Tumor necrosis factor-␣ (TNF) is initially expressed as a 26-kDa membrane-bound precusor protein (pro-TNF) that is shed proteolytically from the cell surface, releasing soluble 17-kDa TNF. We have identified human ADAM 10 (HuAD10) from THP-1 membrane extracts as a metalloprotease that specifically clips a peptide substrate spanning the authentic cleavage site between Ala 76 and Val 77 in pro-TNF. To confirm that HuAD10 has TNF processing activity, we cloned, expressed, and purified an active, truncated form of HuAD10. Characterization of recombinant HuAD10 (rHuAD10) suggests that this enzyme has many of the properties (i.e. substrate specificity, metalloprotease activity, cellular location) expected for a physiologically relevant TNF-processing enzyme. Tumor necrosis factor-␣ (TNF)1 is a cytokine that is produced primarily by activated monocytes and macrophages in response to a variety of physiological stresses such as infection or injury (1). Clinical and experimental evidence has also identified TNF as a mediator of chronic autoimmune diseases such as rheumatoid arthritis (2) and Crohn's disease (3), as well as being involved in the pathology associated with sepsis (1). Accordingly, TNF has become a primary target for therapeutic intervention of several inflammatory diseases.TNF is initially synthesized as a 26-kDa membrane-bound protein (pro-TNF) that is subsequently cleaved to release soluble 17-kDa TNF with an NH 2 terminus of Val 77 (4). The identity of the protease(s) responsible for TNF processing remains controversial. Robache-Gallea et al. (5) detected a serine protease activity (PR3) in monocyte membrane preparations which was able to generate a 17-kDa active TNF with an NH 2 terminus of Arg 78 . In 1994, the partial isolation and characterization of a membrane-bound activity capable of generating the 17-kDa form of TNF were reported (6). The TNF-processing enzyme was thought to be a non-matrix metalloprotease since it was not inhibited by TIMP-1,2 or phosphoramidon, and no calcium requirement was detected. More recently, two members of the ADAM family (TNF-␣ converting enzyme (TACE) and bovine ADAM 10 (BoAD10)) have been shown to possess pro-TNF processing activity (7-9).In this report we describe the isolation, cloning, and characterization of a TNF-processing enzyme from the human monocytic cell line THP-1. The purified recombinant enzyme, rHuAD10, specifically recognizes the authentic cleavage site in pro-TNF and is sensitive to metalloprotease inhibitors that block soluble TNF production (6). MATERIALS AND METHODSReagents-Dinitrophenol-labeled polypeptides were synthesized by the Fmoc (N-(9-fluorenyl)methoxycarbonyl)/t-butyl-based solid phase peptide chemistry method using an Applied Biosystems, Inc. 431A peptide synthesizer (10). All peptides were purified by reversed phase HPLC, and molecular weights were verified by mass spectrometry.HPLC Peptide Assay-TNF processing activity was measured as the ability to cleave a 12-residue peptide spanning the Ala 76 -Val 77 site in pro-TNF. The chromatopho...

It has been shown that bone cells produce and secrete several growth factors (GFs) which are also found in the bone matrix. To investigate the role of these growth factors in bone cell metabolism, we compared the effects of different factors separately and in combination with respect to osteoblastic cell proliferation and differentiation. While basic fibroblast GF (FGF), transforming GF beta-1 (TGF beta), and platelet-derived GF (PDGF) enhance DNA synthesis, they had the opposite effect on alkaline phosphatase (ALP) activity in cell extracts: FGF, TGF beta, and PDGF inhibited cell ALP but strongly stimulated DNA synthesis. The IGFs had little effect on cell ALP but increased the release of ALP into the conditioned medium. In mitogenic tests of combinations of GFs, most had at least additive effects at low concentrations, and FGF, TGF beta, and IGF2 produced synergistic effects. Evidence is presented for (1) the modulation of the effects of one GF by the action of other GF, (2) synergistic interactions between FGF, TGF beta, and IGF2, and (3) a possible role for the observed interactions among GF for the mitogenic effect of human bone extract.

Objective-Erythropoietin (Epo) bioactivity is significantly reduced by modification of lysine residues with amine-reactive reagents, which are the most commonly used reagents for attaching polyethylene glycols (PEGs) to proteins to improve protein half-life in vivo. The aims of this study were to determine whether Epo bioactivity can be preserved by targeting attachment of maleimidePEGs to engineered cysteine analogs of Epo, and to determine whether the PEGylated Epo cysteine analogs have improved pharmacokinetic properties in vivo. Materials and Methods-Thirty-fourEpo cysteine analogs were constructed by site-directed mutagenesis and expressed as secreted proteins in baculovirus-infected insect cells. Following purification, monoPEGylated derivatives of 12 cysteine analogs were prepared using 20 kDamaleimide-PEGs. In vitro biological activities of the proteins were measured in an Epo-dependent cell proliferation assay. Plasma levels of insect cell-expressed wild type Epo (BV Epo) and a PEGylated Epo cysteine analog were quantitated by ELISA following intravenous administration to rats.Results-Biological activities of 17 purified Epo cysteine analogs and 10 purified PEGylated Epo cysteine analogs were comparable to that of BV Epo in the in vitro bioassay. The only PEGylated cysteine analogs that displayed consistently reduced in vitro bioactivities were substitutions for lysine residues, PEG-K45C and PEG-K154C. The PEGylated Epo cysteine analog had a slower initial distribution phase and a longer terminal half-life than BV Epo in rats, but the majority of both proteins were cleared rapidly from the circulation.Conclusions-Targeted attachment of maleimide-PEGs to engineered Epo cysteine analogs permits rational design of monoPEGylated Epo analogs with minimal loss of in vitro biological activity. Insect cell-expressed EPO proteins are cleared rapidly from the circulation in rats, possibly due to improper glycosylation. Site-specific PEGylation appears to improve the pharmacokinetic properties of Epo.

Recent evidence suggests that cytokines, in addition to regulating hematopoiesis and immune functions, may be important paracrine regulators of bone turnover. Interleukin-1 (IL-1) and IL-6 are cytokines that are produced by and affect both hematopoietic and nonhematopoietic cell types. IL-1 stimulates bone resorption and inhibits osteoblast proliferation and collagen production. Previous reports that IL-6 was secreted in murine osteoblast and bone organ cultures in response to IL-1 and PTH suggested that IL-6 has paracrine effects on bone resorption or formation. To determine whether IL-6 has a paracrine function in human bone, IL-6 expression in cells isolated from normal human bone was investigated. IL-6 mRNA levels in untreated cultures were low and variable, and IL-6 secretion was undetectable. PTH had no effect on IL-6 mRNA levels or IL-6 secretion. IL-1 beta increased IL-6 mRNA levels, maximally 40-fold at 12 h. IL-1 beta increased IL-6 secretion to 0.13 nM, more than 80-fold that of untreated controls at 12 h. IL-1 beta also increased IL-1 beta mRNA levels, maximally 9-fold at 12 h, but did not increase cellular levels or secretion of IL-1 beta protein. Recombinant human IL-6 at 0.5-5 nM stimulated resorption in neonatal mouse calvarial organ cultures but had no effect on human bone-derived cell DNA synthesis or type I procollagen mRNA levels. The results suggest that IL-6 production by human osteoblasts may function to enhance osteolytic activity of IL-1 but does not affect proliferative and matrix biosynthetic aspects of bone formation that were tested. Because osteoblasts and bone marrow cells are in close proximity, IL-6 produced by osteoblasts may also function to amplify IL-1 stimulation of immune responses and hematopoiesis in bone marrow.

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