Dystroglycan is not required for maintenance of the luminal epithelial basement membrane or cell polarity in the mouse prostate

Abstract

Background: Dystroglycan is a cell-surface receptor for extracellular matrix proteins including laminins and perlecan. Prior studies have shown its involvement in assembly and/or maintenance of basement membrane structures, cell polarity and tissue morphogenesis; and its expression is often reduced in prostate and other cancers. However, the role of dystroglycan in normal epithelial tissues such as the prostate is unclear.

Methods: To investigate this, we disrupted dystroglycan expression in the prostate via a conditional gene targeting strategy utilizing Cre recombinase expressed in luminal prostate epithelial cells.

Results: Contrary to expectations, deletion of dystroglycan in luminal epithelial cells resulted in no discernable phenotype as judged by histology, basement membrane ultrastructure, localization of dystroglycan ligands, cell polarity or regenerative capacity of the prostate following castration. Dystroglycan expression remains in keratin-5-positive basal cells located in the proximal ducts where dystroglycan expression is elevated in regenerating prostates.

Conclusions: Our results show that dystroglycan in luminal epithelial cells is not required for the maintenance of basement membranes, cell polarity or prostate regeneration. However, it is possible that persistent dystroglycan expression in the basal cell compartment may support these or other functions.

Fig. 1. Dystroglycan localization and expression in the ventral lobe of a prostate specific knockout mouse

DG is expressed on the basal surface of the epithelium (yellow, arrows) in the prostate gland as shown by α-DG (IIH6) and β-DG (8D5)-specific antibodies (A,D). DG expression is lost in DG KO mice (B,E) compared with WT (A,D) although patchy areas of expression remain (arrowheads) (B,E). Tissues stained with secondary antibodies only were used as a negative control (C,F). Muscle surrounding the bladder neck was used as a positive control (inset boxes). Nuclei were stained with DAPI (blue). Scale bar 50 µm. Autofluorescence of luminal secretions (*).

Fig. 2. Histology and ultrastructure of the prostate

Hematoxylin and eosin stain of the ventral (A,D); lateral (B,E) and dorsal (C,F) prostatic lobes in DG WT (n=8) and KO (n=8) mice were similar at six months of age. Scale bar (A–F); 50 µm. Transmission electron microscopy of the basement membrane (arrows) of the lateral lobe of DG WT (G) and KO (J) mice. Epithelial microvilli and tight junctions (arrows) of the lateral lobe of DG WT (H,I) and KO (K,L) mice, respectively. Two WT and two KO mice were analyzed. Epithelial cell (*). Scale bars (H,I,K,L) 1.0 µm (G,J) 0.4 µm.

Fig. 3. Basement membrane and cellular composition of the ventral lobe of the prostate

Extracellular matrix proteins and DG binding partners perlecan and laminin (green) showed a similar localization and staining intensity in DG WT (A,B) and KO mice (D,E) respectively. Likewise, laminins 511/521 (red) were also unchanged (C,F). Basal cell number (G,I) as assessed by anti-K5 staining (red) and proliferation rate (H,J) as assessed by Ki-67 staining (green) were similar. Nuclei were stained with DAPI (blue). 3 WT and 3 KO animals were assessed for each protein. Scale bar 50 µm.

Fig. 4. Involution and regeneration of the lateral and ventral lobes of the mouse prostate

DG WT (A) and KO (B) mice were castrated at 6 to 9 months of age. Testosterone was introduced 21 days post castration and regenerated prostates were analyzed 21 days after reintroduction of testosterone. WT (C) and KO (D) prostates regenerated normally. Scale bar 150 µm.

Fig. 5. DG expression following involution and regeneration of the mouse prostate

DG expression is enriched after involution in both WT and KO mice (A,D) and is reduced after restoration of androgens allowed for regeneration in KO mice. Immunofluorescence staining for DG (orange) was performed on the ventral lobe of castrated WT (A) and KO (D) mice, regenerated ventral lobe (B,E) and normal ventral prostate (C,F) respectively. Nuclei were stained with DAPI (blue). Muscle surrounding the bladder neck was used as a positive control (inset boxes) 6 mice of each genotype were assessed. Scale bar 50 µm. (G) αDG (IIH6) and βDG (8D5) protein expression was enriched in protein lysates of castrated (C1, C2) prostates compared with intact (I) prostates as shown by western blot.

Fig. 6. Expression of DG in the basal cell population of the prostate proximal ducts

Immunofluorescence staining for DG (red) expression in the proximal ducts of WT and KO mice (A,B). Basal cells are indicated by P63, red (C,D,) and Keratin 5, green (E,F) expression. Nuclei are stained with DAPI (blue). 3 mice of each genotype were assessed. Scale bar 50 µm. X-gal staining (blue) detects Cre recombinase activity in the distal ducts (G) but not the proximal ducts (H) of the mouse prostate ventral lobe. Scale bar 50 µm.