Cofilin 1 Is Revealed as an Inhibitor of Glucocorticoid Receptor by Analysis of Hormone-Resistant Cells

Abstract

Significant knowledge about glucocorticoid signaling has accumulated, yet many aspects remain unknown. We aimed to discover novel factors involved in glucocorticoid receptor regulation that do not necessarily require direct receptor interaction. We achieved this by using a functional genetic screen: a stable cell line which cannot survive hormone treatment was engineered, randomly mutated, and selected in the presence of glucocorticoid. A hormone-resistant clone was analyzed by two-dimensional gel electrophoresis. Differentially expressed proteins were identified and tested as candidates for regulation of the glucocorticoid receptor. An unexpected candidate, cofilin 1, inhibited receptor activity. Cofilin is known to promote actin depolymerization and filament severing. Several experiments suggest that this feature of cofilin is involved in its inhibitory action. Both its actin depolymerization activity and its inhibitory action on the receptor are dependent on its phosphorylation state. Treatment of cells with a cytoskeleton-disrupting agent decreased receptor activity, as did overexpression of actin, particularly a mutant actin that does not polymerize. In addition, overexpression of cofilin and actin as well as chemical cytoskeleton disruption changed the subcellular receptor distribution and upregulated c-Jun, which could constitute the inhibitory mechanism of cofilin. In summary, cofilin represents a novel factor that can cause glucocorticoid resistance.

We thank Paul Hill, Dale Milfay, and Isabel Birg for excellent technical assistance; Archana Jacob and Guiseppina Maccarrone for support in mass spectrometry analyses; and Gabriela Wochnik, Amalia Tsolakidou, Marilyn Tirard, and Dietmar Spengler for critical reading of the manuscript. We thank Peter Hutzler and E. Mannweiler (National Research Center for Environment and Health, Munich, Germany) for generous support with laser scanning microscopy.

J.R. was supported by a studentship from the German National Academic Foundation.