RESUMO
Hypertension is a major cause of chronic kidney disease. However, the pathogenesis of hypertensive kidney disease is not fully understood. Recently, we have shown that CXCL16/phosphoinositide-3 kinase γ (PI3Kγ) plays an important role in the development of renal inflammation and fibrosis in angiotensin II (AngII) induced hypertensive nephropathy. In the present study, we examined the role of phosphatase and tensin homolog (PTEN), a major regulator of PI3K signaling, in the pathogenesis of renal inflammation and fibrosis in an experimental model of hypertension induced by AngII. We generated myeloid PTEN conditional knockout mice by crossing PTENflox/flox mice with LysM-driven Cre mice. Littermate LysM-Cre-/- PTENflox/flox mice were used as a control. Both myeloid PTEN knockout mice and their littermate control mice exhibited similar blood pressure at baseline. AngII treatment resulted in an increase in blood pressure that was comparable between myeloid PTEN knockout mice and littermate control mice. Compared with littermate control mice, myeloid PTEN knockout mice developed more severe kidney dysfunction, proteinuria, and fibrosis following AngII treatment. Furthermore, myeloid PTEN deficiency exacerbated total collagen deposition and extracellular matrix protein production and enhanced myeloid fibroblast accumulation and myofibroblast formation in the kidney following AngII treatment. Finally, myeloid PTEN deficiency markedly augmented infiltration of F4/80+ macrophages and CD3+ T cells into the kidneys of AngII-treated mice. Taken together, these results indicate that PTEN plays a crucial role in the pathogenesis of renal inflammation and fibrosis through the regulation of infiltration of myeloid fibroblasts, macrophages, and T lymphocytes into the kidney.
Assuntos
Angiotensina II , Hipertensão , PTEN Fosfo-Hidrolase/metabolismo , Angiotensina II/metabolismo , Animais , Feminino , Fibrose , Humanos , Hipertensão/induzido quimicamente , Hipertensão/genética , Hipertensão/patologia , Hipertensão Renal , Inflamação/patologia , Rim/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nefrite , PTEN Fosfo-Hidrolase/genética , Fosfatidilinositol 3-Quinases/metabolismoRESUMO
Cisplatin, a commonly used anticancer drug, has been shown to induce acute kidney injury, which limits its clinical use in cancer treatment. Emerging evidence has suggested that AMP-activated protein kinase (AMPK), which functions as a cellular energy sensor, is activated by various cellular stresses that deplete cellular ATP. However, the potential role of AMPK in cisplatin-induced apoptosis of renal tubular epithelial cells has not been studied. In this study, we demonstrated that cisplatin activates AMPK (Thr172 phosphorylation) in cultured renal tubular epithelial cells in a time-dependent manner, which was associated with p53 phosphorylation. Compound C, a selective AMPK inhibitor, suppressed cisplatin-induced AMPK activation, p53 phosphorylation, Bax induction, and caspase 3 activation. Furthermore, silencing AMPK expression by siRNA attenuated cisplatin-induced p53 phosphorylation, Bax induction, and caspase 3 activation. In a mouse model of cisplatin-induced kidney injury, compound C inhibited p53 phosphorylation, Bax expression, caspase 3 activation, and apoptosis, protecting the kidney from injury and dysfunction. Taken together, these results suggest that the AMPK-p53-Bax signaling pathway plays a crucial role in cisplatin-induced tubular epithelial cell apoptosis.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Injúria Renal Aguda/induzido quimicamente , Apoptose/efeitos dos fármacos , Cisplatino/toxicidade , Células Epiteliais/efeitos dos fármacos , Túbulos Renais/efeitos dos fármacos , Proteínas Quinases Ativadas por AMP/genética , Injúria Renal Aguda/enzimologia , Injúria Renal Aguda/patologia , Animais , Caspase 3/metabolismo , Linhagem Celular , Células Epiteliais/enzimologia , Células Epiteliais/patologia , Túbulos Renais/enzimologia , Túbulos Renais/patologia , Camundongos , Fosforilação , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
Cisplatin can cause acute kidney injury (AKI), but the molecular mechanisms are not well understood. The objective of the present study was to examine the role of transforming growth factor-ß-activated kinase-1 (TAK1) in the pathogenesis of cisplatin-induced AKI. Wild-type mice and proximal tubule TAK1-deficient mice were treated with vehicle or cisplatin. Compared with wild-type control mice, proximal tubule TAK1-deficient mice had less severe kidney dysfunction, tubular damage, and apoptosis after cisplatin-induced AKI. Furthermore, conditional disruption of TAK1 in proximal tubular epithelial cells reduced caspase-3 activation, proinflammatory molecule expression, and JNK phosphorylation in the kidney in cisplatin-induced AKI. Taken together, cisplatin activates TAK1-JNK signaling pathway to promote tubular epithelial cell apoptosis and inflammation in cisplatin-induced AKI. Targeting TAK1 could be a novel therapeutic strategy against cisplatin-induced AKI.
Assuntos
Injúria Renal Aguda/genética , Antineoplásicos/efeitos adversos , Cisplatino/efeitos adversos , Rim/metabolismo , MAP Quinase Quinase Quinases/genética , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/patologia , Animais , Apoptose/genética , Células Epiteliais/patologia , Rim/patologia , MAP Quinase Quinase Quinases/metabolismo , Camundongos , Camundongos Knockout , FosforilaçãoRESUMO
BACKGROUND: We have shown that the CXCL16/CXCR6 axis plays a critical role in recruiting inflammatory cells and bone marrow-derived fibroblasts into the kidney leading to renal injury and fibrosis. However, the underlying signaling mechanisms are not known. METHODS: In the present study, we examined the role of phosphoinositide-3 kinase γ (PI3Kγ) signaling in the recruitment of inflammatory cells and bone marrow-derived fibroblasts into the kidney and development of renal injury and fibrosis in an experimental model of hypertension induced by angiotensin II. RESULTS: Blood pressure was comparable between wild-type (WT) and PI3Kγ knockout (KO) mice at baseline. Angiotensin II treatment led to an increase in blood pressure that was similar between WT and PI3Kγ KO mice. Compared with WT mice, PI3Kγ KO mice were protected from angiotensin II-induced renal dysfunction and injury and developed less proteinuria. PI3Kγ deficiency suppressed bone marrow-derived fibroblast accumulation and myofibroblast formation in the kidney and inhibited total collagen deposition and extracellular matrix protein production in the kidney in response to angiotensin II. PI3Kγ deficiency inhibited the infiltration of F4/80+ macrophages and CD3+ T cells into the kidney and reduced gene expression levels of pro-inflammatory cytokines in the kidney following angiotensin II treatment. Finally, inhibition of PI3Kγ suppressed CXCL16-induced monocyte migration in vitro. CONCLUSION: These results indicate that PI3Kγ mediates the influx of macrophages, T cells and bone marrow-derived fibroblasts into the kidney resulting in kidney injury and fibrosis.
Assuntos
Injúria Renal Aguda/prevenção & controle , Angiotensina II/toxicidade , Classe Ib de Fosfatidilinositol 3-Quinase/fisiologia , Fibrose/prevenção & controle , Hipertensão/complicações , Injúria Renal Aguda/etiologia , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/patologia , Animais , Citocinas/metabolismo , Modelos Animais de Doenças , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose/etiologia , Fibrose/metabolismo , Fibrose/patologia , Hipertensão/induzido quimicamente , Hipertensão/patologia , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Linfócitos T/metabolismo , Linfócitos T/patologia , Vasoconstritores/toxicidadeRESUMO
Renal fibrosis is a major pathological feature of chronic kidney disease, which is characterized by massive fibroblast activation and excessive production and deposition of extracellular matrix (ECM). Renal fibrosis results in progressive loss of kidney function; however, there is currently no effective therapy available clinically to treat or even reverse renal fibrosis. Although activated fibroblasts/myofibroblasts are responsible for the production and deposition of ECM, their origin has been debatable. Recent studies have provided compelling evidence that bone marrow-derived fibroblast precursors contribute significantly to the population of myofibroblasts and the development of renal fibrosis. Therefore, targeting the molecular signaling mechanisms underlying the recruitment and activation of the bone marrow-derived fibroblast precursors may serve as novel therapeutic strategy for chronic kidney disease. In this review, we appraise recent advances in our understanding of the recruitment and activation of bone marrow-derived fibroblast precursors in the kidney and the development of renal fibrosis and highlight novel molecular signaling pathways that may lead to the development of new therapies for chronic kidney disease.
Assuntos
Medula Óssea , Fibroblastos/citologia , Rim/patologia , Matriz Extracelular , Fibrose , Humanos , Miofibroblastos/citologia , Transdução de SinaisRESUMO
Ca(2+) sensitization of contraction has typically been investigated by bathing muscles in solutions containing agonists. However, it is unknown whether bath-applied agonists and enteric neurotransmission activate similar Ca(2+) sensitization mechanisms. We investigated protein kinase C (PKC)-potentiated phosphatase inhibitor protein of 17 kDa (CPI-17) and myosin phosphatase targeting subunit 1 (MYPT1) phosphorylation in murine gastric fundus muscles stimulated by bath-applied carbachol (CCh) or cholinergic motor neurotransmission. CCh increased MYPT1 phosphorylation at Thr696 (pT696) and Thr853 (pT853), CPI-17 at Thr38 (pT38), and myosin light chain at Ser19 (pS19). Electrical field stimulation (EFS) only increased pT38. In the presence of neostigmine, EFS increased pT38, pT853 and pS19. In fundus muscles of W/W(v) mice, EFS alone increased pT38 and pT853. Atropine blocked all contractions and all increases in pT696, pT853, pT38 and pS19. The Rho kinase (ROCK) inhibitor SAR1x blocked increases in pT853 and pT696. The PKC inhibitors Go6976 and Gf109203x or nicardipine blocked increases in pT38 and pT696. These findings suggest that cholinergic motor neurotransmission activates PKC-dependent CPI-17 phosphorylation. Bath-applied CCh recruits additional ROCK-dependent MYPT1 phosphorylation due to exposure of the agonist to a wider population of muscarinic receptors. Intramuscular interstitial cells of Cajal (ICC-IMs) and cholinesterases restrict ACh accessibility to a select population of muscarinic receptors, possibly only those expressed by ICC-IMs. These results provide the first biochemical evidence for focalized (or synaptic-like) neurotransmission, rather than diffuse 'volume' neurotransmission in a smooth muscle tissue. Furthermore, these findings demonstrate that bath application of contractile agonists to gastrointestinal smooth muscles does not mimic physiological responses to cholinergic neurotransmission.
Assuntos
Cálcio/metabolismo , Fundo Gástrico/fisiologia , Transmissão Sináptica , Animais , Fibras Colinérgicas/efeitos dos fármacos , Fibras Colinérgicas/fisiologia , Estimulação Elétrica , Fundo Gástrico/inervação , Fundo Gástrico/metabolismo , Células Intersticiais de Cajal/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Antagonistas Muscarínicos/farmacologia , Contração Muscular , Proteínas Musculares/metabolismo , Músculo Liso/inervação , Músculo Liso/metabolismo , Músculo Liso/fisiologia , Cadeias Leves de Miosina/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Fosfatase de Miosina-de-Cadeia-Leve , Neostigmina/farmacologia , Fosfoproteínas/metabolismo , Fosforilação , Inibidores de Proteínas Quinases/farmacologiaRESUMO
Diabetic gastroparesis is a common complication of diabetes, adversely affecting quality of life with symptoms of abdominal discomfort, nausea, and vomiting. The pathogenesis of this complex disorder is not well understood, involving abnormalities in the extrinsic and enteric nervous systems, interstitial cells of Cajal (ICCs), smooth muscles and immune cells. The ob/ob mouse model of obesity and diabetes develops delayed gastric emptying, providing an animal model for investigating how gastric smooth muscle dysfunction contributes to the pathophysiology of diabetic gastroparesis. Although ROCK2, MYPT1, and CPI-17 activities are reduced in intestinal motility disorders, their functioning has not been investigated in diabetic gastroparesis. We hypothesized that reduced expression and phosphorylation of the myosin light chain phosphatase (MLCP) inhibitory proteins MYPT1 and CPI-17 in ob/ob gastric antrum smooth muscles could contribute to the impaired antrum smooth muscle function of diabetic gastroparesis. Spontaneous and carbachol- and high K(+)-evoked contractions of gastric antrum smooth muscles from 7 to 12 week old male ob/ob mice were reduced compared to age- and strain-matched controls. There were no differences in spontaneous and agonist-evoked intracellular Ca(2+) transients and myosin light chain kinase expression. The F-actin:G-actin ratios were similar. Rho kinase 2 (ROCK2) expression was decreased at both ages. Basal and agonist-evoked MYPT1 and myosin light chain 20 phosphorylation, but not CPI-17 phosphorylation, was reduced compared to age-matched controls. These findings suggest that reduced MLCP inhibition due to decreased ROCK2 phosphorylation of MYPT1 in gastric antrum smooth muscles contributes to the antral dysmotility of diabetic gastroparesis.
Assuntos
Contração Muscular , Proteínas Musculares/metabolismo , Músculo Liso/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Fosfoproteínas/metabolismo , Antro Pilórico/metabolismo , Quinases Associadas a rho/metabolismo , Animais , Gastroparesia/metabolismo , Gastroparesia/patologia , Gastroparesia/fisiopatologia , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Obesos , Músculo Liso/patologia , Músculo Liso/fisiopatologia , Cadeias Leves de Miosina/metabolismo , Fosforilação , Antro Pilórico/patologia , Antro Pilórico/fisiopatologiaRESUMO
BACKGROUND AND PURPOSE: Renal fibrosis is a common feature of chronic kidney disease. Myeloid fibroblasts and macrophages contribute significantly to the pathogenesis of renal fibrosis. However, the molecular mechanisms underlying myeloid fibroblast activation and macrophage polarization are not fully understood. In this study, we examined the role of Jumonji domain-containing protein-3 (JMJD3) in myeloid fibroblast activation, macrophage polarization, and renal fibrosis development in a preclinical model of obstructive nephropathy. EXPERIMENTAL APPROACH: To examine the role of JMJD3 in renal fibrosis, we generated mice with global or myeloid cell-specific deletion of JMJD3, and we treated wild-type mice with vehicle or GSK-J4 (selective JMJD3 inhibitor). Mice were subjected to unilateral ureteral obstructive injury to induce renal fibrosis. KEY RESULTS: JMJD3 expression was significantly increased in the kidneys during the development of renal fibrosis, which was associated with an increase in H3K27 dimethylation. Mice with either global or myeloid JMJD3 deficiency exhibited significantly reduced total collagen deposition and extracellular matrix protein production, myeloid fibroblast activation and M2 macrophage polarization in the obstructed kidney. Moreover, IFN regulatory factor 4, a mediator of M2 macrophage polarization, was significantly induced in the obstructed kidneys, which was abolished by JMJD3 deficiency. Furthermore, pharmacological inhibition of JMJD3 with GSK-J4 attenuated kidney fibrosis, reduced myeloid fibroblast activation and suppressed M2 macrophage polarization in the obstructed kidney. CONCLUSION AND IMPLICATIONS: Our study identifies JMJD3 as a critical regulator of myeloid fibroblast activation, macrophage polarization, and renal fibrosis development. Therefore, JMJD3 may represent a promising therapeutic target for chronic kidney disease.
Assuntos
Ativação de Macrófagos , Insuficiência Renal Crônica , Camundongos , Animais , Rim/patologia , Macrófagos/metabolismo , Fibrose , Insuficiência Renal Crônica/metabolismo , Fibroblastos/patologia , Camundongos Endogâmicos C57BLRESUMO
A hallmark of chronic kidney disease is renal fibrosis, which can result in progressive loss of kidney function. Currently, there is no effective therapy for renal fibrosis. Therefore, there is an urgent need to identify potential drug targets for renal fibrosis. In this study, we examined the effect of a selective STAT6 inhibitor, AS1517499, on myeloid fibroblast activation, macrophage polarization, and development of renal fibrosis in two experimental murine models. To investigate the effect of STAT6 inhibition on myeloid fibroblast activation, macrophage polarization, and kidney fibrosis, wild-type mice were subjected to unilateral ureteral obstruction or folic acid administration and treated with AS1517499. Mice treated with vehicle were used as control. At the end of experiments, kidneys were harvested for analysis of myeloid fibroblast activation, macrophage polarization, and renal fibrosis and function. Unilateral ureteral obstruction or folic acid administration induced STAT6 activation in interstitial cells of the kidney, which was significantly abolished by AS1517499 treatment. Mice treated with AS1517499 accumulated fewer myeloid fibroblasts and myofibroblasts in the kidney with ureteral obstruction or folic acid nephropathy compared with vehicle-treated mice. Moreover, AS1517499 significantly suppressed M2 macrophage polarization in the injured kidney. Furthermore, AS1517499 markedly reduced the expression levels of extracellular matrix proteins, and development of kidney fibrosis and dysfunction. These findings suggest that AS1517499 inhibits STAT6 activation, suppresses myeloid fibroblast activation, reduces M2 macrophage polarization, attenuates extracellular matrix protein production, and preserves kidney function. Therefore, targeting STAT6 with AS1517499 is a novel therapeutic approach for chronic kidney disease.
Assuntos
Fibroblastos/efeitos dos fármacos , Nefropatias/tratamento farmacológico , Rim/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Pirimidinas/farmacologia , Fator de Transcrição STAT6/antagonistas & inibidores , Animais , Modelos Animais de Doenças , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Proteínas da Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose , Rim/metabolismo , Rim/patologia , Nefropatias/etiologia , Nefropatias/metabolismo , Nefropatias/patologia , Macrófagos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Fenótipo , Fator de Transcrição STAT6/metabolismo , Transdução de Sinais , Obstrução Ureteral/complicaçõesRESUMO
Renal fibrosis is a pathologic feature of chronic kidney disease, which can lead to end-stage kidney disease. Myeloid fibroblasts play a central role in the pathogenesis of renal fibrosis. However, the molecular mechanisms pertaining to myeloid fibroblast activation remain to be elucidated. In the present study, we examine the role of signal transducer and activator of transcription 6 (STAT6) in myeloid fibroblast activation, macrophage polarization, and renal fibrosis development in a mouse model of folic acid nephropathy. STAT6 is activated in the kidney with folic acid nephropathy. Compared with folic-acid-treated wild-type mice, STAT6 knockout mice had markedly reduced myeloid fibroblasts and myofibroblasts in the kidney with folic acid nephropathy. Furthermore, STAT6 knockout mice exhibited significantly less CD206 and PDGFR-ß dual-positive fibroblast accumulation and M2 macrophage polarization in the kidney with folic acid nephropathy. Consistent with these findings, STAT6 knockout mice produced less extracellular matrix protein, exhibited less severe interstitial fibrosis, and preserved kidney function in folic acid nephropathy. Taken together, these results have shown that STAT6 plays a critical role in myeloid fibroblasts activation, M2 macrophage polarization, extracellular matrix protein production, and renal fibrosis development in folic acid nephropathy. Therefore, targeting STAT6 may provide a novel therapeutic strategy for fibrotic kidney disease.
Assuntos
Polaridade Celular , Fibroblastos/metabolismo , Ácido Fólico/metabolismo , Nefropatias/metabolismo , Macrófagos/metabolismo , Células Mieloides/patologia , Fator de Transcrição STAT6/deficiência , Animais , Biomarcadores/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Fibroblastos/patologia , Rim/patologia , Rim/fisiopatologia , Nefropatias/patologia , Macrófagos/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator de Transcrição STAT6/metabolismoRESUMO
Circulating cells have a pathogenic role in the development of hypertensive nephropathy. However, how these cells infiltrate into the kidney are not fully elucidated. In this study, we investigated the role of CXCR6 in deoxycorticosterone acetate (DOCA)/salt-induced inflammation and fibrosis of the kidney. Following uninephrectomy, wild-type and CXCR6 knockout mice were treated with DOCA/salt for 3 weeks. Blood pressure was similar between wild-type and CXCR6 knockout mice at baseline and after treatment with DOCA/salt. Wild-type mice develop significant kidney injury, proteinuria, and kidney fibrosis after three weeks of DOCA/salt treatment. CXCR6 deficiency ameliorated kidney injury, proteinuria, and kidney fibrosis following treatment with DOCA/salt. Moreover, CXCR6 deficiency inhibited accumulation of bone marrow-derived fibroblasts and myofibroblasts in the kidney following treatment with DOCA/salt. Furthermore, CXCR6 deficiency markedly reduced the number of macrophages and T cells in the kidney after DOCA/salt treatment. In summary, our results identify a critical role of CXCR6 in the development of inflammation and fibrosis of the kidney in salt-sensitive hypertension.
Assuntos
Acetato de Desoxicorticosterona/efeitos adversos , Hipertensão/induzido quimicamente , Hipertensão/metabolismo , Rim/patologia , Receptores CXCR6/deficiência , Receptores CXCR6/genética , Cloreto de Sódio na Dieta/efeitos adversos , Animais , Pressão Sanguínea/efeitos dos fármacos , Pressão Sanguínea/genética , Proteínas da Matriz Extracelular/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibrose , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Técnicas de Inativação de Genes , Hipertensão/genética , Hipertensão/patologia , Rim/efeitos dos fármacos , Rim/lesões , Rim/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Cloreto de Sódio na Dieta/administração & dosagemRESUMO
CKD is a global public health problem. Renal fibrosis is a final common pathway leading to progressive loss of function in CKD. The degree of renal fibrosis predicts the prognosis of CKD. Recent studies have shown that bone marrow-derived fibroblasts contribute significantly to the development of renal fibrosis, which may yield novel therapeutic strategy for fibrotic kidney disease. Therefore, it is imperative to accurately assess the degree of renal fibrosis noninvasively to identify those patients who can benefit from antifibrotic therapy. In this review, we summarize recent advances in the assessment of renal fibrosis by magnetic resonance imaging.
Assuntos
Fibrose/diagnóstico por imagem , Rim/diagnóstico por imagem , Rim/patologia , Imageamento por Ressonância Magnética , Humanos , Insuficiência Renal Crônica/diagnóstico por imagem , Insuficiência Renal Crônica/etiologiaRESUMO
Telokin phosphorylation by cyclic GMP-dependent protein kinase facilitates smooth muscle relaxation. In this study we examined the relaxation of gastric fundus smooth muscles from basal tone, or pre-contracted with KCl or carbachol (CCh), and the phosphorylation of telokin S13, myosin light chain (MLC) S19, MYPT1 T853, T696, and CPI-17 T38 in response to 8-Bromo-cGMP, the NO donor sodium nitroprusside (SNP), or nitrergic neurotransmission. We compared MLC phosphorylation and the contraction and relaxation responses of gastric fundus smooth muscles from telokin-/- mice and their wild-type littermates to KCl or CCh, and 8-Bromo-cGMP, SNP, or nitrergic neurotransmission, respectively. We compared the relaxation responses and telokin phosphorylation of gastric fundus smooth muscles from wild-type mice and W/WV mice which lack ICC-IM, to 8-Bromo-cGMP, SNP, or nitrergic neurotransmission. We found that telokin S13 is basally phosphorylated and that 8-Bromo-cGMP and SNP increased basal telokin phosphorylation. In muscles pre-contracted with KCl or CCh, 8-Bromo-cGMP and SNP had no effect on CPI-17 or MYPT1 phosphorylation, but increased telokin phosphorylation and reduced MLC phosphorylation. In telokin-/- gastric fundus smooth muscles, basal tone and constitutive MLC S19 phosphorylation were increased. Pre-contracted telokin-/- gastric fundus smooth muscles have increased contractile responses to KCl, CCh, or cholinergic neurotransmission and reduced relaxation to 8-Bromo-cGMP, SNP, and nitrergic neurotransmission. However, basal telokin phosphorylation was not increased when muscles were stimulated with lower concentrations of SNP or when the muscles were stimulated by nitrergic neurotransmission. SNP, but not nitrergic neurotransmission, increased telokin Ser13 phosphorylation in both wild-type and W/WV gastric fundus smooth muscles. Our findings indicate that telokin may play a role in attenuating constitutive MLC phosphorylation and provide an additional mechanism to augment gastric fundus mechanical responses to inhibitory neurotransmission.
Assuntos
Fundo Gástrico/fisiologia , Músculo Liso/fisiologia , Quinase de Cadeia Leve de Miosina/química , Fragmentos de Peptídeos/química , Animais , Carbacol/química , GMP Cíclico/análogos & derivados , GMP Cíclico/química , Proteínas Quinases Dependentes de GMP Cíclico/química , Fundo Gástrico/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Contração Muscular/efeitos dos fármacos , Relaxamento Muscular/efeitos dos fármacos , Tono Muscular/efeitos dos fármacos , Cadeias Leves de Miosina/química , Neurônios/fisiologia , Óxido Nítrico/química , Doadores de Óxido Nítrico/química , Nitroprussiato/química , Fosforilação , Cloreto de Potássio/químicaRESUMO
Human DNA polymerase (pol) ß is essential for base excision repair. We previously reported a triple somatic mutant of pol ß (p.P261L/T292A/I298T) found in an early onset prostate tumor. This mutation abolishes polymerase activity, and the wild-type allele was not present in the tumor, indicating a complete deficiency in pol ß function. The effect on polymerase activity is unexpected because the point mutations that comprise the triple mutant are not part of the active site. Herein, we demonstrate the mechanism of this loss-of-function. In order to understand the effect of the individual point mutations we biochemically analyzed all single and double mutants that comprise the triple mutant. We found that the p.I298T mutation is responsible for a marked instability of the triple mutant protein at 37ËC. At room temperature the triple mutant's low efficiency is also due to a decrease in the apparent binding affinity for the dNTP substrate, which is due to the p.T292A mutation. Furthermore, the triple mutant displays lower fidelity for transversions in vitro, due to the p.T292A mutation. We conclude that distinct mutations of the triple pol ß mutant are responsible for the loss of activity, lower fidelity, and instability observed in vitro.