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Abstract: Previously we demonstrated that 17β-Estradiol (E2) induced rapid Ca2+ influx via L-type calcium channel activation, which was required for activation of Src/ERK/CREB/Bcl2 signaling cascade and subsequent induction of neuroprotective and neurotrophic responses in rat hippocampal and cortical neurons (Wu et al., 2005; Zhao et al., 2005). The current study determined the presence and specificity of membrane E2 binding sites and the functional consequence of E2 binding to membrane receptors in individual neurons. Using E2-BSA-FITC (fluorescein isothiocyanate) macromolecular complex, membrane E2 binding sites were observed in hippocampal neurons. Punctate FITC signal was observed on plasma membrane of soma and neuronal processes in E2-BSA-FITC binding neurons. No membrane binding was observed with BSA-FITC. Specificity of binding was demonstrated by competition with excess un-conjugated E2. An ERa specific agonist, PPT, and an ERb agonist, DPN, partially competed for E2-BSA-FITC binding. Imaging of intracellular Ca2+ ([Ca2+]i) in live neurons, revealed rapid Ca2+ responses in E2-BSA-FITC binding neurons within minutes that culminated in a greater [Ca2+]i rise and [Ca2+]i spikes at >20min. The same neurons in which E2-BSA-FITC induced a [Ca2+]i rise also exhibited activated pERK (extracellular signal-regulated kinase) that was translocated to the nucleus. Immunofluorescent analyses demonstrated that both excitatory and inhibitory neuronal markers labeled subpopulations of E2-BSA-FITC binding neurons. All E2-BSA-FITC binding neurons expressed L-type calcium channels. These results demonstrate, at a single cell level, that E2 membrane receptors mediate the rapid signaling cascades required for E2 neuroprotective and neurotrophic effects in hippocampal neurons. These results are discussed with respect to therapeutic targets of estrogen therapy in brain. [Copyright &y& Elsevier]

Abstract Background KRAS mutations may predict poor response to radiotherapy. Downstream events from KRAS, such as activation of BRAF, AKT and ERK, may also confer prognostic information but have not been tested in rectal cancer (RC). Our objective was to explore the relationships of KRAS and BRAF mutation status with p-AKT and p-ERK and outcomes in RC. Methods Pre-radiotherapy RC tumor biopsies were evaluated. KRAS and BRAF mutations were assessed by pyrosequencing; p-AKT and p-ERK expression by immunohistochemistry. Results Of 70 patients, mean age was 58; 36% stage II, 56% stage III, and 9% stage IV. Responses to neoadjuvant chemoradiotherapy: 64% limited, 19% major, and 17% pathologic complete response. 64% were KRAS WT, 95% were BRAF WT. High p-ERK levels were associated with improved OS but not for p-AKT. High levels of p-AKT and p-ERK expression were associated with better responses. KRAS WT correlated with lower p-AKT expression but not p-ERK expression. No differences in OS, residual disease, or tumor downstaging were detected by KRAS status. Conclusions KRAS mutation was not associated with lesser response to chemoradiotherapy or worse OS. High p-ERK expression was associated with better OS and response. Higher p-AKT expression was correlated with better response but not OS.

Accumulating evidence indicates that activation of spinal cord astrocytes contributes importantly to nerve injury and inflammation-induced persistent pain and chronic opioid-induced antinociceptive tolerance. Phosphorylation of extracellular signal-regulated kinase (pERK) and induction of interleukin-1 beta (IL-1β) in spinal astrocytes have been implicated in astrocytes-mediated pain. Tissue plasminogen activator (tPA) is a serine protease that has been extensively used to treat stroke. We examined the potential involvement of tPA in chronic opioid-induced antinociceptive tolerance and activation of spinal astrocytes using tPA knockout (tPA(-/-)) mice and astrocyte cultures. tPA(-/-) mice exhibited unaltered nociceptive pain and morphine-induced acute analgesia. However, the antinociceptive tolerance, induced by chronic morphine (10mg/kg/day, s.c.), is abrogated in tPA(-/-) mice. Chronic morphine induces tPA expression in glial fibrillary acidic protein (GFAP)-expressing spinal cord astrocytes. Chronic morphine also increases IL-1β expression in GFAP-expressing astrocytes, which is abolished in tPA-deficient mice. In cultured astrocytes, morphine treatment increases tPA, IL-1β, and pERK expression, and the increased IL-1β and pERK expression is abolished in tPA-deficient astrocytes. tPA is also sufficient to induce IL-1β and pERK expression in astrocyte cultures. Intrathecal injection of tPA results in up-regulation of GFAP and pERK in spinal astrocytes but not up-regulation of ionized calcium binding adapter molecule 1 in spinal microglia. Finally, intrathecal tPA elicits persistent mechanical allodynia, which is inhibited by the astroglial toxin alpha-amino adipate and the MEK (ERK kinase) inhibitor U0126. Collectively, these data suggest an important role of tPA in regulating astrocytic signaling, pain hypersensitivity, and morphine tolerance., (Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.)