TGF-(beta) type I receptor/ALK-5 and Smad proteins mediate epithelial to mesenchymal transdifferentiation in NMuMG breast epithelial cells.
The capacities of different transforming growth factor-(beta) (TGF-(beta)) superfamily members to drive epithelial to mesenchymal transdifferentiation of the murine mammary epithelial cell line NMuMG were investigated. TGF-(beta)1, 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 (beta)-catenin, an effect that was abrogated by the more general serine/threonine kinase and protein kinase C inhibitor, staurosporine. TGF-(beta)1 bound to TGF-(beta) type I receptor (T(beta)R-I)/ALK-5 and T(beta)R-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-(beta)1 and BMP-7 activated the Smad-binding element (SBE)(4) promoter 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-(beta)1 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-(beta)1. In contrast to activin A and BMP-7, TGF-(beta)1 strongly induced Smad2 phosphorylation. Consistent with these findings, TGF-(beta)1 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-(beta)1 signals potently and passes the activation threshold to evoke NMuMG cell transdifferentiation. The TGF-(beta) 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.
Piek
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E
Ludwig Institute for Cancer Research, Box 595, S-75124 Uppsala, Sweden.
Moustakas
A
A
Kurisaki
A
A
Heldin
C H
CH
ten Dijke
P
P
D030201:Activin Receptors, Type I; D028341:Activins; D000818:Animals; D055419:Bone Morphogenetic Protein 7; D019485:Bone Morphogenetic Proteins / Q000502:physiology; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D015536:Down-Regulation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000166:cytology; D007265:Inhibins / Q000502:physiology; D008156:Luciferases / Q000235:genetics; D008321:Mammary Glands, Animal / Q000166:cytology*; D008648:Mesoderm / Q000166:cytology; D051379:Mice; D047428:Protein Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D000077293:Receptor, Transforming Growth Factor-beta Type I; D018125:Receptors, Transforming Growth Factor beta / Q000502:physiology*; D015534:Trans-Activators / Q000502:physiology*; D016212:Transforming Growth Factor beta / Q000502:physiology; D014407:Tumor Cells, Cultured
D055419:Bone Morphogenetic Protein 7; D019485:Bone Morphogenetic Proteins; D004791:Enzyme Inhibitors; D047428:Protein Kinase Inhibitors; D018125:Receptors, Transforming Growth Factor beta; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D028341:Activins; D007265:Inhibins; D008156:Luciferases; D017346:Protein Serine-Threonine Kinases; D030201:Activin Receptors, Type I; D000077293:Receptor, Transforming Growth Factor-beta Type I; C000625842:Tgfbr1 protein, mouse