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 (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.

Effect of glycerol on protein acetylation by acetic anhydride.

We investigated the basis for the previously unexplained stabilization of proteins by glycerol during reaction with acetic anhydride [S. Siegel and W. M. Award, Jr. (1973) J. Biol. Chem. 248, 3233-3240]. Model studies showed that glycerol competes successfully for acetylation against protein hydroxyl groups. In contrast, amino groups are much more potent nucleophiles and their acetylation is not apparently affected. Since alpha-amino and phenolic pKa's did not change significantly in increasing glycerol concentrations, these findings are ascribed to glycerol's lower pKa value as compared to water, leading to the decreased acetylation of tyrosine, threonine, and serine hydroxyl groups in Pronase guanidine-stable chymoelastase. An additional mechanism is important and predominates in the protection against inactivation of bovine delta-chymotrypsin during acetylation and is explained by the recently described basis for protein stabilization in glycerol [K. Gekko and S. N. Timasheff (1981) Biochemistry 20, 4667-4676; 4677-4686]. Those studies demonstrated that glycerol increased the hydrophobicity of nonpolar residues, augmenting their tendency to be removed from protein surfaces. Therefore, the stabilization afforded by glycerol for chymotrypsin is attributed in part to a favoring of the native folded state which forces the side chains of isoleucine-16 and valine-17 to be buried, increasing the apparent pKa of the alpha-amino group of isoleucine-16 as it forms the charge pair with the beta-carboxyl group of aspartate-194. This conclusion was supported by stopped-flow analyses of the interaction of delta-chymotrypsin with proflavin in increasing concentrations of glycerol.

Granulocyte-macrophage colony-stimulating factor from cultured normal rat kidney cell line.

Serum-free medium conditioned by the normal rat kidney (NRK) cell line contains colony-stimulating factors (CSF) that stimulate the in vitro formation of granulocyte and macrophage colonies from rat, mouse, and human marrow. There are two types of CSF: NRK-CSF I stimulates rat and mouse marrow, while NRK-CSF II stimulates the human marrow. The NRK-CSF I has been partially purified to a specific activity of 5 X 10(7) units/mg by employing methods such as preparative isoelectrofocusing, gel filtration chromatography, and preparative gel electrophoresis. It has an isoelectric point of 5.1 and an apparent molecular weight of 35,000 daltons as estimated by gel filtration. It is stable at 50 degrees C for 30 min and relatively resistant to papain, but highly sensitive to trypsin, chymotrypsin, and subtilisin. The secretion of CSF by NRK cells is inhibited by actinomycin D (0.5 micrograms/ml) and cycloheximide (0.5 micrograms/ml), but not by cytosine arabinoside (5 micrograms/ml). Although the CSF activity is inactivated by periodate oxidation (5 mM), thus suggesting its glycoprotein nature, treatment of the CSF with neuraminidase and other glycosidases has no effect on its activity. Anti-NRK-CSF I antibody inhibits the rat/mouse-active CSF from all rat sources, but has no effect on the human-active CSF. Comparative studies with CSF from media conditioned by the transformed cell line (442) and from rat lung and spleen have shown that CSF I from different rat sources share similar isoelectric points, molecular weights, and immunological properties.