EPHB6 controls catecholamine biosynthesis by up-regulating tyrosine hydroxylase transcription in adrenal gland chromaffin cells

Wei Shi,Yujia Wang,Junzheng Peng,Shijie Qi,Nicolas Vitale,Norio Kaneda,Tomiyasu Murata,Hongyu Luo,Jiangping Wu

doi:10.1074/jbc.ra118.005767

Abstract

EPHB6 is a member of the erythropoietin-producing hepatocellular kinase (EPH) family and a receptor tyrosine kinase with a dead kinase domain. It is involved in blood pressure regulation and adrenal gland catecholamine (CAT) secretion, but several facets of EPHB6-mediated CAT regulation are unclear. In this study, using biochemical, quantitative RT-PCR, immunoblotting, and gene microarray assays, we found that EPHB6 up-regulates CAT biosynthesis in adrenal gland chromaffin cells (AGCCs). We observed that epinephrine content is reduced in the AGCCs from male Ephb6-KO mice, caused by decreased expression of tyrosine hydroxylase, the rate-limiting enzyme in CAT biosynthesis. We demonstrate that the signaling pathway from EPHB6 to tyrosine hydroxylase expression in AGCCs involves Rac family small GTPase 1 (RAC1), MAP kinase kinase 7 (MKK7), c-Jun N-terminal kinase (JNK), proto-oncogene c-Jun, activator protein 1 (AP1), and early growth response 1 (EGR1). On the other hand, signaling via extracellular signal-regulated kinase (ERK1/2), p38 mitogen-activated protein kinase, and ELK1, ETS transcription factor (ELK1) was not affected by EPHB6 deletion. We further report that EPHB6's effect on AGCCs was via reverse signaling through ephrin B1 and that EPHB6 acted in concert with the nongenomic effect of testosterone to control CAT biosynthesis. Our findings elucidate the mechanisms by which EPHB6 modulates CAT biosynthesis and identify potential therapeutic targets for diseases, such as hypertension, caused by dysfunctional CAT biosynthesis.

Highlights

EPHB6 is a member of the erythropoietin-producing hepatocellular kinase (EPH) family and a receptor tyrosine kinase with a dead kinase domain
We demonstrate that the signaling pathway from EPHB6 to tyrosine hydroxylase expression in adrenal gland chromaffin cells (AGCCs) involves Rac family small GTPase 1 (RAC1), MAP kinase kinase 7 (MKK7), c-Jun N-terminal kinase (JNK), proto-oncogene c-Jun, activator protein 1 (AP1), and early growth response 1 (EGR1)
To assess whether the KO adrenal glands were compromised in CAT biosynthesis, we measured their CAT content using epinephrine as a representative CAT, as our previous study showed that the levels of three major types of CAT in the 24-h urine were reduced in male KO mice

Summary

Results

Our previous study demonstrated that male but not female Ephb KO mice have reduced CAT secretion [16]. We confirmed by RT-qPCR that Egr mRNA expression was significantly lower in male but not in female KO medullae compared with their WT counterparts (Fig. 2B). According to EMSAs, AP-1 binding to the AP-1-binding sequence in the 5Ј upstream region of the Egr gene was reduced with nuclear extracts of AGCCs from male Ephb KO mice compared with those from WT counterparts (Fig. 4A). The solid-phase anti-EFNB1 Ab but not the anti-EFNB2 Ab augmented nicotine-stimulated TH expression in tsAM5NE chromaffin cells (Fig. 7B), suggesting that EPHB6’s effect on CAT synthesis is mainly via EFNB1 reverse signaling. This indicates that the nongenomic effect of testosterone, but not the simple presence of testicles in adult life or during fetal development, is responsible for the observed diminished AGCC CAT biosynthesis in male KO mice

Discussion

Experimental procedures

Ethics statement