Different signaling activities of 2 type I activin receptors in cells stably expressing Smad7.

(A) Immunoblot showing detectable levels of Smad7 (or Myc-Smad7) in stably infected cells and undetectable levels in parent cells. Clonal cells stably expressing Smad7 were established by retrovector infection and selection. A fixed amount (20 μg each) of total protein was analyzed with gel electrophoresis and subsequent immunoblotting using specific antibody against Smad7. Lane 1 shows results with parent NMuMG cells; lanes 2 and 3, results with clonal progeny cells infected with Smad7 virus and designated NMuMG (clone 2) and NMuMG (clone 3); lane 4, results with NMuMG cells infected with Myc-Smad7 virus and designated NMuMG (clone 4); lanes 5 and 6, results with ST2 progeny cells infected with Smad7 and designated ST2 (clone 5) and ST2 (clone 6); lane 7, results with ST2 progeny cells infected with Myc-Smad7 and designated ST2 (clone 7); and lane 8, results with parent ST2 cells. (B and C) In the cells stably producing Smad7, ActR-I (QD) induced only limited stimulation of the reporter, whereas ActR-IB (TD) enhanced p3TP-Lux activity to the same extent as in the parent cells. ST2 cells, NMuMG cells, and their derivatives were transfected with p3TP-Lux (300 ng), ActR-II vector, and either ActR-I (QD) or ActR-IB (TD) vector (50 ng each) and then cultured without activin treatment. Results are expressed as the increase in induction (x-fold) compared with the value in the vector control in parent cells. These data suggest that in cells expressing an increased amount of Smad7, ActR-IB signaling maintains its activity, whereas ActR-I signaling is prevented from causing the transcriptional response.