Matrix metalloproteases from chondrocytes generate an antiangiogenic 16 kDa prolactin.
The 16 kDa N-terminal fragment of prolactin (16K-prolactin) is a potent antiangiogenic factor. Here, we demonstrate that matrix metalloproteases (MMPs) produced and secreted by chondrocytes generate biologically functional 16K-prolactin from full-length prolactin. When incubated with human prolactin at neutral pH, chondrocyte extracts and conditioned medium, as well as chondrocytes in culture, cleaved the Ser155-Leu156 peptide bond in prolactin, yielding - upon reduction of intramolecular disulfide bonds - a 16 kDa N-terminal fragment. This 16K-prolactin inhibited basic fibroblast growth factor (FGF)-induced endothelial cell proliferation in vitro. The Ser155-Leu156 site is highly conserved, and both human and rat prolactin were cleaved at this site by chondrocytes from either species. Conversion of prolactin to 16K-prolactin by chondrocyte lysates was completely abolished by the MMP inhibitors EDTA, GM6001 or 1,10-phenanthroline. Purified MMP-1, MMP-2, MMP-3, MMP-8, MMP-9 and MMP-13 cleaved human prolactin at Gln157, one residue downstream from the chondrocyte protease cleavage site, with the following relative potency: MMP-8 > MMP-13 > MMP-3 > MMP-1= MMP-2 > MMP-9. Finally, chondrocytes expressed prolactin mRNA (as revealed by RT-PCR) and they contained and released antiangiogenic N-terminal 16 kDa prolactin (detected by western blot and endothelial cell proliferation). These results suggest that several matrix metalloproteases in cartilage generate antiangiogenic 16K-prolactin from systemically derived or locally produced prolactin.
Macotela
Yazmín
Y
Instituto de Neurobiología, Universidad Nacional Autónoma de México, Querétaro, México.
Aguilar
Manuel B
MB
Guzmán-Morales
Jessica
J
Rivera
José C
JC
Zermeño
Consuelo
C
López-Barrera
Fernando
F
Nava
Gabriel
G
Lavalle
Carlos
C
Martínez de la Escalera
Gonzalo
G
Clapp
Carmen
C
D000595:Amino Acid Sequence; D020533:Angiogenesis Inhibitors / Q000378:metabolism*; D000818:Animals; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology / Q000201:enzymology*; D017077:Culture Media, Conditioned; D006801:Humans; D008297:Male; D061965:Matrix Metalloproteinase Inhibitors; D020782:Matrix Metalloproteinases / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011388:Prolactin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar
D020533:Angiogenesis Inhibitors; D017077:Culture Media, Conditioned; D061965:Matrix Metalloproteinase Inhibitors; D010446:Peptide Fragments; D012333:RNA, Messenger; C503760:prolactin 16-kDa fragment, human; D011388:Prolactin; D020782:Matrix Metalloproteinases