PHOSPHORYLATION OF CALCIUM/CALMODULIN-DEPENDENT PROTEIN KINASE II (CAMKII) AND EXTRACELLULAR REGULATED KINASE (ERK) IN STRIATUM MEDIATE NICOTINE DEPENDENCE IN BALB/C MICE

Objective: Nicotine is an active compound in tobacco and has a rewarding effect in the central nervous system (CNS), which may lead to dependence. Although nicotine dependence is elucidated by brain mechanisms, synaptic molecular substrates underlying the dependence remain unclear. We hypothesized that reward signaling is mediated by dopamine and glutamate receptors, in where calcium/calmodulin-dependent kinase II (CaMKII) and extracellular signal-regulated kinase (ERK) may mediate the synaptic signaling of dependence. Methods: To investigate the roles of both CaMKII and ERK on nicotine dependence were assessed by conditioned place preference (CPP) methods followed by dissection. One day after conditioning, preference scores were measured to evaluate nicotine dependence. Mice were sacrificed and their striatum were dissected out for immunoblotting analyses of CaMKII and ERK phosphorylation. Results: Nicotine-induced conditioned place preference as a symptom of nicotine dependence. CaMKII and ERK phosphorylation in striatum significantly increased along with the development of nicotine dependence. Conclusion: We should next apply pharmacological strategies to manipulate CaMKII and ERK signaling. In particular, disruption of reconsolidation by disrupting CaMKII and ERK signaling may propose an attractive therapeutic approach to inhibit nicotine dependence.

Identification of interleukin-16 production on tumor aggravation in hepatocellular carcinoma by a proteomics approach

BACKGROUND: Cytokines play an important role in the immune response, angiogenesis, cell growth, and differentiation in hepatocellular carcinoma (HCC). OBJECTIVE: We performed a comprehensive study to identify tumor-related cytokines and pathways involved in HCC pathogenesis. METHODS: Cytokine production was evaluated in human HCC tissues and adjacent non-tumor tissues using an antibody-based protein array technique. We compared cytokine expression in HCC tissues with that of hepatic hemangioma (HH), liver metastasis of colorectal cancer, and noncancerous liver tissues from transplantation donors. The protein levels and localization of the candidate cytokines were analyzed by western blotting and immunohistochemistry. RESULTS: Increased expression of interleukin (IL)-1 receptor antagonist, macrophage migration inhibitory factor, and IL-16 was observed in HCC and paired adjacent non-tumor tissues compared with noncancerous livers. In addition, there were increased IL-16 levels in HCC tissues compared with HH. IL-16 treatment significantly increased cell proliferation in vitro. The expression of extracellular signal-regulated kinase (ERK)1/2 and cyclin D1 was markedly increased in cells from two HCC cell lines, Huh7 and HepG2, in a dose- and time-dependent manner. Phosphorylated to total ERK1/2 ratio was increased in Huh7 cells following IL-16 50 ng/ml, but not HepG2 cells. ERK phosphorylation have occurred earlier than protein accumulation at 48 h. Pretreatment with the ERK inhibitor, FR18024, or an anti-IL-16 antibody reduced the increase in IL-16 production in HCC cells. CONCLUSIONS: These results suggest that cell proliferation induced by IL-16 is mediated through the ERK pathway, thus, we identified a new factor associated with HCC tumor growth.

MiR-155-5p suppresses SOX1 to promote proliferation of cholangiocarcinoma via RAF/MEK/ERK pathway

Background Accumulating evidence has demonstrated the close relation of SOX1 with tumorigenesis and tumor progression. Upregulation of SOX1 was recently shown to suppress growth of human cancers. However, the expression and role of SOX1 in cholangiocarcinoma (CCA) is not well characterized. Methods Expression levels of SOX1 in CCA tissues and normal bile duct tissues were examined using public GEO database. Western blot and immunohistochemistry were used to confirm the expression levels. Cell proliferation assay (CCK-8) and colony formation assay were performed to assess proliferation of CCA cells. A mouse model of subcutaneous transplantable tumors was used to evaluated proliferation of CCA in vivo. The putative regulating factor of SOX1 were determined using Targetscan and dual-luciferase reporter assay. Results SOX1 was downregulated in CCA tissues. Overexpression of SOX1 significantly inhibited cell proliferation in vitro and suppressed tumor growth in vivo. miR-155-5p directly targeted the 3′-untranslated region (3′UTR) of SOX1 and inhibited expression of SOX1, resulting in the activation of RAF, MEK and ERK phosphorylation, and thus CCA proliferation. However, restoration of SOX1 expression in miR-155-5p overexpressing cell lines decreased the phosphorylation level of RAF, MEK and ERK, as well as the proliferation of CCA cells. Conclusion MiR-155-5p decreased the expression of SOX1 by binding to its 3′UTR, which activated the RAF/MEK/ERK signaling pathway and promoted CCA progression.

5-epi-Sinuleptolide from Soft Corals of the Genus Sinularia Exerts Cytotoxic Effects on Pancreatic Cancer Cell Lines via the Inhibition of JAK2/STAT3, AKT, and ERK Activity

Pancreatic ductal adenocarcinoma is one of the most lethal malignancies: more than half of patients are diagnosed with a metastatic disease, which is associated with a five-year survival rate of only 3%. 5-epi-Sinuleptolide, a norditerpene isolated from Sinularia sp., has been demonstrated to possess cytotoxic activity against cancer cells. However, the cytotoxicity against pancreatic cancer cells and the related mechanisms are unknown. The aim of this study was to evaluate the anti-pancreatic cancer potential of 5-epi-sinuleptolide and to elucidate the underlying mechanisms. The inhibitory effects of 5-epi-sinuleptolide treatment on the proliferation of pancreatic cancer cells were determined and the results showed that 5-epi-sinuleptolide treatment inhibited cell proliferation, induced apoptosis and G2/M cell cycle arrest, and suppressed the invasion of pancreatic cancer cells. The results of western blotting further revealed that 5-epi-sinuleptolide could inhibit JAK2/STAT3, AKT, and ERK phosphorylation, which may account for the diverse cytotoxic effects of 5-epi-sinuleptolide. Taken together, our present investigation unveils a new therapeutic and anti-metastatic potential of 5-epi-sinuleptolide for pancreatic cancer treatment.

CD5L deficiency attenuate acetaminophen-induced liver damage in mice via regulation of JNK and ERK signaling pathway

CD5 molecule like (CD5L), a member of the scavenger receptor cysteine-rich domain superfamily, plays a critical role in immune homeostasis and inflammatory disease. Acetaminophen (APAP) is a safe and effective antipyretic analgesic. However, overdose may cause liver damage or even liver failure. APAP hepatotoxicity is characterized by extensive necrotic cell death and a sterile inflammatory response, in which the role of CD5L remains to be investigated. In this study, we found that the expression of CD5L was increased in the livers of mice after APAP overdose. Furthermore, CD5L deficiency reduced the increase of alanine transaminase (ALT) level, histopathologic lesion area, c-Jun N-terminal kinase (JNK)/extracellular signal-regulated kinase (ERK) phosphorylation level, Transferase-Mediated dUTP Nick End-Labeling positive (TUNEL+) cells proportion, vascular endothelial cell permeability and release of inflammatory cytokines induced by excess APAP. Therefore, our findings reveal that CD5L may be a potential therapeutic target for prevention and treatment of APAP-induced liver injury.

The Q61H mutation decouples KRAS from upstream regulation and renders cancer cells resistant to SHP2 inhibitors

Cancer cells bearing distinct KRAS mutations exhibit variable sensitivity to SHP2 inhibitors (SHP2i). Here we show that cells harboring KRAS Q61H are uniquely resistant to SHP2i, and investigate the underlying mechanisms using biophysics, molecular dynamics, and cell-based approaches. Q61H mutation impairs intrinsic and GAP-mediated GTP hydrolysis, and impedes activation by SOS1, but does not alter tyrosyl phosphorylation. Wild-type and Q61H-mutant KRAS are both phosphorylated by Src on Tyr32 and Tyr64 and dephosphorylated by SHP2, however, SHP2i does not reduce ERK phosphorylation in KRAS Q61H cells. Phosphorylation of wild-type and Gly12-mutant KRAS, which are associated with sensitivity to SHP2i, confers resistance to regulation by GAP and GEF activities and impairs binding to RAF, whereas the near-complete GAP/GEF-resistance of KRAS Q61H remains unaltered, and high-affinity RAF interaction is retained. SHP2 can stimulate KRAS signaling by modulating GEF/GAP activities and dephosphorylating KRAS, processes that fail to regulate signaling of the Q61H mutant.

Rilmenidine mimics caloric restriction via the nischarin I1-imidazoline receptor to extend lifespan in C. elegans

Caloric restriction increases lifespan across species and has health benefits in humans. Because complying with a low-calorie diet is challenging, here we investigated pharmacological interventions mimicking the benefits of caloric restriction. Searching for compounds that elicit a similar gene expression signature to caloric restriction, we identified rilmenidine, an I1-imidazoline receptor agonist and prescription medication for the treatment of hypertension. We then show that treating C. elegans with rilmenidine at young and older ages increases lifespan. We also demonstrate that the stress-resilience, healthspan, and lifespan benefits upon rilmenidine treatment in worms are mediated by the I1-imidazoline receptor nish-1, implicating this receptor as a potential longevity target. Furthermore, we show that rilmenidine treatment increased ERK phosphorylation via NISH-1. Consistent with the shared caloric-restriction-mimicking gene signature, supplementing rilmenidine to caloric restricted C. elegans, genetic reduction of TORC1 function, or rapamycin treatment did not further increase lifespan. The rilmenidine-induced longevity required the transcription factors FOXO/DAF-16 and NRF1,2,3/SKN-1, both important for caloric restriction-mediated longevity. Furthermore, we find that autophagy, but not AMPK signaling, was needed for rilmenidine-induced longevity. Lastly, we find that treating mice with rilmenidine showed transcriptional changes in liver and kidney similar to caloric restriction. Overall, our findings reveal rilmenidine as a caloric restriction mimetic and as a novel geroprotective compound.

The Predominant Role of Arrestin3 in General GPCR Desensitization in Platelets

Arrestins in concert with GPCR kinases (GRKs) function in G protein-coupled receptor (GPCR) desensitization in various cells. Therefore, we characterized the functional differences of arrestin3 versus arrestin2 in the regulation of GPCR signaling and its desensitization in platelets using mice lacking arrestin3 and arrestin2. In contrast to arrestin2, platelet aggregation and dense granule secretion induced by 2-MeSADP, U46619, thrombin, and AYPGKF were significantly potentiated in arrestin3-deficient platelets compared to wild-type (WT) platelets, while non-GPCR agonist CRP-induced platelet aggregation and secretion were not affected. Surprisingly, in contrast to GRK6, platelet aggregation induced by the co-stimulation of serotonin and epinephrine was significantly potentiated in arrestin3-deficient platelets, suggesting the central role of arrestin3 in general GPCR desensitization in platelets. In addition, the second challenge of ADP and AYPGKF restored platelet aggregation in arrestin3-deficient platelets but failed to do so in WT and arrestin2-deficient platelets, confirming that arrestin3 contributes to GPCR desensitization. Furthermore, ADP- and AYPGKF-induced Akt and ERK phosphorylation were significantly increased in arrestin3-deficient platelets. Finally, we found that arrestin3 is critical for thrombus formation in vivo. In conclusion, arrestin3, not arrestin2, plays a central role in the regulation of platelet functional responses and thrombus formation through general GPCR desensitization in platelets.