The Role of TRAPγ/SSR3 in Preproinsulin Translocation Into the Endoplasmic Reticulum.

In the endoplasmic reticulum (ER), the translocation-associated protein complex (TRAP), also called signal sequence receptor (SSR), includes four integral membrane proteins TRAPα/SSR1, TRAPß/SSR2, and TRAPδ/SSR4 with the bulk of their extramembranous portions primarily in the ER lumen, whereas the extramembranous portion of TRAPγ/SSR3 is primarily cytosolic. Individually diminished expression of either TRAPα/SSR1, TRAPß/SSR2, or TRAPδ/SSR4 mRNA is known in each case to lower TRAPα/SSR1 protein levels, leading to impaired proinsulin biosynthesis, whereas forced expression of TRAPα/SSR1 at least partially suppresses the proinsulin biosynthetic defect. Here, we report that diminished TRAPγ/SSR3 expression in pancreatic ß-cells leaves TRAPα/SSR1 levels unaffected while nevertheless inhibiting cotranslational and posttranslational translocation of preproinsulin into the ER. Crucially, acute exposure to high glucose leads to a rapid upregulation of both TRAPγ/SSR3 and proinsulin protein without change in the respective mRNA levels, as observed in cultured rodent ß-cell lines and confirmed in human islets. Strikingly, pancreatic ß-cells with suppressed TRAPγ/SSR3 expression are blocked in glucose-dependent upregulation of proinsulin (or insulin) biosynthesis. Most remarkably, overexpression of TRAPγ/SSR3 in control ß-cells raises proinsulin levels, even without boosting extracellular glucose. The data suggest the possibility that TRAPγ/SSR3 may fulfill a rate-limiting function in preproinsulin translocation across the ER membrane for proinsulin biosynthesis.

Latrophilin GPCR signaling mediates synapse formation.

Neural circuit assembly in the brain requires precise establishment of synaptic connections, but the mechanisms of synapse assembly remain incompletely understood. Latrophilins are postsynaptic adhesion-GPCRs that engage in trans-synaptic complexes with presynaptic teneurins and FLRTs. In mouse CA1-region neurons, Latrophilin-2 and Latrophilin-3 are essential for formation of entorhinal-cortex-derived and Schaffer-collateral-derived synapses, respectively. However, it is unknown whether latrophilins function as GPCRs in synapse formation. Here, we show that Latrophilin-2 and Latrophilin-3 exhibit constitutive GPCR activity that increases cAMP levels, which was blocked by a mutation interfering with G-protein and arrestin interactions of GPCRs. The same mutation impaired the ability of Latrophilin-2 and Latrophilin-3 to rescue the synapse-loss phenotype in Latrophilin-2 and Latrophilin-3 knockout neurons in vivo. Our results suggest that Latrophilin-2 and Latrophilin-3 require GPCR signaling in synapse formation, indicating that latrophilins promote synapse formation in the hippocampus by activating a classical GPCR-signaling pathway.

Differential activities of maize plant elicitor peptides as mediators of immune signaling and herbivore resistance.

Plant elicitor peptides (Peps) are conserved regulators of defense responses and models for the study of damage-associated molecular pattern-induced immunity. Although present as multigene families in most species, the functional relevance of these multigene families remains largely undefined. While Arabidopsis Peps appear largely redundant in function, previous work examining Pep-induced responses in maize (Zm) implied specificity of function. To better define the function of individual ZmPeps and their cognate receptors (ZmPEPRs), activities were examined by assessing changes in defense-associated phytohormones, specialized metabolites and global gene expression patterns, in combination with heterologous expression assays and analyses of CRISPR/Cas9-generated knockout plants. Beyond simply delineating individual ZmPep and ZmPEPR activities, these experiments led to a number of new insights into Pep signaling mechanisms. ZmPROPEP and other poaceous precursors were found to contain multiple active Peps, a phenomenon not previously observed for this family. In all, seven new ZmPeps were identified and the peptides were found to have specific activities defined by the relative magnitude of their response output rather than by uniqueness. A striking correlation was observed between individual ZmPep-elicited changes in levels of jasmonic acid and ethylene and the magnitude of induced defense responses, indicating that ZmPeps may collectively regulate immune output through rheostat-like tuning of phytohormone levels. Peptide structure-function studies and ligand-receptor modeling revealed structural features critical to the function of ZmPeps and led to the identification of ZmPep5a as a potential antagonist peptide able to competitively inhibit the activity of other ZmPeps, a regulatory mechanism not previously observed for this family.

Characterizing relaxin receptor expression and exploring relaxin's effect on tissue remodeling/fibrosis in the human bladder.

BACKGROUND: Relaxin is an endogenous protein that has been shown to have antifibrotic properties in various organ systems. There has been no characterization of relaxin's role in the human bladder. Our objective was to characterize relaxin receptor expression in the human bladder and assess relaxin's effect on tissue remodeling/fibrosis pathways in bladder smooth muscle cells. METHODS: Relaxin family peptide receptor 1 (RXFP1) and RXFP2 expression was assessed using quantitative reverse transcriptase-PCR (qRT-PCR) and immunohistochemistry (IHC) on primary bladder tissue. Primary human smooth muscle bladder cells were cultured and stimulated with various concentrations of relaxin. Western blot, qRTPCR, ELISA, and zymogram assays were used to analyze fibrosis/tissue remodeling pathway proteins. RESULTS: There was universal mRNA transcript detection and protein expression of relaxin receptors in primary bladder specimens. Immunohistochemistry demonstrated RXFP1 and RXFP2 localizing to both urothelial and smooth muscle cell layers of the bladder. 24 h of in vitro relaxin stimulation did not affect mRNA expression of selected proteins in human bladder smooth muscle cells. However, 48 h of in vitro relaxin stimulation resulted in upregulation of active (p = 0.004) and latent (p = 0.027) MMP-2 in cell lysate, and upregulation of active MMP-2 in supernatant (p = 0.04). There was a dose dependent relationship with increasing expression of MMP-2 with increasing relaxin concentration. Relaxin stimulation resulted in decreased levels of active and total TGF-ß1 in supernatant and extracellular matrix (p < 0.005 with 100 ng/mL relaxin stimulation). CONCLUSIONS: In the human bladder, relaxin receptors are expressed at the dome and trigone and localize to the urothelium and smooth muscle cell layers. Stimulation of human bladder SMCs with relaxin in vitro affects expression of MMP-2 and TGF-ß1.

AT1R-AT2R-RXFP1 Functional Crosstalk in Myofibroblasts: Impact on the Therapeutic Targeting of Renal and Cardiac Fibrosis.

BACKGROUND: Recombinant human relaxin-2 (serelaxin), which has organ-protective actions mediated via its cognate G protein-coupled receptor relaxin family peptide receptor 1 (RXFP1), has emerged as a potential agent to treat fibrosis. Studies have shown that serelaxin requires the angiotensin II (AngII) type 2 receptor (AT2R) to ameliorate renal fibrogenesis in vitro and in vivo. Whether its antifibrotic actions are affected by modulation of the AngII type 1 receptor (AT1R), which is expressed on myofibroblasts along with RXFP1 and AT2R, is unknown. METHODS: We examined the signal transduction mechanisms of serelaxin when applied to primary rat renal and human cardiac myofibroblasts in vitro, and in three models of renal- or cardiomyopathy-induced fibrosis in vivo. RESULTS: The AT1R blockers irbesartan and candesartan abrogated antifibrotic signal transduction of serelaxin via RXFP1 in vitro and in vivo. Candesartan also ameliorated serelaxin's antifibrotic actions in the left ventricle of mice with cardiomyopathy, indicating that candesartan's inhibitory effects were not confined to the kidney. We also demonstrated in a transfected cell system that serelaxin did not directly bind to AT1Rs but that constitutive AT1R-RXFP1 interactions could form. To potentially explain these findings, we also demonstrated that renal and cardiac myofibroblasts expressed all three receptors and that antagonists acting at each receptor directly or allosterically blocked the antifibrotic effects of either serelaxin or an AT2R agonist (compound 21). CONCLUSIONS: These findings have significant implications for the concomitant use of RXFP1 or AT2R agonists with AT1R blockers, and suggest that functional interactions between the three receptors on myofibroblasts may represent new targets for controlling fibrosis progression.

Converging physiological roles of the anthrax toxin receptors.

The anthrax toxin receptors-capillary morphogenesis gene 2 (CMG2) and tumor endothelial marker 8 (TEM8)-were identified almost 20 years ago, although few studies have moved beyond their roles as receptors for the anthrax toxins to address their physiological functions. In the last few years, insight into their endogenous roles has come from two rare diseases: hyaline fibromatosis syndrome, caused by mutations in CMG2, and growth retardation, alopecia, pseudo-anodontia, and optic atrophy (GAPO) syndrome, caused by loss-of-function mutations in TEM8. Although CMG2 and TEM8 are highly homologous at the protein level, the difference in disease symptoms points to variations in the physiological roles of the two anthrax receptors. Here, we focus on the similarities between these receptors in their ability to regulate extracellular matrix homeostasis, angiogenesis, cell migration, and skin elasticity. In this way, we shed light on how mutations in these two related proteins cause such seemingly different diseases and we highlight the existing knowledge gaps that could form the focus of future studies.

Transcriptomics of a KDELR1 knockout cell line reveals modulated cell adhesion properties.

KDEL receptors (KDELRs) represent transmembrane proteins of the secretory pathway which regulate the retention of soluble ER-residents as well as retrograde and anterograde vesicle trafficking. In addition, KDELRs are involved in the regulation of cellular stress response and ECM degradation. For a deeper insight into KDELR1 specific functions, we characterised a KDELR1-KO cell line (HAP1) through whole transcriptome analysis by comparing KDELR1-KO cells with its respective HAP1 wild-type. Our data indicate more than 300 significantly and differentially expressed genes whose gene products are mainly involved in developmental processes such as cell adhesion and ECM composition, pointing out to severe cellular disorders due to a loss of KDELR1. Impaired adhesion capacity of KDELR1-KO cells was further demonstrated through in vitro adhesion assays, while collagen- and/or laminin-coating nearly doubled the adhesion property of KDELR1-KO cells compared to wild-type, confirming a transcriptional adaptation to improve or restore the cellular adhesion capability. Perturbations within the secretory pathway were verified by an increased secretion of ER-resident PDI and decreased cell viability under ER stress conditions, suggesting KDELR1-KO cells to be severely impaired in maintaining cellular homeostasis.

Tango knock-ins visualize endogenous activity of G protein-coupled receptors in Drosophila.

G protein-coupled receptors (GPCRs) represent a family of seven-pass transmembrane protein receptors whose ligands include neuropeptides and small-molecule neuromodulators such as dopamine and serotonin. These neurotransmitters act at long distances and are proposed to define the ground state of the nervous system. The Drosophila genome encodes approximately 50 neuropeptides and their functions in physiology and behavior are now under intensive studies. Key information currently lacking in the field is the spatiotemporal activation patterns of endogenous GPCRs. Here we report application of the Tango system, a reporter assay to detect GPCR activity, to endogenous GPCRs in the fly genome. We developed a method to integrate the sensor component of the Tango system to the C-terminus of endogenous genes by using genome editing techniques. We demonstrate that Tango sensors in the Sex-peptide receptor (SPR) locus allow sensitive detection of mating-dependent SPR activity in the female reproductive organ. The method is easily applicable to any GPCR and will provide a way to systematically characterize GPCRs in the fly brain.

Dissociation of impulsivity and aggression in mice deficient for the ADHD risk gene Adgrl3: Evidence for dopamine transporter dysregulation.

Adhesion G protein-coupled receptor L3 (ADGRL3, LPHN3) has putative roles in neuronal migration and synapse function. Various polymorphisms in ADGRL3 have been linked with an increased risk of attention deficit/hyperactivity disorder (ADHD). In this study, we examined the characteristics of Adgrl3-deficient mice in multiple behavioural domains related to ADHD: locomotive activity, impulsivity, gait, visuospatial and recognition memory, sociability, anxiety-like behaviour and aggression. Additionally, we investigated the effect of Adgrl3-depletion at the transcriptomic level by RNA-sequencing three ADHD-relevant brain regions: prefrontal cortex (PFC), hippocampus and striatum. Adgrl3-/- mice show increased locomotive activity across all tests and subtle gait abnormalities. These mice also show impairments across spatial memory and learning domains, alongside increased levels of impulsivity and sociability with decreased aggression. However, these alterations were absent in Adgrl3+/- mice. Across all brain regions tested, the numbers of genes found to exhibit differential expression was relatively small, indicating a specific pathway of action, rather than a broad neurobiological perturbation. Gene-set analysis of differential expression in the PFC detected a number of ADHD-relevant pathways including dopaminergic synapses as well as cocaine and amphetamine addiction. The Slc6a3 gene coding for the dopamine transporter was the most dysregulated gene in the PFC. Unexpectedly, several neurohormone/peptides which are typically only expressed in the hypothamalus were found to be dysregulated in the striatum. Our study further validates Adgrl3 constitutive knockout mice as an experimental model of ADHD while providing neuroanatomical targets for future studies involving ADGRL3 modified models. This article is part of the Special Issue entitled 'Current status of the neurobiology of aggression and impulsivity'.

ADGRL3 rs6551665 as a Common Vulnerability Factor Underlying Attention-Deficit/Hyperactivity Disorder and Autism Spectrum Disorder.

Neurodevelopmental disorders are prevalent, frequently occur in comorbidity and share substantial genetic correlation. Previous evidence has suggested a role for the ADGRL3 gene in Attention-Deficit/Hyperactivity Disorder (ADHD) susceptibility in several samples. Considering ADGRL3 functionality in central nervous system development and its previous association with neurodevelopmental disorders, we aimed to assess ADGRL3 influence in early-onset ADHD (before 7 years of age) and Autism Spectrum Disorder (ASD). The sample comprises 187 men diagnosed with early-onset ADHD, 135 boys diagnosed with ASD and 468 male blood donors. We tested the association of an ADGRL3 variant (rs6551665) with both early-onset ADHD and ASD susceptibility. We observed significant associations between ADGRL3-rs6551665 on ADHD and ASD susceptibilities; we found that G-carriers were at increased risk of ADHD and ASD, in accordance with previous studies. The overall evidence from the literature, corroborated by our results, suggests that ADGRL3 might be involved in brain development, and genetic modifications related to it might be part of a shared vulnerability factor associated with the underlying neurobiology of neurodevelopmental disorders such as ADHD and ASD.

Sex peptide receptor-regulated polyandry modulates the balance of pre- and post-copulatory sexual selection in Drosophila.

Polyandry prolongs sexual selection on males by forcing ejaculates to compete for fertilisation. Recent theory predicts that increasing polyandry may weaken pre-copulatory sexual selection on males and increase the relative importance of post-copulatory sexual selection, but experimental tests of this prediction are lacking. Here, we manipulate the polyandry levels in groups of Drosophila melanogaster by deletion of the female sex peptide receptor. We show that groups in which the sex-peptide-receptor is absent in females (SPR-) have higher polyandry, and - as a result - weaker pre-copulatory sexual selection on male mating success, compared to controls. Post-copulatory selection on male paternity share is relatively more important in SPR- groups, where males gain additional paternity by mating repeatedly with the same females. These results provide experimental evidence that elevated polyandry weakens pre-copulatory sexual selection on males, shifts selection to post-copulatory events, and that the sex peptide pathway can play a key role in modulating this process in Drosophila.

Lect2 Controls Inflammatory Monocytes to Constrain the Growth and Progression of Hepatocellular Carcinoma.

Leukocyte cell-derived chemotaxin-2 (LECT2) was originally identified as a hepatocyte-secreted chemokine-like factor and a positive target of ß-catenin signaling. Here, we dissected out the mechanisms by which LECT2 modulates hepatocellular carcinoma (HCC) development using both HCC mouse models and human HCC samples. We have demonstrated that LECT2 exhibits dual abilities as it has profound repercussions on the tumor phenotype itself and the immune microenvironment. Its absence confers Ctnnb-1-mutated tumor hepatocytes a stronger ability to undergo epithelial to mesenchymal transition and fosters the accumulation of pejorative inflammatory monocytes harboring immunosuppressive properties and strong tumor-promoting potential. Consistent with our HCC mouse model, a low level of LECT2 in human HCC is strongly associated with high tumor grade and the presence of inflammatory infiltrates, emphasizing the clinical value of LECT2 in human liver tumorigenesis. Conclusion: Our findings have demonstrated that LECT2 is a key player in liver tumorigenesis because its absence reshapes the tumor microenvironment and the tumor phenotype, revealing LECT2 as a promising immunotherapeutic option for HCC.

Luteal and hypophyseal expression of the canine relaxin (RLN) system during pregnancy: Implications for luteotropic function.

By acting through its receptors (RXFP1, RXFP2), relaxin (RLN) exerts species-specific effects during pregnancy; possible luteotropic effects through stimulation of prolactin (PRL) release have been suggested. In the domestic dog (Canis lupus familiaris) serum PRL increases in pregnant bitches shortly after RLN appears in the circulation, and a possible functional relationship between the RLN and the PRL systems in regulating progesterone secretion has been implied. Therefore, here (Study 1) the luteal expression and localization of the RLN system was investigated by immunohistochemistry using custom-made antibodies and semi-quantitative PCR, at selected time points during gestation: pre-implantation (d. 8-12), post-implantation (d. 18-25), mid-gestation (d. 35-40) and at normal and antigestagen-induced luteolysis. Further, (Study 2) hypophyseal expression of the RLN system and its spatial association with PRL was assessed. Luteal expression of RLN, but not of its receptors, was time-dependent: it increased significantly following implantation towards mid-gestation and decreased at prepartum. Antigestagen treatment resulted in downregulation of RLN and RXFP2. Whereas RLN was localized in steroidogenic cells, RXFP1 and RXFP2 also stained strongly in macrophages and vascular endothelial cells. The RLN system was detected in the canine adenohypophysis and was co-localized with PRL in hypophyseal lactotrophs. The intraluteal RLN seems to be involved in regulating the canine corpus luteum (CL) in a time-dependent manner. The presence of RLN family members in the adenohypophysis implies their possible involvement in regulating the availability of PRL and other pituitary hormones.

CMG2/ANTXR2 regulates extracellular collagen VI which accumulates in hyaline fibromatosis syndrome.

Loss-of-function mutations in capillary morphogenesis gene 2 (CMG2/ANTXR2), a transmembrane surface protein, cause hyaline fibromatosis syndrome (HFS), a severe genetic disorder that is characterized by large subcutaneous nodules, gingival hypertrophy and severe painful joint contracture. Here we show that CMG2 is an important regulator of collagen VI homoeostasis. CMG2 loss of function promotes accumulation of collagen VI in patients, leading in particular to nodule formation. Similarly, collagen VI accumulates massively in uteri of Antxr2-/- mice, which do not display changes in collagen gene expression, and leads to progressive fibrosis and sterility. Crossing Antxr2-/- with Col6a1-/- mice leads to restoration of uterine structure and reversion of female infertility. We also demonstrate that CMG2 may act as a signalling receptor for collagen VI and mediates its intracellular degradation.

Human relaxins (RLNH1, RLNH2), their receptor (RXFP1) and fetoplacental growth.

Relaxin, a systemic and placental hormone, has potential roles in fetoplacental growth. Human placenta expresses two RLN genes, RLNH1 and RLNH2 Maternal obesity is common and is associated with abnormal fetal growth. Our aims were to relate systemic and cord blood RLNH2, placental RLNs and their receptor (RXFP1) with fetoplacental growth in context of maternal body mass index, and associations with insulin-like growth factor 2 (IGF2) and vascular endothelial growth factor A (VEGFA) in the same placentas. Systemic, cord blood and placental samples were collected prior to term labor, divided by prepregnancy body mass index: underweight/normal (N = 25) and overweight/obese (N = 44). Blood RLNH2 was measured by ELISA; placental RLNH2, RLNH1, RXFP1, IGF2 and VEGFA were measured by quantitative immunohistochemistry and mRNAs were measured by quantitative reverse transcription PCR. Birthweight increased with systemic RLNH2 only in underweight/normal women (P = 0.036). Syncytiotrophoblast RLNH2 was increased in overweight/obese patients (P = 0.017) and was associated with placental weight in all subjects (P = 0.038). RLNH1 had no associations with birthweight or placental weight, but was associated with increased trophoblast and endothelial IGF2 and VEGFA, due to female fetal sex. Thus, while systemic RLNH2 may be involved in birthweight regulation in underweight/normal women, placental RLNH2 in all subjects may be involved in placental weight. A strong association of trophoblast IGF2 with birthweight and placental weight in overweight/obese women suggests its importance. However, an association of only RLNH1 with placental IGF2 and VEGFA was dependent upon female fetal sex. These results suggest that both systemic and placental RLNs may be associated with fetoplacental growth.

Sexual conflict over remating interval is modulated by the sex peptide pathway.

Sexual conflict, in which the evolutionary interests of males and females diverge, shapes the evolution of reproductive systems across diverse taxa. Here, we used the fruit fly to study sexual conflict in natural, three-way interactions comprising a female, her current and previous mates. We manipulated the potential for sexual conflict by using sex peptide receptor (SPR) null females and by varying remating from 3 to 48 h, a period during which natural rematings frequently occur. SPR-lacking females do not respond to sex peptide (SP) transferred during mating and maintain virgin levels of high receptivity and low fecundity. In the absence of SPR, there was a convergence of fitness interests, with all individuals gaining highest productivity at 5 h remating. This suggests that the expression of sexual conflict was reduced. We observed an unexpected second male-specific advantage to early remating, resulting from an increase in the efficiency of second male sperm use. This early window of opportunity for exploitation by second males depended on the presence of SPR The results suggest that the SP pathway can modulate the expression of sexual conflict in this system, and show how variation in the selective forces that shape conflict and cooperation can be maintained.

Overactivation of hedgehog signaling in the developing Müllerian duct interferes with duct regression in males and causes subfertility.

The influence of the hedgehog signaling pathway on reproduction was studied in transgenic mice in which a dominant active allele of the hedgehog signal transducer, smoothened (Smo), was conditionally expressed in the developing Müllerian duct and gonads through recombination mediated by anti-Müllerian hormone receptor 2-cre (Amhr2cre ). Previous studies showed that development of the oviduct and uterus are abnormal in female Amhr2cre/+SmoM2 mice. In the current study, focusing on mutant males, litter size was reduced 53% in crosses with wild-type females. An extra band of undifferentiated tissue extended along each epididymis and vas deferens, a position suggesting derivation from Müllerian ducts that failed to regress fully. Hedgehog signaling was elevated in this tissue, based on mRNA levels of target genes. Amhr2 mRNA was dramatically reduced in the uterus of mutant females and in the extra tissue in the tract of mutant males, suggesting that AMHR2 signaling was inadequate for complete Müllerian duct regression. Spermatogenesis and sperm motility were normal, but testis weight was reduced 37% and epididymal sperm number was reduced 36%. The number of sperm recovered from the uteri of wild-type females after mating with mutant males was reduced 78%. This suggested that sperm transport through the male tract was reduced, resulting in fewer sperm in the ejaculate. Consistent with this, mutant males had unusually tortuous vas deferentia with constrictions within the lumen. We concluded that persistence of a relatively undifferentiated remnant of Müllerian tissue is sufficient to cause subtle changes in the male reproductive tract that reduce fertility.

Loss of BOSS Causes Shortened Lifespan with Mitochondrial Dysfunction in Drosophila.

Aging is a universal process that causes deterioration in biological functions of an organism over its lifetime. There are many risk factors that are thought to contribute to aging rate, with disruption of metabolic homeostasis being one of the main factors that accelerates aging. Previously, we identified a new function for the putative G-protein-coupled receptor, Bride of sevenless (BOSS), in energy metabolism. Since maintaining metabolic homeostasis is a critical factor in aging, we investigated whether BOSS plays a role in the aging process. Here, we show that BOSS affects lifespan regulation. boss null mutants exhibit shortened lifespans, and their locomotor performance and gut lipase activity-two age-sensitive markers-are diminished and similar to those of aged control flies. Reactive oxygen species (ROS) production is also elevated in boss null mutants, and their ROS defense system is impaired. The accumulation of protein adducts (advanced lipoxidation end products [ALEs] and advanced glycation end products [AGEs]) caused by oxidative stress are elevated in boss mutant flies. Furthermore, boss mutant flies are sensitive to oxidative stress challenges, leading to shortened lives under oxidative stress conditions. Expression of superoxide dismutase 2 (SOD2), which is located in mitochondria and normally regulates ROS removal, was decreased in boss mutant flies. Systemic overexpression of SOD2 rescued boss mutant phenotypes. Finally, we observed that mitochondrial mass was greater in boss mutant flies. These results suggest that BOSS affects lifespan by modulating the expression of a set of genes related to oxidative stress resistance and mitochondrial homeostasis.

ANTXR-1 and -2 independent modulation of a cytotoxicity mediated by anthrax toxin in human cells.

Several animal models have shown that anthrax toxin (ATX) elicits a cytotoxic effect on host cells through anthrax toxin receptor (ANTXR) function. In this study, compared with mouse cells, cells obtained from humans exhibited low sensitivity to ATX-mediated cytotoxicity, and the sensitivity was not correlated with expression levels of ANTXRs. ATX treatment also induced a cytotoxic effect in other cultured human cells, human embryonic kidney (HEK) 293 cells, that express ANTXRs at undetectable levels. Furthermore, ectopic expression of ANTXRs in HEK293 cells did not affect the sensitivity to ATX treatment. These findings suggest that there is an ANTXR-independent cytotoxic mechanism in human cells.

Functional Analysis of Novel Candidate Regulators of Insulin Secretion in the MIN6 Mouse Pancreatic ß Cell Line.

Elucidating the regulation of glucose-stimulated insulin secretion (GSIS) in pancreatic ß cells is important for understanding and treating diabetes. The pancreatic ß cell line, MIN6, retains GSIS but gradually loses it in long-term culture. The MIN6 subclone, MIN6c4, exhibits well-regulated GSIS even after prolonged culture. We previously used DNA microarray analysis to compare gene expression in the parental MIN6 cells and MIN6c4 cells and identified several differentially regulated genes that may be involved in maintaining GSIS. Here we investigated the potential roles of six of these genes in GSIS: Tmem59l (Transmembrane protein 59 like), Scgn (Secretagogin), Gucy2c (Guanylate cyclase 2c), Slc29a4 (Solute carrier family 29, member 4), Cdhr1 (Cadherin-related family member 1), and Celsr2 (Cadherin EGF LAG seven-pass G-type receptor 2). These genes were knocked down in MIN6c4 cells using lentivirus vectors expressing gene-specific short hairpin RNAs (shRNAs), and the effects of the knockdown on insulin expression and secretion were analyzed. Suppression of Tmem59l, Scgn, and Gucy2c expression resulted in significantly decreased glucose- and/or KCl-stimulated insulin secretion from MIN6c4 cells, while the suppression of Slc29a4 expression resulted in increased insulin secretion. Tmem59l overexpression rescued the phenotype of the Tmem59l knockdown MIN6c4 cells, and immunostaining analysis indicated that the TMEM59L protein colocalized with insulin and GM130, a Golgi complex marker, in MIN6 cells. Collectively, our findings suggested that the proteins encoded by Tmem59l, Scgn, Gucy2c, and Slc29a4 play important roles in regulating GSIS. Detailed studies of these proteins and their functions are expected to provide new insights into the molecular mechanisms involved in insulin secretion.