Abstract
Canonical TGF-β1 signalling promotes tumor progression by facilitating invasion and metastasis, whereby release of TGF-β1, by (for example) infiltrating immune cells, induces epithelial to mesenchymal transition (EMT). PAX2, a member of the Paired box family of transcriptional regulators, is normally expressed during embryonic development, including in the kidney, where it promotes mesenchymal to epithelial transition (MET). PAX2 expression is silenced in many normal adult tissues. However, in contrast, PAX2 is expressed in several cancer types, including kidney, prostate, breast, and ovarian cancer. While multiple studies have implicated TGF-β superfamily members in modulating expression of Pax genes during embryonic development, few have investigated direct regulation of Pax gene expression by TGF-β1. Here we have investigated direct regulation of PAX2 expression by TGF-β1 in clear cell renal cell carcinoma (CC-RCC) cell lines. Treatment of PAX2-expressing 786-O and A498 CC-RCC cell lines with TGF-β1 resulted in inhibition of endogenous PAX2 mRNA and protein expression, as well as expression from transiently transfected PAX2 promoter constructs; this inhibition was abolished in the presence of expression of the inhibitory SMAD, SMAD7. Using ChIP-PCR we showed TGF-β1 treatment induced SMAD3 protein phosphorylation in 786-O cells, and direct SMAD3 binding to the human PAX2 promoter, which was inhibited by SMAD7 over-expression. Overall, these data suggest that canonical TGF-β signalling suppresses PAX2 transcription in CC-RCC cells due to the direct binding of SMAD proteins to the PAX2 promoter. These studies improve our understanding of tumor progression and epithelial to mesenchyme transition (EMT) in CC-RCC and in other PAX2-expressing cancer types.
Highlights
30 Members of the TGF-β superfamily, including TGF-βs, activins, bone morphogenic proteins (BMPs), anti-Mullerian hormone (MIS/AMH), and growth/differentiation factors (GDFs), such as myostatin, represent multifunctional cytokines secreted by various cell types
To determine whether TGF-β treatment mediates inhibition of PAX2 expression directly or indirectly via regulation of the PAX2 promoter, we investigated whether TGF-β1 treatment could suppress transiently transfected human PAX2 promoter constructs in kidney and renal cell carcinoma (RCC) cell lines
We found that PAX2 promoter-luciferase reporter constructs were transcriptionally active in the HEK293 and TK10 cell lines (Figure 2), suggesting that these cells would be suitable to carry out the reporter assays
Summary
30 Members of the TGF-β superfamily, including TGF-βs, activins, bone morphogenic proteins (BMPs), anti-Mullerian hormone (MIS/AMH), and growth/differentiation factors (GDFs), such as myostatin, represent multifunctional cytokines secreted by various cell types. TGF-β superfamily ligands regulate a vast array of biological processes such as growth, differentiation, migration, extracellular matrix production, angiogenesis, cytokine secretion, apoptosis, setting up of the body plan and organogenesis during embryogenesis, tissue homeostasis and immune regulation in adults [1,2,3,4,5,6,7]. In opposition to Smad, or Smad effector signalling, the vertebrate I-Smads, Smad and Smad act as potent antagonists of TGF-β signalling [18,19,20,21,22,23] through recruitment of either ubiquitin ligases (Smurf1/2), or protein phosphatase I, or by becoming part of the TGF-β receptor complex and thereby interfering in the formation of SMAD2/SMAD3/co-SMAD complexes
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