Role of Thrombospondins in interstitial Extracellular Matrix 3-Dimensional Organisation: Mechanisms and Functions

In our bodies, our skin is supple and our bones are rigid because of the properties of extracellular matrix (ECM) that surrounds the cells in each tissue. ECM is built up from the beginning of development as a suite of large proteins released by cells that bind with each other to form insoluble fibrils and networks. These structures are maintained throughout life, are repaired after injury, and can cause health problems if disrupted in disease. The physical and chemical properties of ECM inform cells whether to be stationary, move, or divide, and modulate the expression of genes. These signals enable cells to function properly throughout life but how the 3-dimensional organisation of ECM is set up and tuned is poorly understood. For all these reasons, research into principles of ECM organisation is important and necessary to identify new therapeutic targets and materials.
The most abundant proteins in ECM are collagens that form elongated fibrils. Many accessory proteins act to maintain the number, sizes, and packing of collagen fibrils, and how the fibrils interact with other ECM components. Accessory proteins are attractive as possible therapeutic targets because they are less abundant (so could be more easy to modulate with a small drug) and yet can act as "levers" to change ECM organisation. Our recent research identified that thrombospondin proteins (TSPs) are conserved with collagens and a few other ECM components throughout animals, from sponges to human. Because of this conservation we consider that the mechanism(s) by which TSPs bind into ECM and modulate collagen fibril organisation are likely to be important and a promising therapeutic target.
In studying how TSPs bind into ECM, we identified a protein sequence motif that is widely conserved between TSPs and is required for them to accumulate into ECM made by cultured cells. TSPs mutated at this site are secreted normally by cells but are not held in the ECM. We discovered recently that this site works by mediating interactions between separate TSP molecules. These interactions cause TSP clusters to form small dot-like structures or "puncta". From this research we developed a model, (a hypothetical diagram), of how TSPs can enter and cluster in ECM to impact on collagen fibril organisation. Our model is novel because neither the molecular identity of the motif or its role in interactions between TSP molecules were known before. Our central goals are:
1), To further understand mechanisms of TSPs within cell culture ECM, to extend the model and strengthen its accuracy.
2), To test how the site we have identified in cell culture works in living animals. To make this test is essential to understand how our discoveries in cell culture relate to protein function in real tissues. With the mouse we can examine the role of the TSP interaction site in the skin of an animal close to human anatomy and physiology that is used widely in biomedical research.
3). From the new genetic strains of mice we will also isolate cells from the skin so that we can closely study their properties in culture and relate the mechanisms that we identify in goal 1 to effects of TSPs on cell functions and ECM properties. These experiments will generate additional materials from the experimental mice and help us understand how our new knowledge might be adapted for clinical materials.

The extracellular matrix (ECM) is an essential component of the tissue environment of cells. Molecular composition, 3-dimensional (3D) organisation and mechanical properties of ECM all provide important cues that control cell phenotype and the assembly of body organs. Standard 2D cell culture does not replicate this environment and the processes by which 3D tissue ECM assembly is maintained through life are poorly understood. This is a major challenge in the ECM field: for example, for in vitro handling of stem cell populations, to maintain cells close to their in vivo differentiation state, or to understand effects of ageing or chronic diseases that are increasing health problems in the UK and worldwide. Progress with advanced cell culture can help reduce and replace animal use in medical research. This application aims to understand molecular systems that support proper assembly of 3D interstitial ECM. We approach this question with a focus on thrombospondins (TSPs), because of recent appreciation that these are very highly-conserved accessory ECM proteins and because of evidence for roles as modulators of collagen fibril organisation in mammals.
Aim 1. Recent data have led us to a novel, unexpected model of a conserved mechanism for how TSPs are deposited into ECM. The mechanistic goal is to understand this process at molecular and cellular levels.
Aim 2. To Identify the specific roles of ECM-bound TSPs in interstitial ECM organisation in vivo. We will examine the roles of ECM-accumulation of TSP1 in the interstitial ECM of mouse dermis, under homeostatic conditions and in the tight skin mouse that has fibrotic excess of dermal ECM.
Aim 3. To elucidate how mechanisms of ECM-accumulation of TSP1 impact on organisation and functions of fibroblast-derived 3D ECM and fibroblast phenotype. Mechanisms identified in Aim 1 will be related to fibroblasts and their native ECM, exploiting fibroblasts from the new genetically-modified mice developed in Aim 2.

Non-academic beneficiaries of our research will include:
1. Clinical researchers working on tissue repair and regeneration, fibrosis, atherosclerosis, arthritis, ageing or pseuodoachondroplasia. Possible impacts: new understanding of the fundamental biology underpinning the conditions they treat, new information about possible therapeutic approaches. New potential collaborations with basic researchers.
2. Biotech industry R+D, specifically companies interested in 3D cell culture, tissue assembly, tissue repair, artificial organs. These contacts will need to be made in the course of the research. We anticipate links may be facilitated by further faculty interactions with Prof. Antony Hollander and his collaborators.
Possible impacts: Impacts: access to information about the basic biology of the ECM. New potential collaborations with basic researchers to develop novel therapeutic approaches. Training possibilities for researchers from my lab. in their labs, and vice versa.

3. Local pupils at primary and secondary school level, prospective undergraduates, teachers.
Impacts: increased knowledge of cutting edge research questions and techniques, increased interest in pursuing science at an advanced level. Increased understanding of mechanisms by which extracellular matrix is built that can inform taught courses on animal cell biology. Research outcomes that will be beneficial to cell biology teaching for demonstrating to students how class materials relate to biomedical research. Increased understanding of the value and limitations of cell culture vs animal use in basic experimental research.

The public will benefit indirectly from the increase in knowledge about basic mechanisms and function of extracellular matrix. Indirectly, and in the long term, people suffering from diseases linked to extracellular matrix pathologies may also benefit.