RESUMEN
Almost all deaths from breast cancer arise from metastasis of the transformed cells to other sites in the body. Hence, uncovering a means of inhibiting breast cancer cell migration would provide a significant advance in the treatment of this disease. Stimulation of the cAMP signaling pathway has been shown to inhibit migration and motility of a number of cell types. A very effective way of selectively stimulating cAMP signaling is through inhibition of cyclic nucleotide phosphodiesterases (PDEs). Therefore, we examined full expression profiles of all known PDE genes at the mRNA and protein levels in four human breast cancer cell lines and eight patients' breast cancer tissues. By these analyses, expression of almost all PDE genes was seen in both cell lines and tissues. In the cell lines, appreciable expression was seen for PDEs 1C, 2A, 3B, 4A, 4B, 4D, 5A, 6B, 6C, 7A, 7B, 8A, 9A, 10A, and 11A. In patients' tissues, appreciable expression was seen for PDEs 1A, 3B, 4A, 4B, 4C, 4D, 5A, 6B, 6C, 7A, 7B, 8A, 8B, and 9A. PDE8A mRNA in particular is prominently expressed in all cell lines and patients' tissue samples examined. We show here that stimulation of cAMP signaling with cAMP analogs, forskolin, and PDE inhibitors, including selective inhibitors of PDE3, PDE4, PDE7, and PDE8, inhibit aggressive triple negative MDA-MB-231 breast cancer cell migration. Under the same conditions, these agents had little effect on breast cancer cell proliferation. This study demonstrates that PDE inhibitors inhibit breast cancer cell migration, and thus may be valuable therapeutic targets for inhibition of breast cancer metastasis. Since PDE8A is expressed in all breast cancer samples, and since dipyridamole, which inhibits PDE8, and PF-04957325, a selective PDE8 inhibitor, both inhibit migration, it suggests that PDE8A may be a valuable novel target for treatment of this disease.
Asunto(s)
Neoplasias de la Mama/metabolismo , Movimiento Celular , AMP Cíclico/metabolismo , Transducción de Señal , Neoplasias de la Mama/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Expresión Génica , Perfilación de la Expresión Génica , Humanos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , ARN Mensajero/genéticaRESUMEN
cAMP signalling is both a major pathway as well as a key therapeutic target for inducing immune tolerance and is involved in Treg cell (regulatory T-cell) function. To achieve potent immunoregulation, cAMP can act through several downstream effectors. One proposed mechanism is that cAMP-mediated suppression, including immunosuppression by Treg cells, results from activation of PKA (protein kinase A) leading to the induction of the transcription factor ICER (inducible cAMP early repressor). In the present study, we examined CD4(+)CD25(-) Teff cell (effector T-cell) and CD4(+)CD25(+) Treg cell immune responses in Crem (cAMP-response-element modulator) gene-deficient mice which lack ICER (Crem(-/-)/ICER-deficient mice). ICER deficiency did not significantly alter the frequency or number of Treg cells and Teff cells. Treg cells or a pharmacological increase in cAMP suppressed Teff cells from Crem(+/+) and Crem(-/-)/ICER-deficient mice to an equivalent degree, demonstrating that ICER is dispensable in these functions. Additionally, activating the cAMP effector Epac (exchange protein directly activated by cAMP) suppressed Teff cells. Treg cells expressed low levels of all cyclic nucleotide Pde (phosphodiesterase) genes tested, but high levels of Epac. These data identify ICER as a redundant mediator of Treg cells and cAMP action on Teff cells and suggest that Epac may function as an alternative effector to promote cAMP-dependent Teff cell suppression.
Asunto(s)
Modulador del Elemento de Respuesta al AMP Cíclico/inmunología , Proteínas Quinasas Dependientes de AMP Cíclico/inmunología , AMP Cíclico/inmunología , Factores de Intercambio de Guanina Nucleótido/inmunología , Tolerancia Inmunológica/fisiología , Linfocitos T Reguladores/inmunología , Animales , Proliferación Celular/fisiología , AMP Cíclico/genética , Modulador del Elemento de Respuesta al AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Femenino , Factores de Intercambio de Guanina Nucleótido/genética , Ratones , Ratones Noqueados , Linfocitos T Reguladores/citologíaRESUMEN
After decades of development, inhibitors targeting cyclic nucleotide phosphodiesterases (PDEs) expressed in leukocytes have entered clinical practice for the treatment of inflammatory disorders, with three PDE4 inhibitors being in clinical use as therapeutics for psoriasis, psoriatic arthritis, chronic obstructive pulmonary disease and atopic dermatitis. In contrast, the PDE8 family that is upregulated in pro-inflammatory T cells is a largely unexplored therapeutic target. We have previously demonstrated a role for the PDE8A-Raf-1 kinase complex in the regulation of myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55) activated CD4+ effector T cell adhesion and locomotion by a mechanism that differs from PDE4 activity. In this study, we explored the in vivo treatment of experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis (MS) induced in mice immunized with MOG using the PDE8-selective inhibitor PF-04957325. For treatment in vivo, mice with EAE were either subcutaneously (s.c.) injected three times daily (10 mg/kg/dose), or were implanted subcutaneously with Alzet mini-osmotic pumps to deliver the PDE8 inhibitor (15.5 mg/kg/day). The mice were scored daily for clinical signs of paresis and paralysis which were characteristic of EAE. We observed the suppression of the clinical signs of EAE and a reduction of inflammatory lesion formation in the CNS by histopathological analysis through the determination of the numbers of mononuclear cells isolated from the spinal cord of mice with EAE. The PDE8 inhibitor treatment reduces the accumulation of both encephalitogenic Th1 and Th17 T cells in the CNS. Our study demonstrates the efficacy of targeting PDE8 as a treatment of autoimmune inflammation in vivo by reducing the inflammatory lesion load.
Asunto(s)
Encefalomielitis Autoinmune Experimental , Animales , Ratones , Ratones Endogámicos C57BL , Glicoproteína Mielina-Oligodendrócito/metabolismo , Inhibidores de Fosfodiesterasa , Hidrolasas Diéster Fosfóricas , Células Th17RESUMEN
Inhibitors targeting cyclic nucleotide phosphodiesterases (PDEs) expressed in leukocytes have entered clinical practice to treat inflammatory disorders, with three PDE4 inhibitors currently in clinical use as therapeutics for psoriasis, psoriatic arthritis, atopic dermatitis and chronic obstructive pulmonary disease. In contrast, the PDE8 family that is upregulated in pro-inflammatory T cells is a largely unexplored therapeutic target. It was shown that PDE8A plays a major role in controlling T cell and breast cancer cell motility, including adhesion to endothelial cells under physiological shear stress and chemotaxis. This is a unique function of PDE8 not shared by PDE4, another cAMP specific PDE, employed, as noted, as an anti-inflammatory therapeutic. Additionally, a regulatory role was shown for the PDE8A-rapidly accelerated fibrosarcoma (Raf)-1 kinase signaling complex in myelin antigen reactive CD4+ effector T cell adhesion and locomotion by a mechanism differing from that of PDE4. The PDE8A-Raf-1 kinase signaling complex affects T cell motility, at least in part, via regulating the LFA-1 integrin mediated adhesion to ICAM-1. The findings that PDE8A and its isoforms are expressed at higher levels in naive and myelin oligodendrocyte glycoprotein (MOG)35 - 55 activated effector T (Teff) cells compared to regulatory T (Treg) cells and that PDE8 inhibition specifically affects MOG35 - 55 activated Teff cell adhesion, indicates that PDE8A could represent a new beneficial target expressed in pathogenic Teff cells in CNS inflammation. The implications of this work for targeting PDE8 in inflammation will be discussed in this review.
RESUMEN
The cAMP signalling pathway has emerged as a key regulator of haematopoietic cell proliferation, differentiation and apoptosis. In parallel, general understanding of the biology of cyclic nucleotide PDEs (phosphodiesterases) has advanced considerably, revealing the remarkable complexity of this enzyme system that regulates the amplitude, kinetics and location of intracellular cAMP-mediated signalling. The development of therapeutic inhibitors of specific PDE gene families has resulted in a growing appreciation of the potential therapeutic application of PDE inhibitors to the treatment of immune-mediated illnesses and haematopoietic malignancies. This review summarizes the expression and function of PDEs in normal haematopoietic cells and the evidence that family-specific inhibitors will be therapeutically useful in myeloid and lymphoid malignancies.
Asunto(s)
Neoplasias Hematológicas/tratamiento farmacológico , Neoplasias Hematológicas/enzimología , Nucleótidos Cíclicos/metabolismo , Inhibidores de Fosfodiesterasa/uso terapéutico , Hidrolasas Diéster Fosfóricas/metabolismo , Humanos , Hidrolasas Diéster Fosfóricas/genética , Sensibilidad y EspecificidadRESUMEN
The levels of cAMP are regulated by phosphodiesterase enzymes (PDEs), which are targets for the treatment of inflammatory disorders. We have previously shown that PDE8 regulates T cell motility. Here, for the first time, we report that PDE8A exerts part of its control of T cell function through the V-raf-1 murine leukemia viral oncogene homolog 1 (Raf-1) kinase signaling pathway. To examine T cell motility under physiologic conditions, we analyzed T cell interactions with endothelial cells and ligands in flow assays. The highly PDE8-selective enzymatic inhibitor PF-04957325 suppresses adhesion of in vivo myelin oligodendrocyte glycoprotein (MOG) activated inflammatory CD4+ T effector (Teff) cells to brain endothelial cells under shear stress. Recently, PDE8A was shown to associate with Raf-1 creating a compartment of low cAMP levels around Raf-1 thereby protecting it from protein kinase A (PKA) mediated inhibitory phosphorylation. To test the function of this complex in Teff cells, we used a cell permeable peptide that selectively disrupts the PDE8A-Raf-1 interaction. The disruptor peptide inhibits the Teff-endothelial cell interaction more potently than the enzymatic inhibitor. Furthermore, the LFA-1/ICAM-1 interaction was identified as a target of disruptor peptide mediated reduction of adhesion, spreading and locomotion of Teff cells under flow. Mechanistically, we observed that disruption of the PDE8A-Raf-1 complex profoundly alters Raf-1 signaling in Teff cells. Collectively, our studies demonstrate that PDE8A inhibition by enzymatic inhibitors or PDE8A-Raf-1 kinase complex disruptors decreases Teff cell adhesion and migration under flow, and represents a novel approach to target T cells in inflammation.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/genética , Linfocitos T CD4-Positivos/metabolismo , Inflamación/genética , Proteínas Proto-Oncogénicas c-raf/genética , 3',5'-AMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , 3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , Animales , Linfocitos T CD4-Positivos/patología , Adhesión Celular/efectos de los fármacos , Permeabilidad de la Membrana Celular/efectos de los fármacos , Movimiento Celular , AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Inhibidores Enzimáticos/administración & dosificación , Humanos , Inflamación/metabolismo , Inflamación/patología , Ratones , Péptidos/genética , Fosforilación , Proteínas Proto-Oncogénicas c-raf/metabolismo , Transducción de SeñalRESUMEN
Abolishing the inhibitory signal of intracellular cAMP is a prerequisite for effector T (Teff) cell function. The regulation of cAMP within leukocytes critically depends on its degradation by cyclic nucleotide phosphodiesterases (PDEs). We have previously shown that PDE8A, a PDE isoform with 40-100-fold greater affinity for cAMP than PDE4, is selectively expressed in Teff vs. regulatory T (Treg) cells and controls CD4(+) Teff cell adhesion and chemotaxis. Here, we determined PDE8A expression and function in CD4(+) Teff cell populations in vivo. Using magnetic bead separation to purify leukocyte populations from the lung draining hilar lymph node (HLN) in a mouse model of ovalbumin-induced allergic airway disease (AAD), we found by Western immunoblot and quantitative (q)RT-PCR that PDE8A protein and gene expression are enhanced in the CD4(+) T cell fraction over the course of the acute inflammatory disease and recede at the late tolerant non-inflammatory stage. To evaluate PDE8A as a potential drug target, we compared the selective and combined effects of the recently characterized highly potent PDE8-selective inhibitor PF-04957325 with the PDE4-selective inhibitor piclamilast (PICL). As previously shown, PF-04957325 suppresses T cell adhesion to endothelial cells. In contrast, we found that PICL alone increased firm T cell adhesion to endothelial cells by ~20% and significantly abrogated the inhibitory effect of PF-04957325 on T cell adhesion by over 50% when cells were co-exposed to PICL and PF-04957325. Despite its robust effect on T cell adhesion, PF-04957325 was over two orders of magnitude less efficient than PICL in suppressing polyclonal Teff cell proliferation, and showed no effect on cytokine gene expression in these cells. More importantly, PDE8 inhibition did not suppress proliferation and cytokine production of myelin-antigen reactive proinflammatory Teff cells in vivo and in vitro. Thus, targeting PDE8 through PF-04957325 selectively regulates Teff cell interactions with endothelial cells without marked immunosuppression of proliferation, while PDE4 inhibition has partially opposing effects. Collectively, our data identify PF-04957325 as a novel function-specific tool for the suppression of Teff cell adhesion and indicate that PDE4 and PDE8 play unique and non-redundant roles in the control of Teff cell functions.
RESUMEN
cAMP-mediated signaling potentiates glucocorticoid-mediated apoptosis in lymphoid cells, but an effective means by which to take advantage of this observation in the treatment of lymphoid malignancies has not been identified. The primary objective of the current study was to determine whether PDE4 inhibitors, a class of compounds in late clinical development that raise intracellular cAMP levels by inhibiting type 4 cyclic nucleotide phosphodiesterases (PDE4), increase the efficacy of glucocorticoid-mediated apoptosis in leukemic cells from patients with B cell chronic lymphocytic leukemia (B-CLL). Rolipram, a prototypic PDE4 inhibitor, synergized with glucocorticoids in inducing B-CLL but not T cell apoptosis. Rolipram also augmented glucocorticoid receptor element (GRE) transactivation in B-CLL cells. In contrast, inhibition of protein kinase A (PKA) with the cAMP antagonist Rp-8Br-cAMPS reversed both glucocorticoid-induced apoptosis and GRE transactivation. CCRF-CEM cells, a well-studied model of glucocorticoid and cAMP-induced apoptosis, differed from B-CLL cells in that stimulation of adenylyl cyclase with the diterpene forskolin was required to increase both glucocorticoid-mediated apoptosis and GRE activation, while PDE4 inhibition had no effect. Consistent with these results, inhibition of PDE4 induced cAMP elevation in B-CLL but not CCRF-CEM cells, while forskolin augmented cAMP levels in CCRF-CEM but not B-CLL cells. While rolipram treatment up-regulated PDE4B in B-CLL, forskolin treatment up-regulated PDE4D in CCRF-CEM cells. These studies suggest that PKA is required for and enhances glucocorticoid-induced apoptosis in B-CLL by modulating glucocorticoid receptor signal transduction. Clinical trials that examine whether PDE4 inhibitors enhance the efficacy of glucocorticoid-containing chemotherapy regimens in B-CLL are indicated.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , Apoptosis/efectos de los fármacos , Glucocorticoides/farmacología , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Inhibidores de Fosfodiesterasa/farmacología , Adenilil Ciclasas/fisiología , Células Cultivadas , Colforsina/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3 , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4 , Sinergismo Farmacológico , Humanos , Leucemia Linfocítica Crónica de Células B/patología , Elementos de Respuesta , Rolipram/farmacología , Activación Transcripcional/efectos de los fármacosRESUMEN
Stimulation of cAMP signaling induces apoptosis in glucocorticoid-sensitive and resistant CEM leukemic and MM.1 multiple myeloma cell lines, and this effect is enhanced by dexamethasone in both glucocorticoid-sensitive cell types and in glucocorticoid-resistant CEM cells. Expression of the mRNA for the glucocorticoid receptor alpha (GR) promoters 1A3, 1B and 1C, expression of mRNA and protein for GR, and the BH3-only proapoptotic proteins, Bim and Bad, and the phosphorylation state of Bad were examined following stimulation of the cAMP and glucocorticoid signaling pathways. Expression levels of GR promoters were increased by cAMP and glucocorticoid signaling, but GR protein expression was little changed in CEM and decreased in MM.1 cells. Stimulation of these two signaling pathways induced Bim in CEM cells, induced Bad in MM.1 cells, and activated Bad, as indicated by its dephosphorylation on ser112, in both cell types. This study shows that leukemic and multiple myeloma cells, including those resistant to glucocorticoids, can be induced to undergo apoptosis by stimulating the cAMP signaling pathway, with enhancement by glucocorticoids, and the mechanism by which this occurs may be related to changes in Bim and Bad expression, and in all cases, to activation of Bad.
RESUMEN
Adenosine appears to be an endogenous somnogen. The lateral dorsal tegmental/pedunculopontine nucleus (LDT/PPT) located in the mesopontine tegmentum is important in the regulation of arousal. Neurons in this nucleus are strongly hyperpolarized by adenosine and express neuronal nitric oxide synthase. Zaprinast is a cyclic nucleotide phosphodiesterase inhibitor, and has been shown in the hippocampal slice to inhibit the field excitatory postsynaptic potential. This action could be blocked by an adenosine receptor antagonist, and therefore is presumably due to adenosine release stimulated by zaprinast. In the present study we tested the effect of zaprinast on extracellular adenosine accumulation in pontine slices containing the LDT. Zaprinast at 10 microM evoked an increase in extracellular adenosine concentration. This effect was blocked by impermeant inhibitors of 5'-nucleotidase, indicating that the extracellular adenosine was derived from extracellular AMP. However, inhibitors of cAMP degradation had little or no effect on zaprinast-evoked adenosine accumulation, suggesting that extracellular cAMP was not the source. Removal of extracellular calcium inhibited the effect of zaprinast. These results demonstrate that a pathway exists by which zaprinast stimulates extracellular adenosine accumulation, and the presence of this pathway in the pontine slice suggests the possibility that it may be relevant for the regulation of behavioral state.
Asunto(s)
Adenosina/metabolismo , Inhibidores de Fosfodiesterasa/farmacología , Puente/efectos de los fármacos , Puente/metabolismo , Purinonas/farmacología , Animales , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Técnicas In Vitro , Ratas , Ratas Sprague-DawleyRESUMEN
Considerable epidemiological evidence demonstrates a positive association between artificial light at night (LAN) levels and incidence rates of breast cancer, suggesting that exposure to LAN is a risk factor for breast cancer. There is a 30-50% higher risk of breast cancer in the highest LAN exposed countries compared to the lowest LAN countries, and studies showing higher incidence of breast cancer among shift workers exposed to more LAN have led the International Agency for Research on Cancer to classify shift work as a probable human carcinogen. Nevertheless, the means by which light can affect breast cancer is still unknown. In this study we examined established human breast cancer cell lines and patients' primary breast cancer tissues for expression of genetic components of phosphodiesterase 6 (PDE6), a cGMP-specific PDE involved in transduction of the light signal, and previously thought to be selectively expressed in photoreceptors. By microarray analysis we find highly significant expression of mRNA for the PDE6B, PDE6C, and PDE6D genes in both the cell lines and patients' tissues, minimal expression of PDE6A and PDE6G and no expression of PDE6H. Using antibody specific for PDE6ß, we find expression of PDE6B protein in a wide range of patients' tissues by immunohistochemistry, and in MCF-7 breast cancer cells by immunofluorescence and Western blot analysis. Considerable expression of key circadian genes, PERIOD 2, CLOCK, TIMELESS, CRYPTOCHROME 1, and CRYPTOCHROME 2 was also seen in all breast cancer cell lines and all patients' breast cancer tissues. These studies indicate that genes for PDE6 and control of circadian rhythm are expressed in human breast cancer cells and tissues and may play a role in transducing the effects of light on breast cancer.
RESUMEN
Stimulation of the cAMP signaling pathway has been shown to induce apoptosis and augment the effects of glucocorticoids in inducing apoptosis in leukemic cells. We recently reported that in primary B cell chronic lymphocytic leukemic (B-CLL) cells, apoptosis could be induced by stimulating the cAMP signaling pathway with a phosphodiesterase4 (PDE4) inhibitor alone; while in contrast, in the CEM T leukemic cell line, PDE4 inhibitors alone were ineffective, and concurrent stimulation of adenylyl cyclase was required to see effects [Tiwari et al. (2005)]. We report here that in the CEM and Jurkat T leukemic cell lines, the most abundantly expressed PDEs are PDE3B, PDE4A, PDE4D, PDE7A, and PDE8A. Selective inhibition of PDE3, PDE4 or PDE7 alone produces little effect on cell viability, but inhibition of all three of these PDEs together dramatically enhances glucocorticoid-induced apoptosis in CEM cells, and overcomes glucocorticoid resistance in a glucocorticoid-resistant CEM cell line. These studies indicate that for some leukemic cell types, a desired therapeutic effect may be achieved by inhibiting more than one form of PDE.
Asunto(s)
Apoptosis/efectos de los fármacos , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 7/antagonistas & inhibidores , Resistencia a Antineoplásicos/efectos de los fármacos , Glucocorticoides/farmacología , Leucemia de Células T/enzimología , Inhibidores de Fosfodiesterasa 3 , Inhibidores de Fosfodiesterasa 4 , Apoptosis/fisiología , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/fisiología , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/fisiología , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 7/fisiología , Resistencia a Antineoplásicos/fisiología , Sinergismo Farmacológico , Humanos , Células Jurkat , Leucemia de Células T/tratamiento farmacológico , Inhibidores de Fosfodiesterasa/farmacología , Células Tumorales CultivadasRESUMEN
BACKGROUND: Abolishing the inhibitory signal of intracellular cAMP by phosphodiesterases (PDEs) is a prerequisite for effector T (Teff) cell function. While PDE4 plays a prominent role, its control of cAMP levels in Teff cells is not exclusive. T cell activation has been shown to induce PDE8, a PDE isoform with 40- to 100-fold greater affinity for cAMP than PDE4. Thus, we postulated that PDE8 is an important regulator of Teff cell functions. METHODOLOGY/PRINCIPAL FINDINGS: We found that Teff cells express PDE8 in vivo. Inhibition of PDE8 by the PDE inhibitor dipyridamole (DP) activates cAMP signaling and suppresses two major integrins involved in Teff cell adhesion. Accordingly, DP as well as the novel PDE8-selective inhibitor PF-4957325-00 suppress firm attachment of Teff cells to endothelial cells. Analysis of downstream signaling shows that DP suppresses proliferation and cytokine expression of Teff cells from Crem-/- mice lacking the inducible cAMP early repressor (ICER). Importantly, endothelial cells also express PDE8. DP treatment decreases vascular adhesion molecule and chemokine expression, while upregulating the tight junction molecule claudin-5. In vivo, DP reduces CXCL12 gene expression as determined by in situ probing of the mouse microvasculature by cell-selective laser-capture microdissection. CONCLUSION/SIGNIFICANCE: Collectively, our data identify PDE8 as a novel target for suppression of Teff cell functions, including adhesion to endothelial cells.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , Modulador del Elemento de Respuesta al AMP Cíclico/metabolismo , Linfocitos T/citología , 3',5'-AMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , 3',5'-AMP Cíclico Fosfodiesterasas/genética , Animales , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/metabolismo , Adhesión Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Quimiocina CXCL12/genética , Claudina-5 , AMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/genética , Citocinas/metabolismo , Dipiridamol/farmacología , Endotelio Vascular/citología , Femenino , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Hidrólisis , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Inhibidores de Fosfodiesterasa/farmacología , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Células TH1/efectos de los fármacos , Células TH1/metabolismo , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/metabolismo , Factores de TiempoRESUMEN
The immune system depends on chemokines to recruit lymphocytes to tissues in inflammatory diseases. This study identifies PDE8 as a new target for inhibition of chemotaxis of activated lymphocytes. Chemotactic responses of unstimulated and concanavalin A-stimulated mouse splenocytes and their modulation by agents that stimulate the cAMP signaling pathway were compared. Dibutyryl cAMP inhibited migration of both cell types. In contrast, forskolin and 3-isobutyl-1-methylxanthine each inhibited migration of unstimulated splenocytes, with little effect on migration of stimulated splenocytes. Only dipyridamole alone, a PDE inhibitor capable of inhibiting PDE8, strongly inhibited migration of stimulated and unstimulated splenocytes and this inhibition was enhanced by forskolin and reversed by a PKA antagonist. Following concanavalin A stimulation, mRNA for PDE8A1 was induced. These results suggest that in employing PDE inhibitor therapy for inflammatory illnesses, inhibition of PDE8 may be required to inhibit migration of activated lymphocytes to achieve a full therapeutic effect.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/fisiología , Quimiotaxis/fisiología , Activación de Linfocitos/fisiología , 1-Metil-3-Isobutilxantina/farmacología , 3',5'-AMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , Animales , Quimiotaxis/efectos de los fármacos , Colforsina/farmacología , Concanavalina A/farmacología , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Dipiridamol/farmacología , Inflamación/tratamiento farmacológico , Activación de Linfocitos/efectos de los fármacos , Ratones , Inhibidores de Fosfodiesterasa/farmacología , ARN Mensajero/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Bazo/citología , Bazo/efectos de los fármacos , Bazo/metabolismo , Factores de TiempoRESUMEN
Adenosine is an important regulator of neuronal excitability. Zaprinast is a cyclic nucleotide phosphodiesterase inhibitor, and has been shown in the hippocampal slice to suppress excitation. This action can be blocked by an adenosine receptor antagonist, and therefore is presumably due to adenosine release stimulated by exposure to zaprinast. To explore the mechanism of this phenomenon further, we examined the effect of zaprinast on adenosine release itself in cultured rat forebrain neurons. Zaprinast significantly stimulated extracellular adenosine accumulation. The effect of zaprinast on adenosine appeared to be mediated by increasing intracellular cyclic adenosine monophosphate (cAMP) and activation of protein kinase A (PKA): (i) zaprinast stimulated intracellular cAMP accumulation; (ii) a cAMP antagonist (Rp-8-Br-cAMP) significantly reduced the zaprinast effect on adenosine; (iii) an inhibitor of phosphodiesterase (PDE)1 (vinpocetine) and an activator of adenylate cyclase (forskolin) mimicked the effect of zaprinast on adenosine. We also found that zaprinast had no effect on adenosine in astrocyte cultures, and tetrodotoxin completely blocked zaprinast-evoked adenosine accumulation in neuronal cultures, suggesting that neuronal activity was likely to be involved. Consistent with a dependence on neuronal activity, NMDA receptor antagonists (MK-801 and D-APV) and removal of extracellular glutamate by glutamate-pyruvate transaminase blocked the effect of zaprinast. In addition, zaprinast was shown to stimulate glutamate release. Thus, our data suggest that zaprinast-evoked adenosine accumulation is likely to be mediated by stimulation of glutamate release by a cAMP- and PKA-dependent mechanism, most likely by inhibition of PDE1 in neurons. Furthermore, regulation of cAMP, either by inhibiting cAMP-PDE activity or by stimulating adenylate cyclase activity, may play an important role in modulating neuronal excitability. These data suggest the existence of a homeostatic negative feedback loop in which increases in neuronal activity are damped by release of adenosine following activation of glutamate receptors.