The Global Phosphorylation Landscape of SARS-CoV-2 Infection.

The causative agent of the coronavirus disease 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has infected millions and killed hundreds of thousands of people worldwide, highlighting an urgent need to develop antiviral therapies. Here we present a quantitative mass spectrometry-based phosphoproteomics survey of SARS-CoV-2 infection in Vero E6 cells, revealing dramatic rewiring of phosphorylation on host and viral proteins. SARS-CoV-2 infection promoted casein kinase II (CK2) and p38 MAPK activation, production of diverse cytokines, and shutdown of mitotic kinases, resulting in cell cycle arrest. Infection also stimulated a marked induction of CK2-containing filopodial protrusions possessing budding viral particles. Eighty-seven drugs and compounds were identified by mapping global phosphorylation profiles to dysregulated kinases and pathways. We found pharmacologic inhibition of the p38, CK2, CDK, AXL, and PIKFYVE kinases to possess antiviral efficacy, representing potential COVID-19 therapies.

Blocking elevated p38 MAPK restores efferocytosis and inflammatory resolution in the elderly.

Increasing age alters innate immune-mediated responses; however, the mechanisms underpinning these changes in humans are not fully understood. Using a human dermal model of acute inflammation, we found that, although inflammatory onset is similar between young and elderly individuals, the resolution phase was substantially impaired in elderly individuals. This arose from a reduction in T cell immunoglobulin mucin receptor-4 (TIM-4), a phosphatidylserine receptor expressed on macrophages that enables the engulfment of apoptotic bodies, so-called efferocytosis. Reduced TIM-4 in elderly individuals was caused by an elevation in macrophage p38 mitogen-activated protein kinase (MAPK) activity. Administering an orally active p38 inhibitor to elderly individuals rescued TIM-4 expression, cleared apoptotic bodies and restored a macrophage resolution phenotype. Thus, inhibiting p38 in elderly individuals rejuvenated their resolution response to be more similar to that of younger people. This is the first resolution defect identified in humans that has been successfully reversed, thereby highlighting the tractability of targeting pro-resolution biology to treat diseases driven by chronic inflammation.

Diverse intracellular pathogens activate type III interferon expression from peroxisomes.

Type I interferon responses are considered the primary means by which viral infections are controlled in mammals. Despite this view, several pathogens activate antiviral responses in the absence of type I interferons. The mechanisms controlling type I interferon-independent responses are undefined. We found that RIG-I like receptors (RLRs) induce type III interferon expression in a variety of human cell types, and identified factors that differentially regulate expression of type I and type III interferons. We identified peroxisomes as a primary site of initiation of type III interferon expression, and revealed that the process of intestinal epithelial cell differentiation upregulates peroxisome biogenesis and promotes robust type III interferon responses in human cells. These findings highlight the importance of different intracellular organelles in specific innate immune responses.

ASK1/ p38 axis inhibition blocks the release of mitochondrial "danger signals" from hepatocytes and suppresses progression to cirrhosis and liver cancer.

BACKGROUND AND AIMS: Apoptosis Signal-regulating Kinase 1 (ASK1) is activated by various pathological stimuli and induces cell apoptosis through downstream p38 activation. We studied the effect of pharmacological ASK1 inhibition on cirrhosis and its sequelae using comprehensive preclinical in vivo and in vitro systems. APPROACH AND RESULTS: Short-term (4-6 wk) and long-term (24-44 wk) ASK1 inhibition using small molecule GS-444217 was tested in thioacetamide-induced and BALB/c. Mdr2-/- murine models of cirrhosis and HCC, and in vitro using primary hepatocyte cell death assays. Short-term GS-444217 therapy in both models strongly reduced phosphorylated p38, hepatocyte death, and fibrosis by up to 50%. Profibrogenic release of mitochondrial DAMP mitochondrial deoxyribonucleic acid from dying hepatocytes was blocked by ASK1 or p38 inhibition. Long-term (24 wk) therapy in BALBc.Mdr2 - / - model resulted in a moderate 25% reduction in bridging fibrosis, but not in net collagen deposition. Despite this, the development of cirrhosis was effectively prevented, with strongly reduced p21 + hepatocyte staining (by 72%), serum ammonia levels (by 46%), and portal pressure (average 6.07 vs. 8.53 mm Hg in controls). Extended ASK1 inhibition for 44 wk in aged BALB/c. Mdr2-/- mice resulted in markedly reduced tumor number and size by ~50% compared to the control group. CONCLUSIONS: ASK1 inhibition suppresses the profibrogenic release of mitochondrial deoxyribonucleic acid from dying hepatocytes in a p38-dependent manner and protects from liver fibrosis. Long-term ASK1 targeting resulted in diminished net antifibrotic effect, but the progression to liver cirrhosis and cancer in BALBc/ Mdr2- / - mice was effectively inhibited. These data support the clinical evaluation of ASK1 inhibitors in fibrotic liver diseases.

Bidirectional regulation of neutrophil migration by mitogen-activated protein kinases.

To kill invading bacteria, neutrophils must interpret spatial cues, migrate and reach target sites. Although the initiation of chemotactic migration has been extensively studied, little is known about its termination. Here we found that two mitogen-activated protein kinases (MAPKs) had opposing roles in neutrophil trafficking. The extracellular signal-regulated kinase Erk potentiated activity of the G protein-coupled receptor kinase GRK2 and inhibited neutrophil migration, whereas the MAPK p38 acted as a noncanonical GRK that phosphorylated the formyl peptide receptor FPR1 and facilitated neutrophil migration by blocking GRK2 function. Therefore, the dynamic balance between Erk and p38 controlled neutrophil 'stop' and 'go' activity, which ensured that neutrophils reached their final destination as the first line of host defense.

Identification of Potent ADCK3 Inhibitors through Structure-Based Virtual Screening.

ADCK3 is a member of the UbiB family of atypical protein kinases in humans, with homologues in archaea, bacteria, and eukaryotes. In lieu of protein kinase activity, ADCK3 plays a role in the biosynthesis of coenzyme Q10 (CoQ10), and inactivating mutations can cause a CoQ10 deficiency and ataxia. However, the exact functions of ADCK3 are still unclear, and small-molecule inhibitors could be useful as chemical probes to elucidate its molecular mechanisms. In this study, we applied structure-based virtual screening (VS) to discover a novel chemical series of ADCK3 inhibitors. Through extensive structural analysis of the active-site residues, we developed a pharmacophore model and applied it to a large-scale VS. Out of ∼170,000 compounds virtually screened, 800 top-ranking candidate compounds were selected and tested in both ADCK3 and p38 biochemical assays for hit validation. In total, 129 compounds were confirmed as ADCK3 inhibitors, and among them, 114 compounds are selective against p38, which was used as a counter-target. Molecular dynamics (MD) simulations were then conducted to predict the binding modes of the most potent compounds within the ADCK3 active site. Through metadynamics analysis, we successfully detected the key amino acid residues that govern intermolecular interactions. The findings provided in this study can serve as a promising starting point for drug development.

p38MAPK builds a hyaluronan cancer niche to drive lung tumorigenesis.

Expansion of neoplastic lesions generates the initial signal that instigates the creation of a tumor niche. Nontransformed cell types within the microenvironment continuously coevolve with tumor cells to promote tumorigenesis. Here, we identify p38MAPK as a key component of human lung cancer, and specifically stromal interactomes, which provides an early, protumorigenic signal in the tissue microenvironment. We found that lung cancer growth depends on short-distance cues produced by the cancer niche in a p38-dependent manner. We identified fibroblast-specific hyaluronan synthesis at the center of p38-driven tumorigenesis, which regulates early stromal fibroblast activation, the conversion to carcinoma-associated fibroblasts (CAFs), and cancer cell proliferation. Systemic down-regulation of p38MAPK signaling in a knock-in model with substitution of activating Tyr182 to phenylalanine or conditional ablation of p38 in fibroblasts has a significant tumor-suppressive effect on K-ras lung tumorigenesis. Furthermore, both Kras-driven mouse lung tumors and orthotopically grown primary human lung cancers show a significant sensitivity to both a chemical p38 inhibitor and an over-the-counter inhibitor of hyaluronan synthesis. We propose that p38MAPK-hyaluronan-dependent reprogramming of the tumor microenvironment plays a critical role in driving lung tumorigenesis, while blocking this process could have far-reaching therapeutic implications.

H19 Promotes HCC Bone Metastasis Through Reducing Osteoprotegerin Expression in a Protein Phosphatase 1 Catalytic Subunit Alpha/p38 Mitogen-Activated Protein Kinase-Dependent Manner and Sponging microRNA 200b-3p.

BACKGROUND AND AIMS: Bone is the second most frequent site of metastasis for HCC, which leads to an extremely poor prognosis. HCC bone metastasis is typically osteolytic, involving the activation of osteoclasts. Long noncoding RNA H19 plays an important role in the pathogenesis of human cancers. Nonetheless, the mechanism underlying the participation of H19 in HCC bone metastasis remains unclear. APPROACH AND RESULTS: The current study established a mouse HCC bone metastasis model by using serial intracardiac injection and cell isolation to obtain cells with distinct bone metastasis ability. H19 was highly expressed in these cells and in clinical HCC bone metastasis specimens. Both osteoclastogenesis in vitro and HCC bone metastasis in vivo were promoted by H19 overexpression, whereas these processes were suppressed by H19 knockdown. H19 overexpression attenuated p38 phosphorylation and further down-regulated the expression of osteoprotegerin (OPG), also known as osteoclastogenesis inhibitory factor. However, up-regulated OPG expression as well as suppressed osteoclastogenesis caused by H19 knockdown were recovered by p38 interference, indicating that p38 mitogen-activated protein kinase (MAPK)-OPG contributed to H19-promoted HCC bone metastasis. Furthermore, we demonstrated that H19 inhibited the expression of OPG by binding with protein phosphatase 1 catalytic subunit alpha (PPP1CA), which dephosphorylates p38. SB-203580-mediated inactivation of p38MAPK reversed the down-regulation of HCC bone metastasis caused by H19 knockdown in vivo. Additionally, H19 enhanced cell migration and invasion by up-regulating zinc finger E-box binding homeobox 1 through the sequestration of microRNA (miR) 200b-3p. CONCLUSIONS: H19 plays a critical role in HCC bone metastasis by reducing OPG expression, which is mediated by the PPP1CA-induced inactivation of the p38MAPK pathway; and H19 also functions as a sponge for miR-200b-3p.

Activation of GATA-binding protein 4 regulates monocyte chemoattractant protein-1 and chemotaxis in periodontal ligament cells.

BACKGROUND AND OBJECTIVES: Periodontitis is a chronic inflammatory disease of periodontal supporting tissues. The persistent inflammatory reaction depends on the release of chemokines to continuously recruit inflammation cells. GATA-binding protein 4 (GATA4) exerts effects on senescence and inflammation, while its role in periodontitis is far from clear. The present study aims to address the effect of GATA4 on regulating chemokines and the chemotaxis in periodontitis. MATERIAL AND METHODS: Periodontitis rat models were constructed to detect the expression of GATA4 and the chemokine monocyte chemoattractant protein-1 (MCP-1) by immunohistochemistry. Lipopolysaccharide (LPS)-stimulated human periodontal ligament (PDL) cells and GATA4-knockdown by siRNA transient transfection PDL cells were used to explore the correlation between GATA4 and chemokines. Transwell assay was performed to detect the role of GATA4 for the recruitment effect of chemokines on macrophages. Mitogen-activated protein kinase (MAPK) inhibitors were scheduled to intervene in LPS-stimulated PDL cells to examine the association between MAPK signaling pathways and GATA4. The expression of GATA4, chemokines, or MAPK signaling molecules was determined by quantitative real-time polymerase chain reaction, western blotting, or cell immunofluorescence. RESULTS: The expression of GATA4 and MCP-1 was significantly increased in periodontitis rat models and in LPS-stimulated PDL cells. Knockdown GATA4 inhibited the expression of GATA4 and MCP-1 as well as suppressed the recruitment of macrophage in LPS-stimulated PDL cells. Inhibitors of p38 and ERK1/2 signaling pathways significantly downregulated the increased expression of GATA4 and MCP-1 induced by LPS in PDL cells. CONCLUSIONS: GATA-binding protein 4 could act as an upstream regulator of MCP-1 and as a downstream regulator of p38 and ERK1/2 signaling pathways to initiate inflammation response and regulate chemotaxis during the progression of periodontitis.

IL-1ß is a key inflammatory cytokine that weakens lactation-specific tight junctions of mammary epithelial cells.

In lactating mammary glands, alveolar mammary epithelial cells (MECs) produce milk and form less-permeable tight junctions (TJs). However, alveolar TJs are weakened with a reduction in milk production in mammary glands due to mastitis or weaning in the presence of high levels of IL-1ß, IL-6, or TNF-α. In this study, using in vitro cultured model of MECs with milk-producing ability and lactation-specific TJs, we investigated whether the aforementioned cytokines affect MEC TJs. The results showed that TNF-α, IL-1ß, and IL-6 affected lactation-specific TJs in different ways. In particular, upon activation of p38 and JNK signalling, IL-1ß caused rapid disruption of TJs at tricellular contact points. IL-1ß treatment led to decreased CLDN3, CLDN4, and OCLN levels and a weakened TJ barrier. The adverse effects of IL-1ß on TJs were mimicked by anisomycin, which is an activator of p38 and JNK signalling, and were blocked by MEC pretreatment with a p38 inhibitor but not a JNK inhibitor. The mislocalization of tricellulin at tricellular contact areas was confirmed in MECs treated with IL-1ß or anisomycin. These results indicate that IL-1ß is a key cytokine that adversely affects the TJs between MECs by activating p38.

Dexmedetomidine Attenuates Hypoxia/Reoxygenation Injury of H9C2 Myocardial Cells by Upregulating miR-146a Expression via the MAPK Signal Pathway.

INTRODUCTION AND OBJECTIVE: Dexmedetomidine (Dex) and a number of miRNAs contribute to ischemia/reperfusion injury. We aimed to explore the role of Dex and miR-146a on myocardial cells injured by hypoxia/reoxygenation (H/R). METHOD: H9C2 cells were injured by H/R. Cell viability was tested using the cell counting kit-8. Lactate dehydrogenase (LDH) activity, superoxide dismutase (SOD) activity, and malondialdehyde (MDA) levels were determined using commercial kits. Flow cytometry was performed to determine apoptosis rate and reactive oxygen species (ROS) level. Protein and mRNA levels were assessed using Western blot and qPCR. RESULTS: miR-146a expression and cell viability of H9C2 cells were downregulated under the circumstance of H/R injury. The tendency could be reversed by Dex, which could also upregulate SOD activity and decrease apoptosis, LDH activity, MDA, 78-kDa glucose-regulated protein (GRP78), and C/EBP homologous protein (CHOP) levels of H9C2 cells. GRP78, CHOP levels, and cell viability were negatively modulated by miR-146a. Dex elevated cell viability, catalase, MnSOD, and NAD(P)H dehydrogenase (NQO1) levels but suppressed apoptosis rate, GRP78, and CHOP levels by increasing miR-146a expression and downregulating ROS, phosphorylation of p38, and extracellular signal-regulated kinases 1/2 levels. By using SB203580 (SB), the p38 mitogen-activated protein kinase (MAPK) inhibitor, Dex or the inhibition of miR-146 upregulated cell viability but downregulated GRP78 and CHOP levels. CONCLUSION: Dex might regulate miR-146a expression, which could further modulate the endoplasmic reticulum stress and oxidative stress and eventually affect the cell viability and apoptosis of myocardial cells injured by H/R via the MAPK signal pathway.

Discovery of novel paeonol-based derivatives against skin inflammation in vitro and in vivo.

T-LAK-cell-originated protein kinase (TOPK), a novel member of the mitogen-activated protein kinase family, is considered an effective therapeutic target for skin inflammation. In this study, a series (A - D) of paeonol derivatives was designed and synthesised using a fragment growing approach, and their anti-inflammatory activities against lipopolysaccharide (LPS)-induced nitric oxide production in RAW264.7 cells were tested. Among them, compound B12 yielded the best results (IC50 = 2.14 µM) with low toxicity (IC50 > 50 µM). Preliminary mechanistic studies indicated that this compound could inhibit the TOPK-p38/JNK signalling pathway and phosphorylate downstream related proteins. A murine psoriasis-like skin inflammation model was used to determine its therapeutic effect.

Functional Roles of JNK and p38 MAPK Signaling in Nasopharyngeal Carcinoma.

c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) family members integrate signals that affect proliferation, differentiation, survival, and migration in a cell context- and cell type-specific way. JNK and p38 MAPK activities are found upregulated in nasopharyngeal carcinoma (NPC). Studies have shown that activation of JNK and p38 MAPK signaling can promote NPC oncogenesis by mechanisms within the cancer cells and interactions with the tumor microenvironment. They regulate multiple transcription activities and contribute to tumor-promoting processes, ranging from cell proliferation to apoptosis, inflammation, metastasis, and angiogenesis. Current literature suggests that JNK and p38 MAPK activation may exert pro-tumorigenic functions in NPC, though the underlying mechanisms are not well documented and have yet to be fully explored. Here, we aim to provide a narrative review of JNK and p38 MAPK pathways in human cancers with a primary focus on NPC. We also discuss the potential therapeutic agents that could be used to target JNK and p38 MAPK signaling in NPC, along with perspectives for future works. We aim to inspire future studies further delineating JNK and p38 MAPK signaling in NPC oncogenesis which might offer important insights for better strategies in diagnosis, prognosis, and treatment decision-making in NPC patients.

Injection of YiQiFuMai powder protects against heart failure via inhibiting p38 and ERK1/2 MAPKs activation.

CONTEXT: Injection of YiQiFuMai (YQFM) powder, a modern Chinese plant-derived medical preparation, has a therapeutic effect in heart failure (HF). However, its therapeutic mechanism remains largely unknown. OBJECTIVE: To investigate the molecular mechanisms of YQFM in HF. MATERIALS AND METHODS: Kinase inhibition profiling assays with 2 mg/mL YQFM were performed against a series of 408 kinases. In addition, the effects of kinase inhibition were validated in cardiomyocyte cell line H9c2. In vivo, HF with reduced ejection fraction (HFrEF) was induced by permanent left anterior descending (LAD) coronary artery ligation for 6 weeks in male Sprague-Dawley rats. Then, HFrEF mice were treated with 0.46 g/kg YQFM or placebo once a day for 2 weeks. Echocardiography, immunohistochemistry, histological staining and Western blotting analysis were performed to assess the myocardial damage and molecular mechanisms. RESULTS: Kinase inhibition profiling analysis demonstrated that mitogen-activated protein kinases (MAPKs) mediated the signalling cascades of YQFM during HF therapy. Meanwhile, p38 and extracellular signal-regulated kinases (ERK1/2) were inhibited after YQFM treatment in H9c2 cells. In rats, the control group had lower left ventricular ejection fraction (LVEF) at 37 ± 1.7% compared with the YQFM group at 54 ± 1.1% (p < 0.0001). Cardiac fibrosis levels in control group rats were significantly higher than YQFM group (30.5 ± 3.0 vs. 14.1 ± 1.0, p < 0.0001). CONCLUSIONS: Our collective in vitro and in vivo experiments demonstrated that YQFM improves left ventricular (LV) function and inhibits fibrosis in HFrEF rats by inhibiting MAPK signalling pathways.

Targeting of BCR-ABL1 and IRE1α induces synthetic lethality in Philadelphia-positive acute lymphoblastic leukemia.

BCR-ABL1-positive acute lymphoblastic leukemia (ALL) cell survival is dependent on the inositol-requiring enzyme 1 alpha (IRE1α) branch of the unfolded protein response. In the current study, we have focused on exploring the efficacy of a simultaneous pharmacological inhibition of BCR-ABL1 and IRE1α in Philadelphia-positive (Ph+) ALL using tyrosine kinase inhibitor (TKI) nilotinib and the IRE1α inhibitor MKC-8866. The combination of 0.5 µM nilotinib and 30 µM MKC-8866 in Ph+ ALL cell lines led to a synergistic effect on cell viability. To mimic this dual inhibition on a genetic level, pre-B-cells from conditional Xbp1+/fl mice were transduced with a BCR-ABL1 construct and with either tamoxifen-inducible cre or empty vector. Cells showed a significant sensitization to the effect of TKIs after the induction of the heterozygous deletion. Finally, we performed a phosphoproteomic analysis on Ph+ ALL cell lines treated with the combination of nilotinib and MKC-8866 to identify potential targets involved in their synergistic effect. An enhanced activation of p38 mitogen-activated protein kinase α (p38α MAPK) was identified. In line with this findings, p38 MAPK and, another important endoplasmic reticulum-stress-related kinase, c-Jun N-terminal kinase (JNK) were found to mediate the potentiated cytotoxic effect induced by the combination of MKC-8866 and nilotinib since the targeting of p38 MAPK with its specific inhibitor BIRB-796 or JNK with JNK-in-8 hindered the synergistic effect observed upon treatment with nilotinib and MKC-8866. In conclusion, the identified combined action of nilotinib and MKC-8866 might represent a successful therapeutic strategy in high-risk Ph+ ALL.

Calpain activation mediates microgravity-induced myocardial abnormalities in mice via p38 and ERK1/2 MAPK pathways.

The human cardiovascular system has adapted to function optimally in Earth's 1G gravity, and microgravity conditions cause myocardial abnormalities, including atrophy and dysfunction. However, the underlying mechanisms linking microgravity and cardiac anomalies are incompletely understood. In this study, we investigated whether and how calpain activation promotes myocardial abnormalities under simulated microgravity conditions. Simulated microgravity was induced by tail suspension in mice with cardiomyocyte-specific deletion of Capns1, which disrupts activity and stability of calpain-1 and calpain-2, and their WT littermates. Tail suspension time-dependently reduced cardiomyocyte size, heart weight, and myocardial function in WT mice, and these changes were accompanied by calpain activation, NADPH oxidase activation, and oxidative stress in heart tissues. The effects of tail suspension were attenuated by deletion of Capns1 Notably, the protective effects of Capns1 deletion were associated with the prevention of phosphorylation of Ser-345 on p47 phox and attenuation of ERK1/2 and p38 activation in hearts of tail-suspended mice. Using a rotary cell culture system, we simulated microgravity in cultured neonatal mouse cardiomyocytes and observed decreased total protein/DNA ratio and induced calpain activation, phosphorylation of Ser-345 on p47 phox , and activation of ERK1/2 and p38, all of which were prevented by calpain inhibitor-III. Furthermore, inhibition of ERK1/2 or p38 attenuated phosphorylation of Ser-345 on p47 phox in cardiomyocytes under simulated microgravity. This study demonstrates for the first time that calpain promotes NADPH oxidase activation and myocardial abnormalities under microgravity by facilitating p47 phox phosphorylation via ERK1/2 and p38 pathways. Thus, calpain inhibition may be an effective therapeutic approach to reduce microgravity-induced myocardial abnormalities.

Optical control of MAP kinase kinase 6 (MKK6) reveals that it has divergent roles in pro-apoptotic and anti-proliferative signaling.

The selective pressure imposed by extrinsic death signals and stressors adds to the challenge of isolating and interpreting the roles of proteins in stress-activated signaling networks. By expressing a kinase with activating mutations and a caged lysine blocking the active site, we can rapidly switch on catalytic activity with light and monitor the ensuing dynamics. Applying this approach to MAP kinase 6 (MKK6), which activates the p38 subfamily of MAPKs, we found that decaging active MKK6 in fibroblasts is sufficient to trigger apoptosis in a p38-dependent manner. Both in fibroblasts and in a murine melanoma cell line expressing mutant B-Raf, MKK6 activation rapidly and potently inhibited the pro-proliferative extracellular signal-regulated kinase (ERK) pathway; to our surprise, this negative cross-regulation was equally robust when all p38 isoforms were inhibited. These results position MKK6 as a new pleiotropic signal transducer that promotes both pro-apoptotic and anti-proliferative signaling, and they highlight the utility of caged, light-activated kinases for dissecting stress-activated signaling networks.

A genome-wide analysis of targets of macrolide antibiotics in mammalian cells.

Macrolide antibiotics, such as erythromycin and josamycin, are natural polyketide products harboring 14- to 16-membered macrocyclic lactone rings to which various sugars are attached. These antibiotics are used extensively in the clinic because of their ability to inhibit bacterial protein synthesis. More recently, some macrolides have been shown to also possess anti-inflammatory and other therapeutic activities in mammalian cells. To better understand the targets and effects of this drug class in mammalian cells, we used a genome-wide shRNA screen in K562 cancer cells to identify genes that modulate cellular sensitivity to josamycin. Among the most sensitizing hits were proteins involved in mitochondrial translation and the mitochondrial unfolded protein response, glycolysis, and the mitogen-activated protein kinase signaling cascade. Further analysis revealed that cells treated with josamycin or other antibacterial agents exhibited impaired oxidative phosphorylation and metabolic shifts to glycolysis. Interestingly, we observed that knockdown of the mitogen-activated protein kinase kinase kinase 4 (MAP3K4) gene, which contributes to p38 mitogen-activated protein kinase signaling, sensitized cells only to josamycin but not to other antibacterial agents. There is a growing interest in better characterizing the therapeutic effects and toxicities of antibiotics in mammalian cells to guide new applications in both cellular and clinical studies. To our knowledge, this is the first report of an unbiased genome-wide screen to investigate the effects of a clinically used antibiotic on human cells.

Tristetraprolin regulates necroptosis during tonic Toll-like receptor 4 (TLR4) signaling in murine macrophages.

The necrosome is a protein complex required for signaling in cells that results in necroptosis, which is also dependent on tumor necrosis factor receptor (TNF-R) signaling. TNFα promotes necroptosis, and its expression is facilitated by mitogen-activated protein (MAP) kinase-activated protein kinase 2 (MK2) but is inhibited by the RNA-binding protein tristetraprolin (TTP, encoded by the Zfp36 gene). We have stimulated murine macrophages from WT, MyD88-/-, Trif-/-, MyD88-/-Trif-/-, MK2-/-, and Zfp36-/- mice with graded doses of lipopolysaccharide (LPS) and various inhibitors to evaluate the role of various genes in Toll-like receptor 4 (TLR4)-induced necroptosis. Necrosome signaling, cytokine production, and cell death were evaluated by immunoblotting, ELISA, and cell death assays, respectively. We observed that during TLR4 signaling, necrosome activation is mediated through the adaptor proteins MyD88 and TRIF, and this is inhibited by MK2. In the absence of MK2-mediated necrosome activation, lipopolysaccharide-induced TNFα expression was drastically reduced, but MK2-deficient cells became highly sensitive to necroptosis even at low TNFα levels. In contrast, during tonic TLR4 signaling, WT cells did not undergo necroptosis, even when MK2 was disabled. Of note, necroptosis occurred only in the absence of TTP and was mediated by the expression of TNFα and activation of JUN N-terminal kinase (JNK). These results reveal that TTP plays an important role in inhibiting TNFα/JNK-induced necrosome signaling and resultant cytotoxicity.

Myostatin regulates the production of fibroblast growth factor 23 (FGF23) in UMR106 osteoblast-like cells.

Myostatin is a signaling molecule produced by skeletal muscle cells (myokine) that inhibits muscle hypertrophy and has further paracrine and endocrine effects in other organs including bone. Myostatin binds to activin receptor type 2B which forms a complex with transforming growth factor-ß type I receptor (TGF-ßRI) and induces intracellular p38MAPK and NFκB signaling. Fibroblast growth factor 23 (FGF23) is a paracrine and endocrine mediator produced by bone cells and regulates phosphate and vitamin D metabolism in the kidney. P38MAPK and NFκB-dependent store-operated Ca2+ entry (SOCE) are positive regulators of FGF23 production. Here, we explored whether myostatin influences the synthesis of FGF23. Fgf23 gene expression was determined by qRT-PCR and FGF23 protein by ELISA in UMR106 osteoblast-like cells. UMR106 cells expressed activin receptor type 2A and B. Myostatin upregulated Fgf23 gene expression and protein production. The myostatin effect on Fgf23 was significantly attenuated by TGF-ßRI inhibitor SB431542, p38MAPK inhibitor SB202190, and NFκB inhibitor withaferin A. Moreover, SOCE inhibitor 2-APB blunted the myostatin effect on Fgf23. Taken together, myostatin is a stimulator of Fgf23 expression in UMR106 cells, an effect at least partially mediated by downstream TGF-ßRI/p38MAPK signaling as well as NFκB-dependent SOCE.