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Glucocorticoid receptors and Cushing's disease
Molecular and Cellular Endocrinology 197 (2002) 69 /72 www.elsevier.com/locate/mce
Review
Glucocorticoid receptors and Cushing’s disease S.W.J. Lamberts 1 Department of Medicine, Erasmus University Rotterdam, Rotterdam, The Netherlands
Abstract Corticotrophinomas are characterized by a relative resistance to the negative feedback action of cortisol on ACTH secretion. In this respect there is a similarity with the clinical syndrome of cortisol resistance (CR). As CR can be caused by genetic abnormalities in the glucocorticoid receptor (GR) gene, we investigated whether the insensitivity of corticotrophinomas to cortisol is also caused by the Novo Mutations in the GR gene. We found that somatic mutations of the GR gene are not a frequent cause of relative CR in these cells, but our observations indicate that loss of heterozygosity (LOH) at the GR gene locus is a relativity frequent phenomenon in pituitary adenomas of patients with Cushing’s disease. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Glucocorticoid receptor; Cortisol; Cushing’s disease; Cortisol resistance
1. Introduction Glucocorticoid (GC) hormones serve a variety of important functions throughout the body. In man, the major circulating GC is cortisol. Under basal conditions, serum cortisol concentrations are under the influence of the hypothalamo /pituitary /adrenal (HPA)-axis to keep a perfect balance between cortisol requirement and cortisol secretion. From clinical observations in patients who are treated with GC therapy for non-endocrine diseases, it is known that there is a considerable variation in the sensitivity to GC in the normal population.
2. Glucocorticoid resistance In extreme cases, this variation may lead to clinical syndromes like cortisol resistance (CR) or cortisol hypersensitivity. In cases of generalized CR, patients present with increased cortisol concentrations compared with the normal population (Chrousos, 1993; Lamberts et al., 1986, 1992). The negative GC feedback on both corticotrophin-releasing hormone (CHR) and adrenoE-mail address: [email protected] (S.W.J. Lamberts). 1 Present address: Department of Medicine, University Hospital Rotterdam, 40 Molenwaterplein, 3015 GD Rotterdam, The Netherlands.
corticotrophin (ACTH) secretion is reduced as a consequence of diminished sensitivity to GC. As a result, the HPA axis is set at a higher level. CRH and ACTH secretion increase, resulting in higher serum cortisol concentrations. In this way, the body tries to achieve a balance between cortisol requirement and cortisol secretion. The increased cortisol concentrations appear to compensate adequately for the reduced sensitivity. However, patients suffering form CR do not show signs or symptoms of cortisol excess, because they simply need these increased serum cortisol concentrations. The sensitivity to GCs is diminished at the hypothalamic and the pituitary level, but also at the peripheral target tissue level. The clinic symptoms seen in-patients with CR are, therefore, not due to GC excess, but secondary to the activation of HPA axis, which results in an increased production of ACTH, resulting in the stimulation of mineralocorticoid and androgen secretion (Lamberts et al., 1992). So in case of CR, the increased ACTH secretion leads to a secondary adrenal overproduction of hormones with mineralocorticoid activity, such as deoxycorticosterone, and with androgen activity such as androstenedione, dehydroepiandrosterone and dehydroepiandro-sterone sulfate. In eight cases of CR described so far, mutations in the hormone-binding domain of the glucocorticoid receptor (GR) gene appeared to be the cause of the disease (Chrousos et al., 1983; Hurley et al., 1991; Karl et al., 1993; Malchoff et al., 1990, 1993; Mendonca et al., 1999; Ruiz et al.,
0303-7207/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 3 - 7 2 0 7 ( 0 2 ) 0 0 2 8 0 - 0
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S.W.J. Lamberts / Molecular and Cellular Endocrinology 197 (2002) 69 /72
2001; Vottero et al., 1999). In an additional five patients with clinical biochemical evidence of CR we did not observe abnormalities in the GR gene (Huizenga et al., 2000). As yet undefined alterations somewhere in the cascade of events starting with ligand binding in the GR protein, and finally resulting in the regulation of the expression of GC responsive genes, or post-receptor defects or interactions with other nuclear factors form the pathophysiologic basis of CR in these patients. Recently two sisters have been described with a combination of GC, mineralocorticoid and androgen resistance (New et al., 1999). It was proposed that these patients represent the first cases of partial resistance to multiple steroids, possibly to a coactivator defect.
3. Glucocorticoid sensitivity in the normal population We studied several aspects of the variability in GC sensitivity in the normal population. In a cohort study in 216 healthy elderly individuals two overnight suppression tests with 1.0 and 0.25 mg of dexamethasone, respectively, were performed at an interval of 2 years and 6 months (Huizenga et al., 1998a). It appeared that serum cortisol concentrations showed a wide range between subjects, and a strong individual stability. This indicates that the HPA-axis is set at a stable and reproducible set point for a given individual (Huizenga et al., 1998a). Subsequently we screened the GR gene for mutations (Koper et al., 1997). This screening revealed the existence of five polymorphisms in the GR gene. It became clear, that a point mutation at cDNA position 1220 resulting in A to G change in codon 363 might be associated with increased sensitivity to GC. The mutation was found in 6% of the normal population (Huizenga et al., 1998b). Subjects carrying this N363S polymorphism were more sensitive to GC, both with respect to short-term effects of exogenously administered GCs (increased cortisol suppression and increased insulin response), and with respect to long-term effects of endogenous CGs (higher body mass index, tendency to a lower bone mineral density). In a recent study in over 300 younger individuals in Australia a high penetrance of obesity was confirmed in a case-control study: heterozygote Ser 363 variants demonstrated increased BMI, but seven homozygote variants were grossly obese (Lin et al., 1999).
4. The pathogenesis of Cushing’s disease Cushing’s disease, pituitary-dependent Cushing’s syndrome, is the hypercortisolemic state secondary to excess or dysregulated ACTH secretion caused by an corticotrophin-secreting adenoma. ACTH production
by the adenoma occurs in a semiautonomous manner, in which one of the principal biochemical features is a resetting of the HPA feedback such that ACTH is secreted in the presence of abnormally high levels of circulating cortisol (Dahia and Grossman, 1999). There has long been debate as to the primary origin of pituitary tumors in general, specifically, as to whether they arise primarily from defects of the hypothalamus or the pituitary. It has been argued that many of the associated endocrine changes seen in Cushing’s disease, particularly abnormalities in the growth, pituitary/ gonadal, and thyroid axes, suggest primary hypothalamic dysfunction. Equally, the occasional lack of an identifiable adenoma at surgery, and instances of tumor recurrence after apparent complete removal of an identified tumor, might also suggest some form of preexisting hypothalamic ‘overdrive’. However, favoring a fundamentally pituitary origin of Cushing’s disease are several lines of evidence: the generally high cure rate after removal of a distinct adenoma, the absence of identifiable corticotroph hyperplasia surrounding the tumor, and, most importantly, the characterization of the monoclonal status of the majority of adenomas assessed (Dahia and Grossman, 1999). It has also become apparent in recent years that most, if not all, of the associated neuroendocrine changes are a manifestation of cortisol excess, and normalize when cortisol levels are medically or surgically brought within the normal range. The great majority of patients with Cushing’s disease are thus thought to harbor distinct ACTH-secreting pituitary adenomas (Dahia and Grossman, 1999).
5. Glucocorticoid resistance and Cushing’s disease Several years ago we reported a unique patient with sporadic generalized CR who, at age 33, presented with infertility and hypertension and, at 38, developed pituitary Cushing’s disease (Karl et al., 1996). Leukocyte-binding studies revealed normal affinity of the GR but a reduction of biding sites by 50%. [3H]thymidine incorporation by this patient’s lymphocytes was not suppressible by dexamethasone. He had a novel heterozygous missense mutation in the GR gene (isoleucine 559 to asparagine 559). The mutant receptor exhibited a strong dominant-negative effect on the ability of the wild-type receptor to induce gene transcription in vitro. The mutation was present in all of the patient’s cultured lymphoblasts and fibroblasts as well as in 50% of his sperm, as demonstrated by single-cell polymerase chain reaction; it was not present in his parents and seven siblings. This novel mutation was thus both de novo and present in the germ line. Immunohistochemical staining of this patient’s pituitary corticotrophinoma revealed accumulation of p53 protein, indicating the presence of
S.W.J. Lamberts / Molecular and Cellular Endocrinology 197 (2002) 69 /72
a putative somatic oncogenic mutation in the p53 gene in the tumor cells. Investigation of the lymphoblast and skin fibroblast cultures for p53 abnormalities did not show any aberration. Thus, a novel de novo germ line mutation of the GR with strong dominant-negative activity caused severe sporadic generalized GC resistance, which preceded corticotroph adenoma formation (Karl et al., 1996). The latter probably was due to the combined effects of chronic corticotroph hyperstimulation, decreased GC negative feedback, and at least one subsequent somatic defect in the control of the cell cycle. Subsequently we screened for the GR gene in leukocyte and tumor DNA from 22 patients with Cushing’s disease for mutations using PCR/single strand conformation polymorphism analysis. In our previous study, we identified five polymorphisms in the GR gene in a normal population. These polymorphisms were used as markers for the possible occurrence of loss of heterozygosity (LOH) at the GR gene locus (Huizenga et al., 1998c). Except for one silent point mutation, we did not identify novel mutations in the GR gene in leukocytes or corticotrophinomas from these patients. Of the 22 patients, 18 were heterozygous for at least one of the polymorphisms. In six of these patients, LOH had occurred in the tumor DNA. Of 21 patients examined for LOH on chromosome 11q13, only one, with a corticotroph carcinoma, showed allelic deletion. As controls we studied 28 pituitary tumors of other subtypes (11 clinically non-functioning, eight prolactinomas, and nine GH-producing adenomas) and found evidence for LOH in only one prolactinoma (Huizenga et al., 1998c). Hypothesizing on the basis of the present data, the following model can be put forward. In a differentiated corticotrophic pituitary cell, a spontaneous genetic alteration occurs at the GR locus. Consequently, this cell is relatively insensitive to cortisol. As the mutated corticotrophic cell is less sensitive to cortisol this could be seen as a relative shortage of inhibitory hormones, and it provides an advantage for clonal selection, as previously proposed by Karl et al. (1996). A striking example of the enormous clonal expansion due to discontinuation of the physiological inhibitory feedback by cortisol is the Nelson tumor, which is an aggressive, growing, ACTH-secreting pituitary adenoma. Nelson tumors appear in 10/30% of patients who have previously undergone bilateral adrenalectomy for Cushing’s disease. Karl et al. (1996),found a somatic frameshift mutation in the GR gene in one of four Nelson tumors investigated. It was concluded that the GR defect might have played a pathophysiological role in the tumorigenesis of this corticotrophinoma. Another example in favor of this is that clonal expansion of a single genetically altered corticothropic cell occurs as a result of diminished negative feedback by cortisol is our
71
patient with CR due to a dominant negative GR gene mutation (Karl et al., 1996). During the treatment for CR with low doses of dexamethasone, the patient developed signs and symptoms of Cushing’s disease. Eventually this patient underwent bilateral adrenalectomy, and subsequently, when a pituitary magnetic resonance scan became positive for a tumor, a corticotrophinoma was removed. It was suggested that in this patient the exposure of the corticotroph to enhanced stimulation by increased production of hypothalamic CRH and arginine vasopressin or a decreased sensitivity of the corticotroph to the negative feedback by cortisol may have led to adenoma formation.
6. Conclusions From the data presented here it can be concluded that simple somatic point mutations in the GR gene in corticotrophinomas do not seem to be the cause of the relative CR in the tumor cells. However, we demonstrate that there is a frequent LOH at the GR gene locus in ACTH-secreting pituitary adenomas. As deletion of (part of) the GR gene leads to GC resistance, this is a possible explanation for their relative resistance to the inhibitory feedback of cortisol in ACTH-secretion. Furthermore, LOH at the GR gene locus may play a pathophysiological role in the initiation of corticotrophinoma formation, although our data are insufficient to relate this to the size of the tumor.
References Chrousos, G.P., 1993. Syndromes of glucocorticoid resistance. Ann. Int. Med. 11, 1113 /1124. Chrousos, G.P., Vingerhoeds, A.C., Loriaux, D.L., Lipsett, M.B., 1983. Primary cortisol resistance: a family study. J. Clin. Endocrinol. Metab. 56, 1243 /1245. Dahia, P.L.M., Grossman, A.B., 1999. The Molecular Pathogenesis of Corticotroph Tumors, vol. 20. The Endocrine Society, pp. 136 / 155. Huizenga, N.A.T.M., Koper, J.W., de Lange, P., Pols, H.A.P., Stolk, R.P., Grobbee, D.E., de Jong, F.H., Lamberts, S.W.J., 1998. Interperson variability but intraperson stability of baseline plasma cortisol concentrations, and its relation to feedback sensitivity of the hypothalamo-pituitary-adrenal axis to a low-dose of dexamethasone in elderly individuals. J. Clin. Endocrinol. Metab. 83, 47 /54. Huizenga, N.A.T.M., Koper, J.W., de Lange, P., Pols, H.A.P., Stolk, R.P., Burger, H., Grobbee, D., Brinkmann, A.O., de Jong, F.H., Lamberts, S.W.J., 1998. A polymorphism in the glucocorticoid receptor may be associated with an increased sensitivity in vivo. J. Clin. Endocrinol. Metab. 83, 144 /151. Huizenga, N.A.T.M., de Lange, P., Koper, J.W., Clayton, R.N., Farrell, W.E., van der Lely, A.J., Brinkmann, A.O., de Jong, F.H., Lamberts, S.W.J., 1998. Human ACTH secreting pituitary adenomas show frequent loss of heterozygosity at the glucocorticoid receptor gene locus. J. Clin. Endocrinol. Metab. 83, 917 /921.
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Huizenga, N.A.T.M., de Lange, P., Koper, J.W., de Herder, W.W., Abs, R., Kasteren, J.H., de Jong, F.H., Lamberts, S.W.J., 2000. Five patients with biochemical and/or clinical generalized glucocorticoid resistance without alterations in the glucocorticoid receptor gene. J. Clin. Endocrinol. Metab. 85, 2076 /2081. Hurley, D.M., Accili, D., Stratakis, C.A., 1991. Point mutation causing a single amino acid substitution in the hormone binding domain of the glucocorticoid receptor in familial glucocorticoid resistance. J. Clin. Invest. 87, 680 /686. Karl, M., Lamberts, S.W.J., Detera-Wadleigh, S.D., 1993. Familial glucocorticoid resistance caused by a splice site deletion in the human glucocorticoid receptor gene. J. Clin. Endocrinol. Metab. 76, 683 /689. Karl, M., Lamberts, S.W.J., Koper, J.W., Katz, D.A., Huizenga, N.E., Kino, T., Haddad, B.R., Hughes, M.R., Chrousos, G.P., 1996. Cushing’s disease preceded by generalized glucocorticoid resistance: clinical consequences of a novel, dominant-neagtive glucocorticoid receptor mutation. Proc. Assoc. Am. Phys. 108, 296 /307. Koper, J.W., Stolk, R.P., de Lange, P., Huizenga, N.A.T.M., Molijn, G.J., Pols, H.A.P., Grobbee, D.E., Karl, M., de Jong, F.H., Lamberts, S.W.J., 1997. Lack of association between five polymorphisms in the human glucocorticoid receptor gene and glucocorticoid resistance. Hum. Genet. 99, 663 /668. Lamberts, S.W.J., Poldermans, D., Zweens, M., de Jong, F.H., 1986. Familial cortisol resistance: differential diagnostic and therapeutic aspects. J. Clin. Endocrinol. Metab. 63, 1328 /1333. Lamberts, S.W.J., Koper, J.W., Biemond, P., den Holder, F.H., de Jong, F.H., 1992. Cortisol receptor resistance: the variability of its clinical presentation and response to treatment. J. Clin. Endocrinol. Metab. 74, 313 /321.
Lin, R.C.Y., Wang, W.Y.S., Morris, B.J., 1999. High penetrance, overweight and glucocorticoid receptor vriant: case /control study. Br. Med. J. 319, 1337 /1338. Malchoff, C.D., Javier, E.C., Malchoff, D.M., Martin, T., Rogol, A., Brandon, D., Loriaux, D.L., Reardon, G.E., 1990. Primary cortisol resistance presenting as isosexual precocity. J. Clin. Endocrinol. Metab. 70, 503 /507. Malchoff, D.M., Brufsky, A., Reardon, G., McDermott, P., Javier, E.C., Bergh, C.H., Rowe, D., Malchoff, C.D., 1993. A mutation of the glucocorticoid receptor in primary cortisol resistance. J. Clin. Invest. 91, 1918 /1925. Mendonca, B.B., Leite, M.V., Bachega, T.A., Karl, M., Chrousos, G.D., 1999. Familial Glucocorticoid Resistance due to a Novel Mutation in the Glucocorticoid Receptor Gene Associated with a Heterozygous Large Conversion of 21-Hydroxylase Gene Causing Female Pseudohermaphroditism (abstract). The Endocrine Society, San Diego, p. 273. New, M.I., Nimkarn, S., Brandon, D.D., Cunningham-Rundles, S., Wilson, R.C., Newfield, R.S., Vandermeulen, J., Barron, N., Russo, C., Loriaux, D.L., O’Malley, B., 1999. Resistance to several steroids in two sisters. J. Clin. Endocrinol. Metab. 84, 4454 /4464. Ruiz, M., Lind, U., Gafvels, M., Eggertsen, G., Carlstedt-Duke, J., Nilsson, L., Holtmann, M., Stierna, P., Wikstrom, A.C., Werner, S., 2001. Characterization of two novel mutations in the glucorticoid receptor gene in patients with primary cortisol resistance. Clin. Endocrinol. 55, 363 /371. Vottero, A., Combe, H., LeComte, P., Longui, C.A., Chrousos, G.P., 1999. A Novel Mutation of the Glucocorticoid Resistance9.90 (abstr). The Endocrine Society, San Diego.
Review
Glucocorticoid receptors and Cushing’s disease S.W.J. Lamberts 1 Department of Medicine, Erasmus University Rotterdam, Rotterdam, The Netherlands
Abstract Corticotrophinomas are characterized by a relative resistance to the negative feedback action of cortisol on ACTH secretion. In this respect there is a similarity with the clinical syndrome of cortisol resistance (CR). As CR can be caused by genetic abnormalities in the glucocorticoid receptor (GR) gene, we investigated whether the insensitivity of corticotrophinomas to cortisol is also caused by the Novo Mutations in the GR gene. We found that somatic mutations of the GR gene are not a frequent cause of relative CR in these cells, but our observations indicate that loss of heterozygosity (LOH) at the GR gene locus is a relativity frequent phenomenon in pituitary adenomas of patients with Cushing’s disease. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Glucocorticoid receptor; Cortisol; Cushing’s disease; Cortisol resistance
1. Introduction Glucocorticoid (GC) hormones serve a variety of important functions throughout the body. In man, the major circulating GC is cortisol. Under basal conditions, serum cortisol concentrations are under the influence of the hypothalamo /pituitary /adrenal (HPA)-axis to keep a perfect balance between cortisol requirement and cortisol secretion. From clinical observations in patients who are treated with GC therapy for non-endocrine diseases, it is known that there is a considerable variation in the sensitivity to GC in the normal population.
2. Glucocorticoid resistance In extreme cases, this variation may lead to clinical syndromes like cortisol resistance (CR) or cortisol hypersensitivity. In cases of generalized CR, patients present with increased cortisol concentrations compared with the normal population (Chrousos, 1993; Lamberts et al., 1986, 1992). The negative GC feedback on both corticotrophin-releasing hormone (CHR) and adrenoE-mail address: [email protected] (S.W.J. Lamberts). 1 Present address: Department of Medicine, University Hospital Rotterdam, 40 Molenwaterplein, 3015 GD Rotterdam, The Netherlands.
corticotrophin (ACTH) secretion is reduced as a consequence of diminished sensitivity to GC. As a result, the HPA axis is set at a higher level. CRH and ACTH secretion increase, resulting in higher serum cortisol concentrations. In this way, the body tries to achieve a balance between cortisol requirement and cortisol secretion. The increased cortisol concentrations appear to compensate adequately for the reduced sensitivity. However, patients suffering form CR do not show signs or symptoms of cortisol excess, because they simply need these increased serum cortisol concentrations. The sensitivity to GCs is diminished at the hypothalamic and the pituitary level, but also at the peripheral target tissue level. The clinic symptoms seen in-patients with CR are, therefore, not due to GC excess, but secondary to the activation of HPA axis, which results in an increased production of ACTH, resulting in the stimulation of mineralocorticoid and androgen secretion (Lamberts et al., 1992). So in case of CR, the increased ACTH secretion leads to a secondary adrenal overproduction of hormones with mineralocorticoid activity, such as deoxycorticosterone, and with androgen activity such as androstenedione, dehydroepiandrosterone and dehydroepiandro-sterone sulfate. In eight cases of CR described so far, mutations in the hormone-binding domain of the glucocorticoid receptor (GR) gene appeared to be the cause of the disease (Chrousos et al., 1983; Hurley et al., 1991; Karl et al., 1993; Malchoff et al., 1990, 1993; Mendonca et al., 1999; Ruiz et al.,
0303-7207/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 3 - 7 2 0 7 ( 0 2 ) 0 0 2 8 0 - 0
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S.W.J. Lamberts / Molecular and Cellular Endocrinology 197 (2002) 69 /72
2001; Vottero et al., 1999). In an additional five patients with clinical biochemical evidence of CR we did not observe abnormalities in the GR gene (Huizenga et al., 2000). As yet undefined alterations somewhere in the cascade of events starting with ligand binding in the GR protein, and finally resulting in the regulation of the expression of GC responsive genes, or post-receptor defects or interactions with other nuclear factors form the pathophysiologic basis of CR in these patients. Recently two sisters have been described with a combination of GC, mineralocorticoid and androgen resistance (New et al., 1999). It was proposed that these patients represent the first cases of partial resistance to multiple steroids, possibly to a coactivator defect.
3. Glucocorticoid sensitivity in the normal population We studied several aspects of the variability in GC sensitivity in the normal population. In a cohort study in 216 healthy elderly individuals two overnight suppression tests with 1.0 and 0.25 mg of dexamethasone, respectively, were performed at an interval of 2 years and 6 months (Huizenga et al., 1998a). It appeared that serum cortisol concentrations showed a wide range between subjects, and a strong individual stability. This indicates that the HPA-axis is set at a stable and reproducible set point for a given individual (Huizenga et al., 1998a). Subsequently we screened the GR gene for mutations (Koper et al., 1997). This screening revealed the existence of five polymorphisms in the GR gene. It became clear, that a point mutation at cDNA position 1220 resulting in A to G change in codon 363 might be associated with increased sensitivity to GC. The mutation was found in 6% of the normal population (Huizenga et al., 1998b). Subjects carrying this N363S polymorphism were more sensitive to GC, both with respect to short-term effects of exogenously administered GCs (increased cortisol suppression and increased insulin response), and with respect to long-term effects of endogenous CGs (higher body mass index, tendency to a lower bone mineral density). In a recent study in over 300 younger individuals in Australia a high penetrance of obesity was confirmed in a case-control study: heterozygote Ser 363 variants demonstrated increased BMI, but seven homozygote variants were grossly obese (Lin et al., 1999).
4. The pathogenesis of Cushing’s disease Cushing’s disease, pituitary-dependent Cushing’s syndrome, is the hypercortisolemic state secondary to excess or dysregulated ACTH secretion caused by an corticotrophin-secreting adenoma. ACTH production
by the adenoma occurs in a semiautonomous manner, in which one of the principal biochemical features is a resetting of the HPA feedback such that ACTH is secreted in the presence of abnormally high levels of circulating cortisol (Dahia and Grossman, 1999). There has long been debate as to the primary origin of pituitary tumors in general, specifically, as to whether they arise primarily from defects of the hypothalamus or the pituitary. It has been argued that many of the associated endocrine changes seen in Cushing’s disease, particularly abnormalities in the growth, pituitary/ gonadal, and thyroid axes, suggest primary hypothalamic dysfunction. Equally, the occasional lack of an identifiable adenoma at surgery, and instances of tumor recurrence after apparent complete removal of an identified tumor, might also suggest some form of preexisting hypothalamic ‘overdrive’. However, favoring a fundamentally pituitary origin of Cushing’s disease are several lines of evidence: the generally high cure rate after removal of a distinct adenoma, the absence of identifiable corticotroph hyperplasia surrounding the tumor, and, most importantly, the characterization of the monoclonal status of the majority of adenomas assessed (Dahia and Grossman, 1999). It has also become apparent in recent years that most, if not all, of the associated neuroendocrine changes are a manifestation of cortisol excess, and normalize when cortisol levels are medically or surgically brought within the normal range. The great majority of patients with Cushing’s disease are thus thought to harbor distinct ACTH-secreting pituitary adenomas (Dahia and Grossman, 1999).
5. Glucocorticoid resistance and Cushing’s disease Several years ago we reported a unique patient with sporadic generalized CR who, at age 33, presented with infertility and hypertension and, at 38, developed pituitary Cushing’s disease (Karl et al., 1996). Leukocyte-binding studies revealed normal affinity of the GR but a reduction of biding sites by 50%. [3H]thymidine incorporation by this patient’s lymphocytes was not suppressible by dexamethasone. He had a novel heterozygous missense mutation in the GR gene (isoleucine 559 to asparagine 559). The mutant receptor exhibited a strong dominant-negative effect on the ability of the wild-type receptor to induce gene transcription in vitro. The mutation was present in all of the patient’s cultured lymphoblasts and fibroblasts as well as in 50% of his sperm, as demonstrated by single-cell polymerase chain reaction; it was not present in his parents and seven siblings. This novel mutation was thus both de novo and present in the germ line. Immunohistochemical staining of this patient’s pituitary corticotrophinoma revealed accumulation of p53 protein, indicating the presence of
S.W.J. Lamberts / Molecular and Cellular Endocrinology 197 (2002) 69 /72
a putative somatic oncogenic mutation in the p53 gene in the tumor cells. Investigation of the lymphoblast and skin fibroblast cultures for p53 abnormalities did not show any aberration. Thus, a novel de novo germ line mutation of the GR with strong dominant-negative activity caused severe sporadic generalized GC resistance, which preceded corticotroph adenoma formation (Karl et al., 1996). The latter probably was due to the combined effects of chronic corticotroph hyperstimulation, decreased GC negative feedback, and at least one subsequent somatic defect in the control of the cell cycle. Subsequently we screened for the GR gene in leukocyte and tumor DNA from 22 patients with Cushing’s disease for mutations using PCR/single strand conformation polymorphism analysis. In our previous study, we identified five polymorphisms in the GR gene in a normal population. These polymorphisms were used as markers for the possible occurrence of loss of heterozygosity (LOH) at the GR gene locus (Huizenga et al., 1998c). Except for one silent point mutation, we did not identify novel mutations in the GR gene in leukocytes or corticotrophinomas from these patients. Of the 22 patients, 18 were heterozygous for at least one of the polymorphisms. In six of these patients, LOH had occurred in the tumor DNA. Of 21 patients examined for LOH on chromosome 11q13, only one, with a corticotroph carcinoma, showed allelic deletion. As controls we studied 28 pituitary tumors of other subtypes (11 clinically non-functioning, eight prolactinomas, and nine GH-producing adenomas) and found evidence for LOH in only one prolactinoma (Huizenga et al., 1998c). Hypothesizing on the basis of the present data, the following model can be put forward. In a differentiated corticotrophic pituitary cell, a spontaneous genetic alteration occurs at the GR locus. Consequently, this cell is relatively insensitive to cortisol. As the mutated corticotrophic cell is less sensitive to cortisol this could be seen as a relative shortage of inhibitory hormones, and it provides an advantage for clonal selection, as previously proposed by Karl et al. (1996). A striking example of the enormous clonal expansion due to discontinuation of the physiological inhibitory feedback by cortisol is the Nelson tumor, which is an aggressive, growing, ACTH-secreting pituitary adenoma. Nelson tumors appear in 10/30% of patients who have previously undergone bilateral adrenalectomy for Cushing’s disease. Karl et al. (1996),found a somatic frameshift mutation in the GR gene in one of four Nelson tumors investigated. It was concluded that the GR defect might have played a pathophysiological role in the tumorigenesis of this corticotrophinoma. Another example in favor of this is that clonal expansion of a single genetically altered corticothropic cell occurs as a result of diminished negative feedback by cortisol is our
71
patient with CR due to a dominant negative GR gene mutation (Karl et al., 1996). During the treatment for CR with low doses of dexamethasone, the patient developed signs and symptoms of Cushing’s disease. Eventually this patient underwent bilateral adrenalectomy, and subsequently, when a pituitary magnetic resonance scan became positive for a tumor, a corticotrophinoma was removed. It was suggested that in this patient the exposure of the corticotroph to enhanced stimulation by increased production of hypothalamic CRH and arginine vasopressin or a decreased sensitivity of the corticotroph to the negative feedback by cortisol may have led to adenoma formation.
6. Conclusions From the data presented here it can be concluded that simple somatic point mutations in the GR gene in corticotrophinomas do not seem to be the cause of the relative CR in the tumor cells. However, we demonstrate that there is a frequent LOH at the GR gene locus in ACTH-secreting pituitary adenomas. As deletion of (part of) the GR gene leads to GC resistance, this is a possible explanation for their relative resistance to the inhibitory feedback of cortisol in ACTH-secretion. Furthermore, LOH at the GR gene locus may play a pathophysiological role in the initiation of corticotrophinoma formation, although our data are insufficient to relate this to the size of the tumor.
References Chrousos, G.P., 1993. Syndromes of glucocorticoid resistance. Ann. Int. Med. 11, 1113 /1124. Chrousos, G.P., Vingerhoeds, A.C., Loriaux, D.L., Lipsett, M.B., 1983. Primary cortisol resistance: a family study. J. Clin. Endocrinol. Metab. 56, 1243 /1245. Dahia, P.L.M., Grossman, A.B., 1999. The Molecular Pathogenesis of Corticotroph Tumors, vol. 20. The Endocrine Society, pp. 136 / 155. Huizenga, N.A.T.M., Koper, J.W., de Lange, P., Pols, H.A.P., Stolk, R.P., Grobbee, D.E., de Jong, F.H., Lamberts, S.W.J., 1998. Interperson variability but intraperson stability of baseline plasma cortisol concentrations, and its relation to feedback sensitivity of the hypothalamo-pituitary-adrenal axis to a low-dose of dexamethasone in elderly individuals. J. Clin. Endocrinol. Metab. 83, 47 /54. Huizenga, N.A.T.M., Koper, J.W., de Lange, P., Pols, H.A.P., Stolk, R.P., Burger, H., Grobbee, D., Brinkmann, A.O., de Jong, F.H., Lamberts, S.W.J., 1998. A polymorphism in the glucocorticoid receptor may be associated with an increased sensitivity in vivo. J. Clin. Endocrinol. Metab. 83, 144 /151. Huizenga, N.A.T.M., de Lange, P., Koper, J.W., Clayton, R.N., Farrell, W.E., van der Lely, A.J., Brinkmann, A.O., de Jong, F.H., Lamberts, S.W.J., 1998. Human ACTH secreting pituitary adenomas show frequent loss of heterozygosity at the glucocorticoid receptor gene locus. J. Clin. Endocrinol. Metab. 83, 917 /921.
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