Pharmacological Inhibition Of P38 MAPK Research Articles

RAD23B mediated proteasomal degradation occurs through p38 MAPK/ATF-2/RAD23B axis under nutrient-deprived conditions in breast cancer.

Metabolic reprogramming in cancer occurs due to interaction of cells with the surrounding tumor microenvironment. In the microenvironment of solid tumors, nutrient deprivation is induced by high consumption of nutrients and insufficient vasculature. Tumor cells alter their metabolic strategies to adapt to the microenvironment. To understand the role of these metabolic changes, in the current study, we have mimicked nutrient deprivation condition in vitro to evaluate the associated signaling pathways in breast cancer cells. In our study, we have shown that nutritional deprivation activated p38 MAPK and activating transcription factor-2 (ATF-2) by increased phosphorylation of Thr180/Tyr182 and Thr71, respectively, in breast cancer cells. Pharmacological inhibition of p38 MAPK showed increased cell viability and reduced expression of ATF-2 and RAD23B under nutrient starvation conditions. Further, silencing of ATF-2 showed increased cell viability and decreased expression of RAD23B under nutrient starvation conditions. This suggests the involvement of p38 MAPK/ATF-2/RAD23B axis as a signaling pathway under nutrition starvation in breast cancer cells. The RAD23B mediated proteasome activity was shown to be much higher under stress conditions indicating a crucial role of RAD23B as a target for breast cancer.

A critical role for Macrophage-derived Cysteinyl-Leukotrienes in HIV-1 induced neuronal injury

Macrophages (MΦ) infected with human immunodeficiency virus (HIV)-1 or activated by its envelope protein gp120 exert neurotoxicity. We found previously that signaling via p38 mitogen-activated protein kinase (p38 MAPK) is essential to the neurotoxicity of HIVgp120-stimulated MΦ. However, the associated downstream pathways remained elusive. Here we show that cysteinyl-leukotrienes (CysLT) released by HIV-infected or HIVgp120 stimulated MΦ downstream of p38 MAPK critically contribute to neurotoxicity. SiRNA-mediated or pharmacological inhibition of p38 MAPK deprives MΦ of CysLT synthase (LTC4S) and, pharmacological inhibition of the cysteinyl-leukotriene receptor 1 (CYSLTR1) protects cerebrocortical neurons against toxicity of both gp120-stimulated and HIV-infected MΦ. Components of the CysLT pathway are differentially regulated in brains of HIV-infected individuals and a transgenic mouse model of NeuroHIV (HIVgp120tg). Moreover, genetic ablation of LTC4S or CysLTR1 prevents neuronal damage and impairment of spatial memory in HIVgp120tg mice. Altogether, our findings suggest a novel critical role for cysteinyl-leukotrienes in HIV-associated brain injury.

PS-BPB05-9: DUAL DELETION OF GUANYLYL CYCLASE-A AND P38 MAPK IN PODOCYTES WITH ALDOSTERONE ADMINISTRATION CAUSES GLOMERULAR INTRACAPILLARY THROMBI

Objective: Previously, we demonstrated that uninephrectomized aldosterone-infused, high salt-fed podocyte-specific guanylyl cyclase-A (GC-A) conditional KO (pod-GC-A cKO) mice exhibit glomerular injury and that pharmacological inhibition of p38 MAPK ameliorates podocyte damage. However, the effects of genetic deletion of p38 MAPK in podocytes of pod-GC-A cKO mice have been unknown. By generating podocyte-specific p38 MAPK and GC-A double cKO mice, the study investigated the effects of p38 MAPK deletion in podocytes on glomerular injury with aldosterone and a high salt diet in mice. Design and method: We generated p38 MAPK(fl/fl);Nephrin-Cre (pod-p38 MA- PK cKO) mice and p38 MAPK(fl/fl);GC-A(fl/fl);Nephrin-Cre (pod-p38MAPK/GC-A double cKO) mice. For induction of glomerular injury, we treated them with aldosterone and high salt at 2 months of age for 3 weeks without nephrectomy (B-ALDO). In vitro, firstly, we examined human podocytes which p38 MAPK was deleted by Crispr/Cas9 system and GC-A was suppressed by siRNA. Secondly, we used a transwell co-culture system to investigate changes of wild-type human umbilical vein endothelial cells (HUVEC) mRNA in the lower layer culture with immortalized p38 MAPK-null podocytes transfected with si-GC-A in the upper layer. Results: Unexpectedly, aldosterone-infused, and high salt-fed (B-ALDO)-treated pod-p38 MAPK/GC-A double cKO mice resulted in significant elevation of serum Cr (0.29 ± 0.04 mg/dL), massive albuminuria (42,0541 ± 8,493 μg/mgCr) and severe foot process effacement in addition to intracapillary fibrin thrombi which indicated endothelial damage. In vitro, knockout of p38 MAPK and suppression of GC-A in human cultured podocytes induced podocyte damage. Deletion of p38 MAPK and inhibition of GC-A in podocytes in the upper layer upregulated TGFB1 and FN1 in the HUVEC in the lower layer, indicating that some humoral factors derived from podocytes could work as cell-to-cell mediators, especially for HUVEC. Conclusions: Genetic dual deletion of p38 MAPK and GC-A in podocytes accelerates both podocyte and endothelium injuries, suggesting that p38 MAPK in podocytes has an essential role in maintaining podocyte-endothelial interactions, especially under diseased conditions.

Inhibition of p38 MAPK decreases hyperglycemia-induced nephrin endocytosis and attenuates albuminuria

Chronic hyperglycemia, as in diabetes mellitus, may cause glomerular damage with microalbuminuria as an early sign. Noteworthy, even acute hyperglycemia can increase glomerular permeability before structural damage of the glomerular filter can be detected. Despite intensive research, specific antiproteinuric therapy is not available so far. Thus, a deeper understanding of the molecular mechanisms of albuminuria is desirable. P38 MAPK signaling is involved in the development of hyperglycemia-induced albuminuria. However, the mechanism of increased p38 MAPK activity leading to increased permeability and albuminuria remained unclear. Recently, we demonstrated that acute hyperglycemia triggers endocytosis of nephrin, the key molecule of the slit diaphragm, and induces albuminuria. Here, we identify p38 MAPK as a pivotal regulator of hyperglycemia-induced nephrin endocytosis. Activated p38 MAPK phosphorylates the nephrin c-terminus at serine 1146, facilitating the interaction of PKCα with nephrin. PKCα phosphorylates nephrin at threonine residues 1120 and 1125, mediating the binding of β-arrestin2 to nephrin. β-arrestin2 triggers endocytosis of nephrin by coupling it to the endocytic machinery, leading to increased glomerular permeability. Pharmacological inhibition of p38 MAPK preserves nephrin surface expression and significantly attenuates albuminuria.Key messagesAcute hyperglycemia triggers endocytosis of nephrin.Activated p38 MAPK phosphorylates the nephrin c-terminus at serine 1146, facilitating the interaction of PKCα with nephrin.PKCα phosphorylates nephrin at threonine residues 1120 and 1125, mediating the binding of β-arrestin2 to nephrin.β-arrestin2 triggers endocytosis of nephrin by coupling it to the endocytic machinery, leading to a leaky glomerular filter.Pharmacological inhibition of p38 MAPK preserves nephrin surface expression and significantly attenuates albuminuria under hyperglycemic conditions.

Pharmacological Inhibition of p38 MAPK Rejuvenates Bone Marrow Derived-Mesenchymal Stromal Cells and Boosts their Hematopoietic Stem Cell-Supportive Ability.

The therapeutic value of mesenchymal stromal cells (MSCs) for various regenerative medicine applications, including hematopoietic stem cell transplantations (HSCT), has been well-established. Owing to their small numbers in vivo, it becomes necessary to expand them in vitro, which leads to a gradual loss of their regenerative capacity. Stress-induced mitogen-activated protein kinase p38 (p38 MAPK) signaling has been shown to compromise the MSC functions. Therefore, we investigated whether pharmacological inhibition of p38 MAPK signaling rejuvenates the cultured MSCs and boosts their functionality. Indeed, we found that the ex vivo expanded MSCs show activated p38 MAPK signaling and exhibit increased oxidative stress. These MSCs show a decreased ability to secrete salutary niche factors, thereby compromising their ability to support hematopoietic stem cell (HSC) self-renewal, proliferation, and differentiation. We, therefore, attempted to rejuvenate the cultured MSCs by pharmacological inhibition of p38 MAPK - a strategy broadly known as "priming of MSCs". We demonstrate that priming of MSCs with a p-38 MAPK inhibitor, PD169316, boosts their niche-supportive functions via upregulation of various HSC-supportive transcription factors. These primed MSCs expand multipotent HSCs having superior homing and long-term reconstitution ability. These findings shed light on the significance of non-cell-autonomous mechanisms operative in the hematopoietic niche and point towards the possible use of pharmacological compounds for rejuvenation ofex vivocultured MSCs. Such approaches could improve the outcome of regenerative therapies involving in vitro cultured MSCs.

Methylation of dual-specificity phosphatase 4 controls cell differentiation.

SUMMARYMitogen-activated protein kinases (MAPKs) are inactivated by dual-specificity phosphatases (DUSPs), the activities of which are tightly regulated during cell differentiation. Using knockdown screening and single-cell transcriptional analysis, we demonstrate that DUSP4 is the phosphatase that specifically inactivates p38 kinase to promote megakaryocyte (Mk) differentiation. Mechanistically, PRMT1-mediated methylation of DUSP4 triggers its ubiquitinylation by an E3 ligase HUWE1. Interestingly, the mechanistic axis of the DUSP4 degradation and p38 activation is also associated with a transcriptional signature of immune activation in Mk cells. In the context of thrombocytopenia observed in myelodysplastic syndrome (MDS), we demonstrate that high levels of p38 MAPK and PRMT1 are associated with low platelet counts and adverse prognosis, while pharmacological inhibition of p38 MAPK or PRMT1 stimulates megakaryopoiesis. These findings provide mechanistic insights into the role of the PRMT1-DUSP4-p38 axis on Mk differentiation and present a strategy for treatment of thrombocytopenia associated with MDS.

Pharmacological targeting of differential DNA repair, radio-sensitizes WRN-deficient cancer cells in vitro and in vivo

Werner (WRN) expression is epigenetically downregulated in various tumors. It is imperative to understand differential repair process in WRN-proficient and WRN-deficient cancers to find pharmacological targets for radio-sensitization of WRN-deficient cancer. In the current investigation, we showed that pharmacological inhibition of CHK1 mediated homologous recombination repair (HRR), but not non-homologous end joining (NHEJ) repair, can causes hyper-radiosensitization of WRN-deficient cancers. This was confirmed in cancer cell lines of different tissue origin (osteosarcoma, colon adenocarcinoma and melanoma) with WRN silencing and overexpression. We established that WRN-depleted cells are dependent on a critical but compromised CHK1-mediated HRR-pathway for repairing ionizing radiation (IR) induced DSBs for their survival. Mechanistically, we unraveled a new finding that the MRE11, CTIP and WRN proteins are largely responsible for resections of late and persistent DSBs. In response to IR-treatment, MRE11 and CTIP-positively and WRN-negatively regulate p38-MAPK reactivation in a CHK1-dependent manner. A degradation resistant WRN protein, mutated at serine 1141, abrogates p38-MAPK activation. We also showed that CHK1-p38-MAPK axis plays important role in RAD51 mediated HRR in WRN-silenced cells. Like CHK1 inhibition, pharmacological-inhibition of p38-MAPK also hyper-radiosensitizes WRN-depleted cells by targeting HR-pathway. Combination treatment of CHK1-inhibitor (currently under various clinical trials) and IR exhibited a strong synergy against WRN-deficient melanoma tumor in vivo. Taken together, our findings suggest that pharmacological targeting of CHK1-p38-MAPK mediated HRR is an attractive strategy for enhancing therapeutic response of radiation treatment of cancer.

The role of MAP-kinase p38 in the m. soleus slow myosin mRNA transcription regulation during short-term functional unloading

The unloading of postural muscles leads to the changes in myosins heavy chains isoforms (MyHCs) mRNAs transcription pattern, that cause severe alterations of muscle functioning. Several transcription factors such as NFATc1 and TEAD1 upregulate slow MyHC mRNA transcription, and p38 MAP kinase can phosphorylate NFAT and TEAD1, causing their inactivation. However, the role p38 MAP kinase plays in MyHCs mRNAs transcription regulation in postural soleus muscle during unloading remains unclear. We aimed to investigate whether pharmacological inhibition of p38 MAPK during rat soleus unloading would prevent the unloading-induced slow-type MyHC mRNA transcription decrease by affecting calcineurin/NFATc1 or TEAD1 signaling. Male Wistar rats were randomly assigned to three groups: cage control (C), 3-day hindlimb suspended group (3HS) and 3-day hindlimb suspended group with the daily oral supplementation of 10 mg/kg p38 MAPK inhibitor VX-745 (3HS + VX-745). 3 days of hindlimb suspension caused the significant decreases of slow MyHC and slow-tonic myh7b mRNAs transcription as well as the decrease of NFATc1-dependent MCIP1.4 mRNA transcription in rat soleus muscles compared to the cage control. P38 MAP-kinase inhibition during hindlimb suspension completely prevented slow MyHC mRNA content decrease and partially prevented slow-tonic myh7b and MCIP1.4 mRNAs transcription decreases compared to the 3HS group. We also observed NFATc1 and TEAD1 myonuclear contents increases in the 3HS + VX-745 group compared to both 3HS and C groups (p < 0.05). Therefore, we found that p38 inhibition counteracts the unloading-induced slow MyHC mRNA transcription downregulation and leads to the activation of calcineurin/NFAT signaling cascade in unloaded rat soleus muscles.

P0143OPPOSING EFFECTS OF P38 MAPK IN PODOCYTES ON ALDOSTERONE-INDUCED GLOMERULAR INJURY IN POD-GC-A CKO MICE

Abstract Background and Aims Previously, we demonstrated that uninephrectomized aldosterone-infused, high salt-fed podocyte-specific guanylyl cyclase-A (natriuretic peptide receptor 1) conditional KO (pod-GC-A cKO) mice exhibited glomerular injury and that pharmacological inhibition of p38 MAPK ameliorates podocyte damage. However, the effects of genetic deletion of p38 MAPK in podocytes of pod-GC-A cKO mice have been unclarified. Method We generated p38 MAPK(fl/fl);Nephrin-Cre (pod-p38 MAPK cKO) mice and p38 MAPK(fl/fl);GC-A(fl/fl);Nephrin-Cre (pod-p38MAPK/GC-A DKO) mice. For induction of glomerular injury, we treated them with aldosterone and high salt at 2 months of age for 3 weeks without nephrectomy (B-ALDO). In vitro, we examined the effect of p38 MAPK inhibitor in cultured human podocytes transfected with GC-A siRNA. Results B-ALDO-treated pod-p38 MAPK/GC-A DKO mice resulted in significant elevation of serum Cr (0.29 ± 0.04 mg/dl), massive albuminuria (42,660 ± 20,200 μg/mgCr) and severe foot process effacement in addition to intracapillary fibrin thrombi which indicated endothelial damage. Vehicle-treated DKO mice, B-ALDO-treated pod-GC-A cKO mice, and B-ALDO-treated pod-p38 MAPK cKO showed normal serum Cr levels (0.14 ± 0.01, 0.18 ± 0.02, 0.20 ± 0.01 mg/dl, respectively), mild increase of albuminuria (223 ± 6.5, 1,496 ± 592, 649 ± 303 μg/mgCr, respectively) and only segmental foot process effacement. Blood pressure was not elevated in either mutant mice compared with that of B-ALDO control mice. Furthermore, glomerular mRNA expressions of MCP-1, PAI-1, and FN were upregulated and that of VEGF-A was downregulated in DKO mice. In vitro, suppression of GC-A mRNA by siRNA in combination with p38 MAPK inhibitor downregulated VEGF mRNA in human cultured podocytes. Our previous works showed that pharmacological inhibition of p38 MAPK in the whole body ameliorated podocyte damage, whereas our current result showed that genetic deletion of p38 MAPK in podocytes aggravated renal injury. In order to explain the discrepancy in these results, we added an analysis of podocyte specific GC-A fl/fl p38 fl/+ cKO mice. Pod GC-A fl/fl p38 fl/+ cKO mice exhibited considerably milder renal damage than pod GC-A fl/fl p38 fl/fl double cKO mice. Conclusion Genetic complete p38 MAPK deletion in GC-A-nul podocytes exacerbated aldosterone-induced glomerular endothelial cell injury as well as podocytes, and resulted in renal dysfunction, probably through VEGF downregulation, whereas partial p38 MAPK inhibition in podocytes ameliorated aldosterone-induced glomerular injury in pod-GC-A cKO mice. These results suggest a certain level of p38 MAPK in podocytes is necessary to protect endothelial and epithelial cells from aldosterone-induced renal injury.

Origanum majorana Essential Oil Triggers p38 MAPK-Mediated Protective Autophagy, Apoptosis, and Caspase-Dependent Cleavage of P70S6K in Colorectal Cancer Cells.

Colorectal cancer (CRC) is the third most common type of cancer in terms of incidence and mortality worldwide. Here we have investigated the anti-colon cancer potential of Origanum majorana essential oil (OMEO) and its underlying mechanisms of action. We showed that OMEO significantly inhibited the cellular viability and colony growth of human HT-29 colorectal cancer cells. OMEO induced protective autophagy, associated with downregulation of the mTOR/p70S6K pathway, and activated caspase-8 and caspase-9-dependent apoptosis. Blockade of autophagy with 3-methyladenine (3-MA) and chloroquine (CQ), two autophagy inhibitors, potentiated the OMEO-induced apoptotic cell death. Inversely, inhibition of apoptosis with the pan-caspase inhibitor, Z-VAD-FMK, significantly reduced cell death, suggesting that apoptosis represents the main mechanism of OMEO-induced cell death. Mechanistically, we found that OMEO induces protective autophagy and apoptotic cells death via the activation of the p38 MAPK signaling pathway. Pharmacological inhibition of p38 MAPK by the p38 inhibitors SB 202190 and SB 203580 not only significantly decreased apoptotic cell death, but also reduced the autophagy level in OMEO treated HT-29 cells. Strikingly, we found that OMEO also induces p38 MAPK-mediated caspase-dependent cleavage of p70S6K, a protein reported to be overexpressed in colon cancer and associated with drug resistance. Our findings suggest that OMEO inhibits colon cancer through p38 MAPK-mediated protective autophagy and apoptosis associated with caspase-dependent cleavage of p70S6K. To the best of our knowledge, this study is the first to report on the implications of the p38 MAPK signaling pathway in targeting p70S6K to caspase cleavage.

Pharmacological Inhibition of p38 MAPK by SB203580 Increases Resistance to Carboplatin in A2780cp Cells and Promotes Growth in Primary Ovarian Cancer Cells.

Chemoresistance renders current chemotherapy regimens ineffective against advanced epithelial ovarian cancer (EOC). Carboplatin (the first-line chemotherapeutic agent to treat EOC) induces cell death by regulating multiple signaling pathways. The objective of this study is to identify the signaling pathways that contribute to carboplatin resistance in EOC. To this end, we performed a proteome profiler human phospho-kinase array experiment and compared the phosphorylation profiles between the cisplatin-sensitive A2780s versus its derivative cisplatin-resistant A2780cp cells. The phospho-kinase array revealed that A2780s and A2780cp cells displayed different profiles in basal and carboplatin-induced phosphorylation. Phosphorylation of p38 MAPK was increased by carboplatin more markedly in A2780s cells compared to A2780cp cells. Inhibition of p38 MAPK activity by its specific inhibitor SB203580 increased resistance to carboplatin in A2780cp cells, but not in A2780s cells or in ascites-derived high-grade serous EOC cells. Interestingly, SB203580 increased the number of viable cells in the primary EOC cells, which was concomitant with an increase in survivin expression. In conclusion, inhibition of p38 MAPK by SB203580 increases resistance to carboplatin in A2780cp cells and the number of viable cells in the primary EOC cells, suggesting that pharmacological inhibition of p38 MAPK might not be an effective therapeutic strategy for EOC.

Matrix metalloproteinase-2: A key regulator in coagulation proteases mediated human breast cancer progression through autocrine signaling.

Cell invasion is attributed to the synthesis and secretion of proteolytically active matrix-metalloproteinases (MMPs) by tumor cells to degrade extracellular matrix (ECM) and promote metastasis. The role of protease-activated receptor 2 (PAR2) in human breast cancer migration/invasion via MMP-2 up-regulation remains ill-defined; hence we investigated whether TF-FVIIa/trypsin-mediated PAR2 activation induces MMP-2 expression in human breast cancer. MMP-2 expression and the signaling mechanisms were analyzed by western blotting and RT-PCR. MMP-2 activity was measured by gelatin zymography. Cell invasion was analyzed by transwell invasion assay whereas; wound healing assay was performed to understand the cell migratory potential. Here, we highlight that TF-FVIIa/trypsin-mediated PAR2 activation leads to enhanced MMP-2 expression in human breast cancer cells contributing to tumor progression. Knock-down of PAR2 abrogated TF-FVIIa/trypsin-induced up-regulation of MMP-2. Again, genetic manipulation of AKT or inhibition of NF-ĸB suggested that PAR2-mediated enhanced MMP-2 expression is dependent on the PI3K-AKT-NF-ĸB pathway. We also reveal that TF, PAR2, and MMP-2 are over-expressed in invasive breast carcinoma tissues as compared to normal. Knock-down of MMP-2 significantly impeded TF-FVIIa/trypsin-induced cell invasion. Further, we report that MMP-2 activates p38 MAPK-MK2-HSP27 signaling axis that leads to actin polymerization and induces cell migration. Pharmacological inhibition of p38 MAPK or MK2 attenuates MMP-2-induced cell migration. The study delineates a novel signaling pathway by which PAR2-induced MMP-2 expression regulates human breast cancer cell migration/invasion. Understanding these mechanistic details will certainly help to identify crucial targets for therapeutic interventions in breast cancer metastasis.

Coralyne, a protoberberine alkaloid, causes robust photosenstization of cancer cells through ATR-p38 MAPK-BAX and JAK2-STAT1-BAX pathways

Photodynamic therapy (PDT) provides an effective cancer treatment option but it requires sufficient cellular oxygen concentration to exert its photosensitizing effects. Due to hypoxic nature of most tumors, widespread clinical application of PDT is restricted and warrants development of photosensitizers which can kill cancer cells in ROS independent manner. Previously, we reported significant enhancement of the anti-cancer property of coralyne in presence of ultraviolet-A (UVA) light exposure against several human carcinoma cell lines. This study aimed at unravelling molecular cascades of events in CUVA treatment (coralyne and UVA light)-mediated photosensitization of human skin cancer. The CUVA-treatment caused robust apoptosis of A431 cancer cells, primarily through mitochondrial and lysosomal dysfunctions. Silencing of BAX conferred a significant protection against CUVA-induced apoptosis. Both lysosomal proteases and caspase-8 activation contributed to BID cleavage. Further, our results revealed that a dual signaling axis e.g., ATR-p38 MAPK and JAK2-STAT1 pathways functioned upstream of BAX activation in apoptosis response. Moreover, transient silencing of ATR and pharmacological inhibition of p38-MAPK or JAK2 significantly abolished the effect of CUVA treatment induced BAX expression and cell death, linking the extrinsic and intrinsic pathways with the observed cell death. Our data suggest that coralyne, which is known topoisomerase-I inhibitor, may be an attractive agent for photo-chemotherapeutic treatment of human skin cancers.

Dopamine directly increases mitochondrial mass and thermogenesis in brown adipocytes.

Brown adipose tissue (BAT) is key to energy homeostasis. By virtue of its thermogenic potential, it may dissipate excessive energy, regulate body weight and increase insulin sensitivity. Catecholamines are critically involved in the regulation of BAT thermogenesis, yet research has focussed on the effects of noradrenaline and adrenaline. Some evidence suggests a role of dopamine (DA) in BAT thermogenesis, but the cellular mechanisms involved have not been addressed. We employed our extensively characterised murine brown adipocyte cells. D1-like and D2-like receptors were detectable at the protein level. Stimulation with DA caused an increase in cAMP concentrations. Oxygen consumption rates (OCR), mitochondrial membrane potential (Δψm) and uncoupling protein 1 (UCP1) levels increased after 24 h of treatment with either DA or a D1-like specific receptor agonist. A D1-like receptor antagonist abolished the DA-mediated effect on OCR, Δψm and UCP1. DA induced the release of fatty acids, which did not additionally alter DA-mediated increases of OCR. Mitochondrial mass (as determined by (i) CCCP- and oligomycin-mediated effects on OCR and (ii) immunoblot analysis of mitochondrial proteins) also increased within 24 h. This was accompanied by an increase in peroxisome proliferator-activated receptor gamma co-activator 1 alpha protein levels. Also, DA caused an increase in p38 MAPK phosphorylation and pharmacological inhibition of p38 MAPK abolished the DA-mediated effect on Δψm In summary, our study is the first to reveal direct D1-like receptor and p38 MAPK-mediated increases of thermogenesis and mitochondrial mass in brown adipocytes. These results expand our understanding of catecholaminergic effects on BAT thermogenesis.

Rottlerin induces cyclooxygenase-2 upregulation through an ATF4 and reactive oxygen species-independent pathway in HEI-OC1 cells

Hearing loss can be caused by infection, inflammation, loud noise and ototoxic drugs. The induction of cyclooxygenase-2 (COX‑2) expression is an important event during the cellular inflammatory response. The present study investigated the effect of rottlerin on CO-2 mRNA and protein expression in HEI-OC1 cells. Cell viability was determined using an MTT assay. Western blotting was used to examine the expression of COX‑2, endoplasmic reticulum stress-associated transcription factors and activation of the MAPK pathway. ROS was measured using the fluorescent probe 2', 7'-dichlorodihydrofluorescein diacetate. Treatment with the natural protein kinaseC δ inhibitor, rottlerin, was shown to increase COX‑2 expression at the protein and mRNA levels in a dose‑dependent manner. Rottlerin was shown to induce increased reactive oxygen species (ROS) generation, however, ROS were not critical for rottlerin‑induced upregulation of COX‑2 expression in HEI‑OC1 cells. In addition, rottlerin was shown to increase the phosphorylation of p38mitogen-activated protein kinase (MAPK). The pharmacological inhibition of p38MAPK and suppression of activating transcription factor4 (an ER stress‑associated transcription factor) expression by small interfering RNA inhibited rottlerin-induced COX‑2 upregulation. Furthermore, COX‑2 expression levels were increased further when cells were treated with rottlerin and interleukin‑1β or protein kinase C activator, PMA. In conclusion, the results of the present study demonstrated that rottlerin is a novel inducer of COX‑2 expression and identified the mechanisms involved in this process. Rottlerin may be considered a potential activator of repair and remodeling.

DEPTOR promotes survival of cervical squamous cell carcinoma cells and its silencing induces apoptosis through downregulating PI3K/AKT and by up-regulating p38 MAP kinase.

DEPTOR is an endogenous inhibitor of mTOR complexes, de-regulated in cancers. The present study reveals a vital role for DEPTOR in survival of cervical squamous cell carcinoma (SCC). DEPTOR was found to be overexpressed in both cervical SCC cells and tissues and it's silencing in cervical SCC cells induced apoptosis, mainly by up-regulation of p38 MAPK and by inhibiting PI3K/AKT pathway via a feed-back inhibition from mTORC1-S6K. DEPTOR silencing resulted in reduced expression of the nitric oxide synthases iNOS and eNOS, as well as increased activation of ERK1/2 and p38 MAP kinases. Activation of AKT signaling by overexpression of constitutively active-AKT (CA-AKT) failed to overcome the apoptosis caused by DEPTOR silencing. Similarly pharmacological inhibition of ERK also failed to control apoptosis. However pharmacological inhibition of p38 MAPK rescued the cells from apoptosis, indicating the major role of p38 MAPK in cell death induced by DEPTOR silencing. DEPTOR was also found to regulate ERK1/2 in an AKT dependent manner. DEPTOR knockdown induced cell death in SiHa cells overexpressing the anti-apoptotic Bcl-2 and Bcl-xL, indicating strong survival role of DEPTOR in these cells. DEPTOR overexpression activated PI3K/AKT by relieving the negative feed-back inhibition from mTORC1-S6K. DEPTOR regulation was also observed to be independent of HPV E6/E7 oncoproteins, but it might be a molecular co-factor contributing to cervical carcinogenesis. In summary, DEPTOR is found to promote survival of cervical SCC cells and its reduction induced apoptosis via differential effects on PI3K/AKT and p38 MAPK and can be a potential target in cervical SCC.

7. Dkk‐1/Ror2 and Wnt5a/Ror2 Production of CDX2: Defining Intestinal Adenocarcinoma

Wnt signaling regulates cell proliferation and differentiation through ahomeostasis of canonical and noncanonical pathways. Aberrant Wnt signaling induces intestinal adenocarcinoma by promoting stem cell hyperproliferation. In the intestine, Wnt5a binds to its receptor Ror2 to increase the caudal homeobox factor CDX2, a transcriptional activator of intestinal differentiation. Contradictorily, it was also shown that Ror2 alone is overexpressed in adenocarcinoma, and that Ror2 alone will mediate CDX2 increase in vitro. The purpose of this research is to determine how CDX2 is increased by Ror2 alone and Wnt5a/Ror2. Human colon adenocarcinoma cells (HT‐29, SW‐480, and RKO) were used as an in vitro epithelial model. HT‐29 cells constitutively express the signaling protein Dickkopf‐1, whereas SW‐480 and RKO do not. Wnt5a/rhDkk‐1 was used to treat Ror2 or siRor2 transfected cells primed with MAP kinase pharmacological inhibitors. The results show that CDX2 was increased after Ror2 overexpression in HT29, but not RKO/SW480 cells. Further addition of Dkk‐1 to RKO/SW480 cells resulted in CDX2 increase. Pharmacological inhibition of p38 MAPK, casein kinase 1, or Src kinase decreased CDX2 protein ~24% in Dkk‐1/Ror2 cells, while inhibition of JNK, casein kinase 1, or Src kinase in Wnt5a/Ror2 cells decreased CDX2 protein ~35%. The conclusion is that Ror2 increase in CDX2 protein is dependent on Dkk‐1 or Wnt5a. Dkk‐1/Ror2 CDX2 production is mediated by the MAP kinases p38, CK1, and Src. Wnt5a/Ror2 increases in CDX2 require JNK activation, but not p38 MAPK. Different signaling pathways, dependent on the presence of Dkk1/Wnt5a, offer an explanation for the seemingly contradictory increase in Ror2 alone seen in intestinal adenocarcinoma.

Wogonin suppresses osteopontin expression in adipocytes by activating PPARα.

Wogonin (5,7-dihydroxy-8-methoxyflavone), a major bioactive compound of the flavonoid family, is commonly extracted from the traditional Chinese medicine Scutellaria baicalensis and possesses antioxidant and anti-inflammatory activities and is assumed to have anti-diabetes function. Indeed, a current study has shown that it can possibly treat metabolic disorders such as those found in db/db mice. However, the underlying molecular mechanism remains largely unclear. The aim of this study was to investigate the impact of wogonin on osteopontin (OPN) expression in adipose tissue from type 1 diabetic mice and in 3T3-L1 adipocytes. Type 1 diabetes was induced by streptozotocin (STZ) injection. 3T3-L1 preadipocytes were converted to 3T3-L1 adipocytes through treatment with insulin, dexamethasone, and 3-isobutyl-1-methylxanthine (IBMX). Western blot analysis and RT-PCR were performed to detect protein expression and mRNA levels, respectively. Wogonin treatment suppressed the increase in serum OPN levels and reduced OPN expression in adipose tissue from STZ-induced type 1 diabetic mice. Administration of wogonin enhanced PPARα expression and activity. Silencing of PPARα diminished the inhibitory effects of wogonin on OPN expression in 3T3-L1 adipocytes. Furthermore, the levels of c-Fos and phosphorylated c-Jun were reduced in wogonin-treated adipose tissue and 3T3-L1 adipocytes. In addition, wogonin treatment dramatically mitigated p38 MAPK phosphorylation. Pharmacological inhibition of p38 MAPK by its specific inhibitor SB203580 increased PPARα activity and decreased OPN expression. Our results suggest that wogonin downregulated OPN expression in adipocytes through the inhibition of p38 MAPK and the sequential activation of the PPARα pathway. Given the adverse effects of high OPN levels on metabolism, our results provide evidence for the potential administration of wogonin as a treatment for diabetes.

P38 MAPK Controls Sensitivity Towards BH3 Mimetics By Regulating Mcl-1 Expression of Chronic Lymphocytic Leukemia in Hypoxia and Acquired Resistance

Background: CLL patients frequently suffer relapse after an initially successful chemotherapy. This distinct resistance towards chemotherapy is thought to be caused by microenvironmental stimulation. Within the tumor microenvironment (TME) cells are not only stimulated by well-known external stimuli like CD40 ligand (CD40L) or activation of the B cell receptor (BCR), but are also exposed to hypoxia, which was found in the bone marrow and lymphatic tissue. Despite the known importance of hypoxia in solid tumors, its impact on survival and treatment response in CLL is still poorly understood.Methods: We have established a novel in vitro model for the CLL microenvironment, which considers both the external stimulation by CD40L and the hypoxic oxygen levels (1% O2). Treatment efficacy of different drugs in normoxia (21% O2) and hypoxia were determined by AnnexinV/7-AAD staining and subsequent FACS analysis. The underlying molecular mechanisms were analyzed via qRT-PCR and immunoblot. Furthermore B-cell lines Raji, Ramos and Mec-1 were continuously exposed to increasing concentrations of fludarabine or the BH3 mimetic ABT-737. After establishment of resistance the molecular adaptation was assessed and correlated to the changes induced by hypoxia.Results: Hypoxia is known to protect solid cancers from chemotherapy. In our model we made similar observations for CLL, since sensitivity to the classical DNA-targeting drugs fludarabine and bendamustine was reduced under hypoxic conditions. Interestingly, the tyrosine kinase inhibitor ibrutinib did not benefit from hypoxia either. However, this resistance was overcome by the mitochondria-targeting BH3 mimetics ABT-199 and ABT-737, whose effect was pronounced under hypoxia.We reveal that this effect was caused by an uncoupling of major signaling pathways. Under hypoxic conditions the activity of Akt, ERK1/2 and NFκB was reduced, while p38 MAPK became hyperphosphorylated. Phospho-p38 (pp38) downregulated Mcl-1 levels, which are the main regulator of sensitivity towards BH3 mimetics. Despite the known heterogeneity in between CLL patients this effect was found in most samples analyzed. The functional importance was underlined by the observation that pharmacological inhibition of p38 MAPK could reconstitute Mcl-1 levels and thereby resistance in hypoxia.The relevance of the pp38-Mcl-1 axis for ABT efficacy was emphasized by findings in B-cell lines with acquired resistance. Each ABT-resistant clone of the three tested cell lines induced p38 activity and decreased Mcl-1 levels. In contrast, in the fludarabine-resistant clones the pp38-Mcl-1 axis was not altered.Conclusion: These are the first experiments providing evidence that hypoxia has a crucial impact on survival and response to chemotherapy in CLL. We show that hypoxia renders CLL cells resistant to classical DNA-targeting agents, while the small molecules ABT-199 and ABT-737, which specifically target mitochondria, efficiently eradicate CLL cells within the microenvironment. Furthermore, we identified the pp38-Mcl-1 axis to be a major determinant of sensitivity to these BH3 mimetics, which warrants further evaluation of p38 as a novel biomarker for prediction of sensitivity to BH3 mimetics. DisclosuresNo relevant conflicts of interest to declare.

The generation of neutrophils in the bone marrow is controlled by autophagy.

Autophagy has been demonstrated to have an essential function in several cellular hematopoietic differentiation processes, for example, the differentiation of reticulocytes. To investigate the role of autophagy in neutrophil granulopoiesis, we studied neutrophils lacking autophagy-related (Atg) 5, a gene encoding a protein essential for autophagosome formation. Using Cre-recombinase mediated gene deletion, Atg5-deficient neutrophils showed no evidence of abnormalities in morphology, granule protein content, apoptosis regulation, migration, or effector functions. In such mice, however, we observed an increased proliferation rate in the neutrophil precursor cells of the bone marrow as well as an accelerated process of neutrophil differentiation, resulting in an accumulation of mature neutrophils in the bone marrow, blood, spleen, and lymph nodes. To directly study the role of autophagy in neutrophils, we employed an in vitro model of differentiating neutrophils that allowed modulating the levels of ATG5 expression, or, alternatively, intervening pharmacologically with autophagy-regulating drugs. We could show that autophagic activity correlated inversely with the rate of neutrophil differentiation. Moreover, pharmacological inhibition of p38 MAPK or mTORC1 induced autophagy in neutrophilic precursor cells and blocked their differentiation, suggesting that autophagy is negatively controlled by the p38 MAPK-mTORC1 signaling pathway. On the other hand, we obtained no evidence for an involvement of the PI3K-AKT or ERK1/2 signaling pathways in the regulation of neutrophil differentiation. Taken together, these findings show that, in contrast to erythropoiesis, autophagy is not essential for neutrophil granulopoiesis, having instead a negative impact on the generation of neutrophils. Thus, autophagy and differentiation exhibit a reciprocal regulation by the p38-mTORC1 axis.