Sanshimao formula inhibits the hypoxia-induced pro-angiogenesis of hepatocellular carcinoma cells partly through regulating MKK6/p38 signaling pathway.

OBJECTIVES: Sanshimao (SSM) is a traditional Chinese medicine formula for advanced hepatocellular carcinoma (HCC). This study was designed to investigate the effect of SSM on HCC-induced angiogenesis and to explore the potential mechanism. METHODS: The endothelial cells were cultured with HCC cells conditioned medium in the 1% oxygen atmosphere to imitate tumor hypoxia microenvironment. EA.hy926 cells migration and tubulogenesis were detected by tube formation and scratch-wound assay. The protein microarray was employed to explore SSM-targeted proteins in Huh7 cells. We also established an animal model to observe the effects of SSM on angiogenesis in vivo. RESULTS: The data indicated that SSM reduced HCC-induced migration and tube formation of EA.hy926 cells at low dose under hypoxic conditions. These effects might be partly owing to suppression of HIF-1α-induced vascular endothelial growth factorα expression in Huh7 cells. Moreover, this inhibition was in an MKK6/P38-dependent way. Besides, Huh7 subcutaneous tumor models in nude mice further demonstrated the inhibition of SSM on tumor weight might be exerted partly by reduction of angiogenesis via blocking MKK6/P38 signaling pathways. CONCLUSION: SSM inhibits HCC-induced pro-angiogenesis under hypoxic conditions via suppression of MKK6/P38 signaling pathways, which is favorable for HCC tumor growth.

APEX1 promotes the oncogenicity of hepatocellular carcinoma via regulation of MAP2K6.

OBJECTIVE: Apurinic/apyrimidinic endonuclease 1 (APEX1), a key enzyme responsible for DNA base excision repair, has been linked to development and progression of cancers. In this work, we aimed to explore the role of APEX1 in hepatocellular carcinoma (HCC) and elucidate its molecular mechanism. METHODS: The expression of APEX1 in HCC tissues and matched adjacent normal tissues (n = 80 cases) was evaluated by immunohistochemistry. Web-based tools UALCAN and the Kaplan-Meier plotter were used to analyze the Cancer Genome Atlas database to compare expression of APEX1 mRNA to 5-year overall survival. APEX1 was stably silenced in two HCC cell lines, Hep 3B and Bel-7402, with shRNA technology. An in vivo tumorigenesis model was established by subcutaneously injecting sh-APEX1-transfected Bel-7402 cells into mice, and tumor growth was determined. We performed high-throughput transcriptome sequencing in sh-APEX1-treated HCC cells to identify the key KEGG signaling pathways induced by silencing of APEX1. RESULTS: APEX1 was significantly upregulated and predicted poor clinical overall survival in HCC patients. Silencing APEX1 inhibited the proliferation of HCC cells in vivo and in vitro, and it repressed invasion and migration and increased apoptosis and the percentage of cells in G1. Differentially expressed genes upon APEX1 silencing included genes involved in TNF signaling. A positive correlation between the expression of APEX1 and MAP2K6 was noted, and overexpressing MAP2K6 overcame cancer-related phenotypes associated with APEX1 silencing. CONCLUSION: APEX1 enhances the malignant properties of HCC via MAP2K6. APEX1 may represent a valuable prognostic biomarker and therapeutic target in HCC.

Fenbendazole and its synthetic analog interfere with HeLa cells' proliferation and energy metabolism via inducing oxidative stress and modulating MEK3/6-p38-MAPK pathway.

Fenbendazole, a broad-spectrum anti-parasitic drug, can be a potential anti-tumor agent. In this study, we synthesized and purified its derivative, analog 6, intending to achieve improved efficacy in cancer cells and decreased toxicity in normal cells. To evaluate in vitro anti-tumor activities of fenbendazole and analog 6 in different cancer cell lines, a CCK-8 assay was performed, and we found that human cervical cancer HeLa cells were more sensitive to analog 6 than to fenbendazole. Furthermore, we explored the associated mechanism, and our results showed that analog 6 and fenbendazole could induce oxidative stress by accumulating ROS. It not only activated the p38-MAPK signaling pathway, thereby inhibiting the proliferation of HeLa cells and enhancing the apoptosis of HeLa cells, but also significantly induced impaired energy metabolism and restrained their migration and invasion. In addition, the modified analog 6 showed reduced toxicity to normal cells without decreased anti-cancer effect. In conclusion, fenbendazole and analog 6 have multiple targets and strong anti-tumor effects on HeLa cells in vitro and in vivo. The optimized analog 6 could inhibit the viability of HeLa cells with lower toxicity than normal human cells, promising to be developed as an antitumor active compound.

MiR-1298-5p level downregulation induced by Helicobacter pylori infection inhibits autophagy and promotes gastric cancer development by targeting MAP2K6.

Helicobacter pylori infection is a leading cause of gastric cancer (GC). However, the underlying mechanisms have not yet been fully elucidated. We aimed to identify microRNAs (miRNAs) regulated by H. pylori infection and their underlying mechanisms in gastric carcinogenesis. Using a mouse model, it was established that H. pylori infection inhibited autophagy in the gastric mucosa. Importantly, H. pylori infection decreased miR-1298-5p levels in human and mouse gastric tissues and human gastric cell lines. Furthermore, the downregulation of miR-1298-5p levels remarkably inhibited autophagy, ultimately increasing the intracellular H. pylori load, which was detected using a gentamicin protection assay. A series of in vitro assays showed that the downregulation of miR-1298-5p expression promoted GC cell proliferation, migration, and invasion. Mechanistically, using bioinformatics prediction, miRNA pull-down assays, and luciferase reporter assays, mitogen-activated protein kinase kinase 6 (MAP2K6) was found to be the direct target of miR-1298-5p, through which miR-1298-5p regulated autophagy and GC cell viability and motility. Moreover, MAP2K6/p38 mitogen-activated protein kinase (MAPK) axis was determined to be the downstream pathway of miR-1298-5p. These findings revealed that H. pylori infection was found to inhibit autophagy and promote tumor growth by regulating miR-1298-5p expression and the miR-1298-5p/MAP2K6/p38 MAPK axis might be a new avenue for the clinical management of H. pylori infection and H. pylori-associated GC.

A novel tumour suppressor protein encoded by circMAPK14 inhibits progression and metastasis of colorectal cancer by competitively binding to MKK6.

BACKGROUND: The mitogen-activated protein kinase (MAPK) pathway is highly associated with the progression and metastasis of various solid tumours. MAPK14, a core molecule of the MAPK pathway, plays vital roles in the colorectal cancer (CRC). Recent studies have shown that circRNAs can affect tumour progression by encoding peptides. However, little is known regarding the potential protein translated from circMAPK14 and whether it plays a role in the carcinogenesis of colorectal cancer. METHODS: The RNA level and translatable potential of circMAPK14 in CRC was verified using qRT-PCR and public databases. RNase R digestion assay, qRT-PCR, sanger sequencing and FISH assays were utilised to verify the circular characteristics and subcellular localisation of circMAPK14. The suppressive role of circMAPK14 on the progression and metastasis of CRC was verified in vivo and in vitro. LC/MS analysis combined with western blotting demonstrated the presence and relative expression of circMAPK14-175aa. The underlying mechanism of circMAPK14-175aa action to inhibit CRC was identified by co-IP analysis. The binding of U2AF2 within the flanking introns of circMAPK14 was evaluated by RNA pull-down assay and RIP assay. Ultimately, luciferase reporter gene assays and ChIP assays confirmed that FOXC1 suppressed transcription of U2AF2 by binding to the U2AF2 promoter in the -400 bp to -100 bp region.　 RESULTS: We identified that hsa_circ_0131663 (termed circMAPK14) showed significantly decreased expression level in cells and tissue samples of CRC, and was primarily localised in the cytoplasm. A series of function experiments demonstrated that circMAPK14 influenced CRC progression and metastasis by encoding a peptide of 175 amino acids (termed circMAPK14-175aa). We also found that circMAPK14-175aa reduced nuclear translocation of MAPK14 by competitively binding to MKK6, thus facilitating ubiquitin-mediated degradation of FOXC1. Moreover, we described a positive feedback loop in CRC in which elevated FOXC1 expression was caused by reduced circMAPK14-175aa expression. This, in turn, decreased circMAPK14 biogenesis by suppressing U2AF2 transcription. CONCLUSION: In summary, we reported for the first time that circMAPK14 functioned as a tumour-suppressor by encoding circMAPK14-175aa, which blocked the progression and metastasis of colorectal cancer.

Quercetin inhibits caerulein-induced acute pancreatitis through regulating miR-216b by targeting MAP2K6 and NEAT1.

Acute pancreatitis (AP) is a common acute abdominal disease with high mortality and mortality rates. Increasing evidences clarified that Traditional Chinese Medicine (TCM) adjuvant therapy for AP can be used and it gives a positive effect. Quercetin (3,3',4',5,7-pentahydroxyflavone, QE) is a type of flavone compound with positive effect on cancer and inflammation prevention. The current study aims to identify the effect of QE on AP and potential molecular effect. In this case, caerulein (CAE) induced AP cell and mice model were used. QE alleviated inflammatory mediators TNF-α, IL-6, and IL-10 in experiments. In addition, miR-216b was increased based on QE treatment. In further study, MAP2K6 of p38/MAPK signaling pathway was identified as a direct target of miR-216b, and QE inhibited p38/MAPK signaling pathway through up-regulating miR-216b. Our study also first confirmed that long non-coding RNA NEAT1 is a direct target of miR-216b and can be suppressed by QE. Because of the target, NEAT1, miR-216b, and MAP2K6 formed a competitive endogenous RNA (ceRNA) network. Besides direct target mediated by QE, it also decreased TNF-α which down-regulated TRAF2 and MAP3K5 located on upstream of p38/MAPK signaling and formed a feedback loop. In conclusion, QE has a protective effect on AP through inhibiting p38/MAPK signaling pathway by up-regulating miR-216b and suppressing TNF-α.

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

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

CCL20 induced by visfatin in macrophages via the NF-κB and MKK3/6-p38 signaling pathways contributes to hepatic stellate cell activation.

Chemokines interact with hepatic resident cells during inflammation and fibrosis. CC chemokine ligand (CCL) 20 has been reported to be important in inflammation and fibrosis in the liver. We hypothesized that visfatin, an adipocytokine, could play a role in hepatic fibrosis via CCL20. We investigated the effect of visfatin on CCL20 in THP-1 human promonocytic cells and examined the molecular mechanisms involved. Following treatment of THP-1 cells with visfatin, CCL20 expression and secretion were assessed. We assessed the intracellular signaling molecules IKK/NF-κB, JAK2/STAT3, MAPKs, and MKK3/6 by western blotting. We treated THP-1 cells with visfatin and signaling inhibitors, and examined CCL20 mRNA and protein levels. To investigate the effect of visfatin-induced CCL20 expression in hepatic stellate cells (HSCs), LX-2 cells were co-cultured with the culture supernatant of THP-1 cells with or without anti-CCL20 neutralizing antibodies, and fibrosis markers were examined by RT-PCR and immunoblotting. In THP-1 cells, visfatin increased the CCL20 mRNA and protein levels. visfatin increased the activities of the NF-κB, p38, and MLK3/6 signaling pathways but not those of the JAK2/STAT3 and ERK pathways. Visfatin treatment together with an NF-κB, p38, or MLK3 inhibitor reduced the mRNA and protein levels of CCL20. The visfatin-induced CCL20 increased the expression of fibrosis markers and CCR6 in HSCs. Following neutralization of CCL20, the levels of fibrosis markers and CCR6 were decreased. Visfatin increases the expression of CCL20 via the NF-κB and MKK3/6-p38 signaling pathways in macrophages, and visfatin-induced CCL20 expression promotes the fibrosis markers in HSCs.

Gut stem cell aging is driven by mTORC1 via a p38 MAPK-p53 pathway.

Nutrients are absorbed solely by the intestinal villi. Aging of this organ causes malabsorption and associated illnesses, yet its aging mechanisms remain unclear. Here, we show that aging-caused intestinal villus structural and functional decline is regulated by mTORC1, a sensor of nutrients and growth factors, which is highly activated in intestinal stem and progenitor cells in geriatric mice. These aging phenotypes are recapitulated in intestinal stem cell-specific Tsc1 knockout mice. Mechanistically, mTORC1 activation increases protein synthesis of MKK6 and augments activation of the p38 MAPK-p53 pathway, leading to decreases in the number and activity of intestinal stem cells as well as villus size and density. Targeting p38 MAPK or p53 prevents or rescues ISC and villus aging and nutrient absorption defects. These findings reveal that mTORC1 drives aging by augmenting a prominent stress response pathway in gut stem cells and identify p38 MAPK as an anti-aging target downstream of mTORC1.

Regulation of stimulus-induced interleukin-8 gene transcription in human adrenocortical carcinoma cells - Role of AP-1 and NF-κB.

Stimulation of H295R adrenocortical carcinoma cells with angiotensin II or cytokines induces the secretion of the chemokine interleukin-8 (IL-8). Here, we have analyzed the molecular mechanism of stimulus-induced IL-8 expression. IL-8 expression and IL-8 promoter activity increased in H295R cells expressing an activated Gαq-coupled designer receptor. H295R cells stimulated with either interleukin-1ß (IL-1ß) or phorbol ester also showed elevated IL-8 mRNA levels and higher IL-8 promoter activities. Deletion and point mutations of the IL-8 promoter revealed that the AP-1 binding site within the IL-8 promoter is essential to connect designer receptor stimulation with the transcriptional activation of the IL-8 gene. Expression of a constitutively active mutant of c-Jun, or expression of constitutively active mutants of the protein kinases MEKK1 and MKK6 confirmed that the IL-8 gene is a bona fide target of AP-1 in adrenocortical carcinoma cells. Upregulation of IL-8 expression in IL-1ß-treated H295R cells required NF-κB while the phorbol ester TPA used both the AP-1 and NF-κB sites of the IL-8 gene to stimulate IL-8 expression. These data were corroborated in experiments with chromatin-embedded AP-1 or NF-κB-responsive reporter genes. While stimulation of Gαq-coupled designer receptors increased the AP-1 activity in the cells, IL-1ß specifically stimulated NF-κB-regulated transcription. Stimulation of the cells with TPA increased both AP-1 and NF-κB activities. We conclude that stimulation of Gαq-coupled designer receptors or IL-1 receptors triggers distinct signaling pathways in H295R cells leading to the activation of either AP-1 or NF-κB. Nevertheless, both signaling cascades converge to the IL-8 gene, inducing IL-8 gene transcription.

Matrine Suppresses Reactive Oxygen Species (ROS)-Mediated MKKs/p38-Induced Inflammation in Oxidized Low-Density Lipoprotein (ox-LDL)-Stimulated Macrophages.

BACKGROUND The objective of this study was to study the anti-inflammatory effect and possibly involved molecular mechanisms of matrine on oxidized low-density lipoprotein (ox-LDL)-exposed macrophages. MATERIAL AND METHODS Cultured human macrophages (THP-1 cell line) were exposed to ox-LDL at final concentrations of 0, 25, 50, and 100 µg/mL. Several cells were then treated with matrine at serial diluted concentrations. 2,7-Dichlorodi-hydrofluorescein diacetate (DCFH-DA) staining was used to evaluate reactive oxygen species (ROS) production; a colorimetric method was used to determine the cellular antioxidant capacity; production of pro-inflammatory cytokines interleukin (IL)18 and tumor necrosis factor (TNF)alpha were determined by enzyme-linked immunosorbent assay (ELISA); and immunoblot assay was used to assess the relative protein phosphorylation and expression. RESULTS ox-LDL exposure significantly elevated intracellular ROS level and supernatant IL18 and TNFalpha concentrations, but impaired total antioxidant capacity (TAC) of macrophages. The relative phosphorylations of MAPK kinase kinases (MKK)6, MKK3, and p38 mitogen-activated protein kinases (MAPK) were increased by ox-LDL exposure. The expression levels of IL18 and TNFalpha were also increased in ox-LDL-treated macrophages. The matrine treatment reduced intracellular ROS level and supernatant IL18 and TNFalpha concentrations and increased TAC in a concentration- dependent manner. The relative phosphorylations of MKK6, MKK3, and p38 MAPK were reduced after matrine administration. Moreover, the expression levels of IL18 and TNFalpha were also decreased by matrine treatment, in a concentration-dependent manner. CONCLUSIONS ox-LDL increases inflammatory response in macrophages by activating the ROS-mediated MKKs/p38 MAPK-induced inflammatory signaling pathway. Matrine suppresses ox-LDL-induced inflammatory by inhibiting the MKKs/p38 MAPK signaling pathway.

Autophagy-induced senescence is regulated by p38α signaling.

Apoptosis and senescence are two mutually exclusive cell fate programs that can be activated by stress. The factors that instruct cells to enter into senescence or apoptosis are not fully understood, but both programs can be regulated by the stress kinase p38α. Using an inducible system that specifically activates this pathway, we show that sustained p38α activation suffices to trigger massive autophagosome formation and to enhance the basal autophagic flux. This requires the concurrent effect of increased mitochondrial reactive oxygen species production and the phosphorylation of the ULK1 kinase on Ser-555 by p38α. Moreover, we demonstrate that macroautophagy induction by p38α signaling determines that cancer cells preferentially enter senescence instead of undergoing apoptosis. In agreement with these results, we present evidence that the induction of autophagy by p38α protects cancer cells from chemotherapy-induced apoptosis by promoting senescence. Our results identify a new mechanism of p38α-regulated basal autophagy that controls the fate of cancer cells in response to stress.

Preparation of Phosphorylated Proteins for NMR Spectroscopy.

Phosphorylation is a ubiquitous posttranslational modification that is essential for the regulation of many cellular processes. The human genome consists of more than 200,000 phosphorylation sites, whose phosphorylation is tightly controlled by ≥500 kinases and ~200 phosphatases. Given the large number of phosphorylation sites and the key role phosphorylation plays in regulating cellular processes, it is essential to characterize the impact of phosphorylation on substrate structure, dynamics, and function. However, a major challenge is the large-scale production of phosphorylated proteins in vitro for these structural, functional, and dynamic studies. Here, we describe an efficient protocol used routinely in our laboratory for the production of phosphorylated proteins. We also describe the methods used for identifying, characterizing, and separating the resulting phosphorylated proteins for subsequent studies.

Gossypetin is a novel MKK3 and MKK6 inhibitor that suppresses esophageal cancer growth in vitro and in vivo.

Gossypetin as a hexahydroxylated flavonoid found in many flowers and Hibiscus. It exerts various pharmacological activities, including antioxidant, antibacterial and anticancer activities. However, the anticancer capacity of gossypetin has not been fully elucidated. In this study, gossypetin was found to inhibit anchorage-dependent and -independent growth of esophageal cancer cells. To identify the molecular target(s) of gossypetin, various signaling protein kinases were screened and results indicate that gossypetin strongly attenuates the MKK3/6-p38 signaling pathway by directly inhibiting MKK3 and MKK6 protein kinase activity in vitro. Mechanistic investigations showed that arginine-61 in MKK6 is critical for binding with gossypetin. Additionally, the inhibition of cell growth by gossypetin is dependent on the expression of MKK3 and MKK6. Gossypetin caused G2 phase cell cycle arrest and induced intrinsic apoptosis by activating caspases 3 and 7 and increasing the expression of BAX and cytochrome c. Notably, gossypetin suppressed patient-derived esophageal xenograft tumor growth in an in vivo mouse model. Our findings suggest that gossypetin is an MKK3 and MKK6 inhibitor that could be useful for preventing or treating esophageal cancer.

Quantitative proteome analysis identifies MAP2K6 as potential regulator of LIFR-induced radioresistance in nasopharyngeal carcinoma cells.

Using Tandem Mass Tags (TMT) labeling and LC-MS/MS analysis of peptides from two cell lines (CNE2 and its radioresistant subclone CNE2-IR), we identified 754 proteins differentially expressed in CNE2-IR compared to CNE2. MAP2K6 was identified as a candidate radioresistance-related protein kinase. In vitro functional analysis revealed that over-expression of MAP2K6 significantly enhanced cell survival and colony formation following irradiation in NPC cells. Further, knockdown of MAP2K6 in radioresistant NPC cells led to decreased colony formation and increased apoptotic cells following irradiation. However, the effect of MAP2K6 in regulating the radioresistance in NPC cells did not seem to depend on p38/MAPK activity. Importantly, MAP2K6 might be required for leukemia inhibitory factor receptor (LIFR)-regulated radioresistance, as the expression levels of MAP2K6 affected LIFR/p70S6K signaling activation in NPC cells. Further, MAP2K6 kinase activity is required to activate LIFR/p70S6K signaling and to confer pro-survival effect on NPC cells. In conclusion, MAP2K6 might be an important regulator of LIFR-induced radioresistance in NPC.

Transcriptional and Epigenetic Changes Influencing Skeletal Muscle Metabolism in Women With Polycystic Ovary Syndrome.

Context: Insulin resistance in skeletal muscle is a major risk factor for the development of type 2 diabetes in women with polycystic ovary syndrome (PCOS). Despite this, the mechanisms underlying insulin resistance in PCOS are largely unknown. Objective: To investigate the genome-wide DNA methylation and gene expression patterns in skeletal muscle from women with PCOS and controls and relate them to phenotypic variations. Design/Participants: In a case-control study, skeletal muscle biopsies from women with PCOS (n = 17) and age-, weight-, and body mass index‒matched controls (n = 14) were analyzed by array-based DNA methylation and mRNA expression profiling. Results: Eighty-five unique transcripts were differentially expressed in muscle from women with PCOS vs controls, including DYRK1A, SYNPO2, SCP2, and NAMPT. Furthermore, women with PCOS had reduced expression of genes involved in immune system pathways. Two CpG sites showed differential DNA methylation after correction for multiple testing. However, an mRNA expression of ∼30% of the differentially expressed genes correlated with DNA methylation levels of CpG sites in or near the gene. Functional follow-up studies demonstrated that KLF10 is under transcriptional control of insulin, where insulin promotes glycogen accumulation in myotubes of human muscle cells. Testosterone downregulates the expression levels of COL1A1 and MAP2K6. Conclusion: PCOS is associated with aberrant skeletal muscle gene expression with dysregulated pathways. Furthermore, we identified specific changes in muscle DNA methylation that may affect gene expression. This study showed that women with PCOS have epigenetic and transcriptional changes in skeletal muscle that, in part, can explain the metabolic abnormalities seen in these women.

Pharmacological targeting of p38 MAP-Kinase 6 (MAP2K6) inhibits the growth of esophageal adenocarcinoma.

Drug repurposing with a better understanding of the underlying mechanism has provided new avenues to find treatment for malignancies. Esophageal adenocarcinoma (EAC) is a rapidly increasing cancer with a dismal 5-year survival rate of <15%. Lack of efficient treatment options contributes to the high mortality rate of EAC. To find new therapy against EAC we performed unbiased drug screening of an FDA-approved drug library and identified that the cardiac glycosides including Ouabain, Digoxin and Digitoxin efficiently inhibit the proliferation of EAC cell lines (OE33 and OE19) both in vitro and in vivo. RNA-Sequencing analysis combined with RNAi screening revealed that Ouabain suppresses the proliferation of EAC cells through downregulation of p38 MAP-Kinase 6 (MAP2K6, also known as MKK6). Consistently, shRNA-mediated knockdown of MKK6 reduced the proliferation of EAC cells and tumor growth. Further analysis demonstrated that MKK6 inhibition leads to the reduced levels of the transcription factor SOX9. In line with this finding, deletion of SOX9 with CRISPR/Cas9 resulted in decreased proliferation of EACs in 3D organoid culture and reduced tumor growth. Together these findings establish a druggable axis that can be harnessed for therapeutic gain against EAC.

MicroRNA-21 prevents excessive inflammation and cardiac dysfunction after myocardial infarction through targeting KBTBD7.

The excessive inflammation triggered by damage-associated molecular patterns (DAMPs) after myocardial infarction (MI) is responsible for the development of cardiac dysfunction and adverse remodeling, while the mechanisms by which inflammation is fine tuned remain to be fully elucidated. MicroRNA-21 (miR-21) has been shown to function in cardiovascular diseases, while its role in inflammatory responses and cardiac function post MI in mice remains unknown. Here, we found that miR-21 expression was markedly increased in border and infarct areas of cardiac tissues during the early inflammatory phase of MI model established by ligating the left-anterior descending coronary artery. MiR-21 knockout mice had decreased survival rates, worse cardiac dysfunction, and increased infarct and scar areas after MI compared with WT mice. MiR-21 knockout mice showed significantly higher levels of inflammatory cytokines including IL-1ß, IL-6, and TNF-α in cardiac tissues, as well as infiltration of CD11b+ monocytes/macrophages with higher expression level of inflammatory cytokines. MI induced the great release of high mobility group protein B1 (HMGB1) and heat shock protein 60 (HSP60) in cardiac tissue. MiR-21 deficiency significantly promoted the inflammatory cytokine production triggered by DAMPs in macrophages, whereas, miR-21 overexpression markedly inhibited the inflammatory cytokine production. Mechanistically, miR-21 deficiency enhanced p38 and NF-κB signaling activation in cardiac tissue post MI and macrophages treated with DAMPs. MiR-21 was found to directly target kelch repeat and BTB (POZ) domain containing 7 (KBTBD7), which promoted DAMP-triggered inflammatory responses in macrophages. Furthermore, KBTBD7 interacted with MKK3/6 and promoted their activation, which in turn enhanced the activation of downstream p38 and NF-κB signaling induced by DAMPs. Therefore, our findings demonstrate that miR-21 attenuates inflammation, cardiac dysfunction, and maladaptive remodeling post MI through targeting KBTBD7 and inhibiting p38 and NF-κB signaling activation, suggesting that miR-21 may function as a novel potential therapeutic target for MI.

PKCα in colon cancer cells promotes M1 macrophage polarization via MKK3/6-P38 MAPK pathway.

Tumor associated macrophages are potential targets of the immune therapy for patients with colon cancer. PKCα acts as a tumor suppressor in the intestine. However, the correlation between PKCα expressed in colon cancer cells and tumor associated macrophages polarization has never been detected. In the present study, the correlation between PKCα expression and level of M1 macrophages was evaluated in human colon cancer tissues. A xenograft mouse model of colon cancer cells with different PKCα expression level was constructed to evaluate the effect of PKCα on M1 macrophages polarization in vivo. Co-culture of colon cancer cells and differentiated macrophages was used to detect the potential interplay in vitro. PKCα regulated production of cytokines which correlated with macrophage polarization and the underlying mechanism was further explored. Our study showed that high PKCα expression in human colon cancer tissues correlated with better prognosis and high M1 macrophage content. PKCα expressed in colon cancer cells inhibited the growth of colon cancer in mice model. PKCα induced macrophages polarized to the M1-like phenotype both in vitro and in vivo. Mechanistically, PKCα targeted P38 via MKK3/6 to promote IL12 and GM-CSF expression which further enhanced M1-like macrophages polarization. In conclusion, this study provided evidence for the first time that PKCα in colon cancer cells play an anticancer action by inducing the polarization of tumor associated macrophages to M1-like phenotype in the tumor microenvironment. PKCα promoted IL12/GM-CSF-mediated M1 polarization through MKK3/6-P38 signaling pathway. Our investigation suggested that modulation of the PKCα signaling pathway might serve as a novel strategy for colon cancer therapy.

Mutual Stabilization between TRIM9 Short Isoform and MKK6 Potentiates p38 Signaling to Synergistically Suppress Glioblastoma Progression.

p38 signaling is broadly involved in controlling inflammation and stress-induced cell death; however, the mechanisms controlling its activity have seldom been studied. Here, we report that TRIM9 short isoform (TRIM9s) potentiates p38 signaling by stabilizing MKK6. Mechanistic studies revealed that TRIM9s promotes the K63-linked ubiquitination of MKK6 at Lys82, thus inhibiting the degradative K48-linked ubiquitination of MKK6 at the same lysine. MKK6 could also stabilize TRIM9s by promoting the phosphorylation of TRIM9s at Ser76/80 via p38, thereby blocking the ubiquitin-proteasome pathway. Further functional analyses showed that p38 signaling plays a critical role in suppressing glioblastoma progression. Co-reduction of MKK6 and TRIM9s is significantly associated with overall poor survival of glioblastoma patients. We identify a positive feedback loop in p38 signaling generated by MKK6-TRIM9s, which suppresses glioblastoma progression, and we provide insights into the mechanisms by which TRIM9s and MKK6 potentiate p38 signaling through mutual stabilization.