Interleukin (IL)-7 is an essential non-redundant cytokine and throughout the life-span
of a T cell signaling via the IL-7 receptor influences cell survival, proliferation and function.
It is therefore no surprise that expression of the IL-7 receptor alpha-chain (CD127) is tightly
regulated. In this study I establish IL-7 down regulates CD127 gene transcription and surface
protein expression in primary human CD8 T cells through two mechanisms.
Upon binding IL-7, surface CD127 is rapidly internalized and phosphorylated at the
critical tyrosine residue Y449. Concurrent activation of the JAK/STAT5 pathway stimulates
expression of CIS, a member of the SOCS family of proteins. CIS protein already expressed
at basal levels and induced by IL-7 bind directly to CD127 as demonstrated by Coimmunoprecipitation
assays and colocalize with both CD127 and the early endosomal
marker EEA1. Subsequent proteasomal degradation of CD127 and CIS is dependent on an
E3 ligase. Through siRNA-mediated knockdowns I confirm CIS plays a predominant role in
the IL-7 mediated degradation of CD127.
The mechanism by which IL-7 suppresses CD127 transcripts in primary human CD8
T cells was also examined. Through qPCR and nuclear run-on assays I illustrate that IL-7
suppresses CD127 gene transcription in a time- and dose-dependent manner. The IL-7
mediated suppression of CD127 transcripts is dependent on JAK/STAT5 signaling. Notably,
cycloheximide blocked IL-7’s ability to down-regulate CD127 transcripts suggesting IL-7
stimulates the de novo synthesis of a transcriptional repressor of the CD127 gene. Through
PCR arrays, qPCR and Western blot analysis the IL-7 inducible transcription factor c-Myb
was identified as a candidate repressor. The region within the CD127 gene promoter required
for IL-7 mediated transcriptional suppression was identified through progressive truncations
using firefly luciferase as a reporter gene and is located from -1760 to -2406 bp upstream of
the TATA box and contains three putative c-Myb binding sites. Using siRNA-mediated
knockdown and transient over-expression, I illustrate c-Myb suppresses CD127 gene
transcription in primary human CD8 T cells. A thorough understanding of the mechanisms
by which IL-7 regulates CD127 expression is imperative and may reveal novel insights into
the contribution of abnormal IL-7 signaling to diseases affecting immune function.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:OOU.#10393/24355 |
| Date | 24 July 2013 |
| Creators | Al-Ghazawi, Feras |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
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