Cyclin-dependent kinase activity is required for type I interferon production

Abstract

Recognition of nucleic acids results in the production of type I IFNs, which activate the JAK/STAT pathway and promote the expression of IFN-stimulated genes. In a search for modulators of this pathway, we discovered an unexpected requirement for cyclin-dependent kinases (CDK) in the production of type I IFN following nucleic acid sensing and virus infection. Inhibition of CDK activity or knockdown of CDK levels leads to a striking block in STAT activation and IFN-stimulated gene expression. CDKs are not required for the initial nucleic acid sensing leading to IFN-β mRNA induction, nor for the response to exogenous IFN-α/β, but are critical for IFN-β release into culture supernatants, suggesting a posttranscriptional role for CDKs in type I IFN production. In the absence of CDK activity, we demonstrate a translational block specific for IFN-β, in which IFN-β mRNA is removed from the actively translating polysomes, while the distribution of other cellular mRNAs or global translation rates are unaffected. Our findings reveal a critical role for CDKs in the translation of IFN-β.

Keywords: CDK inhibitors; IFN-stimulated genes; cyclin-dependent kinases; translational regulation; type I interferon.

Fig. 1.

Inhibition of CDK activity prevents nucleic acid-induced ISG induction and STAT1 activation. (A) THP-1 cells were treated with the CDK inhibitor R547 (10 nM) or DMSO, and transfected with DNA (4 µg/mL) or mock. mRNA levels for the indicated genes were quantified by qRT-PCR 4 h posttransfection, and are presented relative to GAPDH mRNA levels. Values presented are the average of at least three independent experiments, error bars represent the SEM, statistical significance was determined by unpaired Student’s t test: *P < 0.05, **P < 0.01, ***P < 0.001, n.s. not significant. (B) THP-1 and NHDF cells were challenged with DNA (4 µg/mL) or IFN-α (10 U/mL) with R547 or DMSO. Total cell lysates were collected 2 h later and analyzed by Western blot with the indicated antibodies. (C) THP-1 cells were treated with R547 or DMSO and transfected with DNA (4 µg/mL) or mock. Total cell lysates were collected at the indicated time points, and analyzed by Western blot. (D) THP-1 cells were treated with CDK inhibitors R547 (10 nM), dinaciclib (10 nM), AZD-5438 (50 nM), palbociclib (50 nM), SNS-032 (100 nM), or DMSO, and transfected with DNA. Lysates were analyzed as in B. (E) NHDF primary fibroblasts were transfected with siRNAs for CDKs 1, 2, and 4, or a nontargeting control. Knockdown levels of CDKs 1, 2, and 4 were determined at the protein level (Right). Cells were challenged with poly(I:C) (4 µg/mL), and lysates were collected 2.5 h later and analyzed by Western blot (Left) for STAT1 and IRF3 activation.

Fig. 2.

CDK inhibition does not affect IRF3 activation or IFN-induced responses. (A) THP-1 cells were transfected with a Y-form DNA (16) or 5′ triphosphate RNA (5′PPP). Total cell lysates were collected 2 h later and analyzed by Western blot with the indicated antibodies. (B) THP-1 cells were treated with increasing amounts of IFN-β in the presence of 0, 10 or 100 nM R547, as indicated. Lysates were analyzed by Western blot 30 min after treatment. (C) THP-1 cells were transfected with DNA (4 µg/mL) in the presence of R547 (10 nM) or DMSO and assayed by Western blot after 2 h for STAT1, STAT3, and IRF3 phosphorylation using the indicated phospho-specific antibodies. Results are representative of at least two independent experiments.

Fig. 3.

CDK inhibition blocks DNA-induced cytokine production. (A) THP-1 cells were treated with the pan-JAK inhibitor CYT387 (10 µM), and challenged with IFN-α (1, 5, 20 U/mL) or DNA (1, 3, 5 µg/mL). Total cell lysates were collected 2 h later and analyzed by Western blot. (B) THP-1 cells were transfected with DNA (4 µg/mL), in the presence or absence of R547 (10 nM). Supernatants were collected at the indicated time points (1–4 h) and transferred to naïve THP-1 cells. Total cell lysates from the recipient cells were collected 1 h after supernatant transfer and analyzed by Western blot. (C) TE671 cells stably expressing ISRE or NF-κB luciferase reporters were treated with R547 (10 nM) or DMSO and infected with SeV (B). Luciferase activity was measured 24 h later and normalized to total protein levels. (D) NHDF cells were treated with R547 (10 nM) or DMSO and transfected with DNA (4 µg/mL) or mock. Nuclear and cytoplasmic fractions were isolated 3 h after transfection; the purity of the fractions are demonstrated by probing for lamin A/C and tubulin, respectively. NF-κB localization was determined by probing for the p65 subunit. Values presented are the average of at least three independent experiments (A), error bars represent the, statistical significance was determined by unpaired Student’s t test: **P < 0.01, ***P < 0.001.

Fig. 4.

CDK inhibitors prevent the production of type I IFN necessary for JAK/STAT pathway activation. (A) THP-1 cells were transfected with DNA (4 µg/mL) or mock, in the presence of DMSO or R547 (10 nM). IFN-β mRNA levels were quantified 4 h after transfection by qRT-PCR (Left), and IFN-β protein levels were quantified by ELISA in culture supernatants 24 h after transfection (Right). (B) Experiment performed as in A, except THP-1 cells were infected with SeV. (C) THP-1 cells were infected with SeV in the presence of R547 or DMSO. Four hours after infection, mRNA levels for CXCL10 and ISG54 were measured by qRT-PCR; values presented are normalized to GAPDH. (D) Supernatants from DNA-transfected THP-1 cells were preincubated with antibodies against IFN-γ, IL-6, control IgG, or recombinant VACV B18R protein (lanes 3–6) for 1 h and added to recipient cells. Lysates were collected 2 h later and analyzed by Western blot. Alternatively, the recipient cells were preincubated with antibodies against IFNAR2, IFNLR, or a control IgG (lanes 7–9) for 1 h before supernatant transfer. Lysates were collected 2 h later and analyzed as before. Untreated supernatants from DNA or mock-transfected (lanes 1 and 2) cells were included as controls. Values presented are the average of at least three independent experiments, error bars represent the SEM. Statistical significance was determined by unpaired Student’s t test, compared with vehicle treated samples: *P < 0.05, **P < 0.01, ***P < 0.001, n.s. not significant.

Fig. 5.

CDK activity is required for IFN-β mRNA translation. (A) In vitro translation was performed for a control luciferase mRNA in rabbit reticulocyte lysates pretreated with DMSO, R547 (10 nM), or cycloheximide (CHX; 10 µg/mL). Reactions were assayed for luciferase activity 30 min later. (B) THP-1 cells were depleted of endogenous amino acids by incubation with Met/Cys-free media in the presence of DMSO, R547, or CHX for 2 h. ³⁵S-labeled amino acids were added to culture media, allowed to incorporate into newly synthesized proteins for 30 min, and cell lysates were analyzed by SDS/PAGE. Total protein levels were visualized by Coomassie staining (Left), ³⁵S incorporation was determined by autoradiography (Right). (C) THP-1 cells were transfected with DNA (4 µg/mL) in the presence of DMSO or R547. Cytoplasmic extracts were prepared 2 h after transfection, and polysome profiling was performed by sucrose gradient centrifugation. (D) qRT-PCR for the indicated mRNA targets were performed on each fraction. Values are represented as the ratio of mRNAs associated with polysomes over total mRNA for that message. Data are representative of at least two independent experiments.

Fig. 6.

Nucleic acid induced innate immune pathways and their inhibition by CDK inhibitor (CDK_i). Recognition of PAMPs by pattern-recognition receptors (PRRs) triggers innate immune signaling pathways that activate the transcription factors NF-κB and IRF3, which mediate type I IFN production. IFN is released from the cell, interacts with its receptor on the cell surface and activates receptor-associated JAKs. JAKs in turn phosphorylate and activate the STAT family of transcription factors, which stimulate ISG production. In the presence of CDK inhibitors, nucleic acid recognition occurs normally, IRF3 is activated and IFN-β mRNA is synthesized, but the production of IFN-β protein is blocked at the stage of translation. Lack of IFN production prevents receptor signaling, JAK/STAT pathway activation and ISG mRNA induction.