Evidence for the involvement of endothelial cell products in adrenal CITED2 expression

Abstract

The adrenal gland contains a well-organized network of blood vessels, and adrenocortical cells are situated in close proximity to endothelial cells. Recently, several new mechanisms have been characterized that control the release of aldosterone by adrenocortical cells, including the involvement of endothelial-cell-derived factors. Interestingly, a CBP/p300-interacting transactivator with ED-rich tail 2 (CITED2), which is necessary for adrenal development, has been linked to aldosterone synthesis. We have therefore examined the effects of endothelial-cell-conditioned medium (ECCM), as produced during the incubation of human umbilical vein endothelial cells for 24 h, on the promoter activity and mRNA and protein expression of CITED2 in adrenocortical cells as represented by the NCI-H295R cell line. We have found a dose-dependent effect of ECCM on CITED2 promoter activity; this peaks at 480%. Activation of the CITED2 promoter occurs in parallel to an increase in CITED2 messenger RNA (as quantified by real-time polymerase chain reaction) and protein. The stimulatory effect of ECCM can be reversed by blocking mitogen-activated protein kinase activity with the MEK1-inhibitor PD98059. We conclude that products secreted by endothelial cells control not only steroidogenesis, but also factors that are important for adrenocortical development, thereby highlighting the role of cellular interactions within adrenocortical development and physiology.

CITED2 (Cbp/p300-interacting transactivator, with Glu/Asp-rich carboxy-terminal domain, 2)

“…Other studies applying human NCI-H295R adrenal carcinoma cells demonstrate that SF-1 activates the CITED2-promoter (Ferraz- de-Souza et al, 2009). In human NCI-H295R cells CITED2-expression is regulated by basic fibroblast growth factor, endothelial cell products and is dependent on the MAPK-pathway (Haase et al, 2007;Haase et al, 2009). Forskolin could also be identified as a positive regulator of CITED2-expression in NCI-H295R cells (Haase et al, 2007;Romero et al, 2007).…”

“…CITED2 is upregulated by bFGF, Forskolin, endothelial cell products and Angiotensin II in NCI-H295R cells (Haase et al, 2007;Romero et al, 2007;Haase et al, 2009). In part, CITED2-expression in adrenocortical carcinomas could be observed in close proximity to blood vessels (Haase et al, 2009).…”

The Effects of the Endothelium on Adrenal Steroidogenesis and Growth are Mainly Mediated by Proteins Other than Endothelin-1

“…Dispersed cells were grown in DMEM / F12 with supplements (66 nM insulin, 10 nM hydrocortisone, 10 nM 17 β -estradiol, 10 μ g / ml transferrin, 30 nM selenite, 100 U / ml penicillin, 100 μ g / ml streptomycin, 10 μ g / ml ascorbic acid, and 2 % fetal bovine serum) in 75 cm 2 fl asks (Becton Dickinson, Heidelberg, Germany) at 37 ° C in a humidifi ed atmosphere of 5 % CO 2 / 95 % air, as described previously [23] . Human umbilical vein endothelial cells (HUVEC) were obtained as described elsewhere and grown with endothelial cell medium (PromoCell, USA) [23] .…”

“…Dispersed cells were grown in DMEM / F12 with supplements (66 nM insulin, 10 nM hydrocortisone, 10 nM 17 β -estradiol, 10 μ g / ml transferrin, 30 nM selenite, 100 U / ml penicillin, 100 μ g / ml streptomycin, 10 μ g / ml ascorbic acid, and 2 % fetal bovine serum) in 75 cm 2 fl asks (Becton Dickinson, Heidelberg, Germany) at 37 ° C in a humidifi ed atmosphere of 5 % CO 2 / 95 % air, as described previously [23] . Human umbilical vein endothelial cells (HUVEC) were obtained as described elsewhere and grown with endothelial cell medium (PromoCell, USA) [23] . Endothelium-conditioned medium was obtained by exposure of endothelial cells to DMEM / F12 medium for 48 h. For experimental settings, NCI-H295R cells were sub-cultured from 70 % confl uent stock cultures in triplicates into 24-well culture plates (Becton Dickinson Falcon) at a density of 100 000 cells per cm 2 for 48 h. After being washed, cells were exposed to stimulants containing ECCM at concentrations of 5, 10, or 20 % , diluted in DMEM / F12 or ET-1 at concentrations of 10 -9 M, 10 -8 M, or 10 -7 M with or without the addition of BQ123, a selective ET-A receptor antagonist, or BQ788, a salt blocking the AT-B receptor at concentrations of 10 -9 M each.…”

Sporadic solitary aldosterone- and cortisol-co-secreting adenomas: endocrine, histological and genetic findings in a subtype of primary aldosteronism

“…The procedure was carried out according to the manufacturer's instructions and as described previously. 31 All tissues were incubated for 30 min at room temperature. For control, material from APAs, CPAs and normal adrenal glands (NAG) was used.…”