Signal Transduction
The roots of scientific development in China, including aspects that we now call endocrinology, have been documented by Joseph Needham (a Cambridge biochemist who turned his attention to the science and civilization of China).2
mention in a Chinese pharmacopoeia of 725 AD, though their widespread use had to wait another seven centuries until promulgated by Chu ChenHeng. He encouraged their application for the treatment of many conditions. More importantly, the Chinese had, over this period of time, developed a remarkable set of extraction and fractionation techniques, with the aim of obtaining active materials from urine. Pharmacists were engaged ‘almost
on a manufacturing scale’, using up to 200 gallons (almost 1000 L) at a time, to prepare active products by precipitation with calcium sulfate (which sedimented steroid conjugates along with protein), evaporation to dryness, sublimation, and crystallization. According to Chu Chen-Heng, ‘all people suffering from impotence, sexual debility, excess ang of the burning feverish type which no medicine can benefit, will improve. The natural precipitate of urine can also drive out the undue fire element affecting the liver and the bladder’. Joseph Needham had no doubt that the Chinese had, between the 11th and 17th centuries, achieved preparations of androgens and oestrogens that were ‘probably quite effective in the quasi-empirical therapy of the time.’3
The root of these chemical names is the word oestrous (estrus, in American spelling), which derives from the Greek oistros meaning a gadfly. Fritz Spiegl points out that to be stung by a gadfly was thought to send people, especially women, into a frenzy.3
Adolf Friedrich Johann Butenandt (1903–95) was awarded the Nobel Prize in Chemistry in 1939 for his ‘work on sex hormones.’ The Nazis forced him to decline the award, and he was only able to accept it in 1949.
Beginning again
The first steroid hormone to be isolated, purified, and characterized was the oestrous-inducing substance oestrin (estrin), C18H22O2, obtained in gram quantities from the urine of pregnant women.4, 5 Other oestrogenic substances such as oestriol6 soon followed. Stilboestrol, the first synthetic oestrogen, was originally isolated from plants. It lacks the steroid nucleus, characteristic of many hormones; its structure is based on that of stilbene. It was soon followed by ‘dynestrol’, described in 1938.7 Progesterone, first described as a ‘special hormone present in the corpus luteum responsible for preparing the uterus for the reception of embryos’8 required heroic
quantities of material. Adolph Butenandt, who had already isolated the male sex hormone androsterone, used the corpora lutea from 50 000 pigs for the isolation of a few milligrams of ‘progestin’.
The discovery of intracellular hormone receptors
The steroid and thyroid hormones, and other lipophilic messengers (Figure 10.1) are carried by specific binding proteins in the circulation. It has been thought that they penetrate cell membranes and enter cells passively,9 but this is not always the case. For example, there is evidence of a saturable transport mechanism for oestrogens and for thyroid hormone, suggesting that they require the assistance of a protein for this step.10 In the cytosol, most of them bind to intracellular receptor molecules that then migrate to the nucleus, attach to particular regulatory DNA sequences (response elements),
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Nuclear receptors
FIG 10.1 Structures of some nuclear receptor ligands.
Steroid hormones and diethylstilbestrol are shown in green, thyroid hormone is red, retinoic acids are purple, and vitamin D is blue. Chenodeoxycholic acid (in black) is a bile acid.
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Signal Transduction
and activate or inhibit transcription (transactivation or transrepression). Before developing this basic theme, it should be noted that the regulation of transcription is not necessarily the unique function of the intracellular receptors. Some exert effects on cytosolic signalling pathways quite independently of transcription (see pages 290 and 292).
Evidence for intracellular receptors
Although the earlier findings of preferential binding of oestrogens in target tissues (e.g. estradiol in the uterus and vagina) supported a receptor hypothesis, the first actual demonstration of steroid binding to a receptor protein, indeed, the first demonstration of the binding of any hormone to any receptor in a cell-free system, was reported in 1966 by Toft and Gorski.11
They injected female rats with [3H]-estradiol and then carried out subcellular fractionation of the homogenized uteri. This revealed that 50% of the radioactivity was present in the nuclear fraction, while 30% was in the soluble cytosol fraction. After centrifuging the cytosolic material through a sucrose density gradient, they recovered the radioactivity in a component that sedimented at 9.5S, equivalent to a protein of 200 kDa. Binding
of the hormone could be antagonized by diethylstilbestrol, but not by the non-oestrogenic steroids testosterone and corticosterone. Similar highaffinity binding proteins (KD 0.1–10 nmol L 1) are found in soluble fractions prepared from homogenates of various unstimulated hormone-responsive tissues. Following binding of hormone, these receptor proteins are found in the nuclear fraction. The key experimental observation was the accumulation of [3H]-corticosterone in the nuclear fraction of hepatoma cells, at the expense of a loss of radioactivity from the cytosol.12
Studies of the subcellular location of steroid receptors relying on cell fractionation and immunostaining have, however, been controversial, with some conflicting data. To overcome this, it has been essential to visualize receptors in living cells and to follow their translocation by real-time imaging. This became possible with the development of green fluorescent protein (GFP) tagging (see page 196). Cells may be transfected to express functional receptors that are GFP fusion proteins. Trafficking of the tagged receptors within the cell may then be followed using high-resolution fluorescence microscopy (usually laser confocal microscopy: see page 196). This approach has provided the clearest demonstration that cytoplasmic receptors, typified by glucocorticoid receptors (GR), can translocate to the nucleus within an hour of exposure to ligand (Figure 10.2).
The pattern is broadly similar for other steroid hormone receptors, such as mineralocorticoid receptors (MR), androgen receptors (AR), and
progesterone receptors (PR). However, the partition of steroid receptors into either the cytosol or the nucleus is never absolute. Their distribution between
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