The inhibition of the ubiquitin-dependent proteasome system (UPS) via specific drugs is one type of approach used to combat cancer. Although it has been suggested that UPS inhibition prevents the rapid decay of AU-rich element (ARE)-containing messages, very little is known about the cellular mechanisms leading to this effect. Here we establish a link between the inhibition of UPS activity, the formation of cytoplasmic stress granules (SGs), and mRNA metabolism. The assembly of the SGs requires the phosphorylation of the translation initiation factor eIF2α by a mechanism involving the stress kinase GCN2. On prolonged UPS inhibition and despite the maintenance of eIF2α phosphorylation, SGs disassemble and translation recovers in an Hsp72 protein-dependent manner. The formation of these SGs coincides with the disassembly of processing bodies (PBs), known as mRNA decay entities. As soon as the SGs assemble, they recruit ARE-containing messages such as p21^cip1mRNA, which are stabilized under these conditions. Hence, our findings suggest that SGs could be considered as one of the players that mediate the early response of the cell to proteasome inhibitors by interfering temporarily with mRNA decay pathways.

Muscle wasting (cachexia) is a consequence of chronic diseases, such as cancer, and is associated with degradation of muscle proteins such as MyoD. The cytokines tumor necrosis factor alpha and gamma interferon induce muscle degeneration by activating the transcription factor NF-κB and its target genes. Here, we show that a downstream target of NF-κB is the nitric oxide (NO) synthase gene (iNos) and suggest that NO production stimulates MyoD mRNA loss. In fact, although cytokine treatment of iNos^−/− mice activated NF-κB, it did not trigger MyoD mRNA degeneration, demonstrating that NF-κB-mediated muscle wasting requires an active iNOS-NO pathway. The induced expression of iNOS by cytokines relies on both transcriptional activation via NF-κB and increased mRNA stability via the RNA-binding protein HuR. Moreover, we show that HuR regulates iNOS expression in an AMP-activated protein kinase (AMPK)-dependent manner. Furthermore, AMPK activation results in HuR nuclear sequestration, inhibition of iNOS synthesis, and reduction in cytokine-induced MyoD loss. These results define iNOS and HuR as critical players in cytokine-induced cachexia, establishing them as potential therapeutic targets.

Cachexia is a debilitating syndrome characterized by involuntary muscle wasting that is triggered at the late stage of many cancers. While the multifactorial nature of this syndrome and the implication of cytokines such as IL‐6, IFNγ, and TNFα is well established, we still do not know how various effector pathways collaborate together to trigger muscle atrophy. Here, we show that IFNγ/TNFα promotes the phosphorylation of STAT3 on Y705 residue in the cytoplasm of muscle fibers by activating JAK kinases. Unexpectedly, this effect occurs both in vitro and in vivo independently of IL‐6, which is considered as one of the main triggers of STAT3‐mediated muscle wasting. pY‐STAT3 forms a complex with NF‐κB that is rapidly imported to the nucleus where it is recruited to the promoter of the iNos gene to activate the iNOS/NO pathway, a well‐known downstream effector of IFNγ/TNFα‐induced muscle loss. Together, these findings show that STAT3 and NF‐κB respond to the same upstream signal and cooperate to promote the expression of pro‐cachectic genes, the identification of which could provide effective targets to combat this deadly syndrome. Synopsis Proinflammatory cytokines such as IFNγ and TNFα are known to be key mediators of cancer cachexia‐induced muscle loss. Here, they are shown to promote muscle atrophy by directly activating the transcription factor STAT3. IFNγ/TNFα activate the JAKs/STAT3 signaling pathway in an IL‐6‐independent manner in skeletal muscle fibers. IFNγ/TNFα trigger the formation of an NF‐κB/STAT3 complex. NF‐κB is required for the translocation of STAT3 to the nucleus. STAT3 mediates the expression of iNOS to promote IFNγ/TNFα‐induced muscle atrophy.

The RNA-binding protein HuR affects cell fate by regulating the stability and/or the translation of messenger RNAs that encode cell stress response proteins. In this study, we delineate a novel regulatory mechanism by which HuR contributes to stress-induced cell death. Upon lethal stress, HuR translocates into the cytoplasm by a mechanism involving its association with the apoptosome activator pp32/PHAP-I. Depleting the expression of pp32/PHAP-I by RNA interference reduces both HuR cytoplasmic accumulation and the efficiency of caspase activation. In the cytoplasm, HuR undergoes caspase-mediated cleavage at aspartate 226. This cleavage activity is significantly reduced in the absence of pp32/PHAP-I. Substituting aspartate 226 with an alanine creates a noncleavable isoform of HuR that, when overexpressed, maintains its association with pp32/PHAP-I and delays the apoptotic response. Thus, we propose a model in which HuR association with pp32/PHAP-I and its caspase-mediated cleavage constitutes a regulatory step that contributes to an amplified apoptotic response.

A high expression level of the β-actin protein is required for important biological mechanisms, such as maintaining cell shape, growth, and motility. Although the elevated cellular level of the β-actin protein is directly linked to the long half-life of its mRNA, the molecular mechanisms responsible for this effect are unknown. Here we show that the RNA-binding protein HuR stabilizes the β-actin mRNA by associating with a uridine-rich element within its 3′ untranslated region. Using RNA interference to knock down the expression of HuR in HeLa cells, we demonstrate that HuR plays an important role in the stabilization but not in the nuclear/cytoplasmic distribution of the β-actin mRNA. HuR depletion in HeLa cells alters key β-actin-based cytoskeleton functions, such as cell adhesion, migration, and invasion, and these defects correlate with a loss of the actin stress fiber network. Together our data establish that the posttranscriptional event involving HuR-mediated β-actin mRNA stabilization could be a part of the regulatory mechanisms responsible for maintaining cell integrity, which is a prerequisite for avoiding transformation and tumor formation.