Effects of Differentiation-Inducing Agents on Synthesis, Maturation and Secretion of Cathepsin D in U937 and HL-60 Cells

Abstract

Treatment of human monocyte U937 and promyelocyte HL-60 cultures with agents known to induce differentiation (12-0-tetradecanoylphorbol 13-acetate, calcitriol and dimethylsulfoxide) accelerates the maturation of cathepsin D and enhances the incorporation of [ 35 S]methionine into cathepsin D. The most pronounced effects are obtained with calcitriol, which at a concentration of 10~7M increases the incorporation of [ 35 S]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 > 16% of the newly synthesized enzyme. In the presence of 1 OmM NH 4 C1 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 NH 4 C1 and its efficiency is selectively decreased in cells pretreated with calcitriol. Synthese, Reifung und Sekretion von Cathepsin D in differenzierungsinduzierten U937-undHL-60-ZellenZusammenfassung: Induktion der Differenzierung in humanen Monozyten U937 und Promyelozyten HL-60 mit 12-O-Tetradecanoylphorbol-13-acetat, Dimethylsulfoxid und Calcitriol fuhrt zu einer Beschleunigung der Reifung von Cathepsin D. Zusätzlich kommt es zu einer unterschiedlich ausgeprägten Steigerung des Einbaus von [ 35 S]Methionin in Cathepsin D. Eine mehrfache Steigerung der apparenten Synthesegeschwindigkeit wird insbesondere durch Behandlung von U937-Zellen mit Calcitriol erzielt. Der Anteil des Cathepsins D an der gesamten Markierung des extrahierbaren Proteins steigt von etwa 0.08% auf 0.4%. Außerdem steigt der Anteil des sezernierten Cathepsins von etwa 8% auf > 16%. Ammoniumchlorid, l OmM, induziert in unbehandelten sowie in Calcitriolbehandelten Zellen die Sekretion etwa der Hälfte des neu-synthetisierten Cathepsins D. Die Ergebnisse weisen auf die Existenz von zwei Sortierungsmechanismen für Cathepsin D in U937-Zellen hin, von denen der eine NH 4 Cl-sensitiv ist. Behandlung mit Calcitriol reduziert den Anteil des NH 4 Cl-sensitiven Mechanismus am Transport vom Cathepsin D.

The Two Mannose 6-Phosphate Binding Sites of the Insulin-like Growth Factor-II/Mannose 6-Phosphate Receptor Display Different Ligand Binding Properties

“…This is demonstrated in the treatment of cells with the lysosomotropic amines, chloroquine or NH 4 Cl, which raise the intraorganelle pH and impair the receptor-enzyme dissociation. The inability to dissociate ligand results in constantly occupied receptors and thus stimulates the secretion of newly synthesized acid hydrolases (52,53). Our results indicate that the response to low pH is impaired in the Dom3…”

Expression and maturation of human cathepsin D in baby-hamster kidney cells

“…CCL1O) and human skin fibroblasts were maintained at 37°C in a mixture of air and CO2 (19:1) in Dulbecco's modified Eagle medium (Gibco BRL, Eggenstein, Germany) supplemented with penicillin (100 units/ml), streptomycin (100 ,ug/ml), 4.5 mmNaHCO3, 10 in RPMI 1640 (Gibco BRL) medium with 10% (v/v) heatinactivated foetal-bovine serum and treated with 100 nM-1,25-dihydroxyvitamin D3 as described previously [18].…”

“…Immunotitration of cathepsin D activity BHK cells grown to confluency or calcitriol-treated U937 cells [18] were washed three times with 0.14 M-NaCl/10 mM-sodium phosphate, pH 7.4, and taken up in 10 mM-Tris/HCl, pH 7.2. The samples were frozen and thawed three times and sonicated.…”

“…Human cathepsin D was isolated by immunoprecipitation from extracts of calcitriol-treated human promonocytes U937 [18] and of transfected BHK cells. Small and large polypeptide chains were separated from each other and from immunoglobulins by denaturation in the presence of SDS and subsequent PAGE [27].…”

Urokinase receptor is a multifunctional protein: influence of receptor occupancy on macrophage gene expression.

“…ated with cessation of proliferation and acquisition of a differentiated, macrophage-like phenotype (1)(2)(3). Among the many changes in gene expression that accompany the differentiation of these cells are striking changes in the expression of proteases (4)(5)(6)(7)(8). These changes reflect the complex developmental control of proteases and presumably facilitate the functions of migratory progenitors and tissue macrophages.…”