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1.
bioRxiv ; 2023 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-37205366

RESUMO

Background: Lymphangiogenesis is believed to be a protective response in the setting of multiple forms of kidney injury and mitigates the progression of interstitial fibrosis. To augment this protective response, promoting kidney lymphangiogenesis is being investigated as a potential treatment to slow the progression of kidney disease.As injury related lymphangiogenesis is driven by signaling from the receptor VEGFR-3 in response to the cognate growth factor VEGF-C released by tubular epithelial cells, this signaling pathway is a candidate for future kidney therapeutics. However, the consequences to kidney development and function to targeting this signaling pathway remains poorly defined. Methods: We generated a new mouse model expressing Vegf-C under regulation of the nephron progenitor Six2Cre driver strain (Six2Vegf-C). Mice underwent a detailed phenotypic evaluation. Whole kidneys were processed for histology and micro computed tomography 3-dimensional imaging. Results: Six2Vegf-C mice had reduced body weight and kidney function compared to littermate controls. Six2Vegf-C kidneys demonstrated large peripelvic fluid filled lesions with distortion of the pelvicalcyceal system which progressed in severity with age. 3D imaging showed a 3-fold increase in total cortical vascular density. Histology confirmed a substantial increase in LYVE1+/PDPN+/VEGFR3+ lymphatic capillaries extending alongside EMCN+ peritubular capillaries. There was no change in EMCN+ peritubular capillary density. Conclusions: Kidney lymphangiogenesis was robustly induced in the Six2Vegf-C mice. There were no changes in peritubular blood capillary density despite these endothelial cells also expressing VEGFR-3. The model resulted in a severe cystic kidney phenotype that resembled a human condition termed renal lymphangiectasia. This study defines the vascular consequences of augmenting VEGF-C signaling during kidney development and provides new insight into a mimicker of human cystic kidney disease.

2.
J Am Soc Nephrol ; 34(6): 969-987, 2023 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-36787763

RESUMO

SIGNIFICANCE STATEMENT: Ischemia-reperfusion AKI (IR-AKI) is common and causes significant morbidity. Effective treatments are lacking. However, preclinical studies suggest that inhibition of angiopoietin-Tie2 vascular signaling promotes injury, whereas activation of Tie2 is protective. We show that kidney ischemia leads to increased levels of the endothelial-specific phosphatase vascular endothelial protein tyrosine phosphatase (VE-PTP; PTPRB), which inactivates Tie2. Activation of Tie2 through VE-PTP deletion, or delivery of a novel angiopoietin mimetic (Hepta-ANG1), abrogated IR-AKI in mice. Single-cell RNAseq analysis showed Tie2 activation promotes increased Entpd1 expression, downregulation of FOXO1 target genes in the kidney vasculature, and emergence of a new subpopulation of glomerular endothelial cells. Our data provide a molecular basis and identify a candidate therapeutic to improve endothelial integrity and kidney function after IR-AKI. BACKGROUND: Ischemia-reperfusion AKI (IR-AKI) is estimated to affect 2%-7% of all hospitalized patients. The significant morbidity and mortality associated with AKI indicates urgent need for effective treatments. Previous studies have shown activation of the vascular angiopoietin-Tie2 tyrosine kinase signaling pathway abrogates ischemia-reperfusion injury (IRI). We extended previous studies to (1) determine the molecular mechanism(s) underlying kidney injury and protection related to decreased or increased activation of Tie2, respectively, and (2) to test the hypothesis that deletion of the Tie2 inhibitory phosphatase vascular endothelial protein tyrosine phosphatase (VE-PTP) or injection of a new angiopoietin mimetic protects the kidney from IRI by common molecular mechanism(s). METHODS: Bilateral IR-AKI was performed in VE-PTP wild-type or knockout mice and in C57BL/6J mice treated with Hepta-ANG1 or vehicle. Histologic, immunostaining, and single-cell RNA sequencing analyses were performed. RESULTS: The phosphatase VE-PTP, which negatively regulates the angiopoietin-Tie2 pathway, was upregulated in kidney endothelial cells after IRI, and genetic deletion of VE-PTP in mice protected the kidney from IR-AKI. Injection of Hepta-ANG1 potently activated Tie2 and protected the mouse kidney from IRI. Single-cell RNAseq analysis of kidneys from Hepta-ANG1-treated and vehicle-treated mice identified endothelial-specific gene signatures and emergence of a new glomerular endothelial subpopulation associated with improved kidney function. Overlap was found between endothelial-specific genes upregulated by Hepta-ANG1 treatment and those downregulated in HUVECs with constitutive FOXO1 activation, including Entpd1 / ENTPD1 that modulates purinergic receptor signaling. CONCLUSIONS: Our data support a key role of the endothelium in the development of IR-AKI, introduce Hepta-ANG1 as a putative new therapeutic biologic, and report a model to explain how IRI reduces Tie2 signaling and how Tie2 activation protects the kidney. PODCAST: This article contains a podcast at https://dts.podtrac.com/redirect.mp3/www.asn-online.org/media/podcast/JASN/2023_05_23_JSN_Ang_EP23_052323.mp3.


Assuntos
Injúria Renal Aguda , Células Endoteliais , Camundongos , Animais , Células Endoteliais/metabolismo , Angiopoietinas/metabolismo , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/genética , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/metabolismo , Camundongos Endogâmicos C57BL , Endotélio/metabolismo , Rim/metabolismo , Transdução de Sinais , Receptor TIE-2/genética , Angiopoietina-1/uso terapêutico , Camundongos Knockout , Injúria Renal Aguda/prevenção & controle , Injúria Renal Aguda/metabolismo , Isquemia/complicações , Isquemia/metabolismo
3.
Am J Physiol Renal Physiol ; 324(1): F91-F105, 2023 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-36395385

RESUMO

Microvascular dysfunction is a key driver of kidney disease. Pathophysiological changes in the kidney vasculature are regulated by vascular endothelial growth factor receptors (VEGFRs), supporting them as potential therapeutic targets. The tyrosine kinase receptor VEGFR-3, encoded by FLT4 and activated by the ligands VEGF-C and VEGF-D, is best known for its role in lymphangiogenesis. Therapeutically targeting VEGFR-3 to modulate lymphangiogenesis has been proposed as a strategy to treat kidney disease. However, outside the lymphatics, VEGFR-3 is also expressed in blood vascular endothelial cells in several tissues including the kidney. Here, we show that Vegfr-3 is expressed in fenestrated microvascular beds within the developing and adult mouse kidney, which include the glomerular capillary loops. We found that expression levels of VEGFR-3 are dynamic during glomerular capillary loop development, with the highest expression observed during endothelial cell migration into the S-shaped glomerular body. We developed a conditional knockout mouse model for Vegfr-3 and found that loss of Vegfr-3 resulted in a striking glomerular phenotype characterized by aneurysmal dilation of capillary loops, absence of mesangial structure, abnormal interendothelial cell junctions, and poor attachment between glomerular endothelial cells and the basement membrane. In addition, we demonstrated that expression of the VEGFR-3 ligand VEGF-C by podocytes and mesangial cells is dispensable for glomerular development. Instead, VEGFR-3 in glomerular endothelial cells attenuates VEGFR-2 phosphorylation. Together, the results of our study support a VEGF-C-independent functional role for VEGFR-3 in the kidney microvasculature outside of lymphatic vessels, which has implications for clinical therapies that target this receptor.NEW & NOTEWORTHY Targeting VEGFR-3 in kidney lymphatics has been proposed as a method to treat kidney disease. However, expression of VEGFR-3 is not lymphatic-specific. We demonstrated developmental expression of VEGFR-3 in glomerular endothelial cells, with loss of Vegfr-3 leading to malformation of glomerular capillary loops. Furthermore, we showed that VEGFR-3 attenuates VEGFR-2 activity in glomerular endothelial cells independent of paracrine VEGF-C signaling. Together, these data provide valuable information for therapeutic development targeting these pathways.


Assuntos
Nefropatias , Receptor 3 de Fatores de Crescimento do Endotélio Vascular , Camundongos , Animais , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Fator C de Crescimento do Endotélio Vascular/genética , Fator C de Crescimento do Endotélio Vascular/metabolismo , Células Endoteliais/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Capilares/metabolismo
4.
Dev Cell ; 55(6): 737-753.e7, 2020 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-33220174

RESUMO

Bacterial infection triggers a cytokine storm that needs to be resolved to maintain the host's wellbeing. Here, we report that ablation of m6A methyltransferase subunit METTL14 in myeloid cells exacerbates macrophage responses to acute bacterial infection in mice, leading to high mortality due to sustained production of pro-inflammatory cytokines. METTL14 depletion blunts Socs1 m6A methylation and reduces YTHDF1 binding to the m6A sites, which diminishes SOCS1 induction leading to the overactivation of TLR4/NF-κB signaling. Forced expression of SOCS1 in macrophages depleted of METTL14 or YTHDF1 rescues the hyper-responsive phenotype of these macrophages in vitro and in vivo. We further show that LPS treatment induces Socs1 m6A methylation and sustains SOCS1 induction by promoting Fto mRNA degradation, and forced FTO expression in macrophages mimics the phenotype of METTL14-depleted macrophages. We conclude that m6A methylation-mediated SOCS1 induction is required to maintain the negative feedback control of macrophage activation in response to bacterial infection.


Assuntos
Adenosina/análogos & derivados , Ativação de Macrófagos , Metiltransferases/metabolismo , Processamento Pós-Transcricional do RNA , RNA Mensageiro/metabolismo , Proteína 1 Supressora da Sinalização de Citocina/genética , Adenosina/metabolismo , Dioxigenase FTO Dependente de alfa-Cetoglutarato/metabolismo , Animais , Células Cultivadas , Retroalimentação Fisiológica , Feminino , Células HEK293 , Humanos , Masculino , Metilação , Camundongos , Camundongos Endogâmicos C57BL , Células RAW 264.7 , Estabilidade de RNA , RNA Mensageiro/genética , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Proteína 1 Supressora da Sinalização de Citocina/metabolismo , Receptor 4 Toll-Like/metabolismo , Tristetraprolina/metabolismo , Regulação para Cima
5.
Endocrinology ; 159(2): 967-979, 2018 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-29228157

RESUMO

Recent studies show that colonic vitamin D receptor (VDR) signaling protects the mucosal epithelial barrier and suppresses colonic inflammation, but the underlying molecular mechanism remains to be fully understood. To investigate the implication of colonic VDR downregulation seen in patients with inflammatory bowel disease, we assessed the effect of gut epithelial VDR deletion on colonic inflammatory responses in an experimental colitis model. In a 2,4,6-trinitrobenzenesulfonic acid-induced colitis model, mice carrying VDR deletion in gut epithelial cells [VDRflox/flox (VDRf/f);Villin-Cre or VDRΔIEC] or in colonic epithelial cells (VDRf/f;CDX2-Cre or VDRΔCEC) developed more severe clinical colitis than VDRf/f control mice, characterized by more robust T-helper (TH)1 and TH17 responses, with greater increases in mucosal interferon (IFN)-γ+, interleukin (IL)-17+, and IFN-γ+IL-17+ T cells. Accompanying the severe mucosal inflammation was more profound colonic epithelial cell apoptosis in the mutant mice. Treatment with caspase inhibitor Q-VD-OPh dramatically reduced colitis severity and attenuated TH1 and TH17 responses in VDRΔCEC mice. The blockade of cell apoptosis also prevented the increase in mucosal CD11b+CD103+ dendritic cells (DCs), known to be critical for TH17-cell activation. Moreover, depletion of gut commensal bacteria with antibiotics eliminated the robust TH1 and TH17 responses and CD11b+CD103+ DC induction. Taken together, these observations demonstrate that gut epithelial VDR deletion aggravates epithelial cell apoptosis, resulting in increases in mucosal barrier permeability. Consequently, invading luminal bacteria activate CD11b+CD103+ DCs, which promote mucosal TH1 and TH17 responses. Therefore, gut epithelial VDR signaling controls mucosal inflammation by suppressing epithelial cell apoptosis.


Assuntos
Apoptose/genética , Células Epiteliais/fisiologia , Inflamação/genética , Mucosa Intestinal/metabolismo , Microbiota/fisiologia , Receptores de Calcitriol/fisiologia , Animais , Colite/metabolismo , Colite/microbiologia , Colite/patologia , Colo/metabolismo , Colo/microbiologia , Colo/patologia , Células Epiteliais/microbiologia , Inflamação/metabolismo , Inflamação/microbiologia , Inflamação/patologia , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/imunologia , Doenças Inflamatórias Intestinais/metabolismo , Doenças Inflamatórias Intestinais/patologia , Mucosa Intestinal/patologia , Intestinos/patologia , Camundongos , Camundongos Transgênicos , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo
6.
FASEB J ; 31(5): 2065-2075, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28148567

RESUMO

Hyperglycemia is a major pathogenic factor that promotes diabetic nephropathy, but the underlying mechanism remains incompletely understood. Here, we show that high glucose induced cAMP response element-binding protein (CREB)-binding protein (CBP)-mediated H3K9/14 hyperacetylation in approximately 5000 gene promoters in glomerular mesangial cells, including those of Tgfb1, Tgfb3, and Ctgf, the major profibrotic factors that are known to drive diabetic renal fibrogenesis. In these promoters, H3K9/14 hyperacetylation was closely associated with NF-κB or CREB motifs. Chromatin immunoprecipitation assays confirmed that hyperglycemia promoted phospho-p65 or phospho-CREB and CBP bindings and RNA polymerase II recruitment to these promoters in mesangial cells as well as in glomeruli that were purified from type I and type II diabetic mice. Under hyperglycemia, cAMP production and PKA activity were markedly increased as a result of glucose transporter 1-mediated glucose influx that drives glucose metabolism and ATP production, which led to increased phosphorylation of p65 and CREB. Inhibition of adenylyl cyclase or PKA activity blocked p65 and CREB phosphorylation, CBP recruitment, and histone acetylation in these promoters. Collectively, these data demonstrate that the cAMP-PKA pathway plays a key role in epigenetic regulation of key profibrotic factors in diabetes.-Deb, D. K., Bao, R., Li, Y. C. Critical role of the cAMP-PKA pathway in hyperglycemia-induced epigenetic activation of fibrogenic program in the kidney.


Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Epigênese Genética/genética , Hiperglicemia/metabolismo , Rim/metabolismo , Transdução de Sinais , Animais , Diabetes Mellitus Experimental/metabolismo , Camundongos , NF-kappa B/metabolismo , Proteínas Nucleares/metabolismo , Fosforilação , Regiões Promotoras Genéticas/genética
7.
Sci Rep ; 6: 27552, 2016 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-27271344

RESUMO

The renin-angiotensin system (RAS) plays pathogenic roles in renal and cardiovascular disorders, but whether it is involved in colitis is unclear. Here we show that RenTgMK mice that overexpress active renin from the liver developed more severe colitis than wild-type controls. More than 50% RenTgMK mice died whereas all wild-type mice recovered. RenTgMK mice exhibited more robust mucosal TH17 and TH1/TH17 responses and more profound colonic epithelial cell apoptosis compared to wild-type controls. Treatment with aliskiren (a renin inhibitor), but not hydralazine (a smooth muscle relaxant), ameliorated colitis in RenTgMK mice, although both drugs normalized blood pressure. Chronic infusion of angiotensin II into wild-type mice mimicked the severe colitic phenotype of RenTgMK mice, and treatment with losartan [an angiotensin type 1 receptor blocker (ARB)] ameliorated colitis in wild-type mice, confirming a colitogenic role for the endogenous RAS. In human biopsies, pro-inflammatory cytokines were suppressed in patients with inflammatory bowel disease who were on ARB therapy compared to patients not receiving ARB therapy. These observations demonstrate that activation of the RAS promotes colitis in a blood pressure independent manner. Angiotensin II appears to drive colonic mucosal inflammation by promoting intestinal epithelial cell apoptosis and mucosal TH17 responses in colitis development.


Assuntos
Colite/genética , Doenças Inflamatórias Intestinais/genética , Sistema Renina-Angiotensina/genética , Renina/genética , Amidas/administração & dosagem , Angiotensina II/genética , Bloqueadores do Receptor Tipo 1 de Angiotensina II/administração & dosagem , Animais , Apoptose/genética , Colite/fisiopatologia , Colo/metabolismo , Colo/patologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Fumaratos/administração & dosagem , Humanos , Hidralazina/administração & dosagem , Doenças Inflamatórias Intestinais/tratamento farmacológico , Doenças Inflamatórias Intestinais/patologia , Losartan/administração & dosagem , Camundongos , Camundongos Transgênicos , Receptor Tipo 1 de Angiotensina/genética , Células Th17/efeitos dos fármacos
8.
Am J Nephrol ; 38(1): 39-49, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23817135

RESUMO

Diabetic glomerulosclerosis is characterized by accumulation of extracellular matrix proteins, mesangial expansion, and tubulointerstitial fibrosis. Hyperglycemia accelerates development of the disease, a direct result of increased intracellular glucose availability. The facilitative glucose transporter GLUT1 mediates mesangial cell glucose flux which leads to activation of signaling cascades favoring glomerulosclerosis, including pathways mediated by angiotensin II (Ang II), transforming growth factor ß (TGF-ß), connective tissue growth factor (CTGF), and vascular endothelial growth factor (VEGF). Ang II has both hemodynamic and metabolic effects directly inducing GLUT1 and/or matrix protein synthesis through diacyl glycerol (DAG) or protein kinase C (PKC) induction, mesangial cell stretch, and/or through transactivation of the epidermal growth factor receptor, the platelet-derived growth factor receptor, and the insulin-like growth factor-1 receptor, all of which may stimulate GLUT1 synthesis via an ERK-mediated pathway. Conversely, inhibition of Ang II effects suppresses GLUT1 and cellular glucose uptake. GLUT1-mediated glucose flux leads to metabolism of glucose via glycolysis, with induction of DAG, PKC, TGF-ß1, CTGF and VEGF. VEGF in turn triggers both GLUT1 and matrix synthesis. New roles for GLUT1-mTOR and GLUT1-mechano-growth factor interactions in diabetic glomerulosclerosis have also recently been suggested. Recent mouse models confirmed roles for GLUT1 in vivo in stimulating glomerular growth factor expression, growth factor receptors and development of glomerulosclerosis. GLUT1 may therefore act in concert with cytokines and growth factors to induce diabetic glomerulosclerosis. Further clarification of the pathways involved may prove useful for the therapy of diabetic nephropathy. New directions for investigation are discussed.


Assuntos
Nefropatias Diabéticas/fisiopatologia , Transportador de Glucose Tipo 1/fisiologia , Glucose/metabolismo , Hiperglicemia/fisiopatologia , Angiotensina II/fisiologia , Animais , Fator de Crescimento do Tecido Conjuntivo/fisiologia , Nefropatias Diabéticas/etiologia , Nefropatias Diabéticas/metabolismo , Humanos , Hiperglicemia/metabolismo , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta/fisiologia , Fator A de Crescimento do Endotélio Vascular/fisiologia
9.
J Biol Chem ; 288(27): 19450-8, 2013 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-23671281

RESUMO

1,25-Dihydroxyvitamin D (1,25(OH)2D3) is known to suppress NF-κB activity, but the underlying mechanism remains poorly understood. Here we show that the vitamin D receptor (VDR) physically interacts with IκB kinase ß (IKKß) to block NF-κB activation. 1,25(OH)2D3 rapidly attenuates TNFα-induced p65 nuclear translocation and NF-κB activity in a VDR-dependent manner. VDR overexpression inhibits IKKß-induced NF-κB activity. GST pull-down assays and coimmunoprecipitation experiments demonstrated that VDR physically interacts with IKKß and that this interaction is enhanced by 1,25(OH)2D3. Protein mapping reveals that VDR-IKKß interaction occurs between the C-terminal portions of the VDR and IKKß proteins. Reconstitution of VDR(-/-) cells with the VDR C terminus restores the ability to block TNFα-induced NF-κB activation and IL-6 up-regulation. VDR-IKKß interaction disrupts the formation of the IKK complex and, thus, abrogates IKKß phosphorylation at Ser-177 and abolishes IKK activity to phosphorylate IκBα. Consequently, stabilization of IκBα arrests p65/p50 nuclear translocation. Together, these data define a novel mechanism whereby 1,25(OH)2D3-VDR inhibits NF-κB activation.


Assuntos
Núcleo Celular/metabolismo , Quinase I-kappa B/metabolismo , Subunidade p50 de NF-kappa B/metabolismo , Receptores de Calcitriol/metabolismo , Fator de Transcrição RelA/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/genética , Animais , Calcitriol/farmacologia , Núcleo Celular/genética , Células HEK293 , Humanos , Quinase I-kappa B/genética , Camundongos , Camundongos Knockout , Subunidade p50 de NF-kappa B/genética , Mapeamento de Peptídeos , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Estrutura Terciária de Proteína , Receptores de Calcitriol/genética , Fator de Transcrição RelA/genética
10.
J Immunol ; 190(7): 3687-95, 2013 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-23436936

RESUMO

The negative feedback mechanism is essential to maintain effective immunity and tissue homeostasis. 1,25-dihydroxyvitamin D (1,25[OH]2D3) modulates innate immune response, but the mechanism remains poorly understood. In this article, we report that vitamin D receptor signaling attenuates TLR-mediated inflammation by enhancing the negative feedback inhibition. Vitamin D receptor inactivation leads to hyperinflammatory response in mice and macrophage cultures when challenged with LPS, because of microRNA-155 (miR-155) overproduction that excessively suppresses suppressor of cytokine signaling 1, a key regulator that enhances the negative feedback loop. Deletion of miR-155 attenuates vitamin D suppression of LPS-induced inflammation, confirming that 1,25(OH)2D3 stimulates suppressor of cytokine signaling 1 by downregulating miR-155. 1,25(OH)2D3 downregulates bic transcription by inhibiting NF-κB activation, which is mediated by a κB cis-DNA element located within the first intron of the bic gene. Together, these data identify a novel regulatory mechanism for vitamin D to control innate immunity.


Assuntos
Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , MicroRNAs/genética , Transdução de Sinais/efeitos dos fármacos , Proteínas Supressoras da Sinalização de Citocina/genética , Receptores Toll-Like/metabolismo , Vitamina D/análogos & derivados , Animais , Linhagem Celular , Citocinas/imunologia , Citocinas/metabolismo , Ativação Enzimática/efeitos dos fármacos , Retroalimentação Fisiológica/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Hipersensibilidade/genética , Hipersensibilidade/imunologia , Inflamação/imunologia , Inflamação/metabolismo , Mediadores da Inflamação/imunologia , Mediadores da Inflamação/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Lipopolissacarídeos/imunologia , Camundongos , Camundongos Knockout , Modelos Biológicos , NF-kappa B/metabolismo , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Proteína 1 Supressora da Sinalização de Citocina , Transcrição Gênica/efeitos dos fármacos , Vitamina D/farmacologia
11.
Int J Cancer ; 130(1): 10-9, 2012 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-21328347

RESUMO

Emerging evidence supports an inhibitory role for vitamin D in colorectal carcinogenesis; however, the mechanism remains unclear. The adenomatous polyposis coli (APC)/ß-catenin pathway plays a critical role in colorectal carcinogenesis. The purpose of our study is to explore the interactions of vitamin D and APC/ß-catenin pathways in intestinal tumor development. APC(min/+) mice with genetic inactivation of the vitamin D receptor (VDR) were generated through breeding. Intestinal tumorigenesis was compared between APC(min/+) and APC(min/+) VDR(-/-) mice at different ages. No differences were seen in the number of small intestinal and colonic tumors between APC(min/+) and APC(min/+) VDR(-/-) mice aged 3, 4, 6 and 7 months. The size of the tumors, however, was significantly increased in APC(min/+) VDR(-/-) mice in all age groups. Immunostaining showed significant increases in ß-catenin, cyclin D1, phosphorylated Stat-3 and MSH-2 levels and decreases in Stat-1 in APC(min/+) VDR(-/-) tumors compared to APC(min/+) tumors. These observations suggest that VDR signaling inhibits tumor growth rather than tumor initiation in the intestine. Thus, the increased tumor burden in APC(min/+) VDR(-/-) mice is likely due to the loss of the growth-inhibiting effect of VDR. This study provides strong evidence for the in vivo relevance of the interaction demonstrated in vitro between the vitamin D and ß-catenin signaling pathways in intestinal tumorigenesis.


Assuntos
Genes APC/fisiologia , Neoplasias Intestinais/etiologia , Neoplasias Intestinais/patologia , Receptores de Calcitriol/fisiologia , Animais , Western Blotting , Técnicas Imunoenzimáticas , Imunoprecipitação , Neoplasias Intestinais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , Receptores de Calcitriol/antagonistas & inibidores , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , beta Catenina/metabolismo
12.
Arch Biochem Biophys ; 507(2): 241-7, 2011 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-21176770

RESUMO

Plasminogen activator inhibitor (PAI)-1 is a major fibrinolytic inhibitor. High PAI-1 is associated with increased renal and cardiovascular disease risk. Previous studies demonstrated PAI-1 down-regulation by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), but the molecular mechanism remains unknown. Here we show that exposure of mouse embryonic fibroblasts to TNFα or LPS led to a marked induction of PAI-1, which was blunted by 1,25(OH)2D3, NF-κB inhibitor or p65 siRNA, suggesting the involvement of NF-κB in 1,25(OH)2D3-induced repression. In mouse Pai-1 promoter a putative cis-κB element was identified at -299. EMSA and ChIP assays showed that TNF-α increased p50/p65 binding to this κB site, which was disrupted by 1,25(OH)2D3. Luciferase reporter assays showed that PAI-1 promoter activity was induced by TNFα or LPS, and the induction was blocked by 1,25(OH)2D3. Mutation of the κB site blunted TNFα, LPS or 1,25(OH)2D3 effects. 1,25(OH)2D3 blocked IκBα degradation and arrested p50/p65 nuclear translocation. In mice LPS stimulated PAI-1 expression in the heart and macrophages, and the stimulation was blunted by pre-treatment with a vitamin D analog. Together these data demonstrate that 1,25(OH)2D3 down-regulates PAI-1 by blocking NF-κB activation. Inhibition of PAI-1 production may contribute to the reno- and cardio-protective effects of vitamin D.


Assuntos
Calcitriol/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , NF-kappa B/metabolismo , Inibidor 1 de Ativador de Plasminogênio/genética , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Animais , Sequência de Bases , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Regulação para Baixo/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Lipopolissacarídeos/farmacologia , Camundongos , Regiões Promotoras Genéticas/genética , Fator de Necrose Tumoral alfa/farmacologia
13.
Am J Physiol Renal Physiol ; 299(1): F99-F111, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20375117

RESUMO

Previous work identified an important role for hyperglycemia in diabetic nephropathy (The Diabetes Control and Complications Trial Research Group. N Engl J Med 329: 977-986, 1993; UK Prospective Diabetes Study Group. Lancet 352: 837-853, 1998), and increased glomerular GLUT1 has been implicated. However, the roles of GLUT1 and intracellular glucose have not been determined. Here, we developed transgenic GLUT1-overexpressing mice (GT1S) to characterize the roles of GLUT1 and intracellular glucose in the development of glomerular disease without diabetes. GLUT1 was overexpressed in glomerular mesangial cells (MC) of C57BL6 mice, a line relatively resistant to diabetic nephropathy. Blood pressure, blood glucose, glomerular morphometry, matrix proteins, cell signaling, transcription factors, and selected growth factors were examined. Kidneys of GT1S mice overexpressed GLUT1 in glomerular MCs and small vessels, rather than renal tubules. GT1S mice were neither diabetic nor hypertensive. Glomerular GLUT1, glucose uptake, mean capillary diameter, and mean glomerular volume were all increased in the GT1S mice. Moderately severe glomerulosclerosis (GS) was established by 26 wk of age in GT1S mice, with increased glomerular type IV collagen and fibronectin. Modest increases in glomerular basement membrane thickness and albuminuria were detected with podocyte foot processes largely preserved, in the absence of podocyte GLUT1 overexpression. Activation of glomerular PKC, along with increased transforming growth factor-beta1, VEGFR1, VEGFR2, and VEGF were all detected in glomeruli of GT1S mice, likely contributing to GS. The transcription factor NF-kappaB was also activated. Overexpression of glomerular GLUT1, mimicking the diabetic GLUT1 response, produced numerous features typical of diabetic glomerular disease, without diabetes or hypertension. This suggested GLUT1 may play an important role in the development of diabetic GS.


Assuntos
Nefropatias Diabéticas/metabolismo , Transportador de Glucose Tipo 1/metabolismo , Glomérulos Renais/metabolismo , Envelhecimento , Albuminúria/metabolismo , Albuminúria/patologia , Animais , Glicemia/metabolismo , Pressão Sanguínea , Células Cultivadas , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/patologia , Nefropatias Diabéticas/fisiopatologia , Membrana Basal Glomerular/metabolismo , Mesângio Glomerular/metabolismo , Transportador de Glucose Tipo 1/genética , Humanos , Glomérulos Renais/patologia , Células Mesangiais/metabolismo , Células Mesangiais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , NF-kappa B/metabolismo , Podócitos/metabolismo , Proteína Quinase C/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta1/metabolismo , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/metabolismo
14.
Lab Invest ; 90(1): 83-97, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19918242

RESUMO

Reduced nephron numbers may predispose to renal failure. We hypothesized that glucose transporters (GLUTs) may contribute to progression of the renal disease, as GLUTs have been implicated in diabetic glomerulosclerosis and hypertensive renal disease with mesangial cell (MC) stretch. The Os (oligosyndactyly) allele that typically reduces nephron number by approximately 50%, was repeatedly backcrossed from ROP (Ra/+ (ragged), Os/+ (oligosyndactyly), and Pt/+ (pintail)) Os/+ mice more than six times into the Fvb mouse background to obtain Os/+ and +/+ mice with the Fvb background for study. Glomerular function, GLUT1, signaling, albumin excretion, and structural and ultrastructural changes were assessed. The FvbROP Os/+ mice (Fvb background) exhibited increased glomerular GLUT1, glucose uptake, VEGF, glomerular hypertrophy, hyperfiltration, extensive podocyte foot process effacement, marked albuminuria, severe extracellular matrix (ECM) protein deposition, and rapidly progressive renal failure leading to their early demise. Glomerular GLUT1 was increased 2.7-fold in the FvbROP Os/+ mice vs controls at 4 weeks of age, and glucose uptake was increased 2.7-fold. These changes were associated with the activation of glomerular PKCbeta1 and NF-kappaB p50 which contribute to ECM accumulation. The cyclic mechanical stretch of MCs in vitro, used as a model for increased MC stretch in vivo, reproduced increased GLUT1 at 48 h, a stimulus for increased VEGF expression which followed at 72 h. VEGF was also shown to act in a positive feedback manner on MC GLUT1, increasing GLUT1 expression, glucose uptake and fibronectin (FN) accumulation in vitro, whereas antisense suppression of GLUT1 largely blocked FN upregulation by VEGF. The FvbROP Os/+ mice exhibited an early increase in glomerular GLUT1 leading to increased glomerular glucose uptake PKCbeta1, and NF-kappaB activation, with excess ECM accumulation. A GLUT1-VEGF-GLUT1 positive feedback loop may play a key role in contributing to renal disease in this model of nondiabetic glomerulosclerosis.


Assuntos
Albuminúria/etiologia , Transportador de Glucose Tipo 1/metabolismo , Camundongos Mutantes/metabolismo , Néfrons/anormalidades , Insuficiência Renal/etiologia , Insuficiência Renal/fisiopatologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Alelos , Animais , Células Cultivadas , Creatinina/metabolismo , Progressão da Doença , Proteínas da Matriz Extracelular/metabolismo , Mesângio Glomerular/metabolismo , Mesângio Glomerular/patologia , Imuno-Histoquímica , Isoenzimas/metabolismo , Rim/crescimento & desenvolvimento , Rim/metabolismo , Rim/patologia , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes/genética , Microscopia Eletrônica , NF-kappa B/metabolismo , Proteína Quinase C/metabolismo , Estresse Mecânico , Sindactilia/genética , Fator de Crescimento Transformador beta1/metabolismo , Regulação para Cima
15.
Am J Physiol Renal Physiol ; 297(3): F791-801, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19535571

RESUMO

The intrarenal renin-angiotensin system (RAS) plays a key role in the development of diabetic nephropathy. Recently, we showed that combination therapy with an AT(1) receptor blocker (ARB) and an activated vitamin D analog produced excellent synergistic effects against diabetic nephropathy, as a result of blockade of the ARB-induced compensatory renin increase. Given the diversity of vitamin D analogs, here we used a pro-drug vitamin D analog, doxercalciferol (1alpha-hydroxyvitamin D(2)), to further test the efficacy of the combination strategy in long-term treatment. Streptozotocin-induced diabetic DBA/2J mice were treated with vehicle, losartan, doxercalciferol (0.4 and 0.6 microg/kg), or losartan and doxercalciferol combinations for 20 wk. Vehicle-treated diabetic mice developed progressive albuminuria and glomerulosclerosis. Losartan alone moderately ameliorated kidney injury, with renin being drastically upregulated. A similar therapeutic effect was seen with doxercalciferol alone, which markedly suppressed renin and angiotensinogen expression. The losartan and doxercalciferol combination most effectively prevented albuminuria, restored glomerular filtration barrier structure, and dramatically reduced glomerulosclerosis in a dose-dependent manner. These effects were accompanied by blockade of intrarenal renin upregulation and ANG II accumulation. These data demonstrate an excellent therapeutic potential for doxercalciferol in diabetic renal disease and confirm the concept that blockade of the compensatory renin increase enhances the efficacy of RAS inhibition and produces synergistic therapeutic effects in combination therapy.


Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Diabetes Mellitus Experimental/tratamento farmacológico , Nefropatias Diabéticas/prevenção & controle , Ergocalciferóis/farmacologia , Losartan/farmacologia , Sistema Renina-Angiotensina/efeitos dos fármacos , Vitaminas/farmacologia , Albuminúria/etiologia , Albuminúria/prevenção & controle , Angiotensina II/metabolismo , Angiotensinogênio/metabolismo , Animais , Citocinas/metabolismo , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Nefropatias Diabéticas/etiologia , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Quimioterapia Combinada , Fibrose , Membrana Basal Glomerular/efeitos dos fármacos , Membrana Basal Glomerular/patologia , Mediadores da Inflamação/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos DBA , Podócitos/efeitos dos fármacos , Podócitos/patologia , Renina/metabolismo , Sistema Renina-Angiotensina/genética , Fatores de Tempo
16.
Mech Dev ; 125(9-10): 857-64, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18606221

RESUMO

Cell proliferation and cell type specification are coordinately regulated during normal development. Cyclin E, a key G1/S cell cycle regulator, is regulated by multiple tissue-specific enhancers resulting in dynamic expression during Drosophila development. Here, we further characterized the enhancer that regulates cyclin E expression in the developing peripheral nervous system (PNS) and show that multiple sequence elements are required for the full cyclin E PNS enhancer activity. We further show that Wg signaling is important for the expression of cyclin E in the sensory organ precursor (SOP) cells through two conserved TCF binding sites. Blocking Wg signaling does not completely block SOP cell formation but does completely block SOP cell proliferation as well as the subsequent differentiation.


Assuntos
Ciclina E/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Células-Tronco Embrionárias/citologia , Regulação da Expressão Gênica no Desenvolvimento , Sistema Nervoso Periférico/embriologia , Transdução de Sinais , Proteína Wnt1/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Diferenciação Celular , Proliferação de Células , Sequência Conservada , Drosophila melanogaster/citologia , Drosophila melanogaster/embriologia , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Células-Tronco Embrionárias/metabolismo , Elementos Facilitadores Genéticos/genética , Genes Reporter , Proteínas de Fluorescência Verde/metabolismo , Dados de Sequência Molecular , Sistema Nervoso Periférico/citologia , Sistema Nervoso Periférico/metabolismo , Deleção de Sequência , Fatores de Transcrição TCF/metabolismo
17.
Mol Cell Biol ; 27(8): 2987-96, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17296729

RESUMO

Cell differentiation and cell cycle exit are coordinately regulated during development; however, the molecular logic underlying this regulation is not known. The Drosophila cdk inhibitor Dacapo (Dap) is one of the key cell cycle regulators that exhibit dynamic expression during development and contribute to the developmental regulation of the cell cycle. In this study, regulation of Dap expression during cell type specification was investigated. The expression of Dap in the R2 and R5 precursors of the developing eye and in the newly recruited leg disc femoral sense organ precursors was found to be controlled by the epidermal growth factor receptor signaling-regulated transcription factor Pointed (Pnt) and the proneural basic helix-loop-helix proteins Atonal (Ato) and Daughterless (Da). We show that Pnt, Ato, and Da regulate Dap expression directly through their respective binding sites precisely at the time when these transcription factors function to specify neural fates. These results show that Dap expression is directly regulated by developmental mechanisms that simultaneously control cell type specification. This is potentially a general mechanism by which the expression of key cell cycle regulators is coordinated with differentiation during normal development. The direct regulation of key cell cycle regulators by the differentiation factors ensures coordinated regulation of cell cycle and differentiation.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem da Célula , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Receptores ErbB/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Transdução de Sinais , Animais , Sítios de Ligação , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/genética , Elementos Facilitadores Genéticos/genética , Extremidades , Olho/citologia , Olho/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Fluorescência Verde/metabolismo , Proteínas Nucleares/genética , Proteínas Proto-Oncogênicas/metabolismo , Órgãos dos Sentidos/citologia , Órgãos dos Sentidos/metabolismo , Células-Tronco/citologia , Fatores de Transcrição/metabolismo
18.
Cancer Res ; 65(8): 3470-8, 2005 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-15833883

RESUMO

Epidemiologic studies associate consumption of genistein, in the form of dietary soy, with lower rates of metastatic prostate cancer. We have previously shown that genistein inhibits prostate cancer cell detachment in vitro, that it is well tolerated in an older cohort of men with prostate cancer, and that it alters cell signaling in that same cohort. We have also shown that p38 mitogen-activated protein kinase (MAPK) is necessary for transforming growth factor beta (TGF-beta)-mediated increases in prostate cancer adhesion. Although cell invasion is closely linked to metastatic behavior, little is known about how this process is regulated in prostate cancer or what effect, if any, genistein has on associated processes. We now show that genistein inhibits matrix metalloproteinase type 2 (MMP-2) activity in six of seven prostate cell lines tested, blocks MMP-2 induction by TGF-beta, and inhibits cell invasion. Efficacy was seen at low nanomolar concentrations, corresponding to blood concentrations of free genistein attained after dietary consumption. Inhibition of p38 MAPK by either SB203580 or dominant-negative construct blocked induction of MMP-2 and cell invasion by TGF-beta. Genistein exerted similar effects and was found to block activation of p38 MAPK by TGF-beta. This study shows that p38 MAPK is necessary for TGF-beta-mediated induction of MMP-2 and cell invasion in prostate cancer and that genistein blocks activation of p38 MAPK, thereby inhibiting processes closely linked to metastasis, and does so at concentrations associated with dietary consumption. Any potential causal link to epidemiologic findings will require further investigation.


Assuntos
Genisteína/farmacologia , Inibidores de Metaloproteinases de Matriz , Próstata/efeitos dos fármacos , Próstata/enzimologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Linhagem Celular , Ativação Enzimática/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/enzimologia , Humanos , Masculino , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/prevenção & controle
19.
J Biol Chem ; 279(2): 970-9, 2004 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-14578350

RESUMO

Multiple signaling pathways are activated during engagement of the Type I interferon (IFN) receptor to mediate biological responses, including the Jak-Stat and Rac1/p38 Map kinase signaling cascades. In the present study we sought to determine the functional relevance of the p38alpha isoform in IFN signaling, using cells from mouse embryos with targeted disruption of the p38alpha gene. Our data demonstrate that p38alpha activation is essential for Type I IFN-dependent transcriptional regulation via ISRE or GAS elements. On the other hand, the function of p38alpha is not required for IFN-dependent Ser727 or Tyr701 phosphorylation of Stat1 and does not impact on the formation of ISGF3 or SIF nuclear binding complexes. In efforts to identify downstream effectors of p38 that may mediate IFN-dependent transcriptional responses, we found that IFNalpha activates the kinase Msk1, a known regulator of histone phosphorylation and chromatin remodeling. In other studies, we demonstrate that Type I IFN-dependent activation of the kinases MapKapK-2 and MapKapK-3 is defective in the absence of p38alpha, while Type I IFN-dependent antiviral properties are decreased in cells with targeted disruption of the MapKapK-2 gene. Altogether, our data establish that the p38alpha Map kinase pathway regulates activation of downstream effectors that participate in the induction of IFN-dependent gene transcription, to mediate IFN-responses.


Assuntos
Interferon Tipo I/metabolismo , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Transdução de Sinais , Animais , Linhagem Celular Tumoral , Cromatina/metabolismo , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Fibroblastos/metabolismo , Humanos , Immunoblotting , Interferon gama/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Luciferases/metabolismo , Camundongos , Proteína Quinase 14 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Osmose , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes/metabolismo , Serina/química , Estresse Fisiológico , Fatores de Tempo , Transcrição Gênica , Transfecção , Tirosina/química
20.
J Biol Chem ; 278(35): 32544-51, 2003 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-12805378

RESUMO

All-trans-retinoic acid (RA) is a potent inhibitor of leukemia cell proliferation and induces differentiation of acute promyelocytic leukemia cells in vitro and in vivo. For RA to induce its biological effects in target cells, binding to specific retinoic acid nuclear receptors is required. The resulting complexes bind to RA-responsive elements (RAREs) in the promoters of RA-inducible genes to initiate gene transcription and to generate protein products that mediate the biological effects of RA. In this report, we provide evidence that a member of the protein kinase C (PKC) family of proteins, PKC delta, is activated during RA treatment of the NB-4 and HL-60 acute myeloid leukemia cell lines as well as the MCF-7 breast cancer cell line. Such RA-dependent phosphorylation was also observed in primary acute promyelocytic leukemia cells and resulted in activation of the kinase domain of PKC delta. In studies aimed at understanding the functional relevance of PKC delta in the induction of RA responses, we found that pharmacological inhibition of PKC delta (but not of other PKC isoforms) diminished RA-dependent gene transcription via RAREs. On the other hand, overexpression of a constitutively active form of the kinase strongly enhanced RA-dependent gene transcription via RAREs. Gel shift assays and chromatin immunoprecipitation studies demonstrated that PKC delta associated with retinoic acid receptor-alpha and was present in an RA-inducible protein complex that bound to RAREs. Pharmacological inhibition of PKC delta activity abrogated the induction of cell differentiation and growth inhibition of NB-4 blast cells, demonstrating that its function is required for such effects. Altogether, our data provide strong evidence that PKC delta is activated in an RA-dependent manner and plays a critical role in the generation of the biological effects of RA in malignant cells.


Assuntos
Ativação Enzimática , Proteína Quinase C/metabolismo , Tretinoína/metabolismo , Diferenciação Celular , Divisão Celular , Cromatina/metabolismo , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Citometria de Fluxo , Células HL-60 , Humanos , Immunoblotting , Interferon-alfa/metabolismo , Luciferases/metabolismo , Testes de Precipitina , Regiões Promotoras Genéticas , Isoformas de Proteínas , Proteína Quinase C-delta , Estrutura Terciária de Proteína , Receptores do Ácido Retinoico/metabolismo , Receptor alfa de Ácido Retinoico , Transcrição Gênica , Células Tumorais Cultivadas
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