P38 Mitogen-activated Protein Kinase Inhibitor Research Articles

Dual p38MAPK and MEK inhibition disrupts adaptive chemoresistance in mesenchymal glioblastoma to temozolomide.

Precision treatment of glioblastoma is increasingly focused on molecular subtyping, with the mesenchymal subtype particularly resistant to temozolomide. Here, we aim to develop a targeted therapy for temozolomide resensitization in the mesenchymal subtype. We integrated kinomic profiles and kinase inhibitor screens from patient-derived proneural and mesenchymal glioma-propagating cells and public clinical datasets to identify key protein kinases implicated in temozolomide resistance. RNAseq, apoptosis assays, and comet assays were used to examine the role of p38MAPK signaling and adaptive chemoresistance in mesenchymal cells. The efficacy of dual p38MAPK and MEK/ERK inhibition using ralimetinib (selective orally active p38MAPK inhibitor; phase I/II for glioblastoma) and binimetinib (approved MEK1/2 inhibitor for melanoma; phase II for high-grade glioma) in primary and recurrent mesenchymal tumors was evaluated using an intracranial patient-derived tumor xenograft model, focusing on survival analysis. Our transcriptomic-kinomic integrative analysis revealed p38MAPK as the prime target whose gene signature enables patient stratification based on their molecular subtypes and provides prognostic value. Repurposed p38MAPK inhibitors synergize favorably with temozolomide to promote intracellular retention of temozolomide and exacerbate DNA damage. Mesenchymal cells exhibit adaptive chemoresistance to p38MAPK inhibition through a pH-/calcium-mediated MEK/ERK pathway. Dual p38MAPK and MEK inhibition effectively maintain temozolomide sensitivity in primary and recurrent intracranial mesenchymal glioblastoma xenografts. Temozolomide resistance in mesenchymal glioblastoma is associated with p38MAPK activation. Adaptive chemoresistance in p38MAPK-resistant cells is mediated by MEK/ERK signaling. Adjuvant therapy with dual p38MAPK and MEK inhibition prolongs temozolomide sensitivity, which can be developed into a precision therapy for the mesenchymal subtype.

SARS-CoV-2 spike protein-ACE2 interaction increases carbohydrate sulfotransferases and reduces N-acetylgalactosamine-4-sulfatase by p38 MAPK

Immunostaining in lungs of patients who died with COVID-19 infection showed increased intensity and distribution of chondroitin sulfate and decline in N-acetylgalactostamine-4-sulfatase (Arylsulfatase B; ARSB). To explain these findings, human small airway epithelial cells were exposed to the SARS-CoV-2 spike protein receptor binding domain (SPRBD) and transcriptional mechanisms were investigated. Phospho-p38 MAPK and phospho-SMAD3 increased following exposure to the SPRBD, and their inhibition suppressed the promoter activation of the carbohydrate sulfotransferases CHST15 and CHST11, which contributed to chondroitin sulfate biosynthesis. Decline in ARSB was mediated by phospho-38 MAPK-induced N-terminal Rb phosphorylation and an associated increase in Rb-E2F1 binding and decline in E2F1 binding to the ARSB promoter. The increases in chondroitin sulfotransferases were inhibited when treated with phospho-p38-MAPK inhibitors, SMAD3 (SIS3) inhibitors, as well as antihistamine desloratadine and antibiotic monensin. In the mouse model of carrageenan-induced systemic inflammation, increases in phospho-p38 MAPK and expression of CHST15 and CHST11 and declines in DNA-E2F binding and ARSB expression occurred in the lung, similar to the observed effects in this SPRBD model of COVID-19 infection. Since accumulation of chondroitin sulfates is associated with fibrotic lung conditions and diffuse alveolar damage, increased attention to p38-MAPK inhibition may be beneficial in ameliorating Covid-19 infections.

Lactate induces C2C12 myoblasts differentiation by mediating ROS/p38 MAPK signalling pathway

Lactate serves not merely as an energy substrate for skeletal muscle but also regulates myogenic differentiation, leading to an elevation of reactive oxygen species (ROS) levels. The present study was focused on exploring the effects of lactate and ROS/p38 MAPK in promoting C2C12 myoblasts differentiation. Our results demonstrated that lactate increased C2C12 myoblasts differentiation at a range of physiological concentrations, accompanied by enhanced ROS contents. We used n-acetylcysteine (NAC, a ROS scavenger) pretreatment and found that it delayed lactate-induced C2C12 myoblast differentiation by upregulating Myf5 expression on days 5 and 7 and lowering MyoD and MyoG expression. The finding implies that lactate accompanies ROS-dependent manner to promote C2C12 myoblast differentiation. Additionally, lactate significantly increased p38 MAPK phosphorylation to promote C2C12 cell differentiation, but pretreatment with SB203580 (p38 MAPK inhibitor) reduced lactate-induced C2C12 myoblasts differentiation. whereas lactate pretreatment with NAC inhibited p38 MAPK phosphorylation in C2C12 cells, demonstrating that lactate mediated ROS and regulated the p38 MAPK signalling pathway to promote C2C12 cell differentiation. In conclusion, our results suggest that the promotion of C2C12 myoblasts differentiation by lactate is dependent on ROS and the p38 MAPK signalling pathway. These observations reveal a beneficial role for lactate in increasing myogenesis through ROS-sensitive mechanisms as well as providing new ideas regarding the positive impact of ROS in improving the function of skeletal muscle.

Isolation and Identification of a Tibetan Pig Porcine Epidemic Diarrhoea Virus Strain and Its Biological Effects on IPEC-J2 Cells.

Porcine epidemic diarrhoea virus (PEDV) is a coronavirus that can cause severe watery diarrhoea in piglets, with high morbidity and mortality rates, seriously hindering the healthy development of the global swine industry. In this study, we isolated a strain of PEDV from Tibetan pigs and named it CH/GS/2022. Subsequently, we screened the apoptosis signals of PEDV-infected IPEC-J2 cells and studied the correlation between apoptosis signals and cell apoptosis. The results showed that different infections of PEDV induced different degrees of apoptosis in cells, and PEDV-induced cell apoptosis was dose-dependent. We then detected the expression of the p53, p38, JNK, Bax, and Bcl-2 genes in the apoptosis signal pathway. The results showed that 24 h after PEDV infection, the expression of the p53, p38, JNK, and Bax genes in IPEC-J2 cells increased significantly, while the expression of the Bcl-2 gene decreased significantly (p < 0.05). Subsequently, we used Western blot to detect the protein levels of these five genes, and the results showed that PEDV infection upregulated the expression of p53, p38, JNK, and Bax proteins (p < 0.05) while downregulating the expression of Bcl-2 protein (p < 0.05). Thus, it was initially inferred that PEDV infection could regulate cell apoptosis by activating the p53, p38, and JNK signalling pathways. Finally, we further investigated the apoptosis of the cells through the use of inhibitors. The results indicated that the p53 inhibitor Pifithrin-α has a significant inhibitory effect on the expression of the p53 protein after PEDV infection and can reverse the expression levels of Bax and Bcl-2 proteins. This suggested that p53 is involved in PEDV-induced cell apoptosis. Similarly, the p38 MAPK inhibitor SB203580 has an inhibitory effect on the expression of the p38 protein and can reverse the expression levels of Bax and Bcl-2 proteins. This suggested that p38 is also involved in PEDV-induced cell apoptosis. On the other hand, the JNK inhibitor SP600125 has no inhibitory effect on the expression of the JNK protein after PEDV infection, but the expression levels of Bax and Bcl-2 proteins have changed. Furthermore, it is noteworthy that SP600125 can inhibit the activity of apoptotic proteins but not their levels, resulting in reduced cell apoptosis. These preliminary results indicated that JNK may be involved in PEDV-induced IPEC-J2 cell apoptosis.

Formyl Peptide Receptor 2-Dependent cPLA2 and 5-LOX Activation Requires a Functional NADPH Oxidase.

Phospholipases (PL) A2 catalyzes the hydrolysis of membrane phospholipids and mostly generates arachidonic acid (AA). The enzyme 5-lipoxygenase (5-LOX) can metabolize AA to obtain inflammatory leukotrienes, whose biosynthesis highly depends on cPLA2 and 5-LOX activities. Formyl Peptide Receptor 2 (FPR2) belongs to a subfamily of class A GPCRs and is considered the most versatile FPRs isoform. Signaling triggered by FPR2 includes the activation of several downstream kinases and NADPH oxidase (NOX)-dependent ROS generation. In a metabolomic analysis we observed a significant increase in AA concentration in FPR2-stimulated lung cancer cell line CaLu-6. We analyzed cPLA2 phosphorylation and observed a time-dependent increase in cPLA2 Ser505 phosphorylation in FPR2-stimulated cells, which was prevented by the MEK inhibitor (PD098059) and the p38MAPK inhibitor (SB203580) and by blocking NOX function. Similarly, we demonstrated that phosphorylation of 5-LOX at Ser271 and Ser663 residues requires FPR2-dependent p38MAPK and ERKs activation. Moreover, we showed that 5-LOX Ser271 phosphorylation depends on a functional NOX expression. Our overall data demonstrate for the first time that FPR2-induced ERK- and p38MAPK-dependent phosphorylation/activation of cPLA2 and 5-LOX requires a functional NADPH oxidase. These findings represent an important step towards future novel therapeutic possibilities aimed at resolving the inflammatory processes underlying many human diseases.

Vitamin A regulates mitochondrial biogenesis and function through p38 MAPK-PGC-1α signaling pathway and alters the muscle fiber composition of sheep

BackgroundVitamin A (VA) and its metabolite, retinoic acid (RA), are of great interest for their wide range of physiological functions. However, the regulatory contribution of VA to mitochondrial and muscle fiber composition in sheep has not been reported.MethodLambs were injected with 0 (control) or 7,500 IU VA palmitate into the biceps femoris muscle on d 2 after birth. At the age of 3 and 32 weeks, longissimus dorsi (LD) muscle samples were obtained to explore the effect of VA on myofiber type composition. In vitro, we investigated the effects of RA on myofiber type composition and intrinsic mechanisms.ResultsThe proportion of type I myofiber was greatly increased in VA-treated sheep in LD muscle at harvest. VA greatly promoted mitochondrial biogenesis and function in LD muscle of sheep. Further exploration revealed that VA elevated PGC-1α mRNA and protein contents, and enhanced the level of p38 MAPK phosphorylation in LD muscle of sheep. In addition, the number of type I myofibers with RA treatment was significantly increased, and type IIx myofibers was significantly decreased in primary myoblasts. Consistent with in vivo experiment, RA significantly improved mitochondrial biogenesis and function in primary myoblasts of sheep. We then used si-PGC-1α to inhibit PGC-1α expression and found that si-PGC-1α significantly abrogated RA-induced the formation of type I myofibers, mitochondrial biogenesis, MitoTracker staining intensity, UQCRC1 and ATP5A1 expression, SDH activity, and enhanced the level of type IIx muscle fibers. These data suggested that RA improved mitochondrial biogenesis and function by promoting PGC-1α expression, and increased type I myofibers. In order to prove that the effect of RA on the level of PGC-1α is caused by p38 MAPK signaling, we inhibited the p38 MAPK signaling using a p38 MAPK inhibitor, which significantly reduced RA-induced PGC-1α and MyHC I levels.ConclusionVA promoted PGC-1α expression through the p38 MAPK signaling pathway, improved mitochondrial biogenesis, and altered the composition of muscle fiber type.Graphical

Retinoprotective Effect of SkQ1, Visomitin Eye Drops, Is Associated with Suppression of P38 MAPK and ERK1/2 Signaling Pathways Activity.

Visomitin eye drops are the first and, so far, the only drug based on SkQ1 - the mitochondria-targeted antioxidant 10-(6'-plastoquinonyl) decyltriphenylphosphonium, developed in the laboratories of Moscow State University under the leadership of Academician V. P. Skulachev. SkQ1 is considered as a potential tool to combat the aging program. We have previously shown that it is able to prevent and/or suppress development of all manifestations of accelerated senescence in OXYS rats, including retinopathy, similar to the age-related macular degeneration (AMD). Here, we assessed the effect of Visomitin instillations on progression of the AMD-like pathology and p38 MAPK and ERK1/2 activity in the OXYS rat retina (from the age of 9 to 12 months). Wistar and OXYS rats treated with placebo (composition identical to Visomitin with the exception of SkQ1) were used as controls. Ophthalmological examination showed that in the OXYS rats receiving placebo, retinopathy progressed and severity of clinical manifestations did not differ from the intact OXYS rats. Visomitin suppressed progression of the AMD-like pathology in the OXYS rats and significantly improved structural and functional parameters of the retinal pigment epithelium cells and state of microcirculation in the choroid, which, presumably, contributed to preservation of photoreceptors, associative and ganglion neurons. It was found that the activity of p38 MAPK and ERK1/2 in the retina of 12-month-old OXYS rats is higher than that of the Wistar rats of the same age, as indicated by the increased content of phosphorylated forms of p38 MAPK and ERK1/2 and their target protein tau (at position T181 and S396). Visomitin decreased phosphorylation of p38 MAPK, ERK1/2, and tau indicating suppression of activity of these MAPK signaling cascades. Thus, Visomitin eye drops are able to suppress progression of the AMD-like pathology in the OXYS rats and their effect is associated with the decrease in activity of the MAPK signaling cascades.

The CoREST repressor complex mediates phenotype switching and therapy resistance in melanoma.

Virtually all patients with BRAF-mutant melanoma develop resistance to MAPK inhibitors largely through nonmutational events. Although the epigenetic landscape is shown to be altered in therapy-resistant melanomas and other cancers, a specific targetable epigenetic mechanism has not been validated. Here, we evaluated the corepressor for element 1-silencing transcription factor (CoREST) epigenetic repressor complex and the recently developed bivalent inhibitor corin within the context of melanoma phenotype plasticity and therapeutic resistance. We found that CoREST was a critical mediator of the major distinct melanoma phenotypes and that corin treatment of melanoma cells led to phenotype reprogramming. Global assessment of transcript and chromatin changes conferred by corin revealed specific effects on histone marks connected to epithelial-mesenchymal transition-associated (EMT-associated) transcription factors and the dual-specificity phosphatases (DUSPs). Remarkably, treatment of BRAF inhibitor-resistant (BRAFi-R) melanomas with corin promoted resensitization to BRAFi therapy. DUSP1 was consistently downregulated in BRAFi-R melanomas, which was reversed by corin treatment and associated with inhibition of p38 MAPK activity and resensitization to BRAFi therapies. Moreover, this activity was recapitulated by the p38 MAPK inhibitor BIRB 796. These findings identify the CoREST repressor complex as a central mediator of melanoma phenotype plasticity and resistance to targeted therapy and suggest that CoREST inhibitors may prove beneficial for patients with BRAFi-resistant melanoma.

The qualitative and quantitative analysis of Ganoderma lucidum spore powder chemical compounds as p38 MAPK inhibitors by the generation and verification of pharmacophore modelling

Ganoderma lucidum spore powder (GLSP), a high-value health food and medicine, has attracted widespread attention for its biological components and abundant pharmacological activities. However, the chemical component classification, fragmentation patterns, fatty acid quantification and biological activities of GLSP against p38 MAPK have not been clearly articulated. In the present study, a total of 142 chemical compounds were identified in GLSP by UPLC-Orbitrap-HRMS, including 55 lipids (3 glycerides, 21 phospholipids, 4 glycolipids, 5 sterols, 22 fatty acids), 25 triterpenes, 10 alkaloids, 27 organic acids, and 25 others. The characteristic fragmentation patterns of 12 representative components were summarized. As the major compounds, GC-FID quantified 6 unsaturated fatty acids and 8 saturated fatty acids in GLSP and Ganoderma sinense spore powder with little difference. Then, 7 potential p38 MAPK inhibitors were virtually screened out based on the pharmacophore modelling and molecular docking. Finally, Ganoderic acid C6, D, G, AM1, Ganoderenic acid F, Methyl lucidenate E2 were quantified in GLSP samples, and the selected 6 triterpenoids were evaluated and verified as potential inhibitors of p38 MAPK after western blotting in HepG2 cells, which could provide a theoretical basis for new therapeutic opportunities by targeting p38 MAPK signaling with few side effects.

Vitamin D3 inhibits p38 MAPK and senescence-associated inflammatory mediator secretion by senescent fibroblasts that impacts immune responses during ageing.

Vitamin D3 replacement in older insufficient adults significantly improves their antigen-specific varicella zoster virus (VZV) cutaneous immunity. However, the mechanisms involved in this enhancement of cutaneous immunity are not known. Here, we show for the first time that vitamin D3 blocks the senescence-associated secretory phenotype (SASP) production by senescent fibroblasts by partially inhibiting the p38 MAPK pathway. Furthermore, transcriptomic analysis of skin biopsies from older subjects after vitamin D3 supplementation shows that vitamin D3 inhibits the same inflammatory pathways in response to saline as the specific p38 inhibitor, losmapimod, which also enhances immunity in the skin of older subjects. Vitamin D3 supplementation therefore may enhance immunity during ageing in part by blocking p38 MAPK signalling and in turn inhibit SASP production from senescent cells invivo.

Tumor Cell-Intrinsic p38 MAPK Signaling Promotes IL1α-Mediated Stromal Inflammation and Therapeutic Resistance in Pancreatic Cancer.

Inhibition of p38 MAPK suppresses tumor cell-derived IL1α and attenuates the inflammatory stroma and immunosuppressive tumor microenvironment to overcome chemotherapeutic resistance in pancreatic cancer.

Effects of kappa-opioid agonist U-50488 and p38 MAPK inhibitor SB203580 on the spike activity of pyramidal neurons in the basolateral amygdala

Introduction: Kappa-opioid receptor (KOR) signaling in the basolateral amygdala (BLA) underlies KOR agonist-induced aversion. In this study, we aimed to understand the individual and combined effects of KOR agonist U-50488 and p38 MAPK inhibitor SB203580 on the spiking activity of pyramidal neurons in the BLA to shed light on the complex interplay between KORs, the p38 MAPK, and neuronal excitability. Materials and Methods: Electrophysiological experiments were performed using the patch-clamp technique in the whole-cell configuration. Rat brain slices containing the amygdala were prepared, and pyramidal neurons within the BLA were visually patched and recorded in the current clamp mode. The neurons were identified by their accommodation properties and neural activity signals were amplified and analyzed. Using local perfusion, we obtained three dose-response curves for: (a) U-50488 (0.001–10 μM); (b) U-50488 (0.001–10 μM) in the presence of SB203580 (1 μM); and (c) U-50488 (0.01–10 μM) in the presence of SB203580 (5 μM). Results: After the application of U-50488, pyramidal neurons had a higher action potential firing rate in response to a current injection than control neurons (p&lt;0.001). The dose-dependent curves we obtained indicate that the combination of U-50488 and SB203580 results in non-competitive antagonism. This conclusion is supported by the observed change in the curve’s slope with reduction in the maximum effect of U-50488. Thus, it can be assumed that the increase in spike activity of pyramidal neurons of the amygdala is mediated through the beta-arrestin pathway. When this pathway is blocked, the spike activity reverts to its baseline level. Conclusion: Our study found that the KOR agonist-induced spiking activity of the BLA pyramidal neurons is mediated by the beta-arrestin pathway and can be suppressed by the application of the p38 MAPK inhibitor SB203580.

Effect of acupoint catgut embedding on p38 MAPK pathway in the lung tissue of asthmatic rats.

To observe the effect of catgut embedding at "Feishu"(BL13), "Dingchuan" (EX-B1) and "Danzhong" (CV17) on expression of phosphorylated p38 mitogen activated protein kinase (p-p38 MAPK), interleukin-4 (IL-4), interferon-γ (IFN-γ) and changes of airway epithelial cells (AEC) in the lung tissue of bronchial asthma (BA) rats, so as to explore its mechanisms underlying improvement of BA. Forty male Wistar rats were randomly and equally divided into blank control, model, dexamethasone (DEX) and catgut embedding groups. The BA model was established by intraperitoneal injection of suspension of ovalbumin and aluminum hydroxide. Rats of the DEX group received intraperitoneal injection of DEX (1.5 mg/kg), once daily for 2 weeks, and those of the catgut embedding group received catgut embedding at BL13, EX-B1 and CV17 only one time. The rats' sneezing times per miniute in each group were recorded. H.E. staining was used to observe the histopathological changes of the lung tissue under light microscope. A transmission electron microscope (TEM) was used to observe the ultrastructural changes of AEC in the lung tissue, including the thickness of bronchial wall and bronchial smooth muscle by using an image analysis software. The protein expressions of p-p38 MAPK, IL-4 and INF-γ in the lung tissue were determined using Western blot. Morphological observation revealed that in the model group, light microscope showed deformed and swollen bronchial tube wall with increased folds and thickened bronchial smooth muscle；and TEM showed a large number of autophagy vesicles containing swollen and deformed organelles in the AEC, and apparent reduction of intracellular mitochondria, these situations were obviously milder in both DEX and catgut embedding groups. Compared with the blank control group, the sneezing times, thickness of bronchial wall and bronchial smooth muscle in the model group were significantly increased (P<0.01), and the expressions of p-p38 MAPK and IL-4 in lung tissue were significantly increased (P<0.01), while the expression of IFN-γ was significantly decreased (P<0.01) in the model group. In comparison with the model group, the sneezing times, thickness of bronchial wall and bronchial smooth muscle, protein expressions of p-p38 MAPK and IL-4 were significantly decreased (P<0.01), while the expression of IFN-γ was obviously increased (P<0.01) in both the DEX and catgut embedding groups. Acupoint catgut embedding can reduce the expression of IL-4 and increase the expression of IFN-γ by inhibiting p38 MAPK signal pathway of lung tissues in BA rats, which may contribute to its effect in alleviating the degree of airway epithelial cells damage.

Design, Synthesis, in vitro Anti‐inflammatory Activity and in silico Studies of Imidazole‐Based 1,2,3‐Triazole Hybrids Targeting p38 MAP Kinase

Abstractp38 MAP kinases are involved in numerous inflammatory diseases. A series of 14 molecules of 1,2,3 triazole linked 4‐((2‐cyclohexyl‐4,5‐diphenyl‐1H‐imidazol‐1‐yl) methyl)‐1‐phenyl hybrids(7 a–7 n) has been synthesized by click reaction. Spectral data characterized all the synthesized compounds. The final compounds were screened for in vitro p38 MAP kinase inhibitory activity. Three compounds from the series 7 c, 7 f, and 7 n expounded superior activity with IC50 values 234.08±19.65 nM, 222.68±20.69 nM, and 241.70±20.51 nM respectively while two compounds (7 d and 7 e) showed considerable activity compared to prototype drug Adezmapimod (SB203580) (IC50 value 257.13±23.94 nM). The docking study of synthesized molecules revealed that the three compounds (7 f,7 g, and 7 n) showed higher binding affinities than Adezmapimod. The “Desmond v3.6 Program” was utilized to conduct MD simulations. The result revealed that compound 7 f was conformationally stable. The ADME studies were performed using the Swiss ADME algorithm. Studies revealed that all the compound‘s defiance of Lipinski's rule is within acceptable ranges with few violations. Compound 7 f was orally active and showed good gastrointestinal absorption within the acceptable range. The search for newer compounds with increased activity compared to Adezmapimod resulted in identifying a novel subclass of p38 MAP kinase inhibitors.

Discovery of novel NSAID hybrids as cPLA2/COX-2 dual inhibitors alleviating rheumatoid arthritis via inhibiting p38 MAPK pathway

A series of NSAIDs hybrid molecules were synthesized and characterized, and their ability to inhibit NO release in LPS-induced RAW264.7 macrophages was evaluated. Most of the compounds showed significant anti-inflammatory activity in vitro, of which (2E,6Z,9Z,12Z,15Z)-1,1,1-trifluorohenicosa-2,6,9,12,15-pentaen-2-yl 2-(4-benzoylphenyl) propanoate (VI-60) was the most optimal (IC50 = 3.85 ± 0.25 μΜ) and had no cytotoxicity. In addition, VI-60 notably reduced the production of PGE2 in LPS-stimulated RAW264.7 cells compared to ketoprofen. Futhur more, VI-60 significantly inhibited the expression of iNOS, cPLA2, and COX-2 and the phosphorylation of p38 MAPK in LPS-stimulated RAW264.7 cells. The binding of VI-60 to cPLA2 and COX-2 was directly verified by the CETSA technique. In vivo studies illustrated that VI-60 exerted an excellent therapeutic effect on adjuvant-induced arthritis in rats by regulating the balance between Th17 and Treg through inhibiting the p38 MAPK/cPLA2/COX-2/PGE2 pathway. Encouragingly, VI-60 showed a lower ulcerative potential in rats at a dose of 50 mg/kg compared to ketoprofen. In conclusion, the hybrid molecules of NSAIDs and trifluoromethyl enols are promising candidates worthy of further investigation for the treatment of inflammation, pain, and other symptoms in which cPLA2 and COX-2 play a role in their etiology.

Unexpected inhibition of the lipid kinase PIKfyve reveals an epistatic role for p38 MAPKs in endolysosomal fission and volume control

p38 mitogen-activated protein kinases (MAPKs) participate in autophagic signaling; and previous reports suggest that pyridinyl imidazole p38 MAPK inhibitors, including SB203580 and SB202190, induce cell death in some cancer cell-types through unrestrained autophagy. Subsequent studies, however, have suggested that the associated cytoplasmic vacuolation resulted from off-target inhibition of an unidentified enzyme. Herein, we report that SB203580-induced vacuolation is rapid, reversible, and relies on the class III phosphatidylinositol 3-kinase (PIK3C3) complex and the production of phosphatidylinositol 3-phosphate [PI(3)P] but not on autophagy per se. Rather, vacuolation resulted from the accumulation of Rab7 on late endosome and lysosome (LEL) membranes, combined with an osmotic imbalance that triggered severe swelling in these organelles. Inhibition of PIKfyve, the lipid kinase that converts PI(3)P to PI(3,5)P2 on LEL membranes, produced a similar phenotype in cells; therefore, we performed in vitro kinase assays and discovered that both SB203580 and SB202190 directly inhibited recombinant PIKfyve. Cancer cells treated with either drug likewise displayed significant reductions in the endogenous levels of PI(3,5)P2. Despite these results, SB203580-induced vacuolation was not entirely due to off-target inhibition of PIKfyve, as a drug-resistant p38α mutant suppressed vacuolation; and combined genetic deletion of both p38α and p38β dramatically sensitized cells to established PIKfyve inhibitors, including YM201636 and apilimod. The rate of vacuole dissolution (i.e., LEL fission), following the removal of apilimod, was also significantly reduced in cells treated with BIRB-796, a structurally unrelated p38 MAPK inhibitor. Thus, our studies indicate that pyridinyl imidazole p38 MAPK inhibitors induce cytoplasmic vacuolation through the combined inhibition of both PIKfyve and p38 MAPKs, and more generally, that p38 MAPKs act epistatically to PIKfyve, most likely to promote LEL fission.

Abstract C025: Fibroblast-specific IL1R1-p38 MAPK signaling sustains stromal inflammation and contributes to therapeutic resistance in pancreatic cancer

Abstract Introduction: The desmoplastic stroma of pancreatic ductal adenocarcinoma (PDAC) perpetuates therapeutic resistance by impairing drug delivery and effector immune cell infiltration and activation. We have identified tumor cell-derived interleukin-1α (IL1α) as a critical activator of the stroma, comprised of cancer-associated fibroblasts (CAF), towards a pro-inflammatory phenotype via the IL1 receptor, IL1R1, and subsequent activation of p38 MAPK. Our hypothesis is that disruption of the CAF-specific IL1α/p38 MAPK signaling axis can improve chemotherapeutic resistance by remodeling the fibrotic stromal landscape and the overall immune microenvironment in PDAC. Methods: p38 MAPK was pharmacologically inhibited with pexmetinib and genetically with an shRNA lentiviral system. Bulk RNA and ATAC sequencing (RNAseq and ATACseq) were performed on human pancreatic stellate cells (hPSC). Gene set enrichment analyses and motif enrichment analyses were performed on the RNAseq and ATACseq datasets, respectively. Tumor cells (KPC-6694), derived from LSL-KrasG12D/+; LSL-Tp53R172H/+; Pdx1Cre/+ (KPC) murine tumors, were injected orthotopically into Col1a2Cre/+; Mapk14f/f and Col1a2Cre/+; Il1r1f/f mice, and tumors were harvested for downstream analysis. Ptf1aCre/+; LSL-KrasG12D/+; Tgfbr2flox/flox (PKT) mice were treated with pexmetinib (PEX, 30mg/kg, daily PO), anakinra (AKA, 50mg/kg IP, BID) or vehicle control for 2.5 weeks prior to sacrifice for downstream analyses and single-cell RNA sequencing (scRNA) on tumors. Results: In vitro inhibition of p38 MAPK prevented CAF activation into an inflammatory fibroblast, when stimulated with IL1α, demonstrated through bulk RNAseq and ATACseq. Bulk ATACseq revealed epigenetic regulation of the inflammatory CAF phenotype by IL1-mediated activation of p38 MAPK. Stromal-specific deletion of Il1r1 or Mapk14 resulted in signification reduction of tumor weight and intratumoral myeloid-derived suppressor cells. Additionally, pharmacologic blockade of both IL1R1 (AKA) and p38 MAPK inhibition (PEX) altered intratumoral immune cell and CAF populations, by flow cytometry and scRNA. Lastly, p38 inhibition or IL1R1 blockade combined with chemotherapy significantly reduced tumor burden and favorably altered the immune landscape in a PDAC genetically engineered mouse model. Conclusions: These findings provide important mechanistic data in preclinical PDAC models to explore p38 MAPK inhibition in combination therapy to target the fibrotic stroma and immunosuppressive tumor microenvironment. Citation Format: Samara P. Singh, Austin R. Dosch, Siddharth Mehra, Iago de Castro Silva, Vanessa Tonin Garrido, Anna Bianchi, Haleh Amirian, Edmond W. Box, Jashodeep Datta, Nagaraj S. Nagathihalli, Nipun B. Merchant. Fibroblast-specific IL1R1-p38 MAPK signaling sustains stromal inflammation and contributes to therapeutic resistance in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C025.

Transferrin receptor 2 mitigates periodontitis-driven alveolar bone loss.

Periodontitis is associated with significant alveolar bone loss. Patients with iron overload suffer more frequently from periodontitis, however, the underlying mechanisms remain largely elusive. Here, we investigated the role of transferrin receptor 2 (Tfr2), one of the main regulators of iron homeostasis, in the pathogenesis of periodontitis and the dental phenotype under basal conditions in mice. As Tfr2 suppresses osteoclastogenesis, we hypothesized that deficiency of Tfr2 may exacerbate periodontitis-induced bone loss. Mice lacking Tfr2 (Tfr2-/- ) and wild-type (Tfr2+/+ ) littermates were challenged with experimental periodontitis. Mandibles and maxillae were collected for microcomputed tomography and histology analyses. Osteoclast cultures from Tfr2+/+ and Tfr2-/- mice were established and analyzed for differentiation efficiency, by performing messenger RNA expression and protein signaling pathways. After 8 days, Tfr2-deficient mice revealed a more severe course of periodontitis paralleled by higher immune cell infiltration and a higher histological inflammation index than Tfr2+/+ mice. Moreover, Tfr2-deficient mice lost more alveolar bone compared to Tfr2+/+ littermates, an effect that was only partially iron-dependent. Histological analysis revealed a higher number of osteoclasts in the alveolar bone of Tfr2-deficient mice. In line, Tfr2-deficient osteoclastic differentiation ex vivo was faster and more efficient as reflected by a higher number of osteoclasts, a higher expression of osteoclast markers, and an increased resorptive activity. Mechanistically, Tfr2-deficient osteoclasts showed a higher p38-MAPK signaling and inhibition of p38-MAPK signaling in Tfr2-deficient cells reverted osteoclast formation to Tfr2+/+ levels. Taken together, our data indicate that Tfr2 modulates the inflammatory response in periodontitis thereby mitigating effects on alveolar bone loss.

Involvement of Sep38β in the Insecticidal Activity of Bacillus thuringiensis against Beet Armyworm, Spodoptera exigua (Lepidoptera).

The p38 mitogen-activated protein kinases (MAPKs) are associated with insect immunity, tissue repair, and the insecticidal activity of Bacillus thuringiensis (Bt). Here, a p38 MAPK family gene (Sep38β) was identified from Spodoptera exigua. Among the developmental stages, the transcription level of Sep38β was the highest in egg, followed by that in prepupa and pupa. Sep38β expression peaked in Malpighian tubules and the hemolymph of fifth instar larvae. Knockdown of Sep38β or injection of SB203580 (a p38 MAPK inhibitor) significantly downregulated the SeDUOX expression and reactive oxygen species (ROS) level in the midgut, accounting for deterioration of the midgut to scavenge pathogens and enhancement of Bt insecticidal activity. In conclusion, all the results demonstrate that Sep38β regulates the immune-related ROS level in the insect midgut, which suppresses the insecticidal activity of Bt against S. exigua by 17-22%. Our study highlights that Sep38β is essential for insect immunity and the insecticidal activity of Bt to S. exigua and is a potential target for pest control.

Insights into cAMP-dependent molecular mechanisms regulating expression and function of LGALS16 gene in choriocarcinoma JEG-3 cells.

The human choriocarcinoma cell line JEG-3 offers a valuable model to study galectin-16 gene (LGALS16) expression and functions in the context of placental cell differentiation and cancer cell biology. Recent evidence indicates that cAMP-mediated signaling pathways might be responsible for the upregulation of LGALS16; however, the underlying mechanisms are unknown. Here, we employed biochemical inhibitors of the cAMP cascade and CRISPR/Cas9 engineered cells to assess regulatory patterns and associations between cAMP-induced trophoblast differentiation and LGALS16 expression in JEG-3 cells. The expression of LGALS16 was significantly upregulated in parallel with human chorionic gonadotropin beta (CGB), a biomarker of syncytiotrophoblast differentiation, in response to 8-Br-cAMP. Inhibition of p38 MAPK and EPAC significantly altered LGALS16 expression during differentiation, while PKA inhibition failed to change LGALS16 and CGB3/5 expression in our cell model. The CRISPR/Cas9 LGALS16 knockout cell pool expressed a significantly lower amount of CGB3/5, a reduced level of CGB protein, and an unaltered cell growth rate in response to 8-Br-cAMP in comparison with wild-type JEG-3 cells. Collectively, these findings suggest that LGALS16 is required for the trophoblast-like differentiation of JEG-3 cells, and its expression is mediated through p38 MAPK and EPAC signaling pathway branches.