The Modulation of Phospho-Extracellular Signal-Regulated Kinase and Phospho-Protein Kinase B Signaling Pathways plus Activity of Macrophage-Stimulating Protein Contribute to the Protective Effect of Stachydrine on Acetaminophen-Induced Liver Injury.

Stachydrine, a prominent bioactive alkaloid derived from Leonurus heterophyllus, is a significant herb in traditional medicine. It has been noted for its anti-inflammatory and antioxidant characteristics. Consequently, we conducted a study of its hepatoprotective effect and the fundamental mechanisms involved in acetaminophen (APAP)-induced liver injury, utilizing a mouse model. Mice were intraperitoneally administered a hepatotoxic dose of APAP (300 mg/kg). Thirty minutes after APAP administration, mice were treated with different concentrations of stachydrine (0, 2.5, 5, and 10 mg/kg). Animals were sacrificed 16 h after APAP injection for serum and liver tissue assays. APAP overdose significantly elevated the serum alanine transferase levels, hepatic pro-inflammatory cytokines, malondialdehyde activity, phospho-extracellular signal-regulated kinase (ERK), phospho-protein kinase B (AKT), and macrophage-stimulating protein expression. Stachydrine treatment significantly decreased these parameters in mice with APAP-induced liver damage. Our results suggest that stachydrine may be a promising beneficial target in the prevention of APAP-induced liver damage through attenuation of the inflammatory response, inhibition of the ERK and AKT pathways, and expression of macrophage-stimulating proteins.

Sensitivity of eight types of ALK fusion variant to alectinib in ALK-transformed cells.

Tyrosine kinase inhibitors of anaplastic lymphoma kinase (ALK-TKIs) including alectinib have been the standard therapy against ALK fusion gene-positive non-small cell lung cancers (NSCLCs). Many ALK fusion variants have been identified in NSCLCs, and the predominant variants are echinoderm microtubule-associated protein-like 4-ALK (EML4-ALK) variant 1 (V1), V2 and V3a/b. However, there have been conflicting reports on the clinical responses of these variants to ALK-TKIs, and there are few reports on other less common ALK variants. To examine the influence of ALK variants on the efficacy of ALK-TKIs, we analyzed the sensitivity to alectinib of eight types of ALK variant: three major variants (V1, V2 and V3a) and five less common variants (V4; kinesin family member 5-ALK; kinesin light chain 1-ALK; striatin, calmodulin-binding protein-ALK; and tropomyosin-receptor kinase fused gene-ALK). Analysis was done by cell-free kinase assays using the recombinant proteins and by cell, growth assays using murine Ba/F3 cells expressing ALK variants. The kinase activity of each recombinant protein was significantly inhibited by alectinib. Intracellular ALK phosphorylation levels and its downstream signaling mediators, STAT3 and ERK, were suppressed by alectinib in each ALK variant-expressing Ba/F3 cell. Each cellular proliferation was markedly inhibited by alectinib treatment. There was no significant difference in the IC50 values between cells, with a <3.6-fold difference in responsiveness. In conclusion, these eight ALK variants had similar sensitivity to alectinib in vitro, indicating that it may not be possible to predict the response to alectinib just by determination of the ALK variant type in ALK fusion-positive NSCLCs.

2E-Decene-4,6-diyn-1-ol-acetate inhibits osteoclastogenesis through mitogen-activated protein kinase-c-Fos-NFATc1 signalling pathways.

An imbalance of osteoclasts and osteoblasts can result in a variety of bone-related diseases, including osteoporosis. Thus, decreasing the activity of osteoclastic bone resorption is the main therapeutic method for treating osteoporosis. 2E-Decene-4, 6-diyn-1-ol-acetate (DDA) is a natural bioactive compound with anti-inflammatory and anti-cancer properties. However, its effects on osteoclastogenesis are unknown. Murine bone marrow-derived macrophages (BMMs) or RAW264.7 cells were treated with DDA, followed by evaluation of cell viability, RANKL-induced osteoclast differentiation, and pit formation assay. Effects of DDA on RANKL-induced phosphorylation of MAPKs were assayed by western blot analysis. Expression of osteoclast-specific genes was examined with reverse transcription-PCR (RT-PCR) and western blot analysis. In this study, DDA significantly inhibited RANKL-induced osteoclast differentiation in RAW264.7 cells as well as in BMMs without cytotoxicity. DDA also strongly blocked the resorbing capacity of BMM on calcium phosphate-coated plates. DDA inhibited RANKL-induced phosphorylation of ERK, JNK and p38 MAPKs, as well as expression of c-Fos and NFATc1, which are essential transcription factors for osteoclastogenesis. In addition, DDA decreased expression levels of osteoclastogenesis-specific genes, including matrix metalloproteinase-9 (MMP-9), tartrate-resistant acid phosphatase (TRAP), and receptor activator of NF-κB (RANK) in RANKL-induced RAW264.7 cells. Collectively, these findings indicated that DDA attenuates RANKL-induced osteoclast formation by suppressing the MAPKs-c-Fos-NFATc1 signalling pathway and osteoclast-specific genes. These results indicate that DDA may be a potential candidate for bone diseases associated with abnormal osteoclast formation and function.

Pharmaceutical immunoglobulin G impairs anti-carcinoma activity of oxaliplatin in colon cancer cells.

BACKGROUND: Recent evidence proves that intravenous human immunoglobulin G (IgG) can impair cancer cell viability. However, no study evaluated whether IgG application benefits cancer patients receiving chemotherapeutics. METHODS: Influence of pharmaceutical-grade human IgG on the viability of a series of patient-derived colon cancer cell lines with and without chemotherapeutic intervention was determined. Cell death was analysed flow cytometrically. In addition, the influence of oxaliplatin and IgG on the ERK1/2-signalling pathway was evaluated by western blots. RESULTS: We evaluated the effects of pharmaceutical IgG, such as PRIVIGEN® IgG and Tonglu® IgG, in combination with chemotherapeutics. We did not observe any significant effects of IgG on tumour cell viability directly; however, human IgG significantly impaired the anti-tumoral effects of oxaliplatin. Primary cancer cell lines express IgG receptors and accumulate human IgG intracellularly. Moreover, while oxaliplatin induced the activation of ERK1/2, the pharmaceutical IgG inhibited ERK1/2 activity. CONCLUSIONS: The present study demonstrates that pharmaceutical IgG, such as PRIVIGEN® IgG and Tonglu® IgG, can impair the anti-carcinoma activity of oxaliplatin. These data strongly suggest that therapeutic IgG as co-medication might have harmful side effects in cancer patients. The clinical significance of these preclinical observations absolutely advises further preclinical, as well as epidemiological and clinical research.

Mitogen-activated protein kinases are involved in cucurbitacin D-induced antitumor effects on adult T-cell leukemia cells.

Adult T cell leukemia (ATL) is an aggressive and malignant blood disease. We previously reported that steroid-structured cucurbitacin D (CuD) induces apoptosis in ATL cells. In this study, we investigated the effects of mitogen-activated protein kinase (MAPK) signaling inhibitors on CuD-induced cell death in peripheral blood lymphocytes (PBLs) isolated from ATL/acute lymphoblastic leukemia (ALL) patients and two human leukemia cell lines (MT-1 and MT-4). PBLs were isolated from an ATL/ALL patient as well as from a healthy donor. Cell surface markers were examined using flow cytometry. Serum cytokine levels were estimated using LEGENDplex or analyzed at the Center for Clinical and Translational Research of Kyushu University Hospital. Cell proliferation was assessed using the Cell Titer-Glo luminescent cell viability assay. Protein expression was determined by western blotting. PBLs from patients highly expressed CD4 and CD5. Serum from the patient contained high levels of interleukin (IL)-8, IL-10, IL-18, and interferon-γ compared to the healthy donor. CuD-induced cell death was enhanced by the mitogen-activated protein kinase kinase (MEK)1/2 inhibitor U0126. However, a c-Jun N-terminal kinase (JNK) inhibitor prevented CuD-induced cell death. Immunoblot analyses revealed that CuD reduced the phosphorylation of extracellular signal-regulated kinase (ERK), p38, and JNK, and co-treatment with CuD and U0126 did not affect the phosphorylation of ERK. MEK1/2 and p38 inhibitors enhanced CuD-induced cell death, and U0126 enhanced the CuD-induced de-phosphorylation of ERK in MT-1 and MT-4 cells. We conclude that CuD reduces ERK activation, resulting in enhanced antitumor effects on leukemic cells.

MEK inhibition by trametinib overcomes chemoresistance in preclinical nasopharyngeal carcinoma models.

The development of chemoresistance is the major cause of treatment failure in nasopharyngeal carcinoma (NPC). Although 'paradoxical' activation of extracellular signal-regulated kinase (ERK) has been shown to contribute resistance to anticancer treatment, the role of ERK in NPC chemoresistance has not been yet revealed. In this work, we report that trametinib, a clinically available mitogen-activated protein kinase inhibitor for melanoma treatment, overcomes NPC chemoresistance via suppressing ERK activation induced by chemotherapy. We first showed that trametinib at nanomolar concentrations was active against NPC cells and acted synergistically with cisplatin. Trametinib remarkably decreased phosphorylation of ERK and its downstream effector in NPC cells. We next showed that cisplatin treatment stimulates ERK signaling, and furthermore that this can be abolished by trametinib. We finally generated cisplatin-resistant NPC models and demonstrated that trametinib was effective in inhibiting cisplatin-resistant NPC growth, colony formation and survival via suppressing ERK signaling in vitro and in vivo. Our work demonstrates the potential of trametinib in overcoming chemoresistance in preclinical NPC models and provides evidence of initializing clinical trials of using trametinib for NPC treatment.

Repeated nicotine exposure increases the intracellular interaction between ERK-mGluR5 in the nucleus accumbens more in adult than adolescent rats.

Intracellular interactions between protein kinases and metabotropic receptors in the striatum regulate behavioral changes in response to drug exposure. We investigated the difference in the degree of interaction between extracellular signal-regulated kinase (ERK) and metabotropic glutamate receptor subtype 5 (mGluR5) in the nucleus accumbens (NAc) after repeated exposure to nicotine in adult and adolescent rats. The results showed that repeated exposure to nicotine (0.5 mg/kg/day, s.c.) for seven consecutive days increased ERK phosphorylation more in adults than in adolescents. Furthermore, membrane expression of mGluR5 in gamma-aminobutyric acid (GABA) medium spiny neurons was higher in adults than adolescents as a result of repeated exposure to nicotine. Blockade of mGluR5 with MPEP (0.5 nmol/side) decreased the repeated nicotine-induced increase in ERK phosphorylation. Either blockade of mGluR5 or inhibition of ERK with SL327 (150 nmol/side) decreased the repeated nicotine-induced increase in the level of inositol-1,4,5-triphosphate (IP3 ), a key transducer associated with mGluR5-coupled signaling cascades. Similarly, interference of binding between activated ERK and mGluR5 by the blocking peptide, Tat-mGluR5-i (2 nmol/side), decreased the repeated nicotine-induced increases in IP3 and locomotor activity in adults. These findings suggest that the intracellular interaction between ERK and mGluR5 in the NAc is stronger in adult than in adolescent rats, which enhances the understanding of age-associated behavioral changes that occur after repeated exposure to nicotine.

Nicotine abolishes memory-related synaptic strengthening and promotes synaptic depression in the neurogenic dentate gyrus of miR-132/212 knockout mice.

Micro-RNAs (miRNAs) are highly evolutionarily conserved short-length/noncoding RNA molecules that modulate a wide range of cellular functions in many cell types by regulating the expression of a variety of targeted genes. miRNAs have also recently emerged as key regulators of neuronal genes mediating the effects of psychostimulant drugs and memory-related neuroplasticity processes. Smoking is a predominant addictive behaviour associated with millions of deaths worldwide, and nicotine is a potent natural psychoactive agonist of cholinergic receptors, highly abundant in cigarettes. The influence of miRNAs modulation on cholinergic signalling in the nervous system remains however poorly explored. Using miRNA knockout mice and biochemical, electrophysiological and pharmacological approaches, we examined the effects of miR-132/212 gene disruption on the levels of hippocampal nicotinic acetylcholine receptors, total ERK and phosphorylated ERK (pERK) and MeCP2 protein levels, and studied the impact of nicotine stimulation on hippocampal synaptic transmission and synaptic depression and strengthening. miR-132/212 deletion significantly altered α7-nAChR and pERK protein levels, but not total ERK or MeCP2, and resulted in both exacerbated synaptic depression and virtually abolished memory-related synaptic strengthening upon nicotine stimulation. These observations reveal a functional miRNAs/nicotinergic signalling interplay critical for nicotinic-receptor expression and neuroplasticity in brain structures relevant for drug addiction and learning and memory functions.

Antibody-mediated inhibition of syndecan-4 dimerisation reduces interleukin (IL)-1 receptor trafficking and signalling.

OBJECTIVE: Syndecan-4 (sdc4) is a cell-anchored proteoglycan that consists of a transmembrane core protein and glucosaminoglycan (GAG) side chains. Binding of soluble factors to the GAG chains of sdc4 may result in the dimerisation of sdc4 and the initiation of downstream signalling cascades. However, the question of how sdc4 dimerisation and signalling affects the response of cells to inflammatory stimuli is unknown. METHODS: Sdc4 immunostaining was performed on rheumatoid arthritis (RA) tissue sections. Interleukin (IL)-1 induced extracellular signal-regulated kinases (ERK) phosphorylation and matrix metalloproteinase-3 production was investigated. Il-1 binding to sdc4 was investigated using immunoprecipitation. IL-1 receptor (IL1R1) staining on wild-type, sdc4 and IL1R1 knockout fibroblasts was performed in fluorescence-activated cell sorting analyses. A blocking sdc4 antibody was used to investigate sdc4 dimerisation, IL1R1 expression and the histological paw destruction in the human tumour necrosis factor-alpha transgenic mouse. RESULTS: We show that in fibroblasts, the loss of sdc4 or the antibody-mediated inhibition of sdc4 dimerisation reduces the cell surface expression of the IL-1R and regulates the sensitivity of fibroblasts to IL-1. We demonstrate that IL-1 directly binds to sdc4 and in an IL-1R-independent manner leads to its dimerisation. IL-1-induced dimerisation of sdc4 regulates caveolin vesicle-mediated trafficking of the IL1R1, which in turn determines the responsiveness to IL-1. Administration of antibodies (Ab) against the dimerisation domain of sdc4, thus, strongly reduces the expression IL1R1 on arthritic fibroblasts both in vitro and an animal model of human RA. CONCLUSION: Collectively, our data suggest that Ab that specifically inhibit sdc4 dimerisation may support anti-IL-1 strategies in diseases such as inflammatory arthritis.

ß-Arrestin-dependent signaling in GnRH control of hormone secretion from goldfish gonadotrophs and somatotrophs.

In goldfish, two native isoforms of gonadotropin-releasing hormone (GnRH2 and GnRH3) stimulate luteinizing hormone (LH) and growth hormone (GH) release from pituitary cells through activation of cell-surface GnRH-receptors (GnRHRs) on gonadotrophs and somatotrophs. Interestingly, GnRH2 and GnRH3 induce LH and GH release via non-identical post-receptor signal transduction pathways in a ligand- and cell-type-selective manner. In this study, we examined the involvement of ß-arrestins in the control of GnRH-induced LH and GH secretion from dispersed goldfish pituitary cells. Treatment with Barbadin, which interferes with ß-arrestin and ß2-adaptin subunit interaction, reduced LH responses to GnRH2 and GnRH3, as well as GH responses to GnRH2; but enhanced GnRH3-induced GH secretion. Barbadin also had positive influences on basal hormone release, and basal GH release in particular, as well as basal activity of extracellular signal-regulated kinase (ERK) and GnRH-induced ERK activation. These findings indicate that ß-arrestins play permissive roles in the control of GnRH-stimulated LH release. However, in somatotrophs, ß-arrestins, perhaps by mediating agonist-selective endosomal trafficking of engaged GnRHRs, participate in GnRH-isoform-specific GH release responses (stimulatory and inhibitory for GnRH2-GnRHR and GnRH3-GnRHR activation, respectively). The correlative stimulatory influences of Barbadin on basal hormone release and ERK activation suggest that ß-arrestins may negatively regulate basal secretion through modulation of basal ERK activity. These results provide the first direct evidence of a role for ß-arrestins in hormone secretion from an untransformed primary pituitary cell model, and establish these proteins as important receptor-proximal players in mediating functional selectivity downstream of goldfish GnRHRs.

The MEK Inhibitor Trametinib Ameliorates Kidney Fibrosis by Suppressing ERK1/2 and mTORC1 Signaling.

BACKGROUND: During kidney fibrosis, a hallmark and promoter of CKD (regardless of the underlying renal disorder leading to CKD), the extracellular-regulated kinase 1/2 (ERK1/2) pathway, is activated and has been implicated in the detrimental differentiation and expansion of kidney fibroblasts. An ERK1/2 pathway inhibitor, trametinib, is currently used in the treatment of melanoma, but its efficacy in the setting of CKD and renal fibrosis has not been explored. METHODS: We investigated whether trametinib has antifibrotic effects in two mouse models of renal fibrosis-mice subjected to unilateral ureteral obstruction (UUO) or fed an adenine-rich diet-as well as in cultured primary human fibroblasts. We also used immunoblot analysis, immunohistochemical staining, and other tools to study underlying molecular mechanisms for antifibrotic effects. RESULTS: Trametinib significantly attenuated collagen deposition and myofibroblast differentiation and expansion in UUO and adenine-fed mice. We also discovered that in injured kidneys, inhibition of the ERK1/2 pathway by trametinib ameliorated mammalian target of rapamycin complex 1 (mTORC1) activation, another key profibrotic signaling pathway. Trametinib also inhibited the ERK1/2 pathway in cultured primary human renal fibroblasts stimulated by application of TGF-ß1, the major profibrotic cytokine, thereby suppressing downstream mTORC1 pathway activation. Additionally, trametinib reduced the expression of myofibroblast marker α-smooth muscle actin and the proliferation of renal fibroblasts, corroborating our in vivo data. Crucially, trametinib also significantly ameliorated renal fibrosis progression when administered to animals subsequent to myofibroblast activation. CONCLUSIONS: Further study of trametinib as a potential candidate for the treatment of chronic renal fibrotic diseases of diverse etiologies is warranted.

Alzheimer's drug Memantine inhibits metastasis and p-Erk protein expression on 4T1 breast cancer cells.

OBJECTIVES: Drug repurposing studies enable shorter routes to the clinic by skipping the steps like in vitro  in vivo screening, chemical optimization and toxicological studies. In our study, we investigated the potent anti-cancer effect of Alzheimer's drug Memantine on 4T1 breast cancer cells. METHODS: Memantine's effect on proliferation of 4T1 cells was evaluated by using the MTT assay. Memantine inhibited 4T1 cell proliferation in a concentration- dependent manner at 24 and 48 hours. We investigated the drug's effect on the protein expressions of Bax, Bcl-2, Casp-3, Casp-9, E-Cad, Vimentin, B-Cat, GSK3B, p-ERK, ERK, p-GS, GS that are involved in apoptosis, metastasis and cell survival. RESULTS: Memantine altered the Bcl-2, Bax, Casp3, Casp-9 apoptotic protein expression levels. We found that memantine inhibited p-Erk expression and that result suggested a plausible mechanism of action for memantine's antineoplastic effect. Memantine also inhibited wound closure at 24 h, significantly (p = 0.0055). CONCLUSIONS: Memantine inhibited 4T1 breast cancer cell proliferation at significantly lower doses than mostly studied re-purposed drug Metformin. Therefore, we believe that memantine might hold a great promise as a new repositioned drug in cancer treatment and it is our further interest to investigate its effects in vivo (Fig. 3, Ref. 22).

No evidence of attenuation of placental insulin-stimulated Akt phosphorylation and amino acid transport in maternal obesity and gestational diabetes mellitus.

Pregnancies complicated by obesity and/or gestational diabetes (GDM) are associated with peripheral insulin resistance; however, the insulin responsiveness of the placenta in these pregnancy complications remains largely unknown. We tested the hypothesis that primary human trophoblast cells and placental villous explants will be insulin responsive, characterized by amino acid transport, Akt and Erk activity with maternal obesity, and/or GDM. We evaluated term placentas from women with normal body mass index (BMI) (normal; n = 15), obesity (OB; n = 11), normal BMI with GDM (N-GDM; n = 11), and obesity with GDM (OB-GDM; n = 11). In a subgroup, primary human trophoblast cells (PHT) were isolated, and in an independent subgroup placental villous explants were exposed to varying concentrations of insulin. Amino acid transport capacity and insulin signaling activity were determined. Insulin significantly increased amino acid transport activity to a similar degree in PHT cells isolated from normal (+21%), N-GDM (+38%), OB (+37%), and OB-GDM (+35%) pregnancies. Insulin increased Akt and Erk phosphorylation in PHT cells (3-fold) and in villous explants (2-fold) in all groups to a similar degree. In contrast to the peripheral maternal insulin resistance commonly associated with obesity and/or GDM, we found that the placenta is insulin sensitive in these pregnancy complications. We suggest that elevated maternal insulin levels in pregnancies complicated by obesity and/or GDM promote critical placental functions, including amino acid transport. Insulin-stimulated placental nutrient delivery may contribute to the increased risk of fetal overgrowth and adiposity in these pregnancies. Moreover, our findings may inform efforts to optimize insulin regimens for women with GDM.

Sevoflurane Postconditioning Inhibits Autophagy Through Activation of the Extracellular Signal-Regulated Kinase Cascade, Alleviating Hypoxic-Ischemic Brain Injury in Neonatal Rats.

Hypoxic-ischemic brain injury (HIBI) in neonates is one of the major contributors of newborn death and cognitive impairment. Numerous animal studies have demonstrated that autophagy is substantially increased in HIBI and that sevoflurane postconditioning (SPC) can attenuate HIBI. However, if SPC-induced neuroprotection inhibits autophagy in HIBI remains unknown. To investigate if cerebral protection induced by SPC is related to decreased autophagy in the setting of HIBI. Postnatal rats at day 7 (P7) were randomly assigned to 7 different groups: Sham, HIBI, SPC-HIBI, HIBI + rapamycin, SPC-HIBI + rapamycin, HIBI + p-extracellular signal-regulated kinase (p-ERK) inhibitor, and SPC-HIBI + p-ERK inhibitor. To induce HIBI, neonatal rats underwent left common carotid artery ligation, followed by 2 h of hypoxia (8% O2). Rats in the SPC groups were treated with 1 minimum alveolar concentration ([MAC], 2.4%) SPC for 30 min after HIBI induction. Markers of autophagy and expression of ERK cascade components were measured in the rat brains after 24 h. Spatial learning and memory function were examined 29-34 days after administration of an autophagy agonist or a p-ERK inhibitor. The expression of microtubule-associated proteins 1A/1B, light chain 3B II (LC3-II) and tuberous sclerosis complex 2 (TSC2) were decreased in the SPC-HIBI group compared to the HIBI group. Expression of the p62 sequestosome 1 (P62/SQSTM1) protein, p-ERK/ERK, phospho-mammalian target of rapamycin (p-mTOR) and phospho-p70S6 were increased in SPC-HIBI group. Rats within the SPC-HIBI groups that also received the p-ERK inhibitor or autophagy inhibitor demonstrated reduced cross platform times and increased escape latency. Approximately 30 min of 2.4% SPC treatment in the P7 rat HIBI model attenuated excessive autophagy in the brain by elevating the ERK cascade. This finding provides additional insight into HIBI and identifies new targets for therapeutic approaches to treat HIBI.

Silver nanoparticles induce Egr-1-dependent psoriasin expression via the ERK and p38 pathways.

BACKGROUND: Silver nanoparticles (Ag-NPs) can prevent bacterial infection and improve cutaneous wound healing owing to their antimicrobial activity. However, the mechanism of their antimicrobial activity is poorly understood. AIM: To determine the mechanistic relationship between Ag-NP treatment and expression of psoriasin. METHODS: Human epidermal keratinocytes, neonatal (HEKn) were used. Psoriasin mRNA expression was measured by reverse transcription PCR and real-time PCR. Western blotting was performed to verify expression of early growth response-1 (Egr-1) and psoriasin, and phosphorylation of mitogen-activated protein kinase (MAPK). Psoriasin promoter activity by Egr-1 was detected by a luciferase assay. RESULTS: Treatment of HEKn with Ag-NPs induced psoriasin mRNA and protein expression. Upregulation of psoriasin promoter activity was also observed in the luciferase assay. Ag-NPs increased Egr-1 expression, promoter activity and nuclear translocation in HEKn. Psoriasin luciferase activity was increased in HEKn transfected with Egr-1 pcDNA 3.1. Ag-NPs activated MAPK pathways including the extracellular signal-regulated kinase (ERK), p38, and c-Jun-N-terminal kinase (JNK) pathways. The upregulation of Egr-1 expression by Ag-NP stimulation was inhibited by ERK and p38 inhibitors, but not by a JNK inhibitor. Psoriasin expression was reduced in Egr-1 small interfering RNA-transfected HEKn. CONCLUSIONS: Ag-NP treatment induces upregulation of psoriasin expression through Egr-1 expression. We suggest that the ERK and p38 pathways are involved in Egr-1-dependent psoriasin expression.

Inhaled Nitric Oxide Protects Cerebral Autoregulation and Reduces Hippocampal Necrosis After Traumatic Brain Injury Through Inhibition of ET-1, ERK MAPK and IL-6 Upregulation in Pigs.

OBJECTIVE: Traumatic brain injury (TBI) is an important contributor to morbidity and mortality. Cerebral autoregulation is impaired after TBI, contributing to poor outcome. Extracellular signal-related kinase (ERK) mitogen activated protein kinase (MAPK) and ET-1 are upregulated and contribute to impairment of cerebral autoregulation and histopathology after porcine fluid percussion brain injury (FPI). Recent studies show that inhaled nitric oxide (iNO) prevents impairment of cerebral autoregulation and histopathology after FPI in pigs. Unrelated studies indicated an association between ERK and increased IL-6 after FPI. However, the role of IL-6 in central nervous system (CNS) pathology is not well understood. We investigated whether iNO protects autoregulation and limits histopathology after FPI in pigs due to modulation of brain injury associated upregulation of ET-1, ERK MAPK, and IL-6. METHODS: Lateral FPI was produced in anesthetized pigs equipped with a closed cranial window and iNO administered at 30 min or 2 h post injury. RESULTS: CSF ET-1, ERK MAPK, and IL-6 were increased by FPI, but release was blocked by iNO administered at 30 min or 2 h after TBI. The IL-6 antagonist LMT-28 prevented impairment of cerebral autoregulation and hippocampal CA1 and CA3 neuronal necrosis after FPI. Papaverine induced dilation was unchanged by FPI and LMT-28. Protection lasted for at least 2 h after iNO administration was stopped. CONCLUSIONS: These data indicate that iNO protects cerebral autoregulation and reduces hippocampal necrosis after traumatic brain injury through inhibition of ET-1, ERK MAPK, and IL-6 upregulation in pigs.

6,7,4'-Trihydroxyflavone inhibits osteoclast formation and bone resorption in vitro and in vivo.

The balance between the osteoblasts and the osteoclasts is important for the maintenance of the skeleton of the human body. The osteoclasts absorb bone after differentiated into polymorphonuclear cells by the fusion of monocytes/macrophages. We have found that 6,7,4'-Trihydroxyflavone (THF), a compound from the heartwood of Dalbergia Odorifera inhibits receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation, actin ring formation, and bone resorption in RAW 264.7 cells and bone marrow macrophage. THF significantly inhibited the c-Jun-N-terminal kinase signaling pathway without affecting extracellular signal-regulated kinase, p38, and AKT signaling. Moreover, THF inhibited the expression of c-Fos, nuclear factor-activated T cells cytoplasm 1, cathepsin K, and c-src by RANKL. We used a lipopolysaccharide (LPS)-induced bone loss model in mice. Consequently, bone volume per tissue volume, trabecular number's reduction was recovered in THF-treated mice, and trabecular separation's augmentation was also attenuated by THF administration. In summary, THF inhibits RANKL-induced osteoclast differentiation by MAPK signaling pathway and inhibits bone resorption by destroying the actin ring in mature osteoclasts. THF also prevented LPS-induced bone loss in a mice model. Thus, THF may be useful in the treatment of bone diseases associated with excessive osteoclast differentiation and bone resorption.

Salt Induces Adipogenesis/Lipogenesis and Inflammatory Adipocytokines Secretion in Adipocytes.

It is well known that high salt intake is associated with cardiovascular diseases including hypertension. However, the research on the mechanism of obesity due to high salt intake is rare. To evaluate the roles of salt on obesity prevalence, the gene expression of adipogenesis/lipogenesis and adipocytokines secretion according to adipocyte dysfunction were investigated in salt-loading adipocytes. High salt dose-dependently increased the expression of adipogenic/lipogenic genes, such as PPAR-γ, C/EBPα, SREBP1c, ACC, FAS, and aP2, but decreased the gene of lipolysis like AMPK, ultimately resulting in fat accumulation. With SIK-2 and Na⁺/K⁺-ATPase activation, salt increased the metabolites involved in the renin-angiotensin-aldosterone system (RAAS) such as ADD1, CYP11ß2, and MCR. Increasing insulin dependent insulin receptor substrate (IRS)-signaling, resulting in the insulin resistance, mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and Akt-mTOR were activated but AMPK(Thr172) was depressed in salt-loading adipocytes. The expression of pro-inflammatory adipocytokines, TNFα, MCP-1, COX-2, IL-17A, IL-6, leptin, and leptin to adiponectin ratio (LAR) were dose-dependently increased by salt treatment. Using the inhibitors of MAPK/ERK, U0126, we found that the crosstalk among the signaling pathways of MAPK/ERK, Akt-mTOR, and the inflammatory adipogenesis can be the possible mechanism of salt-linked obesity. The possibilities of whether the defense mechanisms against high dose of intracellular salts provoke signaling for adipocytes differentiation or interact with surrounding tissues through other pathways will be explored in future research.

Phosphatidylserine enhances IKBKAP transcription by activating the MAPK/ERK signaling pathway.

Familial dysautonomia (FD) is a genetic disorder manifested due to abnormal development and progressive degeneration of the sensory and autonomic nervous system. FD is caused by a point mutation in the IKBKAP gene encoding the IKAP protein, resulting in decreased protein levels. A promising potential treatment for FD is phosphatidylserine (PS); however, the manner by which PS elevates IKAP levels has yet to be identified. Analysis of ChIP-seq results of the IKBKAP promoter region revealed binding of the transcription factors CREB and ELK1, which are regulated by the mitogen-activated protein kinase (MAPK)/extracellular-regulated kinase (ERK) signaling pathway. We show that PS treatment enhanced ERK phosphorylation in cells derived from FD patients. ERK activation resulted in elevated IKBKAP transcription and IKAP protein levels, whereas pretreatment with the MAPK inhibitor U0126 blocked elevation of the IKAP protein level. Overexpression of either ELK1 or CREB activated the IKBKAP promoter, whereas downregulation of these transcription factors resulted in a decrease of the IKAP protein. Additionally, we show that PS improves cell migration, known to be enhanced by MAPK/ERK activation and abrogated in FD cells. In conclusion, our results demonstrate that PS activates the MAPK/ERK signaling pathway, resulting in activation of transcription factors that bind the promoter region of IKBKAP and thus enhancing its transcription. Therefore, compounds that activate the MAPK/ERK signaling pathway could constitute potential treatments for FD.

Glycyrrhizin Suppresses RANKL-Induced Osteoclastogenesis and Oxidative Stress Through Inhibiting NF-κB and MAPK and Activating AMPK/Nrf2.

The treatment for osteoporosis involves inhibiting bone resorption and osteoclastogenesis. Glycyrrhizin (GLY) is a triterpenoid saponin glycoside known to be as the most medically efficacious component of the licorice plant. It has strong anti-inflammatory, antioxidant, and antitumor properties. We investigated the effect of GLY on osteoclastogenesis, bone resorption, and intracellular oxidative stress and its molecular mechanisms. In vitro osteoclastogenesis assays were performed using bone marrow monocytes with and without glycyrrhizin. We also evaluated the effects of glycyrrhizin on the secretion of TNF-α, IL-1ß, and IL-6 in LPS-stimulated RAW 264.7 cells using ELISA. The effects of glycyrrhizin on the expression of osteoclast-related genes, such as Nfatc1, c-fos, Trap, and cathepsin K (CK), were investigated by RT-PCR. Intracellular reactive oxygen species (ROS) were detected in receptor activator of nuclear factor kappa-Β ligand (RANKL)-stimulated osteoclasts in the presence and absence of glycyrrhizin. During the inhibition of osteoclastogenesis by glycyrrhizin, phosphorylation of AMPK, Nrf2, NF-κB, and MAPK was analyzed using western blotting. Our results showed that glycyrrhizin significantly inhibited RANKL-induced osteoclastogenesis, downregulated the expression of NFATc1, c-fos, TRAP, CK, DC-STAMP, and OSCAR, and inhibited p65, p38, and JNK. Glycyrrhizin was found to significantly decrease the secretion of inflammatory cytokines (TNF-α, IL-1ß, and IL-6). Additionally, glycyrrhizin reduced the formation of ROS in osteoclasts by inducing AMPK phosphorylation and nuclear transfer of NRF2, resulting in an upregulation of antioxidant enzymes, such as HO-1, NQO-1, and GCLC. In summary, we found that glycyrrhizin inhibited RANKL-induced osteoclastogenesis. It was also indicated that glycyrrhizin could reduce oxidative stress by inhibiting the MAPK and NF-κB pathways and activating the AMPK/NRF2 signaling. Therefore, glycyrrhizin may be used as an effective therapeutic agent against osteoporosis and bone resorption.