RESUMEN
Adrenocortical carcinomas (ACCs) overexpress insulin-like growth factor 2 (IGF2), that drives a proliferative autocrine loop by binding to IGF1R and IR, but IGF1R/IR-targeted therapies failed in ACC patients. The cytoskeleton actin-binding protein filamin A (FLNA) impairs IR signalling in melanoma cells. Aims of this study were to test FLNA involvement in regulating IGF1R and IR responsiveness to both IGF2 and inhibitors in ACC. In ACC cells H295R and SW13 and primary cultures (1ACC, 4 adenomas) we found that IGF1R and IR interacted with FLNA, and FLNA silencing increased IGF1R and reduced IR expression, with a downstream effect of increased cell proliferation and ERK phosphorylation. In addition, FLNA knockdown potentiated antiproliferative effects of IGF1R/IR inhibitor Linsitinib and IGF1R inhibitor NVP-ADW742 in H295R. Finally, Western blot showed lower FLNA expression in ACCs (n = 10) than in ACAs (n = 10) and an inverse correlation of FLNA/IGF1R ratio with ERK phosphorylation in ACCs only. In conclusion, we demonstrated that low FLNA levels enhance both IGF2 proliferative effects and IGF1R/IR inhibitors efficacy in ACC cells, suggesting FLNA as a new factor influencing tumor clinical behavior and the response to the therapy with IGF1R/IR-targeted drugs.
Asunto(s)
Neoplasias de la Corteza Suprarrenal/patología , Carcinoma Corticosuprarrenal/patología , Biomarcadores de Tumor/metabolismo , Filaminas/metabolismo , Factor II del Crecimiento Similar a la Insulina/metabolismo , Receptor IGF Tipo 1/antagonistas & inhibidores , Receptor de Insulina/antagonistas & inhibidores , Citoesqueleto de Actina/metabolismo , Neoplasias de la Corteza Suprarrenal/tratamiento farmacológico , Neoplasias de la Corteza Suprarrenal/metabolismo , Carcinoma Corticosuprarrenal/tratamiento farmacológico , Carcinoma Corticosuprarrenal/metabolismo , Apoptosis , Biomarcadores de Tumor/genética , Proliferación Celular , Filaminas/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Imidazoles/farmacología , Factor II del Crecimiento Similar a la Insulina/genética , Mitógenos/farmacología , Pirazinas/farmacología , Pirimidinas/farmacología , Pirroles/farmacología , Transducción de Señal , Células Tumorales CultivadasRESUMEN
Medullary thyroid carcinoma (MTC) is a rare neuroendocrine neoplasm of the parafollicular thyroid C cells. Although somatostatin receptors are expressed by MTCs, treatment with octreotide has shown poor efficacy, whereas recently pasireotide has demonstrated antiproliferative effects in persistent postoperative MTCs. Aim of this study was to test the effects of octreotide and pasireotide on MTC cells proliferation, cell cycle proteins expression, MAPK activation, apoptosis, calcitonin secretion, migration and invasion in TT cell line as well as in primary MTC cultured cells. Our results showed that both octreotide and pasireotide reduced TT cell proliferation (-35.2 ± 12.1%, p < 0.001, and -25.3 ± 24.8%, p < 0.05, at 10-8 M, respectively), with concomitant inhibition of ERK phosphorylation and cyclin D1 expression. This cytostatic effect was accompanied by a proapoptotic action, with an increase of caspase3/7 activity of 1.5-fold. Moreover, both octreotide and pasireotide inhibited cell migration (-50.9 ± 11.3%, p < 0.01, and -40.5 ± 17%, p < 0.05, respectively) and invasion (-61.3 ± 35.1%, p < 0.05, and -49.7 ± 18%, p < 0.01, respectively). No effect was observed on calcitonin secretion. We then tried to extend these observations to primary cultures (n = 5). Octreotide and/or pasireotide were effective in reducing cells proliferation in 3 out of 5 tumors, and to induce cell apoptosis in 1 out of 3 MTCs. Both octreotide and pasireotide were able to reduce cell migration in all MTC tested. SST2, SST3 and SST5 were expressed in all MTC, with a tendency to increased expression of SST2 in RET mutated vs wild type MTCs. In agreement, inhibition of mutated RET in TT cells reduced SST2 expression. In conclusion, we demonstrated that octreotide and pasireotide inhibited cell proliferation and invasiveness in a subset of MTC, supporting their potential use in the control of tumor growth.
Asunto(s)
Carcinoma Neuroendocrino/patología , Octreótido/farmacología , Somatostatina/análogos & derivados , Neoplasias de la Tiroides/patología , Apoptosis/efectos de los fármacos , Calcitonina/metabolismo , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Humanos , Mutación/genética , Invasividad Neoplásica , Proteínas Proto-Oncogénicas c-ret/genética , Somatostatina/metabolismo , Somatostatina/farmacología , Células Tumorales CultivadasRESUMEN
Medullary thyroid carcinoma (MTC) is a rare neuroendocrine tumor that originates from parafollicular thyroid C cells and accounts for 5% of thyroid cancers. In inherited cases of MTC, and in about 40% of sporadic cases, activating mutations of the receptor tyrosine kinase proto-oncogene RET are found. Constitutively active RET triggers signaling pathways involved in cell proliferation, survival and motility, but the mechanisms underlying malignant transformation of C-cells have been only partially elucidated. Cofilin is a key regulator of actin cytoskeleton dynamics. A crucial role of cofilin in tumor development, progression, invasion and metastasis has been demonstrated in different human cancers, but no data are available in MTC. Interestingly, RET activation upregulates cofilin gene expression. The aim of this study was to investigate cofilin contribution in invasiveness and growth of MTC cells, and its relevance in the context of mutant RET signaling. We found that cofilin transfection in human MTC cell line TT significantly increased migration (178⯱â¯44%, pâ¯<â¯0.001), invasion (165⯱â¯28%, pâ¯<â¯0.01) and proliferation (146⯱â¯18%, pâ¯<â¯0.001), accompanied by an increase of ERK1/2 phosphorylation (2.23-fold) and cyclin D1 levels (1.43-fold). Accordingly, all these responses were significantly reduced after genetic silencing of cofilin (-55⯱â¯10% migration, pâ¯<â¯0.001, -41⯱â¯8% invasion, pâ¯<â¯0.001, -17⯱â¯3% proliferation, pâ¯<â¯0.001). These results have been confirmed in primary cells cultures obtained from human MTCs. The inhibition of constitutively active RET in TT cells by both the RET pharmacological inhibitor RPI-1 and the transfection of dominant negative RET mutant (RETΔTK) resulted in a reduction of cofilin expression (-37⯱â¯8%, pâ¯<â¯0.001 and -31⯱â¯16%, pâ¯<â¯0.01, respectively). Furthermore, RPI-1 inhibitory effects on TT cell migration (-57⯱â¯13%, pâ¯<â¯0.01), but not on cell proliferation, were completely abolished in cells transfected with cofilin. In conclusion, these data indicate that an unbalanced cofilin expression, induced by oncogenic RET, contributes to promote MTC invasiveness and growth, suggesting the possibility of targeting cofilin pathway for more effective treatment of MTC.
Asunto(s)
Factores Despolimerizantes de la Actina/metabolismo , Carcinoma Neuroendocrino/metabolismo , Carcinoma Neuroendocrino/patología , Movimiento Celular , Proteínas Proto-Oncogénicas c-ret/metabolismo , Neoplasias de la Tiroides/metabolismo , Neoplasias de la Tiroides/patología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Silenciador del Gen/efectos de los fármacos , Humanos , Mutación/genética , Invasividad Neoplásica , Inhibidores de Proteínas Quinasas/farmacología , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-ret/antagonistas & inhibidoresRESUMEN
Although generally benign, pituitary tumors are frequently locally invasive, with reduced success of neurosurgery and unresponsive to pharmacological treatment with somatostatin or dopamine analogues. The molecular basis of the different biological behavior of pituitary tumors are still poorly identified, but a body of work now suggests that the activity of specific cytoskeleton proteins is a key factor regulating both the invasiveness and drug resistance of these tumors. This review recapitulates the experimental evidence supporting a role for the actin-binding protein filamin A (FLNA) in the regulation of somatostatin and dopamine receptors expression and signaling in pituitary tumors, thus in determining the responsiveness to currently used drugs, somatostatin analogues and dopamine receptor type 2 agonists. Regarding the regulation of invasive behavior of pituitary tumoral cells, we bring evidence to the role of the actin-severing protein cofilin, whose activation status may be modulated by dopaminergic and somatostatinergic drugs, through FLNA involvement. Molecular mechanisms involved in the regulation of FLNA expression and function in pituitary tumors will also be discussed.
Asunto(s)
Filaminas/metabolismo , Neoplasias Hipofisarias/metabolismo , Animales , Citoesqueleto/metabolismo , Humanos , Neoplasias Hipofisarias/tratamiento farmacológico , Neoplasias Hipofisarias/patologíaRESUMEN
An efficient intracellular response to somatostatin analogs (SSA) in pituitary tumors requires filamin A (FLNA). Since cAMP pathway plays an important role in GH-secreting pituitary tumors pathogenesis and FLNA is phosphorylated by PKA on S2152, aim of this study was to investigate in tumoral somatotrophs the impact of cAMP pathway activation and SSA stimulation on FLNA phosphorylation and the consequences on SST2 function. We found a PKA-mediated increase (2-fold) and SST2 agonist-induced decrease (-50%) of FLNA phosphorylation in GH3, GH4C1 and primary somatotroph tumor cells. This modification regulates FLNA function. Indeed, phosphomimetic S2152D FLNA mutant, but not phosphodeficient S2152A, abolished the known SSA antitumoral effects, namely: 1) inhibition of cell proliferation, reduction of cyclin D3 and increase of p27; 2) increase of cell apoptosis; 3) inhibition of cell migration via RhoA activation and cofilin phosphorylation. Coimmunoprecipitation and immunofluorescence assays showed that S2152A FLNA was recruited to activated SST2, whereas S2152D FLNA constitutively bound SST2 on the plasma membrane, but prevented Gαi proteins recruitment to SST2. In conclusion, we demonstrated that FLNA phosphorylation, promoted by cAMP pathway activation and inhibited by SSA, prevented SST2 signaling in GH-secreting tumoral pituitary cells.
Asunto(s)
AMP Cíclico/metabolismo , Filaminas/metabolismo , Adenoma Hipofisario Secretor de Hormona del Crecimiento/metabolismo , Neoplasias Hipofisarias/metabolismo , Proteínas Quinasas/metabolismo , Receptores de Somatostatina/metabolismo , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Adenoma Hipofisario Secretor de Hormona del Crecimiento/genética , Adenoma Hipofisario Secretor de Hormona del Crecimiento/patología , Hormonas/farmacología , Humanos , Fosforilación/efectos de los fármacos , Neoplasias Hipofisarias/genética , Neoplasias Hipofisarias/patología , Ratas , Transducción de Señal/efectos de los fármacos , Somatostatina/farmacología , Somatotrofos/efectos de los fármacos , Somatotrofos/metabolismo , Células Tumorales CultivadasRESUMEN
The pharmacological therapy of GH-secreting pituitary tumors is based on somatostatin (SS) analogs that reduce GH secretion and cell proliferation by binding mainly SS receptors type 2 (SST2). Antimigratory effects of SS have been demonstrated in different cell models, but no data on pituitary tumors are available. Aims of our study were to evaluate SST2 effects on migration and invasion of human and rat tumoral somatotrophs, and to elucidate the molecular mechanism involved focusing on the role of cofilin and filamin A (FLNA). Our data revealed that SST2 agonist BIM23120 significantly reduced GH3 cells migration (-22% ± 3.6%, p < 0.001) and invasion on collagen IV (-31.3% ± 12.2%, p < 0.01), both these effects being reproduced by octreotide and pasireotide. Similar results were obtained in primary cultured cells from human GH-secreting tumors. These inhibitory actions were accompanied by a marked increase in RhoA/ROCK-dependent cofilin phosphorylation (about 2.7-fold in GH3 and 2.1-fold in human primary cells). Accordingly, the anti-invasive effect of the SS analog was mimicked by the overexpression in GH3 cells of the S3D phosphomimetic cofilin mutant, and abolished by both phosphodeficient S3A cofilin and a specific ROCK inhibitor that prevented cofilin phosphorylation. Moreover, FLNA silencing and FLNA dominant-negative mutants FLNA19-20 and FLNA21-24 transfection demonstrated that FLNA plays a scaffold function for SST2-mediated cofilin phosphorylation. Accordingly, cofilin recruitment to agonist-activated SST2 was completely lost in FLNA silenced cells. In conclusion, we demonstrated that SST2 inhibits rat and human tumoral somatotrophs migration and invasion through a molecular mechanism that involves FLNA-dependent cofilin recruitment and phosphorylation.
Asunto(s)
Citoesqueleto/metabolismo , Neoplasias Hipofisarias/tratamiento farmacológico , Neoplasias Hipofisarias/metabolismo , Receptores de Somatostatina/agonistas , Receptores de Somatostatina/metabolismo , Somatostatina/análogos & derivados , Factores Despolimerizantes de la Actina/metabolismo , Animales , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Citoesqueleto/efectos de los fármacos , Filaminas/metabolismo , Humanos , Invasividad Neoplásica , Fosforilación , Neoplasias Hipofisarias/patología , Ratas , Transducción de Señal/efectos de los fármacos , Somatostatina/farmacología , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
cAMP pathway plays a major role in the pathogenesis of cortisol-producing adrenocortical adenomas (CPA). cAMP-induced steroidogenesis is preceded by actin cytoskeleton reorganization, a process regulated by cofilin activity. In this study we investigated cofilin role in mediating cAMP effects on cell morphology and steroidogenesis in adrenocortical tumor cells. We demonstrated that forskolin induced cell rounding and strongly reduced phosphorylated (P)-cofilin/total cofilin ratio in Y1 (-52 ± 16%, p < 0.001) and human CPA cells (-53 ± 18%, p < 0.05). Cofilin silencing significantly reduced both forskolin-induced morphological changes and progesterone production (1.3-fold vs 1.8-fold in controls, p < 0.05), whereas transfection of wild-type or S3A (active), but not S3D (inactive) cofilin, potentiated forskolin effects on cell rounding and increased 3-fold progesterone synthesis with respect to control (p < 0.05). Furthermore, cofilin dephosphorylation by a ROCK inhibitor potentiated forskolin-induced cell rounding and steroidogenesis (2-fold increase vs forskolin alone). Finally, we found a reduced P-cofilin/total cofilin ratio and increased cofilin expression in CPA vs endocrine inactive adenomas by western blot and immunohistochemistry. Overall, these results identified cofilin as a mediator of cAMP effects on both morphological changes and steroidogenesis in mouse and human adrenocortical tumor cells.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Factores Despolimerizantes de la Actina/metabolismo , Neoplasias de la Corteza Suprarrenal/metabolismo , Adenoma Corticosuprarrenal/metabolismo , AMP Cíclico/farmacología , Esteroides/biosíntesis , Factores Despolimerizantes de la Actina/antagonistas & inhibidores , Factores Despolimerizantes de la Actina/genética , Neoplasias de la Corteza Suprarrenal/tratamiento farmacológico , Neoplasias de la Corteza Suprarrenal/patología , Adenoma Corticosuprarrenal/tratamiento farmacológico , Adenoma Corticosuprarrenal/patología , Animales , Colforsina/farmacología , Humanos , Hidrocortisona/metabolismo , Ratones , Fosforilación/efectos de los fármacos , ARN Interferente Pequeño/genética , Células Tumorales Cultivadas , Vasodilatadores/farmacologíaRESUMEN
The role of progenitor/stem cells in pituitary tumorigenesis, resistance to pharmacological treatments and tumor recurrence is still unclear. This study investigated the presence of progenitor/stem cells in non-functioning pituitary tumors (NFPTs) and tested the efficacy of dopamine receptor type 2 (DRD2) and somatostatin receptor type 2 (SSTR2) agonists to inhibit in vitro proliferation. They found that 70% of 46 NFPTs formed spheres co-expressing stem cell markers, transcription factors (DAX1, SF1, ERG1) and gonadotropins. Analysis of tumor behavior showed that spheres formation was associated with tumor invasiveness (OR = 3,96; IC: 1.05-14.88, p = 0.036). The in vitro reduction of cell proliferation by DRD2 and SSTR2 agonists (31 ± 17% and 35 ± 13% inhibition, respectively, p < 0.01 vs. basal) occurring in about a half of NFPTs cells was conserved in the corresponding spheres. Accordingly, these drugs increased cyclin-dependent kinase inhibitor p27 and decreased cyclin D3 expression in spheres. In conclusion, they provided further evidence for the existence of cells with a progenitor/stem cells-like phenotype in the majority of NFPTs, particularly in those with invasive behavior, and demonstrated that the antiproliferative effects of dopaminergic and somatostatinergic drugs were maintained in progenitor/stem-like cells.
Asunto(s)
Carcinogénesis/genética , Recurrencia Local de Neoplasia/tratamiento farmacológico , Neoplasias Hipofisarias/tratamiento farmacológico , Receptores de Dopamina D2/genética , Receptores de Somatostatina/genética , Adulto , Carcinogénesis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Ciclina D3/biosíntesis , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/biosíntesis , Receptor Nuclear Huérfano DAX-1/biosíntesis , Dopaminérgicos/administración & dosificación , Resistencia a Antineoplásicos/genética , Canal de Potasio ERG1/biosíntesis , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Gonadotropinas/biosíntesis , Humanos , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia/genética , Recurrencia Local de Neoplasia/patología , Células Madre Neoplásicas/efectos de los fármacos , Neoplasias Hipofisarias/genética , Neoplasias Hipofisarias/patología , Factores de Empalme de ARN/biosíntesis , Receptores de Dopamina D2/agonistas , Receptores de Somatostatina/agonistas , Esferoides Celulares/efectos de los fármacos , Esferoides Celulares/patologíaRESUMEN
Non-functioning pituitary tumors (NFPTs) frequently present local invasiveness. Dopamine receptor 2 (DRD2) agonists are the only medical therapy that induces tumor shrinkage in some patients. Invasion requires cytoskeleton rearrangements that are tightly regulated by cofilin pathway, whose alterations correlate with invasion in different tumors. We investigated the effect of DR2D agonist on NFPT cells migration/invasion and the molecular mechanisms involved. We demonstrated that DRD2 agonist reduced migration (-44 ± 25%, p < 0.01) and invasion (-34 ± 6%, p < 0.001) and increased about 4-fold Ser3-phosphorylated inactive cofilin (P-cofilin) in NFPT cells. These effects were abolished by inhibiting ROCK, a kinase that phosphorylates cofilin. The overexpression of wild-type or phosphodeficient S3A-cofilin increased HP75 cells migration (+49 ± 6% and +57 ± 9% vs empty vector, respectively, p < 0.05), while phosphomimetic mutant had no effect. Interestingly, P-cofilin levels were lower in invasive vs non-invasive tumors by both western blot (mean P-cofilin/total cofilin ratio 0.77 and 1.93, respectively, p < 0.05) and immunohistochemistry (mean percentage of P-cofilin positive cells 17.6 and 45.7, respectively, p < 0.05). In conclusion, we showed that the invasiveness of pituitary tumors is promoted by the activation of cofilin, which can be regulated by DRD2 and might represent a novel biomarker for pituitary tumors' invasive behavior.
Asunto(s)
Factores Despolimerizantes de la Actina/metabolismo , Movimiento Celular , Neoplasias Hipofisarias/enzimología , Receptores de Dopamina D2/metabolismo , Quinasas Asociadas a rho/metabolismo , Factores Despolimerizantes de la Actina/genética , Adulto , Anciano , Antineoplásicos/farmacología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Agonistas de Dopamina/farmacología , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Masculino , Persona de Mediana Edad , Invasividad Neoplásica , Fosforilación , Neoplasias Hipofisarias/tratamiento farmacológico , Neoplasias Hipofisarias/genética , Neoplasias Hipofisarias/patología , Mutación Puntual , Inhibidores de Proteínas Quinasas/farmacología , Receptores de Dopamina D2/agonistas , Transducción de Señal , Transfección , Células Tumorales Cultivadas , Quinasas Asociadas a rho/antagonistas & inhibidoresRESUMEN
Molecular mechanisms underlying resistance of pituitary tumors to somatostatin (SS) and dopamine (DA) analogues treatment are not completely understood. Resistance has been associated with defective expression of functional somatostatin and dopamine receptors SSTR2, SSTR5, and DRD2, respectively. Recently, a role of cytoskeleton protein filamin A (FLNA) in DRD2 and SSTR receptors expression and signaling in PRL- and GH-secreting tumors, respectively, has been demonstrated, first revealing a link between FLNA expression and responsiveness of pituitary tumors to pharmacological therapy. No molecular events underlying the reduction of FLNA levels in resistant tumors have been so far identified. FLNA can be phosphorylated by PKA on Ser2152, with increased FLNA resistance to cleavage by calpain and conformational changes affecting FLNA regions involved in SSTR2 and DRD2 binding and signal transduction. In this respect, the effect of cAMP/PKA pathway in the regulation of FLNA stability and/or function by modulating its phosphorylation status could assume particular importance in pituitary, where cAMP cascade plays a crucial role in pituitary cell functions and tumorigenesis. This review will discuss the role of FLNA in the regulation of the main GPCRs target of pharmacological treatment of pituitary tumors, that is, SSTR2 and DRD2, focusing on the effects of cAMP/PKA-mediated FLNA phosphorylation on FLNA biological functions.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Filaminas/metabolismo , Hipófisis/metabolismo , Receptores Dopaminérgicos/metabolismo , Somatostatina/metabolismo , Animales , Humanos , FosforilaciónRESUMEN
Somatostatin receptor type 2 (SST2) is the main pharmacological target of medical therapy for GH-secreting pituitary tumors, but molecular mechanisms regulating its expression and signaling are largely unknown. The aim of this study was to investigate the role of cytoskeleton protein filamin A (FLNA) in SST2 expression and signaling in somatotroph tumor cells. We found a highly variable expression of FLNA in human GH-secreting tumors, without a correlation with SST2 levels. FLNA silencing in human tumoral cells did not affect SST2 expression and localization but abolished the SST2-induced reduction of cyclin D1 (-37% ± 15% in control cells, P < .05 vs basal) and caspase-3/7 activation (+63% ± 31% in control cells, P < .05 vs basal). Overexpression of a FLNA dominant-negative mutant that specifically prevents SST2-FLNA binding reduced SST2 expression after prolonged agonist exposure (-55% ± 5%, P < .01 vs untreated cells) in GH3 cells. Moreover, SST2-induced apoptotic effect (77% ± 54% increase of caspase activity, P < .05 vs basal) and SST2-mediated ERK1/2 inhibition (48% ± 17% reduction of ERK1/2 phosphorylation, P < .01 vs basal) were abrogated in cells overexpressing another FLNA mutant that prevents FLNA interaction with partner proteins but not with SST2, suggesting a scaffold function of FLNA in somatotrophs. In conclusion, these data demonstrate that FLNA is involved in SST2 stabilization and signaling in tumoral somatotrophs, playing both a structural and functional role.
Asunto(s)
Filaminas/fisiología , Receptores de Somatostatina/fisiología , Transducción de Señal/fisiología , Somatotrofos/metabolismo , Animales , Apoptosis , Línea Celular Tumoral , Proliferación Celular , Humanos , Estabilidad Proteica , Ratas , Receptores de Somatostatina/agonistasRESUMEN
In the pituitary the activation of cyclic adenosine 3'-5'-monophosphate (cAMP) dependent pathways generates proliferative signals in somatotrophs, whereas in pituitary cells of other lineages its effect remains uncertain. Moreover, the specific role of the two main cAMP effectors, protein kinase A (PKA) and exchange proteins directly activated by cAMP (Epac), has not been defined. Aim of this study was to investigate the effect of cAMP on pituitary adenomatous cells proliferation and to identify PKA and Epac differential involvement. We found that cAMP increased DNA synthesis and cyclin D1 expression in somatotropinomas, whereas it reduced both parameters in prolactinomas and nonfunctioning adenomas, these effects being replicated in corresponding cell lines. Moreover, the divergent cAMP effects were mimicked by Epac and PKA analogs, which activated Rap1 and CREB, respectively. In conclusion, we demonstrated that cAMP exerted opposite effects on different pituitary cell types proliferation, these effects being mediated by both Epac and PKA.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/genética , AMP Cíclico/metabolismo , Regulación Neoplásica de la Expresión Génica , Factores de Intercambio de Guanina Nucleótido/genética , Hipófisis/metabolismo , Subunidades de Proteína/genética , Adenoma/genética , Adenoma/metabolismo , Adenoma/patología , Animales , Proliferación Celular , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Ciclina D1/genética , Ciclina D1/metabolismo , Gonadotrofos/metabolismo , Gonadotrofos/patología , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Lactotrofos/metabolismo , Lactotrofos/patología , Hipófisis/patología , Neoplasias Hipofisarias/genética , Neoplasias Hipofisarias/metabolismo , Neoplasias Hipofisarias/patología , Prolactinoma/genética , Prolactinoma/metabolismo , Prolactinoma/patología , Subunidades de Proteína/metabolismo , Ratas , Transducción de Señal , Somatotrofos/metabolismo , Somatotrofos/patología , Proteínas de Unión al GTP rap1/genética , Proteínas de Unión al GTP rap1/metabolismoRESUMEN
Progressive osseous heteroplasia (POH) is a rare autosomal dominant disorder of mesenchymal differentiation characterized by progressive heterotopic ossification (HO) of dermis, deep connective tissues and skeletal muscle. Usually, initial bone formation occurs during infancy as primary osteoma cutis (OC) then progressively extending into deep connective tissues and skeletal muscle over childhood. Most cases of POH are caused by paternally inherited inactivating mutations of GNAS gene. Maternally inherited mutations as well as epigenetic defects of the same gene lead to pseudohypoparathyroidism (PHP) and Albright's hereditary osteodystrophy (AHO). During the last decade, some reports documented the existence of patients with POH showing additional features characteristic of AHO such as short stature and brachydactyly, previously thought to occur only in other GNAS-associated disorders. Thus, POH can now be considered as part of a wide spectrum of ectopic bone formation disorders caused by inactivating GNAS mutations. Here, we report genetic and epigenetic analyses of GNAS locus in 10 patients affected with POH or primary OC, further expanding the spectrum of mutations associated with this rare disease and indicating that, unlike PHP, methylation alterations at the same locus are absent or uncommon in this disorder.
Asunto(s)
Enfermedades Óseas Metabólicas/diagnóstico , Enfermedades Óseas Metabólicas/genética , Subunidades alfa de la Proteína de Unión al GTP Gs/genética , Osificación Heterotópica/diagnóstico , Osificación Heterotópica/genética , Enfermedades Cutáneas Genéticas/diagnóstico , Enfermedades Cutáneas Genéticas/genética , Adolescente , Niño , Preescolar , Cromograninas , Epigénesis Genética/genética , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mutación/genéticaRESUMEN
RWJ-53308 is a novel nonpeptide glycoprotein IIb/IIIa (GPIIb/IIIa) antagonist that inhibits fibrinogen binding to GPIIb/IIIa with an IC(50) of 0.4+/-0.3 nM. RWJ-53308 inhibits thrombin-induced platelet aggregation in human gel-filtered platelets (IC(50)=60+/-12 nM) and platelet aggregation in human platelet-rich plasma (PRP) in response to collagen, arachidonic acid, ADP, and SFLLRN-NH(2) (IC(50)=60+/-10, 150+/-30, 70+/-4, and 160+/-80 nM, respectively). The potency of RWJ-53308 in dog and guinea pig PRP is similar to human PRP. RWJ-53308 inhibits ex vivo collagen- and ADP-induced platelet aggregation in conscious dogs for up to 4 h following 0.3 mg/kg iv, and through 4 and 6 h following 1 and 3 mg/kg po. Oral bioavailability is 16+/-7%. RWJ-53308 reduces thrombus weight in a canine arteriovenous (AV) shunt model following intravenous (0.01-0.1 mg/kg) and oral (3 mg/kg) administration. In a guinea pig carotid artery pinch-injury model, RWJ-53308 completely suppresses thrombus-induced cyclic flow reductions (CFR) at 0.7 mg/kg iv. RWJ-53308 also blocks thrombus formation in photoactivation- and ferric chloride-induced models of thrombosis in guinea pigs at 0.3 and 1 mg/kg iv, respectively. In summary, RWJ-53308 is a potent orally active GPIIb/IIIa antagonist that may be useful for both acute and chronic treatment of arterial thrombotic disorders.
Asunto(s)
Ácidos Nipecóticos/farmacocinética , Inhibidores de Agregación Plaquetaria/farmacocinética , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/antagonistas & inhibidores , Piridinas/farmacocinética , Animales , Disponibilidad Biológica , Modelos Animales de Enfermedad , Perros , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Fibrinógeno/efectos de los fármacos , Fibrinógeno/metabolismo , Cobayas , Humanos , Concentración 50 Inhibidora , Ácidos Nipecóticos/administración & dosificación , Ácidos Nipecóticos/toxicidad , Agregación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/administración & dosificación , Inhibidores de Agregación Plaquetaria/toxicidad , Unión Proteica/efectos de los fármacos , Piridinas/administración & dosificación , Piridinas/toxicidad , Ratas , Trombosis/tratamiento farmacológicoRESUMEN
Human platelets possess two distinct thrombin-activated receptors, PAR-1 (protease-activated receptor-1) and PAR-4, whereas human vascular smooth muscle cells possess only PAR-1. Although such thrombin receptors have been studied extensively in vitro, their physiological roles are still rather ill-defined. We have now employed a potent, selective PAR-1 antagonist, RWJ-58259, to probe the in vivo significance of PAR-1 in thrombosis and vascular injury. RWJ-58259 was examined in two thrombosis models in guinea pigs: the arteriovenous (A-V) shunt assay (monitoring thrombus weight) and the Rose Bengal intravascular photoactivation assay (monitoring time to occlusion). Administration of RWJ-58259 (10 mg/kg, total i.v. dose) did not inhibit thrombus formation in either thrombosis model, although local, intrashunt delivery in the A-V shunt model did elicit a modest antithrombotic effect (thrombus weight reduction from 35 +/- 2 to 24 +/- 4 mg). These results are consistent with the presence of more than one thrombin-sensitive receptor on guinea pig platelets, in analogy with human platelets. Indeed, we were able to establish that guinea pig platelets express three thrombin receptors, PAR-1, PAR-3, and PAR-4. We also examined RWJ-58259 in a vascular restenosis model involving balloon angioplasty in rats. Perivascular administration of RWJ-58259 (10 mg) significantly reduced neointimal thickness (77 +/- 5 microm to 45 +/- 5 microm, P < 0.05), clearly demonstrating an important role for PAR-1 in vascular injury. From these results, it is evident that a PAR-1 antagonist is not especially effective for treating platelet-dependent thrombosis; however, it could well be beneficial for treating restenosis attendant to arterial injury.
Asunto(s)
Arterias Carótidas/efectos de los fármacos , Arterias Carótidas/metabolismo , Oclusión de Injerto Vascular/tratamiento farmacológico , Indazoles/farmacología , Agregación Plaquetaria/efectos de los fármacos , Receptores de Trombina/antagonistas & inhibidores , Urea/farmacología , Angioplastia de Balón , Animales , Arterias Carótidas/fisiología , Arterias Carótidas/cirugía , Técnicas de Cultivo de Célula , Cobayas , Humanos , Indazoles/química , Indazoles/uso terapéutico , Masculino , Agregación Plaquetaria/fisiología , Ratas , Ratas Sprague-Dawley , Receptor PAR-1 , Receptores de Trombina/fisiología , Trombosis/tratamiento farmacológico , Urea/análogos & derivados , Urea/química , Urea/uso terapéuticoRESUMEN
The antithrombotic, anticoagulant, and kinetic properties of RWJ-50353, a novel, reversible, active-site-directed thrombin inhibitor, were evaluated. RWJ-50353 inhibited the catalytic activity of human alpha-thrombin with a K(i) of 0.19+/-0.02 nM. It showed a 16-fold selectivity relative to inhibition of trypsin and at least 330-fold selectivity relative to inhibition of other biologically important serine proteases. In a gel-filtered platelet preparation, RWJ-50353 inhibited alpha-thrombin-induced aggregation with an IC(50) of 32+/-6 nM. In a canine arteriovenous shunt antithrombotic model, RWJ-50353 demonstrated a significant dose-related (0.1-1.0 mg/kg, i.v.) reduction in thrombus formation with 50% inhibition (ID(50)) obtained at 0.46+/-0.1 mg/kg. In a rabbit deep vein thrombosis model, RWJ-50353 dose-dependently (0.1-1. 0 mg/kg, i.v.) reduced thrombus formation with an ID(50) of 0.25+/-0. 03 mg/kg. The antithrombotic activity in both of these models was associated with only mild prolongations in bleeding time and coagulation parameters. These results demonstrate that RWJ-50353 is a potent, selective thrombin inhibitor that is an effective antithrombotic agent after intravenous administration in models of arterial and venous thrombosis and may be useful in the management of various thrombotic disorders.
Asunto(s)
Plaquetas/efectos de los fármacos , Fibrinolíticos/farmacología , Guanidinas/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Agregación Plaquetaria/efectos de los fármacos , Tiazoles/farmacología , Animales , Plaquetas/fisiología , Perros , Hemostáticos/farmacología , Humanos , Conejos , Trombina/antagonistas & inhibidores , Trombina/farmacologíaRESUMEN
A questionnaire was mailed to nurse transport coordinators at the 76 United States pediatric training programs listing 35 pediatric residents or more, and a related but different questionnaire was mailed to chief pediatric residents at the same centers. Comparisons of responses to a series of questions on the role of the nurse on transport suggest that the pediatric emergency transport team offers an excellent opportunity to implement collaborative practice strategies between nurses and residents. Both groups stated the value of professional discussion and consultation to derive mutually satisfactory resolutions to patient care issues. Physician respondents indicated a genuine respect for the nurses' skill and expanded role in the care of critically ill children during transport. The transport experience offers a window of opportunity for nurses and physicians interested in developing research and practice models aimed at fostering collaboration between nurses and physicians-in-training.
Asunto(s)
Internado y Residencia , Enfermería Pediátrica , Transporte de Pacientes/organización & administración , Niño , Competencia Clínica , Humanos , Investigación en Evaluación de Enfermería , Pediatría/educación , Encuestas y CuestionariosRESUMEN
RWJ-29009, (6S)-trans(-)-1-(6,7-dihydro-6-hydroxy-5,5-dimethyl-2-nitro-5 H-thieno[3,2-b]pyran-7-yl)-2-piperidinone, is a structurally novel and extremely potent potassium channel activator that may be useful for treatment of hypertension and ischemic heart disease. We assessed the cardiovascular profile of RWJ 29009 in anesthetized and conscious dogs. RWJ 29009 (0.1-2 micrograms/kg intravenously, i.v.) dose-relatedly increased coronary blood flow (CBF) and decreased arterial pressure in anesthetized dogs. Total peripheral resistance and coronary vascular resistance were concurrently reduced without significant changes in heart rate (HR) or cardiac output (CO). Left ventricular (LV) dP/dtmax and myocardial contractile force were decreased only at the highest dose of 10 micrograms/kg. Cromakalim (3-100 micrograms/kg), although much less potent, had a qualitatively similar profile. Glyburide pretreatment (5 mg/kg i.v.) shifted the dose response of RWJ 29009 for increasing CBF and decreasing arterial pressure to the right. The dose responses of cromakalim were similarly shifted to the right, whereas the effects of nifedipine on CBF and arterial pressure were not affected by glyburide. RWJ 29009 (0.3 and 1 microgram/kg) had no effect on myocardial O2 consumption (MVO2) except for a transient increase immediately after administration of 1 microgram/kg. MVO2 returned to control 15 min after dosing, although CBF remained significantly increased. In conscious dogs, RWJ 29009 (0.3-10 micrograms/kg, i.v. and orally, p.o.) produced dose-related increases in CBF and decreases in arterial pressure similar to those produced in anesthetized dogs, except that HR was increased concurrently. The i.v. and p.o. potency of RWJ 29009 were comparable, indicating high oral bioavailability. Thus, RWJ 29009 is an extremely potent coronary and peripheral vasodilator with a cardiovascular profile similar to that of other potassium channel activators. Like those of other potassium channel activators, its mechanism of action appears to involve activation of ATP-regulated potassium channels.