HDAC6 preserves BNIP3 expression and mitochondrial integrity by deacetylating p53 at lysine 320.

HDAC6 has been reported as a deacetylase of p53 at multiple lysine residues, associated with the canonical functions of p53, such as apoptosis and tumor suppression. We have previously reported that p53 acetylation at the lysine 320 site accumulates due to the genetic ablation of HDAC6 in mice liver. However, the biological processes affected by K320 acetylation of p53 are yet to be elucidated. In this study, we demonstrate that K320 acetylation of p53 is regulated by HDAC6 deacetylase activity. HDAC6 knockout mouse brains exhibit a significant accumulation of K320 acetylated p53 compared to other tissues. The level of K320 acetylation of p53 inversely correlates with the level of BNIP3, a direct target of p53 and essential for mitophagy. Notably, overexpressing the deacetylation mimic K320R mutant p53 restored BNIP3 expression in HDAC6 knockout MEFs. Furthermore, we observed that neurons are particularly susceptible to the genetic ablation of HDAC6, impacting BNIP3 expression, which inversely correlates with the accumulation of abnormal mitochondria characterized by swollen cristae. Our findings suggest that HDAC6 plays a crucial role in maintaining BNIP3 expression by deacetylating p53 at the K320 site, which is linked to the structural integrity of mitochondria.

SIRT1 suppresses in vitro decidualization of human endometrial stromal cells through the downregulation of forkhead box O1 expression.

SIRT1 regulates survival, DNA repair, and metabolism in human cells and has pleiotropic effects on age-related diseases through either deacetylating target proteins or inhibiting gene transcription. Forkhead box O1 (FOXO1) is one of the most important transcription factors during decidualization. Prolactin (PRL) and insulin-like growth factor-binding protein 1 (IGFBP1) are well-known FOXO1-dependent genes in decidualizing cells. To determine whether SIRT1 plays a role in decidualization, we investigated morphological changes in cells following artificially stimulated decidualization and expression levels of PRL, IGFBP1, and FOXO1 in the immortalized non-neoplastic human endometrial stromal cell line T HESCs. SIRT1 expression decreased in the decidualization condition and SIRT1 inhibited morphological changes caused by decidualization of T HESCs. SIRT1 suppressed PRL, IGFBP1, and FOXO1 expression; inhibited FOXO1, PRL, and IGFBP1 promoter activity; and decreased histone protein acetylation of the FOXO1 promoter. We found that FOXO1 expression increased in the secretory phase compared with the proliferative phase, whereas SIRT1 expression decreased in the secretory phase in the human endometrium. We also revealed that SIRT1 may inhibit embryo implantation according to the blastocyst-like spheroid implantation assay. Collectively, these results indicate that SIRT1 suppresses decidualization of human endometrial stromal cells by inhibiting FOXO1 expression.

APOE4-carrying human astrocytes oversupply cholesterol to promote neuronal lipid raft expansion and Aß generation.

The ε4 allele of APOE-encoding apolipoprotein (ApoE) is one of the strongest genetic risk factors for Alzheimer's disease (AD). One of the overarching questions is whether and how this astrocyte-enriched risk factor initiates AD-associated pathology in neurons such as amyloid-ß (Aß) accumulation. Here, we generate neurons and astrocytes from isogenic human induced pluripotent stem cells (hiPSCs) carrying either APOE ε3 or APOE ε4 allele and investigate the effect of astrocytic ApoE4 on neuronal Aß production. Secretory factors in conditioned media from ApoE4 astrocytes significantly increased amyloid precursor protein (APP) levels and Aß secretion in neurons. We further found that increased cholesterol secretion from ApoE4 astrocytes was necessary and sufficient to induce the formation of lipid rafts that potentially provide a physical platform for APP localization and facilitate its processing. Our study reveals the contribution of ApoE4 astrocytes to amyloidosis in neurons by expanding lipid rafts and facilitating Aß production through an oversupply of cholesterol.

A Synthetic CPP33-Conjugated HOXA9 Active Domain Peptide Inhibits Invasion Ability of Non-Small Lung Cancer Cells.

Homeobox A9 (HOXA9) expression is associated with the aggressive growth of cancer cells and poor prognosis in lung cancer. Previously, we showed that HOXA9 can serve as a potential therapeutic target for the treatment of metastatic non-small cell lung cancer (NSCLC). In the present study, we have carried out additional studies toward the development of a peptide-based therapeutic agent. Vectors expressing partial DNA fragments of HOXA9 were used to identify a unique domain involved in the inhibition of NSCLC cell invasion. Next, we performed in vitro invasion assays and examined the expression of EMT-related genes in transfected NSCLC cells. The C-terminal fragment (HOXA9-C) of HOXA9 inhibited cell invasion and led to upregulation of CDH1 and downregulation of SNAI2 in A549 and NCI-H1299 cells. Reduced SNAI2 expression was consistent with the decreased binding of transcription factor NF-kB to the SNAI2 promoter region in HOXA9-C overexpressing cells. Based on the above results, we synthesized a cell-permeable peptide, CPP33-HADP (HOXA9 active domain peptide), for lung-specific delivery and tested its therapeutic efficiency. CPP33-HADP effectively reduced the invasion ability of NSCLC cells in both in vitro and in vivo mouse models. Our results suggest that CPP33-HADP has significant potential for therapeutic applications in metastatic NSCLC.

Decursinol from Angelica gigas Nakai enhances endometrial receptivity during implantation.

BACKGROUND: Embryo implantation is essential for a successful pregnancy, and an elaborate synchronization between the receptive endometrium and trophoblast is required to achieve this implantation. To increase 'endometrial receptivity', the endometrium undergoes transformation processes including responses of adhesion molecules and cellular and molecular cell to cell communication. Many natural substances from traditional herbs have been studied to aid in the achievement of successful implantation. In this study, we investigated positive effects on embryonic implantation with decursinol that is a major compound extracted from Angelica gigas Nakai known to be associated with promotion of healthy pregnancy in the traditional Korean herbal medicine. METHODS: Expression of cell adhesion molecules after treatment of endometrial epithelial cells by decursinol (40 or 80 µM) was determined using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blot analysis. The alteration of endometrial receptivity by decursinol (40 or 80 µM) was identified with the in vitro implantation model between Ishikawa cells and JAr cell spheroids (diameter, 143 ± 16 µm). Exosomes secreted from Ishikawa cells after treatment of 80 µM decursinol or dimethyl sulfoxide (DMSO) as the vehicle were investigated with invasion of JAr cells and attachment of JAr spheroids to Ishikawa cells. RESULTS: Decursinol significantly (P < 0.05) increased the expression of important endometrial adhesion molecules such as integrin ß1, ß3, ß5 and L-selectin mRNAs and integrin ß5 and L-selectin in protein. The adhesion rate of JAr spheroids to decursinol-treated Ishikawa cells also increased significantly which was 2.4-fold higher than that of the control (P < 0.05). Furthermore, decursinol induced an increase in the release of exosomes from Ishikawa cells and decursinol-induced exosomes showed autocrine (to Ishikawa cells) and paracrine (to JAr cells) positive effects on our implantation model. CONCLUSION: These results propose that decursinol could serve as a new and alternative solution for patients who are infertile.

HDAC6 regulates thermogenesis of brown adipocytes through activating PKA to induce UCP1 expression.

Mitochondrial uncoupling protein 1 (UCP1) is responsible for nonshivering thermogenesis in brown adipose tissue (BAT). UCP1 increases the conductance of the inner mitochondrial membrane (IMM) for protons to make BAT mitochondria generate heat rather than ATP. HDAC6 is a cytosolic deacetylase for non-histone substrates to regulate various cellular processes, including mitochondrial quality control and dynamics. Here, we showed that the body temperature of HDAC6 knockout mice is slightly decreased in normal hosing condition. Interestingly, UCP1 was downregulated in BAT of HDAC6 knockout mice, which extensively linked mitochondrial thermogenesis. Mechanistically, we showed that cAMP-PKA signaling plays a key role in HDAC6-dependent UCP1 expression. Notably, the size of brown adipocytes and lipid droplets in HDAC6 knockout BAT is increased. Taken together, our findings suggested that HDAC6 contributes to mitochondrial thermogenesis in BAT by increasing UCP1 expression through cAMP-PKA signaling pathway.

HDAC6 deficiency induces apoptosis in mesenchymal stem cells through p53 K120 acetylation.

The acetylation of p53 is critical in modulating its pro-apoptotic roles. However, its regulatory mechanism and physiological significance are unclear. Here, we show HDAC6 negatively regulates pro-apoptotic acetylation of p53 at lysine residue 120 (K120) in mesenchymal stem cells (MSCs). The loss of HDAC6 expression in MSCs increases K120 acetylation of p53, which is successfully reversed by the wild-type but not by catalytically dead HDAC6. Deletion of HDAC6 induces caspase-dependent apoptosis by promoting transactivation of Bax and suppression of Bcl-2. Moreover, HDAC6 deficiency leads to mitochondrial dysfunction characterized by aberrant reactive oxygen species production and defective oxidative phosphorylation, which is reversed by ectopic expression of wild-type or acetylation mimetic p53. This study demonstrates that HDAC6 is a critical regulator of a pro-apoptotic p53 K120 acetylation and mitochondrial function in MSCs, suggesting that the modulation of HDAC6 activity could be a novel approach to improve MSC- based therapies.

RAP80 binds p32 to preserve the functional integrity of mitochondria.

RAP80, a member of the BRCA1-A complex, is a well-known crucial regulator of cell cycle checkpoint and DNA damage repair in the nucleus. However, it is still unclear whether Rap80 localizes to another region outside the nucleus and plays different roles with its partners. Here, we found mitochondrial p32 as a novel binding partner of RAP80 by using yeast two-hybrid screening. RAP80 directly binds the internal region of p32 through its arginine rich C-terminal domain. Based on the interaction, we showed that a subset of RAP80 localizes to mitochondria where p32 exists. Loss of function study revealed that RAP80 deficiency reduces the protein level of p32 and p32 dependent mitochondrial translating proteins such as Rieske and COX1. As a result, mitochondrial membrane potential and oxygen consumption are reduced in RAP80 knockdown cells, indicating mitochondrial dysfunction. Our study identifies a novel interaction between RAP80 and p32, which is important for preserving intact mitochondrial function.