Real-time analysis of gene regulation by glucocorticoid hormones

Abstract

There is increasing evidence that temporal factors are important in allowing cells to gain additional information from external factors, such as hormones and cytokines. We sought to discover how cell responses to glucocorticoids develop over time, and how the response kinetics vary according to ligand structure and concentration, and hence have developed a continuous gene transcription measurement system, based on an interleukin-6 (IL-6) luciferase reporter gene. We measured the time to maximal response, maximal response and integrated response, and have compared these results with a conventional, end point glucocorticoid bioassay. We studied natural glucocorticoids (corticosterone and cortisol), synthetic glucocorticoids (dexamethasone) and glucocorticoid precursors with weak, or absent bioactivity. We found a close correlation between half maximal effective concentration (EC50) for maximal response, and for integrated response, but with consistently higher EC50 for the latter. There was no relation between the concentration of ligand and the time to maximal response. A comparison between conventional end point assays and real-time measurement showed similar effects for dexamethasone and hydrocortisone, with a less effective inhibition of IL-6 seen with corticosterone. We profiled the activity of precursor steroids, and found pregnenolone, progesterone, 21-hydroxyprogesterone and 17-hydroxyprogesterone all to be ineffective in the real-time assay, but in contrast, progesterone and 21-hydroxyprogesterone showed an IL-6 inhibitory activity in the end point assay. Taken together, our data show how ligand concentration can alter the amplitude of glucocorticoid response, and also that a comparison between real-time and end point assays reveals an unexpected diversity of the function of glucocorticoid precursor steroids, with implications for human disorders associated with their overproduction.

Glucocorticoids rapidly inhibit cell migration through a novel, non-transcriptional HDAC6 pathway

“…Glucocorticoids (GCs) are steroid hormones that regulate a range of biological functions essential for life, including normal homeostasis, glucose metabolism, resolution of inflammation and development (McMaster et al, 2008;Tu et al, 2018;Tanaka et al, 2017). GCs exert their biological effects through the ubiquitously expressed glucocorticoid receptor (GR; also known as NR3C1), a ligand-inducible transcription factor of the nuclear hormone receptor superfamily (Hollenberg et al, 1985).…”

Serum cholesterol selectively regulates glucocorticoid sensitivity through activation of JNK

“…2011 ). In humans cortisol is the principal circulating Gc, but additional modulatory molecules are present including cortisone and the 5α-reduced cortisol metabolites ( McMaster et al . 2008 , Nixon et al .…”

Does Anesthetic Relief Correspond to Future Pain Relief?

“…Meanwhile, corticosteroids work by binding to glucocorticoid receptors, altering transcription of various genes either directly inducing transcription at the promotor region or by inactivating cofactors required for transcription of proinflammatory proteins 29 . One study concludes that this first step in gene transcription activity alone can take approximately 3 hrs from the time the glucocorticoid comes in contact with a cell 30 . It is well demonstrated that cytokines modulate cellular behavior and play a role in pain signaling and neuronal sensitization 31 .…”

“…29 One study concludes that this first step in gene transcription activity alone can take approximately 3 hrs from the time the glucocorticoid comes in contact with a cell. 30 It is well demonstrated that cytokines modulate cellular behavior and play a role in pain signaling and neuronal sensitization. 31 Thus, corticosteroid pain relief generally takes longer to begin, with the duration of pain relief measured in months.…”