Basic calcium phosphate crystal-induced Egr-1 expression stimulates mitogenesis in human fibroblasts.

Previously, we have reported that basic calcium phosphate (BCP) crystals stimulate mitogenesis and synthesis of matrix metalloproteinases in cultured human foreskin and synovial fibroblasts. However, the detailed mechanisms involved are still unclear. In the present study, using RT-PCR and Egr-1 promoter analysis we showed that BCP crystals could stimulate early growth response gene Egr-1 transcription through a PKCalpha-dependent p44/p42 MAPK pathway.

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.

Telomerase-transduced osteoarthritic fibroblast-like synoviocyte cell line.

To examine whether the life span of fibroblast-like synoviocytes (FLSs) can be extended and to establish FLS cell lines that preserve the characteristics of primary FLSs, we introduced human catalytic subunit of telomerase (hTERT) gene into human osteoarthritic (OA) FLSs. Two hTERT-transduced clonal cell lines were established and one line, hTERT-OA FLS 13A, was characterized. The hTERT-OA FLS 13A cells have a morphology similar to that of the parental untransduced cells and a population-doubling time similar to that of the parental cells of early passages.

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.

Basic calcium phosphate crystals stimulate the endocytotic activity of cells--inhibition by anti-calcification agents.

Pathological calcifications are associated with many medical conditions including diabetes, breast cancer, and crystals-associated osteoarthritis. The deposition of calcium-containing crystals on cells induces detrimental cellular effects and speeds up the progression of associated diseases. We carried out the present study to test the hypotheses that calcium-containing crystals may stimulate the influx of other molecules existing in the extracellular fluid disturbing normal molecular signaling and that anti-calcification agent will inhibit such endocytotic process.

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).

Basic calcium phosphate crystals induce matrix metalloproteinase-1 through the Ras/mitogen-activated protein kinase/c-Fos/AP-1/metalloproteinase 1 pathway. Involvement of transcription factor binding sites AP-1 and PEA-3.

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.