Orexins stimulate steroidogenic acute regulatory protein expression through multiple signaling pathways in human adrenal H295R cells

Orexins mediate a variety of physiological processes, including feeding behavior, the circadian pathway, and cortisol secretion. Steroidogenesis is regulated by a variety of neuropeptides, and one of the key rate-limiting steps is cholesterol transport across the mitochondrial membrane by the steroidogenic acute regulatory protein (StAR). StAR expression can be regulated through several different signaling pathways. Despite the clear link between orexins and steroid production, the actions of the orexin family of hormones on steroid biosynthesis are not fully understood. We present data showing that 100 nM of both orexins A and B for 4 or 24 h significantly up-regulates StAR, in H295R pluripotent adrenocortical cells. We present the dose-dependent and time-dependent characteristics of StAR up-regulation at the protein level, showing significant increases after 4 h at a relatively low agonist concentration (1 nM). We have provided a key analysis of the precise G protein-coupled signaling pathways required for the up-regulation of StAR in response to orexins A and B. This has involved dominant-negative G protein analysis, and the direct inhibition of the protein kinase A, protein kinase C, ERK1/2, and p38 pathways. This shows a fundamental role for multiple G protein-coupled and MAPK-mediated signaling pathways leading to StAR expression. Antagonist analysis also showed that orexin effects on StAR were primarily, but not exclusively, acting through the orexin receptor type 1. This is the first study linking orexin action on StAR expression and comprehensively describes the signaling pathways involved in regulating the complexity of hormone biosynthesis.