Unfolded protein response regulated mirnas in lipotoxic endoplasmic reticulum stress in macrophages

Abstract

The proper functioning and the development of the cell is essential to the fitness of the multicellular organisms - any significant disturbances in cellular mechanisms can lead to a multitude of diseases or death. Among these conditions, the global rise in metabolic diseases like obesity, diabetes and atherosclerosis draw significant research interest focus. Since the prevalence of metabolic disorders in the developed and underdeveloped world is expected to increase further in next decade; understanding the contributing cellular mechanisms is vital for the development of new and effective diagnostic and therapeutic tools against this devastating disease cluster. Among the homeostatic cellular pathways important for health the Unfolded Protein Response (UPR) is highly conserved from yeast to mammals. Aside from most conserved UPR branch Inositol-requiring protein 1(IRE1), the mammalian UPR is composed of three different pathways regulated by IRE1, eukaryotic translation initiation factor 2-alpha kinase 3 (PERK), and activating transcription factor 6 (ATF6). The UPR signaling is activated in response to the accumulation of unfolded or misfolded proteins in ER that leads to endoplasmic reticulum (ER) stress. The goal of the UPR is to re-establish ER homeostasis via inhibition of further protein translation and promoting protein folding. In the case of severe or unresolved ER stress, UPR instead triggers a programmed cell death. Recent studies indicate that noncoding regulatory RNAs such as microRNAs (miRNAs) play important role in both upstream and downstream of the UPR. In this thesis, the regulation of miRNA expression by the different UPR arms are examined in macrophages under lipidinduced or lipotoxic ER stress conditions. The results of PCR array studies of RNA obtained from mouse macrophages stressed with a saturated fatty acid, palmitate (PA) , revealed multiple differentially regulated miRNAs. Among these miRNAs, significantly regulated ones were further examined for their regulation by the different arms of the UPR. Towards this end several complementary approaches were taken: First, significantly regulated microRNAs from microRNA PCR array results were analyzed. Next, macrophages were treated with palmitate after transfection with IRE1 and PERK silencer RNA (siRNA) to assess the role of UPR arms in lipid regulated miRNA regulation and the expression of relevant miRNAs was examined in treated macrophages. As an alternative method, macrophages were treated simultaneously with palmitate and specific inhibitors for IRE1’s endoribonuclease or PERK’s kinase activity. Then miRNA expressions were further examined in IRE1 knock-out mouse embryonic fibroblast (MEF) cell lines transfected with the wild type (WT) IRE or the endoribonuclease domain inactive (RD) mutant of IRE1 to verify the specific regulation of the miRNA by the IRE1’s endoribonuclease activity. As a result, upregulation of miR-2137 expression by palmitate was determined as IRE1-endoribonuclease dependent. Next, potential target mRNAs were examined by the overexpression or knock-down of miR-2137 in macrophages. One possible target mRNA was found to be inositol polyphosphate phosphatase-like 1 (Innpl1) . Aside from miR-2137, miR-33 also showed significant alteration upon PA treatment in macrophages. Since the role of miR-33 in atherosclerosis, obesity and insulin resistance is well established, its expression was studied further in RAW 264.7 macrophage cell line and bone marrow-derived primary macrophages after IRE1 and PERK knock-down with siRNA. ATP-binding cassette, sub-family A (ABC1), member 1 (ABCA1), a known target of miR-33, was investigated as down-stream target of miR-33 in PA treated macrophages, in an IRE1 dependent manner. The results of this study uncovered new UPR regulated miRNAs under lipid stress in macrophages. Excess lipid is one of the prominent causes in metabolic diseases – obesity, atherosclerosis, insulin resistance – and these UPR regulated miRNAs may explain the underlying mechanism behind this set of diseases. Furthermore, the possible gene targets for these miRNAs could be responsible for progression of such conditions. Further studies are needed to reveal the exact mechanisms that can lead to the development of novel therapeutic approaches.