Abstract
Sprouty-related proteins with EVH1 (enabled/vasodilator-stimulated phosphoprotein homology 1) domain (SPREDs) are inhibitors of MAPK signaling. To elucidate SPRED2 in vivo function, we characterized body homeostasis in SPRED2(-/-) mice. They showed a doubled daily water uptake, induced by elevated serum osmolality, originating from increased blood salt load. Accordingly, serum aldosterone was doubled, accompanied by augmented adrenal aldosterone synthase (AS) expression. Surprisingly, serum vasopressin (AVP) was unaltered, and, as evidenced by halved angiotensin II (Ang II) levels, the renin angiotensin system (RAS) was down-regulated. Adrenocorticotropic hormone (ACTH) was significantly elevated in SPRED2(-/-) mice, together with its secretagogue corticotropin-releasing hormone (CRH) and its downstream target corticosterone. ERK phosphorylation in brains was augmented, and hypothalamic CRH mRNA levels were elevated, both contributing to the increased CRH release. Our data were supported by CRH promoter reporter assays in hypothalamic mHypoE-44 cells, revealing a SPRED-dependent inhibition of Ets (ERK/E-twenty-six)-dependent transcription. Furthermore, SPRED suppressed CRH production in these cells. In conclusion, our study suggests that SPRED2 deficiency leads to an increased MAPK signaling, which results in an augmented CRH promoter activity. The subsequent CRH overproduction causes an up-regulation of downstream hypothalamic-pituitary-adrenal (HPA) hormone secretion. This constitutes a possible trigger for the observed compulsive grooming in SPRED2(-/-) mice and may, together with hyperplasia of aldosterone-producing cells, contribute to the hyperaldosteronism and homeostatic imbalances.
Highlights
The HPA3 axis is essential to maintain homeostasis and to manage physical or emotional stress
Expression profiling can be monitored via enzymatic activity of the -galactosidase-neomycin phosphotransferase fusion gene (-geo), which came under control of the endogenous Spred2 promoter
In order to examine if the EVH1-geo fusion protein was present in our SPRED2-deficient mice, we performed Western blot analysis using either an antiSPRED2 antibody, which recognizes an epitope in the coding sequence of exon 4 [28], or an anti--gal antibody (Fig. 1A)
Summary
Animals—SPRED2Ϫ/Ϫ mice were generated by blastocyst injection of the embryonic stem cell line XB228 (International Gene Trap Consortium) [28]. Cloning of EVH1--geo Fusion Protein and -geo Expression Plasmids—RNA was extracted from brains of SPRED2 KO mice using TRIzol reagent (Invitrogen). 24 h prior to transfection, cells were either seeded on 6-well plates for preparation of protein extracts and Western blot analysis or on 2-well chamber slides (Nunc) for X-Gal stainings and grown to 90% confluence. Cells were transiently transfected with Lipofectamine 2000 (Invitrogen) using pcDNA3.1(Ϫ)/Myc-His expression plasmids for SPRED2, EVH1--geo, and -geo or empty vector as negative control. 24 h before transfection, cells were seeded on 96-well plates, grown overnight to 90% confluence, and transfected with Lipofectamine 2000 (Invitrogen) for luciferase reporter assays or CRH ELISA. Western Blotting—After transfection, cells were lysed in 2% SDS in PBS supplemented with Complete protease inhibitor mixture and PhosSTOP phosphatase inhibitor mixture (Roche Applied Science). Values of p Ͻ 0.05 were considered as statistically significant
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