0888-8809/02/$15.00/0 Molecular Endocrinology 16(6):1352–1366 Printed in U.S.A. Copyright © 2002 by The Endocrine Society Activity of the GR in G2 and Mitosis G. ALEXANDER ABEL, GABRIELA M. WOCHNIK, JOE¨ LLE RU¨ EGG, AUDREY ROUYER, FLORIAN HOLSBOER, AND THEO REIN Max Planck Institute of Psychiatry, Munich D-80804, Germany To elucidate the mechanisms mediating the re- DEX-inducible in G2. Even in mitotic cells, only the ported transient physiological glucocorticoid re- stably transfected MMTV promoter was repressed, sistance in G2/M cell cycle phase, we sought to whereas the same promoter transiently trans- Downloaded from https://academic.oup.com/mend/article/16/6/1352/2741824 by guest on 29 September 2021 establish a model system of glucocorticoid-resis- fected was inducible. The use of Hoechst 33342 for tant cells in G2. We synchronized various cell lines synchronization in previous studies probably in G2 to measure dexamethasone (DEX)-induced caused a misinterpretation, because we detected transactivation of either two endogenous promot- interference of this drug with GR-dependent tran- ers (rat tyrosine aminotransferase and mouse me- scription independent of the cell cycle. Finally, GR tallothionein I) or the mouse mammary tumor virus activated a simple promoter in G2, excluding a (MMTV) promoter stably or transiently transfected. functional effect of cell cycle-dependent phos- To circumvent the need for synchronization drugs, phorylation of GR, as implied previously. We con- we stably transfected an MMTV-driven green fluo- clude that GR itself is fully functional throughout rescent protein to directly correlate DEX-induced the entire cell cycle, but GR responsiveness is re- transactivation with the cell cycle position for each pressed in mitosis due to chromatin condensation cell of an asynchronous population using flow cy- rather than to specific modification of GR. tometry. Surprisingly, all promoters tested were (Molecular Endocrinology 16: 1352–1366, 2002) LUCOCORTICOIDS ARE INVOLVED in regula- Conversely, transactivation of GR-responsive promoters Gtion of various biological processes, including is presumed to be cell cycle-dependent since more than development and reproduction, cell growth and pro- 30 yr. One of the first observations was a cell cycle- liferation, and metabolism of carbohydrate, lipids, and dependent induction of tyrosine aminotransferase (TAT) protein (1–3). As a family member of ligand-dependent by glucocorticoids in hepatoma cells (17). While cells transcription factors (4, 5) the GR is activated by hor- were glucocorticoid-responsive during the G1 and S cell mone binding. GR is subsequently translocated into cycle phases, cells in G2 as well as in M were reported to the nucleus, where it binds to specific DNA sequences be completely resistant to glucocorticoids. Moreover, termed glucocorticoid-responsive elements (GREs) induction of endogenous alkaline phosphatase (18) or and increases transcription from nearby promoters. later on of epidermal growth factor receptors (19) in HeLa Several cofactors are important for proper function of cells was shown to be most effective in late G1 and GR. Molecular chaperones in the cytosol keep the S-phase with an apparent lack of glucocorticoid respon- receptor in a conformation capable of binding to hor- siveness during G2/M and early G1. More recently, it was mone with high affinity (6). Chaperones may also be demonstrated that in fibroblasts synchronized in G2 the involved in nuclear translocation of GR (7–9). Several stably transfected mouse mammary tumor-virus (MMTV) nuclear cofactors of GR are also necessary for chro- promoter as well as the endogenous metallothionein-I matin remodeling of nucleosomally organized promot- (MT-I) promoter could not be activated by glucocorti- ers and for efficient interaction with the basal tran- coids (20). However, in accordance with the hypothesis scriptional machinery (10, 11). Important effects of GR of specific glucocorticoid resistance in G2 the MT-I pro- are not mediated by transactivation of target promot- moter was still inducible by heavy metals in G2. Remark- ers, but by transrepression (12, 13). Transrepression is ably, inhibition of GR seemed to be confined to trans- independent of DNA binding (14) but requires interac- activation, because transrepression by GR was not tion with other transcription factors such as activator affected in G2 cells (21). protein 1 or nuclear factor ␬B (15, 16). Despite several observations relating to cell cycle- It is a long-standing observation that glucocorticoids dependent activity of GR, including different hormone exert antiproliferative effects in most cellular contexts. binding during the cell cycle (19, 22, 23) or decreased nuclear translocation of GR in G2 (19, 20), the molec- Abbreviations: CHO, Chinese hamster ovary; CMV, cyto- megaly virus; DEX, dexamethasone; FACS, fluorescence-ac- ular mechanisms leading to G2 silencing of GR func- tivated cell sorter; gal, galactosidase; GFP, green fluorescent tion remained largely unclear. It has been speculated protein; GRE, glucocorticoid-responsive elements; HOE, that differential phosphorylation of GR throughout the Hoechst 33342; Luc, luciferase gene; MTV, mammary tumor cell cycle (24, 25) might contribute to or account for virus; MMTV, mouse mammary tumor virus; MT, metallothio- nein; p, plasmid; PI, propidium iodide; TAT, tyrosine amino- cell cycle-dependent function of GR. Indeed, rat GR transferase; TK, thymidine kinase. was shown to be a target for cyclin-dependent kinases 1352 Abel et al. • GR Activity in G2 and Mitosis Mol Endocrinol, June 2002, 16(6):1352–1366 1353 and mitogen activated protein kinases in vitro (26). However, using site-specific mutations of phosphory- lation sites of GR, it was not possible to identify a distinct phosphorylation pattern of GR that would lead to complete silencing of the receptor (27, 28). Inter- estingly, some functional consequences of GR phos- phorylation were found, but these effects turned out to be promoter specific because they were apparent only at simple promoters containing just one to three GREs, but not at complex promoters like the MMTV promoter (29–31). Complex promoters are able to recruit addi- Downloaded from https://academic.oup.com/mend/article/16/6/1352/2741824 by guest on 29 September 2021 tional cofactors, which themselves might be cell cycle- dependently regulated (32–34). With the aim to identify molecular mechanisms ex- plaining glucocorticoid resistance in G2, we sought to establish an experimental model to measure cell cycle-dependent glucocorticoid resistance. We tested cell cycle-dependent transactivation of endogenous as well as exogenous glucocorticoid-sensitive pro- Fig. 1. Induction of TAT Activity in G2 and Mitosis moters, stably or transiently transfected in various cell At time zero, asynchronously proliferating H4-II-E-C3 cells lines. To our surprise, we found no silencing of GR were stimulated with DEX (5 nM) in the presence of colcemid function in G2 at all. Furthermore, mitotic repression of (300 nM) and 6-3H-thymidine (0.1 ␮Ci). At the indicated times GR-induced transcription apparently is due to general after stimulation, mitotic cells were harvested by shaking chromatin condensation, and not to specific inactiva- them off and TAT activity and 3H-thymidine incorporation tion of GR. were determined. In parallel, aliquots of asynchronously pro- liferating cells were stimulated with DEX in the presence of colcemid and prepared at the same times for control. Gray RESULTS bars represent the DEX-induced TAT activity of G2 cells (relative to vehicle-treated cells) as a function of their position in the cell cycle at the time of DEX induction (i.e. the time Induction of TAT by Dexamethasone (DEX) in before mitosis) compared with asynchronously proliferating Hepatoma Cells in G2 or Mitosis control cells (white bars). Data are given as mean Ϯ SEM of three independent experiments each performed in triplicate. The first systematic investigation of cell cycle-depen- The time course of 3H-thymidine incorporation is given up to dent function of GR reported that induction of endog- 11.5 h after time zero (from right to left). enous TAT in cultured rat hepatoma cells by DEX is completely repressed in the G2 phase of the cell cycle and in mitosis (17). Using the same methodical ap- nonsignificant decrease of TAT induction by DEX com- proach, we first checked whether this original finding pared with asynchronously proliferating cells stimu- is reproducible in our cells endogenously expressing lated under the same conditions in the presence of GR and TAT. Asynchronously proliferating rat hepa- colcemid (Fig. 1). toma H4-II-E-C3 cells were incubated with colcemid, The TAT activities of the noninduced reference cells 6-3H-thymidine and DEX at time zero. Every 90 min, also provide no indication for a decrease of the ligand- mitotic cells were harvested for determination of TAT- independent activity of GR in G2. On the contrary, activity as reporter of GR-induced transcription and there is even a small increase from asynchronously 6-3H-thymidine incorporation as a control marker for proliferating cells to G2 cells (1.5- to 1.6-fold for each cell cycle position of cells at the beginning of induction time point; data used for Fig. 1, but not displayed). (time zero). The rationale of the procedure is that cells The slight decrease in inducibility in G2 cells might collected in mitosis a certain time after stimulation either indicate an only partial repression