ABSTRACT
Studies have shown that disrupting the formation of the ligand-RET-GFRα complex could be an effective way of treating pain and itch. Compared to traditional high-throughput screens, DNA encoded libraries (DELs) have distinguished themselves as a powerful technology for hit identification in recent years. The present work demonstrates the use of DEL technology identifying compound 16 as the first GFRa2/GFRa3 small molecule inhibitor (0.1/0.2 µM respectively) selective over RET. This molecule represents an opportunity to advance the development of small-molecule inhibitors targeting the GFRα-RET interface for the treatment of pain and itch.
Subject(s)
DNA , Glial Cell Line-Derived Neurotrophic Factor Receptors , Small Molecule Libraries , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Small Molecule Libraries/chemical synthesis , Humans , DNA/chemistry , DNA/metabolism , Glial Cell Line-Derived Neurotrophic Factor Receptors/metabolism , Glial Cell Line-Derived Neurotrophic Factor Receptors/antagonists & inhibitors , Drug Discovery , Structure-Activity Relationship , Molecular Structure , Dose-Response Relationship, DrugABSTRACT
Metformin, the world's most prescribed anti-diabetic drug, is also effective in preventing type 2 diabetes in people at high risk1,2. More than 60% of this effect is attributable to the ability of metformin to lower body weight in a sustained manner3. The molecular mechanisms by which metformin lowers body weight are unknown. Here we show-in two independent randomized controlled clinical trials-that metformin increases circulating levels of the peptide hormone growth/differentiation factor 15 (GDF15), which has been shown to reduce food intake and lower body weight through a brain-stem-restricted receptor. In wild-type mice, oral metformin increased circulating GDF15, with GDF15 expression increasing predominantly in the distal intestine and the kidney. Metformin prevented weight gain in response to a high-fat diet in wild-type mice but not in mice lacking GDF15 or its receptor GDNF family receptor α-like (GFRAL). In obese mice on a high-fat diet, the effects of metformin to reduce body weight were reversed by a GFRAL-antagonist antibody. Metformin had effects on both energy intake and energy expenditure that were dependent on GDF15, but retained its ability to lower circulating glucose levels in the absence of GDF15 activity. In summary, metformin elevates circulating levels of GDF15, which is necessary to obtain its beneficial effects on energy balance and body weight, major contributors to its action as a chemopreventive agent.
Subject(s)
Body Weight/drug effects , Energy Metabolism/drug effects , Growth Differentiation Factor 15/metabolism , Metformin/pharmacology , Administration, Oral , Adult , Aged , Animals , Blood Glucose/analysis , Blood Glucose/metabolism , Diet, High-Fat , Double-Blind Method , Energy Intake/drug effects , Enterocytes/cytology , Enterocytes/drug effects , Female , Glial Cell Line-Derived Neurotrophic Factor Receptors/antagonists & inhibitors , Glial Cell Line-Derived Neurotrophic Factor Receptors/deficiency , Glial Cell Line-Derived Neurotrophic Factor Receptors/genetics , Growth Differentiation Factor 15/blood , Growth Differentiation Factor 15/deficiency , Growth Differentiation Factor 15/genetics , Homeostasis/drug effects , Humans , Intestines/cytology , Intestines/drug effects , Male , Metformin/administration & dosage , Mice , Mice, Obese , Middle Aged , Weight Loss/drug effectsABSTRACT
The glycosylphosphatidylinositol-linked GDNF (glial cell derived neurotrophic factor) receptor alpha (GFRA), a coreceptor that recognizes the GDNF family of ligands, has a crucial role in the development and maintenance of the nervous system. Of the four identified GFRA isoforms, GFRA1 specifically recognizes GDNF and is involved in the regulation of proliferation, differentiation, and migration of neuronal cells. GFRA1 has also been implicated in cancer cell progression and metastasis. Recent findings show that GFRA1 can contribute to the development of chemoresistance in osteosarcoma. GFRA1 expression was induced following treatment of osteosarcoma cells with the popular anticancer drug, cisplatin and induction of GFRA1 expression significantly suppressed apoptosis mediated by cisplatin in osteosarcoma cells. GFRA1 expression promotes autophagy by activating the SRC-AMPK signaling axis following cisplatin treatment, resulting in enhanced osteosarcoma cell survival. GFRA1-induced autophagy promoted tumor growth in mouse xenograft models, suggesting a novel function of GFRA1 in osteosarcoma chemoresistance.
Subject(s)
Bone Neoplasms/genetics , Drug Resistance, Neoplasm/genetics , Glial Cell Line-Derived Neurotrophic Factor Receptors/genetics , Glial Cell Line-Derived Neurotrophic Factor Receptors/metabolism , Osteosarcoma/genetics , Animals , Antineoplastic Agents/pharmacology , Biomarkers , Bone Neoplasms/metabolism , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , Disease Susceptibility , Glial Cell Line-Derived Neurotrophic Factor/metabolism , Glial Cell Line-Derived Neurotrophic Factor Receptors/antagonists & inhibitors , Glial Cell Line-Derived Neurotrophic Factor Receptors/chemistry , Humans , Osteosarcoma/metabolism , Protein Binding , Protein Interaction Domains and Motifs , Protein Multimerization , Signal Transduction/drug effectsABSTRACT
Luminal A (hormone receptor-positive) breast cancer constitutes 70% of total breast cancer patients. In an attempt to develop a targeted therapeutic for this cancer indication, we have identified and characterized Glial cell line-Derived Neurotrophic Factor (GDNF) Family Receptor Alpha 1 (GFRA1) antibody-drug conjugates (ADC) using a cleavable valine-citrulline-MMAE (vcMMAE) linker-payload. RNAseq and IHC analysis confirmed the abundant expression of GFRA1 in luminal A breast cancer tissues, whereas minimal or no expression was observed in most normal tissues. Anti-GFRA-vcMMAE ADC internalized to the lysosomes and exhibited target-dependent killing of GFRA1-expressing cells both in vitro and in vivo The ADCs using humanized anti-GFRA1 antibodies displayed robust therapeutic activity in clinically relevant cell line-derived (MCF7 and KPL-1) tumor xenograft models. The lead anti-GFRA1 ADC cross-reacts with rodent and cynomolgus monkey GFRA1 antigen and showed optimal pharmacokinetic properties in both species. These properties subsequently enabled a target-dependent toxicity study in rats. Anti-GFRA1 ADC is well tolerated in rats, as seen with other vcMMAE linker-payload based ADCs. Overall, these data suggest that anti-GFRA1-vcMMAE ADC may provide a targeted therapeutic opportunity for luminal A breast cancer patients. Mol Cancer Ther; 17(3); 638-49. ©2017 AACR.
Subject(s)
Breast Neoplasms/drug therapy , Glial Cell Line-Derived Neurotrophic Factor Receptors/antagonists & inhibitors , Immunoconjugates/pharmacology , Xenograft Model Antitumor Assays , Animals , Antibodies/chemistry , Antibodies/immunology , Antibodies/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Cell Line, Tumor , Cell Survival/drug effects , Cell Survival/genetics , Female , Gene Expression Regulation, Neoplastic/drug effects , Glial Cell Line-Derived Neurotrophic Factor Receptors/genetics , Glial Cell Line-Derived Neurotrophic Factor Receptors/immunology , HEK293 Cells , Humans , Immunoconjugates/immunology , Immunoconjugates/pharmacokinetics , MCF-7 Cells , Macaca fascicularis , Mice, Nude , Mice, SCID , Rats, Sprague-Dawley , Receptors, Steroid/metabolism , Tumor Burden/drug effects , Tumor Burden/geneticsABSTRACT
Tissue injury prompts the release of a number of proalgesic molecules that induce acute and chronic pain by sensitizing pain-sensing neurons (nociceptors) to heat and mechanical stimuli. In contrast, many proalgesics have no effect on cold sensitivity or can inhibit cold-sensitive neurons and diminish cooling-mediated pain relief (analgesia). Nonetheless, cold pain (allodynia) is prevalent in many inflammatory and neuropathic pain settings, with little known of the mechanisms promoting pain vs. those dampening analgesia. Here, we show that cold allodynia induced by inflammation, nerve injury, and chemotherapeutics is abolished in mice lacking the neurotrophic factor receptor glial cell line-derived neurotrophic factor family of receptors-α3 (GFRα3). Furthermore, established cold allodynia is blocked in animals treated with neutralizing antibodies against the GFRα3 ligand, artemin. In contrast, heat and mechanical pain are unchanged, and results show that, in striking contrast to the redundant mechanisms sensitizing other modalities after an insult, cold allodynia is mediated exclusively by a single molecular pathway, suggesting that artemin-GFRα3 signaling can be targeted to selectively treat cold pain.
Subject(s)
Glial Cell Line-Derived Neurotrophic Factor Receptors/metabolism , Hyperalgesia/metabolism , Pain/metabolism , Signal Transduction , Animals , Antibodies, Neutralizing/pharmacology , Disease Models, Animal , Glial Cell Line-Derived Neurotrophic Factor Receptors/antagonists & inhibitors , Glial Cell Line-Derived Neurotrophic Factor Receptors/genetics , Hyperalgesia/genetics , Hyperalgesia/pathology , Inflammation/genetics , Inflammation/metabolism , Inflammation/pathology , Mice , Mice, Knockout , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Pain/genetics , Pain/pathologyABSTRACT
This study aimed to explore the effect of high glucose concentration on the expression of glial cell line-derived neurotrophic factor (GDNF) and its family ligand receptors (GFRs) GFRα1 and GFRα2 in Müller cells and the protective role of GDNF in cultured Müller cells under high glucose circumstance. Cultured Müller cells (untreated or treated with 200 ng/mL of GDNF) were exposed to high glucose conditions (20 mmol/L glucose). We found that the expression levels of GDNF and GFRα1 mRNA and protein increased gradually over time under high glucose and exogenous GDNF-treated conditions, whereas the upregulation in GFRα2 expression was observed only in the early stage of high glucose conditions. Exogenous GDNF not only decreased apoptosis in cultured Müller cells under high glucose circumstance, but also accelerated the levels and speed of synthesis of GDNF and GFRα1 proteins in Müller cells. These results suggest that Müller cells can synthesize GDNF and GFRs under high glucose conditions, and GDNF may play important role in protecting Müller cells during the early stage of diabetic retinopathy. The difference in GFRs expression indicated that GDNF and neurturin may exert different effects on Müller cells under high glucose circumstance.
Subject(s)
Diabetic Retinopathy/metabolism , Glial Cell Line-Derived Neurotrophic Factor Receptors/biosynthesis , Glial Cell Line-Derived Neurotrophic Factor/biosynthesis , Hyperglycemia/metabolism , Hyperglycemia/pathology , Neuroglia/metabolism , Retina/metabolism , Animals , Animals, Newborn , Diabetic Retinopathy/pathology , Diabetic Retinopathy/physiopathology , Disease Models, Animal , Down-Regulation/genetics , Glial Cell Line-Derived Neurotrophic Factor/genetics , Glial Cell Line-Derived Neurotrophic Factor Receptors/antagonists & inhibitors , Glial Cell Line-Derived Neurotrophic Factor Receptors/genetics , Hyperglycemia/physiopathology , Neuroglia/pathology , Primary Cell Culture , Rats , Rats, Sprague-Dawley , Retina/pathology , Retina/physiopathology , Up-Regulation/geneticsABSTRACT
Little is known about the role of neurotrophic growth factors in bone metabolism. This study investigated the short-term effects of glial cell line-derived neurotrophic factor (GDNF) on calvarial-derived MC3T3-E1 osteoblasts. MC3T3-E1 expressed GDNF as well as its canonical receptors, GFRα1 and RET. Addition of recombinant GDNF to cultures in serum-containing medium modestly inhibited cell growth at high concentrations; however, under serum-free culture conditions GDNF dose-dependently increased cell proliferation. GDNF effects on cell growth were inversely correlated with its effect on alkaline phosphatase (AlP) activity showing a significant dose-dependent inhibition of relative AlP activity with increasing concentrations of GDNF in serum-free culture medium. Live/dead and lactate dehydrogenase assays demonstrated that GDNF did not significantly affect cell death or survival under serum-containing and serum-free conditions. The effect of GDNF on cell growth was abolished in the presence of inhibitors to GFRα1 and RET indicating that GDNF stimulated calvarial osteoblasts via its canonical receptors. Finally, this study found that GDNF synergistically increased tumor necrosis factor-α (TNF-α)-stimulated MC3T3-E1 cell growth suggesting that GDNF interacted with TNF-α-induced signaling in osteoblastic cells. In conclusion, this study provides evidence for a direct, receptor-mediated effect of GDNF on osteoblasts highlighting a novel role for GDNF in bone physiology.
Subject(s)
Glial Cell Line-Derived Neurotrophic Factor/pharmacology , Osteoblasts/cytology , Osteoblasts/drug effects , Animals , Bromodeoxyuridine/metabolism , Cell Count , Cell Death/drug effects , Cell Line , Cell Proliferation/drug effects , Cell Survival/drug effects , Glial Cell Line-Derived Neurotrophic Factor/genetics , Glial Cell Line-Derived Neurotrophic Factor/metabolism , Glial Cell Line-Derived Neurotrophic Factor Receptors/antagonists & inhibitors , Glial Cell Line-Derived Neurotrophic Factor Receptors/genetics , Glial Cell Line-Derived Neurotrophic Factor Receptors/metabolism , Humans , Mice , Osteoblasts/metabolism , Protein Binding/drug effects , Proto-Oncogene Proteins c-ret/antagonists & inhibitors , Proto-Oncogene Proteins c-ret/genetics , Proto-Oncogene Proteins c-ret/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Skull/cytologyABSTRACT
Mutations of genes in the renin-angiotensin system are associated with congenital abnormalities of the kidney and urinary tract. The major signaling pathway for branching morphogenesis during kidney development is the c-Ret receptor tyrosine kinase whose ligand is GDNF and whose downstream target is Wnt11. We determined whether angiotensin II, an inducer of ureteric bud branching in vitro, influences the GDNF/c-Ret/Wnt11 pathway. Mouse metanephroi were grown in the presence or absence of angiotensin II or an angiotensin type 1 receptor (AT1R) antagonist and gene expression was measured by whole mount in situ hybridization. Angiotensin II induced the expression of c-Ret and Wnt11 in ureteric bud tip cells. GDNF, a Wnt11-regulated gene expressed in the mesenchyme, was also upregulated by angiotensin II but this downregulated Spry1, an endogenous inhibitor of Ret tyrosine kinase activity in an AT1R-dependent manner. Angiotensin II also decreased Spry1 mRNA levels in cultured ureteric bud cells. Exogenous angiotensin II preferentially stimulated ureteric bud tip cell proliferation in vivo while AT1R blockade increased cell apoptosis. Our findings suggest AT1R-mediated inhibition of the Spry1 gene increases c-Ret tyrosine kinase activity leading to upregulation of its downstream target Wnt11. Enhanced Wnt11 expression induces GDNF in adjacent mesenchyme causing focal bursts of ureteric bud tip cell proliferation, decreased tip cell apoptosis and branching.
Subject(s)
Angiotensin II/physiology , Glial Cell Line-Derived Neurotrophic Factor Receptors/metabolism , Glial Cell Line-Derived Neurotrophic Factor/genetics , Kidney/growth & development , Membrane Proteins/genetics , Morphogenesis , Phosphoproteins/genetics , Receptor, Angiotensin, Type 1/physiology , Wnt Proteins/metabolism , Adaptor Proteins, Signal Transducing , Animals , Apoptosis , Cell Proliferation , Down-Regulation , Gene Expression Regulation , Glial Cell Line-Derived Neurotrophic Factor/antagonists & inhibitors , Glial Cell Line-Derived Neurotrophic Factor Receptors/antagonists & inhibitors , Membrane Proteins/physiology , Mesoderm , Mice , Phosphoproteins/physiology , Urinary Tract/growth & development , Wnt Proteins/geneticsABSTRACT
Spermatogenesis is the process by which spermatogonial stem cells divide and differentiate into sperm. The role of growth factor receptors in regulating self-renewal and differentiation of spermatogonial stem cells remains largely unclear. This study was designed to examine Gfra1 receptor expression in immature and adult mouse testes and determine the effects of Gfra1 knockdown on the proliferation and differentiation of type A spermatogonia. We demonstrated that GFRA1 was expressed in a subpopulation of spermatogonia in immature and adult mice. Neither Gfra1 mRNA nor GFRA1 protein was detected in pachytene spermatocytes and round spermatids. GFRA1 and POU5F1 (also known as OCT4), a marker for spermatogonial stem cells, were co-expressed in a subpopulation of type A spermatogonia from 6-day-old mice. In addition, the spermatogonia expressing GFRA1 exhibited a potential for proliferation and the ability to form colonies in culture, which is a characteristic of stem cells. RNA interference assays showed that Gfra1 small interfering RNAs (siRNAs) knocked down the expression of Gfra1 mRNA and GFRA1 protein in type A spermatogonia. Notably, the reduction of Gfra1 expression by Gfra1 siRNAs induced a phenotypic differentiation, as evidenced by the elevated expression of KIT, as well as the decreased expression of POU5F1 and proliferating cell nuclear antigen (PCNA). Furthermore, Gfra1 silencing resulted in a decrease in RET phosphorylation. Taken together, these data indicate that Gfra1 is expressed dominantly in mouse spermatogonial stem cells and that Gfra1 knockdown leads to their differentiation via the inactivation of RET tyrosine kinase, suggesting an essential role for Gfra1 in spermatogonial stem cell regulation.
Subject(s)
Glial Cell Line-Derived Neurotrophic Factor Receptors/antagonists & inhibitors , Proto-Oncogene Proteins c-ret/antagonists & inhibitors , Spermatogonia/cytology , Stem Cells/cytology , Animals , Cell Differentiation , Glial Cell Line-Derived Neurotrophic Factor Receptors/analysis , Glial Cell Line-Derived Neurotrophic Factor Receptors/genetics , Male , Mice , Mice, Inbred BALB C , Nerve Growth Factors , Octamer Transcription Factor-3/analysis , Octamer Transcription Factor-3/metabolism , RNA, Messenger/analysis , RNA, Messenger/metabolism , RNA, Small Interfering/pharmacology , Sertoli Cells/metabolism , Spermatids/cytology , Spermatids/metabolism , Spermatocytes/cytology , Spermatocytes/metabolism , Spermatogonia/chemistry , Spermatogonia/metabolism , Stem Cells/chemistry , Stem Cells/metabolismABSTRACT
Glial cell line-derived neurotrophic factor (GDNF) has been proved to play an important role in the modulation of nociceptive transmission especially during neuropathic pain. It was reported that electroacupuncture (EA) had potent analgesic effect on neuropathic pain and our previous studies indicated that EA could activate endogenous GDNF signaling system (GDNF and its receptor GFRalpha-1) in dorsal root ganglions (DRGs) of neuropathic pain rats. In order to investigate whether GDNF signaling system was involved in EA analgesia on neuropathic pain, which was induced by chronic constriction injury (CCI) of the sciatic nerve in rats, antisense oligodeoxynucleotide (ODN) specifically against GFRalpha-1 was used in the present study to result in down-regulation of GFRalpha-1 expression. The results showed that: (1) cumulative EA had potent analgesic effect on neuropathic pain in rats; (2) the expression of GFRalpha-1 in DRGs was down-regulated by intrathecal delivery of antisense ODN, but not by normal saline (NS) or mismatch ODN; (3) EA analgesia was significantly attenuated by antisense ODN treatment. The present study demonstrated that endogenous GDNF signaling system was involved in EA analgesia on neuropathic pain in rats, which would deepen our realization of the mechanism of EA analgesia.