RESUMO
Excessive renal TGF-ß production and pro-fibrotic miRNAs are important drivers of kidney fibrosis that lack any efficient treatment. Dysfunctional autophagy might play an important role in the pathogenesis. We aimed to study the yet unknown effects of peroxisome proliferator-activated receptor-γ (PPARγ) agonist pioglitazone (Pio) on renal autophagy and miRNA dysregulation during fibrosis. Mouse primary tubular epithelial cells (PTEC) were isolated, pre-treated with 5 µM pioglitazone, and then stimulated with 10 ng/mL TGF-ß1 for 24 h. Male 10-week-old C57Bl6 control (CTL) and TGF-ß overexpressing mice were fed with regular chow (TGF) or Pio-containing chow (20 mg/kg/day) for 5 weeks (TGF + Pio). PTEC and kidneys were evaluated for mRNA and protein expression. In PTEC, pioglitazone attenuated (p < 0.05) the TGF-ß-induced up-regulation of Col1a1 (1.4-fold), Tgfb1 (2.2-fold), Ctgf (1.5-fold), Egr2 (2.5-fold) mRNAs, miR-130a (1.6-fold), and miR-199a (1.5-fold), inhibited epithelial-to-mesenchymal transition, and rescued autophagy function. In TGF mice, pioglitazone greatly improved kidney fibrosis and related dysfunctional autophagy (increased LC3-II/I ratio and reduced SQSTM1 protein content (p < 0.05)). These were accompanied by 5-fold, 3-fold, 12-fold, and 2-fold suppression (p < 0.05) of renal Ccl2, Il6, C3, and Lgals3 mRNA expression, respectively. Our results implicate that pioglitazone counteracts multiple pro-fibrotic processes in the kidney, including autophagy dysfunction and miRNA dysregulation.
Assuntos
Nefropatias , MicroRNAs , Masculino , Camundongos , Animais , Pioglitazona/farmacologia , Fator de Crescimento Transformador beta/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Nefropatias/tratamento farmacológico , Nefropatias/etiologia , Nefropatias/metabolismo , Rim/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , RNA Mensageiro/genética , Fibrose , Autofagia , Células Epiteliais/metabolismoRESUMO
Patients with chronic kidney disease and experimental animal models of kidney fibrosis manifest diverse progression rates. Genetic susceptibility may contribute to this diversity, but the causes remain largely unknown. We have previously described kidney fibrosis with a mild or severe phenotype in mice expressing transforming growth factor-beta1 (TGF-ß1) under the control of a mouse albumin promoter (Alb/TGF-ß1), on a mixed genetic background with CBAxC57Bl6 mice. Here, we aimed to examine how genetic background may influence kidney fibrosis in TGF-ß1 transgenic mice, and in the unilateral ureteral obstruction (UUO) and subtotal nephrectomy (SNX) mouse models. Congenic C57Bl6(B6)-TGFß and CBAxB6-TGFß (F1) transgenic mice were generated and survival, proteinuria, kidney histology, transcriptome and protein expressions were analyzed. We investigated the kidneys of B6 and CBA mice subjected to UUO and SNX, and the effects of tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) neutralization on the fibrotic process. CBAxB6-TGFß mice developed severe kidney fibrosis and premature death, while B6-TGF-ß mice had mild fibrosis and prolonged survival. Kidney early growth response factor-2 (EGR2) and TIMP-1 expression were induced only in CBAxB6-TGFß mice. Similar strain-dependent early changes in EGR2 and TIMP-1 of mice subjected to UUO or SNX were observed. TIMP-1 neutralization in vivo hindered fibrosis both in transgenic mice and the SNX model. EGR2 over-expression in cultured HEK293 cells induced TIMP-1 while EGR2 silencing hindered TGF-ß induced TIMP-1 production in HK-2 cells and ureteral obstructed kidneys. Finally, EGR2 and TIMP1 was increased in human kidneys manifesting focal segmental glomerulosclerosis suggesting a correlation between animal studies and patient clinical settings. Thus, our observations demonstrate a strong relationship between genetic background and the progression of kidney fibrosis, which might involve early altered EGR2 and TIMP-1 response, but the relationship to patient genetics remains to be explored.
Assuntos
Proteína 2 de Resposta de Crescimento Precoce , Insuficiência Renal Crônica , Inibidor Tecidual de Metaloproteinase-1 , Obstrução Ureteral , Animais , Proteína 2 de Resposta de Crescimento Precoce/genética , Fibrose , Células HEK293 , Humanos , Rim/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Insuficiência Renal Crônica/complicações , Inibidor Tecidual de Metaloproteinase-1/genética , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo , Obstrução Ureteral/complicações , Obstrução Ureteral/genética , Obstrução Ureteral/metabolismoRESUMO
PURPOSE OF REVIEW: Pulmonary arterial hypertension (PAH) is characterized by pulmonary arterial endothelial cell (PAEC) dysfunction and apoptosis, pulmonary arterial smooth muscle cell (PASMC) proliferation, inflammation, vasoconstriction, and metabolic disturbances that include disrupted bone morphogenetic protein receptor (BMPR2)-peroxisome proliferator-activated receptor gamma (PPARγ) axis and DNA damage. Activation of PPARγ improves many of these mechanisms, although erroneous reports on potential adverse effects of thiazolidinedione (TZD)-class PPARγ agonists reduced their clinical use in the past decade. Here, we review recent findings in heart, lung, and kidney research related to the pathobiology of vascular remodeling and tissue fibrosis, and also potential therapeutic effects of the PPARγ agonist pioglitazone. RECENT FINDINGS: Independent of its metabolic effects (improved insulin sensitivity and fatty acid handling), PPARγ activation rescues BMPR2 dysfunction, inhibits TGFß/Smad3/CTGF and TGFß/pSTAT3/pFoxO1 pathways, and induces the PPARγ/apoE axis, inhibiting vascular remodeling. PPARγ activation dampens mtDNA damage via PPARγ/UBR5/ATM pathway, improves function of endothelial progenitor cells (EPCs), and decrease renal fibrosis by repressing TGFß/pSTAT3 and TGFß/EGR1. SUMMARY: Pharmacological PPARγ activation improves many hallmarks of PAH, including dysfunction of BMPR2-PPARγ axis, PAEC, PASMC, EPC, mitochondria/metabolism, and inflammation. Recent randomized controlled trials, including IRIS (Insulin Resistance Intervention After Stroke Trial), emphasize the beneficial effects of PPARγ agonists in PAH patients, leading to recent revival for clinical use.
Assuntos
Matriz Extracelular/fisiologia , Hipertensão Pulmonar/etiologia , Rim/patologia , Músculo Liso Vascular/citologia , Miocárdio/patologia , Miócitos de Músculo Liso/fisiologia , PPAR gama/fisiologia , Fibrose Pulmonar/etiologia , Animais , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/fisiologia , Dano ao DNA , Fibrose , Homeostase , Humanos , PPAR gama/agonistas , Fator de Crescimento Transformador beta1/fisiologiaRESUMO
BACKGROUND: It has been proposed that peroxisome proliferator-activated receptor-γ (PPARγ) agonists might reduce renal fibrosis, however, several studies had contradictory results. Moreover, the possible interaction of TGF-ß1, PPARγ, and transcription factors in renal fibrosis have not been investigated. We hypothesized that oral pioglitazone treatment would inhibit TGF-ß-driven renal fibrosis and its progression, by modulating profibrotic transcription factors in TGF-ß1 transgenic mice. METHODS: Male C57Bl/6 J mice (control, CTL, n = 14) and TGF-ß overexpressing transgenic mice (TGFß, n = 14, having elevated plasma TGF-ß1 level) were divided in two sets at 10 weeks of age. Mice in the first set were fed with regular rodent chow (CTL and TGFß, n = 7/group). Mice in the second set were fed with chow containing pioglitazone (at a dose of 20 mg/kg/day, CTL + Pio and TGFß+Pio, n = 7/group). After 5 weeks of treatment, blood pressure was assessed and urine samples were collected, and the kidneys were analyzed for histology, mRNA and protein expression. RESULTS: TGF-ß1 induced glomerulosclerosis and tubulointerstitial damage were significantly reduced by pioglitazone. Pioglitazone inhibited renal mRNA expression of all the profibrotic effectors: type-III collagen, TGF-ß1, CTGF and TIMP-1, and alike transcription factors cFos/cJun and protein expression of EGR-1, and STAT3 protein phosphorylation. CONCLUSIONS: Oral administration of PPARγ agonist pioglitazone significantly reduces TGF-ß1-driven renal fibrosis, via the attenuation of EGR-1, STAT3 and AP-1. This implies that PPARγ agonists might be effective in the treatment of chronic kidney disease patients.
Assuntos
Proteína 1 de Resposta de Crescimento Precoce/antagonistas & inibidores , Nefropatias/prevenção & controle , PPAR gama/agonistas , Pioglitazona/uso terapêutico , Fator de Transcrição STAT3/antagonistas & inibidores , Fator de Crescimento Transformador beta/antagonistas & inibidores , Animais , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Fibrose , Nefropatias/induzido quimicamente , Nefropatias/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pioglitazona/farmacologia , Fator de Transcrição STAT3/metabolismo , Fator de Crescimento Transformador beta/toxicidadeRESUMO
BACKGROUND/AIMS: Diabetic nephropathy remains a major clinical problem. The effects of prorenin might be adverse, but the literature data are controversial. We compared the renal effects of the (pro)renin receptor ((P)RR) blockade and angiotensin converting enzyme (ACE) inhibition on the progression of diabetic nephropathy in rats. METHODS: Diabetes (DM) was induced by ip. streptozotocin administration in adult male Sprague-Dawley rats, followed by eight weeks of treatment with the (P)RR blocker "handle region" decoy peptide (HRP, 0,1 mg/kg/day) or with the ACE inhibitor Quinapril (Q, 50 mg/kg/day) and grouped as follows: 1. Control (n=10); 2. DM (n=8); 3. DM+HRP (n=6); 4. DM+Q (n=10); 5. DM+Q+HRP (n=10). Renal functional parameters, histology and gene expressions were evaluated. RESULTS: HRP reduced glomerulosclerosis and podocyte desmin expression, but did not affect proteinuria and tubular ERK(1/2) phosphorylation. Both Q and Q+HRP treatment reduced proteinuria, glomerular and tubular damage, tubular TGF-ß1 expression and ERK(1/2) phosphorylation to the same extent. CONCLUSION: The effects of HRP were partially beneficial on diabetic kidney lesions as HRP reduced damage but did not improve tubular damage and failed to reduce ERK(1/2) phosphorylation in rats. The combination of HRP with Quinapril had no additive effects over Quinapril monotherapy on the progression of diabetic nephropathy.
Assuntos
Nefropatias Diabéticas/tratamento farmacológico , Renina/uso terapêutico , Tetra-Hidroisoquinolinas/uso terapêutico , Animais , Diabetes Mellitus Experimental , Interações Medicamentosas , Quimioterapia Combinada , Rim/efeitos dos fármacos , Rim/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Quinapril , Ratos , Ratos Sprague-Dawley , Renina/farmacologia , Tetra-Hidroisoquinolinas/farmacologiaRESUMO
BACKGROUND: Although TGF-ß and the transcription factor Egr-1 play an important role in both kidney fibrosis and in response to acute changes of renal medullary osmolarity, their role under sustained hypo- or hyperosmolar conditions has not been elucidated. We investigated the effects of chronic hypertonicity and hypotonicity on the renal medullary TGF-ß and Egr-1 expression. METHODS: Male adult Sprague Dawley rats (n = 6/group) were treated with 15 mg/day furosemide, or the rats were water restricted to 15 ml/200 g body weight per day. Control rats had free access to water and rodent chow. Kidneys were harvested after 5 days of treament. In cultured inner medullary collecting duct (IMCD) cells, osmolarity was increased from 330 mOsm to 900 mOsm over 6 days. Analyses were performed at 330, 600 and 900 mOsm. RESULTS: Urine osmolarity has not changed due to furosemide treatment but increased 2-fold after water restriction (p < 0.05). Gene expression of TGF-ß and Egr-1 increased by 1.9-fold and 7-fold in the hypertonic medulla, respectively (p < 0.05), accompanied by 6-fold and 2-fold increased c-Fos and TIMP-1 expression, respectively (p < 0.05) and positive immunostaining for TGF-ß and Egr-1 (p < 0.05). Similarly, hyperosmolarity led to overexpression of TGF-ß and Egr-1 mRNA in IMCD cells (2.5-fold and 3.5-fold increase from 330 to 900 mOsm, respectively (p < 0.05)) accompanied by significant c-Fos and c-Jun overexpressions (p < 0.01), and increased Col3a1 and Col4a1 mRNA expression. CONCLUSION: We conclude that both TGF-ß and Egr-1 are upregulated by sustained hyperosmolarity in the rat renal medulla, and it favors the expression of extracellular matrix components.
Assuntos
Ingestão de Líquidos/fisiologia , Proteína 1 de Resposta de Crescimento Precoce/biossíntese , Medula Renal/metabolismo , Fator de Crescimento Transformador beta1/biossíntese , Animais , Sobrevivência Celular/fisiologia , Células Cultivadas , Proteína 1 de Resposta de Crescimento Precoce/genética , Expressão Gênica , Medula Renal/citologia , Masculino , Concentração Osmolar , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Fator de Crescimento Transformador beta1/genéticaAssuntos
Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/metabolismo , Hipertensão Arterial Pulmonar/metabolismo , Transdução de Sinais , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Animais , Humanos , Masculino , Camundongos Transgênicos , Ratos Sprague-DawleyRESUMO
During progressive tubulointerstitial fibrosis, renal tubular epithelial cells transform into α-smooth muscle actin (SMA)-expressing myofibroblasts via epithelial-to-mesenchymal transition (EMT). SMA expression is regulated by transforming growth factor (TGF)-ß1 and cell contact disruption, through signaling events targeting the serum response factor-myocardin-related transcription factor (MRTF) complex. MRTFs are important regulators of fibrosis, tumor cell invasion, and metastasis. Consistent with the role of MRTFs in tumor progression, suppressor of cancer cell invasion (SCAI) was recently identified as a negative regulator of MRTF. Herein, we studied the role of SCAI in a fibrotic EMT model established on LLC-PK1 cells. SCAI overexpression prevented SMA promoter activation induced by TGF-ß1. When co-expressed, it inhibited the stimulatory effects of MRTF-A, MRTF-B or the constitutive active forms of RhoA, Rac1, or Cdc42 on the SMA promoter. SCAI interfered with TGF-ß1-induced SMA, connective tissue growth factor, and calponin protein expression; it rescued TGF-ß1-induced E-cadherin down-regulation. IHC studies on human kidneys showed that SCAI expression is reduced during fibrosis. Kidneys of diabetic rats and mice with unilateral ureteral obstruction depicted significant loss of SCAI expression. In parallel with the decrease of SCAI protein expression, diabetic rat and mouse kidneys with unilateral ureteral obstruction showed SMA expression, as evidenced by using Western blot analysis. Finally, TGF-ß1 treatment of LLC-PK1 cells attenuated SCAI protein expression. These data suggest that SCAI is a novel transcriptional cofactor that regulates EMT and renal fibrosis.
Assuntos
Transição Epitelial-Mesenquimal , Rim/metabolismo , Rim/patologia , Fatores de Transcrição/metabolismo , Actinas/genética , Animais , Caderinas/genética , Caderinas/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Proteínas de Ligação a DNA/metabolismo , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Fibrose , Humanos , Células LLC-PK1 , Camundongos , Proteínas dos Microfilamentos/metabolismo , Regiões Promotoras Genéticas/genética , Transporte Proteico/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Suínos , Fatores de Transcrição/genética , Fator de Crescimento Transformador beta1/farmacologia , Obstrução Ureteral/metabolismo , Obstrução Ureteral/patologia , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , CalponinasRESUMO
BACKGROUND: Diabetic nephropathy (DN) is characterized by podocyte damage and increased phosphodiesterase-5 (PDE-5) activity-exacerbating nitric oxide (NO)-cyclic 3',5' guanosine monophosphate (cGMP) pathway dysfunction. It has been shown that PDE-5 inhibition ameliorates DN. The role of podocytes in this mechanism remains unclear. We investigated how selective PDE-5 inhibition influences podocyte damage in streptozotocin (STZ) diabetic rats. METHODS: Male Sprague-Dawley rats (250-300 g) were injected with STZ and divided into two groups: (i) STZ control (non-treated, STZ, n=6) and (ii) STZ+vardenafil treatment (10 mg/kg/day, STZ-Vard, n=8). Non-diabetic rats served as negative controls (Control, n=7). Following 8 weeks of treatment, immunohistochemical and molecular analysis of the kidneys were performed. RESULTS: Diabetic rats had proteinuria, increased renal transforming growth factor (TGF)-ß1 expression and podocyte damage when compared with controls. Vardenafil treatment resulted in preserved podocyte cGMP levels, less proteinuria, reduced renal TGF-ß1 expression, desmin immunostaining in podocytes and restored both nephrin and podocin mRNA expression. Diabetes led to increased glomerular nitrotyrosine formation and renal neuronal nitric oxide synthase and endothelial nitric oxide synthase mRNA expression, but vardenafil did not influence these parameters. CONCLUSIONS: Our data suggest that a dysfunctional NO-cGMP pathway exacerbates podocyte damage in diabetes. In conclusion, vardenafil treatment preserves podocyte function and reduces glomerular damage, which indicates therapeutic potential in patients with DN.
Assuntos
GMP Cíclico/metabolismo , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Tipo 1/patologia , Nefropatias Diabéticas/prevenção & controle , Imidazóis/farmacologia , Inibidores da Fosfodiesterase 5/farmacologia , Piperazinas/farmacologia , Podócitos/efeitos dos fármacos , Animais , Western Blotting , Células Cultivadas , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/química , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 1/metabolismo , Nefropatias Diabéticas/etiologia , Nefropatias Diabéticas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Estresse Oxidativo , Podócitos/metabolismo , Podócitos/patologia , Proteinúria/etiologia , Proteinúria/metabolismo , Proteinúria/prevenção & controle , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sulfonas/farmacologia , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo , Triazinas/farmacologia , Dicloridrato de VardenafilaRESUMO
Suppressor of cancer cell invasion (SCAI) has been originally characterized as a tumor suppressor inhibiting metastasis in different human cancer cells, and it has been suggested that SCAI expression declines in tumors. The expression patterns and role of SCAI during physiological and pathophysiological processes is still poorly understood. Earlier we demonstrated that SCAI is regulating the epithelial-mesenchymal transition of proximal tubular epithelial cells, it is downregulated during renal fibrosis and it is overexpressed in Wilms' tumors. Here we bring further evidence for the involvement of SCAI during cell plasticity and we examine the prognostic value and expression patterns of SCAI in various tumors. SCAI prevented the activation of the SMA promoter induced by angiotensin II. SCAI expression decreased in a model of endothelial-mesenchymal transition and increased during iPS reprogramming of fibroblasts. During renal fibrosis SCAI expression declined, as evidenced in a rat model of renal transplant rejection and in TGF-ß1 overexpressing transgenic mice. High expression of SCAI correlated with better survival in patients with breast and lung cancers. Intriguingly, in the case of other cancers (gastric, prostate, colorectal) high SCAI expression correlated with poor survival of patients. Finally, we bring evidence for SCAI overexpression in colorectal cancer patients, irrespective of stage or metastatic status of the disease, suggesting a diverse role of SCAI in various diseases and cancer.
Assuntos
Biomarcadores/metabolismo , Plasticidade Celular , Fibrose/patologia , Nefropatias/patologia , Neoplasias/patologia , Fatores de Transcrição/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Estudos de Casos e Controles , Células Cultivadas , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Transição Epitelial-Mesenquimal , Feminino , Fibrose/metabolismo , Seguimentos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Nefropatias/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Pessoa de Meia-Idade , Neoplasias/metabolismo , Prognóstico , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Ratos Endogâmicos BN , Ratos Endogâmicos Lew , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Taxa de SobrevidaRESUMO
BMP2 and TGFß1 are functional antagonists of pathological remodeling in the arteries, heart, and lung; however, the mechanisms in VSMCs, and their disturbance in pulmonary arterial hypertension (PAH), are unclear. We found a pro-proliferative TGFß1-Stat3-FoxO1 axis in VSMCs, and PPARγ as inhibitory regulator of TGFß1-Stat3-FoxO1 and TGFß1-Smad3/4, by physically interacting with Stat3 and Smad3. TGFß1 induces fibrosis-related genes and miR-130a/301b, suppressing PPARγ. Conversely, PPARγ inhibits TGFß1-induced mitochondrial activation and VSMC proliferation, and regulates two glucose metabolism-related enzymes, platelet isoform of phosphofructokinase (PFKP, a PPARγ target, via miR-331-5p) and protein phosphatase 1 regulatory subunit 3G (PPP1R3G, a Smad3 target). PPARγ knockdown/deletion in VSMCs activates TGFß1 signaling. The PPARγ agonist pioglitazone reverses PAH and inhibits the TGFß1-Stat3-FoxO1 axis in TGFß1-overexpressing mice. We identified PPARγ as a missing link between BMP2 and TGFß1 pathways in VSMCs. PPARγ activation can be beneficial in TGFß1-associated diseases, such as PAH, parenchymal lung diseases, and Marfan's syndrome.
Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Proliferação de Células , Glucose/metabolismo , Miócitos de Músculo Liso/citologia , PPAR gama/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta1/metabolismo , Animais , Células Cultivadas , Feminino , Masculino , Camundongos Endogâmicos C57BL , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Artéria Pulmonar/citologia , Artéria Pulmonar/metabolismoRESUMO
Transforming growth factor-beta (TGF-beta) is a bimodal regulator of cellular growth. The cellular effects of TGF-beta depend on the intensity of signals emanating from TGF-beta receptors. Low levels of receptor activity are sufficient to stimulate cell proliferation, while higher degrees of receptor activation are associated with growth inhibition. To study the mechanisms of these effects, a tetracycline-inducible expression system was used to overexpress type II TGF-beta receptors in NIH 3T3 fibroblasts. Overexpressed type II TGF-beta receptors suppressed fibroblast proliferation elicited by TGF-beta1, fibroblast growth factor (FGF) or platelet-derived growth factor (PDGF). Accompanying these anti-proliferative effects, increases in extracellular-signal regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) activity were detected. Furthermore, PDGF alpha-, but not PDGF beta-receptor protein levels were reduced by type II TGF-beta receptor overexpression. In conclusion, our system is an excellent tool to study the molecular mechanisms of growth inhibition by TGF-beta in fibroblasts. Activation of JNK and ERK, or modulation of PDGF receptor expression may be involved in this process.
Assuntos
Fibroblastos/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/biossíntese , Células 3T3 , Animais , Western Blotting , Divisão Celular , Reagentes de Ligações Cruzadas/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno , Sistema de Sinalização das MAP Quinases , Camundongos , Fator de Crescimento Derivado de Plaquetas/metabolismo , Ligação Proteica , Proteínas Serina-Treonina Quinases , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo II , Transdução de Sinais , Transfecção , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
As reports of mice genetically deficient for TGF-beta1 demonstrated aberrant renal class II MHC expression, we investigated inducible class II MHC expression on renal tubular epithelial cells derived from TGF-beta1 knockout (-/-) and wild-type (+/+) mice. IFN-gamma markedly upregulated class II MHC (I-A(b)) expression in both (-/-) and (+/+) tubular epithelial cells. Coincubation studies of (+/+) and (-/-) tubular epithelial cells with IFN-gamma+LPS, or pretreatment of these cells with TGF-beta1, revealed inhibition of IFN-gamma-induced I-A(b) mRNA and cell surface expression that occurred via a decrease in class II transactivator gene expression in both (+/+) and (-/-) tubular epithelial cells. In addition, ICAM-1 was constitutively expressed on both (+/+) and (-/-) tubular epithelial cells and was upregulated by IFN-gamma or IFN-gamma+LPS. ICAM-1 expression in (+/+) and (-/-) tubular epithelial cells, however, was decreased by TGF-beta1. Parallel analysis evaluating B7-1 expression detected low levels of B7-1 in unstimulated (+/+) and (-/-) tubular epithelial cells that were increased by IFN-gamma, LPS, and IFN-gamma+LPS. IFN-gamma+LPS-mediated upregulation of B7-1 was also blocked by pretreatment with TGF-beta1. Cytokine analysis detected significantly higher levels of TNF-alpha and MIP-1alpha mRNA in all treated (-/-) preparations than in (+/+) tubular epithelial cell controls. These studies demonstrate normal patterns of class II MHC, ICAM-1, and B7 expression in TGF-beta1 (-/-) tubular epithelial cells in response to IFN-gamma, LPS, and TGF-beta1. Upregulated cytokine expression at baseline and in response to proinflammatory mediators is apparent in (-/-) tubular epithelial cells, however, and suggests that dysregulation of cytokine expression in inflammatory responses may be a primary event in multifocal inflammation observed in TGF-beta1-deficient animals.