Membrane-type matrix metalloproteinase I (MT1-MMP)-deficient mice were found to have severe defects in skeletal development and angiogenesis. The craniofacial, axial, and appendicular skeletons were severely affected, leading to a short and domed skull, marked deceleration of postnatal growth, and death by 3 wk of age. Shortening of bones is a consequence of decreased chondrocyte proliferation in the proliferative zone of the growth plates. Defective vascular invasion of cartilage leads to enlargement of hypertrophic zones of growth plates and delayed formation of secondary ossification centers in long bones. In an in vivo corneal angiogenesis assay, null mice did not have angiogenic response to implanted FGF-2, suggesting that the defect in angiogenesis is not restricted to cartilage alone. In tissues from null mice, activation of latent matrix metalloproteinase 2 was deficient, suggesting that MT1-MMP is essential for its activation in vivo. Matrix metalloproteinases (MMPs) are a family of Zndependent enzymes that are essential for extracellular matrix (ECM) turnover in normal and pathological conditions (1, 2). The MMPs are produced as latent proenzymes, and can be inhibited by specific tissue inhibitors of metalloproteinases (TIMPs). The enzymes can be divided into two structurally distinct subgroups, i.e., membrane-type (MT-MMP) and secreted MMPs. The secreted MMPs include interstitial collagenases (MMP-1, -8, and -13), which degrade fibrillar collagens, gelatinases (type IV collagenases, MMP-2 and -9) with high activity against gelatin and type IV collagen, and stromelysins (MMP-3, -10, and -11), which degrade a variety of collagenous and noncollagenous ECM proteins. Although the secreted MMPs have different expression patterns, there seems to be considerable redundancy and overlap between them with respect to function. Thus, mice deficient for MMP-2 (3), MMP-3 (4), MMP-7 (5), MMP-9 (6), MMP-10 (7), or MMP-12 (8) are all viable. Of these, only the MMP-9-deficient mice have been reported to show developmental abnormalities, which involve the growth plate and endochondral ossification (6).To date, five genetically distinct MT-MMPs (Mmp14-17, Mmp 21) have been identified (9-14). These enzymes (except MMP-17, which is glycosylphosphatidylinositol anchored) are singlepass type I membrane proteins with an extracellular N terminus containing the catalytic domain and a short C-terminal cytoplasmic domain. The prototypic MT-MMP, MT1-MMP (also termed MMP-14), was first identified as a cellular receptor and activator for pro-MMP-2 (9), but both MT1-MMP and MT2-MMP (MMP-15) have also been shown to have activity against a variety of ECM proteins, including gelatin, fibronectin, vitronectin, fibrillar collagens, and aggrecan (15, 16). MT1-MMP is widely expressed in cultured cells and tissues during development (17), but its strictly regulated spatial and temporal expression indicates more specific roles for this enzyme (17, 18). A crucial role for MT1-MMP for bone growth was recently demonstrated in MT1-MMP-deficient mi...

Premature aging syndromes often result from mutations in nuclear proteins involved in the maintenance of genomic integrity. Lamin A is a major component of the nuclear lamina and nuclear skeleton. Truncation in lamin A causes Hutchinson-Gilford progerial syndrome (HGPS), a severe form of early-onset premature aging. Lack of functional Zmpste24, a metalloproteinase responsible for the maturation of prelamin A, also results in progeroid phenotypes in mice and humans. We found that Zmpste24-deficient mouse embryonic fibroblasts (MEFs) show increased DNA damage and chromosome aberrations and are more sensitive to DNA-damaging agents. Bone marrow cells isolated from Zmpste24-/- mice show increased aneuploidy and the mice are more sensitive to DNA-damaging agents. Recruitment of p53 binding protein 1 (53BP1) and Rad51 to sites of DNA lesion is impaired in Zmpste24-/- MEFs and in HGPS fibroblasts, resulting in delayed checkpoint response and defective DNA repair. Wild-type MEFs ectopically expressing unprocessible prelamin A show similar defects in checkpoint response and DNA repair. Our results indicate that unprocessed prelamin A and truncated lamin A act dominant negatively to perturb DNA damage response and repair, resulting in genomic instability which might contribute to laminopathy-based premature aging.

The mouse ortholog of human FACE-1, Zmpste24, is a multispanning membrane protein widely distributed in mammalian tissues 1,2 and structurally related to Afc1p/ste24p, a yeast metalloproteinase involved in the maturation of fungal pheromones 3 . Disruption of the gene Zmpste24 caused severe growth retardation and premature death in homozygous-null mice. Histopathological analysis of the mutant mice revealed several abnormalities, including dilated cardiomyopathy, muscular dystrophy and lipodystrophy. These alterations are similar to those developed by mice deficient in A-type lamin 4 , a major component of the nuclear lamina 5 , and phenocopy most defects observed in humans with diverse congenital laminopathies 6-8 . In agreement with this finding, Zmpste24-null mice are defective in the proteolytic processing of prelamin A. This deficiency in prelamin A maturation leads to the generation of abnormalities in nuclear architecture that probably underlie the many phenotypes observed in both mice and humans with mutations in the lamin A gene. These results indicate that prelamin A is a specific substrate for Zmpste24 and demonstrate the usefulness of genetic approaches for identifying the in vivo substrates of proteolytic enzymes.To clarify the function of Zmpste24, and to identify in vivo substrates targeted by this proteinase, we generated Zmpste24-null mice (Fig. 1a). After heterozygote intercrossing, we obtained Zmpste24-null, heterozygous and wildtype mice in the expected mendelian ratio. We verified homozygosity with respect to the mutated allele by Southern blot (Fig. 1b), and lack of Zmpste24 Fig. 1 Generation of Zmpste24-deficient mice. a, Schematic representation of the wildtype Zmpste24 locus (WT), targeting vector and targeted allele (KO). Positions of restriction enzyme sites and probes used for Southern-blot analysis are shown. b, Southern-blot analysis of genomic DNA from three littermate progeny of Zmpste24 heterozygote crosses. Probing of EcoRI-digested DNA revealed fragments of 6 kb and 4 kb for wildtype and disrupted alleles, respectively. Probing of BamHI-digested DNA revealed fragments of 10 kb and 8 kb for wildtype and disrupted alleles, respectively. c, Extracts of kidneys from wildtype and Zmpste24 -/-mice were examined with a monoclonal antibody directed against the C terminus of Zmpste24. d, Photograph of two littermate progeny of a Zmpste24 heterozygote cross, at 3 mo. e, Radiograph of a Zmpste24 -/-mouse at 3 mo, compared with a wildtype control. f, Cumulative plot of body weight versus age. Dots represent mean values, and error bars indicate s.e.m.

Zmpste24 (also called FACE-1) is a metalloproteinase involved in the maturation of lamin A (Lmna), an essential component of the nuclear envelope. Both Zmpste24- and Lmna-deficient mice exhibit profound nuclear architecture abnormalities and multiple histopathological defects that phenocopy an accelerated ageing process. Similarly, diverse human progeroid syndromes are caused by mutations in ZMPSTE24 or LMNA genes. To elucidate the molecular mechanisms underlying these devastating diseases, we have analysed the transcriptional alterations occurring in tissues from Zmpste24-deficient mice. We demonstrate that Zmpste24 deficiency elicits a stress signalling pathway that is evidenced by a marked upregulation of p53 target genes, and accompanied by a senescence phenotype at the cellular level and accelerated ageing at the organismal level. These phenotypes are largely rescued in Zmpste24-/-Lmna+/- mice and partially reversed in Zmpste24-/-p53-/- mice. These findings provide evidence for the existence of a checkpoint response activated by the nuclear abnormalities caused by prelamin A accumulation, and support the concept that hyperactivation of the tumour suppressor p53 may cause accelerated ageing.

SUMMARY Many genes that affect replicative lifespan (RLS) in the budding yeast Saccharomyces cerevisiae also affect aging in other organisms such as C. elegans and M. musculus. We performed a systematic analysis of yeast RLS in a set of 4,698 viable single-gene deletion strains. Multiple functional gene clusters were identified, and full genome-to-genome comparison demonstrated a significant conservation in longevity pathways between yeast and C. elegans. Among the mechanisms of aging identified, deletion of tRNA exporter LOS1 robustly extended lifespan. Dietary restriction (DR) and inhibition of mechanistic Target of Rapamycin (mTOR) exclude Los1 from the nucleus in a Rad53-dependent manner. Moreover, lifespan extension from deletion of LOS1 is non-additive with DR or mTOR inhibition, and results in Gcn4 transcription factor activation. Thus, the DNA damage response and mTOR converge on Los1-mediated nuclear tRNA export to regulate Gcn4 activity and aging.

Collagen XVII, a type II transmembrane protein and epithelial adhesion molecule, can be proteolytically shed from the cell surface to generate a soluble collagen. Here we investigated the release of the ectodomain and identi®ed the enzymes involved. After surface biotinylation of keratinocytes, the ectodomain was detectable in the medium within minutes and remained stable for >48 h. Shedding was enhanced by phorbol esters and inhibited by metalloprotease inhibitors, including hydroxamates and TIMP-3, but not by inhibitors of other protease classes or by TIMP-2. This pro®le implicated MMPs or ADAMs as candidate sheddases. MMP-2, MMP-9 and MT1-MMP were excluded, but TACE, ADAM-10 and ADAM-9 were shown to be expressed in keratinocytes and to be actively involved. Transfection with cDNAs for the three ADAMs resulted in increased shedding and, vice versa, in TACE-de®cient cells shedding was signi®cantly reduced, indicating that transmembrane collagen XVII represents a novel class of substrates for ADAMs. Functionally, release of the ectodomain of collagen XVII from the cell surface was associated with altered keratinocyte motility in vitro.

Strontium ralenate is a new anti-osteoporosis agent. The cellular and molecular mechanism underlying the anabolic effect of strontium on bone remains to be elucidated. Osteoblasts, the main bone forming cells are known to be derived from bone marrow mesenchymal stem cells (MSCs). The present study therefore aimed to investigate the possible effects of strontium on MSCs and signaling pathways possibly involved. It was firstly demonstrated that strontium treatment significantly increased osteoblast-related gene expression and alkaline phosphatase (ALP) of osteogenic-differentiating MSCs. Accompanying the enhanced osteogenic differentiation, the increased phosphorylation of mitogen-activated protein kinase (MAPK) ERK1/2 and p38 was detected in strontium-treated MSCs. PD98059 and SB203580, selective inhibitors of ERK1/2 kinase and p38, attenuated the effect of strontium on osteogenesis. Furthermore, it was demonstrated that Rat Sarcoma viral oncogene homolog (RAS), an upstream regulator of ERK1/2 and p38, was activated by strontium treatment and siRNA-mediated Ras knockdown inhibited strontium-stimulated expression of osteogenic markers. Finally, the transcriptional activity and phosphorylation level of Runx2 was significantly increased in response to strontium treatment in MSCs. PD98059 and Ras siRNA inhibited the effect of strontium on Runx2 activation. Taken together, these results indicated that strontium can promote osteogenic differentiation of MSCs through activating the Ras/MAPK signaling pathway and the downstream transcription factor Runx2.

Specific point mutations in lamin A gene have been shown to accelerate aging in humans and mice. Particularly, a de novo mutation at G608G position impairs lamin A processing to produce the mutant protein progerin, which causes the Hutchinson Gilford progeria syndrome. The premature aging phenotype of Hutchinson Gilford progeria syndrome is largely recapitulated in mice deficient for the lamin A-processing enzyme, Zmpste24. We have previously reported that Zmpste24 deficiency results in genomic instability and early cellular senescence due to the delayed recruitment of repair proteins to sites of DNA damage. Here, we further investigate the molecular mechanism underlying delayed DNA damage response and identify a histone acetylation defect in Zmpste24 −/− mice. Specifically, histone H4 was hypoacetylated at a lysine 16 residue (H4K16), and this defect was attributed to the reduced association of a histone acetyltransferase, Mof, to the nuclear matrix. Given the reversible nature of epigenetic changes, rescue experiments performed either by Mof overexpression or by histone deacetylase inhibition promoted repair protein recruitment to DNA damage sites and substantially ameliorated agingassociated phenotypes, both in vitro and in vivo. The life span of Zmpste24 −/− mice was also extended with the supplementation of a histone deacetylase inhibitor, sodium butyrate, to drinking water. Consistent with recent data showing age-dependent buildup of unprocessable lamin A in physiological aging, aged wild-type mice also showed hypoacetylation of H4K16. The above results shed light on how chromatin modifications regulate the DNA damage response and suggest that the reversal of epigenetic marks could make an attractive therapeutic target against laminopathybased progeroid pathologies. E ukaryotic cells are equipped with a surveillance machinery to orchestrate the rapid detection and repair of DNA damage. When DNA damage occurs, chromatin surrounding the doublestrand breaks (DSBs) is altered and histones are modified to facilitate access for repair proteins (1). As a rapid response to DSB induction, the histone H2A variant, H2AX, is phosphorylated at Ser139 (γ-H2AX), which in turn interacts with MDC1, a DSB repair mediator, to facilitate the further recruitment of DNA repair proteins, such as 53BP1 and BRCA1 (2-4). Interestingly, γ-H2AX accumulation has been documented both in human senescent cells and in the fibroblasts of aged mice and primates (5-8). It has been proposed that these age-associated γ-H2AX foci contain nonrepairable DSBs and may have a role in initiating aging, especially because DSBs are very toxic and are one of the most lethal forms of DNA damage. Direct evidence for nonrepairable DNA damage as an inducer of premature aging has been obtained from mouse models that lack DNA repair proteins, such as ATM, Ku70, Ku80, DNA ligase IV, and Ercc1, as well as from humans with premature aging syndromes (9, 10). Together, these studies support the idea that the inability to recruit repair proteins to sites of DNA les...