Kinga Fanni Tóth

Transient receptor potential melastatin 5 (TRPM5) ion channel is expressed in human hair follicles, where its spontaneous activity contributes to the maintenance of the growing, anagen phase of the hair cycle. Because adjacent sebaceous glands also exhibited TRPM5 immunopositivity, topically applied TRPM5 modulators administered to influence hair growth may also affect sebaceous glands. Hence, we aimed to assess expression of TRPM5 as well as effects of TRPM5 modulators [activators: 2,5‐dimethylpyrazine, 2‐heptanone; antagonist: triphenylphosphine oxide (TPPO)] on human SZ95 sebocytes, i.e., on the best available in vitro model to study human sebaceous glands. First, using complementary methods [RNA‐Seq, RT‐qPCR, western blot, siRNA‐mediated gene silencing and fluorescent Na⁺‐ (SBFI AM) and Ca²⁺‐measurements (Fura‐2 AM)], we found that TRPM5 is not expressed in human sebocytes in a functionally active form. Importantly, while non‐cytotoxic (MTT‐assay) concentrations of the activators were ineffective, TPPO promoted sebaceous lipogenesis (Nile Red labelling). This effect was TRPM5‐independent and was found to be mediated in an Akt‐ and epidermal growth factor receptor (EGFR)‐dependent manner, most likely via the Akt‐induced up‐regulation of diacylglycerol O‐acyltransferase (DGAT)‐2. Moreover, TPPO up‐regulated interleukin (IL)‐6 in an EGFR‐ and p38α MAPK‐dependent manner (RT‐qPCR), whereas it decreased the release of IL‐8 (ELISA), and down‐regulated additional pro‐inflammatory cytokines [chemokine (C‐X‐C motif) ligand [CXCL]‐1, CXCL2, CXCL6, colony‐stimulating factor 2, IL‐32; RNA‐Seq]. Collectively, specific TRPM5 modulators are unlikely to exert direct sebaceous gland‐related side effects, while safe TPPO analogues may induce beneficial moderate lipogenic and anti‐inflammatory effects in dry skin dermatoses.

We have recently shown that fluoxetine (FX) suppressed polyinosinic-polycytidylic acid-induced inflammatory response and endothelin release in human epidermal keratinocytes, via the indirect inhibition of the phosphoinositide 3-kinase (PI3K)-pathway. Because PI3K-signaling is a positive regulator of the proliferation, in the current, highly focused follow-up study, we assessed the effects of FX (14 µM) on the proliferation and differentiation of human epidermal keratinocytes. We found that FX exerted anti-proliferative actions in 2D cultures (HaCaT and primary human epidermal keratinocytes [NHEKs]; 48- and 72-h; CyQUANT-assay) as well as in 3D reconstructed epidermal equivalents (48-h; Ki-67 immunohistochemistry). Importantly, FX did not influence epidermal thickness (hematoxylin-eosin staining), and it did not have a major impact on the differentiation-associated alteration of the gene expression pattern (24-h treatments; RNA-Seq). Moreover, neither keratin (K)-1, nor K10 expression was altered by FX in NHEKs (RT-qPCR) or in 3D epidermal equivalents (semi-quantitative immunohistomorphometry). FX did not influence differentiation-induced up-regulation of occludin (RT-qPCR; NHEKs), and did not alter differentiation-associated barrier forming capacity of epidermal keratinocytes (electrical impedance; Lucifer Yellow penetration assay). Our data indicate that, besides the previously reported combined anti-inflammatory and putative anti-pruritic effects, FX may also suppress proliferation of human epidermal keratinocytes without impairing their differentiation and barrier-forming capacity.

Fluoxetine is a safe antidepressant with remarkable anti‐inflammatory actions; therefore, we aimed to investigate its effects on immortalized (HaCaT) as well as primary human epidermal keratinocytes in a polyinosinic‐polycytidylic acid (p(I:C))‐induced inflammatory model. We found that a non‐cytotoxic concentration (MTT‐assay, CyQUANT‐assay) of fluoxetine significantly suppressed p(I:C)‐induced expression and release of several pro‐inflammatory cytokines (Q‐PCR, cytokine array, ELISA), and it decreased the release of the itch mediator endothelins (ELISA). These effects were not mediated by the inhibition of the NF‐κB or p38 MAPK pathways (western blot), or by the suppression of the p(I:C)‐induced elevation of mitochondrial ROS production (MitoSOX Red labeling). Instead, unbiased activity profiling revealed that they were most likely mediated via the inhibition of the phosphoinositide 3‐kinase (PI3K) pathway. Importantly, the PI3K‐inhibitor GDC0941 fully mimicked the effects of fluoxetine (Q‐PCR, ELISA). Although fluoxetine was able to occupy the binding site of GDC0941 (in silico molecular docking), and exerted direct inhibitory effect on PI3K (cell‐free PI3K activity assay), it exhibited much lower potency and efficacy as compared to GDC0941. Finally, RNA‐Seq analysis revealed that fluoxetine deeply influenced the transcriptional alterations induced by p(I:C)‐treatment, and exerted an overall anti‐inflammatory activity. Collectively, our findings demonstrate that fluoxetine exerts potent anti‐inflammatory effects, and suppresses the release of the endogenous itch mediator endothelins in human keratinocytes, most likely via interfering with the PI3K pathway. Thus, clinical studies are encouraged to explore whether the currently reported beneficial effects translate in vivo following its topical administration in inflammatory and pruritic dermatoses.

Atopic dermatitis (AD) is one of the most common skin diseases, the prevalence of which is especially high among children. Although our understanding about its pathogenesis has substantially grown in recent years, and hence, several novel therapeutic targets have been successfully exploited in the management of the disease, we still lack curative treatments for it. Thus, there is an unmet societal demand to identify further details of its pathogenesis to thereby pave the way for novel therapeutic approaches with favorable side effect profiles. It is commonly accepted that dysfunction of the complex cutaneous barrier plays a central role in the development of AD; therefore, the signaling pathways involved in the regulation of this quite complex process are likely to be involved in the pathogenesis of the disease and can provide novel, promising, yet unexplored therapeutic targets. Thus, in the current review, we aim to summarize the available potentially AD-relevant data regarding one such signaling pathway, namely cutaneous opioidergic signaling.

Photodamage-induced and viral keratitis could benefit from treatment with novel nonsteroid anti-inflammatory agents. Therefore, we determined whether human corneal epithelial cells (HCECs) express members of the endocannabinoid system (ECS), and examined how the endocannabinoid anandamide (AEA, N-arachidonoyl ethanolamine) influences the Toll-like receptor 3 (TLR3) agonism- or UVB irradiation-induced inflammatory response of these cells. Other than confirming the presence of cannabinoid receptors, we show that endocannabinoid synthesizing and catabolizing enzymes are also expressed in HCECs in vitro, as well as in the epithelial layer of the human cornea in situ, proving that they are one possible source of endocannabinoids. p(I:C) and UVB irradiation was effective in promoting the transcription and secretion of inflammatory cytokines. Surprisingly, when applied alone in 100 nM and 10 μM, AEA also resulted in increased pro-inflammatory cytokine production. Importantly, AEA further increased levels of these cytokines in the UVB model, whereas its lower concentration partially prevented the transcriptional effect of p(I:C), while not decreasing the p(I:C)-induced cytokine release. HCECs express the enzymatic machinery required to produce endocannabinoids both in vitro and in situ. Moreover, our data show that, despite earlier reports about the anti-inflammatory potential of AEA in murine cornea, its effects on the immune phenotype of human corneal epithelium may be more complex and context dependent.

Nicotinic acid (NA) activates hydroxycarboxylic acid receptor 2 (HCA₂), and it is widely used in treating dyslipidaemias. Since its side effects include skin dryness, whereas its deficiency can be accompanied by dyssebacia, characterized by sebaceous gland enlargement, we asked if HCA₂ is expressed on human sebocytes, and if NA influences sebocyte functions. By using human immortalized SZ95 sebocytes, we found that non‐cytotoxic (≤100 μmol/L; MTT‐assay) concentrations of NA had no effect on the homeostatic sebaceous lipogenesis (SLG; Nile Red), but normalized excessive, acne‐mimicking SLG induced by several lipogenic agents (arachidonic acid, anandamide, linoleic acid + testosterone; Nile Red; 48‐hr treatments). Moreover, it exerted significant anti‐proliferative actions (CyQUANT‐assay), and increased [Ca²⁺]_IC (Fluo‐4 AM‐based Ca²⁺‐measurement). Although NA did not prevent the lipopolysaccharide‐induced pro‐inflammatory response (up‐regulation [Q‐PCR] and release [ELISA] of several pro‐inflammatory cytokines) of the sebocytes, collectively, these data support the concept that NA may be effective in suppressing sebum production in vivo. While exploring the mechanism of the sebostatic actions, we found that sebocytes express HCA₂ (Q‐PCR, immunofluorescent labelling), siRNA‐mediated silencing of which prevented the NA‐induced Ca²⁺‐signal and the lipostatic action. Collectively, our data introduce NA, and HCA₂ activators in general, as novel, potent and most likely safe sebostatic agents, with possible anti‐acne potential.

The endocannabinoid system (ECS) has lately been proven to be an important, multifaceted homeostatic regulator, which influences a wide-variety of physiological processes all over the body. Its members, the endocannabinoids (eCBs; e.g., anandamide), the eCB-responsive receptors (e.g., CB₁, CB₂), as well as the complex enzyme and transporter apparatus involved in the metabolism of the ligands were shown to be expressed in several tissues, including the skin. Although the best studied functions over the ECS are related to the central nervous system and to immune processes, experimental efforts over the last two decades have unambiguously confirmed that cutaneous cannabinoid (“c[ut]annabinoid”) signaling is deeply involved in the maintenance of skin homeostasis, barrier formation and regeneration, and its dysregulation was implicated to contribute to several highly prevalent diseases and disorders, e.g., atopic dermatitis, psoriasis, scleroderma, acne, hair growth and pigmentation disorders, keratin diseases, various tumors, and itch. The current review aims to give an overview of the available skin-relevant endo- and phytocannabinoid literature with a special emphasis on the putative translational potential, and to highlight promising future research directions as well as existing challenges.