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: Sp1 and Sp3 are ubiquitously expressed mammalian transcription factors that activate or repress the expression of a variety of genes and are thought to compete for the same DNA binding site. We used indirect immunofluorescence microscopy and image deconvolution to show that Sp1 and Sp3 are organized into distinct nonoverlapping domains in human breast and ovarian cells. Domains of Sp1 and Sp3 infrequently associate with sites of transcription. Sp3 partitions with the tightly bound nuclear protein fraction of hormone responsive MCF-7 breast cancer cells, whereas only a subpopulation of Sp1 is found in that fraction. Both Sp1 and Sp3 are bound to the nuclear matrix, and the nuclear matrix-associated sites of Sp1 and Sp3 are different. Indirect immunofluorescence studies demonstrate that Sp1 and Sp3 associate with histone deacetylases 1 and 2 and with the estrogen receptor alpha, albeit at low frequencies in MCF-7 cells. Chromatin immunoprecipitation (ChIP) and re-ChIP assays revealed that although both Sp1 and Sp3 bind to the estrogen-responsive trefoil factor 1 promoter in MCF-7 cells, they do not occupy the same promoter. Our results demonstrate the different features of Sp1 and Sp3, providing further evidence that Sp3 is not a functional equivalent of Sp1.Molecular Biology of the Cell 10/2005; 16(9):4073-83. DOI:10.1091/mbc.E05-05-0388 · 4.55 Impact Factor
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ABSTRACT: Regulation of the basal manganese superoxide dismutase (SOD2) promoter depends on the transcriptional activity of the Sp family of transcription factors. Here we report that reduced expression in the presence of Tat is independent of induction with Tumor necrosis factor alpha and that Tat affects the interaction of Sp1 and Sp3 with the basal promoter. Footprinting and electrophoretic mobility shift assay (EMSA) analyses with extracts from HeLa cells showed that Sp1/Sp3 complexes populate the proximal SOD2 promoter, and that Tat leads to an increase in the binding activity of Sp3. In Drosophila S2 cells, both Sp1 and Sp3 activated the basal SOD2 promoter (88.1 +/- 39.4 fold vs. 10.3 +/- 3.5 fold, respectively), demonstrating a positive, yet lower transcriptional regulatory function for Sp3. Additionally, the inability of Sp3 to synergistically affect promoter activity indicates an efficient competition of Sp3 with Sp1 for the multiple Sp binding sites in the SOD2 basal promoter. Tat potentiated both Sp1 and Sp3 activation of the promoter in S2 cells, though the activity of Sp3 was still lower than that of Sp1. Thus, the consequence of a shift by Tat to increased Sp3-containing complexes on the basal SOD2 promoter is decreased SOD2 expression. Together, our studies demonstrate the functional importance of the interaction of Sp1, Sp3, and Tat, revealing a possible mechanism for the attenuation of basal manganese superoxide dismutase expression.Free Radical Biology and Medicine 10/2004; 37(6):869-80. DOI:10.1016/j.freeradbiomed.2004.06.016 · 5.71 Impact Factor