TGF-β type I receptor/ALK-5 and Smad proteins mediate epithelial to mesenchymal transdifferentiation in NMuMG breast epithelial cells

Abstract

The capacities of different transforming growth factor-β (TGF-β) superfamily members to drive epithelial to mesenchymal transdifferentiation of the murine mammary epithelial cell line NMuMG were investigated. TGF-β1, but not activin A or osteogenic protein-1 (OP-1)/bone morphogenetic protein-7 (BMP-7), was able to induce morphological transformation of NMuMG cells as shown by reorganisation of the actin cytoskeleton and relocalisation/downregulation of E-cadherin and β-catenin, an effect that was abrogated by the more general serine/threonine kinase and protein kinase C inhibitor, staurosporine. TGF-β1 bound to TGF-β type I receptor (TβR-I)/ALK-5 and TβR-II, but not to activin type I receptor (ActR-I)/ALK-2. Activin A bound to ActR-IB/ALK-4 and ActR-II, and BMP-7 bound to ActR-I/ALK-2, BMP type I receptor (BMPR-I)/ALK-3, ActR-II and BMPR-II. TGF-β1 and BMP-7 activated the Smad-binding element (SBE)4promoter with equal potency, whereas activin A had no effect. Transfection of constitutively active (CA)-ALK-4 activated the 3TP promoter to the same extent as TGF-β1 and CA-ALK-5 indicating that activin signalling downstream of type I receptors was functional in NMuMG cells. In agreement with this, activin A induced low levels of plasminogen activator inhibitor I expression compared to the high induction by TGF-β1. In contrast to activin A and BMP-7, TGF-β1 strongly induced Smad2 phosphorylation. Consistent with these findings, TGF-β1 induced the nuclear accumulation of Smad2 and/or Smad3. In addition, NMuMG cells transiently infected with adenoviral vectors expressing high level CA-ALK-5 exhibited full transdifferentiation. On the other hand, infections with low level CA-ALK-5, which alone did not result in transdifferentiation, together with Smad2 and Smad4, or with Smad3 and Smad4 led to transdifferentiation. In conclusion, TGF-β1 signals potently and passes the activation threshold to evoke NMuMG cell transdifferentiation. The TGF-β type I receptor (ALK-5) and its effector Smad proteins mediate the epithelial to mesenchymal transition. Activin A does not induce mesenchymal transformation, presumably because the number of activin receptors is limited, while BMP-7-initiated signalling cannot mediate transdifferentiation.