Activation Of P38 Mitogen-activated Protein Kinase Research Articles

Glucocorticoid Receptors Drive Breast Cancer Cell Migration and Metabolic Reprogramming via PDK4.

Corticosteroids act on the glucocorticoid receptor (GR; NR3C1) to resolve inflammation and are routinely prescribed to breast cancer patients undergoing chemotherapy treatment to alleviate side effects. Triple-negative breast cancers (TNBCs) account for 15% to 20% of diagnoses and lack expression of estrogen and progesterone receptors as well as amplified HER2, but they often express high GR levels. GR is a mediator of TNBC progression to advanced metastatic disease; however, the mechanisms underpinning this transition to more aggressive behavior remain elusive. We previously showed that tissue/cellular stress (hypoxia, chemotherapies) as well as factors in the tumor microenvironment (transforming growth factor β [TGF-β], hepatocyte growth factor [HGF]) activate p38 mitogen-activated protein kinase (MAPK), which phosphorylates GR on Ser134. In the absence of ligand, pSer134-GR further upregulates genes important for responses to cellular stress, including key components of the p38 MAPK pathway. Herein, we show that pSer134-GR is required for TNBC metastatic colonization to the lungs of female mice. To understand the mechanisms of pSer134-GR action in the presence of GR agonists, we examined glucocorticoid-driven transcriptomes in CRISPR knock-in models of TNBC cells expressing wild-type or phospho-mutant (S134A) GR. We identified dexamethasone- and pSer134-GR-dependent regulation of specific gene sets controlling TNBC migration (NEDD9, CSF1, RUNX3) and metabolic adaptation (PDK4, PGK1, PFKFB4). TNBC cells harboring S134A-GR displayed metabolic reprogramming that was phenocopied by pyruvate dehydrogenase kinase 4 (PDK4) knockdown. PDK4 knockdown or chemical inhibition also blocked cancer cell migration. Our results reveal a convergence of GR agonists (ie, host stress) with cellular stress signaling whereby pSer134-GR critically regulates TNBC metabolism, an exploitable target for the treatment of this deadly disease.

Role of p38 MAPK, Akt and NFκB in renoprotective effects of nebivolol on renal ischemia-reperfusion injury in rats

ABSTRACT Renal ischemia-reperfusion (I-R) injury is a complex pathophysiologic condition characterized by oxidative stress, inflammation and apoptosis. We investigated the potential renoprotective effect of nebivolol, a β1 adrenergic receptor blocker, against renal I-R injury. We focused on the role of nebivolol in activating p38 mitogen-activated protein kinase (MAPK) signaling, Akt (protein kinase B) and nuclear factor-κB (NFκB) transcription factors, which contribute to oxidative stress, inflammation and apoptosis during renal I-R. We divided 20 adult male Wistar albino rats into three experimental groups. Group 1 was a sham control in which only laparotomy was performed. Group 2 was the I-R group in which both kidneys were made ischemic for 45 min, then reperfused for 24 h. Group 3 was the I-R + nebivolol group in which 10 mg/kg nebivolol was administrated by gavage for 7 days before I-R. We measured Inflammation, oxidative stress and active caspase-3 as well as activation of p38 MAPK, Akt (protein kinase B) and NFκB transcription factor. Nebivolol significantly reduced oxidative stress and increased superoxide dismutase levels during renal I-R. We found that nebivolol significantly decreased interstitial inflammation, and TNF-α and interleukin-1β mRNA expression. Nebivolol significantly reduced active caspase-3 and kidney injury molecule-1 (KIM-1) expressions. Nebivolol also significantly decreased activation of p38 MAPK signaling and NFκB, and induced Akt activation during renal I-R. Our findings suggest that nebivolol may be useful for management of renal I-R injury.

Growth State-Dependent Activation of eNOS in Response to DHA: Involvement of p38 MAPK.

Our laboratory previously reported that docosahexaenoic acid (DHA) differentially activates p38 mitogen-activated protein kinase (MAPK) in growing and quiescent human endothelial cells, which represent the dysfunctional and healthy states in vivo, respectively. Since endothelial nitric oxide synthase (eNOS) activity differs between healthy and dysfunctional endothelial cells, and p38 MAPK reportedly regulates both the activity and expression of eNOS, we hypothesized that the beneficial actions of DHA on endothelial cells are due to eNOS activation by p38 MAPK. The contribution of mitogen- and stress-activated protein kinase (MSK), a p38 MAPK substrate, was also investigated. Growing and quiescent EA.hy926 cells, prepared on Matrigel®-coated plates, were incubated with inhibitors of p38MAPK or MSK before adding DHA. eNOS phosphorylation and levels were quantified by Western blotting. Treatment with 20 µM DHA activated eNOS in both growth states whereas 125 µM DHA suppressed eNOS activation in growing cells. Quiescent cells had higher basal levels of eNOS than growing cells, while 125 µM DHA decreased eNOS levels in both growth states. p38 MAPK inhibition enhanced eNOS activation in quiescent cells but suppressed it in growing cells. Interestingly, 125 µM DHA counteracted these effects of p38 MAPK inhibition in both growth states. MSK was required for eNOS activation in both growth states, but it only mediated eNOS activation by DHA in quiescent cells. MSK thus affects eNOS via a pathway independent of p38MAPK. Quiescent cells were also more resistant to the apoptosis-inducing effect of 125 µM DHA compared to growing cells. The growth state-dependent regulation of p38MAPK and eNOS by DHA provides novel insight into the molecular mechanisms by which DHA influences endothelial cell function.

Effect of the Mitogen-Activated Protein Kinase Pathway on the Erastin-Induced Ferroptosis of Molt-4 Cells.

The role of ferroptosis in human acute lymphoblastic leukemia and its possible molecular mechanisms of action are still unknown. In this study, harvested Molt-4 cells were exposed to different concentrations of erastin, and their proliferation capacity was tested by using the cell counting kit-8 assay. Lipid peroxidation levels were detected through flow cytometry. Mitochondrial alterations were observed through transmission electron microscopy. The expression levels of SLC7A11, glutathione peroxidase 4 (GPX4), and mitogen-activated protein kinase (MAPK) were detected by using quantitative real-time PCR and Western blot analysis. This study found that erastin inhibited the growth of Molt-4 cells. This inhibitory effect could be partially reversed by the ferroptosis inhibitor Ferrostatin-1 and the p38 MAPK inhibitor. The mitochondria of Molt-4 cells treated with erastin shortened and condensed. Compared with those in the control group, the levels of reactive oxygen species and malondialdehyde had increased, whereas the levels of glutathione had decreased in the treatment group. The treatment of Molt-4 cells with erastin decreased the levels of SLC7A11 and GPX4 mRNA and increased the expression levels of p38 MAPK, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase. These findings suggested that erastin caused the ferroptosis of Molt-4 cells. This process may be correlated with the inhibition of the cystine/glutamate antiporter system and GPX4 and the activation of p38 MAPK and ERK1/2.

Cell-free hemoglobin-mediated oxidation of low-density lipoprotein (oxLDL) contributes to lung microvascular endothelial dysfunction during sepsis

Introduction: Disruption of the microvascular endothelial barrier is a critical pathological feature of sepsis-induced acute lung injury. Plasma cell-free hemoglobin (CFH) is elevated in approximately 80% of patients with sepsis and is independently associated with development of acute respiratory distress syndrome (ARDS) and mortality. Oxidized CFH (ferric, 3+) can oxidize low-density lipoprotein (oxLDL), which signals through its major endothelial receptor, the lectin-like oxidized LDL receptor 1 (LOX-1), to cause endothelial dysfunction through several pro-inflammatory pathways including activation of mitogen-activated protein kinases (MAPKs). However, little is known regarding whether LOX-1 receptor signaling leads to microvascular endothelial hyperpermeability or acute lung injury, especially in the context of sepsis. We hypothesized that oxidation of LDL by CFH contributes to lung microvascular endothelial barrier dysfunction and worse outcomes during sepsis through LOX-1 and downstream p38 MAPK. Methods: To test whether generation of oxLDL by CFH is associated with endothelial injury in clinical sepsis, circulating levels of CFH, oxLDL, and vascular injury marker sVE-cadherin were measured in 24 sepsis patients via ELISA and tested for association with development of ARDS. LDL was oxidized by combining LDL with CFH in a test tube overnight at 37°C and oxLDL was quantified by TBARS assay. In primary human lung microvascular endothelial cells (HLMVEC) transendothelial electrical resistance (TER), a measure of barrier dysfunction, was assessed by Electric Cell-substrate Impedance Sensing (ECIS). LOX-1 receptor was blocked using BI-0115 (Boehringer Ingelheim, 20 μM) and p38 MAPK was inhibited using NiPp (100 μM). Results: In sepsis patients, plasma oxLDL levels correlated with CFH (r=0.686, p=0.016) and sVE-cadherin (r=0.603, p=0.012), and tended to be higher in those who developed ARDS (38 U/L [IQR 27, 45] vs. 27 U/L [IQR 19, 35], p=0.1). Oxidation of LDL by CFH exacerbated HLMVEC barrier dysfunction compared to control, LDL, or CFH. Barrier dysfunction induced by CFH or oxLDL was attenuated by blocking the LOX-1 receptor or p38 MAPK. Conclusions: Increased plasma CFH and oxLDL are associated with vascular injury during clinical sepsis; one mechanism by which CFH may cause vascular hyperpermeability and sepsis-mediated lung injury is through oxidation of LDL which can drive signaling through the endothelial LOX-1 receptor and activation of p38 MAPK. NIH R35HL150783, R21GM144915, R01HL158906, R01HL164937, T32HL094296; Parker B. Francis Fellowship This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Connexin 43 dephosphorylation at serine 282 induces spontaneous arrhythmia and increases susceptibility to ischemia/reperfusion injury

BackgroundConnexin 43 (Cx43), the predominant gap junction protein in hearts, is modified by specific (de)phosphorylation events under physiological and pathological states to affect myocardium function and structure. Previously we found that deficiency in Cx43 S282 phosphorylation could impair intercellular communication and contribute to cardiomyocyte apoptosis by activating p38 mitogen-activated protein kinase (p38 MAPK)/factor-associated suicide (Fas)/Fas-associating protein with a novel death domain (FADD) pathway, which is involved in myocardium injury in ischemia/reperfusion (I/R) heart. In addition, mutant at Cx43 S282 substituted with alanine heterozygous mice (S282A+/−) exhibited different degrees of ventricular arrhythmias and only some underwent myocardium apoptosis. In this study, we aimed to investigate the role of Cx43 pS282 in different cardiac pathological phenotypes. MethodsWe examined cardiac function, structure, and relevant protein expression in S282A+/− mice (aged 2, 10 and 30 weeks) by electrocardiograph, echocardiography, histological staining, and co-immunoprecipitation followed by Western blot. Intraperitoneal isoprenaline injection and I/R surgery were applied in S282A+/− mice as external stimulus. 2,3,5-triphenyltetrazolium chloride staining was used for myocardium infarction evaluation. ResultsAdult S282A+/− mice (aged 10 and 30 weeks) still exhibited spontaneous arrhythmia. Unlike neonatal stage (aged around 2 weeks), no apoptosis-related manifestations and the activation of p38 MAPK-Fas-FADD apoptotic pathway were observed in adult S282A+/− hearts. S282A+/− neonatal mice with cardiomyocytes apoptosis exhibited more than 60% dephosphorylation at Cx43 S282 than WT mice, while less than 40% S282 dephosphorylation were found in adult S282A+/− mice. In addition, although S282A+/− mice displayed normal cardiac function, they were highly susceptible to isoproterenol-induced ECG alternans and prone to cardiac injury and deaths upon I/R attack. ConclusionsThese results reinforce that Cx43 S282 dephosphorylation acts as a susceptibility factor in regulating cardiomyocyte survival and cardiac electrical homeostasis in basal conditions and contributes to myocardium injury in the setting of I/R. Cx43 S282 phosphorylation was competent to induce spontaneous arrhythmias, cardiomyocyte apoptosis and deaths based on the degree of S282 dephosphorylation.

Spinal adenosine 2A receptor inhibition restores phrenic long-term facilitation during mild systemic inflammation

Inflammation undermines several forms of neuroplasticity, including phrenic long-term facilitation (pLTF), a form of respiratory motor plasticity elicited by moderate acute intermittent hypoxia (mAIH; 3, 5-min episodes, 5 min intervals; arterial PO2 ~ 40–50 mmHg). Mild inflammation elicited by a low dose of the TLR-4 receptor agonist lipopolysaccharide (LPS; 100 mg/kg, i.p.) abolishes mAIH-induced pLTF by a mechanism that requires spinal p38 MAP kinase and protein phosphatase activity. Neuroinflammation activates microglia and other cells in the CNS, elevating extracellular levels of adenosine—a purine nucleoside known to suppress mAIH-induced pLTF. We hypothesized basal adenosine levels in ventral spinal C 3 -C 5 segments are increased in adult male Sprague-Dawley rats 24 hours post-LPS administration, thereby undermining mAIH-induced pLTF via adenosine 2A receptor (A 2A ) activation. Thus, we predicted that cervical intrathecal administration of the A 2A receptor inhibitor MSX-3 (10 μM, 12 μL) would rescue mAIH-induced pLTF in rats pre-treated with LPS. As predicted, LPS significantly increased basal ventral C 3 -C 5 adenosine levels (p=0.010 vs controls; n=7 per group). In control rats (saline injection, i.p.), MSX-3 enhanced pLTF versus vehicle controls (LPS: 110 ± 16% baseline; controls: 53 ± 6%; p=0.002; each n=6). As expected, mAIH-induced pLTF was abolished in LPS-treated rats (4 ± 6% baseline; n=6). MSX-3 restored mAIH-induced pLTF to levels equivalent to MSX-3 treated control rats (120 ± 14%; p<0.001; n=6; versus LPS controls with MSX-3: p=0.539). Thus, mild LPS-induced inflammation abolishes mAIH-induced pLTF by a mechanism associated with increased spinal adenosine levels and A 2A receptor activation. As we move mAIH forward as a therapeutic modality to improve breathing and non-respiratory movements in people with spinal cord injury or ALS, pretreatment with A 2A receptor inhibitors may offset the undermining effects of systemic inflammation associated with these severe neuromuscular disorders. NIH HL147554, HL148030, T32HL134621 (ABM) and the UF McKnight Brain Institute This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

P38 MAPK activation and STIM1-Orai3 association mediate TRPC6 externalization.

Endothelial cell (EC) migration is critical for the repair of monolayer disruption following angioplasties, but migration is inhibited by lipid oxidation products, including lysophosphatidylcholine (lysoPC), which open canonical transient receptor potential 6 (TRPC6) channels. TRPC6 activation requires an increase in intracellular Ca2+ concentration ([Ca2+]i), the source of which is unknown. LysoPC can activate phospholipase A2 to release arachidonic acid (ArA). ArA can activate arachidonic acid-regulated calcium (ARC) channels that are formed by stromal interaction molecule 1 (STIM1) and Orai1 and Orai3 proteins. Both lysoPC and ArA can activate p38 mitogen-activated protein kinase (MAPK) that induces the phosphorylation required for STIM1-Orai3 association. This is accompanied by an increase in [Ca2+]i and TRPC6 externalization. The effect of lysoPC and ArA is not additive, suggesting activation of the same pathway. The increase in [Ca2+]i activates an Src kinase that leads to TRPC6 activation. Downregulation of Orai3 using siRNA blocks the lysoPC- or ArA-induced increase in [Ca2+]i and TRPC6 externalization and preserves EC migration. These data show that lysoPC induces activation of p38 MAPK, which leads to STIM1-Orai3 association and increased [Ca2+]i. This increase in [Ca2+]i activates an Src kinase leading to TRPC6 externalization, which initiates a cascade of events ending in cytoskeletal changes that disrupt EC migration. Blocking this pathway preserves EC migration in the presence of lipid oxidation products.NEW & NOTEWORTHY The major lysophospholipid component in oxidized LDL, lysophosphatidylcholine (lysoPC), can activate p38 MAP kinase, which in turn promotes externalization of Orai3 and STIM1-Orai3 association, suggesting involvement of arachidonic acid-regulated calcium (ARC) channels. The subsequent increase in intracellular calcium activates an Src kinase required for TRPC6 externalization. TRPC6 activation, which has been shown to inhibit endothelial cell migration, is blocked by p38 MAP kinase or Orai3 downregulation, and this partially preserves endothelial migration in lysoPC.

N-benzyl-N-methyldecan-1-amine and its derivative mitigate 2,4- dinitrobenzenesulfonic acid-induced colitis and collagen-induced rheumatoid arthritis.

As our previous study revealed that N-benzyl-N-methyldecan-1-amine (BMDA), a new molecule originated from Allium sativum, exhibits anti-neoplastic activities, we herein explored other functions of the compound and its derivative [decyl-(4-methoxy-benzyl)-methyl-amine; DMMA] including anti-inflammatory and anti-oxidative activities. Pretreatment of THP-1 cells with BMDA or DMMA inhibited tumor necrosis factor (TNF)-α and interleukin (IL)-1β production, and blocked c-jun terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), MAPKAP kinase (MK)2 and NF-κΒ inflammatory signaling during LPS stimulation. Rectal treatment with BMDA or DMMA reduced the severity of colitis in 2,4-dinitrobenzenesulfonic acid (DNBS)-treated rat. Consistently, administration of the compounds decreased myeloperoxidase (MPO) activity (representing neutrophil infiltration in colonic mucosa), production of inflammatory mediators such as cytokine-induced neutrophil chemoattractant (CINC)-3 and TNF-α, and activation of JNK and p38 MAPK in the colon tissues. In addition, oral administration of these compounds ameliorated collagen-induced rheumatoid arthritis (RA) in mice. The treatment diminished the levels of inflammatory cytokine transcripts, and protected connective tissues through the expression of anti-oxidation proteins such as nuclear factor erythroid-related factor (Nrf)2 and heme oxygenase (HO)1. Additionally, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels did not differ between the BMDA- or DMMA-treated and control animals, indicating that the compounds do not possess liver toxicity. Taken together, these findings propose that BMDA and DMMA could be used as new drugs for curing inflammatory bowel disease (IBD) and RA.

The inhibition of p38 MAPK blocked inflammation to restore the functions of rat meibomian gland epithelial cells

Meibomian glands (MGs) are vital for ocular surface health. However, the roles of inflammation in the progression of meibomian gland dysfunction (MGD) are largely unknown. In this study, the roles of the inflammation factor interleukin-1β (IL-1β) via the p38 mitogen-activated protein kinases (MAPK) signaling pathway on rat meibomian gland epithelial cells (RMGECs) were explored. Eyelids from adult rat mice at 2 months and 2 years of age were stained with specific antibodies against IL-1β to identify inflammation levels. RMGECs were exposed to IL-1β and/or SB203580, a specific inhibitor of p38 MAPK signaling pathway, for 3 days. Cell proliferation, keratinization, lipid accumulation, and matrix metalloproteinases 9 (MMP9) expression were evaluated by MTT assay, polymerase chain reaction (PCR), immunofluorescence staining, apoptosis assay, lipid staining, and Western blot analyses. We found that IL-1β was significantly higher in the terminal ducts of MGs in rats with age-related MGD than in young rats. IL-1β inhibited cell proliferation, suppressed lipid accumulation and peroxisome proliferator activator receptor γ (PPARγ) expression, and promoted apoptosis while activating the p38 MAPK signaling pathway. Cytokeratin 1 (CK1), a marker for complete keratinization, and MMP9 in RMGECs were also up-regulated by IL-1β. SB203580 effectively diminished the effects of IL-1β on differentiation, keratinization, and MMP9 expression by blocking IL-1β-induced p38 MAPK activation, although it also inhibited cell proliferation. The inhibition of the p38 MAPK signaling pathway blocked IL-1β-induced differentiation reduction, hyperkeratinization, and MMP9 overexpression of RMGECs, which provides a potential therapy for MGD.

Oncostatin M stimulates prostaglandin D2-induced osteoprotegerin and interleukin-6 synthesis in osteoblasts

Oncostatin M produced by osteal macrophages plays a significant role in fracture healing. Osteoprotegerin (OPG) secreted by osteoblasts, binds to the receptor activator of nuclear factor-κB (RANK) ligand (RANKL) as a decoy receptor and prevents RANKL from binding to RANK, resulting in bone resorption suppression. Interleukin-6 (IL-6) is a pro-inflammatory cytokine and generally regulates bone resorption. However, accumulating evidence suggests that IL-6 plays pivotal roles in bone formation. We previously showed that prostaglandin D2 (PGD2) induces OPG synthesis by activating p38 mitogen-activated protein (MAP) kinase, stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), and p44/p42 MAP kinase in osteoblast-like MC3T3-E1 cells. Furthermore, we demonstrated that PGD2 stimulates IL-6 synthesis by activating p38 MAP kinase and p44/p42 MAP kinase in MC3T3-E1 cells. In the present study, we investigated whether oncostatin M affects PGD2-stimulated OPG and IL-6 synthesis in MC3T3-E1 cells through MAP kinase activation. The osteoblast-like MC3T3-E1 cells and normal human osteoblasts were treated with oncostatin M and subsequently stimulated with PGD2. Consequently, oncostatin M significantly increased the PGD2-stimulated OPG and IL-6 release in both cells. Oncostatin M significantly enhanced mRNA expression levels of OPG and IL-6 induced by PGD2 similarly in both cells. Regarding the signaling mechanism, oncostatin M did not affect the phosphorylation of p38 MAP kinase, SAPK/JNK, and p44/p42 MAP kinase. Our results suggest that oncostatin M upregulates the PGD2-stimulated OPG and IL-6 synthesis in osteoblasts and therefore affects bone remodeling. However, OPG and IL-6 synthesis are not mediated through p38 MAP kinase, p44/p42 MAP kinase, or SAPK/JNK pathways.

Osteomodulin contributes to keloid development by regulating p38 MAPK signaling.

A keloid is a classic fibrotic skin disease characterized by excessive deposition of extracellular matrix (ECM). Osteomodulin (OMD) is a heterologous protein that is a part of osteoadherin and plays a role in modulating ECM deposition. In this study, we investigated the effects of OMD on ECM synthesis and the tumor-like phenotype of keloid fibroblasts. We enrolled 10 patients with keloids and 10 age- and sex-matched healthy individuals, whose keloid or normal skin tissues were collected during surgery. Real-time quantitative polymerase chain reaction (qRT-PCR), western blotting, and immunohistochemical staining were performed to analyze OMD expression in skin tissues. Cell transfection, CCK-8 assay, EdU staining, Transwell assay, qRT-PCR, western blotting, and immunofluorescence were performed to study the effects of OMD on primary keloid-derived fibroblasts (KFs). OMD exhibited greater expression in human keloid specimens than in normal skin tissues. Consistently, higher expression of OMD was observed in KFs, compared to that in normal fibroblasts. Silencing OMD expression in transforming growth factor (TGF)-β1-treated KFs inhibited cell proliferation and migration, as well as collagen and fibronectin expression; however, overexpression of OMD had the opposite effect. p38 mitogen-activated protein kinase (MAPK) was activated in keloid tissues but not in normal skin. OMD was positively correlated with p38 MAPK activation. Adding SB203580, p38 MAPK inhibitor, significantly reversed the effects of OMD on the regulation of KF phenotype. The high expression of OMD may contribute to hyperproliferation of KFs, their migration, and excess ECM synthesis in KFs via regulation of the p38 MAPK signaling pathway.

Abstract 5831: CD200R signaling contributes to unfavorable tumor microenvironment through regulating production of chemokines by tumor associated macrophages

Abstract CD200 is overexpressed in human solid tumors such as neuroblastoma and melanoma and is considered as an immune checkpoint molecule for dampening cancer immunity. However, how CD200 affects tumor microenvironment (TME) and host immunity is incompletely understood. In the present study, we evaluated the role of the CD200-CD200R pathway in TME using loss-of-function CD200R-/- mice, along with two clinically relevant, CD200-positive murine tumor models: the mouse neuroblastoma NB9464D and melanoma Yummer1.7. We found that comparing with the wild type mice, CD200R-/- mice were significantly more potent in rejecting these CD200+ tumors. Transcriptome analysis by single cell RNA sequencing demonstrated that tumors from CD200R-/- mice had more infiltration of CD45+ immune cells, including CD4+ and CD8+ T cells, and NK cells but less infiltration of neutrophils. Antibody depletion experiments revealed that immune effectors including CD8+ T cells and NK cells in combination, are crucial in inhibiting tumor growth in CD200R-/- mice. Mechanistically, we found that CD200R signaling differentially regulates the expression of chemokines in tumor-associated macrophages (TAMs). In the absence of CD200R, TAMs up-regulate CCL24 and CCL8 and down-regulate CXCL3, CXCL2, and CCL3 via activation of ERK and/or p38 MAP kinases. Increased expression of CCL24 in CD200R-deficient tumors resulted in increased infiltration of eosinophils, which also contributes to anti-tumor activity. Overall, we conclude that CD200R-mediated signaling, via interaction with CD200 on tumor cells, contributes to unfavorable TME primarily through chemokine-dependent recruitment of immune suppressive neutrophils and exclusion of anti-cancer immune effectors. Our study has significant implications in developing CD200-targeted immunotherapy of solid tumors. Citation Format: Cho-Hao Lin, Fatemeh Talebian, Yang Li, Jianmin Zhu, Bolin Zhao, Jin-Qing Liu, Sujit Basu, Xueling Pan, Xi Chen, Pearlly Yan, William E. Carson, Gang Xin, Haitao Wen, Ruoning Wang, Zihai Li, Qin Ma, Xue-Feng Bai. CD200R signaling contributes to unfavorable tumor microenvironment through regulating production of chemokines by tumor associated macrophages [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5831.

Involvement of matrix metalloproteinase 1 and urokinase-type plasminogen activator in the PKCα-p38 MAPK pathway-mediated progression of human liver cancer cells.

Our previous studies have shown that the plasminogen activator (PA) and matrix metalloproteinases (MMPs) proteinase systems were highly expressed in highly malignant liver cancer cells and regulated by PKCα. This study investigates whether the PKCα regulation of PA and MMPs systems is conducted through p38 mitogen-activated protein kinase (MAPK) signaling and the pathway is responsible for promoting cell progression. We found that the expressions of p38 MAPK in both highly malignant HA22T/VGH and SK-Hep-1 liver cancer cells were higher than that in other lower malignancy liver cancer cells. Since PKCα activates p38 MAPK in progression of liver cancer, we suspected the PKCα/p38 MAPK signaling pathway to be involved in the regulation of MMPs and PA systems. When SK-Hep-1 cells were treated with SB203580 or DN-p38, only MMP-1 and u-PA mRNA expressions decreased. The p38 MAPK inhibition also decreased the cell migration and invasion. In addition, the mRNA decay assays showed that the higher expressions of MMP-1 and u-PA mRNA in SK-Hep-1 cells were due to the alteration of mRNA stability by p38 MAPK inhibition. Zymography of SK-Hep-1 cells treated with siPKCα vector also showed the decrease of the activity of MMP-1 and u-PA and confirmed changes in mRNA level. Furthermore, only the transfection of MKK6 to the siPKCα-treated SK-Hep-1 stable clone cell restored the attenuation of MMP-1 and u-PA expressions. The treatment of SK-Hep-1 cells with either inhibitor of MMP-1 or u-PA reduced migration, and the reduction was enhanced with both inhibitors. In addition, tumorigenesis was also reduced with both inhibitors. These data suggest a novel finding that MMP-1 and u-PA are critical components in PKCα/MKK6/p38 MAPK signaling pathway which mediates liver cancer cell progression, and that the targeting of both genes may be a viable approach in liver cancer treatment.

Dual-specificity phosphatase 26-dificient neurons are susceptible to oxygen-glucose deprivation/reoxygenation-evoked apoptosis and proinflammatory response by affecting the TAK1-medaited JNK/P38 MAPK pathway.

Dual-specificity phosphatase 26 (DUSP26) is linked to a broad range of human disorders as it affects numerous signaling cascades. However, the involvement of DUSP26 in ischemic stroke has not been explored. Here, we investigated DUSP26 as a key mediator of oxygen-glucose deprivation/reoxygenation (OGD/R)-associated neuronal injury, an in vitro model for investigating ischemic stroke. A decline in DUSP26 occurred in neurons suffering from OGD/R. A deficiency in DUSP26 rendered neurons more susceptible to OGD/R by aggravating neuronal apoptosis and inflammation, while the overexpression of DUSP26 blocked OGD/R-evoked neuronal apoptosis and inflammation. Mechanistically, enhanced phosphorylation of transforming growth factor-β-activated kinase 1 (TAK1), c-Jun N-terminal kinase (JNK) and P38 mitogen-activated protein kinase (MAPK) was evidenced in DUSP26-deficient neurons suffering from OGD/R, whereas the opposite effects were observed in DUSP26-overexpressed neurons. Moreover, the inhibition of TAK1 abolished the DUSP26-deficiency-elicited activation of JNK and P38 MAPK and exhibited anti-OGD/R injury effects in DUSP26-deficiency neurons. Results from these experiments show that DUSP26 is essential for neurons in defending against OGD/R insult, while neuroprotection is achieved by restraining the TAK1-mediated JNK/P38 MAPK pathway. Therefore, DUSP26 may serve as a therapeutic target for the management of ischemic stroke.

Ionizing Radiation Selectively Increases CXC Ligand 10 Level via the DNA-Damage-Induced p38 MAPK-STAT1 Pathway in Murine J774A.1 Macrophages.

Ionizing radiation (IR) is an important means of tumor treatment in addition to surgery and drugs. Attempts have been made to improve the efficiency of radiotherapy by identifying the various biological effects of IR on cells. Components of the tumor microenvironment, such as macrophages, fibroblasts, and vascular endothelial cells, influence cancer treatment outcomes through communication with tumor cells. In this study, we found that IR selectively increased the production of CXC motif chemokine ligand 10 (CXCL10), which is emerging as an important biomarker for determining the prognosis of anticancer treatments, without changing the levels of CXCL9 and CXCL11 in murine J774A.1 macrophages. Pretreatment with KU55933, an ataxia telangiectasia mutated (ATM) kinase inhibitor, significantly inhibited IR-induced CXCL10 production. In contrast, pretreatment with N-acetyl-cysteine or glutathione, a reactive oxygen species scavenger, did not inhibit IR-induced CXCL10 production. Further, we attempted to identify the intracellular molecular target associated with the IR-induced increase in CXCL10 secretion by J774A.1 macrophages. IR phosphorylated p38 mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 1 (STAT1) in J774A.1 macrophages, and p38 MAPK and STAT1 were involved in CXCL10 via IR using pharmacological inhibitors (SB203580 and fludarabine, respectively) and the siRNA technique.

PROPHYLACTIC n CMT-3 ATTENUATES SEPSIS-INDUCED ACUTE KIDNEY INJURY IN ASSOCIATION WITH NLRP3 INFLAMMASOME ACTIVATION AND APOPTOSIS.

Background: The kidney is the most common extrapulmonary organ injured in sepsis. The current study examines the ability of aerosolized nanochemically modified tetracycline 3 (nCMT-3), a pleiotropic anti-inflammatory agent, to attenuate acute kidney injury (AKI) caused by intratracheal LPS. Methods: C57BL/6 mice received aerosolized intratracheal nCMT-3 (1 mg/kg) or saline, followed by intratracheal LPS (2.5 mg/kg) to induce acute lung injury-induced AKI. Tissues were harvested at 24 h. The effects of nCMT-3 and LPS on AKI were assessed by plasma/tissue levels of serum urea nitrogen, creatinine, neutrophil gelatinase-associated lipocalin, kidney injury molecule 1, and renal histology. Renal matrix metalloproteinase (MMP) level/activity, cytochrome C, Bax, Bcl-2, caspase-3, p38 mitogen-activated protein kinase activation, NLRP3, and caspase-1 were also measured. Apoptotic cells in kidney were determined by TUNEL assay. Renal levels of IL-1β and IL-6 were measured to assess inflammation. Results: Acute lung injury-induced AKI was characterized by increased plasma blood urea nitrogen, creatinine, injury biomarkers (neutrophil gelatinase-associated lipocalin, kidney injury molecule 1), and histologic evidence of renal injury. Lipopolysaccharide-treated mice demonstrated renal injury with increased levels of inflammatory cytokines (IL-1β, IL-6), active MMP-2 and MMP-9, proapoptotic proteins (cytochrome C, Bax/Bcl-2 ratio, cleaved caspase-3), apoptotic cells, inflammasome activation (NLRP3, caspase-1), and p38 signaling. Intratracheal nCMT-3 significantly attenuated all the measured markers of renal injury, inflammation, and apoptosis. Conclusions: Pretreatment with aerosolized nCMT-3 attenuates LPS-induced AKI by inhibiting renal NLRP3 inflammasome activation, renal inflammation, and apoptosis.

Chemical Knockdown of Phosphorylated p38 Mitogen-Activated Protein Kinase (MAPK) as a Novel Approach for the Treatment of Alzheimer's Disease.

Targeted protein degradation (TPD) provides unique advantages over gene knockdown in that it can induce selective degradation of disease-associated proteins attributed to pathological mutations or aberrant post-translational modifications (PTMs). Herein, we report a protein degrader, PRZ-18002, that selectively binds to an active form of p38 MAPK. PRZ-18002 induces degradation of phosphorylated p38 MAPK (p-p38) and a phosphomimetic mutant of p38 MAPK in a proteasome-dependent manner. Given that the activation of p38 MAPK plays pivotal roles in the pathophysiology of Alzheimer's disease (AD), selective degradation of p-p38 may provide an attractive therapeutic option for the treatment of AD. In the 5xFAD transgenic mice model of AD, intranasal treatment of PRZ-18002 reduces p-p38 levels and alleviates microglia activation and amyloid beta (Aβ) deposition, leading to subsequent improvement of spatial learning and memory. Collectively, our findings suggest that PRZ-18002 ameliorates AD pathophysiology via selective degradation of p-p38, highlighting a novel therapeutic TPD modality that targets a specific PTM to induce selective degradation of neurodegenerative disease-associated protein.

IRE1α arm of unfolded protein response in muscle-specific TGF-β signaling-mediated regulation of muscle cell immunological properties

Endoplasmic reticulum stress (ERS) and the unfolded protein response (UPR) are involved in various muscle pathological states. The IRE1α arm of UPR can affect immunological properties of myofiber through restraining p38 mitogen-activated protein kinases (MAPK) activation under inflammatory milieu. However, the relevant pathway molecules regulating the initiation of the IRE1α arm in myofiber remain unclear. In this work, expression of transforming growth factor-beta (TGF-β) and TGF-β receptor II (TGF-βr2), and UPR pathway activation were examined in cardiotoxin (CTX)-damaged mouse muscle, which revealed the activation of TGF-β signaling and UPR in CTX-damaged muscle and in regenerating myofibers. Using control or transgenic mice with TGF-βr2 deleted in skeletal muscle (SM TGF-βr2−/−) and the derived primary differentiating myogenic precursor cells (MPCs) treated with/without ERS activator or inhibitor, IRE1α pathway inhibitor, or TGF-β signaling activator, this study further revealed an essential role of intrinsic TGF-β signaling in regulating muscle cell to express inflammation-related molecules including H-2Kb, H2-Eα, TLR3, and special myokines. TGF-β signaling prompted UPR IRE1α arm and restrained p38 MAPK activation in myofiber under inflammatory milieu. This study uncovers a previously unrecognized function of TGF-β signaling acting as an upstream factor controlling myofiber immune capacities in the inflamed state through the UPR–IRE1α–p38 MAPK pathway.

Proteomic landscape of the extracellular matrix in the fibrotic kidney

The extracellular matrix (ECM) is a complex three-dimensional network of proteins surrounding cells, forming a niche that controls cell adhesion, proliferation, migration and differentiation. The ECM network provides an architectural scaffold for surrounding cells and undergoes dynamic changes in composition and contents during the evolution of chronic kidney disease (CKD). Here, we unveiled the proteomic landscape of the ECM by delineating proteome-wide and ECM-specific alterations in normal and fibrotic kidneys. Decellularized kidney tissue scaffolds were made and subjected to proteomic profiling by liquid chromatography with tandem mass spectrometry. A total of 172 differentially expressed proteins were identified in these scaffolds from mice with CKD. Through bioinformatics analysis and experimental validation, we identified a core set of nine signature proteins, which could play a role in establishing an oxidatively stressed, profibrotic, proinflammatory and antiangiogenetic microenvironment. Among these nine proteins, glutathione peroxidase 3 (GPX3) was the only protein with downregulated expression during CKD. Knockdown of GPX3 invivo augmented ECM expression and aggravated kidney fibrotic lesions after obstructive injury. Transcriptomic profiling revealed that GPX3 depletion resulted in an altered expression of the genes enriched in hypoxia pathway. Knockdown of GPX3 induced NADPH oxidase 2 expression, promoted kidney generation of reactive oxygen species and activated p38 mitogen-activated protein kinase. Conversely, overexpression of exogenous GPX3 alleviated kidney fibrosis, inhibited NADPH oxidase 2 and p38 mitogen-activated protein kinase. These findings suggest that oxidative stress is a pivotal element of the fibrogenic microenvironment. Thus, our studies represent a comprehensive proteomic characterization of the ECM in the fibrotic kidney and provide novel insights into molecular composition of the fibrogenic microenvironment.