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Riboflavin is an essential micronutrient and a precursor of flavin mononucleotide and flavin adenine dinucleotide for maintaining cell homeostasis. Riboflavin deficiency (RD) induces cell apoptosis. Endoplasmic reticulum (ER) stress is considered to induce apoptosis, and C/EBP homologous protein (CHOP) is a key pathway involved in this process. However, whether RD-induced apoptosis is mediated by ER stress and the CHOP pathway remains unclear and needs further investigation. Therefore, the current study presents the effect of RD on ER stress and apoptosis in the human hepatoma cell line (HepG2). Firstly, cells were cultured in a RD medium (4.55 nM riboflavin) and a control (CON) medium (1005 nM riboflavin). We conducted an observation of cell microstructure characterization and determining apoptosis. Subsequently, 4-phenyl butyric acid (4-PBA), an ER stress inhibitor, was used in HepG2 cells to investigate the role of ER stress in RD-induced apoptosis. Finally, CHOP siRNA was transfected into HepG2 cells to validate whether RD triggered ER stress-mediated apoptosis by the CHOP pathway. The results show that RD inhibited cell proliferation and caused ER stress, as well as increased the expression of ER stress markers (CHOP, 78 kDa glucose-regulated protein, activating transcription factor 6) (p < 0.05). Furthermore, RD increased the cell apoptosis rate, enhanced the expression of proapoptotic markers (B-cell lymphoma 2-associated X, Caspase 3), and decreased the expression of the antiapoptotic marker (B-cell lymphoma 2) (p < 0.05). The 4-PBA treatment and CHOP knockdown markedly alleviated RD-induced cell apoptosis. These results demonstrate that RD induces cell apoptosis by triggering ER stress and the CHOP pathway. [ABSTRACT FROM AUTHOR]

Abstract Background Oxidative stress is a common cause of neurodegeneration and plays a central role in retinal degenerative diseases. Heme oxygenase-1 (HMOX1) is a redox-regulated enzyme that is induced in neurodegenerative diseases and acts against oxidative stress but can also promote cell death, a phenomenon that is still unexplained in molecular terms. Here, we test whether HMOX1 has opposing effects during retinal degeneration and investigate the molecular mechanisms behind its pro-apoptotic role. Methods Basal and induced levels of HMOX1 in retinas are examined during light-induced retinal degeneration in mice. Light damage-independent HMOX1 induction at two different expression levels is achieved by intraocular injection of different doses of an adeno-associated virus vector expressing HMOX1. Activation of Müller glial cells, retinal morphology and photoreceptor cell death are examined using hematoxylin-eosin staining, TUNEL assays, immunostaining and retinal function are evaluated with electroretinograms. Downstream gene expression of HMOX1 is analyzed by RNA-seq, qPCR examination and western blotting. The role of one of these genes, the pro-apoptotic DNA damage inducible transcript 3 (Ddit3), is analyzed in a line of knockout mice. Results Light-induced retinal degeneration leads to photoreceptor degeneration and concomitant HMOX1 induction. HMOX1 expression at low levels before light exposure prevents photoreceptor degeneration but expression at high levels directly induces photoreceptor degeneration even without light stress. Photoreceptor degeneration following high level expression of HMOX1 is associated with a mislocalization of rhodopsin in photoreceptors and an increase in the expression of DDIT3. Genetic deletion of Ddit3 in knockout mice prevents photoreceptor cell degeneration normally resulting from high level HMOX1 expression. Conclusion The results reveal that the expression levels determine whether HMOX1 is protective or deleterious in the retina. Furthermore, in contrast to the protective low dose of HMOX1, the deleterious high dose is associated with induction of DDIT3 and endoplasmic reticulum stress as manifested, for instance, in rhodopsin mislocalization. Hence, future applications of HMOX1 or its regulated targets in gene therapy approaches should carefully consider expression levels in order to avoid potentially devastating effects.

Background: Oxidative stress is a common cause of neurodegeneration and plays a central role in retinal degenerative diseases. Heme oxygenase-1 (HMOX1) is a redox-regulated enzyme that is induced in neurodegenerative diseases and acts against oxidative stress but can also promote cell death, a phenomenon that is still unexplained in molecular terms. Here, we test whether HMOX1 has opposing effects during retinal degeneration and investigate the molecular mechanisms behind its pro-apoptotic role. Methods: Basal and induced levels of HMOX1 in retinas are examined during light-induced retinal degeneration in mice. Light damage-independent HMOX1 induction at two different expression levels is achieved by intraocular injection of different doses of an adeno-associated virus vector expressing HMOX1. Activation of Müller glial cells, retinal morphology and photoreceptor cell death are examined using hematoxylin-eosin staining, TUNEL assays, immunostaining and retinal function are evaluated with electroretinograms. Downstream gene expression of HMOX1 is analyzed by RNA-seq, qPCR examination and western blotting. The role of one of these genes, the pro-apoptotic DNA damage inducible transcript 3 (Ddit3), is analyzed in a line of knockout mice. Results: Light-induced retinal degeneration leads to photoreceptor degeneration and concomitant HMOX1 induction. HMOX1 expression at low levels before light exposure prevents photoreceptor degeneration but expression at high levels directly induces photoreceptor degeneration even without light stress. Photoreceptor degeneration following high level expression of HMOX1 is associated with a mislocalization of rhodopsin in photoreceptors and an increase in the expression of DDIT3. Genetic deletion of Ddit3 in knockout mice prevents photoreceptor cell degeneration normally resulting from high level HMOX1 expression. Conclusion: The results reveal that the expression levels determine whether HMOX1 is protective or deleterious in the retina. Furthermore, in contrast to the protective low dose of HMOX1, the deleterious high dose is associated with induction of DDIT3 and endoplasmic reticulum stress as manifested, for instance, in rhodopsin mislocalization. Hence, future applications of HMOX1 or its regulated targets in gene therapy approaches should carefully consider expression levels in order to avoid potentially devastating effects. [ABSTRACT FROM AUTHOR]