10 15 20 25 40 80

Time after addition of Cortisol at 37°C (min)

+ Cycloheximide I 20° C

FIGURE 10-11 Time course in rat thymus cell suspensions at 37°C of cortisol-receptor complex formation, cortisol-induced inhibition of glucose transport, and inhibition of protein synthesis. Kinetics of receptor complex formation was determined with [3H]cortisol at about 0.1 /xM. Inhibitory metabolic effects were produced with about 1 /¿M Cortisol. Glucose transport was measured with 2-min pulses of radioactive hexose initiated at the times indicated. Shaded segments of the horizontal bars in the lower part of the figure roughly indicate the time intervals during which emergence of the Cortisol effect on glucose metabolism can be blocked by treatment with cortexolone (which displaces Cortisol from the glucocorticoid receptors), actinomycin D, and cycloheximide and can be delayed by lowering the temperature. Open bars indicate periods during which these treatments have no effect. At the top of the figure is the sequence of steps by which it is hypothesized that the cortisol-receptor complex leads to the synthesis of a specific protein that inhibits glucose transport. Reproduced from A. Munck and G. R. Crabtree, Glucocorticoid-induced lymphocyte death. Bower, I. D., and Lockshin, R. A. (eds.), (1981). In "Cell Death in Biology and Pathology, pp. 329-357. Chapman and Hall, London and New York.

whole organism. The effects of this hormone are extremely broad, but clearly, it is key to survival and humans cannot live without it.

The mechanism involved in cell death produced by glucocorticoids is intriguing. There have been many advances in understanding cell death by apoptosis, a genetic mechanism that is set in motion by cells susceptible to glucocorticoids, in this case including T and B cells. Other work on the apoptotic mechanism in nematodes has identified critical genes, ced-3, ced-4, and ced-9, in this process, ced-3 encodes a protease that is homologous to the mammalian interleukin-1/3-converting enzyme (ICE), which catalyzes the conversion of the interleukin-1/3 precursor into the active inflammatory cytokine. There are other ICE homologs in mammalian cells, such as ICH (ICE homolog), nedd-3 (mouse homolog), CPP32 (cysteine protease, 32,000 Da, and mch (mammalian ced-3 homologue) to name a few. The three-dimensional structure of ICE has been solved (Figure 10-12). This cysteine protease, which cleaves an aspartyl-X bond, is synthesized in the cell as a proprotein containing two subunits, a p20 and a plO, that are released in free form through the cleavage of aspartyl-X bonds on either end of each subunit. Initital inactive precursors are either activated by a distinct protease, as yet unknown, or autocata-lyzed by a small number of endogenously active ICElike molecules. Because some cells contain respectable amounts of endogenous ICE, autoactivation seems less likely, so that its activity may be controlled by another process, possibly by a highly regulated protease. In fact, ICEe, a splice variant giving rise to only a plO-like protein, can combine with a p20 subunit to form an inactive p30 enzyme, or it could prevent dimerization [(p20)2-(P10)2], So far, it is unlikely that ICE is on the pathway of glucocorticoid-induced apoptosis, but it seems likely that one of the other homologs could be a key member of that pathway. A speculative scheme of glucocorticoid-induced apoptosis in susceptible T or B cells can be contemplated (Figure 10-13).

That the glucocorticoid receptor is a key element in this process is emphasized by the demonstration that a negative regulator of the receptor, called the "modulator," which maintains the receptor in its nonactive oligomeric form, is capable of preventing apoptosis of leukemia cells in the presence of glucocorticoids.



FIGURE 10-12 CPK model of the (p20)2-(pl0)2 tetramer of ICE. Two p20 subunits surround two adjacent plO subunits. The tetrapeptide aldehyde inhibitor is represented by the lighter atoms. The crystallographic 2-fold axis is roughly perpendicular to the plane of drawing and runs through the small hole at the center of the interface between the two plO subunits. /3-Strands 5 and 6 protrude from the top and, by 2-fold symmetry, the bottom of the model. The N- and C-terminal ends of each subunit are labeled. Dynamics simulations suggest that the formation of a tetramer would stabilize the active site of ICE by reducing the mobility of the catalytic Cys285 and other residues near the p20 C-terminus. This is an adaptation of a colored photograph. Reproduced with permission from Wilson, K. P., Black, J. A. F., Thomson, J. A., Kim, E. E., Griffith, J. P., Navia, M. D., Murcko, M. A., Chambers, S. P., Aldape, R. A., Raybuck, S. A., and Livingston, D. J. (1994). "Structure and mechanism of interleukin-1/3 converting enzyme". Nature 370;270-275.

2. Actions of Glucocorticoids on Cells

The cellular mode of action of glucocorticoid hormone is summarized in Figure 10-14, where the target gland is the liver cell, representing the predominant tissue whose overall response is anabolic. Later, the actions of the hormone on the thymus cell, which responds to the hormone catabolically, will be described.

In this model (Figure 10-14), the free hormone is shown permeating the liver cell membrane, as previously discussed. Once inside the cytoplasm of the cell, it can combine with the specific, high-affinity receptor to form a steroid-receptor complex. This com-


- Combines with unoccupied receptor inside target cell

- Transcriptional activation (nucleus)

■ mRNA of Human ced-3 Homolog or regulatory factor or Proenzyme p20

Activation p20

Another specific protease or autoactivation p20


Activation of other proteases?

Activation of other proteases?

Specific endonuclease inactive precursor

Specific endonuclease active

Specific endonuclease inactive precursor

Specific endonuclease active

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