- Email: [email protected]
Autoregulation and crossregulation of nuclear receptor genes
Smith AI, Funder Jw: 1988. Proopiomelanocortin processing in the pituitary, central nervous system, and peripheral tissues. Endocr Rev 9:159-179. Smythe GA, Gnmstein HS, Brad&w JE, Nicholson MW, Compton PJ: 1984. Relationships between brain noradrenergic activity and blood glucose. Nature 308:65-67. Stanley BG, Leibowitz SF: 1984. Neuropeptide Y: stimulation of feeding and drinking by injection into the paraventricular nucleus. Life Sci 35~2635-2642. Stemberg EM, Hill JM, Chrousos GP, et al.: 1989a. Inflammatory mediator-induced hypothalamic-pituitary-adrenal axis activation is defective in streptococcal cell wall arthritis-susceptible Lewis rats. Proc Nat1 Acad Sci USA 86:2174-2178. Stemberg EM, Young III WS, Bernardini R, et al.: 1989b. A central nervous system defect in biosynthesis of corticotropin-releasing hormone is associated with susceptibility to streptococcal cell wall-induced arthritis in Lewis rats. Proc Nat1Acad Sci USA 86:477 l4775. Suda T, Tozawa M, Yamada T, et al.: 1988. Insulin-induced hypoglycemia increases corticotropin-releasing factor messenger ribonucleic acid levels in rat hypothalamus. Endocrinology 123:1371-1375. Swanson LW, Sawchenko PE, Rivier J, Vale Ww: 1983. Organization of ovine corticotropin-releasing factor immunoreactive cells and fibers in the rat brain: an immunohistochemical study. Neuroendocrinology 36:165-186. Thomas GB, Cummins JT, Cavanagh L, Clarke IJ: 1986. Transient increase in prolactin secretion following hypothalamc+ pituitary disconnection in ewes during anoestrus and the breeding season. J Endocrinol 111:425-431. Thomas GB, Cummins JT, Canny BJ, et al.: 1989. The posterior pituitary regulates prolactin, but not adrenocorticotropin orgonadotropin, secretion in the sheep. Endocrinology 125:2204-2211. Ur E, Faria M, Tsagarakis S, et al.: 1991. Atria1 natriuretic peptide in physiological doses does not inhibit the ACTH or cortisol response to corticotrophin-releasing hormone-41 in normal human subjects. J Endocrinol 131:163-167.
Whitnall MH: 1988. Distributions of provasopressin expressing and pro-vasopressin deficient CRH neurons in the paraventricular hypothalamic nucleus of colchicinetreated normal and adrenalectomized rats. J Comp Neuro1275: 13-28.
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Autoregulation and Crossregulation of Nuclear Receptor Genes Jamshed R. Tata
Whereas crossregulation of nuclear receptors has been known for some time, recently several examples of autoregulation have been described, especially during development and specific gene expression. In this review, I discuss both these phenomena, based on some studies from our laboratory on amphibian metamorphosis and egg protein gene expression. These include autoinduction of estrogen receptor (ER) accompanying egg protein gene expression in adult and larval Xenopus; autoinduction of thyroid hormone receptor (TR) during metamorphosis and in adult Xenopus; crossregulation by triiodothyronine (T3) and dexamethasone of autoinduction of ER; and inhibition by PRL of autoinduction and crossinduction of TR and ER genes. A dual receptor threshold model to explain the interplay between T3, estrogen and PRL is presented and its significance to the general question of nuclear receptor autoregulation and crossregulation during development is also discussed. (Trends Endocrinol Metab 1994;5;283-290)
Several investigators
studying nuclear re-
ceptors in response to hormones oping
tissues
or during
in devel-
regulation
of
specific hormonal target gene expression have observed the phenomenon of upregulation
of receptor
number.
The
first
Vance ML, Harris AG: 1991. Long-term treatment of 189 acromegalic patients with the somatostatin analog octreotide: results of the International Multicenter Study Group. Arch Intern Med 151:1573-1578.
in multihormonal systems, whereby one hormone would induce or activate the
Weiner RI, Ganong WF: 1978. Role of brain monoamines and histamine in regulation of anterior pituitary secretion. Physiol Rev 58:905-976.
Jamshed R. Tata is at the Laboratory of Developmental Biochemistry, National Institute for Medical Research, The Ridgeway, London NW7 1AA, England.
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receptor for another, or glucocorticoids such as the upregulation by progesterone of estrogen receptor (ER) or in breast cancer cells, during uterine growth, upon egg protein gene activation in the oviduct or during mammary gland development (Baulieu
and Kelly
1990,
Parker
1991,
Tata 1984, Eriksson and Gustafsson 1983). More recently, the phenomenon of upregulation of members of the nuclear steroid/ thyroid hormone receptor gene family by their own ligands has been described in many growth and developmental systems. Here, I shall consider the phenomenon of
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both ER and ER mRNA was reported for whole Xenopus (Shapiro et al. 1989), whereas coinduction of ER and Vit genes has also been observed for the response to E, of male trout hepatocytes in culture (Pakdel et al. 1989). The same pattern of coupled increase of nuclear ER and the accumulation of FOSP-1 mRNA was observed in primary culture of Xenoptts oviduct cells treated with E, (Varriale and Tata 1990). In both Xenopus hepatocyte and oviduct cell cultures it was possible to show with cycloheximide and antiestrogens that the absolute rate of transcription of Vit and FOSP-1 genes was a direct function of the autoinduction of ER during the early stages of E2 action. In other studies, the developmental competence of Xenopus Vit genes activated by E, was found to be acquired during metamorphosis (Ng et al. 1984). Xenopus tadpoles are insensitive to estrogen during pro-metamorphosis (developmental stages 54 and 53, and even during mid-metamorphosis (until stage 58 or 59), but during metamorphic climax (stages 60-64) they become highly sensitive to the hormone, as seen by the de novo activation of the normally silent Vit genes. Significantly, we have found Figure 1. Upregulation by T, of thyroid hormone receptor (TR) mRNA in different tissues of in recent studies that tadpoles at midpre-metamorphicXenopus tadpoles. The figure shows dark-field imagining of localization by in metamorphosis exhibit a strong ausitu hybridization of W-labeled antisense Xenopus TRa cRNA probe (Kawahara et al. 1991) in toinduction of ER gene when exposed to sagittal sections of brain (a and b), intestine (c and d) and liver (e and f) of stage-52 tadpoles. (a, c, and e) Control (untreated) tadpoles: and (b, d, and f) tadpoles treated for 4 days with 1O-9 estrogen, which again is coupled to Vit M T,. Sense probe gave virtually no hybridization signal and the autoradiographs are thus not gene activation (Rabelo et al. 1994). The shown. Arrows in liver sections (e and f’) indicate an artifact produced in black-and-white thyroid hormone triiodothyronine (T3), photography by pigmentation (Pi) surrounding the bulk of the parenchymal cells (P,) of the but not retinoic acid (RA), also produced liver. The sections were exposed for 1 week for autoradiography. From Rabelo et al. (1994). a small increase in accumulation of ER mRNA in tadpole liver, a finding that will both autoinduction and crossinduction’ question of nuclear receptor autoinducbe considered later when discussing of nuclear receptors of the steroid/thyroid tion when I was studying estrogen2 crossregulation of TR and ER mRNAs by hormone receptor gene family, particuactivation of egg protein genes in XenT, in larval and adult tissues. larly during development and specific opus. In our laboratory, we have intengene expression, and which will be largely sively studied the activation by estrogen ?? Autoinduction of Thyroid Hormone illustrated by investigations from our of both vitellogenin (Vit) and FOSP-1 Receptor (TR) During Amphibian laboratory on amphibian vitellogenesis (frog oviduct specific protein 1) genes in Metamorphosis and metamorphosis. primary cultures of adult male and female Xenopus hepatocytes and oviduct Multiple isoforms of TR, encoded by two cells, respectively (Tata et al. 1986, genes a and j3, have been identified in ?? Autoinduction of Estrogen Perlman et al. 1984, Varriale and Tata diverse mammalian, avian, and amphibReceptor (ER) Accompanying 1990, Tata 1991). ian tissues (Chin 1991, Chatterjee and Egg Protein Gene Expression in In our first studies we observed a close Tata 1992). The relative amounts of differAdult and Developing Xenopus association between the numbers of ent isoforms of TR have been found to My attention was first drawn to the tightly bound ERs in nuclei from privary according to different tissues in the mary male Xenopus hepatocyte cultures same organism, which raises the possibil1 Autoinduction and crossinduction are defined as ity that the well-known tissue-specific and the de novo transcription of Vit the increase in number or concentration of either diversity of responses to thyroid mRNA after the addition of estrogen to nuclear receptor protein or transcripts induced by its own or a different ligand, respectively. the cultures (Perlman et al. 1984). A hormones may reside in these differences. *Estrogen (EJ refers to estradiol 17-B. similar autoinduction in male liver of In Xenopus, although several TR isoform 284
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transcripts have been detected, the two major transcripts correspond to the mammalian al and pl mRNAs that are developmentally regulated (Yaoita and Brown 1990, Kawahara et al. 1991). Amphibian metamorphosis is an ideal model for experimentally exploring postembryonic development and, because the process is obligatorily dependent on thyroid hormones, offers an excellent opportunity to study the developmental significance of TR autoinduction (Tata 1993). Low levels of TR transcripts detected in early tadpole stages (until stage 45) are possibly of maternal origin, but once the tadpole reaches pre-metamorphic stages (53-54) there is a substantial increase in both transcripts, which continue to increase with the onset of metamorphosis, reaching peak values at metamorphic climax (stages 60-64). After this developmental stage the levels decline to low levels seen in adult tissues. Importantly, the two transcripts do not increase to the same extent, nor do they accumulate in parallel in different regions of the developing tadpole. At all developmental stages, TRa mRNA is present in concentrations that are 5-10 times those of TR@ In situ hybridization has revealed high levels of accumulation of TR transcripts in tissues of Xenopus tadpoles, known to be highly sensitive to T,, namely, the brain, liver, intestine, and limb buds (Kawahara et al. 1991). Normal metamorphosis begins at around stages 53154, when the Xenopus tadpole’s thyroid gland begins to secrete thyroid hormone, after which a close relationship exists between the buildup and decline of plasma T, levels and the biphasic developmental regulation of TRa and p transcripts (Yaoita and Brown 1990, Kawahara et al. 1991). It was therefore of some interest to determine how the TR genes would be expressed if metamorphosis is precociously induced with exogenous thyroid hormone. When stage-51/52 Xenopus tadpoles (a few weeks before they would normally begin to metamorphose) were exposed to 2 x 10V9M T,, there was a rapid upregulation of both TRa and B mRNAs (Kawahara et al. 1991, Yaoita and Brown 1990, Tata 1993, Rabelo et al. 1994). Figure 1 illustrates the phenomenon of TR mRNA upregulation in some pre-metamorphic Xenopus tadpole tissues, as seen by in situ hybridization, after the precocious induction of metamorphosis with T, TEA4 Vol. 5, No. 7, 1994
(Rabelo et al. 1994). This autoinduction is one of the most rapid responses to T,, compared with the activation of other genes, such as those encoding albumin and 63-kD keratin and which characterize the acquisition of the adult phenotype (Tata et al. 1993). A major drawback of studies in whole animals is that it is virtually impossible to carry out precise analysis of the early events preceding a given induction, or to interrupt a chain of important cellular events initiated by the hormonal signal, such as protein synthesis or generation of second messengers. For this reason we and others have studied and examined autoinduction of TRs in Xenopus tissue culture cell lines (Machuca and Tata 1992, Kanamori and Brown 1992). In our laboratory we have found that the autoinduction of TRa and l3 mRNA in XTC-2 cells follows the same kinetics as in vivo and that in both cases it is the most rapid response known so far to exogenous thyroid hormone. It was therefore possible to test in cell lines, but not in whole tadpoles, whether or not TR genes represent “immediate early” genes or that their autoinduction required the synthesis of some other “early” protein(s). Experiments with cycloheximide carried out with XTC-2 cells in our laboratory (Machuca and Tata 1992) and with XL177 cells in Brown’s laboratory (Kanamori and Brown 1992) clearly showed that T, failed to fully upregulate TR mRNAs when protein synthesis is inhibited. Thus, one has to consider the involvement of some other “immediate early” protein(s) in the process of receptor autoinduction. Another advantage of transfection of cell cultures is that their use makes it possible to test whether or not the newly induced receptor transcripts produce functional receptor capable of activating its target genes. For example, when we measured the transcription from Xenopus albumin promoter linked to a CAT (chloramphenicol acetyl transferase) reporter transfected into XTC-2 cells, the construct was found to be expressed at a higher than basal level when the cells were exposed to T,, under conditions in which the hormone induces TRa and l3 mRNAs (Tam et al. 1993). Thus, autoinduction of TR mRNA in XTC-2 cells produces more receptor which, in turn, activates the promoter of a gene normally induced by T, in vivo. 01994, Elsevier Science Inc., 1043-2760/94/$7.00
The phenomenon of autoinduction raises the question of whether or not the underlying mechanism involves a direct interaction between the receptor protein and the promoter of its own gene. Recently, our laboratory has found that the Xenopus TRB gene promoter contains two direct-repeat +4 (DR4) and three half-sites of T,-responsive elements (TREs), as well as a DRl site for RXRE, within 1.6 kb upstream of its transcription start site (I. Machuca, G. Esslemont, L. Fairclough, and J.R. Tata unpublished). It is now generally accepted that thyroid hormones act via heterodimers of TR and RXR (Zhang and Pfahl 1993). Significantly, Xenopus TRa and fl heterodimers with RXRa and y (but not TR monomers or homodimers) exhibited high-affinity binding of TRE sites in Xenopus. TRB promoter, while transfection of the latter into XTC-2 cells, or XL-2 cells in which TRg was overexpressed, conferred T, inducibility of transcription from the promoter. Thus, a direct receptor-promoter interaction can explain the autoinduction phenomenon.
??
TR Gene Expression iu Adult Xenopus
That thyroid hormones, and hence TRs, are obligatorily required for the development of amphibian larvae to adults is firmly established. However, we have detected the presence of small amounts of T, in adult Xenopus (Tata et al. 1993), which raises the intriguing question of possible function of thyroid hormones after metamorphosis is completed. If they do have a function in adult amphibia, is the phenomenon of autoinduction of TR maintained throughout adult life? We have recently attempted to answer this question in primary cultures of adult Xenopus hepatocytes (Rabelo and Tata 1993) and find that adult Xenopus tissues express TRa and fi at low levels. More significantly, when adult hepatocytes in primary culture were exposed to very low doses of thyroid hormone (lo-lo to 10-9 M T,), there was an upregulation of TR mRNAs, albeit not as substantial as in pre-metamorphic tadpoles or in XTC-2 cells. This raises the question of physiologic significance of TR autoinduction in adult amphibian tissues.
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5
6
7
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L
autoinduction of estrogen receptor (ER) mRNA by estrogen (EJ in stage 60 (lanes 4-6), but not 56 (kznesl-3), X&opus tadpole liver. ER mRNA was determined by RNase protection assay of 20 p.g of total RNA. The hybridization probe was j2P-labeled 167-bp BgZII-Hind111fragment from Xenopus ER cDNA, whereas a 32P-labeled 5s RNA probe was used as a RNA-loading control. The arrows show the 197-bp free probe, the 167-bp protected band for Xenopus ER, and the smaller 5s RNA bands at the bottom of the gel. Lanes 1 and 4, control (untreated) tadpoles; 2-5, treated with 10-’ M E, alone; 3-6, pretreatment with 10-9M T, for 1 day, followed by T, and E, together for 4 days; and 7, tRNA alone. From Rabelo et al. (1994).
Figure 2. T, enhances the
.
Crossregulation Dexamethasone of ER
by T, and of Autoinduction
As mentioned earlier, E, upregulates its own receptor protein and mRNA, which
in turn is tightly coupled to the activation of Vit genes in primary cultures of adult Xenopus hepatocytes and in tadpoles. In cultures of adult male and female Xenopus hepatocytes, low doses of T, strongly potentiate, within 6-12 h, the induction by E, of Vit mRNA (Rabelo and Tata 1993). In attempting to explain this rapid potentiation effect, it was found that T, also upregulated TR$ (but not TRo) mRNA. At the same time, T, also enhanced the autoinduction of ER mRNA by E,. In Xenoptls tadpoles, this crossinduction by T, was developmental stage specific as shown in Figure 2 (Rabelo et al. 1994). In transient transfection experiments in Xenopus XTC-2 cells, T, was also found to potentiate the transcription by E, of an ERECAT construct, thus suggesting that T, enhances the accumulation of not only ER mRNA but also functional ER. Earlier studies had shown that the developmental competence of Xenoptls 286
hepatocytes to express Vit genes in response to E, was first acquired during late metamorphosis (May and Knowland 1980, Ng et al. 1984). It was therefore of some interest that we recently found T, to enhance the precocious activation of Vit genes by E, in Xenopus tadpoles during metamorphosis (Rabelo et al. 1994). In these studies, low doses of exogenous T, that induce metamorphosis (for example, 10m9M) were found to induce high levels of TR mRNA in several tissues of premetamorphic Xenopus tadpoles, in line with earlier reports (Yaoita and Brown 1990, Baker and Tata 1990 and 1992, Kawahara et al. 1991). Significantly, the same treatment enhanced and accelerated the precocious activation of the silent Vit genes in tadpole liver at metamorphic climax but not before mid-metamorphosis. Under the same experimental conditions, T, fails to influence the autoinduction of ER mRNA at mid-metamorphosis but strongly potentiates this response to E, at metamorphic climax (Figure 2). Thus, a developmentally regulated interplay between thyroid hormone and estrogen determines the kinetics and extent of activation of Vit and ER genes during Xenopus postembryonic development. 01994, Elsevier Science Inc., 1043-2760/94/$7.00
Is this hormonal interplay restricted to T, and E,? In recent studies we have found that T, and dexamethasone (Dex) rapidly enhance the accumulation of ER mRNA and, consequently, the activation of Vit genes if E, is present in primary cultures of adult Xenopus hepatocytes (Ulisse and Tata 1994). The effect of Dex was rapid and produced additive effects with T, on the accumulation of ER mRNA. In examining the kinetics of the crossinduction by the two hormone classes, it was noticed that T, exerted both a rapid and slow action, whereas the effect of Dex was mostly a rapid one. It will be interesting to determine whether or not the other two major steroid hormone classes, androgens and progesterones, also modulate ER mRNA levels, because both are known to modify estrogen action in mammals (Baulieu and Kelly 1990).
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Inhibition by Prolactin of Autoinduction and Crossinduction of TR and ER Genes
Most thyroid hormone-induced developmental changes associated with amphibian metamorphosis are prevented or arrested by PRL or prolactinlike factors, both in vivo and in organ culture (Nicoll 1974). Our laboratory has recently exploited this action of prolactin to explore further the significance of autoinduction of TRs during metamorphosis (Tata 1993) by determining how PRL would affect autoinduction of TR while inhibiting the action of T, in whole tadpoles and in organ cultures of tails and limb buds (Tata et al. 1991, Baker and Tata 1992). As shown in Figure 3, PRL almost completely prevented the upregulation of both TRcl and TRB mRNAs induced by Ts in whole pre-metamorphic Xenopus tadpoles as well as in organ cultures of tails. The inhibition was also visualized by in situ hybridization, particularly in highly T,-sensitive tissues of Xenopus tadpoles (Tata et al. 1993, Rabelo et al. 1994). The conclusion that autoinduction of TR mRNAs (and, most likely, TR proteins) is important for the activation of T, target genes was strongly supported by experiments in which PRL also blocked the activation of albumin and the adult-type 63-kD keratin genes in Xenopus tadpole liver and tail, respectively (Baker and Tata 1992), followed by the abolition of morphologic signs of TEM Vol. 5, No. 7, 1994
T,-induced metamorphosis. earlier that T, potentiates
We have seen the inducibil-
TRa
ity of Vit and ER genes in adult Xenopus hepatocytes, so that the question also arose as to whether or not the larval response to PRL. would be retained after metamorphosis in adult life. Some recent studies from our laboratory suggest that the antagonism between PRL and thyroid hormone that is so characteristic of metamorphosis is retained in adult liver, but we do not know whether this antagonistic action between the two hormones may be brought about by similar mechanisms in larval and adult amphibian tissues (E.M.L. Eabelo and J.R. Tata unpublished). The interplay between PRL and T, and, indirectly, E,, is relevant to the question of “cross-talk” between signals acting via the pathway of receptors located in the plasma membrane and those in the nucleus. Many investigators have recently pointed out the importance of considering this process in integrating individual pathways into intracellular signaling networks (Diamond et al. 1990, Aronica and Katzenellenbogen 1993). Although PFU is known to initiate its action through its plasma membrane receptor, the biochemical pathway and intracellular second messengers have not been definitively established (Rillema 1994). This information would not only be useful in understanding the mechanism of action of PFU but the inhibition of T, action would reveal further characteristics of TR structure and function.
??
A Model for the Interplay Between T,, E,, and PRL
A simple model to account for the autoregulation and crossregulation described previously for T,, Dex, E,, and PRL is presented in Figures 4 and 5. According to this model, there is a low level of functional TR in pre-metamorphic tadpole tissues, which is constitutively expressed in most tissues in early tadpole stages (for example, 52/53) as well as in adult liver. The requirement of upregulation of TR mRNA to activate its target genes suggests some sort of a differential threshold of receptor concentration in order to promote its own induction and, consequently, activation of downstream (TR target) genes. Exogenous T, or increasing levels of endogenous thyroid hormones would
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Figure 3. Inhibition by PRL of autoinduction by T, of TRa and fI mRNA. Where indicated, batches of 18 stage-50 Xenopus tadpoles were immersed for 2 days in 100 mL of medium containing 0.2 IU/mLof ovine PRL before exposure to 1O-9M T, for a further 2 days along with or without PRL. Total RNA was assayed for TRa and fI mRNAs as above. Pr, probe: and Con, control (no hormone). Arrows indicate the protected bands for TRa and g mRNAs (seen as a doublet for B). Adapted from Baker and Tata (1992). activate TR to upregulate its own receptor at a low threshold level via a proteinsynthesis-dependent process. At the same time, T, and Dex would also exert a rapid, protein-synthesis-independent effect in upregulating TR gene expression. PRL, or
PRL-like factor(s) would, by an as yet unknown mechanism, prevent the autoinduction of TR via the relatively slower pathway. Elevated levels of TR, resulting from both pathways, above a higher threshold than that needed for TR autoin-
Figure 4. A model summarizing the essential features of autoregulation and crossregulation of thyroid hormone (TR) and estrogen (ER) receptors by their respective ligands and dexamethasone (Dex) and prolactin (PRL), leading to the activation of vitellogen (Vit) genes in Xenopus. Small amounts of TR expressed constitutively when combined with its ligand T, would amplify itself by a process of autoinduction. Dex would potentiate this action through a rapid, protein-synthesis-independent pathway. E, also causes the upregulation of its own receptor. In the presence of higher levels of liganded TR, the autoinduction of ER would be greatly enhanced. The latter then results in a strong potentiation of activation of the silent Vit genes inXenopus tadpole or adult male hepatocytes. PRL prevents the autoinduction of TR, so that its presence at high concentrations would inhibit the potentiation of autoinduction of ER and Vit gene activation. The difirent sizes of the lettering and arrows denote the relative amount of the receptors or extent of their induction or de-induction. Upward and downward pointing arrows indicate upregulation and downregulation, respectively.
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mented that estrogen induces progesterone receptor as part of its growthThreshold promoting action in the rodent uterus or mammary tissue, whereas the converse occurs during avian egg maturation. Glucocorticoid hormones are also well known to regulate other steroid hormone receptors in various tissues. As regards autoinduction, besides the examples of Xenopus ER and TR discussed in this review, there are recent reports of a similar phenomenon for other receptors. Thus, as listed in Table 1, all three Time t isoforms of the mouse retinoic acid Ligand receptor (RAR) are induced by retinoic acid (de The et al. 1990). Interestingly, a Figure 5. A general dual-threshold pathway to explain the significance of the model in Figure 4 nuclear receptor autoinduction and target gene activation by the ligand. retinoic acid response element (RARE) has been identified in the promoter of duction would activate different sets of an upregulation of ER by its own ligand RARB gene, whose expression is strongly genes in different tissues, depending on E, (Shapiro et al. 1989, Perh-nan et al. enhanced in a tissue-specific and isoformspecific manner during embryonic and 1984), a process that is strongly potentidifferent developmental programs. It is fetal development. More recently, Thumated by T, (Rabelo and Tata 1993, Ulisse also implicit in this model that, directly or mel’s group (Andres and Thummel1992, and Tata 1994). indirectly, TR genes are more sensitive to Karim and Thummel1992) has reported liganded TR than are the “late“ or downthat ecdysone upregulates its own recepstream target genes that determine the ?? Significance of Nuclear Receptor tor transcripts in Drosophila cells, whereas phenotypic changes characteristic of Autoregulation and Crossregulation androgen has also been shown to upregmetamorphosis. The same process of and Future Prospects ulate its own receptor and mRNA in rat antagonism between T, and PRL would accessory sexual tissue (Gonzales-Cadavid apply to adult tissues, except that little is Whereas the phenomenon of crossreguet al., 1993). These examples indicate known about the “downstream” genes lation of nuclear receptors has been that the phenomenon of autoinduction under T, control. Thus, if one considers known for over a decade (Tata 1984), it is of nuclear hormone receptors may be a that two of the ‘downstream” genes in only relatively recently that information general feature of hormone-dependent Xenopus, irrespective of developmental became available about autoregulation postembryonic growth and development. stage, are those encoding ER and Vit, then (Tata et al. 1993). Some examples of The dual-threshold model, indicated in the model in Figure 4 could be further both these processes are summarized in Figure 4 for TR and ER, would then be Table 1. Thus, it has been well docuextended. Thus, Vit genes would require generally applicable to other nuclear receptors in a developmental context of the action of their ligands. Although the previous evidence supTable 1. Auto- and cross-regulation of nuclear receptors by various ports the conclusion that receptor upreghormones and developmental signals is of general occurrence ulation is closely associated with the Receptor(mRNA Up- or cfown- Species and /fonnoJleOf Biokgkal function biological activity of its ligand, more Invohfed fegU/atkrl tissues sional of rxotein) direct proof of a cause-effect relation7 All amphibian Amphibian metamor- ship remains to be obtained. It is also TRy and g Thyroid hormone tissues phosis worth mentioning that much of the current work on developmental aspects Xenopus liver T Vitellogenesis Estrogen ER 7 Drosophila cells Insect metamorphosis of nuclear receptors, and transcription Ecdysone ECR factors generally, is restricted to the t Mouse and chick Morphogenesis Retinoic acid RARa, B, and Y analysis of their transcripts. In view of brain & limbs the importance of not only protein-DNA Rat prostate T Sexual development Androgen AR interactions, but also complex proteinAll amphibian 1 Vitellogenesis Pmlactin TRa and g protein interactions in the context of tissues chromatin organization, future work will Xenopus liver T Vitellogenesis Thyroid ER have to shift its emphasis to the technihormone cally more difficult problem of functionChick oviduct T Egg development Progesterone ER ally active receptor protein molecules. Chick and Xenopus Egg development Glucocorticoids ER T A salient feature of many hormoneliver dependent developmental systems is mulOther receptors: ECR, ecdysone; AR, androgen; and RAR, retinoicacid. tiple hormonal interactions as, for exam-
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ple, during the development gland or in uterine growth Kelly 1990). Our studies on between PRL and thyroid
of mammary (Baulieu and the interplay hormone in
controlling metamorphosis illustrate the importance of receptor autoregulation in the physiologic response to a developmental hormone. The mechanism underlying this type of hormonal interplay and the level of postreceptor events at which it takes place remain unknown. Another factor to be considered is that of heterodimerization of nuclear receptors. For example, the active form of thyroid hormone receptor is a heterodimer with RXR, whose ligand is 94s retinoic acid, whereas heterodimers of RAR-RXR and vitamin D, receptor (VDR)-RXR are also thought to be important for mediating the action of retinoic acid and vitamin D, @hang and Pfahl 1993). In view of the substantial size of the nuclear steroid/thyroid hormone/retinoid receptor gene family, and the suggestion that the functionally active receptors may comprise heterodimers of subunits of different receptors, it will be important to extend studies on autoregulation and crossregulation to other receptors, which would then allow a more precise assessment of how generally important receptor upregulation is for the action of hormones that control developmental or inductive processes. ??
Acknowledgments
The data in Figures 1 and 2 were obtained by Dr. Elida Rabelo. The author thanks Mrs. Ena Heather for the preparation of the manuscript. References Andres AJ, Thummel CS: 1992. Hormones, puffs and flies: the molecular control of metamorphosis by ecdysone. Trends Genet 8132-138. Aronica SM. Katzenellenbogen BS: 1993. Progesterone receptor regulation in uterine cells: stimulation by estrogen, cyclic adenosine 3’,5’-monophosphate, and insulin-like growth factor I and suppression by antiestrogens and protein kinase inhibitors. Endocrinology 128:2045-2052. Baker BS, Tata JR 1990. Accumulation of proto-oncogene c-&-A related transcripts during Xenopus development: association with early acquisition of response to thyroid hormone and estrogen. EMBO J 9:879885. Baker BS, Tata JR: 1992. Prolactin prevents
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Perlman AJ, Wolffe AP, Champion J, Tata JR: 1984. Regulation by estrogen receptor of vitellogenin gene transcription in Xenopus hepatocyte cultures. Mol Cell Endocrinol 38:151-161.
Diamond MI, Miner, JN, Yoshinaga SK, Yamamoto KR: 1990. Transcription factor interactions: selectors of positive or negative regulation from a single DNA element. Science 249:1266-1272.
Rabelo EML, Tata JR: 1993. Thyroid hormone potentiates estrogen activation of vitellogenin genes and autoinduction of estrogen receptor in adult Xenopus hepatocytes. Mol Cell Endocrinol96:3744.
Eriksson H, Gustafsson J-A: 1983. Steroid Hormone Receptors: Structure and Function. Nobel Symposium No. 57. Amsterdam, Elsevier.
Rabelo EML, Baker BS, Tata JR: 1994. Interplay between thyroid hormone and estrogen in modulating expression of their receptor and vitellogenin genes during Xenopus metamorphosis. Mech Dev 45:49-57.
Gonzales-Cadavid NF, Vemet D, Navarro AF, Rodrigues JA, Swerdloff RS, Rajfer J: 1993. Upregulation of the levels of androgen receptor and its mRNA by androgens in smooth-muscle cells from rat penis. Mol Cell Endocrinol90:219-229.
Rillema JA, ed: 1987. Actions of Prolactin on Molecular Processes. Boca Raton, FL, CRC. Rillema JA: 1994. Development of the mammary gland and lactation. Trends Endocrino1 Metab 5:149-154.
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Karim FD, Thummel CS: 1992. Temporal coordination of regulatory gene expression by the steroid hormone ecdysone. EMBO J 11:4083-4093.
Tata JR: 1984. The action of growth and developmental hormones: evolutionary aspects. In Goldberger RF, Yamamoto KR, eds. Biological Regulation and Development, vol3B. New York, Plenum, pp l-58.
Kawahara A, Baker BS, Tata JR: 1991. Developmental and regional expression of thyroid hormone receptor genes during Xenopus metamorphosis. Development 112:933943. Lehmann JM, Zhang x-k, Pfahl M: 1992. RARy2 expression is regulated through a retinoic acid response element embedded in Spl sites. Mol Cell Biol 12:2976-2985. Machuca I, Tata JR: 1992. Autoinduction of thyroid hormone receptor during metamorphosis is reproduced in Xenopus XTC-2 cells. Mol Cell Endocrinol87: 105-l 13. May FEB. Knowland J: 1980. The role of thyroxine in the transition of vitellogenin synthesis from non-inducibility to inducibility during metamorphosis in Xenopus Iuevis. Dev Biol77:419-430. Ng WC, Wolffe AP, Tata JR: 1984. Unequal activation by estrogen of individual Xenopus vitellogenin genes during development. Dev Biol 102:238-247.
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Tata JR: 199 1. Hormonal and developmental regulation of Xenapus estrogen receptor and egg protein gene expression. In Hochberg RB, Naftolin F, eds. The New Biology of Steroid Hormones. New York, Raven, pp 213-225. Tata JR 1993. Gene expression during metamorphosis: an ideal model for postembryonic development. BioEssays 15:239248. Tata JR, Ng WC, Perlman AJ, Wolffe AP: 1986. Activation and regulation of the vitellogenin gene family. In Roy A-K, Clark JH, eds. Gene Regulation by Steroid Hormones III. New York, Springer, pp 205-233. Tata JR, Kawahara A, Baker BS: 1991. Prolactin inhibits both thyroid hormoneinduced morphogenesis and cell death in cultured amphibian larval tissues. Dev Biol 146:72-80. Tata JR, Baker BS, Machuca I, Rabelo EML,
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by a dynamic interplay between the sex hormones and the somatotropic axis. These issues have recently been reviewed at greater length (Kerrigan and Rogol 1992), permitting us to focus here on newer information that has increased the understanding of how gonadal steroids influence the GH axis, particularly during the adolescent growth spurt. Although it is now a burgeoning area of investigation, we will not address the converse effects of GH and the IGFs on the hypothalamicpituitary-gonadal axis.
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Gonadal Steroid Hormone Regulation of the Somatotropic Axis Du&g Puberty in Humans Mechanisms of Androgen and Estrogen Action Daniel L. Metzger, James R. Kerrigan, and Alan D. Rogol
The adolescent growth spurt is associated with a sex steroid hormonedependent rise in GH production; both androgens and estrogens are implicated as positive regulators of the somatotropic axis during puberty. The issue is complicated by the fact that testosterone may act both directly via the androgen receptor and indirectly, after its aromatization to 17#Sestradiol, through the estrogen receptor. Recently, a number of investigators have studied the effects of the administration of androgen and estrogen receptor antagonists, as well as nonaromatizable androgens, on GH secretion. These reports suggest that estrogen receptor-dependent processes play a facilitatory role in the pubertyassociated rise in GH secretion. If androgen receptor-mediated events are involved in the control of the somatotropic axis, their role is likely inhibitory. A hypothalamic site of action of the sex steroids is postulated. (Trends Endocrinol Metab 1994;5:290-296)
The pubertal growth spurt occurs during
lating levels of GH, IGF-I, and the gona-
a period of development
da1 steroid hormones testosterone (T) and 17p-estracliol (Ez ) all increase during
dramatic
neuroendocrine
somatotropic
associated
with
changes in the
and gonadal
axes. Circu-
Daniel L. Metzger is at the Department of Pediatrics, University of British Columbia, Vancouver, BC V6H 3V4, Canada; James R. Kerrigan is at the Department of Pediatrics, East Tennessee State University, Johnson City, TN 37614, USA; and Alan D. Rogol is at the Departments of Pediatrics and Pharmacology, University of Virginia, Charlottesville, VA 22908, USA.
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this transition. These changes are reflected in the acceleration of linear growth velocity, from a mean prepubertal rate of 5.5 cm/year to a maximum of 9.5 cm/year in boys and 8.5 cm/year in girls. Concomitant with this rapid-growth phase is the development of the characteristic physical signs of puberty, which are induced by the gonadal hormones. Evidence is accumulating that suggests that the pubertal growth spurt is orchestrated
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Puberty-Associated Alterations in Somatotropic and GonadaI Steroid Hormones
With the development of RIAs for GH in the 1960s it became apparent that the hormone is normally secreted in an episodic fashion. This knowledge prompted experiments involving repetitive blood sampling, which suggested that mean GH concentrations were higher in adolescents than in prepubertal children or adults. Recently, this finding has been confirmed and elaborated upon in several larger cross-sectional studies (Martha et al. 1989, Rose et al. 1991), which clearly demonstrated that mean GH levels are maximal during midpuberty to late puberty (that is, about Tanner stage III-IV) in both boys and girls. Figure 1 depicts the close relationship between 24-h mean serum GH concentrations and the standard growth velocity curve for North American boys. In these two studies, computer-assisted pulse-detection algorithms were applied to time series of GH levels; the results demonstrated that the late-pubertal rise in mean GH concentrations is a result of an increase primarily in the amplitude, rather than in the frequency, of GH pulses. Deconvolution modeling represents a significant advance in the analysis of endogenous GH kinetics and the mechanism of developmental alterations or induced perturbations of the GH axis. This method enables the simultaneous calculation of subject-specific hormone secretion and elimination functions. To date, deconvolution analysis has been employed in two large cross-sectional studies of children across puberty (AlbertssonW&land et al. 1989, Martha et al. 1992). Both studies report that GH production rates are highest during late puberty and fall toward prepubertal values by adult-
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Whitnall MH: 1988. Distributions of provasopressin expressing and pro-vasopressin deficient CRH neurons in the paraventricular hypothalamic nucleus of colchicinetreated normal and adrenalectomized rats. J Comp Neuro1275: 13-28.
Yang-Yen H-F, Chambard J-C, Sun Y-L, et al.: 1990. Transcriptional interference between c-jun and the glucocorticoid receptor: mutual inhibition of DNA binding due to direct protein-protein interaction. Cell 62: 12051215.
Wick G, Hu Y-H, Gruber J: 1992. The role of the immunoendocrine interaction via the hypothalamc+pituitary-adrenal axis in autoimmune disease: emphasis on the obese strain chicken model. Trends Endocrinol Metab 3:141-146.
Ying S-Y: 1988. Inhibins, activins, and follistatin: gonadal proteins modulating the secretion of follicle-stimulating hormone. Endocr Rev 9:267-293.
Autoregulation and Crossregulation of Nuclear Receptor Genes Jamshed R. Tata
Whereas crossregulation of nuclear receptors has been known for some time, recently several examples of autoregulation have been described, especially during development and specific gene expression. In this review, I discuss both these phenomena, based on some studies from our laboratory on amphibian metamorphosis and egg protein gene expression. These include autoinduction of estrogen receptor (ER) accompanying egg protein gene expression in adult and larval Xenopus; autoinduction of thyroid hormone receptor (TR) during metamorphosis and in adult Xenopus; crossregulation by triiodothyronine (T3) and dexamethasone of autoinduction of ER; and inhibition by PRL of autoinduction and crossinduction of TR and ER genes. A dual receptor threshold model to explain the interplay between T3, estrogen and PRL is presented and its significance to the general question of nuclear receptor autoregulation and crossregulation during development is also discussed. (Trends Endocrinol Metab 1994;5;283-290)
Several investigators
studying nuclear re-
ceptors in response to hormones oping
tissues
or during
in devel-
regulation
of
specific hormonal target gene expression have observed the phenomenon of upregulation
of receptor
number.
The
first
Vance ML, Harris AG: 1991. Long-term treatment of 189 acromegalic patients with the somatostatin analog octreotide: results of the International Multicenter Study Group. Arch Intern Med 151:1573-1578.
in multihormonal systems, whereby one hormone would induce or activate the
Weiner RI, Ganong WF: 1978. Role of brain monoamines and histamine in regulation of anterior pituitary secretion. Physiol Rev 58:905-976.
Jamshed R. Tata is at the Laboratory of Developmental Biochemistry, National Institute for Medical Research, The Ridgeway, London NW7 1AA, England.
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receptor for another, or glucocorticoids such as the upregulation by progesterone of estrogen receptor (ER) or in breast cancer cells, during uterine growth, upon egg protein gene activation in the oviduct or during mammary gland development (Baulieu
and Kelly
1990,
Parker
1991,
Tata 1984, Eriksson and Gustafsson 1983). More recently, the phenomenon of upregulation of members of the nuclear steroid/ thyroid hormone receptor gene family by their own ligands has been described in many growth and developmental systems. Here, I shall consider the phenomenon of
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both ER and ER mRNA was reported for whole Xenopus (Shapiro et al. 1989), whereas coinduction of ER and Vit genes has also been observed for the response to E, of male trout hepatocytes in culture (Pakdel et al. 1989). The same pattern of coupled increase of nuclear ER and the accumulation of FOSP-1 mRNA was observed in primary culture of Xenoptts oviduct cells treated with E, (Varriale and Tata 1990). In both Xenopus hepatocyte and oviduct cell cultures it was possible to show with cycloheximide and antiestrogens that the absolute rate of transcription of Vit and FOSP-1 genes was a direct function of the autoinduction of ER during the early stages of E2 action. In other studies, the developmental competence of Xenopus Vit genes activated by E, was found to be acquired during metamorphosis (Ng et al. 1984). Xenopus tadpoles are insensitive to estrogen during pro-metamorphosis (developmental stages 54 and 53, and even during mid-metamorphosis (until stage 58 or 59), but during metamorphic climax (stages 60-64) they become highly sensitive to the hormone, as seen by the de novo activation of the normally silent Vit genes. Significantly, we have found Figure 1. Upregulation by T, of thyroid hormone receptor (TR) mRNA in different tissues of in recent studies that tadpoles at midpre-metamorphicXenopus tadpoles. The figure shows dark-field imagining of localization by in metamorphosis exhibit a strong ausitu hybridization of W-labeled antisense Xenopus TRa cRNA probe (Kawahara et al. 1991) in toinduction of ER gene when exposed to sagittal sections of brain (a and b), intestine (c and d) and liver (e and f) of stage-52 tadpoles. (a, c, and e) Control (untreated) tadpoles: and (b, d, and f) tadpoles treated for 4 days with 1O-9 estrogen, which again is coupled to Vit M T,. Sense probe gave virtually no hybridization signal and the autoradiographs are thus not gene activation (Rabelo et al. 1994). The shown. Arrows in liver sections (e and f’) indicate an artifact produced in black-and-white thyroid hormone triiodothyronine (T3), photography by pigmentation (Pi) surrounding the bulk of the parenchymal cells (P,) of the but not retinoic acid (RA), also produced liver. The sections were exposed for 1 week for autoradiography. From Rabelo et al. (1994). a small increase in accumulation of ER mRNA in tadpole liver, a finding that will both autoinduction and crossinduction’ question of nuclear receptor autoinducbe considered later when discussing of nuclear receptors of the steroid/thyroid tion when I was studying estrogen2 crossregulation of TR and ER mRNAs by hormone receptor gene family, particuactivation of egg protein genes in XenT, in larval and adult tissues. larly during development and specific opus. In our laboratory, we have intengene expression, and which will be largely sively studied the activation by estrogen ?? Autoinduction of Thyroid Hormone illustrated by investigations from our of both vitellogenin (Vit) and FOSP-1 Receptor (TR) During Amphibian laboratory on amphibian vitellogenesis (frog oviduct specific protein 1) genes in Metamorphosis and metamorphosis. primary cultures of adult male and female Xenopus hepatocytes and oviduct Multiple isoforms of TR, encoded by two cells, respectively (Tata et al. 1986, genes a and j3, have been identified in ?? Autoinduction of Estrogen Perlman et al. 1984, Varriale and Tata diverse mammalian, avian, and amphibReceptor (ER) Accompanying 1990, Tata 1991). ian tissues (Chin 1991, Chatterjee and Egg Protein Gene Expression in In our first studies we observed a close Tata 1992). The relative amounts of differAdult and Developing Xenopus association between the numbers of ent isoforms of TR have been found to My attention was first drawn to the tightly bound ERs in nuclei from privary according to different tissues in the mary male Xenopus hepatocyte cultures same organism, which raises the possibil1 Autoinduction and crossinduction are defined as ity that the well-known tissue-specific and the de novo transcription of Vit the increase in number or concentration of either diversity of responses to thyroid mRNA after the addition of estrogen to nuclear receptor protein or transcripts induced by its own or a different ligand, respectively. the cultures (Perlman et al. 1984). A hormones may reside in these differences. *Estrogen (EJ refers to estradiol 17-B. similar autoinduction in male liver of In Xenopus, although several TR isoform 284
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transcripts have been detected, the two major transcripts correspond to the mammalian al and pl mRNAs that are developmentally regulated (Yaoita and Brown 1990, Kawahara et al. 1991). Amphibian metamorphosis is an ideal model for experimentally exploring postembryonic development and, because the process is obligatorily dependent on thyroid hormones, offers an excellent opportunity to study the developmental significance of TR autoinduction (Tata 1993). Low levels of TR transcripts detected in early tadpole stages (until stage 45) are possibly of maternal origin, but once the tadpole reaches pre-metamorphic stages (53-54) there is a substantial increase in both transcripts, which continue to increase with the onset of metamorphosis, reaching peak values at metamorphic climax (stages 60-64). After this developmental stage the levels decline to low levels seen in adult tissues. Importantly, the two transcripts do not increase to the same extent, nor do they accumulate in parallel in different regions of the developing tadpole. At all developmental stages, TRa mRNA is present in concentrations that are 5-10 times those of TR@ In situ hybridization has revealed high levels of accumulation of TR transcripts in tissues of Xenopus tadpoles, known to be highly sensitive to T,, namely, the brain, liver, intestine, and limb buds (Kawahara et al. 1991). Normal metamorphosis begins at around stages 53154, when the Xenopus tadpole’s thyroid gland begins to secrete thyroid hormone, after which a close relationship exists between the buildup and decline of plasma T, levels and the biphasic developmental regulation of TRa and p transcripts (Yaoita and Brown 1990, Kawahara et al. 1991). It was therefore of some interest to determine how the TR genes would be expressed if metamorphosis is precociously induced with exogenous thyroid hormone. When stage-51/52 Xenopus tadpoles (a few weeks before they would normally begin to metamorphose) were exposed to 2 x 10V9M T,, there was a rapid upregulation of both TRa and B mRNAs (Kawahara et al. 1991, Yaoita and Brown 1990, Tata 1993, Rabelo et al. 1994). Figure 1 illustrates the phenomenon of TR mRNA upregulation in some pre-metamorphic Xenopus tadpole tissues, as seen by in situ hybridization, after the precocious induction of metamorphosis with T, TEA4 Vol. 5, No. 7, 1994
(Rabelo et al. 1994). This autoinduction is one of the most rapid responses to T,, compared with the activation of other genes, such as those encoding albumin and 63-kD keratin and which characterize the acquisition of the adult phenotype (Tata et al. 1993). A major drawback of studies in whole animals is that it is virtually impossible to carry out precise analysis of the early events preceding a given induction, or to interrupt a chain of important cellular events initiated by the hormonal signal, such as protein synthesis or generation of second messengers. For this reason we and others have studied and examined autoinduction of TRs in Xenopus tissue culture cell lines (Machuca and Tata 1992, Kanamori and Brown 1992). In our laboratory we have found that the autoinduction of TRa and l3 mRNA in XTC-2 cells follows the same kinetics as in vivo and that in both cases it is the most rapid response known so far to exogenous thyroid hormone. It was therefore possible to test in cell lines, but not in whole tadpoles, whether or not TR genes represent “immediate early” genes or that their autoinduction required the synthesis of some other “early” protein(s). Experiments with cycloheximide carried out with XTC-2 cells in our laboratory (Machuca and Tata 1992) and with XL177 cells in Brown’s laboratory (Kanamori and Brown 1992) clearly showed that T, failed to fully upregulate TR mRNAs when protein synthesis is inhibited. Thus, one has to consider the involvement of some other “immediate early” protein(s) in the process of receptor autoinduction. Another advantage of transfection of cell cultures is that their use makes it possible to test whether or not the newly induced receptor transcripts produce functional receptor capable of activating its target genes. For example, when we measured the transcription from Xenopus albumin promoter linked to a CAT (chloramphenicol acetyl transferase) reporter transfected into XTC-2 cells, the construct was found to be expressed at a higher than basal level when the cells were exposed to T,, under conditions in which the hormone induces TRa and l3 mRNAs (Tam et al. 1993). Thus, autoinduction of TR mRNA in XTC-2 cells produces more receptor which, in turn, activates the promoter of a gene normally induced by T, in vivo. 01994, Elsevier Science Inc., 1043-2760/94/$7.00
The phenomenon of autoinduction raises the question of whether or not the underlying mechanism involves a direct interaction between the receptor protein and the promoter of its own gene. Recently, our laboratory has found that the Xenopus TRB gene promoter contains two direct-repeat +4 (DR4) and three half-sites of T,-responsive elements (TREs), as well as a DRl site for RXRE, within 1.6 kb upstream of its transcription start site (I. Machuca, G. Esslemont, L. Fairclough, and J.R. Tata unpublished). It is now generally accepted that thyroid hormones act via heterodimers of TR and RXR (Zhang and Pfahl 1993). Significantly, Xenopus TRa and fl heterodimers with RXRa and y (but not TR monomers or homodimers) exhibited high-affinity binding of TRE sites in Xenopus. TRB promoter, while transfection of the latter into XTC-2 cells, or XL-2 cells in which TRg was overexpressed, conferred T, inducibility of transcription from the promoter. Thus, a direct receptor-promoter interaction can explain the autoinduction phenomenon.
??
TR Gene Expression iu Adult Xenopus
That thyroid hormones, and hence TRs, are obligatorily required for the development of amphibian larvae to adults is firmly established. However, we have detected the presence of small amounts of T, in adult Xenopus (Tata et al. 1993), which raises the intriguing question of possible function of thyroid hormones after metamorphosis is completed. If they do have a function in adult amphibia, is the phenomenon of autoinduction of TR maintained throughout adult life? We have recently attempted to answer this question in primary cultures of adult Xenopus hepatocytes (Rabelo and Tata 1993) and find that adult Xenopus tissues express TRa and fi at low levels. More significantly, when adult hepatocytes in primary culture were exposed to very low doses of thyroid hormone (lo-lo to 10-9 M T,), there was an upregulation of TR mRNAs, albeit not as substantial as in pre-metamorphic tadpoles or in XTC-2 cells. This raises the question of physiologic significance of TR autoinduction in adult amphibian tissues.
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4
5
6
7
probe
xER
5s
L
autoinduction of estrogen receptor (ER) mRNA by estrogen (EJ in stage 60 (lanes 4-6), but not 56 (kznesl-3), X&opus tadpole liver. ER mRNA was determined by RNase protection assay of 20 p.g of total RNA. The hybridization probe was j2P-labeled 167-bp BgZII-Hind111fragment from Xenopus ER cDNA, whereas a 32P-labeled 5s RNA probe was used as a RNA-loading control. The arrows show the 197-bp free probe, the 167-bp protected band for Xenopus ER, and the smaller 5s RNA bands at the bottom of the gel. Lanes 1 and 4, control (untreated) tadpoles; 2-5, treated with 10-’ M E, alone; 3-6, pretreatment with 10-9M T, for 1 day, followed by T, and E, together for 4 days; and 7, tRNA alone. From Rabelo et al. (1994).
Figure 2. T, enhances the
.
Crossregulation Dexamethasone of ER
by T, and of Autoinduction
As mentioned earlier, E, upregulates its own receptor protein and mRNA, which
in turn is tightly coupled to the activation of Vit genes in primary cultures of adult Xenopus hepatocytes and in tadpoles. In cultures of adult male and female Xenopus hepatocytes, low doses of T, strongly potentiate, within 6-12 h, the induction by E, of Vit mRNA (Rabelo and Tata 1993). In attempting to explain this rapid potentiation effect, it was found that T, also upregulated TR$ (but not TRo) mRNA. At the same time, T, also enhanced the autoinduction of ER mRNA by E,. In Xenoptls tadpoles, this crossinduction by T, was developmental stage specific as shown in Figure 2 (Rabelo et al. 1994). In transient transfection experiments in Xenopus XTC-2 cells, T, was also found to potentiate the transcription by E, of an ERECAT construct, thus suggesting that T, enhances the accumulation of not only ER mRNA but also functional ER. Earlier studies had shown that the developmental competence of Xenoptls 286
hepatocytes to express Vit genes in response to E, was first acquired during late metamorphosis (May and Knowland 1980, Ng et al. 1984). It was therefore of some interest that we recently found T, to enhance the precocious activation of Vit genes by E, in Xenopus tadpoles during metamorphosis (Rabelo et al. 1994). In these studies, low doses of exogenous T, that induce metamorphosis (for example, 10m9M) were found to induce high levels of TR mRNA in several tissues of premetamorphic Xenopus tadpoles, in line with earlier reports (Yaoita and Brown 1990, Baker and Tata 1990 and 1992, Kawahara et al. 1991). Significantly, the same treatment enhanced and accelerated the precocious activation of the silent Vit genes in tadpole liver at metamorphic climax but not before mid-metamorphosis. Under the same experimental conditions, T, fails to influence the autoinduction of ER mRNA at mid-metamorphosis but strongly potentiates this response to E, at metamorphic climax (Figure 2). Thus, a developmentally regulated interplay between thyroid hormone and estrogen determines the kinetics and extent of activation of Vit and ER genes during Xenopus postembryonic development. 01994, Elsevier Science Inc., 1043-2760/94/$7.00
Is this hormonal interplay restricted to T, and E,? In recent studies we have found that T, and dexamethasone (Dex) rapidly enhance the accumulation of ER mRNA and, consequently, the activation of Vit genes if E, is present in primary cultures of adult Xenopus hepatocytes (Ulisse and Tata 1994). The effect of Dex was rapid and produced additive effects with T, on the accumulation of ER mRNA. In examining the kinetics of the crossinduction by the two hormone classes, it was noticed that T, exerted both a rapid and slow action, whereas the effect of Dex was mostly a rapid one. It will be interesting to determine whether or not the other two major steroid hormone classes, androgens and progesterones, also modulate ER mRNA levels, because both are known to modify estrogen action in mammals (Baulieu and Kelly 1990).
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Inhibition by Prolactin of Autoinduction and Crossinduction of TR and ER Genes
Most thyroid hormone-induced developmental changes associated with amphibian metamorphosis are prevented or arrested by PRL or prolactinlike factors, both in vivo and in organ culture (Nicoll 1974). Our laboratory has recently exploited this action of prolactin to explore further the significance of autoinduction of TRs during metamorphosis (Tata 1993) by determining how PRL would affect autoinduction of TR while inhibiting the action of T, in whole tadpoles and in organ cultures of tails and limb buds (Tata et al. 1991, Baker and Tata 1992). As shown in Figure 3, PRL almost completely prevented the upregulation of both TRcl and TRB mRNAs induced by Ts in whole pre-metamorphic Xenopus tadpoles as well as in organ cultures of tails. The inhibition was also visualized by in situ hybridization, particularly in highly T,-sensitive tissues of Xenopus tadpoles (Tata et al. 1993, Rabelo et al. 1994). The conclusion that autoinduction of TR mRNAs (and, most likely, TR proteins) is important for the activation of T, target genes was strongly supported by experiments in which PRL also blocked the activation of albumin and the adult-type 63-kD keratin genes in Xenopus tadpole liver and tail, respectively (Baker and Tata 1992), followed by the abolition of morphologic signs of TEM Vol. 5, No. 7, 1994
T,-induced metamorphosis. earlier that T, potentiates
We have seen the inducibil-
TRa
ity of Vit and ER genes in adult Xenopus hepatocytes, so that the question also arose as to whether or not the larval response to PRL. would be retained after metamorphosis in adult life. Some recent studies from our laboratory suggest that the antagonism between PRL and thyroid hormone that is so characteristic of metamorphosis is retained in adult liver, but we do not know whether this antagonistic action between the two hormones may be brought about by similar mechanisms in larval and adult amphibian tissues (E.M.L. Eabelo and J.R. Tata unpublished). The interplay between PRL and T, and, indirectly, E,, is relevant to the question of “cross-talk” between signals acting via the pathway of receptors located in the plasma membrane and those in the nucleus. Many investigators have recently pointed out the importance of considering this process in integrating individual pathways into intracellular signaling networks (Diamond et al. 1990, Aronica and Katzenellenbogen 1993). Although PFU is known to initiate its action through its plasma membrane receptor, the biochemical pathway and intracellular second messengers have not been definitively established (Rillema 1994). This information would not only be useful in understanding the mechanism of action of PFU but the inhibition of T, action would reveal further characteristics of TR structure and function.
??
A Model for the Interplay Between T,, E,, and PRL
A simple model to account for the autoregulation and crossregulation described previously for T,, Dex, E,, and PRL is presented in Figures 4 and 5. According to this model, there is a low level of functional TR in pre-metamorphic tadpole tissues, which is constitutively expressed in most tissues in early tadpole stages (for example, 52/53) as well as in adult liver. The requirement of upregulation of TR mRNA to activate its target genes suggests some sort of a differential threshold of receptor concentration in order to promote its own induction and, consequently, activation of downstream (TR target) genes. Exogenous T, or increasing levels of endogenous thyroid hormones would
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Figure 3. Inhibition by PRL of autoinduction by T, of TRa and fI mRNA. Where indicated, batches of 18 stage-50 Xenopus tadpoles were immersed for 2 days in 100 mL of medium containing 0.2 IU/mLof ovine PRL before exposure to 1O-9M T, for a further 2 days along with or without PRL. Total RNA was assayed for TRa and fI mRNAs as above. Pr, probe: and Con, control (no hormone). Arrows indicate the protected bands for TRa and g mRNAs (seen as a doublet for B). Adapted from Baker and Tata (1992). activate TR to upregulate its own receptor at a low threshold level via a proteinsynthesis-dependent process. At the same time, T, and Dex would also exert a rapid, protein-synthesis-independent effect in upregulating TR gene expression. PRL, or
PRL-like factor(s) would, by an as yet unknown mechanism, prevent the autoinduction of TR via the relatively slower pathway. Elevated levels of TR, resulting from both pathways, above a higher threshold than that needed for TR autoin-
Figure 4. A model summarizing the essential features of autoregulation and crossregulation of thyroid hormone (TR) and estrogen (ER) receptors by their respective ligands and dexamethasone (Dex) and prolactin (PRL), leading to the activation of vitellogen (Vit) genes in Xenopus. Small amounts of TR expressed constitutively when combined with its ligand T, would amplify itself by a process of autoinduction. Dex would potentiate this action through a rapid, protein-synthesis-independent pathway. E, also causes the upregulation of its own receptor. In the presence of higher levels of liganded TR, the autoinduction of ER would be greatly enhanced. The latter then results in a strong potentiation of activation of the silent Vit genes inXenopus tadpole or adult male hepatocytes. PRL prevents the autoinduction of TR, so that its presence at high concentrations would inhibit the potentiation of autoinduction of ER and Vit gene activation. The difirent sizes of the lettering and arrows denote the relative amount of the receptors or extent of their induction or de-induction. Upward and downward pointing arrows indicate upregulation and downregulation, respectively.
Dex
Vitt
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PRL
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mented that estrogen induces progesterone receptor as part of its growthThreshold promoting action in the rodent uterus or mammary tissue, whereas the converse occurs during avian egg maturation. Glucocorticoid hormones are also well known to regulate other steroid hormone receptors in various tissues. As regards autoinduction, besides the examples of Xenopus ER and TR discussed in this review, there are recent reports of a similar phenomenon for other receptors. Thus, as listed in Table 1, all three Time t isoforms of the mouse retinoic acid Ligand receptor (RAR) are induced by retinoic acid (de The et al. 1990). Interestingly, a Figure 5. A general dual-threshold pathway to explain the significance of the model in Figure 4 nuclear receptor autoinduction and target gene activation by the ligand. retinoic acid response element (RARE) has been identified in the promoter of duction would activate different sets of an upregulation of ER by its own ligand RARB gene, whose expression is strongly genes in different tissues, depending on E, (Shapiro et al. 1989, Perh-nan et al. enhanced in a tissue-specific and isoformspecific manner during embryonic and 1984), a process that is strongly potentidifferent developmental programs. It is fetal development. More recently, Thumated by T, (Rabelo and Tata 1993, Ulisse also implicit in this model that, directly or mel’s group (Andres and Thummel1992, and Tata 1994). indirectly, TR genes are more sensitive to Karim and Thummel1992) has reported liganded TR than are the “late“ or downthat ecdysone upregulates its own recepstream target genes that determine the ?? Significance of Nuclear Receptor tor transcripts in Drosophila cells, whereas phenotypic changes characteristic of Autoregulation and Crossregulation androgen has also been shown to upregmetamorphosis. The same process of and Future Prospects ulate its own receptor and mRNA in rat antagonism between T, and PRL would accessory sexual tissue (Gonzales-Cadavid apply to adult tissues, except that little is Whereas the phenomenon of crossreguet al., 1993). These examples indicate known about the “downstream” genes lation of nuclear receptors has been that the phenomenon of autoinduction under T, control. Thus, if one considers known for over a decade (Tata 1984), it is of nuclear hormone receptors may be a that two of the ‘downstream” genes in only relatively recently that information general feature of hormone-dependent Xenopus, irrespective of developmental became available about autoregulation postembryonic growth and development. stage, are those encoding ER and Vit, then (Tata et al. 1993). Some examples of The dual-threshold model, indicated in the model in Figure 4 could be further both these processes are summarized in Figure 4 for TR and ER, would then be Table 1. Thus, it has been well docuextended. Thus, Vit genes would require generally applicable to other nuclear receptors in a developmental context of the action of their ligands. Although the previous evidence supTable 1. Auto- and cross-regulation of nuclear receptors by various ports the conclusion that receptor upreghormones and developmental signals is of general occurrence ulation is closely associated with the Receptor(mRNA Up- or cfown- Species and /fonnoJleOf Biokgkal function biological activity of its ligand, more Invohfed fegU/atkrl tissues sional of rxotein) direct proof of a cause-effect relation7 All amphibian Amphibian metamor- ship remains to be obtained. It is also TRy and g Thyroid hormone tissues phosis worth mentioning that much of the current work on developmental aspects Xenopus liver T Vitellogenesis Estrogen ER 7 Drosophila cells Insect metamorphosis of nuclear receptors, and transcription Ecdysone ECR factors generally, is restricted to the t Mouse and chick Morphogenesis Retinoic acid RARa, B, and Y analysis of their transcripts. In view of brain & limbs the importance of not only protein-DNA Rat prostate T Sexual development Androgen AR interactions, but also complex proteinAll amphibian 1 Vitellogenesis Pmlactin TRa and g protein interactions in the context of tissues chromatin organization, future work will Xenopus liver T Vitellogenesis Thyroid ER have to shift its emphasis to the technihormone cally more difficult problem of functionChick oviduct T Egg development Progesterone ER ally active receptor protein molecules. Chick and Xenopus Egg development Glucocorticoids ER T A salient feature of many hormoneliver dependent developmental systems is mulOther receptors: ECR, ecdysone; AR, androgen; and RAR, retinoicacid. tiple hormonal interactions as, for exam-
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ple, during the development gland or in uterine growth Kelly 1990). Our studies on between PRL and thyroid
of mammary (Baulieu and the interplay hormone in
controlling metamorphosis illustrate the importance of receptor autoregulation in the physiologic response to a developmental hormone. The mechanism underlying this type of hormonal interplay and the level of postreceptor events at which it takes place remain unknown. Another factor to be considered is that of heterodimerization of nuclear receptors. For example, the active form of thyroid hormone receptor is a heterodimer with RXR, whose ligand is 94s retinoic acid, whereas heterodimers of RAR-RXR and vitamin D, receptor (VDR)-RXR are also thought to be important for mediating the action of retinoic acid and vitamin D, @hang and Pfahl 1993). In view of the substantial size of the nuclear steroid/thyroid hormone/retinoid receptor gene family, and the suggestion that the functionally active receptors may comprise heterodimers of subunits of different receptors, it will be important to extend studies on autoregulation and crossregulation to other receptors, which would then allow a more precise assessment of how generally important receptor upregulation is for the action of hormones that control developmental or inductive processes. ??
Acknowledgments
The data in Figures 1 and 2 were obtained by Dr. Elida Rabelo. The author thanks Mrs. Ena Heather for the preparation of the manuscript. References Andres AJ, Thummel CS: 1992. Hormones, puffs and flies: the molecular control of metamorphosis by ecdysone. Trends Genet 8132-138. Aronica SM. Katzenellenbogen BS: 1993. Progesterone receptor regulation in uterine cells: stimulation by estrogen, cyclic adenosine 3’,5’-monophosphate, and insulin-like growth factor I and suppression by antiestrogens and protein kinase inhibitors. Endocrinology 128:2045-2052. Baker BS, Tata JR 1990. Accumulation of proto-oncogene c-&-A related transcripts during Xenopus development: association with early acquisition of response to thyroid hormone and estrogen. EMBO J 9:879885. Baker BS, Tata JR: 1992. Prolactin prevents
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the autoinduction of thyroid hormone receptor mRNAs during amphibian metamorphosis. Dev Biol 149:463-467. Baulieu E-E, Kelly P, eds: 1990. Hormones: From Molecules to Disease. Paris, Hermar-m. Chatterjee VKK, Tata JR: 1992. Thyroid hormone receptors and their role in development. Cancer Surv 14: 147-167.
Nicoll CS: 1974. Physiological actions of prolactin. In Knobil E, Sawyer WJH, eds. Handbook of Physiology, sect 7, ~014, part 2. Washington, DC, American Physiological Society, pp 253-292. Pakdel F, Le Guellec C, Vaillant C, Le Roux MG, Valotaire Y: 1989. Identification and estrogen induction of two estrogen receptors (ER) messenger ribonucleic acids in the rainbow trout liver: sequence homology with other ERs. Mol Endocrinol3:445 1.
Chin WW: 1991. Nuclear thyroid hormone receptors. In Parker MC, ed. Nuclear Hormone Receptors. London, Academic, pp 79-102.
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Gonzales-Cadavid NF, Vemet D, Navarro AF, Rodrigues JA, Swerdloff RS, Rajfer J: 1993. Upregulation of the levels of androgen receptor and its mRNA by androgens in smooth-muscle cells from rat penis. Mol Cell Endocrinol90:219-229.
Rillema JA, ed: 1987. Actions of Prolactin on Molecular Processes. Boca Raton, FL, CRC. Rillema JA: 1994. Development of the mammary gland and lactation. Trends Endocrino1 Metab 5:149-154.
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by a dynamic interplay between the sex hormones and the somatotropic axis. These issues have recently been reviewed at greater length (Kerrigan and Rogol 1992), permitting us to focus here on newer information that has increased the understanding of how gonadal steroids influence the GH axis, particularly during the adolescent growth spurt. Although it is now a burgeoning area of investigation, we will not address the converse effects of GH and the IGFs on the hypothalamicpituitary-gonadal axis.
??
Gonadal Steroid Hormone Regulation of the Somatotropic Axis Du&g Puberty in Humans Mechanisms of Androgen and Estrogen Action Daniel L. Metzger, James R. Kerrigan, and Alan D. Rogol
The adolescent growth spurt is associated with a sex steroid hormonedependent rise in GH production; both androgens and estrogens are implicated as positive regulators of the somatotropic axis during puberty. The issue is complicated by the fact that testosterone may act both directly via the androgen receptor and indirectly, after its aromatization to 17#Sestradiol, through the estrogen receptor. Recently, a number of investigators have studied the effects of the administration of androgen and estrogen receptor antagonists, as well as nonaromatizable androgens, on GH secretion. These reports suggest that estrogen receptor-dependent processes play a facilitatory role in the pubertyassociated rise in GH secretion. If androgen receptor-mediated events are involved in the control of the somatotropic axis, their role is likely inhibitory. A hypothalamic site of action of the sex steroids is postulated. (Trends Endocrinol Metab 1994;5:290-296)
The pubertal growth spurt occurs during
lating levels of GH, IGF-I, and the gona-
a period of development
da1 steroid hormones testosterone (T) and 17p-estracliol (Ez ) all increase during
dramatic
neuroendocrine
somatotropic
associated
with
changes in the
and gonadal
axes. Circu-
Daniel L. Metzger is at the Department of Pediatrics, University of British Columbia, Vancouver, BC V6H 3V4, Canada; James R. Kerrigan is at the Department of Pediatrics, East Tennessee State University, Johnson City, TN 37614, USA; and Alan D. Rogol is at the Departments of Pediatrics and Pharmacology, University of Virginia, Charlottesville, VA 22908, USA.
290
this transition. These changes are reflected in the acceleration of linear growth velocity, from a mean prepubertal rate of 5.5 cm/year to a maximum of 9.5 cm/year in boys and 8.5 cm/year in girls. Concomitant with this rapid-growth phase is the development of the characteristic physical signs of puberty, which are induced by the gonadal hormones. Evidence is accumulating that suggests that the pubertal growth spurt is orchestrated
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Puberty-Associated Alterations in Somatotropic and GonadaI Steroid Hormones
With the development of RIAs for GH in the 1960s it became apparent that the hormone is normally secreted in an episodic fashion. This knowledge prompted experiments involving repetitive blood sampling, which suggested that mean GH concentrations were higher in adolescents than in prepubertal children or adults. Recently, this finding has been confirmed and elaborated upon in several larger cross-sectional studies (Martha et al. 1989, Rose et al. 1991), which clearly demonstrated that mean GH levels are maximal during midpuberty to late puberty (that is, about Tanner stage III-IV) in both boys and girls. Figure 1 depicts the close relationship between 24-h mean serum GH concentrations and the standard growth velocity curve for North American boys. In these two studies, computer-assisted pulse-detection algorithms were applied to time series of GH levels; the results demonstrated that the late-pubertal rise in mean GH concentrations is a result of an increase primarily in the amplitude, rather than in the frequency, of GH pulses. Deconvolution modeling represents a significant advance in the analysis of endogenous GH kinetics and the mechanism of developmental alterations or induced perturbations of the GH axis. This method enables the simultaneous calculation of subject-specific hormone secretion and elimination functions. To date, deconvolution analysis has been employed in two large cross-sectional studies of children across puberty (AlbertssonW&land et al. 1989, Martha et al. 1992). Both studies report that GH production rates are highest during late puberty and fall toward prepubertal values by adult-
TEM Vol. 5,No.7,1994