Effects of differentiation-inducing agents on synthesis, maturation and secretion of cathepsin D in U937 and HL-60 cells

Treatment of human monocyte U937 and promyelocyte HL-60 cultures with agents known to induce differentiation (12-O-tetra-decanoylphorbol 13-acetate, calcitriol and dimethylsulfoxide) accelerates the maturation of cathepsin D and enhances the incorporation of [35S]methionine into cathepsin D. The most pronounced effects are obtained with calcitriol, which at a concentration of 10(-7) M increases the incorporation of [35S]methionine into cathepsin D from 0.08% to 0.4% of the detergent-soluble radioactivity. In addition, this treatment enhances the secretion of cathepsin D from about 8% to greater than or equal to 16% of the newly synthesized enzyme. In the presence of 10mM NH4Cl approximately half of the produced cathepsin D is secreted in both control and calcitriol-treated cells. It appears that in U937 cells two mechanisms are involved in sorting of cathepsin D. One of these is sensitive to NH4Cl and its efficiency is selectively decreased in cells pretreated with calcitriol.     

Brefeldin A prevents uncovering but not phosphorylation of the recognition marker in cathepsin D

Brefeldin A (BFA) has been shown to inhibit transiently the subcellular transport of cathepsin D (Oda & Nishimura (1989) Biochem. Biophys. Res. Commun. 163, 220-225). We studied the effect of this antibiotic on processing of the phosphorylated oligosaccharides in cathepsin D in human promonocytes U937. In the presence of the drug the phosphorylation of cathepsin D precursor continued at a diminished rate. The phosphorylated oligosaccharides in cathepsin D comprised mono- and bis-phosphorylated forms. The relative amounts of the two species were not changed in the presence of BFA. The uncovering of the phosphate groups and the proteolytic processing of the phosphorylated precursor were abolished. In an in vitro assay the uncovering enzyme, N-acetylglucosamine-1-phosphodiester N-acetylglucosaminidase was not inhibited by BFA. We suggest that this drug interrupts the traffic between the compartments containing N-acetylglucosaminyl phosphotransferase and N-acetylglucosamine-1-phosphodiester N-acetylglucosaminidase.     

Accelerated secretion of human lysozyme with a disulfide bond mutation.

The mutant human lysozyme, [Ala77, Ala95]lysozyme, in which the disulfide bond Cys77-Cys95 is eliminated, is known to exhibit increased secretion in yeast, compared to wild-type human lysozyme [Taniyama, Y., Yamamoto, Y., Nakao, M., Kikuchi, M. & Ikehara, M. (1988) Biochem. Biophys. Res. Commun. 152, 962-967]. To investigate this phenomenon, mammalian cells were used to analyze the secretion kinetics of [Ala77, Ala95]lysozyme and wild-type human lysozyme. The secretion rate of [Ala77, Ala95]lysozyme during the 150-min chase period was significantly accelerated [half-life (t1/2) = 29 min] compared to that of wild-type human lysozyme (t1/2 = 83 min), when expressed at the same levels within the cells. In contrast, after the 150-min chase, the rates of disappearance of both wild-type and mutant human lysozymes within the cells were similar, and considerably slower (t1/2 = 220 min), respectively. The remaining intracellular wild-type human lysozyme was localized mainly in the endoplasmic reticulum, whereas accelerated transport of the [Ala77, Ala95]lysozyme mutant protein from the endoplasmic reticulum to the Golgi apparatus was observed. Also in yeast cells, similar secretion kinetics and the differences in t1/2 for wild-type and mutant human lysozymes during the early chase period were observed. The two-phase kinetics of disappearance of intracellular human lysozymes suggest that only a proportion of the proteins becomes secretion competent soon after synthesis and is completely secreted during the early chase period, whereas others enter the distinct, slow pathways of intracellular transport and/or degradation. Increased secretion of [Ala77, Ala95]lysozyme is possibly due to enhanced competence for secretion acquired in the endoplasmic reticulum at the early stage of transport events, which is closely connected with the removal of a disulfide bond.     

1,25-Dihydroxyvitamin D3 enhances the generation of nonspecific suppressor cells while inhibiting the induction of cytotoxic cells in a human MLR.

The active vitamin D metabolite, 1,25-dihydroxyvitamin D3 (1,25-D3), has been shown in both in vitro and in vivo experiments to be immunoregulatory. We analyzed the effects of the hormone on the human mixed lymphocyte reaction (MLR), the in vitro model of allograft response. Suppressor-cell activity of MLR-generated effector cells was enhanced by calcitriol (10(-10) to 10(-8) M). This suppressor activity was nonspecific since calcitriol-generated effector cells could suppress a primary MLR with stimulators and/or responders heterologous to the effector-generating MLR. Calcitriol (10(-9) to 10(-7) M) was also effective in preventing the generation of cytotoxic T cells when tested in a 51Cr release assay. While no differences were observed in the phenotypic analyses of the MLR-generated effector cells between 1,25-D3-treated cells and control cells, a significant reduction of class II antigen expression was observed in the presence of the hormone. The effects of calcitriol on human MLR are similar to those observed with cyclosporine.     

Suppression of the 'uncovering' of mannose-6-phosphate residues in lysosomal enzymes in the presence of NH4Cl

The uncovering ratio of phosphate groups in lysosomal enzymes is defined as the percentage of phosphomonoester groups in the oligosaccharide side chains based on the sum of phosphomonoester and phosphodiester groups. Using a new procedure for the specific and complete hydrolysis of uncovered phosphomonoester groups in denatured immunoprecipitates of human cathepsin D, we show that the uncovering ratio varies between different forms of the enzyme and may be used as an indicator of the maturation of its carbohydrate side chains. The uncovering ratio in the total (cellular and secreted) cathepsin D from U937 promonocytes is greater than 95%. It is only slightly decreased in cells incubated in the presence of 1 alpha,25-dihydroxycholecalciferol, in which the rate of synthesis of cathepsin D is several times higher than in the control cells. In U937 cells and also in fibroblasts, the uncovering is nearly complete in intermediate and mature forms of the intracellular cathepsin D but less extensive in the intracellular and secreted precursor. In both cell types, incubation with 10 mM NH4Cl results in a decrease in the uncovering ratio of total cathepsin D. However, the activity of the uncovering enzyme, N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase, as determined with UDP-N-acetylglucosamine is not affected with up to 60 mM NH4Cl. Our results suggest that NH4Cl, in addition to its known effects on the acidic-pH-dependent functions of lysosomal compartments and of mannose-6-phosphate receptors, impairs the processing or transport of lysosomal enzyme precursors at, or proximally to, the site of the uncovering of their mannose-6-phosphate residues.     

Effects of urinary proteins from certain leukemics upon macromolecular synthesis and enzyme levels in bone marrow cultures.

Urinary proteins from human leukemic patients have been found to alter quantitatively macromolecular synthesis in primary mouse bone marrow cultures. Urinary protein-stimulated incorporation of [3H]uridine into RNA was found after 1 day of culture. Increased levels of adenine phosphoribosyltransferase and lysozyme were demonstrable at 3 and 5 days, respectively, with urinary protein-supplemented cultures. The incorporation of 3H-labeled deoxynucleosides into DNA was higher in the presence of urinary proteins after 2 days of culture. The rate of incorporation of [3H]deoxyuridine into DNA was strongly inhibited by 10(-5) M Methotrexate and 10(-6) M 5-fluorodeoxyuridine, however, the effect of urinary proteins on incorporation of [3H]uridine into RNA and lysozyme accumulation were not inhibited. Urinary proteins also stimulated the formation of "colonies" (groups of at least 30 cells) in media containing methylcellulose. This latter phenomenon was also not inhibited by 10(-5) M Methotrexate or 10(-6) M 5-fluorodeoxyuridine. The results of these studies are consistent with the postulate that in the presence of human urinary proteins, mouse bone marrow cells in culture proceed to a phenotype characteristic of circulating peripheral white cells.     

Expression of human cathepsin D in Xenopus oocytes: phosphorylation and intracellular targeting

We have obtained expression of a cDNA clone for human cathepsin D in Xenopus laevis oocytes. Biosynthetic studies with [35S]methionine labeling demonstrated that most of the cathepsin D remained intracellular and underwent proteolytic cleavage, converting a precursor of Mr 47,000 D to a mature form of Mr 39,000 D with processing intermediates of Mr 43,000-41,000 D. greater than 90% of the cathepsin D synthesized by oocytes bound to a mannose 6-phosphate (Man-6-P) receptor affinity column, indicating the presence of phosphomannosyl residues. An analysis of [2-3H]mannose-labeled oligosaccharides directly demonstrated phosphomannosyl residues on cathepsin D. Sucrose-gradient fractionation, performed to define the membranous compartments that cathepsin D traversed during its biosynthesis, demonstrated that cathepsin D is targeted to a subpopulation of yolk platelets, the oocyte equivalent of a lysosome. Xenopus oocytes were able to endocytose lysosomal enzymes from the medium and this uptake was inhibited by Man-6-P, thus demonstrating the presence of Man-6-P receptors in these cells. Therefore, the entire Man-6-P dependent pathway for targeting of lysosomal enzymes is present in the oocytes. Xenopus oocytes should be a useful system for examining signals responsible for the specific targeting of lysosomal enzymes to lysosomes.     

Effect of parathyroid hormone on phospholipid metabolism in osteoblast-like rat osteogenic sarcoma cells.

Previous results have shown that 1,25-dihydroxycholecalciferol [1,25(OH)2D3] enhances the synthesis of phosphatidylserine (PS) and suppresses the synthesis of phosphatidylethanolamine (PE) in osteoblast-like rat osteogenic sarcoma UMR 106 cells [Matsumoto, Kawanobe, Morita & Ogata (1985) J. Biol. Chem. 260, 13704-13709]. In the present study, the effect of parathyroid hormone (PTH) on phospholipid metabolism is examined by using these cells. Treatment of UMR 106 cells with human PTH-(1-34)-peptide suppresses the synthesis of phosphatidylethanolamine in a dose- and time-dependent manner without affecting the synthesis of PS. The maximal effect on PE synthesis is obtained with 2.4 nM-human PTH-(1-34)-peptide when the cells are treated for 48 h or longer. In addition, when human PTH-(1-34)-peptide is added together with the maximal dose of 1,25(OH)2D3, there is a further decline in PE synthesis, whereas the stimulation of PS synthesis by 1,25(OH)2D3 is not altered. Because methylation of PE is suggested to affect hormone receptor-adenylate cyclase coupling, the observed change in PE metabolism by PTH and 1,25(OH)2D3 may be, at least in part, involved in the development of desensitization phenomenon to PTH in these cells.     

Monocyte-derived macrophage and alveolar macrophage fibronectin production and cathepsin D activity

Alveolar macrophages are thought to play an important role in ongoing tissue breakdown and repair processes in the normal lung. The secretion and regulation of cathepsin D (important for the final breakdown of collagen) and fibronectin (involved in the healing process) in human peripheral blood monocytes (PBM) and pulmonary alveolar macrophages (PAM) were investigated. Cathepsin D enzyme activity was measured by quantitating the TCA-soluble fragments of [3H]hemoglobin. Freshly isolated PBM contained less cell-associated cathepsin D activity than did freshly isolated PAM (314 +/- 35 micrograms/10(6) cells vs 381 +/- 35 micrograms/10(6) cells, respectively). After 7-10 days in culture, cell-associated enzyme levels in both PBM and PAM were significantly increased (P less than 0.001 for PBM; P less than 0.0001 for PAM). In addition, freshly isolated PAM secreted more cathepsin D than did freshly isolated PBM (5.8 +/- 3.2 micrograms/10(6) cells vs 0.83 +/- 0.83 micrograms/10(6) cells, P less than 0.02). In the presence of LPS (10 micrograms/ml), cell-associated cathepsin D was inhibited in both PBM and PAM. With the addition of gamma-IFN (500 U/ml), both cell-associated and secreted enzyme were increased in freshly isolated and 10-day-cultured PBM and PAM. In parallel studies, fibronectin secretion (by ELISA assay) in both PBM and PAM increased over time in culture. LPS had no effect on PBM or PAM secretion of human fibronectin while gamma-IFN increased PBM and PAM fibronectin levels. Thus, both macrophage cathepsin D activity and fibronectin secretion are increased by gamma-interferon while macrophage cathepsin D activity, but not fibronectin secretion, is decreased by LPS. These studies demonstrate that human macrophage cathepsin D activity is actively modulated by inflammatory mediators and that macrophage mediators of tissue breakdown and repair are not modulated synchronously.     

Mitogenic effects of certain cathepsins and calciferin on the intact liver in vivo

Calciferin, a new parathyroid hormone stimulating the release of cathepsins D and L (but not B) from isolated lysosomes, or the release of cathepsin D from erythrocytes or ghosts in vitro, elevated free cathepsin D in the blood, and at the same time stimulated DNA synthesis in the intact liver when it was injected into mice. Both calciferin and free cathepsin D in the blood (rats) were elevated concomitantly soon after 70% hepatectomy, reaching a peak around 5 hr. The cathepsin D-elevation was almost proportional to fractional hepatectomies. Cathepsin L (but not B), when injected intraperitoneally into mice, stimulated DNA synthesis and mitosis in the intact liver much like cathepsin D, the effect of which was reported earlier. In contrast to the mitogenic effects of calciferin or cathepsins (D and L) in vivo, only cathepsin L (but not cathepsin D or calciferin) in low concentrations appeared to stimulate DNA synthesis in the cultured liver cells, and also stimulated adenylate cyclase of isolated liver plasma membranes in vitro. Dibutyryl-cyclic AMP in concentrations lower than 10(-5) M also stimulated DNA synthesis in cultured liver cells.     

Measuring the balance between insulin synthesis and insulin release

The absolute rates of hormone synthesis and release were determined in purified pancreatic B cells. Newly synthesized proteins were labeled with L-[3,5-3H]tyrosine or L-[2,5-3H]histidine. When medium glucose was less than or equal to 10 mM, the production of insulin exceeded or equaled its release. Raising the glucose levels above 10 mM did not further increase the rate of insulin synthesis (67 +/- 10 fmol/10(3) cells/2 hour) but elevated that of insulin release up to 3-fold the production rates (181 +/- 10 fmol/10(3) cells/2 hour). In the presence of glucagon or of the phorbol ester 12-O-tetradecanoylphorbol 13-acetate the cells also released 3-fold more hormone that they synthesized; release was however reduced to 25% of the rate of production in the presence of epinephrine. It is concluded that glucose as well as hormonal regulators of islet B cells can influence, bi-directionally, the balance between the rates of insulin synthesis and release.