The synthesis of the nuclear lamina protein lamin A requires the prenylation-dependent processing of its precursor protein, prelamin A. Unlike p2lr", which undergoes similar initial posttranslational modifications, maturation of lamin A results in the proteolytic removal of the prenylated portion of the molecule. We have used an in vitro prenylation system to demonstrate the nature of the prenyl substituent on prelamin A to be a farnesyl group. Further, the in vitro farnesylation of prelamin A requires an intact cysteinealiphatic-aliphatic-other (CAAX) amino acid sequence motif at its carboxyl terminus. The effect of blocking the prenylation of prelamin A on its localization and assembly into the nuclear lamina was investigated by indirect immunofluorescence. Expression of wild-type prelamin A in lovastatin-treated cells showed that nonprenylated prelamin A accumulated as nucleoplasmic particles. Upon addition of mevalonate to lovastatintreated cells, the wild-type lamin A was incorporated into the lamina within 3 hr. Expression of a mutant lamin A in which the carboxyl-terminal 21 amino acids were deleted resulted in a lamin molecule that was directly assembled into the lamina. These results indicate that the carboxyl-terminal peptide of prelamin A blocks its proper assembly into the nuclear lamina and that the prenylation-initiated removal of this peptide can occur in the nucleus.The nuclear lamina is a polymeric protein structure that lines the inner nuclear membrane. In most mammalian cells, it consists of three major proteins, lamins A-C (for review, see ref. 1). Mature lamin A is synthesized from a larger precursor protein (2, 3) and lacks the carboxyl-terminal 18 amino acids predicted by the cDNA sequence (4). Conversion ofthe lamin A precursor, prelamin A, to lamin A is dependent upon the isoprenylation of prelamin A (5). Prelamin A is an example of a class of proteins terminating in the sequence cysteinealiphatic-aliphatic-other (CAAX), which is prenylated at the consensus cysteine (for reviews, see refs. 6 and 7). This motif, shared by such proteins as p215 and the a-type mating factor of Saccharomyces, entrains a series of posttranslational processing steps. These include prenylation at the CAAX cysteine with either a 15-carbon (farnesyl) or 20-carbon (geranylgeranyl) isoprenoid followed by proteolytic removal of the A-A-X amino acids and carboxyl methylation of the now terminal cysteine. Evidence has been reported that the B-type lamins also undergo these prenylationdependent processing reactions (8-10). However, specific steps for the processing of prelamin A have not been directly demonstrated. Activities capable of catalyzing the carboxylterminal processing of a-factor and p2l's in vitro have been described (11,12). For p21'S, these activities were localized to the cytosolic and microsomal compartments (12, 13).Prelamin A and the yeast a-factor share an additional reaction subsequent to these carboxyl-terminal processing events. Both are subject to an endoproteolytic cleavage of their resp...

We hypothesize that the abnormality of p53 seen in RA synovium may contribute to joint degeneration through the regulation of human matrix metalloproteinase-1 (hMMP-1, collagenase-1) gene expression. Transcription assays were performed with luciferase reporters driven by the promoter of the hMMP-1 gene or by a minimal promoter containing tandem repeats of the consensus binding sequence for activator protein-1, cotransfected with p53-expressing plasmids. The results revealed that (i) wild-type (wt) p53 down-regulated the promoter activity of hMMP-1 in a dose-dependent fashion; (ii) four of six p53 mutants (commonly found in human cancers) lost this repression activity; and (iii) this p53 repression activity was mediated at least in part by the activator protein-1 sites found in the hMMP-1 promoter. These findings were further confirmed by Northern analysis. The down-regulation of hMMP-1 gene expression by endogenous wtp53 was shown by treatment of U2-OS cells, a wt-p53-containing osteogenic sarcoma line, and Saos-2 cells, a p53-negative osteogenic sarcoma line, with etoposide, a potent inducer of p53 expression. p53, activated by etoposide, appears to block hMMP-1 promoter activity induced by etoposide in U2-OS cells. In summary, we have shown for the first time that the hMMP-1 gene is a p53 target gene, subject to p53 repression. Because MMP-1 is principally responsible for the irreversible destruction of collagen in articular tissue in RA, abnormality of p53 may contribute to joint degeneration through the regulation of MMP-1 expression.Rheumatoid arthritis (RA) 1 is marked by destruction of the extracellular matrix and it is believed that, among other factors, matrix metalloproteinases (MMPs) play an important role in mediating the degradation of connective tissue matrix components such as collagens and proteoglycans (4, 5). Collagenase-1 (MMP-1), stromelysin (MMP-3), gelatinase A and B (MMP-2 and MMP-9), and collagenase-3 (MMP-13) are all present at significantly elevated levels in cartilage, synovial membranes, and synovial fluid of patients with RA (6 -8). The synovium produces substantial amounts of MMP-1, the major matrix metalloproteinase involved in the degradation of interstitial collagens, specifically, types I-III. MMP-1 expression has been shown to be stimulated by native collagen type I and collagen fragments, phorbol esters, growth factors, and cytokines such as interleukin 1␤ (IL-1␤) and tumor necrosis factor-␣ (9 -12). The activity of MMP-1 is stringently regulated at three levels: the promoter, the activation of proenzyme, and the inhibition of active enzyme. The activator protein-1 (AP-1) binding sites found in the promoters of human collagenase have been shown to be critical to the expression of human collagenase (13-16).The protein product of the p53 tumor suppressor gene plays a very important role in cell growth control, DNA repair, and apoptosis (17). It has been proposed that p53 acts as an "emergency brake" inducing G1 arrest and apoptosis after DNA damage, either by halting cell divi...

Chem. 274, 11535-11540). Here, we report that cotransfection of fibroblast-like synoviocytes with p53 expression and hMMP13CAT reporter plasmids revealed that (i) hMMP13, another member of the human MMP family, was down-regulated by wild type p53, whereas all six of the p53 mutants tested lost the wild type p53 repressor activity in fibroblast-like synoviocytes; (ii) this repression of hMMP-13 gene expression by wild type p53 could be reversed by overexpression of p53 mutants p53-143A, p53-248W, p53-273H, and p53-281G; (iii) the dominant effect of p53 mutants over wild type p53 appears to be a promoter-and mutant-specific effect. An intriguing finding was that p53 mutant p53-281G could conversely stimulate the promoter activity of hMMP13 up to 2-4-fold and that it was dominant over wild type p53. Northern analysis confirmed these findings. Although the significance of these findings is currently unknown, they suggest that in addition to the effect of cytokines activation, the gene expression of hMMP13 could be dysregulated during the disease progression of rheumatoid arthritis (or cancer) associated with p53 inactivation. Since hMMP13 is 5-10 times as active as hMMP1 in its ability to digest type II collagen, the dysregulation or up-modulation of MMP13 gene expression due to the inactivation of p53 may contribute to the joint degeneration in rheumatoid arthritis. Rheumatoid arthritis (RA)1 is a systemic disease of unknown etiology characterized by severe inflammation, abnormal immune response, synovial hyperplasia, and extensive destruction of the articular cartilage (2). The progressive destruction of articular cartilage in RA is partly mediated by matrix metalloproteinases (MMPs) (3, 4). Collagenase-1 (MMP-1), stromelysin (MMP-3), gelatinase A and B (MMP-2 and MMP-9), and collagenase-3 (MMP-13) are all present at significantly elevated levels in cartilage, synovial membranes, and synovial fluid of patients with RA (5-7). MMP-13, a newly discovered human collagenase (6 -9), besides cleaving type II collagen more efficiently than MMP-1 (10), is also active against aggrecan, the major proteoglycan of the hyaline articular cartilage (11). Therefore, it has been suggested that MMP-13 may represent a particularly significant mediator of tissue destruction in arthritis. The p53 tumor suppressor gene has been implicated in the malignant progression of cancers, and the mutational inactivation of p53 is the most frequent genetic alteration in human cancers. Recent studies have linked this powerful tumor suppressor to RA. It has been demonstrated independently by two groups that p53 protein is overexpressed in RA synovium and in synoviocytes cultured from RA patients (12,14), and that mutant p53 transcripts previously identified in human tumors are present in these tissues and cells (13,14).p53 is a transcription factor that recognizes a specific consensus DNA sequence consisting of two copies of a 10-base pair motif, 5Ј-RRRC(A/T)(T/A)GYYY-3Ј (where R indicates a purine nucleoside and Y indicates a pyrimidine nucleoside)...

Synovial fluid basic calcium phosphate (BCP) crystals are common in osteoarthritis and are associated with severe degenerative arthropathy. Besides stimulating synovial fibroblast-like cells to proliferate, BCP crystals are a potent inducer of human matrix metalloproteinases (hMMPs), which can speed up the articular joint tissue degeneration of osteoarthritis patients. Here, we report that transfections with hMMP1 luciferase reporter plasmids in fibroblast-like synoviocytes revealed that the induction of hMMP1 promoter by BCP crystals was mainly mediated through the ؊72AP-1 element. Elimination of the ؊72AP-1 element either by mutation or deletion abolished the induction of hMMP1 promoter activity by BCP crystals almost completely. Interestingly, a mutation at the ؊88PEA-3 site also abolished the induction of hMMP1 promoter. Further mutation at the ؊181AP-1 site resumed the induction, indicating that the ؊181AP-1 element had an effect opposite to the ؊72AP-1 element. The effect of ؊181AP-1 could be inactivated either by a mutation at this ؊181AP-1 site or by the ؊88PEA-3 element. In addition, dominant negative Ras, Raf, and MEK1/2 could block the induction of hMMP1, and a MEK1/2-specific inhibitor (UO126) could block the induction of hMMP1 and c-Fos by BCP crystals. Taken together, these data indicate that multiple elements, including at least AP-1 and PEA-3, are involved in the induction of hMMP1 gene expression by BCP crystals and that the induction follows the Ras/ MAPK/c-Fos/AP-1/MMP1 signaling pathway.

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We have previously reported that human matrix metalloproteinase-1 (MMP1) is a p53 target gene subject to down-regulation (Sun et al. [1999]: J Biol Chem 274:11535-11540]. In the present study, we demonstrate that the down-regulation of the human -83MMP1 promoter fragment by p53 was abolished when the -72AP-1 site was eliminated and that a GAL4-cJun-mediated but not a GAL4-Elk1-mediated induction of pFR-luci was effectively inhibited by p53 suggesting an AP-1 dependent but AP-1 binding independent mechanism. Results from gel mobility shift assays were consistent with an AP-1 binding independent mechanism. We also demonstrate that both p300 and TATA box binding proteins cooperated with the transcription factor AP-1 to induce the promoter of MMP1; however, p53 only inhibited the p300-mediated induction of the MMP1 promoter and the inhibition was -72AP-1 dependent. Furthermore, the down-regulation of the MMP1 promoter and mRNA by p53 could be reversed by p300 and by a p53 binding p300 fragment that had no coactivator activity. Taken together, these results indicate that p53 down-regulates MMP1 mainly by disrupting the communications between the transactivator AP-1 and the basal transcriptional complex, which are partially mediated by p300. Finally, by using p53 truncated mutant constructs, we demonstrate that both the N-terminal activation domain and the C-terminal oligomerization domains of p53 were required for the down-regulation of MMP1 transcription.

Although matrix metalloproteinase-8 (MMP-8) was regarded as the exclusive product of the neutrophils, recent studies have shown that it is also expressed in articular chondrocytes, rheumatoid synovial fibroblasts and endothelial cells. Our aim was to determine the expression of MMP-8 in human fibroblasts (HF) by reverse transcription/polymerase chain reaction (RT/PCR). Northern and Western blotting methods and MMP-8 activity assay. We have shown the expression of MMP-8 in HF and its dose-dependent upregulation by basic calcium phosphate (BCP) and calcium pyrophosphate dihydrate (CPPD) crystals which are markers of severe joint degeneration in osteoarthritis. These effects require new protein synthesis and are reversed by phosphocitrate (PC). The results also show that this fibroblast MMP-8 is distinct from the neutrophil MMP-8 and from the fibroblast MMP-1. These results indicate that MMP-8 may play a significant role in the pathogenic effects of the crystals in osteoarthritis.

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