Identification of novel intracellular functions of the Thrombospondin protein family and characterization of the involvement in the endoplasmic reticulum (ER) protein shuttling, secretion and ER stress activation using in vivo and in vitro models.

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Thrombospondin (TSP) proteins are matricellular proteins typically associated with various extracellular functions. After injury, the heart primarily depends on new extracellular matrix (ECM) deposition and remodeling to maintain its structural and functional integrity. This study will attempt to elucidate the novel functional aspects of the TSP proteins in the mouse heart. However, in contrast to the obvious hypothesis that TSPs function within the ECM to affect cardiac remodeling, our recent studies have suggested that the TSP family of proteins functions in a completely novel manner to affect the ECM protein generation, modification and shuttling and secretion from within the cell. Hence our novel hypothesis is that the TSP family of proteins has a critical intracellular function associated with protein quality control, secretion and organization of ECM components. As an added layer of complexity, we will also investigate the potential differences between the 4 primary TSP family members which have both shared but also unique functional domains. This proposal is organized in two aims: i) investigate the specific role of the 2 primary TSP subfamily members during cardiac disease and ii) determine whether each TSP protein regulates the endoplasmic reticulum (ER) function through the activating transcription factor 6 (ATF6). i) To functionally compare the two TSP subgroups (A: TSP1/2; B: TSP3/4) and elucidate their involvement in cardiac homeostasis and disease, we will focus on TSP1 for group A and TSP3 for group B. The use of cardiac specific TSP overexpressing mice and knockout mice will be used to dissect the functional roles of these 2 proteins in vivo. ii) In contrast to previous findings, the sponsors laboratory has recently shown that TSP proteins have a primary function within the cell, which includes protein shuttling, -quality control and mediation of a protective ER stress response via interaction with ATF6. However, whether the role of ER stress in heart diseases is maladaptive or beneficial is still a controversial topic. In this study, gain and loss of function models of TSP and ATF6 will be used to understand their interplay and functional role in the heart. To study the mechanisms of how TSPs modify protein quality control and intracellular transport and secretion, knockout mouse embryonic fibroblasts (MEF) will serve as an in vitro model. Furthermore, in order to define the specific protein domains responsible for the interplay between TSPs and ATF6 to induce a protective ER stress response, overexpression of single protein domain constructs of TSPs and ATF6 will be used in TSP or ATF6 knockout MEFs.