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.

Insulin and the sebaceous gland function.

Insulin affects metabolic processes in different organs, including the skin. The sebaceous gland (SG) is an important appendage in the skin, which responds to insulin-mediated signals, either directly or through the insulin growth factor 1 (IGF-1) axis. Insulin cues are differently translated into the activation of metabolic processes depending on several factors, including glucose levels, receptor sensitivity, and sebocyte differentiation. The effects of diet on both the physiological function and pathological conditions of the SG have been linked to pathways activated by insulin and IGF-1. Experimental evidence and theoretical speculations support the association of insulin resistance with acne vulgaris, which is a major disorder of the SG. In this review, we examined the effects of insulin on the SG function and their implications in the pathogenesis of acne.

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.

The αMSH-Dependent PI3K Pathway Supports Energy Metabolism, via Glucose Uptake, in Melanoma Cells.

Stimulation of melanocytes and murine melanoma cells with αMSH plus the PI3K inhibitor LY294002 resulted in ROS increase, oxidative DNA damage, and pigment retention. We performed cellular and molecular biology assays (Western blot, FACS, immunofluorescence analysis, scratch assay) on murine and human melanoma cells. Treatment with αMSH plus LY294002 altered cortical actin architecture. Given that cytoskeleton integrity requires energy, we next evaluated ATP levels and we observed a drop in ATP after exposure to αMSH plus LY294002. To evaluate if the αMSH-activated PI3K pathway could modulate energy metabolism, we focused on glucose uptake by analyzing the expression of the Glut-1 glucose translocator. Compared with cells treated with αMSH alone, those exposed to combined treatment showed a reduction of Glut-1 on the plasma membrane. This metabolic alteration was associated with changes in mitochondrial mass. A significant decrease of the cell migratory potential was also observed. We demonstrated that the αMSH-dependent PI3K pathway acts as a regulator of energy metabolism via glucose uptake, influencing the actin cytoskeleton, which is involved in melanosome release and cell motility. Hence, these results could constitute the basis for innovative therapeutical strategies.

Desaturation of sebaceous-type saturated fatty acids through the SCD1 and the FADS2 pathways impacts lipid neosynthesis and inflammatory response in sebocytes in culture.

Sebum is a lipid-rich mixture secreted by the sebaceous gland (SG) onto the skin surface. By penetrating through the epidermis, sebum may be involved in the regulation of epidermal and dermal cells in both healthy and diseased skin conditions. Saturated and monounsaturated fatty acids (FAs), found as free FAs (FFAs) and in bound form in neutral lipids, are essential constituents of sebum and key players of the inflammatory processes occurring in the pilosebaceous unit in acne-prone skin. Little is known on the interplay among uptake of saturated FFAs, their biotransformation, and induction of proinflammatory cytokines in sebocytes. In the human SG, palmitate (C16:0) is the precursor of sapienate (C16:1n-10) formed by insertion of a double bond (DB) at the Δ6 position catalysed by the fatty acid desaturase 2 (FADS2) enzyme. Conversely, palmitoleate (C16:1n-7) is formed by insertion of a DB at the Δ9 position catalysed by the stearoyl coenzyme A desaturase 1 (SCD1) enzyme. Other FFAs processed in the SG, also undergo these main desaturation pathways. We investigated lipogenesis and release of IL-6 and IL-8 pro-inflammatory cytokines in SZ95 sebocytes in vitro after treatment with saturated FFAs, that is, C16:0, margarate (C17:0), and stearate (C18:0) with or without specific inhibitors of SCD1 and FADS2 desaturase enzymes, and a drug with mixed inhibitory effects on FADS1 and FADS2 activities. C16:0 underwent extended desaturation through both SCD1 and FADS2 catalysed pathways and displayed the strongest lipoinflammatory effects. Inhibition of desaturation pathways proved to enhance lipoinflammation induced by SFAs in SZ95 sebocytes. Palmitate (C16:0), margarate (C17:0), and stearate (C18:0) are saturated fatty acids that induce different arrays of neutral lipids (triglycerides) and dissimilar grades of inflammation in sebocytes.

Skin Anti-Inflammatory Potential with Reduced Side Effects of Novel Glucocorticoid Receptor Agonists.

Glucocorticoids (GCs) are commonly used in the treatment of inflammatory skin diseases, although the balance between therapeutic benefits and side effects is still crucial in clinical practice. One of the major and well-known adverse effects of topical GCs is cutaneous atrophy, which seems to be related to the activation of the glucorticoid receptor (GR) genomic pathway. Dissociating anti-inflammatory activity from atrophogenicity represents an important goal to achieve, in order to avoid side effects on keratinocytes and fibroblasts, known target cells of GC action. To this end, we evaluated the biological activity and safety profile of two novel chemical compounds, DE.303 and KL.202, developed as non-transcriptionally acting GR ligands. In primary keratinocytes, both compounds demonstrated anti-inflammatory properties inhibiting NF-κB activity, downregulating inflammatory cytokine release and interfering with pivotal signaling pathways involved in the inflammatory process. Of note, these beneficial actions were not associated with GC-related atrophic effects: treatments of primary keratinocytes and fibroblasts with DE.303 and KL.202 did not induce, contrarily to dexamethasone-a known potent GC-alterations in extracellular matrix components and lipid synthesis, thus confirming their safety profile. These data provide the basis for evaluating these compounds as effective alternatives to the currently used GCs in managing inflammatory skin diseases.

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.

The PI3K pathway induced by αMSH exerts a negative feedback on melanogenesis and contributes to the release of pigment.

The melanocortin-1 receptor (MC1R) belongs to the family of the G protein-coupled receptor (GPCR). Activated GPCRs can promote the phosphoinositide 3-kinase (PI3K) pathway. Few studies deal with the role of the PI3K pathway activation in response to αMSH. On B16-F10 cell line, we investigated the αMSH-dependent modulation of pAKT/AKT, as a key element of the PI3K pathway after rapid and prolonged stimulation. We demonstrated that αMSH triggers a rapid modulation of AKT which culminates in an increase in its phosphorylation. We highlighted a comparable upregulation of pAKT after exposure to αMSH on primary cultures of normal human melanocytes (NHMs) expressing a wild-type MC1R. On B16-F10 cells, NHMs, and an ex vivo model of human skin biopsies, we explored the influence of PI3K/AKT signaling triggered by αMSH, focusing on the control of melanogenesis and pigment release. We showed that the αMSH-dependent PI3K/AKT pathway exerts a negative feedback on melanogenesis and promotes the extracellular release of pigment. We strengthened the role of the PI3K/AKT pathway triggered by αMSH in preserving redox equilibrium and genome integrity, highlighting its role in affecting cell survival.

Acne as an altered dermato-endocrine response problem.

Acne is the most common skin disease in adolescent Westernized populations. Several data support the involvement of the mammalian target of rapamycin complex 1 (mTORC1) signalling in the interplay between androgens, insulin, insulin-like growth factor (IGF1) and high-glycaemic index diet in acne. The peroxisome proliferator-activated receptor γ (PPARγ) is involved in both differentiation and anti-inflammatory response. Low differentiated sebocytes showed decreased expression of PPARγ and increased level of insulin and IGF-1 receptors, resulting in the production of acne-like sebum and inflammatory mediators. In this viewpoint, we discuss how in acne the dysregulation of proliferation and differentiation processes in sebocytes and keratinocytes may be associated with altered response to androgens and other hormones, such as insulin or IGF-1. Moreover, we propose PPARγ modulation as an innovative therapeutic approach to normalize sebocyte differentiation process, interfering with the different mechanisms involved in altered pilosebaceous unit.

Bovine colostrum induces the differentiation of human primary keratinocytes.

Bovine colostrum, the first milk secreted by the mammary glands of cows shortly after they have given birth, provides a natural source of bioactive substances helpful to promote tissue development and repair, and to maintain a healthy immune system. Owing to its properties, the use of colostrum in the treatment of human diseases is under investigation. We evaluated the biological activity of colostrum on human primary keratinocytes, focusing on its effects with regard to a proliferation/differentiation balance. Using cellular and molecular approaches, we showed that colostrum favors a cell cycle withdrawal by increasing the expression of p21/WAF1 and p27/KIP1. It also promotes the transition of keratinocytes from a proliferating to a differentiating state, as assessed by a decrease in keratin 5 and an increase in keratin 16. We demonstrated the ability of colostrum to induce the expression of early and late differentiation markers (keratin 1, involucrin, and filaggrin) and the synthesis of caspase 14 and bleomycin hydrolase, the two main enzymes involved in filaggrin maturation. Moreover, we showed that bovine colostrum is able to promote keratinocyte stratification and terminal differentiation not only in two-dimensional (2D), but also in a more physiological system of three-dimensional (3D) skin equivalents. Finally, we demonstrated that colostrum stimulates cell differentiation through the PI3K/PLC-γ1/PKCα pathways mainly associated to tyrosine kinase receptors. These results suggest the possibility to benefit from colostrum properties for the treatment of skin diseases characterized by altered differentiation and perturbed barrier function.

The α-melanocyte stimulating hormone/peroxisome proliferator activated receptor-γ pathway down-regulates proliferation in melanoma cell lines.

BACKGROUND: The α-Melanocyte Stimulating Hormone (αMSH)/Melanocortin-1 receptor (MC1R) interaction promotes melanogenesis through the cAMP/PKA pathway. The direct induction of this pathway by Forskolin (FSK) is also known to enhance melanocyte proliferation. αMSH acts as a mitogenic agent in melanocytes and its effect on proliferation of melanoma cells is less known. We previously identified the αMSH/Peroxisome Proliferator Activated Receptor (PPARγ) pathway as a new pathway on the B16-F10 mouse melanoma cell line. αMSH induced the translocation of PPARγ into the nucleus as an active transcription factor. This effect was independent of the cAMP/PKA pathway and was mediated by the activation of the PI(4,5)P2/PLC pathway, a pathway which we have described to be triggered by the αMSH-dependent MC1R stimulation. Moreover, in the same study, preliminary experiments showed that mouse melanoma cells responded to αMSH by reducing proliferation and that PPARγ was involved in this effect. Due to its key role in the control of cell proliferation, PPARγ agonists are used in therapeutic models for different forms of cancer, including melanoma. The purpose of this study was: (a) to confirm the different proliferative behavior in response to αMSH in healthy and in melanoma condition; (b) to verify whether the cAMP/PKA pathway and the PLC/PPARγ pathway could exert an antagonistic function in the control of proliferation; (c) to deepen the knowledge of the molecular basis responsible for the down-proliferative response of melanoma cells after exposure to αMSH. METHODS: We employed B16-F10 cell line, a human melanoma cell line (Mel 13) and two primary cultures of human melanocytes (NHM 1 and NHM 2, respectively), all expressing a wild type MC1R and responding to the αMSH in terms of pigmentation. We evaluated cell proliferation through: a) cell counting, b) cell cycle analysis c) protein expression of proliferation modulators (p27, p21, cyclin D1 and cyclin E). RESULTS: The αMSH acted as a mitogenic agent in primary cultures of human melanocytes, whereas it determined a slow down of proliferation in melanoma cell lines. FSK, as an inducer of the cAMP/PKA pathway, reproduced the αMSH mediated effect on proliferation in NHMs but it did not mimic the αMSH effect on proliferation in B16-F10 and Mel 13 melanoma cell lines. Meanwhile, 3 M3-FBS (3 M3), as an inducer of PI(4,5)P2/PLC pathway, reproduced the αMSH proliferative effect. Further experiments, treating melanoma cell lines with αMSH in the presence/absence of GW9662, as an inhibitor of PPARγ, confirmed the key role of this transcription factor in decreasing cell proliferation in response to the hormone exposure. CONCLUSIONS: In both melanoma cell lines, αMSH determined the reduction of proliferation through the PI(4,5)P2/PLC pathway, employing PPARγ as an effector element. These evidence could offer perspectives for new therapeutic approaches for melanoma.

The activation of PPARγ by 2,4,6-Octatrienoic acid protects human keratinocytes from UVR-induced damages.

Increasing attention is addressed to identify products able to enhance skin photoprotection and to prevent skin carcinogenesis. Several studies have demonstrated that the α-melanocyte stimulating hormone (αMSH), acting on a functional MC1R, provides a photoprotective effect by inducing pigmentation, antioxidants and DNA repair. We discovered a link between αMSH and the nuclear receptor Peroxisome Proliferator-Activated Receptor-γ (PPARγ), suggesting that some of the αMSH protective effects may be dependent on PPARγ transcriptional activity. Moreover, we demonstrated that the activation of PPARγ by the parrodiene 2,4,6-octatrienoic acid (Octa) induces melanogenesis and antioxidant defence in human melanocytes and counteracts senescence-like phenotype in human fibroblasts. In this study, we demonstrate that the activation of PPARγ by Octa exerts a protective effect against UVA- and UVB-induced damage on normal human keratinocytes (NHKs), the major target cells of UV radiation. Octa promotes the antioxidant defence, augments DNA repair and reduces the induction of proteins involved in UV-induced DNA damage response. Our results contribute to deepen the analysis of the αMSH/PPARγ connection and suggest perspectives for the development of new molecules and formulations able to prevent cutaneous UV damage by acting on the different skin cell populations through PPARγ activation.

Skin phototype: a new perspective.

Cutaneous phototype is considered mainly related to cutaneous pigmentation and to the eumelanin/pheomelanin ratio, which is mostly genetically determined by the melanocortin 1 receptor (MC1R) polymorphisms. However, data in literature indicate that, in addition to stimulation of eumelanin synthesis, the MC1R signalling activates antioxidant, DNA repair and survival pathways. New emerging aspects regarding photoprotection and skin phototypes are going beyond those features connected to the melanin content in the skin. Important new findings link the MC1R to nuclear receptors activation, shedding light on new extra-melanogenic effects dependent on the α-melanocyte-stimulating hormone (α-MSH) activity and new ways through which such functions are modulated. These evidences indicate that several factors including melanin play a part in defining the basis for individual sun sensitivity, suggesting that the cutaneous phototype represents a 'biochemical fingerprint'.

Modulation of PPARγ provides new insights in a stress induced premature senescence model.

Peroxisome proliferator-activated receptor gamma (PPARγ) may be involved in a key mechanism of the skin aging process, influencing several aspects related to the age-related degeneration of skin cells, including antioxidant unbalance. Therefore, we investigated whether the up-modulation of this nuclear receptor exerts a protective effect in a stress-induced premature senescence (SIPS) model based on a single exposure of human dermal fibroblasts to 8-methoxypsoralen plus + ultraviolet-A-irradiation (PUVA). Among possible PPARγ modulators, we selected 2,4,6-octatrienoic acid (Octa), a member of the parrodiene family, previously reported to promote melanogenesis and antioxidant defense in normal human melanocytes through a mechanism involving PPARγ activation. Exposure to PUVA induced an early and significant decrease in PPARγ expression and activity. PPARγ up-modulation counteracted the antioxidant imbalance induced by PUVA and reduced the expression of stress response genes with a synergistic increase of different components of the cell antioxidant network, such as catalase and reduced glutathione. PUVA-treated fibroblasts grown in the presence of Octa are partially but significantly rescued from the features of the cellular senescence-like phenotype, such as cytoplasmic enlargement, the expression of senescence-associated-ß-galactosidase, matrix-metalloproteinase-1, and cell cycle proteins. Moreover, the alterations in the cell membrane lipids, such as the decrease in the polyunsaturated fatty acid content of phospholipids and the increase in cholesterol levels, which are typical features of cell aging, were prevented. Our data suggest that PPARγ is one of the targets of PUVA-SIPS and that its pharmacological up-modulation may represent a novel therapeutic approach for the photooxidative skin damage.

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 ß-galactosidase (SA-ß-gal). We found that PUVA-treated HDFs grown in the presence of AzA maintained their morphology and reduced MMP-1 release and SA-ß-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.

Linking αMSH with PPARγ in B16-F10 melanoma.

We have discovered a new α-melanocyte stimulating hormone (α-MSH)/peroxisome proliferator activated receptor-γ (PPAR-γ) connection in B16-F10 cells. Both PPAR-γ up-regulation and its induction as an active transcription factor were observed in response to α-MSH. The α-MSH/PPAR-γ connection influenced both pigmentation and proliferation. The forskolin-stimulated cAMP/PKA pathway was not able to induce either PPAR-γ translocation into the nucleus or PPAR-γ transcriptional activity. As the melanocortin-1 receptor, the specific receptor for the α-MSH, is a G-protein coupled receptor, we wondered whether the phosphatidylinositol [PI(4,5)P(2) /PLC(ß) ] signal pathway was involved in mediating the α-MSH-dependent PPAR-γ activation. Employing inhibitors of PI(4,5)P(2) /PLC(ß) pathway, the results of our experiments suggested that this pathway was promoted by α-MSH and that α-MSH played a role in mediating PPAR-γ activation. We have demonstrated, for the first time, that α-MSH induces the PI(4,5)P(2) /PLC(ß) pathway, through analysis of the basic steps of the pathway. The α-MSH effect on PPAR-γ was independent of animal species and was not correlated with the physio-pathological status.

Cystinosin is a melanosomal protein that regulates melanin synthesis.

Cystinosis is a rare autosomal recessive disease characterized by cystine crystal accumulation leading to multiorgan dysfunctions and caused by mutation in CTNS. CTNS encodes cystinosin, a cystine/H(+) symporter that exports cystine out of the lysosomes. Patients with cystinosis frequently exhibit blond hair and fair complexion, suggesting an alteration in melanogenesis. However, the pigmentation singularities of these patients have not been studied, and the role of cystinosin in melanogenesis has remained unknown. In our study, a clinical evaluation of 27 patients with cystinosis showed that 44% had a cutaneous pigmentation dilution compared to their relatives. Analysis of the hair melanin content in these patients by HPLC demonstrated a 50% decrease in eumelanin (4360 vs. 9360 ng/mg), and a 2-fold increase in pheomelanin (53 vs. 20 ng/mg), the yellow/red pigments. Cystinosin-deficient mice also showed a 4-fold increase in hair pheomelanin content. In vitro studies showed that cystinosin was located at melanosomes. CTNS silencing led to a 75% reduction of melanin synthesis that was caused by a degradation of tyrosinase by lysosomal proteases. Our results objectify the pigmentation defect in patients with cystinosis. We also identify the role of CTNS in melanogenesis and add a new gene to the list of the genes involved in the control of skin and hair pigmentation.

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.