Journal: Cell Research

Article Title: Heparanase activates the syndecan-syntenin-ALIX exosome pathway

doi: 10.1038/cr.2015.29

Figure Lengend Snippet: Heparanase stimulates the production of syntenin-1-containing exosomes. (A) Exosome production was evaluated after overnight conditioning of MCF-7 cells with increasing concentrations of proheparanase (0.04-25 nM) and compared to that of cells not receiving proheparanase (0 nM). Exosomes were collected from equivalent amounts of culture medium, conditioned by equal numbers of cells, for equal lengths of time. For each condition both the lysate and exosomal fractions were analyzed by western blot, using cognate antibodies against heparanase, monitoring the conversion of proheparanase (Prohep) into mature heparanase (Hep) and against different exosomal markers: syntenin-1 (Synt1), syndecan-1 (SDC1), syndecan-4 (SDC4), CD63, flotillin-1 (Flo1), CD9 and CD81. Syndecan-1, which is a hybrid heparan sulfate (HS)/chondroitin sulfate proteoglycan, was analyzed using two different approaches. In one approach, the samples were digested with both heparitinase and chondroitinase ABC, removing all glycosaminoglycan chains and enabling visualization of the full-length syndecan core proteins (SDC1 FL) as sharp bands. In the other approach, the samples were digested with chondroitinase ABC only, leaving the HS on the syndecans (SDC1 with HS); comparison of 'SDC1 with HS' and 'SDC1 FL' yields information on the mass of HS on syndecans. Because of the heterogeneity in HS chain length, syndecan-1 with HS is smeared over a wide mass range in the absence of heparanase activity (and is therefore hardly visible in western blot, as illustrated by lane 1 of the lysates). With increasing heparanase activity, the HS chains on syndecan-1 are trimmed to shorter chains of more or less the same length, syndecan-1 with HS migrating as one or a few bands that are readily visualized in western blot (as illustrated by lane 6 of the lysates). Note that cell lysates contain mainly full-length syndecan core proteins; the opposite is true for exosomes, where hardly any full-length syndecan is detected and C-terminal fragments (CTFs) represent the dominant form. β-actin was used as a loading control for the lysates. Western blots are representative of five independent experiments. (B) Histogram representing the quantification of the exosomal levels of syntenin-1 (Synt1), syndecan-1 CTF (SDC1 CTF), CD63, syndecan-4 CTF (SDC4 CTF) and flotillin-1 (Flo1) in response to the addition of increasing concentrations (0 nM till 25 nM) of proheparanase. Values are relative to the exosomal levels measured in absence of exogenously added proheparanase. Bar heights represent mean values, calculated from five independent experiments. Individual data points are shown as white dots on top of the corresponding bars. * P < 0.1, ** P < 0.05, *** P < 0.01 (Student's t -test, assuming normal distribution of the data points). (C) Knockdown of endogenous heparanase reduces the production of syntenin-1-containing exosomes, which can be rescued by the addition of exogenous proheparanase. Duplicate lanes show the results of two independent experiments, run side by side. B16-F10 cells are sham-transfected (−) or stably transfected with a shRNA targeting murine heparanase (+). To rescue the effects of endogenous heparanase knockdown, 10 nM human proheparanase was added to the cells. Heparanase, syntenin-1, syndecan-1 full-length (SDC1 FL), syndecan 1 CTF (SDC1 CTF) and CD63 were analyzed by western blot. Positions of molecular weight markers (in kDa) are indicated on the right of each blot. Note that (because of differences in glycosylation) the Mr of human heparanase is slightly larger than that of mouse heparanase.

Article Snippet: Other antibodies were from commercial sources and were used as recommended by the manufacturer: antibodies against RAB7 (H-50) and β-actin (AC-15) were from Santa Cruz; antibody against the intracellular domain of syndecan 4 was from Abnova; antibodies against human CD63 (MEM-259) was from Abcam; antibody against HA (16B12) was from Covance; anti-flotillin-1 was from BD Biosciences.

Techniques: Western Blot, Activity Assay, Transfection, Stable Transfection, shRNA, Molecular Weight

Journal: Cell Research

Article Title: Heparanase activates the syndecan-syntenin-ALIX exosome pathway

doi: 10.1038/cr.2015.29

Figure Lengend Snippet: The effect of heparanase on exosome production depends on the modification of the heparan sulfate (HS) on syndecans. (A) The role of heparanase enzymatic activity on exosome production was investigated by comparing MCF-7 cells stably expressing wild-type heparanase (WT), catalytically dead heparanase (Cat) or empty vector (Φ) in western blot, in the absence (−) or presence (+) of 10 nM exogenously added proheparanase. (B) The importance of HS was analyzed by treating MCF-7 cells with RNAi targeting EXT1 and EXT2 (KD). Non-targeting RNAi (NT) was used as a control. Cells were challenged with 10 nM proheparanase (+) or left untreated (−). Heparanase activity, reducing the HS on syndecan, was apparent from the migration of chondroitinase ABC-treated syndecan-1 present in cell lysates (SDC1 with HS). EXT1 and EXT2 knockdown leads to the appearance of syndecan-1 that is not substituted with HS (a band running slightly > 70 kDa, after chondroitinase ABC digestion only), not detectable in cells treated with non-targeting RNAi, where all syndecan is substituted with HS (and is larger than SDC FL). A small amount of the syndecan-1 still carried HS and was affected by heparanase addition (yielding a band slightly > 100 kDa, after chondroitinase ABC digestion only), indicating an incomplete knockdown of EXT1 and EXT2. Western blots are representative of three independent experiments. (C) Quantification of the effect of EXT1 and EXT2 knockdown. Histograms representing the exosomal levels of syntenin-1, syndecan-1 CTF, CD63 and flotillin-1 in the different conditions tested (non-targeting RNAi, black bars; non-targeting RNAi and heparanase, dark gray bars; EXT1 and EXT2 RNAi, light gray bars; EXT1 and EXT2 RNAi and heparanase, white bars). Values are relative to the exosomal levels measured in cells treated with non-targeting RNAi and in the absence of exogenously added proheparanase. Bar heights represent mean values, calculated from three independent experiments. Individual data points are shown as white dots on top of the corresponding bars. ** P < 0.05, *** P < 0.01, n.s., not significant (Student's t -test, assuming normal distribution of the data points). (D) Likewise, the role of syndecans was investigated by treating MCF-7 cells with RNAi targeting syndecan-1 and -4 (KD). Non-targeting RNAi (NT) was used as a control. Cells were treated with increasing concentrations of proheparanase (0 to 25 nM) and both the lysates and exosomal fractions were analyzed. Western blots are representative of three independent experiments. Note that lysate and exosome samples derived from NT-and KD-treated cells, separated by a blank space in , were run in the same gel, but not side by side, and that the band intensities in each row are directly comparable. (E) The ability of glypican-1 to rescue the effect of heparanase on exosome production in absence of syndecans was investigated by knocking down both syndecan-1 and -4 (KD) and overexpressing glypican-1 (GPC1) in the same cells. Glypican-1, when deglycanated by heparitinase and chondroitinase ABC treatment (GPC1) or when unmodified (because of overexpression) has a mass of ∼ 60 kDa. Treatment with chondroitinase ABC only reveals 'GPC1 with HS' of a mass of ∼ 90 kDa in heparanase-exposed cells, indicating the HS on glypican-1 is trimmed the same way as the HS on syndecans. Yet, extra glypican fails to rescue the exosomal accumulations of syntenin and CD63. Rescue by expression of mouse syndecan-1 (SDC1) was used as a positive control. Cells treated with non-targeting RNAi (NT) served as reference. Molecular weight markers (in kDa) are indicated on the right of each blot. Western blots are representative of two independent experiments. Note that MCF-7 cells use mainly Man-6-P receptors for heparanase internalization, and that neither the knock down of EXT1 and EXT2 nor the knock down of syndecans has an effect on the uptake of proheparanase and its conversion into mature active form.

Article Snippet: Other antibodies were from commercial sources and were used as recommended by the manufacturer: antibodies against RAB7 (H-50) and β-actin (AC-15) were from Santa Cruz; antibody against the intracellular domain of syndecan 4 was from Abnova; antibodies against human CD63 (MEM-259) was from Abcam; antibody against HA (16B12) was from Covance; anti-flotillin-1 was from BD Biosciences.

Techniques: Modification, Activity Assay, Stable Transfection, Expressing, Plasmid Preparation, Western Blot, Migration, Derivative Assay, Over Expression, Positive Control, Molecular Weight

Journal: Cell Research

Article Title: Heparanase activates the syndecan-syntenin-ALIX exosome pathway

doi: 10.1038/cr.2015.29

Figure Lengend Snippet: Heparanase influences the biogenesis of vesicles of endosomal origin, enhancing intraluminal budding. (A) To investigate whether the extracellular vesicles affected by heparanase were of endosomal origin, RAB7 was knocked down ( RAB7 RNAi) in MCF-7 cells. Non-targeting RNAi (−) served as a control. Cells were left untreated (−) or treated with proheparanase (10 nM). In both experiments, heparanase activity was evaluated using the migration pattern of chondroitinase ABC-, but not heparitinase-treated syndecan-1 present in cell lysates (SDC1 with HS). Molecular weight markers (in kDa) are indicated on the right of each blot. Western blots are representative of three independent experiments. (B) Quantification of the effect of RAB7 knockdown. Histograms representing the exosomal levels of syntenin-1, syndecan-1 CTF and CD63 in the different conditions tested (non-targeting RNAi, black bars; non-targeting RNAi with heparanase, dark gray bars; RAB7 knockdown, light gray bars; RAB7 knockdown with heparanase, white bars). Values are relative to the levels (intensities of the signals) measured in exosomes derived from cells treated with non-targeting RNAi in the absence of proheparanase. Bar heights represent mean values, calculated from three independent experiments. Individual data points are shown as white dots on top of the corresponding bars. ** P < 0.05, n.s., not significant (Student's t -test, assuming normal distribution of the data points). (C) Confocal micrographs of MCF-7 cells co-transfected with mCherry-syntenin-1 (red in merge) and Cerulean-RAB5 Q79L (green in merge). Note the presence of mCherry-syntenin-1, a cytosolic protein, inside the Cerulean-RAB5 Q79L endosomes upon heparanase treatment (50 nM). (D) Confocal micrographs of MCF-7 cells co-transfected with mCherry-syntenin-1 (red in merge), Cerulean-RAB5 Q79L (blue in merge) and syndecan-1 (green in merge), scoring the accumulations (budding) of mCherry-syntenin-1 and of syndecan-1 cytoplasmic domain inside vacuoles outlined by Cerulean-RAB5 Q79L . (E) Quantification of intraluminal budding of mCherry-syntenin-1, as in , by measuring the fluorescence of mCherry-syntenin-1 in the lumen of RAB5 Q79L -positive endosomes, corrected for the size of the RAB5 Q79L -positive endosomes (mean gray value per pixel). Bar heights represent mean values calculated from six independent experiments, scoring at least 30 cells per experiment. Individual data points (mean luminal fluorescence intensity per individual experiment) are shown as white dots on top of the corresponding bars. *** P < 0.01 (Student's t -test, assuming normal distribution of the data points). (F) Quantification of intraluminal budding of mCherry-syntenin-1 and syndecan-1 cytoplasmic domain, as in , by measuring the fluorescence intensity of mCherry-syntenin-1 and syndecan-1 cytoplasmic domain in the lumen of RAB5 Q79L -positive endosomes. Bar heights represent mean values calculated from three independent experiments, scoring at least 30 cells per experiment. Individual data points are shown as white dots on top of the corresponding bars. ** P < 0.05 (Student's t -test, assuming normal distribution of the data points).

Article Snippet: Other antibodies were from commercial sources and were used as recommended by the manufacturer: antibodies against RAB7 (H-50) and β-actin (AC-15) were from Santa Cruz; antibody against the intracellular domain of syndecan 4 was from Abnova; antibodies against human CD63 (MEM-259) was from Abcam; antibody against HA (16B12) was from Covance; anti-flotillin-1 was from BD Biosciences.

Techniques: Activity Assay, Migration, Molecular Weight, Western Blot, Derivative Assay, Transfection, Fluorescence