IGF-1 regulates MMP2 activity in a hypoxia-independent manner in Müller Cells

LORENC VE; LUNA JD; CHIABRANDO GA; SANCHEZ MC

Orlando

Congreso; ARVO Annual Meeting; 2014

Purpose: Proliferative diabetic retinopathy, has been associated with retinal hypoxia and upregulation of VEGF and IGF-1. Under these conditions, Müller cells (MC) acquire migratory abilities, suggesting to be associated with enhanced MMP-2 and MMP-9 activities. However, the biochemical mechanisms regulating MMPs activities and MC migration remain poorly understood. We have previously demonstrated in normoxy, that IGF-1 induces changes in the MMP2 activity in MC. In this work, we investigate the relationship between hypoxia and IGF-1 on the regulation of MMP-2 activity. Methods: The MIO-M1 cell line was stimulated with 10 nM IGF-1,8 hours. All in vitro experiments were performed under normoxic (21% O2) and hypoxic (1% O2) conditions. First, hypoxia was evaluated by analyzing HIF-1α protein by Western blot. To evaluate MMPs activities, MC supernatants were analyzed by zymography where active MMP-2 (66 kDa) as well as pro-MMP-2 (72 kDa) may be clearly identified. In addition, MMP-2 and MMP-14 protein expression were analyzed in MC lysates by Western blot. To block the IGF-1 action, MC were pretreated with αIR3antibody, which specifically inhibits the IGF-1/IGF-1R binding. Also pharmacological inhibitors (PD90058- and LY-294002) for IGF-1-induced intracellular signaling pathways were used. Finally, in OIR model, MMP-2 retinal localization was examined at P7, P15 and P26 by immunofluorescence (IF) microscopy. Results: By zymography, supernatants of MC cultured in normoxy showed, under IGF-1 stimulus, a decreased MMP-2 active form compared with non stimulated IGF-1 cells. This effect was blocked when MC were pretreated with αIR3 or LY-294002, indicating that this was mediated by IGF-1R and PI3K pathway. When MC were cultured in hypoxia protein extracts showed an increasing in HIF- levels with respect to normoxic conditions. In absence of IGF-1, hypoxia produced a slight decrease in proMMP2 activity and an increase in active MMP2 form. The same effect of IGF-1 under normoxic conditions was also observed in the hypoxic ones. In addition, there was a restitution of active MMP2 activity when cells were preincubated with αIR3. We were unable to detect changes in the MMP-14 protein levels, in the presence or absence of IGF-1, under normoxic or hypoxic conditions. Finally, by IF we observed active MMP-2 in the INL and ONL as well as in blood vessels. The MMP-2 expression was highly detected during retinal development (P7) as well as with the induction of retinopathy (P15). Conclusion: These results demonstrated that IGF-1 decreases the active form of MMP-2 in the supernatant of MC, by an oxygen independent pathway. This IGF-1 effect could be related with an enhanced MMP2 activity in OIR model.