Lysosomal cathepsin B participates in the podosome-mediated extracellular matrix degradation and invasion via secreted lysosomes in v-Src fibroblasts.

Biochemical reagents and antibodies CA-074, CA-074Me, E64c and E64d were from Peptide International (Louisville, KY). GM6001, PP2, Bafilomycin A1 and cathepsin B detection kit were from Calbiochem (San Diego, CA). Lysotracker Red DND-99 and Mitotracker Red CMXRos were from Invitrogen (Eugene, OR). Cy3 labeling kit was from Amersham (Piscataway, NJ). The rabbit anti-cathepsin B antibody used here has been described previously ( 33 ). Anti-phosphotyrosine antibody 4G10 was a gift from Dr. Brian Druker (Oregon Health & Science University, Portland, OR). Anti-Cortactin antibody 4F11 was purchased from Upstate Biotechnology Inc. (Lake Placid, NY). Anti-mouse LAMP-1 antibody 1D4B was obtained from the NIH Developmental Studies Hybridoma Bank at the University of Iowa. Secondary antibodies were from Invitrogen (Eugene, OR). DNA constructs v-Src cDNA sequence was PCR-amplified from the pLNCX-v-Src plasmid (gift of Dr. Joan Brugge, Harvard Medical School), and the fragment was digested with Cla I and cloned into Hpa I site of the pMSCV-Hygro vector (Clontech, Mountain View, CA). CFP-β-actin construct pEX-EF1-Actin-b-CFP was purchased from ATCC) Cell lines: NIH3T3 cells (ATCC, Rockville, MD) were retrovirally transduced with MSCV-hygro vector or MSCV-hygro-v-Src and selected in 100 μg/ml hygromycin. All cell lines were maintained in Dulbecco's Modified Eagle's Medium (DMEM, Gibco, Carlsbad, CA) containing 5% fetal bovine serum (HyClone, Logan, UT), 2mM L-glutamine, and penicillin/streptomycin.

Extracellular matrix degradation assay

Cy3-gelatin-coated coverslips were prepared as described (Bowden et al., 2001) with certain modifications. Gelatin was labeled using a Cy3 labeling kit (Amersham, Piscataway, NJ) according to the manufacturer's instructions and dialyzed against PBS. Cy3-gelatin was coated onto glass coverslips and crosslinked with 0.5% glutaraldehyde in PBS for 30 minutes. Coated coverslips were then washed three times each with PBS and 50 mM glycine in PBS. Cells were cultured for various time points to allow ECM degradation, seen as focal loss of fluorescent signal (“holes”) in the labeled gelatin layer.

Immunofluorescence microscopy on fixed cells

Cells grown on coverslips (VWR, Batavia, IL) were fixed in 3.5% paraformaldehyde (PFA) for 30 min at room temperature (RT), permeabilized in 0.5% Triton X-100/PBS, blocked with 2% BSA (Sigma, Saint Louis, MO) in PBS, and incubated with primary antibodies in 2% BSA/PBS. Bound primary antibodies were detected with Alexa 488- or Alexa 647-conjugated goat anti-mouse or goat anti-rabbit secondary antibodies (Invitrogen, Eugene, OR). Polymerized actin was visualized with Alexa 488- or Alexa 647-conjugated phalloidin (Molecular probes, Eugene, OR). Z-stack confocal fluorescence images were obtained with a LSM510 fluorescence confocal microscope (Carl Zeiss, Thornwood, NY) under a 63x oil immersion lens. Other confocal fluorescence images were observed with a Nikon Eclipse microscope (Melville, NY) under a 63x oil immersion lens. Images were processed using the Adobe Photoshop software (Adobe Systems). Areas of degradation were quantified by enumerating total pixels of digested gelatin areas using NIH Image J software 7 . Data were compiled from three independent experiments.

Time-lapse live cell Imaging

The v-Src transformed NIH3T3 cells were plated on glass bottom chamber slides (BD Bioscience, San Jose, CA) and transfected with CFP-β-actin using the Fugene 6 reagent (Roche Diagnostics, Indianapolis, IN) according to the manufacturer's instructions. Twenty-four hours after transfection, cells were placed on the stage of a LSM 510 fluorescence confocal microscope (Carl Zeiss, Thornwood, NY) and maintained at 37 °C in a CO 2 -independent medium (Invitrogen, Eugene, OR) supplemented with 5% FCS. Cells were then loaded with either a cathepsin B selective substrate (crystal violet conjugated [RR] 2 from Calbiochem, San Diego, CA) or Lysotracker Red DND99 (Invitrogen, Eugene, OR). For Bafilomycin A1 experiments, cells were first loaded with lysosomal tracer for more than 15 minutes before adding 20 μM Bafilomycin A1 (Calbiochem, San Diego, CA). Images were collected with a 63x oil immersion objective lens. Data analysis was performed using LSM 510 software. shRNA knockdown experiments Lentiviral shRNA constructs against mouse cathepsins B were purchased from Open Biosystems Inc. (Huntsville, AL). These were transiently co-transfected with packaging plasmids pLP1, pLP2, and pLP/VSVG (Invitrogen, Carlsbad, CA) into 293FT packaging cells (Invitrogen, Carlsbad, CA) using the calcium phosphate precipitation method to generate lentiviral supernatants. The mouse cathepsin B specific shRNA sequences target against 5'-aggtgcaacaagagctgtgaa-3' and 5'-gacttacaaatcaggagtata-3'. The supernatants were used to infect the v-Src-transformed NIH3T3 cells followed by selection and maintenance in medium containing 5 μg/ml puromycin (Sigma, Saint Louis, MO) as described ( 38 ).

Matrigel invasion assay

Matrigel invasion chambers (Becton Dickinson Biosciences, Bedford, MA) were pre-hydrated for 3 hours with serum-free medium. Twenty-five thousand cells in 400 μl serum-free medium were placed in the top chambers and medium containing 5% FCS was placed in the bottom chambers. The chambers were incubated at 37 °C for 24 hours. Non-invading cells were removed from the top surface of filter inserts using cotton swabs. Cells on the bottom surface were then stained with Diff Quick stain (Dade Behring, Newark, DE) and cells in 5 randomly picked fields (at 40x magnification) were enumerated under a light microscope. All assays were carried out in triplicate and repeated three times. Cell migration was assayed as described for invasion assays but using Boyden chambers with uncoated filters (Becton Dickinson Biosciences, Bedford, MA).

Statistical analysis

For analysis of Matrigel invasion and gelatin degradation, differences were analyzed using a two-tailed student t -test with assumed unequal variance. For matrix-degradation difference between LAMP-1 positive and negative rosettes, Chi square test is used to analyze the data. Asterisks in figures represent a P value