Conserved cysteine residues in the mammalian lamin A tail are essential for cellular responses to ROS generation.

Pre-lamin A and progerin have been implicated in normal aging, and the pathogenesis of age-related degenerative diseases is termed 'laminopathies'. Here, we show that mature lamin A has an essential role in cellular fitness and that oxidative damage to lamin A is involved in cellular senescence. Primary human dermal fibroblasts (HDFs) aged replicatively or by pro-oxidants acquire a range of dysmorphic nuclear shapes. We observed that conserved cysteine residues in the lamin A tail domain become hyperoxidized in senescent fibroblasts, which inhibits the formation of lamin A inter- and intramolecular disulfide bonds. Both in the absence of lamin A and in the presence of a lamin A cysteine-to-alanine mutant, which eliminates these cysteine residues (522, 588, and 591), mild oxidative stress induced nuclear disorganization and led to premature senescence as a result of decreased tolerance to ROS stimulators. Human dermal fibroblasts lacking lamin A or expressing the lamin A cysteine-to-alanine mutant displayed a gene expression profile of ROS-responsive genes characteristic of chronic ROS stimulation. Our findings suggest that the conserved C-terminal cysteine residues are essential for lamin A function and that loss or oxidative damage to these cysteine residues promotes cellular senescence.

A flow cytometry-based screen of nuclear envelope transmembrane proteins identifies NET4/Tmem53 as involved in stress-dependent cell cycle withdrawal.

Disruption of cell cycle regulation is one mechanism proposed for how nuclear envelope protein mutation can cause disease. Thus far only a few nuclear envelope proteins have been tested/found to affect cell cycle progression: to identify others, 39 novel nuclear envelope transmembrane proteins were screened for their ability to alter flow cytometry cell cycle/DNA content profiles when exogenously expressed. Eight had notable effects with seven increasing and one decreasing the 4N:2N ratio. We subsequently focused on NET4/Tmem53 that lost its effects in p53(-/-) cells and retinoblastoma protein-deficient cells. NET4/TMEM53 knockdown by siRNA altered flow cytometry cell cycle/DNA content profiles in a similar way as overexpression. NET4/TMEM53 knockdown did not affect total retinoblastoma protein levels, unlike nuclear envelope-associated proteins Lamin A and LAP2α. However, a decrease in phosphorylated retinoblastoma protein was observed along with a doubling of p53 levels and a 7-fold increase in p21. Consequently cells withdrew from the cell cycle, which was confirmed in MRC5 cells by a drop in the percentage of cells expressing Ki-67 antigen and an increase in the number of cells stained for ß-galactosidase. The ß-galactosidase upregulation suggests that cells become prematurely senescent. Finally, the changes in retinoblastoma protein, p53, and p21 resulting from loss of NET4/Tmem53 were dependent upon active p38 MAP kinase. The finding that roughly a fifth of nuclear envelope transmembrane proteins screened yielded alterations in flow cytometry cell cycle/DNA content profiles suggests a much greater influence of the nuclear envelope on the cell cycle than is widely held.

Adult stem cell maintenance and tissue regeneration in the ageing context: the role for A-type lamins as intrinsic modulators of ageing in adult stem cells and their niches.

Adult stem cells have been identified in most mammalian tissues of the adult body and are known to support the continuous repair and regeneration of tissues. A generalized decline in tissue regenerative responses associated with age is believed to result from a depletion and/or a loss of function of adult stem cells, which itself may be a driving cause of many age-related disease pathologies. Here we review the striking similarities between tissue phenotypes seen in many degenerative conditions associated with old age and those reported in age-related nuclear envelope disorders caused by mutations in the LMNA gene. The concept is beginning to emerge that nuclear filament proteins, A-type lamins, may act as signalling receptors in the nucleus required for receiving and/or transducing upstream cytosolic signals in a number of pathways central to adult stem cell maintenance as well as adaptive responses to stress. We propose that during ageing and in diseases caused by lamin A mutations, dysfunction of the A-type lamin stress-resistant signalling network in adult stem cells, their progenitors and/or stem cell niches leads to a loss of protection against growth-related stress. This in turn triggers an inappropriate activation or a complete failure of self-renewal pathways with the consequent initiation of stress-induced senescence. As such, A-type lamins should be regarded as intrinsic modulators of ageing within adult stem cells and their niches that are essential for survival to old age.

The MAGUK-family protein CASK is targeted to nuclei of the basal epidermis and controls keratinocyte proliferation.

The Ca2+/calmodulin-associated Ser/Thr kinase (CASK) binds syndecans and other cell-surface proteins through its PDZ domain and has been implicated in synaptic assembly, epithelial polarity and neuronal gene transcription. We show here that CASK regulates proliferation and adhesion of epidermal keratinocytes. CASK is localised in nuclei of basal keratinocytes in newborn rodent skin and developing hair follicles. Induction of differentiation shifts CASK to the cell membrane, whereas in keratinocytes that have been re-stimulated after serum starvation CASK localisation shifts away from membranes upon entry to S phase. Biochemical fractionation demonstrates that CASK has several subnuclear targets and is found in both nucleoplasmic and nucleoskeletal pools. Knockdown of CASK by RNA interference leads to increased proliferation in cultured keratinocytes and in organotypic skin raft cultures. Accelerated cell cycling in CASK knockdown cells is associated with upregulation of Myc and hyperphosphorylation of Rb. Moreover, CASK-knockdown cells show increased hyperproliferative response to KGF and TGFalpha, and accelerated attachment and spreading to the collagenous matrix. These functions are reflected in wound healing, where CASK is downregulated in migrating and proliferating wound-edge keratinocytes.

Nucleoplasmic LAP2alpha-lamin A complexes are required to maintain a proliferative state in human fibroblasts.

In human diploid fibroblasts (HDFs), expression of lamina-associated polypeptide 2 alpha (LAP2alpha) upon entry and exit from G(0) is tightly correlated with phosphorylation and subnuclear localization of retinoblastoma protein (Rb). Phosphoisoforms of Rb and LAP2alpha are down-regulated in G(0). Although RbS780 phosphoform and LAP2alpha are up-regulated upon reentry into G(1) and colocalize in the nucleoplasm, RbS795 migrates between nucleoplasmic and speckle compartments. In HDFs, which are null for lamins A/C, LAP2alpha is mislocalized within nuclear aggregates, and this is correlated with cell cycle arrest and accumulation of Rb within speckles. Nuclear retention of nucleoplasmic Rb during G(1) phase but not of speckle-associated Rb depends on lamin A/C. siRNA knock down of LAP2alpha or lamin A/C in HDFs leads to accumulation of Rb in speckles and G(1) arrest, probably because of activation of a cell cycle checkpoint. Our results suggest that LAP2alpha and lamin A/C are involved in controlling Rb localization and phosphorylation, and a lack or mislocalization of either protein leads to cell cycle arrest in HDFs.