Fig 2: Negative regulation of LRP2 and megalin proteins by vitamin D in prostate cells and tissue slice explants. A,LRP2 expression following 24 hours of treatment with 50 nmol/L 25D in PrE-AA1, 50 nmol/L 1,25D in LNCaP and 50 nmol/L 1,25D in 22Rv1 cells. B, Immunoblot for megalin after 48 hours of 25D treatment of PrE-AA1 cells. C, Activity of a custom LRP2 promoter luciferase construct after 24 hours of 50 nmol/L 25D treatment in 975E-hTERT cells; RLU, relative luciferase units normalized to the transfection control. D,LRP2 promoter contains the RXR:VDR binding motifs. E,Ex vivo prostate tissue slice workflow. F, Images and quantification of megalin protein by IHC (G) and LRP2 gene expression in tissue slices (H) after 24 hours of treatment with 50 nmol/L 25D. Scale bar, 150 µm. The graph shows the mean pathologist score per gland. Graphs represent the mean ± SEM from triplicate experiments. For tissue slices, graphs show the representative experimental mean ± SD with two replicates per experiment. P values were determined using an unpaired t test.

Fig 3: Megalin expression is dysregulated in cancer and lower with disease aggressiveness. A, Digital quantitation of epithelial megalin expression on a TMA consisting of 118 prostate biopsy cores from 29 patients (EA, n = 9; AA, n = 20). Epithelial regions were segmented by panCK staining, and benign and cancer regions were determined by a board-certified pathologist. Scale bar, 100 µm. Immunofluorescence (IF) intensity per pixel for megalin expressed as mean ± SEM; Ben, benign. B,LRP2 expression in RNAseq dataset of primary prostate cancer tumors by primary Gleason grade from Gerhauser and colleagues DHFZ dataset. Analyses by Gleason was ANOVA with Fisher multicomparisons for Gleason 3 (n = 82) and 4 (n = 24) or 5 (n = 12) combined. C,LRP2 expression in TCGA-PRAD RNAseq dataset with normal (n = 52) and tumor (n = 374) with breakdown by primary Gleason grade (Gleason 3 n = 206, Gleason 4 n = 249, Gleason 5 n = 63). ANOVA with Fisher multicomparisons test. *, P < 0.01; **, P < 0.001; ***, P < 0.001; ****, P < 0.0001.

Fig 4: Prostate cells express megalin and import SHBG-bound T. A, Gene expression of LRP2, AR, VDR, and CYP27B1 in a panel of prostate cell lines as shown by qRT-PCR shown as relative quantitation to HPRT1. Error bars are SEM. (A, inset) Western blot analysis for megalin in prostate cell line panel. B, Regulation of KLK3 (PSA gene) expression after 24 hours following treatment with vehicle control (CTL), 50 nmol/L T alone (T), 50 nmol/L T preincubated with 500 nmol/L SHBG (SHBG), or T + SHBG in cells preincubated with 1 µmol/L MEG-Inh in LNCaP and 22Rv1 prostate cancer lines. C, Visualization (× 63) of SHBG-555 (red) import into cells with DAPI (blue) nuclear and F-actin (green) cytoskeletal counterstains. Statistical analysis was performed using a one-way ANOVA with a two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli for multiple comparisons; *, P < 0.05 for comparison with CTL, all graphs represent mean ± SEM of three or more individual experiments with two replicates per experiment.