RESUMO
We recently identified a previously unidentified 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 subapoptotic levels of endoplasmic reticulum (ER) stress, independent of the well-known vitamin D receptor-dependent mechanism. ER stress increases cellular ceramide and one of its distal metabolites, S1P, which activates NF-κB followed by C/EBPα activation, leading to CAMP production, but in a S1P receptor-independent fashion. We now show that S1P activates 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. Because this ER stress-initiated mechanism is operative in both epithelial cells and macrophages, it appears to be a universal, highly conserved response, broadly protective against diverse external perturbations that lead to increased ER stress. Finally, these studies further 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.
Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Estresse do Retículo Endoplasmático , Lisofosfolipídeos/metabolismo , Transdução de Sinais , Esfingosina/análogos & derivados , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , Proteínas de Choque Térmico/metabolismo , Humanos , Queratinócitos/citologia , Queratinócitos/metabolismo , Camundongos Knockout , Microscopia de Fluorescência , NF-kappa B/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Ligação Proteica , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Receptores de Lisoesfingolipídeo/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esfingosina/metabolismo , Fator 2 Associado a Receptor de TNF/genética , Fator 2 Associado a Receptor de TNF/metabolismo , CatelicidinasRESUMO
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.
Assuntos
Epiderme/metabolismo , Metabolismo dos Lipídeos/fisiologia , Lipídeos , Animais , Células Epidérmicas , HumanosRESUMO
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 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) generates the active GC cortisol from cortisone (or corticosterone from 11-dehydrocorticosterone in rodents). 11ß-HSD1 oxo-reductase activity requires the cofactor NADPH, generated by hexose-6-phosphate dehydrogenase. We previously demonstrated increased 11ß-HSD1 levels in skin obtained from photoexposed versus photoprotected anatomical regions. However, the direct effect of UVR on 11ß-HSD1 expression remains to be elucidated. To investigate the cutaneous regulation of 11ß-HSD1 following UVR in vivo, the dorsal skin of female SKH1 mice was irradiated with 50, 100, 200 and 400 mJ/cm(2) UVB. Measurement of transepidermal water loss, 11ß-HSD1 activity, mRNA/protein expression and histological studies was taken at 1, 3 and 7 days postexposure. 11ß-HSD1 and hexose-6-phosphate dehydrogenase mRNA expression peaked 1 day postexposure to 400 mJ/cm(2) UVB before subsequently declining (days 3 and 7). Corresponding increases in 11ß-HSD1 protein and enzyme activity were observed 3 days postexposure coinciding with reduced GC receptor mRNA expression. Immunofluorescence studies revealed 11ß-HSD1 localization to hyperproliferative epidermal keratinocytes in UVB-exposed skin. 11ß-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 11ß-HSD1 activation represents a novel mechanism that may contribute to the regulation of cutaneous responses to UVR exposure.
Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 1/biossíntese , Epiderme/enzimologia , Epiderme/efeitos da radiação , Raios Ultravioleta/efeitos adversos , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/genética , Animais , Água Corporal/metabolismo , Água Corporal/efeitos da radiação , Proliferação de Células/efeitos da radiação , Relação Dose-Resposta à Radiação , Indução Enzimática/efeitos da radiação , Epiderme/patologia , Feminino , Glucocorticoides/metabolismo , Humanos , Queratinócitos/metabolismo , Queratinócitos/efeitos da radiação , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismoRESUMO
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.
RESUMO
Stress slows cutaneous wound healing (WH) in an endogenous glucocorticoid (GC)-dependent fashion. We investigated whether stress/GC-induced delays in WH require further intracutaneous activation of endogenous GC; and whether blockade or down-regulation of peripheral activation normalizes WH in the face of stress. Delayed WH in our motion-restricted murine model of stress could be attributed to elevated systemic GC, because blockade of GC production (using corticotropin-releasing factor inhibitor, antalarmin), or of peripheral binding to the GC receptor [GCr], with an antagonist, Ru-486, normalized WH. We next investigated whether local blockade or down-regulation of the peripheral GC-activating enzyme, 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1), accelerates cutaneous WH. Topical applications of nonspecific (carbenoxolone) as well as an isoform-specific 11ß-HSD1 inhibitor overcame stress and exogenous GC-induced delays in WH. Moreover, two liver X receptor ligands, TO901317 and GW3695, down-regulated expression of 11ß-HSD1, attenuating stress-induced delays in WH. Combined inhibitor and liver X receptor ligand applications accelerated WH in the face of stress/systemic GC. Thus: (1) intracutaneous conversion of inactive-to-active GC accounts for stress (GC)-induced delays in WH; and (2) blockade or down-regulation of 11ß-HSD1 and/or GCr normalize cutaneous WH in the face of stress/GC. Local blockade or down-regulation of cutaneous GC activation could help enhance WH in various clinical settings.
Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 1/farmacologia , Glucocorticoides/antagonistas & inibidores , Fígado/metabolismo , Receptores de Glucocorticoides/antagonistas & inibidores , Estresse Psicológico/metabolismo , Cicatrização , Animais , Western Blotting , Células Cultivadas , Regulação para Baixo/efeitos dos fármacos , Glucocorticoides/farmacologia , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase em Tempo Real , Cicatrização/efeitos dos fármacosRESUMO
Vitamin D receptor (VDR)-dependent mechanisms regulate human cathelicidin antimicrobial peptide (CAMP)/LL-37 in various cell types, but CAMP expression also increases after external perturbations (such as infection, injuries, UV irradiation, and permeability barrier disruption) in parallel with induction of endoplasmic reticulum (ER) stress. We demonstrate that CAMP mRNA and protein expression increase in epithelial cells (human primary keratinocytes, HaCaT keratinocytes, and HeLa cells), but not in myeloid (U937 and HL-60) cells, following ER stress generated by two mechanistically different, pharmacological stressors, thapsigargin or tunicamycin. The mechanism for increased CAMP following exposure to ER stress involves NF-κB activation leading to CCAAT/enhancer-binding protein α (C/EBPα) activation via MAP kinase-mediated phosphorylation. Furthermore, both increased CAMP secretion and its proteolytic processing to LL-37 are required for antimicrobial activities occur following ER stress. In addition, topical thapsigargin also increases production of the murine homologue of CAMP in mouse epidermis. Finally and paradoxically, ER stress instead suppresses the 1,25(OH)(2) vitamin D(3)-induced activation of VDR, but blockade of VDR activity does not alter ER stress-induced CAMP up-regulation. Hence, ER stress increases CAMP expression via NF-κB-C/EBPα activation, independent of VDR, illuminating a novel VDR-independent role for ER stress in stimulating innate immunity.
Assuntos
Peptídeos Catiônicos Antimicrobianos/biossíntese , Retículo Endoplasmático/metabolismo , Queratinócitos/metabolismo , Receptores de Calcitriol/metabolismo , Transdução de Sinais/fisiologia , Estresse Fisiológico/fisiologia , Resposta a Proteínas não Dobradas/fisiologia , Regulação para Cima/fisiologia , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Retículo Endoplasmático/genética , Células Epidérmicas , Epiderme/metabolismo , Células HL-60 , Células HeLa , Humanos , Queratinócitos/citologia , Masculino , Camundongos , Camundongos Mutantes , NF-kappa B/genética , NF-kappa B/metabolismo , Especificidade de Órgãos/fisiologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores de Calcitriol/genética , Células U937 , CatelicidinasRESUMO
BACKGROUND: Type 2 Gaucher disease is a rare and progressive subtype of this lysosomal storage disorder, marked by rapid, early-onset neurodegeneration. Distinguishing type 2 from types 1 and 3 Gaucher disease has remained challenging, due to the lack of a clear correlation between phenotype and enzymatic activity or genotype. ß-glucocerebrosidase, the enzyme deficient in Gaucher disease, also has an essential role in maintaining epidermal permeability function, by regulating the ratio of ceramides to glucosylceramides in the stratum corneum of the skin. OBJECTIVES: To further assess the diagnostic utility of epidermal evaluations in distinguishing patients with type 2 Gaucher disease in an expanded cohort. STUDY DESIGN: Epidermal samples were evaluated from twenty children with type 2, three patients with type 3 Gaucher disease and two adults with type 1 Gaucher disease with different clinical manifestations and genotypes. Electron microscopy on ruthenium tetroxide post-fixed tissue was performed. RESULTS: Compared to controls and subjects with type 1 and type 3 Gaucher disease, only patients with type 2 Gaucher disease displayed characteristic electron dense, non-lamellar clefts and immature-lamellar membranes. CONCLUSION: The appearance of characteristic alterations in epidermal ultrastructure provides an early and specific diagnostic tool to help in distinguishing type 2 from the other types of Gaucher disease.
Assuntos
Epiderme/patologia , Doença de Gaucher/diagnóstico , Doença de Gaucher/patologia , Adolescente , Biópsia , Criança , Pré-Escolar , Diagnóstico Diferencial , Feminino , Doença de Gaucher/genética , Estudos de Associação Genética , Humanos , Lactente , Masculino , Microscopia Eletrônica , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Gravidez , Diagnóstico Pré-Natal , Prognóstico , Adulto JovemRESUMO
Psychological stress (PS) exerts well-known negative consequences for permeability barrier function in humans and mice, and deterioration of barrier function appears to be attributable largely to excess production of endogenous glucocorticoids (GC). More recently, PS has been shown to compromise antimicrobial defense, also by GC-dependent mechanisms. We assessed here changes in a third antimicrobial peptide (AMP); i.e., the neuropeptide, catestatin (Cst), which also is expressed in the outer epidermis, and previously shown to be regulated by changes in permeability barrier status. In these studies, PS again provoked a decline in both mouse cathelicidin (CAMP) and mouse ß-defensin 3 (mBD3) expression, in a GC-dependent fashion. In contrast, Cst immunostaining instead increased after short-term PS, but then began to decline with more sustained PS. In cultured keratinocytes, we showed further that GC downregulate Cst expression, but ß-adrenergic blockade increased immunostaining for Cst in the face of long-term PS. Furthermore, ß-adrenergic blockade also upregulated CAMP and mBD3 expression. Together, these results suggest that both endogenous GC and ß-adrenergic signaling regulate AMP expression.
Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Cromogranina A/metabolismo , Fragmentos de Peptídeos/metabolismo , Estresse Psicológico/fisiopatologia , Animais , Feminino , Queratinócitos/metabolismo , Camundongos , Dermatopatias Infecciosas/metabolismo , Estresse Psicológico/metabolismo , beta-Defensinas/metabolismoRESUMO
BACKGROUND: Atopic dermatitis (AD) is a chronic inflammatory dermatosis now increasingly linked to mutations that alter the structure and function of the stratum corneum. Activators of peroxisome proliferator-activated receptors (PPARs) alpha, beta/delta, and gamma and liver X receptor (LXR) regulate epidermal protein and lipid production, leading to superior barrier function. Additionally, some of these activators exhibit potent antihyperplastic and anti-inflammatory activity in irritant contact dermatitis and acute allergic contact dermatitis murine models. OBJECTIVE: We evaluated the efficacy of PPAR/LXR activation in a hapten (oxazolone [Ox])-induced AD-like model (Ox-AD) in hairless mice. METHODS: Ox-AD was established with 10 Ox challenges (every other day) on the flank. After the establishment of Ox-AD, twice-daily topical application with individual PPAR/LXR activators was then performed for 4 days, with continued Ox challenges every other day. The efficacy of topical PPAR/LXR activators to reduce parameters of Ox-AD was assessed physiologically, morphologically, and immunologically. RESULTS: Certain topical activators of PPARalpha, PPARbeta/delta, and LXR, but not activators of PPARgamma, reversed the clinical dermatosis, significantly improved barrier function, and increased stratum corneum hydration in Ox-AD mice. In addition, the same activators, but again not PPARgamma, largely reversed the immunologic abnormalities in Ox-AD mice, including the increased T(H)2 markers, such as tissue eosinophil/mast cell density, serum thymus and activation-related chemokine levels, the density of chemoattractant receptor-homologous molecule expressed on T(H)2-positive lymphocytes (but not serum IgE levels), and reduced IL-1alpha and TNF-alpha activation, despite ongoing hapten challenges. CONCLUSION: These results suggest that topical applications of certain activators/ligands of PPARalpha, PPARbeta/delta, and LXR could be useful for the treatment of AD in human subjects.
Assuntos
Dermatite Atópica/tratamento farmacológico , Receptores Nucleares Órfãos/agonistas , PPAR alfa/agonistas , PPAR delta/agonistas , PPAR beta/agonistas , Administração Tópica , Animais , Citocinas/metabolismo , Dermatite Atópica/induzido quimicamente , Dermatite Atópica/imunologia , Dermatite Atópica/patologia , Modelos Animais de Doenças , Epiderme/imunologia , Epiderme/patologia , Feminino , Humanos , Receptores X do Fígado , Camundongos , Camundongos Pelados , Oxazolona/administração & dosagem , Receptores Ativados por Proliferador de Peroxissomo/agonistas , Células Th2/imunologia , Resultado do TratamentoRESUMO
Advanced cancers acquire resistance to chemotherapy, and this results in treatment failure. The cellular mechanisms of chemotherapy resistance are not well understood. Here, for the first time, we show that ceramide contributes to cellular resistance to doxorubicin through up-regulating the gene expression of glucosylceramide synthase (GCS). Ceramide, a cellular lipid messenger, modulates doxorubicin-induced cell death. GCS catalyzes ceramide glycosylation, converting ceramide to glucosylceramide; this process hastens ceramide clearance and limits ceramide-induced apoptosis. In the present study, we evaluated the role of the GCS gene in doxorubicin resistance using several paired wild-type and drug-resistant (doxorubicin-selected) cancer cell lines, including breast, ovary, cervical, and colon. GCS was overexpressed in all drug-resistant counterparts, and suppressing GCS overexpression using antisense oligonucleotide restored doxorubicin sensitivity. Characterizing the effect mechanism showed that doxorubicin exposure increased ceramide levels, enhanced GCS expression, and imparted cellular resistance. Exogenous C(6)-ceramide and sphingomyelinase treatments mimicked the influence of doxorubicin on GCS, activating the GCS promoter and up-regulating GCS gene expression. Fumonisin B(1), an inhibitor of ceramide synthesis, significantly suppressed doxorubicin-up-regulated GCS expression. Promoter truncation, point mutation, gel-shift, and protein-DNA ELISA analysis showed that transcription factor Sp1 was essential for ceramide-induced GCS up-regulation. These data indicate that ceramide-governed GCS gene expression drives cellular resistance to doxorubicin.
Assuntos
Ceramidas/fisiologia , Doxorrubicina/toxicidade , Resistencia a Medicamentos Antineoplásicos/fisiologia , Glucosiltransferases/genética , Adenocarcinoma , Neoplasias da Mama , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Resistência a Múltiplos Medicamentos , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Oligonucleotídeos Antissenso/farmacologiaRESUMO
The prevention of water loss through the skin is critical for terrestrial mammalian species. This function is served by the epidermal permeability barrier, which resides primarily in the extracellular domains of the stratum corneum, the outermost layer of skin, and its highly ordered lamellar membranes composed primarily of free fatty acids, cholesterol, and ceramides (Cer). The dominant lipids in these lamellae are Cer, which comprise a heterogeneous group of chemically distinct species. One particular subfamily of Cer, which is unique to the outer layers of the epidermis of terrestrial mammals, is omega (omega)-O-acylCer (or acylCer). Myriad evidence suggests that these acylCer play critical roles in barrier function. The formation of these epidermal acylCer requires several metabolic steps, including synthesis of very long chain fatty acids, omega-hydroxylation of the fatty acids, and esterification at the omega-hydroxy group with primarily linoleic acid. The authors previously demonstrated that a cytochrome P-450-type enzyme is involved in omega-hydroxylation during acylCer generation and that inhibition of omega-hydroxylation leads to a barrier abnormality in murine epidermis. More recently, we discovered that lack of normal elongation of very long chain fatty acid (or ELOVL) 4 function in mutant ELOVL4 knock-in mice causes acylCer deficiency associated with abnormal barrier formation and neonatal lethality. These results indicate not only that acylCer are critical lipid components for mammalian survival, but also that keratinocytes deploy a complex metabolic pathway leading to the formation of these unique Cer.
Assuntos
Ceramidas/metabolismo , Pele/metabolismo , Água/metabolismo , Animais , Permeabilidade da Membrana Celular/fisiologia , Ceramidas/genética , Ésteres/metabolismo , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Humanos , Queratinócitos/citologia , Queratinócitos/metabolismo , Metabolismo dos Lipídeos/fisiologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Modelos Animais , Pele/citologiaRESUMO
BACKGROUND: Ceramides (Cer) in the stratum corneum are essential for epidermal permeability barrier function. Thus, topical Cer replacement therapy has been employed to improve barrier function in clinical situations associated with Cer deficiency, e.g., atopic dermatitis. Because of the disadvantages of both natural- and skin identical-Cer (central nervous system origins and cost, respectively), synthetic chemical mimics, or pseudoceramides (pseudo-Cer), have been utilized as Cer substitutes. Whereas increased levels of intracellular Cer trigger cell growth inhibition and apoptosis, Cer levels are maintained by metabolic/catabolic pathways protecting cells from Cer-induced apoptosis. However, since the metabolic fates of each pseudo-Cer remain unknown, their widespread deployment in topical agents has raised concern about potential toxicities. OBJECTIVE: We compared the effects of two chemically unrelated commercially available pseudo-Cer to exogenous cell-permeant (C2)- or natural (C18)-Cer on cell growth and apoptosis thresholds in cultured human keratinocytes (CHK). METHODS: Cell growth and cell toxicity of CHK exposed to either C2-Cer or pseudo-Cer were assessed by MTT and lactate dehydrogenase release assays. Mitochondrial membrane potential, an indicator of apoptosis, was measured using membrane permeabilized semi-intact keratinocytes exposed C2-Cer, natural-Cer or pseudo-Cer. RESULTS: While the cell-permeant-Cer inhibits keratinocyte growth and increases cell toxicity, neither of the pseudo-Cer showed these effects. Decreased mitochondrial membrane potential occurred in CHK incubated with cell-permeant- and natural-Cer, but not pseudo-Cer. CONCLUSIONS: Taken together with preclinical safety studies of these pseudo-Cer and their widespread use over the counter without evidence of toxicity, these studies provide further assurance about the safety of these pseudo-Cer for topical use.
Assuntos
Proliferação de Células/efeitos dos fármacos , Ceramidas/toxicidade , Queratinócitos/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Administração Tópica , Morte Celular/efeitos dos fármacos , Células Cultivadas , Ceramidas/administração & dosagem , HumanosRESUMO
Glucocorticoid (GC) excess drives multiple cutaneous adverse effects, including skin thinning and poor wound healing. The ubiquitously expressed enzyme 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) activates mouse corticosterone from 11-dehydrocorticosterone (and human cortisol from cortisone). We previously demonstrated elevated 11ß-HSD1 activity during mouse wound healing, but the interplay between cutaneous 11ß-HSD1 and systemic GC excess is unexplored. Here, we examined effects of 11ß-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. 11ß-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 11ß-HSD1 inhibition. CBX did not regulate skin function or wound healing in the absence of CORT. Our findings demonstrate that 11ß-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.
Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 1/antagonistas & inibidores , Carbenoxolona/uso terapêutico , Corticosterona/intoxicação , Toxidermias/tratamento farmacológico , Inibidores Enzimáticos/uso terapêutico , Glucocorticoides/intoxicação , Pele/efeitos dos fármacos , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/metabolismo , Administração Tópica , Animais , Anti-Inflamatórios/intoxicação , Carbenoxolona/administração & dosagem , Carbenoxolona/efeitos adversos , Corticosterona/sangue , Corticosterona/farmacocinética , Citocinas/antagonistas & inibidores , Citocinas/genética , Citocinas/metabolismo , Toxidermias/etiologia , Toxidermias/metabolismo , Toxidermias/patologia , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/efeitos adversos , Epiderme/efeitos dos fármacos , Epiderme/imunologia , Epiderme/metabolismo , Epiderme/patologia , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/imunologia , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Glucocorticoides/sangue , Glucocorticoides/farmacocinética , Tecido de Granulação/efeitos dos fármacos , Tecido de Granulação/imunologia , Tecido de Granulação/metabolismo , Tecido de Granulação/patologia , Camundongos Pelados , Tamanho do Órgão/efeitos dos fármacos , Pele/lesões , Pele/metabolismo , Pele/patologia , Cicatrização/efeitos dos fármacosRESUMO
Hereditary sensory neuropathy type 1 (HSN1) is a dominantly inherited degenerative disorder of the peripheral nerves. HSN1 is clinically and genetically heterogeneous. One form arises from mutations in the gene SPTLC1 encoding long-chain base 1 (LCB1), one of two subunits of serine palmitoyltransferase (SPT), the enzyme catalyzing the initial step of sphingolipid synthesis. We have examined the effects of the mutations C133Y and C133W, which we have identified in two HSN1 families, on the function of SPT. Although in HSN1 lymphoblasts, the C133Y and C133W mutations do not alter the steady-state levels of LCB1 and LCB2 subunits, they result in reduced SPT activity and sphingolipid synthesis. Moreover, in a mutant Chinese hamster ovary (CHO) cell strain with defective SPT activity due to a lack of the LCB1 subunit, these mutations impair the ability of the LCB1 subunit to complement the SPT deficiency. Furthermore, the overproduction of either the LCB1C133Y or LCB1C133W subunit inhibits SPT activity in CHO cells despite the presence of wild-type LCB1. In addition, we demonstrate that in CHO cells the mutant LCB1 proteins, similar to the normal LCB1, can interact with the wild-type LCB2 subunit. These results indicate that the HSN1-associated mutations in LCB1 confer dominant negative effects on the SPT enzyme.
Assuntos
Aciltransferases/metabolismo , Neuropatias Hereditárias Sensoriais e Autônomas/genética , Mutação , Esfingolipídeos/biossíntese , Aciltransferases/química , Adulto , Animais , Células CHO , Linhagem Celular , Cricetinae , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Subunidades Proteicas , Serina C-PalmitoiltransferaseRESUMO
Previously, we demonstrated that topical applications of peroxisome proliferator-activated receptors (PPARs) and liver X receptor (LXR) activators improve permeability barrier homeostasis. We showed further that stimulation of epidermal differentiation provides one mechanism that could account for such improvement. Here, we studied the effects of these agents on the lipid matrix of the stratum corneum. Hairless mice were treated topically with activators of PPARalpha (WY14643), PPARdelta (GW1514), PPARgamma (ciglitazone), and LXR (22(R)-cholesterol or TO901317) or vehicle twice daily for 3 days. All activators significantly increased epidermal cholesterol, fatty acid, and sphingolipid synthesis, including the production of barrier-specific ceramide species. In addition, lamellar body (LB) formation, secretion, and post-secretory processing accelerated significantly following acute barrier disruption in PPAR/LXR-activator-treated animals. Finally, the activity of epidermal beta-glucocerebrosidase, a key lipid-processing enzyme, increased in PPAR/LXR-activator-treated animals. Thus, topical PPAR and LXR activators stimulate epidermal lipid synthesis, increase LB secretion, and accelerate extracellular lipid processing, providing additional mechanisms that further account for their ability to improve epidermal permeability barrier homeostasis. Since the liposensors are activated by endogenous lipid metabolites, they may serve as unique regulators of barrier homeostasis.
Assuntos
Colesterol/farmacologia , Proteínas de Ligação a DNA/agonistas , Epiderme/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Receptores Ativados por Proliferador de Peroxissomo/agonistas , Proliferadores de Peroxissomos/farmacologia , Receptores Citoplasmáticos e Nucleares/agonistas , Animais , Células Epidérmicas , Epiderme/metabolismo , Homeostase , Receptores X do Fígado , Masculino , Camundongos , Camundongos Pelados , Receptores Nucleares Órfãos , PermeabilidadeRESUMO
Mammalian epidermis produces and delivers large quantities of glucosylceramide and sphingomyelin precursors to stratum corneum extracellular domains, where they are hydrolyzed to corresponding ceramide species. This cycle of lipid precursor formation and subsequent hydrolysis represents a mechanism that protects the epidermis against potentially harmful effects of ceramide accumulation within nucleated cell layers. Prominent skin disorders, such as psoriasis and atopic dermatitis, have diminished epidermal ceramide levels, reflecting altered sphingolipid metabolism, that may contribute to disease severity/progression. Enzymatic processes in the hydrolysis of glucosylceramide and sphingomyelin, and the roles of sphingolipids in skin diseases, are the focus of this review.
Assuntos
Epiderme/metabolismo , Dermatopatias/metabolismo , Esfingolipídeos/metabolismo , Animais , Transporte Biológico , Glicosilação , Humanos , Esfingolipídeos/química , Esfingomielinas/metabolismoRESUMO
Treatment with doxorubicin (DOX) induced apoptosis with an increase of ceramide content in drug-sensitive HL-60 cells, but not in drug-resistant HL-60/ADR cells. In HL-60/ADR cells (but not in HL-60 cells), the levels of mRNA, protein, and activity in glucosylceramide synthase (GCS), which converts ceramide to glucosylceramide, were up-regulated in response to DOX. Thus, abrogation of apoptosis in HL-60/ADR cells might be involved in ceramide reduction through DOX-induced up-regulation of GCS function. Because we reported that a GC-rich/Sp1 promoter binding region was of importance in the regulation of GCS expression, the role of Sp1 in DOX-induced up-regulation of GCS and apoptosis was investigated. DOX induced Sp1 activation in HL-60/ADR cells, as assessed by Sp1 gel shift and promoter-luciferase reporter assays, whereas transfection of double-stranded oligodeoxynucleotides (ODNs) containing a GC-rich/Sp1 region (Sp1 decoy ODNs) inhibited DOX-induced Sp1 activation. In addition, DOX-increased mRNA and enzyme activity in GCS were inhibited by Sp1 decoy, in conjunction with corresponding elevations of ceramide content. Moreover, DOX-induced apoptotic cell death was significantly increased in Sp1 decoy ODN-transfected HL-60/ADR cells over mock-transfected HL-60/ADR cells. Together, the results suggest that transcriptional up-regulation of GCS through DOX-induced activation of Sp1 is one potential mechanism to regulate ceramide increase and apoptosis in HL-60/ADR cells.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Ceramidas/metabolismo , Doxorrubicina/farmacologia , Glucosiltransferases/biossíntese , Fator de Transcrição Sp1/metabolismo , Apoptose/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Ceramidas/biossíntese , Resistencia a Medicamentos Antineoplásicos , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Glucosiltransferases/antagonistas & inibidores , Glucosiltransferases/genética , Células HL-60 , Humanos , Oligonucleotídeos/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Fator de Transcrição Sp1/genética , Ativação Transcricional , Regulação para CimaRESUMO
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.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Ictiose Lamelar/genética , Ictiose Lamelar/patologia , Fenótipo , Pele/metabolismo , Pele/patologia , Alelos , Animais , Sequência de Bases , Ceramidas/metabolismo , Mapeamento Cromossômico , Desmossomos/metabolismo , Modelos Animais de Doenças , Epiderme/metabolismo , Epiderme/patologia , Epiderme/ultraestrutura , Éxons , Genes Recessivos , Glucosilceramidas/metabolismo , Ictiose Lamelar/terapia , Calicreínas/metabolismo , Queratinócitos/metabolismo , Camundongos , Modelos Biológicos , Mutação , Permeabilidade , Análise de Sequência de DNA , Pele/ultraestrutura , Transplante de PeleRESUMO
Ultraviolet irradiation is a major environmental cause of skin cancers, whereas ultraviolet-induced DNA repair and apoptosis are defense mechanisms that rescue and/or protect keratinocytes from this risk. Multiple pathways are involved in ultraviolet-induced keratinocyte apoptosis, including activation of p38-mitogen-activated protein kinase, protein kinase C, and CD95, each of which are associated with caspase activation. Alternatively, ceramides could serve as ultraviolet-induced, second messenger lipids, because they induce cell cycle arrest and apoptosis in a variety of cell types, including keratinocytes. We investigated the role of ceramide versus caspase, and the responsible pathway for ceramide generation in ultraviolet B-induced apoptosis of cultured normal human keratinocytes maintained in low calcium (0.07 mm) medium. Ultraviolet B (40 mJ per cm2) significantly inhibited cultured normal human keratinocyte proliferation, assessed as [3H-methyl]thymidine-thymidine incorporation into DNA, 2 h after irradiation. Terminal nick deoxynucleotide end-labeling-positive apoptotic cells (14.8% at 24 h and 34.4% at 48 h) and trypan blue-positive apoptotic cells (8.4% at 24 h and 28.6% at 48 h) became evident in a time-dependent manner after ultraviolet B irradiation, in parallel with activation of caspase-3. The ceramide content of irradiated cultured normal human keratinocytes increased significantly by 8 h, whereas glucosylceramide only modestly increased, and sphingomyelin content remained unaltered. Metabolic studies with radiolabeled serine, palmitic acid, and phosphorylcholine revealed that the ultraviolet B-induced increase in ceramide results primarily from increased de novo synthesis rather than accelerated sphingomyelin hydrolysis. Increased ceramide synthesis, in turn, could be attributed to increased activity of ceramide synthase (i.e., 1.7-fold increase 8 h after ultraviolet B irradiation), whereas serine palmitoyltransferase activity did not change. Both fumonisin B1, an inhibitor of ceramide synthase, and ISP-1, myriocin an inhibitor of serine palmitoyltransferase, significantly attenuated the ultraviolet B-induced apoptosis in a caspase-3-independent fashion, whereas co-incubation with a caspase-3 inhibitor (Ac-DEVD-chloromethyl-ketone) further attenuated the ultraviolet B-induced apoptosis. Thus, increased de novo ceramide synthesis signals ultraviolet B-induced apoptosis, by a pathway independent of, but in concert with, caspase-3 activation.
Assuntos
Apoptose/fisiologia , Ceramidas/biossíntese , Queratinócitos/citologia , Queratinócitos/efeitos da radiação , Apoptose/efeitos da radiação , Caspase 3 , Caspases/metabolismo , Diferenciação Celular , Células Cultivadas , DNA/biossíntese , Inibidores Enzimáticos/farmacologia , Fumonisinas/farmacologia , Humanos , Queratinócitos/metabolismo , Oxirredutases/metabolismo , Raios UltravioletaRESUMO
We previously demonstrated that the epidermal-specific glycosylphosphatidylinositol (GPI)-anchor-deficient mice, generated by Pig-a gene disruption (Pig-a null mice), exhibited wrinkled and dry skin with hyperkeratosis and abnormal differentiation, and they died within a few days after birth. Here, we investigated the basis for the early demise of these animals, and the potential role of epidermal structural and biochemical abnormalities. The rapid demise of these animals was associated with both diminished epidermal permeability barrier function and decreased stratum corneum (SC) water content. The barrier abnormality could be attributed abnormal internal contents of lamellar bodies, with a downstream failure to generate normal extracellular lamellar bilayers in the SC. Moreover, processing profilaggrin to its monomeric form was impaired in Pig-a null mouse epidermis, while levels of the differentiation-specific proteins, involucrin, loricrin and profilaggrin were normal. Failure of filaggrin processing was accompanied by decreased activity of protein phosphatase 2A, an enzyme involved in profilaggrin to filaggrin processing. Thus, these studies demonstrate a critical role for GPI anchor and GPI-anchored proteins in divergent arms of epidermal terminal differentiation. While the permeability barrier abnormality can be attributed to defects in the lamellar body secretory system, the hydration abnormality is, in part, due to lack of availability of filaggrin-derived proteolytic products. Finally, since the dual abnormalities in the lamellar body secretory system and filaggrin processing resemble two key features of human Harlequin ichthyosis, Pig-a null mice could provide an appropriate analog for further studies of this disease.