RESUMO
Diabetes is the leading cause of end-stage renal disease, resulting in a significant health care burden and loss of economic productivity by affected individuals. Because current therapies for progression of diabetic nephropathy (DN) are only moderately successful, identification of underlying mechanisms of disease is essential to develop more effective therapies. We showed previously that inhibition of arginase using S-(2-boronoethyl)-l-cysteine (BEC) or genetic deficiency of the arginase-2 isozyme was protective against key features of nephropathy in diabetic mouse models. However, those studies did not determine whether all markers of DN were dependent only on arginase-2 expression. The objective of this study was to identify features of DN that are associated specifically with expression of arginase-1 or -2. Elevated urinary albumin excretion rate and plasma urea levels, increases in renal fibronectin mRNA levels, and decreased renal medullary blood flow were associated almost completely and specifically with arginase-2 expression, indicating that arginase-2 selectively mediates major aspects of diabetic renal injury. However, increases in renal macrophage infiltration and renal TNF-α mRNA levels occurred independent of arginase-2 expression but were almost entirely abolished by treatment with BEC, indicating a distinct role for arginase-1. We therefore generated mice with a macrophage-specific deletion of arginase-1 (CD11bCre /Arg1fl/fl ). CD11bCre /Arg1fl/fl mice had significantly reduced macrophage infiltration but had no effect on albuminuria compared with Arg1fl/fl mice after 12 wk of streptozotocin-induced diabetes. These results indicate that selective inhibition of arginase-2 would be effective in preventing or ameliorating major features of diabetic renal injury.
Assuntos
Arginase/metabolismo , Nefropatias Diabéticas/enzimologia , Albuminúria/enzimologia , Albuminúria/etiologia , Animais , Nefropatias Diabéticas/complicações , Fibronectinas/metabolismo , Macrófagos/enzimologia , Masculino , Camundongos , Circulação Renal , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Inflammation is a central pathophysiologic mechanism that contributes to diabetes mellitus and diabetic nephropathy. Recently, we showed that macrophages directly contribute to diabetic renal injury and that pharmacological blockade or genetic deficiency of chemokine (C-C motif) receptor 2 (CCR2) confers kidney protection in diabetic nephropathy. However, the direct role of CCR2 in kidney-derived cells such as podocytes in diabetic nephropathy remains unclear. To study this, we developed a transgenic mouse model expressing CCR2 specifically in podocytes (Tg[NPHS2-Ccr2]) on a nephropathy-prone (DBA/2J) and CCR2-deficient (Ccr2-/-) background with heterozygous Ccr2+/- littermate controls. Diabetes was induced by streptozotocin. As expected, absence of CCR2 conferred kidney protection after nine weeks of diabetes. In contrast, transgenic CCR2 overexpression in the podocytes of Ccr2-/- mice resulted in significantly increased albuminuria, blood urea nitrogen, histopathologic changes, kidney fibronectin and type 1 collagen expression, podocyte loss, and glomerular apoptosis after nine weeks of streptozotocin-induced diabetes. Interestingly, there was no concurrent increase in kidney macrophage recruitment or inflammatory cytokine levels in the mice. These findings support a direct role for CCR2 expression in podocytes to mediate diabetic renal injury, independent of monocyte/macrophage recruitment. Thus, targeting the CCR2 signaling cascade in podocytes could be a novel therapeutic approach for treatment of diabetic nephropathy.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Nefropatias Diabéticas/metabolismo , Podócitos/metabolismo , Receptores CCR2/metabolismo , Albuminúria/genética , Albuminúria/metabolismo , Albuminúria/prevenção & controle , Animais , Apoptose , Nitrogênio da Ureia Sanguínea , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/patologia , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/patologia , Nefropatias Diabéticas/prevenção & controle , Fibronectinas/genética , Fibronectinas/metabolismo , Fibrose , Predisposição Genética para Doença , Mediadores da Inflamação/metabolismo , Macrófagos/metabolismo , Masculino , Camundongos Endogâmicos DBA , Camundongos Knockout , Camundongos Transgênicos , Monócitos/metabolismo , Fenótipo , Podócitos/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores CCR2/deficiência , Receptores CCR2/genética , Transdução de Sinais , Estreptozocina , Regulação para CimaRESUMO
Our previous publication showed that inhibition of arginase prevents the development of diabetic nephropathy (DN). However, identification of targets that retard the progression of established DN-which is more clinically relevant-is lacking. Therefore, we tested the hypothesis that arginase inhibition would prevent the progression of established DN. Effects of arginase inhibition were compared with treatment with the angiotensin-converting enzyme inhibitor captopril, a current standard of care in DN. Experiments were conducted in Ins2(Akita) mice treated with the arginase inhibitor S-(2-boronoethyl)-l-cysteine (BEC) or captopril starting at 6 wk of age for 12 wk (early treatment) or starting at 12 wk of age for 6 wk (late treatment). Early and late treatment with BEC resulted in protection from DN as indicated by reduced albuminuria, histological changes, kidney macrophage infiltration, urinary thiobarbituric acid-reactive substances, and restored nephrin expression, kidney nitrate/nitrite, kidney endothelial nitric oxide synthase phosphorylation, and renal medullary blood flow compared with vehicle-treated Ins2(Akita) mice at 18 wk of age. Interestingly, early treatment with captopril reduced albuminuria, histological changes, and kidney macrophage infiltration without affecting the other parameters, but late treatment with captopril was ineffective. These findings highlight the importance of arginase inhibition as a new potential therapeutic intervention in both early and late stages of diabetic renal injury.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Arginase/antagonistas & inibidores , Ácidos Borônicos/uso terapêutico , Captopril/uso terapêutico , Nefropatias Diabéticas/tratamento farmacológico , Albuminúria/tratamento farmacológico , Albuminúria/metabolismo , Animais , Ácidos Borônicos/farmacologia , Captopril/farmacologia , Nefropatias Diabéticas/metabolismo , Progressão da Doença , Rim/efeitos dos fármacos , Rim/metabolismo , Camundongos , Óxido Nítrico Sintase Tipo III/metabolismo , Fosforilação , Resultado do TratamentoRESUMO
Monocyte/macrophage recruitment correlates strongly with the progression of diabetic nephropathy. Tumor necrosis factor-α (TNF-α) is produced by monocytes/macrophages but the direct role of TNF-α and/or macrophage-derived TNF-α in the progression of diabetic nephropathy remains unclear. Here we tested whether inhibition of TNF-α confers kidney protection in diabetic nephropathy via a macrophage-derived TNF-α-dependent pathway. Compared to vehicle-treated mice, blockade of TNF-α with a murine anti-TNF-α antibody conferred kidney protection in Ins2(Akita) mice as indicated by reductions in albuminuria, plasma creatinine, histopathologic changes, kidney macrophage recruitment, and plasma inflammatory cytokine levels at 18 weeks of age. To assess the direct role of macrophage-derived TNF-α in diabetic nephropathy, we generated macrophage-specific TNF-α-deficient mice (CD11b(Cre)/TNF-α(Flox/Flox)). Conditional ablation of TNF-α in macrophages significantly reduced albuminuria, the increase in plasma creatinine and blood urea nitrogen, histopathologic changes, and kidney macrophage recruitment compared to diabetic TNF-α(Flox/Flox) control mice after 12 weeks of streptozotocin-induced diabetes. Thus, production of TNF-α by macrophages plays a major role in diabetic renal injury. Hence, blocking TNF-α could be a novel therapeutic approach for treatment of diabetic nephropathy.
Assuntos
Nefropatias Diabéticas/metabolismo , Mediadores da Inflamação/metabolismo , Rim/metabolismo , Macrófagos Peritoneais/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Albuminúria/genética , Albuminúria/metabolismo , Albuminúria/prevenção & controle , Animais , Anticorpos Neutralizantes/farmacologia , Biomarcadores/sangue , Nitrogênio da Ureia Sanguínea , Antígeno CD11b/genética , Antígeno CD11b/metabolismo , Quimiotaxia , Creatinina/sangue , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/patologia , Nefropatias Diabéticas/prevenção & controle , Predisposição Genética para Doença , Mediadores da Inflamação/antagonistas & inibidores , Rim/efeitos dos fármacos , Rim/patologia , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , Fenótipo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Transdução de Sinais , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/deficiência , Fator de Necrose Tumoral alfa/genéticaRESUMO
Our recent publication showed that pharmacological blockade of arginases confers kidney protection in diabetic nephropathy via a nitric oxide (NO) synthase (NOS)3-dependent mechanism. Arginase competes with endothelial NOS (eNOS) for the common substrate L-arginine. Lack of L-arginine results in reduced NO production and eNOS uncoupling, which lead to endothelial dysfunction. Therefore, we hypothesized that L-arginine or L-citrulline supplementation would ameliorate diabetic nephropathy. DBA mice injected with multiple low doses of vehicle or streptozotocin (50 mg/kg ip for 5 days) were provided drinking water with or without L-arginine (1.5%, 6.05 g·kg(-1)·day(-1)) or L-citrulline (1.66%, 5.73 g·kg(-1)·day(-1)) for 9 wk. Nonsupplemented diabetic mice showed significant increases in albuminuria, blood urea nitrogen, glomerular histopathological changes, kidney macrophage recruitment, kidney TNF-α and fibronectin mRNA expression, kidney arginase activity, kidney arginase-2 protein expression, and urinary oxidative stress along with a significant reduction of nephrin and eNOS protein expression and kidney nitrite + nitrate compared with normal mice after 9 wk of diabetes. Surprisingly, L-arginine or L-citrulline supplementation in diabetic mice did not affect any of these parameters despite greatly increasing kidney and plasma arginine levels. These findings demonstrate that chronic L-arginine or L-citrulline supplementation does not prevent or reduce renal injury in a model of type 1 diabetes.
Assuntos
Arginina/uso terapêutico , Citrulina/uso terapêutico , Nefropatias Diabéticas/tratamento farmacológico , Suplementos Nutricionais , Aminoácidos/sangue , Aminoácidos/metabolismo , Animais , Arginase/metabolismo , Arginina/sangue , Pressão Sanguínea/fisiologia , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Nefropatias Diabéticas/patologia , Fibronectinas/biossíntese , Rim/patologia , Masculino , Proteínas de Membrana/biossíntese , Camundongos , Camundongos Endogâmicos DBA , Nitratos/metabolismo , Óxido Nítrico Sintase Tipo III/biossíntese , Nitritos/metabolismo , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Monocyte/macrophage recruitment correlates strongly with the progression of renal impairment in diabetic nephropathy (DN), yet their direct role is not clear. We hypothesized that macrophages contribute to direct podocyte injury and/or an abnormal podocyte niche leading to DN. Experiments were conducted in CD11b-DTR mice treated with diphtheria toxin (DT) to deplete macrophages after streptozotocin-induced diabetes. Additional experiments were conducted in bone marrow chimeric (CD11b-DTRâ C57BL6/J) mice. Diabetes was associated with an increase in the M1-to-M2 ratio by 6 wk after the induction of diabetes. Macrophage depletion in diabetic CD11b-DTR mice significantly attenuated albuminuria, kidney macrophage recruitment, and glomerular histological changes and preserved kidney nephrin and podocin expression compared with diabetic CD11b-DTR mice treated with mutant DT. These data were confirmed in chimeric mice indicating a direct role of bone marrow-derived macrophages in DN. In vitro, podocytes grown in high-glucose media significantly increased macrophage migration compared with podocytes grown in normal glucose media. In addition, classically activated M1 macrophages, but not M2 macrophages, induced podocyte permeability. These findings provide evidence showing that macrophages directly contribute to kidney injury in DN, perhaps by altering podocyte integrity through the proinflammatory M1 subset of macrophages. Attenuating the deleterious effects of macrophages on podocytes could provide a new therapeutic approach to the treatment of DN.
Assuntos
Diabetes Mellitus Experimental/complicações , Nefropatias Diabéticas/patologia , Nefropatias Diabéticas/fisiopatologia , Macrófagos/fisiologia , Podócitos/patologia , Animais , Antígeno CD11b/genética , Contagem de Células , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Diabetes Mellitus Experimental/induzido quimicamente , Nefropatias Diabéticas/etiologia , Modelos Animais de Doenças , Glucose/farmacologia , Técnicas In Vitro , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Macrófagos/patologia , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Podócitos/efeitos dos fármacos , Podócitos/metabolismo , Estreptozocina/efeitos adversosRESUMO
Pigment epithelium-derived factor (PEDF) is a multifunctional protein with antiangiogenic, antioxidative, and anti-inflammatory properties. PEDF is involved in the pathogenesis of diabetic retinopathy, but its direct role in the kidneys remains unclear. We hypothesize that a PEDF fragment (P78-PEDF) confers kidney protection in diabetic nephropathy (DN). The localization of the full-length PEDF protein were determined in DBA mice following multiple low doses of streptozotocin. Using immunohistochemistry, PEDF was localized in the kidney vasculature, interstitial space, glomeruli, tubules, and renal medulla. Kidney PEDF protein and mRNA expression were significantly reduced in diabetic mice. Continuous infusion of P78-PEDF for 6 wk resulted in protection from diabetic neuropathy as indicated by reduced albuminuria and blood urea nitrogen, increased nephrin expression, decreased kidney macrophage recruitment and inflammatory cytokines, and reduced histological changes compared with vehicle-treated diabetic mice. In vitro, P78-PEDF blocked the increase in podocyte permeability to albumin and disruption of the actin cytoskeleton induced by puromycin aminonucleoside treatment. These findings highlight the importance of P78-PEDF peptide as a potential therapeutic modality in early phase diabetic renal injury.
Assuntos
Nefropatias Diabéticas/prevenção & controle , Proteínas do Olho/uso terapêutico , Fatores de Crescimento Neural/uso terapêutico , Fragmentos de Peptídeos/uso terapêutico , Serpinas/uso terapêutico , Albuminúria/prevenção & controle , Animais , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Experimental/fisiopatologia , Nefropatias Diabéticas/tratamento farmacológico , Nefropatias Diabéticas/patologia , Proteínas do Olho/fisiologia , Rim/metabolismo , Masculino , Proteínas de Membrana/biossíntese , Camundongos , Camundongos Endogâmicos DBA , Fatores de Crescimento Neural/fisiologia , Podócitos/efeitos dos fármacos , Serpinas/fisiologiaRESUMO
Recently, we showed that pharmacological blockade or genetic deficiency of arginase-2 confers kidney protection in diabetic mouse models. Here, we tested whether the protective effect of arginase inhibition is nitric oxide synthase 3 (eNOS) dependent in diabetic nephropathy. Experiments were conducted in eNOS-knockout and their wild-type littermate mice using multiple low doses of vehicle or streptozotocin, and treated with continuous subcutaneous infusion of vehicle or the arginase inhibitor S-(2-boronoethyl)-L-cysteine by an osmotic pump. Inhibition of arginases for 6 weeks in diabetic wild-type mice significantly attenuated albuminuria, the increase in plasma creatinine and blood urea nitrogen, histopathological changes, kidney fibronectin and TNF-α expression, kidney macrophage recruitment, and oxidative stress compared with vehicle-treated diabetic wild-type mice. Arginase inhibition in diabetic eNOS-knockout mice failed to affect any of these parameters, but reduced kidney macrophage recruitment and kidney TNF-α expression compared with vehicle-treated diabetic eNOS-knockout mice. Furthermore, diabetic wild-type and eNOS-knockout mice exhibited increased kidney arginase-2 protein, arginase activity, and ornithine levels. Thus, arginase inhibition mediates renal tissue protection in diabetic nephropathy by an eNOS-dependent mechanism and has an eNOS-independent effect on kidney macrophage recruitment.
Assuntos
Arginase/antagonistas & inibidores , Ácidos Borônicos/farmacologia , Diabetes Mellitus Experimental/tratamento farmacológico , Nefropatias Diabéticas/prevenção & controle , Inibidores Enzimáticos/farmacologia , Rim/efeitos dos fármacos , Óxido Nítrico Sintase Tipo III/metabolismo , Albuminúria/enzimologia , Albuminúria/prevenção & controle , Animais , Arginase/metabolismo , Biomarcadores/sangue , Nitrogênio da Ureia Sanguínea , Ácidos Borônicos/administração & dosagem , Creatinina/sangue , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/enzimologia , Nefropatias Diabéticas/sangue , Nefropatias Diabéticas/enzimologia , Nefropatias Diabéticas/etiologia , Inibidores Enzimáticos/administração & dosagem , Fibronectinas/metabolismo , Infusões Subcutâneas , Rim/enzimologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico Sintase Tipo III/deficiência , Óxido Nítrico Sintase Tipo III/genética , Ornitina/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Estreptozocina , Fatores de Tempo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
UNLABELLED: c-Met, a high-affinity receptor for hepatocyte growth factor (HGF), plays a critical role in cancer growth, invasion, and metastasis. Hepatocellular carcinoma (HCC) patients with an active HGF/c-Met signaling pathway have a significantly worse prognosis. Although targeting the HGF/c-Met pathway has been proposed for the treatment of multiple cancers, the effect of c-Met inhibition in HCC remains unclear. The human HCC cell lines Huh7, Hep3B, MHCC97-L, and MHCC97-H were used in this study to investigate the effect of c-Met inhibition using the small molecule selective c-Met tyrosine kinase inhibitor PHA665752. MHCC97-L and MHCC97-H cells demonstrate a mesenchymal phenotype with decreased expression of E-cadherin and increased expression of c-Met, fibronectin, and Zeb2 compared with Huh7 and Hep3B cells, which have an epithelial phenotype. PHA665752 treatment blocked phosphorylation of c-Met and downstream phosphoinositide 3-kinase/Akt and mitogen-activated protein kinase/Erk pathways, inhibited cell proliferation, and induced apoptosis in c-Met-positive MHCC97-L and MHCC97-H cells. In xenograft models, administration of PHA665752 significantly inhibited c-Met-positive MHCC97-L and MHCC97-H tumor growth, and PHA665752-treated tumors demonstrated marked reduction of both c-Met phosphorylation and cell proliferation. c-Met-negative Huh7 and Hep3B cells were not affected by c-Met inhibitor treatment in vitro or in vivo. In addition, c-Met-positive MHCC97-L and MHCC97-H cells demonstrated cancer stem cell-like characteristics, such as resistance to chemotherapy, tumor sphere formation, and increased expression of CD44 and ABCG2, and PHA665752 treatment suppressed tumor sphere formation and inhibited CD44 expression. CONCLUSION: c-Met represents a potential target of personalized treatment for HCC with an active HGF/c-Met pathway.
Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Indóis/uso terapêutico , Neoplasias Hepáticas/tratamento farmacológico , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Sulfonas/uso terapêutico , Animais , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Neoplasias Hepáticas/patologia , Camundongos , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Fosforilação , Medicina de Precisão , Proteínas Proto-Oncogênicas c-met/metabolismoRESUMO
UNLABELLED: Hepatocellular carcinoma (HCC) is the third leading cause of cancer mortality worldwide. CD133, a transmembrane glycoprotein, is an important cell surface marker for both stem cells and cancer stem cells in various tissues including liver. CD133 expression has been recently linked to poor prognosis in HCC patients. CD133+ liver cancer cells are characterized by resistance to chemotherapy, self-renewal, multilineage potential, increased colony formation, and in vivo cancer initiation at limited dilution. Recent studies demonstrate that CD133 expression is regulated by DNA methylation. In this study, we explored the role of transforming growth factor beta (TGFbeta), a multifunctional cytokine that plays a critical role in chronic liver injury, in the regulation of CD133 expression. TGFbeta1 is capable of up-regulating CD133 expression specifically within the Huh7 HCC cell line in a time- and dose-dependent manner. Most important, TGFbeta1-induced CD133+ Huh7 cells demonstrate increased tumor initiation in vivo. Forced expression of inhibitory Smads, including Smad6 and Smad7, attenuated TGFbeta1-induced CD133 expression. Within CD133- Huh7 cells, TGFbeta1 stimulation inhibited the expression of DNA methyltransferases (DNMT) 1 and DNMT3beta, which are critical in the maintenance of regional DNA methylation, and global DNMT activity in CD133- Huh7 cells was inhibited by TGFbeta1. DNMT3beta inhibition by TGFbeta1 was partially rescued with overexpression of inhibitory Smads. Lastly, TGFbeta1 treatment led to significant demethylation in CD133 promoter-1 in CD133- Huh7 cells. CONCLUSION: TGFbeta1 is able to regulate CD133 expression through inhibition of DNMT1 and DNMT3beta expression and subsequent demethylation of promoter-1. TGFbeta1-induced CD133+ Huh7 cells are tumorigenic. The mechanism by which TGFbeta induces CD133 expression is partially dependent on the Smads pathway.
Assuntos
Antígenos CD/genética , Carcinoma Hepatocelular/metabolismo , Epigênese Genética/fisiologia , Glicoproteínas/genética , Neoplasias Hepáticas/metabolismo , Peptídeos/genética , Fator de Crescimento Transformador beta/farmacologia , Antígeno AC133 , Linhagem Celular Tumoral , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/biossíntese , Humanos , Células-Tronco Neoplásicas/metabolismo , Proteínas Smad/fisiologia , Regulação para Cima , DNA Metiltransferase 3BRESUMO
UNLABELLED: Epithelial-to-mesenchymal transition (EMT) is predicted to play a critical role in metastatic disease in hepatocellular carcinoma. In this study, we used a novel murine model of EMT to elucidate a mechanism of tumor progression and metastasis. A total of 2 x 10(6) liver cells isolated from Pten(loxp/loxp)/Alb-Cre(+) mice, expanded from a single CD133(+)CD45(-) cell clone, passage 0 (P0), were sequentially transplanted to obtain two passages of tumor cells, P1 and P2. Cells were analyzed for gene expression using microarray and real-time polymerase chain reaction. Functional analysis included cell proliferation, migration, and invasion in vitro and orthotopic tumor metastasis assays in vivo. Although P0, P1, and P2 each formed tumors consistent with mixed liver epithelium, within the P2 cells, two distinct cell types were clearly visible: cells with epithelial morphology similar to P0 cells and cells with fibroblastoid morphology. These P2 mesenchymal cells demonstrated increased locomotion on wound healing; increased cell invasion on Matrigel basement membrane; increased EMT-associated gene expression of Snail1, Zeb1, and Zeb2; and down-regulated E-cadherin. P2 mesenchymal cells demonstrated significantly faster tumor growth in vivo compared with P2 epithelial counterparts, with invasion of intestine, pancreas, spleen, and lymph nodes. Furthermore, P2 mesenchymal cells secreted high levels of hepatocyte growth factor (HGF), which we propose acts in a paracrine fashion to drive epithelial cells to undergo EMT. In addition, a second murine liver cancer stem cell line with methionine adenosyltransferase 1a deficiency acquired EMT after sequential transplantations, indicating that EMT was not restricted to Pten-deleted tumors. CONCLUSION: EMT is associated with a high rate of liver tumor proliferation, invasion, and metastasis in vivo, which is driven by HGF secreted from mesenchymal tumor cells in a feed-forward mechanism.
Assuntos
Carcinoma Hepatocelular/patologia , Transformação Celular Neoplásica/patologia , Células Epiteliais/patologia , Neoplasias Hepáticas/patologia , Mesoderma/patologia , Antígeno AC133 , Animais , Antígenos CD/metabolismo , Carcinoma Hepatocelular/metabolismo , Movimento Celular/fisiologia , Transformação Celular Neoplásica/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Glicoproteínas/metabolismo , Fator de Crescimento de Hepatócito/metabolismo , Neoplasias Hepáticas/metabolismo , Mesoderma/metabolismo , Metionina Adenosiltransferase/genética , Metionina Adenosiltransferase/metabolismo , Camundongos , Camundongos Knockout , Camundongos Nus , Invasividade Neoplásica/patologia , Metástase Neoplásica/patologia , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Peptídeos/metabolismo , Transdução de Sinais/fisiologiaRESUMO
UNLABELLED: Methionine adenosyltransferase (MAT) is an essential enzyme required for S-adenosylmethionine biosynthesis. Hepatic MAT activity falls during chronic liver injury, and mice lacking Mat1a develop spontaneous hepatocellular carcinoma by 18 months. We have previously demonstrated that CD133(+)CD45(-) oval cells isolated from 16-month-old Mat1a(-/-) mice represent a liver cancer stem cell population. The transforming growth factor beta (TGF-beta) pathway constitutes a central signaling network in proliferation, apoptosis, and tumorigenesis. In this study, we tested the response of tumorigenic liver stem cells to TGF-beta. CD133(+)CD45(-) oval cells were isolated from premalignant 16-month-old Mat1a(-/-) mice by flow cytometry and expanded as five clone lines derived from a single cell. All clone lines demonstrated expression of both hepatocyte and cholangiocyte markers and maintained a small population (0.5% to 2%) of CD133(+) cells in vitro, and three of five clone lines produced tumors. Although TGF-beta1 inhibited cell growth equally in CD133(-) and CD133(+) cells from each clone line, the CD133(+) population demonstrated significant resistance to TGF-beta-induced apoptosis compared with CD133(-) cells. Furthermore, CD133(+) cells demonstrated a substantial increase in mitogen-activated protein kinase (MAPK) pathway activation, as demonstrated by phosphorylated extracellular signal-regulated kinase levels before and after TGF-beta stimulation. MAPK inhibition using mitogen-activated protein kinase kinase 1 (MEK1) inhibitor PD98059 led to a significant increase in TGF-beta-induced apoptosis in CD133(+) cells. Conversely, a constitutively active form of MEK1 blocked the apoptotic effects of TGF-beta in CD133(-) cells. CONCLUSION: CD133(+) liver cancer stem cells exhibit relative resistance to TGF-beta-induced apoptosis. One mechanism of resistance to TGF-beta-induced apoptosis in CD133(+) cancer stem cells is an activated mitogen-activated protein kinase/extracellular signal-regulated kinase pathway.
Assuntos
Antígenos CD/metabolismo , Apoptose , Glicoproteínas/metabolismo , Neoplasias Hepáticas Experimentais/etiologia , Metionina Adenosiltransferase/deficiência , Células-Tronco Neoplásicas/fisiologia , Peptídeos/metabolismo , Fator de Crescimento Transformador beta/fisiologia , Antígeno AC133 , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Proliferação de Células , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Fígado/patologia , Neoplasias Hepáticas/patologia , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 1/metabolismo , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Knockout , Transplante HeterólogoRESUMO
BACKGROUND: Protein biomarkers will play a pivotal role in the future of personalized medicine for both diagnosis and treatment decision-making. While the results of several pre-clinical and small-scale clinical studies have demonstrated the value of protein biomarkers, there have been significant challenges to translating these findings into routine clinical care. Challenges to the use of protein biomarkers include inter-sample variability introduced by differences in post-collection handling and ex vivo degradation of proteins and protein modifications. RESULTS: In this report, we re-create laboratory and clinical scenarios for sample collection and test the utility of a new tissue stabilization technique in preserving proteins and protein modifications. In the laboratory setting, tissue stabilization with the Denator Stabilizor T1 resulted in a significantly higher yield of phospho-protein when compared to standard snap freeze preservation. Furthermore, in a clinical scenario, tissue stabilization at collection resulted in a higher yield of total phospho-protein, total phospho-tyrosine, pErkT202/Y204 and pAktS473 when compared to standard methods. Tissue stabilization did not have a significant effect on other post-translational modifications such as acetylation and glycosylation, which are more stable ex-vivo. Tissue stabilization did decrease total RNA quantity and quality. CONCLUSION: Stabilization at the time of collection offers the potential to better preserve tissue protein and protein modification levels, as well as reduce the variability related to tissue processing delays that are often associated with clinical samples.
RESUMO
In adult mouse myocytes, brief exposure to sodium cyanide (CN) in the presence of glucose does not decrease ATP levels, yet produces profound reduction in contractility, intracellular Ca2+ concentration ([Ca2+]i) transient and L-type Ca2+ current (ICa) amplitudes. We analyzed proteomes from myocytes exposed to CN, focusing on ionic currents associated with excitation-contraction coupling. CN induced phosphorylation of α1c subunit of L-type Ca2+ channel and α2 subunit of Na+-K+-ATPase. Methylene blue (MB), a CN antidote that we previously reported to ameliorate CN-induced reduction in contraction, [Ca2+]i transient and ICa amplitudes, was able to reverse this phosphorylation. CN decreased Na+-K+-ATPase current contributed by α2 but not α1 subunit, an effect that was also counteracted by MB. Peptide consensus sequences suggested CN-induced phosphorylation was mediated by protein kinase C epsilon (PKCε). Indeed, CN stimulated PKC kinase activity and induced PKCε membrane translocation, effects that were prevented by MB. Pretreatment with myristoylated PKCε translocation activator or inhibitor peptides mimicked and inhibited the effects of CN on ICa and myocyte contraction, respectively. We conclude that CN activates PKCε, which phosphorylates L-type Ca2+ channel and Na+-K+-ATPase, resulting in depressed cardiac contractility. We hypothesize that this inhibition of ion fluxes represents a novel mechanism by which the cardiomyocyte reduces its ATP demand (decreased ion fluxes and contractility), diminishes ATP turnover and preserves cell viability. However, this cellular protective effect translates into life-threatening cardiogenic shock in vivo, thereby creating a profound disconnect between survival mechanisms at the cardiomyocyte level from those at the level of the whole organism.
RESUMO
CCAAT/enhancer-binding protein-alpha (C/EBPalpha) is a member of the basic leucine zipper transcription factor family and regulates expression of several enzymes in the liver that control glucose and lipid metabolism. Using adenovirus-transduced silent interfering (si)RNA against C/EBPalpha, endogenous liver C/EBPalpha protein was knocked down by 70-80% in 8-wk-old wild-type (WT) and db/db mice. In WT mice, fasting blood glucose concentrations were reduced approximately 24% without changes in plasma free fatty acid and triglycerides, when compared with LacZ adenovirus-treated control mice. Ad-C/EBPalpha siRNA treatment nearly normalized fasting glucose and significantly reduced plasma insulin and free fatty acid content, even though there was no elevation of C/EBPalpha protein in the livers of db/db mice. In parallel with the changes in glucose levels, hepatic glucose production was significantly reduced in C/EBPalpha siRNA-treated WT and db/db mice. mRNA levels of phyosphoenolpyruvate carboxykinase, glucose-6-phosphatase, and liver glycogen synthase were decreased in the C/EBPalpha siRNA-treated WT and db/db mice. Interestingly, the magnitude of reduction in these enzymes was more profound in db/db mice. C/EBPalpha siRNA also decreased mRNA levels of proliferator activator protein-gamma coactivator-1alpha in both the WT and db/db mice but reduced cAMP response element-binding protein only in WT and did not alter hepatic nuclear factor-4alpha and CBP/p300 expression. Expression of genes involved in lipogenesis, such as fatty acid synthase, acetyl-CoA carboxylase, and sterol regulatory element-binding protein-1c was robustly suppressed in the C/EBPalpha siRNA-treated db/db mice. Taken together, these results indicate that C/EBPalpha plays an important role in maintaining glucose and lipid homeostasis in the liver.
Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/antagonistas & inibidores , Diabetes Mellitus/metabolismo , Gluconeogênese , Metabolismo dos Lipídeos , Fígado/metabolismo , RNA Interferente Pequeno/farmacologia , Adenoviridae/genética , Animais , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Proteína beta Intensificadora de Ligação a CCAAT/fisiologia , Proteínas de Ligação a DNA/genética , Glucose/metabolismo , Glucose-6-Fosfatase/genética , Homeostase , Receptores X do Fígado , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores Nucleares Órfãos , PPAR alfa/genética , Fosfoenolpiruvato Carboxiquinase (GTP)/genética , RNA Mensageiro/análise , Receptores Citoplasmáticos e Nucleares/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Fatores de Transcrição/genéticaRESUMO
BACKGROUND: c-Met, a high-affinity receptor for Hepatocyte Growth Factor (HGF), plays a critical role in tumor growth, invasion, and metastasis. Hepatocellular carcinoma (HCC) patients with activated HGF/c-Met signaling have a significantly worse prognosis. Targeted therapies using c-Met tyrosine kinase inhibitors are currently in clinical trials for HCC, although receptor tyrosine kinase inhibition in other cancers has demonstrated early success. Unfortunately, therapeutic effect is frequently not durable due to acquired resistance. METHODS: We utilized the human MHCC97-H c-Met positive (c-Met+) HCC cell line to explore the compensatory survival mechanisms that are acquired after c-Met inhibition. MHCC97-H cells with stable c-Met knockdown (MHCC97-H c-Met KD cells) were generated using a c-Met shRNA vector with puromycin selection and stably transfected scrambled shRNA as a control. Gene expression profiling was conducted, and protein expression was analyzed to characterize MHCC97-H cells after blockade of the c-Met oncogene. A high-throughput siRNA screen was performed to find putative compensatory survival proteins, which could drive HCC growth in the absence of c-Met. Findings from this screen were validated through subsequent analyses. RESULTS: We have previously demonstrated that treatment of MHCC97-H cells with a c-Met inhibitor, PHA665752, results in stasis of tumor growth in vivo. MHCC97-H c-Met KD cells demonstrate slower growth kinetics, similar to c-Met inhibitor treated tumors. Using gene expression profiling and siRNA screening against 873 kinases and phosphatases, we identified ErbB3 and TGF-α as compensatory survival factors that are upregulated after c-Met inhibition. Suppressing these factors in c-Met KD MHCC97-H cells suppresses tumor growth in vitro. In addition, we found that the PI3K/Akt signaling pathway serves as a negative feedback signal responsible for the ErbB3 upregulation after c-Met inhibition. Furthermore, in vitro studies demonstrate that combination therapy with PHA665752 and Gefitinib (an EGFR inhibitor) significantly reduced cell viability and increased apoptosis compared with either PHA665752 or Gefitinib treatment alone. CONCLUSION: c-Met inhibition monotherapy is not sufficient to eliminate c-Met+ HCC tumor growth. Inhibition of both c-Met and EGFR oncogenic pathways provides superior suppression of HCC tumor growth. Thus, combination of c-Met and EGFR inhibition may represent a superior therapeutic regimen for c-Met+ HCC.
Assuntos
Carcinoma Hepatocelular/metabolismo , Sobrevivência Celular/fisiologia , Receptores ErbB/metabolismo , Neoplasias Hepáticas/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Receptor ErbB-3/metabolismo , Transdução de Sinais/fisiologia , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Receptores ErbB/genética , Feminino , Fator de Crescimento de Hepatócito/genética , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Indóis/farmacologia , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia , Masculino , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-met/genética , Receptor ErbB-3/genética , Transdução de Sinais/efeitos dos fármacos , Sulfonas/farmacologiaRESUMO
Our recent publication showed that a small bioactive pigment epithelium derived factor (PEDF) peptide (P78-PEDF) prevents the development of diabetic nephropathy (DN). However, its effects on the progression of established DN were not clear. Therefore, the purpose of this study was to determine the effect of P78-PEDF in the progression of DN and to compare the effects of P78-PEDF and an ACE inhibitor (ACEi), a standard of care in DN. Experiments were conducted in Ins2(Akita) mice treated with P78-PEDF or captopril starting at 6 wks of age for 12 wks (early treatment) or starting at 12 wks of age for 6 wks (late treatment). We first established the optimal dose of the P78-PEDF peptide to ameliorate DN in Ins2(Akita) mouse for a 6 wk study period and found that the peptide was effective at 0.1- 0.5 µg/g/day. We next showed that early or late treatment with P78-PEDF resulted in protection from DN as indicated by reduced albuminuria, kidney macrophage recruitment, histological changes, inflammatory cytokines and fibrotic markers (kidney TNF-α, fibronectin, VEGFA and EGFR), and restored nephrin expression compared with vehicle-treated Ins2(Akita) mice. Interestingly, only early but not late treatment with captopril was as effective as P78-PEDF in reducing most DN complications, despite its lack of effect on nephrin, VEGFA and EGFR expression. These findings highlight the importance of P78-PEDF peptide as a potential therapeutic modality in both the development and progression of diabetic renal injury.
Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Nefropatias Diabéticas/tratamento farmacológico , Proteínas do Olho/uso terapêutico , Rim/efeitos dos fármacos , Fatores de Crescimento Neural/uso terapêutico , Serpinas/uso terapêutico , Animais , Pressão Sanguínea/efeitos dos fármacos , Captopril/farmacologia , Captopril/uso terapêutico , Citocinas/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Progressão da Doença , Proteínas do Olho/farmacologia , Rim/metabolismo , Rim/patologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Fatores de Crescimento Neural/farmacologia , Serpinas/farmacologiaRESUMO
BACKGROUND: Somafostatin receptors (SSTRs) have been suggested to involve in mediating the effect of somatostatin on hepatic stellate cells (HSCs) in an activation-dependent way. We, therefore, try to investigate the relationship between expression of SSTRs and activation of rat HSCs. METHODS: HSCs were isolated from rats by in situ perfusion and single-step density gradient centrifugation. SSTR1-5 mRNA levels in the differentiated first passage HSCs were detected by means of a reverse transcription polymerase chain reaction. On the other hand, hepatic fibrosis was induced in adult male Sprague-Dawley rats by carbon tetrachloride intoxication, and the expression of SSTR1-5 in normal as well as fibrotic livers was measured by immunohistochemical staining. RESULTS: SSTR mRNA and SSTR could not be found in freshly isolated rat HSCs or normal rat liver. However, SSTR1-3 mRNA appeared as HSCs became wholly activated, and could also be identified on the membrane of activated HSCs in the perisinusoid space, fibrous septa, etc. CONCLUSION: The expression of SSTR1-3 in the rat HSC is closely related to its activation. This may reflect one of the main negative regulation mechanisms in the course of HSC activation.