p38 regulates the tumor suppressor PDCD4 via the TSC-mTORC1 pathway

Programmed cell death protein 4 (PDCD4) exerts critical functions as tumor suppressor and in immune cells to regulate inflammatory processes. The phosphoinositide 3-kinase (PI3K) promotes degradation of PDCD4 via mammalian target of rapamycin complex 1 (mTORC1). However, additional pathways that may regulate PDCD4 expression are largely ill-defined. In this study, we have found that activation of the mitogen-activated protein kinase p38 promoted degradation of PDCD4 in macrophages and fibroblasts. Mechanistically, we identified a pathway from p38 and its substrate MAP kinase-activated protein kinase 2 (MK2) to the tuberous sclerosis complex (TSC) to regulate mTORC1-dependent degradation of PDCD4. Moreover, we provide evidence that TSC1 and TSC2 regulate PDCD4 expression via an additional mechanism independent of mTORC1. These novel data extend our knowledge of how PDCD4 expression is regulated by stress- and nutrient-sensing pathways.


INTRODUCTION
Programmed cell death protein 4 (PDCD4) is an RNAbinding tumor suppressor protein that is vital for inhibiting carcinogenesis, tumor progression and invasion [1]. Low PDCD4 expression promotes neoplastic transformation [2]. The activity of the PDCD4 protein seems to be mainly determined by its stabilization [3].
Whether other signal transduction pathways in addition to PI3K regulate PDCD4 expression via mTORC1 is largely unknown. We and others have previously found that the mitogen-activated kinase (MAPK) p38α contributes to the activation of mTORC1 [12,13]. Specifically, the p38 substrate MAP kinase-activated protein kinase 2 (MK2) phosphorylates Ser1210 on the tuberous sclerosis complex 2 (TSC2, Tuberin), a negative regulator of mTORC1 signaling, and contributes to inflammatory cytokine expression in macrophages [12]. In the current study, we wanted to investigate whether p38 controls PDCD4 expression.

p38 negatively regulates PDCD4
To study a potential role of p38 on PDCD4, we used the two well-known p38 activators anisomycin and LPS. Anisomycin has an inhibitory effect on protein translation [14], whereas LPS stimulates inflammatory protein synthesis. In bone marrow-derived macrophages (BMDMs) we found that LPS and anisomycin induced the reduction of PDCD4 (Fig. 1A). Interestingly, chemical inhibition of p38 with BIRB796 [15] prevented the LPS-or anisomycin-induced decrease of PDCD4 (Fig. 1A). To genetically corroborate these findings, we analyzed p38α-deficient BMDMs. We detected higher levels of PDCD4 in unstimulated p38αdeficient BMDMs compared to their control cells (Fig. 1B). Of note, PDCD4 was still partially lost in LPS-or anisomycinstimulated p38α-deficient cells. Moreover, levels of PDCD4 were increased in a macrophage cell line that expressed a catalytic dead mutant of MK2 (K79R) to prevent p38mediated phosphorylation and activation (Fig. 1C). These data suggest that p38 and its substrate MK2 negatively regulate the expression of PDCD4 in macrophages.

p38 controls PDCD4 via TSC1/TSC2
The complex of TSC1 (Hamartin) and TSC2 is a major negative regulator of mTORC1, and its involvement in mTORC1mediated degradation of PDCD4 has been recently suggested [16]. Indeed, deletion of TSC2 in BMDMs strongly abrogated expression of PDCD4 ( Fig. 2A). This effect was reversible by rapamycin and thus dependent on mTORC1 ( Fig. 2A). In addition, serum starvation induced the expression of PDCD4 in Tsc1 +/+ and Tsc2 +/+ fibroblasts ( Fig. 2B and D). In contrast, PDCD4 levels were strongly reduced in either non-starved as well as starved Tsc1 -/and Tsc2 -/fibroblasts similar to macrophages ( Fig. 2B and D). Inhibition of p38 or mTORC1 prevented anisomycin-induced degradation of PDCD4 in Tsc1 +/+ and Tsc2 +/+ fibroblasts ( Fig. 2C and D) and in Tsc2 fl/fl BMDMs stimulated with anisomycin or LPS ( Fig. 2E and F). However, BIRB796 failed to rescue PDCD4 degradation in anisomycin-stimulated Tsc1 -/and Tsc2 -/fibroblasts ( Fig. 2C and D) and in Tsc2 Lyz2 BMDMs (Fig.  2E). These results show that p38 controls PDCD4 expression via TSC1/TSC2. In contrast, rapamycin and the catalytic mTOR inhibitor Torin1 partially restored PDCD4 levels in anisomycin-stimulated Tsc1 -/and Tsc2 -/fibroblasts ( Fig. 2C  and D). As an ATP-competitive inhibitor, Torin1 effectively prevents both mTORC1 and mTORC2 phosphorylation [17]. Interestingly, neither rapamycin nor Torin1 restored PDCD4 in Tsc1 -/and Tsc2 -/cells to a level that is seen in starved Tsc1 +/+ and Tsc2 +/+ fibroblasts, suggesting that TSC1/TSC2 promotes basal expression of PDCD4 that is independent of mTORC1 ( Fig. 2C and D). Similar results were obtained with anisomycin in BMDMs ( Fig. 2E and F). However, we noticed that the inhibitors restored PDCD4 levels in LPS-supplied Tsc2 Lyz2 BMDMs to a comparable level as seen in wild type-representing BMDMs. These results support the concept that anisomycin does not just simply block PDCD4 translation but actively promotes degradation of PDCD4. Previous studies have found that activation of Erk contributes to PDCD4 degradation by enhancing proteasome activity [18]. Our experiments revealed an Erk-independent manner of PDCD4 degradation in Tsc2 -/fibroblasts since Erk expression was even reduced in the TSC2-deficient cells (Fig. 2D). The p90 ribosomal S6 kinases (RSKs) act downstream of Erk [19] and were shown to be promote proteasomal degradation of PDCD4 [20]. However, there was not clear association of p90RSK phosphorylation at Ser380 and PDCD4 levels in Tsc2 fl/fl and Tsc2 Lyz2 BMDMs (Suppl. Fig. 1A). Although PDCD4 can be transcriptionally regulated [21,22], qRT-PCR analysis of PDCD4 mRNA did not reveal significant differences between Tsc2 Lyz2 and Tsc2 fl/fl BMDMs (Suppl. Fig. 1C).

DISCUSSION
The MAPK p38α is ubiquitously expressed in most cell types and regulates diverse functions such as cell proliferation, differentiation, apoptosis, tissue repair, tumorigenesis, or inflammation [25]. Physicochemical stress signals such as heat, osmotic shock, arsenite or anisomycin result in activation of p38 [25]. p38 has been described as either tumor suppressor or oncoprotein depending on the cell type [26]. It will be interesting to evaluate whether PDCD4 contributes to the cell type-specific anti-or protumorigenic functions of p38. PI3K promotes PDCD4 degradation by mTORC1 activity in response to mitogenic signals [11]. Our data now suggests that also p38 induces degradation of PDCD4 via mTORC1 and TSC1/TSC2. We have previously shown that PI3K and p38 coordinately modulate mTORC1 signaling via TSC1/TSC2 in murine macrophages and human monocytes [13]. In agreement, LPS or anisomycin still induced partial degradation of PDCD4 in p38-deficient macrophages suggesting that PI3K and p38 also coordinately control PDCD4 degradation in macrophages (Fig. 3C). PDCD4 is expressed in unstressed, proliferating cells [27] and even though the heterozygous deletion of TSC2 in Tsc2 Lys+/-BMDMs creates a more proliferative macrophage type, degradation of PDCD4 by hyperactive mTORC1 in these cells outweighs healthy upregulation of PDCD4. Interestingly, rapamycin and the catalytic inhibitor Torin1, which fully blocks mTORC1 activity, did not restore PDCD4 expression in Tsc1or Tsc2-deficient fibroblasts to wild-type levels. This indicates an additional positive regulatory role for the TSC complex on PDCD4 expression in fibroblasts independently of mTORC1. The broad PDCD4 network comprises numerous feedback loops, e. g. on PI3K/Akt [28] and dysfunctional recycling of proteins like PDCD4 by the proteasome can be compensated by autophagy [29]. PDCD4 is associated with cell cycle regulation and programmed cell death and is controlled by apoptosis inducers [9,27,30]. Hence, phosphorylation by protein kinases regulating survival pathways, such as casein kinase 2 (CK2), seems plausible. CK2 was already shown to interact with PDCD4 within the nucleus [31,32] with their expression levels being inversely correlated in the tumor setting [33]. This connection would also fit into the overall picture in which PDCD4 acts proapoptotic [34]. Of note, CK2 can directly phosphorylate Akt to promote proliferation via mTORC1 [35]. Since PDCD4 is widely known to be regulated by microRNAs, mainly miR-21, their involvement cannot be ruled out. miR-21 is upregulated in the inflammatory and tumor-associated context [36]. However, we did not find a prominent upregulation of miR-21 in Tsc2-/-fibroblasts (data not shown).
The precise elucidation of the upstream regulatory network that controls PDCD4 in cancer and immune cells may be important to define novel anti-cancer and antiinflammatory strategies. In conclusion, we showed that activation of p38 promotes degradation of PDCD4 via the TSC-mTORC1 pathway (Fig. 4).

SUPPLEMENTAL MATERIAL
All supplemental data for this article are available online at www.cell-stress.com.

CONFLICT OF INTEREST
The authors declare no conflicts of interest.  and mitogen-activated kinase p38 leads to inhibition of the suppressor protein tuberous sclerosis complex 2 (TSC2), followed by activation of mammalian target of rapamycin complex 1 (mTORC1). mTORC1 phosphorylates ribosomal protein S6 kinase beta-1 (S6K1), which in turn phosphorylates programmed cell death protein 4 (PDCD4). PDCD4 is ubiquitinated and degraded by the proteasome.