Identification of SPRED2 (sprouty-related protein with EVH1 domain 2) as a negative regulator of the hypothalamic-pituitary-adrenal axis

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.

FIGURE 1.

Disruption of Spred2 and expression of an EVH1-β-geo fusion protein in SPRED2^−/− mice. A, in SPRED2 KO mice, the Spred2 gene was disrupted by insertion of a gene trap vector between exons 4 and 5. This vector contains a splice acceptor (SA), a reporter gene (β-geo) allowing expression profiling of the endogenous Spred2 promoter via X-Gal stainings, and an SV40 polyadenylation sequence (pA). Upon transcription, β-geo is spliced to the end of exon 4 of Spred2, leading either to a fusion protein coding for the EVH1 domain of SPRED2 and the β-geo reporter, if protein translation starts at the start codon of Spred2 exon1 (first bent arrow), or to the expression of β-geo alone, if translation starts at the start codon of β-geo (second bent arrow). In both cases, translation stops at the stop codon of β-geo (*). The arrows indicate the position of primers used to subclone EVH1-β-geo and β-geo. B, Western blots (IB) with tissue lysates analyzed with anti-SPRED2 (top and middle), and anti-β-gal antibodies (bottom) demonstrated SPRED2 expression in WT organs and the in vivo expression of both β-geo and EVH1-β-geo in SPRED2^−/− mice. C, expression of EVH1-β-geo and β-geo in HEK293 cells after transfection with EVH1-β-geo and β-geo expression plasmids. Lysates were probed with anti-SPRED2 (top and middle) or anti-β-gal antibodies (bottom). D, X-Gal stainings of transfected HEK293 cells confirmed the enzymatic activity of the EVH1-β-geo fusion protein and β-geo. Scale bars, 100 μm.

FIGURE 2.

Excessive water uptake correlates with elevated serum osmolality and increased salt concentrations. A, nearly doubled daily water consumption in KO mice. ***, p < 0.001; n (WT/KO) = 12. B, presumably causative for polydipsia, serum osmolality was significantly increased. **, p < 0.01; n (WT/KO) = 12. In line with hyperosmolality Na⁺ (C) and Cl⁻ (D) levels were significantly elevated. ***, p < 0.001; n (WT) = 12; n (KO) = 17. All values are mean ± S.D. (error bars).

FIGURE 3.

SPRED2-deficiency causes dysregulation of salt and water homeostasis-controlling hormones. A, Ang II, a primary stimulator of aldosterone release, was halved in KO mice. **, p < 0.01; n (WT) = 10; n (KO) = 12. B, in line with hyperosmolality and increased blood salt load, but despite reduced Ang II secretion, serum aldosterone was almost doubled in SPRED2-deficient mice. **, p < 0.01; n (WT/KO) = 14. Immunohistochemistry of AS in adrenal glands of WT (C) and SPRED2^−/− (D) mice exhibited broadened localization of AS in KO tissues, normally restricted to the zona glomerulosa (arrows). Scale bars, 200 μm. E, for AVP, no significant differences could be detected upon water deprivation. n.s., not significant; n (WT) = 10; n (KO) = 17. All values are mean ± S.D. (error bars).

FIGURE 4.

Up-regulation of HPA hormone secretion is accompanied by obsessive overgrooming in SPRED2^−/− mice. Stress hormones secreted successively by organs of the HPA axis were significantly elevated in SPRED2-deficient mice, demonstrated by a 30% increase of CRH in PVN region-containing brain lysates (***, p < 0.001; n (WT/KO) = 20 (A); a 30% increase of serum ACTH (*, p < 0.05; n (WT) = 17; n (KO) = 22) (B); and a more than doubled serum corticosterone level (***, p < 0.001; n (WT/KO) = 12) (C). D, SPRED2^−/− mice developed self-inflicted severe skin lesions on their face, neck, and snout originating from compulsive grooming behavior. All values are mean ± S.D. (error bars).

FIGURE 5.

Spred2 expression profiling in organs of the HPA axis. X-Gal stainings of whole WT and HET organs (arrows) (A, D, and G) and tissue sections of SPRED2^+/− mice (B, E, and H) demonstrated endogenous Spred2 promoter activity. C, F, and I, on protein level, SPRED2 expression was confirmed by immunohistochemistry with tissue sections of WT mice. Uniform expression was observed in hypothalamus (A, B, and C) and pituitary gland (D, E, and F). G–I, in adrenal gland, SPRED2 was predominantly expressed in zona glomerulosa. Scale bars, 500 μm (B, E, and H) and 50 μm (C, F, and I).

FIGURE 6.

In vivo and in vitro suppression of CRH production by SPRED. A, Western blot analysis with anti-ERK and anti-phospho-ERK (P-ERK) antibodies revealed an increased MAPK signaling in PVN-containing brain regions of both 6- and 12-month-old SPRED2^−/− mice. B, quantification of ERK and phospho-ERK signals demonstrated a more than 2.5-fold increase of the phospho-ERK/ERK ratio in young and aged SPRED2 KO mice. ***, p < 0.001; n (WT) ≥ 7; n (KO) ≥ 8. C, top, schematic depiction of the cloned CRH promoter reporter with the approximate position of predicted Ets-binding sites. C, bottom, luciferase reporter assays revealed a 40% reduction of the relative CRH promoter activity by SPRED1 and SPRED2, respectively. No further effect was detected by EVH1-β-geo co-expression. ***, p < 0.001; n.s., not significant; n = 12. D, top, schematic depiction of the 4xEts reporter. D, bottom, suppression of luciferase reporter activity to ∼10% by SPRED1 and SPRED2, respectively. EVH1-β-geo co-expression showed no further effect. ***, p < 0.001; n.s., not significant; n = 12. E, CRH production in mHypoE-44 cells was reduced to 70% by SPRED1 and to 60% by SPRED2, as determined in cell culture supernatants by ELISA. *, p < 0.05 for SPRED1; **, p < 0.01 for SPRED2; n = 12. F, Northern blot analysis with RNA from the PVN-containing hypothalamus of SPRED2 KO mice demonstrated a 60% increase of CRH mRNA. *, p < 0.05; n (WT) = 9; n (KO) = 8. All values are mean ± S.D. (error bars).