Activation of the wt1 Wilms' tumor suppressor gene by NF-kappaB.

The Wilm's tumor suppressor gene, wt1, is expressed in a very defined spatial-temporal fashion and plays a key role in development of the urogenital system. Transacting factors governing wt1 expression are poorly defined. The presence of putative kappa-B binding sites within the wt1 gene prompted us to investigate whether members of the NF-kappaB/Rel family of transcription factors are involved in regulating wt1 expression. In transient transfection assays, ectopic expression of p50 and p65 subunits of NF-kappaB stimulated wt1 promoter activity 10-30-fold. Deletion mutagenesis revealed that NF-kappa-B responsiveness is mediated by a short DNA fragment located within promoter proximal sequences of the major transcription start site. Two kappaB-binding sites are present in this region and form specific complexes with purified NF-kappaB proteins, as revealed by electrophoretic mobility gel shift assays. Ectopic expression of p50 and p65 resulted in increased transcription of the endogenous wt1 gene, as revealed by nuclear run-on experiments. Taken together, these results indicate that members of the NF-kappaB/Rel family are important for activating expression of wt1 and reside upstream of the regulatory cascade leading to wt1 activation.

Overlapping DNA recognition motifs between Sp1 and a novel trans-acting factor within the wt1 tumour suppressor gene promoter.

The Wilms' tumor suppressor gene, wt1 , encodes a zinc finger transcription factor which has been shown to regulate the expression of several genes involved in cellular proliferation and differentiation. Expression of wt1 is developmentally regulated and restricted to a small set of tissues which include the fetal urogenital system, mesothelium and spleen. A highly conserved motif within the wt1 promoter, located between nucleotides -34 and -71 relative to the first transcription start site in the murine promoter, harbors consensus binding sites for Sp1 and members of the paired-box transcription factor family. Pax-2 and Pax-8 are known to enhance expression of wt1 through this conserved regulatory element. In this report, we demonstrate that Sp1 is able to bind to two sites within the 38 bp conserved region (CR). By electrophoretic mobility shift assays (EMSAs), we have identified a novel binding activity, referred to as complex D, which recognizes sequences overlapping one of the Sp1 sites in the CR. EMSA competition experiments indicate that binding of complex D and Sp1 to the CR is mutually exclusive and Sp1 is able to displace complex D binding. In situ UV crosslinking and molecular mass determinations indicate that complex D is a complex of approximately 130 kDa, consisting of at least two proteins of approximately 62 and approximately 70 kDa. Transient transfections suggest that complex D may function as an activator.

Multiple signaling pathways control the activation of the CEF-4/9E3 cytokine gene by pp60v-src.

The CEF-4/9E3 cytokine gene is expressed aberrantly in chicken embryo fibroblasts (CEF) transformed by the Rous sarcoma virus. The expression of CEF-4 is dependent on both transcriptional and post-transcriptional mechanisms of regulation. The characterization of the promoter region indicated that three distinct regulatory elements corresponding to an AP-1 binding site (or TRE), a PRDII/kappaB domain, and a CAAT box are involved in the activation by pp60(v-)src. In this report we investigate the signaling pathways controlling the expression of the TRE and PRDII domain. The expression of a dominant negative mutant of p21(ras) reduced the activity of both elements. In contrast a similar mutant of c-Raf-1 affected modestly the activation dependent on the TRE but not PRDII. The stress-activated protein kinase (SAPK)/Jun N-terminal kinase (JNK) pathway was important for the activity of PRDII and the TRE but was not markedly stimulated by pp60(v-)src. The addition of calphostin C and the inhibition of protein kinase C (PKC) diminished the accumulation of the CEF-4 mRNA and reduced the activity of a TRE-controlled promoter. Likewise, the depletion of PKC by chronic treatment with phorbol esters inhibited the activation of the TRE. Rous sarcoma virus-transformed CEF treated with calphostin C were also flatter, did not display a high degree of criss-crossing, and appeared morphologically normal. Hence PKC was important for the activation of AP-1 and the morphological transformation of CEF. The constitutive expression of CEF-4 was correlated with transformation only when dependent on the TRE. This was not true for PRDII, which was the only element required for the constitutive activation to the CEF-4 promoter in nontransformed cells treated chronically with phorbol esters.

PAX8-mediated activation of the wt1 tumor suppressor gene.

The developing renal system has long been exploited to study the regulation of gene expression during mesenchymal-epithelial transitions. Several transcription factors, including WT1 and PAX8, are expressed early in nephrogenesis and play a key role in this process. The expression of PAX8 occurs in the induced mesenchyme of the developing kidney prior to the upregulation of WT1 levels in the same cells. In this report, we assessed whether the Pax-8 gene product resides upstream of wt1 in a common regulatory pathway. Transfection studies, as well as gel-shift assays, indicate that PAX8 transactivates wt1 through elements within a 38 bp conserved motif, present in human and murine promoters. Two PAX8 isoforms, generated by alternative splicing at the C-terminus and previously thought to lack transactivation potential, were found to be capable of activating wt1 expression. We also demonstrate that the endogenous wt1 promoter can be upregulated by exogenously supplied PAX8, suggesting that a function of PAX8 during mesenchymal--epithelial cell transition in renal development is to induce wt1 gene expression.

The paired-box transcription factor, PAX2, positively modulates expression of the Wilms' tumor suppressor gene (WT1).

The Wilms' tumor suppressor gene, wt1, encodes a zinc finger protein which functions as a transcriptional regulator. Expression of the wt1 gene is developmentally regulated and restricted to a small set of tissues which include the fetal urogenital system, mesothelium, and spleen. In the developing kidney, induction of neprohogenesis by the ureter is accompanied by an increase in expression levels of the Pax-2 gene, a developmentally and spatially regulated paired-box member. This is followed by an increase in wt1 expression as mesenchymal cells condense and differentiate. In this report, we demonstrate that PAX2 isoforms are capable of transactivating the wt1 promoter. Deletion mutagenesis of the wt1 promoter identified an element responsible for mediating PAX2 responsiveness, located between nucleotides -33 and -71 relative to the first wt1 transcription start site. Consistent with its identity as a PAX responsive element, multimerization of this mofit upstream of a heterologous minimal promoter enhanced reporter activity when co-transfected with a Pax-2 expression vector. Finally, we demonstrate that PAX2 can stimulate expression of the endogenous wt1 gene. These results suggest that a role for PAX2 during mesenchyme-to-epithelium transition in renal development is to induce wt1 expression.

Repression of the retinoic acid receptor-alpha gene by the Wilms' tumor suppressor gene product, wt1.

The Wilms' tumor (WT) suppressor gene, WT1, encodes a zinc finger DNA binding protein (wt1) which functions as a transcriptional regulator. Germline WT1 mutations predispose to WTs and in many cases are associated with urogenital anomalies. Identification of wt1 downstream targets is essential to understanding regulatory processes involved in development of this system. In this study, we demonstrate that wt1 can repress transcription of the retinoic acid receptor-alpha 1 (RAR-alpha 1) promoter. Transient transfection, deletion mutagenesis, and mobility shift assays suggest that wt1 mediates repression of the human RAR-alpha 1 promoter through a GC-rich DNA binding motif (5'-GCGGGGGCG-3'), at positions -111 to -120 bp (relative to the transcription initiation site). In contrast, the murine RAR-alpha 1 promoter contains a cryptic binding motif and is not responsive to wt1. These results indicate that some wt1-regulatory pathways are not conserved across species, suggesting a molecular basis for differences in phenotypes between humans and mice harboring WT1 lesions.

Biosynthesis and polarized distribution of neutral endopeptidase in primary cultures of kidney proximal tubule cells.

When cultured in defined medium, kidney proximal convoluted tubule (PCT) cells form a homogeneous population and retain a number of differentiated functions. To characterize this cell system further as a functional model of epithelial polarity, we investigated the biogenic pathway of neutral endopeptidase (NEP), one of the most abundant microvillar membrane proteins in intestinal and kidney cells. We showed that, in contrast with some tumoral cell lines, RNA extracted from PCT cells shows the presence of a single mRNA species encoding NEP. Pulse-chase studies followed by selective immunoprecipitation of NEP molecules present either at the cell surface or in intracellular cell compartments showed that newly synthesized NEP molecules reached the cell surface as early as 30 min after the beginning of the chase with maximum cell surface expression at 60 min. When grown on semipermeable supports, PCT cells were found to target NEP exclusively to the apical plasma membrane. Similar results have been described using MDCK cells to study targeting of recombinant NEP. Thus primary cultures of PCT cells represent a new model with which to investigate the biogenic pathway of endogenous proteins in native epithelial cells.

Activation of human immunodeficiency virus 1 gene expression by the src oncoprotein.

Several genes are induced constitutively in cells transformed by the v-src oncoprotein. This induction is generally dependent on the activation of transcription factors binding to src-responsive elements of the promoter. In previous studies, we showed that the induction of the CEF-4/9E3 cytokine gene by pp60v-src is dependent on the PRDII/kappa B domain of the promoter (Dehbi et al., 1992). In this investigation, we describe the activation of the HIV-1 LTR by pp60v-src and show that a region of 30 bp containing the two NF-kappa B binding sites is critical for activation of the promoter. The induction was dependent on transformation since non-transforming forms of pp60v-src had little or no effect on the promoter. The expression of proviral DNA and the release of p24 antigen were also increased by v-src indicating that viral replication was stimulated in src-transformed cells. The effect of v-src on HIV-1 gene expression occurred in the presence or in the absence of the tat viral trans-activator, in fibroblasts and in Jurkat T lymphocytes. These results indicate that several promoters controlled by PRDII/kappa B may be activated constitutively in v-src transformed cells and suggest that oncogenic tyrosine kinases may play a role in the induction of viruses with a PRDII/kappa B-controlled promoter.

Regulation of gene expression in oncogenically transformed cells.

Several genes expressed in response to growth factors are also regulated aberrantly in oncogenically transformed cells. The constitutive expression of genes encoding extracellular proteases, transcription factors, and cytokines is often correlated with cell transformation. In several instances, the uncontrolled expression of these genes is the result of transcriptional activation. Therefore, much attention has been devoted to the study of promoter function in transformed cells. We now review the results of recent investigations on transformation-dependent gene expression. The activation of several transcription factors in oncogenically-transformed cells is described. Results regarding the regulation of promoters through PRD II/kappa B are presented for cells transformed by a variety of oncogenes. Finally, we discuss the significance of transcription factor activation in the process of cell transformation.

Transcriptional activation of the CEF-4/9E3 cytokine gene by pp60v-src.

The CEF-4/9E3 gene is expressed constitutively in Rous sarcoma virus (RSV)-transformed cells. This expression is largely determined by an increase in transcription of the gene. In this report, we characterize the regulatory elements responsible for the transformation-dependent activation of CEF-4/9E3. Three sequences corresponding to AP-1, PRD II/kappa B, and TAACGCAATT are involved in the process and therefore define the src-responsive unit (SRU) of the CEF-4 promoter. In constructs containing a deletion of the SRU, multiple copies of AP-1 or PRD II/kappa B, but not TAACGCAATT, led to activation of the promoter. Thus, factors interacting with these elements are constitutively activated in RSV-transformed chicken embryo fibroblasts. In agreement with the results of transient expression assays, protein binding to AP-1, PRD II/kappa B, and TAACGCAATT were more abundant in the nuclei of transformed cells. The expression of the CEF-4 promoter was investigated in cells infected by a temperature-sensitive mutant of RSV. No significant increase in CEF-4 promoter activity was detected early after activation of pp60v-src. In contrast, a substantial activation of the CEF-4 promoter was detected late after a temperature shift. Factors interacting with the TAACGCAATT, PRD II/kappa B, and AP-1 elements accumulated gradually over a period of several hours. Therefore, transcriptional activation plays an important role in the late, constitutive expression of the CEF-4 gene in stably transformed cells.

The stringent response blocks DNA replication outside the ori region in Bacillus subtilis and at the origin in Escherichia coli.

When the Bacillus subtilis dnaB37 mutant, defective in initiation, is returned to permissive temperature after growth at 45 degrees C, DNA replication is synchronized. Under these conditions, we have shown previously that DNA replication is inhibited when the Stringent Response is induced by the amino acid analogue, arginine hydroxamate. We have now shown, using DNA-DNA hybridization analysis, that substantial replication of the oriC region nevertheless occurs during the Stringent Response, and that replication inhibition is therefore implemented downstream from the origin. On the left arm, replication continues for at least 190 x 10(3) base-pairs to the gnt gene and for a similar distance on the right arm to the gerD gene. When the Stringent Response is lifted, DNA replication resumed downstream from oriC on both arms, confirming that DNA replication is regulated at a post-initiation level during the Stringent Response in B. subtilis. Resumption of DNA synthesis following the lifting of the Stringent Response did not require protein or RNA synthesis or the initiation protein DnaB. We suggest, therefore, that a specific control region, involving Stringent Control sites, facilitate reversible inhibition of fork movement downstream from the origin via modifications of a replisome component during the Stringent Response. In contrast, in Escherichia coli, induction of the Stringent Response appears to block initiation of DNA replication at oriC itself. No DNA synthesis was detected in the oriC region and, upon lifting the Stringent Response, replication occurred from oriC. Post-initiation control in B. subtilis therefore results in duplication of many key genes involved in growth and sporulation. We discuss the possibility that such a control might be linked to differentiation in this organism.