Identification of Arhgap28, a New Regulator of Stress Fibre Formation in Cells Assembling a Fibrous Extracellular Matrix

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Abstract

The motivation for this PhD thesis was to understand the molecular basis of how cells regulate the formation of an organised and mechanically strong extracellular matrix (ECM). In tendon this process begins during embryogenesis with the appearance of bundles of narrow-diameter (~30 nm) collagen fibrils that are parallel to the tendon long axis. At the onset of collagen fibrillogenesis, the cells elongate, the fibrils are constrained within plasma membrane channels with their ends contained in tension-sensitive actin-stabilised plasma membrane protrusions. The mechanism by which actin is reorganised during cell elongation and the formation of tension-sensitive plasma membrane protrusions is poorly understood. The small GTPase RhoA is the major regulator of actin reorganisation into stress fibres, which have been implicated in mechanotransduction, ECM assembly and remodelling. The hypothesis tested by this PhD thesis was that the organisation and tensioning of extracellular collagen fibrils is generated on a blueprint of tensioned actin filaments within the cell. Rho activity is regulated specifically by Rho GTPase activating proteins (RhoGAPs). By comparing the global gene expression of tendon tissues at different developmental stages, Arhgap28, a novel RhoGAP, which is expressed during tendon development but not during postnatal maturation, was identified.Arhgap28 belongs to a large family of RhoGAPs containing the closely related members, Arhgap6 and Arhgap18, which have previously been shown to regulate RhoA and stress fibre formation. Arhgap28 expression was upregulated in embryonic fibroblasts cultured in a 3D, tensioned embryonic tendon-like construct compared to monolayer culture. Arhgap28 expression was further enhanced during the development of mechanical strength and stiffness of the tendon constructs, but downregulated when the tension in tendon constructs was released. Overexpression of a C-terminal V5-tagged Arhgap28 protein caused a reduction in RhoA activation and disruption of stress fibre assembly. Modulation of Rho signalling using lysophosphatidic acid and Y27632 showed that collagen remodelling by cells in collagen gels and tendon constructs is regulated by RhoA signalling. A tissue-wide qPCR analysis identified Arhgap28 in several tissues including tendon, bone, and skin. An Arhgap28 reporter mouse (Arhgap28gt) and an Arhgap28 knockout mouse (Arhgap28del) were also studied to investigate the role of Arhgap28 in tissue organisation in vivo. Arhgap28gt mice showed Arhgap28 expression in bones at E18.5. Homozygous Arhgap28del mice were viable, appeared normal but expressed a truncated Arhgap28 transcript, which if translated, would produce a protein lacking the RhoGAP domain. Therefore, it was hypothesised that knockout mice were normal due to compensation from another RhoGAP. Overexpression of Arhgap6 in Arhgap28-null bone tissues was confirmed. Upregulation in RhoA expression was also detected, further suggesting that Arhgap28 regulates RhoA. Interestingly, a microarray comparison of bone tissues from wild type and Arhgap28-null mice showed that genes linked to bone dysplasia are downregulated in Arhgap28-null bone. Together, these results suggest that formation of a strong and organised collagen ECM is mediated by RhoA-generated cellular tension and that Arhgap28 and Arhgap6 might be co-regulators of this process.

Layman's Abstract

Discovery of Arhgap28, a Molecule Involved in Controlling Cell Forces Important in Making a Strong TendonMovements of the body such as running or gripping a door handle require strong tissue types such as muscle, bone, tendon, ligament and cartilage. These tissues make up the musculoskeletal system. Their ability to transmit and withstand mechanical loading is because of a rope-like protein called collagen, which surrounds the cells. Collagen is important as it allows tissues of the body to endure the constant forces of being pulled, pushed and squashed. In bone tissue, these fibrils are woven together to allow calcium to bind, which creates strong bone that can bear the weight of the body. The collagen ropes or ‘fibrils’ in tendon are bundled together like a rope so that tendons can be stretched repetitively without breaking. How the collagen fibrils are organised is linked to the function of the tissue. A disadvantage of having such a unique and strong tissue structure is that once damaged, the collagen fibrils do not fully repair, often resulting in poor healing and long-term recovery. It is estimated that the annual cost of tendon injury in the UK is £25 billion. Scientists led by Professor Karl Kadler at the University of Manchester want to understand how organisation of collagen fibrils is established during the formation of musculoskeletal tissues, so that new medical treatments to improve tissue healing may be developed. As a starting point, the scientists began studying tendon tissue. Since 2004, they have discovered that the cells that live in tendon are responsible for organising the collagen fibrils into non-crisscrossing, straight strands. They found that the cell scaffolding plays important roles in the organisation of collagen fibrils and in generating pulling forces used to make tendons strong. This PhD project investigated the idea that the organisation of the scaffolding inside the cell is responsible for determining the arrangement of the collagen fibrils outside the cell. Scientists found a gene called Arhgap28 that is more prevalent in cells when they make tendon tissues. Experiments showed that Arhgap28 could prevent the cell scaffolding from generating pulling forces. Scientists now think that Arhgap28 controls how the cell is able to touch and pull on fibrils so that they can be arranged correctly and produce a tissue that is not too stiff and not too loose. Further understanding of Arhgap28 would pave the way for improved therapeutics for musculoskeletal injuries in the future.

Keyword(s)

Arhgap28; Rho GTPase activating protein; extracellular matrix; tendon; tendon

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