MCPIP1, alias Regnase-1 binds and cleaves mRNA of C/EBP/beta

CCAAT/enhancer-binding protein beta (C/EBPβ) is a transcription factor controlling a broad range of genes essential for homeostasis, including genes related to immune functions, inflammation, metabolism and growth. Monocyte chemoattractant protein-1-induced protein 1 (MCPIP1) also called as Regnase-1 is an RNase and has been shown to decrease the stability of short-lived transcripts coding for inflammation-related proteins, including IL-1β, IL-6, IL-2, IL-8, IL-12b, IER-3, c-Rel. We found previously that the half-life of the C/EBPβ transcript is regulated by MCPIP. To understand the mechanism driving down-regulation of C/EBPβ by MCPIP1, we applied an in vitro cleavage assay, followed by a luciferase-reporter assay and RNA immunoprecipitation (RIP). We demonstrated that MCPIP1 recognizes regions of the 3'UTR of C/EBPβ mRNA and promotes its decay by introducing direct endonucleolytic cleavage

Depletion of Mcpip1 in murine myeloid cells results in intestinal dysbiosis followed by allergic inflammation

MCPIP1 (called also Regnase-1) is a negative regulator of inflammation. Knockout of the Zc3h12a gene, encoding Mcpip1 in cells of myeloid origin ($Mcpip1^{MKO}$), has a pathological effect on many organs. The aim of this study was to comprehensively analyze pathological changes in the skin caused by Mcpip1 deficiency in phagocytes with an emphasis on its molecular mechanism associated with microbiome dysbiosis. $Mcpip1^{MKO}$ mice exhibited spontaneous wound formation on the skin. On a molecular level, the Th2-type immune response was predominantly characterized by an increase in Il5 and Il13 transcript levels, as well as eosinophil and mast cell infiltration. Irritation by DNFB led to a more severe skin contact allergy in $Mcpip1^{MKO}$ mice. Allergic reactions on the skin were strongly influenced by gut dysbiosis and enhanced systemic dissemination of bacteria. This process was followed by activation of the C/EBP pathway in peripheral macrophages, leading to local changes in the cytokine microenvironment that promoted the Th2 response. A reduced bacterial load inhibited allergic inflammation, indicating the role of intestinal dysbiosis in the development of skin diseases. Our results clearly show that MCPIP1 in phagocytes is an essential negative regulator that controls the gut-skin axis

A search for genes modulated by interleukin-6 alone or with interleukin-1β in HepG2 cells using differential display analysis

AbstractInterleukin-1 and interleukin-6 are principal cytokines involved in regulation of expression of acute-phase proteins. In the joint action of both cytokines IL-1 can suppress or enhance the IL-6-dependent induction of gene expression. Here, we report changes in the transcriptome profile of HepG2 cells exposed to IL-6 alone, or IL-1 and IL-6. Cytokine-responsive genes were identified by differential display analysis. Validation of observed changes in the transcript level was carried out using the slot blot method. Out of 88 cDNA species modulated by IL-6, only 38 represent different known genes whereas 18 clones match genomic clones in NCBI data with hypothetical cDNA sequences (the remaining 32 clones showed no homology with the database or represented several clones of the same gene). In the experiments with HepG2 cells prestimulated for 3 h with IL-1 and then stimulated with IL-6, 43 cDNA fragments were amplified. Twenty-three of them represent known genes while 10 clones have inserts matching hypothetical cDNA sequences in NCBI data. The identified transcripts modulated by IL-6 or both cytokines in HepG2 cells code for intracellular proteins of various function. The largest groups represent genes engaged in metabolism, protein synthesis and signaling pathways. Among all genes identified as differentially regulated under stimulation by IL-6, or IL-1/IL-6, six were detected in both types of stimulation. None of the typical genes coding for plasma acute phase proteins was identified in our experiments. This indicates that differential display cannot be used to characterize the profile of a given transcriptome. On the other hand, it is a useful technique for detection of new genes responding to IL-6 alone or IL-6 in combination with IL-1

Substrate specificity of human MCPIP1 endoribonuclease

AbstractMCPIP1, also known as Regnase-1, is a ribonuclease crucial for regulation of stability of transcripts related to inflammatory processes. Here, we report that MCPIP1 acts as an endonuclease by degrading several stem-loop RNA structures and single-stranded RNAs. Our studies revealed cleavage sites present in the stem-loops derived from the 3′ untranslated region of the interleukin-6 transcript. Furthermore, MCPIP1 induced endonuclease cleavage at the loop motif of stem-loop structures. Additionally, we observed that MCPIP1 could cleave single-stranded RNA fragments. However, RNA substrates shorter than 6 nucleotides were not further affected by MCPIP1 nucleolytic activity. In this study, we also determined the dissociation constants of full-length MCPIP1D141N and its ribonuclease domain PIN D141N with twelve oligonucleotides substrates. The equilibrium binding constants (Kd) for MCPIP1D141N and the RNA targets were approximately 10 nM. Interestingly, we observed that the presence of a zinc finger in the PIN domain increases the affinity of this protein fragment to 25-nucleotide-long stem-loop RNA but not to shorter ones. Furthermore, size exclusion chromatography of the MCPIP1 and PIN proteins suggested that MCPIP1 undergoes homooligomerization during interaction with RNA substrates. Our results provide insight into the mechanism of MCPIP1 substrate recognition and its affinity towards various oligonucleotides.</jats:p

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

<p>Abstract</p> <p>Background</p> <p>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 - <it>ZC3H12A </it>is poorly explored.</p> <p>Results</p> <p>Here 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 <it>ZC3H12A </it>reporter construct we have shown that a <it>ZC3H12A </it>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 <it>in vivo </it>binding of both transcription factors to this region of the <it>ZC3H12A </it>promoter.</p> <p>Conclusions</p> <p>We conclude that the transcription factor Elk-1 plays an important role in the activation of <it>ZC3H12A </it>expression in response to IL-1β stimulation.</p