Nesprin-2-dependent ERK1/2 compartmentalisation regulates the DNA damage response in vascular smooth muscle cell ageing

Prelamin A accumulation and persistent DNA damage response (DDR) are hallmarks of vascular smooth muscle cell (VSMC) ageing and dysfunction. Although prelamin A is proposed to interfere with DNA repair, our understanding of the crosstalk between prelamin A and the repair process remains limited. The extracellular signal-regulated kinases 1 and 2 (ERK1/2) have emerged as key players in the DDR and are known to enhance ataxia telangiectasia-mutated protein (ATM) activity at DNA lesions, and in this study, we identified a novel relationship between prelamin A accumulation and ERK1/2 nuclear compartmentalisation during VSMC ageing. We show both prelamin A accumulation and increased DNA damage occur concomitantly, before VSMC replicative senescence, and induce the localisation of ERK1/2 to promyelocytic leukaemia protein nuclear bodies (PML NBs) at the sites of DNA damage via nesprin-2 and lamin A interactions. Importantly, VSMCs treated with DNA damaging agents also displayed prelamin A accumulation and ERK compartmentalisation at PML NBs, suggesting that prelamin A and nesprin-2 are novel components of the DDR. In support of this, disruption of ERK compartmentalisation at PML NBs, by either depletion of nesprin-2 or lamins A/C, resulted in the loss of ATM from DNA lesions. However, ATM signalling and DNA repair remained intact after lamins A/C depletion, whereas nesprin-2 disruption ablated downstream Chk2 activation and induced genomic instability. We conclude that lamins A/C and PML act as scaffolds to organise DNA-repair foci and compartmentalise nesprin-2/ERK signalling. However, nesprin-2/ERK signalling fidelity, but not their compartmentalisation at PML NBs, is essential for efficient DDR in VSMCs.DNA damage is a major driving force during cellular ageing, and it has been implicated in hastening the development of cardiovascular diseases, including atherosclerosis where the accumulation of senescent cells has been shown to accelerate disease.¹ Normally, DNA damage is efficiently repaired by the DNA damage response (DDR), a complex signalling cascade of proteins that include sensors (NBS1/MRE11), transducers (ataxia telangiectasia-mutated protein (ATM)/ataxia telangiectasia- and Rad3-related protein (ATR)) and effectors (p53/p21). However, if damage is overwhelming or repair is inefficient, the accumulation of unrepaired DNA damage leads to persistent DNA damage signalling and premature senescence.² Recently, the nuclear lamina has been implicated in the DDR, and its disruption is associated with accelerated cardiovascular ageing.³The nuclear lamina is composed of A-type (lamins A/C) and B-type (lamins B1/B2) lamins that underlie the nuclear envelope (NE) and extend throughout the nucleoplasm⁴ to form a scaffold that is essential for the compartmentalisation and the integrity of nuclear signalling.⁴ The pathological accumulation of lamin A precursors such as prelamin A or progerin causes Hutchinson Gilford Progeria Syndrome (HGPS), a disease of accelerated ageing where patients develop severe early-onset arteriosclerosis characterised by vascular smooth muscle cell (VSMC) attrition.⁴ During the DDR, lamin A interacts with Ku70 and γH2AX, and forms a framework that is essential for the positional stability of repair foci.^{5, 6} Prelamin A accumulation interferes with DNA-repair processes and has been shown to delay the recruitment of DNA-repair proteins such as 53BP1 to double strand breaks (DSBs)⁷ and cause mislocalisation of DNA PK_cs.⁸ Fibroblasts and induced pluripotent stem (iPS) cells derived from HGPS patients show persistent DNA damage signalling and premature senescence.⁸ Importantly, aged VSMCs, both in vitro and in vivo, also accumulate prelamin A and activate persistent DDR, suggesting a key role for the nuclear lamina in vascular ageing.²The nesprin family of spectrin-repeat (SR) proteins were first identified as NE lamin A-binding proteins. However, both nesprin-1 and nesprin-2 show extensive alternate splicing, and variants have been shown to localise to multiple nuclear and cytoplasmic compartments.⁹ One such variant, nesprin-2βΔKASH1, retains a lamin A-binding region and localises to promyelocytic leukaemia protein nuclear bodies (PML NBs) in VSMCs.¹⁰ Previously, we demonstrated that nesprin-2βΔKASH1 scaffolds extracellular signal-regulated kinases 1 and 2 (ERK1/2) at PML NBs and acts to regulate nuclear ERK1/2 activity and downstream VSMC proliferation.¹⁰ Importantly, both ERK and PML NBs have also been implicated in the DDR; PML and ERK1/2 localise at DNA lesions where ERK1/2 enhance ATM- and ATR-mediated repair.^{11, 12, 13} In addition, PML and ERK1/2 are essential for regulating the cell cycle in response to DNA damage; PML forms nucleolar cap structures that sequester MDM2 and activate DNA damage-mediated p53 signalling, while ERK1/2 are essential for efficient G2/M checkpoint activation.^{14, 15, 16} Similar to nesprin-2, PML and ERK1/2 have also been shown to associate with the nuclear lamina suggesting that the nuclear lamina, potentially via nesprin-2βΔKASH1, may regulate ERK compartmentalisation during the DDR.^{17, 18}In this study, we identify a novel signalling complex that regulates compartmentalisation of ERK1/2 during the DDR in VSMCs. We show that the nuclear lamina tethers PML NBs and spatially organises nuclear signalling events. Disruption of this organisation results in ATM mislocalisation from DNA-repair foci and impairs downstream DNA-repair signalling, ultimately leading to genomic instability.