title: Molecular imaging of TGFβ-induced Smad2/3 phosphorylation reveals a role for receptor tyrosine kinases in modulating TGFβ signaling.
abstract: OBJECTIVE
  The dual modality of TGFβ, both as a potent tumor suppressor and a stimulator of tumor progression, invasion, and metastasis, make it a critical target for therapeutic intervention in human cancers. The ability to carry out real-time, noninvasive imaging of TGFβ-activated Smad signaling in live cells and animal models would significantly improve our understanding of the regulation of this unique signaling cascade. To advance these efforts, we developed a highly sensitive molecular imaging tool that repetitively, noninvasively, and dynamically reports on TGFBR1 kinase activity.
  
  
  METHODS
  The bioluminescent TGFβR1 reporter construct was developed using a split firefly luciferase gene containing a functional sensor of Smad2 phosphorylation, wherein inhibition of TGFβ receptor1 kinase activity leads to an increase in reporter signaling. The reporter was stably transfected into mammalian cells and used to image in vivo and in vitro bioluminescent activity as a surrogate for monitoring TGFBR1 kinase activity.
  
  
  RESULTS
  The reporter was successfully used to monitor direct and indirect inhibition of TGFβ-induced Smad2 and SMAD3 phosphorylation in live cells and tumor xenografts and adapted for high-throughput screening, to identify a role for receptor tyrosine kinase inhibitors as modulators of TGFβ signaling.
  
  
  CONCLUSIONS
  The reporter is a dynamic, noninvasive imaging modality for monitoring TGFβ-induced Smad2 signaling in live cells and tumor xenografts. It has immense potential for identifying novel effectors of R-Smad phosphorylation, for validating drug-target interaction, and for studying TGFβ signaling in different metastasis models.
authors.affiliation: Center for Molecular Imaging, University of Michigan, Ann Arbor, USA.
authors.forename: Shyam
authors.initials: S
authors.lastname: Nyati
authors.forename: Katrina
authors.initials: K
authors.lastname: Schinske
authors.forename: Dipankar
authors.initials: D
authors.lastname: Ray
authors.forename: Mukesh K
authors.initials: MK
authors.lastname: Nyati
authors.forename: Mukesh
authors.initials: M
authors.lastname: Nyati
authors.forename: Brian Dale
authors.initials: BD
authors.lastname: Ross
authors.forename: Alnawaz
authors.initials: A
authors.lastname: Rehemtulla
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D005260:Female; D017930:Genes, Reporter; D016147:Genes, Tumor Suppressor; D059014:High-Throughput Nucleotide Sequencing; D057166:High-Throughput Screening Assays; D006801:Humans; D008163:Luminescent Measurements; D051379:Mice; D008819:Mice, Nude; D057054:Molecular Imaging; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D034622:RNA Interference; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism; D000077293:Receptor, Transforming Growth Factor-beta Type I; D018125:Receptors, Transforming Growth Factor beta / Q000378:metabolism*; D015398:Signal Transduction; D051899:Smad2 Protein / Q000235:genetics / Q000378:metabolism*; D051900:Smad3 Protein / Q000235:genetics / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism
chemical_list: D018125:Receptors, Transforming Growth Factor beta; C497782:SMAD2 protein, human; C497790:SMAD3 protein, human; D051899:Smad2 Protein; D051900:Smad3 Protein; D016212:Transforming Growth Factor beta; D020794:Receptor Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D000077293:Receptor, Transforming Growth Factor-beta Type I; C000625841:TGFBR1 protein, human; C000625842:Tgfbr1 protein, mouse