Elucidating the Contribution of the BAF A12T Mutation to Cellular Mechanisms of Premature Aging

The nuclear envelope is a dynamic structure and organizing hub for cell structure and function. The underlying nuclear lamina is composed of a thick network of intermediate filaments, Lamin A/C and B, and inner nuclear membrane-associated proteins such as Lap2, Emerin and Man1. BAF, a central organizing protein, is a small, dimeric, essential protein that binds to dsDNA, proteins of the inner nuclear membrane and Lamin A. Cells with perturbations of the nuclear lamina display dysmorphic nuclei which have the potential to lead to altered gene expression, increased DNA damage events, and premature cellular senescence. In this study, we address a single amino acid substitution from Alanine 12 to Threonine (A12T) in BAF that gives rise to a nuclear morphology defect. At the clinical level, patients who inherit a homozygous BAF A12T mutation have a rare premature aging syndrome called Nestor Guillermo Progeria Syndrome (NGPS) with symptoms such as osteolysis, adipolysis and craniofacial defects that present at approximately two years of age and progress rapidly, ultimately shortening patient lifespan significantly. Tissues of mesenchymal origin appear to be most severely impacted in this disease, leading to the hypothesis that mesenchymal stem cells are preferentially affected. To address the cellular and organismal pathology of NGPS, we used CRISPR/Cas9 technology to establish BAFA12T/BAFA12T human induced pluripotent stem cells (hiPSCs). A12T BAF iPSCs appeared normal, however when differentiated to induced mesenchymal stem cells (iMSCs), A12T BAF cells exhibited a marked increase in cellular senescence and an elevated cellular stress response. The mechanism of A12T BAF’s contribution of this cell-type specific pathology was not well understood. Our data indicate that A12T BAF ‘s binding to Lamin A is greatly diminished, thereby weakening the Lamin A network of the nuclear lamina and the structural integrity of the nuclear envelope. Mechanosensitive iMSCs are unable to withstand this weakened network and the nuclei appear to rupture, thereby causing cellular stress and ultimately, senescence. Our results indicate that the A12T BAF mutation is necessary but not sufficient to cause premature cellular aging. All cell types expressing BAFA12T/BAFA12T have dysmorphic nuclei, however cells derived from BAFA12T/BAFA12T mice are viable and primary dermal fibroblasts are not senescent, nor do they demonstrate elevated levels of DNA damage. Furthermore, A12T BAF mice do not have shortened lifespans nor do they have defects in mesenchymal derived tissue. These data highlight the nuances of this mutation’s effect both at the cellular and organismal level and elucidates a new role for BAF at the nuclear envelope as a key player in cell mechanosensitivity and the network of the nucleoskeleton and cytoskeleton

IL-1 stimulates ceramide accumulation without inducing apoptosis in intestinal epithelial cells.

BACKGROUND: In inflammatory bowel disease (IBD), cytokine levels (such as interleukin-1 (IL-1)) are elevated. We have shown previously that IL-1 activates phospholipid signaling pathways in intestinal epithelial cells (EEC), leading to increased ceramide levels. AIM: To determine whether ceramide induces apoptosis in IEC. METHODS: Apoptosis was evaluated by annexin-V binding or Hoechst nuclear staining. Levels of bcl-2, bcl-x, bax, p53 and p21 were determined by Western blotting, and celi cycle analysis was determined by flow cytometry. RESULTS: IL-1 increased ceramide accumulation in a time-dependent and concentration-dependent manner with a peak response at 4 h, with [IL-1] = 30 ng/ml. Neither IL-1 nor ceramide induced apoptosis in EEC, but they increased bcl-2 levels and decreased bax and p21 levels without affecting bcl-x and p53 levels. They also caused a slight but significant increase in the G2/M phase. These data suggest a role for ceramide in IBD and suggest a possible mechanism for the enhanced tumorigenic activity in IBD patients

Connexins: a myriad of functions extending beyond assembly of gap junction channels

Connexins constitute a large family of trans-membrane proteins that allow intercellular communication and the transfer of ions and small signaling molecules between cells. Recent studies have revealed complex translational and post-translational mechanisms that regulate connexin synthesis, maturation, membrane transport and degradation that in turn modulate gap junction intercellular communication. With the growing myriad of connexin interacting proteins, including cytoskeletal elements, junctional proteins, and enzymes, gap junctions are now perceived, not only as channels between neighboring cells, but as signaling complexes that regulate cell function and transformation. Connexins have also been shown to form functional hemichannels and have roles altogether independent of channel functions, where they exert their effects on proliferation and other aspects of life and death of the cell through mostly-undefined mechanisms. This review provides an updated overview of current knowledge of connexins and their interacting proteins, and it describes connexin modulation in disease and tumorigenesis

Pomegranate Juice Prevents the Formation of Lung Nodules Secondary to Chronic Cigarette Smoke Exposure in an Animal Model

Background. Cigarette smoke (CS) induces an oxidative stress, DNA damage, and lung cancer. Pomegranate juice (PJ) possess potent antioxidant activity attributed to its polyphenols. We investigated whether PJ supplementation would prevent the formation of lung nodules, attenuate mitotic activity, and reduce hypoxia-inducible factor-1α (HIF-1α) expression secondary to CS exposure in an animal model. Methods. Mice were divided into: Control group, CS group, CS + PJ group, and PJ-only group. CS and CS + PJ were exposed to CS, 5 days per week, for a total of 5 months. Animals were then housed for additional four months. CS + PJ and PJ groups received PJ throughout the experiment period while others received placebo. At the end of the experiment, the incidence of lung nodules was assessed by (1) histological analysis, (2) mitotic activity [measurement of PHH3 antibodies], and (3) measurement of HIF-1α expression. Results. The incidence of lung nodules was significantly increased in CS. CS exposure significantly increased PHH3 and HIF-1α expression. PJ supplementation attenuated the formation of lung nodules and reduced PHH3 and HIF-1α expression. Conclusion. PJ supplementation significantly decreased the incidence of lung cancer, secondary to CS, prevented the formation of lung nodules, and reduced mitotic activity and HIF-1α expression in an animal model

The quinoxaline di-N-oxide DCQ blocks breast cancer metastasis in vitro and in vivo by targeting the hypoxia inducible factor-1 pathway

BACKGROUND: Although tumor hypoxia poses challenges against conventional cancer treatments, it provides a therapeutic target for hypoxia-activated drugs. Here, we studied the effect of the hypoxia-activated synthetic quinoxaline di-N-oxide DCQ against breast cancer metastasis and identified the underlying mechanisms. METHODS: The human breast cancer cell lines MCF-7 (p53 wildtype) and MDA-MB-231 (p53 mutant) were treated with DCQ under normoxia or hypoxia. Drug toxicity on non-cancerous MCF-10A breast cells was also determined. In vitro cellular responses were investigated by flow cytometry, transfection, western blotting, ELISA and migration assays. The anti-metastatic effect of DCQ was validated in the MDA-MB-231 xenograft mouse model. RESULTS: DCQ selectively induced apoptosis in both human breast cancer cells preferentially under hypoxia without affecting the viability of non-cancerous MCF-10A. Cancer cell death was associated with an increase in reactive oxygen species (ROS) independently of p53 and was inhibited by antioxidants. DCQ-induced ROS was associated with DNA damage, the downregulation of hypoxia inducible factor-1 alpha (HIF-1α), and inhibition of vascular endothelial growth factor (VEGF) secretion. In MCF-7, HIF-1α inhibition was partially via p53-activation and was accompanied by a decrease in p-mTOR protein, suggesting interference with HIF-1α translation. In MDA-MB-231, DCQ reduced HIF-1α through proteasomal-dependent degradation mechanisms. HIF-1α inhibition by DCQ blocked VEGF secretion and invasion in MCF-7 and led to the inhibition of TWIST in MDA-MB-231. Consistently, DCQ exhibited robust antitumor activity in MDA-MB-231 breast cancer mouse xenografts, enhanced animal survival, and reduced metastatic dissemination to lungs and liver. CONCLUSION: DCQ is the first hypoxia-activated drug showing anti-metastatic effects against breast cancer, suggesting its potential use for breast cancer therapy

Tax ubiquitylation and SUMOylation control the dynamic shuttling of Tax and NEMO between Ubc9 nuclear bodies and the centrosome

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The regulation of RhoA at focal adhesions by StarD13 is important for astrocytoma cell motility

Malignant astrocytomas are highly invasive into adjacent and distant regions of the normal brain. Rho GTPases are small monomeric G proteins that play important roles in cytoskeleton rearrangement, cell motility, and tumor invasion. In the present study, we show that the knock down of StarD13, a GTPase activating protein (GAP) for RhoA and Cdc42, inhibits astrocytoma cell migration through modulating focal adhesion dynamics and cell adhesion. This effect is mediated by the resulting constitutive activation of RhoA and the subsequent indirect inhibition of Rac. Using Total Internal Reflection Fluorescence (TIRF)-based Förster Resonance Energy Transfer (FRET), we show that RhoA activity localizes with focal adhesions at the basal surface of astrocytoma cells. Moreover, the knock down of StarD13 inhibits the cycling of RhoA activation at the rear edge of cells, which makes them defective in retracting their tail. This study highlights the importance of the regulation of RhoA activity in focal adhesions of astrocytoma cells and establishes StarD13 as a GAP playing a major role in this process