Altered cathepsin D metabolism in PHEX antisense human osteoblast cells

X-linked hypophosphatemia (XLH), the most common form of hereditary rickets, is caused by loss-of-function mutations of PHEX gene in osteoblast cells, leading to rachitic bone disease and hypophosphatemia. Available evidence today indicates that the bone defect in XLH is caused not only by hypophosphatemia and altered vitamin D metabolism, but also by locally released osteoblastic mineralization inhibitory factor(s), referred to as minhibin. In our present study, we found that suppression of PHEX expression by PHEX antisense in human osteoblast cells caused an increase in cathepsin D expression at protein, but not mRNA, levels. This was associated with a decrease in cathepsin D degradation and an increased cathepsin D release into culture media. Our results also showed that lowering cathepsin D activity in antisense cell conditioned media abolished their inhibitory effect on osteoblast cell calcification, suggesting the involvement of cathepsin D in mediating the minhibin activity of the antisense cell conditioned media.     

Acetate-induced apoptosis in colorectal carcinoma cells involves lysosomal membrane permeabilization and cathepsin D release

Colorectal carcinoma (CRC) is one of the most common causes of cancer-related mortality. Short-chain fatty acids secreted by dietary propionibacteria from the intestine, such as acetate, induce apoptosis in CRC cells and may therefore be relevant in CRC prevention and therapy. We previously reported that acetic acid-induced apoptosis in Saccharomyces cerevisiae cells involves partial vacuole permeabilization and release of Pep4p, the yeast cathepsin D (CatD), which has a protective role in this process. In cancer cells, lysosomes have emerged as key players in apoptosis through selective lysosomal membrane permeabilization (LMP) and release of cathepsins. However, the role of CatD in CRC survival is controversial and has not been assessed in response to acetate. We aimed to ascertain whether LMP and CatD are involved in acetate-induced apoptosis in CRC cells. We showed that acetate per se inhibits proliferation and induces apoptosis. More importantly, we uncovered that acetate triggers LMP and CatD release to the cytosol. Pepstatin A (a CatD inhibitor) but not E64d (a cathepsin B and L inhibitor) increased acetate-induced apoptosis of CRC cells, suggesting that CatD has a protective role in this process. Our data indicate that acetate induces LMP and subsequent release of CatD in CRC cells undergoing apoptosis, and suggest exploiting novel strategies using acetate as a prevention/therapeutic agent in CRC, through simultaneous treatment with CatD inhibitors.     

Angiostatin generation by cathepsin D secreted by human prostate carcinoma cells

Angiostatin, a potent endogenous inhibitor of angiogenesis, is generated by cancer-mediated proteolysis of plasminogen. The culture medium of human prostate carcinoma cells, when incubated with plasminogen at a variety of pH values, generated angiostatic peptides and miniplasminogen. The enzyme(s) responsible for this reaction was purified and identified as procathepsin D. The purified procathepsin D, as well as cathepsin D, generated two angiostatic peptides having the same NH(2)-terminal amino acid sequences and comprising kringles 1-4 of plasminogen in the pH range of 3.0-6.8, most strongly at pH 4.0 in vitro. This reaction required the concomitant conversion of procathepsin D to catalytically active pseudocathepsin D. The conversion of pseudocathepsin D to the mature cathepsin D was not observed by the prolonged incubation. The affinity-purified angiostatic peptides inhibited angiogenesis both in vitro and in vivo. Importantly, procathepsin D secreted by human breast carcinoma cells showed a significantly lower angiostatin-generating activity than that by human prostate carcinoma cells. Since deglycosylated procathepsin D from both prostate and breast carcinoma cells exhibited a similar low angiostatin-generating activity, this discrepancy appeared to be attributed to the difference in carbohydrate structures of procathepsin D molecules between the two cell types. The seminal vesicle fluid from patients with prostate carcinoma contained the mature cathepsin D and procathepsin D, but not pseudocathepsin D, suggesting that pseudocathepsin D is not a normal intermediate of procathepsin D processing in vivo. The present study provides evidence for the first time that cathepsin D secreted by human prostate carcinoma cells is responsible for angiostatin generation, thereby causing the prevention of tumor growth and angiogenesis-dependent growth of metastases.     

Human cathepsin D

A literature survey was performed of human cathepsin D gene, cathepsin D biosynthesis, posttranslatory modifications, transport within the cell, substrate specificity and catalytic effect. Methods used to determine the activity and level of this proteinase as well as its role in the biochemistry and pathobiochemistry of cells, tissues and organs were considered.     

Testosterone increases osteoprotegerin mRNA expression in mouse osteoblast cells.

The role that androgens play in the regulation of bone metabolism has been substantiated in animals and humans. We previously demonstrated that testosterone inhibits osteoclast differentiation stimulated by parathyroid hormone through the androgen receptor in mouse bone-cell cultures. However, the details of this mechanism are still unknown. The present study was aimed at examining whether testosterone would affect the mRNA levels of osteoprotegerin (OPG) and receptor activator of Nf kappa B ligand (RANKL) in mouse bone-cell cultures as well as mouse osteoblastic cell-line, MC3T3-E1 cells by employing semi-quantitative RT-PCR. Testosterone increased OPG mRNA expression in both mouse bone-cell cultures and MC3T3-E1 cells. 10-8 M PTH-(1-34) as well as 10-8M 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] inhibited OPG mRNA expression in mouse bone cells. 10-8 M testosterone antagonized OPG mRNA expression inhibited by 10-8 M PTH-(1-34), but failed to affect OPG mRNA expression inhibited by 10-8 M 1,25(OH)2D3. 10-8 M alpha-dehydrotestosterone, a non-aromatizable androgen, increased OPG mRNA expression. On the other hand, testosterone did not affect RANKL mRNA expression in MC3T3-E1 or mouse bone cells. In conclusion, the present study demonstrated that testosterone increased OPG mRNA expression in mouse bone-cell cultures and the osteoblastic cell line. These effects are likely to take place through the androgen receptor.     

Role of abnormal neutral endopeptidase-like activities in Hyp mouse bone cells in renal phosphate transport

We investigated whether the absence of Phex (phosphate-regulating gene with homologies to endopeptidases on the X chromosome) in the Hyp mouse affects the expression and activity of neprilysin (NEP) and of endothelin-converting enzyme-like endopeptidase (ECEL1/DINE) in bone marrow stromal cells (BMSC) and osteoblasts (Ob). Total NEP-like activity was higher in Ob than in BMSC regardless of genotype, and Hyp cells showed higher activities than normal. Conditioned media (CM) from Hyp BMSC and Ob inhibited inorganic phosphate (P(i)) uptake by mouse proximal tubule cells, and incubating Hyp Ob with phosphoramidon prevented the production of the inhibitor of renal P(i) uptake. A linear relationship was observed between the NEP-like activity of Hyp and normal cells and the inhibition of P(i) uptake. NEP and ECEL1/DINE mRNA levels were higher in Hyp cells than in normal cells, and in situ hybridization of ECEL1/DINE confirmed higher levels of expression in the Hyp mouse than in normal cells. In conclusion, we observed a correlation between the inhibition of P(i) uptake by CM from Hyp cells and elevated NEP-like activities.     

Breakdown of brain tubulin by cerebral cathepsin D

The breakdown of cytoplasmic tubulin from brain (purified by ammonium sulfate fractionation and DEAE cellulose chromatography) by cathepsin D from brain (purified by ammonium sulfate fractionation and pepstatin Sepharose chromatography) was studied; changes in the intensity of tubulin gel bands were determined. The pH optimum of hemoglobin breakdown by cathepsin D was 3.2; the pH optimum for tubulin breakdown was 5.8; at pH 5.8 there was no significant hemoglobin breakdown by the enzyme. Tubulin breakdown had an apparent K_m of 1.8 × 10⁻⁵ M and a V_max of 0.56 μg tubulin (μg enzyme per min). The rate of breakdown was heterogeneous and studied on length of incubation; the major portion of tubulin was rapidly broken down and a smaller portion was more stable. The rate under our experimental conditions was 18%/h in the 1–4 h period and 2%/h after 4 h. This was not due to enzyme instability: after 4 h of inhibition freshly added tubulin was rapidly broken down, whereas freshly added enzyme did not increase the rate of breakdown. Thus breakdown heterogeneity was due to substrate (tubulin) heterogeneity. Pepstatin inhibited cathepsin D breakdown of tubulin at acid pH; at pH 7.6 it had no effect. Leupeptin was not inhibitory. We calculated that the cathepsin D content in brain, if fully active, could break down cytoplasmic tubulin with a half-life of 24 h, but it is likely that under in vivo conditions enzyme activity is greatly modified.     

Phospholipids activate cathepsin D

Total lipids as well as phospholipids extracted from the mitochondrial-lysosomal fraction of porcine adrenal cortex activated the lysosomal cathepsin D of this tissue 30- and 40-fold, respectively, with bovine serum albumin as the substrate. Phosphatidic acid, phosphatidyl ethanolamine, phosphatidyl serine, phosphatidyl inositol, phosphatidyl glycerol and cardiolipin were found to activate greatly the cathepsin D. The degree of activation ranged from 6-fold by phosphatidyl ethanolamine to 40-fold by cardiolipin at 1 mM, respectively. These results strongly point to the importance of phospholipids in intracellular protein degradation by lysosomal cathepsin D.     

Structural differences between rabbit cathepsin E and cathepsin D

Rabbit cathepsins D and E were isolated from bone marrow. Both enzymes were purified by affinity chromatography on pepstatin-Sepharose 4B and Con A-Sepharose 4B. Purity of the enzymes was ascertained by two-dimensional gel electrophoresis after iodination. The isoelectric point of cathepsin D was found to be 6.95. Cathepsin E was shown to consist of two subunits having molecular masses each of 40 kDa and isoelectric points of 4.60 and 4.65, respectively. The amino-acid composition of cathepsin E was found to be different from that of cathepsin D.     

Increased carcinogenic potential of myeloid tumor cells induced by aberrant TGF-beta1-signaling and upregulation of cathepsin B

The TGF-beta signaling pathways are implicated in cancer. Cysteine cathepsins can contribute to the carcinogenic potential of tumor cells. The aim of this study was to investigate the regulation of cysteine cathepsin expression by TGF-beta1 and the functional implications in tumor cells. We found an upregulation of cathepsin B (CathB, 2- to 5-fold) in different myeloid tumor cells (THP-1, MonoMac-1, MonoMac-6) after incubation with TGF-beta1. No upregulation was found in monocytes, and there was suppression of CathB expression in epithelial tumor cells (A549). Increased cathepsin B activity led to enhanced carcinogenic potential, which was reflected by increased migration and invasion of the cells and resistance to inhibitor-induced apoptosis. Analysis of the TGF-beta signaling pathways showed no alterations in TGF-beta/BMP receptor expression or SMAD2/3 phosphorylation, and no influence of MAP kinase pathways. However, a reduction in SMAD1 expression was detected. The lack of BMP action on cysteine cathepsin expression in myeloid tumor cells, but not in epithelial tumor cells, suggests a defect in the Smad1/Smad5 pathway. We located a related TGF-beta1-responsive element within the first intron of the CathB gene. In conclusion, alterations in the TGF-beta1 signaling pathway lead to upregulation of CathB, which contributes to the carcinogenic potential of tumor cells.     

Side-chain oxidation of vitamin D3 in mouse kidney mitochondria: effect of the Hyp mutation and 1,25-dihydroxyvitamin D3 treatment

Side-chain oxidation of vitamin D is an important degradative pathway. In the present study we compared the enzymes involved in side-chain oxidation in normal and Hyp mouse kidney. Homogenates of normal mouse kidney catalyze the conversion of 25-hydroxyvitamin D3 to 24,25-dihydroxyvitamin D3, 24-oxo-25-hydroxyvitamin D3 and 24-oxo-23,25-dihydroxyvitamin D3. After subcellular fractionation, total side-chain oxidative activity, estimated by the sum of the three products synthesized per milligram protein under initial rate conditions, coincided with the mitochondrial enzyme marker succinate-cytochrome-c reductase. Treatment of normal mice with 1,25-dihydroxyvitamin D3 (1.5 ng/g) resulted in an eightfold increase in mitochondrial enzyme activity, with no change in apparent Km but a significant rise in Vmax. With 24,25-dihydroxyvitamin D3 as the substrate, normal renal mitochondria produced 24-oxo-25-hydroxyvitamin D3 and 24-oxo-23,25-dihydroxyvitamin D3, and the synthesis of these metabolites could be increased sixfold by pretreatment with 1,25-dihydroxyvitamin D3. In the Hyp mouse, the side-chain oxidation pathway showed similar subcellular distribution of enzyme activity. However, product formation from 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 was twofold greater in mutant than in normal mitochondria. Furthermore, 1,25-dihydroxyvitamin D3 pretreatment of Hyp mice resulted in a 3.4-fold increase over basal metabolism of both 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3. These results demonstrate that (i) kidneys from normal and Hyp mice possess basal and 1,25-dihydroxyvitamin D3 inducible enzyme system(s) in the mitochondrial fraction, which catalyze the side-chain oxidation of 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3, and (ii) the Hyp mutation appears to perturb the renal metabolism of both substrates only in the basal state.     

Expression of human cathepsin L or human cathepsin V in mouse thymus mediates positive selection of T helper cells in cathepsin L knock-out mice

A genetic deficiency of the cysteine protease cathepsin L (Ctsl) in mice results in impaired positive selection of conventional CD4+ T helper cells as a result of an incomplete processing of the MHC class II associated invariant chain or incomplete proteolytic generation of positively selecting peptide ligands. The human genome encodes, in contrast to the mouse genome, for two cathepsin L proteases, namely cathepsin L (CTSL) and cathepsin V (CTSV; alternatively cathepsin L2). In the human thymic cortex, CTSV is the predominately expressed protease as compared to CTSL or other cysteine cathepsins. In order to analyze the functions of CTSL and CTSV in the positive selection of CD4+ T cells we employed Ctsl knock-out mice crossed either with transgenic mice expressing CTSL under the control of its genuine human promoter or with transgenic mice expressing CTSV under the control of the keratin 14 (K14) promoter, which drives expression to the cortical epithelium. Both human proteases are expressed in the thymus of the transgenic mice, and independent expression of both CTSL and CTSV rescues the reduced frequency of CD4+ T cells in Ctsl-deficient mice. Moreover, the expression of the human cathepsins does not change the number of CD4+CD25+Foxp3+ regulatory T cells, but the normalization of the frequency of conventional CD4+ T cell in the transgenic mice results in a rebalancing of conventional T cells and regulatory T cells. We conclude that the functional differences of CTSL and CTSV in vivo are not mainly determined by their inherent biochemical properties, but rather by their tissue specific expression pattern.     

Activation of cathepsin D by glycine ethyl ester.

Cathepsin D purified from bovine spleen is activated by glycine ethyl ester. A maximum of 70% activation was observed at a glycine ethyl ester concentration of 0.1 M and at pH 4.5. The activation effect appears to be reversible.     

Analysis of big endothelin-1 digestion by cathepsin D

Digestion of big endothelin (ET)-1 by cathepsin D, which is the only substantially identified protease showing ET converting enzyme activity, was characterized. Increased doses of cathepsin D showed decrease of immunoreactive (ir-) ET produced from big ET-1. Time course of big ET-1 conversion showed marked increase of ir-ET in a relatively short period, but further incubation resulted in the decrease of ir-ET. Incubation at various pHs with different doses of cathepsin D revealed that low doses of cathepsin D yielded the maximum production of ir-ET at pH 3.5-4.0, but higher doses of cathepsin D showed a bimodal curve of ir-ET production, which may be due to degradation of ir-ET. HPLC analysis revealed that cathepsin D cleaves Asn18-Ile19 bond in addition to Trp21-Val22 bond of big ET-1. These data suggests cathepsin D is not a physiological endothelin converting enzyme.     

Characterization of parathyroid hormone fragments produced by cathepsin D

Cleavage of parathyroid hormone by cathepsin D was studied. Four primary products were detected and separated by high performance liquid chromatography. Two of the fragments are fluorescent and therefore contain residue 23 (tryptophan). These fragments are NH2-terminal in origin. The other two cross-react with antisera directed against COOH-terminal portions of the hormone; they are the complementary COOH-terminal fragments. Microsequencing and amino acid analysis showed that the two COOH-terminal fragments are 35-84 and 38-84 bovine parathyroid hormone. By CNBr cleavage and amino acid analysis, the two NH2-terminal fragments were shown to be the complementary 1-37 and 1-34 fragments. The 1-37 fragment is transitory and is rapidly hydrolyzed to 1-34, so that only relatively small amounts are detected at any one time. However, 34-84 was not converted to 38-84, although cleavage at other sites in the COOH-terminal fragments was observed with more exhaustive digestion. The 1-34 fragment appears to be the final product of the action of cathepsin D on parathyroid hormone. Both enzymatically produced NH2-terminal fragments were fully active in the renal membrane adenylyl cyclase assay system.