C-terminal mechano-growth factor induces heme oxygenase-1-mediated neuroprotection of SH-SY5Y cells via the protein kinase Cϵ/Nrf2 pathway.

Recently, a variant of insulin-like growth factor-1, mechano-growth factor (MGF), has been discovered whose 24-amino-acid carboxy end is protective in models of stroke, nerve injury, and amyotrophic lateral sclerosis, suggesting broad-spectrum neuroprotective properties. Moreover, we recently demonstrated in vitro and in vivo that a modified protease-resistant 24-amino-acid MGF derivative (MGF24) protects dopaminergic neurons from oxidative stress-induced apoptosis via induction of the stress response protein heme oxygenase-1. However, the underlying mechanism by which MGF24 up-regulates heme oxygenase-1 expression is unknown. In this study, we demonstrate that MGF24-induced heme oxygenase-1 up-regulation is dependent on activation of protein kinase Cϵ and NF-E2-related factor-2 (Nrf2). MGF24 induces nuclear translocation of Nrf2, and siRNA knockdown of Nrf2 or of heme oxygenase-1 prevents MGF24-induced heme oxygenase-1 up-regulation and neuroprotection of SH-SY5Y cells against 6-hydroxydopamine-induced cell death. Pharmacological inhibition of ERK, p38 MAPK, PI3K/Akt, or PKC signaling revealed that only PKC inhibition by GF109203X prevents MGF24's ability to protect against 6-hydroxydopamine-induced cell death. GF109203X also prevented MGF24-induced Nrf2 nuclear translocation and heme oxygenase-1 up-regulation. siRNA knockdown of protein kinase Cϵ blocks MGF24-induced Nfr2 nuclear translocation, heme oxygenase-1 expression, and neuroprotection. Taken together, these results demonstrate that PKC activity is needed for MGF24's activation of Nrf2, which in turn increases heme oxygenase-1 expression, a critical event in mediating MGF24's neuroprotection against 6-hydroxydopamine-induced apoptosis.

Estradiol stimulates progesterone synthesis in hypothalamic astrocyte cultures.

The brain synthesizes steroids de novo, especially progesterone. Recently estradiol has been shown to stimulate progesterone synthesis in the hypothalamus and enriched astrocyte cultures derived from neonatal cortex. Estradiol-induced hypothalamic progesterone has been implicated in the control of the LH surge. The present studies were undertaken to determine whether hypothalamic astrocytes derived from female neonatal or female postpubertal rats increased production of progesterone in response to an estradiol challenge. Estradiol induced progesterone synthesis in postpubertal astrocytes but not neonatal astrocytes. This estradiol action was blocked by the estrogen receptor antagonist ICI 182,780. Previously we had demonstrated that estradiol stimulates a rapid increase in free cytosolic Ca(2+) ([Ca(2+)](i)) spikes in neonatal cortical astrocytes acting through a membrane estrogen receptor. We now report that estradiol also rapidly increased [Ca(2+)](i) spikes in hypothalamic astrocytes. The membrane-impermeable estradiol-BSA construct also induced [Ca(2+)](i) spikes. Both estradiol-BSA and estradiol were blocked by ICI 182,780. Depleting intracellular Ca(2+) stores prevented the estradiol-induced increased [Ca(2+)](i) spikes, whereas removing extracellular Ca(2+) did not prevent estradiol-induced [Ca(2+)](i) spikes. Together these results indicate that estradiol acts through a membrane-associated receptor to release intracellular stores of Ca(2+). Thapsigargin, used to mimicked the intracellular release of Ca(2+) by estradiol, increased progesterone synthesis, suggesting that estradiol-induced progesterone synthesis involves increases in [Ca(2+)](i). Estradiol treatment did not change levels of steroid acute regulatory protein, P450 side chain cleavage, 3beta-hydroxysteroid dehydrogenase, and sterol carrier protein-2 mRNAs as measured by quantitative RT-PCR, suggesting that in vitro, estradiol regulation of progesterone synthesis in astrocytes does not depend on transcription of new steroidogenic proteins. The present results are consistent with our hypothesis that estrogen-positive feedback regulating the LH surge involves stimulating local progesterone synthesis by hypothalamic astrocytes.

Manganese superoxide dismutase-mediated gene expression in radiation-induced adaptive responses.

Antioxidant enzymes are critical in oxidative stress responses. Radioresistant variants isolated from MCF-7 human carcinoma cells following fractionated ionizing radiation (MCF+FIR cells) or overexpression of manganese superoxide dismutase (MCF+SOD cells) demonstrated dose-modifying factors at 10% isosurvival of 1.8 and 2.3, respectively. MCF+FIR and MCF-7 cells (exposed to single-dose radiation) demonstrated 5- to 10-fold increases in MnSOD activity, mRNA, and immunoreactive protein. Radioresistance in MCF+FIR and MCF+SOD cells was reduced following expression of antisense MnSOD. DNA microarray analysis and immunoblotting identified p21, Myc, 14-3-3 zeta, cyclin A, cyclin B1, and GADD153 as genes constitutively overexpressed (2- to 10-fold) in both MCF+FIR and MCF+SOD cells. Radiation-induced expression of these six genes was suppressed in fibroblasts from Sod2 knockout mice (-/-) as well as in MCF+FIR and MCF+SOD cells expressing antisense MnSOD. Inhibiting NF-kappa B transcriptional activity in MCF+FIR cells, by using mutant I kappa B alpha, inhibited radioresistance as well as reducing steady-state levels of MnSOD, 14-3-3 zeta, GADD153, cyclin A, and cyclin B1 mRNA. In contrast, mutant I kappa B alpha was unable to inhibit radioresistance or reduce 14-3-3 zeta, GADD153, cyclin A, and cyclin B1 mRNAs in MCF+SOD cells, where MnSOD overexpression was independent of NF-kappa B. These results support the hypothesis that NF-kappa B is capable of regulating the expression of MnSOD, which in turn is capable of increasing the expression of genes that participate in radiation-induced adaptive responses.

Membrane estrogen receptor-alpha interacts with metabotropic glutamate receptor type 1a to mobilize intracellular calcium in hypothalamic astrocytes.

Estradiol, acting on a membrane-associated estrogen receptor-alpha (mERalpha), induces an increase in free cytoplasmic calcium concentration ([Ca(2+)](i)) needed for progesterone synthesis in hypothalamic astrocytes. To determine whether rapid estradiol signaling involves an interaction of mERalpha with metabotropic glutamate receptor type 1a (mGluR1a), changes in [Ca(2+)](i) were monitored with the calcium indicator, Fluo-4 AM, in primary cultures of female postpubertal hypothalamic astrocytes. 17beta-Estradiol over a range of 1 nm to 100 nm induced a maximal increase in [Ca(2+)](i) flux measured as a change in relative fluorescence [DeltaF Ca(2+) = 615 +/- 36 to 641 +/- 47 relative fluorescent units (RFU)], whereas 0.1 nm of estradiol stimulated a moderate [Ca(2+)](i) increase (275 +/- 16 RFU). The rapid estradiol-induced [Ca(2+)](i) flux was blocked with 1 microm of the estrogen receptor antagonist ICI 182,780 (635 +/- 24 vs. 102 +/- 11 RFU, P < 0.001) and 20 nmof the mGluR1a antagonist LY 367385 (617 +/- 35 vs. 133 +/- 20 RFU, P < 0.001). Whereas the mGluR1a receptor agonist (RS)-3,5-dihydroxyphenyl-glycine (50 microm) also stimulated a robust [Ca(2+)](i) flux (626 +/- 23 RFU), combined treatment of estradiol (1 nm) plus (RS)-3,5-dihydroxyphenyl-glycine (50 microm) augmented the [Ca(2+)](i) response (762 +/- 17 RFU) compared with either compound alone (P < 0.001). Coimmunoprecipitation demonstrated a direct physical interaction between mERalpha and mGluR1a in the plasma membrane of hypothalamic astrocytes. These results indicate that mERalpha acts through mGluR1a, and mGluR1a activation facilitates the estradiol response, suggesting that neural activity can modify estradiol-induced membrane signaling in astrocytes.