Alberto M. Pendás

Genomic instability in laminopathy-based premature aging

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.

Loss of collagenase-2 confers increased skin tumor susceptibility to male mice

Matrix metalloproteinases (MMPs) have fundamental roles in tumor progression, but most clinical trials with MMP inhibitors have not shown improvements in individuals with cancer. This may be partly because broad-range inhibitors also reduce host-protective antitumor properties of individual MMPs. We generated mice deficient in collagenase-2 (Mmp8), an MMP mainly produced by neutrophils in inflammatory reactions and detected in some malignant tumors. Loss of Mmp8 did not cause abnormalities during embryonic development or in adult mice. Contrary to previous studies with MMP-deficient mice, however, the absence of Mmp8 strongly increased the incidence of skin tumors in male Mmp8−/−mice. Female Mmp8−/−mice whose ovaries were removed or were treated with tamoxifen were also more susceptible to tumors compared with wild-type mice. Bone marrow transplantation experiments confirmed that Mmp8 supplied by neutrophils was sufficient to restore the natural protection against tumor development mediated by this protease in male mice. Histopathological analysis showed that mutant mice had abnormalities in the inflammatory response induced by carcinogens. Our study identifies a paradoxical protective role for Mmp8 in cancer and provides a genetic model to evaluate the molecular basis of gender differences in cancer susceptibility.

Defective prelamin A processing and muscular and adipocyte alterations in Zmpste24 metalloproteinase–deficient mice

The mouse ortholog of human FACE-1, Zmpste24, is a multispanning membrane protein widely distributed in mammalian tissues and structurally related to Afc1p/ste24p, a yeast metalloproteinase involved in the maturation of fungal pheromones. 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, a major component of the nuclear lamina, and phenocopy most defects observed in humans with diverse congenital laminopathies. 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.

Accelerated ageing in mice deficient in Zmpste24 protease is linked to p53 signalling activation.

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.

Chromosomal mapping and nucleotide sequence of two tandem repeats of Atlantic salmon 5S rDNA

Atlantic salmon 5S ribosomal DNA (5S rDNA) was amplified by the polymerase chain reaction, using as primers conserved sequences from the coding region of rainbow trout 5S rRNA. Two amplified products of different molecular weights were obtained, cloned, and sequenced, revealing them to be tandemly arranged. The nucleotide sequences differed between the two clones in the length of the nontranscribed spacer (NTS) and in three nucleotides of the coding sequence. By means of fluorescence in situ hybridization the 5S rDNA was chromosomally located in the heterochromatic arm of the pair bearing the satellite, adjacent to the major ribosomal DNA locus (rDNA).

Identification and Characterization of a Novel Human Matrix Metalloproteinase with Unique Structural Characteristics, Chromosomal Location, and Tissue Distribution

We have cloned a novel member of the matrix metalloproteinase (MMP) family of proteins from a human liver cDNA library. The isolated cDNA contains an open reading frame coding for a polypeptide of 508 amino acids, which has been tentatively called MMP-19. This protein exhibits the domain structure characteristic of previously described MMPs, including a signal sequence, a prodomain with the cysteine residue essential for maintaining the latency of these enzymes, an activation locus with the zinc-binding site, and a COOH-terminal fragment with sequence similarity to hemopexin. However, it lacks a series of structural features distinctive of the diverse MMP subclasses, including the Asp, Tyr, and Gly residues located close to the zinc-binding site in collagenases, the fibronectin-like domain of gelatinases, the transmembrane domain of membrane-type (MT) MMPs, and the furin-activation sequence common to stromelysin-3 and MT-MMPs. In addition, the 9-residue insertion rich in hydrophobic amino acids present at the hinge region in stromelysins is replaced in MMP-19 by a longer insertion very rich in acidic residues. On the basis of these structural characteristics, we propose that MMP-19 does not belong to any of the previously defined MMP-subclasses and may represent the first member of a new MMP subfamily. Chromosomal location of the MMP-19 gene revealed that it maps to chromosome 12q14, which is also a unique location for any MMPs mapped to date. The cDNA encoding a full-length MMP-19 was expressed in Escherichia coli, and after purification and refolding, the recombinant protein was able to degrade synthetic substrates for MMPs. MMP-19 proteolytic activity was abolished by TIMP-2 and EDTA, thus providing additional evidence that the isolated cDNA codes for an authentic MMP. Northern blot analysis of polyadenylated RNAs isolated from a variety of human tissues revealed that MMP-19 is mainly expressed in placenta, lung, pancreas, ovary, spleen, and intestine, suggesting that it may play a specialized role in these tissues.

Cloning and Characterization of Human MMP-23, a New Matrix Metalloproteinase Predominantly Expressed in Reproductive Tissues and Lacking Conserved Domains in Other Family Members

A cDNA encoding a new human matrix metalloproteinase (MMP), tentatively called MMP-23, has been cloned from an ovary cDNA library. This protein exhibits sequence similarity with MMPs, but displays a different domain structure. Thus, MMP-23 lacks a recognizable signal sequence and has a short prodomain, although it contains a single cysteine residue that can be part of the cysteine-switch mechanism operating for maintaining enzyme latency. The C-terminal domain is considerably shortened and shows no sequence similarity to hemopexin, whereas all human MMPs, with the exception of matrilysin, contain four hemopexin-like repeats. Furthermore, MMP-23 is devoid of structural features distinctive of the diverse MMP subclasses, including the specific residues located close to the zinc-binding site in collagenases, the transmembrane domain of membrane-type MMPs, or the fibronectin-like domain of gelatinases. Fluorescent in situ hybridization experiments showed that the human MMP-23 gene maps to 1p36, a location which differs from all MMP genes mapped to date. Recombinant MMP-23 produced inEscherichia coli exhibits low, but significant proteolytic activity against a synthetic substrate commonly used for assaying MMPs. Northern blot analysis demonstrated that MMP-23 is predominantly expressed in ovary, testis, and prostate, suggesting that this new MMP may play a specialized role in reproductive processes.

Matrix metalloproteinases and tumor progression.

The matrix metalloproteinases (MMPs) are a family of more than 20 distinct enzymes that are frequently overexpressed in human tumors. Functional studies have shown that MMPs play an important role in the proteolytic destruction of extracellular matrix and basement membranes, thereby facilitating tumor invasion and metastasis. In addition, these enzymes may also be important in other steps of tumor evolution including neoplastic cell proliferation and angiogenesis stimulation. On the basis of the relevance of MMPs in tumor progression, a number of different strategies aimed to block the unwanted activity of these enzymes in cancer have been developed. Unfortunately, most clinical trials with the first series of MMP inhibitors have failed to show clear benefit in patients with advanced cancer. Explanations for this lack of success include the failure to recognize the role of these enzymes in early stages of the disease as well as inadequacy of either the employed inhibitors or the proteases to be targeted. The introduction of novel concepts such as tumor degradome, and global approaches to protease analysis, may facilitate the identification of the relevant MMPs that must be targeted in each individual cancer patient. On the other hand, the finding that MMPs are enzymes whose effects on biologically active substrates can have profound consequences on cell behaviour, suggests that selective inhibition of a limited set of MMPs at early stages of tumor evolution might be much more effective than using wide-spectrum inhibitors active against most family members, and administered to patients at late stages of the disease. Further studies directed to elucidate these questions will be necessary to clarify whether any of the multiple strategies of MMP inhibition may be part of future therapeutic approaches to control tumor progression.

Sex chromosome linkage of 5S rDNA in rainbow trout (Oncorhynchus mykiss)

The karyotype of the rainbow trout is characterized by a primitive XX/XY sex-determining chromosomal system. (Thorgaard et al., 1977). In the present study using FISH we have physically linked the 5S rRNA genes to the partially undifferentiated X chromosome pair. PCR amplified 5S rDNA was used for FISH and hybridization signals indicated that the genes were duplicated, present in one acrocentric and one metacentric pair of chromosomes. After analyzing several individuals, the female metaphases showed four fluorescent signals whereas males presented only three signals. Two of the three signals obtained in males corresponded to the metacentric pair whereas the single signal was mapped to the heterochromatin that cytologically differentiates the X chromosome from the Y chromosome. Double FISH experiments demonstrated that the 5S rDNA which is not sex linked is located at the NOR bearing arm close to the major ribosomal RNA genes (5.8S, 18S and 28S), similar to the situation observed in Atlantic salmon (Pendas et al., 1994a).

Ribosomal RNA genes are interspersed throughout a heterochromatic chromosome arm in Atlantic salmon

The ribosomal RNA genes (rDNA) have been mapped by fluorescent in situ hybridization (FISH) using four rDNA probes (rDNA/FISH) to Atlantic salmon chromosomes. Signals appeared over the whole heterochromatic chromosome arm displaying the secondary constriction and satellites. The size polymorphism of this sort arm, revealed by C-banding, was confirmed by rDNA/FISH, supporting large interindividual differences in the number of rDNA copies. Conventional techniques for the detection of nucleolar organizer regions are discussed, and their results are compared with those of rDNA/FISH.