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. 2017 Dec:64:27-39.
doi: 10.1016/j.matbio.2017.04.001. Epub 2017 Apr 20.

The perlecan-interacting growth factor progranulin regulates ubiquitination, sorting, and lysosomal degradation of sortilin

Ryuta Tanimoto  1 , Chiara Palladino  2 , Shi-Qiong Xu  1 , Simone Buraschi  3 , Thomas Neill  3 , Leonard G Gomella  1 , Stephen C Peiper  3 , Antonino Belfiore  4 , Renato V Iozzo  3 , Andrea Morrione  5
Affiliations

The perlecan-interacting growth factor progranulin regulates ubiquitination, sorting, and lysosomal degradation of sortilin

Ryuta Tanimoto et al. Matrix Biol. 2017 Dec.

Abstract

Despite extensive clinical and experimental studies over the past decades, the pathogenesis and progression to the castration-resistant stage of prostate cancer remains largely unknown. Progranulin, a secreted growth factor, strongly binds the heparin-sulfate proteoglycan perlecan, and counteracts its biological activity. We established that progranulin acts as an autocrine growth factor and promotes prostate cancer cell motility, invasion, and anchorage-independent growth. Progranulin was overexpressed in prostate cancer tissues vis-à-vis non-neoplastic tissues supporting the hypothesis that progranulin may play a key role in prostate cancer progression. However, progranulin's mode of action is not well understood and proteins regulating progranulin signaling have not been identified. Sortilin, a single-pass type I transmembrane protein of the Vps10 family, binds progranulin in neurons and targets progranulin for lysosomal degradation. Significantly, in DU145 and PC3 cells, we detected very low levels of sortilin associated with high levels of progranulin production and enhanced motility. Restoring sortilin expression decreased progranulin levels, inhibited motility and anchorage-independent growth and destabilized Akt. These results demonstrated a critical role for sortilin in regulating progranulin and suggest that sortilin loss may contribute to prostate cancer progression. Here, we provide the novel observation that progranulin downregulated sortilin protein levels independent of transcription. Progranulin induced sortilin ubiquitination, internalization via clathrin-dependent endocytosis and sorting into early endosomes for lysosomal degradation. Collectively, these results constitute a regulatory feed-back mechanism whereby sortilin downregulation ensures sustained progranulin-mediated oncogenesis.

Keywords: Castration-resistant prostate cancer cells; Progranulin; Prostate cancer; Sortilin.

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Figures

Fig. 1.
Fig. 1.
Progranulin regulates sortilin stability. (A–B), PC3 and DU145 cells were serum-starved for 24 h and then stimulated for the indicated time points with progranulin (40 nM). Samples were lysed and sortilin levels were assessed by immunoblot with anti-sortilin antibodies. (C–D) Cyclohexamide (CHX, 100 μM) was supplemented with progranulin. Sortilin levels were normalized to β-actin. Densitometric analysis is expressed as arbitrary units and is the average of two independent experiments.
Fig. 2.
Fig. 2.
Progranulin depletion enhances sortilin stability. (A–B), Controls (shSCR) and progranulin-depleted (shPGRN) PC3 and DU145 cells were plated in serum-rich media overnight and then transferred to serum-free media for the indicated time points. Samples were lysed and sortilin protein levels were assessed by immunoblot with anti-sortilin antibodies. (C–D) Cyclohexamide (CHX, 100 μM) was incubated for the indicated time points. Sortilin levels were quantified over β-actin. Densitometric analysis is expressed as arbitrary units and represents the average of two independent experiments.
Fig. 3.
Fig. 3.
Progranulin promotes sortilin ubiquitination. (A) PC3 and (B) DU145 cells were transiently transfected with a HA-Ubiquitin plasmids as described in Experimental procedures. After 24 h, cells were serum-starved for an additional 24 h followed by stimulation with 40 nM progranulin (PGRN) for 1 h. Both samples were supplemented with proteosomal and lysosomal inhibitors to accumulate ubiquitinated species. Lysates were immunoprecipitated with anti-sortilin antibodies, resolved via SDS PAGE and membranes incubated with anti-HA and anti-sortilin antibodies. Experiments shown are representative of two independent experiments from four pooled independent samples.
Fig. 4.
Fig. 4.
Progranulin depletion inhibits sortilin ubiquitination. (A) PC3/shSCR and PC3/shPGRN cells were grown overnight in serum-rich media, transiently transfected with an HA-Ubiquitin plasmid and then transferred after extensive washing to serum-free conditions supplemented with proteosomal and lysosomal inhibitors for 1 h. Lysates were immunoprecipitated with anti-sortilin antibodies, run on SDS PAGE and filters incubated with anti-HA and anti-sortilin antibodies. (B) To detect sortilin ubiquitination at endogenous ubiquitin levels, PC3/shSCR and PC3/shPGRN cells were grown overnight in serum-containing media and then transferred after extensive washing to serum-free condition supplemented with proteosomal and lysosomal inhibitors for 1 h. Lysates were immunoprecipitated with anti-sortilin antibodies, run on SDS PAGE and membranes incubated with anti-ubiquitin and anti-sortilin antibodies. Experiments shown are representative of two independent experiments from four pooled independent samples.
Fig. 5.
Fig. 5.
Progranulin promotes sortilin internalization via a clathrin-dependent but caveolin-1-independent pathway. (A) Sortilin co-localization (yellow) with caveolin-1 (red) or (B) clathrin (red) was assessed in PC3/GFP-sortilin cells by confocal laser microscopy. Pictures are representative of at least ten fields from three independent experiments. An average of 100 cells was examined for each condition. Scale bar ~10 μm. Nuclei were stained with DAPI. Insets were digitally magnified 3×. Co-localization index is expressed in arbitrary units and was calculated using ImageJ (*P < 0.05).
Fig. 6.
Fig. 6.
Progranulin promotes sortilin trafficking into early endosomes and lysosomes. (A) Sortilin (green) co-localization with EEA1 (red) or (B) lysosomes (red, Lysotracker) was assessed in PC3/GFP-sortilin cells by confocal microscopy. Images are representative of at least ten fields from three independent experiments. An average of 100 cells was examined for each condition. Scale bar ~10 μm. Nuclei were stained with DAPI. Insets were digitally enlarged 3×. The co-localization index is expressed as arbitrary units and was calculated using ImageJ (*P < 0.05, **P < 0.01).
Fig. 7.
Fig. 7.
Sortilin levels are stabilized by lysosomal inhibition. Serum-starved PC3/GFP-Sortilin cells were incubated with recombinant progranulin (40 nM) with or without leupeptin (100 μM) on ice for 1 h. After washing, cells were shifted to 37 °C and imaged at the indicated time points (seconds). Images were acquired every 12 s for 14 min using live cell confocal microscopy. The background-corrected fluorescence intensity relative to time 0 was quantified and plotted using the software Zeiss AIM 4.2 SP1 and is expressed as a function of time. Error bars represent standard deviation for n = 4 (*P < 0.05, **P < 0.01).
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