Establishment of a tagged variant of Lgr4 receptor suitable for functional and expression studies in the mouse

Leucine-rich repeat-containing G-protein-coupled receptor 4 (LGR4) is produced in a broad spectrum of mouse embryonic and adult tissues and its deficiency results in embryonal or perinatal lethality. The LGR4 function was mainly related to its potentiation of canonical Wnt signaling; however, several recent studies associate LGR4 with additional signaling pathways. To obtain a suitable tool for studying the signaling properties of Lgr4, we generated a tagged variant of the Lgr4 receptor using gene targeting in the mouse oocyte. The modified Lgr4 allele expresses the Lgr4 protein fused with a triple hemagglutinin (3HA) tag located at the extracellular part of the protein. The allele is fully functional, enabling tracking of Lgr4 expression in the mouse tissues. We also show that via surface labeling, the 3HA tag allows direct isolation and analysis of living Lgr4-positive cells obtained from the small intestinal crypts. Finally, the HA tag-specific antibody can be employed to characterize the biochemical features of Lgr4 and to identify possible biding partners of the protein in cells derived from various mouse tissues.     

Immunohistochemical Demonstration of Leptin in Pancreatic Islets of Non-Obese Diabetic and CD-1 mice: Co-localization in Glucagon Cells and its Attenuation at the Onset of Diabetes

Leptin is a 16 kD polypeptide hormone produced predominantly by white adipose tissue and exerts profound effects on food intake and energy balance. More recent studies have shown extra sites of leptin production in human and rodent tissues and have ascribed additional roles for the hormone, e.g., in immune and reproductive functions. A role for the hormone has also been implicated in insulin-dependent diabetes mellitus in the non-obese diabetic (NOD) mouse. However, whether leptin originates from islet cells of the mouse is not known. Here dual-label immunohistochemistry was employed to examine leptin expression in islet cells, and its distribution and cellular sources in pancreatic sections of female NOD/Ak and CD-1 mice of various ages. For comparison, leptin immunolabelling was examined in adult pancreatic sections from male NOD/Ak CD-1, Balb/c and FVB/N mice and female severe combined immunodeficient CB. 17 mice. Pancreatic tissues from adult female guinea pig, sheep and cattle and neonatal pigs were also studied. Our results show that in the day 1 NOD and CD-1 mice, leptin immunolabelling was observed in selective glucagon cells within the developing islets while at days 15 and 22, it became more intense and co-incident. This pattern of staining was maintained at days 40, 90, 150 and 250. In the female NOD mouse, leptin was absent in intra-islet immune cells. Its expression was variable in islets from male NOD and CD-1 mice. In spontaneously diabetic female NOD mice and following acceleration of diabetes with cyclophosphamide, despite the persistence of strong immunolabelling for glucagon in the re-distributed alpha cells, leptin expression was either absent, diminished or present in only a proportion of alpha cells. The reduction in leptin labelling was often associated with diabetic islets which had insulitis in association with only a small number of residual beta cells. Leptin expression was absent in guinea pig, ovine, bovine and neonatal porcine islet cells, despite the expression of intensely labelled glucagon cells. The present results demonstrate leptin co-localization in glucagon cells of the mouse islet. Its expression diminishes in the presence of inadequate insulin. Leptin produced within the mouse islet may have bi-directional influences on leptin and insulin regulation and may play local functions in islet development and metabolism.     

Defective postnatal development of the male reproductive tract in LGR4 knockout mice

The final outcome of tube elongation and branching is to maximize the epithelial exchange surfaces in tubular organs. The molecular and cellular basis of these processes is actively studied in model organs such as mammary glands, liver and kidney, but they remain almost unexplored in the male reproductive tract. Here, we report that the orphan G protein-coupled receptor LGR4/GPR48 plays a role in the postnatal tissue remodeling needed for elongation and convolution of the efferent ducts and epididymis. In LGR4 knockout male mice, tube elongation fails, resulting in a hypoplastic and poorly convoluted tract. Cell proliferation is dramatically reduced in KO affected tissues, providing an explanation to the observed phenotype. Detailed analysis showed that LGR4 inactivation manifests differently in the affected organs. In efferent ducts, immune cells infiltrate the epithelium and reach the lumen, blocking the transit of sperm and testicular fluid. In addition, the hypoplasia and low convolution result in a reduction of the epithelial area involved in liquid reabsorption. Both phenomena contribute in tissue swelling upstream the blockade due to liquid and sperm accumulation, with secondary damaging effects on the germinal epithelium. In the epididymis, the thin and highly convoluted duct is replaced by a large cystic tube which is surrounded by a thick condensation of mesenchymal cells. The abnormal organization of the cellular compartments in and around the ducts suggests that LGR4 might play a role in epithelial-mesenchymal interactions. Altogether, our data identify LGR4 as an important signaling molecule implicated in the tube morphogenesis of the male reproductive tract.     

Expression pattern of the orphan receptor LGR4/GPR48 gene in the mouse

Leucine-rich G-protein-coupled Receptors (LGR) constitute a subfamily of receptors related to glycoprotein hormone receptors. Amongst them, LGR4, LGR5 and LGR6 form a cluster for which natural agonists are still unknown. By an extensive gene trapping approach, Leighton et al. (2001) obtained a mouse line in which the LGR4 gene is disrupted by a trap vector carrying two biological markers, beta-geo (a fusion between bacterial beta-galactosidase and neomycin phosphotransferase) and a placental alkaline phosphatase (PLAP). Due to perinatal lethality, characterization of adult mice homozygous for this insertion has been impaired. In the present study we have investigated LacZ and PLAP activity patterns in heterozygous mice as a marker for LGR4 natural expression at both macroscopic and histological levels. We present a detailed atlas of LGR4 expression, which displays very wide expression with particularly strong activity in cartilages, kidneys, reproductive tracts and nervous system cells.     

G protein-coupled receptor signaling and sphingosine-1-phosphate play a phylogenetically conserved role in endocrine pancreas morphogenesis

During development pancreatic endocrine cells migrate in a coordinated fashion. This migration is necessary to form fully functional islets, but the mechanisms involved remain unknown. Therapeutic strategies to restore β-cell mass and islet functionality by reprogramming endogenous exocrine cells would be strengthened from simultaneous treatments that enhance endocrine cell clustering. We found that endocrine progenitors respond to and regulate G protein-coupled receptor (GPCR) signaling in order to cluster in islets. Rgs4, a dedicated regulator of GPCR signaling, was specifically expressed in early epithelial endocrine progenitors of both zebrafish and mouse, and its expression in the mouse endocrine progenitors was strictly dependent upon Ngn3, the key specification gene of the endocrine lineage. Rgs4 loss of function resulted in defects in islet cell aggregation. By genetically inactivating Gα(i)-mediated GPCR signaling in endocrine progenitors, we established its role in islet cell aggregation in both mouse and zebrafish. Finally, we identified sphingosine-1-phosphate (S1P) as a ligand mediating islet cell aggregation in both species acting through distinct but closely related receptors.     

Modulation of endocrine pancreas development but not beta-cell carcinogenesis by Sprouty4

Sprouty (Spry) proteins modulate signal transduction pathways elicited by receptor tyrosine kinases (RTK). Depending on cell type and the particular RTK, Spry proteins exert dual functions: They can either repress RTK-mediated signaling pathways, mainly by interfering with the Ras/Raf/mitogen-activated protein kinase pathway or sustaining RTK signal transduction, for example by sequestering the E3 ubiquitin-ligase c-Cbl and thus preventing ubiquitylation, internalization, and degradation of RTKs. Here, by the inducible expression of murine Spry4 in pancreatic beta cells, we have assessed the functional role of Spry proteins in the development of pancreatic islets of Langerhans in normal mice and in the Rip1Tag2 transgenic mouse model of beta-cell carcinogenesis. beta cell-specific expression of mSpry4 provokes a significant reduction in islet size, an increased number of alpha cells per islet area, and impaired islet cell type segregation. Functional analysis of islet cell differentiation in cultured PANC-1 cells shows that mSpry4 represses adhesion and migration of differentiating pancreatic endocrine cells, most likely by affecting the subcellular localization of the protein tyrosine phosphatase PTP1B. In contrast, transgenic expression of mSpry4 during beta-cell carcinogenesis does not significantly affect tumor outgrowth and progression to tumor malignancy. Rather, tumor cells seem to escape mSpry4 transgene expression.     

Identification of the Ubiquitin-like Domain of Midnolin as a New Glucokinase Interaction Partner

Glucokinase acts as a glucose sensor in pancreatic beta cells. Its posttranslational regulation is important but not yet fully understood. Therefore, a pancreatic islet yeast two-hybrid library was produced and searched for glucokinase-binding proteins. A protein sequence containing a full-length ubiquitin-like domain was identified to interact with glucokinase. Mammalian two-hybrid and fluorescence resonance energy transfer analyses confirmed the interaction between glucokinase and the ubiquitin-like domain in insulin-secreting MIN6 cells and revealed the highest binding affinity at low glucose. Overexpression of parkin, an ubiquitin E3 ligase exhibiting an ubiquitin-like domain with high homology to the identified, diminished insulin secretion in MIN6 cells but had only some effect on glucokinase activity. Overexpression of the elucidated ubiquitin-like domain or midnolin, containing exactly this ubiquitin-like domain, significantly reduced both intrinsic glucokinase activity and glucose-induced insulin secretion. Midnolin has been to date classified as a nucleolar protein regulating mouse development. However, we could not confirm localization of midnolin in nucleoli. Fluorescence microscopy analyses revealed localization of midnolin in nucleus and cytoplasm and co-localization with glucokinase in pancreatic beta cells. In addition we could show that midnolin gene expression in pancreatic islets is up-regulated at low glucose and that the midnolin protein is highly expressed in pancreatic beta cells and also in liver, muscle, and brain of the adult mouse and cell lines of human and rat origin. Thus, the results of our study suggest that midnolin plays a role in cellular signaling of adult tissues and regulates glucokinase enzyme activity in pancreatic beta cells.     

Androgen receptor expression in human tissues: an immunohistochemical study.

The cellular localization of the human androgen receptor was visualized immunohistochemically using a mouse monoclonal antibody (MAb) F39.4, directed against a fragment of the N-terminal domain of the androgen receptor. The nuclear immunoreactivity of various human tissues with F39.4 was generally consistent with earlier biochemical and autoradiographic data. However, previously suggested androgen receptor expression in thyroid, pancreatic, gastrointestinal, and bladder tissues was not confirmed immunohistochemically. Stratified squamous epithelia of vagina and cervix showed selective immunostaining of the basal cell layer, whereas in the preputial epithelium the intensity of immunoreactivity decreased gradually with maturation. In contrast, glandular epithelia of the sweat glands, male accessory sex organs, and female breast showed nearly exclusive F39.4 staining of the inner cylindric layer. In the testis, Sertoli cells, peritubular myoid cells, and interstitial cells were immunoreactive with MAb F39.4. Expression of the androgen receptor by smooth muscle tissue was largely confined to the male reproductive organs. The specificity and sensitivity of this simple and rapidly performed immunohistochemical technique in the detection of the human androgen receptor at the cellular and subcellular level makes it worthwhile to study tissue androgen receptor expression by immunohistochemistry in physiological and pathological states.     

Conditional expression of Smad7 in pancreatic beta cells disrupts TGF-beta signaling and induces reversible diabetes mellitus

Identification of signaling pathways that maintain and promote adult pancreatic islet functions will accelerate our understanding of organogenesis and improve strategies for treating diseases like diabetes mellitus. Previous work has implicated transforming growth factor-beta (TGF-beta) signaling as an important regulator of pancreatic islet development, but has not established whether this signaling pathway is required for essential islet functions in the adult pancreas. Here we describe a conditional system for expressing Smad7, a potent inhibitor of TGF-beta signaling, to identify distinct roles for this pathway in adult and embryonic beta cells. Smad7 expression in Pdx1+ embryonic pancreas cells resulted in striking embryonic beta cell hypoplasia and neonatal lethality. Conditional expression of Smad7 in adult Pdx1+ cells reduced detectable beta cell expression of MafA, menin, and other factors that regulate beta cell function. Reduced pancreatic insulin content and hypoinsulinemia produced overt diabetes that was fully reversed upon resumption of islet TGF-beta signaling. Thus, our studies reveal that TGF-beta signaling is crucial for establishing and maintaining defining features of mature pancreatic beta cells.     

R-Spondin potentiates Wnt/β-catenin signaling through orphan receptors LGR4 and LGR5

The Wnt/β-catenin signaling pathbway controls many important biological processes. R-Spondin (RSPO) proteins are a family of secreted molecules that strongly potentiate Wnt/β-catenin signaling, however, the molecular mechanism of RSPO action is not yet fully understood. We performed an unbiased siRNA screen to identify molecules specifically required for RSPO, but not Wnt, induced β-catenin signaling. From this screen, we identified LGR4, then an orphan G protein-coupled receptor (GPCR), as the cognate receptor of RSPO. Depletion of LGR4 completely abolished RSPO-induced β-catenin signaling. The loss of LGR4 could be compensated by overexpression of LGR5, suggesting that LGR4 and LGR5 are functional homologs. We further demonstrated that RSPO binds to the extracellular domain of LGR4 and LGR5, and that overexpression of LGR4 strongly sensitizes cells to RSPO-activated β-catenin signaling. Supporting the physiological significance of RSPO-LGR4 interaction, Lgr4-/- crypt cultures failed to grow in RSPO-containing intestinal crypt culture medium. No coupling between LGR4 and heterotrimeric G proteins could be detected in RSPO-treated cells, suggesting that LGR4 mediates RSPO signaling through a novel mechanism. Identification of LGR4 and its relative LGR5, an adult stem cell marker, as the receptors of RSPO will facilitate the further characterization of these receptor/ligand pairs in regenerative medicine applications.     

LGR4/GPR48 Inactivation Leads to Aniridia-Genitourinary Anomalies-Mental Retardation Syndrome Defects

AGR syndrome (the clinical triad of aniridia, genitourinary anomalies, and mental retardation, a subgroup of WAGR syndrome for Wilm''s tumor, aniridia, genitourinary anomalies, and mental retardation) is a rare syndrome caused by a contiguous gene deletion in the 11p13–14 region. However, the mechanisms of WAGR syndrome pathogenesis are elusive. In this study we provide evidence that LGR4 (also named GPR48), the only G-protein-coupled receptor gene in the human chromosome 11p12–11p14.4 fragment, is the key gene responsible for the diseases of AGR syndrome. Deletion of Lgr4 in mouse led to aniridia, polycystic kidney disease, genitourinary anomalies, and mental retardation, similar to the pathological defects of AGR syndrome. Furthermore, Lgr4 inactivation significantly increased cell apoptosis and decreased the expression of multiple important genes involved in the development of WAGR syndrome related organs. Specifically, deletion of Lgr4 down-regulated the expression of histone demethylases Jmjd2a and Fbxl10 through cAMP-CREB signaling pathways both in mouse embryonic fibroblast cells and in urinary and reproductive system mouse tissues. Our data suggest that Lgr4, which regulates eye, kidney, testis, ovary, and uterine organ development as well as mental development through genetic and epigenetic surveillance, is a novel candidate gene for the pathogenesis of AGR syndrome.     

Expression of estrogen receptor alpha and beta during mouse embryogenesis.

In adult mammals numerous target tissues and organs for estrogens exist. Little is known about possible target organs during embryogenesis other than the reproductive tract and the gonads. This is the first report on the expression of estrogen receptor beta (ERbeta) in comparison with ERalpha mRNA during mouse embryogenesis. We found expression of estrogen receptor mRNA in the reproductive tract, but also in the atrial wall, brain, kidney, urethra, bladder neck, mammary gland primordium, midgut, cartilage primordia and perichondria.     

Distribution of GDNF family receptor α3 and RET in rat and human non-neural tissues

The neurotrophic growth factor artemin binds selectively to GDNF family receptor α3 (GFRα3), forming a molecular complex with the co-receptor RET which mediates downstream signaling. This signaling pathway has been demonstrated to play an important role in the survival and maintenance of nociceptive sensory neurons and in the development of sympathetic neurons. However, the presence and potential role of this artemin-responsive pathway in non-neural tissues has not been fully explored to-date. To study the distribution of GFRα3 and RET in adult rat and human non-neural tissues, we carried out a comprehensive immunohistochemical study. We stained major organs from the digestive, urinary, reproductive, immune, respiratory and endocrine systems, and from other systems (cardiovascular, skeletal muscle), as well as regions of the nervous system for comparison. In both rat and human, the majority of non-neural cells did not exhibit detectable GFRα3-like immunoreactivity. In the rat, GFRα3- and RET-like staining were found in the same non-neural cell type only in kidney. In the human digestive and reproductive systems, a subset of epithelial cells exhibited GFRα3- and RET-like staining, suggesting co-localization. In other tissues, sub-populations of cells expressed either GFRα3- or RET-like immunoreactivity. The functional consequences of GFRα3 expression in non-neural cells remain to be determined.     

G protein-coupled receptors as regulators of pancreatic islet functionality

Glucose homeostasis is maintained by hormones secreted from different types of pancreatic islets and its dysregulation can result in diseases including diabetes mellitus. The secretion of hormones from pancreatic islets is highly complex and tightly controlled by G protein-coupled receptors (GPCRs). Moreover, GPCR signaling may play a role in enhancing islet cell replication and proliferation. Thus, targeting GPCRs offers a promising strategy for regulating the functionality of pancreatic islets. Here, available RNAseq datasets from human and mouse islets were used to identify the GPCR expression profile and the impact of GPCR signaling for normal islet functionality is discussed.     

Distribution of CIAPIN1 in normal fetal and adult human tissues.

CIAPIN1, a newly identified antiapoptotic molecule that plays an essential role in mouse definitive hematopoiesis, is considered a downstream effector of the receptor tyrosine kinase-Ras signaling pathway. Our previous studies have indicated that CIAPIN1 is involved in the development of multidrug resistance (MDR) in gastric cancer cells. However, the mechanism of CIAPIN1-mediated antiapoptosis and MDR has not been fully elucidated. To reveal the possible physiological role of CIAPIN1, we examined the expression and distribution of CIAPIN1 in fetal and adult human tissues using immunohistochemistry. We found that CIAPIN1 was ubiquitously distributed in fetal and adult tissues, and was localized in both the cytoplasm and the nucleus. The expression patterns of CIAPIN1 were similar in fetal and adult tissues, and was correlated with the previously described expression pattern of p21ras. These observations suggest that CIAPIN1 expression appears to be involved in cell differentiation, and that it might exert universal and possibly important physiological functions under the regulation of Ras in humans.     

LGR5 regulates survival through mitochondria-mediated apoptosis and by targeting the Wnt/β-catenin signaling pathway in colorectal cancer cells

Colorectal cancer (CRC) is one of the most common causes of cancer-related death worldwide. The leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) is a newly identified surface marker of colorectal cancer stem cells (CSCs). Expression level of LGR5 is commonly elevated in human CRCs. Our previous study demonstrated that the elevated expression of LGR5 is associated with CRC initiation and progression. However, the role of LGR5 in CRC pathogenesis has not been sufficiently established. In this study, we aimed to characterize the role of LGR5 in CRC pathogenesis using the loss-of-function approach. Depletion of LGR5 suppressed the growth of several cultured CRC cells and caused an increase in the fraction of apoptotic cells, which were analyzed using Annexin V/PI staining and DNA fragmentation assay. Furthermore, depleting LGR5 induced apoptosis through the loss of mitochondrial membrane potential. Additionally, depletion of LGR5 suppressed β-catenin nuclear translocation and blocked the activity of Wnt/β-catenin signaling as manifested in the reduced expression of c-myc and cyclin D, two Wnt/β-catenin targets in CRC cells. Treatment with Wnt3a considerably alleviated the growth inhibition and apoptotic cell death induced by LGR5 depletion in CRC cells. These data suggested that LGR5 regulates cell proliferation and survival by targeting the Wnt/β-catenin signaling pathway. Thus, the findings of this study suggest that LGR5 plays a vital role in CRC pathogenesis and has the potential to serve as a diagnostic marker and a therapeutic target for CRC patients.     

Conditional Expression of Smad7 in Pancreatic β Cells Disrupts TGF-β Signaling and Induces Reversible Diabetes Mellitus

Identification of signaling pathways that maintain and promote adult pancreatic islet functions will accelerate our understanding of organogenesis and improve strategies for treating diseases like diabetes mellitus. Previous work has implicated transforming growth factor-β (TGF-β) signaling as an important regulator of pancreatic islet development, but has not established whether this signaling pathway is required for essential islet functions in the adult pancreas. Here we describe a conditional system for expressing Smad7, a potent inhibitor of TGF-β signaling, to identify distinct roles for this pathway in adult and embryonic β cells. Smad7 expression in Pdx1 ⁺ embryonic pancreas cells resulted in striking embryonic β cell hypoplasia and neonatal lethality. Conditional expression of Smad7 in adult Pdx1 ⁺ cells reduced detectable β cell expression of MafA, menin, and other factors that regulate β cell function. Reduced pancreatic insulin content and hypoinsulinemia produced overt diabetes that was fully reversed upon resumption of islet TGF-β signaling. Thus, our studies reveal that TGF-β signaling is crucial for establishing and maintaining defining features of mature pancreatic β cells.     

胰岛类器官研究进展

类器官是将具有多向分化潜能的干细胞或组织细胞在特定环境下培养分化成为能够模拟原生器官结构和功能的三维结构.类器官在各种疾病模型研究及药物筛选中发挥至关重要的作用.近年来,通过体外诱导胰腺组织或多能干细胞分化形成具有胰岛细胞功能的胰岛类器官研究成为热点,为胰岛相关疾病模型、药物研究以及糖尿病的治疗提供了新的手段.本文针对...