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Progerin and Telomere Dysfunction Trigger Cellular Senescence in Normal Human Fibroblasts
Posted on: July 21, 2011

Cellular senescence, the finite division capacity of normal somatic cells in vitro, has long been used as a cellular model for understanding mechanisms underlying normal aging. A popular hypothesis of cellular senescence is that progressive attrition of telomeric DNA results in loss of telomere capping proteins, exposing DNA breaks that activate cell cycle arrest and senescence. Telomeres are the specialized structures at the chromosome ends, made up of many kilobases of a simple DNA repeat (TTAGGG)n bound by a multiprotein complex known as shelterin. Telomeres cannot be copied to their extreme termini by DNA polymerase, and so undergo progressive shortening unless elongated by a ribonucleoprotein named telomerase. Telomerase is composed of a reverse transcriptase (TERT) and an RNA component (TERC) that serves as a template for telomere elongation. In somatic cells lacking telomerase, gradual telomere loss and ultimate senescence are inevitable. Consistent with this model, there is a strong association between cell immortalization and persistent telomerase expression. Besides telomere shortening, cellular senescence can be triggered by a variety of environmental and intracellular stimuli, including γ-irradiation, oxidative stress, and overexpression of certain oncogenes, such as H-rasV12. Moreover, mutations in certain genes, such as lamin A (LMNA; accession no. NM_170707), have been linked to premature senescence.

LMNA encodes 2 intermediate filament proteins, lamin A and lamin C. Together with lamin B, they form a dynamic meshwork located just inside the nuclear inner membrane, named the nuclear lamina. The lamina provides important mechanical support to the nuclear structure and also influences chromatin organization, gene expression, and DNA replication. To date, at least 13 human diseases (referred to as the laminopathies) have been associated with the mutations in lamin genes. Among them, Hutchinson-Gilford progeria syndrome (HGPS) has received the most attention because of its striking premature aging phenotype, including alopecia, diminished subcutaneous fat, premature atherosclerosis, and skeletal abnormalities. Children with HGPS die at an average age of 12 years, usually from heart attack or stroke. The vast majority of HGPS cases are associated with a de novo nucleotide substitution at position 1824 (C→T) in the LMNA gene. This mutation does not affect the coded amino acid (and is thus generally referred to as G608G), but partially activates a cryptic splice donor site in exon 11 of LMNA, leading to the production of a prelamin A mRNA that contains an internal deletion of 150 base pairs. This transcript is then translated into a protein known as progerin, which lacks 50 amino acids near the C terminus. Indeed, progerin has been found to accumulate in multiple tissues in biopsies from HGPS patients, including skin, tongue, breast, heart, liver, kidney, stomach, bladder, diaphragm, pancreas, spleen, thyroid, adipose tissue, joint cartilage, bone, skeletal muscle, heart, and large and small arteries.

The cellular phenotypes in HGPS include blebbing (i.e., abnormal shape) of nuclei, thickening of the nuclear lamina, loss of peripheral heterochromatin, clustering of nuclear pores, and premature senescence. Interestingly, prior to developing these obvious nuclear morphological changes, fibroblasts from HGPS patients exhibit broad abnormalities in histone modification patterns. HGPS fibroblasts also show global changes in gene expression and a delayed response in DNA-damage repair.

Gene transfer experiments have left no doubt that progerin acts as a dominant negative. Progerin expression induces multiple defects during mitosis: cytokinesis delay, abnormal chromosome segregation, and binucleation. Several LMNA mutant mouse models have been created. A transgenic model carrying the G608G mutated human LMNA in a bacterial artificial chromosome (BAC) shows progressive loss of vascular smooth muscle cells in the medial layer of large arteries, closely resembling the most lethal aspect of the human phenotype.

The molecular mechanism of progerin toxicity is at least partially understood. Normal lamin A is farnesylated at its C terminus, and that posttranslational modification is thought to play a role in targeting lamin A to the inner nuclear membrane. But subsequently, the ZMPSTE24 endoprotease cleaves off the last 18 amino acids at the C terminus of lamin A, including the farnesyl tail. This releases lamin A from its membrane anchor and allows it to take its place in the nuclear scaffold. The 50-amino acid internal deletion in progerin includes the ZMPSTE24 cleavage site, and thus progerin remains permanently farnesylated. Recently, treatment with farnesyl transferase inhibitors (FTIs) has been shown to improve progerin-induced cellular phenotypes in vitro and in several HGPS mouse models. A clinical trial with an FTI in children with HGPS was initiated in May 2007.

It has always been of great interest to determine the biological relevance of HGPS to normal aging. This interest was heightened by the detection of progerin mRNA and progerin protein in cells obtained from healthy individuals, which indicates that the cryptic splice site activated by the HGPS mutation is also capable of being used in the presence of the normal sequence of exon 11. Recently, progerin transcripts and progerin were identified in vivo in skin biopsies from healthy individuals, and a recent analysis suggested that progerin transcript levels increase in late-passage cells from HGPS patients and parental controls.

Using a reporter construct, Scaffidi et al. showed that the normal LMNA sequence at G608G could also be used as a weak splice donor to produce progerin in normal individuals. Collins et al have shown that a significant percentage of progerin-positive cells from normal individuals are morphologically abnormal, exhibiting phenotypes that resemble HGPS cells. This suggests that activation of progerin expression in normal cells, triggered by some unknown signal, may contribute to senescence.

However, the cause-and-effect relationship between normal aging and progerin production in normal individuals has not been determined. Collins et al. assayed the effects of cellular passage and donor age on the activation of progerin production and found that the cryptic splice donor site that produces progerin is activated in senescent cells. Screening various primary and transformed cell lines revealed an interesting inverse correlation between cell immortalization and progerin transcription, and ectopic expression of telomerase in normal fibroblast cells resulted in a significant decrease in progerin production. In addition, it has been found that progerin production was not induced in telomere-independent, oncogene-driven senescence, further supporting a potential causal relationship between telomere-induced senescence and progerin production. Consistent with this model, it was found that elevated levels of progerin were induced in fibroblast cells whose telomeres had been uncapped. With splicing-sensitive exon microarrays, it was further shown that extensive changes in alternative splicing of multiple genes, including LMNA, occurred as telomeres shortened and cells approached senescence. Taken together, these findings are suggestive of synergism between telomere damage and progerin production in induction of cellular aging.

Source: Kan Cao, Cecilia D. Blair, Dina A. Faddah, Julia E. Kieckhaefer, Michelle Olive, Michael R. Erdos, Elizabeth G. Nabel and Francis S. Collins; Progerin and telomere dysfunction collaborate to trigger cellular senescence in normal human fibroblasts.; J Clin Invest. 2011;121(7):2833-2844.doi:10.1172/JCI43578.
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