Cytokine-induced expression of SOCS (suppressor of cytokine signalling) molecules is important for the negative regulatory control of STAT (signal transduction and activators of transcription)-dependent cytokine signalling, e.g. for the signal transduction of IL-6 (interleukin-6)-type cytokines through the JAK (Janus kinase)/STAT cascade. STAT activation itself represents an important step in the transcriptional activation of SOCS3 gene expression. However, downstream of the STAT-responsive element, the SOCS3 gene contains a GC-rich element in its 5′-upstream region. The aim of the present study was to investigate the implications of this GC-rich element in the transcriptional control of SOCS3 gene expression. In the present study, we show that mutation of this GC-rich element abolishes IL-6-dependent transcriptional activation of the SOCS3 promoter and that Sp3 (specificity protein 3), a ubiquitously expressed transcription factor, but not Sp1 binds to this GC-rich motif, suggesting that Sp3 is involved in the regulation of SOCS3 expression. The results suggest that Sp3 is important for IL-6-induced transcriptional activation of the SOCS3 (gene) promoter and acts as an enhancer of basal as well as induced transcriptional activity, resulting in enhanced SOCS3 mRNA and protein expression. Mutation of Lys-483, a potential target for Sp3 acetylation, inhibited Sp3-mediated enhancement of SOCS3 mRNA expression and SOCS3 promoter activation, indicating that the acetylation of this lysine residue of Sp3 is important for the enhancing effect of Sp3 on SOCS3 expression.

The DNMT3A (DNA methyltransferase 3A) and DNMT3B genes encode putative de novo methyltransferases and show complex transcriptional regulation in the presence of three and two different promoters respectively. All promoters of DNMT3A and DNMT3B lack typical TATA sequences adjacent to their transcription start sites and contain several Sp1-binding sites. The importance of these Sp1-binding sites was demonstrated by using a GC-rich DNA-binding protein inhibitor, mithramycin A, i.e. on the basis of decrease in the promoter activities and mRNA expression levels of DNMT3A and DNMT3B . Overexpression of Sp1 and Sp3 up-regulated the promoter activities of these two genes. The physical binding of Sp1 and Sp3 to DNMT3A and DNMT3B promoters was confirmed by a gel shift assay. Interestingly, Sp3 overexpression in HEK-293T cells (human embryonic kidney 293T cells) resulted in 3.3- and 4.0-fold increase in DNMT3A and DNMT3B mRNA expression levels respectively by quantitative reverse transcriptase–PCR, whereas Sp1 overexpression did not. Furthermore, an antisense oligonucleotide to Sp3 significantly decreased the mRNA levels of DNMT3A and DNMT3B . These results indicate the functional importance of Sp proteins, particularly Sp3, in the regulation of DNMT3A and DNMT3B gene expression.

20α-Hydroxysteroid dehydrogenase (20α-HSD), which metabolizes progesterone to an inactive steroid in the corpus luteum of mice and rats but not of humans, is thought to play a crucial role in shortening the oestrous cycles in these rodent species. We determined the nucleotide sequence of the 5′-flanking region of the mouse 20α-HSD gene, and examined its promoter activity using a rat luteinized granulosa cell culture. A reporter assay, using reporter constructs of various lengths of the 5′-flanking region, revealed that the region between −83 and 60 bp upstream of the transcription start site was essential for transcriptional activity. Furthermore, mutational analysis demonstrated that a putative Sp1 site in this region was critical to the expression of the reporter gene. Electrophoretic mobility-shift assays showed that the interaction of proteins in a nuclear extract from rat luteinized granulosa cells with this region was inhibited by a competitor having the wild-type Sp1 sequence in its promoter, but not a mutated Sp1 sequence. Supershift analysis confirmed that Sp1 and Sp3 were present in the nuclear extract of these cells, and that these factors bound to the element. Finally, promoter activity was elevated by the co-transfection of an Sp1 expression vector, and, to a lesser extent, by an Sp3 expression vector, supporting further the involvement of these factors in the expression of the 20α-HSD gene.

The transition of normally quiescent glomerular MCs (mesangial cells) to a highly proliferative phenotype with characteristics of myofibroblasts is a process commonly observed in inflammatory diseases affecting the renal glomerulus, the ultimate result of which is glomerulosclerosis. Generation of proteolytically active MMP (matrix metalloproteinase)-2 by the membrane-associated membrane type 1 (MT1)-MMP is responsible for the transition of mesangial cells to the myofibroblast phenotype [Turck, Pollock, Lee, Marti and Lovett (1996) J. Biol. Chem. 271 , 15074–15083]. In the present study, we show that the expression of MT1-MMP within the context of MCs is mediated by three discrete cis -acting elements: a proximal non-canonical Sp1 site that preferentially binds Sp1; an overlapping Sp1/Egr-1-binding site that preferentially binds Egr-1; and a more distal binding site for the NFAT (nuclear factor of activated T cells) that binds the NFAT c1 isoform present in MC nuclear extracts. Transfection with an NFAT c1 expression plasmid, or activation of calcineurin with a calcium ionophore, yielded major increases in NFAT c1 nuclear DNA-binding activity, MT1-MMP transcription and protein synthesis, which were additive with the lower levels of transactivation provided by the proximal Sp1 and the overlapping Sp1/Egr-1 sites. Specific binding of NFAT c1 to the MT1-MMP promoter was confirmed by chromatin immunoprecipitation studies, while MT1-MMP expression was suppressed by treatment with the calcineurin inhibitor, cyclosporin A. These studies are the first demonstration that a specific NFAT isoform enhances transcription of an MMP (MT1-MMP) that plays a major role in the proteolytic events that are a dominant feature of acute glomerular inflammation. Suppression of MT1-MMP by commonly used calcineurin inhibitors may play a role in the development of renal fibrosis following renal transplantation.

Mitogen-activated protein kinases (MAPKs) are inactivated by a dual specificity phosphatase, MAPK phosphatase-1 (MKP-1). MKP-1 is transcribed as an immediate early response gene (IEG) following various stimuli. In the pituitary cell line GH4C1, MKP-1 gene transcription is strongly induced by thyrotropin-releasing hormone (TRH) as well as by epidermal growth factor (EGF) as a consequence of activated MAPK/extracellular-signal-regulated kinase (ERK) signalling. Intriguingly, reporter gene analysis with the MKP-1 promoter showed strong basal transcription, but only limited induction by TRH and EGF. Site-directed mutagenesis of the reporter construct combined with band-shift and in vivo studies revealed that part of the constitutive activity of the MKP-1 promoter resides in two GC boxes bound by Sp1 and Sp3 transcription factors in the minimal promoter. Basal transcription of transiently transfected luciferase reporter can be initiated by either of the two GC boxes or also by either of the two cAMP/Ca 2+ responsive elements or by the E-box present in the proximal promoter. On the other hand, when analysed by stable transfection, the five responsive elements are acting in synergy to transactivate the MKP-1 proximal promoter. We show in this study that the MKP-1 promoter can function as a constitutive promoter or as a rapid and transient sensor for the activation state of MAPKs/ERKs. This dual mode of transcription initiation may have different consequences for the control of a block to elongation situated in the first exon of the MKP-1 gene, as described previously [Ryser, Tortola, van Haasteren, Muda, Li and Schlegel (2001) J. Biol. Chem. 276 , 33319–33327].

The activator protein-2 (AP-2) family of DNA-binding transcription factors are developmentally regulated and also play a role in human neoplasia. In particular, the AP-2γ protein has been shown to be overexpressed in a high percentage of breast tumours. In the present study, we report the complete sequence determination of the human TFAP2C gene encoding the AP-2γ transcription factor plus the mapping of the transcription start site used in breast tumour-derived cells. The 5′-end of the gene lies within a CpG island and transcription is initiated at a single site within a classical initiator motif. We have gone on to investigate why some breast tumour-derived cell lines readily express AP-2γ, whereas others do not, and show that the proximal promoter (+191 to −312) is differentially active in the two cell phenotypes. DNase footprinting led to the identification of three Sp1/Sp3-binding sites within this region, two of which are absolutely required both for promoter function and cell-type-specific activity. By Western blotting a panel of expressing and non-expressing breast tumour lines we show that the latter have higher levels of Sp3. Furthermore, increasing Sp3 levels in AP-2γ-expressing cells led to the repression of AP-2γ promoter activity, particularly when Sp3 inhibitory function was maximized through sumoylation. We propose that differences in the level and activity of Sp3 between breast tumour lines can determine the expression level of their AP-2γ gene.

We analysed in detail the minimal promoter of transcription factor Sp1, which extends 217bp from the initiation of transcription. Within this sequence we identified putative binding sites for Sp1, nuclear factor Y (NF-Y), activator protein 2 ('AP-2'), CCAAT/enhancer-binding protein ('C/EBP') and E2F transcription factors. In one case, the boxes for Sp1 and NF-Y are overlapping. Gel-shift and supershift assays demonstrated specific binding of Sp1, Sp3 and NF-Y proteins. Transient transfections and luciferase assays revealed activation of the Sp1 minimal promoter upon overexpression of Sp1 itself, NF-Y and E2F. Whereas overexpression of NF-Y or E2F had an additive effect on Sp1 overexpression, the activation of Sp1 transcription due to Sp1 was counteracted by Sp3 overexpression. Mutagenesis analysis of the NFY/Sp1-overlapping box revealed that both factors compete for this box, and that when the NF-Y site of this overlapping box is specifically mutated there is an increase in Sp1 binding, thus increasing transcriptional activity. These results help to explain the complex regulation of the Sp1 gene, which depends on the relative amounts of Sp1, Sp3, E2F and NF-Y proteins in the cell.

MN/CA IX (MN) is a tumour-associated isoenzyme of the carbonic anhydrase family. Previous deletion analysis of the MN promoter established that protected regions (PRs) 1 and 2 are crucial for its transcriptional activity. Computer-assisted searching indicated putative binding sites for activator protein (AP) 2 and specificity protein (SP) 1 transcription factors, plus a CACCC box in PR1 and an AP1 site in PR2. PR1 produced four complexes in electrophoretic mobility-shift assay (EMSA) with HeLa nuclear extracts. Of these, three were completely competed with the SP1 and transforming growth factor-β retinoblastoma control-element CACCC box (RCE) probes, whereas the AP2 probe competed against the same three complexes partially. Supershift EMSA identified SP1 in the complex 1 and SP3 in the complexes 2 and 4. Point mutations in the SP1 site abrogated the PR1 function, while mutations affecting the overlapping CACCC box/AP2 site in PR1had minor effect on MN promoter activity. Block-replaced MN promoter mutants that had a consensus binding site (SP1 or AP2) or the RCE in place of PR1 demonstrated the stringent selectivity of the PR1 position as only the SP1 mutant reconstituted the MN promoter activity. The consensus SP1 probe generated the same SP1 and SP3 complexes as PR1 in EMSA; therefore we conclude that SP activity is both necessary and sufficient in the PR1 position. The critical role of AP1 in the PR2 position was confirmed by supershift of the PR2 complex with c-Fos antibody and markedly decreased activity of the construct with a mutated AP1 site. Detailed deletion analysis proved that PR1+PR2 account for 90% of the MN promoter activity, while neither PR1 nor PR2 on their own are sufficient for transactivation. Thus, synergistic co-operation between SP and AP1 factors bound to the adjacent PR1 and PR2, respectively, is necessary for MN transcriptional activity. The PR1+PR2 module also stimulated transcription from a heterologous promoter. The modulation of AP1 activity with PMA stimulated MN expression and activated the MN promoter, whereas inhibition of protein kinase C activity had no effect on MN expression in HeLa cells.