Impaired Signal Transduction Research Articles

Extracellular bimolecular fluorescence complementation for investigating membrane protein dimerization: a proof of concept using class B GPCRs.

Bimolecular fluorescence complementation (BiFC) methodology uses split fluorescent proteins to detect interactions between proteins in living cells. To date, BiFC has been used to investigate receptor dimerization by splitting the fluorescent protein between the intracellular portions of different receptor components. We reasoned that attaching these split proteins to the extracellular N-terminus instead may improve the flexibility of this methodology and reduce the likelihood of impaired intracellular signal transduction. As a proof-of-concept we used receptors for calcitonin gene-related peptide, which comprise heterodimers of either the calcitonin or calcitonin receptor-like receptor in complex with an accessory protein (receptor activity-modifying protein 1). We created fusion constructs in which split mVenus fragments were attached to either the C-termini or N-termini of receptor subunits. The resulting constructs were transfected into Cos7 and HEK293S cells, where we measured cAMP production in response to ligand stimulation, cell surface expression of receptor complexes, and BiFC fluorescence. Additionally, we investigated ligand-dependent internalization in HEK293S cells. We found N-terminal fusions were better tolerated with regards to cAMP signaling and receptor internalization. N-terminal fusions also allowed reconstitution of functional fluorescent mVenus proteins, however fluorescence yields were lower than with C-terminal fusion. Our results suggest that BiFC methodologies can be applied to the receptor N-terminus, thereby increasing the flexibility of this approach, and enabling further insights into receptor dimerization.

Vitronectin promotes insulin resistance in trophoblast cells by activating JNK in gestational diabetes mellitus.

Gestational diabetes mellitus (GDM) is a common disorder in the clinic, which may lead to severe detrimental outcomes both for mothers and infants. However, the underlying mechanisms for GDM are still not clear. In the present study, we performed label-free proteomics using placentas from GDM patients and normal controls. Vitronectin caused our attention among differentially expressed proteins due to its potential role in the pathological progression of GDM. Vitronectin was increased in the placentas of GDM patients, which was confirmed by Western blot analysis. Vitronectin represses insulin signal transduction in trophoblast cells, whereas the knockdown of vitronectin further potentiates insulin-evoked events. Neutralization of CD51/61 abolishes the repressed insulin signal transduction in vitronectin-treated trophoblast cells. Moreover, vitronectin activates JNK in a CD51/61-depedent manner. Inhibition of JNK rescues impaired insulin signal transduction induced by vitronectin. Overall, our data indicate that vitronectin binds CD51/61 in trophoblast cells to activate JNK, and thus induces insulin resistance. In this regard, increased expression of vitronectin is likely a risk factor for the pathological progression of GDM. Moreover, blockade of vitronectin production or its receptors (CD51/61) may have therapeutic potential for dealing with GDM.

Water channel Aquaporin 4 is required for T cell activation.

Abstract Aquaporins are a family of ubiquitously expressed transmembrane water channels. We have previously reported that Aquaporin 4 (AQP4) is expressed on T cells and that treatment with a small molecule AQP4 inhibitor significantly delays T cell mediated heart allograft rejection. The goal of this study was to identify mechanisms of prolonged graft survival following AQP4 blockade. Stimulation of murine WT T cells + AER-270 or AQP4 −/−T cells resulted in decreased expression of T cell activation markers CD25 and CD44, and reduced cell volume when compared to control WT T cells. In addition, the absence or inhibition of AQP4 also limited IL-2 production by T cells following stimulation. Intact AQP4 was also required for optimal IFNg production by T cells. Analogously, AQP4 inhibition significantly limited the ability of human T cells to proliferate following TCR stimulation. We then examined the role of AQP4 in TCR signal transduction and found that AQP4 was required for nuclear translocation of NF-KB and NFAT and for optimal calcium flux following TCR stimulation. Either the absence or inhibition of AQP4 impaired TCR signal transduction as early as phosphorylation of Lck (pY394), ZAP70 (pY319) and LAT (p171). AQP4 was previously shown to associate with the actin cytoskeleton in other cell types, we next tested its involvement in TCR polarization/capping. We found that loss of AQP4 resulted in significantly impaired TCR capping and dysfunctional post-TCR stimulation actin remodeling kinetics. Our findings reveal novel mechanisms underlying the importance of AQP4 in optimal T lymphocyte activation and identify AQP4 as a potential therapeutic target for preventing TCR-mediated T cell activation in solid organ transplantation and other diseases.

The Uterine Melatonergic Systems of AANAT and Melatonin Membrane Receptor 2 (MT2) Are Essential for Endometrial Receptivity and Early Implantation in Mice.

In the current study, using Aanat and Mt2 KO mice, we observed that the preservation of the melatonergic system is essential for successful early pregnancy in mice. We identified that aralkylamine N-acetyltransferase (AANAT), melatonin receptor 1A (MT1), and melatonin receptor 1B (MT2) were all expressed in the uterus. Due to the relatively weak expression of MT1 compared to AANAT and MT2, this study focused on AANAT and MT2. Aanat and Mt2 KO significantly reduced the early implantation sites and the abnormal morphology of the endometrium of the uterus. Mechanistical analysis indicated that the melatonergic system is the key player in the induction of the normal nidatory estrogen (E2) response for endometrial receptivity and functions by activating the STAT signaling pathway. Its deficiency impaired the interactions between the endometrium, the placenta, and the embryo. The reduction in melatonin production caused by Aanat KO and the impairment of signal transduction caused by Mt2 KO reduced the uterine MMP-2 and MMP-9 activity, resulting in a hyperproliferative endometrial epithelium. In addition, melatonergic system deficiency also increased the local immunoinflammatory reaction with elevated local proinflammatory cytokines leading to early abortion in the Mt2 KO mice compared to the WT mice. We believe that the novel data obtained from the mice might apply to other animals including humans. Further investigation into the interaction between the melatonergic system and reproductive effects in different species would be worthwhile.

Advanced Glycation End Products Effects on Adipocyte Niche Stiffness and Cell Signaling.

Adipose tissue metabolism under hyperglycemia results in Type II diabetes (T2D). To better understand how the adipocytes function, we used a cell culture that was exposed to glycation by adding intermediate carbonyl products, which caused chemical cross-linking and led to the formation of advanced glycation end products (AGEs). The AGEs increased the cells and their niche stiffness and altered the rheological viscoelastic properties of the cultured cells leading to altered cell signaling. The AGEs formed concomitant with changes in protein structure, quantified by spectroscopy using the 8-ANS and Nile red probes. The AGE effects on adipocyte differentiation were viewed by imaging and evidenced in a reduction in cellular motility and membrane dynamics. Importantly, the alteration led to reduced adipogenesis, that is also measured by qPCR for expression of adipogenic genes and cell signaling. The evidence of alteration in the plasma membrane (PM) dynamics (measured by CTxB binding and NP endocytosis), also led to the impairment of signal transduction and a decrease in AKT phosphorylation, which hindered downstream insulin signaling. The study, therefore, presents a new interpretation of how AGEs affect the cell niche, PM stiffness, and cell signaling leading to an impairment of insulin signaling.

Current Studies on Molecular Mechanisms of Insulin Resistance.

Diabetes is a metabolic disease that raises the risk of microvascular and neurological disorders. Insensitivity to insulin is a characteristic of type II diabetes, which accounts for 85-90 percent of all diabetic patients. The fundamental molecular factor of insulin resistance may be impaired cell signal transduction mediated by the insulin receptor (IR). Several cell-signaling proteins, including IR, insulin receptor substrate (IRS), and phosphatidylinositol 3-kinase (PI3K), have been recognized as being important in the impaired insulin signaling pathway since they are associated with a large number of proteins that are strictly regulated and interact with other signaling pathways. Many studies have found a correlation between IR alternative splicing, IRS gene polymorphism, the complicated regulatory function of IRS serine/threonine phosphorylation, and the negative regulatory role of p85 in insulin resistance and diabetes mellitus. This review brings up-to-date knowledge of the roles of signaling proteins in insulin resistance in order to aid in the discovery of prospective targets for insulin resistance treatment.

Sphingosine-1-phosphate controls endothelial sphingolipid homeostasis via ORMDL.

Disruption of sphingolipid homeostasis and signaling has been implicated in diabetes, cancer, cardiometabolic, and neurodegenerative disorders. Yet, mechanisms governing cellular sensing and regulation of sphingolipid homeostasis remain largely unknown. In yeast, serine palmitoyltransferase, catalyzing the first and rate-limiting step of sphingolipid de novo biosynthesis, is negatively regulated by Orm1 and 2. Lowering sphingolipids triggers Orms phosphorylation, upregulation of serine palmitoyltransferase activity and sphingolipid de novo biosynthesis. However, mammalian orthologs ORMDLs lack the N-terminus hosting the phosphosites. Thus, which sphingolipid(s) are sensed by the cells, and mechanisms of homeostasis remain largely unknown. Here, we identify sphingosine-1-phosphate (S1P) as key sphingolipid sensed by cells via S1PRs to maintain homeostasis. The increase in S1P-S1PR signaling stabilizes ORMDLs, restraining SPT activity. Mechanistically, the hydroxylation of ORMDLs at Pro137 allows a constitutive degradation of ORMDLs via ubiquitin-proteasome pathway, preserving SPT activity. Disrupting S1PR/ORMDL axis results in ceramide accrual, mitochondrial dysfunction, impaired signal transduction, all underlying endothelial dysfunction, early event in the onset of cardio- and cerebrovascular diseases. Our discovery may provide the molecular basis for therapeutic intervention restoring sphingolipid homeostasis.

The N-Linked Glycosylation Site N191 Is Necessary for PKA Signal Transduction in Eel Follicle-Stimulating Hormone Receptor.

The follicle-stimulating hormone receptor (FSHR) contains several N-linked glycosylation sites in its extracellular region. We conducted the present study to determine whether conserved glycosylated sites in eel FSHR are necessary for cyclic adenosine monophosphate (cAMP) signal transduction. We used site-directed mutagenesis to induce four mutations (N120Q, N191Q, N272Q, and N288Q) in the N-linked glycosylation sites of eel FSHR. In the eel FSHR wild-type (wt), the cAMP response was gradually increased in a dose-dependent manner (0.01-1500 ng/mL), displaying a high response (approximately 57.5 nM/104 cells) at the Rmax level. Three mutants (N120Q, N272Q, and N288Q) showed a considerably decreased signal transduction as a result of high-ligand treatment, whereas one mutant (N191Q) exhibited a completely impaired signal transduction. The expression level of the N191Q mutant was only 9.2% relative to that of the eel FSHR-wt, indicating a negligible expression level. The expression levels of the N120Q and N272Q mutants were approximately 35.9% and 24% of the FSHG-wt, respectively. The N288Q mutant had an expression level similar to that of the eel FSHR-wt, despite the mostly impaired cAMP responsiveness. The loss of the cell surface agonist-receptor complexes was very rapid in the cells expressing eel FSHR-wt and FSHR-N288Q mutants. Specifically, the N191Q mutant was completely impaired by the loss of cell surface receptors, despite treatment with a high concentration of the agonist. Therefore, we suggest that the N191 site is necessary for cAMP signal transduction. This finding implies that the cAMP response, mediated by G proteins, is directly related to the loss of cell surface receptors as a result of high-agonist treatment.

Skin Microhemodynamics and Mechanisms of Its Regulation in Type 2 Diabetes Mellitus.

The review presents modern ideas about peripheral microhemodynamics, approaches to the ana-lysis of skin blood flow oscillations and their diagnostic significance. Disorders of skin microhemodynamics in type 2 diabetes mellitus (DM) and the possibility of their interpretation from the standpoint of external and internal interactions between systems of skin blood flow regulation, based on a comparison of couplings in normal and pathological conditions, including models of pathologies on animals, are considered. The factors and mechanisms of vasomotor regulation, among them receptors and signaling events in endothelial and smooth muscle cells considered as models of microvessels are discussed. Attention was drawn to the disturbance of Ca2+-dependent regulation of coupling between vascular cells and NO-dependent regulation of vasodilation in diabetes mellitus. The main mechanisms of insulin resistance in type 2 DM are considered to be a defect in the number of insulin receptors and impaired signal transduction from the receptor to phosphatidylinositol-3-kinase and downstream targets. Reactive oxygen species plays an important role in vascular dysfunction in hyperglycemia. It is assumed that the considered molecular and cellular mechanisms of microhemodynamics regulation are involved in the formation of skin blood flow oscillations. Parameters of skin blood microcirculation can be used as diagnostic and prognostic markers for assessing the state of the body.

St. John's wort extract STW3-VI affects the binding behaviour, signal transduction and lateral mobility of 5-HT2A receptors observed by single molecule detection and the use of a novel fluorescence-labeled ligand

Background5-HT2A receptors play an important role in the development and therapy of depression. St. John's wort (SJW) extracts are used in the treatment of depression. The effects of pre-treatment with SJW extract on the behaviour of 5-HT2A receptors in the plasma membrane have not been well described at the molecular level. MethodsIn this work, the influence of the SJW extract STW3-VI on 5-HT2A receptor internalization and calcium response under stimulating conditions are determined using different reporter cell lines. Influences on 5-HT2A receptor binding as well as on the lateral diffusion behaviour of receptor-ligand complexes (RLC) in the plasma membrane are investigated by fluorescence correlation spectroscopy (FCS) and single particle tracking (SPT) in real time on living cells. Therefore, a selective fluorescently labeled ligand was first developed. ResultsSTW3-VI pre-treatment inhibited both the 5-HT2A receptor internalization and the calcium response after agonistic stimulation with (±)-TCB-2, which suggests an inhibitory effect. Receptor binding studies using FCS surprisingly showed a dose-dependent increase in 5-HT2A receptor binding under STW3-VI. However, it is noticeable that there was a clear redistribution towards faster diffusing RLC, who possibly contribute less to signal transduction. The diffusive states of 5-HT2A receptors identified by SPT changed also under STW3-VI. Remarkably, there was a redistribution from faster diffusing to immobile RLC, while at the same time the transition probabilities for a receptor to change the diffusive state decreased clearly compared to control cells. This particular lateral diffusion behaviour likely characterizes 5-HT2A receptors with impaired signal transduction. ConclusionThe present data indicate a correlation between receptor status and lateral mobility, with STW3-VI apparently having an inhibitory effect on 5-HT2A receptor-mediated signaling in the cell models used here. Single-molecule techniques such as FCS and SPT provide important data that contribute to a better understanding of therapeutically relevant drug-target interactions and downstream regulatory processes.

Constitutively Activating Mutants of Equine LH/CGR Constitutively Induce Signal Transduction and Inactivating Mutations Impair Biological Activity and Cell-Surface Receptor Loss In Vitro.

The signal transduction of the equine lutropin/choriogonadotropin receptor (eLH/CGR) is unclear in naturally occurring activating/inactivating mutants of this receptor, which plays an important role in reproductive physiology. We undertook the present study to determine whether conserved structurally related mutations in eLH/CGR exhibit similar mechanisms of signal transduction. We constructed four constitutively activating mutants (M398T, L457R, D564G, and D578Y) and three inactivating mutants (D405N, R464H, and Y546F); measured cyclic adenosine monophosphate (cAMP) accumulation via homogeneous time-resolved fluorescence assays in Chinese hamster ovary cells; and investigated cell-surface receptor loss using an enzyme-linked immunosorbent assay in human embryonic kidney 293 cells. The eLH/CGR-L457R-, -D564G-, and -D578Y-expressing cells exhibited 16.9-, 16.4-, and 11.2-fold increases in basal cAMP response, respectively. The eLH/CGR-D405N- and R464H-expressing cells presented a completely impaired signal transduction, whereas the Y546F-expressing cells exhibited a small increase in cAMP response. The cell-surface receptor loss was 1.4- to 2.4-fold greater in the activating-mutant-expressing cells than in wild-type eLH/CGR-expressing cells, but was completely impaired in the D405N- and Y546F-expressing cells, despite treatment with a high concentration of agonist. In summary, the state of activation of eLH/CGR influenced agonist-induced cell-surface receptor loss, which was directly related to the signal transduction of constitutively activating mutants.

Constitutive Activating Eel Luteinizing Hormone Receptors Induce Constitutively Signal Transduction and Inactivating Mutants Impair Biological Activity.

In contrast to the human lutropin receptor (hLHR) and rat LHR (rLHR), very few naturally occurring mutants in other mammalian species have been identified. The present study aimed to delineate the mechanism of signal transduction by three constitutively activating mutants (designated M410T, L469R, and D590Y) and two inactivating mutants (D383N and Y546F) of the eel LHR, known to be naturally occurring in human LHR transmembrane domains. The mutants were constructed and measured cyclic adenosine monophosphate (cAMP) accumulation via homogeneous time-resolved fluorescence assays in Chinese hamster ovary (CHO)-K1 cells. The activating mutant cells expressing eel LHR-M410T, L469R, and D590Y exhibited a 4.0-, 19.1-, and 7.8-fold increase in basal cAMP response without agonist treatment, respectively. However, inactivating mutant cells expressing D417N and Y558F did not completely impaired signal transduction. Specifically, signal transduction in the cells expressing activating mutant L469R was not occurred with a further ligand stimulation, showing that the maximal response exhibited approximately 53% of those of wild type receptor. Our results suggested that the constitutively activating mutants of the eel LHR consistently occurred without agonist treatment. These results provide important information of LHR function in fish and regulation with regard to mutations of highly conserved amino acids in glycoprotein hormone receptors.

From hair colour to diagnosis

Hair pigmentation contributes extremely to the visual appearance and human-to-human communication, thus exerting enormous sexual and social impact. Follicular melanogenesis depends on genetic, biochemical and physical phenomena as well as proper epithelial-melanocytic interaction. Shades of grey, yellow, brown, red, and black produced by melanin define the exact determination of hair colour. Versatile clinical conditions depend on genetic melanogenetic changes, pigment transfer to bulb keratinocytes defects and impaired signal transduction pathways. Herein, an update of recent scientific advances on follicular melanogenesis and its pathological-driven compartmental changes reflecting specific disease phenotypes, are presented.

Role of Chitinase 3-Like 1 Protein in the Pathogenesis of Hepatic Insulin Resistance in Nonalcoholic Fatty Liver Disease.

A recently discovered human glycoprotein, chitinase 3-like 1 (Chi3L1), may play a role in inflammation, tissue remodeling, and visceral fat accumulation. We hypothesize that Chi3L1 gene expression is important in the development of hepatic insulin resistance characterized by the generation of pAKT, pGSK, and pERK in wild type and Chi3L1 knockout (KO) murine liver following insulin stimulation. The Chi3L1 gene and protein expression was evaluated by Real Time PCR and ELISA; lipid accumulation in hepatocytes was also assessed. To alter Chi3L1 function, three different anti-Chi3L1 monoclonal antibodies (mAbs) were administered in vivo and effects on the insulin signaling cascade and hepatic lipid deposition were determined. Transmission of the hepatic insulin signal was substantially improved following KO of the CHi3L1 gene and there was reduced lipid deposition produced by a HFD. The HFD-fed mice exhibited increased Chi3L1 expression in the liver and there was impaired insulin signal transduction. All three anti-Chi3L1 mAbs partially restored hepatic insulin sensitivity which was associated with reduced lipid accumulation in hepatocytes as well. A KO of the Chi3L1 gene reduced lipid accumulation and improved insulin signaling. Therefore, Chi3L1 gene upregulation may be an important factor in the generation of NAFLD/NASH phenotype.

Constitutive Activation and Inactivation of Mutations Inducing Cell Surface Loss of Receptor and Impairing of Signal Transduction of Agonist-Stimulated Eel Follicle-Stimulating Hormone Receptor.

In the present study, we investigated the signal transduction of mutants of the eel follicle-stimulating hormone receptor (eelFSHR). Specifically, we examined the constitutively activating mutant D540G in the third intracellular loop, and four inactivating mutants (A193V, N195I, R546C, and A548V). To directly assess functional effects, we conducted site-directed mutagenesis to generate mutant receptors. We measured cyclic adenosine monophosphate (cAMP) accumulation via homogeneous time-resolved fluorescence assays in Chinese hamster ovary (CHO-K1) cells and investigated cell surface receptor loss using an enzyme-linked immunosorbent assay in human embryonic kidney (HEK) 293 cells. The cells expressing eelFSHR-D540G exhibited a 23-fold increase in the basal cAMP response without agonist treatment. The cells expressing A193V, N195I, and A548V mutants had completely impaired signal transduction, whereas those expressing the R546C mutant exhibited little increase in cAMP responsiveness and a small increase in signal transduction. Cell surface receptor loss in the cells expressing inactivating mutants A193V, R546C, and A548V was clearly slower than in the cell expressing the wild-type eelFSHR. However, cell surface receptor loss in the cells expressing inactivating mutant N195I decreased in a similar manner to that of the cells expressing the wild-type eelFSHR or the activating mutant D540G, despite the completely impaired cAMP response. These results provide important information regarding the structure–function relationships of G protein-coupled receptors during signal transduction.

A missense mutation sheds light on a novel structure-function relationship of RANKL.

The tumor necrosis factor (TNF)-like core domain of receptor activator of nuclear factor-κB ligand (RANKL) is a functional domain critical for osteoclast differentiation. One of the missense mutations identified in patients with osteoclast-poor autosomal recessive osteopetrosis (ARO) is located in residue methionine 199 that is replaced with lysine (M199K) amid the TNF-like core domain. However, the structure-function relationship of this mutation is not clear. Sequence-based alignment revealed that the fragment containing human M199 is highly conserved and equivalent to M200 in rat. Using site-directed mutagenesis, we generated three recombinant RANKL mutants M200K/A/E (M200s) by replacing the methionine 200 with lysine (M200K), alanine (M200A), and glutamic acid (M200E), representative of distinct physical properties. TRAcPstaining and bone pit assay showed that M200s failed to support osteoclast formation and bone resorption, accompanied by impaired osteoclast-related signal transduction. However, no antagonistic effect was found in M200s against wild-type rat RANKL. Analysis of the crystal structure of RANKL predicted that this methionine residue is located within the hydrophobic core of the protein, thus, likely to be crucial for protein folding and stability. Consistently, differential scanning fluorimetry analysis suggested that M200s were less stable. Western blot analysis analyses further revealed impaired RANKL trimerization by M200s. Furthermore, receptor-ligand binding assay displayed interrupted interaction of M200s to its intrinsic receptors. Collectively, our studies revealed the molecular basis of human M199-induced ARO and elucidated the indispensable role of rodent residue M200 (equivalent to human M199) for the RANKL function.

Yellow-fruited phenotype is caused by 573 bp insertion at 5' UTR of YFT1 allele in yft1 mutant tomato

The yft1 tomato mutant has a yellow-fruited phenotype controlled by a recessive gene of YFT1 allele, which has been shown by map-based cloning to be a homolog of ETHYLENE INSENSITIVE 2 (EIN2). Genetic lesion of YFT1 allele in yft1 is attributed to a 573 bp DNA fragment (IF573) insertion at 1,200 bp downstream of the transcription start site. Transcriptomic analysis revealed that YFT1 lesion resulted in 5,053 differentially expressed genes (DEGs) in yft1 pericarp compared with the M82 wild type cultivar. These were annotated as being involved in ethylene synthesis, chromoplast development, and carotenoid synthesis. The YFT1 lesion caused a reduction in its own transcript levels in yft1 and impaired ethylene emission and signal transduction, delayed chromoplast development and decreased carotenoid accumulation. The molecular mechanism underlying the downregulated YFT1 allele in yft1 was examined at both RNA and DNA levels. The IF573 event appeared to introduce two negative regulatory sequences located at -272 to -173 bp and -172 to -73 bp in the YFT1 allele promoter, causing alterative splicing due to introduction of aberrant splicing sites, and breaking upstream open reading frames (uORF) structure in the 5'-UTR. Those results a new provided insight into molecular regulation of color formation in tomato fruit.

Abstract 135: Increased expression of ganglioside GM2 correlates with aggressiveness of human pancreatic ductal adenocarcinoma

Abstract (a) Purpose: Gangliosides are molecules composed of glycosphingolipids linked with one or more sialic acids, and are known to be key components of lipid rafts, which act as platforms for signal transduction. Changes in ganglioside levels affect the expression of raft-associated proteins on the cell surface and lead to reduced membrane fluidity, thus, resulting in cellular dysfunction, such as impaired signal transduction. The ganglioside GM2 is one of the major series of gangliosides that has several biological functions, such as in cell adhesion and signal transduction. GM2 is highly expressed in several types of human malignant tumors, such as melanomas, gliomas, and neuroblastomas, but is absent or weakly expressed in normal tissues. However, the association between gangliosides and pancreatic ductal adenocarcinoma (PDAC) has not been elucidated yet. Thus, in this study, we aimed to clarify the expression and role of ganglioside GM2 in PDAC. (b) Experimental procedures: GM2 expression in eight human PDAC cell lines was examined by fluorescence-activated cell sorter (FACS) analysis. The morphology, growth, and stemness of GM2- and GM2+ sorted-cells were compared by transmission electron microscopy, growth assays, real-time PCR analysis of the markers of stemness, and sphere forming assays. Cell motility was evaluated by invasion assay. For comparing tumorigenicity between the GM2- and GM2+ sorted-cells, heterotopic implantations were performed. The expression levels and clinico-pathological roles of GM2 in patients with PDAC were examined by immunohistochemical analysis of 117 pancreatic tissue samples. (c) Results: The fraction of GM2+ cells was the highest (21.4%) in MIA PaCa-2 from among the eight cell lines, as revealed by FACS analysis. GM2-expressing MIA PaCa-2 cells had higher growth rates under adherent growth conditions. In 3D-culture, which enriches cancer stem cells (CSCs), most MIA PaCa-2 cells expressed GM2 and the cells responded to TGF-β1 treatment to promote invasion. Transplantation of the GM2-expressing cells into nude mice resulted in development of larger tumors as compared to that for control cells that did not express GM2. In the PDAC cases, GM2 expression was significantly associated with younger age, larger tumor size, advanced stage, and higher histological grade. (d) Conclusions: We show that a PDAC cell line overexpressing GM2 exhibits high growth rate and high tumor initiation in 2D-culture. Further, pancreatic CSC-like cells expressing GM2 exhibit responsiveness against TGF-β1, resulting in enhanced invasion in 3D-culture. Furthermore, GM2 expression is associated with the growth and advanced stage of human PDAC. Further studies will be required for the development of an early detection method for GM2-overexpressing pancreatic cancers and to develop novel therapeutic strategies targeting GM2. Citation Format: Norihiko Sasaki, Kenichi Hirabayashi, Masaki Michishita, Kimimasa Takahashi, Fumio Hasegawa, Fujiya Gomi, Yoko Itakura, Naoya Nakamura, Masashi Toyoda, Toshiyuki Ishiwata. Increased expression of ganglioside GM2 correlates with aggressiveness of human pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 135.

A Novel Glucocorticoid Receptor Mutation in Primary Generalized Glucocorticoid Resistance Disease

Objective: Primary generalized glucocorticoid resistance (PGGR) is a rare hereditary disease characterized by generalized partial target-tissue insensitivity to glucocorticoids. To date, few cases have been reported, and more cases, especially involving other races, are needed to fully understand this disease. Methods: This study presented a novel glucocorticoid receptor mutation in a PGGR pedigree. The index patient was a 14-year-old male with fatigue, hypokalemia, hypertension, and polyuria. Eleven family members were available for the genetic screen. Next-generation sequencing and Sanger sequencing were used to identify the mutation. We systematically investigated the molecular mechanism through which the mutation impaired glucocorticoid signal transduction in COS-7 cells. Results: The index patient carried a de novo homo-zygous mutation within exon 6 (c.1652C>A, p.551S>Y), whereas eight family members carrying a heterozygous mutation were all phenotypically silent. The affinity of the human glucocorticoid receptor (hGR) for the ligand was 1.97-fold lower in the patient than in the family members. Mutant hGRα (551Y) displayed a 3.2-fold reduction in its ability to transactivate glucocorticoid-responsive genes. When exposed to the same concentration of dexamethasone, hGRα (551Y) displayed a reduced ability to trans-locate into the nucleus and decreased levels of hGR dimer formation and could not effectively induce the glucocorticoid response element to regulate the transcription of related genes. After 2 years of dexamethasone treatment, the volume of the left and right adrenal glands of the index subject decreased by 55.6% and 32.4%, respectively. The pituitary volume decreased by 18.9%. During the 2-year follow-up, none of the heterozygous carriers developed hypertension or hypokalemia. Conclusion: We described a novel homozygous glucocorticoid receptor mutation causing PGGR. This homozygous mutation leads to hypertension and hypokalemia, but its heterozygous mutation has no relevant clinical symptoms. Abbreviations: ACTH = adrenocorticotropic hormone; DBD = DNA-binding domain; GR = glucocorticoid receptor; GRE = glucocorticoid response element; hGR = human glucocorticoid receptor; LBD = ligand-binding domain; PGGR = primary generalized glucocorticoid resistance.

Tumor Necrosis Factor‐α Activates Integrin Signaling in Vascular Smooth Muscle Cells via α5 Receptor

Tumor necrosis factor‐α (TNFα) signaling requires superoxide (O2•−) to be produced extracellularly by NADPH oxidase 1 (Nox1) in vascular smooth muscle cells (VSMCs). Extracellular superoxide dismutase (ecSOD) converts O2•− to hydrogen peroxide (H2O2). It binds to the cell surface by association with either heparin‐sulfate or fibulin‐5, an extracellular matrix protein that interacts with integrin receptors. We previously found that ecSOD knockdown (siRNA) impaired TNFα signal transduction as reflected by MAPK and NF‐κB activation. Targeting of ecSOD also inhibited TNFα‐dependent activation of focal adhesion kinase (FAK) which reflects integrin receptor activation. Here we investigated the mechanism by which TNFα activates integrin signaling in VSMC. We analyzed NF‐κB activation in the presence of selective blocking antibodies (Ab) for integrin receptor subtypes previously shown to be expressed in VSMCs including α4, α5, αv, β1, and β3. TNFα‐induced NF‐κB activation was not affected by blockade of integrin receptors α4, αv, β1, and β3. However blocking of α5 reduced TNFα‐induced NF‐κB activation (control IgG + TNFα 7.36 ± 0.7, anti‐α5 Ab + TNFα 4.78 ± 0.3 fold change, p < 0.05, n = 5). We next sought to determine if α5 integrin is associated with upstream steps of integrin signaling in VSMCs. FAK and integrin‐linked kinase (ILK) are downstream effectors of integrin receptor activation. After 10 min, TNFα induced phosphorylation of both FAK and Protein Kinase B (Akt) which was used as a surrogate for ILK activation. Blockade of α5 receptors decreased TNFα‐induced phosphorylation of both FAK and Akt. Thus, short term exposure of VSMCs to TNFα transactivates α5 integrins and subsequently FAK and ILK‐Akt. We speculate that ecSOD localizes TNFα‐induced H2O2 production to the vicinity of α5 integrins via physical association with fibulin‐5 and this redox environment affects activation of the receptor.