RESUMEN
(E)-3,4-Dihydroxybenzylideneacetone (compound 1) inhibited receptor activator of NF-κB ligand-induced osteoclastogenesis of C57BL/6 bone marrow monocyte/macrophages with IC50 of 7.8 µM (IC50 of alendronate, 3.7 µM) while stimulating the differentiation of MC3T3-E1 osteoblastic cells, accompanied by the induction of Runt-related transcription factor 2, alkaline phosphatase, and osteocalcin. (E)-4-(3-Hydroxy-4-methoxyphenyl)-3-buten-2-one (compound 2c) showed a dramatically increased osteoclast-inhibitory potency with IC50 of 0.11 µM while sustaining osteoblast-stimulatory activity. (E)-4-(4-Hydroxy-3-methoxyphenyl)-3-buten-2-one (compound 2g) stimulated alkaline phosphatase production 2-fold at 50 µM without changing osteoclast-inhibitory activity, compared with compound 1. Oral administration of compounds 1, 2c, and 2g prevented ovariectomy-induced osteoporosis in ddY mice to a degree proportional to their osteoclastogenesis-inhibitory potencies. The administration of 1 (mg/kg)/d compound 2c ameliorated histomorphometry of osteoporotic bone to a degree comparable with 10 (mg/kg)/d alendronate. Conclusively, the in vitro capacity of a few benzylideneacetone derivatives to inhibit osteoclastogenesis supported by independent osteoblastogenesis activation was convincingly reflected in in vivo management of osteoporosis, suggesting a potential novel therapeutics for osteopenic diseases.
Asunto(s)
Compuestos de Bencilideno/uso terapéutico , Butanonas/uso terapéutico , Osteogénesis/efectos de los fármacos , Fosfatasa Alcalina/metabolismo , Animales , Antineoplásicos/síntesis química , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapéutico , Compuestos de Bencilideno/síntesis química , Compuestos de Bencilideno/farmacocinética , Butanonas/síntesis química , Butanonas/farmacocinética , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Femenino , Fémur/patología , Humanos , Ratones , Estructura Molecular , Subunidad p50 de NF-kappa B/metabolismo , Factores de Transcripción NFATC/metabolismo , Osteoblastos/metabolismo , Osteocalcina/metabolismo , Osteoclastos/metabolismo , Osteoporosis/tratamiento farmacológico , Células RAW 264.7 , Relación Estructura-Actividad , Tibia/patologíaRESUMEN
Whereas high-dose ultraviolet B (UVB) is detrimental to the epidermal permeability barrier, suberythemal doses of UVB are used to treat atopic dermatitis (AD), which is characterized by defective permeability barrier and antimicrobial function. As epidermal permeability barrier and antimicrobial peptide (AMP) expression are coregulated and interdependent functions, we hypothesized that suberythemal doses of UVB exposure could regulate AMP expression in parallel with permeability barrier function. Hairless mice were exposed to 40 mJ cm(-2) UVB (about 1/2 minimal erythema dose) daily for 1 or 3 days. Twenty-four hours after the last exposure, epidermal barrier function was assessed and skin specimens were taken for western blotting, immunohistochemistry, and quantitative reverse transcription-PCR for mouse beta-defensin (mBD)-2, mBD3 and cathelin-related antimicrobial peptide (CRAMP). mRNA levels of the vitamin D receptor (VDR), 1alpha-hydroxylase and key epidermal lipid synthetic enzymes were also quantified. After 3 days of UVB exposure, acceleration of barrier recovery and augmentation in expression of epidermal differentiation markers (for example, involucrin and filaggrin) occurred in parallel with increased mBD2, mBD3, and CRAMP expression at both the mRNA and protein level. VDR, 1alpha-hydroxylase, and the major epidermal lipid synthetic enzymes were also upregulated. When an inhibitor of 1alpha, 25 dihydroxyvitamin D(3) formation, ketoconazole, was applied immediately after UVB exposure, the cutaneous vitamin D system was inhibited, which in turn blocked epidermal lipid synthesis, AMP expression, and permeability barrier homeostasis, suggesting that the beneficial effect of low-dose UVB depends, at least in part, on activation of the cutaneous vitamin D system. Our results provide new insights into the mechanisms whereby low-dose UVB comprises effective therapy for AD.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/farmacología , Epidermis/inmunología , Epidermis/efectos de la radiación , Animales , Catelicidinas , Diferenciación Celular , Colecalciferol/metabolismo , Femenino , Inmunohistoquímica , Cetoconazol/farmacología , Lípidos/química , Ratones , Ratones Pelados , Modelos Biológicos , Permeabilidad , Receptores de Calcitriol/metabolismo , Rayos UltravioletaRESUMEN
A-kinase-anchoring protein 149 (AKAP149) is a member of a structurally diverse, though functionally similar anchoring protein family and is localized to the outer membrane of mitochondria and in the endoplasmic reticulum-nuclear envelope network. AKAP149 plays an important role in controlling the subcellular localization and temporal specificity of protein phosphorylation and mRNA metabolism by tethering kinases and phosphatases, such as protein kinase A and type I protein phosphatase, through its N-terminal protein-binding motifs and mRNAs via its C-terminal RNA-binding motifs. It is well recognized that caspases play a central role in transducing and amplifying the intracellular death signal and that apoptosis is executed as a consequence of caspase-mediated cleavage of multiple cellular substrates. The identification of novel death substrates and elucidation of the consequences of their proteolytic cleavages by caspases are therefore crucial for our understanding of cell death and other biological processes. Herein, we demonstrated that AKAP149 is a direct substrate of active caspase-3, -8 -and -10 in vitro and in vivo. 35S-labeled full-length AKAP149 was completely cleaved in vitro by active caspase-3, -8 and -10 into two fragments of approximately 105 and 45 kDa, while caspase-2 cleaved it partially and caspase-1 did not cleave it at all. AKAP149 was also cleaved by caspases during Fas- and staurosporine-induced apoptosis in Jurkat T and HeLa cells, which were blocked by specific inhibitors of caspase-3 and -8. The specific cleavage site for these caspases was mapped in vitro and in vivo to Asp582 at AKAP149, which is located between the protein kinase A regulatory subunit anchoring and KH RNA-binding domains. In addition, HeLa cells transiently overexpressing AKAP149 D582E mutant were resistant to staurosporine-induced HeLa cell apoptosis. Taken together, these data suggest that AKAP149 activity may be deregulated by caspase-dependent proteolysis during apoptotic cell death and may provide useful information for elucidating the apoptosis signaling pathways in detail.
Asunto(s)
Proteínas de Anclaje a la Quinasa A/metabolismo , Apoptosis/fisiología , Caspasas/metabolismo , Proteínas de Anclaje a la Quinasa A/genética , Ácido Aspártico/química , Ácido Aspártico/genética , Células HeLa , Humanos , Células Jurkat , Mutación/genética , Estaurosporina/farmacología , Receptor fas/farmacologíaRESUMEN
Apoptosis executed by the mammalian caspase family plays a fundamental role in cellular homeostasis. Deregulation of this process is associated with several human diseases. The multimerization of ligand-induced death receptors results in the recruitment of the death inducing signaling complex and autocatalytic activation of initiator caspases, including caspase-8 and -10. However, it is still unclear how initiator caspases trigger and control the early apoptotic signaling pathways, partly because the downstream proteolytic cleavage targets of the initiator caspases are not completely known. Although it is known that a number of proteins are cleaved by various members of the caspase family, the identification of specific cleavage substrates of the initiator caspases 8 and 10, has been hindered by a lack of systematic and broadly applicable strategies for substrate identification. In the present study we constructed a mouse cDNA library and used it to perform a systematic, genome-wide screen for novel in vitro substrates of caspase-8 and -10. From this, we successfully identified six putative caspase substrates, including five novel proteins (ABCF1, AKAP1, CPE, DOPEY1 and GOPC1) that may be targeted specifically by the initiator caspases 8 and 10 during the early stages of apoptosis. These findings may provide useful information for elucidating the apoptotic signaling pathways downstream of the death receptors.
Asunto(s)
Caspasa 10/metabolismo , Caspasa 8/metabolismo , Genoma/genética , Procesamiento Proteico-Postraduccional , Animales , Células Clonales , Biología Computacional , ADN Complementario/aislamiento & purificación , Biblioteca de Genes , Hígado/enzimología , Ratones , Reproducibilidad de los Resultados , Especificidad por SustratoRESUMEN
Several signals have been suggested in maintaining skin barrier homeostasis, but epidermal calcium ions are currently thought to be a main signaling factor. It is not clear, however, exactly how an intracellular calcium level decreases in response to the loss of an extracellular calcium gradient. In this study, we investigated the effects of several broad-type and isozyme-specific protein kinase C (PKC) inhibitors on epidermal permeability barrier recovery. Topical application of chelerythrine chloride, a broad-type PKC inhibitor, and rottlerin, a PKCdelta-specific inhibitor, significantly impeded the barrier recovery rate at 3 and 6 hours after barrier disruption. A significant decrease in the number and secretion of lamellar bodies was also observed at the inhibitor-treated site. Calcium ion-capture cytochemistry showed that the epidermal calcium gradient was rapidly reformed in inhibitor-treated skin, though recovery of the corresponding barrier function was not observed. In cultured keratinocytes treated with either inhibitor, increased intracellular calcium did not return to the baseline concentration after extracellular calcium decreased. These results suggest that PKC inhibitors, especially a PKCdelta-specific inhibitor, delay barrier recovery by affecting the intracellular calcium concentration after a loss of the extracellular calcium gradient. Furthermore, PKCdelta is important in controlling a decrease in intracellular calcium concentration.
Asunto(s)
Acetofenonas/farmacología , Benzopiranos/farmacología , Calcio/metabolismo , Epidermis/efectos de los fármacos , Proteína Quinasa C-delta/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Cicatrización de Heridas/efectos de los fármacos , Animales , Calcio/análisis , Células Cultivadas , Epidermis/química , Epidermis/metabolismo , Isoenzimas/antagonistas & inhibidores , Lípidos/análisis , Lípidos/biosíntesis , Masculino , Ratones , Ratones PeladosRESUMEN
Skin atrophy is one of the most frequent side-effects of the topical glucocorticoid. Skin barrier impairment has also been reported as a steroid-induced side effect. Although there have been various studies on preventing or minimizing this atrophogenic effect, little has been reported about preventing barrier impairment. This study was performed to determine the effects of a multilamellar emulsion (MLE) that had a well-ordered lamellar structure on the steroid-induced barrier impairment and epidermal atrophy. To confirm these effects of MLE, 0.05% clobetasol-17-propionate (CP) and 0.05% clobetasol-17-propionate in MLE (MLE/CP) were topically applied to both flanks of hairless mice for 9 days. The topically applied CP induced a significant impairment of the epidermal permeability barrier, and MLE/CP also did not have a preventive effect on this change. However, skinfold thickness studies and histological studies showed that MLE/CP significantly reduced the steroid-induced atrophy. The topical application of MLE/CP was also shown to have a preventive effect on the steroid-induced increase of the stratum corneum (SC) surface pH. In addition, the electron microscopic findings showed relatively well-conserved lamellar bilayers in the skin treated with MLE, as compared to CP only. The results showed that the topical application of MLE immediately after CP treatment prevented the glucocorticoid-induced transepidermal water loss values increase. Light microscopy measurements showed that the skin treated with MLE immediately after CP treatment for 1 week had a slightly lower decline of skin thickness than did the CP-treated skin. These results suggest that MLE should be effective for preventing glucocorticoid-induced epidermal atrophy and for repairing the barrier impairment.
Asunto(s)
Clobetasol/análogos & derivados , Epidermis/efectos de los fármacos , Epidermis/patología , Absorción Cutánea/efectos de los fármacos , Administración Tópica , Animales , Atrofia , Biopsia con Aguja , Clobetasol/farmacología , Modelos Animales de Enfermedad , Emulsiones , Femenino , Glucocorticoides , Inmunoquímica , Masculino , Ratones , Ratones Pelados , Permeabilidad/efectos de los fármacos , Probabilidad , Valores de Referencia , Factores de Riesgo , Sensibilidad y Especificidad , Absorción Cutánea/fisiología , Enfermedades de la Piel/tratamiento farmacológico , Enfermedades de la Piel/patologíaRESUMEN
Vascular endothelium, situated at the boundary between blood and tissues, is now known to play a critical role in the inflammatory process through recruiting immune cells into tissues and sites of inflammation. Lipopolysaccharide (LPS), endotoxic component extracted from the cell wall of gram-negative bacteria, stimulates endothelial cells to activate the nuclear transcription factor NF-kappaB and induce various adhesion molecules and inflammatory mediators. Among the anti-apoptotic genes activated by NF-kappaB, transcripts for inhibitor of apoptosis proteins (IAPs) are rapidly induced in response to LPS and delay apoptosis through direct and indirect inhibition of caspase activity. In the present study we carried out cDNA microarray analysis to elucidate how LPS alters program of gene expression of human umbilical vein endothelial cells (HUVECs) and to identify genes that are differentially expressed in HUVECs cultured with LPS as a mimickappaing of pathologic and physiologic inflammatory conditions in vitro. From the analysis of cDNA microarray together with Northern blotting and semi-quantitative RT-PCR, we identified dramatically upregulated both previously reported and undiscovered transcripts for adhesion molecules, inflammation/chemokappaines, transcription factors and anti-apoptotic proteins in LPS-stimulated HUVECs. In addition, we simultaneously identified anti-inflammatory, anti-oxidative stress and pro-apoptotic genes highly upregulated by LPS treatment in HUVECs. Surprisingly, although cIAP-1, cIAP-2 and XIAP transcripts were highly upregulated, their expression of endogenous proteins were not increased in HUVECs stimulated with LPS indicating the existence of yet undiscovered transcriptional or translational mechanisms may control expression and regulation of IAPs. The data presented here therefore suggest that when endothelial cells are challenged by inflammatory stimuli such as LPS, they undergo functional changes not only for the proinflammatory but also for the anti-inflammatory states and these may further be controlled by particular cellular or environmental signals in vascular pathological and physiological diseases.
Asunto(s)
Endotelio Vascular/fisiología , Perfilación de la Expresión Génica , Lipopolisacáridos/farmacología , Venas Umbilicales/fisiología , Células Cultivadas , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Humanos , Proteínas Inhibidoras de la Apoptosis , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas/efectos de los fármacos , Proteínas/genética , Proteínas/metabolismo , Venas Umbilicales/citología , Venas Umbilicales/efectos de los fármacos , Proteína Inhibidora de la Apoptosis Ligada a XRESUMEN
Together with the explosion in the availability of genome data of a number of organisms including human and mouse, various methods and programs for computational prediction of protein-coding genes and annotation of functional proteins have dramatically increased. For the last decade there has been intense interest in the role of protein phosphorylation which is involved in post-translation modification mechanisms critically regulating inter/intracellular communication, patho/physiological responses and homeostasis during many biological processes. In the present study a total of 202 functionally uncharacterized human full-coding cDNA sequences were investigated using a bioinformatics-based approach. Ten novel potential substrates for protein kinases have been identified which may play multiple roles in regulating intracellular phosphorylation signalling pathways. In addition, 5 of those may be involved in the human-only post-translation mechanism regulated by specific protein kinases. The data presented here therefore would greatly contribute toward the understanding of human molecular basis and cellular signalling networks.
Asunto(s)
Biología Computacional , Biología Molecular , Proteínas Quinasas/fisiología , Proteínas Serina-Treonina Quinasas , Transducción de Señal/fisiología , Secuencias de Aminoácidos , Animales , Humanos , Ratones , Fosforilación , Proteínas Quinasas/química , Proteínas Quinasas/genética , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/fisiología , Proteínas Proto-Oncogénicas c-aktRESUMEN
Prolonged exposure of human epidermis to excess endogenous or exogenous glucocorticoids can result in well-recognized cutaneous abnormalities. Here, we determined whether short-term glucocorticoid treatment would also display adverse effects, specifically on two key epidermal functions, permeability barrier homeostasis and stratum corneum integrity and cohesion, and the basis for such changes. In humans 3 d of treatment with a potent, commonly employed topical glucocorticoid (clobetasol), applied topically, produced a deterioration in barrier homeostasis, characterized by delayed barrier recovery and abnormal stratum corneum integrity (rate of barrier disruption with tape strippings) and stratum corneum cohesion (microg protein removed per stripping). Short-term systemic and topical glucocorticoid produced similar functional defects in mice, where the basis for these abnormalities was explored further. Both the production and secretion of lamellar bodies were profoundly decreased in topical glucocorticoid-treated mice resulting in decreased extracellular lamellar bilayers. These structural changes, in turn, were attributable to a profound global inhibition of lipid synthesis, demonstrated both in epidermis and in cultured human keratinocytes. The basis for the abnormality in stratum corneum integrity and cohesion was a diminution in the density of corneodesmosomes in the lower stratum corneum. We next performed topical replacement studies to determine whether lipid deficiency accounts for the glucocorticoid-induced functional abnormalities. The abnormalities in both permeability barrier homeostasis and stratum corneum integrity were corrected by topical applications of an equimolar distribution of free fatty acids, cholesterol, and ceramides, indicating that glucocorticoid-induced inhibition of epidermal lipid synthesis accounts for the derangements in both cutaneous barrier function and stratum corneum integrity/cohesion. These studies indicate that even short-term exposure to potent glucocorticosteroids can exert profound negative effects on cutaneous structure and function. Finally, topical replenishment with epidermal physiologic lipids could represent a potential method to reduce the adverse cutaneous effects of both topical glucocorticoid treatment and Cushing's syndrome.
Asunto(s)
Antiinflamatorios/administración & dosificación , Clobetasol/administración & dosificación , Epidermis/efectos de los fármacos , Epidermis/metabolismo , Homeostasis/efectos de los fármacos , Administración Tópica , Adulto , Animales , Desmosomas/ultraestructura , Esquema de Medicación , Epidermis/fisiopatología , Epidermis/ultraestructura , Glucocorticoides , Humanos , Lípidos/antagonistas & inhibidores , Lípidos/biosíntesis , Lípidos/farmacología , Masculino , Ratones , Ratones Pelados , Microscopía Electrónica , Persona de Mediana Edad , Permeabilidad/efectos de los fármacosRESUMEN
Mammalian epidermis normally displays a distinctive calcium gradient, with low levels in the basal/spinous layers and high levels in the stratum granulosum. Although changes in stratum granulosum calcium regulate the lamellar body secretory response to permeability barrier alterations, whether modulations in calcium also regulate the expression of differentiation-specific proteins in vivo remains unknown. As acute barrier perturbations reduce calcium levels in stratum granulosum, we studied the regulation of murine epidermal differentiation after loss of calcium accompanying acute barrier disruption and by exposure of such acutely perturbed skin sites to either low (0.03 M) or high (1.8 M) calcium. Three hours after acute barrier disruption, coincident with reduced calcium and ultrastructural evidence of accelerated lamellar body secretion, both northern analyses and in situ hybridization revealed decreased mRNA levels for loricrin, profilaggrin, and involucrin in the outer epidermis, but protein levels did not change significantly. Moreover, exposure of acutely disrupted skin sites to low calcium solutions sustained the reduction in mRNA levels, whereas exposure to high calcium solutions restored normal mRNA levels (blocked by the L-type calcium channel inhibitor, nifedipine). Finally, with prolonged exposure to a low (<10% relative humidity) or high (>80% relative humidity) humidity, calcium levels increased and declined, respectively. Accordingly, mRNA and protein levels of the differentiation-specific markers increased and decreased at low and high relative humidity, respectively. These results provide direct evidence that acute and sustained fluctuations in epidermal calcium regulate expression of differentiation-specific proteins in vivo, and demonstrate that modulations in epidermal calcium coordinately regulate events late in epidermal differentiation that together form the barrier.