MEF Cells Research Articles

AMPK Reprograms Gene Expression and Promotes Adaptation/Survival in Response to Metabolic Stress through Binding to a Chromatin-Associated Transcription Complex in Acute Lymphoblastic Leukemia

Survival rates for relapsed/refractory acute lymphoblastic leukemia (ALL), the most common cancer in children and adolescents, remain dismal. We and others have reported that ALL cells are vulnerable to conditions inducing energy/ER-stress mediated by AMP-activated protein kinase (AMPK) activation. AMPK has been reported to interact with chromatin-associated proteins (e.g., histone H2B) in MEF cells to epigenetically regulate gene expression in response to environmental/cellular stress (Bungard et al. Science, 2010; 329:1201). To identify genome-wide genes regulated by direct association of AMPK to chromatin in response to energy/metabolic stress, we first constructed Bp-ALL (NALM6, REH) and T-ALL (CCRF-CEM, KE-37) stable cell lines expressing HA-AMPKα1 or HA-AMPKα2. Next, using HA and RNA pol II antibodies, we performed ChIP-seq assays in CCRF-CEM/HA-AMPKα2 (CN2) grown in glucose-free RPMI for 24 h. ChIP-seq differentially identified 171 candidate genes in CN2 treated with no-glucose vs. 431 genes in untreated controls. Data analysis using the Encode and ChEA database identified the TATA-Box Binding Protein Associated Factor, CCAAT Enhancer Binding Protein Delta (CEBPD), the negative elongation factor complex member E (NELFE), and the Promyelocytic leukemia protein (PML) among highly ranked transcription factors (TFs) may associated with AMPKα2 on chromatin. To correlate the level of gene mRNA expression and recruitment of AMPKα2 to chromatin gene loci regulated in response to energy/metabolic stress, we performed RNA-seq assays in CN2 cells treated with or without glucose deprivation for 24 h. RNA-seq data analysis indicated that of the 3497 genes altered by AMPK activation, two thirds were downregulated whereas the remaining were upregulated. Kyoto Encyclopedia of Genes and Genomes gene set and BioPlanet 2019 gene set analysis identified metabolic pathways, DNA replication/metabolism, and cell cycle as the main biological processes altered in CN2 cells in response to metabolic stress. Among downregulated genes in response to metabolic stress, we uncovered a cluster of histone genes. To confirm and validate our data, we used RT-qPCR and ChIP-qPCR assays on selected histone gene candidates (H1-2/ HIST1H1C, H1-3/HIST1H1D, H4C4/HIST1H4D) which exhibited both decreased recruitment of HA-AMPKα2 to chromatin and mRNA downregulation in response to metabolic stress. Further ChIP-qPCR assays using an AMPKα2 antibody confirmed these data in KASUMI-2 cells (Bp-ALL). Similar data were also observed in CN2 cells treated with AICAR, another AMPK activator. Additional experiments were conducted using the allosteric AMPK activators compound 991 and PF-06409577. Using Co-IP experiments, we uncovered that AMPKα2 interacted with putative members of an AMPK/chromatin-associated transcription factor complex which included the TATA-Box Binding Protein Associated Factor (TAF), integrator (INT), and RNA polymerase II. To investigate the role of AMPK kinase activity on AMPKα2-associated chromatin regulated gene targets, we determined the effect of genetic constructs encoding a constitutively active (CA) form of AMPKα2 on histone gene mRNA expression in CCRF-CEM and MEF AMPKα1/AMPKα2 double knockout (DKO) cells, and found that in both cell types the expression of the full-length CA-AMPKα2 (T172D) lead to decreased mRNA expression of the histone genes examined, suggesting AMPK kinase activity is required to regulate histone genes in response to energy/metabolic stress. In conclusion, our data show that in response to metabolic stress, AMPK binds directly to a multi-protein complex on chromatin to reprogram gene expression to promote cellular adaptation/survival in ALL. Further elucidation of AMPK's interactions with members of the putative AMPK/chromatin-associated transcription complex may lead to unique opportunities for epigenetic-based therapeutic interventions and combination strategies to exploit synthetic lethality in relapse/refractory ALL and other hematological malignancies. DisclosuresNo relevant conflicts of interest to declare.

NudCL2 is an autophagy receptor that mediates selective autophagic degradation of CP110 at mother centrioles to promote ciliogenesis

Primary cilia extending from mother centrioles are essential for vertebrate development and homeostasis maintenance. Centriolar coiled-coil protein 110 (CP110) has been reported to suppress ciliogenesis initiation by capping the distal ends of mother centrioles. However, the mechanism underlying the specific degradation of mother centriole-capping CP110 to promote cilia initiation remains unknown. Here, we find that autophagy is crucial for CP110 degradation at mother centrioles after serum starvation in MEF cells. We further identify NudC-like protein 2 (NudCL2) as a novel selective autophagy receptor at mother centrioles, which contains an LC3-interacting region (LIR) motif mediating the association of CP110 and the autophagosome marker LC3. Knockout of NudCL2 induces defects in the removal of CP110 from mother centrioles and ciliogenesis, which are rescued by wild-type NudCL2 but not its LIR motif mutant. Knockdown of CP110 significantly attenuates ciliogenesis defects in NudCL2-deficient cells. In addition, NudCL2 morphants exhibit ciliation-related phenotypes in zebrafish, which are reversed by wild-type NudCL2, but not its LIR motif mutant. Importantly, CP110 depletion significantly reverses these ciliary phenotypes in NudCL2 morphants. Taken together, our data suggest that NudCL2 functions as an autophagy receptor mediating the selective degradation of mother centriole-capping CP110 to promote ciliogenesis, which is indispensable for embryo development in vertebrates.

Antioxidant, α-Glucosidase Inhibitory, and Anti-Inflammatory Activities and Cell Toxicity of Waxy and Normal Wheat Sprouts at Various Germination Time

Germination is an effective process to improve the bioactivities including antioxidant and hypoglycemic activities of grains, but its effect on waxy wheat has not yet been actively studied. This study, therefore, examined the effect of germination time on the activities of Korean waxy and normal wheat sprouts. The total phenolic and flavonoid contents, and antioxidant activities of the waxy and normal wheat sprouts increased with germination time. Flavonoid content and antioxidant activity were higher in waxy wheat sprouts than in normal ones, but the total phenolic content and α-glucosidase inhibitory activity were not significantly different. The NO production ratio of MEF cells was higher for waxy wheat sprout than for normal ones, thereby indicating lower anti-inflammatory activity of waxy wheat sprouts. The viabilities of Caco-2 cells treated with waxy wheat sprouts was higher than that of cells treated with normal ones for the water extract. These results imply that waxy wheat sprouts exhibit better antioxidant activity and less cell toxicity for water extract, and therefore, could be used as a health-promoting food.

Cover Image, Volume 138, Issue 43

The cover image by Seda Ceylan shows an SEM photograph representing cell interaction on H. perforatum oil incorporated membrane scaffold. Immediately after a scaffold is implanted, protein adsorption to its surface occurs, mediating cell adhesion and providing signals to the cell through cell adhesion receptors, mainly integrins. After cell adhering on tissue engineered scaffold, cells are able to release active compounds for signaling, extra-cellular matrix deposition, cell proliferation and differentiation. In this research study, H. perforatum oil incorporation increased the viability of MEF cells and this membrane can be used for tissue engineering. DOI: 10.1002/app.51385

DAXX ameliorates metabolic dysfunction in mice with diet-induced obesity by activating the AMP-activated protein kinase-related kinase MPK38/MELK

Death domain-associated protein (DAXX) is involved in the activation of adipocyte apoptosis and is downregulated in response to a high-fat diet (HFD), which implies that the inhibition of adipocyte apoptosis may cause obesity. However, the anti-obesity effects of DAXX in diet-induced obesity (DIO) remain to be characterized. Here, we identified DAXX as an interacting partner of murine protein serine-threonine kinase 38 (MPK38). This interaction was mediated by the C-terminal (amino acids 270–643) domain of MPK38 and the N-terminal (amino acids 1–440) domain of DAXX and was increased by diverse signals that activate ASK1/TGF-β/p53 signaling. MPK38 phosphorylated DAXX at Thr578. Wild-type DAXX, but not a DAXX T578A mutant, stimulated MPK38-dependent ASK1/TGF-β/p53 signaling by increasing the stability of MPK38 and complex formation between MPK38 and its downstream targets, such as ASK1, Smad3, and p53. This mechanism was also shown in MEF cells that were null (−/−) for DAXX. Furthermore, the adenovirally-mediated reinstatement of DAXX expression activated MPK38 and ameliorated diet-induced defects in glucose and lipid metabolism in mice. These results indicate that DAXX limits obesity-induced metabolic abnormalities in DIO mice by activating MPK38.

Deficiency of heat shock factor 4 promotes lens epithelial cell senescence through upregulating p21cip1 expression

Genetic mutations in heat shock factor 4 (Hsf4) is associated with both congenital and age-related cataracts. Hsf4 regulates lens development through its ability to both activate and inhibit transcription. Previous studies suggested Hsf4 is involved in modulating cellular senescence depending on p21cip1 and p27 kip1 expression in MEF cells. Here, we found that Hsf4 acts as a suppressor of p21cip1 expression and plays an anti-senescence role during lens development. Knocking out Hsf4 facilitated UVB-induced cellular senescence in mouse lens epithelial cells (mLECs). p21cip1 was upregulated at both the mRNA and protein levels in HSF4−/− mLECs under control and UVB-treated conditions, and knockdown of p21cip1 by siRNA alleviated UVB-induced cellular senescence. HSF4 directly bound to the p21cip1 promoter and increased H3K27m3 levels at the p21cip1 proximal promoter region by recruiting the methyltransferase EZH2. In animal models, p21cip1 was gradually upregulated in wild-type mouse lenses with increasing age, while Hsf4 levels decreased. We generated a Hsf4 mutant mice line (Hsf4del-42) which displayed obvious congenital cataract phenotype. The expression of p21cip1 and senescence-associated cytokines were induced in the cataractous lenses of Hsf4del-42 mice. H3K27m3 and EZH2 levels decreased in p21cip1 promoters in the lenses of Hsf4del-42 mice. The SA-β-Gal activities were positive in lens epithelia of aged Hsf4null zebrafish compared to wild-type lenses. p21cip1 and senescence-associated cytokines levels were also upregulated in lenses of Hsf4null zebrafish. Accordingly, we propose that HSF4 plays a protective role in lens epithelial cells against cellular senescence during lens development and aging, partly by fine-tuning p21cip1 expression.

Abstract 1984: Crosstalk between sirtuins and TGF-β signaling suppresses liver cancer

Abstract Background: Cancer stem cells that escape suppression by key pathways such as TGF-β represent one of the mechanisms for poor prognosis in hepatocellular cancer (HCC). Our mouse models with disruption of TGF-β signaling (β2SP+/-/Smad3+/-) develop HCC and phenocopy an epigenetically human cancer stem cell disorder, providing clues towards the role of TGF-β in the switch in stem cell transformation to HCC. An examination of HCC genomics revealed a surprising and strong correlation between TGF-β and Sirtuins (SIRTs) expression. SIRTs are also implicated in cancer stem cell and HCC suppression. Here we hypothesized that the TGF-β pathway harnesses Sirtuin members to maintain stem cell homeostasis, which may mediate the progression of HCC. Methods: Overall Survival (OS) analysis was carried out by using early-stage HCC (TCGA, Firehose Legacy, stage N1, n=186) from cBioportal. ChIP assay was performed in HepG2 cells using anti-Smad3 and normal rabbit IgG. Biotinylated SIRT6 promoter with/without SMAD binding sites was used as probes to pull down the DNA-interacting proteins in HepG2 cells treated with/without TGF-β for 2h. Results: Analysis of TCGA HCC dataset (cBioportal, stage N1, n=186) indicated reduced OS for HCC patients with decreased SIRT6 together with loss of SPTBN1 in the early stage (Median survival months: 40.37 vs 83.18, p=0.0417). • Chip-qPCR and the DNA-protein pulldown assay revealed that Smad3 positively upregulates the SIRT6 mRNA expression (Smad3 vs IgG, SBE1: 3.27±0.53 vs 0.99±0.01, p<0.05; SBE2: 3.32±0.74 vs 1.12±0.3, p<0.05) through direct binding to the SIRT6 promoter in HepG2 cells after TGF-β treatment. • Co-IP analysis revealed directly binding of SIRT6 with β2sp and Smad3. • Overexpression of SIRT6 represses Smad3 and β2sp expression (p<0.01) and results in inhibition of the TGF-β target genes (p<0.05) such as MMP9 (68.2% inhibited), HMGA2 (53.4% inhibited), and PAI-1 (35.4% inhibited); whereas knockdown of SIRT6 increases β2sp expression in HepG2 cells (3 fold, p<0.05). • Similar to mice with disruption of SIRT6 (SIRT6-/-) that develop liver inflammation, steatosis with progression to tumorigenesis, mice with TGF-β signaling defect (Smad3+/-Sptbn1+/-) also develop fatty liver with progression to tumorigenesis under HFD. Interestingly, we found that SIRT6 was significantly downregulated in MEF cells from mice with loss of TGF-β signaling (Smad3+/-Sptbn1+/-). Conclusions: Our findings suggest that the TGF-β signaling participates in positively regulating the expression of SIRT6. In turn, SIRT6 acts as a negative regulator of TGF-β signaling through de-acetylating Smad3 and β2sp. SIRT6 and TGF-β signaling exert integrative yet complex roles in inhibiting hepatic steatosis, inflammation, and HCC that when disrupted lead to poor prognosis in HCC. The data provide new combined driving roles for the two pathways in suppressing NASH and HCC. Citation Format: Xiyan Xiang, Sobia Zaidi, Shuyun Rao, Kazufumi Ohshiro, Zhanhuai Wang, Wilma Jogunoori, Chuxia Deng, Patricia Latham, Lopa Mishra. Crosstalk between sirtuins and TGF-β signaling suppresses liver cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1984.

Abstract 89: A TGF-beta Pathway-SREBP1 axis controls liver diseases from nonalcoholic steatohepatitis to hepatocellular carcinoma

Abstract Background/Aims: The global epidemic of obesity has led to an alarming rise in nonalcoholic steatohepatitis (NASH) and hepatocellular cancer (HCC). In addition to abnormal fat accumulation and liver injury, fibrosis is a key predictive factor for progression and transformation. Yet, our understanding of how a major fibrosis pathway, transforming growth factor β (TGF-β), and how members, SMAD3 with its adaptor SPTBN 1 -contribute to the progression of NASH-driven HCC remains unclear. Here, we sought to better understand the role of TGF-β1/SMAD3/SPTBN1 in regulating the switch between NASH and HCC. Methods: We generated liver-specific SPTBN1 knockout mice (Albumin Cre+Sptbn1loxp/loxp, LKO). LKO and controls were given a high-fat diet (HFD) or DEN; Phenotypic analyses and mechanistic insight were obtained through RNA-seq, mass spectrometry, structure modeling, cell fractionation, and imaging-immunofluorescence, immunohistochemistry, and interactions studies were performed in human HCC cell lines (Huh7, Hep3B). Results: Strikingly, LKO did not develop HFD induced obesity, NASH, and cancer, that occurred in wild type, Flox control, NASH, and DEN/HCC models. Compared to controls, LKO liver tissues displayed 2-7-fold decreases in NASH associated pro-fibrotic genes (Col1a1, Col1a2) and lipid metabolism genes (CD36, Slc27a1, Plin4, Plin2) (p<0.05), and over 4-fold increases in Fgf21 mRNA levels (LKO vs control: 3.67±1.9 vs 0.84±0.16). Analysis of key regulators of lipogenesis revealed decreased SREBP1 (0.33±0.11 VS 1.35±0.22, p=0.006) and targets as well as PPARG (3.15±1.24 VS 19.03±4.98, p=0.02) in LKO compared to controls. Functionally, Sptbn1 MEF cells show marked reduction in SREBP1 reporter activity compared to WT MEFs (LDLR-luc: 0.25±0.04 vs 1.0±0.08, p<0.05) with decreased nuclear SREBP1 labeling in LKO tissues. Depletion of Sptbn1 leads to impaired TGF-β1/SMAD3 signaling and decreased TNF-α induced ER stress/caspase-3 activated SREBP1. N-terminal SPTBN1 (D50-T975) binds SREBP1 (Q295-K374) and induces its nuclear translocation. HFD -driven caspase-3 activation and SREBP1-SPTBN1 cleavage and binding occur independently of insulin induced gene (INSIG) and SREBP cleavage-activating protein (SCAP). We observe raised levels of SPTBN1 together with Caspase-3, and lipogenic pathways (e.g., FASN, SCD1) in human NASH/HCC progression. Additional human HCC TCGA analysis revealed poor prognosis in patients with high SREBP1 activity. Finally, siRNA targeting Sptbn1 in vivo protects mice from HFD-induced NASH and reduces NASH-associated transcriptional changes in a human 3D-culture NASH model. Conclusions: We uncovered a surprising and promising role of SPTBN1 siRNA in inhibiting NASH and HCC, through modulation of its binding partners, SMAD3 and SREBP1. These data provide new insights into the switch in obesity driven liver cancer as well as new therapeutic targets. Citation Format: Shuyun Rao, Zhanhuai Wang, Kazufumi Ohshiro, Sobia Zaidi, Xiaochun Yang, Patricia Latham, Wilma Jogunoori, Xianyan Xiang, Inhee Chung, Kirti Shetty, Michele Vacca, Antonio Vidal-Puig, Lopa Mishra. A TGF-beta Pathway-SREBP1 axis controls liver diseases from nonalcoholic steatohepatitis to hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 89.

Automated Cell Mechanical Characterization by On-Chip Sequential Squeezing: From Static to Dynamic.

The mechanical properties of cells are harmless biomarkers for cell identification and disease diagnosis. Although many systems have been developed to evaluate the static mechanical properties of cells for biomedical research, their robustness, effectiveness, and cost do not meet clinical requirements or the experiments with a large number of cell samples. In this paper, we propose an approach for on-chip cell mechanical characterization by analyzing the dynamic behavior of cells as they pass through multiple constrictions. The proposed serpentine microfluidic channel consisted of 20 constrictions connected in series and divided into five rows for tracking cell dynamic behavior. Assisted by computer vision, the squeezing time of each cell through five rows of constrictions was automatically collected and filtered to evaluate the cell's mechanical deformability. We observed a decreasing passage time and increasing dynamic deformability of the cells as they passed through the multiple constrictions. The deformability increase rate of the HeLa cells was eight times greater than that of MEF cells. Moreover, the weak correlation between the deformability increase rate and the cell size indicated that cell recognition based on measuring the deformability increase rate could hardly be affected by the cell size variation. These findings showed that the deformability increase rate of the cell under on-chip sequential squeezing as a new index has great potential in cancer cell recognition.

CUL5-ASB6 Complex Promotes p62/SQSTM1 Ubiquitination and Degradation to Regulate Cell Proliferation and Autophagy.

p62/SQSTM1 (sequestosome-1) is a key protein involved in multiple cellular bioprocesses including autophagy, nutrient sensing, cell growth, cell death, and survival. Therefore, it is implicated in human diseases such as obesity and cancer. Here, we show that the CUL5–ASB6 complex is a ubiquitin E3 ligase complex mediating p62 ubiquitination and degradation. Depletion of CUL5 or ASB6 induced p62 accumulation, and overexpression of ASB6 promoted ubiquitination and degradation of p62. Functionally, ASB6 overexpression can inhibit the proliferation of MEF and hepatocellular carcinoma cells by reducing p62 protein level, and impair the occurrence of autophagy. Overall, our study identified a new molecular mechanism regulating p62 stability, which may provide additional insights for understanding the delicate control of p62 and cell proliferation–autophagy control in physiological and pathological settings.

35-OR: Epigenetic Regulation of White Adipose Tissue Browning by HMGN Proteins

Chromatin architecture proteins HMGNs (High Mobility Group Nucleosome-binding) affect local and global chromatin structure and colocalize with cell-specific regulatory sites and regulate cell identity. The role of HMGN proteins in adipocyte function and energy metabolism is yet to be investigated. Here we report that HMGN1 and HMGN2 double knock-out (DKO) mice on chow diet exhibited decreased weight gain (10%) and total activity with increased food intake and energy expenditure compared to control mice. DKO mice on a high-fat diet also displayed increased food intake and decreased weight gain. We have observed that DKO mice possess less fat mass, which is correlated with decreased WAT size, but not BAT. Histological analysis of WAT from mice revealed smaller adipocytes and increased numbers of cells in DKO, while immunofluorescence analysis indicated more UCP1 in DKO, suggestive of increased WAT browning in DKO mice. Investigations on white preadipocytes or MEF cells differentiation by adipogenic cocktail revealed faster differentiation and elevated UCP1 in DKO cells. Time course RNA seq analysis on in-vitro differentiated adipocytes also confirmed enhanced browning in DKO cells. We further investigated the distribution of active histone marks such as H3K9 acetylation and H3K27 acetylation in adipocytes differentiated from preadipocytes. Our time-course ChIP seq analyses indicated that HMGN’s absence altered both H3K9 and H3K27 acetylation patterns in DKO cells compared to WT. Interestingly, correlation analysis of ChIP seq and RNA seq suggests that DKO preadipocytes and adipocytes lost active chromatin marks at certain white adipocyte-specific enhancers. In summary, loss of HMGN attributes thermogenic character to WAT in DKO mice, thereby contributing to increased energy expenditure, which might protect mice from metabolic disorders—increased white adipocyte browning in DKO is partly by reducing the active histone marks at white specific enhancers, thereby altering white adipocyte identity. Disclosure R. Nanduri: None. T. Furusawa: None. A. Lobanov: None. O. Gavrilova: None. M. Bustin: None. Funding National Cancer Institute

When multiple caveolins make the difference: Cav1 partly compensates Cav3 alterations and rescues ion channels expression

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Fondazione Cariplo Caveolae are small-membrane invagination that contribute both to buffering excessive contraction-dependent membrane strain and to initiation of membrane repair. Moreover, they constitute micro-domains where receptors and ion channels are clustered, favouring their functional interaction. Caveolin-3 (Cav3) is the key structural component of muscular caveolae. Mutations in Cav3 gene are associated with alterations of the skeletal muscle architecture leading to some rare forms of hereditary skeletal myopathies and/or cardiomyopathies called caveolinopathies. Notably, skeletal muscle dysfunctions usually precede cardiac dysfunctions, even though the mutated Cav3 is expressed in both cell types. An important difference between skeletal fibers and cardiomyocytes is that in the latter, caveolin-1 (Cav1) participates with Cav3 to form caveolae; skeletal myotubes instead do not express Cav1.The delay or lack of onset of cardiac alterations in caveolinopathies may depend on a preserved micro-domains organization in the heart compared to skeletal muscle, due to Cav1 expression. We decided to focus on a specific mutation T78K found in heterozygous in a patient with Ripple muscle disease and hyperCKemia. We have characterized human cardiomyocytes (CM) differentiated from induced pluripotent stem cells (iPSC) derived from this patient and one healthy control. In particular, we have investigated which caveolin isoforms are expressed at day 30 of differentiation, finding both Cav1 and Cav3, with a significant decrease of Cav3 isoform in T78K-CM. Their different expressions significantly increase T78K membrane resistance (3.27 ± 0.6 GΩ versus 1.64 ± 0.4 GΩ in the CTRL-CM), and consequently membrane excitability. The T78K_CM showed an increase spontaneous beating rate compared to CTRL (1,75± 0,08 Hz and 0,89 ± 0,4 Hz, respectively). Previous laboratory analysis conducted in caveolin-free MEF cells, co-transfected with WT and T78K Cav3 mutation, revealed that the T78K mutant is dominant, inducing the retention of WT Cav3 in the perinuclear areas and causes significant reduction in current density of three ion channels (HCN4, Kv1.5 and Kir2.1) known to interact with caveolins. The dominant decreased in cav3 expression is in line with previous data in skeletal muscle biopsy, however, electrophysiological data would be likely incompatible with life. For this reason, we decided to compare the impact of this mutation in CHO cells that exhibit high levels of Cav1, and in cav-1 expressing MEF line. In these systems, the membrane localization of Cav3 T78K is rescued both in heterozygous and homozygous conditions. In line with caveolin membrane expression, HCN4, Kv1.5 and Kir2.1 density is also rescued to normal levels. These results constitute the first evidence of a possible role of Cav1 in compensating membrane disorganization and disfunction due to Cav3 mutations in the heart, making this organ less susceptible to caveolinopathies.

Propolis loaded and genipin-crosslinked PVA/chitosan membranes; characterization properties and cytocompatibility/genotoxicity response for wound dressing applications

Loading propolis by a simple process using genipin as a crosslinking agent and fabrication of a novel PVA/Chitosan-Propolis membrane scaffolds were reported for wound dressing applications. The research is focused on the effects of propolis on characterization properties of membrane such as chemical structure, surface morphology, degradation ratio, crystallinity, hydrophilicity, water uptake capacity, water vapour transmission rate and mechanical aspect. It was noticed that water uptake capacity and hydrophilicity properties of membrane considerably affected by the propolis. By addition of (0.50, % v/v) propolis, the contact angle of the PVA/Chitosan membrane was remarkably decreased from 86.29° ± 3 to 45 ± 2°. 3-(4,5-dimethylthiazoyl-2-yl)-2,5-diphenylte-trazolium (MTT) bromide test and SEM were used to analyse the cytocompatibility of the membranes and morphology of cells on membrane. The propolis incorporated membrane showed cell proliferation rate 176 ± 13%, 775 ± 1%, and 853 ± 23%, at 24 h, 27 h and 120 h, respectively. SEM images also supported the cell behaviour on membrane. DNA fragmentation was also investigated with genotoxicity test. The studies on the interactions between membranes and MEF cells revealed that the incorporation of propolis into membrane promoted cell proliferation. These overall results presented that propolis incorporated membranes could have potentially appealing application as scaffolds for wound healing applications.

Caspase-9 acts as a regulator of necroptotic cell death.

Necroptosis is a regulated necrotic-like cell death modality which has come into the focus of attention since it is known to contribute to the pathogenesis of many inflammatory and degenerative diseases as well as to tumor regulation. Based on current data, necroptosis serves as a backup mechanism when death receptor-induced apoptosis is inhibited or absent. However, the necroptotic role of the proteins involved in mitochondrial apoptosis has not been investigated. Here, we demonstrated that the stimulation of several death and pattern recognition receptors induced necroptosis under caspase-compromised conditions in wild-type, but not in caspase-9-negative human Jurkat and murine MEF cells. Cerulein-induced pancreatitis was significantly reduced in mice with acinar cell-restricted caspase-9 gene knockout. The absence of caspase-9 led to impaired association of receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and RIPK3 and resulted in decreased phosphorylation of RIP kinases, but the overexpression of RIPK1 or RIPK3 rescued the effect of caspase-9 deficiency. Inhibition of either Aurora kinase A (AURKA) or its known substrate, glycogen synthase kinase 3β (GSK3ß) restored necroptosis sensitivity of caspase-9-deficient cells, indicating an interplay between caspase-9 and AURKA-mediated pathways to regulate necroptosis. Our findings suggest that caspase-9 acts as a newly identified regulator of necroptosis, and thus, caspase-9 provides a promising therapeutic target to manipulate the immunological outcome of cell death.

Novel function of Nanos2 in expression of innate immunity genes and its probable roles in maintenance of pluripotency state.

Objective(s):Cell-based therapeutic approaches have witnessed significant developments during the last decade especially after approval of MSCs based treatment of graft versus host disease. Several cell-based approaches have shown immunomodulatory behavior during regeneration following the unknown cascade of events but the exact mechanisms are yet to be defined. Clinical applications of cell-based drugs are hampered all over the world because of incomplete understanding of molecular mechanisms requiring the application of mechanistic approaches to solving the mystery. Current work has given us the idea that Nanos2 enhances the cellular pluripotency characteristics while down-regulating the innate immunity genes, simultaneously.Materials and Methods:The immunomodulatory behavior of cells was studied against cells carrying the ectopic expression of Nanos2 in comparison with Stella and Oct4 individually and simultaneously using SON vector (Stella, Nanos2 and Oct4). Results:It was observed that overexpression of Nanos2 leads to down-regulation of Interferon-Stimulated Genes (ISGs)-mRNAs such as Ifitm1, lsg15, Oas2, and Oas12. Nanos2 overexpressing MEF cells have shown restrictive inflammatory effects when cells were treated with inflammatory stimuli such as LPS and Poly (I:C). Conclusion:From our recent findings in line with many others, it can be concluded that Nanos2 acts as a coin with two sides, regulating pluripotency and immunity together which enhances resistance against inflammatory stimuli. Nanos2 could be a potential candidate as a molecular drug for management of inflammation and immunomodulation but it requires a comprehensive comparative expression analysis of innate immunity genes in vitro and in vivo.

Protein kinase D, ubiquitin and proteasome pathways are involved in adenosine receptor-stimulated NR4A expression in myeloid cells

Adenosine is a purine nucleoside pivotal for homeostasis in cells and tissues. Stimulation of the adenosine receptors (AR) has been shown to regulate the nuclear orphan receptor 4A (NR4A1-3) family, resulting in attenuation of hyper-inflammatory responses in myeloid cells. The NR4A1-3 orphan receptors are early immediate response genes and transcriptional regulators of cell and tissue homeostasis. The signal transduction and transcriptional mechanism(s) of how AR-stimulation promotes NR4A expression in myeloid cells is unknown and is the focus of this study. We confirm that adenosine and the stable analogue, 5′-N-Ethylcarboxamidoadenosine (NECA), enhance NR4A1-3 expression in THP-1 cells. Pharmacological approaches identified that protein kinase D (PKD) mediates AR-stimulated NR4A expression in myeloid cells and reveals no involvement of PKA nor PKC. The role of NF-κB, a principal regulator of NR4A expression in myeloid cells, was examined as a possible transcriptional regulator downstream of PKD. Utilising BAY11-7082 and MG-132, inhibitors of the respective ubiquitin and proteasome pathways essential for NF-κB activation, suggested a prospective role for NF-κB, or more specifically signalling via IKKα/β. However, biological interventional studies using overexpression of IκBα in myeloid cells and MEF cells lacking IKKα and IKKβ (IKKα/β−/-) revealed the NF-κB pathway is not utilised in mediating AR-stimulated NR4A expression. Thus, this study contributes mechanistic insight into how AR signalling modulates the expression of NR4A receptors, pivotal regulators of inflammatory responses in myeloid cells.

Pathogenic LRRK2 regulates ciliation probability upstream of tau tubulin kinase 2 via Rab10 and RILPL1 proteins

Mutations that activate LRRK2 protein kinase cause Parkinson's disease. We showed previously that Rab10 phosphorylation by LRRK2 enhances its binding to RILPL1, and together, these proteins block cilia formation in a variety of cell types, including patient derived iPS cells. We have used live-cell fluorescence microscopy to identify, more precisely, the effect of LRRK2 kinase activity on both the formation of cilia triggered by serum starvation and the loss of cilia seen upon serum readdition. LRRK2 activity decreases the overall probability of ciliation without changing the rates of cilia formation in R1441C LRRK2 MEF cells. Cilia loss in these cells is accompanied by ciliary decapitation, and kinase activity does not change the timing or frequency of decapitation or the rate of cilia loss but increases the percent of cilia that are lost upon serum addition. LRRK2 activity, or overexpression of RILPL1 protein, blocks release of CP110 from the mother centriole, a step normally required for early ciliogenesis; LRRK2 blockade of CP110 uncapping requires Rab10 and RILPL1 proteins and is due to failure to recruit TTBK2, a kinase needed for CP110 release. In contrast, deciliation probability does not change in cells lacking Rab10 or RILPL1 and relies on a distinct LRRK2 pathway. These experiments provide critical detail to our understanding of the cellular consequences of pathogenic LRRK2 mutation and indicate that LRRK2 blocks ciliogenesis upstream of TTBK2 and enhances the deciliation process in response to serum addition.

Semi-Automated Method for Image Analysis of mtDNA Nucleoids Dynamics

Mitochondrial DNA is tightly packed into nucleoprotein complexes, called nucleoids, formed by mtDNA and mitochondrial transcription factor TFAM. Nucleoids inhabit the organelle, associated to inner mitochondrial membrane. The dynamics of mitochondria involves constant reshaping, and the relative dynamic localization of nucleoids and mitochondrial membranes, remains unexplored.We applied a machine-learning semi-automated method to study the simultaneous dynamic re-distribution of nucleoids and mitochondria. MEF cells were transfected with cDNA codifying mCherry-OMP25 (targeting the outer mitochondrial membrane, OMM) and Tfam-GFP (to visualize the nucleoids), and imaged the cells by high-resolution confocal microscopy at a Zeiss 880 Airyscan confocal microscope. Three-dimensional segmentation of Z-stacks series by means of trainable Weka protocol, allowed us to determine mitochondrial and nucleoid dimensions, shape descriptors, distribution and relative positioning over time, including speed and directionality. Our data show that the semi-automated method, trainable Weka, is a powerful tool to study mitochondrial nucleoids dynamics in the context of permanent mitochondria reshaping.

The N-terminal BRCT domain determines MCPH1 function in brain development and fertility

MCPH1 is a causal gene for the neurodevelopmental disorder, human primary microcephaly (MCPH1, OMIM251200). Most pathogenic mutations are located in the N-terminal region of the gene, which encodes a BRCT domain, suggesting an important function of this domain in brain size determination. To investigate the specific function of the N-terminal BRCT domain in vivo, we generated a mouse model lacking the N’-BRCT domain of MCPH1 (referred as Mcph1-ΔBR1). These mutant mice are viable, but exhibit reduced brain size, with a thinner cortex due to a reduction of neuroprogenitor populations and premature neurogenic differentiation. Mcph1-ΔBR1 mice (both male and female) are infertile; however, almost all female mutants develop ovary tumours. Mcph1-ΔBR1 MEF cells exhibit a defect in DNA damage response and DNA repair, and show the premature chromosome condensation (PCC) phenotype, a hallmark of MCPH1 patient cells and also Mcph1 knockout cells. In comparison with Mcph1 complete knockout mice, Mcph1-ΔBR1 mice faithfully reproduce all phenotypes, indicating an essential role of the N-terminal BRCT domain for the physiological function of MCPH1 in the control of brain size and gonad development as well as in multiple cellular processes.

Mitochondrial transfer from induced pluripotent stem cells rescues developmental potential of in vitro fertilized embryos from aging females†.

Conventional heterologous mitochondrial replacement therapy is clinically complicated by "tri-parental" ethical concerns and limited source of healthy donor oocytes or zygotes. Autologous mitochondrial transfer is a promising alternative in rescuing poor oocyte quality and impaired embryo developmental potential associated with mitochondrial disorders, including aging. However, the efficacy and safety of mitochondrial transfer from somatic cells remains largely controversial, and unsatisfying outcomes may be due to distinct mitochondrial state in somatic cells from that in oocytes. Here, we propose a potential strategy for improving in vitro fertilization (IVF) outcomes of aging female patients via mitochondrial transfer from induced pluripotent stem (iPS) cells. Using naturally aging mice and well-established cell lines as models, we found iPS cells and oocytes share similar mitochondrial morphology and functions, whereas the mitochondrial state in differentiated somatic cells is substantially different. By microinjection of isolated mitochondria into fertilized oocytes following IVF, our results indicate that mitochondrial transfer from iPS, but not MEF cells, can rescue the impaired developmental potential of embryos from aging female mice and obtain an enhanced implantation rate following embryo transfer. The beneficial effect may be explained by the fact that mitochondrial transfer from iPS cells not only compensates for aging-associated loss of mtDNA, but also rescues mitochondrial metabolism of subsequent preimplantation embryos. Using mitochondria from iPS cells as the donor, our study not only proposes a promising strategy for improving IVF outcomes of aging females, but also highlights the importance of synchronous mitochondrial state in supporting embryo developmental potential.