OBJECTIVE

To compare the clinical characteristics of triple-negative (TN) breast cancer and non-triple-negative (NTN) breast cancer, enrich the information of TN patients, and provide evidences for individualized combined treatment.

METHODS

The data of 408 cases received operation in the year of 2002 was enrolled in this study. TN patients were confirmed according to the immunohistochemical (IHC) test of estrogen receptor (ER), progesterone receptor (PR) and HER-2/neu. The clinical characteristics, recurrence, metastasis and survival were compared between the two groups.

RESULTS

Seventy-seven patients (18.9%) were confirmed TN cases. The median follow-up was 64 months (range, 3-79 months). Of all the cases, 58 occurred local recurrence or metastasis and 51 died, it was 19 and 12 in TN group. Compared with the NTN group, the TN patient tended to be younger and the tumor mass larger (P=0.015 and 0.011). However, axillary lymph nodes metastasis occurred more often in NTN patients than in TN patients (P=0.001). The rate of local recurrence and metastasis in TN group was significantly higher than in NTN group (P=0.005 and 0.025), and TN cases were more likely to develop lung metastasis than NTN patients (P<0.01). The 3-year and 5-year overall survival rate in TN group were significantly lower than in NTN group (86.4% vs. 93.4%, P=0.0205; 77.7% vs. 87.9%, P=0.0215). The 3-year and 5-year disease-free survival rate in TN group were also significantly lower than in NTN group (78.4% vs. 92.4%, P=0.0038; 72.8% vs. 85.8%, P=0.0041). Tumor size, lymph node status and triple-negative were the most important factors influencing the prognosis on multivariate Cox regression analysis.

CONCLUSIONS

TN breast cancer haa some specific clinical characteristics. The prognosis of TN patients is worse than that of NTN patients. Further study is needed to find individualized treatment for TN breast cancer patients.

Cryptosporidium parvum is a waterborne protozoan parasite that is found intracellularly in host animals, including humans, and causes severe diarrhea, which can lead to the death of an immunocompromised individual. Previously, we found that this organism is highly radioresistant as it can productively infect mice after exposure to a 10-kGy dose of γ-radiation. To understand how C. parvum avoids radiation damage, we characterized its protein expression patterns 6, 24, and 48 h after a 10-kGy dose of γ-radiation using two-dimensional PAGE. The gels showed 10 silver-stained spots that increased or decreased in size following γ-irradiation. Five proteins contained in these spots were identified using MALDI-TOF MS peptide fingerprinting, and two of these showed an increase in expression after γ-irradiation. These proteins were identified by LC-MS/MS as proteasome subunit alpha type 4 (NTN hydrolase fold) and thioredoxin peroxidase-like protein. The roles of these two upregulated proteins as related to the radioresistance of C. parvum remain to be evaluated.

Penicillin V acylases (PVA) catalyze the deacylation of the beta-lactam antibiotic phenoxymethylpenicillin (Pen V). They are members of the Ntn hydrolase family and possess an N-terminal cysteine as the main catalytic nucleophile residue. They form the evolutionarily related cholylglycine hydrolase (CGH) group which includes bile salt hydrolases (BSH) responsible for bile deconjugation. Even though a few PVA and BSH structures have been reported, no structure of a functional PVA from Gram-negative bacteria is available. Here, we report the crystal structure of a highly active PVA from Gram-negative Pectobacterium atrosepticum (PaPVA) at 2.5Å resolution. Structural comparison with PVAs from Gram-positive bacteria revealed that PaPVA had a distinctive tetrameric structure and active site organization. In addition, mutagenesis of key active site residues and biochemical characterization of the resultant variants elucidated the role of these residues in substrate binding and catalysis. The importance of residue Trp23 and Trp87 side chains in binding and correct positioning of Pen V by PVAs was confirmed using mutagenesis and substrate docking with a 15ns molecular dynamics simulation. These results establish the unique nature of Gram-negative CGHs and necessitate further research about their substrate spectrum.

γ-Glutamyltranspeptidases (γ-GTs), ubiquitous in glutathione metabolism for γ-glutamyl transfer/hydrolysis, are N-terminal nucleophile (Ntn)-hydrolase fold proteins that share an autoproteolytic process for self-activation. γ-GT homologues are widely present in Gram-positive actinobacteria where their Ntn-hydrolase activities, however, are not involved in glutathione metabolism. Herein, we demonstrate that the formation of 4-Alkyl-L-(dehydro)proline (ALDP) residues, the non-proteinogenic α-amino acids that serve as vital components of many bioactive metabolites found in actinobacteria, involves unprecedented Ntn-hydrolase activity of γ-GT homologue for C-C bond cleavage. The related enzymes share a key Thr residue, which acts as an internal nucleophile for protein hydrolysis and then as a newly released N-terminal nucleophile for carboxylate side-chain processing likely through the generation of an oxalyl-Thr enzyme intermediate. These findings provide mechanistic insights into the biosynthesis of various ALDP residues/associated natural products, highlight the versatile functions of Ntn-hydrolase fold proteins, and particularly generate interest in thus far less-appreciated γ-GT homologues in actinobacteria.

Neurturin (NTN) was previously described for its neuronal activities, but recently, we have shown that this factor is also involved in asthma physiopathology. However, the underlying mechanisms of NTN are unclear. The aim of this study was to investigate NTN involvement in acute bronchial Th2 responses, to analyze its interaction with airway structural cells, and to study its implication in remodeling during acute and chronic bronchial inflammation in C57BL/6 mice. We analyzed the features of allergic airway inflammation in wild-type and NTN(-/-) mice after sensitization with two different allergens, OVA and house dust mite. We showed that NTN(-/-) dendritic cells and T cells had a stronger tendency to activate the Th2 pathway in vitro than similar wild-type cells. Furthermore, NTN(-/-) mice had significantly increased markers of airway remodeling like collagen deposition. NTN(-/-) lung tissues showed higher levels of neutrophils, cytokine-induced neutrophil chemoattractant, matrix metalloproteinase 9, TNF-α, and IL-6. Finally, NTN had the capacity to decrease IL-6 and TNF-α production by immune and epithelial cells, showing a direct anti-inflammatory activity on these cells. Our findings support the hypothesis that NTN could modulate the allergic inflammation in different mouse asthma models.

OBJECTIVE

Neurturin (NTN) is a potent neuronal survival factor in the central and peripheral nervous systems. We previously described altered expression of mRNAs for NTN and one of its receptor components, GFRa-2 in degenerative retinas of rd/rd mice. Towards assessing the potential for transfer of these genes to counteract retinal degeneration, we examined recombinant adeno-associated virus (rAAV) constructs for expression of NTN and GFRa-2 transgenes in retinal cells in vitro and for the effect of transgene expression on retinal function following intraocular delivery in rd/rd mice.

METHODS

The rAAV constructs incorporated epitope tags to facilitate discrimination between transgenic and endogenous expression. Expression of murine NTN was driven by a CMV promoter and a partial murine opsin promoter was used to drive expression of human GFRa-2. rAAV preparations were used to infect mouse retinal cell cultures and for intraocular injection of predegenerative rd/rd mice. Endogenous and transgene expression was analyzed by immunofluorescence. Photoreceptor function in treated mice was assessed by electroretinography.

RESULTS

Both vectors delivered and expressed their transgenes in vitro and in vivo. Differential targeting was achieved in vivo through the use of alternative promoters. Under the conditions examined, no functional rescue of rd photoreceptors was observed.

CONCLUSIONS

Therapeutic treatment of the rd model of retinal degeneration does not appear to be effected by simple modulation of the expression of NTN or GFRa-2, and may therefore depend on additional synergistic factors. Our AAV constructs will facilitate the development of combinatorial approaches to the treatment of central and peripheral neurodegenerations.

Faster or stronger response to pathogen occurs if plants undergo prior priming. Cytokinins seem to be also involved in plant priming and in response to pathogens. Susceptibility to Potato virus Y(NTN) (PVY(NTN)) was studied in transgenic cytokinin overproducing (Pssu-ipt) tobacco and compared with nontransgenic plants. Since cytokinin overproduction inhibits development of plant roots and grafting overcomes this limitation, both types were grown as rooted and/or grafted plants to check also the effect of grafting. Control rooted tobacco (C), the most susceptible to PVY(NTN), showed always symptoms during the infection together with the rising virus content and a systemic response, such as accumulation of H2O2, salicylic acid (SA) and other phenolic acids, and stress-induced enzyme activities. In transgenic and grafted plants, the response to PVY(NTN) was dependent on protective mechanisms activated prior to the inoculation. In Pssu-ipt tobacco, cytokinin active forms and SA contents exceeded manifold their content in C. Grafting promoted the accumulation of phenolics, but SA, and stimulated peroxidase activities. Thus, the pre-infection barrier established in both transgenic and grafted plants helped to suppress partly the virus multiplication and resulted in milder symptom development. However, only the synergic effect of both grafting and the high cytokinins led to PVY(NTN) tolerance in transgenic grafts. Possible mechanisms were discussed.

A full-length infectious cDNA clone of potato virus Y (PVY) was constructed based on an isolate of the PVY(NTN-NW) strain, SYR-II-2-8, which is able to cause potato tuber necrotic ringspot disease (PTNRD). Silent point mutations were introduced to modify sequences similar to the prokaryotic promoter elements detected at the 5' terminus of the P3 coding region of SYR-II-2-8. This, along with modification of the growing conditions of E. coli, led to the successful construction of a stable full-length infectious cDNA clone, named p2-8C3. The biological properties of p2-8C3 were identical to those of SYR-II-2-8, both of which induced PTNRD in potato, veinal necrosis in tobacco, and similar symptoms in the potato cultivars inoculated.

The activity and presence of isoforms of NADP-dependent malic enzyme (NADP-ME, EC 1.1.1.40) were studied in non-transgenic and transgenic Nicotiana benthamiana plants containing potyviral gene for helper component protease (HC-pro) and in plants infected by Potato virus Y strain NTN (PVY(NTN)). No significant changes in enzyme activities and isoenzyme pattern were observed due to foreign gene introduction and PVY(NTN) infection. However, the activity and isoenzyme composition of NADP-ME measured in extracts from different parts of the plants showed significant differences. Non-denaturating electrophoresis followed by specific detection of NADP-ME activity in polyacrylamide gel detected the presence of only one isoform in roots and younger leaves. Two isoforms of NADP-ME were detected in older leaves and stem (relative molecular mass approximately 248,000 and approximately 280,000) and three isoforms corresponding to tetramer, dimer and monomer were found in flowers. The activity of NADP-ME and the isoenzyme pattern was discussed in relation to its role in plant metabolism within distinct plant parts.

The effect of indomethacin on protein excretion and on the synthesis of glomerular basement membrane (GBM) was studied during various stages of nephrotoxic nephritis (NTN) in the rat. Daily administration of indomethacin (4 mg/kg) was instituted 1, 6, or 21 days after the induction of NTN with 210, 240, or 268 microgram kidney-fixing antibodies (KFAb). Proteinuria in rats with nephritis induced by 210 microgram KFAb decreased under treatment with indomethacin regardless of the day on which treatment was started but was not affected by indomethacin in rats with clinically more severe nephritis induced with higher doses of KFAb. GBM synthesis was measured in vivo and in vitro by determination of the incorporation of 3H-proline into the GBM. NTN rats treated with indomethacin showed increased GBM synthesis early in the course of NTN, over and above an already increased synthesis. In the later phase of NTN indomethacin treatment did not affect GBM synthesis. The absence of a relationship between the effect of indomethacin on proteinuria and its effect on GBM synthesis clearly shows that the reduction of protein excretion occurring under indomethacin treatment is not mediated by alterations in the rate of GBM synthesis.

We showed previously that 17β estradiol (E2) led to improved survival in nephrotoxic serum induced nephritis (NTN) in male mice. In this study we determined whether E2 regulates vascular cell adhesion molecule (VCAM)-1, an adhesion molecule that is upregulated in kidney during autoimmune nephritis, in mesangial cells (MC). We show that E2 inhibited VCAM-1 up-regulation in kidneys in vivo during NTN, and in MCs upon TNFα stimulation. VCAM-1 up-regulation in MCs was controlled by the transcription factor NFκB. E2 inhibited RNA polymerase II recruitment to the VCAM-1 promoter, but not p65 recruitment. Interestingly E2 inhibited TNFα stimulated interaction between poly (ADP-ribose) polymerase-1 (PARP-1) and p65. As PARP-1 is required for VCAM-1 upregulation in MCs, our data suggest that E2 may inhibit pre-initiation complex formation at VCAM-1 promoter by inhibiting PARP-1 recruitment to p65. We propose that E2 plays an important role in regulating renal inflammation locally.

The National Clinician Scientist Scheme was devised to enable future leaders of academic medicine to continue post-doctoral research and at the same time complete their specialist registrar training. The awards are generously funded by the major grant-giving bodies and are held in a variety of specialties in academic departments across the UK. Award winners are given the suffix A to add to their existing national training number (NTN) achieved in open competition in the relevant specialty. The deanery whence they came is permitted an additional NTN to be awarded to the clinician scientist's successor in the deanery specialty training programme. The clinician scientist's further clinical training is arranged on an individual basis to dovetail with their ongoing research.

In 2011-2014, ELISA or nucleic acid spot hybridization (NASH) testing for common potato viruses or Potato spindle tuber viroid (PSTVd) was performed on 500 leaf samples collected in potato fields in the northeast provinces Heilongjiang and Inner Mongolia, China. The results revealed that 38.4% (Heilongjiang) and 27.7% (Inner Mongolia) were positive for Potato virus Y (PVY). To unveil the strain composition and population structure of PVY in the region, the multiplex RT-PCR described by Chikh-Ali et al. was performed on all of the ELISA-PVY-positive samples. Of the 158 samples whose PVY strain scenarios could be determined, PVY^NTN-NW-SYR-II and PVY^N-Wi were the most abundant strains, occurring in 58.9 and 47.5% samples, followed by PVY^NTN-NW-SYR-I (31.0%), PVY^N:O (19.6%), Eu-PVY^NTN (7.6%), NA-PVY^N (1.3%), and PVY^O (0.6%). In the 84 single-strain-infected samples, PVY^N-Wi accounted for 41.7%, PVY^NTN-NW-SYR-II for 40.5%, PVY^NTN-NW-SYR-I for 14.3%, and PVY^N:O and Eu-PVY^NTN for 3.6% each. Seven isolates representing PVY^NTN-NW-SYR-I (HLJ-6-1 and HLJ-9-4), PVY^NTN-NW-SYR-II (INM-W-369-12 and SC-1-1-2), PVY^N:O (HLJ-30-2), and PVY^N-Wi (HLJ-BDH-2 and HLJ-C-429) were sequenced and analyzed molecularly. Whereas the sequence identities for isolates belonging to the same strain group were >98.5%, they fell for isolates belonging to different strain groups to 92.7-98.1% at the genome level and 96.1-98.4% at the polyprotein level. Interestingly, the exact location of the recombination events varied among isolates within a strain group. Phylogenetic analysis of all 42 full length PVY sequences from China indicated that most clustered to various recombinant groups, despite the fact that the PVY isolates were isolated from at least five host species. Pathological analysis of four isolates representing PVY^N:O, PVY^N-Wi, PVY^NTN-NW-SYR-I, and PVY^NTN-NW-SYR-II revealed that the PVY^NTN-NW-SYR-II isolate incited the most severe symptoms on potato cultivar Kexin 13, followed by PVY^NTN-NW-SYR-I, PVY^N:O and PVY^N-Wi. The PVY^NTN-NW-SYR-I and PVY^NTN-NW-SYR-II isolates also caused necrotic ringspots on the tubers of Kexin 13, indicating their ability to induce the potato tuber necrotic ringspot disease in potato.

Isolates of potato virus Y (PVY) have been divided into several strains. We determined the genomic sequences of PVY isolates AQ4 and FZ10 from tobacco in China. AQ4 and FZ10 had genome of 9700 and 9698 nucleotides, respectively. In phylogenetic analysis of complete genome sequences, AQ4 was clustered with strain N-Wi, and FZ10 with NTN. AQ4 had two recombination sites within the P1 and P3 genes, while FZ10 had three within the P1, P3 and NIa-Pro genes. When compared to typical NTN isolates, FZ10 lacked a recombination site within the CP gene and, thus, represents a novel recombination type of PVY.

Neurturin (NTN) belongs to a structurally related family of bioactive molecules which include glial cell-line derived neutrotrophic factor (GDNF) and perserphin (PSP). NTN exerts its effects through a multicomponent receptor system which include a receptor (GFRalpha-2) and the proto-oncogene c-RET. We report here the identification of three splice isoforms of the GFRalpha-2 receptors (GFRalpha-2a, GFRalpha-2b and GFRalpha-2c) by reverse transcription-PCR (RT-PCR). GFRalpha-2b is a novel splice variant. All three isoforms were found to be expressed in various adult murine tissues as well as in the brain of the newborn human. The identity of these isoforms were further confirmed by the isolation of the gene and the characterisation of the splice junctions.

Glomerulonephritis (GN) encompasses a range of immune-mediated disorders that cause inflammation within the glomerulus of the kidney. The pathogenesis of GN is complex. Intricacy arises from factors such as autoimmunity, cancer and structural abnormalities within the kidney. Studies using animal models have highlighted crucial interaction between inflammatory cells and cells intrinsic to the kidney, both of which are fundamental to the pathogenesis of GN. This review aims to provide insight on a 'suitable' model for nephrotoxic nephritis and glomerulonephritis (NTN GN) and relate its experimental validity to humans. The BALB/c NTN murine model and Wistar Kyoto (WKY) rat have held experimental validity in the study of GN in humans. The chemokine receptor CXCR3 also mediates renal T-cell recruitment and subsequent tissue injury in NTN. It is noteworthy to consider CXCR3 blockade in Th1-mediated renal inflammation as future therapeutic options for patients with GN and subsets thereof. Currently used immunosuppressive therapies for GN are not always uniformly effective and are frequently associated with serious side-effects. Corticosteroids are effective in several types of GN owing to their ability to inhibit the pro-inflammatory effects of cytokines known to promote glomerular inflammation. Differences between experimental and human GN complicate translation of experimental therapies into practice. More research is required to translate animal model research into a better comprehension of human GN disease. However, the complexity of GN research makes findings a challenge to replicate.

A technique that couples lead (Pb) isotopes and multi-element concentrations with meteorological analysis was used to assess source contributions to precipitation samples at the Bondville, Illinois USA National Trends Network (NTN) site. Precipitation samples collected over a 16month period (July 1994-October 1995) at Bondville were parsed into six unique meteorological flow regimes using a minimum variance clustering technique on back trajectory endpoints. Pb isotope ratios and multi-element concentrations were measured using high resolution inductively coupled plasma-sector field mass spectrometry (ICP-SFMS) on the archived precipitation samples. Bondville is located in central Illinois, ~250km downwind from smelters in southeast Missouri. The Mississippi Valley Type ore deposits in Missouri provided a unique multi-element and Pb isotope fingerprint for smelter emissions which could be contrasted to industrial emissions from the Chicago and Indianapolis urban areas (~125km north and east, of Bondville respectively) and regional emissions from electric utility facilities. Differences in Pb isotopes and element concentrations in precipitation corresponded to flow regime. Industrial sources from urban areas, and thorogenic Pb from coal use, could be differentiated from smelter emissions from Missouri by coupling Pb isotopes with variations in element ratios and relative mass factors. Using a three endmember mixing model based on Pb isotope ratio differences, industrial processes in urban airsheds contributed 56±19%, smelters in southeast Missouri 26±13%, and coal combustion 18±7%, of the Pb in precipitation collected in Bondville in the mid-1990s.

Kidney development involves a series of complex interactions between the ureteric bud and undifferentiated mesenchyme, resulting in the production of the nephron unit. Among locally derived soluble factors, a particular relevance has been recognized to glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) for the mesenchyme-to-epithelial conversion of a metanephron. Nephroblastoma is a developmental tumor of the kidney deriving from metanephric blastema that mimics renal development and may offer an adequate model of human nephrogenesis. We investigated the immunohistochemical expression of GDNF, NTN, and their receptors (GFRalpha1, 2, and 3, and Ret) in normal human kidney and in 42 nephroblastomas, 20 of which were associated with nephrogenic rests (group A) and 22 were not (group B). We compared the immunostaining pattern in group A vs. group B and correlated clinical course with stage, grade, presence of nephrogenic rests, and immunohistochemical findings. GDNF, NTN, and their receptors were expressed in mature kidney and in 67% (GDNF) and 33% (NTN) of tumors, particularly in the epithelial component; precursor lesions were negative. No significant differences of expression were observed between groups A and B tumors. Low stage (P = 0.012), absence of nephrogenic rests (P = 0.016), intense expression of GDNF (P = 0.034), and NTN (P = 0.05) were associated with a more favorable outcome. Besides inductive activity in nephrogenesis, GDNF and NTN may play a role in maintaining differentiation and survival functions in mature kidney and may contribute to induce differentiation of nephroblastoma cells toward the less aggressive epithelial component. The pathway of activation seems to follow an autocrine/paracrine mechanism, as neurotrophic factors, GFRalpha1-2-3 receptors and Ret are coexpressed.

Precipitation chemistry and depth measurements obtained by the Canadian Air and Precipitation Monitoring Network (CAPMoN) and the US National Atmospheric Deposition Program/National Trends Network (NADP/NTN) were compared for the 10-year period 1995-2004. Colocated sets of CAPMoN and NADP instrumentation, consisting of precipitation collectors and rain gages, were operated simultaneously per standard protocols for each network at Sutton, Ontario and Frelighsburg, Ontario, Canada and at State College, PA, USA. CAPMoN samples were collected daily, and NADP samples were collected weekly, and samples were analyzed exclusively by each network's laboratory for pH, H(+), Ca(2+), Mg(2+), Na(+), K(+), NH4(+), Cl(-), NO3(-), and SO4(2-). Weekly and annual precipitation-weighted mean concentrations for each network were compared. This study is a follow-up to an earlier internetwork comparison for the period 1986-1993, published by Alain Sirois, Robert Vet, and Dennis Lamb in 2000. Median weekly internetwork differences for 1995-2004 data were the same to slightly lower than for data for the previous study period (1986-1993) for all analytes except NO3(-), SO4(2-), and sample depth. A 1994 NADP sampling protocol change and a 1998 change in the types of filters used to process NADP samples reversed the previously identified negative bias in NADP data for hydrogen-ion and sodium concentrations. Statistically significant biases (alpha = 0.10) for sodium and hydrogen-ion concentrations observed in the 1986-1993 data were not significant for 1995-2004. Weekly CAPMoN measurements generally are higher than weekly NADP measurements due to differences in sample filtration and field instrumentation, not sample evaporation, contamination, or analytical laboratory differences.

The receptor tyrosine kinase RET is part of a functional receptor for glial cell derived neurotrophic factor (GDNF) and neurturin (NTN) which are potent neurotrophic factors for motoneurons. Here, we have studied RET-like immunoreactivity of motoneurons in post-mortem spinal cords of patients with amyotrophic lateral sclerosis (ALS) and in controls. We report that the intensity of RET-like immunostaining of motoneurons in ALS is decreased significantly to 81% of control values. Despite this change, the proportion of all large (>40 micron diameter) motoneurons showing RET-like immunoreactivity in ALS remains high (82-85%) and is not significantly different to controls. The persistence of RET-like immunoreactivity in the majority of large motoneurons in ALS could be important in the design of clinical trials of GDNF and NTN.

BACKGROUND

Neuroblastoma (NBL) is one of the most common solid malignancies in childhood and is derived from the sympathetic precursor cells. Although p53, a tumor suppressor, has been reported to be rarely mutated in NBLs, it is sequestered abnormally in the cytoplasm of the NBL cell. The mechanism and functional role of the abnormal intracellular localization of p53 remain unclear.

METHODS

Here, we established an in vitro system of apoptosis model using a NBL cell line CHP134 which also showed a cytoplasmic sequestration of p53. The treatment of the cells with 1 or 5 microM all-trans retinoic acid (RA) induced moderate neurite outgrowth followed by massive death of CHP134 cells by days 5 to 6.

RESULTS

TUNEL staining showed that the cell death was due to apoptosis. Immunofluorescent stain demonstrated that p53 was strongly positive in the nucleus on day 5, which was accompanied with induction of p21WAF1. In addition, expression of caspase-3 was also increased during the cell death. Intriguingly, the RA treatment induced expression of Ret tyrosine kinase receptor in CHP134 cells.

CONCLUSIONS

The addition of ligands, glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN), inhibited apoptosis as well as nuclear accumulation of p53 in the cell. The present results suggest that the RA-induced apoptosis of NBL cells is associated with activation of both the caspase cascade and the p53-mediated pathway with its nuclear translocation. The neurotrophic signal through the GDNF-Ret system may prevent the neuronal cell death.

Glial-cell-line-derived neurotrophic factor (GDNF) and neurturin (NTN) are structurally related to TGF-beta and are survival factors for sympathetic, sensory, and central nervous system neurons. GDNF transmits its signal primarily through a receptor complex containing the receptor tyrosine kinase Ret and a glycosyl-phosphatidylinositol (GPI)-linked receptor, GDNFR alpha. NTN utilizes a receptor complex system that consists of Ret and another GPI-linked receptor, NTNR alpha. We have identified a mouse cDNA, termed GFR alpha-3, that encodes a putative GPI-linked receptor. At the protein level, mouse GFR alpha-3 is 35% identical to mouse GDNFR alpha and 36% identical to mouse NTNR alpha. Northern blot analysis showed that GFR alpha-3 is expressed in fetal mouse heart, brain, lung, and kidney and adult heart. These results indicate that the tissue distribution of GFR alpha-3 mRNA is different from that of GDNFR alpha or NTNR alpha mRNA, and suggest that GFR alpha-3 may function in differentiation of embryonic cells expressing its mRNA.

Cyclic AMP (cAMP) and neurotrophic factors are known to interact closely to promote neurite outgrowth and neuronal regeneration. Glial cell line-derived neurotrophic factor (GDNF) and its family member neurturin (NTN) transduce signal through a multi-component receptor complex consisting of GDNF family receptor alpha 2 (GFRα2) and Ret receptor tyrosine kinase. Neurons from GFRα2-deficient mice do not promote axonal initiation when stimulated by NTN, consistent with the role of GFRα2 in neuronal outgrowth. Multiple alternatively spliced isoforms of GFRα2 are known to be expressed in the nervous system. GFRα2a and GFRα2c but not GFRα2b promoted neurite outgrowth. It is currently unknown if cAMP signalling is differentially regulated by these isoforms. In this study, NTN activation of GFRα2a and GFRα2c but not GFRα2b induced biphasic ERK1/2 activation and phosphorylation of the major cAMP target CREB. Interestingly, inhibition of cAMP signalling significantly impaired GFRα2a and GFRα2c-mediated neurite outgrowth while cAMP agonists cooperated with GFRα2b to induce neurite outgrowth. Importantly, the specific cAMP effector PKA but not Epac was essential for NTN-induced neurite outgrowth, through transcription and translation-dependent activation of late phase ERK1/2. Taken together, these results not only demonstrated the essential role of cAMP-PKA signalling in NTN-induced biphasic ERK1/2 activation and neurite outgrowth, but also suggested cAMP-PKA signalling as a hitherto unrecognized underlying mechanism contributing to the differential neuritogenic activities of GFRα2 isoforms.