Inhibition of cathepsin D by tripeptides containing statine analogs.

Various analogs of statine, a remarkable amino acid component of the protease inhibitor pepstatine, were synthesized and evaluated as tripeptide derivatives for their activity against cathepsin D and HIV-1 protease.

Tissue inhibitor of metalloproteinase-1 levels in bronchoalveolar lavage fluid from asthmatic subjects.

Airway remodeling is a well-recognized feature in patients with chronic asthma. The accumulation in the submucosa of fibrous proteins that are substrates of matrix metalloproteinases (MMP), and the demonstration of increased levels of MMP-9 in bronchoalveolar lavage fluid, prompted us to determine whether there was an imbalance between MMPs and tissue inhibitors of metalloproteinase (TIMP) in such patients. We investigated the presence of TIMPs and other MMPs. TIMP levels were compared with those of all MMPs and inflammatory cytokines. Adults with stable asthma, either untreated or treated with glucocorticoids (GCs), were enrolled. Healthy nonsmokers served as a control population. MMPs and TIMPs were identified through zymography or immunoblotting. TIMPs, MMPs, and cytokines were measured with enzyme immunoassays. TIMP-1 levels were significantly higher in untreated asthmatic subjects than in GC-treated subjects or controls (p < 0.0001), and were far greater than those of MMP-1, MMP-2, MMP-3, and MMP-9 combined. TIMP-2 was undetectable. TIMP-1 levels were correlated with levels of interleukin-6 (p < 0.012) and the number of alveolar macrophages recovered (p < 0.005). This observation has important implications, since an excess of TIMP-1 could lead to airway fibrosis, a hallmark of airway remodelling in patients with chronic asthma.

Increased expression of tissue inhibitor of metalloproteinase-1 and loss of correlation with matrix metalloproteinase-9 by macrophages in asthma.

Alveolar macrophages (AMs) can mediate tissue destruction and repair by synthesizing matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) as well as inflammatory cytokines, which regulate their production. Imbalances between these enzymes and inhibitors may contribute to the tissue damage and remodeling seen in inflammatory diseases. In this study, we examined the role of AMs in chronic asthma. We have previously demonstrated an increased production of MMP-9 by AMs in untreated asthmatic patients as compared with healthy subjects, and in asthmatics treated with inhaled corticosteroids and patients with chronic bronchitis. We now report on the expression of TIMP-1, the inhibitor of MMP-9, and compare the levels and the regulation by cytokines of both MMP-9 and TIMP-1. Enzyme and inhibitor were measured using an enzyme immunoassay and immunoprecipitation. TIMP-1 steady-state mRNA levels were measured using the RNase protection assay. AMs from untreated asthmatics were found to produce more TIMP-1 both at protein and mRNA levels than AMs from other groups. The release of TIMP-1 and MMP-9 from individual AMs was significantly correlated in control populations and the molecules mainly complexed to each other, whereas this was not true for untreated asthmatics, indicating an imbalance between MMP-9 and TIMP-1 production. In the latter population, TIMP-1 release was inhibited by an anti-IL-6 antibody and MMP-9 release by anti-TNF-alpha, anti-IL-6, and anti-IL-1/beta antibodies. The imbalance of MMP-9 and TIMP-1 production, via the involvement of different cytokines, suggests that AMs may be involved in the abnormal repair observed in chronic asthma.

Increased release of matrix metalloproteinase-9 in bronchoalveolar lavage fluid and by alveolar macrophages of asthmatics.

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.

Identification of prometalloproteinase-3 as a major protein secreted by human endometrial fibroblasts and inhibited by coculture with trophoblast cells.

To assess endometrial fibroblast-cytotrophoblast interactions, we used a coculture system allowing analysis of the potential cell morphology modifications and protein secretion variations possibly involved in endometrial invasion arrest. Stromal cells and cytotrophoblasts were isolated from endometrial biopsies and first-trimester placental villi, respectively. In our culture conditions, a 57-kDa protein that was secreted by cultured fibroblasts but was absent in the 4-day coculture medium was found to be identical to prometalloproteinase-3 (proMMP-3) through determination of amino acid sequences of NH2-terminal and internal peptides. Northern blotting analysis of endometrial fibroblast total RNA showed a 38.6% metalloproteinase-3 (MMP-3) mRNA inhibition by 4-day 10(-6) M R5020 treatment. Inhibition of proMMP-3 secretion was weak when cytotrophoblasts were cultured for 4 days in a polycarbonate membrane insert over cultured fibroblasts without possible cell contact in spite of high levels of progesterone produced by cytotrophoblasts. Furthermore, cytotrophoblasts cultured on a monolayer of endometrial fibroblasts became syncytia, and most of the fibroblasts were decidualized. The closeness of the two cell types allowed paracrine relationships that might facilitate the progesterone action. Since MMP-3 is known to activate collagenases, inhibition of its secretion by cell contact might be a mechanism of invasion arrest for trophoblast cell migration.

Expression of cathepsin D in primary and metastatic human melanoma and dysplastic nevi.

High levels of cytosolic cathepsin D expression have been associated with poor prognosis in breast cancer node-negative patients. In this work, we provide evidence that three cell lines established from human metastatic melanomas--IIB-MEL-J, IIB-MEL-LES, and IIB-MEL-IAN--express high levels of procathepsin D mRNA. IIB-MEL-J cells secreted into the conditioned media about 30% of the newly synthesized protein, which was active at acidic pH. Melanoma tumors arising in nude mice after injection of the three different cell lines expressed high levels of procathepsin D mRNA. Moreover, 13 human metastatic melanomas expressed variable levels of procathepsin D mRNA. To study the possible association between cathepsin D expression and melanoma development, samples corresponding to 10 primary tumors, 11 metastatic melanomas, 10 dysplastic nevi, 27 nevocellular nevi, and normal melanocytes were studied by immunohistochemistry for cathepsin D-specific staining. We found that cathepsin D was expressed in all of the dysplastic nevi and primary and metastatic melanomas tested but in only 18% of nevocellular nevi (five of 27), whereas normal melanocytes showed no cathepsin D expression. The overall data indicate that cathepsin D is expressed at a high level by melanoma cells, and because of its expression in preneoplastic lesions, it may be associated with melanoma development.

Specific mannose-6-phosphate receptor-independent sorting of pro-cathepsin D in breast cancer cells.

The secretion of pro-cathepsin D (pro-cath-D) in some human metastatic breast cancer cells (MCF7, MDA/MB231), contrary to normal mammary cells, is not increased by ammonium chloride treatment, indicating a mannose-6-phosphate-independent sorting to lysosomes. By studying a variety of cell lines and lysosomal enzymes, we show that secretion of newly synthesized pro-cath-D was not mediated by the 46-kDa mannose-6-phosphate receptor (MPR) and that its resistance to NH4Cl for secretion was specific to cath-D and not to other lysosomal enzymes. This resistance appeared to be correlated with the basal hypersecretion of pro-cath-D, but not with its overexpression. By contrast, pro-cath-D secretion was increased by NH4Cl in fibroblasts and nontumoral epithelial mammary cells, suggesting a specificity for cancer cells. Immunofluorescence staining showed that pro-cath-D, but neither cathepsin B nor beta-hexosaminidase, accumulated in intracytoplasmic vesicles of cells treated with ammonium chloride. In pulse--chase experiments and by subcellular fractionation on Percoll gradient, cath-D was found to be sorted into dense lysosomes whether cells were treated or not by NH4Cl. Treatment of cells with NH4Cl, however, inhibited processing and maturation of pro-cath-D, which was also observed in light vesicles in the absence of NH4Cl. Part of pro-cath-D, but not processed enzyme, was also found to be membrane associated in saponin-permeabilized cells. We conclude that in breast cancer cells, the MPR-independent pathway of pro-cath-D to lysosome is predominant compared to normal cells and other lysosomal enzymes. This alternative pathway should therefore be considered, in addition to MPR, to explain pro-cath-D sorting and activation in breast cancer cells.

Missense polymorphism (C/T224) in the human cathepsin D pro-fragment determined by polymerase chain reaction--single strand conformational polymorphism analysis and possible consequences in cancer cells.

Overexpression of cathepsin D in human breast cancers is associated with a higher risk of relapse and metastasis. Also, pro-enzyme routing is altered in several tumoral mammary cell lines, leading to its hypersecretion. MCF7 cells compared to normal kidney carry a C-->T transition at position 224 in the cathepsin D gene which converts Ala to valine in its pro-fragment. Using polymerase chain reaction-single strand conformational polymorphism analysis (PCR-SSCP), the variant T allele frequency was found to be 23-30%, and equally distributed in cancer and normal cells. Six to nine per cent of genotypes were homozygous T/T, 34-41% were heterozygous T/C and 50-59% were homozygous C/C. Moreover, genotypes were identical in 19 out of 20 matched sets of tumoral mammary cells and normal white blood cells from the same patients. Loss of heterozygosity was noted in 1 case. C/T224 transition is thus not due to a somatic event. However, this missense polymorphism might modify procathepsin D secretion and/or maturation in breast cancer cells.

Estradiol stimulates cell growth and secretion of procathepsin D and a 120-kilodalton protein in the human ovarian cancer cell line BG-1.

Ovarian cancers are highly invasive. In a first attempt to define the hormones and factors involved in the control of tumor invasion and metastasis, we have used the human ovarian cancer cell line BG-1 which contains both estrogen and progesterone receptors. Protein synthesis and secretion was assayed by [35S]methionine incorporation and polyacrylamide gel electrophoresis followed by fluorography. Three responses to estradiol were found: 1) procathepsin D secretion was increased, whereas the corresponding intracellular proteins were not significantly affected; 2) an abundant but nonidentified 120-kilodalton (kDa) estrogen-induced secreted glycoprotein, different from CA125, was detected for the first time; and 3) the number of cells as determined by DNA assay was markedly stimulated, reaching a higher level of confluency. The antiestrogen OH-tamoxifen was weakly agonist at low concentrations to stimulate cell growth but was a pure antagonist on the 120-kDa protein. The steroid specificity of these responses strongly suggests that they are mediated by the estrogen receptor. We conclude that cathepsin D secretion is specifically stimulated by estrogen in this ovarian cancer cell line as it is in estrogen receptor-positive breast cancer cells. Both cathepsin D and a newly described 120-kDa secreted glycoprotein are potential markers of hormone responsiveness and/or aggressiveness which deserve to be further studied in clinical ovarian cancers.

Estradiol down-regulates the mannose-6-phosphate/insulin-like growth factor-II receptor gene and induces cathepsin-D in breast cancer cells: a receptor saturation mechanism to increase the secretion of lysosomal proenzymes.

We have studied the regulation by estradiol of the mannose-6-phosphate (Man-6-P)/insulin-like growth factor-II (IGF-II) receptor concentration in different breast cancer cell lines. The mRNA level was assayed by Northern blot using the H5.1 cDNA probe. The protein level was assayed by Western ligand blot, by binding saturation with [125I]procathepsin-D on total membrane preparations, and by immunoprecipitation of 35S-labeled proteins. In three estrogen receptor-positive cell lines (MCF7, T47D, and ZR75-1), estradiol specifically decreased the steady state level of the Man-6-P/IGF-II receptor protein and mRNA. Moreover, in different cell lines and in primary culture of normal mammary cells, the secretion of procathepsin-D was inversely correlated with the level of Man-6-P/IGF-II receptor protein and mRNA. We conclude that estradiol down-regulates the Man-6-P/IGF-II receptor in breast cancer cells. Since two of its ligands, procathepsin-D and IGF-II, are induced by estrogen, we propose that the Man-6-P/IGF-II receptor becomes saturated after estrogen treatment. This model might explain the previously described estrogen-induced secretion of procathepsin-D and other lysosomal proenzymes routed by the same transport system.

MCF7 mammary cancer cells respond to bFGF and internalize it following its release from extracellular matrix: a permissive role of cathepsin D.

High and low affinity receptors for basic fibroblast growth factor (bFGF) were detected by binding experiments on MCF7 breast cancer cells. These cells were stimulated for growth by physiological concentrations of bFGF. However, in contrast to endothelial cells, these MCF7 cells did not produce detectable amounts of biologically active bFGF or related heparin-binding growth factor(s) of the FGF family. In vitro, the cathepsin D (cath-D) secreted by MCF7 cells was able to digest extracellular matrix (ECM) and to release ECM-bound 125I-bFGF. When MCF7 cells were cultured on ECM containing bound bFGF, they internalized bFGF, which was slowly and partially proteolyzed in the cells. Processing occurred in acidic compartments and was inhibited by leupeptin. Pepstatin A, an inhibitor of aspartyl proteases, had no effect on the processing but reduced internalization of matrix-bound bFGF by MCF7 cells. Taken together, these results suggest a cooperation between cath-D and bFGF, by which the protease could facilitate the release of bFGF from ECM and its subsequent use by breast cancer cells and/or adjacent cells involved in angiogenesis.

Cathepsin D in breast cancer: from molecular and cellular biology to clinical applications.

Cathepsin D is a ubiquitously expressed lysosomal protease. Initially synthesized as an inactive precursor of 52 kD (pro-cathepsin D), the enzyme is subsequently converted to its active forms by proteolytic processing. Breast cancer cells, unlike normal cells, secrete high levels of pro-cathepsin D; this abnormal secretion is due to both overexpression of the gene and altered processing of the protein. Recent transfection experiments indicate that overexpression of cathepsin D can increase the metastatic potential of tumor cells in nude mice. This study complements clinical studies, which have shown than high cathepsin D concentrations in the cytosol of primary breast cancers may be predictive of subsequent metastasis, particularly for patients with axillary node-negative tumors. These results, and the potential mechanisms by which cathepsin D may promote metastasis, are considered here.

Cathepsin D: a protease involved in breast cancer metastasis.

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.

Estradiol increases the secretion by MCF7 cells of several lysosomal pro-enzymes.

The synthesis and secretion of pro-cathepsin D is increased by estrogens in MCF7 cells. We quantified the effect of estradiol on other lysosomal enzymes in order to investigate the mechanism of this hypersecretion. Precursors of beta-hexosaminidase, cathepsin B and beta-galactosidase, which are routed to lysosomes via the mannose-6-phosphate (Man-6-P) receptor, were secreted in much lower amounts than pro-cathepsin D, but their secretion was also increased by estradiol. The activity of acid phosphatase, which is routed to lysosomes via a different transmembrane mechanism, was not altered by estradiol. While estradiol stimulated gene expression of pro-cathepsin D, it had no effect on that of pro-cathepsin B. We conclude that estradiol stimulates the secretion of several lysosomal pro-enzymes in MCF7 cells, suggesting that a general mechanism is responsible for this derouting rather than a specific alteration of cathepsin D structure.

Regulation, clinical and biological significance of cathepsin D in breast cancer.

The lysosomal protease, pro-cathepsin D, is overexpressed and secreted by human breast cancers. In estrogen-responsive breast cancer cell lines, estrogens and growth factors stimulate cathepsin D expression through distinct mechanisms. Clinical studies indicate that high cathepsin D concentration in primary breast cancers is correlated with an increased risk of metastasis and particularly useful to orientate node-negative tumors towards an adjuvant therapy.

Increased secretion, altered processing, and glycosylation of pro-cathepsin D in human mammary cancer cells.

In human mammary cancer cells, pro-cathepsin D (pro-Cath-D) is induced by estrogens and 50% of it is secreted. To determine whether its secretion is characteristic of mammary cells or transformed cells, we compared its production, processing, and glycosylation in primary cultures of normal mammary epithelial cells to those found in breast cancer cell lines. The cytosolic concentration of total cathepsin D (precursor and mature enzyme) measured by enzyme-linked immunosorbent assay was 8 times higher in cancer cells. Its mRNA level estimated by Northern blot analysis was 8 to 50 times higher and its secretion was 30 times higher in cancer cells. Using pulse-chase labeling, the cellular processing of pro-Cath-D was altered in hormone-dependent and -independent breast cancer cells in comparison to normal cells. This alteration resulted in a lower accumulation of mature enzyme, while the secretion and cytoplasmic accumulation of pro-Cath-D was greater in breast cancer cells than in normal cells. NH4Cl increased secretion of the proenzyme in normal cells but not in cancer cells. The secreted proenzyme was markedly heterogeneous and had a more acidic pI in MCF7 cells than in normal mammary cells. These acidic forms disappeared following endo-beta-N-acetylglucosaminidase H treatment indicating that the structural difference between pro-Cath-D of normal and of cancer mammary cells was located on high mannose or hybrid N-linked oligosaccharides. This difference may be responsible for the altered routing of the pro-Cath-D in breast cancer cells.

[Pro-cathepsin D can activate in vitro pro-cathepsin B secreted by ovarian cancers]. / La pro-cathepsine D peut activer in vitro la pro-cathepsine B sécrétée par les cancers ovariens.

A precursor form of cathepsin B (Mr 45-47 kd) was purified from ascitic fluids of patients with ovarian adenocarcinomas. Following pepsin activation, this precursor produced a 33 kd cathepsin B-like proteinase closely related to lysosomal cathepsin B. A similar activation was found using the 52 kd pro-cathepsin D secreted by the MCF7 human breast cancer cells. This activation was a time, dose and pH dependent process. These results suggest that the 52 kd pro-cathepsin D may be involved in the early steps of the "metastatic cascade", activating pro-cathepsin B in an acidic environment.

Overexpression and hormonal regulation of pro-cathepsin D in mammary and endometrial cancer.

Pro-cathepsin D is overexpressed in breast cancer cells compared to normal mammary epithelial cells. Moreover, its processing and maturation are altered resulting in increased secretion. In estrogen-responsive breast cancer cell lines such as MCF7 cells and ZR75-1 cells, the 2.2-kb cathepsin D mRNA is accumulated specifically by estrogens and growth factors. Estrogen regulation is mostly transcriptional, while growth factors stabilize the mRNA and act indirectly. In estrogen-receptor-negative cell lines, there is a constitutive high production of cathepsin D mRNA. Moreover in uterine cells, progesterone is the inducer rather than estrogen, indicating the complexity of regulation by steroids, depending on the tissue. The increased production of cathepsin D appears to be correlated with the aggressiveness of the tumour, as shown by retrospective clinical studies, suggesting a role in mammary carcinogenesis.