Patsharaporn Techasintana

Posttranscriptional Gene Regulation of IL-17 by the RNA-Binding Protein HuR Is Required for Initiation of Experimental Autoimmune Encephalomyelitis

IL-17 is a proinflammatory cytokine produced by activated Th17 cells and other immune cells. IL-17–producing Th17 cells are major contributors to chronic inflammatory and autoimmune diseases, such as multiple sclerosis, rheumatoid arthritis, and inflammatory bowel disease. Although the transcriptional regulation of Th17 cells is well understood, the posttranscriptional regulation of IL-17 gene expression remains unknown. The RNA-binding protein HuR positively regulates the stability of many target mRNAs via binding the AU-rich elements present in the 3′ untranslated region of many inflammatory cytokines including IL-4, IL-13, and TNF-α. However, the regulation of IL-17 expression by HuR has not been established. CD4+ Th17 cells from HuR knockout mice had decreased IL-17 steady-state mRNA and protein levels compared with wild-type Th17 cells, as well as decreases in frequency of IL-17+ cells. Moreover, we demonstrated that HuR directly binds to the IL-17 mRNA 3′ untranslated region by using RNA immunoprecipitation and biotin pulldown assays. In addition, the knockout of HuR decreased cellular proliferation of CD4+ T cells. Mice with adoptively transferred HuR KO Th17 cells had delayed initiation and reduced disease severity in the onset of experimental autoimmune encephalomyelitis compared with wild-type Th17 cells. Our results reveal a HuR-induced posttranscriptional regulatory mechanism of Th17 differentiation that influences IL-17 expression. These findings may provide novel therapeutic targets for the treatment of Th17-mediated autoimmune neuroinflammation.

Conditional knockout of the RNA-binding protein HuR in CD4⁺ T cells reveals a gene dosage effect on cytokine production.

The posttranscriptional mechanisms by which RNA binding proteins (RBPs) regulate T-cell differentiation and cytokine production in vivo remain unclear. The RBP HuR binds to labile mRNAs, usually leading to increases in mRNA stability and/or translation. Previous work demonstrated that HuR binds to the mRNAs encoding the Th2 transcription factor trans-acting T-cell-specific transcription factor (GATA-3) and Th2 cytokines interleukin (IL)-4 and IL-13, thereby regulating their expression. By using a novel conditional HuR knockout (KO) mouse in which HuR is deleted in activated T cells, we show that Th2-polarized cells from heterozygous HuR conditional (OX40-Cre HuRfl/+) KO mice had decreased steady-state levels of Gata3, Il4 and Il13 mRNAs with little changes at the protein level. Surprisingly, Th2-polarized cells from homozygous HuR conditional (OX40-Cre HuRfl/fl) KO mice showed increased Il2, Il4 and Il13 mRNA and protein via different mechanisms. Specifically, Il4 was transcriptionally upregulated in HuR KO T cells, whereas Il2 and Il13 mRNA stabilities increased. Additionally, when using the standard ovalbumin model of allergic airway inflammation, HuR conditional KO mice mounted a robust inflammatory response similar to mice with wild-type HuR levels. These results reveal a complex differential posttranscriptional regulation of cytokines by HuR in which gene dosage plays an important role. These findings may have significant implications in allergies and asthma, as well as autoimmune diseases and infection.

Transcriptomic-Wide Discovery of Direct and Indirect HuR RNA Targets in Activated CD4+ T Cells.

Due to poor correlation between steady state mRNA levels and protein product, purely transcriptomic profiling methods may miss genes posttranscriptionally regulated by RNA binding proteins (RBPs) and microRNAs (miRNAs). RNA immunoprecipitation (RIP) methods developed to identify in vivo targets of RBPs have greatly elucidated those mRNAs which may be regulated via transcript stability and translation. The RBP HuR (ELAVL1) and family members are major stabilizers of mRNA. Many labs have identified HuR mRNA targets; however, many of these analyses have been performed in cell lines and oftentimes are not independent biological replicates. Little is known about how HuR target mRNAs behave in conditional knock-out models. In the present work, we performed HuR RIP-Seq and RNA-Seq to investigate HuR direct and indirect targets using a novel conditional knock-out model of HuR genetic ablation during CD4+ T activation and Th2 differentiation. Using independent biological replicates, we generated a high coverage RIP-Seq data set (>160 million reads) that was analyzed using bioinformatics methods specifically designed to find direct mRNA targets in RIP-Seq data. Simultaneously, another set of independent biological replicates were sequenced by RNA-Seq (>425 million reads) to identify indirect HuR targets. These direct and indirect targets were combined to determine canonical pathways in CD4+ T cell activation and differentiation for which HuR plays an important role. We show that HuR may regulate genes in multiple canonical pathways involved in T cell activation especially the CD28 family signaling pathway. These data provide insights into potential HuR-regulated genes during T cell activation and immune mechanisms.

The RNA-Binding Protein HuR Posttranscriptionally Regulates IL-2 Homeostasis and CD4+ Th2 Differentiation

Posttranscriptional gene regulation by RNA-binding proteins, such as HuR (elavl1), fine-tune gene expression in T cells, leading to powerful effects on immune responses. HuR can stabilize target mRNAs and/or promote translation by interacting with their 3′ untranslated region adenylate and uridylate–rich elements. It was previously demonstrated that HuR facilitates Th2 cytokine expression by mRNA stabilization. However, its effects upon IL-2 homeostasis and CD4+ Th2 differentiation are not as well understood. We found that optimal translation of Il2ra (CD25) required interaction of its mRNA with HuR. Conditional HuR knockout in CD4+ T cells resulted in loss of IL-2 homeostasis and defects in JAK–STAT signaling, Th2 differentiation, and cytokine production. HuR-knockout CD4+ T cells from OVA-immunized mice also failed to proliferate in response to Ag. These results demonstrate that HuR plays a pivotal role in maintaining normal IL-2 homeostasis and initiating CD4+ Th2 differentiation.

Abstract 3084: The role of RNA-binding protein HuR in lung metastasis of triple-negative breast cancer

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Breast cancer is the second most common cancer among women worldwide. It is estimated that approximately one million women are diagnosed with breast cancer annually. Furthermore, more than 410,000 women will die each year from the disease primarily due to tumor metastasis. Currently, even the most effective treatments often result in recurrence and metastasis, in part due to genetic factors. Therefore, understanding the mechanism of breast cancer related gene regulation is crucial for the development of novel treatments and preventive strategies to those who are predisposed to developing breast cancer. Previously, we have shown that the RNA-binding protein HuR posttranscriptionally regulates various proto-oncogenes by stabilizing their mRNAs and facilitating their translation into proteins. Additionally, HuR upregulation and cytoplasmic localization has been associated with invasive cancer progression and poor prognosis. HuR has been described to control genes in multiple areas of the acquired capabilities model of cancer and has been hypothesized to be a tumor-maintenance gene, allowing for cancers to proliferate once they are established. HuR regulates genes involved in angiogenesis, cell growth and cell cycle regulation including VEGFα, TSP1, HIF1α, CDKN1A(p21) and β-catenin. In this study, we investigated the role of HuR in an aggressive triple-negative (estrogen receptor ER-, human epidermal growth factor receptor 2 (HER2)- and progesterone receptor (PR)-) breast cancer cell subline LM-2. The LM-2 cells were derived from triple-negative breast cancer cells MDA-MB-231 which were isolated from two rounds of in vivo selection of the cancer cells which metastasized to the lungs. The LM-2 cells were retrovirally transduced with triple-fusion protein reporter construct encoding thymidine kinase1, green fluorescent protein (GFP) and firefly luciferase in order to obtain nuclear imaging, fluorescent and bioluminescent properties for in vivo and in vitro tracking. LM2 cells were further transfected with a plasmid containing HA-HuR or empty vector control to investigate the function of HuR in LM2 cells. Two clones of HuR overexpressing LM2 cells were shown to grow faster in vitro compared to the empty vector control. Also, HuR overexpression significantly facilitates tumor invasion in vitro by matrigel invasion assay. Further analysis of HuR overexpressing LM2 cells using in vivo imaging system (IVIS) will reveal the role of HuR in breast cancer growth and metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3084. doi:10.1158/1538-7445.AM2011-3084