PURPOSE: Müller cells (MC) are known to undergo functional and morphological changes and to produce matrix metalloproteinases (MMPs) during retinal proliferative diseases. Alpha 2-macroglobulin (α2-M) receptor (LRP-1) is a large endocytic receptor expressed in most cell types. In previous studies we have demonstrated that MC express LRP-1 and its ligand, α2-M, is able to induce MMP-2 activity. However, the biochemical mechanism by which α2-M/LRP-1 system may control this activity is unclear, which could involve both endocytic and intracellular pathways activation. Herein we investigate the putative mechanisms of a2-M/LRP-1 system on the MMPs regulation and cell migration in a Müller cell line.
MATERIALS AND METHODS:Müller cells culture: The spontaneously immortalized human Müller cell line, MIO-M1, was a kind gift from Dr GA Limb, Institute of Ophthalmology and Moorfields Eye Hospital, London, UK. The MIO-M1 cells were cultured at 37 ºC, 5% CO2, 95% air in Dulbecco´s minimal essential medium (DMEM) with high glucose and Glutamax I supplemented with 10% fetal bovine serum (FBS) and 100 U/ml penicillin/100 µg/ml streptomycin. Müller cells were starved with DMEM without FBS for at least 4 h prior to the incubation with 60 nM a2M for different time periods.
Zymographic analysis: The effect of a2M on MMPs activity was evaluated in aliquots of MIO-M1 supernatants by zymographic analysis. To measure the MMPs activity, supernatants were electrophoresed through 7.5% polyacrylamide gels copolimerized with 1.5% gelatin as substrate (gelatin SDS-PAGE). After electrophoresis, gels were soaked for 30 min with 2.5% (v/v) Triton X-100 and the MMPs activity was developed at 37 °C in the enzyme buffer (50 mM of Tris, 0.2 M sodium chloride, and 5 mM of calcium chloride pH 7.6) for 48 h. After incubation, gels were stained overnight with Coomassie brilliant blue R-250 0.125% (w/v) in 45% (v/v) methanol, 10% (v/v) acetic acid, and the stain was removed by the same solution without the dye. MMP-2 and MMP-9 were identified by molecular size using high-molecular mass (14.5–200 kDa) standards (Bio-Rad, Hercules, CA).
RNA purification: Total cellular RNA was extracted using TRIzol reagent, according to the manufacturer´s instructions (Invitrogen) .
Reverse Transcription PCR (RT-PCR): The expression of MMP-2, MT1-MMP and TIMP-2 was analyzed by RT-PCR. Five µg of total RNA was reverse-transcribed into cDNA. Then 2 µl of cDNA were added to 20 µl of reaction buffer. PCR products were size fractioned on a 1% agarose gel and detected by ethidium bromide staining and identified using molecular markers.
Western blot analysis: To evaluate the MAPK (ERK ½) activation and the expression of MT1-MMP, cell lysates from MIO-M1 were applied on 10% SDS-PAGE, electrotransferred to a nitrocellulose membranes and incubated overnight at 4 ºC with a mouse monoclonal anti p- ERK ½ or MT1-MMP antibodies, respectively. HRP-conjugated secondary antibody was added prior to detection with ECL reagent.
Immunofluorescence Staining: Cells were plated on glass coverslips and cultured in the presence or absence of a2M for different times at 37 ºC. Cells were fixed with methanol, then blocked in PBS-Tween 20 with 5% NGS and incubated with monoclonal anti MT1-MMP or monoclonal anti TIMP-2. Finally the secondary donkey anti-mouse Alexa-488 or donkey anti-mouse Alexa-594 (Molecular Probes), respectively were added. Nuclei were counterstained with Hoechst 33258 Cells were visualized by fluorescence microscopy (Nikon Eclipse TE 2000-U). Images were captured using a cooled digital CCD Camera and imaging software.
Wound-healing assay: Cell migration was determined by wound-healing assay. MIO-M1 cells were seeded in laminin- or collagen-coated dishes, and after 18 h of quiescence, a wound was created across the surface with a tip. The cells tha moved into the scraped area were quantified by light microscopy.
