Tris DBA ameliorates IgA nephropathy by blunting the activating signal of NLRP3 inflammasome through SIRT1‐ and SIRT3‐mediated autophagy induction

Tris (dibenzylideneacetone) dipalladium (Tris DBA), a small‐molecule palladium complex, can inhibit cell growth and proliferation in pancreatic cancer, lymphocytic leukaemia and multiple myeloma. Given that this compound is particularly active against B‐cell malignancies, we have been suggested that it can alleviate immune complexes (ICs)–mediated conditions, especially IgA nephropathy (IgAN). The therapeutic effects of Tris DBA on glomerular cell proliferation and renal inflammation and mechanism of action were examined in a mouse model of IgAN. Treatment of IgAN mice with Tris DBA resulted in markedly improved renal function, albuminuria and renal pathology, including glomerular cell proliferation, neutrophil infiltration, sclerosis and periglomerular inflammation in the renal interstitium, together with (Clin J Am Soc Nephrol. 2011, 6, 1301‐1307) reduced mitochondrial ROS generation; (Am J Physiol‐Renal Physiol. 2011. 301, F1218‐F1230) differentially regulated autophagy and NLRP3 inflammasome; (Clin J Am Soc Nephrol. 2012, 7, 427‐436) inhibited phosphorylation of JNK, ERK and p38 MAPK signalling pathways, and priming signal of the NLRP3 inflammasome; and (Free Radic Biol Med. 2013, 61, 285‐297) blunted NLRP3 inflammasome activation through SIRT1‐ and SIRT3‐mediated autophagy induction, in renal tissues or cultured macrophages. In conclusion, Tris DBA effectively ameliorated the mouse IgAN model and targeted signalling pathways downstream of ICs‐mediated interaction, which is a novel immunomodulatory strategy. Further development of Tris DBA as a therapeutic candidate for IgAN is warranted.     

MAPK14/p38α confers irinotecan resistance to TP53-defective cells by inducing survival autophagy

Recently we have shown that the mitogen-activated protein kinase (MAPK) MAPK14/p38α is involved in resistance of colon cancer cells to camptothecin-related drugs. Here we further investigated the cellular mechanisms involved in such drug resistance and showed that, in HCT116 human colorectal adenocarcinoma cells in which TP53 was genetically ablated (HCT116-TP53KO), overexpression of constitutively active MAPK14/p38α decreases cell sensitivity to SN-38 (the active metabolite of irinotecan), inhibits cell proliferation and induces survival-autophagy. Since autophagy is known to facilitate cancer cell resistance to chemotherapy and radiation treatment, we then investigated the relationship between MAPK14/p38α, autophagy and resistance to irinotecan. We demonstrated that induction of autophagy by SN38 is dependent on MAPK14/p38α activation. Finally, we showed that inhibition of MAPK14/p38α or autophagy both sensitizes HCT116-TP53KO cells to drug therapy. Our data proved that the two effects are interrelated, since the role of autophagy in drug resistance required the MAPK14/p38α. Our results highlight the existence of a new mechanism of resistance to camptothecin-related drugs: upon SN38 induction, MAPK14/p38α is activated and triggers survival-promoting autophagy to protect tumor cells against the cytotoxic effects of the drug. Colon cancer cells could thus be sensitized to drug therapy by inhibiting either MAPK14/p38 or autophagy.     

Dysregulated lipolysis and lipophagy in lipid droplets of macrophages from high fat diet‐fed obese mice

Obesity is associated with lipid droplet (LD) accumulation, dysregulated lipolysis and chronic inflammation. Previously, the caspase recruitment domain‐containing protein 9 (CARD9) has been identified as a potential contributor to obesity‐associated abnormalities including cardiac dysfunction. In the current study, we explored a positive feedback signalling cycle of dysregulated lipolysis, CARD9‐associated inflammation, impaired lipophagy and excessive LD accumulation in sustaining the chronic inflammation associated with obesity. C57BL/6 WT and CARD9^−/− mice were fed with normal diet (ND, 12% fat) or a high fat diet (HFD, 45% fat) for 5 months. Staining of LDs from peritoneal macrophages (PMs) revealed a significant increase in the number of cells with LD and the number of LD per cell in the HFD‐fed WT but not CARD9^−/− obese mice. Rather, CARD9 KO significantly increased the mean LD size. WT obese mice showed down regulation of lipolytic proteins with increased diacylglycerol (DAG) content, and CARD9 KO normalized DAG with restored lipolytic protein expression. The build‐up of DAG in the WT obese mice is further associated with activation of PKCδ, NF‐κB and p38 MAPK inflammatory signalling in a CARDD9‐dependent manner. Inhibition of adipose triglyceride lipase (ATGL) by Atglistatin (Atg) resulted in similar effects as in CARD9^−/− mice. Interestingly, CARD9 KO and Atg treatment enhanced lipophagy. In conclusion, HFD feeding likely initiated a positive feedback signalling loop from dysregulated lipolysis, CARD9‐dependent inflammation, impaired lipophagy, to excessive LD accumulation and sustained inflammation. CARD9 KO and Atg treatment protected against the chronic inflammation by interrupting this feedforward cycle.     

The problem of pyridinyl imidazole class inhibitors of MAPK14/p38α and MAPK11/p38β in autophagy research

In addition to its established role in inflammation, the stress-activated p38 MAP kinase pathway plays major roles in the regulation of cell cycle, senescence, and autophagy. Robust studies could establish mechanistic links between MAPK11-MAPK14/p38 signaling and macroautophagy converging at ATG9-trafficking and BECN1 phosphorylation. However, several reports seem to monitor MAPK11-MAPK14/p38-dependence of autophagy exclusively by the use of the SB203580/SB202190 class of MAPK14/MAPK11/p38α/β inhibitors. In this “Letter to the editor” we present data to support our claim that these inhibitors interfere with autophagic flux in a MAPK11-MAPK14/p38-independent manner and hence should no longer be used as pharmacological tools in the analysis of MAPK11-MAPK14/p38-dependence of autophagy. We propose a general guideline from Autophagy with regard to this issue to avoid such misinterpretations in the future.     

MAPK14/p38α confers irinotecan resistance to TP53-defective cells by inducing survival autophagy

Recently we have shown that the mitogen-activated protein kinase (MAPK) MAPK14/p38α is involved in resistance of colon cancer cells to camptothecin-related drugs. Here we further investigated the cellular mechanisms involved in such drug resistance and showed that, in HCT116 human colorectal adenocarcinoma cells in which TP53 was genetically ablated (HCT116-TP53KO), overexpression of constitutively active MAPK14/p38α decreases cell sensitivity to SN-38 (the active metabolite of irinotecan), inhibits cell proliferation and induces survival-autophagy. Since autophagy is known to facilitate cancer cell resistance to chemotherapy and radiation treatment, we then investigated the relationship between MAPK14/p38α, autophagy and resistance to irinotecan. We demonstrated that induction of autophagy by SN38 is dependent on MAPK14/p38α activation. Finally, we showed that inhibition of MAPK14/p38α or autophagy both sensitizes HCT116-TP53KO cells to drug therapy. Our data proved that the two effects are interrelated, since the role of autophagy in drug resistance required the MAPK14/p38α. Our results highlight the existence of a new mechanism of resistance to camptothecin-related drugs: upon SN38 induction, MAPK14/p38α is activated and triggers survival-promoting autophagy to protect tumor cells against the cytotoxic effects of the drug. Colon cancer cells could thus be sensitized to drug therapy by inhibiting either MAPK14/p38 or autophagy.     

Trigonelline induces autophagy to protect mesangial cells in response to high glucose via activating the miR-5189-5p-AMPK pathway

BackgroundDiabetic nephropathy (DN) is a primary cause of end‐stage renal disease. Increasing evidence indicates that microRNAs (miRNAs) are involved in DN pathogenesis. Trigonelline (TRL) has been shown to lower blood sugar and cholesterol levels, promote nerve regeneration, and exert anti-cancer and sedative properties.MethodThe effect of TRL on human mesangial cell (HMC) growth was assessed using the MTT assay. Differentially expressed miRNAs were validated using real-time quantitative polymerase chain reaction (real-time PCR). Bioinformatics, cell transfection, and Western blot analyses were utilized to confirm the binding of miR-5189-5p to HIF1AN. The effects of miR-5189-5 expression on cell proliferation were also assessed. Western blot analysis was used to determine the activation of multiple signaling molecules including phosphorylated-(p)-AMPK, SIRT1, LC3B, p62, and Beclin-1 in the autophagy pathway.ResultsTRL improved proliferation, increased the expression of miR-5189-5p, reduced HIF1AN, and restored the inhibition of autophagy in HMCs induced by high glucose. MiR-5189-5p mimics inhibited HIF1AN expression, and the miR-5189-5p inhibitor increased HIF1AN expression. MiR-5189-5p mimics significantly improved the proliferation of HMCs induced by high glucose, reduced the relative protein expression of p-AMPK, SIRT1, LC3B, and Beclin-1, and significantly increased the relative protein expression of p62.ConclusionWe showed that TRL up-regulated miR-5189-5p expression, activated the AMPK pathway, and activated autophagy in HMCs. Our study demonstrates that TRL could be a new treatment strategy to protect mesangial cells in response to high glucose.     

Activated mesangial cells induce glomerular endothelial cells proliferation in rat anti‐Thy‐1 nephritis through VEGFA/VEGFR2 and Angpt2/Tie2 pathway

ObjectivesWe aimed to investigate the underlying mechanism of endothelial cells (ECs) proliferation in anti‐Thy‐1 nephritis.Materials and methodsWe established anti‐Thy‐1 nephritis and co‐culture system to explore the underlying mechanism of ECs proliferation in vivo and in vitro. EdU assay kit was used for measuring cell proliferation. Immunohistochemical staining and immunofluorescence staining were used to detect protein expression. ELISA was used to measure the concentration of protein in serum and medium. RT‐qPCR and Western blot were used to qualify the mRNA and protein expression. siRNA was used to knock down specific protein expression.ResultsIn anti‐Thy‐1 nephritis, ECs proliferation was associated with mesangial cells (MCs)‐derived vascular endothelial growth factor A (VEGFA) and ECs‐derived angiopoietin2 (Angpt2). In vitro co‐culture system activated MCs‐expressed VEGFA to promote vascular endothelial growth factor receptor2 (VEGFR2) activation, Angpt2 expression and ECs proliferation, but inhibit TEK tyrosine kinase (Tie2) phosphorylation. MCs‐derived VEGFA stimulated Angpt2 expression in ECs, which inhibited Tie2 phosphorylation and promoted ECs proliferation. And decline of Tie2 phosphorylation induced ECs proliferation. In anti‐Thy‐1 nephritis, promoting Tie2 phosphorylation could alleviate ECs proliferation.ConclusionsOur study showed that activated MCs promoted ECs proliferation through VEGFA/VEGFR2 and Angpt2/Tie2 pathway in experimental mesangial proliferative glomerulonephritis (MPGN) and in vitro co‐culture system. And enhancing Tie2 phosphorylation could alleviate ECs proliferation, which will provide a new idea for MPGN treatment.     

The essential function of CARD9 in diet‐induced inflammation and metabolic disorders in mice

Inflammation and metabolic disorder are common pathophysiological conditions, which play a vital role in the development of obesity and type 2 diabetes. The purpose of this study was to explore the effects of caspase recruitment domain (CARD) 9 in the high fat diet (HFD)‐treated mice and attempt to find a molecular therapeutic target for obesity development and treatment. Sixteen male CARD9^?/? and corresponding male WT mice were fed with normal diet or high fat diet, respectively, for 12 weeks. Glucose tolerance, insulin resistance, oxygen consumption and heat production of the mice were detected. The CARD9/MAPK pathway‐related gene and protein were determined in insulin‐responsive organs using Western blotting and quantitative PCR. The results showed that HFD‐induced insulin resistance and impairment of glucose tolerance were more severe in WT mice than that in the CARD9^?/? mice. CARD9 absence significantly modified O₂ consumption, CO₂ production and heat production. CARD9^?/? mice displayed the lower expression of p38 MAPK, JNK and ERK when compared to the WT mice in both HFD‐ and ND‐treated groups. HFD induced the increase of p38 MAPK, JNK and ERK in WT mice but not in the CARD9^?/? mice. The results indicated that CARD9 absence could be a vital protective factor in diet‐induced obesity via the CARD9/MAPK pathway, which may provide new insights into the development of gene knockout to improving diet‐induced obesity and metabolism disorder.     

CARD9 Regulation and its Role in Cardiovascular Diseases

Caspase recruitment domain-containing protein 9 (CARD9) is an adaptor protein expressed on myeloid cells and located downstream of pattern recognition receptors (PRRs), which transduces signals involved in innate immunity. CARD9 deficiency is associated with increased susceptibility to various fungal diseases. Increasing evidence shows that CARD9 mediates the activation of p38 MAPK, NF-κB, and NLRP3 inflammasome in various CVDs and then promotes the production of proinflammatory cytokines and chemokines, which contribute to cardiac remodeling and cardiac dysfunction in certain cardiovascular diseases (CVDs). Moreover, CARD9-mediated anti-apoptosis and autophagy are implicated in the progression of CVDs. Here, we summarize the structure and function of CARD9 in innate immunity and its various roles in inflammation, apoptosis, and autophagy in the pathogenesis of CVDs. Furthermore, we discuss the potential therapies targeting CARD9 to prevent CVDs and raise some issues for further exploring the role of CARD9 in CVDs.     

FGF9 promotes mouse spermatogonial stem cell proliferation mediated by p38 MAPK signalling

ObjectivesFibroblast growth factor 9 (FGF9) is expressed by somatic cells in the seminiferous tubules, yet little information exists about its role in regulating spermatogonial stem cells (SSCs).Materials and Methods Fgf9 overexpression lentivirus was injected into mouse testes, and PLZF immunostaining was performed to investigate the effect of FGF9 on spermatogonia in vivo. Effect of FGF9 on SSCs was detected by transplanting cultured germ cells into tubules of testes. RNA‐seq of bulk RNA and single cell was performed to explore FGF9 working mechanisms. SB203580 was used to disrupt p38 MAPK pathway. p38 MAPK protein expression was detected by Western blot and qPCR was performed to determine different gene expression. Small interfering RNA (siRNA) was used to knock down Etv5 gene expression in germ cells.ResultsOverexpression of Fgf9 in vivo resulted in arrested spermatogenesis and accumulation of undifferentiated spermatogonia. Exposure of germ cell cultures to FGF9 resulted in larger numbers of SSCs over time. Inhibition of p38 MAPK phosphorylation negated the SSC growth advantage provided by FGF9. Etv5 and Bcl6b gene expressions were enhanced by FGF9 treatment. Gene knockdown of Etv5 disrupted the growth effect of FGF9 in cultured SSCs along with downstream expression of Bcl6b.ConclusionsTaken together, these data indicate that FGF9 is an important regulator of SSC proliferation, operating through p38 MAPK phosphorylation and upregulating Etv5 and Bcl6b in turn.     

Acetylshikonin induces autophagy‐dependent apoptosis through the key LKB1‐AMPK and PI3K/Akt‐regulated mTOR signalling pathways in HL‐60 cells

Acetylshikonin (ASK) is a natural naphthoquinone derivative of traditional Chinese medicine Lithospermum erythrorhyzon. It has been reported that ASK has bactericidal, anti‐inflammatory and antitumour effects. However, whether ASK induces apoptosis and autophagy in acute myeloid leukaemia (AML) cells and the underlying mechanism are still unclear. Here, we explored the roles of apoptosis and autophagy in ASK‐induced cell death and the potential molecular mechanisms in human AML HL‐60 cells. The results demonstrated that ASK remarkably inhibited the cell proliferation, viability and induced apoptosis in HL‐60 cells through the mitochondrial pathway, and ASK promoted cell cycle arrest in the S‐phase. In addition, the increased formation of autophagosomes, the turnover from light chain 3B (LC3B) I to LC3B II and decrease of P62 suggested the induction of autophagy by ASK. Furthermore, ASK significantly decreased PI3K, phospho‐Akt and p‐p70S6K expression, while enhanced phospho‐AMP‐activated protein kinase (AMPK) and phospho‐liver kinase B1(LKB1) expression. The suppression of ASK‐induced the conversion from LC3B I to LC3B II caused by the application of inhibitors of AMPK (compound C) demonstrated that ASK‐induced autophagy depends on the LKB1/AMPK pathway. These data suggested that the autophagy induced by ASK were dependent on the activation of LKB1/AMPK signalling and suppression of PI3K/Akt/mTOR pathways. The cleavage of the apoptosis‐related markers caspase‐3 and caspase‐9 and the activity of caspase‐3 induced by ASK were markedly reduced by inhibitor of AMPK (compound C), an autophagy inhibitor 3‐methyladenine (3‐MA) and another autophagy inhibitor chloroquine (CQ). Taken together, our data reveal that ASK‐induced HL‐60 cell apoptosis is dependent on the activation of autophagy via the LKB1/AMPK and PI3K/Akt‐regulated mTOR signalling pathways.     

Protein-tyrosine kinase Pyk2 mediates endothelin-induced p38 MAPK activation in glomerular mesangial cells

Endothelin-1 (ET-1), a member of a family of 21 amino acid peptides possessing vasoconstrictor properties, is known to stimulate mesangial cell proliferation. In this study, ET-1 (100 nm) induced a rapid activation of p21(ras) in human glomerular mesangial cells (HMC). Inhibition of Src family tyrosine kinase activation with [4-Amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine] or chelation of intracellular free calcium with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester significantly decreased ET-1dependent p21(ras) activation and suggested the involvement of the cytoplasmic proline-rich tyrosine kinase Pyk2. We have observed that Pyk2 was expressed in HMC and was tyrosine-phosphorylated within 5 min of ET-1 treatment. ET-1-induced activation of Pyk2 was further confirmed using phospho-specific anti-Pyk2 antibodies. Surprisingly, Src kinase activity was required upstream of ET-1-induced autophosphorylation of Pyk2. To determine whether Pyk2 autophosphorylation mediated ET-1-dependent p21(ras) activation, adenovirus-mediated transfer was employed to express a dominant-negative form of Pyk2 (CRNK). CRNK expression inhibited ET-1-induced endogenous Pyk2 autophosphorylation, but did not abolish ET-1-mediated increases in GTP-bound p21(ras) levels. ET-1-induced activation of the p38 MAPK (but not ERK) pathway was inhibited in HMC and in rat glomerular mesangial cells expressing the dominant-negative form of Pyk2. These findings suggest that the engagement of Pyk2 is important for ET-1-mediated p38 MAPK activation and hence the biological effect of this peptide in mesangial cells.     

Dual Involvement of Growth Arrest-Specific Gene 6 in the Early Phase of Human IgA Nephropathy

Background

Gas6 is a growth factor that causes proliferation of mesangial cells in the development of glomerulonephritis. Gas6 can bind to three kinds of receptors; Axl, Dtk, and Mer. However, their expression and functions are not entirely clear in the different glomerular cell types. Meanwhile, representative cell cycle regulatory protein p27 has been reported to be expressed in podocytes in normal glomeruli with decreased expression in proliferating glomeruli, which inversely correlated with mesangial proliferation in human IgA nephropathy (IgAN).

Methods

The aim of this study is to clarify Gas6 involvement in the progression of IgAN. Expression of Gas6/Axl/Dtk was examined in 31 biopsy proven IgAN cases. We compared the expression levels with histological severity or clinical data. Moreover, we investigated the expression of Gas6 and its receptors in cultured podocytes.

Results

In 28 of 31 cases, Gas6 was upregulated mainly in podocytes. In the other 3 cases, Gas6 expression was induced in endothelial and mesangial cells, which was similar to animal nephritis models. Among 28 podocyte type cases, the expression level of Gas6 correlated with the mesangial hypercellularity score of IgAN Oxford classification and urine protein excretion. It also inversely correlated with p27 expression in glomeruli. As for the receptors, Axl was mainly expressed in endothelial and mesangial cells, while Dtk was expressed in podocytes. In vitro, Dtk was expressed in cultured murine podocytes, and the expression of p27 was decreased by Gas6 stimulation.

Conclusions

Gas6 was uniquely upregulated in either endothelial/mesangial cells or podocytes in IgAN. The expression pattern can be used as a marker to classify IgAN. Gas6 has a possibility to be involved in not only mesangial proliferation via Axl, but also podocyte injury via Dtk in IgAN.     

Falcarindiol inhibits LPS-induced inflammation via attenuating MAPK and JAK-STAT signaling pathways in murine macrophage RAW 264.7 cells

The purpose of the study was to investigate the mechanism of total flavone of Desmodium styracifolium (TFDS) in regulating the formation of urinary calculi. Protein levels of KIM-1, LC3-II, p-p38 were measured by Western blot. The effect of different COM concentrations, different TFDS concentrations, SB203580 (specific inhibitor of p38/MAPK), and overexpression of KIM-1 on cell viability were detected by WST-1 assay. The apoptotic cells and FITC positive cells were detected by flow cytometry. HK-2 cell viability decreased with the increase of COM concentration, and the protein levels of KIM-1, LC3-II, and p-p38 increased with the time. Blocking the p38/MAPK pathway or co-cultured with TFDS inhibited the effects of COM on apoptosis and autophagy of HK-2 cells. In addition, blocking the p38/MAPK pathway inhibited the expression of KIM-1. In COM-induced cells, after treated with SB203580, overexpression of KIM-1 could reverse the protection effect of SB203580 on COM-induced cell damage and the inhibition of SB203580 on COM-induced excessive autophagy, suggesting p38/MAPK regulated KIM-1 to regulate COM-induced cell apoptosis and autophagy. Finally, we proved that TFDS inhibited p38/MAPK pathway. And the protection effect of COM-induced cell injury increased with the increase of TFDS concentration, and the adhesion between COM and cells decreased with the increase of TFDS concentration. With the increase of the concentration of TFDS, p38/MAPK pathway was gradually inhibited, and KIM-1 and autophagy related proteins were decreased. TFDS inhibited HK-2 cell apoptosis and autophagy by regulating KIM-1 via p38/MAPK pathway.     

Rapamycin ameliorates IgA nephropathy via cell cycle-dependent mechanisms

IgA nephropathy is the most frequent type of glomerulonephritis worldwide. The role of cell cycle regulation in the pathogenesis of IgA nephropathy has been studied. The present study was designed to explore whether rapamycin ameliorates IgA nephropathy via cell cycle-dependent mechanisms. After establishing an IgA nephropathy model, rats were randomly divided into four groups. Coomassie Brilliant Blue was used to measure the 24-h urinary protein levels. Renal function was determined using an autoanalyzer. Proliferation was assayed via Proliferating Cell Nuclear Antigen (PCNA) immunohistochemistry. Rat mesangial cells were cultured and divided into the six groups. Methylthiazolyldiphenyl-tetrazolium bromide (MTT) and flow cytometry were used to detect cell proliferation and the cell cycle phase. Western blotting was performed to determine cyclin E, cyclin-dependent kinase 2, p27^Kip1, p70S6K/p-p70S6K, and extracellular signal-regulated kinase 1/2/p- extracellular signal-regulated kinase 1/2 protein expression. A low dose of the mammalian target of rapamycin (mTOR) inhibitor rapamycin prevented an additional increase in proteinuria, protected kidney function, and reduced IgA deposition in a model of IgA nephropathy. Rapamycin inhibited mesangial cell proliferation and arrested the cell cycle in the G1 phase. Rapamycin did not affect the expression of cyclin E and cyclin-dependent kinase 2. However, rapamycin upregulated p27^Kip1 at least in part via AKT (also known as protein kinase B)/mTOR. In conclusion, rapamycin can affect cell cycle regulation to inhibit mesangial cell proliferation, thereby reduce IgA deposition, and slow the progression of IgAN.     

RBP-J promotes cell growth and metastasis through regulating miR-182-5p-mediated Tiam1/Rac1/p38 MAPK axis in colorectal cancer

BackgroundRBP-J is involved in number of cellular processes. However, the potential mechanisms of RBP-J on colorectal cancer (CRC) development have not been clearly defined. In this study, we aimed to investigate the role and molecular mechanism of RBP-J in CRC.MethodsThe expression levels of RBP-J and Tiam1 in CRC tissues and cells were evaluated by RT-qPCR or western blot. RBP-J was knocked down with sh-RBP-J or overexpressed by pcDNA3.1-RBP-J in CRC cells. Cell proliferation, migration and invasion abilities were analyzed by MTT, wound healing, and transwell assay, respectively. CHIP-qPCR, RIP and dual luciferase reporter assays were performed to confirm the interaction between miR-182-5p and RBP-J or Tiam1. Expression levels of p-p38 MAPK, p38 MAPK, Slug-1, Twist1 and MMP-9 were analyzed by western blot. G-LISA test was used to detect Rac1 activity.ResultsOur results showed that the expression of RBP-J and Tiam1 was significantly up-regulated in CRC tissues and cells. RBP-J overexpression promoted proliferation, migration and invasion of CRC cells. Moreover, RBP-J was found to directly target miR-182-5p promoter and positively regulate the Tiam1/Rac1/p38 MAPK signaling pathway in CRC cells. It was also proved that miR-182-5p can bind Tiam1 directly. Furthermore, experiments revealed that RBP-J could promote CRC cell proliferation, migration and invasion via miR-182-5p-mediated Tiam1/Rac1/p38 MAPK axis. In addition, knockdown of RBP-J reduced tumor growth and metastasis in CRC mice.ConclusionRBP-J regulates CRC cell growth and metastasis through miR-182-5p mediated Tiam1/Rac1/p38 MAPK signaling pathway, implying potential novel therapeutic targets for CRC patients.     

p38α‐MAPK‐deficient myeloid cells ameliorate symptoms and pathology of APP‐transgenic Alzheimer's disease mice

Alzheimer''s disease (AD), the most common cause of dementia in the elderly, is pathologically characterized by extracellular deposition of amyloid‐β peptides (Aβ) and microglia‐dominated inflammatory activation in the brain. p38α‐MAPK is activated in both neurons and microglia. How p38α‐MAPK in microglia contributes to AD pathogenesis remains unclear. In this study, we conditionally knocked out p38α‐MAPK in all myeloid cells or specifically in microglia of APP‐transgenic mice, and examined animals for AD‐associated pathologies (i.e., cognitive deficits, Aβ pathology, and neuroinflammation) and individual microglia for their inflammatory activation and Aβ internalization at different disease stages (e.g., at 4 and 9 months of age). Our experiments showed that p38α‐MAPK‐deficient myeloid cells were more effective than p38α‐MAPK‐deficient microglia in reducing cerebral Aβ and neuronal impairment in APP‐transgenic mice. Deficiency of p38α‐MAPK in myeloid cells inhibited inflammatory activation of individual microglia at 4 months but enhanced it at 9 months. Inflammatory activation promoted microglial internalization of Aβ. Interestingly, p38α‐MAPK‐deficient myeloid cells reduced IL‐17a‐expressing CD4‐positive lymphocytes in 9 but not 4‐month‐old APP‐transgenic mice. By cross‐breeding APP‐transgenic mice with Il‐17a‐knockout mice, we observed that IL‐17a deficiency potentially activated microglia and reduced Aβ deposition in the brain as shown in 9‐month‐old myeloid p38α‐MAPK‐deficient AD mice. Thus, p38α‐MAPK deficiency in all myeloid cells, but not only in microglia, prevents AD progression. IL‐17a‐expressing lymphocytes may partially mediate the pathogenic role of p38α‐MAPK in peripheral myeloid cells. Our study supports p38α‐MAPK as a therapeutic target for AD patients.     

Downregulation of miR‑214-3p attenuates mesangial hypercellularity by targeting PTEN‑mediated JNK/c-Jun signaling in IgA nephropathy

Mesangial cell (MC) proliferation and matrix expansion are basic pathological characteristics of IgA nephropathy (IgAN). However, the stepwise mechanism of MC proliferation and the exact set of related signaling molecules remain largely unclear. In this study, we found a significant upregulation of miR-214-3p in the renal cortex of IgAN mice by miRNA sequencing. In situ hybridization analysis showed that miR-214-3p expression was obviously elevated in MCs in the renal cortex in IgAN. Functionally, knockdown of miR-214-3p alleviated mesangial hypercellularity and renal lesions in IgAN mice. In vitro, the inhibition of miR-214-3p suppressed MC proliferation and arrested G1-S cell cycle pSrogression in IgAN. Mechanistically, a luciferase reporter assay verified PTEN as a direct target of miR-214-3p. Downregulation of miR-214-3p increased PTEN expression and reduced p-JNK and p-c-Jun levels, thereby inhibiting MC proliferation and ameliorating renal lesions in IgAN. Moreover, these changes could be attenuated by co-transfection with PTEN siRNA. Collectively, these results illustrated that miR-214-3p accelerated MC proliferation in IgAN by directly targeting PTEN to modulate JNK/c-Jun signaling. Therefore, miR-214-3p may represent a novel therapeutic target for IgAN.     

Resveratrol protects H9c2 embryonic rat heart derived cells from oxidative stress by inducing autophagy: role of p38 mitogen-activated protein kinase

Recently, many studies have attempted to illustrate the mechanism of autophagy in protection against oxidative stress to the heart induced by H(2)O(2). However, whether resveratrol-induced autophagy involves the p38 mitogen-activated protein kinase (MAPK) pathway is still unknown. This study aimed to investigate whether treating H9c2 cells with resveratrol increases autophagy and attenuates the cell death and apoptosis induced by oxidative stress via the p38 MAPK pathway. Resveratrol with or without SB202190, an inhibitor of the p38 MAPK pathway, was added 30 min before H(2)O(2). After H(2)O(2) treatment, the cells were incubated under 5% CO(2) at 37 °C for 24 h to assess cell survival and death or incubated for 20 min for Western blot and transmission electron microscopy. Flow cytometry was used to detect apoptosis after 6 h of H(2)O(2) treatment. Resveratrol at 20 μmol/L protected H9c2 cells treated with 100 μmol/L H(2)O(2) from oxidative damage. It increased cell survival and markedly decrease lactate dehydrogenase release. In addition, resveratrol increased autophagy and decreased H(2)O(2)-induced apoptosis. Furthermore, the protective effects of resveratrol were inhibited by 10 μmol/L SB202190. Thus, resveratrol protected H(2)O(2)-treated H9c2 cells by upregulating autophagy via the p38 MAPK pathway.     

Zinc rescues obesity‐induced cardiac hypertrophy via stimulating metallothionein to suppress oxidative stress‐activated BCL10/CARD9/p38 MAPK pathway

Obesity often leads to obesity‐related cardiac hypertrophy (ORCH), which is suppressed by zinc‐induced inactivation of p38 mitogen‐activated protein kinase (p38 MAPK). In this study, we investigated the mechanisms by which zinc inactivates p38 MAPK to prevent ORCH. Mice (4‐week old) were fed either high fat diet (HFD, 60% kcal fat) or normal diet (ND, 10% kcal fat) containing variable amounts of zinc (deficiency, normal and supplement) for 3 and 6 months. P38 MAPK siRNA and the p38 MAPK inhibitor SB203580 were used to suppress p38 MAPK activity in vitro and in vivo, respectively. HFD activated p38 MAPK and increased expression of B‐cell lymphoma/CLL 10 (BCL10) and caspase recruitment domain family member 9 (CARD9). These responses were enhanced by zinc deficiency and attenuated by zinc supplement. Administration of SB203580 to HFD mice or specific siRNA in palmitate‐treated cardiomyocytes eliminated the HFD and zinc deficiency activation of p38 MAPK, but did not significantly impact the expression of BCL10 and CARD9. In cultured cardiomyocytes, inhibition of BCL10 expression by siRNA prevented palmitate‐induced increased p38 MAPK activation and atrial natriuretic peptide (ANP) expression. In contrast, inhibition of p38 MAPK prevented ANP expression, but did not affect BCL10 expression. Deletion of metallothionein abolished the protective effect of zinc on palmitate‐induced up‐regulation of BCL10 and phospho‐p38 MAPK. HFD and zinc deficiency synergistically induce ORCH by increasing oxidative stress‐mediated activation of BCL10/CARD9/p38 MAPK signalling. Zinc supplement ameliorates ORCH through activation of metallothionein to repress oxidative stress‐activated BCL10 expression and p38 MAPK activation.