The effects of overexpression of lamin a and two mutants associated with premature aging on stem cell differentiation and proliferation.

Abstract

The nuclear lamina is composed of A and B-type lamins and performs a number of functions within the nucleus. Mutations within lamins give rise to a group of diseases called laminopathies including autosomal dominant Emery-Dreifuss muscular dystrophy and lipodystrophy. Hutchinson Gilford Progeria Syndrome (HOPS), or so-called premature aging, is a rare and devastating laminopathy disorder caused by mutations in LMNA that leads to the production of a truncated mutant form of prelamin A, called progerin or LAA50, that cannot undergo proteolytic processing to yield mature lamin A. In an atypical form of HOPS, a point mutation at amino acid 644 alters the Zmpste24 endoproteolytic cleavage site, causing the production of mutant prelamin A. Why and how these prelamin A mutants cause such devastating phenotypes is not folly understood. It has been suggested that the mutant prelamin A may affect the ability of adult stem cells to self-renewal and differentiate, which are essential processes in order to replace damage of old tissues, critical for organismal longevity. Herein, expression plasmids containing two mutations found in classical and atypical HOPS were transiently overexpressed in clonal rat dermal papilla (DP) 9 cells, which, in vivo, reside at the base of the hair follicle and play an important role in hair follicle cycling. DP9 cells showed an increased number of nuclear abnormalities compared to control cells, which have previously been shown to be characteristic of cells from HGPS patients. DP9 cell lines stably overexpressing either FLAG-prelamin A， FLAG-prelamin ΑΔ50 or FLAG-prelamm A(R644C) were created. To assess the ability of the stem cells to self-renew, stable cell lines were routinely passaged and counted. Results indicate that stable overexpression of FLAG-prelamin ΑΔ50 and FLAG-prelamin A(R644C) inhibits the ability of stem cells to self-renew in vitro. Previous reports have shown that DP9 cells may be directed towards both adipogenic and osteogenic lineages. Therefore, in this study, stable DP9 cell lines expressing FLAG- prelamin A， FLAG-prelamin ΑΔ50 or FLAG-prelamin A(R644C) were exposed to an adipogenic medium for six days. Results showed that overexpression of FLAG-prelamin A, FLAG-prelamm ΑΔ50 and FLAG-prelamm A(R644C) inhibited the accumulation of intracellular lipids, reflecting a decreased ability to differentiate in vitro. Taken together, these results suggest the lipodystrophy and alopecia associated with HGPS may be due to the failure of adult stem cell populations within each tissue to both self-renew and differentiate, and may underpin the disease pathogenesis. Furthermore, these results have implications for other laminopathies that produce mutant prelamin A.