Knocking out the Stress Response

NEWS & VIEWS Knocking out the stress response

Transgenic animals and knockout mice have been generated with deﬁned defects in various components of the hypothalamic-pituitary-adrenal axis and the autonomic nervous system. These models provide valuable and novel insights into the development, crosstalk, organization, and functioning of the stress system.

Life is a dynamic homeostatic process that is con- Deletion and/or overexpression of the genes of stantly challenged by disturbing forces or stressors. corticotropin releasing hormone (CRH), the central Hence, a state of threatened homeostasis or stress is an regulator of the HPA axis and its receptors and binding essential feature of life. The complex adaptive response protein, of the pituitary hormones and their receptors, of living organisms is the product of profound selective of the steroid biosynthetic enzymes and regulatory pro- pressures throughout their evolutionary history; this is teins and of the glucocorticoid and mineralocorticoid true for their ontogeny, prime and decline. Before we receptors, have allowed an in vivo dissection of the are born and, definitely, when we take our first breath, HPA axis at both central and peripheral levels (Figure we experience enormous stress. During our life-span, 1). Likewise, knockout and overexpression of the genes stress in excess and out of control becomes harmful, of catecholamine biosynthetic enzymes, membrane causing and aggravating metabolic, cardiovascular, and transporters and receptors have helped define their immune system disorders.1 Therefore, stress contrib- individual in vivo roles, also at the central and utes to the leading causes of morbidity and mortality peripheral levels. in modern societies. The hypothalamic-pituitary-adrenal (HPA) axis and What can be learned from knocking out or the autonomic nervous system are key components of overexpressing genes related to the stress system? the mammalian stress system. They regulate behavioral, neuroendocrine, metabolic, visceral and The first step is to define the key components and immune functions, which involve the maintenance of revisit the classic concepts of homeostasis and stress. homeostasis and the adaptation of the organism to Before the era of animal transgenesis, the stressful conditions. The appropriate functioning of positive/negative feedback regulations of the HPA axis these systems depends on intact intricate and finely and the autonomic nervous system were established tuned regulatory mechanisms, which act on several through physiologic in vivo experiments involving levels within the central nervous system and the often crude surgical interventions, including removal periphery.1 of the hormone-producing glands, destroying brain In recent years, fundamental principles and key nuclei or sectioning nerves. Later, isolated, perfused cellular and molecular components of the stress system organs, cell cultures and cell lines of the hypothala- and its two peripheral axes have been discovered, as a mus, the pituitary and the adrenal cortex and medulla result of intensive basic science studies. To understand were used to study and elucidate the mechanisms of the integrated functions of this system in vivo, how- hormone synthesis and receptor action. The creation of ever, this knowledge must be combined with physio- knockout models has allowed a return to the assess- logic and clinical insights. Transgenic animals have ment of in vivo physiology, which has the potential to been and are being used to analyze the regulation and reveal the integrative roles, pleiotropy and redundancy in vivo function of genes.2 Thus, recent advances that of each component of the neuroendocrine and neural resulted from the generation of transgenic and knock- limbs of the stress system. out mice have provided the opportunity to elucidate Knocking out the genes for CRH and ACTH led to the physiologic importance of each of the components adrenal atrophy and low glucocorticoid levels in mice of the HPA axis and autonomic nervous system. and to an impaired stress response, which confirmed their known physiologic roles as the main regulators of the HPA axis.3,4 In turn, overproduction of CRH led Correspondence: SR Bornstein, MD, National Institute of Child to excess glucocorticoid production, with growth retar- Health and Human Development, Developmental Endocrinology Branch, National Institutes of Health, Building 10, Room 10N262, dation, excess fat accumulation, muscle atrophy and 9000 Rockville Pike, Bethesda, Maryland 20892, USA. E-mail: thin skin, similar to the manifestations of Cushing syn- bornstesȰmail.nih.gov drome in humans.5 As expected, knocking out the gene News & Views 404

Figure 1 The stress system. The hypothalmic pituitary-adrenal axis interacts with the autonomic nervous system and the immune system in both directions. Corticotropin-producing pituitary cells are immuno-stained with antibodies to ACTH. The electromicrograph, as highlighted with the magniﬁer lens, demonstrates a cortisol-producing cortical cell in direct contact with a catecholamine-producing chromafﬁn cell. News & Views 405 Table 1 Animal models for altered gene expression in the stress system

Stress Altered Gene Expression Phenotype system Overexpression Deﬁciency

HPA CRH CRH5,19–21 CRH3,25,26 Overexpression is accompanied by phenotypic axis CRH-BP22,23 CRH-R9,10,15 alterations, such as: ACTH POMC4 • Cushing like appearance, Steroidogenic SF-124 SF-127 • increased anxiety and activity, enzymes, regulatory StAR28,29 • impaired metabolic and proteins and 21OHase11,30 • immunological functions. receptors 11␤-HSD-131 Deﬁcient function of components of the HPA axis results in: GR-II32,39 • high rate of perinatal death, whereas rescued animals GR6,40 are viable and apparently healthy, • impaired HPA axis function accompanied by alterations in the diurnal rhythm, altered adrenal architecture and an impaired stress response, • impaired function of the sympathoadrenal system, • reduced anxiety • alterations with respect to immune function, metabolism

SNS Catecholaminergic TH41 TH12,45 Overexpression appears to be well compensated with enzymes DBH42 DBH8,46–50 normal catecholamine levels and phenotype, viability, PNMT43,44 PNMT7 behavior and metabolism, whereas depletion of catecholamines results in perinatal death and severe developmental, physiological, metabolic, immunity and behavioral alterations if rescued.

IS Cytokines LIF13 LIF52,53 Altered gene expression results in profound effects on Il-614 the HPA axis function at the central and peripheral Il-1 R51 level.

Abbreviations: CRH: Corticotropin Releasing Hormone; CRH-BP: Corticotropin Releasing Hormone Binding Protein; CRH-R: Corticotropin releasing factor receptor; POMC: Pre-Proopiomelanocortin; StAR: Steroidogenic Acute Regulatory Protein; SF- 1; Steroidogenic Factor 1; 21OHase: 21-Hydroxylase; 11␤-HSD-1: 11␤-Hydroxysteroid dehydrogenase type 1: GR-II: Type II glucocorticoid receptor; GR: Glucocorticoid receptor knockout; TH: Tyrosine hydroxylase; DBH: Dopamine-␤-hydroxylase; PNMT: Phenylethanolamine-N-methyl transferase; LIF: Leukemia Inhibitory Factor; Il-6: Interleukin 6, IL-1 R: Interleukin 1 receptor. for the glucocorticoid receptor eliminated glucocort- natal development, as well as for movement and feed- icoid action and was not compatible with postnatal life ing behaviors. Indeed, depletion of catecholamines in due to insufficient maturation of the fetal lung.6 On the dopamine beta-hydroxylase deficient mice revealed other hand, deletion or overexpression of the enzyme unanticipated retarded growth, an impaired thermo- responsible for the production of epinephrine resulted regulatory response, altered locomotive activity and in no major changes in body weight, plasma glucose, impaired maternal behavior.8 In contrast, selective blood pressure or behavior, which confirmed earlier ablation of pituitary ACTH production led to adreno- conclusions that deficiency of adrenomedullary epi- cortical insufficiency in older and adult but not in very nephrine could be compensated for by systemic young animals in whom adrenal function remained norepinephrine (Table 1).7 basically unchanged (Table 1).4

Besides confirming what we have known before, We now also have the ability to define who is what are the new insights that we have gained talking to whom from these models? There are new insights into the interaction between the We now have the ability to examine effects on matu- two stress axes and other related systems, such as the ration and aging as well as the long-term sequelae of noradrenergic arousal, dopaminergic meso-corticolim- changes in the stress system. There is an astonishing bic and immune systems.1 Definitely, knocking out temporal plasticity in the stress system which is seen components of the HPA axis affects the autonomic ner- more in early life and less in adulthood. Thus, while vous system. Indeed, CRH-receptor knockout mice adult life is possible with catecholamine deficiency, exhibit both adrenocortical dysfunction9 and an atro- catecholamines seem to be essential for fetal and post- phied adrenal medulla,10 while glucocorticoid receptor News & Views 406 knockout mice are incapable of epinephrine biosyn- upon whether compensatory redundant mechanisms thesis;6 the latter because the activity of the enzyme are activated during these times. Also, different effects catecholamine methyl transferase is dependent upon are expected when the deficit introduced to the animal the presence of glucocorticoids. Mice deficient in the is generalized or tissue-specific or when it is expressed adrenocortical 21-hydroxylase enzyme have low early or late in life. New advances in creating trans- plasma corticosterone levels but high levels of steroid genic models and knockout mice have started to help precursors; in addition to their glucocorticoid overcome these limitations; now targeting of transgene deficiency, they have a marked suppression of epi- expression to specific tissues or time-restricted inacti- nephrine production.11 It will now be of interest to find vation or expression of genes, allow differentiation out how the previously unsuspected epinephrine between early vs late or tissue-limited vs generalized deficiency in this common genetic disorder influences acute or chronic pathologic states.2 the clinical picture of the patients. The stress system is crucial for the survival of the In a reciprocal fashion, adrenocortical function is alt- self and species and has evolved over time to a highly ered in transgenic animals overexpressing phenyl- plastic, flexible and interactive biological system that is ethanolamin-N-methyl transferase (PNMT) with affected by and influences most functions of an intact increased production of epinephrine or in the tyrosine organism. Deletion and/or overexpression of genes hydroxylase (TH) knockout mice in parallel with from within this system demonstrate relative redun- reduced catecholamine production (Bornstein et al, dancy, but also pervasive permissiveness that influ- unpublished observation).12 These results demonstrate ences a large number of vital functions. Thus, these a mutual interdependence of the sympathetic system models provide valuable insights into the develop- and the HPA axis at the level of the adrenal gland. ment, cross-talk, organization and functioning of the Therefore, it will be important to study the function of stress system. the adrenal medulla in disorders of the adrenal cortex Recent research has elucidated mechanisms of how and vice versa. early life events and variations in maternal physiology Furthermore, animal transgenesis provides evidence or behavior influence the expression of genes in the for the close interaction between the HPA physiology and behavior of the progeny over the entire axis/sympathetic system and the immune system. life-span.18 The knock-out/transgenic models that have Transgenic expression of cytokines, such as interleu- specific defects in the stress system will allow us to kin-6 and leukemia inhibiting factor (LIF), lead to identify the relative importance of each component in modulation of HPA and autonomic system function at this early programming of the organism’s physical and various levels. Pituitary-specific overexpression of LIF behavioral traits. Future studies based on these find- causes Cushing’s syndrome, while IL-6 overexpressing ings will hopefully allow the investigation of the mech- transgenic mice have hyperplasia of both the adrenal anisms of stress system plasticity further. We will cortex and medulla and increased plasma cortico- hopefully find out if we can influence, train or block sterone levels.13,14 Interestingly, in the latter model, stress plasticity as a preventive treatment option. Also, ACTH levels were decreased suggesting a direct some of these mutant animals will be ideal for preclinical immune-adrenal regulation mediated by IL-6. In con- protocols of gene therapy strategies and the testing of trast, in normal mice the pituitary-adrenal response to novel small molecule receptor agonists and antagonists. local inflammation is mediated largely by hypotha- 1 2 1 15 SR Bornstein ,ABo¨ttner and GP Chrousos lamic CRH and vasopressin. Mice deficient in CRF- 1Developmental Endocrinology Branch R1 are also able to stimulate their pituitary and adrenal National Institute of Child Health glands, apparently bypassing CRH/AVP action; this and Human Development suggests direct stimulation of pituitary ACTH and adre- 15 Bethesda, MD 20892, USA nal cortisol secretion by inflammatory cytokines. 2University of Leipzig, Department of Internal Medicine These findings are essential for the understanding of Leipzig 04103, Germany the complex interaction of our stress system and inflammation in critical illness, and may explain the References maintenance of high glucocorticoid levels while pituitary ACTH is low in patients with this condition.16 1 Chrousos GP. Stressors, Stress, and Neuroendocrine Integration of the Adaptive Response: The 1997 Hans Selye Memorial Lecture. 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