Vasoinhibins, N-terminal mouse prolactin fragments, participate in mammary gland involution

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   Figure 1

   mPRL protease cleavage and N-terminal sequences. (A) Effect of Cath D concentration on mPRL cleavage. (Mr) molecular mass marker (masses given in kDa at left and right of blot) (1) mPRL 2 μg, (2) mPRL 2 μg+Cath D 80 ng+Pep A 8 ng, (3) mPRL 2 μg+Cath D 80 ng, (4) mPRL 2 μg+Cath D 120 ng, and (5) mPRL 2 μg+Cath D 150 ng. (B) Effects of thrombin and MMP8 on mPRL cleavage. (Mr) molecular mass marker (masses given in kDa at left and right of blot) (1) mPRL 2 μg+thrombin 0.05 units, (2) mPRL 2 μg +thrombin 0.5 units, (3) mPRL 2 μg+thrombin 1 unit, (4) mPRL 2 μg+MMP8 0.4 ng, (5) mPRL 2 μg+MMP8 4 ng, and (6) mPRL 2 μg+MMP8 8 ng. (C) Amino acid sequence analysis of the site of mPRL cleavage by Cath D. mPRL consisted of 197 amino acids and was cleaved between residues 148 (Trp) and 149 (Ser) by Cath D. (A) and (B) shows a representative result from three independent experiments.
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   Figure 2

   Immunoblotting of PRL, N-terminal mPRL fragment, and Cath D in MG. (A) Immunoblot analysis of various physiological states of mouse MG with N-PRL Ab. The molecular sizes of the protein bands show that the reactants were full-length PRL (23 kDa) and the N-terminal mPRL fragment. (B) Immunoblots of various physiological states of mouse MG tissue reacted with Cath D Ab. The molecular sizes of the protein bands show that the reactants were mature Cath D (33 kDa) and intermediate Cath D (48 kDa). Mr, molecular mass marker (masses given in kDa at left and right of blots); P10, day 10 of pregnancy; L10, day 10 of lactation; W0, delivery day; W1–5, day 1–5 of weaning; Pit, mouse pituitary (female L10); in (A) the positive control (PC) was mPRL incubated with Cath D, at 37 °C for 10 min; in (B) the PC was mouse liver (female L10). In (A) and (B), total protein for each column was quantified and equivalent amounts were applied. (A) and (B) each show a representative result from three independent experiments.
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   Figure 3

   Effects of 1–145mPRL on MG fragments and MCF7. Representative images of three independent experiments shown for DNA fragmentation. (A) The blue signals in (a) and (c) show DAPI staining of each right-side panel. The green signals in (b) (−1–145mPRL) and (d) (+1–145mPRL) show TUNEL-positive cells after 8 h incubation with day 14 lactation MG fragments. Bars=50 μm. −1–145mPRL, 8 h after addition of 1–145mPRL buffer; +1–145mPRL, 8 h after addition of 1–145mPRL. (B) The blue signals in (a) and (c) show DAPI staining of each right-side panel. The green signals in (b) (−1–145mPRL) and d (+1–145mPRL) show TUNEL-positive cells after 3 h incubation with MCF cells. Bars=100 μm. −1–145mPRL, 3 h after addition of 1–145mPRL buffer; +1–145mPRL, 3 h after addition of 1–145mPRL. Each graph column shows the percentage TUNEL-positive MG fragment cells/DAPI (−1–145mPRL 33.69% and+1–145mPRL 75.39%) or TUNEL-positive MCF7 cells/DAPI (1–145mPRL buffer 20.44% and 1–145mPRL 63.3%). Cell counts were examined in five fields per section and in triplicate. Data were analyzed by Student's t-test. *P<0.01 vs respective control without 1–145mPRL.
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   Figure 4

   Prl, Prlr, and Cath D mRNA expression. Mr, molecular mass marker; VG, virgin MG; P18, pregnancy day 18 MG; L0, delivery day; L8, lactation day 8; L18, lactation day 18; W3, weaning day 3; A pit, anterior pituitary gland; P pit, posterior pituitary gland; NC, no template control. A result representative of three independent experiments was shown.
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   Figure 5

   mPRL cleavage by intracellular organelles or extracellular products of MG. (A) mPRL was cleaved by VG and W3 MG microsomal fractions dose-dependently. (Mr) molecular mass marker (masses given in kDa at left and right of blot); (1–3) VG; (4–6) W3. (1, 4) MG microsomal fraction 1 μg+mPRL 2 μg. (2, 5) MG microsomal fraction 5 μg+mPRL 2 μg. (3, 6) MG microsomal fraction 10 μg+mPRL 2 μg. PC, positive control Cath D 200 ng+mPRL 2 μg. (7) MG microsomal fraction 10 μg+mPRL 2 μg+Pep A 30 ng. (8) MG microsomal fraction 10 μg+mPRL 2 μg (pH 7.4). NC, negative control Cath D 200 ng+mPRL 2 μg. (B, left panel) Detection of biotin of cleaved mPRL from MG fragments incubated with mPRL^biot. Mr: molecular mass marker (masses given in kDa at left and right of blot), −mPRL^biot: addition of phosphate buffer (dilutions of mPRL^biot), +mPRL^biot: addition of biotinylated mPRL, Positive control (PC): mPRL^biot was cleaved by purified Cath D, negative control (NC): mouse pituitary gland. (B, right panel) Detection of Cath D by immunoblotting from MG fragments incubated with mPRL^biot. Mr: molecular mass marker (masses given in kDa at left and right of blot), +mPRL^biot: addition of biotinylated mPRL, Liver: positive control for Cath D. (C, left panel) Detection of biotin of cleaved mPRL from MG fragment culture medium incubated with mPRL^biot. Mr: molecular mass marker (masses given in kDa at left and right of blot), −mPRL^biot: addition of phosphate buffer (dilutions of mPRL^biot), +mPRL^biot: addition of biotinylated mPRL, PC: mPRL^biot was cleaved by purified Cath D, NC: mouse pituitary gland. Pep A⁺: addition of an aspartic proteinase inhibitor. (C, right panel) Detection of Cath D by immunoblotting of MG fragment culture medium incubated with mPRL^biot. Mr: molecular mass marker (masses given in kDa at left and right of blot); +mPRL^biot: addition of biotinylated mPRL, Liver: positive control for Cath D. (A) and (B) each show a sample representative of three independent experiments.

• © 2014 Society for Endocrinology