Vitamin D and Wnt3A have additive and partially overlapping modulatory effects on gene expression and phenotype in human colon fibroblasts - PubMed (original) (raw)
Vitamin D and Wnt3A have additive and partially overlapping modulatory effects on gene expression and phenotype in human colon fibroblasts
Gemma Ferrer-Mayorga et al. Sci Rep. 2019.
Abstract
The Wnt/β-catenin signalling pathway is essential for intestinal epithelium homeostasis, but its aberrant activation is a hallmark of colorectal cancer (CRC). Several studies indicate that the bioactive vitamin D metabolite 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) inhibits proliferation and promotes epithelial differentiation of colon carcinoma cells in part through antagonism of the Wnt/β-catenin pathway. It is now accepted that stromal fibroblasts are crucial in healthy and pathologic intestine: pericryptal myofibroblasts are constituents of the stem cell niche and cancer-associated fibroblasts (CAFs) contribute to CRC progression. However, studies on the combined action of 1,25(OH)2D3 and Wnt factors in colon fibroblasts are lacking. Here we show by global transcriptomic studies that 1,25(OH)2D3 and Wnt3A have profound, additive, partially overlapping effects on the gene expression profile of CCD-18Co human colon myofibroblasts. Moreover, 1,25(OH)2D3 and Wnt3A inhibit CCD-18Co cell proliferation and migration, while 1,25(OH)2D3 reduces, but Wnt3A increases, their capacity to contract collagen gels (a marker of fibroblast activation). These data were largely confirmed in patient-derived primary colon normal fibroblasts and CAFs, and in fibroblasts from other origins. Our results indicate that 1,25(OH)2D3 and Wnt3A are strong regulators of colon fibroblast biology and contribute to a better knowledge of intestinal homeostasis and stromal fibroblast action in CRC.
Conflict of interest statement
The authors declare no competing interests.
Figures
Figure 1
1,25(OH)2D3 and Wnt3A drastically regulate the gene expression program of CCD-18Co human colon myofibroblasts. (a) Western blot analysis of VDR and CYP24A1 protein levels in CCD-18Co cells treated with 1,25(OH)2D3 and/or Wnt3A for 24 h. β-Actin was used as a loading control. Images of a representative experiment (left) and the quantification (mean ± SEM) of three independent experiments (right) are shown. Full-length blots are presented in Supplementary Fig. S6. (b) RT-qPCR analysis of CYP24A1 and AXIN2 RNA levels in CCD-18Co cells treated as in (a). The mean ± SEM of three independent experiments is shown. (c) Volcano plots showing the RNA-seq results (FDR-adjusted P value vs. fold-change) obtained in CCD-18Co cells treated for 24 h with 1,25(OH)2D3 (left), Wnt3A (middle), or both (right) compared with vehicle-treated cells. Each dot represents a gene. Genes above the red line are considered differentially expressed (FDR-adjusted P value < 0.05). Some previously known target genes of 1,25(OH)2D3 (THBD, CYP24A1, and CYP3A4) or Wnt3A (APCDD1, AXIN2, and TCF7) are labelled. (d,e) Left, Venn diagram showing the overlap between the genes identified as significantly regulated by each single treatment (1,25(OH)2D3 or Wnt3A). The number of genes included in each group is depicted. Right, scattergram and simple linear regression analysis of the relationship between the observed effect of the combined treatment (log2 of the fold-change) and the expected additive effect (calculated by adding up the log2 of the fold-change of the single treatments) for the group of genes highlighted in the adjacent Venn diagram. Each dot represents a gene. (f) Left, Venn diagram showing the overlap among the genes identified as significantly regulated by the single (1,25(OH)2D3 or Wnt3A) or the combined (1,25(OH)2D3 + Wnt3A) treatment. The number of genes included in each group is depicted. Right, scattergram and simple linear regression analysis of the relationship between the observed effect of the combined treatment (log2 of the fold-change) and the expected additive effect (calculated by adding up the log2 of the fold-change of the single treatments) for the group of genes highlighted in the adjacent Venn diagram. Each dot represents a gene.
Figure 2
Validation by RT-qPCR of several genes identified as differentially expressed in the RNA-seq study. (a) RT-qPCR analysis of the RNA levels of the indicated genes in CCD-18Co cells treated with 1,25(OH)2D3 and/or Wnt3A for 24 h. Genes are grouped according to the single stimulus that regulates each gene in the RNA-seq data. The mean ± SEM of the fold-change vs. vehicle-treated cells in three independent experiments is depicted. (b) Scattergram and simple linear regression analysis of the relationship between the effect of the combined treatment (log2 of the fold-change vs. vehicle-treated cells) and the expected additive effect (calculated by adding up the log2 of the fold-change vs. vehicle-treated cells of the single treatments) for the genes analysed by RT-qPCR in (a). Each dot represents a gene.
Figure 3
Effects of 1,25(OH)2D3 and Wnt3A on the phenotype of CCD-18Co human colon myofibroblasts. (a) Collagen gel contraction assay of CCD-18Co cells in the presence of 1,25(OH)2D3 and/or Wnt3A. The gel area was measured at 0 and 96 h. Results are shown as the fold-change in the gel area at 96 h calculated vs. vehicle-treated cells in three independent experiments (mean ± SEM) (left). Representative stereomicroscope images of collagen gels are depicted (right). Bar, 4 mm. (b) Proliferation of CCD-18Co cells treated with 1,25(OH)2D3 and/or Wnt3A. Left, cell proliferation was assessed at the indicated times and normalized vs. time 0 h (mean ± SEM of three independent experiments). Right, the graph depicts cell proliferation at 120 h calculated as percentage vs. vehicle-treated cells (mean ± SEM of three independent experiments). (c) Wound healing assay of CCD-18Co cells in the presence of 1,25(OH)2D3 and/or Wnt3A. The scratch area was measured at 0 and 24 h. Results are shown as the fold-change in the scratch area at 24 h calculated vs. vehicle-treated cells in three independent experiments (mean ± SEM) (left). Representative bright-field images of the scratches are depicted (right). Bar, 300 µm.
Figure 4
1,25(OH)2D3 and Wnt3A action in patient-derived primary human colon fibroblasts. (a) RT-qPCR analysis of the RNA expression of three genes identified in the RNA-seq study as significantly regulated by the single treatment with 1,25(OH)2D3 (OSR2, OSR1, and PGD) or Wnt3A (OSR1 and PGD) was performed in five paired NF and CAF primary cultures derived from CRC patients (#60, #62, #63, #65, and #66) and treated with 1,25(OH)2D3 and/or Wnt3A for 24 h. Data are shown as log2 of the fold-change vs. vehicle-treated cells and the horizontal bars indicate the median values. (b) Collagen gel contraction assay of five primary cultures of NFs from the indicated patients in the presence of 1,25(OH)2D3 and/or Wnt3A. The gel area was measured at 0 and 96 h. Results are depicted as the fold-change in the gel area at 96 h calculated vs. vehicle-treated fibroblasts. Data from each patient (mean ± SD) (left) and the mean ± SEM of all patients (right) are shown.
Figure 5
1,25(OH)2D3 and Wnt3A effects in IMR-90 human lung fibroblasts and BJ-hTERT human foreskin fibroblasts. (a,b) Western blot analysis of VDR and AXIN2 protein levels in IMR-90 (a) and BJ-hTERT (b) cells treated with 1,25(OH)2D3 and/or Wnt3A for 24 h. β-Tubulin was used as a loading control. Images of a representative experiment (upper panels) and the quantification (mean ± SEM) of three independent experiments (lower panels) are shown. Full-length blots are presented in Supplementary Figs. S7 and S8. (c) RT-qPCR analysis of CYP24A1 and AXIN2 RNA levels in IMR-90 fibroblasts treated as in (a). The mean ± SEM of three independent experiments is shown. (d,e) Wound healing assay of IMR-90 (d) and BJ-hTERT (e) fibroblasts in the presence of 1,25(OH)2D3 and/or Wnt3A. The scratch area was measured at 0 and 30 h (d) or at 0 and 16 h (e). Results are shown as the fold-change in the scratch area at 30 h (d) or at 16 h (e) calculated vs. vehicle-treated cells in three independent experiments (mean ± SEM) (left). Representative bright-field images of the scratches are depicted (right). Bar, 300 µm.
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