Mechanism of raloxifene-induced upregulation of glutamate transporters in rat primary astrocytes

Abstract

Raloxifene (RX), a selective estrogen receptor modulator (SERM), exerts neuroprotection in multiple clinical and experimental settings. Astrocytic glutamate transporters GLT-1 (EAAT2) and GLAST (EAAT1) are the main glutamate transporters in the central nervous system, taking up most of excess glutamate from the synaptic cleft to prevent excitotoxic neuronal death. Since drugs targeting astrocytic glutamate transporters to enhance their expression and function represent potential therapeutics for neurodegenerative disorders associated with excitotoxicity, we tested if RX modulates the expression and function of GLT-1 and GLAST in rat primary astrocytes. The results showed that RX significantly increased glutamate uptake and expression of GLT-1 mRNA and protein levels. RX enhanced GLT-1 expression by the activation of multiple signaling pathways including ERK, EGFR and CREB mediated by estrogen receptors (ERs) ER-α, ER-β and GPR30. At the transcriptional level, NF-κB played a critical role in RX-induced GLT-1 expression as RX increased NF-κB reporter activity and induced binding of NF-κB p65 and p50 to the GLT-1 promoter. RX attenuated the reduction of GLT-1 expression and glutamate uptake induced by manganese (Mn) whose chronic high levels of exposure cause manganism. RX also upregulated GLAST by increasing its promoter activity and protein levels via the NF-κB pathway and ERs. Our findings provide new insight into the mechanism of RX-induced enhancement of GLT-1 and GLAST expression, as well as the attenuation of Mn-reduced expression of these transporters. These findings will be highly valuable for developing therapeutics of neurodegenerative diseases associated with impaired astrocytic glutamate transporters.

Chronic Hyponatremia Causes Neurologic and Psychologic Impairments

“…Glutamate uptake assays were performed as described previously 63 to the reaction media to provide a final concentration of 100 nM glutamate. The reaction was continued for 10 minutes at 37°C and terminated by washing three times with ice-cold PBS, immediately followed by cell lysis in 1 ml of 1 N sodium hydroxide.…”

Transcriptional Regulation of the Astrocytic Excitatory Amino Acid Transporter 1 (EAAT1) via NF-κB and Yin Yang 1 (YY1)

“…Earlier studies have also suggested that transcriptional regulation of EAAT1 might be different from that of EAAT2 because the mTORPI3k/Akt signaling cascade induced only EAAT2 expression (27,28). Some studies show that NF-B might also play a critical role in the regulation of EAAT1 expression induced by EGF, TGF-␣, and raloxifene in rat primary astrocytes (19,21). However, the precise relationship between NF-B and EAAT1 has yet to be characterized.…”

“…The EAAT1 promoter is stimulated by growth factors such as EGF and TGF-␣, leading to enhancement of EAAT1 mRNA expression (17,18), as well as protein levels (19,20). Estrogen and selective estrogen receptor modulators, such as tamoxifen and raloxifene, also enhance EAAT1 mRNA and protein levels (21,22). The transcription factor NF-B has been reported to be a critical positive regulator of GLT-1/EAAT2 and mediates the stimulatory effects of various modulators such as ceftriaxone (23), EGF (24,25), tamoxifen, and raloxifene (21,26) on EAAT2 expression.…”

“…Estrogen and selective estrogen receptor modulators, such as tamoxifen and raloxifene, also enhance EAAT1 mRNA and protein levels (21,22). The transcription factor NF-B has been reported to be a critical positive regulator of GLT-1/EAAT2 and mediates the stimulatory effects of various modulators such as ceftriaxone (23), EGF (24,25), tamoxifen, and raloxifene (21,26) on EAAT2 expression. Earlier studies have also suggested that transcriptional regulation of EAAT1 might be different from that of EAAT2 because the mTORPI3k/Akt signaling cascade induced only EAAT2 expression (27,28).…”

“…The same group showed that the anti-epileptic drug valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, increases EAAT1 promoter activity, mRNA/protein levels, and uptake activity in chick Bergmann glia cells while decreasing YY1 binding to the EAAT1 promoter (29), indicating a role for YY1 in repressing EAAT1. TNF-␣ decreases EAAT1 promoter activity, mRNA, and protein levels (18,21). Manganese, an environmental toxin that causes Parkinson disease-like pathological signs and symptoms, referred to as manganism, decreases EAAT1 expression and function in astrocytes (21,22,36).…”