T-cell activation decreases miRNA-15a/16 levels to promote MEK1–ERK1/2–Elk1 signaling and proliferative capacity

Abstract

While miRs have been extensively studied in the context of malignancy and tumor progression, their functions in regulating T-cell activation are less clear. In initial studies, we found reduced levels of miR-15a/16 at 3 to 18 h post–T-cell receptor (TCR) stimulation, suggesting a role for decreased levels of this miR pair in shaping T-cell activation. To further explore this, we developed an inducible miR15a/16 transgenic mouse model to determine how elevating miR-15a/16 levels during early stages of activation would affect T-cell proliferation and to identify TCR signaling pathways regulated by this miR pair. Doxycycline (DOX)-induced expression of miR-15a/16 from 0 to 18 h post-TCR stimulation decreased ex vivo T-cell proliferation as well as in vivo antigen-specific T-cell proliferation. We also combined bioinformatics and proteomics approaches to identify the mitogen-activated protein kinase kinase 1 (MEK1) (Map2k1) as a target of miR-15a/16. MEK1 targeting by miR-15a/16 was confirmed using miR mimics that decreased Map2k1 mRNA containing the 3′-UTR target nucleotide sequence (UGCUGCUA) but did not decrease Map2k1 containing a mutated control sequence (AAAAAAAA). Phosphorylation of downstream signaling molecules, extracellular signal–regulated protein kinase 1/2 (ERK1/2) and Elk1, was also decreased by DOX-induced miR-15a/16 expression. In addition to MEK1, ERK1 was subsequently found to be targeted by miR-15a/16, with DOX-induced miR-15a/16 reducing total ERK1 levels in T cells. These findings show that TCR stimulation reduces miR-15a/16 levels at early stages of T-cell activation to facilitate increased MEK1 and ERK1, which promotes the sustained MEK1–ERK1/2–Elk1 signaling required for optimal proliferation. While miRs have been extensively studied in the context of malignancy and tumor progression, their functions in regulating T-cell activation are less clear. In initial studies, we found reduced levels of miR-15a/16 at 3 to 18 h post–T-cell receptor (TCR) stimulation, suggesting a role for decreased levels of this miR pair in shaping T-cell activation. To further explore this, we developed an inducible miR15a/16 transgenic mouse model to determine how elevating miR-15a/16 levels during early stages of activation would affect T-cell proliferation and to identify TCR signaling pathways regulated by this miR pair. Doxycycline (DOX)-induced expression of miR-15a/16 from 0 to 18 h post-TCR stimulation decreased ex vivo T-cell proliferation as well as in vivo antigen-specific T-cell proliferation. We also combined bioinformatics and proteomics approaches to identify the mitogen-activated protein kinase kinase 1 (MEK1) (Map2k1) as a target of miR-15a/16. MEK1 targeting by miR-15a/16 was confirmed using miR mimics that decreased Map2k1 mRNA containing the 3′-UTR target nucleotide sequence (UGCUGCUA) but did not decrease Map2k1 containing a mutated control sequence (AAAAAAAA). Phosphorylation of downstream signaling molecules, extracellular signal–regulated protein kinase 1/2 (ERK1/2) and Elk1, was also decreased by DOX-induced miR-15a/16 expression. In addition to MEK1, ERK1 was subsequently found to be targeted by miR-15a/16, with DOX-induced miR-15a/16 reducing total ERK1 levels in T cells. These findings show that TCR stimulation reduces miR-15a/16 levels at early stages of T-cell activation to facilitate increased MEK1 and ERK1, which promotes the sustained MEK1–ERK1/2–Elk1 signaling required for optimal proliferation. T-cell lymphocytes are activated through the T-cell receptor (TCR) together with coreceptors such as CD28, which triggers cellular proliferation as a key step in promoting adaptive immunity (1Jones R.G. Thompson C.B. Revving the engine: Signal transduction fuels T cell activation.Immunity. 2007; 27: 173-178Google Scholar). Mitogen-activated protein kinase pathways are rapidly activated downstream of TCR engagement, and one of these pathways of particular importance for promoting proliferative capacity is the Ras/Raf/mitogen-activated protein kinase kinase (MEK)/extracellular signal–regulated protein kinase (ERK) pathway (2Franklin R.A. Tordai A. Patel H. Gardner A.M. Johnson G.L. Gelfand E.W. Ligation of the T cell receptor complex results in activation of the Ras/Raf-1/MEK/MAPK cascade in human T lymphocytes.J. Clin. Invest. 1994; 93: 2134-2140Google Scholar). TCR engagement leads to rapid activation of Ras, which in turn activates RAF kinase, which in turn phosphorylates and activates MEK1 and MEK2 (mitogen-activated protein kinase kinase 1 and 2). MEK1/2 phosphorylates and activates ERK1/2, which subsequently phosphorylates cytoplasmic and nuclear substrates involved in the regulation of cell proliferation. Even though this pathway is activated within minutes of TCR engagement to initiate T-cell activation, some degree of sustained MEK–ERK activity in later stages is required for cells to upregulate gene transcription that facilitates cell cycle entry and suppresses negative regulators of the cell cycle (3Tsukamoto H. Irie A. Nishimura Y. B-Raf contributes to sustained extracellular signal-regulated kinase activation associated with interleukin-2 production stimulated through the T cell receptor.J. Biol. Chem. 2004; 279: 48457-48465Google Scholar). In fact, interleukin 2 that is secreted after initial MEK–ERK pathway activation has been shown to stimulate MEK1 expression in later stages of activation (4Liang Q. Guo L. Gogate S. Karim Z. Hanifi A. Leung D.Y. Gorska M.M. Alam R. IL-2 and IL-4 stimulate MEK1 expression and contribute to T cell resistance against suppression by TGF-beta and IL-10 in asthma.J. Immunol. 2010; 185: 5704-5713Google Scholar). This suggests that MEK1 levels increasing well after initial TCR signaling may be an important contributor to the sustained activation of T cells that promote cell cycling and proliferation. An important role in regulating T-cell activation is emerging for miRs, which are short noncoding RNAs that mostly bind through base pairing to 3′-UTRs of target mRNAs to decrease target protein levels (5Dzafo E. Bianchi N. Monticelli S. Cell-intrinsic mechanisms to restrain inflammatory responses in T lymphocytes.Immunol. Rev. 2021; 300: 181-193Google Scholar, 6Emamgolizadeh Gurt Tapeh B. Mosayyebi B. Samei M. Beyrampour Basmenj H. Mohammadi A. Alivand M.R. Hassanpour P. Solali S. microRNAs involved in T-cell development, selection, activation, and hemostasis.J. Cell Physiol. 2020; 235: 8461-8471Google Scholar, 7Scherm M.G. Daniel C. miRNA regulation of T cells in islet autoimmunity and type 1 diabetes.Curr. Diab. Rep. 2020; 20: 41Google Scholar). The magnitude of target protein reduction is usually modest, but the accumulated effects on signaling pathways are often important for shaping cellular functions (8Guo H. Ingolia N.T. Weissman J.S. Bartel D.P. Mammalian microRNAs predominantly act to decrease target mRNA levels.Nature. 2010; 466: 835-840Google Scholar). T-cell activation has been shown to induce dynamic changes in the expression of miRs, some of which increase, whereas others decrease to shape T-cell activation and differentiation (9Teteloshvili N. Smigielska-Czepiel K. Kroesen B.J. Brouwer E. Kluiver J. Boots A.M. van den Berg A. T-cell activation induces dynamic changes in miRNA expression patterns in CD4 and CD8 T-cell subsets.Microrna. 2015; 4: 117-122Google Scholar). The miR-15/16 family of miRNAs has been extensively studied for their roles as tumor suppressors (10Pekarsky Y. Croce C.M. Role of miR-15/16 in CLL.Cell Death Differ. 2015; 22: 6-11Google Scholar), but evidence is emerging for this miR pair as regulators of T-cell activation (11Gagnon J.D. Kageyama R. Shehata H.M. Fassett M.S. Mar D.J. Wigton E.J. Johansson K. Litterman A.J. Odorizzi P. Simeonov D. Laidlaw B.J. Panduro M. Patel S. Jeker L.T. Feeney M.E. et al.miR-15/16 restrain memory T cell differentiation, cell cycle, and survival.Cell Rep. 2019; 28: 2169-2181.e4Google Scholar). The miR-15/16 family consists of two loci present in the human and mouse genome, one of which is located on chromosome 13 in humans and 14 in mice, which is comprised of a contiguous DNA sequence encoding immature mir-15a and mir-16-1 separated by a 54-nucleotide spacer. RNA transcribed from this DNA sequence generates two immature hairpin RNAs (mir-15a; mir-16-1) that are processed into four mature miRs (miR-15a-3p and miR-15a-5p; miR-16-1-3p and miR-16-1-5p). The two mature -5p miRs (referred to as miR-15a/16 hereafter) share sequence homology and simultaneously target several mRNAs (12Kozomara A. Griffiths-Jones S. miRBase: Annotating high confidence microRNAs using deep sequencing data.Nucleic Acids Res. 2014; 42: D68-D73Google Scholar). A recent study using a loss-of-function approach showed that miR-15a/16 directly targeted mRNA that were part of an extensive network of pathways that influence T-cell differentiation, survival, and memory (11Gagnon J.D. Kageyama R. Shehata H.M. Fassett M.S. Mar D.J. Wigton E.J. Johansson K. Litterman A.J. Odorizzi P. Simeonov D. Laidlaw B.J. Panduro M. Patel S. Jeker L.T. Feeney M.E. et al.miR-15/16 restrain memory T cell differentiation, cell cycle, and survival.Cell Rep. 2019; 28: 2169-2181.e4Google Scholar). Interestingly, decreased miR15a/16 levels were detected 1 to 4 days post-TCR stimulation, but it was not determined how impaired downregulation of this miR cluster may affect T-cell activation occurring during the critical early stages as these cells prepare to proliferate. In other words, if miR-15a/16 levels cannot sufficiently decrease during early T-cell activation, how would proliferation be impacted and which signaling pathways would be most affected? To address these questions, we developed a gain-of-function model: an inducible miR15a/16 transgenic mouse in which levels of these miRs in T cells are elevated compared with endogenous levels during early stages of TCR-induced activation. Higher miR-15a/16 levels during TCR activation were found to impair T-cell proliferation ex vivo and in vivo. Proteomics, bioinformatics, and Western blot analyses identified MEK1 and ERK1 as direct targets of miR15/a16, and further studies showed that the MEK1–ERK1/2–Elk1 pathway was regulated by miR15a/16 during T-cell activation. These findings reveal a new mechanism by which decreased miR-15a/16 during TCR activation promotes sustained signals important for T-cell proliferation. Because a recent report showed that miR-15a/16 levels decreased over several days after T-cell activation (11Gagnon J.D. Kageyama R. Shehata H.M. Fassett M.S. Mar D.J. Wigton E.J. Johansson K. Litterman A.J. Odorizzi P. Simeonov D. Laidlaw B.J. Panduro M. Patel S. Jeker L.T. Feeney M.E. et al.miR-15/16 restrain memory T cell differentiation, cell cycle, and survival.Cell Rep. 2019; 28: 2169-2181.e4Google Scholar), we set out to determine how interfering with this decrease would affect T-cell proliferation. To induce miR-15a/16 levels higher than endogenous levels, we generated a transgenic mouse model with miR-15a/16 expression under the control of doxycycline (DOX) treatment. These transgenic mice were injected once with DOX or PBS as a control, and after 24 h, splenic CD3+ T cells were purified and either frozen at −20 °C (nonactivated) or TCR activated for 18 h in media containing DOX or PBS followed by freezing at −20 °C (Fig. 1A). Real-time PCR was used to measure relative levels of miR-15a and miR-16, and results showed that DOX induced ∼35% and ∼70% higher levels of both miRs in nonactivated and activated T cells, respectively (Fig. 1B). Carboxyfluorescein succinimidyl ester (CFSE) assays were conducted on ex vivo T cells to analyze the proliferative capacity in conditions of elevated miR-15a/16 compared with controls. Results showed that the division index (average number of cell divisions, including the undivided peak) and the proliferation index (total number of divisions divided by the number of cells that went into division) were both decreased in the DOX-treated group (Fig. 1C). As expected, DOX treatment did not affect the proliferative capacity of T cells (Fig. suggesting that the elevated miR-15a/16 expression induced by DOX in the transgenic T cells, and not DOX to reduced proliferation. To the effects of elevated miR-15a/16 on in vivo T-cell a transgenic mouse model was generated by the miR-15a/16 mice with transgenic mice The T cells from these transgenic mice a TCR the to in the context of with the miR-15a/16 of purified T cells from these transgenic mice that the of the into mice followed by with the to of in vivo of the antigen-specific T cells or with DOX-induced miR-15/16 expression (Fig. Results showed that DOX treatment to of the antigen-specific T cells in to (Fig. and with the ex vivo these in vivo findings that elevated miR-15/16 levels in T cells with proliferation in to We to identify signaling and pathways that on decreased miR-15a/16 levels in T cells during TCR-induced activation. the we identified mRNAs targeted by miR-15a and with a of the that were in the of and T-cell signaling pathway (Fig. In with this we proteomics to measure levels of in activated T cells from the transgenic mice with DOX to induce expression of miR-15a/16 compared with PBS the from these two approaches identified with levels by DOX treatment that also were to be directly targeted by miR-15a/16 were but MEK1 was decreased by DOX treatment with an that Interestingly, the that MEK1 was not an target of miR-15a/16, we the direct targeting of MEK1 by signaling identified as miR-15a/16 targets bioinformatics and T cells, of activated cytoplasmic of of activated cytoplasmic of in a new of the 3′-UTRs of human and mouse MEK1 mRNA a miR-15a/16 for both (Fig. Western blot of activated T cells purified from the miR-15a/16 transgenic mice showed that DOX-induced miR-15a/16 expression to decreased MEK1 (Fig. with these the decreased levels of miR-15a/16 TCR-induced T-cell activation both and increased MEK1 mRNA and protein levels (Fig. To determine if the mRNA as the target for the miR-15a/16, we two of MEK1 encoding DNA one containing the 3′-UTR target sequence and containing this sequence mutated to 3′-UTR which were into expression (Fig. cells were with these with mimics for miR-15a/16 or control of MEK1 were by Western and results showed that addition of miR-15a/16 mimics to decreased MEK1 levels for cells with MEK1 but on MEK1 levels for the mutated MEK1 (Fig. While the reduction was this is with reduction by miRs and with from 1 and of the miR mimics was confirmed by and in are in these that the of MEK1 mRNA (UGCUGCUA) functions as a miR-15a/16 target for with levels of during T-cell activation. MEK1 is a kinase that phosphorylates and activates ERK1 and by on and C.M. A. The of a protein kinase that phosphorylates the gene Scholar), we the of DOX-induced miR-15a/16 expression on during T-cell activation. Western blot using an both and showed that DOX-induced expression of miR-15a/16 reduced of both in activated T cells (Fig. A and control using T cells showed that DOX treatment (Fig. total ERK1 also decreased with DOX treatment for and T cells. bioinformatics analyses shown in ERK1 (mitogen-activated protein kinase as a target for miR-15a and The proteomics in showed that DOX-induced miR-15a/16 decreased ERK1 by with a for the but at the for DOX-induced expression of miR15a/16 on in the early stages and of TCR stimulation (Fig. which is with the in from increased MEK1 protein These results combined with the Western blot that the reduced miR-15a/16 with TCR stimulation is for levels of MEK1 and ERK1 with a on the pathway by this miR pair. We out an signaling to simultaneously analyze on signaling for signaling downstream from that may be by DOX-induced miR-15a/16 expression. We identified and that were or by with DOX treatment in activated miR-15a/16 transgenic T cells (Fig. The in this was of particular that of this on by activated promotes proliferation of cells M. S. mitogen-activated protein kinase (ERK) 1/2 and kinase pathways activation of transcription 1 in J. Cell Biol. Scholar). Western blot confirmed that DOX-induced miR-15a/16 decreased during T-cell activation (Fig. showed that DOX treatment on T cells with miR-15a/16 (Fig. was not by and miR-15a/16 target sequence was found in the mRNA suggesting that the effects on are to reduced activation by elevated miR-15a/16 The role of miR15a/16 in regulating tumor or malignancy for a of has been well E. R. Y. As tumor suppressors and 2015; Scholar, H. S. K. Role of in the of Scholar). that miR-15/16 as a tumor by directly targeting 2 and Y. Croce C.M. and gene to Death Differ. Scholar), but signaling and pathways regulated by this miR pair in activated T cells may be the recent report that miR-15a/16 levels decreased over several days after T-cell activation (11Gagnon J.D. Kageyama R. Shehata H.M. Fassett M.S. Mar D.J. Wigton E.J. Johansson K. Litterman A.J. Odorizzi P. Simeonov D. Laidlaw B.J. Panduro M. Patel S. Jeker L.T. Feeney M.E. et al.miR-15/16 restrain memory T cell differentiation, cell cycle, and survival.Cell Rep. 2019; 28: 2169-2181.e4Google Scholar), this study set out to determine how this set of miRs may target key signaling in T cells. a new transgenic mouse model in which we induce higher levels of miR-15a/16 with DOX we were to show that elevated levels of these miRs decreased the proliferative capacity of T cells. by and approaches to signaling pathways regulated during T-cell activation, we that MEK1 and ERK1 are directly targeted by miR15a/16 and the MEK1–ERK1/2–Elk1 pathway as an important pathway that is regulated by this miR in T cells that miR-15a/16 decreased after TCR activation the results of et (11Gagnon J.D. Kageyama R. Shehata H.M. Fassett M.S. Mar D.J. Wigton E.J. Johansson K. Litterman A.J. Odorizzi P. Simeonov D. Laidlaw B.J. Panduro M. Patel S. Jeker L.T. Feeney M.E. et al.miR-15/16 restrain memory T cell differentiation, cell cycle, and survival.Cell Rep. 2019; 28: 2169-2181.e4Google and their by a early decrease in these miRs at h post-TCR activation. The increased MEK1 is not 18 h post-TCR stimulation, suggesting that it several for the effects of miR levels to MEK1 protein The post-TCR stimulation is well after the of that TCR-induced pathway is for promoting optimal T-cell activation. MEK1 such as are most often 1 h to TCR stimulation in to roles for this kinase in activation N. M. M.E. receptor 1 expression in activated T cells is regulated by the kinase signaling Cell Scholar, K. M.M. T-cell receptor induces signaling pathways in T S. A. Scholar). the role of TCR-induced miR-15a/16 reduction in MEK1 protein to 18 h post-TCR activation is a sustained MEK–ERK that is important for optimal T-cell proliferation. has been to be a key signaling downstream of a role in the regulation of a of cellular functions N. Y. N. 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