RESUMEN
Prolonged inflammation and impaired re-epithelization are major contributing factors to chronic non-healing diabetic wounds; diabetes is also characterized by xerosis. Advanced glycation end products (AGEs), and the activation of toll-like receptors (TLRs), can trigger inflammatory responses. Aquaporin-3 (AQP3) plays essential roles in keratinocyte function and skin wound re-epithelialization/re-generation and hydration. Suberanilohydroxamic acid (SAHA), a histone deacetylase inhibitor, mimics the increased acetylation observed in diabetes. We investigated the effects of TLR2/TLR4 activators and AGEs on keratinocyte AQP3 expression in the presence and absence of SAHA. Primary mouse keratinocytes were treated with or without TLR2 agonist Pam3Cys-Ser-(Lys)4 (PAM), TLR4 agonist lipopolysaccharide (LPS), or AGEs, with or without SAHA. We found that (1) PAM and LPS significantly upregulated AQP3 protein basally (without SAHA) and PAM downregulated AQP3 protein with SAHA; and (2) AGEs (100 µg/mL) increased AQP3 protein expression basally and decreased AQP3 levels with SAHA. PAM and AGEs produced similar changes in AQP3 expression, suggesting a common pathway or potential crosstalk between TLR2 and AGEs signaling. Our findings suggest that TLR2 activation and AGEs may be beneficial for wound healing and skin hydration under normal conditions via AQP3 upregulation, but that these pathways are likely deleterious in diabetes chronically through decreased AQP3 expression.
Asunto(s)
Acuaporina 3 , Receptor Toll-Like 2 , Ratones , Animales , Acuaporina 3/genética , Acuaporina 3/metabolismo , Receptor Toll-Like 2/genética , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 4/metabolismo , Lipopolisacáridos/farmacología , Lipopolisacáridos/metabolismo , Queratinocitos/metabolismo , Vorinostat/metabolismo , Productos Finales de Glicación Avanzada/farmacología , Productos Finales de Glicación Avanzada/metabolismoRESUMEN
Our previous work shows that dioleoylphosphatidylglycerol (DOPG) accelerates corneal epithelial healing in vitro and in vivo by unknown mechanisms. Prior data demonstrate that DOPG inhibits toll-like receptor (TLR) activation and inflammation induced by microbial components (pathogen-associated molecular patterns, PAMPs) and by endogenous molecules upregulated in psoriatic skin, which act as danger-associated molecular patterns (DAMPs) to activate TLRs and promote inflammation. In the injured cornea, sterile inflammation can result from the release of the DAMP molecule, heat shock protein B4 (HSPB4), to contribute to delayed wound healing. Here, we show in vitro that DOPG inhibits TLR2 activation induced in response to HSPB4, as well as DAMPs that are elevated in diabetes, a disease that also slows corneal wound healing. Further, we show that the co-receptor, cluster of differentiation-14 (CD14), is necessary for PAMP/DAMP-induced activation of TLR2, as well as of TLR4. Finally, we simulated the high-glucose environment of diabetes to show that elevated glucose levels enhance TLR4 activation by a DAMP known to be upregulated in diabetes. Together, our results demonstrate the anti-inflammatory actions of DOPG and support further investigation into its development as a possible therapy for corneal injury, especially in diabetic patients at high risk of vision-threatening complications.
Asunto(s)
Proteína HMGB1 , Receptor Toll-Like 2 , Humanos , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Alarminas , Antígenos CD19 , Glucosa , Proteínas de Choque Térmico/metabolismo , Proteína HMGB1/metabolismo , Inflamación/metabolismo , Receptor Toll-Like 2/genética , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 4/metabolismo , Fosfatidilgliceroles/farmacologíaRESUMEN
Glycerol is used in many skin care products because it improves skin function. Anecdotal reports by patients on the National Psoriasis Foundation website also suggest that glycerol may be helpful for the treatment of psoriasis, although to date no experimental data confirm this idea. Glycerol entry into epidermal keratinocytes is facilitated by aquaglyceroporins like aquaporin-3 (AQP3), and its conversion to phosphatidylglycerol, a lipid messenger that promotes keratinocyte differentiation, requires the lipid-metabolizing enzyme phospholipase-D2 (PLD2). To evaluate whether glycerol inhibits inflammation and psoriasiform lesion development in the imiquimod (IMQ)-induced mouse model of psoriasis, glycerol's effect on psoriasiform skin lesions was determined in IMQ-treated wild-type and PLD2 knockout mice, with glycerol provided either in drinking water or applied topically. Psoriasis area and severity index, ear thickness and ear biopsy weight, epidermal thickness, and inflammatory markers were quantified. Topical and oral glycerol ameliorated psoriasiform lesion development in wild-type mice. Topical glycerol appeared to act as an emollient to induce beneficial effects, since even in PLD2 knockout mice topical glycerol application improved skin lesions. In contrast, the beneficial effects of oral glycerol required PLD2, with no improvement in psoriasiform lesions observed in PLD2 knockout mice. Our findings suggest that the ability of oral glycerol to improve psoriasiform lesions requires its PLD2-mediated conversion to phosphatidylglycerol, consistent with our previous report that phosphatidylglycerol itself improves psoriasiform lesions in this model. Our data also support anecdotal evidence that glycerol can ameliorate psoriasis symptoms and therefore might be a useful therapy alone or in conjunction with other treatments.
Asunto(s)
Glicerol/farmacología , Imiquimod/efectos adversos , Psoriasis/tratamiento farmacológico , Piel/metabolismo , Animales , Acuaporina 3/genética , Acuaporina 3/metabolismo , Modelos Animales de Enfermedad , Humanos , Imiquimod/farmacología , Ratones , Ratones Noqueados , Fosfolipasa D/deficiencia , Fosfolipasa D/metabolismo , Psoriasis/inducido químicamente , Psoriasis/genética , Psoriasis/metabolismoRESUMEN
Skin serves not only as a protective barrier to microbial entry into the body but also as an immune organ. The outer layer, the epidermis, is composed predominantly of keratinocytes, which can be stimulated to produce proinflammatory mediators. Although some inflammation is useful to defend against infection, excessive or persistent inflammation can lead to the development of inflammatory skin diseases, such as psoriasis, a common skin disorder affecting approximately 2% of the US population. We have previously found that phosphatidylglycerol (PG) derived from soy can inhibit inflammation in a contact irritant ear edema mouse model. Here, we investigated the ability of soy PG to inhibit inflammatory mediator expression in response to activators of the pattern recognition receptors, toll-like receptor-2 (TLR2) and -4 (TLR4). We found that in epidermal keratinocytes, soy PG inhibited TLR2 and TLR4 activation and inflammatory mediator expression in response to a synthetic triacylated lipopeptide and lipopolysaccharide, respectively, as well as an endogenous danger-associated molecular pattern. However, at higher concentrations, soy PG alone enhanced the expression of some proinflammatory cytokines, suggesting a narrow therapeutic window for this lipid. Dioleoylphosphatidylglycerol (DOPG), but not dioleoylphosphatidylcholine, exerted a similar inhibitory effect, completely blocking keratinocyte inflammatory mediator expression induced by TLR2 and TLR4 activators as well as NFκB activation in a macrophage cell line (RAW264.7); however, DOPG was not itself proinflammatory even at high concentrations. Furthermore, DOPG had no effect on NFκB activation in response to a TLR7/8 agonist. Our results suggest that DOPG could be used to inhibit excessive skin inflammation. SIGNIFICANCE STATEMENT: Although inflammation is beneficial for clearing an infection, in some cases, the infection can be excessive and/or become chronic, thereby resulting in considerable tissue damage and pathological conditions. We show here that the phospholipid phosphatidylglycerol can inhibit the activation of toll-like receptors 2 and 4 of the innate immune system as well as the downstream inflammatory mediator expression in response to microbial component-mimicking agents in epidermal keratinocytes that form the physical barrier of the skin.
Asunto(s)
Mediadores de Inflamación/metabolismo , Queratinocitos/metabolismo , Moléculas de Patrón Molecular Asociado a Patógenos/farmacología , Fosfatidilgliceroles/farmacología , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 4/metabolismo , Animales , Calgranulina B/farmacología , Humanos , Imidazoles/farmacología , Lipopéptidos/farmacología , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , FN-kappa B/metabolismo , Células RAW 264.7 , Receptores de Reconocimiento de Patrones/metabolismo , Proteínas Recombinantes/farmacología , Glycine max/químicaRESUMEN
The water and glycerol channel, aquaporin-3 (AQP3), plays an important role in the skin epidermis, with effects on hydration, permeability barrier repair and wound healing; therefore, information about the mechanisms regulating its expression is important for a complete understanding of skin function physiologically and in disease conditions. We previously demonstrated that histone deacetylase inhibitors (HDACi) induce the mRNA and protein expression of AQP3, in part through the p53 family, transcription factors for which acetylation is known to affect their regulatory activity. Another set of transcription factors previously shown to induce AQP3 expression and/or regulate skin function are the peroxisome proliferator-activated receptors (PPARs). Since there are reports that PPARs are also acetylated, we examined the involvement of these nuclear hormone receptors in HDACi-induced AQP3 expression. We first verified that a PPARγ agonist upregulated AQP3 mRNA and protein levels and that this increase was blocked by a PPARγ antagonist. We then showed that the PPARγ antagonist also inhibited AQP3 expression induced both by a broad-spectrum HDACi and an HDAC3-selective inhibitor. Interestingly, a PPARα antagonist also inhibited HDACi-induced AQP3 expression. These antagonist effects were observed in both primary mouse and normal human keratinocytes. Furthermore, PPARγ overexpression enhanced HDACi-stimulated AQP3 mRNA levels. Thus, our results suggest that PPARγ and/or PPARα may play a role in regulating AQP3 levels in the skin; based on the ability of PPAR agonists to promote epidermal differentiation and/or inhibit proliferation, topical PPAR agonists might be considered as a therapy for hyperproliferative skin disorders, such as psoriasis.
Asunto(s)
Acuaporina 3/biosíntesis , Inhibidores de Histona Desacetilasas/farmacología , Queratinocitos/citología , PPAR alfa/metabolismo , Adenoviridae/metabolismo , Animales , Animales Recién Nacidos , Diferenciación Celular , Proliferación Celular , Sistemas de Liberación de Medicamentos , Células Epidérmicas/metabolismo , Epidermis/metabolismo , Humanos , Queratinocitos/metabolismo , Ratones , PPAR alfa/antagonistas & inhibidores , Permeabilidad , Fenotipo , Piel/metabolismo , Enfermedades de la Piel/metabolismoRESUMEN
Oxidative stress contributes to inflammatory skin diseases, including psoriasis. Monomethylfumarate (MMF) is an antipsoriatic agent with a poorly understood mechanism of action. In other cell types MMF increases the expression of nuclear factor erythroid-derived 2-like 2 (Nrf2), a transcription factor that regulates cellular antioxidant responses, to reduce oxidative stress like that observed in inflammatory disorders such as multiple sclerosis. We tested the hypothesis that MMF enhances Nrf2 activity in keratinocytes, thereby improving their capacity to counteract environmental stresses. We used Western analysis, immunofluorescence, and real-time quantitative reverse-transcription polymerase chain reaction to examine the effect of MMF on the expression of Nrf2 and its targets. We also measured intracellular reactive oxygen species (ROS) levels following MMF treatment. Our data show that MMF increased total and nuclear Nrf2 levels in primary mouse keratinocytes and enhanced mRNA expression of several Nrf2-downstream effectors, including heme oxygenase-1 and peroxiredoxin-6. Moreover, MMF treatment attenuated the generation of ROS following hydrogen peroxide treatment. On the other hand, the expression and membranous localization of aquaporin-3 (AQP3), a glycerol channel implicated in keratinocyte differentiation, was stimulated by MMF, which also enhanced keratinocyte glycerol uptake. The Nrf2 activator sulforaphane also increased AQP3 levels, suggesting that AQP3 expression may be regulated by Nrf2. We show for the first time that MMF stimulates Nrf2 and AQP3 expression and function/activity in keratinocytes. This effect may account, in part, for the previously observed ability of MMF to inhibit proliferation and inflammatory mediator production and promote differentiation in keratinocytes and to treat psoriasis.
Asunto(s)
Acuaporina 3/biosíntesis , Fumaratos/farmacología , Maleatos/farmacología , Factor 2 Relacionado con NF-E2/biosíntesis , Psoriasis , ARN Mensajero/biosíntesis , Animales , Animales Recién Nacidos , Acuaporina 3/agonistas , Acuaporina 3/genética , Secuencia de Bases , Células Cultivadas , Expresión Génica , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Ratones , Factor 2 Relacionado con NF-E2/agonistas , ARN Mensajero/agonistas , ARN Mensajero/genéticaRESUMEN
The skin is the largest organ of the body, serving as an important barrier between the internal milieu and the external environment. The skin is also one of the first lines of defense against microbial infection and other hazards, and thus, the skin has important immune functions . This organ is composed of many cell types, including immune-active dendritic cells (epidermal Langerhans cells and dermal dendritic cells), connective tissue-generating dermal fibroblasts and pigment-producing melanocytes. Comprising the outer skin layer are the epidermal keratinocytes, the predominant cell of this layer, the epidermis , which provides both a mechanical barrier and a water -permeability barrier. Recent data suggest that aquaporins, a family of barrel-shaped proteins surrounding internal pores that allow the passage of water and, in some family members, small solutes such as glycerol , play critical roles in regulating various skin parameters. The involvement of different aquaporin family members in skin function is discussed.
Asunto(s)
Acuaporina 1/metabolismo , Dermatitis por Contacto/metabolismo , Queratinocitos/metabolismo , Psoriasis/metabolismo , Piel/metabolismo , Agua/metabolismo , Animales , Acuaporina 1/genética , Transporte Biológico , Dermatitis por Contacto/genética , Dermatitis por Contacto/patología , Fibroblastos/citología , Fibroblastos/metabolismo , Regulación de la Expresión Génica , Glicerol/metabolismo , Humanos , Queratinocitos/citología , Células de Langerhans/citología , Células de Langerhans/metabolismo , Melanocitos/citología , Melanocitos/metabolismo , Permeabilidad , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Psoriasis/genética , Psoriasis/patología , Piel/citologíaRESUMEN
Indoleamine-2,3-dioxygenase (IDO) degrades the essential amino acid tryptophan resulting in tryptophan depletion and the accumulation of catabolites such as kynurenine. The expression/activity of IDO in various cells, including macrophages and dendritic cells, results in an inhibition of T-cell responses in a number of situations, such as toward allogeneic fetuses and tissue grafts. Psoriasis is an immune-mediated skin disease involving T cells; kynureninase and its generation of catabolites downstream of IDO are reported to play an important role in this disease. We hypothesized that mice lacking the IDO1 gene would exhibit a hyperactive immune response and an exacerbation of skin lesions in the imiquimod-induced mouse model of psoriasis. Littermate wild-type and IDO1-knockout mice were treated with imiquimod for 5 days, and the severity of psoriasiform skin lesions assessed using the psoriasis area and severity index (PASI), ear edema measured using a digital caliper, and thickness of the epidermis determined by histology. Expression of pro-inflammatory mediators and tryptophan-metabolizing enzymes was monitored using quantitative RT-PCR. Imiquimod increased ear edema, PASI scores, and epidermal thickness in both WT and IDO1 knockout mice; however, there were no differences observed between the 2 genotypes. There were also no differences in imiquimod's induction of skin inflammatory mediators, indicating no effect of IDO1 gene loss in this psoriasis model. Although these data suggest a lack of involvement of IDO1 in psoriatic skin inflammation, other possible mechanisms, such as compensatory changes in other pathways and the involvement of the IDO2 isoform, must also be considered.
RESUMEN
Huntingtin interacting protein 1 related (Hip1r) is an F-actin- and clathrin-binding protein involved in vesicular trafficking. In this study, we demonstrate that Hip1r is abundantly expressed in the gastric parietal cell, predominantly localizing with F-actin to canalicular membranes. Hip1r may provide a critical function in vivo, as demonstrated by extensive changes to parietal cells and the gastric epithelium in Hip1r-deficient mice. Electron microscopy revealed abnormal apical canalicular membranes and loss of tubulovesicles in mutant parietal cells, suggesting that Hip1r is necessary for the normal trafficking of these secretory membranes. Accordingly, acid secretory dynamics were altered in mutant parietal cells, with enhanced activation and acid trapping, as measured in isolated gastric glands. At the whole-organ level, gastric acidity was reduced in Hip1r-deficient mice, and the gastric mucosa was grossly transformed, with fewer parietal cells due to enhanced apoptotic cell death and glandular hypertrophy associated with cellular transformation. Hip1r-deficient mice had increased expression of the gastric growth factor gastrin, and mice mutant for both gastrin and Hip1r exhibited normalization of both proliferation and gland height. Taken together, these studies demonstrate that Hip1r plays a significant role in gastric physiology, mucosal architecture, and secretory membrane dynamics in parietal cells.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Células Parietales Gástricas/fisiología , Vesículas Secretoras/fisiología , Proteínas Adaptadoras Transductoras de Señales , Animales , Apoptosis/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Principales Gástricas/metabolismo , Células Principales Gástricas/patología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Ácido Gástrico/metabolismo , Determinación de la Acidez Gástrica , Mucosa Gástrica/metabolismo , Mucosa Gástrica/patología , Gastrinas/sangre , Gastrinas/genética , Expresión Génica/efectos de los fármacos , ATPasa Intercambiadora de Hidrógeno-Potásio/genética , ATPasa Intercambiadora de Hidrógeno-Potásio/metabolismo , Histamina/farmacología , Factor Intrinseco/genética , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Ratones Noqueados , Proteínas de Microfilamentos , Microscopía Electrónica , Proteína Relacionada con la Hormona Paratiroidea/metabolismo , Células Parietales Gástricas/efectos de los fármacos , Conejos , Ranitidina/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Vesículas Secretoras/ultraestructuraRESUMEN
Lasp1 is an actin-binding, signaling pathway-regulated phosphoprotein that is overexpressed in several cancers. siRNA knockdown in cell lines retards cell migration, suggesting the possibility that Lasp1 upregulation influences cancer metastasis. Herein, we utilized a recently developed gene knockout model to assess the role of Lasp1 in modulating nontransformed cell functions. Wound healing and tumor initiation progressed more rapidly in Lasp1(-/-) mice compared with Lasp1(+/+) controls. Embryonic fibroblasts (MEFs) derived from Lasp1(-/-) mice also migrated more rapidly in vitro. These MEFs characteristically possessed increased focal adhesion numbers and displayed more rapid attachment compared with wild-type MEFs. Differential microarray analyses revealed alterations in message expression for proteins implicated in cell migration, adhesion, and cytoskeletal organization. Notably, the focal adhesion protein, lipoma preferred partner (LPP), a zyxin family member and putative Lasp1 binding protein, was increased about twofold. Because LPP gene disruption reduces cell migration, we hypothesize that LPP plays a role in enhancing the migratory capacity of Lasp1(-/-) MEFs, perhaps by modifying the subcellular localization of other motility-associated proteins. The striking contrast in the functional effects of loss of Lasp1 in innate cells compared with cell lines reveals distinct differences in mechanisms of motility and attachment in these models.
Asunto(s)
Movimiento Celular/fisiología , Proteínas de Homeodominio/fisiología , Proteínas de Neoplasias/fisiología , Neoplasias Experimentales/patología , 9,10-Dimetil-1,2-benzantraceno , Animales , Western Blotting , Adhesión Celular , Proliferación Celular , Células Cultivadas , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Adhesiones Focales/metabolismo , Perfilación de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Proteínas con Dominio LIM , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias Experimentales/inducido químicamente , Neoplasias Experimentales/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Psoriasis is a common skin disorder characterized by hyperproliferation and aberrant differentiation of epidermal keratinocytes and inflammation. We previously showed that phosphatidylglycerol (PG) can regulate keratinocyte function and suppress skin inflammation. Based on data suggesting that PG can inhibit toll-like receptor (TLR) activation induced by microorganisms and their components, we determined whether PG can inhibit TLR activation in response to antimicrobial peptides. These peptides, which are up-regulated in psoriasis, are known to function as danger-associated molecular patterns (i.e., DAMPs) to activate TLRs and the innate immune system. Because S100A9 is elevated in psoriatic skin and in animal models of psoriasis, we selected S100A9 as a representative antimicrobial peptide DAMP. We showed that in primary keratinocytes and a macrophage cell line, PG suppressed inflammatory mediator production induced by recombinant S100A9 functioning through both TLR2 and TLR4. In addition, PG, but not phosphatidylcholine, inhibited downstream S100A9-elicited TLR2 and NF-κB activation. These results, to our knowledge previously unreported, show PG's ability to inhibit DAMP-induced TLR activation, thereby reducing inflammatory signals. In addition, topical PG ameliorated skin lesions and inflammation in a mouse model of psoriasis. Together, these results suggest the possibility of developing PG as a therapy for psoriasis.
Asunto(s)
Alarminas/metabolismo , Regulación de la Expresión Génica , Fosfatidilgliceroles/farmacología , Psoriasis/genética , ARN/genética , Receptores Toll-Like/genética , Animales , Animales Recién Nacidos , Western Blotting , Calgranulina B/biosíntesis , Calgranulina B/efectos de los fármacos , Calgranulina B/genética , Células Cultivadas , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Inmunohistoquímica , Queratinocitos/metabolismo , Queratinocitos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Psoriasis/metabolismo , Psoriasis/patología , Transducción de Señal , Receptores Toll-Like/biosíntesisRESUMEN
Osteoporosis, low bone mass that increases fracture susceptibility, affects approximately 75 million individuals in the United States, Europe and Japan, with the number of osteoporotic fractures expected to increase by more than 3-fold over the next 50 years. Bone mass declines with age, although the mechanisms for this decrease are unclear. Aging enhances production of reactive oxygen species, which can affect bone formation and breakdown. The multiple sclerosis drug Tecfidera contains dimethylfumarate, which is rapidly metabolized to monomethylfumarate (MMF); MMF is thought to function through nuclear factor erythroid-derived-2-like-2 (Nrf2), a transcription factor activated by oxidative stress which induces the expression of endogenous anti-oxidant systems. We hypothesized that MMF-elicited increases in anti-oxidants would inhibit osteopenia induced by ovariectomy, as a model of aging-related osteoporosis and high oxidative stress. We demonstrated that MMF activated Nrf2 and induced anti-oxidant Nrf2 target gene expression in bone marrow-derived mesenchymal stem cells. Sham-operated or ovariectomized adult female mice were fed chow with or without MMF and various parameters monitored. Ovariectomy produced the expected effects, decreasing bone mineral density and increasing body weight, fat mass, bone marrow adiposity and serum receptor activator of nuclear factor-kappa-B ligand (RANKL) levels. MMF decreased fat but not lean mass. MMF improved trabecular bone microarchitecture after adjustment for body weight, although the unadjusted data showed few differences; MMF also tended to increase adjusted cortical bone and to reduce bone marrow adiposity and serum RANKL levels. Because these results suggest the possibility that MMF might be beneficial for bone, further investigation seems warranted.
RESUMEN
Aquaporin- (AQP) 3, a water and glycerol channel, plays an important role in epidermal function, with studies showing its involvement in keratinocyte proliferation, differentiation, and migration and in epidermal wound healing and barrier repair. Increasing speculation about the use of histone deacetylase (HDAC) inhibitors to treat skin diseases led us to investigate HDAC's role in the regulation of AQP3. The broad-spectrum HDAC inhibitor suberoylanilide hydroxamic acid induced AQP3 mRNA and protein expression in a dose- and time-dependent manner in normal keratinocytes. The SAHA-induced increase in AQP3 levels resulted in enhanced [3H]glycerol uptake in normal but not in AQP3-knockout keratinocytes, confirming that the expressed AQP3 was functional. Use of HDAC inhibitors with different specificities limited our exploration of the responsible HDAC member to HDAC1, HDAC2, or HDAC3. Cre-recombinase-mediated knockdown and overexpression of HDAC3 suggested a role for HDAC3 in suppressing AQP3 expression basally. Further investigation implicated p53 as a transcription factor involved in regulating HDAC inhibitor-induced AQP3 expression. Thus, our study supports the regulation of AQP3 expression by HDAC3 and p53. Because suberoylanilide hydroxamic acid is already approved to treat cutaneous T-cell lymphoma, it could potentially be used as a therapy for skin diseases like psoriasis, where AQP3 is abnormally expressed.
Asunto(s)
Acuaporina 3/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Glicerol/metabolismo , Histona Desacetilasas/farmacología , Queratinocitos/metabolismo , Animales , Animales Recién Nacidos , Acuaporina 3/metabolismo , Transporte Biológico/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Proliferación Celular/fisiología , Células Cultivadas , Epidermis/metabolismo , Humanos , Técnicas In Vitro , Queratinocitos/citología , Ratones , Ratones Noqueados , Sensibilidad y Especificidad , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Heterozygous endothelial nitric oxide synthase (eNOS) deficiency is associated with normal endothelium-dependent responses, however, little is known regarding the mechanisms that maintain or impair endothelial function with heterozygous eNOS deficiency. The goals of this study were to (1) determine mechanism(s) which serve to maintain normal endothelial function in the absence of a single eNOS gene; and (2) to determine whether heterozygous eNOS deficiency predisposes blood vessels to endothelial dysfunction in response to a high-fat diet (HFD). Responses of carotid arteries were examined in wild-type (eNOS(+/+)) and heterozygous eNOS-deficient (eNOS(+/-)) treated with either vehicle (saline), N(G)-nitro-L-arginine (L-NNA, 100 µmol/L), an inhibitor of nitric oxide synthase, or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 1 µmol/L), an inhibitor of soluble guanylyl cyclase (sGC), and in eNOS(+/+) and eNOS(+/-) mice fed a control (10%) or a 45% HFD (kcal from fat). Responses to acetylcholine (ACh) were similar in vehicle-treated arteries from eNOS(+/+) and eNOS(+/-) mice, and were equally inhibited by L-NNA and ODQ. Phosphorylation of eNOS Ser1176, a site associated with increased eNOS activity, was significantly greater in eNOS(+/-) mice most likely as a compensatory response for the loss of a single eNOS gene. In contrast, responses to ACh were markedly impaired in carotid arteries from eNOS(+/-), but not eNOS(+/+), mice fed a HFD. Vascular superoxide levels as well as plasma levels of the pro-inflammatory cytokine interleukin-6 (IL-6) were selectively increased in HFD-fed eNOS(+/-) mice. In reconstitution experiments, IL-6 produced concentration-dependent impairment of endothelial responses as well as greater increases in NADPH-stimulated superoxide levels in arteries from eNOS(+/-) mice fed a control diet compared to eNOS(+/+) mice. Our findings of increased Ser1176-phosphorylation reveal a mechanism by which NOS- and sGC-dependent endothelial function can be maintained with heterozygous eNOS deficiency. In addition, heterozygous eNOS deficiency predisposes blood vessels to developing endothelial dysfunction in response to a HFD. The impairment produced by a HFD in eNOS(+/-) mice appears to be mediated by IL-6-induced increases in vascular superoxide. These findings serve as an important example of eNOS haploinsufficiency, one that may contribute to the development of carotid artery disease in obese humans.
RESUMEN
Pioglitazone belongs to the class of drugs called thiazolidinediones (TZDs), which are widely used as insulin sensitizers in the treatment of diabetes. A major side effect of TZDs is fluid retention. The steroid hormone aldosterone also promotes sodium and fluid retention; however, the effect of pioglitazone on aldosterone production is controversial. We analyzed the effect of pioglitazone alone and in combination with angiotensin II (AngII) on the late rate-limiting step of adrenocortical steroidogenesis in human adrenocortical carcinoma HAC15 cells. Treatment with pioglitazone for 24 h significantly increased the expression of CYP11B2 and enhanced AngII-induced CYP11B2 expression. Despite the observed changes in mRNA levels, pioglitazone significantly inhibited AngII-induced aldosterone production and CYP11B2 protein levels. On the other hand, pioglitazone stimulated the expression of the unfolded protein response (UPR) marker DDIT3, with this effect occurring at early times and inhibitable by the PPARγ antagonist GW9962. The levels of DDIT3 (CHOP) and phospho-eIF2α (Ser51), a UPR-induced event that inhibits protein translation, were also increased. Thus, pioglitazone promotes CYP11B2 expression but nevertheless inhibits aldosterone production in AngII-treated HAC15 cells, likely by blocking global protein translation initiation through DDIT3 and phospho-eIF2α. In contrast, pioglitazone promoted AngII-induced CYP11B1 expression and cortisol production. Since cortisol enhances lipolysis, this result suggests the possibility that PPARs, activated by products of fatty acid oxidation, stimulate cortisol secretion to promote utilization of fatty acids during fasting. In turn, the ability of pioglitazone to stimulate cortisol production could potentially underlie the effects of this drug on fluid retention.
Asunto(s)
Corteza Suprarrenal/efectos de los fármacos , Aldosterona/biosíntesis , Angiotensina II/farmacología , Hidrocortisona/biosíntesis , Tiazolidinedionas/farmacología , Corteza Suprarrenal/citología , Corteza Suprarrenal/metabolismo , Aldosterona/metabolismo , Anilidas/farmacología , Línea Celular Tumoral , Citocromo P-450 CYP11B2/genética , Citocromo P-450 CYP11B2/metabolismo , Combinación de Medicamentos , Ácidos Grasos/metabolismo , Regulación de la Expresión Génica , Humanos , Hidrocortisona/metabolismo , PPAR gamma/antagonistas & inhibidores , PPAR gamma/genética , PPAR gamma/metabolismo , Fosforilación/efectos de los fármacos , Pioglitazona , Biosíntesis de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal , Factor de Transcripción CHOP/genética , Factor de Transcripción CHOP/metabolismo , eIF-2 Quinasa/genética , eIF-2 Quinasa/metabolismoRESUMEN
Lasp-1 (LIM and SH3 domain protein 1) is a multidomain actin-binding protein that is differentially expressed within epithelial tissues and brain. In the gastric mucosa, Lasp-1 is highly expressed in the HCl-secreting parietal cell, where it is prominently localized within the F-actin-rich subcellular regions. Histamine-induced elevation of parietal cell [cAMP]i increases Lasp-1 phosphorylation, which is correlated with activation of HCl secretion. To determine whether Lasp-1 is involved in the regulation of HCl secretion in vivo, we generated a murine model with a targeted disruption of the Lasp-1 gene. Lasp-1-null mice had slightly lower body weights but developed normally and had no overt phenotypic abnormalities. Basal HCl secretion was unaffected by loss of Lasp-1, but histamine stimulation induced a more robust acid secretory response in Lasp-1-null mice compared with wild-type littermates. A similar effect of histamine was observed in isolated gastric glands on the basis of measurements of accumulation of the weak base [14C]aminopyrine. In addition, inhibition of the acid secretory response to histamine by H2 receptor blockade with ranitidine proceeded more slowly in glands from Lasp-1-null mice. These findings support the conclusion that Lasp-1 is involved in the regulation of parietal HCl secretion. We speculate that cAMP-dependent phosphorylation of Lasp-1 alters interactions with F-actin and/or endocytic proteins that interact with Lasp-1, thereby regulating the trafficking/activation of the H+, K+-ATPase (proton pump).
Asunto(s)
Ácido Gástrico/metabolismo , Mucosa Gástrica/metabolismo , Histamina/farmacología , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Envejecimiento , Animales , Proteínas del Citoesqueleto , Femenino , Mucosa Gástrica/citología , Mucosa Gástrica/efectos de los fármacos , Eliminación de Gen , Proteínas con Dominio LIM , Masculino , Ratones , Ratones Endogámicos C57BL , Osteoclastos/metabolismoRESUMEN
Tumor protein D52 is expressed at relatively high levels in cells within the gastrointestinal tract that undergo classical exocytosis and is overexpressed in several cancers. Current evidence supports a role for D52 in the regulation of vesicular trafficking. D52 function(s) are regulated by calcium-dependent phosphorylation; however, the intracellular mechanisms that mediate this process are not well characterized. The goal of this study was to identify the calcium-dependent phosphorylation site(s) in D52 and to characterize the protein kinase(s) that mediate this phosphorylation. Using mass spectrometry and site-directed mutagenesis, we identified a single amino acid residue, S(136), that undergoes increased phosphorylation upon elevation of intracellular Ca(2+) concentration. A phosphospecific antibody (pS(136)) was produced and used to characterize D52 kinase activity in gastric mucosal, colonic T84, and HEK293 cells. By using D52 as a substrate, a protein kinase with a molecular weight (M(r)) of approximately 50 kDa was identified with "in gel" assays. This kinase comigrated with rat brain calcium/calmodulin-dependent protein kinase (CAMK2)alpha cross-reacted with pan-specific CAMK2 antibodies as well as with anti-active CAMK2 (pT(286/287)) antibody when activated. Carbachol-stimulated phosphorylation of S(136) was inhibited by the CAMK2 inhibitor KN93 (IC(50) 38 microM) and by the calmodulin antagonist W7 (IC(50) 3.3 nM). A previously uncharacterized CAMK2 isoform, CAMK2delta6, which has the same domain structure and M(r) as CAM2alpha, was identified in gastric mucosa by RT-PCR. The cloned, expressed protein comigrated with D52 kinase and colocalized with D52 protein in T84 and HEK293 cells. These findings support a role for CAMK2delta6 in the mediation of D52 phosphorylation.
Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Proteínas de Neoplasias/metabolismo , Serina/metabolismo , Secuencia de Aminoácidos , Animales , Calcio/farmacología , Carbacol/farmacología , Línea Celular , Mucosa Gástrica/citología , Humanos , Ratones , Mutagénesis Sitio-Dirigida , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , ConejosRESUMEN
IQGAPs, GTPase-activating proteins with an IQ motif, are thought to regulate many actin cytoskeleton-based activities through interactions with Cdc42 and Rac. Recently, Cdc42 was implicated in regulation of gastric parietal cell HCl secretion, and IQGAP2 was immunolocalized with Cdc42 to F-actin-rich intracellular canalicular membranes of isolated gastric parietal cells in primary culture. Here we sought to define distribution and localization of IQGAP1 and IQGAP2 in major oxyntic (acid-secreting) gastric mucosal cell types and to determine whether secretory agonists modulate these proteins. Differential staining protocols were used to identify different cell populations (parietal, chief, surface/pit, and mucous neck cells) in semi-intact glands isolated from rabbit gastric mucosae and to characterize these same cells after dispersion and fractionation on isopycnic density gradients with simultaneous staining for F-actin, H+-K+-ATPase, and GSII lectin-binding sites. There was a pronounced increase in intracellular F-actin staining in dispersed chief cells, apparently from internalization of F-actin-rich apical membranes that normally abut the gland lumen. Therefore, other membrane-associated proteins might also be redistributed by disruption of cell-cell contacts. Western blot analyses were used to quantitate relative concentrations of IQGAPs in defined mucosal cell fractions, and gastric glands were used for in situ localizations. We detected uniform levels of IQGAP2 expression in oxyntic mucosal cells with predominant targeting to regions of cell-cell contact and nuclei of all cell types. IQGAP2 was not detected in parietal cell intracellular canaliculi. IQGAP1 expression was variable and targeted predominantly to the cortex of chief and mucous neck cells. Parietal cells expressed little or no IQGAP1 vs. other mucosal cell types. Phosphoprotein affinity chromatography, isoelectric focusing, and phosphorylation site analyses indicated that both IQGAP1 and IQGAP2 are phosphoproteins potentially regulated by [Ca2+]i/PKC and cAMP signaling pathways, respectively. Stimulation of glands with carbachol, which elevates [Ca2+]i and activates PKC, induced apparent translocation of IQGAP1, but not IQGAP2, to apical poles of chief (zymogen) and mucous neck cells. This response was mimicked by PMA but not by ionomycin or by elevation of [cAMP]i with forskolin. Our observations support a novel, PKC-dependent role for IQGAP1 in regulated exocytosis and suggest that IQGAP2 may play a more general role in regulating cell-cell interactions and possibly migration within the gastric mucosa.
Asunto(s)
Células Epiteliales/metabolismo , Mucosa Gástrica/metabolismo , Proteínas Activadoras de ras GTPasa/metabolismo , Secuencia de Aminoácidos , Animales , Células Cultivadas , Mucosa Gástrica/citología , Expresión Génica/fisiología , Regulación de la Expresión Génica/fisiología , Datos de Secuencia Molecular , Células Parietales Gástricas/metabolismo , Fosforilación , Conejos , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transducción de Señal , Fracciones Subcelulares/metabolismo , Proteínas Activadoras de ras GTPasa/fisiologíaRESUMEN
Developmentally regulated brain proteins (drebrins) are highly expressed in brain where they may regulate actin filament formation in dendritic spines. Recently, the drebrin E2 isoform was detected in certain epithelial cell types including the gastric parietal cell. In gastric parietal cells, activation of HCl secretion is correlated with actin filament formation and elongation within intracellular canaliculi, which are the sites of acid secretion. The aim of this study was to define the pattern of drebrin expression in gland units in the intact rabbit oxyntic gastric mucosa and to initiate approaches to define the functions of this protein in parietal cells. Drebrin E2 expression was limited entirely or almost entirely to parietal cells and depended upon the localization of parietal cells along the gland axis. Rabbit drebrin E2 was cloned and found to share 86% identity with human drebrin 1a and to possess a number of cross-species conserved protein-protein interaction and phosphorylation consensus sites. Two-dimensional Western blot and phosphoaffinity column analyses confirmed that drebrin is phosphorylated in parietal cells, and several candidate phosphorylation sites were identified by mass spectrometry. Overexpression of epitope-tagged drebrin E2 led to the formation of microspikes and F-actin-rich ring-like structures in cultured parietal cells and suppressed cAMP-dependent acid secretory responses. In Madin-Darby canine kidney cells, coexpression of epitope-tagged drebrin and the Rho family GTPase Cdc42, which induces filopodial extension, produced an additive increase in the length of microspike projections. Coexpression of dominant negative Cdc42 with drebrin E2 did not prevent drebrin-induced microspike formation. These findings suggest that 1) drebrin can induce the formation of F-actin-rich membrane projections by Cdc42-dependent and -independent mechanisms; and that 2) drebrin plays an active role in directing the secretagogue-dependent formation of F-actin-rich filaments on the parietal cell canalicular membrane. Finally, the differential distribution of drebrin in parietal cells along the gland axis suggests that drebrin E2 may be an important marker of parietal cell differentiation and functionality.