A blood atlas of COVID-19 defines hallmarks of disease severity and specificity.

Treatment of severe COVID-19 is currently limited by clinical heterogeneity and incomplete description of specific immune biomarkers. We present here a comprehensive multi-omic blood atlas for patients with varying COVID-19 severity in an integrated comparison with influenza and sepsis patients versus healthy volunteers. We identify immune signatures and correlates of host response. Hallmarks of disease severity involved cells, their inflammatory mediators and networks, including progenitor cells and specific myeloid and lymphocyte subsets, features of the immune repertoire, acute phase response, metabolism, and coagulation. Persisting immune activation involving AP-1/p38MAPK was a specific feature of COVID-19. The plasma proteome enabled sub-phenotyping into patient clusters, predictive of severity and outcome. Systems-based integrative analyses including tensor and matrix decomposition of all modalities revealed feature groupings linked with severity and specificity compared to influenza and sepsis. Our approach and blood atlas will support future drug development, clinical trial design, and personalized medicine approaches for COVID-19.

Proline Hydroxylation Primes Protein Kinases for Autophosphorylation and Activation.

Activation of dual-specificity tyrosine-phosphorylation-regulated kinases 1A and 1B (DYRK1A and DYRK1B) requires prolyl hydroxylation by PHD1 prolyl hydroxylase. Prolyl hydroxylation of DYRK1 initiates a cascade of events leading to the release of molecular constraints on von Hippel-Lindau (VHL) ubiquitin ligase tumor suppressor function. However, the proline residue of DYRK1 targeted by hydroxylation and the role of prolyl hydroxylation in tyrosine autophosphorylation of DYRK1 are unknown. We found that a highly conserved proline in the CMGC insert of the DYRK1 kinase domain is hydroxylated by PHD1, and this event precedes tyrosine autophosphorylation. Mutation of the hydroxylation acceptor proline precludes tyrosine autophosphorylation and folding of DYRK1, resulting in a kinase unable to preserve VHL function and lacking glioma suppression activity. The consensus proline sequence is shared by most CMGC kinases, and prolyl hydroxylation is essential for catalytic activation. Thus, formation of prolyl-hydroxylated intermediates is a novel mechanism of kinase maturation and likely a general mechanism of regulation of CMGC kinases in eukaryotes.

Signaling via the kinase p38α programs dendritic cells to drive TH17 differentiation and autoimmune inflammation.

Dendritic cells (DCs) bridge innate and adaptive immunity, but how DC-derived signals regulate T cell lineage choices remains unclear. We report here that the mitogen-activated protein kinase p38α programmed DCs to drive the differentiation of the T(H)17 subset of helper T cells. Deletion of p38α in DCs protected mice from T(H)17 cell-mediated autoimmune neuroinflammation, but deletion of p38α in macrophages or T cells did not. We also found that p38α orchestrated the expression of cytokines and costimulatory molecules in DCs and further 'imprinted' signaling via the receptor for interleukin 23 (IL-23R) in responding T cells to promote T(H)17 differentiation. Moreover, p38α was required for tissue-infiltrating DCs to sustain T(H)17 responses. This activity of p38α was conserved in mouse and human DCs and was dynamically regulated by pattern recognition and fungal infection. Our results identify p38α signaling as a central pathway for the integration of instructive signals in DCs for T(H)17 differentiation and inflammation.

MK2 degradation as a sensor of signal intensity that controls stress-induced cell fate.

Cell survival in response to stress is determined by the coordination of various signaling pathways. The kinase p38α is activated by many stresses, but the intensity and duration of the signal depends on the stimuli. How different p38α-activation dynamics may impact cell life/death decisions is unclear. Here, we show that the p38α-signaling output in response to stress is modulated by the expression levels of the downstream kinase MK2. We demonstrate that p38α forms a complex with MK2 in nonstimulated mammalian cells. Upon pathway activation, p38α phosphorylates MK2, the complex dissociates, and MK2 is degraded. Interestingly, transient p38α activation allows MK2 reexpression, reassembly of the p38α-MK2 complex, and cell survival. In contrast, sustained p38α activation induced by severe stress interferes with p38α-MK2 interaction, resulting in irreversible MK2 loss and cell death. MK2 degradation is mediated by the E3 ubiquitin ligase MDM2, and we identify four lysine residues in MK2 that are directly ubiquitinated by MDM2. Expression of an MK2 mutant that cannot be ubiquitinated by MDM2 enhances the survival of stressed cells. Our results indicate that MK2 reexpression and binding to p38α is critical for cell viability in response to stress and illustrate how particular p38α-activation patterns induced by different signals shape the stress-induced cell fate.

P38α MAPK Coordinates Mitochondrial Adaptation to Caloric Surplus in Skeletal Muscle.

Excessive calorie intake leads to mitochondrial overload and triggers metabolic inflexibility and insulin resistance. In this study, we examined how attenuated p38α activity affects glucose and fat metabolism in the skeletal muscles of mice on a high-fat diet (HFD). Mice exhibiting diminished p38α activity (referred to as p38αAF) gained more weight and displayed elevated serum insulin levels, as well as a compromised response in the insulin tolerance test, compared to the control mice. Additionally, their skeletal muscle tissue manifested impaired insulin signaling, leading to resistance in insulin-mediated glucose uptake. Examination of muscle metabolites in p38αAF mice revealed lower levels of glycolytic intermediates and decreased levels of acyl-carnitine metabolites, suggesting reduced glycolysis and ß-oxidation compared to the controls. Additionally, muscles of p38αAF mice exhibited severe abnormalities in their mitochondria. Analysis of myotubes derived from p38αAF mice revealed reduced mitochondrial respiratory capacity relative to the myotubes of the control mice. Furthermore, these myotubes showed decreased expression of Acetyl CoA Carboxylase 2 (ACC2), leading to increased fatty acid oxidation and diminished inhibitory phosphorylation of pyruvate dehydrogenase (PDH), which resulted in elevated mitochondrial pyruvate oxidation. The expected consequence of reduced mitochondrial respiratory function and uncontrolled nutrient oxidation observed in p38αAF myotubes mitochondrial overload and metabolic inflexibility. This scenario explains the increased likelihood of insulin resistance development in the muscles of p38αAF mice compared to the control mice on a high-fat diet. In summary, within skeletal muscles, p38α assumes a crucial role in orchestrating the mitochondrial adaptation to caloric surplus by promoting mitochondrial biogenesis and regulating the selective oxidation of nutrients, thereby preventing mitochondrial overload, metabolic inflexibility, and insulin resistance.

Hepatic p38α MAPK controls gluconeogenesis via FOXO1 phosphorylation at S273 during glucagon signalling in mice.

AIMS/HYPOTHESIS: Hyperglucagonaemia-stimulated hepatic glucose production (HGP) contributes to hyperglycaemia during type 2 diabetes. A better understanding of glucagon action is important to enable efficient therapies to be developed for the treatment of diabetes. Here, we aimed to investigate the role of p38 MAPK family members in glucagon-induced HGP and determine the underlying mechanisms by which p38 MAPK regulates glucagon action. METHODS: p38α, ß, γ and δ MAPK siRNAs were transfected into primary hepatocytes, followed by measurement of glucagon-induced HGP. Adeno-associated virus serotype 8 carrying p38α MAPK short hairpin RNA (shRNA) was injected into liver-specific Foxo1 knockout, liver-specific Irs1/Irs2 double knockout and Foxo1S273D knockin mice. Foxo1S273A knockin mice were fed a high-fat diet for 10 weeks. Pyruvate tolerance tests, glucose tolerance tests, glucagon tolerance tests and insulin tolerance tests were carried out in mice, liver gene expression profiles were analysed and serum triglyceride, insulin and cholesterol levels were measured. Phosphorylation of forkhead box protein O1 (FOXO1) by p38α MAPK in vitro was analysed by LC-MS. RESULTS: We found that p38α MAPK, but not the other p38 isoforms, stimulates FOXO1-S273 phosphorylation and increases FOXO1 protein stability, promoting HGP in response to glucagon stimulation. In hepatocytes and mouse models, inhibition of p38α MAPK blocked FOXO1-S273 phosphorylation, decreased FOXO1 levels and significantly impaired glucagon- and fasting-induced HGP. However, the effect of p38α MAPK inhibition on HGP was abolished by FOXO1 deficiency or a Foxo1 point mutation at position 273 from serine to aspartic acid (Foxo1S273D) in both hepatocytes and mice. Moreover, an alanine mutation at position 273 (Foxo1S273A) decreased glucose production, improved glucose tolerance and increased insulin sensitivity in diet-induced obese mice. Finally, we found that glucagon activates p38α through exchange protein activated by cAMP 2 (EPAC2) signalling in hepatocytes. CONCLUSIONS/INTERPRETATION: This study found that p38α MAPK stimulates FOXO1-S273 phosphorylation to mediate the action of glucagon on glucose homeostasis in both health and disease. The glucagon-induced EPAC2-p38α MAPK-pFOXO1-S273 signalling pathway is a potential therapeutic target for the treatment of type 2 diabetes.

Requirement for epithelial p38α in KRAS-driven lung tumor progression.

Malignant transformation entails important changes in the control of cell proliferation through the rewiring of selected signaling pathways. Cancer cells then become very dependent on the proper function of those pathways, and their inhibition offers therapeutic opportunities. Here we identify the stress kinase p38α as a nononcogenic signaling molecule that enables the progression of KrasG12V-driven lung cancer. We demonstrate in vivo that, despite acting as a tumor suppressor in healthy alveolar progenitor cells, p38α contributes to the proliferation and malignization of lung cancer epithelial cells. We show that high expression levels of p38α correlate with poor survival in lung adenocarcinoma patients, and that genetic or chemical inhibition of p38α halts tumor growth in lung cancer mouse models. Moreover, we reveal a lung cancer epithelial cell-autonomous function for p38α promoting the expression of TIMP-1, which in turn stimulates cell proliferation in an autocrine manner. Altogether, our results suggest that epithelial p38α promotes KrasG12V-driven lung cancer progression via maintenance of cellular self-growth stimulatory signals.

Differential Modulation of the Phosphoproteome by the MAP Kinases Isoforms p38α and p38ß.

The p38 members of the mitogen-activated protein kinases (MAPKs) family mediate various cellular responses to stress conditions, inflammatory signals, and differentiation factors. They are constitutively active in chronic inflammatory diseases and some cancers. The differences between their transient effects in response to signals and the chronic effect in diseases are not known. The family is composed of four isoforms, of which p38α seems to be abnormally activated in diseases. p38α and p38ß are almost identical in sequence, structure, and biochemical and pharmacological properties, and the specific unique effects of each of them, if any, have not yet been revealed. This study aimed to reveal the specific effects induced by p38α and p38ß, both when transiently activated in response to stress and when chronically active. This was achieved via large-scale proteomics and phosphoproteomics analyses using stable isotope labeling of two experimental systems: one, mouse embryonic fibroblasts (MEFs) deficient in each of these p38 kinases and harboring either an empty vector or vectors expressing p38αWT, p38ßWT, or intrinsically active variants of these MAPKs; second, induction of transient stress by exposure of MEFs, p38α-/-, and p38ß-/- MEFs to anisomycin. Significant differences in the repertoire of the proteome and phosphoproteome between cells expressing active p38α and p38ß suggest distinct roles for each kinase. Interestingly, in both cases, the constitutive activation induced adaptations of the cells to the chronic activity so that known substrates of p38 were downregulated. Within the dramatic effect of p38s on the proteome and phosphoproteome, some interesting affected phosphorylation sites were those found in cancer-associated p53 and Hspb1 (HSP27) proteins and in cytoskeleton-associated proteins. Among these, was the stronger direct phosphorylation by p38α of p53-Ser309, which was validated on the Ser315 in human p53. In summary, this study sheds new light on the differences between chronic and transient p38α and p38ß signaling and on the specific targets of these two kinases.

Biophysical insights into OR2T7: Investigation of a potential prognostic marker for glioblastoma.

Glioblastoma multiforme (GBM) is the most aggressive and prevalent form of brain cancer, with an expected survival of 12-15 months following diagnosis. GBM affects the glial cells of the central nervous system, which impairs regular brain function including memory, hearing, and vision. GBM has virtually no long-term survival even with treatment, requiring novel strategies to understand disease progression. Here, we identified a somatic mutation in OR2T7, a G-protein-coupled receptor (GPCR), that correlates with reduced progression-free survival for glioblastoma (log rank p-value = 0.05), suggesting a possible role in tumor progression. The mutation, D125V, occurred in 10% of 396 glioblastoma samples in The Cancer Genome Atlas, but not in any of the 2504 DNA sequences in the 1000 Genomes Project, suggesting that the mutation may have a deleterious functional effect. In addition, transcriptome analysis showed that the p38α mitogen-activated protein kinase (MAPK), c-Fos, c-Jun, and JunB proto-oncogenes, and putative tumor suppressors RhoB and caspase-14 were underexpressed in glioblastoma samples with the D125V mutation (false discovery rate < 0.05). Molecular modeling and molecular dynamics simulations have provided preliminary structural insight and indicate a dynamic helical movement network that is influenced by the membrane-embedded, cytofacial-facing residue 125, demonstrating a possible obstruction of G-protein binding on the cytofacial exposed region. We show that the mutation impacts the "open" GPCR conformation, potentially affecting Gα-subunit binding and associated downstream activity. Overall, our findings suggest that the Val125 mutation in OR2T7 could affect glioblastoma progression by downregulating GPCR-p38 MAPK tumor-suppression pathways and impacting the biophysical characteristics of the structure that facilitates Gα-subunit binding. This study provides the theoretical basis for further experimental investigation required to confirm that the D125V mutation in OR2T7 is not a passenger mutation. With validation, the aforementioned mutation could represent an important prognostic marker and a potential therapeutic target for glioblastoma.

Identification of the TF-miRNA-mRNA co-regulatory networks involved in sepsis.

Sepsis is a life-threatening medical condition caused by a dysregulated host response to infection. Recent studies have found that the expression of miRNAs is associated with the pathogenesis of sepsis and septic shock. Our study aimed to reveal which miRNAs may be involved in the dysregulated immune response in sepsis and how these miRNAs interact with transcription factors (TFs) using a computational approach with in vitro validation studies. To determine the network of TFs, miRNAs, and target genes involved in sepsis, GEO datasets GSE94717 and GSE131761 were used to identify differentially expressed miRNAs and DEGs. TargetScan and miRWalk databases were used to predict biological targets that overlap with the identified DEGs of differentially expressed miRNAs. The TransmiR database was used to predict the differential miRNA TFs that overlap with the identified DEGs. The TF-miRNA-mRNA network was constructed and visualized. Finally, qRT-PCR was used to verify the expression of TFs and miRNA in HUVECs. Between the healthy and sepsis groups, there were 146 upregulated and 98 downregulated DEGs in the GSE131761 dataset, and there were 1 upregulated and 183 downregulated DEMs in the GSE94717 dataset. A regulatory network of the TF-miRna target genes was established. According to the experimental results, RUNX3 was found to be downregulated while MAPK14 was upregulated, which corroborates the result of the computational expression analysis. In a HUVECs model, miR-19b-1-5p and miR-5009-5p were found to be significantly downregulated. Other TFs and miRNAs did not correlate with our bioinformatics expression analysis. We constructed a TF-miRNA-target gene regulatory network and identified potential treatment targets RUNX3, MAPK14, miR-19b-1-5p, and miR-5009-5p. This information provides an initial basis for understanding the complex sepsis regulatory mechanisms.

P38α-MAPK phosphorylates Snapin and reduces Snapin-mediated BACE1 transportation in APP-transgenic mice.

Amyloid ß peptide (Aß) is the major pathogenic molecule in Alzheimer's disease (AD). BACE1 enzyme is essential for the generation of Aß. Deficiency of p38α-MAPK in neurons increases lysosomal degradation of BACE1 and decreases Aß deposition in the brain of APP-transgenic mice. However, the mechanisms mediating effects of p38α-MAPK are largely unknown. In this study, we used APP-transgenic mice and cultured neurons and observed that deletion of p38α-MAPK specifically in neurons decreased phosphorylation of Snapin at serine, increased retrograde transportation of BACE1 in axons and reduced BACE1 at synaptic terminals, which suggests that p38α-MAPK deficiency promotes axonal transportation of BACE1 from its predominant locations, axonal terminals, to lysosomes in the cell body. In vitro kinase assay revealed that p38α-MAPK directly phosphorylates Snapin. By further performing mass spectrometry analysis and site-directed mutagenic experiments in SH-SY5Y cell lines, we identified serine residue 112 as a p38α-MAPK-phosphorylating site on Snapin. Replacement of serine 112 with alanine did abolish p38α-MAPK knockdown-induced reduction of BACE1 activity and protein level, and transportation to lysosomes in SH-SY5Y cells. Taken together, our study suggests that activation of p38α-MAPK phosphorylates Snapin and inhibits the retrograde transportation of BACE1 in axons, which might exaggerate amyloid pathology in AD brain.

Virtual Screening and Molecular Docking to Study the Mechanism of Chinese Medicines in the Treatment of Coronavirus Infection.

BACKGROUND Heat-clearing and detoxifying herbs (HDHs) play an important role in the prevention and treatment of coronavirus infection. However, their mechanism of action needs further study. This study aimed to explore the anti-coronavirus basis and mechanism of HDHs. MATERIAL AND METHODS Database mining was performed on 7 HDHs. Core ingredients and targets were screened according to ADME rules combined with Neighborhood, Co-occurrence, Co-expression, and other algorithms. GO enrichment and KEGG pathway analyses were performed using the R language. Finally, high-throughput molecular docking was used for verification. RESULTS HDHs mainly acts on NOS3, EGFR, IL-6, MAPK8, PTGS2, MAPK14, NFKB1, and CASP3 through quercetin, luteolin, wogonin, indirubin alkaloids, ß-sitosterol, and isolariciresinol. These targets are mainly involved in the regulation of biological processes such as inflammation, activation of MAPK activity, and positive regulation of NF-kappaB transcription factor activity. Pathway analysis further revealed that the pathways regulated by these targets mainly include: signaling pathways related to viral and bacterial infections such as tuberculosis, influenza A, Ras signaling pathways; inflammation-related pathways such as the TLR, TNF, MAPK, and HIF-1 signaling pathways; and immune-related pathways such as NOD receptor signaling pathways. These pathways play a synergistic role in inhibiting lung inflammation and regulating immunity and antiviral activity. CONCLUSIONS HDHs play a role in the treatment of coronavirus infection by regulating the body's immunity, fighting inflammation, and antiviral activities, suggesting a molecular basis and new strategies for the treatment of COVID-19 and a foundation for the screening of new antiviral drugs.

Filamentous nestin and nonmuscle myosin IIB are associated with a migratory phenotype in neonatal rat cardiomyocytes.

Cardiomyocyte migration represents a requisite event of cardiogenesis and the regenerative response of the injured adult zebrafish and neonatal rodent heart. The present study tested the hypothesis that the appearance of the intermediate filament protein nestin in neonatal rat ventricular cardiomyocytes (NNVMs) was associated in part with the acquisition of a migratory phenotype. The cotreatment of NNVMs with phorbol 12,13-dibutyrate (PDBu) and the p38α/ß mitogen-activated protein kinase inhibitor SB203580 led to the de novo synthesis of nestin. The intermediate filament protein was subsequently reorganized into a filamentous pattern and redistributed to the leading edge of elongated membrane protrusions translating to significant lengthening of NNVMs. PDBu/SB203580 treatment concomitantly promoted the reorganization of nonmuscle myosin IIB (NMIIB) located predominantly at the periphery of the plasma membrane of NNVMs to a filamentous phenotype extending to the leading edge of elongated membrane protrusions. Coimmunoprecipitation assay revealed a physical interaction between NMIIB and nestin after PDBu/SB203580 treatment of NNVMs. In wild-type and heterozygous NMIIB embryonic hearts at E11.5-E14.5 days, nestin immunoreactivity was identified in a subpopulation of cardiomyocytes elongating perpendicular to the compact myocardium, at the leading edge of nascent trabeculae and during thickening of the compact myocardium. In mutant embryonic hearts lacking NMIIB protein expression, trabeculae formation was reduced, the compact myocardium significantly thinner and nestin immunoreactivity undetectable in cardiomyocytes at E14.5 days. These data suggest that NMIIB and nestin may act in a coordinated fashion to facilitate the acquisition of a migratory phenotype in neonatal and embryonic cardiomyocytes.

Screening of DNA-Encoded Small Molecule Libraries inside a Living Cell.

DNA-encoded small molecule libraries (DELs) have facilitated the discovery of novel modulators of many different therapeutic protein targets. We report the first successful screening of a multimillion membered DEL inside a living cell. We demonstrate a novel method using oocytes from the South African clawed frog Xenopus laevis. The large size of the oocytes of 1 µL, or 100 000 times bigger than a normal somatic cell, permits simple injection of DELs, thus resolving the fundamental problem of delivering DELs across cell membranes for in vivo screening. The target protein was expressed in the oocytes fused to a prey protein, to allow specific DNA labeling and hereby discriminate between DEL members binding to the target protein and the endogenous cell proteins. The 194 million member DEL was screened against three pharmaceutically relevant protein targets, p38α, ACSS2, and DOCK5. For all three targets multiple chemical clusters were identified. For p38α, validated hits with single digit nanomolar potencies were obtained. This work demonstrates a powerful new approach to DEL screening, which eliminates the need for highly purified active target protein and which performs the screening under physiological relevant conditions and thus is poised to increase the DEL amenable target space and reduce the attrition rates.

Metabolic benefits of inhibition of p38α in white adipose tissue in obesity.

p38 has long been known as a central mediator of protein kinase A (PKA) signaling in brown adipocytes, which positively regulate the transcription of uncoupling protein 1 (UCP-1). However, the physiological role of p38 in adipose tissues, especially the white adipose tissue (WAT), is largely unknown. Here, we show that mice lacking p38α in adipose tissues display a lean phenotype, improved metabolism, and resistance to diet-induced obesity. Surprisingly, ablation of p38α causes minimal effects on brown adipose tissue (BAT) in adult mice, as evident from undetectable changes in UCP-1 expression, mitochondrial function, body temperature (BT), and energy expenditure. In contrast, genetic ablation of p38α in adipose tissues not only markedly facilitates the browning in WAT upon cold stress but also prevents diet-induced obesity. Consistently, pharmaceutical inhibition of p38α remarkably enhances the browning of WAT and has metabolic benefits. Furthermore, our data suggest that p38α deficiency promotes white-to-beige adipocyte reprogramming in a cell-autonomous manner. Mechanistically, inhibition of p38α stimulates the UCP-1 transcription through PKA and its downstream cAMP-response element binding protein (CREB), which form a positive feedback loop that functions to reinforce the white-to-beige phenotypic switch during cold exposure. Together, our study reveals that inhibition of p38α is able to promote WAT browning and confer metabolic benefits. Our study also indicates that p38α in WAT represents an exciting pharmacological target to combat obesity and metabolic diseases.

LncRNA XIST knockdown alleviates LPS-induced acute lung injury by inactivation of XIST/miR-132-3p/MAPK14 pathway : XIST promotes ALI via miR-132-3p/MAPK14 axis.

Acute lung injury (ALI) is a fatal inflammatory response syndrome. LncRNA XIST (XIST) is a lung cancer-related gene and participates in pneumonia. However, whether XIST participates in lipopolysaccharides (LPS)-induced ALI remains unclear. LPS-induced inflammation model was constructed in vitro, then cell viability, cytokines, cell apoptosis, protein, and mRNA expressions were individually detected by cell counting kit-8, enzyme-linked immunosorbent assay and flow cytometry, Western blot, and qRT-PCR. A dual-luciferase reporter assay confirmed the relationships among XIST, miR-132-3p, and MAPK14. Furthermore, inflammation and conditions after knockdown of XIST were assessed by hematoxylin and eosin staining, lung wet-to-dry weight ratio, PaO2/FiO2 ratio, and malondialdehyde (MDA) contents using LPS-induced in vivo model. Our findings indicated that the LPS challenge decreased cell viability, increased cell apoptosis, and caused secretions of pro-inflammatory cytokines. Noticeably, LPS significantly upregulated XIST, MAPK14, and downregulated miR-132-3p. Mechanistically, XIST acted as a molecular sponge to suppress miR-132-3p, and MAPK14 was identified as a target of miR-132-3p. Functional analyses demonstrated that XIST silencing remarkably increased cell survival and alleviated cell death and lung injury through decreasing TNF-α, IL-1ß, IL-6, accumulation of inflammatory cells, alveolar hemorrhage, MDA release, and increased PaO2/FiO2 ratio, as well as upregulating Bcl-2, and downregulating Bax, MAPK14, and p-extracellular signal-regulated kinases ½. In contrast, inhibition of the miR-132-3p antagonized the effects of XIST silencing. In conclusion, inhibition of XIST exhibited a protective role in LPS-induced ALI through modulating the miR-132-3p/MAPK14 axis.

The role of MAPK11/12/13/14 (p38 MAPK) protein in dopamine agonist-resistant prolactinomas.

BACKGROUND: Prolactinoma is a functional pituitary adenoma that secretes excessive prolactin. Dopamine agonists (DAs) such as bromocriptine (BRC) are the first-line treatment for prolactinomas, but the resistance rate is increasing year by year, creating a clinical challenge. Therefore, it is urgent to explore the molecular mechanism of bromocriptine resistance in prolactinomas. Activation of the P38 MAPK pathway affects multidrug resistance in tumours. Our previous studies have demonstrated that inhibiting MAPK14 can suppress the occurrence of prolactinoma, but the role of MAPK11/12/13/14 (p38 MAPK) signalling in dopamine agonist-resistant prolactinomas is still unclear. METHODS: A prolactinoma rat model was established to determine the effect of bromocriptine on MAPK11/12/13/14 signalling. DA-resistant GH3 cells and DA-sensitive MMQ cells were used, and the role of MAPK11/12/13/14 in bromocriptine-resistant prolactinomas was preliminarily verified by western blot, RT-qPCR, ELISA, flow cytometry and CCK-8 experiments. The effects of MAPK11 or MAPK14 on bromocriptine-resistant prolactinomas were further verified by siRNA transfection experiments. RESULTS: Bromocriptine was used to treat rat prolactinoma by upregulating DRD2 expression and downregulating the expression level of MAPK11/12/13/14 in vivo experiments. The in vitro experiments showed that GH3 cells are resistant to bromocriptine and that MMQ cells are sensitive to bromocriptine. Bromocriptine could significantly reduce the expression of MAPK12 and MAPK13 in GH3 cells and MMQ cells. Bromocriptine could significantly reduce the expression of MAPK11, MAPK14, NF-κB p65 and Bcl2 in MMQ but had no effect on MAPK11, MAPK14, NF-κB p65 and Bcl2 in GH3 cells. In addition, knockdown of MAPK11 and MAPK14 in GH3 cells by siRNA transfection reversed the resistance of GH3 cells to bromocriptine, and haloperidol (HAL) blocked the inhibitory effect of bromocriptine on MAPK14, MAPK11, and PRL in MMQ cells. Our findings show that MAPK11 and MAPK14 proteins are involved in bromocriptine resistance in prolactinomas. CONCLUSION: Bromocriptine reduces the expression of MAPK11/12/13/14 in prolactinomas, and MAPK11 and MAPK14 are involved in bromocriptine resistance in prolactinomas by regulating apoptosis. Reducing the expression of MAPK11 or MAPK14 can reverse bromocriptine resistance in prolactinomas.

Protein kinase p38α signaling in dendritic cells regulates colon inflammation and tumorigenesis.

Dendritic cells (DCs) play pivotal roles in maintaining intestinal homeostasis, but how the DCs regulate diverse immune networks on homeostasis breakdown remains largely unknown. Here, we report that, in response to epithelial barrier disruption, colonic DCs regulate the differentiation of type 1 regulatory T (Tr1) cells through p38α-dependent IL-27 production to initiate an effective immune response. Deletion of p38α in DCs, but not in T cells, led to increased Tr1 and protected mice from dextran sodium sulfate-induced acute colitis and chronic colitis-associated colorectal cancer. We show that higher levels of IL-27 in p38α-deficient colonic cDC1s, but not cDC2s, were responsible for the increase of Tr1 cells. Moreover, p38α-dependent IL-27 enhanced IL-22 secretion from intestinal group 3 innate lymphoid cells and protected epithelial barrier function. In p38α-deficient DCs, the TAK1-MKK4/7-JNK-c-Jun axis was hyperactivated, leading to high IL-27 levels, and inhibition of the JNK-c-Jun axis suppressed IL-27 expression. ChIP assay revealed direct binding of c-Jun to the promoter of Il27p28, which was further enhanced in p38α-deficient DCs. In summary, here we identify a key role for p38α signaling in DCs in regulating intestinal inflammatory response and tumorigenesis, and our finding may provide targets for the treatment of inflammatory intestinal diseases.

Perturbation of p38α MAPK as a Novel Strategy to Effectively Sensitize Chronic Myeloid Leukemia Cells to Therapeutic BCR-ABL Inhibitors.

Chronic myeloid leukemia (CML) is a hematopoietic malignancy characterized by the presence of the BCR-ABL oncogene. Therapeutic regimens with tyrosine kinase inhibitors (TKIs) specifically targeting BCR-ABL have greatly improved overall survival of CML. However, drug intolerance and related toxicity remain. Combined therapy is effective in reducing drug magnitude while increasing therapeutic efficacy and, thus, lowers undesired adverse side effects. The p38 MAPK activity is critically linked to the pathogenesis of a number of diseases including hematopoietic diseases; however, the role of each isozyme in CML and TKI-mediated effects is still elusive. In this study, we used specific gene knockdown to clearly demonstrate that the deficiency of p38α greatly enhanced the therapeutic efficacy in growth suppression and cytotoxicity of TKIs, first-generation imatinib, and second generation dasatinib by approximately 2.5-3.0-fold in BCR-ABL-positive CML-derived leukemia K562 and KMB5 cells. Knockdown of p38ß, which displays the most sequence similarity to p38α, exerted distinct and opposite effects on the TKI-mediated therapeutic efficacy. These results show the importance of isotype-specific intervention in enhancing the therapeutic efficacy of TKI. A highly specific p38α inhibitor, TAK715, also significantly enhanced the imatinib- and dasatinib-mediated therapeutic efficacy, supporting the feasibility of p38α deficiency in future clinic application. Taken together, our results demonstrated that p38α is a promising target for combined therapy with BCR-ABL-targeting tyrosine kinase inhibitors for future application to increase therapeutic efficacy.

Chlorella vulgaris modulates the expression of senescence-associated genes in replicative senescence of human diploid fibroblasts.

Human diploid fibroblasts (HDFs) cultured in vitro have limited capacity to proliferate after population doubling is repeated several times, and they enter into a state known as replicative senescence or cellular senescence. This study aimed to investigate the effect of Chlorella vulgaris on the replicative senescence of HDFs by determining the expression of senescence-associated genes. Young and senescent HDFs were divided into untreated control and C. vulgaris-treated groups. A senescence-associated gene transcription analysis was carried out with qRT-PCR. Treatment of young HDFs with C. vulgaris reduced the expression of SOD1, CAT and CCS (p < 0.05). In addition, the expression of the SOD2 gene was increased with C. vulgaris treatment in young, pre-senescent and senescent HDFs (p < 0.05). Treatment of senescent HDFs with C. vulgaris resulted in the downregulation of TP53 gene expression. The expression of the CDKN2A gene was significantly decreased upon C. vulgaris treatment in young and senescent HDFs. C. vulgaris treatment was also found to significantly upregulate the expression of the MAPK14 gene in pre-senescent HDFs. In addition, the expression of MAPK14 was significantly upregulated compared to that in the untreated senescent HDFs (p < 0.05). In summary, the expression of senescence-associated genes related to antioxidants and the insulin/insulin-like growth factor-1 signalling, DNA damage-associated signalling, cell differentiation and cell proliferation pathways was modulated by C. vulgaris during replicative senescence of human diploid fibroblasts.