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MiR-485 inhibits histone deacetylase HDAC5, HIF1α and PFKFB3 expression to alleviate epilepsy in cellular and rodent models.

ABSTRACT: We investigated the role of microRNA (miR)-485 and its downstream signaling molecules on mediating epilepsy in cellular and rat models. We established a cellular epilepsy model by exposing hippocampal neurons to magnesium and a rat model by treating ICR mice with lithium chloride (127 mg/kg) and pilocarpine (30 mg/kg). We confirmed that miR-485 could bind and inhibit histone deacetylase 5 (HDAC5) and then measured expression of miR-485 and in mice and cells. Cells were transfected with overexpression or knockdown of miR-485, HDAC5, hypoxia-inducible factor-1alpha (HIF1α), or 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 enzyme (PFKFB3) to verify their roles in apoptosis, oxidative stress, and inflammation in epileptic hippocampal neurons. Binding relationship between miR-485, HDAC5, HIF1α, and PFKFB3 was verified. Oxidative stress and inflammation marker levels in epilepsy model mice were assessed. miR-485 was downregulated and HDAC5 was upregulated in cell and animal model of epilepsy. Seizure, neuronal apoptosis, oxidative stress (increased SOD and GSH-Px expression and decreased MDA and 8-OHdG expression) and inflammation (reduced IL-1β, TNF-α, and IL-6 expression) were reduced by miR-485 in epileptic cells. HIF1α and PFKFB3 expression was reduced by HDAC5 knockdown in cells, which was recapitulated in vivo. Thus, miR-485 alleviates neuronal damage and epilepsy by inhibiting HDAC5, HIF1α, and PFKFB3.

SUBMITTER: Pan W

PROVIDER: S-EPMC8202868 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Class IIa histone deacetylase (HDAC) isoforms such as HDAC5 are critical signal-responsive repressors of maladaptive cardiomyocyte hypertrophy, through nuclear interactions with transcription factors including myocyte enhancer factor-2. β-Adrenoceptor (β-AR) stimulation, a signal of fundamental importance in regulating cardiac function, has been proposed to induce both phosphorylation-independent nuclear export and phosphorylation-dependent nuclear accumulation of cardiomyocyte HDAC5. The relative importance of phosphorylation at Ser259/Ser498 versus Ser279 in HDAC5 regulation is also controversial. We aimed to determine the impact of β-AR stimulation on the phosphorylation, localization, and function of cardiomyocyte HDAC5 and delineate underlying molecular mechanisms. A novel 3-dimensional confocal microscopy method that objectively quantifies the whole-cell nuclear/cytoplasmic distribution of green fluorescent protein tagged HDAC5 revealed the β-AR agonist isoproterenol to induce β1-AR-mediated and protein kinase A-dependent HDAC5 nuclear accumulation in adult rat cardiomyocytes, which was accompanied by dephosphorylation at Ser259/279/498. Mutation of Ser259/Ser498 to Ala promoted HDAC5 nuclear accumulation and myocyte enhancer factor-2 inhibition, whereas Ser279 ablation had no such effect and did not block isoproterenol-induced nuclear accumulation. Inhibition of the Ser/Thr phosphatase PP2A blocked isoproterenol-induced HDAC5 dephosphorylation. Co-immunoprecipitation revealed a specific interaction of HDAC5 with the PP2A targeting subunit B55α, as well as catalytic and scaffolding subunits, which increased >3-fold with isoproterenol. Knockdown of B55α in neonatal cardiomyocytes attenuated isoproterenol-induced HDAC5 dephosphorylation. β-AR stimulation induces HDAC5 nuclear accumulation in cardiomyocytes by a mechanism that is protein kinase A-dependent but requires B55α-PP2A-mediated dephosphorylation of Ser259/Ser498 rather than protein kinase A-mediated phosphorylation of Ser279.

Project description:We have previously demonstrated that crosstalk between lysine-specific demethylase 1 (LSD1) and histone deacetylases (HDACs) facilitates breast cancer proliferation. However, the underlying mechanisms are largely unknown. Here, we report that expression of HDAC5 and LSD1 proteins were positively correlated in human breast cancer cell lines and tissue specimens of primary breast tumors. Protein expression of HDAC5 and LSD1 was significantly increased in primary breast cancer specimens in comparison with matched-normal adjacent tissues. Using HDAC5 deletion mutants and co-immunoprecipitation studies, we showed that HDAC5 physically interacted with the LSD1 complex through its domain containing nuclear localization sequence and phosphorylation sites. Although the in vitro acetylation assays revealed that HDAC5 decreased LSD1 protein acetylation, small interfering RNA (siRNA)-mediated HDAC5 knockdown did not alter the acetylation level of LSD1 in MDA-MB-231 cells. Overexpression of HDAC5 stabilized LSD1 protein and decreased the nuclear level of H3K4me1/me2 in MDA-MB-231 cells, whereas loss of HDAC5 by siRNA diminished LSD1 protein stability and demethylation activity. We further demonstrated that HDAC5 promoted the protein stability of USP28, a bona fide deubiquitinase of LSD1. Overexpression of USP28 largely reversed HDAC5-KD-induced LSD1 protein degradation, suggesting a role of HDAC5 as a positive regulator of LSD1 through upregulation of USP28 protein. Depletion of HDAC5 by shRNA hindered cellular proliferation, induced G1 cell cycle arrest, and attenuated migration and colony formation of breast cancer cells. A rescue study showed that increased growth of MDA-MB-231 cells by HDAC5 overexpression was reversed by concurrent LSD1 depletion, indicating that tumor-promoting activity of HDAC5 is an LSD1 dependent function. Moreover, overexpression of HDAC5 accelerated cellular proliferation and promoted acridine mutagen ICR191-induced transformation of MCF10A cells. Taken together, these results suggest that HDAC5 is critical in regulating LSD1 protein stability through post-translational modification, and the HDAC5-LSD1 axis has an important role in promoting breast cancer development and progression.

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MiR-485 inhibits histone deacetylase HDAC5, HIF1α and PFKFB3 expression to alleviate epilepsy in cellular and rodent models.

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