Regnase-2 inhibits glioblastoma cell proliferation.

Regnase-2 (Reg-2/MCPIP2/ZC3H12B) is uniquely expressed at a high level in the healthy brain and down-regulated in samples from patients with glioma, reaching the lowest level in high-grade glioblastoma multiforme (GBM). This RNase is involved in the regulation of neuroinflammation through the degradation of IL-6 and IL-1 mRNAs, key pro-inflammatory cytokines for GBM pathology. Reg-2 is a strong inhibitor of the proliferation of human glioblastoma cell lines and blocks their potential to form colonies. Here, we describe that overexpression of Reg-2 stalls glioblastoma cells in the G1 phase of the cell cycle and reduces the level of transcripts implicated in cell cycle progression. These newly identified targets include CCND1, CCNE1, CCNE2, CCNA2, CCNB1, and CCNB2, encoding the cyclins as well as AURKA and PLK1, encoding two important mitosis regulators. By RNA immunoprecipitation we confirmed the direct interaction of Reg-2 with the investigated transcripts. We also tested mRNA regions involved in their interaction with Reg-2 on the example of CCNE2. Reg-2 interacts with the 3'UTR of CCNE2 in a dose-dependent manner. In conclusion, our results indicate that Reg-2 controls key elements in GBM biology by restricting neuroinflammation and inhibiting cancer cell proliferation.

The homeostatic function of Regnase-2 restricts neuroinflammation.

The precise physiological functions and mechanisms regulating RNase Regnase-2 (Reg-2/ZC3H12B/MCPIP2) activity remain enigmatic. We found that Reg-2 actively modulates neuroinflammation in nontransformed cells, including primary astrocytes. Downregulation of Reg-2 in these cells results in increased mRNA levels of proinflammatory cytokines IL-1ß and IL-6. In primary astrocytes, Reg-2 also regulates the mRNA level of Regnase-1 (Reg-1/ZC3H12A/MCPIP1). Reg-2 is expressed at high levels in the healthy brain, but its expression is reduced during neuroinflammation as well as glioblastoma progression. This process is associated with the upregulation of Reg-1. Conversely, overexpression of Reg-2 is accompanied by the downregulation of Reg-1 in glioma cells in a nucleolytic NYN/PIN domain-dependent manner. Interestingly, low levels of Reg-2 and high levels of Reg-1 correlate with poor-glioblastoma patients' prognoses. While Reg-2 restricts the basal levels of proinflammatory cytokines in resting astrocytes, its expression is reduced in IL-1ß-activated astrocytes. Following IL-1ß exposure, Reg-2 is phosphorylated, ubiquitinated, and degraded by proteasomes. Simultaneously, the Reg-2 transcript is destabilized by tristetraprolin (TTP) and Reg-1 through the AREs elements and conservative stem-loop structure present in its 3'UTR. Thus, the peer-control loop, of Reg-1 and Reg-2 opposing each other, exists. The involvement of TTP in Reg-2 mRNA turnover is confirmed by the observation that high TTP levels correlate with the downregulation of the Reg-2 expression in high-grade human gliomas. Additionally, obtained results reveal the importance of Reg-2 in inhibiting human and mouse glioma cell proliferation. Our current studies identify Reg-2 as a critical regulator of homeostasis in the brain.

Molecular Mechanisms of ZC3H12C/Reg-3 Biological Activity and Its Involvement in Psoriasis Pathology.

The members of the ZC3H12/MCPIP/Regnase family of RNases have emerged as important regulators of inflammation. In contrast to Regnase-1, -2 and -4, a thorough characterization of Regnase-3 (Reg-3) has not yet been explored. Here we demonstrate that Reg-3 differs from other family members in terms of NYN/PIN domain features, cellular localization pattern and substrate specificity. Together with Reg-1, the most comprehensively characterized family member, Reg-3 shared IL-6, IER-3 and Reg-1 mRNAs, but not IL-1ß mRNA, as substrates. In addition, Reg-3 was found to be the only family member which regulates transcript levels of TNF, a cytokine implicated in chronic inflammatory diseases including psoriasis. Previous meta-analysis of genome-wide association studies revealed Reg-3 to be among new psoriasis susceptibility loci. Here we demonstrate that Reg-3 transcript levels are increased in psoriasis patient skin tissue and in an experimental model of psoriasis, supporting the immunomodulatory role of Reg-3 in psoriasis, possibly through degradation of mRNA for TNF and other factors such as Reg-1. On the other hand, Reg-1 was found to destabilize Reg-3 transcripts, suggesting reciprocal regulation between Reg-3 and Reg-1 in the skin. We found that either Reg-1 or Reg-3 were expressed in human keratinocytes in vitro. However, in contrast to robustly upregulated Reg-1 mRNA levels, Reg-3 expression was not affected in the epidermis of psoriasis patients. Taken together, these data suggest that epidermal levels of Reg-3 are negatively regulated by Reg-1 in psoriasis, and that Reg-1 and Reg-3 are both involved in psoriasis pathophysiology through controlling, at least in part different transcripts.

Influence of the reporter vector backbone on 2-deoxyglucose dependent promoter activation.

Reporter vectors are very often used to investigate mechanisms responsible for regulation of promoter activity. Since their first generation, many new variants were constructed to increase sensitivity and reduce background signal. However, these tools are still imperfect and can generate false results. We have found that depending on the backbone of the reporter vector, pGL3 or pGL2, different results are obtained for a eukaryotic promoter's activation by metabolic changes. These observations were done in the course of investigation of the MMP2 (matrix metalloproteinase-2) promoter regulation in response to inhibition of glycolysis.

Potential limitations of the Sleeping Beauty transposon use in gene expression studies.

MCPIP2 is the least known member of the MCPIP family of proteins. Recently we have found that it is a new RNase involved in transcript turnover. However, the full spectrum of its cellular targets is still unidentified. To discover transcripts which are regulated by this protein we have employed Sleeping Beauty transposons. This tool allows for rapid generation of a stable transgenic cell line with inducible expression of the desired gene. In this study, we analysed how the Sleeping Beauty system itself influences expression of chosen genes, namely IL-6, Regnase-1 and VEGF. We found that the system alone may influence expression of IL-6. Our results indicate that Sleeping Beauty transposons should be used with caution in studies that are focused on changes in the transcript level.

ZC3H12B/MCPIP2, a new active member of the ZC3H12 family.

ZC3H12B is the most enigmatic member of the ZC3H12 protein family. The founding member of this family, Regnase-1/MCPIP1/ZC3H12A, is a well-known modulator of inflammation and is involved in the degradation of inflammatory mRNAs. In this study, for the first time, we characterized the properties of the ZC3H12B protein. We show that the biological role of ZC3H12B depends on an intact NYN/PIN RNase domain. Using RNA immunoprecipitation, experiments utilizing actinomycin D and ELISA, we show that ZC3H12B binds interleukin-6 (IL-6) mRNA in vivo, regulates its turnover, and results in reduced production of IL-6 protein upon stimulation with IL-1ß. We verified that regulation of IL-6 mRNA stability occurs via interaction of ZC3H12B with the stem-loop structure present in the IL-6 3'UTR. The IL-6 transcript is not the only target of ZC3H12B. ZC3H12B also interacts with other known substrates of Regnase-1 and ZC3H12D, such as the 3'UTRs of IER3 and Regnase-1, and binds IER3 mRNA in vivo. Using immunofluorescence, we examined the localization of ZC3H12B within the cell. ZC3H12B forms small, granule-like structures in the cytoplasm that are characteristic of proteins involved in mRNA turnover. The overexpression of ZC3H12B inhibits proliferation by stalling the cell cycle in the G2 phase. This effect of ZC3H12B is also NYN/PIN dependent. The analysis of the ZC3H12B mRNA level reveals its highest expression in the human brain and the neuroblastoma cell line SH-SY5Y, although the factors regulating its expression remain elusive. Down-regulation of ZC3H12B in SH-SY5Y cells by specific shRNAs results in up-regulation of ZC3H12B-target mRNAs.

Intact NYN/PIN-Like Domain is Crucial for the Degradation of Inflammation-Related Transcripts by ZC3H12D.

ZC3H12D belongs to a recently discovered family of proteins containing four members of which the most studied and best described is the RNase ZC3H12A (MCPIP1/Regnase-1). ZC3H12A is a crucial negative regulator of inflammation. It accelerates the turnover of transcripts of a spectrum of proinflammatory cytokines, as well as its own mRNA. The biological role of ZC3H12D is less clear, although it was shown that this member of ZC3H12 family is also involved in the regulation of inflammation. Here, we show that ZC3H12A and ZC3H12D recognize a set of common target mRNAs encoding proteins that play important roles in the course of the inflammation. Similarly to ZC3H12A, ZC3H12D participates in the 3'UTR-dependent regulation of the turnover of mRNAs encoding interleukin-6 (IL-6), tumor necrosis factor (TNF), and immediate early response 3 gene (IER3). The ZC3H12A mRNA is also among the identified ZC3H12D targets. Using the combination of immunofluorescence with single molecule RNA fluorescence in situ hybridization (smRNA FISH) we have shown that ZC3H12D protein interacts with the ZC3H12A transcript. The direct binding of these two molecules in vivo was further confirmed by RNA immunoprecipitation. Simultaneously, overexpression of ZC3H12D increases the turnover rate of transcripts containing ZC3H12A 3'UTR. Using reporter gene assays we have confirmed that the Asp95 residue present in the NYN/PIN-like domain is crucial for ZC3H12D biological activity. We have also revealed that ZC3H12D recognizes the same structural elements present in the 3'UTRs of the investigated transcripts, as ZC3H12A. J. Cell. Biochem. 118: 487-498, 2017. © 2016 Wiley Periodicals, Inc.

The perplexities of the ZC3H12A self-mRNA regulation.

The mechanisms regulating transcript turnover are key processes in the regulation of gene expression. The list of proteins involved in mRNAs' degradation is still growing, however, the details of RNase-mRNAs interactions are not fully understood. ZC3H12A is a recently discovered inflammation-related RNase engaged in the control of proinflammatory cytokine transcript turnover. ZC3H12A also regulates its own transcript half-live. Here, we studied the details of this regulation. Our results confirm the importance of the 3'UTR in ZC3H12A-dependent ZC3H12A mRNA degradation. We compared the mouse and human stemloop structures present in this region and discovered that the human conserved stem-loop structure is not sufficient for ZC3H12A-dependent degradation. However, this structure is important for the ZC3H12A mRNA post-transcriptional regulation. Our studies emphasize the importance of the neighboring features of the identified stem-loop structure for its biological activity. Removal of this region together with the stem-loop structure greatly inhibits the ZC3H12A regulation of the investigated 3'-untranslated region (3'UTR).

IF-combined smRNA FISH reveals interaction of MCPIP1 protein with IER3 mRNA.

MCPIP1 and IER3 are recently described proteins essential for maintenance of immune homeostasis. IER3 is involved in the regulation of apoptosis and differentiation and has been shown lately to protect activated T cells and macrophages from apoptosis. MCPIP1 is an RNase critical for controlling inflammation-related mRNAs. MCPIP1 interacts with and degrades a set of stem-loop-containing mRNAs (including IL-6). Our results demonstrate the involvement of MCPIP1 in the regulation of IER3 mRNA levels. A dual luciferase assay revealed that over-expression of MCPIP1 resulted in a decrease of luciferase activity in the samples co-transfected with constructs containing luciferase CDS attached to IER3 3'UTR. We identified a stem-loop structure similar to that described to be important for destabilization of the IL-6 mRNA by MCPIP1. Examination of IER3 3'UTR sequence, structure and evolutionary conservation revealed that the identified stem-loop is buried within a bigger element. Deletion of this fragment abolished the regulation of IER3 3'UTR-containing transcript by MCPIP1. Finally, using immunofluorescence-combined single-molecule RNA FISH we have shown that the MCPIP1 protein co-localizes with IER3 mRNA. By this method we also proved that the presence of the wild-type NYN/PIN-like domain of MCPIP1 correlated with the decreased level of IER3 mRNA. RNA immunoprecipitation further confirmed the interaction of MCPIP1 with IER3 transcripts in vivo.

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.

Transcription factor Elk-1 participates in the interleukin-1ß-dependent regulation of expression of immediate early response gene 3 (IER3).

Immediate early response gene 3 (IER3) encodes a protein involved in the regulation of apoptosis and differentiation. Recently the role of IER3 in the regulation of extracellular signal-regulated kinases (ERKs) was discovered. IER3 prolongs ERKs activation by inhibition of phosphatase PP2A. Here we show that interleukin-1ß (IL-1ß)-induced IER3 expression is mediated by the ERK1/2 target, transcription factor Elk-1. We identified sequences in the IER3 promoter responsible for its ERKs-dependent activation, namely ETS5/6. Elk-1 binds to these sequences and is phosphorylated following IL-1ß stimulation. Mutation of ETS5/6 binding site abolishes activation of IER3 promoter by IL-1ß as well as by the constitutively active form of Elk-1 (Elk-VP16). Thus IER3 acts not only as a regulator of ERKs activation, but also as a ERKs-Elk-1-dependent downstream effector.

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.

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)-1ß 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-1ß. The function of IL-1ß 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.

EGF activates TTP expression by activation of ELK-1 and EGR-1 transcription factors.

BACKGROUND: Tristetraprolin (TTP) is a key mediator of processes such as inflammation resolution, the inhibition of autoimmunity and in cancer. It carries out this role by the binding and degradation of mRNA transcripts, thereby decreasing their half-life. Transcripts modulated by TTP encode proteins such as cytokines, pro-inflammatory agents and immediate-early response proteins. TTP can also modulate neoplastic phenotypes in many cancers. TTP is induced and functionally regulated by a spectrum of both pro- and anti-inflammatory cytokines, mitogens and drugs in a MAPK-dependent manner. So far the contribution of p38 MAPK to the regulation of TTP expression and function has been best described. RESULTS: Our results demonstrate the induction of the gene coding TTP (ZFP36) by EGF through the ERK1/2-dependent pathway and implicates the transcription factor ELK-1 in this process. We show that ELK-1 regulates ZFP36 expression by two mechanisms: by binding the ZFP36 promoter directly through ETS-binding site (+ 883 to +905 bp) and by inducing expression of EGR-1, which in turn increases ZFP36 expression through sequences located between -111 and -103 bp. CONCLUSIONS: EGF activates TTP expression via ELK-1 and EGR-1 transcription factors.

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.

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

BACKGROUND: MCPIP 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. RESULTS: Here we report that the proinflammatory cytokine IL-1beta 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-1beta. 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. CONCLUSIONS: We conclude that the transcription factor Elk-1 plays an important role in the activation of ZC3H12A expression in response to IL-1beta stimulation.

[Degradation of eukaryotic transcripts. Role and regulation of tristetraprolin activity]. / Degradacja transkryptów eukariotycznych. Funkcja i regulacja tristetraproliny.

The kinetics of transcripts turnover is a very important aspect of the regulation of the amount of newly synthesized proteins. Transcripts can serve as a template for protein synthesis as long as they remain in the cytoplasm, bound to the ribosomes. Degradation of mRNAs effectively influences quantity of transcripts in this pool. The process of mRNA degradation, similarly to transcription, is precisely regulated, mainly by proteins which interact with mRNA. These proteins are responsible both for transcripts stabilization and degradation. In this article we have summarized known pathways of mRNA degradation. We have also reviewed the present state of knowledge on tristetraprolin, one of the best characterized proteins that takes part in the transcripts turnover.

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

In human monocyte-derived macrophages, the MCPIP gene (monocyte chemoattractant protein-induced protein) is strongly activated by interleukin-1beta (IL-1beta). 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-1beta 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-1beta 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-1beta mRNA are due to shortening of the IL-1beta 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-1beta and MCPIP observed in an RNA immunoprecipitation assay suggests that this novel RNase may be involved in the regulation of expression of several genes.

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.

[Regulation of PAI-1 expression]. / Regulacja ekspresji genu PAI-1.

PAI-1 (plasminogen activator inhibitor-1) is a member of plasminogen cascade with an inhibitory role in plasmin activation. Plasmin is a protease capable of acting on wide range of substrates and, together with metaloproteinases, is a main proteolytic enzyme. Except its role in plasminogen cascade, PAI-1 has an affinity to vitronectin and uPA/uPAR what involves PAI-1 in cell's motility. PAI-1 gene is regulated in response to cytokines, hormones and many growth factors among which TGFbeta is the most important one. The PAI-1 promoter contains SBE, CAGA box, HRE, ERE, NFkB - binding sites, Sp-1, AP-1 and other. Cooperation between transcription factors bound to promoter and cross-talks between kinases and other upstream proteins decide about gene expression. This work describes the present knowledge in this field.