High mobility group 1 (HMGB1) protein is both a nuclear factor and a secreted protein. In the cell nucleus it acts as an architectural chromatin-binding factor that bends DNA and promotes protein assembly on specific DNA targets. Outside the cell, it binds with high affinity to RAGE (the receptor for advanced glycation end products) and is a potent mediator of inflammation. HMGB1 is secreted by activated monocytes and macrophages, and is passively released by necrotic or damaged cells. Here we report that Hmgb1(-/-) necrotic cells have a greatly reduced ability to promote inflammation, which proves that the release of HMGB1 can signal the demise of a cell to its neighbours. Apoptotic cells do not release HMGB1 even after undergoing secondary necrosis and partial autolysis, and thus fail to promote inflammation even if not cleared promptly by phagocytic cells. In apoptotic cells, HMGB1 is bound firmly to chromatin because of generalized underacetylation of histone and is released in the extracellular medium (promoting inflammation) if chromatin deacetylation is prevented. Thus, cells undergoing apoptosis are programmed to withhold the signal that is broadcast by cells that have been damaged or killed by trauma.

High Mobility Group 1 protein (HMGB1) is a chromatin component that, when leaked out by necrotic cells, triggers in¯ammation. HMGB1 can also be secreted by activated monocytes and macrophages, and functions as a late mediator of in¯ammation. Secretion of a nuclear protein requires a tightly controlled relocation program. We show here that in all cells HMGB1 shuttles actively between the nucleus and cytoplasm. Monocytes and macrophages acetylate HMGB1 extensively upon activation with lipopolysaccharide; moreover, forced hyperacetylation of HMGB1 in resting macrophages causes its relocalization to the cytosol. Cytosolic HMGB1 is then concentrated by default into secretory lysosomes, and secreted when monocytic cells receive an appropriate second signal.

Mutations in the nuclear structural protein lamin A cause the premature aging syndrome Hutchinson-Gilford progeria (HGPS). Whether lamin A plays any role in normal aging is unknown. We show that the same molecular mechanism responsible for HGPS is active in healthy cells. Cell nuclei from old individuals acquire defects similar to those of HGPS patient cells, including changes in histone modifications and increased DNA damage. Age-related nuclear defects are caused by sporadic use, in healthy individuals, of the same cryptic splice site in lamin A whose constitutive activation causes HGPS. Inhibition of this splice site reverses the nuclear defects associated with aging. These observations implicate lamin A in physiological aging.

Hutchinson-Gilford progeria syndrome (HGPS) is a childhood premature aging disease caused by a spontaneous point mutation in lamin A, one of the major architectural elements of the mammalian cell nucleus 1-4 . The HGPS mutation activates an aberrant cryptic splice site in the lamin A premRNA, leading to synthesis of a truncated lamin A protein and concomitant reduction of wild type lamin A levels 3,4 . Fibroblasts from HGPS patients have severe morphological abnormalities in nuclear envelope structure. Here we show that the cellular disease phenotype is reversible in cells from HGPS patients. Introduction of wild type lamin A protein does not rescue the cellular disease symptoms. The mutant lamin A mRNA and protein can be efficiently eliminated by correction of the aberrant splicing event using a modified oligonucleotide targeted to the activated cryptic splice site. Upon splicing correction, HGPS fibroblasts assume normal nuclear morphology, the aberrant nuclear distribution and cellular levels of lamina-associated proteins are rescued, defects in heterochromatin-specific histone modifications are corrected, the dynamic properties of lamin A are restored, and proper expression of several misregulated genes is reestablished. Our results establish proof of principle for the application of small molecules to the correction of the premature aging phenotype in HGPS patients.Hutchinson-Gilford Progeria Syndrome is most commonly caused by a de novo heterozygous silent substitution at codon 608 (G608G: GGC>GGT) of the lamin A/C (LMNA) gene 3,4 . The mutation resides in exon 11 of LMNA and activates an exonic cryptic donor splice site four nucleotides upstream. The pre-mRNA derived from the mutated allele is spliced using this aberrant donor splice site and the correct exon 12 acceptor splice site, giving rise to a truncated lamin A mRNA lacking the terminal 150 nucleotides of exon 11 3,4 . As a consequence of the aberrant splicing event a mutant protein, Δ50 lamin A, containing a 50 aa internal deletion in its globular tail domain is generated 3,4 . As previously reported 3-6 , we observed that fibroblasts from HGPS patients are characterized by the presence of dysmorphic nuclei with altered size and shape, exhibiting lobules, wrinkles and herniations as evident by staining for lamin A (Fig. 1a,b). We found aberrant morphology (as assessed by a nuclear envelope contour ratio of < 0.7; see Methods for definition) in typically ~ 70% of cells in multiple HGPS cell lines (Supplementary Table 1 online). In addition to these well-established morphological changes, we found several novel defects in HGPS fibroblasts. In ~ 70% of the cells, lamin B, the major lamin A-interacting partner, and multiple members of the family of lamina-associated polypeptide (LAP2s) were depleted from their typical localization at the nuclear envelope and the global cellular amount of these proteins was reduced up to 6-fold compared to the control cells when assessed by total cellular fluorescence intensity measurements (See Methods) (Fig ...