Inhibitor PD98059 Research Articles

Olive polyphenols attenuate TNF-α-stimulated M-CSF and IL-6 synthesis in osteoblasts: Suppression of Akt and p44/p42 MAP kinase signaling pathways

BackgroundOlive oil polyphenols, which possess cytoprotective activities like anti-oxidant and anti-inflammatory effects, could modulate osteoblast functions. The aim of this study is to elucidate the effects and the underlying mechanisms of hydroxytyrosol and oleuropein on the tumor necrosis factor-α (TNF-α)-induced macrophage colony-stimulating factor (M-CSF) and interleukin-6 (IL-6) synthesis in osteoblasts. MethodsOsteoblast-like MC3T3-E1 cells were pretreated with hydroxytyrosol, oleuropein, deguelin, PD98059 or wedelolactone, and then stimulated by TNF-α. The levels of M-CSF and IL-6 in the conditioned medium were determined with ELISA. The mRNA expression levels of M-CSF or IL-6 were determined with real-time RT-PCR. The phosphorylation levels of Akt, p44/p42 mitogen-activated protein (MAP) kinase or NF-κB in the cell lysates were determined with Western blot analysis. ResultsHydroxytyrosol and oleuropein attenuated the TNF-α-stimulated M-CSF release. Deguelin, an inhibitor of Akt, significantly suppressed the TNF-α-stimulated M-CSF release, which failed to be affected by the MEK1/2 inhibitor PD98059 or the IκB inhibitor wedelolactone. Hydroxytyrosol and oleuropein suppressed the TNF-α-induced phosphorylation of Akt and p44/p42 MAP kinase. Hydroxytyrosol and oleuropein attenuated the TNF-α-stimulated IL-6 release. Hydroxytyrosol suppressed the TNF-α-induced mRNA expressions of M-CSF and IL-6. Hydroxytyrosol or oleuropein failed to affect the cell viability. ConclusionOur present findings strongly suggest that olive oil polyphenols hydroxytyrosol and oleuropein down-regulates TNF-α signaling at the points upstream of Akt and p44/p42 MAP kinase in osteoblasts, leading to the attenuation of M-CSF and IL-6 synthesis.

Circulating fibroblast growth factor-2 precipitates HIV nephropathy in mice.

ABSTRACTPeople of African ancestry living with the human immunodeficiency virus-1 (HIV-1) are at risk of developing HIV-associated nephropathy (HIVAN). Children with HIVAN frequently show high plasma fibroblast growth factor-2 (FGF-2) levels; however, the role of circulating FGF-2 in the pathogenesis of childhood HIVAN is unclear. Here, we explored how circulating FGF-2 affected the outcome of HIVAN in young HIV-Tg26 mice. Briefly, we demonstrated that FGF-2 was preferentially recruited in the kidneys of mice without pre-existing kidney disease, precipitating HIVAN by activating phosphorylated extracellular signal-regulated kinase (pERK) in renal epithelial cells, without inducing the expression of HIV-1 genes. Wild-type mice injected with recombinant adenoviral FGF-2 (rAd-FGF-2) vectors carrying a secreted form of human FGF-2 developed transient and reversible HIVAN-like lesions, including proteinuria and glomerular enlargement. HIV-Tg26 mice injected with rAd-FGF-2 vectors developed more-significant proliferative and pro-fibrotic inflammatory lesions, similar to those seen in childhood HIVAN. These lesions were partially reversed by treating mice with the FGF/VEGF receptor tyrosine kinase inhibitor PD173074. These findings suggest that high plasma FGF-2 levels may be an independent risk factor for precipitating HIVAN in young children.

Helicobacter pylori promotes gastric cancer progression by upregulating semaphorin 5A expression via ERK/MMP9 signaling

Helicobacter pylori (H. pylori) infection is the strongest risk factor for the occurrence and development of gastric carcinoma. However, the molecular mechanism underlying H. pylori-induced pathogenesis has not yet been fully characterized. Here, we explored whether H. pylori upregulates semaphorin 5A to promote gastric cancer progression via the extracellular regulated protein kinases/matrix metalloproteinase (ERK/MMP9) signaling pathway. In this study, H. pylori upregulated semaphorin 5A expression in vitro and in vivo. Using the human gastric carcinoma cell lines SGC7901, SGC7901-siScrambled, and SGC7901-siSema 5A, our studies showed that H. pylori increased the proliferation, growth, migration, and invasiveness of gastric cancer cells via its effects on semaphorin 5A and that H. pylori increased the expression of MMP9 in gastric cancer cells via the semaphorin 5A-mediated ERK signaling pathway. Further analysis revealed that the ERK inhibitor PD98059 and MMP9 antibody (Ab) attenuated H. pylori-induced gastric cancer cell invasion and metastasis in vitro through a semaphorin 5A-dependent mechanism. In conclusion, H. pylori could promote gastric cancer progression in a semaphorin 5A-dependent manner via the ERK/MMP9 signaling pathway. Semaphorin 5A and its related signaling molecules potentially represent latent targets for H. pylori-related gastric cancer therapy.

The dipeptide prolyl-hydroxyproline promotes cellular homeostasis and lamellipodia-driven motility via active β1-integrin in adult tendon cells

Collagen-derived hydroxyproline (Hyp)-containing peptides have a variety of biological effects on cells. These bioactive collagen peptides are locally generated by the degradation of endogenous collagen in response to injury. However, no comprehensive study has yet explored the functional links between Hyp-containing peptides and cellular behavior. Here, we show that the dipeptide prolyl-4-hydroxyproline (Pro-Hyp) exhibits pronounced effects on mouse tendon cells. Pro-Hyp promotes differentiation/maturation of tendon cells with modulation of lineage-specific factors and induces significant chemotactic activity in vitro. In addition, Pro-Hyp has profound effects on cell proliferation, with significantly upregulated extracellular signal–regulated kinase phosphorylation and extracellular matrix production and increased type I collagen network organization. Using proteomics, we have predicted molecular transport, cellular assembly and organization, and cellular movement as potential linked-network pathways that could be altered in response to Pro-Hyp. Mechanistically, cells treated with Pro-Hyp demonstrate increased directional persistence and significantly increased directed motility and migration velocity. They are accompanied by elongated lamellipodial protrusions with increased levels of active β1-integrin–containing focal contacts, as well as reorganization of thicker peripheral F-actin fibrils. Pro-Hyp–mediated chemotactic activity is significantly reduced (p < 0.001) in cells treated with the mitogen-activated protein kinase kinase 1/2 inhibitor PD98059 or the α5β1-integrin antagonist ATN-161. Furthermore, ATN-161 significantly inhibits uptake of Pro-Hyp into adult tenocytes. Thus, our findings document the molecular basis of the functional benefits of the Pro-Hyp dipeptide in cellular behavior. These dynamic properties of collagen-derived Pro-Hyp dipeptide could lead the way to its application in translational medicine.

Glucose-impaired Corneal Re-epithelialization Is Promoted by a Novel Derivate of Dimethyl Fumarate.

Glucose induces corneal epithelial dysfunctions characterized by delayed wound repair. Nuclear erythroid 2-related factor 2 (Nrf2) mediates cell protection mechanisms even through the Heme oxygenase-1 (HO-1) up-regulation. Here, we synthesized new HO-1 inducers by modifying dimethyl fumarate (DMF) and used docking studies to select VP13/126 as a promising compound with the best binding energy to Kelch-like ECH-associated protein 1 (keap1), which is the the regulator of Nrf2 nuclear translocation. We verified if VP13/126 protects SIRC cells from hyperglycemia compared to DMF. SIRC were cultured in normal (5 mM) or high glucose (25 mM, HG) in presence of DMF (1–25 μM) or VP13/126 (0.1–5 μM) with or without ERK1/2 inhibitor PD98059 (15 μM). VP13/126 was more effective than DMF in the prevention of HG-induced reduction of cell viability and proliferation. Reduction of wound closure induced by HG was similarly counteracted by 1 μM VP13/126 and 10 μM DMF. VP13/126 strongly increased phospho/total ERK1/2 and restored HO-1 protein in HG-treated SIRC; these effects are completely counteracted by PD98059. Moreover, high-content screening analysis showed a higher rate of Nrf2 nuclear translocation induced by VP13/126 than DMF in HG-stimulated SIRC. These data indicate that VP13/126 exerts remarkable pro-survival properties in HG-stimulated SIRC, promoting the Nrf2/HO-1 axis.

Neurons and Astrocytes in Ventrolateral Periaqueductal Gray Contribute to Restraint Water Immersion Stress-Induced Gastric Mucosal Damage via the ERK1/2 Signaling Pathway

BackgroundThe restraint water immersion stress (RWIS) model includes both psychological and physical stimulation, which may lead to gastrointestinal disorders and cause gastric mucosal damage. The ventrolateral periaqueductal gray (VLPAG) contributes to gastrointestinal function, but whether it is involved in RWIS-induced gastric mucosal damage has not yet been reported.MethodsThe expression of glial fibrillary acidic protein, neuronal c-Fos, and phosphorylated extracellular signal regulated kinase 1/2 in the VLPAG after RWIS was assessed using western blotting and immunocytochemical staining methods. Lateral ventricle injection of astrocytic toxin L-a-aminoadipate and treatment with extracellular signal-regulated kinase (ERK)1/2 signaling pathway inhibitor PD98059 were further used to study protein expression and distribution in the VLPAG after RWIS.ResultsThe expression of c-Fos, glial fibrillary acidic protein, and phosphorylated extracellular signal regulated kinase 1/2 in the VLPAG significantly increased following RWIS and peaked at 1 hour after RWIS. Lateral ventricle injection of the astrocytic toxin L-a-aminoadipate significantly alleviated gastric mucosal injury and decreased the activation of neurons and astrocytes. Treatment with the ERK1/2 signaling pathway inhibitor PD98059 obviously suppressed gastric mucosal damage as well as the RWIS-induced activation of neurons and astrocytes in the VLPAG.ConclusionsThese results suggested that activation of VLPAG neurons and astrocytes induced by RWIS through the ERK1/2 signaling pathway may play a critical role in RWIS-induced gastric mucosa damage.

Epitranscriptomic Reader HNRNPA2B1 Confers Endocrine Resistance to Breast Cancer Cells

Despite new combination therapies improving survival of breast cancer patients with estrogen receptor α (ER+) tumors, the molecular mechanisms for endocrine-resistant metastatic disease remain unresolved. HNRNPA2B1 (Heterogeneous Nuclear Ribonucleoprotein A2/B1), an RNA binding protein that functions as reader of the N(6)-methyladenosine (m6A) mark in transcribed RNA, is upregualted in tamoxifen- and fulvestrant-resistant, estrogen receptor (ERα)+ LCC9 and LY2 cells derived from MCF-7 endocrine-sensitive luminal A breast cancer cells (1). The miRNA-seq transcriptome of MCF-7 cells transiently overexpressing HNRNPA2B1 (A2B1) identified gene ontology (GO) pathways including “cellular response to steroid hormone signaling and estradiol” and “positive regulation of protein ser/thr kinase activity”. Modest (~ 4.5-fold) stable HNRNPA2B1 overexpression in MCF-7 cells (MCF-7-A2B1) results in ablation of growth inhibition by 4-hydroxytamoxifen (4-OHT) and fulvestrant. This was not due to loss or decrease of ERα; in fact, ERα was increased. Conversely, transient knockdown of HNRNPA2B1 in LCC9 and LY2 cells sensitized the cells to growth inhibition by 4-OHT and fulvestrant while reducing ERα. MCF-7-A2B1 cells showed increased migration, invasion, clonogenicity, soft agar colony size, and markers of epithelial-to-mesenchymal transition. Like LCC9 cells, MCF-7-A2B1 cells showed activation of AKT and MAPK and high androgen receptor (AR). Treatment of MCF-7-A2B1 cells with either PI3K inhibitor Wortmannin or MEK inhibitor PD98059 inhibited soft agar colony formation and reduced colony size. Knockdown of HNRNPA2B1 in MCF-7-A2B1 reduces clonogenicity, but had no effect on the clonogenicity of either LCC9 or LY2 cells. These data suggest a role for HNRNPA2B1 in promoting the initiation of acquired endocrine-resistance by activating ser/thr kinase growth factor signaling pathways. Selective inhibition of HNRNPA2B1 may be a target to prevent acquistion of endocrine therapy resistance, but not treat established metastatic disease. Reference: (1) Klinge CM, Piell KM, Tooley CS, Rouchka EC. HNRNPA2/B1 is upregulated in endocrine-resistant LCC9 breast cancer cells and alters the miRNA transcriptome when overexpressed in MCF-7 cells. Scientific reports 2019; 9:9430

Triclosan Increases Production of the Pro‐inflammatory Cytokine Interleukin‐1β in Human Immune Cells: The role of ERK1/2 pathway

Triclosan (TCS) is an antimicrobial agent that is incorporated into medical and personal care products such as topical antiseptics, deodorants, toothpastes, antibacterial soaps, dishwashing liquids, acne treatments, makeup, and lotions. TCS is readily absorbed through the skin. The usage of these products, which rely upon direct contact with skin, is a source of exposure to the compound. Studies have found approximately 1 μM of TCS in human blood plasma upon ingestion of TCS-containing mouthwash. Interleukin-1 beta (IL-1β) is an important pro-inflammatory cytokine produced by immune cells such as monocytes and lymphocytes, and plays a critical role in immune response regulation, tissue repair, and cellular growth. Overproduction of IL-1β can contribute to chronic inflammation and inflammatory diseases such as rheumatoid arthritis and multiple sclerosis. IL-1β has also been shown to stimulate tumor development. A previous study in our lab showed that when peripheral blood mononuclear cells (PBMCs) were exposed to 0.05-5 μM of TCS for a period of 24 hours, secretion of IL-1β increased in an ERK 1/2 dependent manner. Mitogen activated protein kinases (MAPKs), such as ERK 1/2, are known to regulate both the secretion and production of IL-1β. The purpose of the current study is to determine whether TCS is stimulating the immune cell's ability to produce IL-1β (or solely releasing pre-existing stores of this cytokine) as well as the role of ERK 1/2 pathway in any TCS-induced elevation of IL-1β production. PBMCs were exposed to TCS concentrations ranging from 0.05-5 μM for time periods of 30 minutes, 6 hours, or 24 hours. The cellular production (combination of secreted and intracellular levels of IL-1β) in response to this treatment was investigated by using Enzyme Linked Immunosorbent Assays to measure secretion and Western Blots to measure intracellular levels. The results indicate that production of IL-1β was increased at each length of exposure, with the greatest TCS-induced increases seen at 6 h. To investigate the mechanism of this increased production of IL-1β, PBMCs were exposed to MEK inhibitor PD98059 to block ERK1/2 pathway activity. Prior exposure of PBMCs to the MEK inhibitor diminished TCS-induced increases in IL-1β production. Thus, TCS not only stimulates the secretion of IL-1β from immune cells, but also increases production of this potent inflammatory cytokine, which can contribute to several pathologies. TCS-induced increases in IL-1β were shown to be dependent on the ERK 1/2 MAPK pathway.

Effect of Whole Tissue Culture and Basic Fibroblast Growth Factor on Maintenance of Tie2 Molecule Expression in Human Nucleus Pulposus Cells.

Previous work showed a link between Tie2+ nucleus pulposus progenitor cells (NPPC) and disc degeneration. However, NPPC remain difficult to maintain in culture. Here, we report whole tissue culture (WTC) combined with fibroblast growth factor 2 (FGF2) and chimeric FGF (cFGF) supplementation to support and enhance NPPC and Tie2 expression. We also examined the role of PI3K/Akt and MEK/ERK pathways in FGF2 and cFGF-induced Tie2 expression. Young herniating nucleus pulposus tissue was used. We compared WTC and standard primary cell culture, with or without 10 ng/mL FGF2. PI3K/Akt and MEK/ERK signaling pathways were examined through western blotting. Using WTC and primary cell culture, Tie2 positivity rates were 7.0 ± 2.6% and 1.9 ± 0.3% (p = 0.004), respectively. Addition of FGF2 in WTC increased Tie2 positivity rates to 14.2 ± 5.4% (p = 0.01). FGF2-stimulated expression of Tie2 was reduced 3-fold with the addition of the MEK inhibitor PD98059 (p = 0.01). However, the addition of 1 μM Akt inhibitor, 124015-1MGCN, only reduced small Tie2 expression (p = 0.42). cFGF similarly increased the Tie2 expression, but did not result in significant phosphorylation in both the MEK/ERK and PI3K/Akt pathways. WTC with FGF2 addition significantly increased Tie2 maintenance of human NPPC. Moreover, FGF2 supports Tie2 expression via MEK/ERK and PI3K/Akt signals. These findings offer promising tools and insights for the development of NPPC-based therapeutics.

Nuclear FGFR2 Interacts with the MLL-AF4 Oncogenic Chimera and Positively Regulates HOXA9 Gene Expression in t(4;11) Leukemia Cells.

The chromosomal translocation t(4;11) marks an infant acute lymphoblastic leukemia associated with dismal prognosis. This rearrangement leads to the synthesis of the MLL-AF4 chimera, which exerts its oncogenic activity by upregulating transcription of genes involved in hematopoietic differentiation. Crucial for chimera’s aberrant activity is the recruitment of the AF4/ENL/P-TEFb protein complex. Interestingly, a molecular interactor of AF4 is fibroblast growth factor receptor 2 (FGFR2). We herein analyze the role of FGFR2 in the context of leukemia using t(4;11) leukemia cell lines. We revealed the interaction between MLL-AF4 and FGFR2 by immunoprecipitation, western blot, and immunofluorescence experiments; we also tested the effects of FGFR2 knockdown, FGFR2 inhibition, and FGFR2 stimulation on the expression of the main MLL-AF4 target genes, i.e., HOXA9 and MEIS1. Our results show that FGFR2 and MLL-AF4 interact in the nucleus of leukemia cells and that FGFR2 knockdown, which is associated with decreased expression of HOXA9 and MEIS1, impairs the binding of MLL-AF4 to the HOXA9 promoter. We also show that stimulation of leukemia cells with FGF2 increases nuclear level of FGFR2 in its phosphorylated form, as well as HOXA9 and MEIS1 expression. In contrast, preincubation with the ATP-mimetic inhibitor PD173074, before FGF2 stimulation, reduced FGFR2 nuclear amount and HOXA9 and MEIS1 transcript level, thereby indicating that MLL-AF4 aberrant activity depends on the nuclear availability of FGFR2. Overall, our study identifies FGFR2 as a new and promising therapeutic target in t(4;11) leukemia.

Hypoxia-inducible factor-1α-mediated upregulation of CD99 promotes the proliferation of placental mesenchymal stem cells by regulating ERK1/2.

BACKGROUNDAs human placenta-derived mesenchymal stem cells (hP-MSCs) exist in a physiologically hypoxic microenvironment, various studies have focused on the influence of hypoxia. However, the underlying mechanisms remain to be further explored.AIMThe aim was to reveal the possible mechanisms by which hypoxia enhances the proliferation of hP-MSCs.METHODSA hypoxic cell incubator (2.5% O2) was used to mimic a hypoxic microenvironment. Cell counting kit-8 and 5-ethynyl-20-deoxyuridine incorporation assays were used to assay the proliferation of hP-MSCs. The cell cycle was profiled by flow cytometry. Transcriptome profiling of hP-MSCs under hypoxia was performed by RNA sequencing. CD99 mRNA expression was assayed by reverse transcription-polymerase chain reaction. Small interfering RNA-mediated hypoxia-inducible factor 1α (HIF-1α) or CD99 knockdown of hP-MSCs, luciferase reporter assays, and the ERK1/2 signaling inhibitor PD98059 were used in the mechanistic analysis. Protein expression was assayed by western blotting; immunofluorescence assays were conducted to evaluate changes in expression levels.RESULTSHypoxia enhanced hP-MSC proliferation, increased the expression of cyclin E1, cyclin-dependent kinase 2, and cyclin A2, and decreased the expression of p21. Under hypoxia, CD99 expression was increased by HIF-1α. CD99-specific small interfering RNA or the ERK1/2 signaling inhibitor PD98059 abrogated the hypoxia-induced increase in cell proliferation.CONCLUSIONHypoxia promoted hP-MSCs proliferation in a manner dependent on CD99 regulation of the MAPK/ERK signaling pathway in vitro.

Warfarin Induces Calcification of the Aortic Valve Through Extracellular Signal-regulated Kinase 1/2 and β-catenin Signaling

BackgroundRecent clinical evidence suggests an association between warfarin use and calcification of the aortic valve. We sought to determine the effect of warfarin on aortic valve interstitial cell (AVIC) osteogenic protein expression and the signaling pathways by which this effect is mediated. MethodsHuman AVICs were isolated from normal aortic valves of patients undergoing cardiac transplantation, whereas diseased AVICs were isolated from patients undergoing aortic valve replacement for aortic stenosis. AVICs were treated with various anticoagulants, and osteogenic protein expression was evaluated using immunoblotting. Phosphorylation of lipoprotein receptor-related protein 6 (LRP6) and extracellular signal-regulated kinase 1/2 (ERK1/2) was evaluated after treatment with warfarin. AVICs were pretreated with LRP6 inhibitor dkk1 and ERK1/2 inhibitor PD98059 followed by treatment with warfarin, and osteogenic protein expression was evaluated. ResultsWarfarin, but not heparin or dabigatran, significantly increased Runx-2 and Osx expression in both normal and diseased human AVICs. Upregulation of β-catenin protein expression and nuclear translocation occurred in diseased AVICs but not normal AVICs after warfarin treatment. Warfarin induced phosphorylation of LRP6 in diseased AVICs only and phosphorylation of ERK1/2 in both normal and diseased AVICs. LRP6 inhibition attenuated warfarin-induced Runx-2 expression in diseased AVICs. ERK1/2 inhibition attenuated warfarin-induced Runx-2 expression in both normal and diseased AVICs. ConclusionsWarfarin induces osteogenic activity in normal and diseased isolated human AVICs. This effect is mediated by ERK1/2 in both diseased and normal AVICs, but in diseased AVICs β-catenin signaling also plays a role. These results implicate the role of warfarin in aortic valve calcification and highlight potential mechanisms for warfarin-induced aortic stenosis.

The In Vitro Effect of Steroid Hormones, Arachidonic Acid, and Kinases Inhibitors on Aquaporin 1, 2, 5, and 7 Gene Expression in the Porcine Uterine Luminal Epithelial Cells during the Estrous Cycle.

Aquaporins (AQPs) are integral membrane proteins, which play an important role in water homeostasis in the uterus. According to the literature, the expression of aquaporins in reproductive structures depends on the local hormonal milieu. The current study investigated the effect of selected PKA kinase inhibitor H89 and MAPK kinase inhibitor PD98059, on the expression of AQP1, 2, 5, and 7, and steroid hormones (E2), progesterone (P4), and arachidonic acid (AA) in the porcine endometrium on days 18–20 and 2–4 of the estrous cycle (the follicular phase where estrogen and follicle-stimulating hormone (FSH) are secreted increasingly in preparation for estrus and the luteal phase where the ovarian follicles begin the process of luteinization with the formation of the corpus luteum and progesterone secretion, respectively). The luminal epithelial cells were incubated in vitro in the presence of the aforementioned factors. The expression of mRNA was determined by the quantitative real-time PCR technique. In general, in Experiment 1, steroid hormones significantly increased expression of AQP1, 2, and 5 while arachidonic acid increased expression of AQP2 and AQP7. On the other hand, MAPK kinase inhibitor significantly decreased the expression of AQP1 and 5. In Experiment 2, E2, P4, or AA combined with kinase inhibitors differentially affected on AQPs expression. E2 in combination with PKA inhibitor significantly decreased expression of AQP1 but E2 or P4 combined with this inhibitor increased the expression of AQP5 and 7. On the contrary, E2 with PD98059 significantly increased AQP5 and AQP7 expression. Progesterone in combination with MAPK kinase inhibitor significantly downregulated the expression of AQP5 and upregulated AQP7. Arachidonic acid mixed with H89 or PD98059 caused a decrease in the expression of AQP5 and an increase of AQP7. The obtained results indicate that estradiol, progesterone, and arachidonic acid through PKA and MAPK signaling pathways regulate the expression of AQP1 and AQP5 in the porcine luminal epithelial cells in the periovulatory period.

TELMISARTAN IMPROVES MYOCARDIAL REMODELING IN RATS WITH HYPERTENSIVE LEFT VENTRICULAR HYPERTROPHY THROUGH INHIBITING LEPTIN AUTOCRINE INDUCED BY ANGII AND ACTIVATING PPARGAMMA

Objective: The mechanism of left ventricular hypertrophy (LVH) in hypertension is complex. This study was to investigate the role of telmisartan in improving myocardial remodeling in hypertensive LVH and explore the potential molecular mechanism. Design and method: Eight-week male Sprague-Dawley (SD) rats were used to establish hypertensive LVH model through abdominal aortic constriction. Telmisartan was intragastrically administered 6weeks after surgery. Cardiac fibroblasts were used in vitro experiments under the treatment of angiotensin II (AngII), leptin or different inhibitors. The cardiac function and myocardial remodeling level were evaluated in rats, and the AngII-AT1R-ROS-ERK1/2-AP-1 pathway, leptin-JAK2/STAT3-TGF-β1/Smad3 pathway, and the metabolism-related parameters of collagen fibers were detected in cardiac fibroblasts. Results: Telmisartan treatment could alleviate left ventricular diastolic dysfunction (Table 1), reduce myocardial local RAAS and leptin level (Table 2), improve myocardial fibrosis and inhibit the activation of leptin-JAK2/STAT3-TGF-β1/Smad3 pathway in hypertensive LVH rats (Figure 1). AngII could increase the expression of leptin and induce AP-1 nuclear transposition in cardiac fibroblasts, and AT1R antagonist telmisartan, ROS scavenger NAC, ERK1/2 inhibitor PD98059 or AP-1 inhibitor SR11302 could inhibit leptin synthesis and secretion induced by AngII (Figure 2). Leptin could induce collagen metabolic disorders in cardiac fibroblasts, which could be partly restored by JAK2 inhibitor AG-490, STAT3 inhibitor S3I-201 or telmisartan. Telmisartan could reduce leptin synthesis and secretion as good as leptin antagonist, and improve collagen metabolism disorder induced by AngII better than leptin antagonist (Figure 3). Telmisartan could increase the expression of PPAR-γ to almost 3-fold of the control group. When pretreated with PPAR-γ inhibitor GW9662, telmisartan couldn’t restore the collagen metabolic disorders and the increased expression of P-STAT3 stimulated by leptin (Figure 4). Conclusions: Telmisartan can improve myocardial remodeling in hypertensive LVH rats via two ways: one is inhibiting leptin autocrine induced by local AngII as AT1R antagonist, the other is inhibiting leptin downstream signaling molecular STAT3 phosphorylation as PPAR-γ agonist (Figure 5). These findings demonstrate the crosstalk between myocardial local RAAS and leptin, and reveal a new molecular mechanism of telmisartan in improving myocardial remodeling in hypertensive LVH.

USP18-deficiency in cervical carcinoma is crucial for the malignant behavior of tumor cells in an ERK signal-dependent manner.

Ubiquitin-specific peptidase (USP)18 belongs to the USP family, and is involved in cleaving and removing ubiquitin or ubiquitin-like molecules from their target molecules. Recently, increasing evidence has suggested that USP18 is constitutively expressed in different types of human tumors, and ectopic expression or downregulation of USP18 expression may contribute to tumorigenesis. However, the role of USP18 in uterine cervical cancer (UCC) remains unclear. Thus, the present study aimed to investigate USP18 expression in a human tissue microarray constructed using UCC and non-cancer cervical tissues, and to determine the potential role and molecular mechanism by which USP18 is implicated in the tumor biology of human UCC HeLa cells. Microarray analysis demonstrated that USP18 protein expression was downregulated in tumor tissues compared with in normal tissues. In addition, in vitro analysis revealed that USP18-knockdown markedly promoted the proliferation, colony formation, migration and aggressiveness of HeLa cells. Mechanistic analysis demonstrated that USP18-knockdown increased the levels of Bcl-2, STAT3 and phosphorylated-ERK in HeLa cells. Notably, USP18 silencing-induced malignant phenotypes were interrupted following exogenous administration of the ERK1/2 inhibitor PD98059. Overall, the results of the present study suggested that USP18 may be a potent inhibitor involved in UCC tumor-associated biological behaviors, which are associated with the ERK signaling pathway.

Discovering the role of VEGF signaling pathway in mesendodermal induction of human embryonic stem cells

Human embryonic stem cells (hESCs) have the unique feature of unlimited self-renewal and differentiation into derivatives of all three germ layers in human body, providing a powerful in vitro model for studying cell differentiation. FGF2, BMP4 and TGF-β signaling have been shown to play crucial roles in mesendodermal differentiation of hESCs. However, their underlying molecular mechanisms and other signaling pathways potentially involved in mesendodermal differentiation of hESCs remain to be further investigated. In this study, we uncover that VEGF signaling pathway plays a critical role in the mesendodermal induction of hESCs. Treating hESCs with Lenvatinib, a pan-inhibitor of VEGF receptors (VEGFRs), impedes their mesendodermal induction. Conversely, overexpression of VEGFA165, a major human VEGF isoform, promotes the mesendodermal differentiation. Similar to the VEGFR inhibitor, MEK inhibitor PD0325901 hinders mesendodermal induction of hESCs. In contrast, overexpression of ERK2GOF, an intrinsically active ERK2 mutant, markedly reduces the inhibitory effect of the VEGFR inhibitor. Thus, the MEK-ERK cascade plays an important role for the function of VEGF signaling pathway in the mesendodermal induction of hESCs. All together, this study identifies the critical role of VEGF signaling pathway as well as potential crosstalk of VEGF signaling pathway with other known signaling pathways in mesendodermal differentiation of hESCs.

Ginsenoside Compound K Promotes Proliferation, Migration and Differentiation of Schwann Cells via the Activation of MEK/ERK1/2 and PI3K/AKT Pathways.

The proliferation and differentiation of Schwann cells are critical for the remyelination of injured peripheral nerve. Ginsenoside compound K (CK) is a metabolite produced from ginsenoside Rb1 which has strong anti-inflammatory effects. However, the potential effects of CK on Schwann cells have not been studied systematically before. Therefore, this study was aimed to explore the functions of CK in Schwann cell proliferation, migration and differentiation and its potential regulatory mechanism. Primary Schwann cells and RSC96 cells were treated with or without CK at different doses. The proliferation and migration of primary Schwann cells and RSC96 cells were examined by Cell Counting Kit-8 (CCK-8) and Transwell assays, respectively. The mRNA expression of myelin-associated glycoprotein (MAG) and myelin basic protein (MBP) was tested by quantitative real-time polymerase chain reaction (qRT-PCR). The levels of all proteins were examined by Western blot. CK could promote cell proliferation, migration and induce MAG and MBP expression in primary Schwann cells and RSC96 cells. Furthermore, CK activated MEK/ERK1/2 and PI3K/AKT pathways, and the beneficial effects of CK on primary Schwann cells and RSC96 cells were distinctly suppressed by inhibitor PD98059 or LY294002. Ginsenoside compound K induced cell proliferation, migration and differentiation via the activation of MEK/ERK1/2 and PI3K/AKT pathways in cultured primary Schwann cells and RSC96 cells.

PRDX2 Protects Against Atherosclerosis by Regulating the Phenotype and Function of the Vascular Smooth Muscle Cell.

Peroxiredoxin 2 (PRDX2), an inhibitor of reactive oxygen species (ROS), is potentially involved in the progression of atherosclerosis (AS). The aim of this study was to explore the role and mechanism of PRDX2 in AS. The expression of PRDX2 was evaluated in 14 human carotid artery tissues with or without AS. The results showed that the positive reaction of PRDX2 was observed in the carotid artery vascular smooth muscle cells (CAVSMCs). To assess the mechanism by which PRDX2 may function in AS, the CAVSMCs were transfected with pEX4-PRDX2 and si-PRDX2. The catalase, hydrogen peroxide (H2O2) scavenger, was used to further confirm that PRDX2-induced inhibitory effects might be mediated through reducing ROS levels. Phenotype alteration and functional testing included transcription testing, immunostaining, and expression studies. The drug of MAPK signaling pathway inhibitors SB203580, SP600125, and PD98059 was used to evaluate the underlying mechanism. In this study, we found that the protein level of PRDX2 and the level of H2O2 were higher in the human AS carotid artery tissues than in the normal carotid artery tissues, accompanied with the activation of MAPK signaling pathway. The up-regulation of PRDX2 in the CAVSMCs significantly decreased the expression of ROS, collagen type I (COL I), collagen type III (COL III), vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) and inhibited the proliferation, migration, and transformation of the CAVSMCs. The up-regulation of PRDX2 reversed the effect of the CAVSMCs treated with tumor necrosis factor-α (TNF-α). In addition, PRDX2 down-regulation promoted the protein levels of p-p38, p-JNK, and p-ERK, which was confirmed in relevant MAPK inhibitor treatment experiments. Our results suggest a protective role of PRDX2, as a scavenger of ROS, in AS progression through inhibiting the VSMC phenotype alteration and function via MAPK signaling pathway.

Impaired instrumental learning in Spred1-/- mice, a model for a rare RASopathy.

RASopathies are neuro-cardio-facio-cutaneous disorders stemming from mutations in genes regulating the RAS-MAPK pathway. Legius syndrome is a rare RASopathy disorder caused by mutations in the SPRED1 gene. SPRED1 protein negatively regulates activation of Ras by inhibiting RAS/RAF and by its interaction with neurofibromin, a Ras GTPase-activating protein (RAS-GAP). Cognitive impairments have been reported in Legius syndrome as well as in other RASopathy disorders. Modelling these cognitive deficits in a Spred1 mouse model for Legius syndrome has demonstrated spatial learning and memory deficits, but other cognitive domains remained unexplored. Here, we attempted to utilize a cognitive touchscreen battery to investigate if Spred1-/- mice exhibit deficits in other cognitive domains. We show that Spred1-/- mice had heterogeneous performance in instrumental operant learning, with a large subgroup (n= 9/20) failing to reach the standard criterion on touchscreen operant pretraining, precluding further cognitive testing. To examine whether targeting the RAS-MAPK signalling pathway could rescue these cognitive impairments, Spred1-/- mice were acutely treated with the clinically relevant mitogen-activated protein kinase (MEK) inhibitor PD325901. However, MEK inhibition did not improve their instrumental learning. We conclude that Spred1-/- mice can model severe cognitive impairments that cannot be reversed in adulthood.

GLI1 reduces drug sensitivity by regulating cell cycle through PI3K/AKT/GSK3/CDK pathway in acute myeloid leukemia

Acute myeloid leukemia (AML) is a hematological malignancy with high incidence and recurrence rates. Gene expression profiling has revealed that transcriptional overexpression of glioma‐associated oncogene 1 (GLI1), a vital gene in the Hedgehog (Hh) signaling pathway, occurs in poor-prognosis AML, and high levels of phosphoinositide-3-kinase, regulatory subunit 1 (PIK3R1) and AKT3 predict shorter overall survival in AML patients. In this study, we discovered that GLI1 overexpression promotes cell proliferation and reduces chemotherapy sensitivity in AML cells while knocking down GLI1 has the opposite effect. Moreover, GLI1 promoted cell cycle progression and led to elevated protein levels of cyclins and cyclin-dependent kinases (CDKs) in AML cells. By luciferase assays and co-immunoprecipitation, we demonstrated that the PI3K/AKT pathway is directly activated by GLI1. GLI1 overexpression significantly accelerates tumor growth and upregulated p-AKT, CDK4, and cyclinD3 in vivo. Notably, the GLI1 inhibitor GANT61 and the CDK4/6 inhibitor PD 0332991 had synergistic effects in promoting Ara-c sensitivity in AML cell lines and patient samples. Collectively, our data demonstrate that GLI1 reduces drug sensitivity by regulating cell cycle through the PI3K/AKT/GSK3/CDK pathway, providing a new perspective for involving GLI1 and CDK4/6 inhibitors in relapsed/refractory (RR) patient treatment.