RESUMO
The RET tyrosine kinase receptor and its ligand, glial cell line-derived neurotrophic factor (GDNF) are critical regulators of renal and neural development. It has been demonstrated that RET activates a variety of downstream signaling cascades, including the RAS/mitogen-activated protein kinase and phosphatidylinositol-3-kinase(PI3-K)/AKT pathways. However, nuclear targets specific to RET-triggered signaling still remain elusive. We have previously identified a novel zinc finger protein, GZF1, whose expression is induced during GDNF/RET signaling and may play a role in renal branching morphogenesis. Here, we report the DNA binding property of GZF1 and its potential target gene. Using the cyclic amplification and selection of targets technique, the consensus DNA sequence to which GZF1 binds was determined. This sequence was found in the 5' regulatory region of the HOXA10 gene. Electrophoretic mobility shift assay revealed that GZF1 specifically binds to the determined consensus sequence and suppresses transcription of the luciferase gene from the HOXA10 gene regulatory element. These findings thus suggest that GZF1 may regulate the spatial and temporal expression of the HOXA10 gene which plays a role in morphogenesis.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Homeodomínio/genética , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Elementos de Resposta , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Sequência Consenso , Análise Mutacional de DNA , Proteínas de Ligação a DNA/química , Proteínas Homeobox A10 , Humanos , Fatores de Transcrição Kruppel-Like , Camundongos , Dados de Sequência Molecular , Proteínas Nucleares/química , Proteínas Repressoras/química , Fatores de Transcrição/química , Dedos de ZincoRESUMO
The glial cell line-derived neurotrophic factor (GDNF)-RET signaling pathway plays an important role in kidney development. We have previously identified a novel zinc finger protein, glial cell line-derived neurotrophic factor-inducible zinc finger protein 1 (GZF1), whose expression was induced in the human neuroblastoma cell line TGW expressing RET by GDNF stimulation and was also detected in mouse metanephric kidney. In the present study, we examined the immunohistochemical expression of GZF1 in normal human kidney and various kidney diseases including chronic kidney disease, acute kidney injury, and cancers, and assessed the clinical significance of GZF1 expression. In the normal kidney, GZF1 was highly expressed only in the proximal tubular epithelial cells that were also positive for angiotensin-converting enzyme. We also evaluated GZF1 expression in various kidney diseases including membranous nephropathy, minimal change nephrotic syndrome with or without acute kidney injury, immunoglobulin A nephropathy, diabetic nephropathy, acute tubular necrosis, and antineutrophil cytoplasmic antibody-related glomerulonephritis. We found that decreased expression of GZF1 was associated with an increase in tubulointerstitial damage and serum creatinine levels. In addition, GZF1 expression was undetectable or very low in most cases of renal cell carcinomas and Wilms tumors. These findings suggest that GZF1 represents a new marker for renal proximal tubules and that there is an inverse correlation between the expression level of GZF1 and tubular function.
Assuntos
Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Nefropatias/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores , Western Blotting , Criança , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Células HeLa , Humanos , Técnicas Imunoenzimáticas , Nefropatias/patologia , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
GZF1 is a zinc finger protein induced by glial cell-line-derived neurotrophic factor (GDNF). It is a sequence-specific transcriptional repressor with a BTB/POZ (Broad complex, Tramtrack, Bric a brac/Poxvirus and zinc finger) domain and ten zinc finger motifs. In the present study, we used immunoprecipitation and mass spectrometry to identify nucleolin as a GZF1-binding protein. Deletion analysis revealed that zinc finger motifs 1-4 of GZF1 mediate its association with nucleolin. When zinc fingers 1-4 were deleted from GZF1 or nucleolin expression was knocked down by short interference RNA (siRNA), nuclear localization of GZF1 was impaired. These results suggest that nucleolin is involved in the proper subcellular distribution of GZF1. In addition, overexpression of nucleolin moderately inhibited the transcriptional repressive activity of GZF1 whereas knockdown of nucleolin expression by siRNA enhanced its activity. Thus, the repressive activity of GZF1 is modulated by the level at which nucleolin is expressed. Finally, we found that knockdown of GZF1 and nucleolin expression markedly impaired cell proliferation. These findings suggest that the physiological functions of GZF1 may be regulated by the protein's association with nucleolin.