p38 (MAPK) stress signalling in replicative senescence in fibroblasts from progeroid and genomic instability syndromes

Abstract

Werner Syndrome (WS) is a human segmental progeria resulting from mutations in a DNA helicase. WS fibroblasts have a shortened replicative capacity, an aged appearance, and activated p38 MAPK, features that can be modulated by inhibition of the p38 pathway. Loss of the WRNp RecQ helicase has been shown to result in replicative stress, suggesting that a link between faulty DNA repair and stress-induced premature cellular senescence may lead to premature ageing in WS. Other progeroid syndromes that share overlapping pathophysiological features with WS also show defects in DNA processing, raising the possibility that faulty DNA repair, leading to replicative stress and premature cellular senescence, might be a more widespread feature of premature ageing syndromes. We therefore analysed replicative capacity, cellular morphology and p38 activation, and the effects of p38 inhibition, in fibroblasts from a range of progeroid syndromes. In general, populations of young fibroblasts from non-WS progeroid syndromes do not have a high level of cells with an enlarged morphology and F-actin stress fibres, unlike young WS cells, although this varies between strains. p38 activation and phosphorylated HSP27 levels generally correlate well with cellular morphology, and treatment with the p38 inhibitor SB203580 effects cellular morphology only in strains with enlarged cells and phosphorylated HSP27. For some syndromes fibroblast replicative capacity was within the normal range, whereas for others it was significantly shorter (e.g. HGPS and DKC). However, although in most cases SB203580 extended replicative capacity, with the exception of WS and DKC the magnitude of the effect was not significantly different from normal dermal fibroblasts. This suggests that stress-induced premature cellular senescence via p38 activation is restricted to a small subset of progeroid syndromes.

Fig. 1

Bar chart showing mean percentage increase in experimental replicative capacity using SB203580 for each syndrome; the probability that the increase seen is within the range for normal cells is given for those that are statistically significant

Fig. 2

Observation of cellular morphology and F-actin stress fibres in progeroid and genomic instability syndrome fibroblasts. Cells were fixed and stained with phalloidin-FITC. For each panel, the cell type and PD values are given. For a and b a suffix.c are control cells and a suffix.sb are cells treated with SB203580: for the other syndromes the effects of SB203580 are not shown as the effects are small. For syndromes where the cellular morphology differs little from normal cells only representative strains are shown. Bars are 100 μm

Fig. 3

Immunoblot analysis of stress signalling proteins. Protein lysates were prepared from primary cells and from hTERT-immortalized HCA2 cells. Expression levels were compared for phosphorylated p38 (p-p38), p38, phosphorylated HSP27 (p-HSP27) and HSP27. HCA2^tert cells and HCA2^tert cells treated with anisomycin were used as negative and positive controls respectively for antibody efficacy (shown in a). Three sets of immunoblots were done and are shown as a, b, and c, for each immunoblot HCA2 cells were used as an internal standard (note that the black lines in b and c indicate that the single lanes have been cut and pasted from the same gels as the rest of the samples for each panel, however, the images have been handled in the same manner otherwise). Protein levels were normalised with respect to total p38

Fig. 4

Assessment of stress p38 activity in TERT-transduced lines. Immunoblot for phosphorylated p38 (p-p38) and p38 [N(AG16409)^tert cells ± anisomycin treatment were used as controls]