Recombinant R-spondin2 and Wnt3a up- and down-regulate novel target genes in C57MG mouse mammary epithelial cells

Abstract

R-spondins (Rspos) comprise a family of four secreted proteins that have important roles in cell proliferation, cell fate determination and organogenesis. Rspos typically exert their effects by potentiating the Wnt/β-catenin signaling pathway. To systematically investigate the impact of Rspo/Wnt on gene expression, we performed a microarray analysis using C57MG mouse mammary epithelial cells treated with recombinant Rspo2 and/or Wnt3a. We observed the up- and down-regulation of several previously unidentified target genes, including ones that encode proteins involved in immune responses, effectors of other growth factor signaling pathways and transcription factors. Dozens of these changes were validated by quantitative real time RT-PCR. Time course experiments showed that Rspo2 typically had little or no effect on Wnt-dependent gene expression at 3 or 6 h, but enhanced expression at 24 h, consistent with biochemical data indicating that Rspo2 acts primarily to sustain rather than acutely increase Wnt pathway activation. Up-regulation of gene expression was inhibited by pre-treatment with Dickkopf1, a Wnt/β-catenin pathway antagonist, and by siRNA knockdown of β-catenin expression. While Dickkopf1 blocked Rspo2/Wnt3a-dependent down-regulation, a number of down-regulated genes were not affected by β-catenin knockdown, suggesting that in these instances down-regulation was mediated by a β-catenin-independent mechanism.

Figure 1. Schematic summary of Rspo2/Wnt3a-dependent gene expression in C57MG cells.

(A) Heat map shows genes that were up-regulated (red) or down-regulated (blue) at least two-fold relative to the normal value (yellow). Microarray analysis was performed with RNA harvested and processed individually from three monolayers for each treatment group [B = BSA (16 ug/ml); R = Rspo2 (100 ng/ml); W = Wnt3a (100 ng/ml); R/W = Rspo2 (100 ng/ml) + Wnt3a (100 ng/ml)]. (B) Venn diagram indicates the number of genes that were up-regulated uniquely or in common by Rspo2, Wnt3a or the combination of both. (C) Venn diagram indicates the number of genes that were down-regulated uniquely or in common by Rspo2, Wnt3a or the combination of both.

Figure 2. Time-course of Rspo2- and Wnt3a-dependent gene up-regulation in C57MG cells.

(A) Quantitative RT-PCR analysis of specific gene expression in response to Rspo2 (100 ng/ml) alone (blue), Wnt3a (100 ng/ml) alone (green) or the two together (red) at the indicated time points. Data are the means +/− S.D. (error bars, sometimes too small to see) of triplicate measurements from one of several experiments with similar results. (B) Immunoblot analysis of Klf5 and Ahr protein expression following treatment with BSA carrier, Rspo2 (100 ng/ml), Wnt3a (100 ng/ml) alone or in combination for the indicated periods. Membranes were re-probed for Hsp70 as a loading control.

Figure 3. Time-course of Rspo2- and Wnt3a-dependent LRP5/6 phosphorylation and β-catenin stabilization.

C57MG cells were treated with BSA, Rspo2 (100 ng/ml), Wnt3a (100 ng/ml) alone or in combination for the indicated periods. Cell lysates (30 µg protein/lane) were immunoblotted for phosphorylated LRP5/6, total LRP5, total LRP6 and Hsp70, the latter serving as a loading control. Soluble β-catenin was measured by immunoblotting after pull-down with GST-E-cadherin.

Figure 4. Dkk1 inhibited up-regulation of genes by Rspo2 and Wnt3a.

(A) Immunoblot analysis of total β-catenin (cell lysate, 30 µg protein/lane) and soluble β-catenin (GST-E-cadherin pull-down) from C57MG cells incubated for 3 h with BSA or Rspo2 (10 ng/ml) plus Wnt3a (100 ng/ml), with or without 20 min Dkk1 (1 µg/ml) pre-treatment. Western blot of Hsp70 was loading control. (B–D) Quantitative PCR analysis of Axin2, Ahr and CXCR6 expression by C57MG cells after 24 h incubation with BSA or Rspo2 plus Wnt3a, with or without Dkk1 pre-treatment [experiment performed in parallel with (A), using the same reagent concentrations]. Results are means +/− S.D. of triplicate measurements from one of four representative experiments.

Figure 5. Knockdown of β-catenin inhibited up-regulation of genes by Rspo2 and Wnt3a.

(A) C57MG cells were pre-treated with either luciferase or pooled Ctnnb1 siRNA reagent for 24 h prior to additional 24 h incubation with BSA or Rspo2 (10 ng/ml) plus Wnt3a (100 ng/ml). Total and soluble β-catenin and Hsp70 were detected as described in Fig. 4 legend. (B–D) Quantitative PCR analysis of Axin2, Ahr and CXCR6 expression by C57MG cells after 24 h incubation with luciferase or pooled Ctnnb1 siRNA reagent followed by 24 h treatment with BSA or Rspo2 plus Wnt3a [experiment performed in parallel with (A), using the same reagent concentrations]. Results are means +/− S.D. of triplicate measurements from one of three representative experiments.

Figure 6. Time-course of Rspo2- and Wnt3a-dependent gene down-regulation in C57MG cells.

Quantitative PCR analysis of specific gene expression in response to Rspo2 (100 ng/ml) alone (blue), Wnt3a (100 ng/ml) alone (green) or the two together (red) at the indicated time points. Data are the means +/− S.D. (error bars, sometimes too small to be seen) of triplicate measurements from one of several experiments with similar results.

Figure 7. Contrasting effects of Dkk1 and β-catenin knockdown on representative genes down-regulated by Rspo2 and Wnt3a.

(A) Quantitative RT-PCR analysis of Angpt1 and CCL7 expression following 24 h incubation with BSA or Rspo2 (10 ng/ml) plus Wnt3a (100 ng/ml), with or without Dkk1 (1 µg/ml) pre-treatment. (B) Quantitative RT-PCR analysis of Angpt1 and CCL7 expression following 24 h incubation with BSA or Rspo2 (10 ng/ml) plus Wnt3a (100 ng/ml) after pre-treatment with siLuc vs. siCtnnb1. Results in (A) and (B) are means +/− S.D. of triplicate measurements from one of three representative experiments performed simultaneously with experiments illustrated in Figures 4 and 5.