Neutrophil collagenase (matrix metalloproteinase-8 or MMP-8) is regarded as being synthesized exclusively by polymorphonuclear neutrophils (PMN). However, in vivo MMP-8 expression was observed in mononuclear fibroblast-like cells in the rheumatoid synovial membrane. In addition, we detected MMP-8 mRNA expression in cultured rheumatoid synovial fibroblasts and human endothelial cells. Up-regulation of MMP-8 was observed after treatment of the cells with either tumor necrosis factor-␣ (10 ng/ml) or phorbol 12-myristate 13-acetate (10 nM). Western analysis showed a similar regulation at the protein level. The size of secreted MMP-8 was 50 kDa, which is about 30 kDa smaller than MMP-8 from PMN. Conditioned media from rheumatoid synovial fibroblasts contained both type I and II collagen degrading activity. However, degradation of type II collagen, but not that of type I collagen, was completely inhibited by 50 M doxycycline, suggesting specific MMP-8 activity. In addition, doxycycline down-regulated MMP-8 induction, at both the mRNA and protein levels. Thus MMP-8 exerts markedly wider expression in human cells than had been thought previously, implying that PMN are not the only source of cartilage degrading activity at arthritic sites. The inhibition of both MMP-8 activity and synthesis by doxycycline provides an incentive for further studies on the clinical effects of doxycycline in the treatment of rheumatoid arthritis.Extracellular matrix degradation is fundamental to connective tissue remodeling during physiological processes as well as during the progress of several pathological phenomena. Matrix turnover is regulated by a delicate balance among the production, activation, and inhibition of proteolytic enzymes. The matrix metalloproteinases (MMPs) 1 form a gene family of at least 14 enzymes participating in extracellular matrix remodeling. MMPs, together with the factors associated with their regulation, are reported to be highly implicated in various diseases such as rheumatoid arthritis, osteoarthritis, corneal ulceration, atherosclerosis, and tumor invasion and metastasis (for reviews, see Refs. 1-3). Previous studies have demonstrated that neutrophil-derived MMPs such as collagenase (MMP-8) and gelatinase B (MMP-9, 92-kDa type IV collagenase), play a key role in the degradation of extracellular matrix constituents i.e. during the course of inflammatory diseases (4 -7). Collagenases exist as three distinct molecules, namely the fibroblast type (MMP-1, collagenase-1) (8), the neutrophil type (MMP-8) (9), and collagenase-3 (MMP-13) (10). They all are able to degrade specifically the fibrillar collagen types I, II, and III as well as type VII and X collagens (11, 12), serpins (4, 13), ␤-casein, and human ␣ 2 -macroglobulin (14). Among collagenases, MMP-8 most effectively hydrolyzes the native type I and II collagens, whereas MMP-1 prefers type III collagen. MMP-8 is a considerably more efficient enzyme than MMP-1 with respect to almost all substrates except for type III collagen (9). MMP-1 is transcribed and exp...

Prolyl 4‐hydroxylase (EC 1.14.11.2), an alpha 2 beta 2 tetramer, catalyses the formation of 4‐hydroxyproline in collagens by the hydroxylation of proline residues in peptide linkages. We report here the isolation of cDNA clones coding for the beta‐subunit of prolyl 4‐hydroxylase from a human hepatoma lambda gt11 library and a corresponding human placenta library. Five overlapping clones covering all the coding sequences and almost all the non‐coding sequences were characterized. The size of the mRNA hybridizing with these clones in Northern blotting is approximately 2.5 kb. The clones encode a polypeptide of 508 amino acid residues, including a signal peptide of 17 amino acids. These human sequences were found to be very similar to those recently reported for rat protein disulphide isomerase (EC 5.3.4.1). The degree of homology between these two proteins was 84% at the level of nucleotide sequences or 94% at the level of amino acid sequences. Southern blot analyses of human genomic DNA with a cDNA probe for the beta‐subunit indicated the presence of only one gene containing these sequences. The product of a single gene thus appears to possess two different enzymatic functions depending on whether it is present in cells in monomer form or in the prolyl 4‐hydroxylase tetramer.

Prolyl 4-hydroxylase (proline hydroxylase, EC 1.14.11.2) catalyzes the formation of 4-hydroxyproline in collagens. The vertebrate enzyme is an ␣ 2 ␤ 2 tetramer, the ␤ subunit of which is identical to protein disulfideisomerase (PDI, EC 5.3.4.1). We report here on cloning of the recently discovered ␣(II) subunit from human sources. The mRNA for the ␣(II) subunit was found to be expressed in a variety of human tissues, and the presence of the corresponding polypeptide and the (␣(II)) 2 Prolyl 4-hydroxylase (proline hydroxylase, EC 1.14.11.2) catalyzes the hydroxylation of proline in -Xaa-Pro-Gly-triplets in collagens and other proteins with collagen-like sequences. The enzyme plays a central role in the synthesis of all collagens, as the 4-hydroxyproline residues formed in the reaction are essential for the folding of the newly synthesized collagen polypeptide chains into triple helical molecules. The vertebrate enzyme is an ␣ 2 ␤ 2 tetramer in which the ␣ subunits contribute to most parts of the two catalytic sites (for reviews, see Refs.

Prolyl 4-hydroxylase [procollagen-proline, 2-oxoglutarate 4-dioxygenase; procollagen-L-proline, 2-oxoglutarate:oxygen oxidoreductase (4-hydroxylating), EC 1.14.11.2], an a212 tetramer, catalyzes the formation of 4-hydroxyproline in collagens by the hydroxylation of proline residues in peptide linkages. We report here on the isolation of cDNA clones encoding the a-subunit of the enzyme from human tumor HT-1080, placenta, and fibroblast cDNA libraries. Eight overlapping clones covering almost all of the corresponding 3000-nucleotide mRNA, including all the coding sequences, were characterized. These clones encode a polypeptide of 517 amino acid residues and a signal peptide of 17 amino acids. Previous characterization of cDNA clones for the f8-subunit of prolyl 4-hydroxylase has indicated that its C terminus has the amino acid sequence Lys-Asp-Glu-Leu, which, it has been suggested, is necessary for the retention of a polypeptide within the lumen of the endoplasmic reticulum. The a-subunit does not have this C-terminal sequence, and thus one function of the fl-subunit in the prolyl 4-hydroxylase tetramer appears to be to retain the enzyme within this cell organelle. Interestingly, three of the cDNA clones for the a-subunit contained a 64-nucleotide sequence homologous but not identical to the corresponding 64-nucleotide sequence found in four other cDNA clones. Nuclease S1 mapping experiments demonstrated that this difference was due to the existence of two types of mRNA present in approximately equal amounts. Southern blot analyses of human genomic DNA with a cDNA probe for the a-subunit suggested the presence of only one gene encoding the two types of mRNA, which appear to result from mutually exclusive alternative splicing of primary transcripts of one gene.Prolyl 4-hydroxylase [procollagen-proline, 2-oxoglutarate 4-dioxygenase; procollagen-L-proline, 2-oxoglutarate:oxygen oxidoreductase (4-hydroxylating), EC 1.14.11.2] catalyzes the formation of 4-hydroxyproline in collagens by the hydroxylation of proline residues in peptide linkages. This enzyme plays a central role in collagen synthesis, as the 4-hydroxyproline residues formed in the reaction are essential for the folding of the newly synthesized procollagen polypeptide chains into triple-helical molecules. The active prolyl 4-hydroxylase is a tetramer (a2/32) with a molecular weight of -240,000 and consisting of two different types of enzymically inactive monomer with molecular weights of =64,000 (a-subunit) and -60,000 (,3-subunit) (for a recent review, see ref. 1). Complete cDNA-derived amino acid sequences have recently been determined for the 13-subunit of human (2) and chicken (3, 4) prolyl 4-hydroxylases. Surprisingly, this 13-subunit has been found to be identical to the enzyme protein disulfide isomerase (2, 5, 6) and a major cellular thyroid hormone binding protein (7,8) and highly similar to a glycosylation site binding protein of oligosaccharyl transferase (9).The a-subunit of prolyl 4-hydroxylase probably contributes a major part of the ...

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