Intracellular Trafficking and Secretion of Matrix Metalloproteinases During Macrophage Migration
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INTRACELLULAR TRAFFICKING AND SECRETION OF MATRIX METALLOPROTEINASES DURING MACROPHAGE MIGRATION Joan Rohl Bachelor of Science, Master of Science Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy School of Biomedical Sciences Faculty of Health Queensland University of Technology 2016 KEYWORDS Collagen Chronic wounds Extracellular matrix Degradation Inflammation Intracellular trafficking Macrophages Matrigel Matrix metalloproteinases Migration MMP14 MMP9 Secretion SNAP23 Soluble NSF attachment protein receptor Syntaxin 4 Trafficking machinery Trafficking pathways VAMP4 VAMP7 VAMP8 i ABSTRACT The skin, the largest organ of the human body, serves as a physical barrier and as such, has many important biological functions. If compromised, timely repair is vital to ensure that the skin can fulfil these roles. During the healing process, the wounded area is cleared from pathogens and debris, a provisional matrix is formed to assist wound closure, the vascular network is restored and ultimately, the tissue is remodelled resulting in scar formation. Cutaneous wound healing is characterised by four overlapping and interdependent phases – haemostasis, inflammation, proliferation, maturation – and involves multiple different cell types. Any disturbances in this highly complex process can delay the healing process and lead to chronic wounds. They are defined as wounds that show no significant healing within four weeks. Chronic wounds affect around 450,000 Australians every year and treatment of these is estimated to cost the health care system $3 billion annually. Persistent and elevated inflammation contributes to the formation of non‐ healing wounds and the number of macrophages within a wound can determine the level and duration of inflammation. Macrophages are recruited from the bloodstream and infiltrate the tissue during wound healing. They create a path by degrading the extracellular matrix (ECM), the acellular component that comprises the dermal tissue and plays many important roles in wounded and unwounded skin. The ECM acts not only as a scaffold for the cells, but also regulates cell adhesion, chemotaxis and migration, as well as storing growth factors and cytokines. Matrix metalloproteinases (MMPs) cleave the ECM and thus help remodel the tissue during the healing process. During normal wound healing, MMP activity is tightly controlled. However, in non‐healing wounds increased levels of certain MMPs, such as MMP9 and MMP14, cause extensive tissue damage and contribute to sustained inflammation. MMP14 facilitates macrophage tissue infiltration, which then amplifies the inflammatory response within the wound. Current treatment strategies to reduce MMP levels in the wound fluid or to inhibit their activity have not been effective. For these enzymes to access their substrates, they first have to be secreted as is the case of MMP9 or incorporated into the plasma membrane as is the case with MM14. Preventing the delivery of MMP9 and MMP14 to the cell surface might reduce tissue damage and macrophage infiltration leading to an overall reduction of inflammation. In order to inhibit MMP cell surface delivery, it is necessary to elucidate their intracellular transport route and identify proteins that regulate these pathways in macrophages. ii At least two major pathways exist in macrophages by which proteins can be delivered to the cell surface. One is a classical pathway where proteins are synthesised in the endoplasmic reticulum (ER), shuttled through the Golgi apparatus and transported to the surface either directly or indirectly through the recycling endosome. One other major secretory pathway is the lysosomal pathway, in which proteins are delivered to the cell surface from the cytosol or the Golgi complex through a lysosome or lysosome‐ related organelle. Transport of proteins between these organelles and the cell surface occurs within membrane‐bound vesicles. Delivery of vesicles to target organelles or the plasma membrane requires membrane fusion, which is facilitated by soluble N‐ ethylmaleimide‐sensitive‐factor attachment protein receptor (SNARE) proteins at all points in the trafficking pathway. These proteins form a complex of one R‐SNARE on one membrane and two or three Q‐SNAREs on the opposing membrane, which not only brings the membranes into close proximity, but also provides the energy and specificity required for membrane fusion. Each SNARE protein has a precise subcellular distribution and regulates a distinct step in a pathway. The intracellular trafficking pathways and the SNARE machinery responsible for cell surface delivery of MMP9 and MMP14 proteins are mostly unclear in macrophages. It was therefore the aim of this thesis to identify the critical SNARE proteins and pathways required for MMP9 secretion and for MMP14 cell surface delivery in macrophages. Targeting these proteins could reduce MMP activity, macrophage infiltration and inflammation in chronic wounds. The work reported herein shows that the expression and cell surface delivery of MMP9 and MMP14 is upregulated upon activation of macrophages with the bacterial cell wall component lipopolysaccharide (LPS). This is in contrast to neutrophils, which store their MMP9 in tertiary granules prior to secretion and to some cancer cell lines that have been shown to constitutive express MMP14 use a recycling mechanism to regulate the level at the cell surface. Hence, trafficking pathways for individual MMPs can be cell type specific. It was found that in macrophages, the newly made enzymes are trafficked via the Golgi complex prior to MMP9 secretion or incorporation of MMP14 into the plasma membrane. Immunofluorescence microscopy showed that in addition to their localisation in the Golgi complex they both were found in late endosomes/lysosomes. Using targeted siRNA knockdown of SNARE proteins that regulate distinct pathways in macrophages, the responsible trafficking pathways were further investigated. Disrupting the key MMP intracellular trafficking pathways by reducing the levels of a key SNARE involved in their exocytosis should lead to compromised cell surface delivery of the protein of interest. Surprisingly, for MMP9 multiple SNAREs iii involved in the classical or lysosomal pathways were targeted with siRNA but no reduction in MMP9 secretion was found. However, targeting some of these SNAREs, including VAMP3, VAMP4, VAMP7, VAMP8, Stx2 and SNAP23, led to an increase in extracellular MMP9 levels suggesting that an endocytic clearance mechanism might be influencing extracellular MMP9 levels and masking the exocytic pathways. The trafficking of MMP14 was also tested. MMP14 trafficking from the Golgi complex to late endosomes/lysosomes prior to the cell surface was found to be regulated by Golgi R‐SNARE VAMP4, the late endosomal/lysosomal R‐SNAREs VAMP7/8 and the surface Q‐SNARE complex Stx4/SNAP23. In macrophages MMP14 was found to be delivered to podosomes, actin‐rich cell membrane structures implicated in cell migration and matrix degradation. Targeting any one of these SNARE proteins lead to a reduction in gelatin matrix degradation in the area of the podosomes. Accordingly, disrupting this SNARE machinery also significantly reduced the ability of macrophages to effectively invade into both 3D Matrigel™ and collagen I gels. Thus, SNARE proteins that reduce surface MMP14 delivery have been identified providing new potential targets to reduce MMP14 surface activity, attenuate macrophage tissue infiltration, dampen inflammation and improve wound healing outcomes. Overall, this thesis advances the knowledge of trafficking pathways for cell surface delivery of MMP9 and MMP14 in macrophages and identifies key proteins involved in these pathways. For MMP9, further investigation into its endocytic mechanisms of clearance might allow better manipulation of MMP9 levels in the environment to reduce excessive tissue damage in chronic wounds. SNARE machinery responsible for MMP14 cell surface delivery, matrix degradation and macrophage invasion was identified and could represent novel therapeutic targets for the treatment of chronic wounds. iv TABLE OF CONTENTS KEYWORDS ......................................................................................................................................... I ABSTRACT ......................................................................................................................................... II TABLE OF CONTENTS ..................................................................................................................... V LIST OF FIGURES ......................................................................................................................... VIII LIST OF TABLES ................................................................................................................................ X LIST OF ABBREVIATIONS ............................................................................................................ XI STATEMENT OF ORIGINAL AUTHORSHIP .......................................................................... XIII LIST OF PUBLICATIONS ............................................................................................................ XIV ACKNOWLEDGEMENTS .............................................................................................................. XV CHAPTER 1: LITERATURE REVIEW .......................................................................................... 1 1.1 INTRODUCTION .......................................................................................................................................