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

Monocyte chemoattractant protein-induced protein 1 (MCPIP1) enhances angiogenic and cardiomyogenic potential of murine bone marrow-derived mesenchymal stem cells

The current evidence suggests that beneficial effects of mesenchymal stem cells (MSCs) toward myocardial repair are largely due to paracrine actions of several factors. Although Monocyte chemoattractant protein-induced protein 1 (MCPIP1) is involved in the regulation of inflammatory response, apoptosis and angiogenesis, whether MCPIP1 plays any role in stem cell-induced cardiac repair has never been examined. By employing retroviral (RV)-transduced overexpression of MCPIP1, we investigated the impact of MCPIP1 on viability, apoptosis, proliferation, metabolic activity, proteome, secretome and differentiation capacity of murine bone marrow (BM) - derived MSCs. MCPIP1 overexpression enhanced angiogenic and cardiac differentiation of MSCs compared with controls as indicated by elevated expression of genes accompanying angiogenesis and cardiomyogenesis in vitro. The proangiogenic activity of MCPIP1-overexpressing MSCs (MCPIP1-MSCs) was also confirmed by increased capillary-like structure formation under several culture conditions. This increase in differentiation capacity was associated with decreased proliferation of MCPIP1-MSCs when compared with controls. MCPIP1-MSCs also expressed increased levels of proteins involved in angiogenesis, autophagy, and induction of differentiation, but not adverse inflammatory agents. We conclude that MCPIP1 enhances endothelial and cardiac differentiation of MSCs. Thus, modulating MCPIP1 expression may be a novel approach useful for enhancing the immune-regulatory, anti-apoptotic, anti-inflammatory and regenerative capacity of BM-derived MSCs for myocardial repair and regeneration of ischemic tissues

Highly conserved regions of C/EBPβ mRNA might fold into hairpins.

<p>Clustal Omega alignment of the C/EBPβ mRNA 3'UTR of eight mammalian species revealed regions of high conservation status. Within these, a set of potential hairpins is marked in yellow and green.</p

Primers used for amplification of 3’UTR C/EBPβ mRNA.

<p>Primers used for amplification of 3’UTR C/EBPβ mRNA.</p

MCPIP1-D141N with completely abolished RNase activity retains interaction ability against the 3'UTR of C/EBPβ mRNA.

<p>Results of electrophoretic mobility shift assays performed with increasing amounts (0–200 pmol) of recombinant MCPIP1-D141N <b>A</b>. or PIN domain alone, MCPIP1 PIN-D141N <b>B</b>. incubated with 2 pmol of <i>in vitro</i>–transcribed the 3'UTR fragments of C/EBPβ mRNA.</p

MCPIP1 binds C/EBPβ transcript <i>in vivo</i>.

<p>HepG2 cells were transfected with an empty plasmid or plasmid coding for enzymatically inactive MCPIP1-D141N with linked Flag tag at N-terminus. Cell lysates were immunoprecipitated with anti-Flag antibodies followed by RNA extraction. <b>A</b>. Western blot showing expression of a MCPIP1 and tubulin as a loading control. <b>B</b>. Isolated RNA was reverse transcribed and analyzed by the real-time PCR using primers specific to C/EBPβ or 28S RNA with actin specific primers as an internal reference. These are representative results of two independent experiments.</p

MCPIP1 directly cleaves the 3’UTR of C/EBPβ mRNA.

<p><b>A</b>. Cleavage assay of the 3'UTR fragments of C/EBPβ mRNA combined with recombinant MCPIP1 (residues 1–599) or MCPIP1 PIN (residues 134–327). <b>B</b>. Cleavage assay of the synthetized 3'UTR of C/EBPβ mRNA (-3-19) stem loop structure combined with recombinant MCPIP1-WT or MCPIP1-D141N as a control.</p