Cell-derived Neurotrophic Factor Family Receptor Research Articles

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.

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.

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.

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.

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.

Immunolocalization of proteins in the spermatogenesis process of canine.

Spermatogenesis is a process in which differentiated cells are produced and the adult stem cell population-known as spermatogonial stem cells (SSCs)-is continuously replenished. However, the molecular mechanisms underlying these processes are not fully understood in the canine species. We addressed this in this study by analysing the expression of specific markers in spermatogonia of seminiferous tubules of canine testes. SSCs at different stages of reproductive development (prepubertal and adult) were examined by immunohistochemistry and flow cytometry. Glial cell-derived neurotrophic factor family receptor alpha-1 (GFRA1), deleted in azoospermia-like (DAZL) and promyelocytic leukaemia zinc finger (PLZF) were expressed in SSCs, while stimulated by retinoic acid gene 8 (STRA8) was detected only in undifferentiated spermatogonia in prepubertal testis and differentiated spermatogonia and spermatocytes in adult canine. Octamer-binding transcription factor 4 (OCT4) showed an expression pattern, and the levels did not differ between the groups examined. However, C-kit expression varied as a function of reproductive developmental stage. Our results demonstrate that these proteins play critical roles in the self-renewal and differentiation of SSCs and can serve as markers to identify canine spermatogonia at specific stages of development.

Antinociceptive activity of astragaloside IV in the animal model of chronic constriction injury.

To investigate the applicability of astragaloside IV (AG) for the treatment of refractory neuropathic pain, we systemically evaluated the antinociceptive activity of AG in the animal model of chronic constriction injury. We studied behaviors, electrophysiology, and biochemistry from day 2 to day 23 after the surgery. We found that when administered intraperitoneally at the dose of 60 mg/kg, AG caused significant inhibition of allodynia and hyperalgesia induced by mechanic and thermal stimuli as well as downregulation of the expressions of a series of proteins involved in mediating neuropathic pain in the dorsal root ganglia, such as P2X purinoceptor 3, glial cell-derived neurotrophic factor, glial cell-derived neurotrophic factor family receptor α1, and transient receptor potential cation channel subtypes A1 and V1. Further investigation showed that AG restored the nerve conduction velocity and the histological structure of the damaged sciatic nerve on day 23 after the surgery. Moreover, results from immunoelectron microscope showed that glial cell-derived neurotrophic factor family receptor α1 induced by AG could form a circular band in the myelin debris between the injured axons and Schwann cells, contributing toward restoration of the damaged nerve. In conclusion, in our animal model, AG effectively inhibited the neuropathic pain induced by chronic constriction injury.

Characterization of spermatogonial markers in the mature testis of the dogfish (Scyliorhinus canicula L.)

In dogfish, spermatogenesis progresses from a restricted germinative zone, which lines the dorsal testicular vessel. Single spermatogonia (A(s)), including the spermatogonial stem cells (SSCs), produce successively paired (A(p)), undifferentiated (A(u4) to A(u512)), and differentiated (A(d1) to A(d8)) spermatogonia and preleptotene (PL) spermatocytes through 13 mitoses. Dogfish spermatogonial subpopulations present classical morphological characteristics but cannot be distinguished on the basis of molecular markers. This characterization has been initiated in mammals despite the difficulty to separate each spermatogonial subpopulation. For instance, both glial cell-derived neurotrophic factor family receptor alpha 1 (GFRα1) and promyelocytic leukemia zinc finger protein (PLZF) are markers of undifferentiated spermatogonia, whereas receptor tyrosine kinase C-kit is a marker of differentiated spermatogonia. The aim of this study is to characterize spermatogonial markers and to differentiate several spermatogonial subpopulations. Dogfish cDNA sequences have been identified and validated by phylogenetic analyses for gfrα1, plzf, pou2, as well as for high-mobility group box proteins 2 and 3 (hmgb2 and 3) and for mini-chromosome maintenance protein 6 (mcm6). We have used the anatomical advantage of the polarized dogfish testis to analyze the expression of those markers by RT-PCR and in situ hybridization. gfrα1, pou2, and plzf have been detected in the testicular germinative zone, suggesting that spermatogonial markers are relatively well conserved among vertebrates but with a less restricted expression for plzf. Moreover, hmgb3 and mcm6 have been identified as new markers of differentiated spermatogonia. Finally, this first molecular characterization of spermatogonial subpopulations in a chondrichthyan model will be useful for further studies on the SSC niche evolution.

Magnetic activated cell sorting allows isolation of spermatogonia from adult primate testes and reveals distinct GFRa1‐positive subpopulations in men

Isolation of spermatogonial stem cells (SSCs) could enable in vitro approaches for exploration of spermatogonial physiology and therapeutic approaches for fertility preservation. SSC isolation from adult testes is difficult due to low cell numbers and lacking cell surface markers. Glial cell-derived neurotrophic factor family receptor alpha-1 (GFRalpha1) plays a crucial role for the maintenance of SSCs in rodents and is expressed in monkey spermatogonia. Magnetic activated cell sorting was employed for the enrichment of GFRalpha1+ spermatogonia from adult primate testes. Magnetic activated cell sorting of monkey cells enriched GFRalpha1+ cells threefold. 11.4% of GFRalpha1+ cells were recovered. 42.9% of GFRalpha1+ cells were recovered in sorted fractions of human testicular cells, representing a fivefold enrichment. Interestingly, a high degree of morphological heterogeneity among the GFRalpha1+ cells from human testes was observed. Magnetic activated cell sorting using anti-GFRalpha1 antibodies provides an enrichment strategy for spermatogonia from monkey and human testes.

Vascular-derived artemin: a determinant of vascular sympathetic innervation?

Vascular sympathetic innervation is an important determinant of blood pressure and blood flow. The mechanisms that determine vascular sympathetic innervation are not well understood. The present study tests the hypothesis that vascular-derived artemin promotes the development of sympathetic innervation to blood vessels by promoting sympathetic axon growth. RT-PCR and Western analyses indicate that artemin is expressed by cultured vascular smooth muscle and arteries, and artemin coreceptors, glial cell-derived neurotrophic factor family receptor alpha3 and ret, are expressed by postganglionic sympathetic neurons. The effects of artemin on axon growth were assessed on explants of neonatal rat sympathetic ganglia. In the presence, but not in the absence, of nerve growth factor, exogenous artemin stimulated neurite growth. Femoral arteries (FA) from adult rats contain artemin, and these arteries stimulated sympathetic neurite growth. Growth in the presence of FA was 92.2 +/- 11.9 mm, and that in the absence of FA was 26.3 +/- 5.4 mm (P < 0.05). FA stimulation of axon growth was reduced by an antibody that neutralized the activity of artemin (P < 0.05). These data indicate that artemin is expressed in arteries, and its receptors are expressed and functional in the postganglionic sympathetic neurons that innervate them. This suggests that artemin may be a determinant of vascular sympathetic innervation.

Morphological Characterization of the Spermatogonial Subtypes in the Neonatal Mouse Testis1

Spermatogenesis is the process of differentiation of diploid type A spermatogonia to haploid spermatozoa. Several subtypes of A spermatogonia have been characterized in the adult mouse testis. These include A-single (A(s)), A-paired (A(pr)), A-aligned (A(al)), and A1-A4. However, in the immature testis, very little information is available on subtypes and morphological features of type A spermatogonia. Six-day-old mouse testes, fixed either in Bouin solution or 5% glutaraldehyde, were embedded in paraffin and Epon, respectively. Thick sections (approximately 1 microm) of Epon-embedded tissue were stained with toluidine blue and revealed three subtypes of spermatogonia by light microscopy. The smallest spermatogonia (subtype I) appeared as single cells and exhibited a round or oval flattened nucleus with one or two prominent dense nucleoli and a characteristic unstained round and centrally located vacuole. These cells bound toluidine blue more avidly and appeared darker in comparison with the other cell types. Electron microscopy of thin sections (90 nm) revealed a finely granulated chromatin homogeneously distributed in the nucleus and sparse organelles in the cytoplasm. The second subtype of spermatogonia (subtype II) also displayed dark staining but was larger than subtype I; there was no central vacuole in the nucleus and heterochromatin clumps were observed. The largest subtype of spermatogonia (subtype III) showed large heterochromatin clumps and a pale staining nucleus. Intercellular bridges were noted between subtypes II and III. Based on the dye avidity, the three subtypes were classified as dark, transitional, and pale spermatogonia, respectively. Image analyses of 30 different cells of each subtype revealed a decline in gray-scale intensity from subtype I to III. Five-micrometer sections of paraffin-embedded tissue were immunoassayed with an antibody against the glial cell-derived neurotrophic factor family receptor alpha-1 (GFRalpha-1) receptor, a putative marker for undifferentiated spermatogonia, showing positive reaction only in germ cells. The pattern of GFRalpha-1 expression, coupled to the overall morphology of the cells, indicates that at this stage of development, mouse seminiferous tubules contain essentially A(s), A(pr), and possibly A(al) spermatogonia. Thus, the present study indicates the presence of subtypes of type A spermatogonia in the immature mouse testis similar to that described previously in adult monkey and man.