Novel Action of Activin and Bone Morphogenetic Protein in Regulating Aldosterone Production by Human Adrenocortical Cells

Abstract

We have uncovered a functional bone morphogenetic protein (BMP) and activin system complete with ligands (BMP-6 and activin betaA/betaB), receptors (activin receptor-like kinase receptors 2, 3, and 4; activin type-II receptor; and BMP type-II receptor), and the binding protein follistatin in the human adrenocortical cell line H295R. Administration of activin and BMP-6 to cultures of H295R cells caused concentration-responsive increases in aldosterone production. The mRNA levels of steroidogenic acute regulatory protein or P450 steroid side-chain cleavage enzyme, the rate-limiting steps of adrenocortical steroidogenesis, were enhanced by activin and BMP-6. Activin and BMP-6 also activated the transcription of steroidogenic acute regulatory protein as well as the late-step steriodogenic enzyme CYP11B2. Activin enhanced ACTH-, forskolin-, or dibutyryl-cAMP- but not angiotensin II (Ang II)-induced aldosterone production, whereas BMP-6 specifically augmented Ang II-induced aldosterone production. Activin and ACTH but not BMP-6 increased cAMP production. Follistatin, which inhibits activin actions by binding, suppressed basal and ACTH-induced aldosterone secretion but failed to affect the Ang II-induced aldosterone level. Furthermore, MAPK signaling appeared to be involved in aldosterone production induced by Ang II and BMP-6 because an inhibitor of MAPK activation, U0126, reduced the level of aldosterone synthesis stimulated by Ang II and BMP-6 but not activin. In addition, Ang II reduced the expression levels of BMP-6 but increased that of activin betaB, whereas ACTH had no effect on these levels. Collectively, the present data suggest that activin acts to regulate adrenal aldosterone synthesis predominantly by modulating the ACTH-cAMP-protein kinase A signaling cascade, whereas BMP-6 works primarily by modulating the Ang II-MAPK cascade in human adrenal cortex in an autocrine/paracrine fashion.

cAMP-dependent protein kinase activation inhibits proliferation and enhances apoptotic effect of tumor necrosis factor-α in NCI-H295R adrenocortical cells

“…Previous reports have described that adrenocortical cell apoptosis is affected by the in vitro culture system, species and differentiation status (Negoescu et al 1995, Carsia et al 1998, Spencer et al 1999. Although this cell line was recently reported to respond to ACTH (Suzuki et al 2004), previous studies showed no response in either the H295R cell line or in its parental H295 cells, probably because of low ACTH receptor expression (Staels et al 1993, Rainey et al 1994, Denner et al 1996. Similarly, our H295R cells did not show significant response to ACTH treatment.…”

Expression of N-type calcium channels in human adrenocortical cells and their contribution to corticosteroid synthesis

“…The cells were maintained at 37 1C in a humidified atmosphere containing air and carbon dioxide (95%/5%, vol/vol). 24 Assay for steroid production H295R cells were cultured in 12-well plates until 80% confluence. Next, the medium was replaced with fresh medium containing 0.2% Nu-serum, and the cells were incubated for an additional 24 h. Then, the culture supernatants were replaced with medium containing Ang II, including vehicle (0.1% dimethyl sulfoxide) or each calcium channel inhibitor.…”

Mechanisms of adrenal gland growth: signal integration by extracellular signal regulated kinases1/2

“…In addition to its tropic effects angiotensin II dependent activation of ERK1/2 resulted in an activation of the hormonesensitive lipase (cholesterol ester hydrolase) in adrenal zona glomerulosa cells, which supports a role of ERK1/2 for steroidogenesis, as activation of this lipase initializes cholesterol mobilization to the outer mitochondrial membrane (Cherradi et al 2003). The function of angiotensin II as one important regulator of adrenal ERK1/2 activity is further substantiated by the existence of a number of co-effectors of angiotensin II dependent ERK1/2 phosphorylation like ACTH (Chabre et al 1995), integrins (Campbell et al 2003, Otis et al 2007b or BMP-6 (Suzuki et al 2004, Inagaki et al 2006. The effect of BMP-6 is thought to be exerted in an autocrine fashion via SMAD proteins, which are known modulators of ERK1/2 activity (Kano et al 2005).…”