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Increased glycolysis in the lung vasculature has been connected to the development of pulmonary hypertension (PH). We therefore investigated whether glycolytic regulator 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase (PFKFB3)-mediated endothelial glycolysis plays a critical role in the development of PH. Heterozygous global deficiency of Pfkfb3 protected mice from developing hypoxia-induced PH, and administration of the PFKFB3 inhibitor 3PO almost completely prevented PH in rats treated with Sugen 5416/hypoxia, indicating a causative role of PFKFB3 in the development of PH. Immunostaining of lung sections and Western blot with isolated lung endothelial cells showed a dramatic increase in PFKFB3 expression and activity in pulmonary endothelial cells of rodents and humans with PH. We generated mice that were constitutively or inducibly deficient in endothelial Pfkfb3 and found that these mice were incapable of developing PH or showed slowed PH progression. Compared with control mice, endothelial Pfkfb3-knockout mice exhibited less severity of vascular smooth muscle cell proliferation, endothelial inflammation, and leukocyte recruitment in the lungs. In the absence of PFKFB3, lung endothelial cells from rodents and humans with PH produced lower levels of growth factors (such as PDGFB and FGF2) and proinflammatory factors (such as CXCL12 and IL1β). This is mechanistically linked to decreased levels of HIF2A in lung ECs following PFKFB3 knockdown. Taken together, these results suggest that targeting PFKFB3 is a promising strategy for the treatment of PH. [ABSTRACT FROM AUTHOR]

Histone methyltransferase G9a has critical roles in promoting cancer-cell growth and gene suppression, but whether it is also associated with the DNA damage response is rarely studied. Here, we report that loss of G9a impairs DNA damage repair and enhances the sensitivity of cancer cells to radiation and chemotherapeutics. In response to DNA double-strand breaks (DSBs), G9a is phosphorylated at serine 211 by casein kinase 2 (CK2) and recruited to chromatin. The chromatin-enriched G9a can then directly interact with replication protein A (RPA) and promote loading of the RPA and Rad51 recombinase to DSBs. This mechanism facilitates homologous recombination (HR) and cell survival. We confirmed the interaction between RPA and G9a to be critical for RPA foci formation and HR upon DNA damage. Collectively, our findings demonstrate a regulatory pathway based on CK2-G9a-RPA that permits HR in cancer cells and provide further rationale for the use of G9a inhibitors as a cancer therapeutic. [ABSTRACT FROM AUTHOR]

The function of α-synuclein (α-syn) has been long debated, and two seemingly divergent views have emerged. In one, α-syn binds to VAMP2, acting as a SNARE chaperone--but with no effect on neurotransmission--while another posits that α-syn attenuates neurotransmitter release by restricting synaptic vesicle mobilization and recycling. Here, we show that α-syn-VAMP2 interactions are necessary for α-syn-induced synaptic attenuation. Our data connect divergent views and suggest a unified model of α-syn function. [ABSTRACT FROM AUTHOR]

In mammalian cells, tumor suppressor p53 plays critical roles in the regulation of glucose metabolism, including glycolysis and oxidative phosphorylation, but whether and how p53 also regulates gluconeogenesis is less clear. Here, we report that p53 efficiently down-regulates the expression of phosphoenolpyruvate carboxy-kinase (PCK1) and glucose-6-phosphatase (G6PC), which encode rate-limiting enzymes in gluconeogenesis. Cell-based assays demonstrate the p53-dependent nuclear exclusion of forkhead box protein 01 (FoxOI), a key transcription factor that mediates activation of PCK1 and G6PC, with consequent alleviation of Fox01-dependent gluconeogenesis. Further mechanistic studies show that p53 directly activates expression of the NAD+-dependent histone deacetylase sirtuin 6 (SIRT6), whose interaction with Fox01 leads to Fox01 deacetylation and export to the cytoplasm. In support of these observations, p53-mediated Fox01 nuclear exclusion, down-regulation of PCK1 and G6PC expression, and regulation of glucose levels were confirmed in C57BL/J6 mice and in liver-specific Sirt6 conditional knockout mice. Our results provide insights into mechanisms of metabolism-related p53 functions that may be relevant to tumor suppression. [ABSTRACT FROM AUTHOR]

Suppressor of variegation 3-9 homolog 1 (SUV39H1), a histone methyltransferase, catalyzes histone 3 lysine 9 trimethylation and is involved in heterochromatin organization and genome stability. However, the mechanism for regulation of the enzymatic activity of SUV39H1 in cancer cells is not yet well known. In this study, we identified SET domain-containing protein 7 (SET7/9), a protein methyltransferase, as a unique regulator of SUV39H1 activity. In response to treatment with adriamycin, a DNA damage inducer, SET7/9 interacted with SUV39H1 in vivo, and a GST pull-down assay confirmed that the chromodomain-containing region of SUV39H1 bound to SET7/9. Western blot using antibodies specific for antimethylated SUV39H1 and mass spectrometry demonstrated that SUV39H1 was specifically methylated at lysines 105 and 123 by SET7/9. Although the half-life and localization of methylated SUV39H1 were not noticeably changed, the methyltransferase activity of SUV39H1 was dramatically down-regulated when SUV39H1 was methylated by SET7/ 9. Consequently, H3K9 trimethylation in the heterochromatin decreased significantly, which, in turn, led to a significant increase in the expression of satellite 2 (Sat2) and α-satellite (α-Sat), indicators of heterochromatin relaxation. Furthermore, a micrococcal nuclease sensitivity assay and an immunofluorescence assay demonstrated that methylation of SUV39H1 facilitated genome instability and ultimately inhibited cell proliferation. Together, our data reveal a unique interplay between SET7/9 and SUV39H1-two histone methyltransferases-that results in heterochromatin relaxation and genome instability in response to DNA damage in cancer cells. [ABSTRACT FROM AUTHOR]

Maternal separation (MS), which can lead to hypothalamic pituitary adrenal axis dysfunction and behavioral abnormalities in rhesus monkeys, is frequently used to model early adversity. Whether this deleterious effect on monkeys is reversible by later experience is unknown. In this study, we assessed the basal hair cortisol in rhesus monkeys after 1.5 and 3 y of normal social life following an early separation. These results showed that peer-reared monkeys had significantly lower basal hair cortisol levels than the mother-reared monkeys at both years examined. The plasma cortisol was assessed in the monkeys after 1.5 y of normal social life, and the results indicated that the peak in the peer-reared cortisol response to acute stressors was substantially delayed. In addition, after 3 y of normal social life, abnormal behavioral patterns were identified in the peer-reared monkeys. They showed decreases in locomotion and initiated sitting together, as well as increases in stereotypical behaviors compared with the mother-reared monkeys. These results demonstrate that the deleterious effects of MS on rhesus monkeys cannot be compensated by a later normal social life, suggesting that the effects of MS are long-lasting and that the maternal-separated rhesus monkeys are a good animal model to study early adversity and to investigate the development of psychiatric disorders induced by exposure to early adversity. [ABSTRACT FROM AUTHOR]

Numerous studies indicate that Sirtuin 1 (SIRT1), a mammalian nicotinamide adenine dinucleotide (NAD+)-dependent histone deacetylase (HDAC), plays a crucial role in p53-mediated stress responses by deacetylating p53. Nevertheless, the acetylation levels of p53 are dramatically increased upon DNA damage, and it is not well understood how the SIRT1-p53 interaction is regulated during the stress responses. Here, we identified Set7/9 as a unique regulator of SIRT1. SIRT1 interacts with Set7/9 both in vitro and in vivo. In response to DNA damage in human cells, the interaction between Set7/9 and SIRT1 is significantly enhanced and coincident with an increase in p53 acetylation levels. Importantly, the interaction of SIRT1 and p53 is strongly suppressed in the presence of Set7/9. Consequently, SIRT1-mediated deacetylation of p53 is abrogated by Set7/9, and p53-mediated transactivation is increased during the DNA damage response. Of note, whereas SIRT1 can be methylated at multiple sites within its N terminus by Set7/9, a methylation-defective mutant of SIRT1 still retains its ability to inhibit p53 activity. Taken together, our results reveal that Set7/9 is a critical regulator of the SIRT1-p53 interaction and suggest that Set7/9 can modulate p53 function indirectly in addition to acting through a methylation-dependent mechanism. [ABSTRACT FROM AUTHOR]