PRIMARY CILIA OF SYSTEMIC SCLEROSIS (SSC) DERMAL FIBROBLASTS ARE DISRUPTED BY DOWNREGULATION OF CAVEOLIN-1 AND ABERRANT ACTIVITY OF AURORA A KINASE, INDEPENDENT OF TRANSFORMING GROWTH FACTOR BETA.

Wells, R.; Ross, R.; Timmis, A.; Georgiou, I.; Johnson, C.; Del Galdo, N.; Del Galdo, F.

PRIMARY CILIA OF SYSTEMIC SCLEROSIS (SSC) DERMAL FIBROBLASTS ARE DISRUPTED BY DOWNREGULATION OF CAVEOLIN-1 AND ABERRANT ACTIVITY OF AURORA A KINASE, INDEPENDENT OF TRANSFORMING GROWTH FACTOR BETA.

All Authors

Wells, R.

Ross, R.

Timmis, A.

Georgiou, I.

Johnson, C.

Del Galdo, N.

Del Galdo, F.

LTHT Author

Ross, Rebecca
Del Galdo, Francesco

LTHT Department

NIHR Leeds Biomedical Research Centre
Rheumatology

Publication Date

2024

Item Type

Conference Abstract

Abstract

Introduction: Systemic Sclerosis (SSc) is characterised by abnormal activation of tissue fibroblasts. The cellular and molecular mechanisms driving this activation remain unclear, but aberrant TGFbeta signalling activation and downregulation of cell membrane component Caveolin-1 are implicated. Both TGFbeta and Caveolin-1 have roles at the primary cilium (PC), an antenna-like organelle which forms a signalling hub for pathways involved in cellular homeostasis. Several of these pathways are dysregulated in SSc. This study aimed to examine PC structure in SSc dermal fibroblasts to determine its potential contribution to SSc pathogenesis via signalling dysregulation, and investigate TGFbeta activation and Caveolin-1 downregulation in any abnormal ciliary phenotype. Material(s) and Method(s): Dermal fibroblasts from healthy control (HC) and SSc patients (limited, diffuse, and VEDOSS) were isolated. PC were visualised by fluorescent confocal microscopy. Average PC length was quantified from at least 100 PC across 3 fields per condition. Fibroblasts were treated with exogenous TGFbeta, TGFbetaR1 inhibitor SD208, ROCK2 inhibitor KD025, and Aurora A kinase (AURKA) inhibitor MLN8054. Canonical TGFbeta signalling was inhibited with siRNA targeting SMAD3. HC fibroblasts were treated with shRNA against Caveolin-1 (shCAV) to mimic SSc-associated downregulation, or Scramble control shRNA (shSCR). Result(s): SSc patient fibroblast PC were, on average, 45% shorter than HC (2.5+/-0.6mum vs 4.6+/-1.3mum (P<0.0001)), regardless of SSc subtype. HC shCAV fibroblasts displayed significantly shorter PC (3.0+/-0.8mum) together with already described increased fibrotic markers and sensitivity to TGFbeta. TGFbeta treatment significantly reduced PC length in both HC and SSc. The effect of TGFbeta was fully abolished by TGFbetaR1 inhibition, partially abolished by ROCK2 inhibition, and unaffected by SMAD3 knockdown, but none of these treatments were able to 'rescue' SSc or shCAV cilia length to that of HC. Inhibition of AURKA, known to be involved in ciliary disassembly, did not prevent TGFbeta-induced shortening, but did restore PC length in SSc and shCAV fibroblasts to lengths resembling HC and shSCR controls, leaving HC and shSCR cilia unaffected. Conclusion(s): PC length is stably reduced in SSc dermal fibroblasts, early in disease, with Caveolin-1 downregulation and aberrant AURKA activity contributing to the shorter cilium. While TGFbeta activation can shorten PC via a non-canonical pathway involving ROCK2, this is not responsible for the short cilia phenotype in SSc. These findings support the notion that SSc profibrotic activation may involve mechanisms beyond TGFbeta, and highlight the potential significance of Caveolin-1 and AURKA in modulating PC length as contributors to profibrotic activation, and potential therapeutic targets for tissue fibrosis.