Sp1 and Sp3 transcription factors synergistically regulate HGF receptor gene expression in kidney.
ABSTRACT We investigated the expression pattern and underlying mechanism that controls hepatocyte growth factor (HGF) receptor (c-met) expression in normal kidney and a variety of kidney cells. Immunohistochemical staining showed widespread expression of c-met in mouse kidney, a pattern closely correlated with renal expression of Sp1 and Sp3 transcription factors. In vitro, all types of kidney cells tested expressed different levels of c-met, which was tightly proportional to the cellular abundances of Sp1 and Sp3. Both Sp1 and Sp3 bound to the multiple GC boxes in the promoter region of the c-met gene. Coimmunoprecipitation suggested a physical interaction between Sp1 and Sp3. Functionally, Sp1 markedly stimulated c-met promoter activity. Although Sp3 only weakly activated the c-met promoter, its combination with Sp1 synergistically stimulated c-met transcription. Conversely, deprivation of Sp proteins by transfection of decoy Sp1 oligonucleotide or blockade of Sp1 binding with mithramycin A inhibited c-met expression. The c-met receptor in all types of kidney cells was functional and induced protein kinase B/Akt phosphorylation in a distinctly dynamic pattern after HGF stimulation. These results indicate that members of the Sp family of transcription factors play an important role in regulating constitutive expression of the c-met gene in all types of renal cells. Our findings suggest that HGF may have a broader spectrum of target cells and possess wider implications in kidney structure and function than originally thought.
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ABSTRACT: The primary objective of this study is to identify small molecules that target critical transcription factors for potential application in the chemoprevention of esophageal cancer. Specificity proteins (Sp) play a critical role in the growth and metastasis of several malignancies including esophageal cancer. Researchers at the M. D. Anderson Cancer Center Orlando Cancer Research Institute have reported previously that tolfenamic acid (TA) inhibits cancer cell proliferation and tumor growth through the degradation of Sp1, Sp3, and Sp4. We evaluated the chemopreventive properties of TA against esophageal tumorigenesis in N-nitrosomethylbenzylamine (NMBA)-induced murine tumor model. Fischer-344 rats were treated with NMBA (0.5 mg/kg s.c. 3 times a week) for 5 weeks to initiate the tumor formation, and then treated with 50 mg/kg TA from week 6 through week 25. Tumor incidence, tumor multiplicity (number of papilloma per rat), and tumor volume were evaluated after 25 weeks. All rats in the control group that received only NMBA developed lesions (100% incidence), while the TA-treated group showed significantly lower (33%) tumor incidence and tumor multiplicity. Furthermore, the tumor volume was significantly diminished in the TA-treated group when compared with the control group. Using small molecules such as TA to target key transcription factors associated with tumorigenesis for the prevention of esophageal malignancies is a new and promising strategy. Results of the current study provide evidence that TA, when given orally after tumor initiation, can significantly suppress tumorigenesis induced by carcinogenic nitrosamines in rats. These appealing results demonstrate that TA may potentially serve as an effective chemopreventive agent in patient populations vulnerable to esophageal cancer.Investigational New Drugs 01/2011; 30(3):853-61. · 3.50 Impact Factor
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ABSTRACT: rs143383 is a C to T transition SNP located in the 5'untranslated region (5'UTR) of the growth differentiation factor 5 gene GDF5. The T allele of the SNP is associated with increased risk of osteoarthritis (OA) in Europeans and in Asians. This susceptibility is mediated by the T allele producing less GDF5 transcript relative to the C allele, a phenomenon known as differential allelic expression (DAE). The aim of this study was to identify trans-acting factors that bind to rs143383 and which regulate this GDF5 DAE. Protein binding to the gene was investigated by two experimental approaches: 1) competition and supershift electrophoretic mobility shift assays (EMSAs) and 2) an oligonucleotide pull down assay followed by quantitative mass spectrometry. Binding was then confirmed in vivo by chromatin immunoprecipitation (ChIP), and the functional effects of candidate proteins investigated by RNA interference (RNAi) and over expression. Using these approaches the trans-acting factors Sp1, Sp3, P15, and DEAF-1 were identified as interacting with the GDF5 5'UTR. Knockdown and over expression of the factors demonstrated that Sp1, Sp3, and DEAF-1 are repressors of GDF5 expression. Depletion of DEAF-1 modulated the DAE of GDF5 and this differential allelic effect was confirmed following over expression, with the rs143383 T allele being repressed to a significantly greater extent than the rs143383 C allele. In combination, Sp1 and DEAF-1 had the greatest repressive activity. In conclusion, we have identified four trans-acting factors that are binding to GDF5, three of which are modulating GDF5 expression via the OA susceptibility locus rs143383.PLoS Genetics 06/2013; 9(6):e1003557. · 8.52 Impact Factor
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ABSTRACT: Recent observations suggest that p53 mutations are responsible not only for growth of primary tumors but also for their dissemination. However, mechanisms involved in p53-mediated control of cell motility and invasion remain poorly understood. By using the primary ovarian surface epithelium cell culture, we show that conditional inactivation of p53 or expression of its mutant forms results in overexpression of MET receptor tyrosine kinase, a crucial regulator of invasive growth. At the same time, cells acquire increased MET-dependent motility and invasion. Wild-type p53 negatively regulates MET expression by two mechanisms: (i) transactivation of MET-targeting miR-34, and (ii) inhibition of SP1 binding to MET promoter. Both mechanisms are not functional in p53 absence, but mutant p53 proteins retain partial MET promoter suppression. Accordingly, MET overexpression, cell motility, and invasion are particularly high in p53-null cells. These results identify MET as a critical effector of p53 and suggest that inhibition of MET may be an effective antimetastatic approach to treat cancers with p53 mutations. These results also show that the extent of advanced cancer traits, such as invasion, may be determined by alterations in individual components of p53/MET regulatory network.Proceedings of the National Academy of Sciences 08/2011; 108(34):14240-5. · 9.74 Impact Factor