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. 2003 Aug 19;100(17):9791-6.
doi: 10.1073/pnas.1733973100. Epub 2003 Aug 6.

Repression of Smad transcriptional activity by PIASy, an inhibitor of activated STAT

Jianyin Long  1 , Isao Matsuura, Dongming He, Guannan Wang, Ke Shuai, Fang Liu
Affiliations

Repression of Smad transcriptional activity by PIASy, an inhibitor of activated STAT

Jianyin Long et al. Proc Natl Acad Sci U S A. .

Abstract

Smad proteins mediate transforming growth factor beta (TGF-beta)-inducible transcriptional responses. Protein inhibitor of activated signal transducer and activator of transcription (PIAS) represents a family of proteins that inhibits signal transducer and activator of transcription and also regulates other transcriptional responses. In an effort to identify Smad-interacting proteins by a yeast three-hybrid screen with Smad3 and Smad4 as baits, we identified PIASy, a member of the PIAS family. In yeast, PIASy interacts strongly with Smad4 and also with receptor-regulated Smads. In mammalian cells, PIASy binds most strongly with Smad3 and also associates with other receptor-regulated Smads and Smad4. The interaction between Smad3 and PIASy is increased in the presence of TGF-beta and occurs through the C-terminal domain of Smad3. Moreover, Smad3, Smad4, and PIASy can form a ternary complex. PIASy does not inhibit Smad complex binding to DNA, but it represses Smad transcriptional activity. Interestingly, conditional overexpression of PIASy selectively inhibits a subset of endogenous TGF-beta-responsive genes, which includes the cyclin-dependent kinase inhibitor p15, and the plasminogen activator inhibitor 1. We further show that PIASy can interact constitutively with histone deacetylase 1 (HDAC1) and that addition of HDAC inhibitor trichostatin A (TSA) can prevent the inhibitory function of PIASy. Taken together, our studies indicate that PIASy can inhibit TGF-beta/Smad transcriptional responses through interactions with Smad proteins and HDAC.

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Figures

Fig. 1.
Fig. 1.
Interaction of PIASy with Smad3 and other Smad proteins. (A) Yeast two-hybrid assay of PIASy interaction with Smads1–4. (B) Interaction of PIASy with Smads1–4 in 293T cells. (C) TGF-β treatment increases the interactions between PIASy with Smad3 or Smad2, but has little effect on PIASy–Smad4 interaction. TβRI (T204D) was cotransfected for TGF-β stimulation in 293T cells. T7 Ab-coupled agarose beads were used in the immunoprecipitation. (D) PIASy interacts with the C-terminal domain of Smad3. FL, full length; N, N-terminal domain; LC, linker and C-terminal domain; and C, C-terminal domain. Smad3 (FL) comigrates with a nonspecific band above the IgG (heavy chain). (E) 35S-labeled-PIASy synthesized by in vitro translation (IVT) binds to GST-Smad3. (F) Endogenous interaction between Smad3 and PIASy. HaCaT cells were treated with or without TGF-β for 1 h. Cell lysates were immunoprecipitated with a Smad3 Ab and were immunoblotted with a PIASy Ab.
Fig. 2.
Fig. 2.
PIASy can form a ternary complex with Smad3 and Smad4. (A) Detection of the Smad3–Smad4–PIASy ternary complex. Epitope-tagged Smad3, Smad4, and PIASy, and TβRI (T204D) for TGF-β stimulation were cotransfected into COS cells and analyzed as indicated. (B) Smad4 can increase the interaction between Smad3 and PIASy. (C) Smad3 can increase the interaction between Smad4 and PIASy. TβRI (T204D) was cotransfected for TGF-β stimulation in B and C.
Fig. 3.
Fig. 3.
PIASy inhibits TGF-β/Smad transcriptional responses. Mv1Lu/L17 cells were cotransfected with the A3-Lux reporter gene, FAST-1, and various amounts of T7-PIASy plasmid (Left) or with the 3TP-Lux reporter gene and increasing doses of T7-PIASy plasmid (Right). Cells were treated with or without TGF-β and analyzed for luciferase activity.
Fig. 4.
Fig. 4.
Conditional overexpression of PIASy selectively inhibits a subset of TGF-β/Smad-responsive genes. (A) Time course of PIASy protein induction after removal of tet. A representative PIASy Mv1Lu tTA stable clone was analyzed by immunoblot with the PIASy Ab. (B) Northern blot analysis of TGF-β induction of p15, PAI-1, JunB, and Smad7 transcripts in the parental Mv1Lu tTA cells as well as in the PIASy clone with or without tet. Poly(A)+ RNA (4 μg) of each sample was used. GAPDH served as a loading control.
Fig. 5.
Fig. 5.
(A) PIASy does not inhibit Smad DNA-binding activity. Cell extracts from TGF-β-treated or untreated HaCaT cells were incubated with 2× SBE DNA probe in the absence or presence of increasing amount of purified Flag-PIASy protein (50, 100, 200, and 400 ng) and were then subjected to electrophoresis. (B) PIASy can be recruited to the SBE through interaction with Smads in DNA-affinity precipitation assay. (C) PIASy inhibits GAL4-Smad3 transcriptional activity. Mv1Lu/L17 cells were cotransfected with GAL4-Luc reporter gene, GAL4 DNA-binding domain GAL4 (DBD), or GAL4-Smad3, and increasing amount of T7-PIASy plasmid, treated with or without TGF-β, and analyzed for luciferase activity.
Fig. 6.
Fig. 6.
PIASy can inhibit Smad-mediated transcriptional activation in an HDAC-dependent manner. (A) Interaction of PIASy and HDAC1. Flag-HDAC1, T7-PIASy, and TβRI (T204D) for TGF-β stimulation were cotransfected into COS cells and analyzed as indicated. (B) TSA can disrupt the inhibitory effect of PIASy. Mv1Lu/L17 cells were cotransfected with the A3-Lux reporter gene and FAST-1 in the absence or presence of 300 ng of PIASy DNA. Cells were treated with TGF-β and increasing amounts of TSA at the same time for 18–24 h and then were analyzed for luciferase activity. (Left) One representative experiment. (Right) Summary of several experiments. For each dose of TSA, the fold increase refers to the luciferase activity in the presence of TSA divided by the luciferase activity in the absence of TSA. The fold increase in the presence of cotransfected PIASy divided by the fold increase in the absence of PIASy then was calculated and plotted against each dose of TSA. This ratio in the absence of TSA is set as 1.
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