Transforming growth factor b-1 emhances Smad transcriptional activity through activation of p8 gene expression

ANDRÉS C. GARCIA-MONTERO; SOPHIE VASSEUR; LUCIANA E. GIONO; EDUARDO T. CÁNEPA; SILVIA MORENO; JEAN CHARLES DAGORN; JUAN LUCIO IOVANNA

BIOCHEMICAL JOURNAL

The Biochemical Society

Lugar: Londres; Año: 2001 vol. 357 p. 249 - 253

0264-6021

We report that exposure of mouse embryonic ®broblasts to transforming growth factor b-1 (TGFb-1) (5 ng}ml) results in a strong activation of p8 mRNA expression that precedes the induction of cell growth. Involvement of the p8 promoter in the regulation was demonstrated by using a p8±chloramphenicol acetyltransferase construct. We therefore speculated that p8 might be a mediator of TGFb-1 in these cells. The incorporation of [$H]thymidine on treatment with TGFb-1 was indeed signi®- cantly higher in p8+/+ ®broblasts than in p8−/− ®broblasts. Smad transcriptional activity was used as marker of the TGFb-1 signalling pathway, to probe the lower p8−/− response to TGFb-1. Two Smad-binding elements (SBEs)±luciferase constructs were transfected into p8−/− and p8+/+ embryonic ®broblastsb-1 (TGFb-1) (5 ng}ml) results in a strong activation of p8 mRNA expression that precedes the induction of cell growth. Involvement of the p8 promoter in the regulation was demonstrated by using a p8±chloramphenicol acetyltransferase construct. We therefore speculated that p8 might be a mediator of TGFb-1 in these cells. The incorporation of [$H]thymidine on treatment with TGFb-1 was indeed signi®- cantly higher in p8+/+ ®broblasts than in p8−/− ®broblasts. Smad transcriptional activity was used as marker of the TGFb-1 signalling pathway, to probe the lower p8−/− response to TGFb-1. Two Smad-binding elements (SBEs)±luciferase constructs were transfected into p8−/− and p8+/+ embryonic ®broblastsb-1 in these cells. The incorporation of [$H]thymidine on treatment with TGFb-1 was indeed signi®- cantly higher in p8+/+ ®broblasts than in p8−/− ®broblasts. Smad transcriptional activity was used as marker of the TGFb-1 signalling pathway, to probe the lower p8−/− response to TGFb-1. Two Smad-binding elements (SBEs)±luciferase constructs were transfected into p8−/− and p8+/+ embryonic ®broblasts$H]thymidine on treatment with TGFb-1 was indeed signi®- cantly higher in p8+/+ ®broblasts than in p8−/− ®broblasts. Smad transcriptional activity was used as marker of the TGFb-1 signalling pathway, to probe the lower p8−/− response to TGFb-1. Two Smad-binding elements (SBEs)±luciferase constructs were transfected into p8−/− and p8+/+ embryonic ®broblasts+/+ ®broblasts than in p8−/− ®broblasts. Smad transcriptional activity was used as marker of the TGFb-1 signalling pathway, to probe the lower p8−/− response to TGFb-1. Two Smad-binding elements (SBEs)±luciferase constructs were transfected into p8−/− and p8+/+ embryonic ®broblastsb-1 signalling pathway, to probe the lower p8−/− response to TGFb-1. Two Smad-binding elements (SBEs)±luciferase constructs were transfected into p8−/− and p8+/+ embryonic ®broblasts−/− response to TGFb-1. Two Smad-binding elements (SBEs)±luciferase constructs were transfected into p8−/− and p8+/+ embryonic ®broblastsb-1. Two Smad-binding elements (SBEs)±luciferase constructs were transfected into p8−/− and p8+/+ embryonic ®broblasts−/− and p8+/+ embryonic ®broblasts before treatment with TGFb-1. A lower level of Smad transactivation was observed in p8−/− embryonic ®broblasts, under basal conditions and after stimulation with TGFb-1. To test whether Smad underexpression in p8−/− cells was actually due to p8 depletion, p8−/− embryonic ®broblasts were transfected with a human p8 expression plasmid together with an SBE±luciferase construct. The expression of p8 restored Smad transactivation in unstimulated and TGFb-1-treated cells to the level found in p8+/+b-1. A lower level of Smad transactivation was observed in p8−/− embryonic ®broblasts, under basal conditions and after stimulation with TGFb-1. To test whether Smad underexpression in p8−/− cells was actually due to p8 depletion, p8−/− embryonic ®broblasts were transfected with a human p8 expression plasmid together with an SBE±luciferase construct. The expression of p8 restored Smad transactivation in unstimulated and TGFb-1-treated cells to the level found in p8+/+−/− embryonic ®broblasts, under basal conditions and after stimulation with TGFb-1. To test whether Smad underexpression in p8−/− cells was actually due to p8 depletion, p8−/− embryonic ®broblasts were transfected with a human p8 expression plasmid together with an SBE±luciferase construct. The expression of p8 restored Smad transactivation in unstimulated and TGFb-1-treated cells to the level found in p8+/+b-1. To test whether Smad underexpression in p8−/− cells was actually due to p8 depletion, p8−/− embryonic ®broblasts were transfected with a human p8 expression plasmid together with an SBE±luciferase construct. The expression of p8 restored Smad transactivation in unstimulated and TGFb-1-treated cells to the level found in p8+/+−/− cells was actually due to p8 depletion, p8−/− embryonic ®broblasts were transfected with a human p8 expression plasmid together with an SBE±luciferase construct. The expression of p8 restored Smad transactivation in unstimulated and TGFb-1-treated cells to the level found in p8+/+−/− embryonic ®broblasts were transfected with a human p8 expression plasmid together with an SBE±luciferase construct. The expression of p8 restored Smad transactivation in unstimulated and TGFb-1-treated cells to the level found in p8+/+b-1-treated cells to the level found in p8+/+ cells. We concluded that TGFb-1 activates p8 expression, which in turn enhances the Smad-transactivating function responsible for TGFb-1 activity.b-1 activates p8 expression, which in turn enhances the Smad-transactivating function responsible for TGFb-1 activity.b-1 activity.