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Please use this identifier to cite or link to this item: http://hdl.handle.net/10277/239

Authors: Clerici, Moira
Title: AMP-activated protein kinase regulates hyaluronan biosynthesis.
Abstract: HA is an atypical GAG because, although it is composed by GlcUA and GlcNAc, it does not contain sulfatation and it is not linked to any PG core protein. Moreover, HA synthesis is on plasma membrane by HASs, instead of Golgi. HA, as hydrophilic and viscous polysaccharide, increases the volume of extracellular space, thus contributing to tissue remodeling and facilitating cell mobility. This classical view of HA as a space filler and swelling agent has been complemented recently by interesting findings indicating that it mediates homing of stem cells, leukocyte adherence to endothelial cells, monocyte binding to virally infected or stressed mesenchymal cells and bacterial adherence. Furthermore, high molecular mass HA and its oligosaccharide degradation products create specific intracellular signals that promote cell locomotion, influence cell division, block apoptosis and induce membrane transporters (Rahmanian et al., 1997; Takahashi et al., 2005). Moreover, HA has a proatherosclerotic role in vascular diseases, because it promotes cellular migration and proliferation and because it’s involved in immune cells recruitment. As the synthesis of HA precursors requires ATP, UTP and other critical metabolic molecules (as glucose, glutamine and acetyl-CoA) making the HA production an energy consuming process, we hypnotized that in condition of low energy charge this anabolic process could be controlled by AMPK, a serine/threonine kinase which works as an energy sensor in all eukaryotic cells. In condition of low ATP content, this enzyme is activated and subsequently it switches off several anabolic processes (i.e.: gluconeogenesis, fatty acids synthesis, lipolysis, glycogen synthesis) and switches on some catabolic events (i.e.: glycolisis, fatty acids -oxidation) inside the cell. Moreover AMPK activation can be regulated not only by a decrement of ATP/AMP ratio, but also by physiological stimuli independent of the energy status of the cell, including hormones, nutrient depletion, heat shock and some drugs as metformin and AICAR. In the present study, we have used human aortic smooth muscle cells (hAoSMCs) because they produce an elevate amount of HA, essential for their proliferation and migration and for immune cells recruitment. All these processes are the most critical events involved in the onset of neointima formation, which is a pivotal step for the development and progression of all vascular diseases (including atherosclerosis), a main complication in diabetes. In fact, physiologically, SMCs resides in the tunica media in a contractile quiescent phenotype determining the vessel tone, whereas the neointima-forming SMCs posses a synthetic phenotype that induces cell proliferation and migration. Moreover, synthetic SMCs produce many ECM molecules, including HA, and ECM modifying enzymes, as metalloproteinases (Vigetti et al., 2006). The increment of HA in the media induces SMC motility through the interaction with CD44 and RHAMM (Savani et al., 1995). SMCs produced HA enhances immune cell recruitment and SMC dedifferentiation (Cuff et al., 2001). Therefore the ability to control and regulate HA biosynthesis by AMP-activated protein kinase could be a new good strategy to arrest atherosclerotic progression. In this work, AMPK-activation has been induced by AICAR, metformin and 2-deoxyglucose (2-DG) treatments, observing a significant and dose-dependent reduction of HA biosynthesis and an unchanged production of the other GAGs. These observations has been confirmed using different AICAR inhibitors (AMDA and Dipyridamole) and specific AMPK inhibitor (Compound C), and by transient transfections with constitutive active form of AMPK (CAAMPK) in AMPK-/- Mef-t cells. As the reduction of HA synthesis induced by AMPK activation was not due to an alteration of expression of genes involved in HA metabolism (as verified by quantitative RT-PCR analyses), we hypothesized that AMPK could act at post-transcriptional level, modifying the activity of HA synthetic enzymes (i.e.: Has1, Has2 and Has3). Cotransfecting each HAS isoform with (CA)AMPK in COS1 cells we found that AMPK specifically inhibits HAS2 activity. As AMPK is a kinase, we assayed whether a phosphorylative event could control HAS2 activity: by treatments in COS1 membrane fractions with SAP and CIP we observed that the removal of the hypothetic phosphorylation site fully restores HAS2 activity. For these reasons we hypothesized that AMPK could downregulate HA-synthesis inhibiting HAS2 activity by phosphorylation. In order to confirm this results, we have performed an immunoprecipitation assay in transfected COS1 cells, following by mass spectrometry analysis (MS-analysis). As HA has a pivotal role in some processes involved in the onset of atherosclerotic plaque as cell proliferation, migration and immune cells recruitment, we assayed the involvement of AMPK in those. Using hAoSMCs, we have demonstrated the ability of AMPK (activated by transfection as well as by treatments with AICAR and metformin) to arrest cell-cycle in G0/G1 phase, without causing apoptosis, to inhibit cell migration and to reduce monocytes adhesion. Taken together these results demonstrate that AICAR and metformin could have a new inhibitory effect on HA synthesis in hAoSMCs and an additional vasoprotective effect. Moreover, the increasing number of processes in which HA is involved suggest that to know the way to control and regulate its metabolism could be an important finding for a large number of medical applications as cancer, diabetes and atherosclerosis.
Issue Date: 2009
Doctoral course: Biotecnologie
Publisher: Università degli Studi dell'Insubria
Citation: Clerici, M.AMP-activated protein kinase regulates hyaluronan biosynthesis. (Doctoral Thesis, Università degli Studi dell'Insubria, 2009).

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