observations in transgenic IKKγ defective mice, it was suggested that IKK-deficient cells may undergo rapid hyper-proliferation and apoptosis/necrosis, leading to increased pro-inflammatory cytokine production in the neighboring IKK-positive cells. The objective of this study was to ascertain if beta HPV 15 can alter apoptosis and NF-κB pathway in normal and IKKγ-deficient keratinocytes. The human immortalized keratinocyte cell line (HaCaT), and human primary keratinocyte (HPK) cells were transduced with a retrovirus expressing E6-E7 proteins of HPV 15 and IKKγ was successful silenced mimicking the HPV15 infection and IP. HPV15 E6-E7 gene expression improved NF-κB activity in human keratinocytes even when IKKγ was silenced by siRNA. In IKKγ silenced keratinocyte cells, TNF-α-induced apoptosis was strongly reduced by the expression of HPV15 E6-E7 genes. Beta HPV15 exerted this anti-apoptotic activity by decreasing pro-apoptotic BAK and cleaved Caspase 3 proteins. In conclusion, we can speculate that presence of persistent infection by beta papillomavirus might influence the biological fate of IP by altering NF-κB activation and apoptosis in IKKγ mutated cells, favoring their survival and possibly the development of tumors in the late stage of disease. Taken together, our data reinforce the importance of host genetic background in the pathogenesis of HPV-associated skin lesions.

Another point of view on p16 and Ki67 expression in melanocytic and non-melanocytic cutaneous lesions.

The new analysis of the expression of cell cycle regulators (used in various neoplasms) and the nominal immunohistologic assays still represent valid and feasible diagnostic methods in most pathology practice. We examined 114 paraffin-embedded histologic specimens of melanocytic cutaneous lesions. The primary objective of this study was to explore diagnostic potential of the two important cell cycle regulators, p16 and Ki67, also evaluating the variations of expression by use of a semi quantitative graded scale. Another aim was to study the hypothetical correlation between p16 expression (in melanocytic and non-melanocytic lesions) and two independent variables, i.e. patient age and anatomical sites (sun exposed or non-sun exposed) of the lesions. Cell population was considered positive for antibody-specific p16 and Ki67 when at least 33% of the cells showed well-defined nuclear and/or cytoplasmic staining. A special p16 and Ki67 trend was found only in Spitz nevus, atypical Spitz nevus and invasive malignant melanoma (MM). No discriminative values were found regarding other lesions (junctional melanocytic nevus, MM in situ, superficially spreading MM, non-melanoma skin cancers). p16 was over-expressed on sun exposed sites and hypo-expressed on non-sun exposed areas, yielding a statistically significant correlation (p<0.03). According to patient age, p16 was over-expressed in patients aged ≥61 and hypo-expressed in patients aged ≤60 (p=0.09).

Marked inhibition of retinal neovascularization in rats following soluble-flt-1 gene transfer.

BACKGROUND: In mouse models of retinopathy of prematurity (ROP) inhibitors of vascular endothelial growth factor (VEGF) functions administered systemically completely block retinal neovascularization. In contrast, selective ocular VEGF depletion has achieved an approx. 50% inhibition of retinal neovascular growth. It is unclear whether a more complete inhibition of new blood vessel development can be obtained with an anti-VEGF therapy localized to the eye. Therefore, the objective of the present study was to determine the effect of local anti-VEGF therapy in a different animal model which closely mimics human ROP. METHODS: Rats were exposed to alternating cycles of high and low levels of oxygen for 14 days immediately after birth; thereafter, they were intravitreally injected with an adenoviral vector expressing a secreted form of the VEGF receptor flt-1 (Ad.sflt), which acts by sequestering VEGF. Contralateral eyes were injected with the control vector carrying the reporter gene expressing beta-galactosidase (Ad.betaGal). RESULTS: At the peak of retinal neovascular growth, i.e. post-natal day 21 (P21), we observed up to 97.5% decrease in retinal neovascularization in animals injected with Ad.sflt. At the end of observation (P28), no significant difference in retinal vessel number was detected in both oxygen-injured and normoxic Ad.sflt-treated retinas compared with untreated or Ad.betaGal-treated retinas. CONCLUSION: Adenoviral-mediated sflt-1 gene transfer induces a near-complete inhibition of ischemia-induced retinal neovascularization in rats without affecting pre-existing retinal vessels.