RESUMO
Molecular signalling mediated by the phosphatidylinositol-3-kinase (PI3K)-Akt axis is a key regulator of cellular functions. Importantly, alteration of the PI3K-Akt signalling underlies the development of different human diseases, thus prompting the investigation of the pathway as a molecular target for pharmacologic intervention. In this regard, recent studies showed that small molecule inhibitors of PI3K, the upstream regulator of the pathway, reduced the development of inflammation during acute pancreatitis, a highly debilitating and potentially lethal disease. Here we investigated whether a specific reduction of Akt activity, by using either pharmacologic Akt inhibition, or genetic inactivation of the Akt1 isoform selectively in pancreatic acinar cells, is effective in ameliorating the onset and progression of the disease. We discovered that systemic reduction of Akt activity did not protect the pancreas from initial damage and only transiently delayed leukocyte recruitment. However, reduction of Akt activity decreased acinar proliferation and exacerbated acinar-to-ductal metaplasia (ADM) formation, two critical events in the progression of pancreatitis. These phenotypes were recapitulated upon conditional inactivation of Akt1 in acinar cells, which resulted in reduced expression of 4E-BP1, a multifunctional protein of key importance in cell proliferation and metaplasia formation. Collectively, our results highlight the critical role played by Akt1 during the development of acute pancreatitis in the control of acinar cell proliferation and ADM formation. In addition, these results harbour important translational implications as they raise the concern that inhibitors of PI3K-Akt signalling pathways may negatively affect the regeneration of the pancreas. Finally, this work provides the basis for further investigating the potential of Akt1 activators to boost pancreatic regeneration following inflammatory insults. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Assuntos
Células Acinares/enzimologia , Proliferação de Células , Pâncreas Exócrino/enzimologia , Ductos Pancreáticos/enzimologia , Pancreatite/enzimologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células Acinares/efeitos dos fármacos , Células Acinares/patologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ceruletídeo , Modelos Animais de Doenças , Masculino , Metaplasia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pâncreas Exócrino/efeitos dos fármacos , Pâncreas Exócrino/patologia , Ductos Pancreáticos/efeitos dos fármacos , Ductos Pancreáticos/patologia , Pancreatite/induzido quimicamente , Pancreatite/genética , Pancreatite/patologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/análise , Proteínas Proto-Oncogênicas c-akt/deficiência , Proteínas Proto-Oncogênicas c-akt/genética , Ratos , Transdução de SinaisRESUMO
Proliferation of pancreatic acinar cells is a critical process in the pathophysiology of pancreatic diseases, because limited or defective proliferation is associated with organ dysfunction and patient morbidity. In this context, elucidating the signalling pathways that trigger and sustain acinar proliferation is pivotal to develop therapeutic interventions promoting the regenerative process of the organ. In this study we used genetic and pharmacological approaches to manipulate both local and systemic levels of thyroid hormones to elucidate their role in acinar proliferation following caerulein-mediated acute pancreatitis in mice. In addition, molecular mechanisms mediating the effects of thyroid hormones were identified by genetic and pharmacological inactivation of selected signalling pathways.In this study we demonstrated that levels of the thyroid hormone 3,3',5-triiodo-l-thyronine (T3) transiently increased in the pancreas during acute pancreatitis. Moreover, by using genetic and pharmacological approaches to manipulate both local and systemic levels of thyroid hormones, we showed that T3 was required to promote proliferation of pancreatic acinar cells, without affecting the extent of tissue damage or inflammatory infiltration.Finally, upon genetic and pharmacological inactivation of selected signalling pathways, we demonstrated that T3 exerted its mitogenic effect on acinar cells via a tightly controlled action on different molecular effectors, including histone deacetylase, AKT, and TGFß signalling.In conclusion, our data suggest that local availability of T3 in the pancreas is required to promote acinar cell proliferation and provide the rationale to exploit thyroid hormone signalling to enhance pancreatic regeneration. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Assuntos
Células Acinares/metabolismo , Proliferação de Células , Hipertireoidismo/metabolismo , Pâncreas Exócrino/metabolismo , Pancreatite/metabolismo , Tri-Iodotironina/metabolismo , Células Acinares/patologia , Animais , Ceruletídeo , Modelos Animais de Doenças , Histona Desacetilases/metabolismo , Hipertireoidismo/genética , Hipertireoidismo/patologia , Iodeto Peroxidase/deficiência , Iodeto Peroxidase/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pâncreas Exócrino/patologia , Pancreatite/induzido quimicamente , Pancreatite/genética , Pancreatite/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo II/deficiência , Receptor do Fator de Crescimento Transformador beta Tipo II/genética , Transdução de Sinais , Tiroxina/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Regulação para CimaRESUMO
Pancreatic ductal adenocarcinoma (PDAC), which is the primary cause of pancreatic cancer mortality, is poorly responsive to currently available interventions. Identifying new targets that drive PDAC formation and progression is critical for developing alternative therapeutic strategies to treat this lethal malignancy. Using genetic and pharmacological approaches, we investigated in vivo and in vitro whether uptake of the monoamine serotonin [5-hydroxytryptamine (5-HT)] is required for PDAC development. We demonstrated that pancreatic acinar cells have the ability to readily take up 5-HT in a transport-mediated manner. 5-HT uptake promoted activation of the small GTPase Ras-related C3 botulinum toxin substrate 1 (Rac1), which is required for transdifferentiation of acinar cells into acinar-to-ductal metaplasia (ADM), a key determinant in PDAC development. Consistent with the central role played by Rac1 in ADM formation, inhibition of the 5-HT transporter Sert (Slc6a4) with fluoxetine reduced ADM formation both in vitro and in vivo in a cell-autonomous manner. In addition, fluoxetine treatment profoundly compromised the stromal reaction and affected the proliferation and lipid metabolism of malignant PDAC cells. We propose that Sert is a promising therapeutic target to counteract the early event of ADM, with the potential to stall the initiation and progression of pancreatic carcinogenesis. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Assuntos
Carcinoma Ductal Pancreático/enzimologia , Proliferação de Células , Genes ras , Neuropeptídeos/metabolismo , Pâncreas/enzimologia , Neoplasias Pancreáticas/enzimologia , Serotonina/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/prevenção & controle , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Transdiferenciação Celular , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Embrião de Galinha , Modelos Animais de Doenças , Ativação Enzimática , Fluoxetina/farmacologia , Predisposição Genética para Doença , Humanos , Metaplasia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neovascularização Patológica , Pâncreas/efeitos dos fármacos , Pâncreas/patologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/prevenção & controle , Fenótipo , Ratos , Proteínas da Membrana Plasmática de Transporte de Serotonina/efeitos dos fármacos , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Transdução de Sinais , Triptofano Hidroxilase/genética , Triptofano Hidroxilase/metabolismoRESUMO
Adult pancreatic acinar cells have the ability to re-enter the cell cycle and proliferate upon injury or tissue loss. Despite this mitotic ability, the extent of acinar proliferation is often limited and unable to completely regenerate the injured tissue or restore the initial volume of the organ, thus leading to pancreatic dysfunction. Identifying molecular determinants of enhanced proliferation is critical to overcome this issue. In this study, we discovered that Murphy Roths Large (MRL/MpJ) mice can be exploited to identify molecular effectors promoting acinar proliferation upon injury, with the ultimate goal to develop therapeutic regimens to boost pancreatic regeneration. Our results show that, upon cerulein-induced acinar injury, cell proliferation was enhanced and cell cycle components up-regulated in the pancreas of MRL/MpJ mice compared to the control strain C57BL/6. Initial damage of acinar cells was exacerbated in these mice, manifested by increased serum levels of pancreatic enzymes, intra-pancreatic trypsinogen activation and acinar cell apoptosis. In addition, MRL/MpJ pancreata presented enhanced inflammation, de-differentiation of acinar cells and acinar-to-ductal metaplasia. Manipulation of inflammatory levels and mitogenic stimulation with the thyroid hormone 5,3-L-tri-iodothyronine revealed that factors derived from initial acinar injury rather than inflammatory injury promote the replicative advantage in MRL/MpJ mice.
Assuntos
Células Acinares/citologia , Inflamação/metabolismo , Pâncreas/metabolismo , Células Acinares/efeitos dos fármacos , Animais , Proliferação de Células/efeitos dos fármacos , Ceruletídeo/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pâncreas/efeitos dos fármacos , Pâncreas/imunologiaRESUMO
Pancreatic fibrosis is the hallmark of chronic pancreatitis, a highly debilitating disease for which there is currently no cure. The key event at the basis of pancreatic fibrosis is the deposition of extracellular matrix proteins by activated pancreatic stellate cells (PSCs). Transforming growth factor ß (TGFß) is a potent profibrotic factor in the pancreas as it promotes the activation of PSC; thus, pharmacologic interventions that effectively reduce TGFß expression harbor considerable therapeutic potential in the treatment of chronic pancreatitis. In this study, we investigated whether TGFß expression is reduced by pharmacologic inhibition of the epigenetic modifiers histone deacetylases (HDACs). To address this aim, chronic pancreatitis was induced in C57BL/6 mice with serial injections of cerulein, and the selective class 1 HDAC inhibitor MS-275 was administered in vivo in a preventive and therapeutic manner. Both MS-275 regimens potently reduced deposition of extracellular matrix and development of fibrosis in the pancreas after 4 weeks of chronic pancreatitis. Reduced pancreatic fibrosis was concomitant with lower expression of pancreatic TGFß and consequent reduced PSC activation. In search of the cell types targeted by the inhibitor, we found that MS-275 treatment abrogated the expression of TGFß in acinar cells stimulated by cerulein treatment. Our study demonstrates that MS-275 is an effective antifibrotic agent in the context of experimental chronic pancreatitis and thus may constitute a valid therapeutic intervention for this severe disease.
Assuntos
Benzamidas/administração & dosagem , Inibidores de Histona Desacetilases/administração & dosagem , Pâncreas/efeitos dos fármacos , Pancreatite Crônica/induzido quimicamente , Pancreatite Crônica/tratamento farmacológico , Piridinas/administração & dosagem , Fator de Crescimento Transformador beta/metabolismo , Animais , Benzamidas/farmacologia , Linhagem Celular , Ceruletídeo/efeitos adversos , Modelos Animais de Doenças , Fibrose/prevenção & controle , Regulação da Expressão Gênica , Inibidores de Histona Desacetilases/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Pâncreas/patologia , Células Estreladas do Pâncreas/citologia , Células Estreladas do Pâncreas/efeitos dos fármacos , Células Estreladas do Pâncreas/metabolismo , Pancreatite Crônica/metabolismo , Piridinas/farmacologia , RatosRESUMO
BACKGROUND AND PURPOSE: Nonsteroidal anti-inflammatory drugs (NSAIDs) are administered to manage the pain typically found in patients suffering from pancreatitis. NSAIDs also display anti-proliferative activity against cancer cells; however, their effects on normal, untransformed cells are poorly understood. Here, we evaluated whether NSAIDs inhibit the proliferation of pancreatic acinar cells during the development of acute pancreatitis. EXPERIMENTAL APPROACH: The NSAIDs ibuprofen and diclofenac were administered to C57BL/6 mice after induction of pancreatitis with serial injections of cerulein. In addition, ibuprofen was administered concomitantly with 3,5,3-L-tri-iodothyronine (T3), which induces acinar cell proliferation in the absence of tissue inflammation. The development of pancreatic inflammation, acinar de-differentiation into metaplastic lesions and acinar proliferation were quantified by histochemical, biochemical and RT-PCR approaches. KEY RESULTS: Therapeutic ibuprofen treatment selectively reduced pancreatic infiltration of activated macrophages in vivo, and M1 macrophage polarization and pro-inflammatory cytokine expression both in vivo and in vitro. Reduced macrophage activation was accompanied by reduced acinar de-differentiation into acinar-to-ductal metaplasia. Acinar proliferation was significantly impaired in the presence of ibuprofen and diclofenac, as demonstrated at both the level of proliferation markers and expression of cell cycle regulators. Ibuprofen also reduced acinar cell proliferation induced by mitogenic stimulation with T3, a treatment that does not elicit pancreatic inflammation. CONCLUSIONS AND IMPLICATIONS: Our study provides evidence that the NSAIDs ibuprofen and diclofenac inhibit pancreatic acinar cell division. This suggests that prolonged treatment with these NSAIDs may negatively affect the regeneration of the pancreas and further studies are needed to confirm these findings in a clinical setting. LINKED ARTICLES: This article is part of a themed section on Inventing New Therapies Without Reinventing the Wheel: The Power of Drug Repurposing. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.2/issuetoc.
Assuntos
Células Acinares/efeitos dos fármacos , Diclofenaco/farmacologia , Ibuprofeno/farmacologia , Mitógenos/farmacologia , Pâncreas/citologia , Pâncreas/efeitos dos fármacos , Pancreatite/tratamento farmacológico , Pancreatite/patologia , Células Acinares/citologia , Células Acinares/patologia , Animais , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ceruletídeo , Citocinas/metabolismo , Diclofenaco/uso terapêutico , Ibuprofeno/uso terapêutico , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Masculino , Camundongos , Infiltração de Neutrófilos/efeitos dos fármacos , Pâncreas/patologia , Pancreatite/induzido quimicamente , Tri-Iodotironina/antagonistas & inibidores , Tri-Iodotironina/farmacologiaRESUMO
BACKGROUND AND PURPOSE: Pancreatitis is a common inflammation of the pancreas with rising incidence in many countries. Despite improvements in diagnostic techniques, the disease is associated with high risk of severe morbidity and mortality and there is an urgent need for new therapeutic interventions. In this study, we evaluated whether histone deacetylases (HDACs), key epigenetic regulators of gene transcription, are involved in the development of the disease. EXPERIMENTAL APPROACH: We analysed HDAC regulation during cerulein-induced acute, chronic and autoimmune pancreatitis using different transgenic mouse models. The functional relevance of class I HDACs was tested with the selective inhibitor MS-275 in vivo upon pancreatitis induction and in vitro in activated macrophages and primary acinar cell explants. KEY RESULTS: HDAC expression and activity were up-regulated in a time-dependent manner following induction of pancreatitis, with the highest abundance observed for class I HDACs. Class I HDAC inhibition did not prevent the initial acinar cell damage. However, it effectively reduced the infiltration of inflammatory cells, including macrophages and T cells, in both acute and chronic phases of the disease, and directly disrupted macrophage activation. In addition, MS-275 treatment reduced DNA damage in acinar cells and limited acinar de-differentiation into acinar-to-ductal metaplasia in a cell-autonomous manner by impeding the EGF receptor signalling axis. CONCLUSIONS AND IMPLICATIONS: These results demonstrate that class I HDACs are critically involved in the development of acute and chronic forms of pancreatitis and suggest that blockade of class I HDAC isoforms is a promising target to improve the outcome of the disease.
Assuntos
Doenças Autoimunes/tratamento farmacológico , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/efeitos dos fármacos , Pancreatite/tratamento farmacológico , Células Acinares/metabolismo , Doença Aguda , Animais , Doenças Autoimunes/fisiopatologia , Benzamidas/farmacologia , Modelos Animais de Doenças , Receptores ErbB/metabolismo , Histona Desacetilases/metabolismo , Leucócitos/metabolismo , Masculino , Metaplasia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pancreatite/fisiopatologia , Pancreatite Crônica/tratamento farmacológico , Pancreatite Crônica/fisiopatologia , Piridinas/farmacologia , Fatores de TempoRESUMO
Determining signalling pathways that regulate pancreatic regeneration following pancreatitis is critical for implementing therapeutic interventions. In this study we elucidated the molecular mechanisms underlying the effects of transforming growth factor-ß (TGFß) in pancreatic epithelial cells during tissue regeneration. To this end, we conditionally inactivated TGFß receptor II (TGFß-RII) using a Cre-LoxP system under the control of pancreas transcription factor 1a (PTF1a) promoter, specific for the pancreatic epithelium, and evaluated the molecular and cellular changes in a mouse model of cerulein-induced pancreatitis. We show that TGFß-RII signalling does not mediate the initial acinar cell damage observed at the onset of pancreatitis. However, TGFß-RII signalling not only restricts acinar cell replication during the regenerative phase of the disease but also limits ADM formation in vivo and in vitro in a cell-autonomous manner. Analyses of molecular mechanisms underlying the observed phenotype revealed that TGFß-RII signalling stimulates the expression of cyclin-dependent kinase inhibitors and intersects with the EGFR signalling axis. Finally, TGFß-RII ablation in epithelial cells resulted in increased infiltration of inflammatory cells in the early phases of pancreatitis and increased activation of pancreatic stellate cells in the later stages of pancreatitis, thus highlighting a TGFß-based crosstalk between epithelial and stromal cells regulating the development of pancreatic inflammation and fibrosis. Collectively, our data not only contribute to clarifying the cellular processes governing pancreatic tissue regeneration, but also emphasize the conserved role of TGFß as a tumour suppressor, both in the regenerative process following pancreatitis and in the initial phases of pancreatic cancer.
Assuntos
Células Acinares/patologia , Pâncreas/patologia , Pancreatite/patologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Amilases/metabolismo , Animais , Carcinoma Ductal Pancreático/patologia , Pontos de Checagem do Ciclo Celular/fisiologia , Proliferação de Células/fisiologia , Transformação Celular Neoplásica/patologia , Células Cultivadas , Ceruletídeo/toxicidade , Células Epiteliais/patologia , Fibrose/patologia , Irritantes/toxicidade , Lipase/metabolismo , Masculino , Metaplasia/patologia , Camundongos Knockout , Camundongos Transgênicos , Pâncreas/enzimologia , Neoplasias Pancreáticas/patologia , Pancreatite/enzimologia , Receptor do Fator de Crescimento Transformador beta Tipo IIRESUMO
The exocrine pancreas exhibits a distinctive capacity for tissue regeneration and renewal following injury. This regenerative ability has important implications for a variety of disorders, including pancreatitis and pancreatic cancer, diseases associated with high morbidity and mortality. Thus, understanding its underlying mechanisms may help in developing therapeutic interventions. Serotonin has been recognized as a potent mitogen for a variety of cells and tissues. Here we investigated whether serotonin exerts a mitogenic effect in pancreatic acinar cells in three regenerative models, inflammatory tissue injury following pancreatitis, tissue loss following partial pancreatectomy, and thyroid hormone-stimulated acinar proliferation. Genetic and pharmacological techniques were used to modulate serotonin levels in vivo. Acinar dedifferentiation and cell cycle progression during the regenerative phase were investigated over the course of 2 weeks. By comparing acinar proliferation in the different murine models of regeneration, we found that serotonin did not affect the clonal regeneration of mature acinar cells. Serotonin was, however, required for acinar dedifferentiation following inflammation-mediated tissue injury. Specifically, lack of serotonin resulted in delayed up-regulation of progenitor genes and delayed the formation of acinar-to-ductal metaplasia and defective acinar cell proliferation. We identified serotonin-dependent acinar secretion as a key step in progenitor-based regeneration, as it promoted acinar cell dedifferentiation and the recruitment of type 2 macrophages. Finally, we identified a regulatory Hes1-Ptfa axis in the uninjured adult pancreas, activated by zymogen secretion. Our findings indicated that serotonin plays a critical role in the regeneration of the adult pancreas following pancreatitis by promoting the dedifferentiation of acinar cells.