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. 2014 Mar 20;20(1):93-108.
doi: 10.2119/molmed.2013.00127.

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

Matthew M Gubin  1 , Patsharaporn Techasintana  1 , Joseph D Magee  1 , Garrett M Dahm  1 , Robert Calaluce  1 , Jennifer L Martindale  2 , Maryln S Whitney  1 , Craig L Franklin  1 , Cindy Besch-Williford  1 , John W Hollingsworth  3 , Kotb Abdelmohsen  2 , Myriam Gorospe  2 , Ulus Atasoy  1
Affiliations

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

Matthew M Gubin et al. Mol Med. .

Abstract

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 HuR(fl/+)) 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 HuR(fl/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.

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Figures

Figure 1
Figure 1
OX40-Cre HuRfl/+ and OX40-Cre HuRfl/fl knockout mice display significant decreases in HuR protein levels. (A) Western blot analysis of HuR expression (and β-tubulin as loading control) in activated T cells from OX40-Cre HuRfl/+ compared with HuRfl/+ control mice and (C) in activated T cells from OX40-Cre HuRfl/fl compared with HuRfl/fl control mice. (B) Intracellular HuR staining assessed by fluorescence-activated cell sorter analysis in activated T cells from OX40-Cre HuRfl/+ and HuRfl/+ control mice and (D) from OX40-Cre HuRfl/fl and HuRfl/fl control mice. (E) Proliferation assay reveals HuR knockout does not affect T-cell proliferation (mean ± standard error of the mean [SEM]; n = 3). Western blot and intracellular HuR staining representative of n = 3.
Figure 2
Figure 2
Th2-polarized cells from OX40-Cre HuRfl/+ heterozygous knockout mice display decreases in Il4, Il13 and Gata3 steady-state mRNA levels. (A) Real-time PCR analysis of Gata3, Il2, Il4 and Il13 mRNA in Th2-polarized cells. (B) Gata3 mRNA decay as assessed by real-time PCR in OX40-Cre HuRfl/+ and HuRfl/+ T cells (n = 3 for half-life). (C) ELISA of supernatants from Th2-polarized OX40-Cre HuRfl/+ mice. (D) Real-time PCR analysis of Ifng in Th1-polarized cells from OX40-Cre HuRfl/+ compared with HuRfl/+ control mice. (E) IFN-γ ELISA of supernatants from Th1-polarized OX40-Cre HuRfl/+ mice (error bars represent mean ± SEM; n = 3; *p < 0.05). Act. D, actinomycin D; N.S., not significant.
Figure 3
Figure 3
Th2-polarized cells from OX40-Cre HuRfl/fl homozygous knockout mice display increases in Il2, Il4 and Il13 steady-state mRNA and protein levels. Real-time PCR analysis of Gata3, Il2, Il4 and Il13 mRNA in Th2-polarized cells. (B) Gata3 mRNA decay as assessed by real-time PCR in OX40-Cre HuRfl/fl and HuRfl/fl T cells (n = 3 for half-life). (C) ELISA of supernatants from Th2-polarized OX40-Cre HuRfl/fl mice. (D) Real-time PCR analysis of Ifng in Th1-polarized cells from OX40-Cre HuRfl/fl compared with HuRfl/fl control mice. (E) IFN-γ ELISA of supernatants from Th1-polarized OX40-Cre HuRfl/fl mice (error bars represent mean ± SEM; n = 3; *p < 0.05). N.S., not significant.
Figure 4
Figure 4
Il4 is transcriptionally upregulated in Th2-polarized cells from OX40-Cre HuRfl/fl homozygous knockout mice while Il13 and Il2 mRNA stabilities are increased. (A) Transcriptional assay using nascent RNA capture assay and real-time PCR analysis of Il13, Il4 and Il2 mRNA in Th2-polarized cells from OX40-Cre HuRfl/fl and HuRfl/fl mice. (B) Il2 mRNA decay as assessed by real-time PCR in OX40-Cre HuRfl/fl and HuRfl/fl T cells. (C) Il13 mRNA decay as assessed by real-time PCR in OX40-Cre HuRfl/fl and HuRfl/fl T cells. (D) Il4 mRNA decay as assessed by real-time PCR in OX40-Cre HuRfl/fl and HuRfl/fl T cells (error bars represent mean ± SEM; n = 3; *p < 0.05; n = 3 for half-life).
Figure 5
Figure 5
Polysomal distribution of Il2 and Il13 mRNA is unaltered in OX40-Cre HuRfl/fl homozygous knockout mice. (A) Absorbance profile for RNA separated by velocity sedimentation through a sucrose gradient. RNA was extracted from each fraction (B), and the levels of Il13 and Il2 mRNA (C) in each fraction from each population (OX40-Cre HuRfl/fl or HuRfl/fl control) was measured by RT-qPCR. (D) The levels of Gapdh mRNA were also measured in each fraction. The data shown are representative of two independent experiments. LMWP, low-molecular-weight polysomes; HMWP, heavy-molecular-weight polysomes.
Figure 6
Figure 6
BALF cellularity and serum IgE titers in HuR knockout mice with allergic airway inflammation. (A) Number of cells recovered from the BALF of immunized HuR knockout and control mice and nonimmunized mice. (B) Cellular composition represented as a percent of total cells recovered from the BALF from immunized HuR knockout mice and control mice. (C) Serum IgE levels as measured by ELISA from immunized HuR knockout or control mice and nonimmunized knockout mice. (D) BALF IL-4 and IL-13 levels were measured by using ELISA comparing immunized HuR knockout and control mice with nonimmunized knockout mice (mean ± SEM of n = 8 mice in each group; three independent experiments; *p < 0.05). N.S., not significant.
Figure 7
Figure 7
Lung histology and airway hyperresponsiveness. (A) Lungs recovered from non-immunized HuRfl/fl control mice display normal lung architecture with little inflammation. (B) Lungs from immunized HuRfl/fl control mice display significant inflammation consistent with allergic airway inflammation. (C, D) Lungs from immunized HuR knockout mice have significant lung inflammation consistent with an allergic airway inflammatory response (representative hematoxylin and eosin staining of lungs; n = 3 nonimmunized control mice, n = 5 immunized knockout, and n = 5 immunized control mice). (E) Lung airway resistance in response to methacholine exposure (mean ± SEM of n = 6 mice in each group; two independent experiments; *p < 0.05). N.S., not significant. Scale bars, left panels, 200 μm × 10; right panels; 200 μm × 100.
Figure 7
Figure 7
Lung histology and airway hyperresponsiveness. (A) Lungs recovered from non-immunized HuRfl/fl control mice display normal lung architecture with little inflammation. (B) Lungs from immunized HuRfl/fl control mice display significant inflammation consistent with allergic airway inflammation. (C, D) Lungs from immunized HuR knockout mice have significant lung inflammation consistent with an allergic airway inflammatory response (representative hematoxylin and eosin staining of lungs; n = 3 nonimmunized control mice, n = 5 immunized knockout, and n = 5 immunized control mice). (E) Lung airway resistance in response to methacholine exposure (mean ± SEM of n = 6 mice in each group; two independent experiments; *p < 0.05). N.S., not significant. Scale bars, left panels, 200 μm × 10; right panels; 200 μm × 100.
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