Glial Cell-derived Neurotrophic Factor Family Research Articles

H3K9me3 Levels Affect the Proliferation of Bovine Spermatogonial Stem Cells.

Spermatogonial stem cells (SSCs) possess the characteristics of self-renewal and differentiation, as well as the ability to generate functional sperm. Their unique stemness has broad applications in male infertility treatment and species preservation. In rodents, research on SSCs has been widely reported, but progress is slow in large livestock such as cattle and pigs due to long growth cycles, difficult proliferation in vitro, and significant species differences. Previously, we showed that histone 3 (H3) lysine 9 (K9) trimethylation (H3K9me3) is associated with the proliferation of bovine SSCs. Here, we isolated and purified SSCs from calf testicular tissues and investigated the impact of different H3K9me3 levels on the in vitro proliferation of bovine SSCs. The enriched SSCs eventually formed classical stem cell clones in vitro in our feeder-free culture system. These clones expressed glial cell-derived neurotrophic factor family receptor alpha-1 (GFRα1, specific marker for SSCs), NANOG (pluripotency protein), C-KIT (germ cell marker), and strong alkaline phosphatase (AKP) positivity. qRT-PCR analysis further showed that these clones expressed the pluripotency genes NANOG and SOX2, and the SSC-specific marker gene GFRα1. To investigate the dynamic relationship between H3K9me3 levels and SSC proliferation, H3K9me3 levels in bovine SSCs were first downregulated using the methyltransferase inhibitor, chaetocin, or transfection with the siRNA of H3K9 methyltransferase suppressor of variegation 3-9 homologue 1 (SUV39H1). The EDU (5-Ethynyl-2'-deoxyuridine) assay revealed that SSC proliferation was inhibited. Conversely, when H3K9me3 levels in bovine SSCs were upregulated by transfecting lysine demethylase 4D (KDM4D) siRNA, the EDU assay showed a promotion of cell proliferation. In summary, this study established a feeder-free culture system to obtain bovine SSCs and explored its effects on the proliferation of bovine SSCs by regulating H3K9me3 levels, laying the foundation for elucidating the regulatory mechanism underlying histone methylation modification in the proliferation of bovine SSCs.

Growth differentiation factor-15 stimulates the synthesis of corticotropin-releasing factor in hypothalamic 4B cells

Growth differentiation factor-15 (GDF15) is a stress-activated cytokine that regulates cell growth and inflammatory and stress responses. We previously reported the role and regulation of GDF15 in pituitary corticotrophs. Dexamethasone increases Gdf15 gene expression levels and production. GDF15 suppresses adrenocorticotropic hormone synthesis in pituitary corticotrophs and subsequently mediates the negative feedback effect of glucocorticoids. Here, we analyzed corticotropin-releasing factor (Crf) promoter activity in hypothalamic 4B cells transfected with promoter-driven luciferase reporter constructs. The effects of time and GDF15 concentration on Crf mRNA levels were analyzed using quantitative real-time polymerase chain reaction. Glial cell-derived neurotrophic factor family receptor α-like (GFRAL) protein is expressed in 4B cells. GDF15 increased Crf promoter activity and Crf mRNA levels in 4B cells. The protein kinase A and C pathways also contributed to the GDF15-induced increase in Crf gene expression. GDF15 stimulates GFRAL, subsequently increasing the phosphorylation of AKT, an extracellular signal-related kinase, and the cAMP response element-binding protein. Therefore, GDF15-dependent pathways may be involved in regulating Crf expression under stressful conditions in hypothalamic cells.

Identification of GDF15 peptide fragments inhibiting GFRAL receptor signaling

Growth differentiation factor 15 (GDF15) is believed to be a major causative factor for cancer-induced cachexia. Recent elucidation of the central circuits involved in GDF15 function and its signaling through the glial cell-derived neurotrophic factor family receptor α-like (GFRAL) has prompted the interest of targeting the GDF15-GFRAL signaling for energy homeostasis and body weight regulation. Here, we applied advanced peptide technologies to identify GDF15 peptide fragments inhibiting GFRAL signaling. SPOT peptide arrays revealed binding of GDF15 C-terminal peptide fragments to the extracellular domain of GFRAL. Parallel solid-phase peptide synthesis allowed for generation of complementary GDF15 peptide libraries and their subsequent functional evaluation in cells expressing the GFRAL/RET receptor complex. We identified a series of C-terminal fragments of GDF15 inhibiting GFRAL activity in the micromolar range. These novel GFRAL peptide inhibitors could serve as valuable tools for further development of peptide therapeutics towards the treatment of cachexia and other wasting disorders.

Growth differentiation factor 15 induces cisplatin resistance through upregulation of xCT expression and glutathione synthesis in gastric cancer.

Gastric cancer is a common cancer worldwide, particularly in East Asia. Chemotherapy is used in adjuvant or palliative therapies for gastric cancer. However, subsequent chemoresistance often develops. Growth differentiation factor 15 (GDF15) links to several cancers, but its effect on chemoresistance in gastric cancer remains unclear. Here, we analyzed clinical samples from genetic databases and included patients with gastric cancer. We dissected the regulatory mechanism underlying GDF15-mediated resistance of cisplatin in human gastric cancer cells. We showed that GDF15 serum levels might be a valuable biomarker for predicting prognosis in gastric cancer. The expressions of GDF15 and its receptor glial cell-derived neurotrophic factor family receptor a-like (GFRAL) in gastric tumors are important for malignant progression. Moreover, GDF15 expression is increased in gastric cancer cells with cisplatin resistance, resulting from elevated intracellular glutathione (GSH) and antioxidant activities. Upregulated GDF15 could increase intracellular GSH content by activating the GFRAL-GCN2-eIF2α-ATF4 signaling, enhancing cystine-uptake transporter xCT expression, and contributing biosynthesis of GSH in human gastric cancer cells. In conclusion, our results indicate that GDF15 could induce chemoresistance by upregulating xCT expression and GSH biosynthesis in human gastric cancer cells. Targeting GDF15 could be a promising treatment method for gastric cancer progression.

Inhibition of RET signaling attenuates renal hypertrophic growth, proximal tubule dilation, and kidney damage in the 5/6Nx rat model of chronic kidney disease

Progression of chronic kidney disease (CKD) involves compensatory hypertrophy of remaining nephrons leading to additional nephron loss. Understanding the precise mechanisms responsible for hypertrophy may help develop novel strategies to preserve renal function in people living with CKD. Our laboratory utilized the 5/6 nephrectomy (5/6Nx) rat model of renal insufficiency, where 2/3 of the left kidney and the entire right kidney was surgically excised in 10-week-old male Sprague Dawley rats. The remnant kidney underwent substantial compensatory hypertrophy between 5- and 7-weeks post-surgery. We then performed transcriptomic analysis of kidneys collected 6 weeks after 5/6Nx or sham surgery (n=6/group) to gain an unbiased perspective of molecular pathways involved with hypertrophic remodeling. Through transcriptomic analysis, we found that transcript expression of glial cell derived Neurotrophic factor (GDNF) was 7.64-fold higher in 5/6Nx remnant kidneys compared to sham-operated control. GDNF is an essential kidney morphogen in the developmental process that had not been implicated in renal compensatory hypertrophic remodeling. Immunohistochemistry (IHC) showed increased GDNF protein expression in the 5/6Nx remnant kidney. In the kidney GDNF elicits biological function through binding of its co-receptor GDNF family receptor α (GFRα) which then forms a complex with the tyrosine kinase RET resulting in downstream signaling. We hypothesized that GDNF signaling promotes tubular hypertrophic growth response in the 5/6Nx. To test this, we pharmacologically inhibited the GDNF/GFRα/RET complex using pralsetinib (3mg/kg/bw in corn oil; 1x daily oral gavage) from 5- to 7-weeks post-5/6Nx or sham surgery. Treatment control animals received vehicle. During this time we collected blood, urine, and conducted ultrasound analysis of the remnant kidney transverse cross sectional-area. Analysis of left kidney CSA revealed a decrease in kidney size between weeks 5 and 7 (1.58 ± 0.89 cm2, treatment vs 2.06 ± 0.071 cm2, vehicle; p=0.02; a two-way RM ANOVA), kidney wet weight and kidney/body weight were lower in pralsetinib vs. vehicle treated animals (66.85 ± 4.47% and 67.19 ± 2.30%, respectively; mean ± SEM, p < 0.05, student’s t-test). IHC analysis of renal cortex showed pralestinib treatment decreased proximal tubule diameter compared to vehicle (61.51 ± 3.45 μm, treatment vs 71.33 ± 9.82 μm, vehicle; p=0.01, student’s t-test). Additionally, plasma creatinine and blood urea nitrogen levels were decreased when compared to vehicle treated animals (0.88 ± 0.51 mg/dL, treatment vs 1.4 ± 0.21 mg/dL, vehicle; mean ± SEM, p < 0.03, student’s t-test and 37.63 ± 10.62 mg/dL, treatment vs 71.13 ± 33.50 mg/dL, vehicle; mean ±SEM, p<0.001, student’s t-test, respectively). Taken together, our data suggests inhibition of RET signaling prevented whole kidney hypertrophic growth and proximal tubule remodeling while preserving kidney function following 5/6Nx in rats. NIH-T32 Training Grant This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

GDF15 as a key disease target and biomarker: linking chronic lung diseases and ageing.

Growth differentiation factor 15 (GDF15), a member of the transforming growth factor-beta superfamily, is expressed in several human organs. In particular, it is highly expressed in the placenta, prostate, and liver. The expression of GDF15 increases under cellular stress and pathological conditions. Although numerous transcription factors directly up-regulate the expression of GDF15, the receptors and downstream mediators of GDF15 signal transduction in most tissues have not yet been determined. Glial cell-derived neurotrophic factor family receptor α-like protein was recently identified as a specific receptor that plays a mediating role in anorexia. However, the specific receptors of GDF15 in other tissues and organs remain unclear. As a marker of cell stress, GDF15 appears to exert different effects under different pathological conditions. Cell senescence may be an important pathogenetic process and could be used to assess the progression of various lung diseases, including COVID-19. As a key member of the senescence-associated secretory phenotype protein repertoire, GDF15 seems to be associated with mitochondrial dysfunction, although the specific molecular mechanism linking GDF15 expression with ageing remains to be elucidated. Here, we focus on research progress linking GDF15 expression with the pathogenesis of various chronic lung diseases, including neonatal bronchopulmonary dysplasia, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, and pulmonary hypertension, suggesting that GDF15 may be a key biomarker for diagnosis and prognosis. Thus, in this review, we aimed to provide new insights into the molecular biological mechanism and emerging clinical data associated with GDF15 in lung-related diseases, while highlighting promising research and clinical prospects.

Overview of growth differentiation factor 15 in metabolic syndrome.

Growth and differentiation factor 15 (GDF15) is a member of the transforming growth factor-β (TGF-β) superfamily. GDF15 has been linked with several metabolic syndrome pathologies such as obesity and cardiovascular diseases. GDF15 is considered to be a metabolic regulator, although its precise mechanisms of action remain to be determined. Glial cell-derived neurotrophic factor family receptor alpha-like (GRAL), located in the hindbrain, has been identified as the receptor for GDF15 and signals through the coreceptor receptor tyrosine kinase (RET). Administration of GDF15 analogues in preclinical studies using various animal models has consistently been shown to induce weight loss through a reduction in food intake. GDF15, therefore, represents an attractive target to combat the current global obesity epidemic. In this article, we review current knowledge on GDF15 and its involvement in metabolic syndrome.

Tumor-associated macrophage-derived GDNF promotes gastric cancer liver metastasis via a GFRA1-modulated autophagy flux

PurposeLiver metastasis, a lethal malignancy of gastric cancer (GC) patients, execrably impairs their prognosis. As yet, however, few studies have been designed to identify the driving molecules during its formation, except screening evidence pausing before their functions or mechanisms. Here, we aimed to survey a key driving event within the invasive margin of liver metastases.MethodsA metastatic GC tissue microarray was used for exploring malignant events during liver-metastasis formation, followed by assessing the expression patterns of glial cell-derived neurotrophic factor (GDNF) and GDNF family receptor alpha 1 (GFRA1). Their oncogenic functions were determined by both loss- and gain-of-function studies in vitro and in vivo, and validated by rescue experiments. Multiple cell biological studies were performed to identify the underlying mechanisms.ResultsIn the invasive margin, GFRA1 was identified as a pivotal molecule involved in cellular survival during liver metastasis formation, and we found that its oncogenic role depends on tumor associated macrophage (TAM)-derived GDNF. In addition, we found that the GDNF-GFRA1 axis protects tumor cells from apoptosis under metabolic stress via regulating lysosomal functions and autophagy flux, and participates in the regulation of cytosolic calcium ion signalling in a RET-independent and non-canonical way.ConclusionFrom our data we conclude that TAMs, homing around metastatic nests, induce the autophagy flux of GC cells and promote the development of liver metastasis via GDNF-GFRA1 signalling. This is expected to improve the comprehension of metastatic pathogenesis and to provide a novel direction of research and translational strategies for the treatment of metastatic GC patients.

Role of GDNF, GFRα1 and GFAP in a Bifidobacterium-Intervention Induced Mouse Model of Intestinal Neuronal Dysplasia.

ObjectiveTo investigate the effects of glial cell-derived neurotrophic factor (GDNF), GDNF family receptor alpha 1 (GFRα1), and glial fibrillary acidic protein (GFAP) on colonic motility in a mouse model of intestinal neuronal dysplasia by intervention with Bifidobacterium and to explore the influence of Bifidobacterium on enteric glial cells (EGCs).MethodsWestern blotting and qRT-PCR were employed to detect the expression of GFRα1 and GFAP in colonic tissues of mice with or without Tlx2 mutations, and ELISA was used to detect the expression of GDNF in serum. IHC was used to detect the appearance of the ganglion cells. Subsequently, Tlx2 homozygous mutant (Tlx2−/−) mice were treated with Bifidobacterium. Colonic motility was measured before and after intervention by measuring the glass bead expelling time. The variations in abdominal circumference and GDNF, GFRα1, and GFAP expression were measured. In addition, 16SrRNA gene sequencing was performed to detect the abundance of the intestinal microbiota.ResultsThe mRNA and protein expression of GFRα1 and GFAP was decreased in the colonic tissues of Tlx2−/− mice and GDNF expression was decreased in serum compared with Tlx2+/− and WT mice. After confirming the colonization of Bifidobacterium by 16S rRNA gene sequencing, the expelling time and abdominal distension were ameliorated, and the expression of GFAP, GDNF, and GFRα1 was increased.ConclusionsThe expression of GDNF, GFRα1, and GFAP is associated with colonic motility. The altered expression of EGC-related factors suggested that Bifidobacterium may be involved in the EGC activation process. The amelioration of IND symptoms after intervention with Bifidobacterium prompted the elicitation of adjuvant therapy.

PD39-10 A XENO-FREE CULTURE METHOD FOR THE IN VITRO EXPANSION OF HUMAN SPERMATOGONIAL STEM CELLS

You have accessJournal of UrologyInfertility: Basic Research & Pathophysiology (PD39)1 Sep 2021PD39-10 A XENO-FREE CULTURE METHOD FOR THE IN VITRO EXPANSION OF HUMAN SPERMATOGONIAL STEM CELLS Meghan Robinson, Ryan Flannigan, and Luke Witherspoon Meghan RobinsonMeghan Robinson More articles by this author , Ryan FlanniganRyan Flannigan More articles by this author , and Luke WitherspoonLuke Witherspoon More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002049.10AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: In vitro expansion of spermatogonial stem cells (SSCs) has been established using animal-derived fetal bovine serum (FBS), however, animal components introduce the risk of contaminating with pathogens, making them unsuitable for medical use. This study set out to develop xeno-free culture conditions for the expansion of human SSCs. METHODS: SSCs were derived from human induced pluripotent stem cells (hiPSCs) and tested in various xeno-free media conditions in combination with growth factors Prostaglandin D2 (PDG2) and Insulin-Like Growth Factor 1 (IGF1). Primary SSCs then underwent 3 passages in the best condition, were cryopreserved and thawed, and then analysed for changes in gene and protein expression by real time polymerase chain reaction (RT-qPCR) and immunocytochemistry. RESULTS: 10 ng/mL PDG2 with 10 ng/mL IGF1 was found to replace FBS and BSA without loss of viability or growth. ROCK inhibitor Y-27632 was determined to be necessary for viability upon thawing. Compared to established conditions, the SSCs shared identical protein expression profiles for the SSC markers Glial Cell-Derived Neurotrophic Factor Family Receptor Alpha 1 (GFRA1), G-Protein Coupled Receptor 125 (GPR125), Thy-1 Cell Surface Antigen (CD90), and Stage-Specific Embryonic Antigen 4 (SSEA4) (Figure 1A-B). RT-qPCR analyses revealed no significant variation in gene expression of undifferentiated germ cell markers and Inhibitor Of Differentiation 4 (ID4), and Fibroblast Growth Factor Receptor 3 (FGFR3). The greatest change seen was an increase in Deleted In Azoospermia Like (DAZL), a regulator of both SSC proliferation and differentiation (23-fold) (Figure 1C). CONCLUSIONS: This study identified a combination of growth factors capable of replacing FBS and BSA components in established SSC expansion cell culture. This xeno-free defined formulation allows standardized SSC culture and removes the risk of animal pathogens. Source of Funding: VCHRI, CUASF, CIHR, UBC © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e671-e672 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Meghan Robinson More articles by this author Ryan Flannigan More articles by this author Luke Witherspoon More articles by this author Expand All Advertisement Loading ...

GDF15: emerging biology and therapeutic applications for obesity and cardiometabolic disease.

Growth differentiation factor 15 (GDF15) is a member of the TGFβ superfamily whose expression is increased in response to cellular stress and disease as well as by metformin. Elevations in GDF15 reduce food intake and body mass in animal models through binding to glial cell-derived neurotrophic factor family receptor alpha-like (GFRAL) and the recruitment of the receptor tyrosine kinase RET in the hindbrain. This effect is largely independent of other appetite-regulating hormones (for example, leptin, ghrelin or glucagon-like peptide 1). Consistent with an important role for the GDF15-GFRAL signalling axis, some human genetic studies support an interrelationship with human obesity. Furthermore, findings in both mice and humans have shown that metformin and exercise increase circulating levels of GDF15. GDF15 might also exert anti-inflammatory effects through mechanisms that are not fully understood. These unique and distinct mechanisms for suppressing food intake and inflammation makes GDF15 an appealing candidate to treat many metabolic diseases, including obesity, type 2 diabetes mellitus, non-alcoholic fatty liver disease, cardiovascular disease and cancer cachexia. Here, we review the mechanisms regulating GDF15 production and secretion, GDF15 signalling in different cell types, and how GDF15-targeted pharmaceutical approaches might be effective in the treatment of metabolic diseases.

Glial-derived neurotrophic factor in human airway smooth muscle.

Airway smooth muscle (ASM) cells modulate the local airway milieu via production of inflammatory mediators and growth factors including classical neurotrophins, such as brain-derived neurotrophic factor (BDNF). The glial cell-derived neurotrophic factor (GDNF) family of ligands (GFLs) are nonclassical neurotrophins and their role in the airway is barely understood. The major GFLs, GDNF and Neurturin (NRTN) bind to GDNF family receptor (GFR) α1 and α2 respectively that pair with Ret receptor to accomplish signaling. In this study, we found GDNF is expressed in human lung and increased in adult asthma, while human ASM expresses GDNF and its receptors. Accordingly, we used human ASM cells to test the hypothesis that ASM expression and autocrine signaling by GFLs regulate [Ca2+ ]i . Serum-deprived ASM cells from non-asthmatics were exposed to 10 ng/ml GDNF or NRTN for 15 min (acute) or 24 h (chronic). In fura-2 loaded cells, acute GDNF or NRTN alone induced [Ca2+ ]i responses, and further enhanced responses to 1 µM ACh or 10 µM histamine. Ret inhibitor (SPP86; 10 µM) or specific GDNF chelator GFRα1-Fc (1 µg/ml) showed roles of these receptors in GDNF effects. In contrast, NRTN did not enhance [Ca2+ ]i response to histamine. Furthermore, conditioned media of nonasthmatic and asthmatic ASM cells showed GDNF secretion. SPP86, Ret inhibitor and GFRα1-Fc chelator markedly decreased [Ca2+ ]i response compared with vehicle, highlighting autocrine effects of secreted GDNF. Chronic GDNF treatment increased histamine-induced myosin light chain phosphorylation. These novel data demonstrate GFLs particularly GDNF/GFRα1 influence ASM [Ca2+ ]i and raise the possibility that GFLs are potential targets of airway hyperresponsiveness.

GDF-15 as a Weight Watcher for Diabetic and Non-Diabetic People Treated With Metformin.

Weight gain and obesity are global health concerns contributing to morbidity with increased risks of cardiovascular disease, diabetes, liver steatohepatitis and cancer. Pharmacological therapies or bariatric surgery are often required for those who fail to adhere to diet and lifestyle modifications. Metformin, a widely used antidiabetic agent, seems to have a health benefit beyond its anti-hyperglycemic properties, with few side effects. Emerging evidence shows weight loss to be associated with metformin in both diabetic and non-diabetic individuals. Recently, the growth differentiation factor 15 (GDF-15), a member of the transforming growth factor beta superfamily, has been identified as a key mediator of metformin-induced weight loss. Metformin increases the secretion of GDF-15, which binds exclusively to glial cell-derived neurotrophic factor family receptor alpha-like (GFRAL). This gut-brain cytokine works as a prominent player in reducing food intake and body weight in health and disease, like anorexia nervosa and cancer. Herein, we critically review advances in the understanding of the weight-reducing effects of metformin via the GDF-15 pathway.

GDNF promotes hair formation and cutaneous wound healing by targeting bulge stem cells

Glial-cell-derived neurotrophic factor (GDNF) is a well-studied neuroregenerative factor; however, the degree to which it supports hair formation and skin wound repair is not known. By using a Gfra1 (GDNF family receptor alpha 1) knock-in reporter mouse line, GDNF signaling was found to occur within hair bulge stem cells (BSCs) during the initiation of the hair cycle and early stages of hair formation after depilation. Both recombinant and transgene overexpression of GDNF promoted BSC colony growth, hair formation, and skin repair after wounding through enhanced self-renewal of BSCs and commitment of BSC-derived progenitors into becoming epidermal cells at the injury site. Conditional ablation of Gfra1 among BSCs impaired the onset of the hair cycle, while conditional ablation of the GDNF family member signal transducer, Ret, within BSCs prevented the onset of the hair cycle and depilation-induced anagen development of hair follicles. Our findings reveal that GDNF promotes hair formation and wound repair and that bulge stem cells are critical mediators of both.

Revisiting the Role of Neurotrophic Factors in Inflammation.

The neurotrophic factors are well known for their implication in the growth and the survival of the central, sensory, enteric and parasympathetic nervous systems. Due to these properties, neurturin (NRTN) and Glial cell-derived neurotrophic factor (GDNF), which belong to the GDNF family ligands (GFLs), have been assessed in clinical trials as a treatment for neurodegenerative diseases like Parkinson’s disease. In addition, studies in favor of a functional role for GFLs outside the nervous system are accumulating. Thus, GFLs are present in several peripheral tissues, including digestive, respiratory, hematopoietic and urogenital systems, heart, blood, muscles and skin. More precisely, recent data have highlighted that different types of immune and epithelial cells (macrophages, T cells, such as, for example, mucosal-associated invariant T (MAIT) cells, innate lymphoid cells (ILC) 3, dendritic cells, mast cells, monocytes, bronchial epithelial cells, keratinocytes) have the capacity to release GFLs and express their receptors, leading to the participation in the repair of epithelial barrier damage after inflammation. Some of these mechanisms pass on to ILCs to produce cytokines (such as IL-22) that can impact gut microbiota. In addition, there are indications that NRTN could be used in the treatment of inflammatory airway diseases and it prevents the development of hyperglycemia in the diabetic rat model. On the other hand, it is suspected that the dysregulation of GFLs produces oncogenic effects. This review proposes the discussion of the biological understanding and the potential new opportunities of the GFLs, in the perspective of developing new treatments within a broad range of human diseases.

Expression and functional analyses of ephrin type-A receptor 2 in mouse spermatogonial stem cells†.

Spermatogonial stem cells (SSCs) undergo continuous self-renewal division in response to self-renewal factors. The present study identified ephrin type-A receptor 2 (EPHA2) on mouse SSCs and showed that supplementation of glial cell-derived neurotrophic factor (GDNF) and fibroblast growth factor 2 (FGF2), which are both SSC self-renewal factors, induced EPHA2 expression in cultured SSCs. Spermatogonial transplantation combined with magnetic-activated cell sorting or fluorescence-activated cell sorting also revealed that EPHA2 was expressed in SSCs. Additionally, ret proto-oncogene (RET) phosphorylation levels decreased following the knockdown (KD) of Epha2 expression via short hairpin ribonucleic acid (RNA). Although the present immunoprecipitation experiments did not reveal an association between RET with EPHA2, RET interacted with FGFR2. The Epha2 KD decreased the proliferation of cultured SSCs and inhibited the binding of cultured SSCs to laminin-coated plates. The Epha2 KD also significantly reduced the colonization of testis cells by spermatogonial transplantation. EPHA2 was also expressed in human GDNF family receptor alpha 1-positive spermatogonia. The present results indicate that SSCs express EPHA2 and suggest that it is a critical modifier of self-renewal signals in SSCs.

Identification of differentially expressed genes regulated by methylation in colon cancer based on bioinformatics analysis.

BACKGROUNDDNA methylation, acknowledged as a key modification in the field of epigenetics, regulates gene expression at the transcriptional level. Aberrant methylation in DNA regulatory regions could upregulate oncogenes and downregulate tumor suppressor genes without changing the sequences. However, studies of methylation in the control of gene expression are still inadequate. In the present research, we performed bioinformatics analysis to clarify the function of methylation and supply candidate methylation-related biomarkers and drivers for colon cancer.AIMTo identify and analyze methylation-regulated differentially expressed genes (MeDEGs) in colon cancer by bioinformatics analysis.METHODSWe downloaded RNA expression profiles, Illumina Human Methylation 450K BeadChip data, and clinical data of colon cancer from The Cancer Genome Atlas project. MeDEGs were identified by analyzing the gene expression and methylation levels using the edgeR and limma package in R software. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed in the DAVID database and KEGG Orthology-Based Annotation System 3.0, respectively. We then conducted Kaplan–Meier survival analysis to explore the relationship between methylation and expression and prognosis. Gene set enrichment analysis (GSEA) and investigation of protein-protein interactions (PPI) were performed to clarify the function of prognosis-related genes.RESULTSA total of 5 up-regulated and 81 down-regulated genes were identified as MeDEGs. GO and KEGG pathway analyses indicated that MeDEGs were enriched in multiple cancer-related terms. Furthermore, Kaplan–Meier survival analysis showed that the prognosis was negatively associated with the methylation status of glial cell-derived neurotrophic factor (GDNF) and reelin (RELN). In PPI networks, GDNF and RELN interact with neural cell adhesion molecule 1. Besides, GDNF can interact with GDNF family receptor alpha (GFRA1), GFRA2, GFRA3, and RET. RELN can interact with RAFAH1B1, disabled homolog 1, very low-density lipoprotein receptor, lipoprotein receptor-related protein 8, and NMDA 2B. Based on GSEA, hypermethylation of GDNF and RELN were both significantly associated with pathways including “RNA degradation,” “ribosome,” “mismatch repair,” “cell cycle” and “base excision repair.”CONCLUSIONAberrant DNA methylation plays an important role in colon cancer progression. MeDEGs that are associated with the overall survival of patients may be potential targets in tumor diagnosis and treatment.

Enhancement of HGF-induced tubulogenesis by endothelial cell-derived GDNF.

Tubulogenesis, the organization of epithelial cells into tubular structures, is an essential step during renal organogenesis as well as during the regeneration process of renal tubules after injury. In the present study, endothelial cell-derived factors that modulate tubule formation were examined using an in vitro human tubulogenesis system. When human renal proximal tubular epithelial cells (RPTECs) were cultured in gels, tubular structures with lumens were induced in the presence of hepatocyte growth factor (HGF). Aquaporin 1 was localized in the apical membrane of these tubular structures, suggesting that these structures are morphologically equivalent to renal tubules in vivo. HGF-induced tubule formation was significantly enhanced when co-cultured with human umbilical vein endothelial cells (HUVECs) or in the presence of HUVEC-conditioned medium (HUVEC-CM). Co-culture with HUVECs did not induce tubular structures in the absence of HGF. A phospho-receptor tyrosine kinase array revealed that HUVEC-CM markedly enhanced phosphorylation of Ret, glial cell-derived neurotrophic factor (GDNF) receptor, in HGF-induced tubular structures compared to those without HUVEC-CM. HUVECs produced GDNF, and RPTECs expressed both Ret and GDNF family receptor alpha1 (co-receptor). HGF-induced tubule formation was significantly enhanced by addition of GDNF. Interestingly, not only HGF but also GDNF significantly induced phosphorylation of the HGF receptor, Met. These data indicate that endothelial cell-derived GDNF potentiates the tubulogenic properties of HGF and may play a critical role in the epithelial-endothelial crosstalk during renal tubulogenesis as well as tubular regeneration after injury.

Preserved Expression of Skin Neurotrophic Factors in Advanced Diabetic Neuropathy Does Not Lead to Neural Regeneration despite Pancreas and Kidney Transplantation.

Diabetic peripheral neuropathy (DPN) is a common complication of diabetes with potential severe consequences. Its pathogenesis involves hyperglycemia-linked mechanisms, which may include changes in the expression of neurotrophic growth factors. We analyzed the expression of 29 factors potentially related to nerve degeneration and regeneration in skin biopsies from 13 type 1 diabetic pancreas and kidney recipients with severe DPN including severe depletion of intraepidermal nerve fibers (IENF) in lower limb skin biopsies (group Tx1 1st examination). The investigation was repeated after a median 28-month period of normoglycemia achieved by pancreas transplantation (group Tx1 2nd examination). The same tests were performed in 13 stable normoglycemic pancreas and kidney recipients 6–12 years posttransplantation (group Tx2), in 12 matched healthy controls (group HC), and in 12 type 1 diabetic subjects without severe DPN (group DM). Compared to DM and HC groups, we found a significantly higher (p < 0.05–0.001) expression of NGF (nerve growth factor), NGFR (NGF receptor), NTRK1 (neurotrophic receptor tyrosine kinase 1), GDNF (glial cell-derived neurotrophic factor), GFRA1 (GDNF family receptor alpha 1), and GFAP (glial fibrillary acidic protein) in both transplant groups (Tx1 and Tx2). Enhanced expression of these factors was not normalized following the median 28-month period of normoglycemia (Tx1 2nd examination) and negatively correlated with IENF density and with electrophysiological indices of DPN (vibration perception threshold, electromyography, and autonomic tests). In contrast to our expectation, the expression of most of 29 selected factors related to neural regeneration was comparable in subjects with severe peripheral nerve fiber depletion and healthy controls and the expression of six factors was significantly upregulated. These findings may be important for better understanding the pathophysiology of nerve regeneration and for the development of intervention strategies.

Both Gfrα1a and Gfrα1b Are Involved in the Self-renewal and Maintenance of Spermatogonial Stem Cells in Medaka.

Glial cell-derived neurotrophic factor family receptor alpha-1 (GFRα1) plays a crucial role in the self-renewal and maintenance of spermatogonial stem cells (SSCs) from mammals. However, to date, our knowledge about its role in fish SSCs is limited. In the present study, the medaka (Oryzias latipes) gfrα1 duplicate genes, Olgfrα1a and Olgfrα1b, were cloned and characterized. Furthermore, their expression profile and biological activity were investigated. OlGfrα1a and OlGfrα1b predict 524 and 466 amino acid residues, respectively. Both are orthologous to mammalian Gfrα1 by sequence analyses and appear high in spermatogonia by in situ hybridization assay. The knockdown of OlGfrα1a and/or OlGfrα1b via Vivo-Morpholino oligos significantly inhibited the self-renewal and maintenance of SSCs, as evidenced by the decreased proliferation activity of SG3 cells (a spermatogonial stem cell line derived from adult medaka testis) as well as spermatogonia in the testicular organ culture and by the decreased survival rate and expression levels of pluripotency-related genes (klf4, lin28b, bcl6b, and etv5) in SG3 cells. Additionally, our study indicates that OlGfrα1a might function by binding either Gdnfa or Gdnfb (the two medaka Gdnf homologs), whereas OlGfrα1b function by binding Gdnfa not Gdnfb. Taken together, our study indicates that both OlGfrα1a and OlGfrα1b are involved in the self-renewal and maintenance of SSCs by binding Gdnfa and/or Gdnfb, respectively. These findings suggest that the GDNF/GFRα1 signaling pathway might be conserved from mammals to fish species.