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. 2019 Mar:180:200-207.
doi: 10.1016/j.exer.2019.01.001. Epub 2019 Jan 3.

Decorin antagonizes corneal fibroblast migration via caveolae-mediated endocytosis of epidermal growth factor receptor

Rajiv R Mohan  1 , Ratnakar Tripathi  2 , Ajay Sharma  2 , Prashant R Sinha  2 , Elizabeth A Giuliano  3 , Nathan P Hesemann  4 , Shyam S Chaurasia  2
Affiliations

Decorin antagonizes corneal fibroblast migration via caveolae-mediated endocytosis of epidermal growth factor receptor

Rajiv R Mohan et al. Exp Eye Res. 2019 Mar.

Abstract

Decorin (Dcn), a small leucine-rich proteoglycan, is involved in the regulation of corneal wound healing. Epidermal growth factor receptor (EGFR) plays a critical role in corneal fibroblasts proliferation, migration and extracellular matrix (ECM) modulation upon injury or infection. The present study aimed to investigate the mechanistic role of Dcn in EGFR internalization to the regulation of corneal stromal fibroblasts (CSFs) migration, a key step in the corneal wound healing. Human corneal stromal fibroblasts (hCSF) cultures were generated from donor corneas. At 70% confluence, cells were switched to serum-free conditions for 48 h and then treated with decorin (250 nM) in the presence or absence of EGF (100 ng/ml) for various time points (10-60 min). Cell lysates were subjected to proteome array analysis screening for 42 different phosphorylated human receptor tyrosine kinases (RTKs), immunocytochemistry, and western blots to analyze EGFR phosphorylation. The scratch-wound assay was performed to evaluate the effects of decorin on EGF-mediated hCSF migration. Dcn caused a rapid EGFR phosphorylation within 10 min of exposure in RTK blot defining its role as a biological ligand for EGFR in hCSFs. Prolonged exposure to Dcn caused complete disappearance of EGFR and inhibition of the hCSF migration in the scratch wound assay suggesting Dcn binding to EGFR causes EGFR down-regulation. Immunostaining studies indicated that Dcn-treatment to hCSFs internalizes Dcn-EGFR complex, which does not require tyrosine kinase activity when treated with the AG1478 inhibitor and co-localizes the complex to the perinuclear region. Next, we found that Dcn-EGFR complex does not follow canonical early endosome internalization as revealed by the EEA1 antibody instead binds to the CD63 antibody directed for degradation by the late endosome. We also found that Dcn regulates the EGFR recycling by preventing its binding to Rab11, a specific antibody for recycling endosome. Further, hCSFs-pretreated with pharmacological inhibitors, methyl-β-cyclodextrin and chlorpromazine and supplemented with Dcn suggested EGFR trafficking via the caveolae-mediated pathway. These results suggest that Dcn acts as a biological ligand for EGFR and modulates hCSF migration via EGFR down-regulation, thus playing a vital role in corneal wound healing.

Keywords: Caveolae; Corneal wound healing; Decorin; Endocytosis; Epidermal growth factor receptor.

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Figures

Fig. 1.
Fig. 1.. Decorin increases phosphorylated EGFR in hCSF cells.
Proteome Profiler Human Phospho-receptor tyrosine kinase (RTK) kit was used to test 42 phosphorylated RTKs in hCSF cells under serum-free conditions treated with ± rhDcn (250 nM) for 10 min. EGFR band was observed in the Dcn-treated culture (B) shown by arrow compared to the control (vehicle only) group (A). Panel C shows quantification data of three assays. Data were presented as mean ± SEM. * represents the reference spots on the array blot as described in the RTK kit.
Fig. 2.
Fig. 2.. Prolonged exposure to decorin decreases EGFR expression.
Human CSF cultures were incubated in rhDcn (250 nM) for 15, 30, or 60 min. EGFR expression was increased at 15 min timepoint, but decreases at 30 min and completely disappeared at 60 min time point (A). On the other hand, hCSF cultures incubated in rhEGF (100 ng/ml) showed significantly increased expression of EGFR throughout the duration of the experiment (B). Panel C shows quantification data of three experiments. Data were presented as mean ± SEM.
Fig. 3.
Fig. 3.. Decorin impedes EGF-mediated migration of hCSFs.
In vitro wound scratch assay was performed in hCSF cultures with ± rhEGF and/or rhDcn. In this assay, EGF (100 ng/ml) profoundly stimulated hCSF migration (B; 91% ± 7.2%, p < 0.001) than vehicle (A). The Dcn (250 nM) treatment significantly reduced EGF-mediated hCSF migration (D; 89% ± 9.4%, p < 0.001). The inhibitory effects of decorin were similar to the effects observed with the EGF antibody (C; 92% ± 8.2%, p < 0.001). Data were presented as mean ± SEM.
Fig. 4.
Fig. 4.. Decorin induces internalization of EGFR and co-localizes with decorin to the perinuclear region.
EGFR was localized on the plasma membrane (green, arrowhead) in the non-treated hCSF cells (A). Incubation of hCSF cultures for 1 h with rhDcn (250 nM) translocated EGFR to the perinuclear region (B) as evident from the co-localized double EGFR-Dcn stained cells (yellow; arrows) which was absent in vehicle-treated (-dcn) cultures (A). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 5.
Fig. 5.. Decorin-induced internalization of EGFR does not require tyrosine kinase activity.
Confocal microscopy of hCSF cells pretreated with AG1478 (a specific tyrosine kinase activity inhibitor at 5 μM for 60 min) and incubated with rhDcn (250 nM) do not inhibit internalization of EGFR and colocalize with Dcn (yellow; arrows) in the perinuclear space (C) similar to the non-treated cells (A). On the contrary, rhEGF-treatment (100 ng/ml) internalizes of EGFR was inhibited by AG1478 and EGFR expression (D) was seen only at the cell surface (green, arrowheads) while non-treated cells show internalization of EGFR + EGF (yellow; arrows). Merged image shows co-localization of EGFR + EGF observed near peri-nuclear region (B). Bar = 25 μM. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 6.
Fig. 6.. Decorin-mediated EGFR internalization does not involve early endosome.
Human CSFs were incubated with rhDcn (250 nM, A-D) or rhEGF (100 ng/ml, E-H). Double immunostaining of hCSFs with EGFR (green, A and E) and early endosome marker, EEA1, (red, B and F) showed EGFR and EEA1 co-localization vesicle only with rhEGF treatment (G, arrows). On the other hand, rhDcn-treatment to hCSF cells showed insignificant co-localization of EGFR and EEA1 (C). Schematic diagram shows internalization does not target early endosome (EEA1) upon decorin treatment (D) but it targets EEA1 when treated with EGF (H). Bar = 10 μM. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 7.
Fig. 7.. Decorin-EGFR complex degradation involves late endosome.
Human CSFs incubated with rhDcn (A–D) or rhEGF (E–H) and double-immunostained with EGFR (green) and late endosome marker, CD63, (red). Decorin treatment showed high co-localization of EGFR and CD63 in an overlay image (C; yellow, arrows) compared to the rhEGF (G, yellow, arrows). The EGFR staining (green) is shown in panels A and E, and CD63 staining (red) is shown in panels B and F. Schematic diagram shows EGFR internalization target late endosome upon decorin treatment (D) compared to EGF treatment (H). Bar = 10 μM. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 8.
Fig. 8.. Decorin prevents recycling of EGFR.
Human CSF cells incubated with rhDcn (A–D) or rhEGF (E–H) and double immunostained with EGFR (green) and recycling endosome marker, Rab11 (red). The overlay images of double-immunostaining showed their co-localization with rhEGF treatment (G; yellow; arrows), which was absent in the rhDcn-treated hCSF cells (D) indicating attenuation of EGFR recycling. Schematic diagram shows internalization does not target late endosome (Rab11) upon decorin treatment (D) while it targets Rab11 when treated with EGF (H). Bar = 10 μM. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 9.
Fig. 9.. Decorin-mediated internalization of EGFR ensues via caveolar-mediated endocytosis.
Confocal photomicrograph showing inhibition of caveolae formation in hCSF cells using methyl-β-cyclodextrin (MβCD) that depletes cholesterol from the plasma membrane, rhDcn-induced internalization of EGFR was completely blocked (B) and EGFR was localized only in the plasma membrane (arrowheads). Contrary to this, rhEGF-induced internalization of EGFR was not affected (E, arrowheads) when treated with MβCD. When clathrin assembly was inhibited by chlorpromazine, a cationic amphiphilic drug that preferentially blocks the receptor recycling by preventing the adapter protein AP-2 of clathrin assembly on clathrin-coated pits, rhDcn-induced internalization of EGFR was observed (C, arrowheads) while rhEGF-induced internalization of EGFR was disrupted (F, arrowheads). The vehicle-treated (-dcn) showed no EGFR internalization as shown in Fig. 4A. Bar = 5 μM.
Fig. 10.
Fig. 10.. Schematic representation describing the internalization of EGFR by decorin and EGF induction.
Decorin is taken up by the EGFR and the Dcn-EGFR complex is internalized and transferred to the perinuclear vesicles, commonly known as caveosome bypassing the early endosome intake. At this stage, Dcn-EGFR complexes are sent to late endosome (CD63) for final degradation. Conversely, EGF-induces stepwise EGFR endocytosis primarily in early endosome (EEA1), where some part of the EGF-EGFR complex enters the recycling endosome (Rab11) and some portion enters into the late endosome (CD63) for final degradation.
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