pADP-ribosylation regulates the cytoplasmic localization, cleavage, and pro-apoptotic function of HuR.

HuR (ElavL1) is one of the main post-transcriptional regulators that determines cell fate. Although the role of HuR in apoptosis is well established, the post-translational modifications that govern this function remain elusive. In this study, we show that PARP1/2-mediated poly(ADP)-ribosylation (PARylation) is instrumental in the pro-apoptotic function of HuR. During apoptosis, a substantial reduction in HuR PARylation is observed. This results in the cytoplasmic accumulation and the cleavage of HuR, both of which are essential events for apoptosis. These effects are mediated by a pADP-ribose-binding motif within the HuR-HNS region (HuR PAR-binding site). Under normal conditions, the association of the HuR PAR-binding site with pADP-ribose is responsible for the nuclear retention of HuR. Mutations within this motif prevent the binding of HuR to its import factor TRN2, leading to its cytoplasmic accumulation and cleavage. Collectively, our findings underscore the role of PARylation in controlling the pro-apoptotic function of HuR, offering insight into the mechanism by which PARP1/2 enzymes regulate cell fate and adaptation to various assaults.

Tankyrase-1 regulates RBP-mediated mRNA turnover to promote muscle fiber formation.

Poly(ADP-ribosylation) (PARylation) is a post-translational modification mediated by a subset of ADP-ribosyl transferases (ARTs). Although PARylation-inhibition based therapies are considered as an avenue to combat debilitating diseases such as cancer and myopathies, the role of this modification in physiological processes such as cell differentiation remains unclear. Here, we show that Tankyrase1 (TNKS1), a PARylating ART, plays a major role in myogenesis, a vital process known to drive muscle fiber formation and regeneration. Although all bona fide PARPs are expressed in muscle cells, experiments using siRNA-mediated knockdown or pharmacological inhibition show that TNKS1 is the enzyme responsible of catalyzing PARylation during myogenesis. Via this activity, TNKS1 controls the turnover of mRNAs encoding myogenic regulatory factors such as nucleophosmin (NPM) and myogenin. TNKS1 mediates these effects by targeting RNA-binding proteins such as Human Antigen R (HuR). HuR harbors a conserved TNKS-binding motif (TBM), the mutation of which not only prevents the association of HuR with TNKS1 and its PARylation, but also precludes HuR from regulating the turnover of NPM and myogenin mRNAs as well as from promoting myogenesis. Therefore, our data uncover a new role for TNKS1 as a key modulator of RBP-mediated post-transcriptional events required for vital processes such as myogenesis.

The formation of HuR/YB1 complex is required for the stabilization of target mRNA to promote myogenesis.

mRNA stability is the mechanism by which cells protect transcripts allowing their expression to execute various functions that affect cell metabolism and fate. It is well-established that RNA binding proteins (RBPs) such as HuR use their ability to stabilize mRNA targets to modulate vital processes such as muscle fiber formation (myogenesis). However, the machinery and the mechanisms regulating mRNA stabilization are still elusive. Here, we identified Y-Box binding protein 1 (YB1) as an indispensable HuR binding partner for mRNA stabilization and promotion of myogenesis. Both HuR and YB1 bind to 409 common mRNA targets, 147 of which contain a U-rich consensus motif in their 3' untranslated region (3'UTR) that can also be found in mRNA targets in other cell systems. YB1 and HuR form a heterodimer that associates with the U-rich consensus motif to stabilize key promyogenic mRNAs. The formation of this complex involves a small domain in HuR (227-234) that if mutated prevents HuR from reestablishing myogenesis in siHuR-treated muscle cells. Together our data uncover that YB1 is a key player in HuR-mediated stabilization of pro-myogenic mRNAs and provide the first indication that the mRNA stability mechanism is as complex as other key cellular processes such as mRNA decay and translation.

Pharmacological or genetic inhibition of iNOS prevents cachexia-mediated muscle wasting and its associated metabolism defects.

Cachexia syndrome develops in patients with diseases such as cancer and sepsis and is characterized by progressive muscle wasting. While iNOS is one of the main effectors of cachexia, its mechanism of action and whether it could be targeted for therapy remains unexplored. Here, we show that iNOS knockout mice and mice treated with the clinically tested iNOS inhibitor GW274150 are protected against muscle wasting in models of both septic and cancer cachexia. We demonstrate that iNOS triggers muscle wasting by disrupting mitochondrial content, morphology, and energy production processes such as the TCA cycle and acylcarnitine transport. Notably, iNOS inhibits oxidative phosphorylation through impairment of complexes II and IV of the electron transport chain and reduces ATP production, leading to energetic stress, activation of AMPK, suppression of mTOR, and, ultimately, muscle atrophy. Importantly, all these effects were reversed by GW274150. Therefore, our data establish how iNOS induces muscle wasting under cachectic conditions and provide a proof of principle for the repurposing of iNOS inhibitors, such as GW274150 for the treatment of cachexia.

Human antigen R promotes lung fibroblast differentiation to myofibroblasts and increases extracellular matrix production.

Idiopathic pulmonary fibrosis (IPF) is a disease of progressive scarring caused by excessive extracellular matrix (ECM) deposition and activation of α-SMA-expressing myofibroblasts. Human antigen R (HuR) is an RNA binding protein that promotes protein translation. Upon translocation from the nucleus to the cytoplasm, HuR functions to stabilize messenger RNA (mRNA) to increase protein levels. However, the role of HuR in promoting ECM production, myofibroblast differentiation, and lung fibrosis is unknown. Human lung fibroblasts (HLFs) treated with transforming growth factor ß1 (TGF-ß1) showed a significant increase in translocation of HuR from the nucleus to the cytoplasm. TGF-ß-treated HLFs that were transfected with HuR small interfering RNA had a significant reduction in α-SMA protein as well as the ECM proteins COL1A1, COL3A, and FN1. HuR was also bound to mRNA for ACTA2, COL1A1, COL3A1, and FN. HuR knockdown affected the mRNA stability of ACTA2 but not that of the ECM genes COL1A1, COL3A1, or FN. In mouse models of pulmonary fibrosis, there was higher cytoplasmic HuR in lung structural cells compared to control mice. In human IPF lungs, there was also more cytoplasmic HuR. This study is the first to show that HuR in lung fibroblasts controls their differentiation to myofibroblasts and consequent ECM production. Further research on HuR could assist in establishing the basis for the development of new target therapy for fibrotic diseases, such as IPF.

The senescence-associated secretory phenotype as a driver of tumor growth: does G3BP1 hold the key?

Cellular senescence is a double-edged sword that, depending on the context, acts as either a potent tumor protective mechanism or an age-related driver of diseases such as cancer. Our recent findings show that the rasGAP SH3-binding protein 1 (G3BP1) activates the senescent-associated secretory phenotype (SASP) that, in turn, mediates cancer growth/progression.

Role of Human Antigen R (HuR) in the Regulation of Pulmonary ACE2 Expression.

Patients with COPD may be at an increased risk for severe illness from COVID-19 because of ACE2 upregulation, the entry receptor for SARS-CoV-2. Chronic exposure to cigarette smoke, the main risk factor for COPD, increases pulmonary ACE2. How ACE2 expression is controlled is not known but may involve HuR, an RNA binding protein that increases protein expression by stabilizing mRNA. We hypothesized that HuR would increase ACE2 protein expression. We analyzed scRNA-seq data to profile ELAVL1 expression in distinct respiratory cell populations in COVID-19 and COPD patients. HuR expression and cellular localization was evaluated in COPD lung tissue by multiplex immunohistochemistry and in human lung cells by imaging flow cytometry. The regulation of ACE2 expression was evaluated using siRNA-mediated knockdown of HuR. There is a significant positive correlation between ELAVL1 and ACE2 in COPD cells. HuR cytoplasmic localization is higher in smoker and COPD lung tissue; there were also higher levels of cleaved HuR (CP-1). HuR binds to ACE2 mRNA but knockdown of HuR does not change ACE2 protein levels in primary human lung fibroblasts (HLFs). Our work is the first to investigate the association between ACE2 and HuR. Further investigation is needed to understand the mechanistic underpinning behind the regulation of ACE2 expression.

G3BP1 controls the senescence-associated secretome and its impact on cancer progression.

Cellular senescence is a known driver of carcinogenesis and age-related diseases, yet senescence is required for various physiological processes. However, the mechanisms and factors that control the negative effects of senescence while retaining its benefits are still elusive. Here, we show that the rasGAP SH3-binding protein 1 (G3BP1) is required for the activation of the senescent-associated secretory phenotype (SASP). During senescence, G3BP1 achieves this effect by promoting the association of the cyclic GMP-AMP synthase (cGAS) with cytosolic chromatin fragments. In turn, G3BP1, through cGAS, activates the NF-κB and STAT3 pathways, promoting SASP expression and secretion. G3BP1 depletion or pharmacological inhibition impairs the cGAS-pathway preventing the expression of SASP factors without affecting cell commitment to senescence. These SASPless senescent cells impair senescence-mediated growth of cancer cells in vitro and tumor growth in vivo. Our data reveal that G3BP1 is required for SASP expression and that SASP secretion is a primary mediator of senescence-associated tumor growth.

Autophagy and heat-shock response impair stress granule assembly during cellular senescence.

Stress granules (SGs) are membraneless organelles formed in response to insult. These granules are related to pathological granules found in age-related neurogenerative diseases such as Parkinson's and Alzheimer's. Previously, we demonstrated that senescent cells, which accumulate with age, exposed to chronic oxidative stress, are unable to form SGs. Here, we show that the senescent cells' inability to form SGs correlates with an upregulation in both the heat-shock response and autophagy pathways, both of which are well-established promoters of SG disassembly. Our data also reveals that the knockdown of HSP70 and ATG5, important components of the heat-shock response and autophagy pathways, respectively, restores the number of SGs formed in senescent cells exposed to chronic oxidative stress. Surprisingly, under these conditions, the depletion of HSP70 or ATG5 did not affect the clearance of these SGs during their recovery from chronic stress. These data reveal that senescent cells possess a unique heat-shock and autophagy-dependent ability to impair the formation of SGs in response to chronic stress, thereby expanding the existing understanding of SG dynamics in senescent cells and their potential contribution to age-related neurodegenerative diseases.

Depletion of HuR in murine skeletal muscle enhances exercise endurance and prevents cancer-induced muscle atrophy.

The master posttranscriptional regulator HuR promotes muscle fiber formation in cultured muscle cells. However, its impact on muscle physiology and function in vivo is still unclear. Here, we show that muscle-specific HuR knockout (muHuR-KO) mice have high exercise endurance that is associated with enhanced oxygen consumption and carbon dioxide production. muHuR-KO mice exhibit a significant increase in the proportion of oxidative type I fibers in several skeletal muscles. HuR mediates these effects by collaborating with the mRNA decay factor KSRP to destabilize the PGC-1α mRNA. The type I fiber-enriched phenotype of muHuR-KO mice protects against cancer cachexia-induced muscle loss. Therefore, our study uncovers that under normal conditions HuR modulates muscle fiber type specification by promoting the formation of glycolytic type II fibers. We also provide a proof-of-principle that HuR expression can be targeted therapeutically in skeletal muscles to combat cancer-induced muscle wasting.

HuR counteracts miR-330 to promote STAT3 translation during inflammation-induced muscle wasting.

Debilitating cancer-induced muscle wasting, a syndrome known as cachexia, is lethal. Here we report a posttranscriptional pathway involving the RNA-binding protein HuR as a key player in the onset of this syndrome. Under these conditions, HuR switches its function from a promoter of muscle fiber formation to become an inducer of muscle loss. HuR binds to the STAT3 (signal transducer and activator of transcription 3) mRNA, which encodes one of the main effectors of this condition, promoting its expression both in vitro and in vivo. While HuR does not affect the stability and the cellular movement of this transcript, HuR promotes the translation of the STAT3 mRNA by preventing miR-330 (microRNA 330)-mediated translation inhibition. To achieve this effect, HuR directly binds to a U-rich element in the STAT3 mRNA-3'untranslated region (UTR) located within the vicinity of the miR-330 seed element. Even though the binding sites of HuR and miR-330 do not overlap, the recruitment of either one of them to the STAT3-3'UTR negatively impacts the binding and the function of the other factor. Therefore, together, our data establish the competitive interplay between HuR and miR-330 as a mechanism via which muscle fibers modulate, in part, STAT3 expression to determine their fate in response to promoters of muscle wasting.

eIF4A inhibition prevents the onset of cytokine-induced muscle wasting by blocking the STAT3 and iNOS pathways.

Cachexia is a deadly muscle wasting syndrome that arises under conditions linked to chronic inflammation, such as cancer. Cytokines, including interferon γ (IFNγ), tumor necrosis factor α (TNFα) and interleukin-6 (IL-6), and their downstream effectors such as Signal Transducer and Activator of Transcription 3 (STAT3), have been shown to play a prominent role in muscle wasting. Previously, we demonstrated that Pateamine A (PatA), a compound that targets eukaryotic initiation factor 4A (eIF4A), could prevent muscle wasting by modulating the translation of the inducible Nitric Oxide Synthase (iNOS) mRNA. Here we show that hippuristanol, a compound that impedes eIF4A in a manner distinct from PatA, similarly inhibits the iNOS/NO pathway and cytokine-induced muscle wasting. Furthermore, we show that hippuristanol perturbs the activation of the STAT3 pathway and expression of STAT3-gene targets such as IL-6. The decreased activation of STAT3, which resulted from a decrease in STAT3 protein expression, was due to the inhibition of STAT3 translation as there were no changes in STAT3 mRNA levels. These effects are likely dependent on the inhibition of eIF4A activity since we observed similar results using PatA. Our results identify the inhibition of eIF4A-responsive transcripts, such as STAT3, as a viable approach to alleviate cachexia.

Post-transcriptional regulation of Pabpn1 by the RNA binding protein HuR.

RNA processing is critical for proper spatial and temporal control of gene expression. The ubiquitous nuclear polyadenosine RNA binding protein, PABPN1, post-transcriptionally regulates multiple steps of gene expression. Mutations in the PABPN1 gene expanding an N-terminal alanine tract in the PABPN1 protein from 10 alanines to 11-18 alanines cause the muscle-specific disease oculopharyngeal muscular dystrophy (OPMD), which affects eyelid, pharynx, and proximal limb muscles. Previous work revealed that the Pabpn1 transcript is unstable, contributing to low steady-state Pabpn1 mRNA and protein levels in vivo, specifically in skeletal muscle, with even lower levels in muscles affected in OPMD. Thus, low levels of PABPN1 protein could predispose specific tissues to pathology in OPMD. However, no studies have defined the mechanisms that regulate Pabpn1 expression. Here, we define multiple cis-regulatory elements and a trans-acting factor, HuR, which regulate Pabpn1 expression specifically in mature muscle in vitro and in vivo. We exploit multiple models including C2C12 myotubes, primary muscle cells, and mice to determine that HuR decreases Pabpn1 expression. Overall, we have uncovered a mechanism in mature muscle that negatively regulates Pabpn1 expression in vitro and in vivo, which could provide insight to future studies investigating therapeutic strategies for OPMD treatment.

The AMPK agonist 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), but not metformin, prevents inflammation-associated cachectic muscle wasting.

Activation of AMPK has been associated with pro-atrophic signaling in muscle. However, AMPK also has anti-inflammatory effects, suggesting that in cachexia, a syndrome of inflammatory-driven muscle wasting, AMPK activation could be beneficial. Here we show that the AMPK agonist AICAR suppresses IFNγ/TNFα-induced atrophy, while the mitochondrial inhibitor metformin does not. IFNγ/TNFα impair mitochondrial oxidative respiration in myotubes and promote a metabolic shift to aerobic glycolysis, similarly to metformin. In contrast, AICAR partially restored metabolic function. The effects of AICAR were prevented by the AMPK inhibitor Compound C and were reproduced with A-769662, a specific AMPK activator. AICAR and A-769662 co-treatment was found to be synergistic, suggesting that the anti-cachectic effects of these drugs are mediated through AMPK activation. AICAR spared muscle mass in mouse models of cancer and LPS induced atrophy. Together, our findings suggest a dual function for AMPK during inflammation-driven atrophy, wherein it can play a protective role when activated exogenously early in disease progression, but may contribute to anabolic suppression and atrophy when activated later through mitochondrial dysfunction and subsequent metabolic stress.

Stress granules counteract senescence by sequestration of PAI-1.

Cellular senescence is a physiological response by which an organism halts the proliferation of potentially harmful and damaged cells. However, the accumulation of senescent cells over time can become deleterious leading to diseases and physiological decline. Our data reveal a novel interplay between senescence and the stress response that affects both the progression of senescence and the behavior of senescent cells. We show that constitutive exposure to stress induces the formation of stress granules (SGs) in proliferative and presenescent cells, but not in fully senescent cells. Stress granule assembly alone is sufficient to decrease the number of senescent cells without affecting the expression of bona fide senescence markers. SG-mediated inhibition of senescence is associated with the recruitment of the plasminogen activator inhibitor-1 (PAI-1), a known promoter of senescence, to these entities. PAI-1 localization to SGs increases the translocation of cyclin D1 to the nucleus, promotes RB phosphorylation, and maintains a proliferative, non-senescent state. Together, our data indicate that SGs may be targets of intervention to modulate senescence in order to impair or prevent its deleterious effects.

STAT3 promotes IFNγ/TNFα-induced muscle wasting in an NF-κB-dependent and IL-6-independent manner.

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.

Destabilization of nucleophosmin mRNA by the HuR/KSRP complex is required for muscle fibre formation.

HuR promotes myogenesis by stabilizing the MyoD, myogenin and p21 mRNAs during the fusion of muscle cells to form myotubes. Here we show that HuR, via a novel mRNA destabilizing activity, promotes the early steps of myogenesis by reducing the expression of the cell cycle promoter nucleophosmin (NPM). Depletion of HuR stabilizes the NPM mRNA, increases NPM protein levels and inhibits myogenesis, while its overexpression elicits the opposite effects. NPM mRNA destabilization involves the association of HuR with the decay factor KSRP as well as the ribonuclease PARN and the exosome. The C terminus of HuR mediates the formation of the HuR-KSRP complex and is sufficient for maintaining a low level of the NPM mRNA as well as promoting the commitment of muscle cells to myogenesis. We therefore propose a model whereby the downregulation of the NPM mRNA, mediated by HuR, KSRP and its associated ribonucleases, is required for proper myogenesis.

Regulation of eukaryotic initiation factor 4AII by MyoD during murine myogenic cell differentiation.

Gene expression during muscle cell differentiation is tightly regulated at multiple levels, including translation initiation. The PI3K/mTOR signalling pathway exerts control over protein synthesis by regulating assembly of eukaryotic initiation factor (eIF) 4F, a heterotrimeric complex that stimulates recruitment of ribosomes to mRNA templates. One of the subunits of eIF4F, eIF4A, supplies essential helicase function during this phase of translation. The presence of two cellular eIF4A isoforms, eIF4AI and eIF4AII, has long thought to impart equivalent functions to eIF4F. However, recent experiments have alluded to distinct activities between them. Herein, we characterize distinct regulatory mechanisms between the eIF4A isoforms during muscle cell differentiation. We find that eIF4AI levels decrease during differentiation whereas eIF4AII levels increase during myofiber formation in a MyoD-dependent manner. This study characterizes a previously undefined mechanism for eIF4AII regulation in differentiation and highlights functional differences between eIF4AI and eIF4AII. Finally, RNAi-mediated alterations in eIF4AI and eIF4AII levels indicate that the myogenic process can tolerate short term reductions in eIF4AI or eIF4AII levels, but not both.

Aryl hydrocarbon receptor-dependent retention of nuclear HuR suppresses cigarette smoke-induced cyclooxygenase-2 expression independent of DNA-binding.

The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor that responds to man-made environmental toxicants, has emerged as an endogenous regulator of cyclooxygenase-2 (Cox-2) by a mechanism that is poorly understood. In this study, we first used AhR-deficient (AhR(-/-) ) primary pulmonary cells, together with pharmacological tools to inhibit new RNA synthesis, to show that the AhR is a prominent factor in the destabilization of Cox-2 mRNA. The destabilization of Cox-2 mRNA and subsequent suppression of cigarette smoke-induced COX-2 protein expression by the AhR was independent of its ability to bind the dioxin response element (DRE), thereby differentiating the DRE-driven toxicological AhR pathway from its anti-inflammatory abilities. We further describe that the AhR destabilizes Cox-2 mRNA by sequestering HuR within the nucleus. The role of HuR in AhR stabilization of Cox-2 mRNA was confirmed by knockdown of HuR, which resulted in rapid Cox-2 mRNA degradation. Finally, in the lungs of AhR(-/-) mice exposed to cigarette smoke, there was little Cox-2 mRNA despite robust COX-2 protein expression, a finding that correlates with almost exclusive cytoplasmic HuR within the lungs of AhR(-/-) mice. Therefore, we propose that the AhR plays an important role in suppressing the expression of inflammatory proteins, a function that extends beyond the ability of the AhR to respond to man-made toxicants. These findings open the possibility that a DRE-independent AhR pathway may be exploited therapeutically as an anti-inflammatory target.