In view of the role interleukin (IL)-7 plays in T cell survival, homeostasis and function it is no surprise expression of the IL-7 receptor-α (CD127) is tightly regulated. Dysregulation of IL-7 signaling and CD127 expression has been implicated in a number of diseases such as multiple sclerosis, breast cancer, arthritis as well as in HIV infection. Thus it is important to understand how IL-7 regulates its own receptor expression. Here we elucidate the mechanism by which IL-7 suppresses CD127 transcripts in primary human CD8 T cells. We show by qPCR and nuclear run-on assays 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 which in turn suppresses CD127 gene transcription. Through DNA microarrays and PCR arrays, the IL-7 inducible transcription factor c-Myb was identified. 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 c-Myb binding sites. Using siRNA-mediated knockdown and transient over-expression, we illustrate c-Myb suppresses CD127 gene transcription in primary human CD8 T cells.
Interleukin (IL)-7 is an essential non-redundant cytokine influencing cell survival and proliferation throughout the life-span of a T cell. IL-7 is currently being investigated as a potential therapy in HIV+ individuals with poor immunological response to antiretroviral therapy. In order to optimize the use of IL-7 in therapeutic settings, it is crucial to understand how expression of the IL-7 receptor-α (CD127) is regulated. We elucidate in this study the mechanism by which IL-7 targets surface CD127 protein for degradation and describe the role of SOCS proteins, a family of eight proteins (CIS and SOCS1-7), in this process. We show here that in primary human CD8 T cells, upon binding IL-7, surface CD127 is rapidly internalized and phosphorylated at Y449 within 20 minutes of IL-7 stimulation, while activation of the JAK/STAT5 pathway stimulates expression of CIS transcripts and proteins. Following IL-7 stimulation, CIS binds directly to CD127 as demonstrated by Co-IP and co-localizes with both CD127 and the early endosomal marker EEA1, then with the proteasomal marker MCP20, as demonstrated by confocal microscopy. Subsequent proteasomal degradation of CD127 and CIS is dependent on an E3 ligase and can be blocked with the E3 ligase inhibitor SMER3. Through siRNA-mediated knockdowns we confirm roles of CIS in the IL-7 mediated degradation of the CD127 protein.
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