TGF-β Decreases the Stability of IL-18-Induced IFN-γ mRNA through the Expression of TGF-β-Induced Tristetraprolin in KG-1 Cells

Abstract

We have previously reported that transforming growth factor-β (TGF-β) down-regulates interferon-γ (IFN-γ) production in an interleukin-18 (IL-18) treated mouse natural killer (NK) cell line, LNK5E6. In LNK5E6 cells, TGF-β exhibited no inhibition of the IL-18-induced transcription of IFN-γ, but did stimulate the degradation of IFN-γ mRNA induced by IL-18. In the present study, we investigated the mechanism of the down-regulatory effects of TGF-β on IFN-γ mRNA expression in a human myelomonocytic cell line, KG-1, which produces IFN-γ in response to IL-18 alone. Interestingly, IL-18 induced the production of the IFN-γ through the stabilization of IFN-γ mRNA, but not the enhanced transcription of IFN-γ gene. The stability of IFN-γ mRNA was regulated by mRNA destabilizing elements in the 3′untranslated region (UTR) of IFN-γ mRNA, especially adenylate-uridylate (AU)-rich elements (AREs) in the 5′ half of 3′UTR. Tristetraprolin (TTP), one of the ARE-binding proteins, destabilizes IFN-γ mRNA, and IL-18 repressed the expression of TTP mRNA. Moreover, TGF-β repressed the IL-18-induced expression of IFN-γ mRNA through the induction of TTP mRNA to destabilize IFN-γ mRNA. Our data is the first to reveal that the crosstalk between IL-18 and TGF-β through the expression of TTP regulates the production of IFN-γ.

Transforming growth factor-β (TGF-β) family members are critical regulators for tissue development and homeostasis in metazoan organisms, including proliferation, differentiation, migration, and apoptosis.¹⁾ They also have unique and potent immunoregulatory properties.²⁾ In an immune system, TGF-β is produced by lymphocytes, macrophages, and dendritic cells, and its expression serves in both autocrine and paracrine modes to control the proliferation, differentiation, and the state of activation of these immune cells. Excessive production and activation of latent TGF-β has been linked to immuno-defects associated with malignancy and autoimmune disorders, to susceptibility to opportunistic infection, and to the fibrotic complications associated with chronic inflammatory conditions. In contrast, deficiency of TGF-β₁ resulted in severe pathology, leading to death associated with dysfunction of the immune and inflammatory systems.³⁾

Interleukin-18 (IL-18) was originally identified as a factor promoting interferon-γ (IFN-γ) production and it was originally called IFN-γ inducing factor.⁴⁾ IL-18 had a structure related to IL-1, and IL-18 receptor resembles that for IL-1.⁵⁾ IL-18, like IL-1 and agents interacting with Toll-like receptors, signals via MyD88 which activates tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6) and ultimately nuclear factor-κB (NF-κB).⁶⁾ IL-18 is secreted mainly from macrophages, and primarily acts in concert with IL-12, IL-2, microbial agents, or mitogens to produce IFN-γ, particularly in NK cells, T cells, and macrophages.^7–9) It enhances the cytotoxic activity of natural killer (NK) and T cells by mechanisms possibly involving the perforin-dependent and death receptor-dependent pathways. IL-18-deficient mice exhibit diminished NK cell activity and markedly reduced IFN-γ production in response to bacterial and lipopolysaccharide (LPS) challenges.¹⁰⁾

An issue of great importance is how the immunoregulatory effects of TGF-β are mediated. Although there is ample documentation of inhibitory effects of TGF-β, an understanding of the mechanism by which effects on immune cell function occur is not clearly understood. We have previously reported that TGF-β down-regulates IFN-γ production in IL-18-treated mouse NK cell line LNK5E6.¹¹⁾ In LNK5E6 cells, TGF-β exhibited no inhibition of the IL-18-induced transcription of IFN-γ, but accelerated the degradation of IFN-γ transcript. In addition, TGF-β has been shown to inhibit the expression of a number of genes in cultured cells as consequence of reducing mRNA stability, including inducible nitrite oxide synthase (iNOS),¹²⁾ and CXC chemokine CXCL1 (KC).¹³⁾ However, the mechanisms of regulation of mRNA stability by TGF-β are not so clear.

In this report, we investigated the mechanism of the suppressive effect of TGF-β on IFN-γ mRNA expression in a human myelomonocytic cell line KG-1, which produced IFN-γ in response to IL-18 alone. Interestingly, IL-18 induced the production of IFN-γ through the stabilization of its mRNA. The stability of IFN-γ mRNA was regulated by mRNA destabilizing elements in the 3′untranslated regions (UTRs) of its mRNA, especially adenylate-uridylate (AU)-rich elements (AREs) in the 5′ half of 3′UTR. Tristetraprolin (TTP, also known as ZFP36 or Tis11), the best-known member of ARE-binding proteins, destabilizes the IFN-γ mRNA, and IL-18 repressed the expression of TTP mRNA. Moreover, TGF-β up-regulated the expression of TTP mRNA, and repressed the expression of IL-18-induced IFN-γ mRNA through its destabilization. Our data first reveals the crosstalk between IL-18 and TGF-β through expression of TTP regulates the production of IFN-γ.

MATERIALS AND METHODS

Reagents

RPMI 1640, fetal bovine serum (FBS), phorbol 12-myristate 13-acetate (PMA) and SB203580 were purchased from Sigma (St. Louis, MO, U.S.A.); Human recombinant TGF-β₁ was purchased from R&D Systems (Minneapolis, MN, U.S.A.). Human recombinant IL-18 was purchased from Medical & Biological Laboratories Co. (Nagoya, Japan).

Plasmids

pIFN-γ (−2.7 kb)-Luc¹⁴⁾ and pGL3-4κB-Luc¹⁵⁾ were kindly provided from Dr. Howard A. Young and Dr. Takashi Okamoto, respectively. pRL-TK and pcDNA3/TRAF6 were previously described.^15,16) pEGFP-C1 was purchased from Clontech (Mountain View, CA, U.S.A.). pIFN-γ (−791–+76)-Luc was prepared by polymerase chain reaction (PCR) amplification. Luciferase reporter plasmids were constructed using the pGL3-control vector (Promega, Madison, WI, U.S.A.) carrying a SV40 promoter/luciferase expression unit. Three constructs, pGL3/IFN-γ (+577–+1181), pGL3/IFN-γ (+577–+786) and pGL3/IFN-γ (+787–+1181), containing the 3′UTR sequences of mouse IFN-γ cDNA were amplified by PCR and cloned into an XbaI site (+1934) located between the luciferase gene and the poly(A) signal in the pGL3-control vector. To generate human TTP expression vectors, fragments of the corresponding cDNAs were amplified by reverse transcription (RT)-PCR using mRNA derived from KG-1 cells. The amplified fragments were subcloned into pCMV5/Flag.¹⁶⁾ The fragments amplified by PCR and the presence of missense or deleted mutations were confirmed by sequencing.

Cell Lines and Transfections and Reporter Assay

KG-1, Jurkat and EL-4 cells were cultured in RPMI 1640 supplemented with 10% heat-inactivated FBS, 100 U/mL of penicillin G, 100 µg/mL of streptomycin. KG-1 and Jurkat cells were electroporated using a Bio-Rad Gene Pulser (BioRad, Hercules, CA, U.S.A.). Cells (1×10⁷) suspended in RPMI1640 were transfected by electroporation (246 V, 975 µF) with 20 µg of DNA. EL-4 cells were transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, U.S.A.) following the manufacturer’s instructions. Luciferase activity was measured and standardized as previously described.¹⁶⁾

RNA Extraction and Northern Blot Analysis and RT-PCR

RNA extraction and Northern blotting analysis have been described previously.^11,16) The following probes were used for hybridization: green fluorescent protein (GFP) gene (758 bp), NheI and BglII-digested fragment of pEGFP-C1, and TTP gene (980 bp), SalI and SmaI-digested fragment of pCMV5/Flag-TTP. For detection of human IFN-γ mRNA, a 550-bp fragment of the cDNA was amplified by PCR with primers: sense 5′-CTC GGA AAC GAT GAA ATA TAC-3′; antisense 5′-TTA CTG GGA TGC TCT TCG-3′, and used as template for a [³²P]dCTP labeled. The probes for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA have been described previously.¹⁶⁾

RT-PCR analysis was performed as described previously.¹⁷⁾ PCR programs and primers used for PCR were as follows: a 94°C, 9 min initial denaturation step, 35 cycles (for IFN-γ, 5′-CTC GGA AAC GAT GAA ATA TAC-3′ and 5′-TTA CTG GGA TGC TCT TCG-3′), 35–36 cycles (for TTP, 5′-TCA TCC ACA ACC CTA GCG AA-3′ and 5′-GAT GCG ATT GAA GAT GGG GA-3′), 33 cycles (for HuR, 5′-GGT TAT GAA GAC CAC ATG GCC G-3′ and 5′-CTG GCG AGT GGT ACA GCT GCG-3′), 26 cycles (for GAPDH, 5′-TGA AGG TCG GAG TCA ACG GAT TTG GT-3′ and 5′-CAT GTG GGC CAT GAG GTC CAC CAC-3′) at 94°C for 1 min, 52–60°C for 1 min and 72°C for 1 min on a 2720 Thermal Cycler (Applied Biosystems, Foster City, CA, U.S.A.). PCR amplification was performed in the linear range and PCR products were solved by 1.5–2% agarose gel electrophoresis.¹⁸⁾

RESULTS

IL-18 Induced IFN-γ mRNA without Upregulation of the IFN-γ Promoter Activity

A human myelomonocytic cell line KG-1, produces IFN-γ in response to IL-18 even in the absence of any co-stimulatory signals.¹⁹⁾ As shown in Fig. 1A, IL-18 induced IFN-γ mRNA time-dependently in KG-1 cells. IFN-γ mRNA expression was induced at 2 h and peaked at 6 h after IL-18 stimulation. To analyze the signaling pathway involved in the IFN-γ mRNA up-regulation, we treated KG-1 cells with SB203580 (a p38 Mitogen-activated protein kinase (MAPK) inhibitor), PD98059 (a MEK1/2 inhibitor), SP600125 (a c-Jun N-terminal kinase (JNK) inhibitor), LY294002 or wortmannin (PI3K inhibitors) and then examined IFN-γ mRNA up-regulation in KG-1 cells in response to IL-18 (Fig. 1B and data not shown). SB203580 completely suppressed the IFN-γ mRNA expression by IL-18 (lane 3). In contrast, other inhibitors hardly affect the IFN-γ mRNA expression by IL-18 (data not shown). These results suggest that p38 MAPK pathway may play the important role in the IFN-γ mRNA up-regulation by IL-18.

Fig. 1. IL-18 Induced IFN-γ mRNA without Up-Regulation of the IFN-γ Promoter Activity

(A) KG-1 cells were stimulated with 50 ng/mL of IL-18 for the indicated periods. Total RNA was extracted and the expression levels of IFN-γ and GAPDH mRNA were determined by RT-PCR. (B) KG-1 cells were pretreated with 10 µM SB203580 for 1 h, and then cells were stimulated with 50 ng/mL of IL-18 for 6 h. RT-PCR analyses of the IFN-γ and GAPDH mRNA levels in KG-1 cells as in (A). (C) Schematic diagram of IFN-γ gene promoter. (D, E) KG-1 cells were transiently transfected with the indicated IFN-γ promoter containing reporter plasmids. After 36 h, the transfected cells were stimulated with 50 ng/mL of IL-18 for 6 h and examined for luciferase activities. All firefly luciferase activities were normalized with Renilla luciferase activities. The experiment was performed in triplicate, and the data are represented as the mean fold activations±S.D. (F, G) KG-1 cells were transiently transfected with the IFN-γ promoter reporter (−791–+76) or 4κB-Luc with or without TRAF6 expression plasmid. After 36 h, the luciferase activity was measured as in (D, E).

The previous reports demonstrated that IL-18 induces the recruitment of NF-κB to the region −786 to −776 site of the IFN-γ promoter in KG-1 cells,¹⁹⁾ and showed that IL-18 activated directly the AP-1 site (−190) of the IFN-γ promoter in primary CD4⁺ T cells²⁰⁾ (Fig. 1C). We next evaluated whether IL-18 activates the IFN-γ promoter (−2.7 kb and −791–+76) using luciferase reporter assays (Figs. 1D, E). Surprisingly, IL-18 could not up-regulate each promoter activity. Although TRAF6-induced NF-κB activation was observed in KG-1 cells (Fig. 1G), overexpression of TRAF6 did not enhance the IFN-γ promoter activity (−791–+76) (Fig. 1F). These results suggest that the increase in IFN-γ mRNA level in response to IL-18 is not regulated at the transcriptional level in KG-1 cells.

Identification of RNA Destabilizing Activities in the 3′UTR of IFN-γ mRNA

Gene expression may be regulated, at least in part, at post-transcriptional level by factors promoting rapid degradation of mRNA.²¹⁾ Although the role of micro RNAs (miRNAs) in regulating mRNA translation and stability is well established, RNA-binding proteins (RBPs) also play significant roles in posttranscriptional regulation. Recruitment of RBPs to the transcripts modulates diverse processes such as splicing, polyadenylation, mRNA translation, stability and subcellular localization. For example, AUF1 and TTP are well known to up-regulate the ARE-dependent mRNA turnover.^22,23) In contrast, ELAV/Hu family proteins stabilize and/or activate translation of target mRNAs.²⁴⁾ Potential mRNA destabilizing motifs AUUUA and related sequences (AUUUUA, AUUUUUA) are located in the 3′UTR of human and mouse IFN-γ mRNA. The positions of these sequences are indicated in Table 1. There are eight typical AREs in the 3′UTR of mouse IFN-γ mRNA, and seven of eight AREs are located in the 5′ half of 3′UTR. To investigate whether these sequences regulate stability of IFN-γ mRNA, 3′UTR of IFN-γ mRNA or its 5′ half or 3′ half were placed downstream of the luciferase coding region of pGL3-control vector carrying an SV40 promoter/luciferase expression unit.²⁵⁾ Due to the low transfection efficiency in KG-1 cells, we used a murine T-lymphoma cell line EL-4, which is easily transduced with plasmid DNA by lipofection. Luciferase activities in transfected EL-4 cells using these plasmids were compared with that using an original pGL3-control vector. If the inserted fragments from the IFN-γ 3′UTR contain RNA-destabilizing activity, the luciferase activity generated will be less than that of the control plasmid. In non-stimulated EL-4 cells, reporter activity fell by ca. 80% when whole 3′UTR inserted version of reporter gene was used (Fig. 2; +577–+1181). We also observed a reduction of reporter activity when the 5′ half of 3′UTR (+577–+786), as well as whole 3′UTR, were inserted, but did not when the 3′ half of 3′UTR (+787–+1181) was inserted. Unfortunately, EL-4 produced less IFN-γ in response to IL-18 (our unpublished observation). Therefore, we treated cells with PMA, which was demonstrated to stabilize the ARE-containing IL-10 mRNA in EL-4 cells.²⁵⁾ PMA was also reported to stabilize other ARE carrying mRNAs, such as TNF-α and IL-2.^26,27) In PMA-stimulated EL-4 cells, reduced reporter activities using whole 3′UTR and the 5′ half of 3′UTR inserted plasmids were largely restored, however, a reporter activity of the 3′ half of 3′UTR inserted version was almost unchanged. These results suggest that the stability of IFN-γ mRNA is regulated by mRNA-destabilizing elements in the 3′UTR of IFN-γ mRNA, especially ARE in the 5′ half of 3′UTR.

Table 1. The Homology of AU Motif between Human and Mouse IFN-γ mRNA 3′UTR

The 3′UTR sequences of human and mouse IFN-γ mRNA are shown. The AU-motifs in 3′UTR are shown in bold letters, and the polyA signal is underlined.

Fig. 2. Identification of RNA Destabilizing Activities in the 3′UTR of IFN-γ mRNA

EL-4 cells were transiently transfected with the indicated reporter plasmids and pRL-TK. After 24 h, cells were stimulated with 50 nM PMA for 12 h or left untreated. The luciferase activity in cell lysates was measured. All firefly luciferase activities were normalized with Renilla luciferase activities.

TTP Is a Crucial Factor for the IFN-γ mRNA Stability

Our data suggest that IL-18 up-regulates the expression of IFN-γ mRNA through the stabilization of IFN-γ mRNA but not the enhanced transcription of IFN-γ gene. Therefore, IL-18 can be involved in the expression of ARE-binding proteins that either stabilize or destabilize mRNA. Tristetraprolin (TTP) is the prototype of a family of zinc finger proteins carrying a pair of closely spaced CCCH class zinc fingers.²⁸⁾ It has been reported that TTP is able to promote the turnover of certain mRNAs containing AREs in their 3′UTR.²⁹⁾ The mRNA of several cytokines (TNF-α, granulocyte macrophage colony stimulating factor (GM-CSF), and IL-3) essential for immune response and inflammation contain this class of AREs (class II AREs) in their 3′UTR.^29,30) However, the stability of IFN-γ mRNA has not well investigated in detail. Therefore, we investigated whether IL-18 changes the expression level of ARE-binding proteins, TTP and HuR, in KG-1 cells. As shown in Fig. 3A, IL-18 did not affect the expression level of HuR mRNA, but immediately down-regulated the TTP mRNA level. Moreover, upon IL-18 treatment, the expression level of IFN-γ mRNA is time dependently up-regulated in reverse proportion to the TTP mRNA reduction.

Fig. 3. TTP Is a Crucial Factor for the IFN-γ mRNA Instability

(A) KG-1 cells were treated with 50 ng/mL of IL-18 for the indicated periods, and then total RNA was prepared. The expression levels of TTP, HuR, IFN-γ, and GAPDH mRNA were determined by RT-PCR. (B, C) KG-1 cells (B) or EL-4 cells (C) were transiently transfected with the indicated reporter plasmids and pRL-TK in the presence or absence of TTP expression plasmid. After 36 h, the luciferase activity in cell lysates was measured. All firefly luciferase activities were normalized with Renilla luciferase activities. (D) Jurkat cells were transiently transfected with the indicated reporter plasmids and pEGFP-C1 with or without wild-type or dominant negative TTP expression plasmids. After 40 h, total RNA was prepared and analyzed by Northern blotting using luciferase and GFP cDNA as probes.

Next, we generated a TTP expression plasmid to investigate whether TTP actually affects the expression of IFN-γ mRNA. As shown in Figs. 3B and C, ectopic expression of TTP in KG-1 cells and EL-4 cells results in the suppressing the reporter activity of whole IFN-γ 3′UTR inserted reporter plasmid (pGL3 IFN-γ 3′UTR (+577–+1181)). We ectopically expressed wild-type TTP (TTP wt) or its dominant negative mutant (TTP C124R)²⁸⁾ with pGL3 IFN-γ 3′UTR (+577–+1181) in Jurkat cells to determine the effect of TTP on the decay of the reporter transcripts by Northern blot analysis (Fig. 3D). Jurkat cells were efficiently transfected by electroporation than KG-1 cells. Insertion of IFN-γ 3′UTR just after a luciferase gene results in the remarkable reduction of the luciferase mRNA expression (Fig. 3D, lanes 1, 2). Co-transfection of wild-type TTP resulted in a further reduction of the luciferase mRNA expression, while co-transfection of TTP C124R largely restored its expression. These results suggest that the decay of IFN-γ mRNA is promoted by TTP, and IL-18 stabilizes the IFN-γ mRNA by repressing TTP mRNA expression.

TGF-β Represses the IL-18-Induced the Expression of IFN-γ mRNA in KG-1 Cells

We have previously reported that TGF-β down-regulates IFN-γ production in the IL-18 treated mouse NK cell line LNK5E6.¹¹⁾ We therefore examined the effect of TGF-β on the IL-18-induced IFN-γ mRNA expression in KG-1 cells. KG-1 cells were pretreated with TGF-β for 1 h and stimulated with IL-18, and then IFN-γ mRNA levels were measured by RT-PCR. As shown in Fig. 4A, TGF-β clearly suppressed the IFN-γ mRNA expression induced by IL-18 in KG-1 cells as well as in LNK5E6 cells.

Fig. 4. Requirement of TTP on the Suppression of IFN-γ mRNA Expression by TGF-β

(A) KG-1 cells were pretreated with or without 100 pM TGF-β for 1 h and stimulated with 50 ng/mL of IL-18 for 6 h. Total RNA was extracted and the expression levels of IFN-γ and GAPDH mRNA were determined by RT-PCR. (B, C) KG-1 cells were treated with 100 pM TGF-β for the indicated periods (B), or treated with the indicated concentration of TGF-β for 1 h (C). Total RNA was extracted and the expression levels of TTP and GAPDH mRNA were examined by RT-PCR. (D, E) KG-1 cells were pretreated with or without 100 pM TGF-β (D) or 10 µM SB203580 (E) for 1 h and stimulated with 50 ng/mL of IL-18 for 6 h. Total RNA was extracted and the expression levels of IFN-γ and GAPDH mRNA were determined by RT-PCR. (F) KG-1 cells were incubated with or without 50 ng/mL of IL-18. After 5 h, cells were treated with 100 pM TGF-β for 1 h or left untreated. PolyA⁺ RNA was prepared and then analyzed by Northern blotting using human IFN-γ and GAPDH cDNA as probes (left). The autoradiogram was measured by densitometry and relative level of IFN-γ was calculated as the ratio between IFN-γ and GAPDH mRNA in the same experimental sample (right). (G) A putative model for the IFN-γ production regulated by the TTP expression level. IL-18 induces IFN-γ mRNA expression by repressing TTP mRNA expression (black T-bar). Alternatively, IL-18 induces p38-mediated phosphorylation and inactivation of TTP (gray arrow and T-bar), results in stabilizing IFN-γ mRNA. On the other hand, TGF-β represses IFN-γ mRNA expression by inducing TTP mRNA expression (black arrow).

Involvement of TTP on the Suppression of IFN-γ mRNA Expression by TGF-β

It was reported that TTP was induced by TGF-β in a human T cell line HuT78.³¹⁾ Therefore, we investigated whether TGF-β also induced the expression of TTP in KG-1 cells. As shown in Fig. 4B, the expression of TTP mRNA was induced and peaked at 1 h after TGF-β stimulation, and gradually declined. We also observed that the expression of TTP mRNA was elevated in response to TGF-β in a dose dependent manner (Fig. 4C). In addition, TGF-β antagonized the down-regulation of TTP mRNA by IL-18, but SB203580 had little effect (Figs. 4D, E).

To investigate whether TTP involved in the suppression of IFN-γ mRNA expression induced by TGF-β, KG-1 cells were stimulated with IL-18 for 5 h, and incubated with or without TGF-β for another 1 h. And then polyA⁺ RNA was isolated, and the expression of IFN-γ mRNA was examined by Northern blot analysis (Fig. 4F). Although IL-18 induced the expression of IFN-γ mRNA, treatment of TGF-β for only 1 h prior to harvest reduced the elevation of IFN-γ mRNA level induced by IL-18 to 50%. These results suggest that TTP is involved in inducing IFN-γ mRNA in response to IL-18 and also in preventing its induction by TGF-β³²⁾ (Fig. 4G).

DISCUSSION

In this study, we demonstrated that TGF-β inhibited the up-regulation of IFN-γ mRNA expression in response to IL-18 in a human myelomonocytic cell line KG-1. We have previously demonstrated that in a mouse NK cell line LNK5E6, TGF-β exhibited no inhibition of the IL-18-induced IFN-γ transcription, but that stimulated the degradation of IFN-γ mRNA induced by IL-18.¹¹⁾ We have now observed a similar phenotype in KG-1 cells, and revealed an involvement of TTP induced by TGF-β in the destabilization of IFN-γ mRNA (Fig. 4G).

Mechanisms of how TTP destabilizes the target mRNAs are becoming clarified. TTP directly interacts with the class II ARE, which are destabilizing elements located at the 3′UTR responsible for rapid degradation of transcripts, followed by stimulating deadenylation and subsequent further destruction of the mRNA.^23,28) A single Cys-to-Arg mutation in the zinc-finger domain of TTP (TTP C124R) prevented binding of the mutant protein to the TNFα AREs and lost its promoting activity for deadenylation.^28,33) p38 MAPK pathway has been reported to be responsible for the TTP inactivation.³⁴⁾ Phosphorylation of TTP was not caused by p38 MAPK itself but did by p38 activated MAPKAPK2, and created a substrate for the phospho-serine/threonine binding protein 14-3-3 to localize TTP to the cytoplasm.^35,36) Recently it was shown that the phosphorylated TTP associated with 14-3-3 protein resulted in losing the ability of TTP to trigger the deadenylation of tethered mRNA by preventing the recruitment of cytoplasmic deadenylases.^37,38) In our study, pre-treatment of SB203580, a p38 MAPK inhibitor, inhibited the induction of IFN-γ mRNA by IL-18 (Fig. 1B), indicating that p38 MAPK pathway is necessary to stabilize IFN-γ mRNA. SB203580 probably suppressed the IL-18-induced TTP phosphorylation and inactivation, resulted in destabilizing IFN-γ mRNA by active TTP (Fig. 4G). Our findings is consistent with the report that the synergistic induction of IFN-γ protein by IL-12 and IL-18 in human NK cells involves the stabilization of IFN-γ mRNA by p38 pathway.³⁹⁾ Our study also first revealed that the suppression of TTP expression by IL-18 in KG-1 cells (Fig. 3A). The activity of TTP is regulated by its expression level and by its phosphorylation.

TTP-deficient mice manifested inflammatory disorders characterized by cachexia, arthritis and auto immunities.⁴⁰⁾ The mRNA of several cytokines responsible for the immune response, inflammation and hematopoiesis, such as TNF-α, IL-1β and GM-CSF, contain ARE motifs in their 3′UTR, and TTP is able to promote their turnover. One of the proinflammatory cytokines, IFN-γ has also ARE motifs in the 3′UTR of its mRNA (Table 1), and TTP has been shown to play an important role in promoting the destabilization of IFN-γ mRNA in our study. TGF-β-deficient mice also exhibit a wasting syndrome accompanied by multifocal, mixed inflammatory cell response and tissue necrosis.^3,41) In addition, previous reports showed a role for TGF-β in inhibiting macrophage activation, as evidenced by the suppression of a number of activation markers including iNOS, TNF-α, and IL-1β.^42,43) As shown in Table 2, multiple copies of ARE motifs have been observed frequently in downregulated genes by TGF-β.^{2,11–13,43–45)} Among of them, it has been reported that mRNAs of TNF-α, GM-CSF, CXCL1, IL-1β and IL-2 are targeted for degradation by TTP.⁴⁶⁾ Very recently, TTP was also reported to regulate the expression of IFN-γ mRNA.⁴⁷⁾ In the present study, we showed that TGF-β induces the expression of TTP mRNA, which is consistent with previous reports.³¹⁾ Because the TGF-β-deficient mice exhibit a quite similar phenotype with TTP-deficient mice, it is intriguing to speculate that the anti-inflammatory responses of TGF-β may be, at least partly, caused by TGF-β induced TTP. Collectively, TGF-β may act to suppress a number of immunostimulatory genes not only by inhibiting transcription but also by decreasing mRNA stability through TTP induction.

Table 2. Genes Post-transcriptionally Regulated by TGF-β

mRNA	Number of ARE motifs	TGF-β-mediated gene repression	Reference
IFN-γ	7 (human), 8 (mouse)	yes	11,43)
iNOS	6 (human), 5 (mouse)	yes	12)
CXCL1 (KC)	8 (human), 8 (mouse)	yes	13)
IL-1β	6 (human), 4 (mouse)	yes	2)
TNF-α	9 (human), 8 (mouse)	yes	2,43)
GM-CSF	10 (human), 10 (mouse)	yes	44)
IL-2	8 (human), 8 (mouse)	yes	45)

It has been reported that IL-4-STAT6 signaling induced TTP to inhibit TNF-α production in mast cells.⁴⁸⁾ Kishore et al. demonstrated that, in mouse macrophages, IL-10-mediated inhibition of inflammatory gene expression can be mediated by an ARE cluster present in the 3′UTR of sensitive genes.⁴⁹⁾ TGF-β also induces TTP expression through Smad3/4 activation in a human T cell line HuT78.³¹⁾ Consequently, immunosuppressive cytokines may have common characteristics to induce TTP or other ARE binding proteins to destabilize the mRNA of immunostimulatory molecules.

Evaluating the collective evidences, our data first reveal that the crosstalk between IL-18 and TGF-β through regulating the TTP expression to control IFN-γ production (Fig. 4G). Further study for the crosstalk between pro-inflammatory cytokine IL-18 and immunosuppressive cytokine TGF-β is essential in preventing immune dysregulation and diseases.

Acknowledgments

This work was supported by a Grant-in-Aid for Scientific Research (C) (No. 24590085) from Japan Society for the Promotion of Science (JSPS), a Grant-in-Aid for Young Scientists (B) (No. 24700983) from JSPS and a Grant-in-Aid for Research in Nagoya City University. YI was supported by the Kowa Life Science Foundation, the Research Foundation for Pharmaceutical Sciences, the Japan Rheumatism Foundation, the Mochida Memorial Foundation for Medical and Pharmaceutical Research, the Nakatomi Foundation, the Suzuken Memorial Foundation, the Hori Sciences & Arts Foundation and the Takeda Science Foundation.

Conflict of Interest

The authors declare no conflict of interest.

REFERENCES

• 1) Massagué J, Blain SW, Lo RS. TGFβ signaling in growth control, cancer, and heritable disorders. Cell, 103, 295–309 (2000).
• 2) Letterio JJ, Roberts AB. Regulation of immune responses by TGF-β. Annu. Rev. Immunol., 16, 137–161 (1998).
• 3) Shull MM, Ormsby I, Kier AB, Pawlowski S, Diebold RJ, Yin M, Allen R, Sidman C, Proetzel G, Calvin D, Annunziata N, Doetschman T. Targeted disruption of the mouse transforming growth factor-beta 1 gene results in multifocal inflammatory disease. Nature, 359, 693–699 (1992).
• 4) Okamura H, Tsutsui H, Komatsu T, Yutsudo M, Hakura A, Tanimoto T, Torigoe K, Okura T, Nukada Y, Hattori K, Akita K, Namba M, Tanabe F, Konishi K, Fukuda S, Kurimoto M. Cloning of a new cytokine that induces IFN-γ production by T cells. Nature, 378, 88–91 (1995).
• 5) Garlanda C, Dinarello CA, Mantovani A. The interleukin-1 family: back to the future. Immunity, 39, 1003–1018 (2013).
• 6) Adachi O, Kawai T, Takeda K, Matsumoto M, Tsutsui H, Sakagami M, Nakanishi K, Akira S. Targeted disruption of the MyD88 gene results in loss of IL-1- and IL-18-mediated function. Immunity, 9, 143–150 (1998).
• 7) Stoll S, Jonuleit H, Schmitt E, Muller G, Yamauchi H, Kurimoto M, Knop J, Enk AH. Production of functional IL-18 by different subtypes of murine and human dendritic cells (DC): DC-derived IL-18 enhances IL-12-dependent Th1 development. Eur. J. Immunol., 28, 3231–3239 (1998).
• 8) Micallef MJ, Ohtsuki T, Kohno K, Tanabe F, Ushio S, Namba M, Tanimoto T, Torigoe K, Fujii M, Ikeda M, Fukuda S, Kurimoto M. Interferon-gamma-inducing factor enhances T helper 1 cytokine production by stimulated human T cells: synergism with interleukin-12 for interferon-gamma production. Eur. J. Immunol., 26, 1647–1651 (1996).
• 9) Hunter CA, Timans J, Pisacane P, Menon S, Cai G, Walker W, Aste-Amezaga M, Chizzonite R, Bazan JF, Kastelein RA. Comparison of the effects of interleukin-1 alpha, interleukin-1 beta and interferon-gamma-inducing factor on the production of interferon-gamma by natural killer. Eur. J. Immunol., 27, 2787–2792 (1997).
• 10) Tsutsui H, Nakanishi K, Matsui K, Higashino K, Okamura H, Miyazawa Y, Kaneda K. IFN-gamma-inducing factor up-regulates Fas ligand-mediated cytotoxic activity of murine natural killer cell clones. J. Immunol., 157, 3967–3973 (1996).
• 11) Hayashi H, Inoue Y, Tsutsui H, Okamura H, Nakanishi K, Onozaki K. TGFβ down-regulates IFN-γ production in IL-18 treated NK cell line LNK5E6. Biochem. Biophys. Res. Commun., 300, 980–985 (2003).
• 12) Vodovotz Y, Bogdan C, Paik J, Xie QW, Nathan C. Mechanisms of suppression of macrophage nitric oxide release by transforming growth factor β. J. Exp. Med., 178, 605–613 (1993).
• 13) Dai Y, Datta S, Novotny M, Hamilton TA. TGFβ inhibits LPS-induced chemokine mRNA stabilization. Blood, 102, 1178–1185 (2003).
• 14) Gonsky R, Deem RL, Young HA, Targan SR. D2 mediates activation of the IFN-γ intronic STAT binding region in mucosal T cells. Eur. J. Immunol., 33, 1152–1162 (2003).
• 15) Matsumura T, Degawa T, Takii T, Hayashi H, Okamoto T, Inoue JI, Onozaki K. TRAF6-NF-κB pathway is essential for interleukin-1-induced TLR2 expression and its functional response to TLR2 ligand in murine hepatocytes. Immunology, 109, 127–136 (2003).
• 16) Inoue Y, Kitagawa M, Onozaki K, Hayashi H. Contribution of the constitutive and inducible degradation of Smad3 by the ubiquitin-proteasome pathway to transforming growth factor-beta signaling. J. Interferon Cytokine Res., 24, 43–54 (2004).
• 17) Matsumura T, Ito A, Takii T, Hayashi H, Onozaki K. Endotoxin and cytokine regulation of toll-like receptot (TLR) 2 and TLR4 gene expression in murine liver and hepatocytes. J. Interferon Cytokine Res., 20, 915–921 (2000).
• 18) Inoue Y, Itoh Y, Abe K, Okamoto T, Daitoku A, Fukamizu A, Onozaki K, Hayashi H. Smad3 is acetylated by p300/CBP to regulate its transactivation activity. Oncogene, 26, 500–508 (2007).
• 19) Kojima H, Aizawa Y, Yanai Y, Nagaoka K, Takeuchi M, Ohta T, Ikegami H, Ikeda M, Kurimoto M. An essential role for NF-kappa B in IL-18-induced IFN-gamma expression in KG-1 cells. J. Immunol., 162, 5063–5069 (1999).
• 20) Barbulescu K, Becker C, Schlaak JF, Schmitt E, Meyer Zum Büschenfelde KH, Neurath MF. IL-12 and IL-18 differentially regulate the transcriptional activity of the human IFN-gamma promoter in primary CD4⁺ T lymphocytes. J. Immunol., 160, 3642–3647 (1998).
• 21) Bevilacqua A, Ceriani MC, Capaccioli S, Nicolin A. Post-transcriptional regulation of gene expression by degradation of messenger RNAs. J. Cell. Physiol., 195, 356–372 (2003).
• 22) Zhang W, Wagner BJ, Ehrenman K, Schaefer AW, DeMaria CT, Crater D, DeHaven K, Long L, Brewer G. Purification, characterization, and cDNA cloning of an AU-rich element RNA-binding protein, AUF1. Mol. Cell. Biol., 13, 7652–7665 (1993).
• 23) Carballo E, Lai WS, Blackshear PJ. Feedback inhibition of macrophage tumor necrosis factor-alpha production by tristetraprolin. Science, 281, 1001–1005 (1998).
• 24) Fan XC, Steitz JA. Overexpression of HuR, a nuclear-cytoplasmic shuttling protein, increases the in vivo stability of ARE-containing mRNAs. EMBO J., 17, 3448–3460 (1998).
• 25) Powell MJ, Thompson SA, Tone Y, Waldmann H, Tone M. Posttranscriptional regulation of IL-10 gene expression through sequences in the 3′-untranslated region. J. Immunol., 165, 292–296 (2000).
• 26) Stoecklin G, Lu M, Rattenbacher B, Moroni S. A constitutive decay elements promotes tumor necrosis factor alpha mRNA degradation via an AU-rich element-independent pathway. Mol. Cell. Biol., 23, 3506–3515 (2003).
• 27) Shim J, Lim H, Yates JR III, Karin M. Nuclear export of NF90 is required for interleukin-2 mRNA stabilization. Mol. Cell, 10, 1331–1344 (2002).
• 28) Lai WS, Carballo E, Strum JR, Kennington EA, Phillips RS, Blackshear PJ. Evidence that tristetraprolin bind to AU-rich elements and promotes the deadenylation and destabilization of tumor necrosis factor alpha mRNA. Mol. Cell. Biol., 19, 4311–4323 (1999).
• 29) Lai WS, Carballo E, Thorn JM, Kennington EA, Blackshear PJ. Interaction of CCCH zinc finger proteins with mRNA. Binding of tristetraprolin-related zinc finger proteins to Au-rich elements and destabilization of mRNA. J. Biol. Chem., 275, 17827–17837 (2000).
• 30) Carballo E, Lai WS, Blackshear PJ. Evidence that tristetraprolin is a physiological regulator of granulocyte-macrophage colony-stimulating factor messenger RNA deadenylation and stability. Blood, 95, 1891–1899 (2000).
• 31) Ogawa K, Chen F, Kim YJ, Chen Y. Transcriptional regulation of tristetraprolin by transforming growth factor-beta in human T cells. J. Biol. Chem., 278, 30373–30381 (2003).
• 32) Schaljo B, Kratochvill F, Gratz N, Sadzak I, Sauer I, Hammer M, Vogl C, Strobl B, Müller M, Blackshear PJ, Poli V, Lang R, Murray PJ, Kovarik P. Tristetraprolin is required for full anti-inflammatory response of murine macrophages to IL-10. J. Immunol., 183, 1197–1206 (2009).
• 33) Lai WS, Kennington EA, Blackshear PJ. Interaction of CCCH zinc finger proteins with mRNA: non-binding tristetraprolin mutants exert an inhibitory effect on degradation of AU-rich element-containing mRNAs. J. Biol. Chem., 277, 9606–9613 (2002).
• 34) Carballo E, Cao H, Lai WS, Kennington EA, Campbell D, Blackshear PJ. Decreased sensitivity of tristetraprolin-deficient cells to p38 inhibitors suggests the involvement of tristetraprolin in the p38 signaling pathway. J. Biol. Chem., 276, 42580–42587 (2001).
• 35) Johnson BA, Stehn JR, Yaffe MB, Blackwell TK. Cytoplasmic localization of tristetraprolin involves 14–3-3-dependent and -independent mechanisms. J. Biol. Chem., 277, 18029–18036 (2002).
• 36) Stoecklin G, Stubbs T, Kedersha N, Wax S, Rigby WF, Blackwell TK, Anderson P. MK2-induced tristetraprolin: 14-3-3 complexes prevent stress granule association and ARE-mRNA decay. EMBO J., 23, 1313–1324 (2004).
• 37) Marchese FP, Aubareda A, Tudor C, Saklatvala J, Clark AR, Dean JL. MAPKAP kinase 2 blocks tristetraprolin-directed mRNA decay by inhibiting CAF1 deadenylase recruitment. J. Biol. Chem., 285, 27590–27600 (2010).
• 38) Clement SL, Scheckel C, Stoecklin G, Lykke-Andersen J. Phosphorylation of tristetraprolin by MK2 impairs AU-rich element mRNA decay by preventing deadenylase recruitment. Mol. Cell. Biol., 31, 256–266 (2011).
• 39) Mavropoulos A, Sully G, Cope AP, Clark AR. Stabilization of IFN-γ mRNA by MAPKp38 in IL-12 and IL-18-stimulated human NK cells. Blood, 105, 282–288 (2005).
• 40) Taylor GA, Carballo E, Lee DM, Lai WS, Thompson MJ, Patel DD, Schenkman DI, Gilkeson GS, Broxmeyer HE, Haynes BF, Blackshear PJ. A pathogenetic role for TNF alpha in the syndrome of cachexia, arthritis, and autoimmunity resulting from tristetraprolin (TTP) deficiency. Immunity, 4, 445–454 (1996).
• 41) Kulkarni AB, Huh CG, Becker D, Geiser A, Lyght M, Flanders KC, Roberts AB, Sporn MB, Ward JM, Karlsson S. Transforming growth factor beta 1 null mutation in mice causes excessive inflammatory response and early death. Proc. Natl. Acad. Sci. U.S.A., 90, 770–774 (1993).
• 42) Tsunawaki S, Sporn M, Ding A, Nathan C. Deactivation of macrophages by transforming growth factor-β. Nature, 334, 260–262 (1988).
• 43) Espevik T, Figari IS, Shalaby MR, Lackides GA, Lewis GD, Shepard HM, Palladino MA Jr. Inhibition of cytokine production by cyclosporine A and transforming growth factor β. J. Exp. Med., 166, 571–576 (1987).
• 44) Alam R, Forsythe P, Stafford S, Fukuda Y. Transforming growth factor β abrogates the effects of hematopoietins on eosinophils and induces their apoptosis. J. Exp. Med., 179, 1041–1045 (1994).
• 45) Brabletz T, Pfeuffer I, Schorr E, Siebelt F, Wirth T, Serfling E. Transforming growth factor β and cyclosporine A inhibit the inducible activity of the interleukin-2 gene in T cells through a noncanonical octamer-binding site. Mol. Cell. Biol., 13, 1155–1162 (1993).
• 46) Brooks SA, Blackshear PJ. Tristetraprolin (TTP): Interactions with mRNA and proteins, and current thoughts on mechanisms of action. Biochim. Biophys. Acta, 1829, 666–679 (2013).
• 47) Ogilvie RL, Sternjohn JR, Rattenbacher B, Vlasova IA, Williams DA, Hau HH, Blackshear PJ, Bohjanen PR. Tristetraprolin mediates interferon-γ mRNA decay. J. Biol. Chem., 284, 11216–11223 (2009).
• 48) Suzuki K, Nakajima H, Ikeda K, Maezawa Y, Suto A, Takatori H, Saito Y, Iwamoto I. IL-4-Stat6 signaling induces tristetraprolin expression and inhibits TNF-alpha production in mast cells. J. Exp. Med., 198, 1717–1727 (2003).
• 49) Kishore R, Tebo JM, Kolosov M, Hamilton TA. Clustered AU-rich elements are the target of IL-10-mediated mRNA destabilization in mouse macrophages. J. Immunol., 162, 2457–2461 (1999).