Aneta Kasza

Interleukin-1-inducible MCPIP protein has structural and functional properties of RNase and participates in degradation of IL-1β mRNA

In human monocyte‐derived macrophages, the MCPIP gene (monocyte chemoattractant protein‐induced protein) is strongly activated by interleukin‐1β (IL‐1β). Using bioinformatics, a PIN domain was identified, spanning amino acids 130‐280; such domains are known to possess structural features of RNases. Recently, RNase properties of MCPIP were confirmed on transcripts coding for interleukins IL‐6 and IL‐12p40. Here we present evidence that siRNA‐mediated inhibition of the MCPIP gene expression increases the level of the IL‐1β transcript in cells stimulated with LPS, whereas overexpression of MCPIP exerts opposite effects. Cells with an increased level of wild‐type MCPIP showed lower levels of IL‐1β mRNA. However, this was not observed when mutant forms of MCPIP, either entirely lacking the PIN domain or with point mutations in this domain, were used. The results of experiments with actinomycin D indicate that lower levels of IL‐1β mRNA are due to shortening of the IL‐1β transcript half‐life, and are not related to the presence of AU‐rich elements in the 3′ UTR. The interaction of the MCPIP with transcripts of both IL‐1β and MCPIP observed in an RNA immunoprecipitation assay suggests that this novel RNase may be involved in the regulation of expression of several genes.

Ternary complex factor-serum response factor complex-regulated gene activity is required for cellular proliferation and inhibition of apoptotic cell death.

ABSTRACT Members of the ternary complex factor (TCF) subfamily of the ETS-domain transcription factors are activated through phosphorylation by mitogen-activated protein kinases (MAPKs) in response to a variety of mitogenic and stress stimuli. The TCFs bind and activate serum response elements (SREs) in the promoters of target genes in a ternary complex with a second transcription factor, serum response factor (SRF). The association of TCFs with SREs within immediate-early gene promoters is suggestive of a role for the ternary TCF-SRF complex in promoting cell cycle entry and proliferation in response to mitogenic signaling. Here we have investigated the downstream gene regulatory and phenotypic effects of inhibiting the activity of genes regulated by TCFs by expressing a dominantly acting repressive form of the TCF, Elk-1. Inhibition of ternary complex activity leads to the downregulation of several immediate-early genes. Furthermore, blocking TCF-mediated gene expression leads to growth arrest and triggers apoptosis. By using mutant Elk-1 alleles, we demonstrated that these effects are via an SRF-dependent mechanism. The antiapoptotic gene Mcl-1 is identified as a key target for the TCF-SRF complex in this system. Thus, our data confirm a role for TCF-SRF-regulated gene activity in regulating proliferation and provide further evidence to indicate a role in protecting cells from apoptotic cell death.

Transcription factors Elk-1 and SRF are engaged in IL1-dependent regulation of ZC3H12A expression

BackgroundMCPIP is a novel CCCH zinc finger protein described as an RNase engaged in the regulation of immune responses. The regulation of expression of the gene coding for MCPIP - ZC3H12A is poorly explored.ResultsHere we report that the proinflammatory cytokine IL-1β rapidly induces the synthesis of MCPIP in primary monocyte-derived macrophages and HepG2 cells. This up-regulation takes place through the MAP kinase pathway and following activation of the transcription factor Elk-1. Using a ZC3H12A reporter construct we have shown that a ZC3H12A promoter region, stretching from -76 to +60, mediates activation by IL-1β. This region contains binding sites for Elk-1 and its partner SRF. Chromatin immunoprecipitation analysis confirms in vivo binding of both transcription factors to this region of the ZC3H12A promoter.ConclusionsWe conclude that the transcription factor Elk-1 plays an important role in the activation of ZC3H12A expression in response to IL-1β stimulation.

A Novel Mechanism of Tissue Inhibitor of Metalloproteinases-1 Activation by Interleukin-1 in Primary Human Astrocytes

Reactive astrogliosis is the gliotic response to brain injury with activated astrocytes and microglia being the major effector cells. These cells secrete inflammatory cytokines, proteinases, and proteinase inhibitors that influence extracellular matrix (ECM) remodeling. In astrocytes, the expression of tissue inhibitor of metalloproteinases-1 (TIMP-1) is up-regulated by interleukin-1 (IL-1), which is a major neuroinflammatory cytokine. We report that IL-1 activates TIMP-1 expression via both the IKK/NF-κB and MEK3/6/p38/ATF-2 pathways in astrocytes. The activation of the TIMP-1 gene can be blocked by using pharmacological inhibitors, including BAY11-7082 and SB202190, overexpression of the dominant-negative inhibitor of NF-κB (IκBαSR), or by the knock-down of p65 subunit of NF-κB. Binding of activated NF-κB (p50/p65 heterodimer) and ATF-2 (homodimer) to two novel regulatory elements located –2.7 and –2.2 kb upstream of the TIMP-1 transcription start site, respectively, is required for full IL-1-responsiveness. Mutational analysis of these regulatory elements and their weak activity when linked to the minimal tk promoter suggest that cooperative binding is required to activate transcription. In contrast to astrocytes, we observed that TIMP-1 is expressed at lower levels in gliomas and is not regulated by IL-1. We provide evidence that the lack of TIMP-1 activation in gliomas results from either dysfunctional IKK/NF-κB or MEK3/6/p38/ATF-2 activation by IL-1. In summary, we propose a novel mechanism of TIMP-1 regulation, which ensures an increased supply of the inhibitor after brain injury, and limits ECM degradation. This mechanism does not function in gliomas, and may in part explain the increased invasiveness of glioma cells.

The ETS Domain Transcription Factor Elk-1 Regulates the Expression of Its Partner Protein, SRF

The ternary complex factors (TCF) are a subfamily of ETS domain transcription factors that bind and activate serum response elements (SREs) in the promoters of target genes in a ternary complex with a second transcription factor, serum response factor (SRF). Here, we have identified the SRF gene as a target for the TCFs, thereby providing a positive feedback loop whereby TCF activation leads to the enhancement of the expression of its partner protein SRF. The binding of the TCF Elk-1 to the SRF promoter and subsequent regulation of SRF expression occurs in a ternary complex-dependent manner. Our data therefore reveal that SRF is an important target for the ERK and Rho signaling pathways that converge on a ternary TCF-SRF complex at the SRE on the SRF promoter.

Overlapping promoter targeting by Elk-1 and other divergent ETS-domain transcription factor family members

ETS-domain transcription factors play important roles in controlling gene expression in a variety of different contexts; however, these proteins bind to very similar sites and it is unclear how in vivo specificity is achieved. In silico analysis is unlikely to reveal specific targets for individual family members and direct experimental approaches are therefore required. Here, we take advantage of an inducible dominant-negative expression system to identify a group of novel target genes for the ETS-domain transcription factor Elk-1. Elk-1 is thought to mainly function through cooperation with a second transcription factor SRF, but the targets we identify are largely SRF-independent. Furthermore, we demonstrate that there is a high degree of overlapping, cell type-specific, target gene binding by Elk-1 and other ETS-domain transcription factors. Our results are therefore consistent with the notion that there is a high degree of functional redundancy in target gene regulation by ETS-domain transcription factors in addition to the specific target gene regulation that can be dictated through heterotypic interactions exemplified by the Elk-1-SRF complex.

IL-1 and EGF regulate expression of genes important in inflammation and cancer

This review focuses on the mechanisms by which the expression of specific genes is regulated by two proteins that are important in inflammation and cancer, namely the pro-inflammatory cytokine interleukin (IL)-1β and epidermal growth factor (EGF). In the review the receptors that recognize factors that cause inflammation are described with main focus on the receptors associated with activation of IL-1β. The function of IL-1β and pathways leading to activation of transcription factors, particularly NFκB and Elk-1 are analyzed. Then the mechanisms of EGF action, with particular emphasis of the activation of Elk-1 are illustrated. The link between aberrant signaling of EGF receptor family members and cancer development is explained. The relationship between inflammation and tumorigenesis is discussed.

Signal-dependent Elk-1 target genes involved in transcript processing and cell migration

Elk-1 was regarded as a transcription factor engaged mainly in the regulation of cell growth, differentiation, and survival. Recent findings show the engagement of Elk-1 in the control of expression of genes encoding proteins involved in transcript turnover, such as MCPIP1/ZC3H12A and tristetraprolin (TTP/ZFP36). Thus, Elk-1 plays an important role in the control of gene expression not only through the stimulation of expression of transcription factors, but also through regulation of transcript half-live. Moreover, Elk-1 is engaged in the regulation of expression of genes encoding proteins that control proteolytic activity, such as inhibitor of plasminogen activator-1 (PAI-1) and metalloproteinases-2 and -9 (MMP-2 and MMP-9). This review summarizes the biological roles of proteins with expression regulated by Elk-1, involved in transcripts turnover or in cell migration. The broad range of function of these proteins illustrates the complex role of Elk-1 in the regulation of cancer and inflammation.

Simultaneous detection of mRNA and protein in single cells using immunofluorescence-combined single-molecule RNA FISH

Although the concept of combining immunofluorescence (IF) with single-molecule RNA fluorescence in situ hybridization (smRNA FISH) seems obvious, the specific materials used during IF and smRNA FISH make it difficult to perform these procedures simultaneously on the same specimen. Even though there are reports where IF and smRNA FISH were combined with success, these were insufficient in terms of signal intensities, staining patterns, and GFP-compatibility, and a detailed exploration of the various factors that influence IF and smRNA FISH outcome has not been published yet. Here, we report a detailed study of conditions and reagents used in classic IF and smRNA FISH that allowed us to establish an easy, robust, and GFP-compatible procedure. Our protocol enables simultaneous detection of mRNA and protein quantity as well as the subcellular distribution of these molecules in single cells by combining an RNase-free modification of the IF technique and the more recent smRNA FISH method. Using this procedure, we have shown the direct interaction of RNase MCPIP1 with IL-6 mRNA. We also demonstrate the use of our protocol in heterogeneous cell population analysis, revealing cell-to-cell differences in mRNA and protein content.

Epidermal growth factor regulates PAI-1 expression via activation of the transcription factor Elk-1

PAI-1 (plasminogen activator inhibitor-1) in breast cancer cells is involved in tumour development and metastasis of breast cancer cells. PAI-1 function and the regulation of its expression have been precisely investigated. Here we report that EGF, which promotes breast cancer tumour growth and survival, rapidly induces PAI-1 expression in the breast adenocarcinoma cell line MCF-7 through the activation of the transcription factor Elk-1. We have found that the PAI-1 promoter fragment (-140 to +173) containing the Ets consensus binding site is activated by Elk-1. Chromatin immunoprecipitation analysis confirms in vivo binding of Elk-1 to the PAI-1 promoter and demonstrates that Elk-1 phosphorylation on the Ets binding site is EGF-dependent.