Research ArticleTissue Repair

Galectin-3 initiates epithelial-stromal paracrine signaling to shape the proteolytic microenvironment during corneal repair

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Science Signaling  16 Jul 2019:
Vol. 12, Issue 590, eaaw7095
DOI: 10.1126/scisignal.aaw7095
  • Fig. 1 MMP9 is locally activated in epithelial cells as a result of direct contact with fibroblasts.

    (A) Time-lapse phase-contrast and fluorescence images of immortalized human corneal epithelial cells carrying the MMP9-eGFP reporter construct and primary human corneal fibroblasts. (B) Gelatin zymography of culture media from epithelial cells and fibroblasts grown alone or in a mixed coculture system in time course experiments. n = 4 independent experiments. (C) Quantitative polymerase chain reaction (qPCR) analysis of MMP9 expression in cells grown alone or in coculture for 24 hours. n = 8 independent experiments. (D) Gelatin zymography showing MMP activity with increasing numbers of fibroblasts relative to epithelial cells in coculture for 24 hours. n = 3 independent experiments. (E) Gelatin zymography of culture media from cells grown alone or in coculture using immortalized human corneal epithelial cells and either primary human corneal fibroblasts or telomerase-immortalized human corneal fibroblasts (hTERT). n = 4 independent experiments. The box and whisker plots show the 25 and 75 percentiles (box), the median (horizontal line in box), and the minimum and maximum data values (whiskers). Significance was determined using the Wilcoxon test. *P < 0.05. Scale bar, 250 μm.

  • Fig. 2 Galectin-3 stimulates reciprocal signaling and influences epithelial MMP9 secretion.

    (A) Fluorescence intensity measured in human corneal epithelial cells carrying the MMP9 reporter construct (EpiGFP). The serum-free conditioned media (CM) of fibroblasts grown alone or in coculture with epithelial cells for 24 hours were diluted in a 1:1 ratio with fresh media and used to stimulate homotypic cultures of EpiGFP cells for an additional 24 hours. n = 3 independent experiments. (B) Exogenous soluble galectin-3 (rhGal3) or BSA in serum-free media were incubated with fibroblasts for 24 hours. The conditioned media were precleared with α-lactose agarose beads to remove galectin-3 before incubation with epithelial cells. After an additional 24 hours, the epithelial cell culture supernatants were analyzed by gelatin zymography to determine the abundance of MMP9. The presence of galectin-3 in precleared conditioned media was evaluated by immunoblotting. n = 4 independent experiments. (C) Epithelial cells were transfected with scrambled (siScr) or galectin-3 (siGal3) siRNA, and the presence of galectin-3 in epithelial cell lysates was assessed by immunoblotting. After 48 hours of transfection, cells were incubated with fibroblasts in a mixed coculture system for an additional 24 hours. MMP9 abundance in the media was quantified by gelatin zymography and shown as a scatter plot. n = 7 independent experiments. (D) Same as in (C) except that galectin-3 was knocked down in fibroblasts (siGal3) that were then cocultured with epithelial cells. n = 4 independent experiments. The data represent the mean ± SD. Significance was determined using one-way analysis of variance (ANOVA) with Tukey’s post hoc test (A and B) or Wilcoxon test (C and D). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

  • Fig. 3 IL-1β acts as a galectin-3–induced paracrine signal in fibroblasts.

    (A) Relative gene expression from PCR arrays on a mixed coculture system (Epi + fibro) or fibroblasts exposed to exogenous soluble galectin-3 (rhGal3 + fibro) for 24 hours. Data were normalized to cultures of fibroblasts grown alone with no stimulation. The green and red dots indicate significant increases or decreases compared with the control, respectively. n = 3 independent experiments. (B) Venn diagram representation of genes showing increased and decreased expression in coculture conditions or in fibroblasts exposed to galectin-3, as determined by PCR array. The expression amounts of the two overlapping genes are shown for each individual condition. n = 3 independent experiments. (C) ELISA showing the amounts of IL-1β and CXCL10 in culture supernatants obtained from the conditions in (A). n = 4 (IL-1β) or n = 3 (CXCL10) independent experiments. (D) Epithelial cells were transfected with scrambled or galectin-3 siRNA. After 48 hours of transfection, cells were incubated with fibroblasts in a mixed coculture system for an additional 24 hours. IL-1β and CXCL10 amounts in culture supernatants were quantified by ELISA. n = 6 independent experiments. (E) Fibroblasts were transfected with siScr or IL-1β siRNA (siIL1). After 48 hours of transfection, cells were incubated with BSA or rhGal3 in serum-free media for an additional 24 hours. IL-1β amounts in culture supernatants were quantified by ELISA. n = 3 independent experiments. (F) Culture supernatants obtained from the conditions in (E) were precleared twice with α-lactose agarose beads to remove endogenous galectin-3 before incubation with epithelial cells. After 24 hours, the gelatinase (MMP9) activity of epithelial cell culture supernatants was quantified by gelatin zymography. n = 4 independent experiments. The box and whisker plots show the 25 and 75 percentiles (box), the median, and the minimum and maximum data values (whiskers). The data in (C) and (E) represent the mean ± SD. Significance was determined using one-way ANOVA with Tukey’s post hoc test. **P < 0.01.

  • Fig. 4 The basement membrane protects against galectin-3–induced IL-1β signaling.

    (A) Schematic representation of the two models of corneal injury used in this study. (B) The corneal thickness was determined immediately after wounding by anterior segment optical coherence tomography. n = at least 19 eyes from 19 mice per group. (C) Re-epithelialization was monitored after 14 to 16 hours using fluorescein, which stains areas where the epithelium is absent or damaged. (D) Control and wounded corneas were harvested 14 to 16 hours after injury and subjected to immunofluorescence for laminin-α1 (red; localizes to basement membranes) and vimentin (green; present in mesenchymal cells). Nuclei were stained by 4′,6-diamidino-2-phenylindole (DAPI) (blue). Fibroblasts in close proximity to the epithelium are noted with arrowheads. The gelatinolytic activity in these samples was assessed by in situ zymography. Data are representative of two independent experiments. (E) The presence of IL-1β, galectin-3, and β-actin in de-epithelialized corneas from wild-type (WT) and galectin-3–deficient (Gal3−/−) mice was evaluated by immunoblotting. n = 3 independent experiments using three corneas per group. A representative blot is shown. (F) ImageJ quantification of IL-1β staining shown in (E). n = 3 independent experiments. (G) The gelatinolytic activity in WT and galectin-3–deficient mice was assessed by in situ zymography. The box and whisker plots show the 25 and 75 percentiles (box), the median, and the minimum and maximum data values (whiskers). The data in (F) represent the mean ± SD. Significance was determined using one-way ANOVA with Tukey’s post hoc test. **P < 0.01; ***P < 0.001; ****P < 0.0001. Scale bars, 100 μm.

  • Fig. 5 Mechanism of galectin-3–induced paracrine signaling in epithelial-stromal interactions.

    Multiple functions have been ascribed to the basement membrane, a thin and highly cross-linked extracellular matrix that underlies all epithelia. During homeostatic conditions, these include promotion of mechanical strength and the maintenance of tissue architecture. Disruption of the basement membrane by trauma allows for direct interactions between epithelial cells and stromal fibroblasts, resulting in a cascade of signaling events critical for tissue repair. We propose that galectin-3 of epithelial origin initiates such events by physically interacting with fibroblasts through an as yet unidentified receptor. As a consequence, the stromal fibroblasts begin to synthesize and secrete abundant amounts of IL-1β, which act in a paracrine fashion to regulate the proteolytic microenvironment by activating the MMP9 promoter in epithelial cells. This reciprocal form of communication would be expected to lead to the remodeling of the collagenous extracellular matrix.

Supplementary Materials

  • stke.sciencemag.org/cgi/content/full/12/590/eaaw7095/DC1

    Fig. S1. Characterization of the epithelial MMP9 promoter reporter cell line.

    Fig. S2. Effect of fibroblast fractions on epithelial MMP9 secretion.

    Fig. S3. Effect of deoxymannojirimycin on MMP9 secretion during heterotypic cell-cell contacts.

    Fig. S4. Galectin-3 multimerization is necessary to induce IL-1β secretion by corneal fibroblasts.

    Table S1. Gene expression analyses by PCR array.

    Movie S1. Time-lapse video microscopy of fibroblasts interacting with the epithelial MMP9 promoter reporter cell line.

  • The PDF file includes:

    • Fig. S1. Characterization of the epithelial MMP9 promoter reporter cell line.
    • Fig. S2. Effect of fibroblast fractions on epithelial MMP9 secretion.
    • Fig. S3. Effect of deoxymannojirimycin on MMP9 secretion during heterotypic cell-cell contacts.
    • Fig. S4. Galectin-3 multimerization is necessary to induce IL-1β secretion by corneal fibroblasts.
    • Table S1. Gene expression analyses by PCR array.
    • Legend of movie S1

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.avi format). Time-lapse video microscopy of fibroblasts interacting with the epithelial MMP9 promoter reporter cell line.

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