Unknown,Transcriptomics,Genomics,Proteomics

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Smad2 and Smad3 have differential sensitivity in relaying TGF b signaling and inversely regulate early lineage specification

ABSTRACT: The transforming growth factor beta (TGFÎ²) related signaling is one of the most important signaling pathways regulating early developmental events. Smad2 and Smad3 are structurally similar and it is mostly considered that they are equally important in mediating TGFÎ² signals. Here, we show that Smad3 is an insensitive TGFÎ² transducer as compared with Smad2. Smad3 preferentially localizes within the nucleus and is thus sequestered from membrane signaling. The ability of Smad3 in oligomerization with Smad4 upon agonist stimulation is also impaired given its unique linker region. Smad2 mediated TGFÎ² signaling plays a crucial role in epiblast development and patterning of three germ layers. However, signaling unrelated nuclear localized Smad3 is dispensable for TGFÎ² signaling-mediated epiblast specification, but important for early neural development, an event blocked by TGFÎ²/Smad2 signaling. Both Smad2 and Smad3 bind to the conserved Smads binding element (SBE), but they show nonoverlapped target gene binding specificity. We conclude that Smad2 and Smad3 possess differential sensitivities in relaying TGFÎ² signaling and have distinct roles in regulating early developmental events. GFP, GFP-Smad2 and GFP-Smad3 constitutively expressed Smad3-/- mouse ESCs were differentiated to day6 neuroepithelia and collected for Chip-Seq with an anti-GFP antibody.

ORGANISM(S): Mus musculus

SUBMITTER: yanhua du

PROVIDER: E-GEOD-76557 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:TGF-b is an important pleiotropic cytokine with potent immunoregulatory properties. Although many previous reports have been proposed for the immunoregulatory functions of TGF-b on T cells, such as the suppression of cell proliferation, cytokine production and cytokine signaling, as well as the induction of apoptosis, it is not well elucidated whether the each effect of TGF-b on T cells is dependent on Smad signaling or Smad-independent other signaling pathways. The aim of the study was to clarify the involvement of Smad signaling and to investigate the redundancy of Smad2 and Smad3 on various TGF-b-mediated regulation of gene expression in CD4+ T cells. We used microarrays to detail the global program of gene expression regulated by TGF-b in CD4+ T cells, and identified distinct classes of up/down-regulated genes which are dependent on or independent of TGF-b-Smad signaling. Most of genes regulated by TGF-b were redundantly dependent on Smad2 and Smad3, including Foxp3 and IL-2. In addition, some genes were sufficiently regulated via Smad2 or Smad3 signaling alone. In contrast, TGF-b-mediated RORgt induction was independent of Smad signaling. CD4+CD25-CD44loCD62Lhi T cells (naive) were isolated from the spleens in wild-type (WT), T cell-specific Smad2 conditional knockout (Smad2KO or Smad2del/del), Smad3 knockout (Smad3KO or Smad3-/-) or Smad2del/delSmad3+/- mice by using a BD FACS ariaTM cell sorter (BD Bioscience) (purity: >98%). Freshly purified cells were then stimulated with anti-TCR stimuli in the absence or presence of TGF-b for 24 hr, respectively. A complete and precise experimental procedure is given in the "treatment protocol". It was very difficult to obtain the enough number of CD4+CD25-CD44loCD62Lhi naive T cells from Smad2del/delSmad3-/- mice because alomost all of CD4+ T cells were activated in Smad2del/delSmad3-/- mice. We confirmed that the several known Smad-regulated genes were almost out of control in Smad2del/delSmad3+/- CD4+ T cells by using quantitative RT-PCR. Furthermore, previous studies have reported the similar results in other cell types deficit in two alleles of Smad2 and one allele of Smad3. For these reasons, we substituted Smad2del/delSmad3+/- naive T cells for Smad2/3-deficient naive T cells. Cells were quickly collected 24 hr after culture for RNA extraction and hybridization on Affymetrix microarrays.

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Smad2 and Smad3 have differential sensitivity in relaying TGF b signaling and inversely regulate early lineage specification

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