The adhesion protein E-cadherin plays a central part in the process of epithelial morphogenesis. Expression of this protein is downregulated during the acquisition of metastatic potential at late stages of epithelial tumour progression. There is evidence for a transcriptional blockage of E-cadherin gene expression in this process. Here we show that the transcription factor Snail, which is expressed by fibroblasts and some E-cadherin-negative epithelial tumour cell lines, binds to three E-boxes present in the human E-cadherin promoter and represses transcription of E-cadherin. Inhibition of Snail function in epithelial cancer cell lines lacking E-cadherin protein restores the expression of the E-cadherin gene.

The transcription factor snail is a repressor of E-cadherin gene expression in epithelial tumour cells.

β-Catenin plays a dual role in the cell: one in linking the cytoplasmic side of cadherin-mediated cell–cell contacts to the actin cytoskeleton and an additional role in signaling that involves transactivation in complex with transcription factors of the lymphoid enhancing factor (LEF-1) family. Elevated β-catenin levels in colorectal cancer caused by mutations in β-catenin or by the adenomatous polyposis coli molecule, which regulates β-catenin degradation, result in the binding of β-catenin to LEF-1 and increased transcriptional activation of mostly unknown target genes. Here, we show that the cyclin D1 gene is a direct target for transactivation by the β-catenin/LEF-1 pathway through a LEF-1 binding site in the cyclin D1 promoter. Inhibitors of β-catenin activation, wild-type adenomatous polyposis coli, axin, and the cytoplasmic tail of cadherin suppressed cyclin D1 promoter activity in colon cancer cells. Cyclin D1 protein levels were induced by β-catenin overexpression and reduced in cells overexpressing the cadherin cytoplasmic domain. Increased β-catenin levels may thus promote neoplastic conversion by triggering cyclin D1 gene expression and, consequently, uncontrolled progression into the cell cycle.

https://www.pnas.org/content/pnas/96/10/5522.full.pdf

The cyclin D1 gene is a target of the beta-catenin/LEF-1 pathway

Role of transcription factor NF-kappa B/Rel in induction of nitric oxide synthase.

A family of cyclin-dependent kinases (CDKs) cooperatively regulates mammalian cell cycle progression (for review, see Sherr 1993). During G1 phase, D-type cyclins (D1, D2, and D3) are synthesized and assemble with either CDK4 or CDK6 in response to growth factor stimulation, thereby generating active holoenzymes that help inactivate the growth-suppressive function of the retinoblastoma protein (Rb) through its phosphorylation (for review, see Weinberg 1995). Cyclin D holoenzyme complexes also titrate CDK inhibitors, such as p27Kip1 and p21Cip1, facilitating the activation of cyclin E-CDK2 and subsequent entry into the DNA synthetic phase of the cell cycle (for review, see Sherr and Roberts 1995).

Ras-mediated pathways are important for cyclin D1 induction and its assembly with CDKs. Overexpression of activated oncogenic Ras alleles, but not wild-type Ras, initiates DNA synthesis independently of growth factor stimulation (Feramisco et al. 1984). Conversely, microinjection of antibodies that inactivate Ras or introduction of certain dominant-negative Ras alleles can block S-phase entry induced by mitogens (Mulcahy et al. 1985; Mittnacht et al. 1997; Peeper et al. 1997). Both cyclin D1 expression and assembly require the sequential activities of Raf1, mitogen-activated protein kinase-kinases (MEK1 and MEK2), and the sustained activation of extracellular signal-regulated protein kinases (ERKs; Albanese et al. 1995; Lavoie et al. 1996; Winston et al. 1996; Aktas et al. 1997; Kerkhoff and Rapp 1997; Weber et al. 1997; Cheng et al. 1998).

In turn, cyclin D1 degradation is mediated by phosphorylation-triggered, ubiquitin-dependent proteolysis (Diehl et al. 1997). Polyubiquitination of protein substrates involves the sequential action of three distinct enzymes termed E1, E2 (UBC; ubiquitin-conjugating enzyme), and E3 (ubiquitin ligase; Ciechanover 1994; King et al. 1996). Specificity of substrate recognition is dependent on several factors including E2 and E3 selectivity (King et al. 1996; Skowyra et al. 1997; Renny-Feldman et al. 1997), recognition motifs within the target proteins themselves (Glotzer et al. 1991), and, in some cases, a requirement for phosphorylation of specific residues within the substrate (Deshaies et al. 1995; Clurman et al. 1996; Lanker et al. 1996; Won et al. 1996). Ubiquitin-dependent degradation of cyclin D1 requires phosphorylation of a specific threonine residue (Thr-286) located near the protein carboxyl terminus, and this phosphorylation is not mediated by cyclin D-dependent kinases themselves (Diehl et al. 1997). Because the kinase that phosphorylates this residue has not yet been identified, it remains unclear whether cyclin D1 proteolysis, like its synthesis and assembly, is subject to mitogen regulation.

The subcellular distribution of D-type cyclins is also likely to be regulated by cell cycle-dependent events. Cyclin D1 accumulates in the nuclei of cells during G1 phase, but once DNA replication begins, it disappears from the nucleus (Baldin et al. 1993), despite the fact that its level of synthesis does not decrease markedly during S phase (Matsushime et al. 1991). The mechanisms that regulate the periodic subcellular redistribution of cyclin D1 during the cell division cycle have also not been defined.

We now demonstrate that glycogen synthase kinase-3β (GSK-3β) catalyzes the phosphorylation of cyclin D1 on Thr-286, thereby regulating cyclin D1 turnover in response to mitogenic signals. In turn, GSK-3β-mediated phosphorylation of cyclin D1 redirects the protein from the nucleus to the cytoplasm. Our results support a model in which phosphorylation of cyclin D1 on Thr-286 by GSK-3β links processes governing cyclin D1 subcellular localization with its proteasomal degradation.

/pdf/glycogen-synthase-kinase-3b-regulates-cyclin-d1-proteolysis-yriv13q4t5.pdf

Glycogen synthase kinase-3β regulates cyclin D1 proteolysis and subcellular localization

The transcription factor nuclear factor-kappa B (NF-kappa B) is critical for the inducible expression of multiple cellular and viral genes involved in inflammation and infection including interleukin-1 (IL-1), IL-6, and adhesion molecules. The anti-inflammatory drugs sodium salicylate and aspirin inhibited the activation of NF-kappa B, which further explains the mechanism of action of these drugs. This inhibition prevented the degradation of the NF-kappa B inhibitor, I kappa B, and therefore NF-kappa B was retained in the cytosol. Sodium salicylate and aspirin also inhibited NF-kappa B-dependent transcription from the Ig kappa enhancer and the human immunodeficiency virus (HIV) long terminal repeat (LTR) in transfected T cells.

https://science.sciencemag.org/content/sci/265/5174/956.full.pdf

Inhibition of NF-kappa B by sodium salicylate and aspirin

Rapid detection of octamer binding proteins with ‘mini extracts’, prepared from a small number of cells

The octamer sequence ATGCAAAT is found in the promoters of immunoglobulin (Ig) heavy and light chain genes and in the heavy chain enhancer and is a major determinant of the cell type specific expression of Ig genes in B cells. An apparent paradox is that the same sequence serves as an upstream promoter or enhancer element in a variety of housekeeping genes such as the histone H2B and U snRNA genes. The differential usage of this regulatory sequence motif is thought to be mediated by different species of octamer binding proteins. One species of 100 kd, designated OTF-1, is present in all cell types and may exert its activating function only when it can interact with additional adjacent transcription factors. The lymphoid cell specific octamer binding protein of 60 kd (OTF-2A) specifically stimulates Ig promoters which consist essentially of a TATA-box and an octamer sequence upstream of it. Here we present evidence for yet another B cell specific octamer binding protein of 75 kd (OTF-2B). From several findings, including the absence of OTF-2B (but not OTF-2A) from a lymphocyte line that cannot respond to the IgH enhancer, we propose a role of the novel octamer factor in the long range activation by the IgH enhancer. We have used the proteolytic clipping bandshift assay (PCBA) technique to distinguish the three different forms found in B cells. This analysis indicates that the 75 kd-species OTF-2B is closely related to the 60 kd species OTF-2A.

Identification of a novel lymphoid specific octamer binding protein (OTF-2B) by proteolytic clipping bandshift assay (PCBA)

The 'octamer' sequence, ATGCAAAT or its complement ATTTGCAT, is a key element for the transcriptional regulation of immunoglobulin genes in B-lymphocytes as well as a number of housekeeping genes in all cell types. In lymphocytes, the octamer-binding protein Oct-2A and variants thereof are thought to contribute to the B-cell specific gene expression, while the ubiquitous protein Oct-1 seems to control general octamer site-dependent transcription. Various other genes, for example interleukin-1 and MHC class II genes, contain an octamer sequence in the promoter and are expressed in cells of both the immune and nervous systems. This prompted us to analyze the octamer-binding proteins in the latter cells. Using the electrophoretic mobility shift assay, at least six novel octamer binding proteins were detected in nuclear extracts of cultured mouse astrocytes. These proteins are differentially expressed in human glioblastoma and neuroblastoma cell lines. The nervous system-derived (N-Oct) proteins bound to the octamer DNA sequence in a manner which is indistinguishable from the Oct-1 and Oct-2A proteins. The relationship of the N-Oct proteins to Oct-1 and Oct-2A was analyzed by proteolytic clipping bandshift assays and by their reactivity towards antisera raised against recombinant Oct-1 and Oct-2A proteins. On the basis of these assays, all N-Oct-factors were found to be distinct from the ubiquitous Oct-1 and the lymphoid-specific Oct-2A proteins. In melanoma cells that contain the N-Oct-3 factor, a transfected lymphocyte-specific promoter was neither activated nor was it repressed upon contransfection with an Oct-2A expression vector. We therefore speculate that N-Oct-3 and other N-Oct factors have a specific role in gene expression in cells of the nervous system.

Astrocytes and glioblastoma cells express novel octamer-DNA binding proteins distinct from the ubiquitous Oct-1 and B cell type Oct-2 proteins

Eukaryotic expression vectors for the analysis of mutant proteins

All promoters of immunoglobulin heavy chain genes contain three conserved sequence motifs: a heptamer motif CTCATGA, an octamer motif ATGCAAAT, and a TATA box. We show that, despite their different sequences, both the heptamer and the octamer motif are bound by the same octamer transcription factors (Oct factors, also referred to as OTFs), namely the lymphoid-specific proteins Oct-2A and Oct-2B, as well as the ubiquitous protein Oct-1. Even though binding to the octamer motif is stronger, a single heptamer motif can bind Oct proteins and mediate transcriptional activity in lymphoid cells. Furthermore, factor binding to the octamer motif facilitates binding to the nearby heptamer motif. We propose that the heptamer element plays a role early in B-cell differentiation to ensure that the heavy chain promoters are transcriptionally activated before the light chain promoters, which do not contain the heptamer motif.

Edgar Schreiber

Papers

Rapid detection of octamer binding proteins with ‘mini extracts’, prepared from a small number of cells

Identification of a novel lymphoid specific octamer binding protein (OTF-2B) by proteolytic clipping bandshift assay (PCBA)

Astrocytes and glioblastoma cells express novel octamer-DNA binding proteins distinct from the ubiquitous Oct-1 and B cell type Oct-2 proteins

Eukaryotic expression vectors for the analysis of mutant proteins

Octamer transcription factors bind to two different sequence motifs of the immunoglobulin heavy chain promoter.