RESUMEN
Mechanical disturbance is directly implicated in the development of osteoarthritis (OA) but the precise mode for degenerative changes is still largely unknown because of the complexity of the biomechanical and biochemical milieu in the articular joint. To investigate the effects of tensile strain on articular cartilage, cyclic equibiaxial tensile strain (CTS, 0.5 Hz, 10% strain) was applied to monolayer cultures of porcine articular chondrocytes by using a Flexercell strain unit. Overproduction of proinflammatory mediators and imbalanced expression of anabolic and catabolic genes were induced. The cellular secretion of nitric oxide (NO) and prostaglandin E(2) (PGE(2)), as well as the mRNA level of cyclooxygenase-2 (COX-2) were up-regulated in response to mechanical stimuli. Additionally, CTS resulted in an initial peak of anabolic response at 3 h of stretch with respect to the expression of type II collagen and aggrecan. After 12 h of CTS, the expression for these two cartilage-specific matrix proteins fell to control levels. A distinct catabolic response developed after 24 h of stretch with an increase in matrix metalloproteinase-1 (MMP-1). Interestingly, a parallel increase in transforming growth factor (TGF) beta3 was associated with the anabolic changes while an increase in expression of TGF beta1, the predominant isoform of the TGF family, appeared at 24 h. The expression at 24 h of MMP-1, an enzyme that degrades interstitial collagens as well as other cartilage matrix proteins and TGF beta1, may signify a shift towards matrix remodeling and potentially a change in matrix composition as a consequence of continuous CTS.
Asunto(s)
Cartílago Articular/metabolismo , Condrocitos/metabolismo , Expresión Génica , Agrecanos/genética , Agrecanos/metabolismo , Animales , Cartílago Articular/citología , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Dinoprostona/genética , Dinoprostona/metabolismo , Metaloproteinasa 1 de la Matriz/genética , Metaloproteinasa 1 de la Matriz/metabolismo , Óxido Nítrico/metabolismo , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Resistencia a la Tracción , Regulación hacia ArribaRESUMEN
Prostaglandin E(2) (PGE(2)) plays a critical role in skeletal physiology and bone loss. PGE(2) production is regulated in vivo by at least two cyclooxygenase (COX) isozymes, COX-1 and COX-2. The purpose of this study was to investigate the in vivo effects of the selective deletion of COX-1 or COX-2 on bone mineral density (BMD), bone microarchitecture and bone strength in wild type (WT), COX-1(-/-) and COX-2(-/-) mice. Using a LUNAR PIXImus, BMD was measured in 18 (WT), 18 COX-1(-/-) and 16 COX-2(-/-) mice. COX-1(-/-) mice exhibited significantly higher BMD (0.0506 g/cm(2) +/- 0.0014 g/cm(2)) than either WT (0.0493 g/cm(2) +/- 0.0019, P < or = 0.05) or COX-2(-/-) (0.0473 g/cm(2) +/- 0.0034, P < or = 0.01) mice. COX-2(-/-) mice had significantly lower BMD than WT (P < or = 0.01) or COX-1(-/-) (P < or = 0.01). Flexure stress of the femurs, determined by breaking the bones with three-point bending, correlated with bone density. Although plasma levels of both Ca(2+) and PTH were comparable in wild type and COX-1(-/-) mice, both were elevated in COX-2(-/-) mice consistent with primary hyperparathyroidism. These studies suggest that COX enzymes are important regulators of BMD and bone strength in mice. The beneficial effect of absence of the COX-1 enzyme on skeletal parameters may be secondary to decreases in PGE(2). On the other hand, primary hyperparathyroidism and lower bone magnesium content may account for the lower BMD and impairments in bone strength of COX-2(-/-) mice. Further elucidation of the effects of the COX pathway on bone remodeling may provide important information on potential therapeutic targets for preventing and/or treating osteoporosis.
Asunto(s)
Densidad Ósea/fisiología , Huesos/fisiología , Ciclooxigenasa 1/deficiencia , Ciclooxigenasa 2/deficiencia , Animales , Fenómenos Biomecánicos , Índice de Masa Corporal , Densidad Ósea/genética , Huesos/metabolismo , Calcio/sangre , Ciclooxigenasa 1/genética , Ciclooxigenasa 2/genética , Dinoprostona/metabolismo , Femenino , Fémur/metabolismo , Fémur/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Noqueados , Hormona Paratiroidea/sangreRESUMEN
1. Prenatal patency of the ductus arteriosus is maintained by prostaglandin (PG) E(2), conceivably in concert with nitric oxide (NO). Local PGE(2) formation is sustained by cyclooxygenase-1 (COX1) and cyclooxygenase-2 (COX2), a possible exception being the mouse in which COX1, or both COXs, are reportedly absent. Here, we have examined the occurrence of functional COX isoforms in the near-term mouse ductus and the possibility of COX deletion causing NO upregulation. 2. COX1 and COX2 were detected in smooth muscle cells by immunogold electronmicroscopy, both being located primarily in the perinuclear region. Cytosolic and microsomal PGE synthases (cPGES and mPGES) were also found, but they occurred diffusely across the cytosol. COX1 and, far more frequently, COX2 were colocalised with mPGES, while neither COX appeared to be colocalized with cPGES. 3. The isolated ductus from wild-type and COX1-/- mice contracted promptly to indomethacin (2.8 micro M). Conversely, the contraction of COX2-/- ductus to the same inhibitor started only after a delay and was slower. 4. N(G)-nitro-L-arginine methyl ester (L-NAME, 100 micro M) weakly contracted the isolated wild-type ductus. Its effect, however, increased three- to four-fold after deleting either COX, hence equalling that of indomethacin. 5. In vivo, the ductus was patent in all mice foetuses, whether wild-type or COX-deleted. Likewise, no genotype-related difference was noted in its postnatal closure. 6. We conclude that the mouse ductus has a complete system for PGE(2) synthesis comprising both COX1 and COX2. The two enzymes respond differently to indomethacin but, nevertheless, deletion of either one results in NO upregulation. PGE(2) and NO can function synergistically in keeping the ductus patent.
Asunto(s)
Conducto Arterial/enzimología , Isoenzimas/biosíntesis , Óxido Nítrico Sintasa/biosíntesis , Prostaglandina-Endoperóxido Sintasas/biosíntesis , Animales , Ciclooxigenasa 1 , Ciclooxigenasa 2 , Conducto Arterial/embriología , Inmunohistoquímica , Isoenzimas/genética , Proteínas de la Membrana , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Electrónica , Prostaglandina-Endoperóxido Sintasas/genética , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Various lines of evidence have implicated cyclooxygenase (COX)-2 as a modulator of the fever induced by the exogenous pyrogen lipopolysaccharide (LPS). Thus, treatment with specific inhibitors of COX-2 suppresses the febrile response without affecting basal body (core) temperature (T(c)). Furthermore, COX-2 gene-ablated mice are unable to develop a febrile response to intraperitoneal (i.p.) LPS, whereas their COX-1-deficient counterparts produce fevers not different from their wild-type (WT) controls. To extend the apparently critical role of COX-2 for LPS-induced fevers to fevers produced by endogenous pyrogens, we studied the thermal responses of COX-1- and COX-2 congenitally deficient mice to i.p. and intracerebroventricular (i.c.v.) injections of recombinant murine (rm) interleukin (IL)-1beta. We also assessed the effects of one selective COX-1 inhibitor, SC-560, and two selective COX-2 inhibitors, nimesulide (NIM) and dimethylfuranone (DFU), on the febrile responses of WT and COX-1(-/-) mice to LPS and rmIL-1beta, i.p. Finally, we verified the integrity of the animals' responses to PGE2, i.c.v. I.p. and i.c.v. rmIL-1beta induced similar fevers in WT and COX-1 knockout mice, but provoked no rise in the T(c)s of COX-2 null mutants. The fever produced in WT mice by i.p. LPS was not affected by SC-560, but it was attenuated and abolished by NIM and DFU, respectively, while that caused by i.p. rmIL-1beta was converted into a T(c) fall by DFU. There were no differences in the responses to i.c.v. PGE2 among the WT and COX knockout mice. These results, therefore, further support the notion that the production of PGE2 in response to pyrogens is critically dependent on COX-2 expression.
Asunto(s)
Encéfalo/efectos de los fármacos , Dinoprostona/metabolismo , Fiebre/enzimología , Interleucina-1/farmacología , Isoenzimas/metabolismo , Fragmentos de Péptidos/farmacología , Prostaglandina-Endoperóxido Sintasas/metabolismo , Pirógenos/metabolismo , Animales , Temperatura Corporal/efectos de los fármacos , Temperatura Corporal/fisiología , Encéfalo/enzimología , Ciclooxigenasa 1 , Ciclooxigenasa 2 , Fiebre/inducido químicamente , Fiebre/fisiopatología , Inflamación/metabolismo , Inflamación/fisiopatología , Inyecciones Intraventriculares , Interleucina-1/metabolismo , Interleucina-1beta , Isoenzimas/antagonistas & inhibidores , Isoenzimas/genética , Lipopolisacáridos/metabolismo , Lipopolisacáridos/farmacología , Proteínas de la Membrana , Ratones , Ratones Noqueados , Neuroinmunomodulación/efectos de los fármacos , Neuroinmunomodulación/fisiología , Fragmentos de Péptidos/metabolismo , Prostaglandina-Endoperóxido Sintasas/genética , Pirógenos/farmacologíaRESUMEN
OBJECTIVES: Nonsteroidal antiinflammatory drugs (NSAIDs) that are more selective for PGHS-2 maintain their antiinflammatory properties but exhibit fewer unfavorable gastrointestinal side effects. To evaluate the usefulness of the murine PGHS-null cell system in analyzing PGHS-2 selective inhibitors, we tested PGHS-2 non-selective NSAIDs and two PGHS-2 specific compounds using either endogenous or exogenous sources of substrate, arachidonic acid. MATERIALS AND METHODS: A whole-cell assay system for testing the efficacy of PGHS isozyme-specific inhibitors using murine PGHS-1 or PGHS-2-null fibroblast cell lines derived from lung tissues of PGHS-2(-/-) and PGHS-(-/-) mice, respectively, was employed. PGHS-1 and PGHS-2 null cell lines were exposed to three widely used NSAIDs, ibuprofen, indomethacin and aspirin, and two PGHS-2 specific inhibitors, MK-966 (rofecoxib) and NS-398. Excess arachidonic acid (AA) was provided externally and internally via calcium ionophore A23187. PGHS-1 and PGHS-2 activity were determined by measuring the prostaglandin E2 production by radioimmunoassay. IC50 and IC80 values of each compound were determined from the reduction of PGE2 levels as a measure of inhibition of existing PGHS isozyme in the PGHS-null cells. RESULTS: In our murine PGHS-null cell systems, both PGHS-1 and PGHS-2 null cells can use both externally provided AA and endogenous, A23187-derived AA. Both NS-398 and MK-966 were potent inhibitors and demonstrated strong selectivity for PGHS-2. Among the non-selective NSAIDs, based on the PGHS-2 IC50/PGHS-1 IC50 ratio ranking, ibuprofen is more selective for PGHS-2 than indomethacin while aspirin is the least selective inhibitor regardless of the arachidonic acid source. Indomethacin and MK-966 IC50 values for PGHS-2 were in the range of 10(-9)-10(-8) M while the IC50 values for aspirin were in the range of 10(-5) M. There were differences in the ranking of indomethacin and ibuprofen when the IC80 ratios were used. CONCLUSION: Here, we report on a whole cell assay system for testing the efficacy of PGHS isozyme-specific inhibitors using murine PGHS-1 or PGHS-2-null cell lines. This system, using cells that express either PGHS-1 or PGHS-2, offers a convenient and reliable method to determine IC50 and IC80 values of the two PGHS isoforms, entirely independent of each other, in the same cell type. The results of our evaluation using a panel of NSAIDs, both PGHS-2 selective and non-selective inhibitors, correlate well with previously published clinical and laboratory data, demonstrating the usefulness of the whole-cell assay system described here.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Inhibidores Enzimáticos/farmacología , Isoenzimas/antagonistas & inhibidores , Animales , Ácido Araquidónico/metabolismo , Células Cultivadas , Ciclooxigenasa 1 , Ciclooxigenasa 2 , Dinoprostona/biosíntesis , Proteínas de la Membrana , Ratones , Prostaglandina-Endoperóxido SintasasRESUMEN
In mice heterozygous for the cyclooxygenase-2 gene (COX-2+/-) the body weight was enhanced by 33% as compared to homozygous COX-2-/- mice. The weights of the gonadal fat pads in COX-2+/- mice were enhanced by 3.5 to 4.7 fold as compared to COX-2-/- mice and by 1.5 to 3.5 fold as compared to wild-type controls+/+ Serum leptin levels and leptin release by cultured adipose tissue of COX-2+/- mice were both elevated as compared to either control or COX-2-/- animals. The basal release of PGE2 or 6 keto PGF1alpha per fat pad over a 24 h incubation of adipose tissue was reduced by 80% and 95% respectively in tissue from COX-2-/- mice. NS-398, a specific COX-2 inhibitor, inhibited leptin release by 27% in adipose tissue from control mice, 31% in tissue from COX-1-/- mice and by 23% in tissue from COX-2+/- mice while having no effect on leptin release by adipose tissue from COX-2-/- mice. These data indicate that heterozygous COX-2 mice develop obesity which is not secondary to a defect in leptin release by adipose tissue.
Asunto(s)
Tejido Adiposo/metabolismo , Isoenzimas/genética , Obesidad/genética , Prostaglandina-Endoperóxido Sintasas/genética , 6-Cetoprostaglandina F1 alfa/metabolismo , Tejido Adiposo/efectos de los fármacos , Animales , Peso Corporal , Ciclooxigenasa 1 , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa/farmacología , Dinoprostona/metabolismo , Epidídimo/anatomía & histología , Femenino , Heterocigoto , Humanos , Isoenzimas/metabolismo , Leptina/sangre , Leptina/metabolismo , Masculino , Proteínas de la Membrana , Ratones , Ratones Noqueados , Nitrobencenos/farmacología , Obesidad/etiología , Obesidad/fisiopatología , Tamaño de los Órganos , Prostaglandina-Endoperóxido Sintasas/metabolismo , Estadística como Asunto , Sulfonamidas/farmacologíaRESUMEN
Prostaglandins formed by cyclooxygenase-1 (COX-1) or COX-2 produce hyperalgesia in sensory nerve endings. To assess the relative roles of the two enzymes in pain processing, we compared responses of COX-1- or COX-2-deficient homozygous and heterozygous mice with wild-type controls in the hot plate and stretching tests for analgesia. Preliminary observational studies determined that there were no differences in gross parameters of behavior between the different groups. Surprisingly, on the hot plate (55 degrees C), the COX-1-deficient heterozygous groups showed less nociception, because mean reaction time was longer than that for controls. All other groups showed similar reaction times. In the stretching test, there was less nociception in COX-1-null and COX-1-deficient heterozygotes and also, unexpectedly, in female COX-2-deficient heterozygotes, as shown by a decreased number of writhes. Measurements of mRNA levels by reverse transcription-PCR demonstrated a compensatory increase of COX-1 mRNA in spinal cords of COX-2-null mice but no increase in COX-2 mRNA in spinal cords of COX-1-null animals. Thus, compensation for the absence of COX-1 may not involve increased expression of COX-2, whereas up-regulation of COX-1 in the spinal cord may compensate for the absence of COX-2. The longer reaction times on the hot plate of COX-1-deficient heterozygotes are difficult to explain, because nonsteroid anti-inflammatory drugs have no analgesic action in this test. Reduction in the number of writhes of the COX-1-null and COX-1-deficient heterozygotes may be due to low levels of COX-1 at the site of stimulation with acetic acid. Thus, prostaglandins made by COX-1 mainly are involved in pain transmission in the stretching test in both male and female mice, whereas those made by COX-2 also may play a role in the stretching response in female mice.
Asunto(s)
Isoenzimas/metabolismo , Dolor/fisiopatología , Prostaglandina-Endoperóxido Sintasas/metabolismo , Animales , Conducta Animal , Cruzamientos Genéticos , Ciclooxigenasa 1 , Ciclooxigenasa 2 , Femenino , Homocigoto , Calor , Isoenzimas/deficiencia , Isoenzimas/genética , Masculino , Proteínas de la Membrana , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Noqueados , Dolor/genética , Prostaglandina-Endoperóxido Sintasas/deficiencia , Prostaglandina-Endoperóxido Sintasas/genética , Tiempo de Reacción , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Caracteres SexualesAsunto(s)
Fiebre/etiología , Isoenzimas/fisiología , Prostaglandina-Endoperóxido Sintasas/fisiología , Animales , Ácido Araquidónico/metabolismo , Encéfalo/enzimología , Ciclooxigenasa 1 , Ciclooxigenasa 2 , Citocinas/farmacología , Dinoprostona/biosíntesis , Dinoprostona/fisiología , Fiebre/fisiopatología , Interleucina-1/farmacología , Isoenzimas/genética , Lipopolisacáridos/toxicidad , Proteínas de la Membrana , Ratones , Ratones Noqueados , Fosfolipasas A/metabolismo , Prostaglandina-Endoperóxido Sintasas/genética , Especificidad por Sustrato , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
We investigated the molecular mechanisms by which treatment of the human osteoblast-like cell line MG-63 with interleukin 1beta (IL-1) and/or fibroblast growth factor 1 (FGF-1) elicited prostaglandin biosynthesis. IL-1 induced a 5-fold increase in PGE(2) production compared to controls. While treatment with FGF-1 alone did not affect PGE(2) biosynthesis, it enhanced the formation of PGE(2) by IL-1 by an additional 3- to 5-fold. IL-1-induced PGE(2) biosynthesis accompanied increases in steady-state levels of mRNAs encoding cPLA(2) (10- to 15-fold) and PGHS-2 (>3-fold) and concomitant increases in cPLA(2) protein (>3-fold) and PGHS-2 protein (>1. 5-fold). FGF-1 treatment did not affect PGHS-2 gene expression, but enhanced the effect of IL-1 on PGHS-2 expression by an additional 2- to 3-fold. FGF-1 alone enhanced cPLA(2) expression (5-fold), and the combined effects of FGF-1 and IL-1 on cPLA(2) expression were additive. There was no measurable effect of either agonist on PGHS-1 expression. We also discovered that induction of PGE(2) biosynthesis in response to IL-1 or IL-1/FGF-1 was affected by the density of MG-63 cells in culture. Subconfluent cultures displayed a 3- to 10-fold greater response to IL-1 or IL-1/FGF-1 than confluent cultures. The decreased PGE(2) induction by IL-1 in confluent cultures was associated with reduced IL-1 receptor expression. We conclude that the signaling pathways resulting in PGE(2) biosynthesis in response to proinflammatory agents like IL-1 are subject to complex regulation by additional soluble mediators as well as cell-cell or cell-extracellular matrix interactions.
Asunto(s)
Dinoprostona/biosíntesis , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Interleucina-1/metabolismo , Animales , Bovinos , Recuento de Células , Células Cultivadas , Ciclooxigenasa 2 , Relación Dosis-Respuesta a Droga , Inducción Enzimática , Factor 1 de Crecimiento de Fibroblastos , Factor 2 de Crecimiento de Fibroblastos/farmacología , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Interleucina-1/farmacología , Isoenzimas/biosíntesis , Isoenzimas/genética , Proteínas de la Membrana , Osteosarcoma , Fosfolipasas A/biosíntesis , Fosfolipasas A/genética , Prostaglandina-Endoperóxido Sintasas/biosíntesis , Prostaglandina-Endoperóxido Sintasas/genética , ARN Mensajero , Conejos , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Receptores de Interleucina-1/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Células Tumorales CultivadasRESUMEN
Interleukin-1beta (IL-1) is a potent inducer of cyclooxygenase-2 (COX-2) and prostaglandin biosynthesis in many types of cells, yet little is known about the molecular mechanisms regulating IL-1 mediated prostanoid biosynthesis in the endothelium of the microvasculature. Therefore, we examined the cis- and trans-acting factors regulating IL-1-induced COX-2 expression in the human microvascular endothelial cell line, HMEC-1. IL-1 enhanced steady state levels of COX-2 protein and mRNA synthesis by approximately 2-fold which preceded a 2-fold increase in PGF(alpha) biosynthesis. Expression of a series of COX-2 promoter-luciferase constructs in IL-1 treated HMEC-1 cells revealed that the 'full length' (-1432/+59 bp) promoter was 10 times more active than the SV-40 promoter/enhancer and that it could be further activated by IL-1. Surprisingly however, all except for the shortest COX-2 promoter construct retained the ability to respond to IL-1 and luciferase activity driven by -191/+59 bp COX-2 promoter was as responsive to IL-1 as the full-length promoter. Moreover, site-directed promoter mutagenesis and electophoretic mobility shift assays (EMSA) indicate that the combinatorial actions of AP2, NF-IL6, and CRE elements are critical for both constitutive and IL-1-inducible COX-2 promoter activity. Understanding the mechanism(s) regulating COX-2 gene expression and prostaglandin biosynthesis in the microvasculature has important implications with regard to inflammation and angiogenesis in vivo.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT , AMP Cíclico/metabolismo , Proteínas de Unión al ADN/genética , Endotelio Vascular/enzimología , Regulación Enzimológica de la Expresión Génica/genética , Isoenzimas/genética , Proteínas Nucleares/genética , Prostaglandina-Endoperóxido Sintasas/genética , Factores de Transcripción/genética , Transcripción Genética/genética , Secuencia de Bases , Proteína delta de Unión al Potenciador CCAAT , Línea Celular , Ciclooxigenasa 2 , Cartilla de ADN , Dinoprost/biosíntesis , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Humanos , Proteínas de la Membrana , Mutación , Regiones Promotoras Genéticas , Factor de Transcripción AP-2RESUMEN
OBJECTIVE: To determine the effects of cyclooxygenase 1 (COX-1) and COX-2 gene deletion on collagen-induced arthritis (CIA). METHODS: Mice that were susceptible to CIA but lacked either the COX-1 or the COX-2 gene were immunized with type II collagen (CII), and the incidence and severity of arthritis were compared with findings in wild-type animals, by clinical and histologic examination. The immune response was assessed by measuring total CII IgG, IgG1, and IgG2 antibody production in sera from immunized mice. The passive transfer of arthritis, accomplished using anti-CII monoclonal antibodies, was tested in wild-type and COX-deficient (-/-) mice. Splenocytes cultured from CII-immunized wild-type and COX-/- mice were challenged with bovine alpha1(II), and cytokine production was assessed. RESULTS: COX-2 gene deletion reduced the incidence and severity of CIA compared with findings in wild-type and COX-1-/- mice. Histologic examination of joints after the onset of clinical arthritis revealed cartilage erosions, proliferation of the synovial lining, and inflammatory cell infiltration in wild-type and COX-1-/- mice, but not in COX-2-/- mice. COX-2-/- mice exhibited reduced anti-CII IgG antibody levels, indicating a decreased immune response. However, cytokine production by spleen cells from immunized mice indicated no cytokine deficiencies in COX-2-/- mice compared with wild-type or COX-1-/- mice. More important, arthritis could not be passively transferred to naive COX-2-/- mice, indicating a requirement for COX-2 in the pathogenesis of arthritis, independent of the immune response. CONCLUSION: COX-2-/- mice exhibit at least 2 defects resulting in down-modulation of the development of CIA: a reduced immune response to CII demonstrated by a markedly reduced antibody titer, and an "inflammatory" defect reflected by the inability to passively transfer arthritis to COX-2-/- mice.
Asunto(s)
Artritis/inmunología , Enfermedades Autoinmunes/prevención & control , Isoenzimas/genética , Prostaglandina-Endoperóxido Sintasas/genética , Animales , Anticuerpos Monoclonales/inmunología , Formación de Anticuerpos , Artritis/inducido químicamente , Colágeno/inmunología , Ciclooxigenasa 2 , Citocinas/fisiología , Eliminación de Gen , Inmunización , Inmunización Pasiva , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Bazo/química , Bazo/citologíaRESUMEN
Various lines of evidence have implicated inducible cyclooxygenase-2 (COX-2) in fever production. Thus, its expression is selectively enhanced in brain after peripheral exogenous (e.g., lipopolysaccharide [LPS]) or endogenous (e.g., interleukin-1) pyrogen administration, while selective COX-2 inhibitors suppress the fever induced by these pyrogens. In this study, we assessed the febrile response to LPS of congenitally constitutive COX-1 (COX-1-/-) and COX-2 (COX-2-/-)-deficient C57BL/6J-derived mice. COX-1+/- and COX-2+/- mice were also evaluated; controls were wild-type C57BL/6J mice (Jackson Labs.). All the animals were pretrained daily for two weeks to the experimental procedures. LPS was injected intraperitoneally at 1 microgram/mouse; pyrogen-free saline (PFS) was the vehicle and control solution. Core temperatures (Tcs) were recorded using thermocouples inserted 2 cm into the colon. The presence of the COX isoforms was determined in cerebral blood vessels immunocytochemically after the experiments, without knowledge of the functional results. The data showed that the wild-type, COX-1+/-, and COX-1-/- mice all responded to LPS with a 1 degrees C rise in Tc within 1 h; the fever gradually abated over the next 4 h. By contrast, COX-2+/- and COX-2-/- mice displayed no Tc rise after LPS. PFS did not affect the Tc of any animal. It would appear therefore that COX-2 is necessary for LPS-induced fever production.
Asunto(s)
Fiebre/fisiopatología , Isoenzimas/genética , Prostaglandina-Endoperóxido Sintasas/genética , Animales , Southern Blotting , Encéfalo/enzimología , Encéfalo/inmunología , Ciclooxigenasa 1 , Ciclooxigenasa 2 , Dinoprostona/fisiología , Fiebre/inducido químicamente , Fiebre/inmunología , Regulación Enzimológica de la Expresión Génica/inmunología , Genotipo , Lipopolisacáridos/farmacología , Proteínas de la Membrana , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuroinmunomodulación/fisiologíaRESUMEN
In this study, we examined the effects of cholecalciferol, a primary keratinocyte metabolite and precursor of the hydroxylated form of vitamin D3, 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3], on prostaglandin E2 (PGE2) production in human keratinocytes by examining its respective effects on cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), and cytosolic phospholipase A2 (cPLA2) expression, the rate-limiting enzymes regulating PGE2 biosynthesis and differentiation of keratinocytes. Cholecalciferol induced PGE2 production, whereas 1alpha,25(OH)2D3 had no effect on PGE2 production both in normal human epidermal keratinocytes and in the immortalized human keratinocyte cell line, HaCaT. In HaCaT cells, neither COX-1 mRNA nor protein was detectable without stimulation and COX-1 expression did not increase in response to cholecalciferol treatment. Although cPLA2 mRNA and protein were constitutively expressed in untreated HaCaT cells, expression levels did not increase in response to cholecalciferol treatment; however, unlike COX-1 and cPLA2 expression, COX-2 mRNA and COX-2 protein expression increased in response to cholecalciferol treatment. Calphostin C, a potent protein kinase C inhibitor, significantly reduced cholecalciferol-induced PGE2 production by inhibiting cholecalciferol-enhanced COX-2 mRNA and protein expression. These results indicate that (i) 1alpha,25(OH)2D3 does not induce PGE2 biosynthesis in keratinocytes, (ii) cholecalciferol-induced PGE2 production is primarily COX-2 dependent, and (iii) cholecalciferol enhances both COX-2 mRNA and protein expression, via a protein kinase C-dependent mechanism in human keratinocytes. Furthermore, cholecalciferol increased total cellular transglutaminase activity dose dependently, suggesting a potential role for cholecalciferol in regulating the differentiation of human keratinocytes.
Asunto(s)
Colecalciferol/farmacología , Dinoprostona/biosíntesis , Queratinocitos/metabolismo , Transglutaminasas/metabolismo , Ácido Araquidónico/farmacología , Diferenciación Celular/efectos de los fármacos , Línea Celular , Inhibidores Enzimáticos/farmacología , Humanos , Queratinocitos/citología , Queratinocitos/enzimología , Naftalenos/farmacología , Prostaglandina-Endoperóxido Sintasas/genética , Proteína Quinasa C/antagonistas & inhibidores , ARN Mensajero/análisisRESUMEN
Interleukin-1beta (IL-1) is a potent inducer of prostaglandin E2 (PGE2) synthesis. We previously showed that ceramide accumulates in fibroblasts treated with IL-1 and that it enhances IL-1-induced PGE2 production. The present study was undertaken to determine the mechanism(s) by which ceramide and IL-1 interact to enhance PGE2 production by examining their respective effects on the rate-limiting enzymes in PGE2 synthesis, cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), and cytosolic phospholipase A2 (cPLA2). IL-1-induced PGE2 synthesis required approximately 8 h even though COX-1 was constitutively expressed (both mRNA and protein) and enzymatically active in untreated cells. Conversely, COX-2 mRNA was barely detectable in untreated cells but within 2 h, ceramide or IL-1 alone induced a 5 and 20 fold increase in COX-2 mRNA, respectively. However, IL-1 induced COX-2 protein synthesis was only detectable 6-7 h after maximal COX-2 mRNA induction; COX-2 protein accumulation was not induced by ceramide alone. Ceramide however, reduced the length of time required for IL-1 to induce COX-2 protein accumulation and increased COX-2 protein accumulation. IL-1 induced a 15 fold increase in COX-1 mRNA including an alternatively spliced form of COX-1. IL-1, but not ceramide induced cPLA2 mRNA and protein expression which corresponded with the initiation of PGE2 synthesis. These observations indicate that, (1) while either ceramide or IL-1 rapidly induced COX-2 mRNA, COX-2 protein only accumulated in IL-1 treated cells after a delay of 6-7 h, (2) IL-1-induced PGE2 synthesis required both COX-2 and cPLA2 protein synthesis and, (3) ceramide enhanced (temporally and quantitatively) IL-1-induced COX-2 protein
Asunto(s)
Dinoprostona/biosíntesis , Interleucina-1/farmacología , Esfingosina/análogos & derivados , Empalme Alternativo , Ácido Araquidónico/farmacología , Células Cultivadas , Ciclooxigenasa 1 , Ciclooxigenasa 2 , Fibroblastos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Isoenzimas/biosíntesis , Isoenzimas/genética , Proteínas de la Membrana , Fosfolipasas A/biosíntesis , Fosfolipasas A/genética , Fosfolipasas A2 , Prostaglandina-Endoperóxido Sintasas/biosíntesis , Prostaglandina-Endoperóxido Sintasas/genética , Esfingosina/farmacologíaRESUMEN
Prostaglandin E2 (PGE2) production in immortalized, nontransformed cells derived from wild-type, cyclooxygenase 1-deficient (COX-1(-/-)) or cyclooxygenase 2-deficient (COX-2(-/-)) mice was examined after treatment with interleukin (IL)-1beta, tumor necrosis factor alpha, acidic fibroblast growth factor, and phorbol ester (phorbol myristate acetate). Compared with their wild-type counterparts, COX-1(-/-) or COX-2(-/-) cells exhibited substantially enhanced expression of the remaining functional COX gene. Furthermore, both basal and IL-1-induced expression of cytosolic phospholipase A2 (cPLA2), a key enzyme-regulating substrate mobilization for PGE2 biosynthesis, was also more pronounced in both COX-1(-/-) and COX-2(-/-) cells. Thus, COX-1(-/-) and COX-2(-/-) cells have the ability to coordinate the upregulation of the alternate COX isozyme as well as cPLA2 genes to overcome defects in prostaglandin biosynthetic machinery. The potential for cells to alter and thereby compensate for defects in the expression of specific genes such as COX has significant clinical implications given the central role of COX in a variety of disease processes and the widespread use of COX inhibitors as therapeutic agents.
Asunto(s)
Dinoprostona/biosíntesis , Isoenzimas/fisiología , Prostaglandina-Endoperóxido Sintasas/fisiología , Animales , Células Cultivadas , Ciclooxigenasa 1 , Ciclooxigenasa 2 , Inducción Enzimática , Interleucina-1/farmacología , Isoenzimas/biosíntesis , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Proteínas de la Membrana , Ratones , Ratones Endogámicos C57BL , Fosfolipasas A/biosíntesis , Fosfolipasas A2 , Prostaglandina-Endoperóxido Sintasas/biosíntesisRESUMEN
This study shows the induction of HLA-DR (DR) in fibroblasts by IFN-gamma and investigates the molecular mechanisms involved in the further DR down-regulation by TGF-beta 1. Kinetics of DR induction on human dermal fibroblasts by IFN-gamma showed that 1 hr of exposure was required to induce detectable levels of DR, and maximal DR expression was achieved only after 2 days of exposure to IFN-gamma. TGF-beta 1 inhibited DR induction by IFN-gamma, although complete inhibition never could be achieved, even with high concentrations of TGF-beta 1 and low concentrations of IFN-gamma. Inhibition was not accounted for by reduction in cell numbers, as TGF-beta 1 stimulated growth of the fibroblasts. Inhibition of DR induction was seen only if TGF-beta 1 was added during the first 24 hr of IFN-gamma treatment. TGF-beta 1 inhibited equally well if the cells were pretreated for as little as 1 hr and then washed before addition of IFN-gamma. TGF-beta 1 did not cause an overall suppression of protein synthesis. Northern blot analysis revealed that TGF-beta 1 greatly reduced the steady-state level of DR beta mRNA induced by IFN-gamma at 24 hr, and then DRP transcripts became undetectable at later stages. It is concluded that early intracellular signals must build up to stimulate maximum DR synthesis, which, later on, are inactivated or degraded by the action of TGF-beta 1. We suggest that these mechanisms regulating DR gene transcription involve the action of genes coding for specific IFN-gamma-inducible transcriptional factors that are turned on and off in an expeditious manner.
Asunto(s)
Antígenos HLA-DR/biosíntesis , Interferón gamma/farmacología , Factor de Crecimiento Transformador beta/farmacología , Células Cultivadas , Antagonismo de Drogas , Fibroblastos/inmunología , Humanos , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Proteínas Recombinantes/farmacologíaRESUMEN
Ceramide, produced through either the induction of SM hydrolysis or synthesized de novo transduces signals mediating differentiation, growth, growth arrest, apoptosis, cytokine biosynthesis and secretion, and a variety of other cellular functions. A generalized ceramide signal transduction scheme is shown in Fig. 2 in which ceramide is generated through the activation of distinct SMases residing in separate subcellular compartments in response to specific stimuli. Clearly, specificity of cellular responses to ceramide depends upon many factors which include the nature of the stimulus, co-stimulatory signals and the cell type involved. Ceramide derived from neutral SMase activation is thought to be involved in modulating CAPK and MAP kinases, PLA2 (arachidonic acid mobilization), and CAPP while ceramide generated through acid SMase activation appears to be primarily involved in NF-kappa B activation. While there is no apparent cross-talk between these two ceramide-mediated signalling pathways, there is likely to be significant cross-talk between ceramide signalling and other signal transduction pathways (e.g., the PKC and MAP kinase pathways). Other downstream targets for ceramide action include Cox, IL-6 and IL-2 gene expression, PKC zeta, Vav, Rb, c-Myc, c-Fos, c-Jun and other transcriptional regulators. Many, if not all, of these ceramide-mediated signalling events have been identified in the various cells comprising the immune system and are integral to the optimal functioning of the immune system. Although the role of the SM pathway and the generation of ceramide in T and B lymphocytes have only recently been recognized, it is clear from these studies that signal transduction through SM and ceramide can strongly affect the immune response, either directly through cell signalling events, or indirectly through cytokines produced by other cells as the result of signalling through the SM pathway. An overview of the signalling mechanisms coupling ceramide to the modulation of the immune response is depicted in Fig. 3 and shows how ceramide may play pivotal roles in regulating a number of complex processes. The SM pathway represents a potentially valuable focal point for therapeutic control of immune responses, perhaps for either enhancement of the activity of T cells in the elimination of tumors, or the down-regulation of lymphocyte function in instances of autoimmune disease. The recent explosion of knowledge regarding ceramide signalling notwithstanding, a number of critical questions need to be answered before a comprehensive, mechanistic understanding can be formulated relative to the incredibly varied effects of ceramide on cell function. For example, (i) how is a structurally simple molecule like ceramide able to mediate so many different, and sometimes paradoxical, physiological responses ranging from cell proliferation and differentiation to inhibition of cell growth and apoptosis, (ii) what are the molecular identities and modes of activation of the various SMase isoforms, (iii) what determines the distribution of the unique isoforms of SMase in cells of different lineages or at different stages of differentiation, (iv) what is the relative contribution of ceramide generated through SM hydrolysis versus de novo synthesis, and (v) by what means does ceramide interact with specific intracellular targets? Although a number of ceramide-activatable kinases, phosphatases, and their protein substrates have been identified, a more extensive search for additional cellular targets will be indispensable in determining the phosphorylation cascades linking the activation of the SM pathway to the regulation of nuclear events. Clearly, cross-talk between ceramide-induced signal transduction cascades and other signalling pathways adds to the inherent difficulty in distinguishing the specific effects of complex, intertwining signalling pathways.
Asunto(s)
Ceramidas/inmunología , Ceramidas/fisiología , Inmunidad , Transducción de Señal , Fenómenos Fisiológicos Celulares , Humanos , Esfingomielina Fosfodiesterasa/metabolismo , Esfingomielinas/metabolismoRESUMEN
We previously reported that ceramide, the immediate product of sphingomyelin hydrolysis, increases in response to interleukin (IL)-1 beta and plays a role in modulating IL-1 beta-mediated prostaglandin E2 production and cyclooxygenase gene expression in human fibroblasts (Ballou, L. R., C. P. Chao, M. A. Holness, S. C. Barker, and R. Raghow. 1992. J. Biol. Chem. 267:20044-20050). Here we describe the effects of ceramide in another IL-1 beta-mediated process in these cells, the induction of IL-6 production. We found that submicromolar concentrations of C2-ceramide induced IL-6 gene expression and protein production as effectively as IL-1 beta. Both D-erythro-C2-ceramide (a cell-permeable analogue of natural ceramide) and D-threo-C2-ceramide were potent inducers of IL-6 production, while neither L isomer of ceramide was effective. Compared with IL-1 beta-induced IL-6 production, cells treated with ceramide or exogenous sphingomyelinase induced 82 and 50% of maximal IL-1 beta-induced IL-6 levels by 6 h, respectively; by 24 h all three treatments induced similar levels of IL-6 production. Ceramide-induced IL-6 messenger RNA could be detected within 1 h of treatment and reached maximal levels by 24 h. These findings suggest that ceramide may play a role in the regulation of IL-6 gene expression.
Asunto(s)
Ceramidas/farmacología , Interleucina-6/genética , Secuencia de Bases , Células Cultivadas , Cartilla de ADN/química , Fibroblastos , Expresión Génica/efectos de los fármacos , Humanos , Técnicas In Vitro , Datos de Secuencia Molecular , ARN Mensajero/genética , Esfingomielina Fosfodiesterasa/farmacología , Factores de TiempoRESUMEN
The cytokine-mediated stimulation of sphingomyelin (SM) metabolism is emerging as an important signal transduction pathway via the generation of ceramide and sphingosine, products which have been shown to affect a wide variety of biological processes. Because SM-mediated signal transduction is initiated via the hydrolysis of an integral membrane phospholipid by a phospholipase C-like enzyme (sphingomyelinase) to yield lipids which modulate protein kinase C activity, the SM and phosphatidylinositol (PI) signaling pathways share certain similarities. The present study was undertaken to examine the potential for interplay between SM and PI turnover by testing the effects of sphingosine, sphingosine-1-phosphate, and ceramide on PI turnover. In dermal fibroblasts, sphingosine stimulated a rapid dose-dependent hydrolysis of PI, yielding inositol 1,4,5-triphosphate, followed by increased levels of intracellular calcium. Sphingosine-induced inositol phosphate (IP) accumulation was observed between 5 and 30 microM sphingosine with a maximal accumulation of 2.7-fold over control levels. Enhanced IP formation was measured as early as 5 s following sphingosine treatment and IP levels remained elevated for more than 60 min. Intracellular calcium mobilization accompanied the dose-dependent accumulation of IPs in response to sphingosine, although this effect was not apparent until after a 30-40-s lag period. Interestingly, sphingosine-1-phosphate stimulated a more rapid release of intracellular Ca2+ than sphingosine, but it had no effect on PI turnover. DL-threo-Dihydrosphingosine, a competitive inhibitor of sphingosine kinase, stimulates both PI turnover and Ca2+ flux, but does not block the action of sphingosine relative to those two processes. Ceramide (added as C2-ceramide), N-stearylamine, and stearoyl-D-sphingosine did not affect PI turnover or Ca2+ mobilization. Pretreatment of intact cells with pertussis toxin partially inhibited sphingosine-mediated IP accumulation, suggesting a role for guanine nucleotide binding protein(s) (G protein) in sphingosine-stimulated PI turnover. Furthermore, guanosine 5'-O-(3-thiotriphosphate) stimulated, whereas guanosine 5'-O-(2-thiodiphosphate) inhibited, sphingosine-induced IP accumulation in permeabilized cells. Collectively, these data suggest that sphingosine enhances PI turnover by stimulating phospholipase C activity, and the activation of this process may be modulated by G protein interactions. Thus, the regulation of PI turnover and Ca2+ mobilization by sphingosine may represent another mechanism by which sphingosine modulates cell function and that these effects can be distinguished from those of ceramide.
Asunto(s)
Calcio/metabolismo , Fosfatidilinositoles/metabolismo , Esfingosina/metabolismo , Células Cultivadas , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Nucleótidos de Guanina/farmacología , Humanos , Cinética , Toxina del Pertussis , Fosforilación , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
To examine the possible role of basic fibroblast growth factor (FGF) in regulating the effects of TNF alpha, we tested the effect of FGF on TNF alpha-mediated PGE2 production and TNF alpha receptor expression in human fibroblasts. We found that, while FGF alone had no effect on PGE2 production, it enhanced the amount of PGE2 produced in response to TNF alpha between 3 and 11-fold. FGF stimulated TNF alpha-induced PGE2 production independent of potential TNF alpha-mediated IL-1 production, as neither anti-IL-1 mAbs nor IL-1 receptor antagonist protein (IRAP) inhibited TNF alpha induced-PGE2 production or the stimulatory effect of FGF. A one minute exposure of cells to FGF prior to removal was sufficient to significantly enhance TNF alpha-induced PGE2 production; the maximal FGF effect was reached after a 6 h preincubation. We also found that FGF significantly enhanced TNF alpha receptor expression. Untreated fibroblasts expressed approximately 3,900 receptors/cell, while cells treated with FGF for 6 h expressed approximately 9,500 receptors/cell, a 2.4-fold increase in receptor number; there was no apparent change in affinity for TNF alpha (Kd 3.8 x 10(-11) M). The FGF-mediated increase in TNF alpha receptor expression and TNF alpha-mediated PGE2 production could be abolished by FGF mAbs, indicating a specific FGF effect. These results show that FGF increases TNF alpha receptor expression and suggest that this may account, at least in part, for the ability of FGF to enhance TNF alpha-mediated PGE2 production in human fibroblasts.