In order to determine whether matrix metalloproteinases (MMPs) contribute to inflammation in asthma, we have examined the release of MMPs in bronchoalveolar lavage (BAL) fluids and their production and regulation by alveolar macrophages (AM), in short-term culture. BAL was collected from 38 asthmatic subjects (24 untreated and 14 treated with inhaled corticosteroids), 26 healthy nonsmokers, and 18 patients with chronic bronchitis used as a control group for another inflammation. The profile of MMPs present in BAL fluid and AM supernatant, determined by zymographic analysis, was found to be similar in all populations. The main enzyme released was identified immunologically as MMP-9, a potent collagenolytic and elastolytic enzyme. Its release, measured using enzyme immunoassay, was significantly enhanced in fluids and in AM supernatants from untreated asthmatics compared with those from the other populations. Enhanced MMP-9 levels, in asthma, could not be explained by a different sensitivity of AM to interleukin-4, interferon-gamma, or dexamethasone, compounds that have been shown to inhibit MMP-9. The phorbol ester phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, significantly increased MMP-9 in AM from healthy control subjects but not in those from untreated asthmatics. Calphostin C and H7, PKC inhibitors, significantly reduced PMA-stimulated MMP-9 release in AM from healthy control subjects and spontaneous MMP-9 release in AM from untreated asthmatics. H8, a PKA inhibitor, was inactive in both populations. These data suggest that the stimulation of MMP-9 release in AM from untreated asthmatic patients occurs, at least partly, via signals activating PKC.

The growth of MCF 7 human breast cancer cells is stimulated in vitro by estradiol (E2) and we have previously shown that estrogen-regulated glycoproteins released into the culture medium can partly mimic this effect. In this paper, we evaluate the mitogenic activity of the 52 K glycoprotein, which is a major E2-stimulated protein released by MCF 7 cells. The 52 K protein was purified 600-fold by affinity chromatography on Concanavalin A and an anti-52 K monoclonal antibody Sepharose columns. The 99% purified 52 K protein fraction stimulated the growth of estrogen-deprived MCF 7 cells. A mean 1.7-fold increase was obtained with nanomolar concentrations of seven different preparations of 52 K protein. This stimulation represented 40% of the mitogenic effect of E2. Both the 52 K protein and E2 induced microvilli at the cell surface but the effect of the 52 K protein occurred earlier. Other putative growth factors which are also stimulated by E2 and observed by [35S]cysteine labeling did not comigrate with the purified 52 K protein. Finally, the labeled 52 K protein was found to enter MCF 7 cells and to be processed into an immunoreactive 34 K protein. These data indicate that the E2-regulated 52 K glycoprotein is an autocrine mitogen on MCF 7 cells in culture and support the hypothesis that estrogens stimulate the growth of mammary cancer via this (and possibly other) secreted protein(s) acting as autocrine (and paracrine?) growth factors.

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Cathepsin D is an acidic lysosomal protease present in all cells. In estrogen receptor positive and negative breast cancer cell lines, the mRNA coding for pro-cathepsin D is overexpressed and sorting and maturation of the pro-enzyme are altered, via possibly saturation of the Man-6-P/IGF-II receptor, leading to accumulation of the active proteinase in large endosomes and to secretion of the precursor (52K protein). In MCF7 cells, the cathepsin D mRNA is induced directly and transcriptionally by estrogens and indirectly by growth factors. In patients, there is a significant correlation between high cathepsin D concentrations in the cytosol of primary breast cancer and development of metastasis. This marker is independent of other prognostic factors and appears to be particularly useful in axillary node-negative tumors. Transfection of a human cDNA cathepsin D expression vector under the control of SV40 promoter increases the metastatic potential of 3YA1-Ad12 rat tumorigenic cells when intravenously injected into nude mice. The mechanism of cathepsin D-induced metastasis is currently unknown. These results indicate that overexpression of cathepsin D might facilitate breast cancer metastasis, suggesting new possible therapeutic approaches.