Altered expression of AKT1 and P38A in the colons of patients with Hirschsprung's disease.

PURPOSE: Hirschsprung's disease (HSCR) is a functional obstruction of the gastrointestinal tract due to the congenital absence of enteric ganglion cells. The proto-oncogene RET is one of the primary genes implicated in the aetiology of HSCR. We designed this study to investigate the expression of 10 RET regulatory network genes in the colons of patients with HSCR. METHODS: HSCR tissue specimens (n = 28) were collected at the time of pull-through surgery. qPCR analysis was applied to compare the expression levels of 10 genes in the RET regulatory network. Western blot analysis was performed to quantify the protein expression. Immunohistochemistry was performed to determine the localization of AKT1 and P38A in HSCR colon tissue. RESULTS: AKT1 (p = 0.015) and P38A (p = 0.039) were both significantly downregulated in the aganglionic segment compared to those in the ganglionic segment in HSCR patients (n = 28). Western blot analysis revealed the decreasing protein expression of AKT1 and P38A in the aganglionic segment compared to ganglionic segment and control colon tissues (p < 0.05). Immunohistochemistry staining revealed that both AKT1 and P38A were localized in the colonic mucosa and were significantly decreased in the aganglionic segment. CONCLUSION: To our knowledge, we report for the first time the expression of RET regulatory network genes in the colons of patients with HSCR. The markedly decreased expression of AKT1 and P38A suggested a possible role in HSCR pathogenesis.

Transforming Growth Factor ß/NR4A1-Inducible Breast Cancer Cell Migration and Epithelial-to-Mesenchymal Transition Is p38α (Mitogen-Activated Protein Kinase 14) Dependent.

Transforming growth factor ß (TGF-ß)-induced migration of triple-negative breast cancer (TNBC) cells is dependent on nuclear export of the orphan receptor NR4A1, which plays a role in proteasome-dependent degradation of SMAD7. In this study, we show that TGF-ß induces p38α (mitogen-activated protein kinase 14 [MAPK14]), which in turn phosphorylates NR4A1, resulting in nuclear export of the receptor. TGF-ß/p38α and NR4A1 also play essential roles in the induction of epithelial-to-mesenchymal transition (EMT) and induction of ß-catenin in TNBC cells, and these TGF-ß-induced responses and nuclear export of NR4A1 are blocked by NR4A1 antagonists, the p38 inhibitor SB202190, and kinase-dead [p38(KD)] and dominant-negative [p38(DN)] forms of p38α. Inhibition of NR4A1 nuclear export results in nuclear export of TGF-ß-induced ß-catenin, which then undergoes proteasome-dependent degradation. TGF-ß-induced ß-catenin also regulates NR4A1 expression through formation of the ß-catenin-TCF-3/TCF-4/LEF-1 complex on the NR4A1 promoter. Thus, TGF-ß-induced nuclear export of NR4A1 in TNBC cells plays an essential role in cell migration, SMAD7 degradation, EMT, and induction of ß-catenin, and all of these pathways are inhibited by bis-indole-derived NR4A1 antagonists that inhibit nuclear export of the receptor and thereby block TGF-ß-induced migration and EMT.

The alkyllysophospholipid edelfosine enhances TRAIL-mediated apoptosis in gastric cancer cells through death receptor 5 and the mitochondrial pathway.

The ether phospholipid edelfosine is the prototype of a group of synthetic antitumor alkyllysophospholipid (ALP) compounds that exert pro-apoptotic effects in various types of cancer cells through cell type-dependent mechanisms. In this study, we examined the antitumor effect of edelfosine in human gastric cancer cells. Edelfosine decreased cell viability and induced autophagic death at a moderate concentration (~30 µM), whereas it induced apoptotic cell death at concentrations over 30 µM. Interestingly, low concentrations of edelfosine (5-10 µM) effectively enhanced recombinant human tumor necrosis factor (TNF)-related apoptosis-inducing ligand (rhTRAIL/TNFSF10)-induced apoptosis and clonogenicity in gastric cancer cells, including TRAIL-resistant AGS cells. Edelfosine upregulated the protein level of death receptor 5 (DR5/TNFRSF10B) and/or increased DR5 upregulation in lipid rafts. In addition, edelfosine-mediated rhTRAIL sensitization was regulated by the DR5 pathway. Edelfosine also activated p38MAPK (MAPK14), and edelfosine-mediated rhTRAIL sensitization was partially regulated by a p38-mediated decrease in mitochondrial membrane potential. This study suggests a novel therapeutic strategy targeting gastric cancer cells by using the combination of edelfosine and TRAIL.

NEK9-dependent proliferation of cancer cells lacking functional p53.

Dysfunction of the p53 network is a major cause of cancer development, and selective elimination of p53-inactivated cancer cells therefore represents an ideal therapeutic strategy. In this study, we performed a microRNA target screen that identified NEK9 (NIMA-related kinase 9) as a crucial regulator of cell-cycle progression in p53-inactivated cancer cells. NEK9 depletion selectively inhibited proliferation in p53-deficient cancer cells both in vitro and in vivo. The resultant cell-cycle arrest occurred predominantly in G1 phase, and exhibited senescence-like features. Furthermore, NEK9 repression affected expression of a broad range of genes encoding cell-cycle regulators and factors involved in mRNA processing, suggesting a novel role for NEK9 in p53-deficient cells. Lung adenocarcinoma patients with positive staining for NEK9 and mutant p53 proteins exhibited significantly poorer prognoses, suggesting that expression of both proteins promotes tumor growth. Our findings demonstrate that a novel NEK9 network regulates the growth of cancer cells lacking functional p53.

Loss of p38δ mitogen-activated protein kinase expression promotes oesophageal squamous cell carcinoma proliferation, migration and anchorage-independent growth.

Oesophageal cancer is an aggressive tumour which responds poorly to both chemotherapy and radiation therapy and has a poor prognosis. Thus, a greater understanding of the biology of oesophageal cancer is needed in order to identify novel therapeutic targets. Among these targets p38 MAPK isoforms are becoming increasingly important for a variety of cellular functions. The physiological functions of p38α and -ß are now well documented in contrast to -γ and -δ which are comparatively under-studied and ill-defined. A major obstacle to deciphering the role(s) of the latter two p38 isoforms is the lack of specific chemical activators and inhibitors. In this study, we analysed p38 MAPK isoform expression in oesophageal cancer cell lines as well as human normal and tumour tissue. We observed specifically differential p38δ expression. The role(s) of p38δ and active (phosphorylated) p38δ (p-p38δ) in oesophageal squamous cell carcinoma (OESCC) was delineated using wild-type p38δ as well as active p-p38δ, generated by fusing p38δ to its upstream activator MKK6b(E) via a decapeptide (Gly-Glu)5 linker. OESCC cell lines which are p38δ-negative (KE-3 and -8) grew more quickly than cell lines (KE-6 and -10) which express endogenous p38δ. Re-introduction of p38δ resulted in a time-dependent decrease in OESCC cell proliferation which was exacerbated with p-p38δ. In addition, we observed that p38δ and p-p38δ negatively regulated OESCC cell migration in vitro. Finally both p38δ and p-p38δ altered OESCC anchorage-independent growth. Our results suggest that p38δ and p-p38δ have a role in the suppression of OESCC. Our research may provide a new potential target for the treatment of oesophageal cancer.

Anti-inflammatory effect of Sanshuibaihu decoction may be associated with nuclear factor-kappa B and p38 MAPK alpha in collagen-induced arthritis in rat.

Sanshuibaihu decoction (SSBH) is an anti-arthritic Chinese herbal formula which has been used in the treatment of rheumatoid arthritis (RA) for many years. We herein aimed to confirm its anti-arthritic effect and explore the potential mechanism of action on collagen-induced arthritis (CIA) in rats. CIA was induced by immunizing 50 female Wistar rats with bovine type II collagen. 13 days following the immunization rats with CIA were treated with SSBH (50mg/kg), leflunomide (LEF) (10mg/kg) and physiological saline for 30 days, and rats without CIA were left untreated. After the treatment, paw edema was obviously improved in SSBH-treated rats, with the significant difference of arthritis score (F=6.032, P=0.006) observed between the three treated groups. In pathological observation, SSBH-treated rats showed a significant improvement of inflammatory infiltration, synovial hyperplasia, cartilage and bone destruction and joint fusion. After the treatment of SSBH, radiological score of knee (t=11.504, P=0.000) and ankle joints (t=9.250, P=0.000) was decreased significantly. In situ hybridization on joint tissue section indicated only slight synovial hyperblastosis and expression of NF-kappaB in SSBH-treated rats. Image analysis indicated a significant difference of means of integrated optical density (MIOD) (F=3.956, P=0.040) and means of stained area (MSA) (F=3.867, P=0.032) of NF-kappaB between the three treated groups. MIOD and MSA of SSBH-treated group were significantly lower vs control. Enzyme linked immunosorbent assay (ELISA) showed a significant difference (F=10.167, P=0.000) of the amount of p-p38 MAPKalpha in the three treated groups. The detected amount of p-p38 MAPKalpha in SSBH-treated group was significantly lower vs control. These results show SSBH has an inhibiting effect on CIA, which may be associated with NF-kappaB and p38 MAPKalpha.

The alpha-isoform of p38 MAPK specifically regulates arthritic bone loss.

Pharmacological inhibitors have provided evidence for the key role of p38 MAPK in osteoclast differentiation and in inflammation-induced bone loss. However, these inhibitors block more than one of the four p38 isoforms, usually p38alpha and p38beta, and sometimes also other kinases such as JNK3. We show in this study that p38alpha is the main p38 isoenzyme expressed in the osteoclast precursors and in the mature osteoclasts. p38alpha as well as its downstream substrates were phosphorylated in osteoclast progenitors stimulated by TNF-alpha. Using Mx-cre-mediated conditional gene inactivation we demonstrated that mice lacking p38alpha were protected against TNF-alpha-induced bone destruction at the site of inflammation as well as against TNF-alpha-mediated systemic bone loss. The bone protection was associated to decreased osteoclast numbers in vivo as well as a decreased IL-1beta expression in the inflamed tissue and in the isolated monocytes. The phenotype was cell autonomous because, similarly to p38alpha-deficient cells, knockdown of p38alpha in monocytes resulted in a decreased osteoclast differentiation in vitro. It was not caused by major changes in RANKL-mediated ERK or JNK activation but rather associated to an increased NF-kappaB activation caused by a decrease in IkappaBalpha recovery. Thus, our data show that developing specific inhibitors of the alpha-isoenzyme of p38 would be beneficial for the treatment of inflammation-induced bone destruction as observed in rheumatoid arthritis.

Enhanced soluble production of biologically active recombinant human p38 mitogen-activated-protein kinase (MAPK) in Escherichia coli.

The conditions were optimized for maximum soluble yield of biologically active recombinant p38alpha mitogen activated protein kinase (MAPK) vis-à-vis insoluble fraction (inclusion body formation). This study reports a rapid, economical and single step purification process for the overproduction of GST tagged p38alpha MAPK. A yield of 18 mg of highly purified and soluble protein per liter of bacterial culture within 6 h timeframe was achieved. The purified protein was found to be biologically suitable for phosphorylation by upstream kinases and was catalytically active. We further demonstrated that our in-house p38alpha MAPK is more potent (>30%) than a commercially available enzyme.

A novel p38 alpha MAPK inhibitor suppresses brain proinflammatory cytokine up-regulation and attenuates synaptic dysfunction and behavioral deficits in an Alzheimer's disease mouse model.

BACKGROUND: An accumulating body of evidence is consistent with the hypothesis that excessive or prolonged increases in proinflammatory cytokine production by activated glia is a contributor to the progression of pathophysiology that is causally linked to synaptic dysfunction and hippocampal behavior deficits in neurodegenerative diseases such as Alzheimer's disease (AD). This raises the opportunity for the development of new classes of potentially disease-modifying therapeutics. A logical candidate CNS target is p38 alpha MAPK, a well-established drug discovery molecular target for altering proinflammatory cytokine cascades in peripheral tissue disorders. Activated p38 MAPK is seen in human AD brain tissue and in AD-relevant animal models, and cell culture studies strongly implicate p38 MAPK in the increased production of proinflammatory cytokines by glia activated with human amyloid-beta (A beta) and other disease-relevant stressors. However, the vast majority of small molecule drugs do not have sufficient penetrance of the blood-brain barrier to allow their use as in vivo research tools or as therapeutics for neurodegenerative disorders. The goal of this study was to test the hypothesis that brain p38 alpha MAPK is a potential in vivo target for orally bioavailable, small molecules capable of suppressing excessive cytokine production by activated glia back towards homeostasis, allowing an improvement in neurologic outcomes. METHODS: A novel synthetic small molecule based on a molecular scaffold used previously was designed, synthesized, and subjected to analyses to demonstrate its potential in vivo bioavailability, metabolic stability, safety and brain uptake. Testing for in vivo efficacy used an AD-relevant mouse model. RESULTS: A novel, CNS-penetrant, non-toxic, orally bioavailable, small molecule inhibitor of p38 alpha MAPK (MW01-2-069A-SRM) was developed. Oral administration of the compound at a low dose (2.5 mg/kg) resulted in attenuation of excessive proinflammatory cytokine production in the hippocampus back towards normal in the animal model. Animals with attenuated cytokine production had reductions in synaptic dysfunction and hippocampus-dependent behavioral deficits. CONCLUSION: The p38 alpha MAPK pathway is quantitatively important in the A beta-induced production of proinflammatory cytokines in hippocampus, and brain p38 alpha MAPK is a viable molecular target for future development of potential disease-modifying therapeutics in AD and related neurodegenerative disorders.

The antiapoptotic effect of heme oxygenase-1 in endothelial cells involves the degradation of p38 alpha MAPK isoform.

Heme oxygenase-1 (HO-1) protects endothelial cells (EC) from undergoing apoptosis. This effect is mimicked by CO, generated via the catabolism of heme by HO-1. The antiapoptotic effect of CO in EC was abrogated when activation of the p38alpha and p38beta MAPKs was inhibited by the pyridinyl imidazole SB202190. Using small interfering RNA, p38beta was found to be cytoprotective in EC, whereas p38alpha was not. When overexpressed in EC, HO-1 targeted specifically the p38alpha but not the p38beta MAPK isoform for degradation by the 26S proteasome, an effect reversed by the 26S proteasome inhibitors MG-132 or lactacystin. Inhibition of p38alpha expression was also observed when HO-1 was induced physiologically by iron protoporphyrin IX (hemin). Inhibition of p38alpha no longer occurred when HO activity was inhibited by tin protoporphyrin IX, suggesting that p38alpha degradation was mediated by an end product of heme catabolism. Exogenous CO inhibited p38alpha expression in EC, suggesting that CO is the end product that mediates this effect. The antiapoptotic effect of HO-1 was impaired when p38alpha expression was restored ectopically or when its degradation by the 26S proteasome was inhibited by MG-132. Furthermore, the antiapoptotic effect of HO-1 was lost when p38beta expression was targeted by a specific p38beta small interfering RNA. In conclusion, the antiapoptotic effect of HO-1 in EC is dependent on the degradation of p38alpha by the 26S proteasome and on the expression of p38beta.