Cutaneous squamous cell carcinoma (cSCC) is a common skin cancer, caused by mutagenesis resulting from excess ultraviolet radiation or other types of oxidative stress. These stressors also upregulate production of a cutaneous innate immune element, cathelicidin antimicrobial peptide (CAMP), via endoplasmic reticulum (ER) stress-initiated, sphingosine-1-phosphate (S1P) signaling pathway. While CAMP has beneficial antimicrobial activities, it also can be pro-inflammatory and pro-carcinogenic. We addressed whether and how S1P-induced CAMP production leads to cSCC development. Our study demonstrated that: 1) CAMP expression is increased in cSCC cells and skin from cSCC patients; 2) S1P levels are elevated in cSCC cells, while inhibition of S1P production attenuates CAMP-stimulated cSCC growth; 3) exogenous CAMP stimulates cSCC, but not normal human keratinocyte growth; 4) blockade of formyl peptide receptor-like (FPRL) 1 protein, a CAMP receptor, attenuates cSCC growth as well as the growth and invasion of cSCC cells mediated by CAMP into an extracellular matrix-containing fibroblast substrate; 5) Foxp3+ regulatory T cell (which decreases anti-tumor immunity) levels increase in cSCC skin; and 6) CAMP induces ER stress in cSCC cells. Together, the ER stress-S1P-CAMP axis forms a vicious circle, creating a favorable environment for cSCC development, i.e., cSCC growth and invasion impedes anti-cancer immunity.

Cutaneous squamous cell carcinoma (cSCC) is a common cancer, caused by mutagenesis from excess ultraviolet radiation (UVR) or other types of oxidative stress. These stressors also upregulate production of a key innate immune element, cathelicidin antimicrobial peptide (CAMP), via endoplasmic reticulum (ER) stress-initiated, sphingosine-1-phosphate (S1P) signaling. While CAMP has beneficial antimicrobial activities, it also can be pro-inflammatory and pro-carcinogenic. We addressed whether/how S1P-induced CAMP production leads to cSCC development. We found that: 1) CAMP expression was markedly increased in cSCC cells and in cSCC patients' skin; 2) inhibition of S1P production attenuated CAMP-stimulated cSCC growth; 3) exogenous CAMP stimulates cSCC growth, but not growth of normal human keratinocytes; 4) blockade of formyl peptide receptor-like (FPRL) 1 protein (a receptor of CAMP) activation attenuated cSCC growth as well as the growth-promoting effects of exogenously applied CAMP on cSCC cells, and also slowed invasion of cSCC cells into an extracellular matrix-containing fibroblast substrate; and 5) Foxp3+ regulatory T (Treg) cells (which diminish anti-tumor immunity) levels were increased in cSCC skin. These results suggest that the relationship of S1P signaling to CAMP overproduction is crucial for cSCC development. Since Treg cells in a cancer microenvironment weaken anti-cancer immunity, we will also address roles of CAMP in Treg function in cSCC.

Cathelicidin antimicrobial peptide (CAMP) is a key antimicrobial peptide in skin. CAMP production is increased during epidermal differentiation and enriched in the stratum corneum. We recently identified a novel endoplasmic reticulum (ER) stress-mediated sphingosine-1-phosphate (S1P)-dependent mechanism of CAMP synthesis. In this study, we found that S1P synthesized by an isoform of sphingosine kinase (SPHK), SPHK1, serves as a signal for CAMP synthesis; and conversely, another isoform SPHK2 likely has a suppressor or no role in CAMP synthesis. Pertinently, prior studies showed that physiological ER stress is essential for normal epidermal differentiation. We here show that: increased ER stress occurs in differentiated cultured keratinocytes (KC); 2) increases in both CAMP and S1P production depend upon differentiation level of KC (proliferated

Glucocorticoid (GC) excess drives multiple cutaneous adverse effects, including skin thinning and poor wound healing. The ubiquitously expressed enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) activates mouse corticosterone from 11-dehydrocorticosterone (and human cortisol from cortisone). We previously demonstrated elevated 11β-HSD1 activity during mouse wound healing, but the interplay between cutaneous 11β-HSD1 and systemic GC excess is unexplored. Here, we examined effects of 11β-HSD1 inhibition by carbenoxolone (CBX) in mice treated with corticosterone (CORT) or vehicle for 6 weeks. Mice were treated bidaily with topical CBX or vehicle (VEH) 7 days before wounding and during wound healing. CORT mice displayed skin thinning and impaired wound healing but also increased epidermal integrity. 11β-HSD1 activity was elevated in unwounded CORT skin and was inhibited by CBX. CORT mice treated with CBX displayed 51%, 59%, and 100% normalization of wound healing, epidermal thickness, and epidermal integrity, respectively. Gene expression studies revealed normalization of interleukin 6, keratinocyte growth factor, collagen 1, collagen 3, matrix metalloproteinase 9, and tissue inhibitor of matrix metalloproteinase 4 by CBX during wound healing. Importantly, proinflammatory cytokine expression and resolution of inflammation were unaffected by 11β-HSD1 inhibition. CBX did not regulate skin function or wound healing in the absence of CORT. Our findings demonstrate that 11β-HSD1 inhibition can limit the cutaneous effects of GC excess, which may improve the safety profile of systemic steroids and the prognosis of chronic wounds.

The innate immune element, cathelicidin antimicrobial peptide (CAMP), is a vital antimicrobial peptide needed for the formation of the antimicrobial skin barrier. We recently identified a novel endoplasmic reticulum (ER) stress-mediated sphingosine-1-phosphate (S1P)-dependent mechanism of CAMP synthesis. Interestingly, in this study, we found that S1P synthesized by an isoform of sphingosine kinase (SPHK), SPHK1, serves as a signal for CAMP synthesis and conversely, another isoform (SPHK2) likely has a suppressor role in CAMP production. CAMP production is increased during epidermal differentiation and enriched in the stratum corneum. Pertinently, prior studies showed that physiological ER stress is essential for normal epidermal differentiation. We here investigated how CAMP production is increased during epidermal differentiation. We found that 1) increased ER stress is evident in differentiated cultured keratinocytes; 2) increases in both CAMP and S1P production depend upon differentiation level of keratinocyte (proliferatedwt, but not dominant negative SPHK2 suppresses CAMP production in both proliferated and differentiated KC. Our current study suggests that both an increase in SPHK1 and a decrease in SPHK2 expression coordinately stimulate CAMP production during epidermal differentiation.

This study aimed to elucidate the cross-talk between immune/non-immune cutaneous cells in response to Staphylococcus (S) aureus stress, including the influence of the secretome of S. aureus-challenged, non-immune skin cells (keratinocytes and fibroblasts) on the functional specialization of skin-resident dendritic cells (DCs). For this purpose, we generated conditioned media (CM) from S. aureus-challenged (strain USA 300) human keratinocytes and fibroblasts, and investigated whether they would influence the functional maturation of Langerhans cells (LCs) and DCs prepared from blood monocytes. This phenotypical and functional maturation of DCs was assessed by FACS, qPCR and ELISA analyses. CM from S. aureus-challenged keratinocytes and fibroblasts efficiently increased HLA-DR and CD86 surface expression (P<0.05) and mRNA expression of several inflammatory markers (P<0.05) in both LCs and DCs. Importantly, we identified certain variations in both the reactivity and the functional response of LCs/DCs in response to the type of secretome to which they were exposed. Interestingly, keratinocyte CM increased the expression of IL-1β, TNF-α and GM-CSF, which are involved in LCs/DCs maintenance and maturation. In contrast, fibroblast CM triggered a dual profile of both Th1 cytokines (IL-12 and IFN-γ) and IL-10, suggesting the functional specialization of two different DCs subsets and/or the induction of tolerogenic DCs. This study provides new insights into the dynamics between different cutaneous cell types, and how they can drive immune responses in response to S. aureus challenges. Further studies will be required to identify the full spectrum of effector molecular species from keratinocyte and fibroblast secretomes that contribute to the pathogenesis and immune responses following colonization by S. aureus.

We recently identified a novel sphingosine-1-phosphate (S1P) signaling mechanism that stimulates production of a key innate immune element, cathelicidin antimicrobial peptide (CAMP), in mammalian cells exposed to external perturbations, such as UVB irradiation and other oxidative stressors that provoke sub-apoptotic levels of endoplasmic reticulum (ER) stress, independent of the well-known vitamin D receptor-dependent mechanism (Park K., et al., J Biol Chem, 2011 and Mol Cell Biol, 2014). ER stress increases cellular ceramide and one of its distal metabolites, S1 P, which activates NF-κB followed by C/EBPα activation, leading to CAMP production, but in a S1P receptor-independent fashion. Yet, how S 1P activates NF-κB remains unresolved. We elucidated that S1P-mediated activation of NF-κB through formation of a previously-unidentified signaling complex, consisting of S1P, TRAF2, and RIP1 that further associates with three stress-responsive proteins; i.e., heat shock proteins (GRP94 and HSP90α) and IRE1α. S1P specifically interacts with the N-terminal domain of heat shock proteins. This ER stress-initiated mechanism is operative in both epithelial cells and macrophages, suggesting that our identified pathway is universal, highly-conserved response to enhance innate immunity in response to external stress. Our studies illuminate how ER stress and S1P orchestrate critical stress-specific signals that regulate production of one protective response by stimulating production of the key innate immune element, CAMP.

Staphylococcus (S) aureus is a transient microbe of normal skin, but when the epidermal permeability barrier is compromised, it can invade with virulent and inflammatory consequences. Prior studies have demonstrated a direct effect of S. aureus on epidermal cytokine production, leading to widespread responses in other cutaneous cells types. We assessed here how S. aureus provokes downstream inflammation. Specifically, we investigated the effects of conditioned medium from normal cultured human keratinocytes (KC) or fibroblasts, after treatment with heat-killed S. aureus (HKSA) on cytokine/chemokine production by dendritic cells (DC), prepared from normal human monocytes by GM-CSF and IL-4-induced differentiation. Following incubation with KC conditioned medium, mRNA production of IL-1β and IL-8 by DC increased markedly (by 9.1±3.5-fold and 5.0±2.7-fold, respectively). IL-12 expression increased in DC following incubation with fibroblast conditioned medium (14.4±7.6-fold). It is well known that IL-1β and IL-8 recruit innate immune cells (e.g., neutrophils and monocytes), while IL-12 activates Th1 cells. Hence, these results indicate that KC and fibroblasts play divergent, potentially complementary roles in provoking inflammatory responses following exposure to S. aureus.

We recently discovered a novel sphingosine-1-phosphate (S1P)-induced signaling mechanism of cathelicidin antimicrobial peptide (CAMP) production in response to external perturbant-mediated endoplasmic reticulum (ER) stress. Activation occurs by an S1P receptor (S1PR)–independent activation of NF-κB, followed by C/EBPα transactivation (rather than through the well-established vitamin D receptor-mediated mechanism). Yet, how S1P activates NF-κB remains unresolved. In addition to using both specific activators and inhibitors of each S1PR isomers, we have employed a gene silencing approach and showed that ER stress-induced CAMP upregulation is not changed in S1PR1, 2 and 3 knockout mouse skin and in cultured human keratinocytes treated with siRNA against S1PR4 and S1PR5, further confirming that ER stress-induced CAMP production occurs in an S1PR-independent fashion. We next assessed whether S1P binds to specific cytosolic proteins instead of binding to S1PR, followed by activation of NF-κB. Proteomics approach using MALDI-TOF-MS analysis revealed that S1P binds to specific stress-responsive proteins. S1P conjugated agarose beads study characterized S1P binding to stress responsive proteins, which form a signaling complex that activates NF-κB. Finally, the ER stress-mediated increases in CAMP production is reduced in cells by inhibiting or knocking down each protein component of the signaling complex, as well as by approached that reduce S1P production, further supporting our theory that a S1P-mediated signaling complex serves to upregulate CAMP production in response to ER stress. Our study illuminates how ER stress and S1P coordinately enhance innate immunity in response to external perturbations that induce ER stress.

We have asked how different external perturbations, i.e. epidermal permeability disruption and oxidative stress stimulate antimicrobial peptide productions (AMP). We demonstrated that these external perturbations induce subtoxic levels of endoplasmic reticulum (ER) stress that stimulates a major epidermal AMP, cathelicidin antimicrobial peptide (CAMP), via a newly-identified NF-κB-to-c/EBPα- (rather than the well-established vitamin D receptor-) dependent mechanism, and we recently elucidated that ER stress also increases production of the pro-apoptotic lipid ceramide (Cer) and its anti-apoptotic metabolite, sphingosine-1-phosphate (S1P), which activates NF-κB, leading to increased CAMP production in human keratinocytes (KC). We characterize here how ER stress initiates the increase of another major AMP, human beta-defensin(s) (hBDs). Cultured KC incubated with a pharmacological ER stressor, thapsigargin, increased hBD2/hBD3 and CAMP (but not hBD1) production. Inhibition of S1P production by an inhibitor of ceramidase attenuated the expected increase in CAMP expression, while blockade of Cer-1-phosphate (C1P), another Cer metabolite, using siRNA or an inhibitor of Cer kinase, suppressed hBD2/hBD3, but not CAMP expression following ER stress. Exogenous C1P also increased hBD2/hBD3 production, indicating that C1P stimulates hBD2/hBD3 expression. We then elucidated a novel pathway of C1P-induced hBD2/hBD3 expression, in which C1P stimulates downstream hBDs via cPLA2-to-15d-PGJ2-to-PPARα/PPARβ/δ-to-STAT1/STAT3 transcriptional mechanism. Our studies discovered two new regulatory mechanisms of key epidermal AMP, CAMP and hBD2/hBD3 synthesis by S1P and C1P, respectively, signals that enhance innate immunity in response to ER stress induced by external perturbations.

Harlequin Ichthyosis is a severe skin disease caused by mutations in the human gene encoding ABCA12. Here, we characterize a novel mutation in intron 29 of the mouse Abca12 gene that leads to the loss of a 5' splice donor site and truncation of the Abca12 RNA transcript. Homozygous mutants of this smooth skin or smsk allele die perinatally with shiny translucent skin, typical of animal models of Harlequin Ichthyosis. Characterization of smsk mutant skin showed that the delivery of glucosylceramides and CORNEODESMOSIN was defective, while ultrastructural analysis revealed abnormal lamellar bodies and the absence of lipid lamellae in smsk epidermis. Unexpectedly, mutant stratum corneum remained intact when subjected to harsh chemical dissociation procedures. Moreover, both KALLIKREIN 5 and -7 were drastically decreased, with retention of desmoplakin in mutant SC. In cultured wild type keratinocytes, both KALLIKREIN 5 and -7 colocalized with ceramide metabolites following calcium-induced differentiation. Reducing the intracellular levels of glucosylceramide with a glucosylceramide synthase inhibitor resulted in decreased secretion of KALLIKREIN proteases by wild type keratinocytes, but not by smsk mutant keratinocytes. Together, these findings suggest an essential role for ABCA12 in transferring not only lipids, which are required for the formation of multilamellar structures in the stratum corneum, but also proteolytic enzymes that are required for normal desquamation. Smsk mutant mice recapitulate many of the pathological features of HI and can be used to explore novel topical therapies against a potentially lethal and debilitating neonatal disease.

Cutaneous squamous cell carcinoma (cSCC) is a common cancer, often initiated by oxidative stress, particularly ultraviolet irradiation (UVR). We recently demonstrated that oxidative stressors upregulate a key innate immune element, cathelicidin antimicrobial peptide (CAMP/LL-37) via endoplasmic reticulum (ER)-mediated, sphingosine-1-phosphate (S1P) signaling mechanism. Pertinently, prior studies showed that CAMP/LL-37 production also increases in certain cancers, while it remains unresolved why CAMP overproduction occurs and how CAMP could increase tumorigenesis. Here we investigated whether and how CAMP contributes to the development of cSCC. CAMP mRNA/peptide production was increased significantly higher in cSCC cell lines) than in normal human keratinocytes (KC). Moreover, exogenous CAMP significantly stimulated the growth of cSCC, but not normal KC. The CAMP receptor that is localized in the plasma membranes mRNA/protein levels are higher in cSCC compared with KC. Blockade of formyl peptide receptor-like (FPRL) 1 protein (FPRL1) by a specific receptor antagonist (WRW4) attenuated cSCC growth. Moreover, cSCC, but not KC, invaded into a dermal equivalent, and exogenous CAMP further stimulated cSCC invasion into an in vitro dermal equivalent, but WRW4 suppressed cSCC invasion. Next, elevated CAMP expression and cell growth in SCC were significantly suppressed by specific inhibitors of sphingosine kinase 1. Finally, consistent with cultured cSCC, both CAMP and FPRL1 protein levels are elevated in cSCC skin vs. normal skin. Together, these studies suggest that S1P signaling stimulates CAMP overproduction leading to activation of a FPRL1 and results in enhancing cSCC growth and invasion. Excessive production of an innate immune response facilitates cSCC tumorigenesis.

Significance The cathelicidin antimicrobial peptide (CAMP) is an innate immune element that promotes antimicrobial defense, but excessive CAMP can stimulate inflammation and tumorigenesis. We recently discovered that external perturbations that induce subtoxic levels of endoplasmic reticulum (ER) stress increase sphingosine-1-phosphate (S1P) production, in turn activating NF-κB–mediated CAMP synthesis. We report here that S1P interacts with the heat shock proteins (HSP90α and GRP94) through a previously unidentified S1P receptor-independent intracellular mechanism, followed by the activation of NF-κB leading to stimulation of CAMP production. These studies illuminate the critical role of both ER stress and S1P in orchestrating stress-specific signals that enhance innate immunity.

Detrimental consequences of ultraviolet radiation (UVR) in skin include photoageing, immunosuppression and photocarcinogenesis, processes also significantly regulated by local glucocorticoid (GC) availability. In man, the enzyme 11-hydroxysteroid dehydrogenase type 1 (11-HSD1) generates the active GC cortisol from cortisone (or corticosterone from 11-dehydrocorticosterone in rodents). 11-HSD1 oxo-reductase activity requires the cofactor NADPH, generated by hexose-6-phosphate dehydrogenase. We previously demonstrated increased 11-HSD1 levels in skin obtained from photoexposed versus photoprotected anatomical regions. However, the direct effect of UVR on 11-HSD1 expression remains to be elucidated. To investigate the cutaneous regulation of 11-HSD1 following UVR in vivo, the dorsal skin of female SKH1 mice was irradiated with 50, 100, 200 and 400mJ/cm(2) UVB. Measurement of transepidermal water loss, 11-HSD1 activity, mRNA/protein expression and histological studies was taken at 1, 3 and 7days postexposure. 11-HSD1 and hexose-6-phosphate dehydrogenase mRNA expression peaked 1day postexposure to 400mJ/cm(2) UVB before subsequently declining (days 3 and 7). Corresponding increases in 11-HSD1 protein and enzyme activity were observed 3days postexposure coinciding with reduced GC receptor mRNA expression. Immunofluorescence studies revealed 11-HSD1 localization to hyperproliferative epidermal keratinocytes in UVB-exposed skin. 11-HSD1 expression and activity were also induced by 200 and 100 (but not 50)mJ/cm(2) UVB and correlated with increased transepidermal water loss (indicative of barrier disruption). UVB-induced 11-HSD1 activation represents a novel mechanism that may contribute to the regulation of cutaneous responses to UVR exposure.

Corneocytes in mammalian stratum corneum are surrounded by a monolayer of covalently bound ω-OH-ceramides that form the corneocyte (-bound) lipid envelope (CLE). We review here the structure, composition, and possible functions of this structure, with insights provided by inherited and acquired disorders of lipid metabolism. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier. Guest Editors: Kenneth R. Feingold and Peter Elias.