Immortalized Mouse Embryonic Fibroblasts Research Articles

DDAH1 deficiency promotes intracellular oxidative stress and cell apoptosis via a miR-21-dependent pathway in mouse embryonic fibroblasts

Asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase (NOS) inhibitor, is degraded by dimethylarginine dimethylaminohydrolase 1 (DDAH1). Emerging evidence suggests that plasma ADMA accumulation, DDAH1 activity/expression reduction, and microRNA-21 (miR-21) upregulation are linked to disease pathology, but the mechanisms remain largely unknown. In the present study, we assessed the potential role of the ADMA–DDAH1–miR-21 pathway in the regulation of the cellular redox state and apoptosis using wild-type (WT) and DDAH1-knockout (KO) immortalized mouse embryonic fibroblasts (MEFs). DDAH1 deficiency significantly increased ADMA levels, enhanced cellular oxidative stress, and rendered cells more vulnerable to apoptosis induced by tert-butyl hydroperoxide (tBHP) or A23187. However, treatment with exogenous ADMA (1–80μM) for 24h or for a prolonged period (10μM, 10 passages) in WT MEFs had no marked effect on intracellular reactive oxygen species (ROS) and apoptosis sensitivity. Interestingly, miR-21 expression was significantly increased, by 4 fold, in DDAH1−/− MEFs, and the induction of miR-21 by DDAH1 deficiency was dependent on oxidative stress and NF-κB activation. Inhibition of DDAH1 activity by PD 404182 also increased miR-21 expression. Furthermore, inhibition of miR-21 with a lentiviral vector in DDAH1−/− MEFs significantly upregulated SOD2 expression and the attenuated oxidative stress and apoptosis induced by tBHP or A23187. Taken together, our results suggest that DDAH1 not only acts as an enzyme degrading ADMA but also controls cellular oxidative stress and apoptosis via a miR-21-dependent pathway.

Chrysin inhibits adipogenic differentiation of mouse embryonic fibroblasts

Chrysin is an active flavonoid wildly presented in many herbs. It has the effect to reduce serum lipid. To investigate the effect of chrysin on the adipogenic differentiation of mouse embryonic fibroblasts, methyl thiazolyl tetrazolium (MTT) and crystal violet were used to detect the cytotoxic effect of chrysin on Immortalized mouse embryonic fibroblasts (iMEFs). Propidium iodide (PI) staining combined with flow cytometry (FCM) was employed to detect the effects of different concentrations of chrysin on iMEFs cell cycle. The effect of chrysin on adipogenic differentiation ability of iMEFs was determined by oil red O staining. Semi-quantitative PCR was employed to detect the effect of chrysin on mRNA transcriptional levels of adipogenic differentiation markers, including perilipin 2, adiponectin (adipoq), Fabp4, LPL, MCP-1 and adipogenic differentiation key transcription factor peroxisome proliferators-actiated receptor-gamma 2(PPAR-γ2). Results indicated that chrysin had certain cytotoxic effect for iMEFs in a dose-dependent manner, and the IC₅₀ was identified nearly to 30 μmol•L⁻¹. FCM analysis showed that chrysin could affect the cell-cycle distribution of iMEFs, increasing the ratio of cells in G1 phase. Adipogenic differentiation inducing experiment showed that 30 μmol•L⁻¹ chrysin significantly reduced lipid drops accumulation induced by insulin and dexamethasone. In addition, the mRNA transcriptional levels of PPAR-γ2 and LPL were significantly decreased and mRNA levels of fabp 4, MCP-1, adipoq were also affected after chrysin treatment. The experiment results suggest that chrysin attenuates the adipogenic differentiation capacity of mesenchymal stem cells.

Abstract 2026: G0S2 functions as a tumor suppressor gene through inhibition of c-Myc

Abstract G0/G1 switch gene 2 (G0S2) is a direct retinoic acid target gene that is widely expressed in diverse organs and has been reported to have a role in adipose lipolysis by negatively regulating adipose triglyceride lipase (ATGL). G0S2 is also commonly silenced by DNA methylation in a variety of solid tumors including glioma, head and neck and lung cancers, and G0S2 induction is associated with retinoic acid-mediated clinical remissions in acute promyelocytic leukemia. This evidence suggests that G0S2 may possess tumor suppressor activity although definitive proof and mechanistic details are lacking. We now clearly demonstrate using G0S2 null immortalized mouse embryonic fibroblasts (MEFs) that G0S2 can function to oppose oncogene-induced transformation. G0S2 null MEFs were readily transformed with H-RAS or EGFR while wild-type MEFs were not. Importantly, re-introduction of G0S2 reversed H-RAS transformation of G0S2 null MEFs. Although G0S2 is a known regulator of fat metabolism through attenuating ATGL activity, we found that G0S2 repression of oncogene-induced transformation was independent of ATGL inhibition. Detailed microarray analysis demonstrated that G0S2 knockout alone upregulated gene signatures associated with transformation, proliferation and Myc transcriptional responses. G0S2 knockout also induced c-Myc expression at the transcript and protein level. Finally, transient and stable shRNA-mediated repression of c-Myc completely abrogated oncogene-induced transformation of G0S2 null MEFs. Taken together, these findings indicate that G0S2 can function as a tumor suppressor by opposing Myc. Studies to elucidate the mechanism underlying G0S2 regulation of Myc are ongoing. Understanding the functions and mechanisms of this novel tumor suppressor pathway may lead to new pharmacologic strategies for many cancers where G0S2 is silenced. Citation Format: Christina Y. Yim, David J. Sekula, Sarah J. Freemantle, Ethan Dmitrovsky, Michael J. Spinella. G0S2 functions as a tumor suppressor gene through inhibition of c-Myc. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2026. doi:10.1158/1538-7445.AM2015-2026

Hypoxia Inducible Factors Modulate Mitochondrial Oxygen Consumption and Transcriptional Regulation of Nuclear-Encoded Electron Transport Chain Genes.

Hypoxia inducible factor-1 (HIF1) is a stress-responsive nuclear transcription factor that is activated with a decrease in oxygen availability. HIF1 regulates the expression of genes involved in a cell's adaptation to hypoxic stress, including those with mitochondrial specific function. To gain a more comprehensive understanding of the role of HIF1 in mitochondrial homeostasis, we studied the link between hypoxia, HIF1 transactivation, and electron transport chain (ETC) function. We established immortalized mouse embryonic fibroblasts (MEFs) for HIF1α wild-type (WT) and null cells and tested whether HIF1α regulates mitochondrial respiration by modulating gene expressions of nuclear-encoded ETC components. High-throughput quantitative real-time polymerase chain reaction was performed to screen nuclear-encoded mitochondrial genes related to the ETC to identify those whose regulation was HIF1α-dependent. Our data suggest that HIF1α regulates transcription of cytochrome c oxidase (CcO) heart/muscle isoform 7a1 (Cox7a1) under hypoxia, where it is induced 1.5-2.5-fold, whereas Cox4i2 hypoxic induction was HIF1α-independent. We propose that adaptation to hypoxic stress of CcO as the main cellular oxygen consumer is mediated by induction of hypoxia-sensitive tissue-specific isoforms. We suggest that HIF1 plays a central role in maintaining homeostasis in cellular respiration during hypoxic stress via regulation of CcO activity.

Evaluation of the bioactive properties of avenanthramide analogs produced in recombinant yeast.

Saccharomyces cerevisiae has been proven to be a valuable tool for the expression of plant metabolic pathways. By engineering a S. cerevisiae strain with two plant genes (4cl-2 from tobacco and hct from globe artichoke) we previously set up a system for the production of two novel phenolic compounds, N-(E)-p-coumaroyl-3-hydroxyanthranilic acid (Yeast avenanthramide I, Yav I) and N-(E)-caffeoyl-3-hydroxyanthranilic acid (Yeast avenanthramide II, Yav II). These compounds have a structural similarity with a class of bioactive oat compounds called avenanthramides. By developing a fermentation process for the engineered S. cerevisiae strain, we obtained a high-yield production of Yav I and Yav II. To examine the biological relevance of these compounds, we tested their potential antioxidant and antiproliferative properties upon treatment of widely used cell models, including immortalized mouse embryonic fibroblast cell lines and HeLa cancer cells. The outcomes of our experiments showed that both Yav I and Yav II enter the cell and trigger a significant up-regulation of master regulators of cell antioxidant responses, including the major antioxidant protein SOD2 and its transcriptional regulator FoxO1 as well as the down-regulation of Cyclin D1. Intriguingly, these effects were also demonstrated in cellular models of the human genetic disease Cerebral Cavernous Malformation, suggesting that the novel phenolic compounds Yav I and Yav II are endowed with bioactive properties relevant to biomedical applications. Taken together, our data demonstrate the feasibility of biotechnological production of yeast avenanthramides and underline a biologically relevant antioxidant activity of these molecules.

Inactivation of Sag/Rbx2/Roc2 E3 Ubiquitin Ligase Triggers Senescence and Inhibits Kras-Induced Immortalization

Our recent study showed that SAG/RBX2 E3 ubiquitin ligase regulates apoptosis and vasculogenesis by promoting degradation of NOXA and NF1, and co-operates with Kras to promote lung tumorigenesis by activating NFκB and mTOR pathways via targeted degradation of tumor suppressive substrates including IκB, DEPTOR, p21 and p27. Here we investigated the role of Sag/Rbx2 E3 ligase in cellular senescence and immortalization of mouse embryonic fibroblasts (MEFs) and report that Sag is required for proper cell proliferation and KrasG12D-induced immortalization. Sag inactivation by genetic deletion remarkably suppresses cell proliferation by inducing senescence, which is associated with accumulation of p16, but not p53. Mechanistically, Sag deletion caused accumulation of Jun-B, a substrate of Sag-Fbxw7 E3 ligase and a transcription factor that drives p16 transcription. Importantly, senescence triggered by Sag deletion can be largely rescued by simultaneous deletion of Cdkn2a, the p16 encoding gene, indicating its causal role. Furthermore, KrasG12D-induced immortalization can also be abrogated by Sag deletion via senescence induction, which is again rescued by simultaneous deletion of Cdkn2a. Finally, we found that Sag deletion inactivates KrasG12D activity and block the MAPK signaling pathway, together with accumulated p16, to induce senescence. Taken together, our results demonstrated that Sag is a KrasG12D-cooperating oncogene required for KrasG12D-induced immortalization and transformation, and targeting SAG-SCF E3 ligase may, therefore, have therapeutic value for senescence-based cancer treatment.

Abstract SY06-04: Regulation of cancer cell proliferation and survival by NF-κB

Abstract SY06-04: Regulation of cancer cell proliferation and survival by NF-κB

PF-4708671 activates AMPK independently of p70S6K1 inhibition.

The P70 ribosomal protein S6 kinase 1 (P70S6K1) is activated by the mammalian target of rapamycin (mTORC1) and regulates proliferation, growth, and metabolism. PF-4708671 is a novel, cell-permeable, has been proposed to be a highly specific inhibitor of p70S6K1. It is used in micromolar concentration range to dissect signaling pathways downstream of mTORC1 and to study the function of p70S6K1. Here we show that PF-4708671 induces AMP-activated protein kinase (AMPK) phosphorylation and activation in immortalized mouse embryonic fibroblasts (MEF) independently of p70S6K1, due to specific inhibition of mitochondrial respiratory chain Complex I.

Serotonin regulates calcium homeostasis in lactation by epigenetic activation of hedgehog signaling.

Calcium homeostasis during lactation is critical for maternal and neonatal health. We previously showed that nonneuronal/peripheral serotonin [5-hydroxytryptamine (5-HT)] causes the lactating mammary gland to synthesize and secrete PTHrP in an acute fashion. Here, using a mouse model, we found that genetic inactivation of tryptophan hydroxylase 1 (Tph1), which catalyzes the rate-limiting step in peripheral 5-HT synthesis, reduced circulating and mammary PTHrP expression, osteoclast activity, and maternal circulating calcium concentrations during the transition from pregnancy to lactation. Tph1 inactivation also reduced sonic hedgehog signaling in the mammary gland during lactation. Each of these deficiencies was rescued by daily injections of 5-hydroxy-L-tryptophan (an immediate precursor of 5-HT) to Tph1-deficient dams. We used immortalized mouse embryonic fibroblasts to demonstrate that 5-HT induces PTHrP through a sonic hedgehog-dependent signal transduction mechanism. We also found that 5-HT altered DNA methylation of the Shh gene locus, leading to transcriptional initiation at an alternate start site and formation of a variant transcript in mouse embryonic fibroblasts in vitro and in mammary tissue in vivo. These results support a new paradigm of 5-HT-mediated Shh regulation involving DNA methylation remodeling and promoter switching. In addition to having immediate implications for lactation biology, identification and characterization of a novel functional regulatory relationship between nonneuronal 5-HT, hedgehog signaling, and PTHrP offers new avenues for the study of these important factors in development and disease.

Reductive detoxification of acrolein as a potential role for aldehyde reductase (AKR1A) in mammals

Aldehyde reductase (AKR1A), a member of the aldo–keto reductase superfamily, suppresses diabetic complications via a reduction in metabolic intermediates; it also plays a role in ascorbic acid biosynthesis in mice. Because primates cannot synthesize ascorbic acid, a principle role of AKR1A appears to be the reductive detoxification of aldehydes. In this study, we isolated and immortalized mouse embryonic fibroblasts (MEFs) from wild-type (WT) and human Akr1a-transgenic (Tg) mice and used them to investigate the potential roles of AKR1A under culture conditions. Tg MEFs showed higher methylglyoxal- and acrolein-reducing activities than WT MEFs and also were more resistant to cytotoxicity. Enzymatic analyses of purified rat AKR1A showed that the efficiency of the acrolein reduction was about 20% that of glyceraldehyde. Ascorbic acid levels were quite low in the MEFs, and while the administration of ascorbic acid to the cells increased the intracellular levels of ascorbic acid, it had no affect on the resistance to acrolein. Endoplasmic reticulum stress and protein carbonylation induced by acrolein treatment were less evident in Tg MEFs than in WT MEFs. These data collectively indicate that one of the principle roles of AKR1A in primates is the reductive detoxification of aldehydes, notably acrolein, and protection from its detrimental effects.

The piggyBac transposon-mediated expression of SV40 T antigen efficiently immortalizes mouse embryonic fibroblasts (MEFs).

Mouse embryonic fibroblasts (MEFs) are mesenchymal stem cell (MSC)-like multipotent progenitor cells and can undergo self-renewal and differentiate into to multiple lineages, including bone, cartilage and adipose. Primary MEFs have limited life span in culture, which thus hampers MEFs’ basic research and translational applications. To overcome this challenge, we investigate if piggyBac transposon-mediated expression of SV40 T antigen can effectively immortalize mouse MEFs and that the immortalized MEFs can maintain long-term cell proliferation without compromising their multipotency. Using the piggyBac vector MPH86 which expresses SV40 T antigen flanked with flippase (FLP) recognition target (FRT) sites, we demonstrate that mouse embryonic fibroblasts (MEFs) can be efficiently immortalized. The immortalized MEFs (piMEFs) exhibit an enhanced proliferative activity and maintain long-term cell proliferation, which can be reversed by FLP recombinase. The piMEFs express most MEF markers and retain multipotency as they can differentiate into osteogenic, chondrogenic and adipogenic lineages upon BMP9 stimulation in vitro. Stem cell implantation studies indicate that piMEFs can form bone, cartilage and adipose tissues upon BMP9 stimulation, whereas FLP-mediated removal of SV40 T antigen diminishes the ability of piMEFs to differentiate into these lineages, possibly due to the reduced expansion of progenitor populations. Our results demonstrate that piggyBac transposon-mediated expression of SV40 T can effectively immortalize MEFs and that the reversibly immortalized piMEFs not only maintain long-term cell proliferation but also retain their multipotency. Thus, the high transposition efficiency and the potential footprint-free natures may render piggyBac transposition an effective and safe strategy to immortalize progenitor cells isolated from limited tissue supplies, which is essential for basic and translational studies.

RB in glutamine metabolism.

RB in glutamine metabolism.

Overexpression of Ad5 precursor terminal protein accelerates recombinant adenovirus packaging and amplification in HEK-293 packaging cells.

Recombinant adenoviruses are one of the most common vehicles for efficient in vitro and in vivo gene deliveries. Here, we investigate whether exogenous precursor terminal protein (pTP) expression in 293 cells improves the efficiency of adenovirus packaging and amplification. We used a piggyBac transposon-based vector and engineered a stable 293 line that expresses high level of Ad5 pTP, designated as 293pTP. Using the AdBMP6-GLuc that expresses green fluorescent protein (GFP), BMP6 and Gaussia luciferase, we found that the infectivity of AdBMP6-GLuc viral samples packaged in 293pTP cells was titrated up to 19.3 times higher than that packaged in parental 293 cells. AdBMP6-GLuc viral samples packaged in 293pTP cells exhibited significantly higher transduction efficiency in 143B and immortalized mouse embryonic fibroblast (iMEF) cells, as assessed by fluorescence-activated cell sorting analysis of GFP-positive cells, the luciferase activity assay and BMP6-induced osteogenic marker alkaline phosphatase activities in iMEFs. When adenovirus amplification efficiency was analyzed, we found that 293pTP cells infected with AdBMP6-GLuc yielded up to 12.6 times higher titer than that in parental 293 cells, especially at lower multiplicities of infection. These results strongly suggest that exogenous pTP expression may accelerate the packaging and amplification of recombinant adenoviruses. Thus, the engineered 293pTP cells should be a superior packaging line for efficient adenovirus production.

Monocyte chemotactic protein induced protein 1 (MCPIP1) fails to affect adipogenesis in immortalized mouse embryonic fibroblasts (iMEF) (LB149)

Background: Increased adipogenesis is associated with chronic inflammation. MCPIP1 has been shown to suppress inflammation through regulating pro‐inflammatory cytokines in the immune system. Recent studies found that MCPIP1 is involved in the process of adipogenesis in 3T3‐L1 cells but the results are controversial. Our preliminary data indicates that MCPIP1 knockout impaired hepatic insulin signaling. The current study was designed to deterimine if MCPIP1 affects adipogenesis by regulating the insulin signal transduction pathway. Methods: MEFs were isolated from both MCPIP1 heterozygous (no phenotypic changes) and knockout C57/BL6 mice and immortalized. At 2 days post‐confluency, the cells were treated with or without a differentiation cocktail containing dexamethasone, IBMX, trogliterazone, and different concentrations of insulin. After 48 hours, cells were switched to medium containing 10% FBS with or without the different concentrations of insulin for the remainder of the differentiation period. Adipocyte differentiation was measured using Oil Red‐O staining by visualizing the intracellular lipid droplets. Lipids were extracted with isopropanol. The concentration of lipid was measured, calculated, and expressed by normalizing to the protein concentration. Results: Although a slight increase in adipogenesis was seen in the MCPIP1 KO model compared to the heterozygous model, overall there was no significant difference between these groups (p>0.05). Also, no difference in adipogenesis was observed among the different concentrations of insulin groups in cells from either heterozygous or MCPIP1 KO mice (p>0.05). Conclusion: From the data we collected thus far, we conclude that MCPIP1 might not play a key role in adipogenesis in iMEFs. Insulin may not be required to stimulate adipogenesis in iMEFs isolated from MCPIP1 knockout and heterozygous mice.Grant Funding Source: Supported by ATSU‐KCOM Biomedical Sciences Program

The Parkinson’s gene PINK1 regulates cell cycle progression and promotes cancer-associated phenotypes

PINK1 (phosphatase and tensin homolog deleted on chromosome 10 (PTEN)-induced kinase 1), a Parkinson's disease-associated gene, was identified originally because of its induction by the tumor-suppressor PTEN. PINK1 promotes cell survival and potentially metastatic functions and protects against cell stressors including chemotherapeutic agents. However, the mechanisms underlying PINK1 function in cancer cell biology are unclear. Here, using several model systems, we show that PINK1 deletion significantly reduced cancer-associated phenotypes including cell proliferation, colony formation and invasiveness, which were restored by human PINK1 overexpression. Results show that PINK1 deletion causes major defects in cell cycle progression in immortalized mouse embryonic fibroblasts (MEFs) from PINK1(-/-) mice, and in BE(2)-M17 cells stably transduced with short hairpin RNA against PINK1. Detailed cell cycle analyses of MEF cell lines from several PINK1(-/-) mice demonstrate an increased proportion of cells in G2/M and decreased number of cells in G1 following release from nocodazole block. This was concomitant with increased double and multi-nucleated cells, a reduced ability to undergo cytokinesis and to re-enter G1, and significant alterations in cell cycle markers, including failure to increase cyclin D1, all indicative of mitotic arrest. PINK1(-/-) cells also demonstrated ineffective cell cycle exit following serum deprivation. Cell cycle defects associated with PINK1 deficiency occur at points critical for cell division, growth and stress resistance in cancer cells were rescued by ectopic expression of human PINK1 and demonstrated PINK1 kinase dependence. The importance of PINK1 for cell cycle control is further supported by results showing that cell cycle deficits induced by PINK1 deletion were linked mechanistically to aberrant mitochondrial fission and its regulation by dynamin-related protein-1 (Drp1), known to be critical for progression of mitosis. Our data indicate that PINK1 has tumor-promoting properties and demonstrates a new function for PINK1 as a regulator of the cell cycle.

Nuclear Structures Surrounding Internal Lamin Invaginations

A- and C-type lamins are intermediate filament proteins responsible for the maintenance of nuclear shape and most likely nuclear architecture. Here, we propose that pronounced invaginations of A/C-type lamins into the nuclear interior represent channels for the transport of regulatory molecules to and from nuclear and nucleolar regions. Using fluorescent protein technology and immunofluorescence, we show that A-type lamin channels interact with several nuclear components, including fibrillarin- and UBF-positive regions of nucleoli, foci of heterochromatin protein 1 β, polycomb group bodies, and genomic regions associated with DNA repair. Similar associations were observed between A/C-type lamin channels and nuclear pores, lamin-associated protein LAP2α, and promyelocytic leukemia nuclear bodies. Interestingly, regions with high levels of A/C-type lamins had low levels of B-type lamins, and vice versa. These characteristics were observed in primary and immortalized mouse embryonic fibroblasts as well as human and mouse embryonic stem cell colonies exhibiting stem cell-specific lamin positivity. Our findings indicate that internal channels formed by nuclear lamins likely contribute to normal cellular processes through association with various nuclear and nucleolar structures.

Cellular Mechanisms For Transferrin Receptor Endocytosis Vital For Terminal Erythroid Differentiation

Clathrin-mediated endocytosis (CME) is a key endocytic portal that regulates transferrin receptor (TfR) endocytosis. Although CME is among the most-studied cellular systems in cell biology, little is known regarding how this key system physiologically functions in vivo.The CALM gene (for Clathrin Assembly Lymphoid Myeloid, also known as PICALM), located on human chromosome 11q14, encodes a 652 aa protein containing multiple domains functioning in clathrin-coated vesicle formation. Since CALM is predominantly expressed in erythroblasts, and hematopoietic-specific Calmconditional knockout mice, which we developed, exhibit a severe iron-deficient anemia, we hypothesized that CALM functions to increase efficiency of TfR endocytosis in erythroblasts so that iron uptake is not rate-limiting. To test this hypothesis, we performed FACS-based endocytosis assay in primary erythroblasts, in vitro erythroid differentiation assay, live-cell imaging and CALM add-back rescue experiments.TfR endocytosis was severely attenuated in Calm-deficient basophilic- and polychromatophilic erythroblasts: efficiency of TfR endocytosis was less than 25% of that of control, as revealed by FACS-based endocytosis assay. Unexpectedly, TfR endocytosis was not active in orthochromatophilic erythroblasts regardless of genotype, while they express TfR at high levels.For live cell imaging, we established immortalized mouse embryonic fibroblasts (MEFs), in which the Calm gene can be deleted in an inducible manner (CalmF/F ERT2-Cre+ MEFs). The MEFs were then stably transduced with the retrovirus encoding an EGFP-tagged AP2-bound adaptin σ2. The dynamics of clathrin-coated vesicle formation was determined using fast live cell fluorescence spinning disk confocal microscopy. Clathrin-coat dynamics was barely affected in the absence of Calm under regular culture condition; however, it was completely stalled under high membrane tension (e.g. hypo-osmotic medium, jasplakinolide treatment). To determine functional domain(s) of CALM, a series of CALM mutants were generated, “added-back” to Calm-deficient MEFs and coat dynamics examined by live cell imaging. Mutant that does not bind to phosphatidylinositol 4,5-bisphosphate of the plasma membrane (PIP2-mutant CALM) failed to rescue TfR endocytosis in Calm-deficient MEFs, suggesting that CALM binding to PIP2, but not to v-SNAREs or EPS15, is essential for CALM-mediated TfR endocytosis.Splenic erythroblasts were significantly expanded in Calm conditional knockout mice. In contrast, Calm-deficient hematopoietic progenitors cannot give rise to immature erythroblasts in vitro, suggesting that Calm-deficiency is partially compensated in vivo via non-cell autonomous mechanisms (e.g. central macrophages). Iron supplement treatment could significantly rescue erythroid development of Calm-deficient progenitors in vitro, indicating that iron-deficiency is a primary cause of developmental defects seen in Calm-deficient erythroblasts. As expected, retrovirus-mediated expression of WT-Calm, but not PIP2-mutant, completely rescued erythroid development in Calm-deficient erythroblasts in vitro.Our data indicate that CALM plays a key role in clathrin-mediated endocytosis in a context-dependent (high membrane tension) and cell-type-specific (erythroblasts) manner. We propose that CALM is essential for transferrin uptake in erythroblasts by functioning as an erythroid-specific clathrin adaptor. Disclosures:No relevant conflicts of interest to declare.

Abstract 1673: Adiponectin induces autophagic cell death in breast cancer cells through SIRT1 mediated deacetylation of LKB1 leading to ATG1 activation.

Abstract Introduction: Obesity is an independent risk factor for the development of breast cancer. Molecular effects of obesity are mediated via perturbations in the adipocytokine profile. Adiponectin is widely considered as an adipocytokine with therapeutic potential as it inhibits growth of breast cancer cells but the mechanisms underlying growth inhibitory effects of adiponectin are still elusive. The present study was designed to systematically elucidate the underlying mechanisms by which adiponectin inhibits growth of breast cancer cells. Methods: Characteristic of autophagy were evaluated using transmission electron microscopy, acridine orange staining and western blot analysis of key effector molecules of autophagy. RT-PCR, western blot and immunofluorescence analysis were used to examine SIRT1 (Sirtuin 1), liver kinase B1 (LKB1), and AMP-activated protein kinase (AMPK) axes. Functional importance of AMPK activation and LKB1 overexpression in the biologic effects of adiponectin was examined by using AMPK-null and AMPK-wild type (WT) immortalized mouse embryonic fibroblasts (MEFs) and isogenic LKB1-knockdown cell line pairs. LKB1 immunoprecipitates were used to examine LKB1 deacetylation in response to adiponectin. Results: We provide strong evidence that adiponectin causes autophagy in breast cancer cells. Adiponectin treated breast cancer cells exhibit autophagosomes and major autophagosomal protein level changes including ATG1 (autophagy related 1) activation, cleavage of LC3 and suppression of p62 (p62/SQSTM1) expression. Adiponectin-induced autophagy leads to breast cancer cell death as evident by increased levels of cleaved-caspase 9, cleaved-PARP, and Tunel positivity. Adiponectin-mediated autophagy-induction as well as autophagic cell death is attenuated in the presence of autophagy inhibitors. Analysis of the underlying molecular mechanisms reveals that adiponectin treatment increases AMPK activation that is required for adiponectin-mediated ATG1 overexpression and phosphorylation. Intriguingly, we discover that adiponectin increases SIRT1 expression which deacetylates LKB1 leading to its activation. LKB1 knock-down inhibits adiponectin mediated autophagy induction as evidenced by modulations in major autophagosomal protein levels including ATG1. Analysis of breast tumors treated with adiponectin reveals significant increase in the levels of SIRT1, LKB1, phospho AMPK, phospho ATG1 as well as decreased p62. Conclusions: These data uncover a novel role of adiponectin as an inducer of autophagic cell death and provide the first in vitro and in vivo evidence of the integral role of the SIRT1-LKB1-ATG1 axis in adiponectin mediated autophagic cell death of breast cancer cells. Citation Format: Seung J. Chung, Neeraj Saxena, Dipali Sharma. Adiponectin induces autophagic cell death in breast cancer cells through SIRT1 mediated deacetylation of LKB1 leading to ATG1 activation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1673. doi:10.1158/1538-7445.AM2013-1673

Unraveling Complex Interplay between Heat Shock Factor 1 and 2 Splicing Isoforms

Chaperone synthesis in response to proteotoxic stress is dependent on a family of transcription factors named heat shock factors (HSFs). The two main factors in this family, HSF1 and HSF2, are co-expressed in numerous tissues where they can interact and form heterotrimers in response to proteasome inhibition. HSF1 and HSF2 exhibit two alternative splicing isoforms, called α and β, which contribute to additional complexity in HSF transcriptional regulation, but remain poorly examined in the literature. In this work, we studied the transcriptional activity of HSF1 and HSF2 splicing isoforms transfected into immortalized Mouse Embryonic Fibroblasts (iMEFs) deleted for both Hsf1 and Hsf2, under normal conditions and after proteasome inhibition. We found that HSF1α is significantly more active than the β isoform after exposure to the proteasome inhibitor MG132. Furthermore, we clearly established that, while HSF2 had no transcriptional activity by itself, short β isoform of HSF2 exerts a negative role on HSF1β-dependent transactivation. To further assess the impact of HSF2β inhibition on HSF1 activity, we developed a mathematical modelling approach which revealed that the balance between each HSF isoform in the cell regulated the strength of the transcriptional response. Moreover, we found that cellular stress such as proteasome inhibition could regulate the splicing of Hsf2 mRNA. All together, our results suggest that relative amounts of each HSF1 and HSF2 isoforms quantitatively determine the cellular level of the proteotoxic stress response.

Immobilization of Heparan Sulfate on Electrospun Meshes to Support Embryonic Stem Cell Culture and Differentiation *

As our understanding of what guides the behavior of multi- and pluripotent stem cells deepens, so too does our ability to utilize certain cues to manipulate their behavior and maximize their therapeutic potential. Engineered, biologically functionalized materials have the capacity to influence stem cell behavior through a powerful combination of biological, mechanical, and topographical cues. Here, we present the development of a novel electrospun scaffold, functionalized with glycosaminoglycans (GAGs) ionically immobilized onto the fiber surface. Bound GAGs retained the ability to interact with GAG-binding molecules and, crucially, presented GAG sulfation motifs fundamental to mediating stem cell behavior. Bound GAG proved to be biologically active, rescuing the neural differentiation capacity of heparan sulfate-deficient mouse embryonic stem cells and functioning in concert with FGF4 to facilitate the formation of extensive neural processes across the scaffold surface. The combination of GAGs with electrospun scaffolds creates a biomaterial with potent applicability for the propagation and effective differentiation of pluripotent stem cells.