Daniela Kovacs

Melanins and melanogenesis: methods, standards, protocols

Despite considerable advances in the past decade, melanin research still suffers from the lack of universally accepted and shared nomenclature, methodologies, and structural models. This paper stems from the joint efforts of chemists, biochemists, physicists, biologists, and physicians with recognized and consolidated expertise in the field of melanins and melanogenesis, who critically reviewed and experimentally revisited methods, standards, and protocols to provide for the first time a consensus set of recommended procedures to be adopted and shared by researchers involved in pigment cell research. The aim of the paper was to define an unprecedented frame of reference built on cutting‐edge knowledge and state‐of‐the‐art methodology, to enable reliable comparison of results among laboratories and new progress in the field based on standardized methods and shared information.

Integrative Analysis of Epigenetic Modulation in Melanoma Cell Response to Decitabine: Clinical Implications

Decitabine, an epigenetic modifier that reactivates genes otherwise suppressed by DNA promoter methylation, is effective for some, but not all cancer patients, especially those with solid tumors. It is commonly recognized that to overcome resistance and improve outcome, treatment should be guided by tumor biology, which includes genotype, epigenotype, and gene expression profile. We therefore took an integrative approach to better understand melanoma cell response to clinically relevant dose of decitabine and identify complementary targets for combined therapy. We employed eight different melanoma cell strains, determined their growth, apoptotic and DNA damage responses to increasing doses of decitabine, and chose a low, clinically relevant drug dose to perform whole-genome differential gene expression, bioinformatic analysis, and protein validation studies. The data ruled out the DNA damage response, demonstrated the involvement of p21Cip1 in a p53-independent manner, identified the TGFβ pathway genes CLU and TGFBI as markers of sensitivity to decitabine and revealed an effect on histone modification as part of decitabine-induced gene expression. Mutation analysis and knockdown by siRNA implicated activated β-catenin/MITF, but not BRAF, NRAS or PTEN mutations as a source for resistance. The importance of protein stability predicted from the results was validated by the synergistic effect of Bortezomib, a proteasome inhibitor, in enhancing the growth arrest of decitabine in otherwise resistant melanoma cells. Our integrative analysis show that improved therapy can be achieved by comprehensive analysis of cancer cells, identified biomarkers for patients selection and monitoring response, as well as targets for improved combination therapy.

Differential response to keratinocyte growth factor receptor and epidermal growth factor receptor ligands of proliferating and differentiating intestinal epithelial cells

The expression of the keratinocyte growth factor receptor (KGFR) has been analyzed on intestinal epithelial Caco‐2 cells upon confluence‐induced spontaneous differentiation. Western blot and immunofluorescence analysis showed that the expression of functional KGFRs, differently from that of epidermal growth factor receptor (EGFR), was up‐modulated in post‐confluent differentiated cultures compared with the pre‐confluent cells. Confocal microscopy and immunoelectron microscopy revealed that the up‐regulated KGFRs displayed a basolateral polarized distribution on the cell surfaces in the monolayer. In vivo immunohistochemical analysis on normal human colon tissue sections showed that KGFRs, differently from EGFRs, were mostly distributed on the more differentiated cells located on the upper portion of the intestinal crypt. Bromodeoxyuridine incorporation assay and Ki67 labeling indicated that the differentiated cells were able to proliferate in response to the two ligands of KGFR, KGF and FGF‐10, whereas they were not stimulated by the EGFR ligands TGFα and EGF. Western blot and quantitative immunofluorescence analysis of the expression of carcinoembryonic antigen (CEA) in post‐confluent cells revealed that incubation with KGF induced an increase of cell differentiation. Taken together these results indicate that up‐modulation of KGFR may be required to promote proliferation and differentiation in differentiating cells and that, among the cells componing the intestinal epithelial monolayer, the target cells for KGFR ligands appear to be different during differentiation from those responsive to EGFR ligands.

The Eumelanin Intermediate 5,6-Dihydroxyindole-2-Carboxylic Acid Is a Messenger in the Cross-Talk among Epidermal Cells

Interest in colorless intermediates of melanocyte metabolism has traditionally been related to their role as melanin precursors, though several lines of evidence scattered in the literature suggested that these compounds may exert an antioxidant and protective function per se unrelated to pigment synthesis. Herein, we disclose the remarkable protective and differentiating effects of 5,6-dihydroxyindole-2-carboxylic acid (DHICA), a diffusible dopachrome tautomerase (DCT)-dependent eumelanin intermediate, on primary cultures of human keratinocytes. At micromolar concentrations, DHICA induced: (a) time- and dose-dependent reduction of cell proliferation without concomitant toxicity; (b) enhanced expression of early (spinous keratins K1 and K10 and envelope protein involucrin) and late (loricrin and filaggrin) differentiation markers; (c) increased activities and expression of antioxidant enzymes; and (d) decreased cell damage and apoptosis following UVA exposure. The hitherto unrecognized role of DHICA as an antiproliferative, protective, and antiapoptotic endogenous cell messenger points to a reappraisal of the biological functions of melanocytes and DCT in skin homeostasis and photoprotection beyond the mere provision of melanin pigments, and provides, to our knowledge, a previously unreported possible explanation to the higher resistance of the dark-skinned eumelanic phenotypes to sunburn and skin cancer.

Immunohistochemical analysis of keratinocyte growth factor and fibroblast growth factor 10 expression in psoriasis.

Abstract:  The pathogenic mechanism underlying the hyperproliferation of keratinocytes in psoriasis is still not completely clarified. The production of cytokines released by activated T lymphocytes infiltrating the upper dermis probably has a crucial role. Even dermal fibroblasts can participate in the process through the secretion of growth factors, and some studies have reported an increased expression of the insulin‐like growth factor 1. Few studies, however, have focused on the possible involvement of the keratinocyte growth factor (KGF/FGF‐7) and the fibroblast growth factor 10 (FGF‐10/KGF‐2), which are secreted by fibroblasts and stimulate keratinocyte proliferation acting through a receptor specifically expressed by epithelial cells. The aim of this study was to investigate the expression of KGF and FGF‐10 on the skin of patients with psoriasis by immunohistochemical analysis and to evaluate the correlation with the lymphocyte infiltrate and the epidermal proliferation. Immunostaining for KGF and FGF‐10 showed that both the growth factors are upregulated in the upper dermis of psoriatic skin, and that the expression is correlated with the presence of T‐cell infiltrate and with keratinocyte proliferation. Our data suggest that in psoriatic lesions activated lymphocytes can stimulate fibroblasts to produce KGF and FGF‐10, which in turn contribute to sustain the hyperproliferative status of the keratinocytes.

MC1R stimulation by α-MSH induces catalase and promotes its re-distribution to the cell periphery and dendrites

We demonstrated a direct correlation between melanogenic and catalase activities on in vitro and ex vivo models. Here, we investigated whether the stimulation of Melanocortin‐1 Receptor (MC1R) could influence catalase expression, activity and cellular localization. For this purpose, we treated B16‐F0 melanoma cells with α‐Melanocyte Stimulating Hormone (α‐MSH) and we showed a rapid induction of catalase through a cAMP/PKA‐dependent, microphthalmia‐associated transcription factor (MITF) independent mechanism, acting at post‐transcriptional level. Moreover, α‐MSH promoted a partial re‐distribution of catalase to the cell periphery and dendrites. This work strengthens the correlation between melanogenesis and anti‐oxidants, demonstrating the induction of catalase in response to a melanogenic stimulation, such as α‐MSH‐dependent MC1R activation. Moreover, this study highlights catalase regulatory mechanisms poorly known, and attributes to α‐MSH a protective role in defending melanocytes, and possibly keratinocytes, not only on the basis of its pigmentary action, but also for its capacity to stimulate a quick anti‐oxidant defence.

Expression of Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand Receptors 1 and 2 in Melanoma

Purpose: The proapoptotic receptors tumor necrosis factor–related apoptosis-inducing ligand receptor 1 (TRAIL-R1) and TRAIL-R2 are targets of drugs in clinical development, and receptor expression levels may be important determinants of sensitivity to receptor agonists. We assessed TRAIL-R1 and TRAIL-R2 expression patterns in a large cohort of melanomas and benign nevi. Experimental Design: We analyzed tissue microarrays containing 546 melanomas and 540 nevi using our automated quantitative method to measure protein levels in situ (AQUA). The system uses S100 to define pixels as melanoma (tumor mask) within the array spot and measures intensity of TRAIL-receptor expression using Cy5-conjugated antibodies within the mask. AQUA scores were correlated with clinical and pathologic variables. Results: TRAIL-R1 and TRAIL-R2 expression was higher in melanomas than in nevi (P < 0.0001), and higher in primary than in metastatic specimens (P = 0.0031 and P < 0.0001, respectively). TRAIL-R1 and TRAIL-R2 expression exceeding the 95th percentile for nevi was found in 19% and 74% of melanoma specimens, respectively. Although on univariate analysis, high TRAIL-R2 expression correlated with increased survival (P = 0.0439), it was not associated with survival within the primary or metastatic subcohorts. TRAIL-R1 expression was not associated with survival. Conclusions: TRAIL-R1 and TRAIL-R2 expression is higher in malignant melanocytes than in their benign counterparts, suggesting that these receptors might be effective therapeutic targets in melanoma. Expression is higher in early-stage disease than in metastatic specimens, and expression exceeding that found in nevi is found in a substantially larger fraction of melanomas for TRAIL-R2 compared with TRAIL-R1. Assessment of baseline tumor TRAIL receptor expression may be important in analysis of clinical trials involving TRAIL receptor agonists.

2,4,6-Octatrienoic acid is a novel promoter of melanogenesis and antioxidant defence in normal human melanocytes via PPAR-γ activation.

Given the importance of the tanning response in protecting human skin from the harmful effects of UV radiation, one important research priority is to identify novel molecules that are capable of promoting pigmentation and/or antioxidant defence. Parrodienes share some structural features with carotenoids and retinoids, stimulate cell antioxidant defence and counteract senescence‐like phenotype in fibroblasts. We selected the parrodiene‐derivative 2,4,6‐octatrienoic acid (Octa) to study its impact on key parameters of melanogenesis and antioxidant defence in organ‐cultured human skin and in normal human melanocytes. Octa promoted melanogenesis by up‐regulating tyrosinase and microphthalmia‐associated transcription factor expression. This correlated with an increase of melanin content in both human epidermis in situ and cultured human epidermal melanocytes. Moreover, Octa increased the biological antioxidant potential content and the expression and activity of catalase. Activation of peroxisome proliferator‐activated receptor (PPAR)‐γ was necessary to evoke these effects. These data strongly encourage the systematic study of Octa as a novel candidate promoter of human skin photoprotection.

Azelaic acid reduced senescence-like phenotype in photo-irradiated human dermal fibroblasts: possible implication of PPARγ.

Azelaic acid (AzA) has been used for the treatment for inflammatory skin diseases, such as acne and rosacea. Interestingly, an improvement in skin texture has been observed after long‐time treatment with AzA. We previously unrevealed that anti‐inflammatory activity of AzA involves a specific activation of PPARγ, a nuclear receptor that plays a relevant role in inflammation and even in ageing processes. As rosacea has been considered as a photo‐aggravated disease, we investigated the ability of AzA to counteract stress‐induced premature cell senescence (SIPS). We employed a SIPS model based on single exposure of human dermal fibroblasts (HDFs) to UVA and 8‐methoxypsoralen (PUVA), previously reported to activate a senescence‐like phenotype, including long‐term growth arrest, flattened morphology and increased synthesis of matrix metalloproteinases (MMPs) and senescence‐associated β‐galactosidase (SA‐β‐gal). We found that PUVA‐treated HDFs grown in the presence of AzA maintained their morphology and reduced MMP‐1 release and SA‐β‐galactosidase‐positive cells. Moreover, AzA induced a reduction in ROS generation, an up‐modulation of antioxidant enzymes and a decrease in cell membrane lipid damages in PUVA‐treated HDFs. Further evidences of AzA anti‐senescence effect were repression of p53 and p21, increase in type I pro‐collagen and abrogation of the enhanced expression of growth factors, such as HGF and SCF. Interestingly, PUVA‐SIPS showed a decreased activation of PPARγ and AzA counteracted this effect, suggesting that AzA effect involves PPARγ modulation. All together these data showed that AzA interferes with PUVA‐induced senescence‐like phenotype and its ability to activate PPAR‐γ provides relevant insights into the anti‐senescence mechanism.

Preclinical Studies of a Specific PPARγ Modulator in the Control of Skin Inflammation

Peroxisome proliferator-activated receptor γ (PPARγ) antagonizes inflammatory signals by interfering with NF-κB nuclear translocation. Consistently, PPARγ agonists have been proposed in various inflammatory skin disorders, but their wide use has been limited by severe side effects. Classes of compounds with specific PPARγ agonism have been designed to selectively target inflammatory pathways. Among these compounds, GED-0507-34L has been developed and recently used in phase II clinical trials for inflammatory bowel diseases. This study was aimed at assessing the role of GED-0507-34L in preclinical models of inflammatory skin diseases. The compound modulated PPARγ function and suppressed the inflammatory process inhibiting NF-κB nuclear translocation with the consequent reduction of inflammatory cytokines expression, such as IL-6, IL-8, IL-12, IL-21, IL-23, tumor necrosis factor-α (TNF-α), and cyclooxygenase-2 (COX-2) in normal human keratinocytes and lymphocytes treated with lipopolysaccharide (LPS) or TNF-α. Moreover, an altered proliferation and expression of differentiation markers induced by TNF-α were also counteracted. In psoriasis-like skin lesions elicited in mice by IL-21, topical application of GED-0507-34L reduced cellular infiltrate and epidermal hyperplasia, normalizing the differentiation process. The results indicate that GED-0507-34L possesses anti-inflammatory properties useful for the management of patients with inflammatory skin diseases including psoriasis. Phase I trial on patients is ongoing.