Wild-type Mouse Embryonic Fibroblasts Research Articles

Deletion of the neural tube defect–associated gene Mthfd1l disrupts one-carbon and central energy metabolism in mouse embryos

One-carbon (1C) metabolism is a universal folate-dependent pathway essential for de novo purine and thymidylate synthesis, amino acid interconversion, universal methyl-donor production, and regeneration of redox cofactors. Homozygous deletion of the 1C pathway gene Mthfd1l encoding methylenetetrahydrofolate dehydrogenase (NADP+-dependent) 1-like, which catalyzes mitochondrial formate production from 10-formyltetrahydrofolate, results in 100% penetrant embryonic neural tube defects (NTDs), underscoring the central role of mitochondrially derived formate in embryonic development and providing a mechanistic link between folate and NTDs. However, the specific metabolic processes that are perturbed by Mthfd1l deletion are not known. Here, we performed untargeted metabolomics on whole Mthfd1l-null and wildtype mouse embryos in combination with isotope tracer analysis in mouse embryonic fibroblast (MEF) cell lines to identify Mthfd1l deletion-induced disruptions in 1C metabolism, glycolysis, and the TCA cycle. We found that maternal formate supplementation largely corrects these disruptions in Mthfd1l-null embryos. Serine tracer experiments revealed that Mthfd1l-null MEFs have altered methionine synthesis, indicating that Mthfd1l deletion impairs the methyl cycle. Supplementation of Mthfd1l-null MEFs with formate, hypoxanthine, or combined hypoxanthine and thymidine restored their growth to wildtype levels. Thymidine addition alone was ineffective, suggesting a purine synthesis defect in Mthfd1l-null MEFs. Tracer experiments also revealed lower proportions of labeled hypoxanthine and inosine monophosphate in Mthfd1l-null than in wildtype MEFs, suggesting that Mthfd1l deletion results in increased reliance on the purine salvage pathway. These results indicate that disruptions of mitochondrial 1C metabolism have wide-ranging consequences for many metabolic processes, including those that may not directly interact with 1C metabolism.

Potential role for Ext1-dependent heparan sulfate in regulating P311 gene expression in A549 carcinoma cells

BackgroundExostosin-1 (EXT1), a member of the EXT protein family, is indispensable for synthesis of heparan sulfate (HS) chains that bind to and modulate the signaling efficiency of numerous growth factor activities. We have previously shown that Ext1 mutated mouse embryonic fibroblasts produce short sulfated HS chains which dramatically influence tumor cell behavior in a 3-dimensional (3D) heterospheroid system composed of tumor cells and fibroblasts. MethodsIn this study, we have used both 2D co-culture and 3D heterospheroid models, consisting of human A549 carcinoma cells co-cultured with wild-type or Ext1-mutated mouse embryonic fibroblasts. Results and conclusionsGene expression profiling of differentially expressed genes in fibroblast/A549 heterospheroids identified P311 as a gene substantially down-regulated in A549 cells co-cultured with Ext1-mutated fibroblasts. In addition, we observed that the Ext1 mutants displayed reduced Tgf-β1 mRNA levels and lower levels of secreted active TGF-β protein. Re-introduction of Ext1 in the Ext1 mutant fibroblasts rescued the levels of Tgf-β1 mRNA, increased the amounts of secreted active TGF-β in these cells, as well as P311 mRNA levels in adjacent A549 cells. Accordingly, small interfering RNAs (siRNAs) against fibroblast Tgf-β1 reduced P311 expression in neighboring A549 tumor cells. Our data raises the possibility that fibroblast Ext1 levels play a role in P311 expression in A549/fibroblast co-culture through TGF-β1. General significanceThis study considers a possible novel mechanism of Ext1-regulated heparan sulfate structure in modifying tumor-stroma interactions through altering stromal tgf-ß1 expression.

ATG5 Promotes Death Signaling in Response to the Cyclic Depsipeptides Coibamide A and Apratoxin A.

Our understanding of autophagy and lysosomal function has been greatly enhanced by the discovery of natural product structures that can serve as chemical probes to reveal new patterns of signal transduction in cells. Coibamide A is a cytotoxic marine natural product that induces mTOR-independent autophagy as an adaptive stress response that precedes cell death. Autophagy-related (ATG) protein 5 (ATG5) is required for coibamide-induced autophagy but not required for coibamide-induced apoptosis. Using wild-type and autophagy-deficient mouse embryonic fibroblasts (MEFs) we demonstrate that coibamide-induced toxicity is delayed in ATG5−/− cells relative to ATG5+/+ cells. Time-dependent changes in annexin V staining, membrane integrity, metabolic capacity and caspase activation indicated that MEFs with a functional autophagy pathway are more sensitive to coibamide A. This pattern could be distinguished from autophagy modulators that induce acute ER stress (thapsigargin, tunicamycin), ATP depletion (oligomycin A) or mTORC1 inhibition (rapamycin), but was shared with the Sec61 inhibitor apratoxin A. Coibamide- or apratoxin-induced cell stress was further distinguished from the action of thapsigargin by a pattern of early LC3-II accumulation in the absence of CHOP or BiP expression. Time-dependent changes in ATG5-ATG12, PARP1 and caspase-3 expression patterns were consistent with the conversion of ATG5 to a pro-death signal in response to both compounds.

Cleavage of Arpin by Calpain as a Potential Regulation Mechanism of the Arp2/3 Complex and Cell Motility

Cell movement is mediated by cycles of actin polymerisation. A novel protein, Arpin, was discovered that has been suggested to decrease cell motility through competitive inhibition of WASP family proteins, the activators of the complex driving actin polymerisation. In preliminary studies, Arpin was found to inhibit the Arp2/3 complex (Gautreau and Blanchoin, personal communication). Arpin contains sequence homology to the Arp2/3-binding site of WASP proteins. Calpains, a family of intracellular calcium-dependent cysteine proteases, can be found near the plasma membrane and the concentration of calcium ion required for activation is decreased when calpain is bound to the plasma membrane in the presence of phospholipids (Leloup et al. 2010). The common localization of calpain and Arpin, along with the known contributions calpain has in regulating other cell motility proteins, makes calpain a likely candidate for Arpin regulation. By transfecting calpain wild-type (pz/pz), knockdown (p/p) and lentivirus rescue mouse embryonic fibroblasts with a plasmid containing the Arpin gene (c15orf38), I hope to show the presence of differential cleavage of Arpin by calpain in the wild-type cells compared to the calpain knock-down cells through Western blot analysis. Understanding how Arpin is regulated will provide a basis for further research in cell motility, which has contributions to cancer metastasis and other diseases.

Reciprocity of Action of Increasing Oct4 and Repressing p53 in Transdifferentiation of Mouse Embryonic Fibroblasts into Cardiac Myocytes.

p53 is a barrier to somatic cell reprogramming. Deletion or transient suppression of p53 increases the efficiency of reprogramming of somatic cells into induced pluripotent stem cells. Whether p53 represents an obstacle to a similar process transdifferentiation of somatic cells is unknown. However, it is predicted that inhibition of p53 would promote transdifferentiation of fibroblasts into cardiomyocytes. In this study, the effect of p53 on the capacity of cardiogenic transdifferentiation is evaluated using p53 wild-type (p53+/+), p53 heterozygous mutant (p53+/-), and p53 homozygous mutant (p53-/-) mouse embryonic fibroblasts (MEFs). Repression of p53 in MEFs increases the expression level of mesoderm transcription factors Brachyury (T) and MESP1. The cardiac-specific markers, Myh6 (Myosin, Heavy Chain 6), Myh7 (Myosin, Heavy Chain 7), and cTnI (cardiac muscle troponin I), show elevated expression in p53+/- and p53-/- MEFs compared with wild-type MEFs, but cardiac muscle troponin T (cTnT) showed a lower expression level when p53 was inhibited. After induction to cardiac differentiation, cTnT expression increased and markers of endoderm and ectoderm decreased in p53+/- and p53-/- MEFs. The effect of an important reprogramming factor Oct4 on cardiac transdifferentiation was also evaluated in the allelic series of p53 MEFs. We found that overexpression of Oct4 significantly enhanced Mesp1, Tbx5, and Isl1 expression in p53+/+ and p53+/- MEFs. Oct4 also enhanced cTnT expression in all three cell lines, especially in p53+/- MEFs. Thus, inhibition of p53 expression and viral expression of Oct4 both promote transdifferentiation of MEFs into cardiomyocytes, establishing reciprocity of action in the process.

DOCK1 inhibition suppresses cancer cell invasion and macropinocytosis induced by self-activating Rac1P29S mutation

Rac1 is a member of the Rho family of small GTPases that regulates cytoskeletal reorganization, membrane polarization, cell migration and proliferation. Recently, a self-activating mutation of Rac1, Rac1P29S, has been identified as a recurrent somatic mutation frequently found in sun-exposed melanomas, which possesses increased inherent GDP/GTP exchange activity and cell transforming ability. However, the role of cellular Rac1-interacting proteins in the transforming potential of Rac1P29S remains unclear. We found that the catalytic domain of DOCK1, a Rac-specific guanine nucleotide exchange factor (GEF) implicated in malignancy of a variety of cancers, can greatly accelerate the GDP/GTP exchange of Rac1P29S. Enforced expression of Rac1P29S induced matrix invasion and macropinocytosis in wild-type (WT) mouse embryonic fibroblasts (MEFs), but not in DOCK1-deficient MEFs. Consistently, a selective inhibitor of DOCK1 that blocks its GEF function suppressed the invasion and macropinocytosis in WT MEFs expressing Rac1P29S. Human melanoma IGR-1 and breast cancer MDA-MB-157 cells harbor Rac1P29S mutation and express DOCK1 endogenously. Genetic inactivation and pharmacological inhibition of DOCK1 suppressed their invasion and macropinocytosis. Taken together, these results indicate that DOCK1 is a critical regulator of the malignant phenotypes induced by Rac1P29S, and suggest that targeting DOCK1 might be an effective approach to treat cancers associated with Rac1P29S mutation.

Cold-inducible RNA-binding protein (CIRP) induces translation of the cell-cycle inhibitor p27Kip1.

The CDK inhibitor p27Kip1 plays a central role in controlling cell proliferation and cell-cycle exit. p27Kip1 protein levels oscillate during cell-cycle progression and are regulated by mitogen or anti-proliferative signaling. The abundance of the protein is frequently determined by post-transcriptional mechanisms including ubiquitin-mediated proteolysis and translational control. Here, we report that the cold-inducible RNA-binding protein (CIRP) selectively binds to the 5′ untranslated region of the p27Kip1 mRNA. CIRP is induced, modified and relocalized in response to various stress stimuli and can regulate cell survival and cell proliferation particularly during stress. Binding of CIRP to the 5′UTR of the p27Kip1 mRNA significantly enhanced reporter translation. In cells exposed to mild hypothermia, the induction of CIRP correlated with increased translation of a p27Kip1 5′UTR reporter and with the accumulation of p27Kip1 protein. shRNA-mediated CIRP knockdown could prevent the induction of translation. We found that p27Kip1 is central for the decreased proliferation at lower temperature, since p27Kip1 KO mouse embryonic fibroblasts (MEFs) hardly increased their doubling time in hypothermic conditions, whereas wild-type MEFs significantly delayed proliferation in response to cold stress. This suggests that the CIRP-dependent p27Kip1 upregulation during mild hypothermia contributes to the cold shock-induced inhibition of cell proliferation.

Inactivation of DNA-PK by knockdown DNA-PKcs or NU7441 impairs non-homologous end-joining of radiation-induced double strand break repair.

The DNA-dependent protein kinase (DNA-PK) complex plays a pivotal role in non-homologous end-joining (NHEJ) repair. We investigated the mechanism of NU7441, a highly selective DNA-PK inhibitor, in NHEJ-competent mouse embryonic fibroblast (MEF) cells and NHEJ-deficient cells and explored the feasibility of its application in radiosensitizing nasopharyngeal carcinoma (NPC) cells. We generated wild-type and DNA-PKcs−/− MEF cells. Clonogenic survival assays, flow cytometry, and immunoblotting were performed to study the effect of NU7441 on survival, cell cycle, and DNA repair. NU7441 profoundly radiosensitized wild-type MEF cells and SUNE-1 cells, but not DNA-PKcs−/− MEF cells. NU7441 significantly suppressed radiation-induced DSB repair post-irradiation through unrepaired and lethal DNA damage, the cell cycle arrest. The effect was associated with the activation of cell cycle checkpoints. The present study revealed a mechanism by which inhibition of DNA-PK sensitizes cells to irradiation suggesting that radiotherapy in combination with DNA-PK inhibitor is a promising paradigm for the management of NPC which merits further investigation.

Wound healing-related properties detected in an experimental model with a collagen gel contraction assay are affected in the absence of tenascin-X

Patients with tenascin-X (TNX)-deficient type Ehlers-Danlos syndrome (EDS) do not exhibit delayed wound healing, unlike classic type EDS patients, who exhibit mutations in collagen genes. Similarly, in TNX-knockout (KO) mice, wound closure of the skin is normal even though these mice exhibit a reduced breaking strength. Therefore, we speculated that the wound healing process may be affected in the absence of TNX. In this study, to investigate the effects of TNX absence on wound healing-related properties, we performed collagen gel contraction assays with wild-type (WT) and TNX-KO mouse embryonic fibroblasts (MEFs). Collagen gels with embedded TNX-KO MEFs showed significantly greater contraction than those containing WT MEFs. Subsequently, we assessed collagen gel contraction-related properties, such as the activities of matrix metalloproteinase (MMP)-2 and MMP-9 and the protein and mRNA expression levels of transforming growth factor β1 (TGF-β1) in the collagen gels. The activities of MMP-2 and MMP-9 and the expression level of TGF-β1 were elevated in the absence of TNX. Furthermore, filopodia-like protrusion formation, cell proliferation, migration, and collagen expression in MEFs were promoted in the absence of TNX. These results indicate that these wound healing-related properties are affected in a TNX-deficient extracellular environment.

Sequestosome1/p62 protects mouse embryonic fibroblasts against low-dose methylercury-induced cytotoxicity and is involved in clearance of ubiquitinated proteins

Methylmercury (MeHg) is a widely distributed environmental pollutant that causes a series of cytotoxic effects. However, molecular mechanisms underlying MeHg toxicity are not fully understood. Here, we report that sequestosome1/p62 protects mouse embryonic fibroblasts (MEFs) against low-dose MeHg cytotoxicity via clearance of MeHg-induced ubiquitinated proteins. p62 mRNA and protein expression in MEFs were temporally induced by MeHg exposure p62-deficient MEFs exhibited higher sensitivity to MeHg exposure compared to their wild-type (WT) counterparts. An earlier and higher level of accumulation of ubiquitinated proteins was detected in p62-deficient cells compared with WT MEFs. Confocal microscopy revealed that p62 and ubiquitinated proteins co-localized in the perinuclear region of MEFs following MeHg treatment. Further analysis of MEFs revealed that ubiquitinated proteins co-localized with LC3-positive puncta upon co-treatment with MeHg and chloroquine, an autophagy inhibitor. In contrast, there was minimal co-localization in p62-deficient MEFs. The present study, for the first time, examined the expression and distribution of p62 and ubiquitinated proteins in cells exposed to low-dose MeHg. Our findings suggest that p62 is crucial for cytoprotection against MeHg-induced toxicity and is required for MeHg-induced ubiquitinated protein clearance.

Peroxiredoxin I participates in the protection of reactive oxygen species-mediated cellular senescence.

Peroxiredoxin I (Prx I) plays an important role as a reactive oxygen species (ROS) scavenger in protecting and maintaining cellular homeostasis; however, the underlying mechanisms are not well understood. Here, we identified a critical role of Prx I in protecting cells against ROS-mediated cellular senescence by suppression of p16INK4a expression. Compared to wild-type mouse embryonic fibroblasts (WT-MEFs), Prx I−/− MEFs exhibited senescence-associated phenotypes. Moreover, the aged Prx I−/− mice showed an increased number of cells with senescence associated-β-galactosidase (SA-β-gal) activity in a variety of tissues. Increased ROS levels and SA-β-gal activity, and reduction of chemical antioxidant in Prx I−/− MEF further supported an essential role of Prx I peroxidase activity in cellular senescence that is mediated by oxidative stress. The up-regulation of p16INK4a expression in Prx I−/− and suppression by overexpression of Prx I indicate that Prx I possibly modulate cellular senescence through ROS/p16INK4a pathway.

MiR‑342‑5p promotes Zmpste24‑deficient mouse embryonic fibroblasts proliferation by suppressing GAS2.

Cellular senescence is an irreversible growth arrest of cells that maintain their metabolic activities. Premature senescence can be induced by different stress factors and occurs in mouse embryonic fibroblasts (MEFs) derived from Zmpste24 metalloproteinase-deficient mice, a progeria mouse model of Hutchinson-Gilford Progeria Syndrome. Previous studies have shown that miR-342-5p, an intronic microRNA (miRNA/miR) reportedly involved in ageing associated diseases, is downregulated in Zmpste24−/− MEFs. However, whether miR-342-5p is associated with the premature senescence phenotype of Zmpste24−/− MEFs remains unclear. Thus, the present study investigated the effects of miR-342-5p on cellular senescence and cell proliferation in Zmpste24−/− MEFs. The results showed that miR-342-5p overexpression ameliorated the cellular senescence phenotype to a certain extent, promoted cell proliferation and increased the G2+M cell cycle phase in Zmpste24−/− MEFs. Nonetheless, it was difficult to observe the opposite cell phenotypes in wild-type (WT) MEFs transfected with the miR-342-5p inhibitor. Growth-arrest-specific 2 (GAS2) was identified as a target gene of miR-342-5p in Zmpste24−/− MEFs. In addition, miR-342-5p was identified to be downregulated in WT MEFs during replicative senescence, while Gas2 was upregulated. Taken together, these findings suggest that downregulated miR-342-5p is involved in regulating cell proliferation and cell cycles in Zmpste24−/− MEFs by suppressing GAS2 in vitro.

Core fucose is critical for CD14-dependent Toll-like receptor 4 signaling.

Core fucosylation, a posttranslational modification of N-glycans, modifies several growth factor receptors and impacts on their ligand binding affinity. Core-fucose-deficient mice generated by ablating the α1,6 fucosyltransferase enzyme, Fut8, exhibit severe pulmonary emphysema, partly due to impaired macrophage function, similar to aged Toll-like receptor 4 (Tlr4)-deficient mice. We therefore suspect that a lack of core fucose affects the TLR4-dependent signaling pathway. Indeed, upon lipopolysaccharide stimulation, Fut8-deficient mouse embryonic fibroblasts (MEFs) produced similar levels of interleukin-6 but markedly reduced levels of interferon-β (IFN-β) compared with wild-type MEFs. Lectin blot analysis of the TLR4 signaling complex revealed that core fucosylation was specifically found on CD14. Even though similar levels of TLR4/myeloid differentiation factor 2 (MD2) activation and dimerization were observed in Fut8-deficient cells after lipopolysaccharide stimulation, internalization of TLR4 and CD14 was significantly impaired. Given that internalized TLR4/MD2 induces IFN-β production, impaired IFN-β production in Fut8-deficient cells is ascribed to impaired TLR4/MD2 internalization. These data show for the first time that glycosylation critically regulates TLR4 signaling.

NAC1 Regulates Somatic Cell Reprogramming by Controlling Zeb1 and E-cadherin Expression.

SummaryReprogramming somatic cells to induced pluripotent stem cells (iPSCs) is a long and inefficient process. A thorough understanding of the molecular mechanisms underlying reprogramming is paramount for efficient generation and safe application of iPSCs in medicine. While intensive efforts have been devoted to identifying reprogramming facilitators and barriers, a full repertoire of such factors, as well as their mechanistic actions, is poorly defined. Here, we report that NAC1, a pluripotency-associated factor and NANOG partner, is required for establishment of pluripotency during reprogramming. Mechanistically, NAC1 is essential for proper expression of E-cadherin by a dual regulatory mechanism: it facilitates NANOG binding to the E-cadherin promoter and fine-tunes its expression; most importantly, it downregulates the E-cadherin repressor ZEB1 directly via transcriptional repression and indirectly via post-transcriptional activation of the miR-200 miRNAs. Our study thus uncovers a previously unappreciated role for the pluripotency regulator NAC1 in promoting efficient somatic cell reprogramming.

The histone acetylation mediated by Gcn5 regulates the Hoxc11 gene expression in MEFs.

Hox genes are responsible for encoding transcription factors that are essential for anterior-posterior body patterning at early stages of embryogenesis. However, detailed mechanisms of Hox genes are yet to be defined. Protein kinase B alpha (Akt1) was previously identified as a possible upstream regulator of Hox genes. Furthermore, the Hoxc11 gene has been upregulated in Akt1 null (Akt1-/-) mouse embryonic fibroblasts (MEFs), while repressed in wild-type MEFs. In this study, we propose to investigate the role of Gcn5, a histone acetyltransferase, in the regulation of Hoxc11 expression in MEFs. We showed that the H3 lysine 9 acetylation (H3K9ac) status has the same correlation with Hoxc11 expression and reported that Gcn5 is associated with the upregulation of Hoxc11 expression through H3K9ac in Akt1-/- MEFs. Since Hoxc11 was upregulated through histone acetylation in Akt1-/- MEFs, a functional role of Gcn5 on Hoxc11 expression was analyzed in Akt1-/- MEFs treated with Gcn5 specific inhibitor or transfected with Gcn5-small interfering RNA (Gcn5-siRNA). When the expression of Hoxc11 was analyzed using RT-PCR and real-time PCR, the Hoxc11 mRNA level was found to be similar in both Akt1-/- MEFs and control-siRNA transfected Akt1-/- MEFs. However, the Hoxc11 expression level was decreased in Gcn5-inhibited or Gcn5-knockdown Akt1-/- MEFs. Additionally, to analyze Gcn5-mediated histone acetylation status, chromatin immunoprecipitation assay was carried out in Gcn5-siRNA-transfected Akt1-/- MEFs. The H3K9ac at the Hoxc11 locus was decreased in Gcn5-knockdown Akt1-/- MEFs compared to controls. Based on these findings, we conclude that Gcn5 regulates Hoxc11 gene expression through mediating site-specific H3K9 acetylation in Akt1-/- MEFs.

Abstract 3463: Mitotic regulation of the tumor suppressor phosphatase PHLPP1

Abstract The PH domain Leucine-rich repeat Protein Phosphatases (PHLPP) are tumor suppressors originally discovered for their ability to directly dephosphorylate and inactivate the pro-survival kinase Akt, a key transducer of growth factor signaling. In the decade since this discovery, a number of other PHLPP targets have been elucidated, but still little is known about the molecular mechanisms governing the function and regulation of PHLPP itself. Here we report that PHLPP1 is hyperphosphorylated during mitosis and that, additionally, PHLPP1 suppresses the phosphorylation of the mitotic kinase CDK1. Specifically, we show that PHLPP1 undergoes an electrophoretic mobility shift in mitotic cells that is lost when lysates are treated in vitro with lambda phosphatase, suggesting that PHLPP1 is phosphorylated during mitosis. In addition to a cell cycle-dependent change in its post-translational modifications, PHLPP1 suppresses the phosphorylation of the main mitotic cyclin dependent kinase, CDK1, at Tyr15, an inhibitory phosphorylation that must be removed in order for cells to progress through mitosis. First, Tyr15 phosphorylation is increased in phlpp1-/- mouse embryonic fibroblasts (MEFs) compared to wild-type MEFs. Second, pharmacological inhibition of PHLPP1 in wild-type MEFs results in an increase in Tyr15 phosphorylation. Activation of key mitotic kinases, such as CDK1, and inactivation of phosphatases is required for the large increase in protein phosphorylation that occurs during mitosis. As such, reversible protein phosphorylation plays a role in regulating proper progression through mitosis, which is essential as errors in mitosis can result in aneuploidy, a hallmark of cancer. Our data support a previously undescribed role for PHLPP1 in regulating mitotic progression, potentially providing novel insight into how PHLPP suppresses tumorigenesis. Citation Format: Agnieszka T. Grzechnik, Alexandra C. Newton. Mitotic regulation of the tumor suppressor phosphatase PHLPP1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3463. doi:10.1158/1538-7445.AM2017-3463

Abstract 5539: The phenotypic characterization of Lrig null mouse embryonic fibroblasts

Abstract The LRIG gene family consists of LRIG1, LRIG2 and LRIG3. The most studied of these is LRIG1, which is a tumor suppressor gene. The LRIG1 protein is downregulated in various types of cancer and it negatively regulates receptor tyrosine kinases including the EGF- and PDGF-receptors. Lrig1 also regulates stem cell homeostasis in mouse skin and intestine. The functions of LRIG2 and LRIG3 are less known, but some findings imply that they might antagonize LRIG1. Intriguingly, the sole LRIG homolog in C. elegans, Sma-10, positively regulates BMP signaling. Thus, it is not known to what degree the LRIG proteins have the same, overlapping, or opposing functions, and the molecular mechanisms involved are poorly characterized. To elucidate the functions of the three LRIG proteins we generated a series of wildtype and LRIG-null (Lrig1-/-;Lrig2-/-;Lrig3-/-) mouse embryonic fibroblast (MEF) lines. These cell lines were created from homozygous Lrig1flox/flox;Lrig2flox/flox;Lrig3flox/flox mouse embryos. The FLOXed Lrig genes were then deleted with CRE-recombinase, thereby creating Lrig-null MEFs. ddPCR analysis showed that the Lrig-null MEF lines were contaminated with less than 1% of cells carrying wildtype Lrig3 alleles. Lrig-null MEFs showed a higher proliferation rate and migrated more in trans-well chambers than the wildtype cells. These cell lines will also be useful to investigate the importance of Lrig proteins for other cellular functions such as, signal transduction, lipidomics, transcriptomics, secretomics, metabolomics, and oxidative stress sensitivity. We also transduced the Lrig-null cells with individual inducible Lrig-genes thus creating a powerful system for the examination of the biology of the specific Lrig proteins. Citation Format: Carl Herdenberg, Håkan Hedman. The phenotypic characterization of Lrig null mouse embryonic fibroblasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5539. doi:10.1158/1538-7445.AM2017-5539

The deubiquitinating enzyme Usp14 allosterically inhibits multiple proteasomal activities and ubiquitin-independent proteolysis

The proteasome-associated deubiquitinating enzyme Usp14/Ubp6 inhibits protein degradation by catalyzing substrate deubiquitination and by poorly understood allosteric actions. However, upon binding a ubiquitin chain, Usp14 enhances proteasomal degradation by stimulating ATP and peptide degradation. These studies were undertaken to clarify these seemingly opposite regulatory roles of Usp14 and their importance. To learn how the presence of Usp14 on 26S proteasomes influences its different activities, we compared enzymatic and regulatory properties of 26S proteasomes purified from wild-type mouse embryonic fibroblast cells and those lacking Usp14. The proteasomes lacking Usp14 had higher basal peptidase activity than WT 26S, and this activity was stimulated to a greater extent by adenosine 5'-O-(thiotriphosphate) (ATPγS) than with WT particles. These differences were clear even though Usp14 is present on only a minor fraction (30-40%) of the 26S in WT mouse embryonic fibroblast cells. Addition of purified Usp14 to the WT and Usp14-defficient proteasomes reduced both their basal peptidase activity and the stimulation by ATPγS. Usp14 inhibits these processes allosterically because a catalytically inactive Usp14 mutant also inhibited them. Proteasomes lacking Usp14 also exhibited greater deubiquitinating activity by Rpn11 and greater basal ATPase activity than WT particles. ATP hydrolysis by WT proteasomes is activated if they bind a ubiquitinated protein, which is loosely folded. Surprisingly, proteasomes lacking Usp14 could be activated by such proteins even without a ubiquitin chain present. Furthermore, proteasomes lacking Usp14 are much more active in degrading non-ubiquitinated proteins (e.g. Sic1) than WT particles. Thus, without a ubiquitinated substrate present, Usp14 suppresses multiple proteasomal activities, especially basal ATP consumption and degradation of non-ubiquitinated proteins. These allosteric effects thus reduce ATP hydrolysis by inactive proteasomes and nonspecific proteolysis and enhance proteasomal specificity for ubiquitinated proteins.

The Ubiquitin Ligase RNF125 Targets Innate Immune Adaptor Protein TRIM14 for Ubiquitination and Degradation.

Tripartite motif-containing 14 (TRIM14) is a mitochondrial adaptor that facilitates innate immune signaling. Upon virus infection, the expression of TRIM14 is significantly induced, which stimulates the production of type-I IFNs and proinflammatory cytokines. As excessive immune responses lead to harmful consequences, TRIM14-mediated signaling needs to be tightly balanced. In this study, we identify really interesting new gene-type zinc finger protein 125 (RNF125) as a negative regulator of TRIM14 in the innate antiviral immune response. Overexpression of RNF125 inhibits TRIM14-mediated antiviral response, whereas knockdown of RNF125 has the opposite effect. RNF125 interacts with TRIM14 and acts as an E3 ubiquitin ligase that catalyzes TRIM14 ubiquitination. RNF125 promotes K48-linked polyubiquitination of TRIM14 and mediates its degradation via the ubiquitin-proteasome pathway. Consequently, wild-type mouse embryonic fibroblasts show significantly reduced TRIM14 protein levels in late time points of viral infection, whereas TRIM14 protein is retained in RNF125-deficient mouse embryonic fibroblasts. Collectively, our data suggest that RNF125 plays a new role in innate immune response by regulating TRIM14 ubiquitination and degradation.

Identification of CD146 as a novel molecular actor involved in systemic sclerosis

We highlight for the first time that CD146/sCD146 is involved in fibrotic process during SSc. sCD146 could thus constitute a new biomarker to assess disease activity, and potentially a new target for therapeutic applications.