|
| Status |
Public on Jun 22, 2010 |
| Title |
Gene expression profiling in murine Smad-deficient CD4+ T cells stimulated with TGF-b |
| Organism |
Mus musculus |
| Experiment type |
Expression profiling by array
|
| Summary |
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.
|
| |
|
| Overall design |
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.
|
| |
|
| Contributor(s) |
Takimoto T, Yoshimura A |
| Citation(s) |
20548029 |
| Submission date |
Dec 22, 2009 |
| Last update date |
Feb 11, 2019 |
| Contact name |
Tomohito Takimoto |
| E-mail(s) |
takimoto@bioreg.kyushu-u.ac.jp
|
| Phone |
+81-3-5363-3483
|
| Fax |
+81-3-5360-1508
|
| Organization name |
Keio University School of Medicine
|
| Department |
Microbiology and Immunology
|
| Street address |
35 Shinanomachi
|
| City |
Shinjyuku-ku |
| State/province |
Tokyo |
| ZIP/Postal code |
160-8582 |
| Country |
Japan |
| |
|
| Platforms (1) |
| GPL1261 |
[Mouse430_2] Affymetrix Mouse Genome 430 2.0 Array |
|
| Samples (8)
|
|
| Relations |
| BioProject |
PRJNA122433 |
Less...
More...