ZIA BC 011214 (ZIA) | |||
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Title | Post-Transcriptional Regulation of Interleukin-7 Receptor Expression | ||
Institution | NCI, Bethesda, MD | ||
Principal Investigator | Park, Jung-Hyun | NCI Program Director | N/A |
Cancer Activity | N/A | Division | CCR |
Funded Amount | $560,973 | Project Dates | 00/00/0000 - 00/00/0000 |
Fiscal Year | 2016 | Project Type | Intramural |
Research Topics w/ Percent Relevance | Cancer Types w/ Percent Relevance | ||
Autoimmune Diseases (35.0%) Cancer (100.0%) |
Hodgkins disease (20.0%) Leukemia (20.0%) Non Hodgkins Lymphoma (20.0%) |
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Research Type | |||
Normal Functioning Development and Characterization of Model Systems |
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Abstract | |||
The molecular mechanisms that control gc expression is still largely veiled. Previously, we reported alternative splicing of gc pre-mRNA as a new mechanism to downregulate surface gc protein expression which also resulted in the production of bioactive soluble gc proteins. Because the soluble form of gc (sgc) is generated at the expense of membrane gc protein expression, sgc expression inversely correlates with the amount of surface gc expression. Therefore, sgc expression represents a novel mechanism to suppress gc expression on cell surface. We have now identified DP thymocytes as a major source of soluble gc protein expression in the thymus. Because sgc proteins suppress signaling of IL-2 and IL-7, sgc production by DP thymocytes would create an overall suppressive milieu for gc cytokine signaling in the thymus. In fact, we found that transgenic overexpression of sgc resulted in impaired thymopoiesis and diminished thymic NKT cell generation, a process dependent on IL-15 signaling. Generation of IL-2-dependent Foxp3+ T regulatory cells or IL-7-dependent CD8SP thymocytes, however, were not affected. Collectively, these results demonstrate a previously unappreciated role for sgc in downregulating surface gc expression and also in dampening gc cytokine signaling in thymocytes, which can inhibit the generation and differentiation of specific T cell subsets in the thymus. In parallel to soluble gc receptors, we also found soluble IL-7Ra proteins in serum of human and mice. In humans, soluble IL-7Ra has been previously described and it is known that they are produced by alternative splicing of the IL-7Ra pre-mRNA. In contrast, soluble IL-7Ra proteins have not been reported in mice, and there is no molecular evidence for an alternative IL-7Ra splice isoform in mice. Nonetheless, we were able to detect soluble IL-7Ra proteins in wildtype mouse sera by ELISA. Currently, the molecular basis of soluble IL-7Ra proteins in mice is not known to us, and we do not exclude the possibility of membrane protein shedding. We are currently addressing this question using newly generate ELISA assays and by immunoprecipitation to clarify the source of soluble IL-7Ra and what mechanisms would induce their expression. Additionally, we wished to understand the regulatory mechanisms of IL-7Ra and gc expression at transcriptional level. Currently, there is no molecular explanation how distinct IL-7Ra expression is achieved on T cell and thymocyte subsets and there is also no information available how gc transcription is controlled during T cell activation and differentiation. Upon analyzing surface gc expression, we found that gc expression is highly induced on immature DN and mature SP thymocytes but significantly suppressed on pre-selection DP thymocytes. We found that the deficiency of the transcription factor RORgt reversed this defect, and that gc expression was restored in DP thymocytes of RORgt-KO mice. These data suggested that RORgt could act as a potential suppressor of gc expression. To directly address this questions, we generated transgenic mice expressing RORgt under the control of human CD2 promoter/enhancer elements. We confirmed transgenic RORgt expression by intracellular staining and Western blot analysis. Interestingly, RORgt overexpression did not affect T cell development in the thymus, and it only showed marginal effects on CD4/CD8 lineage commitment. To confirm that the RORgt transgene is functional, we are currently in the process of backcrossing the RORgt transgene into RORgt-deficient mice with the aim to demonstrate that it can rescue RORgt-deficiency in T cells. After confirmation, we will assess gc surface and protein expression in RORgt-transgenic and RORgt-transgenic/KO mice to examine the potential role of RORgt on gc regulation. In addition to RORgt, we have previously shown that the zinc finger protein Gfi1 is involved in regulating cytokine receptor expression in thymocytes. Using germline Gfi1-deficient mice, we show |