A dominant negative mutation uncovers cooperative control of caudal Wolffian duct development by Sprouty genes.

The Wolffian ducts (WD) are paired epithelial tubules central to the development of the mammalian genitourinary tract. Outgrowths from the WD known as the ureteric buds (UB) generate the collecting ducts of the kidney. Later during development, the caudal portion of the WD will form the vas deferens, epididymis and seminal vesicle in males, and will degenerate in females. While the genetic pathways controlling the development of the UB are firmly established, less is known about those governing development of WD portions caudal to the UB. Sprouty proteins are inhibitors of receptor tyrosine kinase (RTK) signaling in vivo. We have recently shown that homozygous mutation of a conserved tyrosine (Tyr53) of Spry1 results in UB defects indistinguishable from that of Spry1 null mice. Here, we show that heterozygosity for the Spry1 Y53A allele causes caudal WD developmental defects consisting of ectopically branched seminal vesicles in males and persistent WD in females, without affecting kidney development. Detailed analysis reveals that this phenotype also occurs in Spry1+/- mice but with a much lower penetrance, indicating that removal of tyrosine 53 generates a dominant negative mutation in vivo. Supporting this notion, concomitant deletion of one allele of Spry1 and Spry2 also recapitulates the genital phenotype of Spry1Y53A/+ mice with high penetrance. Mechanistically, we show that unlike the effects of Spry1 in kidney development, these caudal WD defects are independent of Ret signaling, but can be completely rescued by lowering the genetic dosage of Fgf10. In conclusion, mutation of tyrosine 53 of Spry1 generates a dominant negative allele that uncovers fine-tuning of caudal WD development by Sprouty genes.

Evaluation of blood cell viability rate, gene expression, and O-GlcNAcylation profiles as indicative signatures for fungal stimulation of salmonid cell models.

Fungal diseases of fish are a significant economic problem in aquaculture. Using high-throughput expression analysis, we identified potential transcript markers in primary head kidney and secondary embryonic cells from salmonid fish after stimulation with the inactivated fungi Mucor hiemalis and Fusarium aveneacium and with purified fungal molecular patterns. The transcript levels of most of the 45 selected genes were altered in head-kidney cells after 24 h of stimulation with fungal antigens. Stimulation with the inactivated fungus M. hiemalis induced the most pronounced transcriptional changes, including the pathogen receptor-encoding genes CLEC18A and TLR22, the cytokine-encoding genes IL6 and TNF, and the gene encoding the antimicrobial peptide LEAP2. In parallel, we analyzed the total GlcNAcylation status of embryonic salmonid cells with or without stimulation with inactivated fungi. O-GlcNAcylation modulates gene expression, intracellular protein, and signal activity, but we detected no significant differences after a 3-h stimulation. A pathway analysis tool identified the "apoptosis of leukocytes" based on the expression profile 24 h after fungal stimulation. Fluorescence microscopy combined with flow cytometry revealed apoptosis in 50 % of head-kidney leukocytes after 3 h stimulation with M. hiemalis, but this level decreased by > 5% after 24 h of stimulation. The number of apoptotic cells significantly increased in all blood cells after a 3-h stimulation with fungal molecular patterns compared to unstimulated controls. This in vitro approach identified transcript-based parameters that were strongly modulated by fungal infections of salmonid fish.

Sprouty1 Controls Genitourinary Development via its N-Terminal Tyrosine.

BACKGROUND: Studies in mice suggest that perturbations of the GDNF-Ret signaling pathway are a major genetic cause of congenital anomalies of the kidney and urinary tract (CAKUT). Mutations in Sprouty1, an intracellular Ret inhibitor, results in supernumerary kidneys, megaureters, and hydronephrosis in mice. But the underlying molecular mechanisms involved and which structural domains are essential for Sprouty1 function are a matter of controversy, partly because studies have so far relied on ectopic overexpression of the gene in cell lines. A conserved N-terminal tyrosine has been frequently, but not always, identified as critical for the function of Sprouty1 in vitro. METHODS: We generated Sprouty1 knockin mice bearing a tyrosine-to-alanine substitution in position 53, corresponding to the conserved N-terminal tyrosine of Sprouty1. We characterized the development of the genitourinary systems in these mice via different methods, including the use of reporter mice expressing EGFP from the Ret locus, and whole-mount cytokeratin staining. RESULTS: Mice lacking this tyrosine grow ectopic ureteric buds that will ultimately form supernumerary kidneys, a phenotype indistinguishable to that of Sprouty1 knockout mice. Sprouty1 knockin mice also present megaureters and vesicoureteral reflux, caused by failure of ureters to separate from Wolffian ducts and migrate to their definitive position. CONCLUSIONS: Tyrosine 53 is absolutely necessary for Sprouty1 function during genitourinary development in mice.

Autophagy orchestrates adaptive responses to targeted therapy in endometrial cancer.

Targeted therapies in endometrial cancer (EC) using kinase inhibitors rarely result in complete tumor remission and are frequently challenged by the appearance of refractory cell clones, eventually resulting in disease relapse. Dissecting adaptive mechanisms is of vital importance to circumvent clinical drug resistance and improve the efficacy of targeted agents in EC. Sorafenib is an FDA-approved multitarget tyrosine and serine/threonine kinase inhibitor currently used to treat hepatocellular carcinoma, advanced renal carcinoma and radioactive iodine-resistant thyroid carcinoma. Unfortunately, sorafenib showed very modest effects in a multi-institutional phase II trial in advanced uterine carcinoma patients. Here, by leveraging RNA-sequencing data from the Cancer Cell Line Encyclopedia and cell survival studies from compound-based high-throughput screenings we have identified the lysosomal pathway as a potential compartment involved in the resistance to sorafenib. By performing additional functional biology studies we have demonstrated that this resistance could be related to macroautophagy/autophagy. Specifically, our results indicate that sorafenib triggers a mechanistic MAPK/JNK-dependent early protective autophagic response in EC cells, providing an adaptive response to therapeutic stress. By generating in vivo subcutaneous EC cell line tumors, lung metastatic assays and primary EC orthoxenografts experiments, we demonstrate that targeting autophagy enhances sorafenib cytotoxicity and suppresses tumor growth and pulmonary metastasis progression. In conclusion, sorafenib induces the activation of a protective autophagic response in EC cells. These results provide insights into the unopposed resistance of advanced EC to sorafenib and highlight a new strategy for therapeutic intervention in recurrent EC.

7-dehydrocholesterol efficiently supports Ret signaling in a mouse model of Smith-Opitz-Lemli syndrome.

Smith-Lemli-Opitz syndrome (SLOS) is a rare disorder of cholesterol synthesis. Affected individuals exhibit growth failure, intellectual disability and a broad spectrum of developmental malformations. Among them, renal agenesis or hypoplasia, decreased innervation of the gut, and ptosis are consistent with impaired Ret signaling. Ret is a receptor tyrosine kinase that achieves full activity when recruited to lipid rafts. Mice mutant for Ret are born with no kidneys and enteric neurons, and display sympathetic nervous system defects causing ptosis. Since cholesterol is a critical component of lipid rafts, here we tested the hypothesis of whether the cause of the above malformations found in SLOS is defective Ret signaling owing to improper lipid raft composition or function. No defects consistent with decreased Ret signaling were found in newborn Dhcr7(-/-) mice, or in Dhcr7(-/-) mice lacking one copy of Ret. Although kidneys from Dhcr7(-/-) mice showed a mild branching defect in vitro, GDNF was able to support survival and downstream signaling of sympathetic neurons. Consistently, GFRα1 correctly partitioned to lipid rafts in brain tissue. Finally, replacement experiments demonstrated that 7-DHC efficiently supports Ret signaling in vitro. Taken together, our findings do not support a role of Ret signaling in the pathogenesis of SLOS.

2-phenylethynesulphonamide (PFT-µ) enhances the anticancer effect of the novel hsp90 inhibitor NVP-AUY922 in melanoma, by reducing GSH levels.

Heat shock proteins (HSPs), are molecular chaperones that assist the proper folding of nascent proteins. This study aims to evaluate the antitumour effects of the hsp90 inhibitor NVP-AUY922 in melanoma, both in vitro and in vivo. Our results show that NVP-AUY922 inhibits melanoma cell growth in vitro, with down regulation of multiple signalling pathways involved in melanoma progression such as NF-ĸB and MAPK/ERK. However, NVP-AUY922 was unable to limit tumour growth in vivo. Cotreatment of A375M xenografts with NVP-AUY922 and PFT-µ, a dual inhibitor of both hsp70 and autophagy, induced a synergistic increase of cell death in vitro, and delayed tumour formation in A375M xenografts. PFT-µ depleted cells from the reduced form of glutathione (GSH) and increased oxidative stress. The oxidative stress induced by PFT-µ further enhanced NVP-AUY922-induced cytotoxic effects. These data suggest a potential therapeutic role for NVP-AUY922 used in combination with PFT-µ, in melanoma.

Involvement of c-Jun-JNK pathways in the regulation of programmed cell death of developing chick embryo spinal cord motoneurons.

Key features of developmentally regulated programmed cell death (PCD) have been described for the first time in the chick nervous system. JNK/c-Jun pathway was involved in early events determining normal and pathological neuronal death as shown in experimental models. In the chick embryo, PCD of motoneurons (MNs) in ovo occurs within a well-defined temporal window and can be subjected to experimental manipulation. Taking advantage of this in vivo system, we explored the role of c-Jun and JNK pathway in the regulation of PCD in MNs. By using specific antibodies against phospho-c-Jun (Ser 63, 73) and JNK we demonstrated that before MNs acquire apoptotic phenotype there is an increase in c-Jun. Blockage of neuromuscular activity by the GABA agonist muscimol reduces PCD and diminishes c-Jun immunoreactivity in MNs. Extensive induction of PCD, either due to injection of beta-bungarotoxin or limb bud removal, is also preceded by an increase in c-Jun immunoreactivity that is also associated with upregulation of phospho-c-Jun and JNK. Translocation of JNK from cytoplasm to MN nuclei was also detected. After acute application of beta-bungarotoxin, which is a strong apoptotic stimulus for MNs, c-Jun phosphorylation occurs on serine 73, whereas serine 63 is the main site for c-Jun phosphorylation after limb bud removal. These results demonstrated that the JNK/c-Jun pathway is involved in the decision phase of normal and induced apoptosis in MNs. Pharmacological interventions involving this pathway should be explored as a potential therapeutic target for promoting MN survival.

c-Jun-like immunoreactivity in apoptosis is the result of a crossreaction with neoantigenic sites exposed by caspase-3-mediated proteolysis.

Previous reports in various cells and species have shown that apoptotic cells are specifically and strongly labeled by certain c-Jun/N-terminal antibodies, such as c-Jun/sc45. This kind of immunoreactivity is confined to the cytoplasm. It is not due to c-Jun but appears to be related to c-Jun-like neoepitopes generated during apoptosis. This study was planned to gain further information about c-Jun-like immunostaining during apoptosis and to evaluate these antibodies as possible tools for characterizing cell death. Most of the experiments were performed in chick embryo spinal cord. When the apoptotic c-Jun-like immunoreactivity and caspase-3 immunostaining patterns were compared, we found that both antibodies immunostained the same dying cells in a similar pattern. In contrast to TUNEL staining, which reveals a positive reaction in both apoptotic and necrotic dying cells, active caspase-3 and c-Jun/sc45 antibodies are more selective because they stained only apoptotic cells. When cytosolic extracts from normal tissues were digested in vitro with caspase-3, c-Jun/sc45 immunoreactivity was strongly induced in several proteins, as demonstrated by Western blotting. Similar results were found when normal tissue sections were treated with caspase-3. Our results show that c-Jun/sc45 antibodies react with neoepitopes generated from cell proteins cleaved by activated caspases during apoptosis. We conclude that c-Jun/sc45 antibodies may be useful for detecting apoptosis. They can even be used in archival paraffin-embedded tissue samples.