Matrix metalloproteases from chondrocytes generate an antiangiogenic 16 kDa prolactin

Yazmn Macotela 2 Manuel B. Aguilar 2 Jessica Guzmn-Morales 2 Jos C. Rivera 2 Consuelo Zermeo 1 Fernando Lpez-Barrera 2 Gabriel Nava 2 Carlos Lavalle 0 Gonzalo Martnez de la Escalera 2 Carmen Clapp ) 2 0 Hospital General Xoco, Secretaria de Salud , Gobierno del Distrito Federal , Mexico 1 Escuela Superior de Medicina, Instituto Politecnico Nacional , Mexico DF , Mexico 2 Instituto de Neurobiologia, Universidad Nacional Autonoma de Mexico , Queretaro , Mexico - The 16 kDa N-terminal fragment of prolactin (16Kprolactin) 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 e pH, chondrocyte extracts and conditioned medium, as well c n as chondrocytes in culture, cleaved the Ser155-Leu156 ice peptide bond in prolactin, yielding upon reduction of S intramolecular disulfide bonds a 16 kDa N-terminal l fragment. This 16K-prolactin inhibited basic fibroblast l e C growth factor (FGF)-induced endothelial cell proliferation f in vitro. The Ser155-Leu156 site is highly conserved, and lo both human and rat prolactin were cleaved at this site by n chondrocytes from either species. Conversion of prolactin a ru to 16K-prolactin by chondrocyte lysates was completely o J Introduction Cartilage is an avascular tissue where blood vessel invasion is highly restricted, except during endochondral bone formation (Harper and Klagsbrun, 1999; Erlebacher et al., 1995) or in degenerative joint diseases, such as osteoarthritis and rheumatoid arthritis (Walsh, 1999). Blood vessel invasion from the underlying bone can lead to cartilage resorption by triggering apoptosis of chondrocytes and degradation of the extracellular matrix (Gerber et al., 1999; Zelzer et al., 2004). Matrix metalloproteinases (MMPs) are a family of extracellular matrix-degrading enzymes that share common functional domains and activation mechanisms (Sternlicht and Werb, 2001). MMPs produced by chondrocytes are upregulated in association with blood vessel invasion (Ortega et al., 2003), and they are required for endochondral bone formation (Stickens et al., 2004) and for cartilage destruction in osteoarthritis (Billinghurst et al., 1997) and in rheumatoid arthritis (Tetlow and Woolley, 1995). Identification of factors controlling angiogenesis in cartilage can lead to the discovery of new pathways regulating cartilage remodeling and repair in both healthy and diseased states. Because a variety of angiogenic factors are present in cartilage (Gelb et al., 1990; Twal et al., 1994; Harada et al., 1994), its avascularity probably results from naturally abolished by the MMP inhibitors EDTA, GM6001 or 1,10phenanthroline. Purified MMP-1, MMP-2, MMP-3, MMP8, 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. occurring inhibitors that prevent new vessel growth. Antiangiogenic factors identified in cartilage include inhibitors of proteases (DiMuzio et al., 1987; Folkman and Shing, 1992; Moses and Langer, 1991), chondromodulin-I (Hiraki et al., 1997), troponin-I (Moses et al., 1999), and metastatin (Liu et al., 2001). In addition, cartilage contains a group of angiogenesis inhibitors derived by proteolysis from larger proteins that are not themselves inhibitors of angiogenesis (Cao, 2001). Two such inhibitors are endostatin, a C-terminal fragment of collagen XVIII (OReilly et al., 1997), and angiostatin, an internal fragment of plasminogen (OReilly et al., 1994). Endostatin is expressed in cartilage (Pufe et al., 2004), and both endostatin and angiostatin are generated by the proteolytic action of MMPs and cathepsins present in cartilage (Felbor et al., 2000; Lijnen et al., 1998; Patterson and Sang, 1997; OReilly et al., 1999; Morikawa et al., 2000). Another antiangiogenic domain is the 16 kDa N-terminal fragment of the hormone prolactin (PRL) (hereafter referred to as 16K-PRL). The 23 kDa parent molecule PRL (also referred to as 23K-PRL, full-length PRL or PRL) lacks inhibitory activity on endothelial cells (Clapp et al., 1993), whereas 16KPRL inhibits angiogenesis in vivo and in vitro by suppressing growth-factor-induced endothelial cell proliferation (Clapp et al., 1993, Tabruyn et al., 2005), and stimulating the expression of the type 1 plasminogen activator inhibitor (Lee et al., 1998) and endothelial cell apoptosis (Martini et al., 2000). The acidic-aspartyl endoprotease cathepsin-D has been claimed to be the protease responsible for cleaving full-length PRL to 16K-PRL (Baldocchi et al., 1993). However, the relevance of this protease is debatable because human PRL, unlike rat PRL, is resistant (Khurana et al., 1999a) or much less susceptible (Piwnica et al., 2004) to cleavage by cathepsin-D. In search of the biologically relevant PRL-cleaving protease, we reasoned that a likely source for such an enzyme would be an avascular tissue rich in antiangiogenic factors, such as cartilage. Here, we present the first report that MMPs produced by chondrocytes cleave PRL at a highly conserved site and generate biologically active 16K-PRL. We also provide evidence that MMPs may cleave PRL derived from circulation and PRL produced by chondrocytes. Results Proteolysis of human PRL by chondrocyte proteases Incubation of human PRL with lysates from rat chondrocytes resulted in its partial conversion to fragments with apparent molecular masses of 17, 16, 15 and 14 kDa, as revealed by reducing SDS-PAGE (Fig. 1). The proportion of PRL fragments varied according to the pH of the incubation buffer. ce At pH 5, only the 14 kDa PRL fragment was produced; as the n pH increased, the amount of this fragment declined and others e ic became apparent, including the 16 kDa fragment which S predominated at pH 7 (Fig. 1A). There was no proteolysis lle when PRL was incubated at neutral or acid pH in the absence C of chondrocyte lysates (Fig. 1A,B). Addition of pepstatin-A, fo an inhibitor of cathepsin-D, to the incubation mixture l completely abolished proteolytic cleavage by chondrocyte a n lysates at pH 5 but not at pH 7 (Fig. 1B). These results indicate r u o J 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 g of protein from rat chondrocyte lysates at various pH values. Arrows indicate 16 (...truncated)