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First published online September 29, 2004
doi: 10.1242/10.1242/jcs.01396

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Cathepsin D released by lactating rat mammary epithelial cells is involved in prolactin cleavage under physiological conditions

¹ Faculté des Sciences, Université Chouaib Doukkali, B.P. 20 El Jadida, Morocco
² Dipartimento di Scienze Mediche, Università `Amedeo Avogadro', via Solaroli 17-28100, Novara, Italy
³ INRA, Unité Génomique et Physiologie de la Lactation, 78352 Jouy-en-Josas CEDEX, France

View larger version (81K): [in a new window]   Fig. 1. Detection of rPRL cleaved forms in vesicular fractions and incubation media from acini. (A) Scanning electron micrograph of acinus obtained after enzymatic dissociation of lactating mammary tissue. The 3D organisation and the morphological integrity are well preserved. Bar, 13.8 µm. (B) Electrophoresis under reducing conditions and immunoblotting analysis of rPRL molecular forms detected in tissue and in incubation medium of acini incubated in the presence of rPRL. Incubation media and acini were treated for immunoblotting as described in Materials and Methods. Immunoblotting analysis of: rPRL forms in Hanks' medium, incubated for 60 minutes at 37°C in the presence of rPRL (lane 1); vesicular content, obtained as described in Materials and Methods, of acini incubated in the presence of 5 µg/ml rPRL for 15 minutes at 20°C, washed and then chased for 5 minutes at 37°C, a time interval corresponding to the presence of PRL inside vesicles (lane 2); medium from acini incubated without exogenous rPRL (lane 3); medium from acini incubated for 60 minutes at 37°C in the presence of 5 µg/ml rPRL (lane 4). The PRL forms generated in vitro by incubating 1.5 µg/ml rPRL in citrate-phosphate buffer pH 3.2 containing bovine cathepsin D (enzyme to protein ratio 1:200) are also shown (lane 5). Positions of the molecular mass markers (kDa) are indicated on the left.

View larger version (35K): [in a new window]   Fig. 2. Effect of ammonium chloride and of chloroquine on rPRL cleavage in rat MECs. Electrophoresis under reducing conditions and immunoblotting analysis of rPRL forms detectable in mammary epithelial cells and in their media upon incubation with 23 kDa rPRL in the presence or absence of 10 mM ammonium chloride (NH4Cl) or 10 µM chloroquine (ClQ) as indicated. Mammary fragments were preincubated for 30 minutes at 37°C in the presence or absence of the drug, then 5 µg/ml rPRL was added or not for 30 minutes at 20°C in order to accumulate the hormone intracellularly and further incubated for 60 minutes at 37°C. Incubation media and fragments were treated for immunoblotting as described in Materials and Methods. (A) Immunoblotting analysis of rPRL forms in homogenates from tissue fragments (cells) incubated in the presence of weak bases. The 23 kDa form of rPRL was detectable in tissue incubated in the presence of exogenous PRL. (B) Immunoblotting analysis of rPRL forms in the incubation medium of tissues fragments (medium) incubated in the presence of weak bases. In addition to the 23 kDa form of rPRL, a form with an Mr of about 18 kDa and a much more abundant form with an Mr of 16 kDa were detectable. (C) Immunoblotting analysis of rPRL forms in homogenate and medium of tissue fragments incubated in Hanks' medium in the presence of exogenous rPRL. In tissue fragments (cells) the 23 kDa was detectable. In the medium, the 23 kDa and the 16 kDa forms were detectable. Positions of the molecular mass markers (kDa) are indicated on the left.

View larger version (41K): [in a new window]   Fig. 3. Mammary acini-conditioned medium contains a PRL-cleaving activity. (A) Electrophoresis under reducing conditions and immunoblotting analysis of rPRL forms detectable in acini-conditioned medium upon incubation of rPRL. We used acini-conditioned medium, pre-heated to 100°C or not, in which 2.5 µg/ml rPRL was incubated for 60 minutes at 37°C or at 4°C. The experiment reveals the formation of 16 kDa PRL in acini-conditioned medium at 37°C, but not in those conditions (4°C, pre-heating at 100°C) that are known to abolish enzymatic activities. (B) In vitro proteolysis of PRL. Immunoblotting analysis of rPRL after incubation for 60 minutes at 37°C in citrate buffer pH 7.2 (lane 1); in citrate buffer at pH 3.2 containing 0.1 U/ml CD (lane 2); or in conditioned medium at pH 7.4 (lane 3). The lower band of the doublet (lane 3) aligns with the 16 kDa form generated by total digestion of CD at pH 3.2 (lane 2). (C) Time course (from 0 minute to 24 hours) of PRL cleavage at 37°C in acini-conditioned medium. (D) Immunoblotting analysis of rPRL forms incubated in Hanks' medium for 6 hours at 37°C in the presence of rPRL. Positions of the molecular mass markers (kDa) are indicated on the left.

View larger version (98K): [in a new window]   Fig. 4. Effect of BFA on milk protein secretion in MECs. Acini were incubated in the absence or presence of 5 µM BFA for 60 minutes at 37°C, cytospun, fixed, permeabilised with Triton X-100 and treated for immunofluorescence. (A) In control acinus (CO) numerous secretory vesicles labelled with the RAM/MSP antibody are detectable in the cytoplasm (arrows). In the presence of BFA, cytoplasmic secretory vesicles decorated with the antibody RAM/MSP were no more detectable. It is notable that in BFA-treated cells huge lipid globules, whose periphery is strongly labelled with RAM/MSP antibody, accumulate at the apical region (arrowheads) (BM, basal membrane; L, lumen). Bar, 10 µm. (B) Media from control and treated acini were analysed by immunoblotting to reveal the presence of milk proteins. In the control medium (lane -) immunoreactive bands correspond to the numerous milk proteins. The number of milk proteins found in the medium was drastically reduced in the presence of BFA (lane +).

View larger version (44K): [in a new window]   Fig. 5. BFA does not abrogate the PRL cleaving activity in acini-conditioned medium. Acini were incubated in the absence (lane -) or presence (lane +) of 5 µM BFA for 60 minutes at 37°C as described in Materials and Methods and the conditioned media used for assaying the PRL-cleaving activity. To this end, 2.5 µg/ml rPRL was added to the conditioned medium and after a 60 minute incubation at 37°C the samples were analysed by electrophoresis under reducing conditions and immunoblotting to reveal the PRL forms. The experiment demonstrates that the PRL-cleaving enzyme is present in the conditioned medium of BFA-treated acini. Positions of the molecular mass markers (kDa) are indicated on the left.

View larger version (31K): [in a new window]   Fig. 6. Cathepsin D activity is required in the conditioned medium for an efficient cleavage of rPRL. (A) Proteolytic effect of purified thrombin and cathepsin D on rPRL. Immunoblotting analysis of rPRL forms when 1.5 µg/ml rPRL was incubated in Tris-buffer pH 7.4 for 60 minutes at 37°C in the absence (lane 1) or presence of purified enzymes and their inhibitors: 12 U/ml thrombin (lane 2); 0.1 U/ml CD (lane 3); 0.1 U/ml CD and 25 µM pepstatin A (lane 4); or 12 U/ml thrombin and 15 U/ml hirudin (lane 5). (B) Effect of hirudin on the cleaving activity of cathepsin D in Tris-buffer at pH 7.4 for 60 minutes at 37°C. Immunoblotting analysis of rPRL forms in the presence of 0.1 U/ml CD alone (lane 2) or with 15 U/ml hirudin (lane 1). (C) Effect of hirudin on the cleaving activity of the conditioned medium. Immunoblotting analysis of rPRL forms when 1 µg/ml PRL was incubated for 60 minutes at 37°C in conditioned medium in the absence (lane 1) or presence of 15 U/ml hirudin. (lane 2). The doublet is detectable in both lanes 1 and 2. (D) Effect of pepstatin A on the cleaving activity of the conditioned medium. Immunoblotting analysis of rPRL forms when 1 µg/ml rPRL was incubated for 60 minutes at 37°C in conditioned medium in the absence (lane 1) or presence of 10 µM pepstatin A (lane 2). The doublet is hardly detectable in lane 2. (E) Acini-conditioned medium was depleted of CD by immunoprecipitation. In native medium (lane 1) the following rat CD molecular forms can be detected: ProCD, the 51 kDa precursor form; Msc, the 44 kDa single-chain mature form; LM, the 31 kDa large chain of the double-chain mature form. Almost all molecular forms of CD were cleared from the medium after immunoprecipitation with anti-CD serum (lane 2). (F) Immunoblotting analysis of rPRL molecular forms obtained by incubation for 60 minutes at 37°C of 2.5 µg/ml rPRL in conditioned medium (lane 1) or CD-immunodepleted mammary acini-conditioned medium (lane 2). No cleaved forms of rPRL were detectable in the latter case. At the end of the incubation periods media were treated for immunoblotting as described in Materials and Methods.

View larger version (75K): [in a new window]   Fig. 7. Immunofluorescence localisation of cathepsin D in mammary acini of lactating rat. Fragments of the mammary glands of rat were fixed and immunolabelled with anti-cathepsin D (1:100) and FITC-conjugated goat anti-rabbit IgG. (a) Control section treated as described with omission of the primary antibody. (b) DAPI staining of the control section. (c) Cathepsin D is located in cytoplasmic spots (arrow), in the lumen of the acini (L) and strongly accumulated in the basal region of the acini (arrowheads) in close proximity of the basal membrane (BM). Stroma cells are also stained (double arrow). (d) DAPI staining of the section showed in panel c. Bar, 10 µm.

View larger version (57K): [in a new window]   Fig. 8. MECs secrete immature and mature forms of CD; BFA does not completely abolish this secretion. (A) Immunofluorescence localisation of CD and TGN38 (as a marker of Golgi apparatus) in acini incubated for 60 minutes at 37°C in the absence (control, Co) or the presence (BFA) of 5 µM BFA. CD was detectable as red spots at the basal region of cells incubated in control medium (Co, arrows). A strong red labelling was also detectable in the basal region of acini incubated in the presence of BFA (arrows). In acini incubated in control medium, TGN38 (green) was located in the supranuclear region of cells (arrowheads) surrounding the aperture of the acinus which corresponds to the connection with the ductules (L). When acini were incubated in the presence of BFA, TGN38 was dispersed in the cells (arrowhead) confirming the disassembly effect of the toxin on the Golgi complex (BM, basal membrane; L, lumen). Bar, 10 µm. (B) Immunoblotting analysis of CD in cell homogenates (H) and conditioned media (CM) from acini incubated as above. The CD molecular forms are indicated (P, proCD; Msc, mature single-chain; LM, large chain of the mature double-chain). The enzymatic active mature single-chain CD form is detectable in medium from BFA-treated acini.

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