Neuroprotective action of raloxifene against hypoxia-induced damage in mouse hippocampal cells depends on ERα but not ERβ or GPR30 signalling.
Raloxifene is the selective estrogen receptor modulator (SERM) currently used in clinical practice to activate estrogen receptors (ERs) in bone tissue and to antagonise ERs in breast and uterine cancers. Little is known, however, about mechanisms of action of raloxifene on hypoxia-induced neuronal cell damage. The aim of the present study was to investigate the neuroprotective potential of raloxifene against hypoxia-induced damage of mouse hippocampal cells in primary cultures, with a particular focus on raloxifene interactions with the classical nuclear ERs (ERα, ERβ) and the recently identified membrane ER G-protein-coupled receptor 30 (GPR30). In this study, 18 h of hypoxia increased hypoxia inducible factor 1 alpha (Hif1α) mRNA expression and induced apoptotic processes, such as loss of the mitochondrial membrane potential, activation of caspase-3 and fragmentation of cell nuclei based on Hoechst 33342 staining. These effects were accompanied by reduced ATPase and intracellular esterase activities as well as substantial lactate dehydrogenase (LDH) release from cells exposed to hypoxia. Our study demonstrated strong neuroprotective and anti-apoptotic caspase-3-independent actions of raloxifene in hippocampal cells exposed to hypoxia. Raloxifene also inhibited the hypoxia-induced decrease in Erα mRNA expression and attenuated the hypoxia-induced rise in Erβ and Gpr30 mRNA expression levels. Impact of raloxifene on hypoxia-affected Erα mRNA was mirrored by fluctuations in the protein level of the receptor as demonstrated by Western blot and immunofluorescent labelling. Raloxifene-induced changes in Erβ mRNA expression level were in parallel with ERβ immunofluorescent labeling. However, changes in Gpr30 mRNA level were not reflected by changes in the protein levels measured either by ELISA, Western blot or immunofluorescent staining at 24h post-treatment. Using specific siRNAs, we provided evidence for a key involvement of ERα, but not ERβ or GPR30 in neuroprotective action of raloxifene against hypoxia-induced cell damage. This study may have implications for the treatment or prevention of hypoxic brain injury and the administration of current or new generations of SERMs specific to ERα. This article is part of a Special Issue entitled "Sex steroids and brain disorders".
Department of Experimental Neuroendocrinology, Institute of Pharmacology, Polish Academy of Sciences, 12 Smetna Street, 31-343 Krakow, Poland.
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Rzemieniec
Department of Experimental Neuroendocrinology, Institute of Pharmacology, Polish Academy of Sciences, 12 Smetna Street, 31-343 Krakow, Poland.
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Litwa
Department of Experimental Neuroendocrinology, Institute of Pharmacology, Polish Academy of Sciences, 12 Smetna Street, 31-343 Krakow, Poland.
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Wnuk
Department of Experimental Neuroendocrinology, Institute of Pharmacology, Polish Academy of Sciences, 12 Smetna Street, 31-343 Krakow, Poland.
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Lason
Department of Genetics and Evolution, Institute of Zoology, Jagiellonian University, 9 Gronostajowa Street, 30-387 Krakow, Poland.
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Gołas
Department of Cell Biology and Imaging, Confocal Microscopy Laboratory, Institute of Zoology, Jagiellonian University, 9 Gronostajowa Street, 30-387 Krakow, Poland.
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Krzeptowski
Department of Experimental Neuroendocrinology, Institute of Pharmacology, Polish Academy of Sciences, 12 Smetna Street, 31-343 Krakow, Poland. Electronic address: kajta@if-pan.krakow.pl.
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Kajta
D000818:Animals; D053148:Caspase 3 / Q000378:metabolism; D015687:Cell Hypoxia / Q000187:drug effects / Q000235:genetics; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D047628:Estrogen Receptor alpha / Q000235:genetics / Q000378:metabolism*; D047629:Estrogen Receptor beta / Q000235:genetics / Q000378:metabolism*; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects; D006624:Hippocampus / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D000860:Hypoxia / Q000188:drug therapy* / Q000235:genetics / Q000503:physiopathology; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D051379:Mice; D018696:Neuroprotective Agents / Q000494:pharmacology*; D020849:Raloxifene Hydrochloride / Q000494:pharmacology*; D011960:Receptors, Estrogen; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism*; D020845:Selective Estrogen Receptor Modulators / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects
D047628:Estrogen Receptor alpha; D047629:Estrogen Receptor beta; C532507:GPER1 protein, mouse; C497443:Hif1a protein, mouse; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D018696:Neuroprotective Agents; D011960:Receptors, Estrogen; D043562:Receptors, G-Protein-Coupled; D020845:Selective Estrogen Receptor Modulators; D020849:Raloxifene Hydrochloride; D007770:L-Lactate Dehydrogenase; D053148:Caspase 3
Apoptosis; Immunoblots; Primary neuronal cell cultures; SERMs; siRNAs