Leonor Wenger

p53 Down-regulates Human Matrix Metalloproteinase-1 (Collagenase-1) Gene Expression

Recent studies show that the p53 tumor suppressor protein is overexpressed in rheumatoid arthritis (RA) synovium and that somatic mutations previously identified in human tumors are present in RA synovium (Firestein, G. S., Echeverri, F., Yeo, M., Zvaifler, N. J., and Green, D. R. (1997)Proc. Natl. Acad. Sci. U. S. A. 94, 10895–10900; Firestein, G. S., Nguyen, K., Aupperle, K. R., Yeo, M., Boyle, D. L., and Zvaifler, N. J. (1996) Am. J. Pathol. 149, 2143–2151; Reme, T., Travaglio, A., Gueydon, E., Adla, L., Jorgensen, C., and Sany, J. (1998) Clin. Exp. Immunol. 111, 353–3581). 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 wt-p53 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.

p53 down‐regulates matrix metalloproteinase‐1 by targeting the communications between AP‐1 and the basal transcription complex

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.

Induction of Matrix Metalloproteinase-8 in Human Fibroblasts by Basic Calcium Phosphate and Calcium Pyrophosphate Dihydrate Crystals: Effect of Phosphocitrate

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 (RTIPCR), 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 arc markers of severe joint degeneration in ostcoarthritis. 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 ostcoarthritis.

Hepatocyte growth factor and its actions in growth plate chondrocytes.

Hepatocyte growth factor (HGF) has been implicated as a paracrine regulator of organogenesis and repair in many tissues. Here we have studied the expression and actions of HGF in intact rachitic rat growth plate and derived cultures of proliferative zone chondrocytes. In vivo and in vitro chondrocytes express HGF mRNA; 1,25(OH)2 has a three-fold maximal stimulatory effect, which can be blocked by H-7, an inhibitor of protein kinase C. Although HGF elaboration and action generally follow a paracrine model, chondrocytes appear capable of both expressing and responding to HGF. mRNA encoding the HGF receptor (c-met) was detected in both growth cartilage and derived chondrocyte cultures. HGF addition to chondrocyte cultures increased collagen II mRNA and alkaline phosphatase enzymatic activity to degrees comparable to that observed for active vitamin D metabolites. Combining HGF and 1,25-D evoked a synergistic response (ninefold) of alkaline phosphatase activity. To assess whether a similar stimulatory effect might be seen with bioactive peptides and HGF, we investigated the effect of HGF pretreatment on acute responses of chondrocytes to synthetic human calcitonin, an anabolic chondrocyte regulator whose skeletal action are mediated principally by cAMP elevation and subsequent protein kinase A activation. CTs maximal activation of protein kinase A was increased by prior HGF treatment from 56% to 78%. In concert, our findings indicate that in addition to HGFs classical paracrine role during skeletal growth, this growth factor may modulate hormonal sensitivity of the chondrocyte during proliferation, differentiation, and/or apoptosis.

Induction of Early Growth Response Gene Egr2 by Basic Calcium Phosphate Crystals through a Calcium-Dependent Protein Kinase C-Independent p44/42 Mitogen-Activated Protein Kinase Pathway

Using the reverse transcriptase-polymerase chain reaction we examined the effect of basic calcium phosphate (BCP) crystals on the induction of the early growth response gene Egr2 transcription and the signal transduction pathway involved. The results showed that BCP crystals induced Egr2 transcription up to 8-fold, peaking at 24 h after treatment. The induction of Egr2 was confirmed by transient transfection assays using an Egr2 promoter/luciferase reporter construct and could be inhibited by the p44/42 mitogen-activated protein kinase (MAPK)-specific inhibitor U0126, or by calcium chelator TMB-8, but not by the SAPK2/p38 MAPK inhibitor SB202190 or by the protein kinase C inhibitor bisindolylmaleimide I (Bis-I). Using the Mercury Pathway Profiling System (Clontech, Palo Alto, Calif., USA) we further showed that induced Egr2 could stimulate the activities of several transcription factors that are associated with cell proliferation, such as c-fos, SRF and c-myc.

Human metalloproteinase-1 (collagenase-1) is a tumor suppressor protein p53 target gene.

Matrix metalloproteinases (MMPs) are a family of secreted or transmembrane proteins that can degrade all the proteins of the extracellular matrix and have been implicated in many abnormal physiological conditions, including arthritis and cancer metastasis. 1,2 Rheumatoid arthritis (RA) is marked by the destruction of the extracellular matrix and the degradation of native collagen, which are initiated mainly by the action of one type of metalloproteinase, the collagenases: collagenase-1 (MMP-1), neutrophil collagenase (MMP-8), and a recently discovered one, collagenase-3 (MMP-13) 1,3 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. Moreover, recent studies have linked this powerful tumor suppressor to RA. 4–6 It was demonstrated that p53 protein was overexpressed in RA synovium, 4 and that mutant p53 transcripts are present. 5,6 Because p53 protein can function directly as a transcriptional regulator, we speculated that the overexpression of MMP-1 seen in cancer or RA might be associated with the inactivation of p53. We tested this hypothesis directly by cotransfecting Saos-2 cells with a fulllength hMMP1 promoter/luciferase construct 7 (hMMP-1/luci) plus wild-type (wt) and mutant p53 expression plasmids. Transcription assays revealed that wt-p53 downregulated the promoter activity of hMMP-1 in a dose-dependent fashion (F IG . 1, top). Clearly, if this repression activity was crucial to the tumor suppressor activity of wt-p53, naturally occurring p53 mutants might be defective in this function. As expected, most of tumor-derived p53 mutants examined lost this repression activity (F IG . 1, top). Northern blot assays give similar results (F IG . 1, bottom). Two lines of evidence suggest that the repression of hMMP-1 by wt-p53 is a physiologically relevant response. First of all, serum-stimulated expression of endogenous hMMP1 messenger was completely repressed by wt-p53, but not mutant p53 (F IG . 1, bottom). Second, hMMP-1/luci reporter activities were stimulated by etoposide only

Inhibition of calcium phosphate-DNA coprecipitates induced cell death by phosphocitrates.

Phosphocitrate [PC] is a powerful inhibitor of biological crystallization and a potential disease modifying drug for crystal associated diseases such as crystals associated osteoarthritis [OA]. Recently, it has been reported that a new PC complex salt, calcium sodium PC [CaNaPC], is much more powerful than its precursor, sodium PC [NaPC], in reducing the size of chemically-induced calcified plaques in rat when examined using a calcergy assay (1). The molecular mechanisms underlying such a superior activity as a calcification inhibitor over its precursor NaPC are currently unknown. In order to evaluate the potential of CaNaPC as a disease modifying drug for crystals associated OA, we examined and compared CaNaPC and its precusor NaPC using several cell- based assays. CaNaPC was found to have an inhibitory potency similar to that of NaPC toward preventing the stimulating effects of basic calcium phosphate [BCP] crystals on the induction of MMP1, thymidine uptake and endocytosis. However, CaNaPC proved much more powerful than NaPC in the inhibition of amorphous calcium phosphate-DNA coprecipitates-induced cell death. These results suggest that the superior anti-calcification activity of NaCaPC over NaPC observed in rat is probably due to its superior activity in the inhibition of the effects associated with amorphous calcium phosphate clusters/aggregates/precipitates but not the effects associated with BCP crystals. Since amorphous calcium phosphate clusters/aggregates/precipitates are precursors of BCP crystals and coexist with calcium-containing crystals in calcified tissues (2-6), these amorphous clusters/aggregates/precipitates, similar to BCP crystals, may have played a significant role in pathological calcifications and in the development of crystals associated diseases such as crystals associated OA. The superior activity of CaNaPC over its precursor NaPC in the inhibition of amorphous calcium phosphate-DNA coprecipitates-induced cell death may, at least in part, explain its powerful anti-calcification activity in vivo. The findings suggest that CaNaPC through a dual action of inhibiting both the detrimental biological effects of formed BCP crystals and preforming amorphous calcium phosphate clusters/aggregates/precipitates, could present as a better disease-modifying drug for crystals associated OA than its parent NaPC.

Loss of decorin from the surface zone of articular cartilage in a chick model of osteoarthritis

The objective of this study was to immunolocalize decorin and to assess changes as a result of pyridoxine (PN) deficiency in chick articular cartilage from femoral condyles. After maintenance on a normal diet for the first two weeks after hatching, 15 broiler chickens were deprived of this vitamin for 6 weeks. It was previously shown that the ankle joints of PN-deficient animals are swollen with effusions. They also present an abnormal gait, enlarged bony margins, and fissuring of the articular cartilages. Milder changes (no fissures) were also shown in the knee joints. Data from a previous study were suggestive that sulfated glycosaminoglycans are lost from the knee cartilage surface into synovial fluid. The current study was focused on the small proteoglycan, decorin, which coats the surface of collagen fibrils and may regulate their morphology. To examine decorin in normal and PN-deficient articular cartilage, a monoclonal antibody to an epitope on the protein core of decorin was used for immunohistochemical staining of tissue sections and for Western Blot analysis of cartilage extracts. Reduction of staining with the antibody was demonstrated in the tangential surface zone of PN-deficient cartilage, and Western Blot analysis showed reduced intensity of decorin bands compared to normal controls. These data suggest that a lack of decorin may play a role in the enlargement of collagen bundles in the tangential zone of PN-deficient articular cartilage as observed in a previous electron microscopic study.