Rapid membrane effect of estrogens on stimulation of corticotropin-releasing hormone.

BACKGROUND: Classic nuclear-initiated estrogen signaling stimulates corticotropin-releasing hormone (CRH) gene expression as a transcription factor. However, the possible mechanism by which membrane-initiated estrogen signaling (MIES) influences CRH expression remains unclear. There are indications that MIES may upregulate nitric oxide (NO) production through the phosphatidylinositol 3-hydroxy kinase (PI3K) and potentially through the mitogen-activated protein kinase (MAPK) pathway. OBJECTIVES: We investigated the effect of MIES-mediated kinase pathways on CRH expression with or without NO synthesis. METHOD: In SK-N-SH cell culture, estradiol-bovine serum albumin (E2-BSA) was used as the specific membrane estrogen receptor activator, with a specific NO donor, and/or inhibitors for NO synthase (NOS), PI3K, MAPK, protein kinase A (PKA), and protein kinase C (PKC). RESULTS: E2-BSA significantly increased NO and CRH levels in the medium and NOS1-mRNA levels in the cells. In addition, NO donor up-regulated CRH expression, while NOS-inhibitor down-regulated it. When the inhibitor of MAPK and/or the inhibitor of PI3K was added to the medium, only the latter appeared to significantly block the stimulating effect of E2-BSA on NO synthesis, and this was accompanied by an increased CRH expression in the medium. We further studied the effect of the MIES-PKC-mediated pathway on CRH expression, with or without NOS-inhibitor, while the MIES-PKA(-PI3K) pathway served as a control. We found that MIES-PKC upregulated CRH expression independent of NO synthesis. CONCLUSION: MIES can efficiently upregulate CRH expression via various intracellular kinase pathways and may thus be a crucial component in the stress response.

α-Cyperone Inhibits PMA-Induced EPCR Shedding through PKC Pathway.

α-Cyperone, a sesquiterpene compound represents 25.23% of the total oil and is the most abundant compound in Cyperus rotundus oil. Endothelial cell protein C receptor (EPCR) is a main member in protein C (PC) anti-coagulation system. EPCR could be shed from cell surface, and is mediated by tumor necrosis factor-α converting enzyme (TACE). Nothing that EPCR is a marker of vascular barrier integrity in vascular inflammatory disease and takes part in systemic inflammatory disease. In this study, we investigated whether α-cyperone could inhibit EPCR shedding. To observe the effect, we investigated this issue by detection the effect of α-cyperone on phorbol-12-myristate 13-acetate (PMA)-induced EPCR shedding in human umbilical vein endothelial cells (HUVECs). The cells were pretreated with α-cyperone for 12 h, and then stimulated by PMA for 1 h. The solute EPCR (sEPCR) and expression of membrane EPCR (mEPCR) were measured by enzyme-linked immunosorbent assay (ELISA) and Western blot. The mRNA, protein level and activity of TACE were tested by quantitative (q)RT-PCR, Western blot and InnoZyme TACE activity assay kit. Furthermore, we measured the protein level of mitogen-activated protein kinase (MAPK) signaling and protein kinase C (PKC) pathway under this condition by Western blot. The results showed that α-cyperone could suppress PMA-induced EPCR shedding through inhibiting the expression and activity of TACE. In addition, α-cyperone could inhibit PKC translocation, but not have an effect on phosphorylation of c-Jun N-terminal kinase (JNK), p38 and extracellular regulated protein kinases (ERK) 1/2. Given these results, α-cyperone inhibits PMA-induced EPCR shedding through PKC pathway, which will provide an experimental basis for further research on α-cyperone.

Inhibition of δ-opioid receptors induces brain glioma cell apoptosis through the mitochondrial and protein kinase C pathways.

Brain glioma is a malignant tumor with a high incidence rate and poor prognosis that has become a focus of studies of central nervous system diseases. Previous studies have suggested that δ-opioid receptors may affect the proliferation and apoptosis of numerous types of tumor cells. However, to date, their precise mechanism(s) of action have not been elucidated. The present study aimed to investigate the effects of inhibiting δ-opioid receptors in brain glioma cell proliferation and apoptosis and their relevant molecular mechanisms. Various doses of naltrindole were supplied to treat brain glioma cells using the MTT method to assess the proliferation index. Flow cytometry was used to investigate the changes in cell apoptosis and mitochondrial membrane potential. The expression levels of Bax, Bcl-2, Bcl-xL, cytochrome c, caspase-9, caspase-3 and protein kinase C (PKC) were measured using western blotting. Naltrindole was observed to inhibit brain glioma cell proliferation and promote apoptosis in a dose- and time-dependent manner. Furthermore, the addition of naltrindole lead to changes in the brain glioma cell membrane potential and regulated Bax translocation to the mitochondrial membrane, consequently promoting the release of cytochrome c into the cytoplasm, followed by the activation of caspase-9 and -3, which caused cell apoptosis. In addition, naltrindole was able to regulate the expression levels of the cellular internal phosphorylated PKC proteins, which are closely associated with the inhibition of cell proliferation. In conclusion, the inhibition of δ-opioid receptors may inhibit brain glioma cell proliferation and lead to apoptosis, which is closely associated with the mitochondrial and PKC pathways.