RESUMO
Autosomal dominant polycystic kidney disease results from the loss of the PKD1 gene product, polycystin 1. Regulatory mechanisms are unresolved, but an apparent G/C sequence bias in the gene is consistent with co-transcriptional R-loop formation. R-loops regulate gene expression and stability, and they form when newly synthesized RNA extensively pairs with the template DNA to displace the non-template strand. In this study, we tested two human PKD1 sequences for co-transcriptional R-loop formation in vitro. We observed RNase H-sensitive R-loop formation in intron 1 and 22 sequences, but only in one transcriptional orientation. Therefore, R-loops may participate in PKD1 expression or stability.
RESUMO
The transcription factor CUX1 has been implicated in either tumor suppression or progression, depending on the cancer entity and the prevalent CUX1 isoform. Previously, we could show that CUX1 acts as an important mediator of tumor cell proliferation and resistance to apoptosis in pancreatic cancer cell lines. However, in vivo evidence for its impact on pancreatic carcinogenesis, isoform-specific effects and downstream signaling cascades are missing. We crossbred two different CUX1 isoform mouse models (p200 CUX1 and p110 CUX1) with KC (KrasLSL-G12D/+; Ptf1aCre/+) mice, a genetic model for pancreatic precursor lesions (PanIN). In the context of oncogenic KRASs, both mice KCCux1p200 and KCCux1p110 led to increased PanIN formation and development of invasive pancreatic ductal adenocarcinomata (PDAC). In KCCux1p110 mice, tumor development was dramatically more accelerated, leading to formation of invasive PDAC within 4 weeks. In vitro and in vivo, we could show that CUX1 enhanced proliferation by activating MEK-ERK signaling via an upstream increase of ADAM17 protein, which in turn led to an activation of EGFR. Additionally, CUX1 further enhanced MEK-ERK activation through upregulation of the serine/threonine kinase MOS, phosphorylating MEK in a KRAS-independent manner. We identified p110 CUX1 as major driver of pancreatic cancer formation in the context of mutant KRAS. These results provide the first in vivo evidence for the importance of CUX1 in the development of pancreatic cancer, and highlight the importance of CUX1-dependent signaling pathways as potential therapeutic targets.
RESUMO
Systemic inflammation in pregnant obese women is associated with 1.5- to 2-fold increase in serum Interleukin-6 (IL-6) and newborns with lower kidney/body weight ratio but the role of IL-6 in increased susceptibility to chronic kidney (CKD) in adult progeny is not known. Since IL-6 crosses the placental barrier, we administered recombinant IL-6 (10 pg/g) to pregnant mice starting at mid-gestation yielded newborns with lower body (p < 0.001) and kidney (p < 0.001) weights. Histomorphometry indicated decreased nephrogenic zone width (p = 0.039) with increased numbers of mature glomeruli (p = 0.002) and pre-tubular aggregates (p = 0.041). Accelerated maturation in IL-6 newborns was suggested by early expression of podocyte-specific protein podocin in glomeruli, increased 5-methyl-cytosine (LC-MS analysis for CpG DNA methylation) and altered expression of certain genes of cell-cycle and apoptosis (RT-qPCR array-analysis). Western blotting showed upregulated pJAK2/pSTAT3. Thus, treating dams with IL-6 as a surrogate provides newborns to study effects of maternal systemic inflammation on future susceptibility to CKD in adulthood.
Assuntos
Interleucina-6/efeitos adversos , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamente , Animais , Animais Recém-Nascidos/crescimento & desenvolvimento , Apoptose/efeitos dos fármacos , Peso ao Nascer/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Feminino , Rim/crescimento & desenvolvimento , Rim/metabolismo , Rim/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Tamanho do Órgão/efeitos dos fármacos , Gravidez , Efeitos Tardios da Exposição Pré-Natal/patologiaRESUMO
Notch pathway activation plays a central role in the pathogenesis of many glomerular diseases. We have previously shown that Notch4 expression was upregulated in various renal cells in human immunodeficiency virus (HIV)-associated nephropathy (HIVAN) patients and rodent models of HIVAN. In this study, we examined whether the Notch pathway can be distinctly activated by HIV-1 gene products and whether Notch4, in particular, can influence disease progression. Using luciferase reporter assays, we did not observe activation of the NOTCH4 promoter with the HIV protein Nef in podocytes. Further, we observed upregulated expression of a gamma secretase complex protein, presenilin 1, but not Notch4, in podocytes infected with an HIV-1 expression construct. To assess the effects of Notch4 on HIVAN disease progression, we engineered Tg26 mice with global deletion of the Notch4 intracellular domain (Notch4dl ), which is required for signaling function. These mice (Notch4d1/Tg26+ ) showed a significant improvement in renal function and a significant decrease in mortality compared to Tg26 mice. Histological examination of kidneys showed that Notch4d1/Tg26+ mice had overall glomerular, tubulointerstitial injury and a marked decrease in interstitial inflammation. A significant decrease in the proliferating cells was observed in the tubulointerstitial compartments of Notch4d1/Tg26+ mice. Consistent with the diminished inflammation, kidneys from Notch4d1/Tg26+ mice also showed a significant decrease in expression of the inflammatory cytokine transcripts Il-6 and Ccl2, as well as the master inflammatory transcription factor NF-κB (Nfkb1 transcripts and p65 protein). These data identify Notch4 as an important mediator of tubulointerstitial injury and inflammation in HIVAN and a potential therapeutic target.
Assuntos
Nefropatia Associada a AIDS/metabolismo , Inflamação/metabolismo , Subunidade p50 de NF-kappa B/metabolismo , Receptor Notch4/metabolismo , Animais , Proliferação de Células , Cruzamentos Genéticos , Modelos Animais de Doenças , Progressão da Doença , Feminino , Deleção de Genes , Células HEK293 , Humanos , Rim/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Podócitos/metabolismo , Transdução de Sinais , Fator de Transcrição RelA/metabolismo , Resultado do Tratamento , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismoRESUMO
Cux1 is a homeodomain protein involved in cell cycle regulation and kidney development. Cux1 represses the cyclin kinase inhibitor p27 during early kidney development, promoting cell proliferation in the nephrogenic zone. Promoter reporter analysis of p27 revealed that Cux1 represses p27 in a concentration dependent manner, and immunoprecipitation showed that Cux1 interacts with the co-repressor Grg4 and the histone deacetylases HDAC1 and HDAC3. Chromatin immunoprecipitation (ChIP) identified the interaction of Cux1, Grg4, HDAC1, and HDAC3 at two different sites in the p27 promoter. To determine whether there was an interaction between these two loci in the developing kidney, we performed chromatin conformation capture (3C) assay. Analysis of newborn kidney tissue with 3C and ChIP-loop showed that the p27 promoter forms a loop intersecting at these two loci and that Cux1 bridges these two sites. To determine whether HDACs are required for Cux1 repression of p27 we analyzed p27 promoter activity in the presence of the HDAC inhibitor trichostatin A (TSA). TSA treatment completely relieved the repression of p27 by Cux1 and Grg4, demonstrating that Cux1 represses p27 in an HDAC dependent manner. To begin to test whether HDAC inhibitors could be used to target Cux1 repression of p27 for the treatment of PKD, we treated Pkd1 targeted pregnant mice with TSA or vehicle beginning at embryonic day 10.5 until embryonic day 18.5. Newborn Pkd1 mutant mice that received vehicle exhibited extensive collecting duct cysts, while newborn Pkd1 mutant mice that received TSA showed a significant reduction in cysts. Moreover, p27 expression was upregulated in TSA treated Pkd1 mice. Taken together, these results suggest that HDACs are required for cyst growth, and further support studies indicating that HDAC inhibitors may be an effective treatment for PKD.
RESUMO
Cux1 is a homeodomain protein involved in cell cycle regulation and kidney development. Cux1 represses the cyclin kinase inhibitor p27 during early kidney development, promoting cell proliferation in the nephrogenic zone. Promoter reporter analysis of p27 revealed that Cux1 represses p27 in a concentration dependent manner, and immunoprecipitation showed that Cux1 interacts with the co-repressor Grg4 and the histone deacetylases HDAC1 and HDAC3. Chromatin immunoprecipitation (ChIP) identified the interaction of Cux1, Grg4, HDAC1, and HDAC3 at two different sites in the p27 promoter. To determine whether there was an interaction between these two loci in the developing kidney, we performed chromatin conformation capture (3C) assay. Analysis of newborn kidney tissue with 3C and ChIP-loop showed that the p27 promoter forms a loop intersecting at these two loci and that Cux1 bridges these two sites. To determine whether HDACs are required for Cux1 repression of p27 we analyzed p27 promoter activity in the presence of the HDAC inhibitor trichostatin A (TSA). TSA treatment completely relieved the repression of p27 by Cux1 and Grg4, demonstrating that Cux1 represses p27 in an HDAC dependent manner. To begin to test whether HDAC inhibitors could be used to target Cux1 repression of p27 for the treatment of PKD, we treated Pkd1 targeted pregnant mice with TSA or vehicle beginning at embryonic day 10.5 until embryonic day 18.5. Newborn Pkd1 mutant mice that received vehicle exhibited extensive collecting duct cysts, while newborn Pkd1 mutant mice that received TSA showed a significant reduction in cysts. Moreover, p27 expression was upregulated in TSA treated Pkd1 mice. Taken together, these results suggest that HDACs are required for cyst growth, and further support studies indicating that HDAC inhibitors may be an effective treatment for PKD.
RESUMO
Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common monogenic hereditary disorders in humans characterized by fluid-filled cysts, primarily in the kidneys. Cux1, a cell cycle regulatory gene highly expressed during kidney development, is elevated in the cyst-lining cells of Pkd1 mutant mice, and in human ADPKD cells. However, forced expression of Cux1 is insufficient to induce cystic disease in transgenic mice or to induce rapid cyst formation after cilia disruption in the kidneys of adult mice. Here we report a double mutant mouse model that has a conditional deletion of the Pkd1 gene in the renal collecting ducts together with a targeted mutation in the Cux1 gene (Pkd1CD;Cux1tm2Ejn). While kidneys isolated from newborn Pkd1CD mice exhibit cortical and medullary cysts, kidneys isolated from newborn Pkd1CD;Cux1tm2Ejn-/- mice did not show any cysts. Because Cux1tm2Ejn-/- are perinatal lethal, we evaluated Pkd1CD mice that were heterozygote for the Cux1 mutation. Similar to the newborn Pkd1CD;Cux1tm2Ejn-/- mice, newborn Pkd1CD;Cux1tm2Ejn+/- mice did not show any cysts. Comparison of Pkd1CD and Pkd1CD;Cux1tm2Ejn+/- mice at later stages of development showed a reduction in the severity of PKD in the Pkd1CD;Cux1tm2Ejn+/- mice. Moreover, we observed an increase in expression of the cyclin kinase inhibitor p27, a target of Cux1 repression, in the rescued collecting ducts. Taken together, our results suggest that Cux1 expression in PKD is not directly involved in cystogenesis but promotes cell proliferation required for expansion of existing cysts, primarily by repression of p27.
Assuntos
Proliferação de Células , Proteínas de Homeodomínio/metabolismo , Túbulos Renais Coletores/metabolismo , Proteínas Nucleares/metabolismo , Rim Policístico Autossômico Dominante/metabolismo , Proteínas Repressoras/metabolismo , Fatores Etários , Animais , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Predisposição Genética para Doença , Proteínas de Homeodomínio/genética , Túbulos Renais Coletores/patologia , Camundongos Knockout , Mutação , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Fenótipo , Rim Policístico Autossômico Dominante/genética , Rim Policístico Autossômico Dominante/patologia , Rim Policístico Autossômico Dominante/prevenção & controle , Proteínas Repressoras/deficiência , Proteínas Repressoras/genética , Índice de Gravidade de Doença , Transdução de Sinais , Canais de Cátion TRPP/genética , Canais de Cátion TRPP/metabolismoRESUMO
Polycystic kidney disease (PKD) is a life-threatening genetic disorder characterized by the presence of fluid-filled cysts primarily in the kidneys. PKD can be inherited as autosomal recessive (ARPKD) or autosomal dominant (ADPKD) traits. Mutations in either the PKD1 or PKD2 genes, which encode polycystin 1 and polycystin 2, are the underlying cause of ADPKD. Progressive cyst formation and renal enlargement lead to renal insufficiency in these patients, which need to be managed by lifelong dialysis or renal transplantation. While characteristic features of PKD are abnormalities in epithelial cell proliferation, fluid secretion, extracellular matrix and differentiation, the molecular mechanisms underlying these events are not understood. Here we review the progress that has been made in defining the function of the polycystins, and how disruption of these functions may be involved in cystogenesis.
Assuntos
Rim/patologia , Doenças Renais Policísticas/patologia , Animais , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Rim/embriologia , Mecanotransdução Celular/genética , Modelos Biológicos , Doenças Renais Policísticas/embriologia , Doenças Renais Policísticas/genética , Via de Sinalização Wnt/genéticaRESUMO
The Notch pathway is an evolutionarily conserved signaling cascade that is critical in kidney development and has also been shown to play a pathogenetic role in a variety of kidney diseases. We have previously shown that the Notch signaling pathway is activated in human immunodeficiency virus-associated nephropathy (HIVAN) as well as in a rat model of the disease. In this study, we examined Notch signaling in the well established Tg26 mouse model of HIVAN. Notch signaling components were distinctly upregulated in the kidneys of these mice as well as in immortalized podocytes derived from these mice. Notch1 and Notch4 were upregulated in the Tg26 glomeruli, and Notch4 was also expressed in tubules. Notch ligands Jagged1, Jagged2, Delta-like1, and Delta-like 4 were all upregulated in the tubules of Tg26 mice, but glomeruli showed minimal expression of Notch ligands. To examine a potential pathogenetic role for Notch in HIVAN, Tg26 mice were treated with GSIXX, a gamma secretase inhibitor that blocks Notch signaling. Strikingly, GSIXX treatment resulted in significant improvement in both histological kidney injury scores and renal function. GSIXX-treated Tg26 mice also showed diminished podocyte proliferation and dedifferentiation, cellular hallmarks of the disease. Moreover, GSIXX blocked podocyte proliferation in vitro induced by HIV proteins Nef and Tat. These studies suggest that Notch signaling can promote HIVAN progression and that Notch inhibition may be a viable treatment strategy for HIVAN.
Assuntos
Nefropatia Associada a AIDS/metabolismo , Podócitos/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptor Notch1/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais/fisiologia , Nefropatia Associada a AIDS/tratamento farmacológico , Nefropatia Associada a AIDS/patologia , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Animais , Desdiferenciação Celular/efeitos dos fármacos , Desdiferenciação Celular/fisiologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Linhagem Celular Transformada , Dibenzazepinas/farmacologia , Dipeptídeos/farmacologia , Modelos Animais de Doenças , Progressão da Doença , Humanos , Rim/efeitos dos fármacos , Rim/patologia , Rim/fisiologia , Ligantes , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos , Podócitos/citologia , Podócitos/efeitos dos fármacos , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Receptor Notch1/antagonistas & inibidores , Receptor Notch4 , Receptores Notch/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologiaRESUMO
Disrupting the function of cilia in mouse kidneys results in rapid or slow progression of cystic disease depending on whether the animals are juveniles or adults, respectively. Renal injury can also markedly accelerate the renal cyst formation that occurs after disruption of cilia in adult mice. Rates of cell proliferation are markedly higher in juvenile than adult kidneys and increase after renal injury, suggesting that cell proliferation may enhance the development of cysts. Here, we induced cilia loss in the kidneys of adult mice in the presence or absence of a Cux-1 transgene, which maintains cell proliferation. By using this model, we were able to avoid additional factors such as inflammation and dedifferentiation, which associate with renal injury and may also influence the rate of cystogenesis. After induction of cilia loss, cystic disease was not more pronounced in adult mice with the Cux-1 transgene compared with those without the transgene. In conclusion, these data suggest that proliferation is unlikely to be the sole mechanism underlying the rapid cystogenesis observed after injury in mice that lose cilia function in adulthood.
Assuntos
Cílios/patologia , Doenças Renais Císticas/etiologia , Doenças Renais Císticas/patologia , Túbulos Renais Proximais/patologia , Animais , Proliferação de Células , Cílios/fisiologia , Expressão Gênica/efeitos dos fármacos , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/fisiologia , Camundongos , Camundongos Mutantes , Camundongos Transgênicos , Proteínas Nucleares/genética , Proteínas Nucleares/fisiologia , Proteínas Repressoras/genética , Proteínas Repressoras/fisiologia , Canais de Cátion TRPP/genética , Canais de Cátion TRPP/fisiologia , Tamoxifeno/farmacologia , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/fisiologiaRESUMO
Polycystic kidney diseases (PKD) are inherited disorders characterized by fluid-filled cysts primarily in the kidneys. We previously reported differences between the expression of Cux1, p21, and p27 in the cpk and Pkd1 null mouse models of PKD. Embryonic lethality of Pkd1 null mice limits its study to early stages of kidney development. Therefore, we examined mice with a collecting duct specific deletion in the Pkd1 gene. Cux1 was ectopically expressed in the cyst lining epithelial cells of newborn, P7 and P15 Pkd1(CD) mice. Cux1 expression correlated with cell proliferation in early stages of cystogenesis, however, as the disease progressed, fewer cyst lining cells showed increased cell proliferation. Rather, Cux1 expression in late stage cystogenesis was associated with increased apoptosis. Taken together, our results suggest that increased Cux1 expression associated with apoptosis is a common feature of late stage cyst progression in both the cpk and Pkd1(CD) mouse models of PKD.
Assuntos
Apoptose/genética , Inibidor de Quinase Dependente de Ciclina p27/genética , Proteínas de Homeodomínio/genética , Doenças Renais Císticas/genética , Proteínas Nucleares/genética , Proteínas Repressoras/genética , Canais de Cátion TRPP/genética , Animais , Animais Recém-Nascidos , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Progressão da Doença , Regulação para Baixo , Ativação Enzimática/genética , Feminino , Regulação da Expressão Gênica , Inativação Gênica , Doenças Renais Císticas/metabolismo , Doenças Renais Císticas/patologia , Túbulos Renais Coletores/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Canais de Cátion TRPP/metabolismo , Transfecção , Regulação para Cima/genéticaRESUMO
The homeodomain CUX1 protein exists as multiple isoforms that arise from proteolytic processing of a 200-kDa protein or an alternate splicing or from the use of an alternate promoter. The 200-kDa CUX1 protein is highly expressed in the developing kidney, where it functions to regulate cell proliferation. Transgenic mice ectopically expressing the 200-kDa CUX1 protein develop renal hyperplasia associated with reduced expression of the cyclin kinase inhibitor p27. A 55-kDa CUX1 isoform is expressed exclusively in the testes. We determined the pattern and timing of CUX1 protein expression in developing testes. CUX1 expression was continuous in Sertoli cells from prepubertal testes but became cyclic when spermatids appeared. In testes from mature mice, CUX1 was highly expressed only in round spermatids at stages IV-V of spermatogenesis, in both spermatids and Sertoli cells at stages VI-X of spermatogenesis, and only in Sertoli cells at stage XI of spermatogenesis. While most of the seminiferous tubules in wild-type mice were between stages VI and X of spermatogenesis, there was a significant reduction in the percentage of seminiferous tubules between stages VI and X in Cux1 transgenic mice and a significant increase in the percentage of seminiferous tubules in stages IV-V and XI. Moreover, CUX1 was not expressed in proliferating cells in testes from either wild-type or transgenic mice. Thus, unlike the somatic form of CUX1, which has a role in cell proliferation, the testis-specific form of CUX1 is not involved in cell division and appears to play a role in signaling between Sertoli cells and spermatids.
Assuntos
Proteínas de Homeodomínio/genética , Proteínas Nucleares/genética , Proteínas Repressoras/genética , Células de Sertoli/metabolismo , Espermátides/metabolismo , Espermatogênese/genética , Animais , Comunicação Celular/genética , Comunicação Celular/fisiologia , Divisão Celular/genética , Divisão Celular/fisiologia , Regulação da Expressão Gênica/fisiologia , Proteínas de Homeodomínio/metabolismo , Proteínas de Homeodomínio/fisiologia , Masculino , Camundongos , Camundongos Transgênicos , Proteínas Nucleares/metabolismo , Proteínas Nucleares/fisiologia , Proteínas Repressoras/metabolismo , Proteínas Repressoras/fisiologia , Células de Sertoli/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Espermátides/fisiologia , Espermatogênese/fisiologia , Testículo/citologia , Testículo/metabolismo , Testículo/fisiologiaRESUMO
Zhou et al. validate the expression of markers of the innate immune system in the cpk mouse model of polycystic kidney disease (PKD), in human recessive PKD, and in human autosomal dominant PKD and show that CD14 expression correlates with PKD progression, even from very early stages of disease. Moreover, they show that CD14 is expressed from the renal tubule epithelial cells, suggesting a mechanism of Toll-like receptor-4 activation in PKD prior to the infiltration of inflammatory cells.
Assuntos
Receptores de Lipopolissacarídeos/análise , Doenças Renais Policísticas/diagnóstico , Animais , Biomarcadores/análise , Progressão da Doença , Humanos , Inflamação/patologia , Túbulos Renais/patologia , Camundongos , Receptor 4 Toll-Like/metabolismoRESUMO
OBJECTIVE: HIV-associated nephropathy (HIVAN) is characterized by the development of glomerulosclerosis and is associated with glomerular epithelial cell proliferation. It has recently been shown that activation of the Notch signaling pathway in podocytes results in glomerulosclerosis and podocyte proliferation. To determine whether Notch signaling is involved in renal disorder associated with HIVAN, we evaluated the expression of Notch receptors in HIVAN. DESIGN: We evaluated the expression of the Notch signaling pathway using an HIV-transgenic (HIV-Tg) rat model of HIVAN, and biopsy samples from HIVAN and normal controls. METHODS: Paraffin sections and kidney lysates were used for immunohistochemistry, immunofluorescence and western blot analysis. RESULTS: A collapsing variant of glomerulosclerosis and focal segmental sclerosis was observed in HIV-Tg rats. Glomeruli of HIV-Tg rats demonstrated activation of Notch1 and Notch4, as determined by the presence of the intracellular domains. In addition, we observed increased expression of the Notch target protein, hairy enhancer of split homolog-1 in glomeruli of these animals. The expression of the Groucho homolog transducin-like enhancer protein 4, a Notch effector protein, and the homeodomain protein cut homeobox 1 were also significantly increased in glomeruli of HIV-Tg rats, and this was associated with decreased expression of the cyclin kinase inhibitor p27. Intriguingly, renal biopsy samples from HIVAN patients also showed upregulation of cleaved Notch1 and Notch4 in the glomeruli compared with the expression in normal kidneys. CONCLUSION: Our results demonstrate activation of Notch signaling pathway in HIVAN, thereby underscoring its role in disease pathogenesis.
Assuntos
Nefropatia Associada a AIDS/patologia , Infecções por HIV/patologia , Glomérulos Renais/patologia , Podócitos/metabolismo , Receptores Notch/metabolismo , Animais , Proliferação de Células , Infecções por HIV/genética , Imuno-Histoquímica , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/genéticaRESUMO
The mammalian cut proteins are a broadly expressed family of nuclear transcription factors related to the Drosophila protein cut. One member of the cut family, Cux1, has been shown to function as a cell cycle dependent transcription factor, regulating the expression of a number of cell cycle regulatory proteins. Cux1 expression is developmentally regulated in multiple tissues suggesting an important regulatory function. Cux1 exists as multiple isoforms that arise from proteolytic processing of a 200 kD protein or use of an alternate promoter. Several mouse models of Cux1 have been generated that suggest important roles for this gene in cell cycle regulation during hair growth, lung development and maturation, and genitourinary tract development. Moreover, the aberrant expression of Cux1 may contribute to diseases such as polycystic kidney disease and cancer. In this review, we will focus on the phenotypes observed in the five existing transgenic mouse models of Cux1, and discuss the role of Cux1 in kidney development and disease.
Assuntos
Rim/citologia , Animais , Calcineurina/genética , Ciclo Celular/genética , Ciclo Celular/fisiologia , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Proliferação de Células , Modelos Animais de Doenças , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/fisiologia , Rim/crescimento & desenvolvimento , Rim/fisiologia , Camundongos , Camundongos Mutantes , Camundongos Transgênicos , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/fisiologia , Fenótipo , Doenças Renais Policísticas/genética , Doenças Renais Policísticas/fisiopatologia , Proteínas Repressoras/química , Proteínas Repressoras/genética , Proteínas Repressoras/fisiologiaRESUMO
The homeodomain protein Cux1 is highly expressed in the nephrogenic zone of the developing kidney where it functions to regulate cell proliferation. Here we show that Cux1 directly interacts with the co-repressor Grg4 (Groucho 4), a known effector of Notch signaling. Promoter reporter based luciferase assays revealed enhanced repression of p27(kip1) promoter activity by Cux1 in the presence of Grg4. Chromatin immunoprecipitation (ChIP) assays demonstrated the direct interaction of Cux1 with p27(kip1) in newborn kidney tissue in vivo. ChIP assays also identified interactions of Cux1, Grg4, HDAC1, and HDAC3 with p27(kip1) at two separate sites in the p27(kip1) promoter. DNAse1 footprinting experiments revealed that Cux1 binds to the p27(kip1) promoter on the sequence containing two Sp1 sites and a CCAAT box approximately 500 bp from the transcriptional start site, and to an AT rich sequence approximately 1.5 kb from the transcriptional start site. Taken together, these results identify Grg4 as an interacting partner for Cux1 and suggest a mechanism of p27(kip1) repression by Cux1 during kidney development.
Assuntos
Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Proteínas de Homeodomínio/metabolismo , Rim/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Animais , Animais Recém-Nascidos , Sítios de Ligação , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Linhagem Celular , Imunoprecipitação da Cromatina , Inibidor de Quinase Dependente de Ciclina p27/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Humanos , Rim/crescimento & desenvolvimento , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Repressoras/genéticaRESUMO
Polycystic kidney diseases (PKD) are inherited as autosomal dominant (ADPKD) or autosomal recessive (ARPKD) traits and are characterized by progressive enlargement of renal cysts. Aberrant cell proliferation is a key feature in the progression of PKD. Cux1 is a homeobox gene that is related to Drosophila cut and is the murine homolog of human CDP (CCAAT Displacement Protein). Cux1 represses the cyclin kinase inhibitors p21 and p27, and transgenic mice ectopically expressing Cux1 develop renal hyperplasia. However, Cux1 transgenic mice do not develop PKD. Here, we show that a 246 amino acid deletion in Cux1 accelerates PKD progression in cpk mice. Cystic kidneys isolated from 10-day-old cpk/Cux1 double mutant mice were significantly larger than kidneys from 10-day-old cpk mice. Moreover, renal function was significantly reduced in the Cux1 mutant cpk mice, compared with cpk mice. The mutant Cux1 protein was ectopically expressed in cyst-lining cells, where expression corresponded to increased cell proliferation and apoptosis, and a decrease in expression of the cyclin kinase inhibitors p27 and p21. While the mutant Cux1 protein altered PKD progression, kidneys from mice carrying the mutant Cux1 protein alone were phenotypically normal, suggesting the Cux1 mutation modifies PKD progression in cpk mice. During cell cycle progression, Cux1 is proteolytically processed by a nuclear isoform of the cysteine protease cathepsin-L. Analysis of the deleted sequences reveals that a cathepsin-L processing site in Cux1 is deleted. Moreover, nuclear cathepsin-L is significantly reduced in both human ADPKD cells and in Pkd1 null kidneys, corresponding to increased levels of Cux1 protein in the cystic cells and kidneys. These results suggest a mechanism in which reduced Cux1 processing by cathepsin-L results in the accumulation of Cux1, downregulation of p21/p27, and increased cell proliferation in PKD.
Assuntos
Proteínas Nucleares/deficiência , Doenças Renais Policísticas/genética , Doenças Renais Policísticas/fisiopatologia , Animais , Catepsina L , Catepsinas/genética , Cruzamentos Genéticos , Cisteína Endopeptidases/genética , Progressão da Doença , Proteínas de Homeodomínio/genética , Humanos , Camundongos , Camundongos Knockout , Mutação , Proteínas Nucleares/genética , Tamanho do Órgão , Doenças Renais Policísticas/patologia , Rim Policístico Autossômico Dominante/genética , Proteínas Repressoras/genética , Deleção de SequênciaRESUMO
Cux-1 is a murine homeobox gene structurally related to Drosophila cut. Cux-1 is highly expressed in the nephrogenic zone of the developing kidney, where its expression coincides with cell proliferation. Cux-1 functions as a transcriptional repressor of the cyclin kinase inhibitors (CKI) p21 and p27. Cux-1 DNA binding activity is negatively regulated by phosphorylation, and dephosphorylation of Cux-1 results in increased DNA binding. Transgenic mice ectopically expressing Cux-1 develop renal hyperplasia associated with the down-regulation of the CKI p27. Calcineurin A (CnA) alpha (-/-) mice display renal hypoplasia associated with the ectopic expression of p27. CnA is a serine/threonine phosphatase activated by intracellular calcium. Inhibiting CnA with cyclosporin A (CsA) leads to nephron deficit in rat metanephric organ cultures and apoptosis in various renal cell lines. To determine whether the ectopic expression of p27 in CnA-alpha -/- kidneys results from the down-regulation of Cux-1, metanephroi from embryonic Cux-1 transgenic and wild-type mice were harvested and cultured with CsA for 5 days. CsA treatment significantly inhibited growth of wild-type metanephroi. In contrast, CsA-treated Cux-1 transgenic kidney cultures were not growth inhibited, but showed high levels of cell proliferation in the nephrogenic zone. Moreover, in CsA-treated Cux-1 transgenic kidney cultures, p27 was not expressed in the nephrogenic zone, but only up-regulated in maturing glomeruli and tubules. Taken together, our results demonstrate that ectopic expression of Cux-1 can rescue the effects of CsA inhibition of CnA and suggest that Cux-1 may be regulated by calcineurin A.
Assuntos
Inibidores de Calcineurina , Proteínas de Homeodomínio/metabolismo , Rim/embriologia , Rim/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Animais , Calcineurina/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Ciclosporina/farmacologia , Feminino , Expressão Gênica , Genes Homeobox , Proteínas de Homeodomínio/genética , Imunossupressores/farmacologia , Rim/enzimologia , Camundongos , Camundongos Transgênicos , Modelos Genéticos , Proteínas Nucleares/genética , Técnicas de Cultura de Órgãos , Gravidez , Proteínas Repressoras/genéticaRESUMO
We examined the expression of Slc12a2 (NKCC1) transcripts in the developing mouse by Northern blot analysis and in situ hybridization (ISH) using riboprobes transcribed from a cDNA encoding the transmembrane domain of human Slc12a2. In developing kidney, the 7.5-kb Slc12a2 transcript was expressed at all stages examined (13.5 d.p.c. to adult) but was more abundant in immature metanephroi. ISH revealed that NKCC1 was expressed in both mesenchymal cells and early nephric structures, but not branching ureteric buds, of developing metanephroi. A marked increase in expression was observed in the endocapillary cells of capillary loop stage glomeruli, and high expression was observed in the glomeruli of more mature nephrons. This was in contrast to Slc12a1 (NKCC2), where expression was excluded from the glomerulus. Extra-renal expression of Slc12a2 was examined in 13.5, 15.5, and 16.5 d.p.c. mouse embryos. Slc12a2 was highly expressed in the developing lung, gut, submandibular gland, tooth bud, and nasal epithelium. Slc12a2 expression was also observed in the developing central and peripheral nervous systems, including choroid plexus and trigeminal and dorsal root ganglia.
Assuntos
Desenvolvimento Embrionário/fisiologia , Expressão Gênica , Rim/embriologia , Organogênese/fisiologia , Simportadores de Cloreto de Sódio-Potássio/metabolismo , Animais , Membrana Basal/metabolismo , Encéfalo/embriologia , Encéfalo/metabolismo , Embrião de Mamíferos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Mucosa Intestinal/metabolismo , Intestinos/embriologia , Rim/metabolismo , Pulmão/embriologia , Pulmão/metabolismo , Camundongos , Membro 2 da Família 12 de Carreador de Soluto , Glândula Submandibular/embriologia , Glândula Submandibular/metabolismo , Distribuição TecidualRESUMO
Cux-1 is a member of a family of homeobox genes structurally related to Drosophila Cut. Mammalian Cut proteins function as transcriptional repressors of genes specifying terminal differentiation in multiple cell lineages. In addition, mammalian Cut proteins serve as cell-cycle-dependent transcriptional factors in proliferating cells, where they function to repress expression of the cyclin kinase inhibitors p21 and p27. Previously we showed that transgenic mice expressing Cux-1 under control of the CMV immediate early gene promoter develop multiorgan hyperplasia. Here we show that mice constitutively expressing Cux-1 exhibit hepatomegaly correlating with an increase in cell proliferation. In addition, the increase in Cux-1 expression in transgenic livers was associated with a decrease in p21, but not p27, expression. Within transgenic livers, Cux-1 was ectopically expressed in a population of small cells, but not in mature hepatocytes, and many of these small cells expressed markers of proliferation. Transgenic livers showed an increase in alpha-smooth muscle actin, indicating activation of hepatic stellate cells, and an increase in cells expressing chromogranin-A, a marker for hepatocyte precursor cells. Morphological analysis of transgenic livers revealed inflammation, hepatocyte swelling, mixed cell foci, and biliary cell hyperplasia. These results suggest that increased expression of Cux-1 may play a role in the activation of hepatic stem cells, possibly through the repression of the cyclin kinase inhibitor p21.