Ong, V.H.; (2009) Expression and regulation of monocyte chemoattractant protein-3 (MCP-3) in fibrosis. Doctoral thesis , UCL (University College London).

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Abstract

Systemic sclerosis is a multisystem connective tissue disease characterised by skin thickening and widespread, but variable, visceral fibrosis. The aetiopathogenesis is likely to involve immunological activation and microvascular dysfunction leading to excessive accumulation of extracellular matrix (ECM) with increased production of collagen type I in lesional tissues. This implies a dysregulated repair process probably as a consequence of aberrant crosstalk between fibroblasts and inflammatory cells. It has been proposed that a hierarchical cascade of soluble mediators in which initial induction of proinflammatory cytokines expressed by the inflammatory infiltrate may lead to expression of profibrotic mediators including TGFβ. A salient feature of the inflammatory response is directional migration of leucocytes into subendothelial tissues orchestrated by chemokines in a spatially and temporally-regulated multistep process. Work described in this thesis explores the expression of chemokine, monocyte chemoattractant protein-3 (MCP-3/CCL7) in SSc and in murine models for SSc: type 1 tight skin mouse (Tsk1) and a transgenic mouse strain (TβRIIΔk) in which there is fibroblast-directed disruption of TGFβ signalling. The hypothesis that crosstalk between MCP-3 and TGFβ may modulate the signalling response in the fibrotic microenvironment was also explored. Overexpression of MCP-3 was demonstrated on cDNA expression profiling and protein analysis of neonatal Tsk1 and TβRIIΔk fibroblasts. This was supported by immunohistochemical studies on dermal tissues. Similar upregulation dermal patterns of MCP-3 protein expression were observed in the early stage of diffuse cutaneous SSc. Activation of collagen reporter genes by MCP-3 in transgenic mouse fibroblasts and wildtype neonatal mouse fibroblasts harbouring proα2(Ι)collagen promoter reporter gene constructs is mediated via sequences within the proximal promoter and is partly dependent on TGFβ. This coinduction between the two factors in the fibrotic response is also demonstrated by activation of TGFβ signalling pathways by MCP-3 leading to type I collagen secretion. In addition, MCP-3 gene expression is stimulated by TGFβ. Comparison of downstream signalling pathways that regulate collagen gene activation by both cytokines confirms the central role of MAPK pathway activation in mediating the effects of both factors. An additive effect of these two agonists was demonstrated for key TGFβ-regulated genes on comparative microarray analysis. Overall, these results demonstrate that overexpression of MCP-3 is a key biochemical feature of early stage SSc and murine models of SSc, and suggest a novel role for this chemokine as a profibrotic mediator in addition to its role in regulating leucocyte recruitment. Furthermore, there is a potentially important interplay between MCP-3 and TGFβ in modulation of the signalling response in the fibrotic microenvironment.

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