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Published on: February 27, 2020
Recombinant R-spondin2 and Wnt3a up- and down-regulate novel target genes in C57MG mouse mammary epithelial cells
Bolormaa Baljinnyam1, Malgorzata Klauzinska, Saad Saffo
1Laboratory of Cellular and Molecular Biology, National Cancer Institute, Bethesda, Maryland, United States of America.
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.
Insights
R-spondins (Rspos) sustain Wnt/β-catenin signaling, impacting gene expression over time. This study identified new Rspo/Wnt target genes involved in various cellular processes.
Area of Science:
- Molecular Biology
- Cell Biology
- Developmental Biology
Background:
- R-spondins (Rspos) are secreted proteins crucial for cell proliferation, fate determination, and organogenesis.
- Rspos primarily function by potentiating the Wnt/β-catenin signaling pathway.
Purpose of the Study:
- To systematically investigate the impact of Rspo/Wnt signaling on gene expression.
- To identify novel target genes regulated by Rspo2 and Wnt3a.
Main Methods:
- Microarray analysis of C57MG mouse mammary epithelial cells treated with Rspo2 and/or Wnt3a.
- Quantitative real-time RT-PCR for validation of gene expression changes.
- Time course experiments to analyze the temporal effects of Rspo2 on Wnt-dependent gene expression.
Main Results:
- Identified numerous previously unknown Rspo/Wnt target genes, including those involved in immune responses, growth factor signaling, and transcription.
- Rspo2 enhanced Wnt-dependent gene expression primarily at 24 hours, suggesting a sustaining role rather than acute activation.
- Gene up-regulation was blocked by Dickkopf1 and β-catenin knockdown, confirming Wnt/β-catenin pathway involvement.
- Some gene down-regulation was independent of β-catenin, indicating alternative mechanisms.
Conclusions:
- Rspo/Wnt signaling regulates a broad spectrum of genes, including novel targets.
- Rspo2's primary role is to sustain Wnt pathway activation, with delayed effects on gene expression.
- Both β-catenin-dependent and -independent mechanisms mediate Rspo/Wnt-induced gene expression changes.
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