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First published online 11 April 2006
doi: 10.1242/jcs.02887

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Matrix metalloproteases from chondrocytes generate an antiangiogenic 16 kDa prolactin

¹ Instituto de Neurobiología, Universidad Nacional Autónoma de México, Querétaro, México
² Escuela Superior de Medicina, Instituto Politécnico Nacional, México DF, México
³ Hospital General "Xoco", Secretaría de Salud, Gobierno del Distrito Federal, México

View larger version (28K): [in a new window]   Fig. 1. Cleavage of human prolactin by chondrocyte lysates at different pHs. (A) Reducing western blot analysis of proteolytic products generated from PRL by incubation of 200 ng of human PRL with 2 µg of protein from rat chondrocyte lysates at various pH values. Arrows indicate 16 kDa and 14 kDa PRL fragments. (B) Effect of the cathepsin-D inhibitor, pepstatin-A on the acidic or neutral proteolytic activity of chondrocyte lysates. Migration of PRL isoforms of various relative molecular mass are shown on the left.

View larger version (28K): [in a new window]   Fig. 2. Comparison of PRL-cleaving activity of lysates from rat and human chondrocytes and other human cells. (A,B) Reducing western blot analysis of the PRL proteolytic products obtained at pH 7 when 200 ng of rat or human PRL were incubated in the absence (-) or presence (+) of 2 µg of protein from lysates of rat chondrocytes (A), or when 200 ng of human PRL were incubated with the indicated protein concentrations of lysates from human or rat chondrocytes (B). Arrow indicates the 16K-PRL proteolytic product. (C) The PRL (23K-PRL) and 16K-PRL bands obtained after incubation with various protein concentrations of lysates from human or rat chondrocytes were evaluated densitometrically and the results are expressed as a percentage of the 23K-PRL band evaluated after incubation in the absence of chondrocytes. (D) Reducing western blot analysis of proteolytic cleavage products resulting from the incubation of 200 ng of human PRL with increasing concentrations of lysates from human chondrocytes, skin fibroblasts, U937 lymphoma cells, and A549 lung carcinoma cells. Positions of the PRL isoforms of the indicated relative molecular mass are shown on the left of western blots.

View larger version (22K): [in a new window]   Fig. 3. Identification of PRL fragments generated by neutral proteases of chondrocytes. (A) Reducing western blot analysis of proteolytic products generated from human PRL by incubation with lysates from rat chondrocytes at pH 7 as revealed by the anti-human PRL antiserum (anti-PRL) or by monoclonal antibody INN-368 directed against the C-terminal region of PRL (C-Term). Black arrows indicate the PRL fragments detected by anti-PRL antiserum, and white arrows the PRL fragments detected by INN-368. (B) Western blot analysis under reducing (R) and non-reducing (NR) conditions illustrating the composition of proteolytic products generated from human PRL as revealed by their N-terminal sequencing. Under reducing conditions, the 16 and 14 kDa fragments gave the sequence LPICPGGA, corresponding to the N-terminus of undigested PRL, whereas the 5 kDa fragment had the N-terminal sequence LQMADEE starting at residue Leu156. Under non-reducing conditions, the 25 kDa PRL had both the N-terminal sequence of PRL and a second N-terminus starting at residue Leu156, indicating that it corresponds to a PRL cleaved between Ser155 and Leu156, which upon ß-mercaptoethanol reduction (ß-ME) yields the observed 16 and 5 kDa PRL products. Positions of the PRL isoforms of the indicated relative molecular mass are shown on the left of western blots. (C) Human and rat PRL amino acid sequences neighboring the chondrocyte cleavage site in the human (S155-L156) and the rat (S153-L154) hormones. The predicted chondrocyte cleavage site and its adjacent amino acids are shown for PRLs of different vertebrates.

View larger version (27K): [in a new window]   Fig. 4. The 16K-PRL generated by chondrocytes inhibits endothelial cell proliferation. (A) Coomassie-Blue-stained, non-reducing (NR) SDS-PAGE showing the PRL preparations tested for bioactivity. The preparation containing 16K-PRL was obtained by the incubation of human recombinant PRL with chondrocyte lysates, reduction, and carbamidomethylation as indicated in the methods section. In the preparation with no 16K-PRL, human PRL was added at the end of the incubation of chondrocyte lysates to avoid cleavage, and then the preparation was subjected to reduction and carbamidomethylation. The concentration of PRL in the two preparations was 200 nM and that of 16K-PRL was 100 nM as determined by non-reducing SDS-PAGE densitometry. (B) 16K-PRL inhibition of bFGF-induced proliferation of bovine umbilical vein endothelial cells. Endothelial cells were starved of serum for 24 hours and then allowed to proliferate in complete medium for an additional 24-hour period with or without 2 ng/ml bFGF and the mixture of PRL and chondrocyte proteins that did or did not contain 16K-PRL. Values are the mean ± s.e.m. of triplicate determinations *P<0.05 vs bFGF without 16K-PRL. Data are representative of three independent experiments.

View larger version (24K): [in a new window]   Fig. 5. The neutral PRL-cleaving proteases are secreted by chondrocytes and identified as MMPs. (A) Reducing western blot analysis of proteolytic products generated from PRL by incubation of 200 ng of human PRL with 5 µl of conditioned medium (CM) or non-conditioned medium (NCM), obtained after incubation with or without rat chondrocytes, respectively. Arrows indicate the 16 kDa fragment. (B) Reducing western blot analysis of proteolytic products generated from PRL by incubation of human PRL with non-conditioned medium (lane 1) or with chondrocyte-conditioned medium either alone (lane 2) or together with the serine protease inhibitor aprotinin (10 µg/ml), cysteine protease inhibitor E-64 (25 µg/ml), aspartyl protease inhibitor pepstatin A (Pepst A, 1.4 µM), MMP inhibitors EDTA (5 mM) and 1,10-phenanthroline (Phenant, 10 mM), or after heat inactivation (90°C for 30 min) of conditioned medium (). (C) Reducing western blot analysis of proteolytic products generated from PRL by incubation of human PRL with chondrocyte conditioned medium in the absence or presence of the MMP inhibitor GM6001 (10 µg/ml). (D,E) Reducing (R) and non-reducing (NR) western blot analysis of proteolytic products generated from PRL by incubation of 8 µg of human PRL with or without 106 chondrocytes in the absence or presence of the MMP inhibitor GM6001 (10 µg/ml). Arrows indicate 16 and 25 kDa fragments. Positions of the PRL isoforms of the indicated relative molecular mass are shown on the left of western blots.

View larger version (34K): [in a new window]   Fig. 6. Purified MMPs cleave PRL. Reducing (A) and non-reducing (B) western blot analysis of proteolytic products generated from PRL by incubation of 200 ng of human PRL with 2 µg of protein of rat chondrocyte lysate (L), rat chondrocyte-conditioned medium (CM), or with the indicated amount (ng of protein) of purified MMP-8, MMP-13, MMP-3, MMP-1, MMP-2 or MMP-9. The non-incubated human PRL standard (PRL std) is shown. Arrows indicate the various PRL proteolytic products and their relative molecular mass is indicated on the left of western blots. (C) The densitometric values of the 16-kDa-PRL and 17-kDa-PRL fragments generated by incubation with various concentrations of the purified MMPs were expressed as percentage of that of the 23-kDa-PRL band in the non-incubated PRL standard.

View larger version (41K): [in a new window]   Fig. 7. PRL mRNA, PRL, and N-terminal 16K-PRL are detected in chondrocytes and in chondrocyte-conditioned medium. (A) RT-PCR product obtained from rat chondrocytes using primers specific for PRL (lane 3). PRL cDNA was used as a positive control (lane 1), and omission of reverse transcriptase served as a negative control (lane 2). (B) Reducing western blot analysis of immunoreactive PRL-like proteins in chondrocytes. Western blots with a mixture of purified standards (stds) of rat PRL, N-terminal 16K-PRL, and C-terminal 6K-PRL were run in parallel with 200 µg of chondrocyte lysate and were first probed with anti-rat PRL antiserum (anti-PRL), then stripped and reprobed with monoclonal antibody INN-368 directed against the C-terminal end of PRL (C-Term), and subsequently stripped and reprobed with monclonal antibody INN-1 directed against PRL N-terminal region (N-Term). (C) Western blot analysis of chondrocyte-conditioned medium immunoprecipitated with anti-PRL or normal rabbit serum (NRS), and blotted with either anti-PRL or INN-1. Arrows indicate immunoreactive proteins of 17 and 16 kDa. The relative molecular masses of PRL standards are indicated. (D) Proliferation of bovine umbilical vein endothelial cells incubated in the absence (basal) or presence of 10 µl of chondrocyte-conditioned medium alone or together with a 1:400 dilution of anti-PRL or NRS. Values are the mean ± s.e.m. of three independent experiments. *P<0.05 vs basal.

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