Sexually dimorphic actions of glucocorticoids: beyond chromosomes and sex hormones

Abstract

Sexual dimorphism is a well-documented phenomenon that is observed at all levels of the animal kingdom. Historically, sex hormones (testosterone and estrogen) have been implicated as key players in a wide array of pathologies displaying sexual dimorphism in their etiology and progression. While these hormones clearly contribute to sexually dimorphic diseases, other factors may be involved in this phenomenon as well. In particular, the stress hormone cortisol exerts differential effects in both males and females. The underlying molecular basis for the sexually dimorphic actions of glucocorticoids is unknown but clearly important to understand, since synthetic glucocorticoids are the most widely prescribed medication for the treatment of chronic inflammatory diseases and hematological cancers in humans.

Introduction

It is widely appreciated that there are gender-based differences in many species of the animal kingdom, referred to as sexual dimorphism. Sexual dimorphism is attributed to a difference in genetic landscape between males and females, due to the presence of different sexual reproductive organs. Gonad development is dictated by the sex chromosomes, with women having two X chromosomes, and males having one X and one Y chromosome. The SRY gene is located on the Y chromosome, which leads to testis formation and male sexual differentiation.¹ This genetic variation leads to gender-specific differences in organs, such as the brain, kidney, liver, and adipose tissue,² with downstream physiological consequences on lipid and lipoprotein metabolism,³ antioxidant defense,^4,5 and immune function.⁶ Differential regulation of key physiological processes account for the sexual dimorphism observed in an array of pathologies. For example, women are more prone to osteoporosis,⁷ Cushing’s disease,⁸ and a number of autoimmune disorders,⁹ while men are more affected by diseases such as hepatocellular carcinoma,¹⁰ cardiovascular disease,¹¹ chronic kidney disease,¹² and certain neurological disorders such as Parkinson’s disease.¹³ While the biological basis underpinning the susceptibility of one gender versus another to certain diseases has been studied, our current understanding of this phenomenon is limited. From a mechanistic perspective, this review will discuss our current understanding of the biological basis for sexual dimorphism and highlight new advances made in understanding the role of corticosteroids, which expand the current concepts of sexual dimorphism.

Sex chromosomes

The core difference between the genders lies within the chromosomes we possess. The typical human has 23 pairs of chromosomes in each cell, with the first 22 being autosomes, and the last being an allosome (sex chromosome). The allosome is what make men and women biologically unique from one another. Since women have two X chromosomes and men only have one, an X chromosome must be silenced in order to ensure proper gene dosage, known as X chromosome inactivation.¹⁴ Allosome differences indeed predispose males to a variety of diseases that women are not afflicted by, such as X-linked lymphoproliferative disease,¹⁵ hemophilia A, and hemophilia B.¹⁶ The preponderance of males with X-linked diseases is due to the presence of only one X chromosome, leaving only one copy of a mutated gene, whereas women have two copies and can silence the mutated chromosome and still have a functional copy. While having two X chromosomes appears to protect women against certain X-linked diseases, there are still certain diseases that afflict women due to their X chromosome, typically due to impaired inactivation. An example of this is that women with a tiny ring X chromosome have impaired dosage compensation, leading to mental retardation.¹⁷

Sex hormones

Gonadal differences between men and women lead to variation in sex hormones produced (testosterone in men and estrogen in women), which are responsible for differential gene expression leading to unique cellular environments. Sexually dimorphic diseases are mostly attributed to differences in sex hormones between males and females; these differences are presumed to be the underlying basis by which some diseases show a higher prevalence in one gender over another.

Estrogens are a group of molecules including estradiol (E2), estrone, and estriol, which are the primary sex hormones in females. E2 is the most abundant and potent endogenous estrogen and exerts its biological effects through the estrogen receptor α (ERα) and estrogen receptor β (ERβ). ERα and ERβ are highly expressed in female reproductive tissues, including the ovaries and uterus; however, the receptors for estrogens are also expressed in other tissues of the body, including the liver, bone, vascular endothelium, various regions of the brain, and adipose tissue,¹⁴ indicating that estrogens play a much broader biological role than simply affecting reproduction. Indeed, knockout studies of the ER have revealed that estrogens play a role in regulating lipid storage¹⁸ and bone resorption.¹⁹ In fact, estrogen serves a protective role in preventing cardiovascular disease in premenopausal women compared to age-matched males.^20–22 This protective effect is lost when menopause is reached and estrogen levels drop, predisposing postmenopausal women to cardiovascular disease. A fine balance of estrogen signaling needs to be maintained and, if not maintained, different pathologies are observed. For example, estrogen has been implicated in the progression of certain reproductive cancers, such as ovarian,¹⁵ endometrial,¹⁶ cervical,¹⁷ and breast cancer.²³

With estrogen serving a protective role and dysregulation of its signaling leading to pathologies, it has been a prime target for therapeutic interventions. Hormone replacement therapy,¹⁹ ER antagonists,²⁴ and, more recently, the generation of selective ER modulators (SERMS)—molecules that alter ER signaling in the absence of ligand²⁵—are all used clinically to treat an array of diseases. These therapies have proven efficacious to an extent; however, some diseases such as breast cancer become resistant to these therapies, indicating that this pathway may not be the only driving force behind some sexually dimorphic diseases.

The predominant sex hormone in men is testosterone and 5α-dihydrotestosterone. Testosterone is a cholesterol metabolite synthesized in the primary reproductive organ of males, the testis. Similar to estrogens, testosterone exerts its actions through a nuclear receptor, the androgen receptor (AR). Activation of ARs not only contributes to male sexual maturation, but also serves a physiological purpose outside of the gonads, such as regulating muscle growth.²³ While regulating normal physiological functions, testosterone has been recently shown through meta-analysis to be positively correlated with a risk for the development of cardiovascular disease.²⁶

Although there is strong evidence that sex hormones have a major role in either the contribution to or protection from certain sexually dimorphic diseases, the presence or absence of sex steroids cannot fully explain the sexual dimorphism observed in others. For example, the differences observed in lipid metabolism between males and females have classically been thought to be the result of the differences in sex steroids, namely estrogen, which has been linked to lower levels of circulating triglycerides and cholesterol.^27,28 In recent years, however, the effects of estrogen on lowering circulating lipids has been challenged by the notion that the increase in low-density lipoprotein observed in postmenopausal women could possibly be due to the process of aging instead of the loss of ovarian function.²⁹ More substantial evidence that sex hormones may not be the sole contributor to sexually dimorphic diseases comes from experiments in which ovariectomy or castration do not alter disease onset or progression. For example, with regard to amyotrophic lateral sclerosis (ALS), a disease with a higher prevalence in males, no alteration in disease onset or survival is observed when male SOD1-G93A rats (a transgenic rat model of ALS) are castrated.³⁰ Since free testosterone is dramatically reduced in the castrated males, yet no alterations in the progression of ALS were observed, it has been concluded that testosterone is not the key determinant in ALS onset and progression. It suggests that factors other than sex steroids contribute to this sexually dimorphic disease.

Nonsex hormone pathways displaying sexual dimorphism

If sex steroids and chromosomes are not the sole contributors to sexually dimorphic diseases, what other factors could contribute to this phenomenon? Recent evidence shows that there are other signaling pathways independent of direct sex steroid signaling that exert sexually dimorphic effects between men and women. For example, women are more effective at suppressing hepatic gluconeogenesis in response to insulin, indicating, at least in the liver, that women are more sensitive to the actions of insulin than males.^31,32 Furthermore, women have a higher glucose effectiveness (the ability of glucose to stimulate glucose uptake) than men; however, no differences between pre- and postmenopausal women are observed, suggesting the sexually dimorphic effects of glucose uptake are independent of the direct actions of estrogens.³² Of note, however, sex steroids do exert both reversible and nonreversible effects during development, and these nonreversible programming effects cannot be excluded when interpreting data from aging studies.

Another biological pathway that displays distinct regulation between males and females is growth hormone (GH) signaling. GH is of particular interest because it regulates a class of genes involved in drug metabolism, known as the cytochrome P450 family. It was found that the sexually dimorphic differences in hepatic drug metabolism are due to differences in circulating GH, not androgens, between male and female rats.³³

Glucocorticoids

Inflammation is a balance between the initiation of an inflammatory response and the abatement of that response. When there is dysregulation of this balance it can lead to tissue damage and destruction; therefore, the body has a number of mechanisms to keep this response in check. One important endogenous regulator of the inflammatory response are glucocorticoids, steroid hormones that exert potent anti-inflammatory effects in the body³⁴ through activation of the glucocorticoid receptor (GR), a ligand-activated transcription factor. Since glucocorticoids serve as such potent anti-inflammatory molecules in the body, they are widely prescribed for the treatment of inflammatory conditions and hematological cancers.³⁴

Since inflammation is a core component of a variety of sexually dimorphic diseases, such as autoimmunity,⁹ it was postulated by Duma et al. that glucocorticoids exert their anti-inflammatory effects in a sexually dimorphic manner and this may be the underlying mechanism by which men and women respond to inflammation differently.³⁵ By using the liver as a target organ for glucocorticoids, whole-genome microarray analysis was performed in adrenalectomized male and female Sprague Dawley rats, revealing a number of genes differentially regulated in a gender-specific manner, a phenomenon previously documented.^36–39 Unexpected, however, was the differential gene expression in male and female rats in response to synthetic glucocorticoid treatment. On a genome-wide scale, males differentially regulated approximately 5000 genes and females regulated approximately 8000 genes in response to glucocorticoid treatment. Moreover, the direction in which genes are regulated displayed a sexually dimorphic pattern, with glucocorticoids mainly repressing genes in the male liver while inducing genes in the female liver. Intriguingly, there are many genes that appear to be anti-correlated in response to glucocorticoid treatment. For example, Crtb1 decreases in females while being induced in males. Other genes, such as Egfr, are downregulated in males and upregulated in females. Downstream of this differential gene expression is a divergence in the physiological functions of glucocorticoids between male and female rats. Through Ingenuity Pathway Analysis, Duma et al. found that in response to glucocorticoid administration several different biological pathways were altered, such as endoplasmic reticulum stress and apoptosis in male rats, while a higher degree of regulation of the circadian rhythm and IL-6 signaling pathway was seen in female rats.

Functionally, dexamethasone treatment at several different doses could rescue male rats challenged with lipopolysaccharide compared to female rats, who only survived when treated with the two highest doses of dexamethasone.³⁵ Ovariectomy could only partially rescue the female rats in response to dexamethasone treatment, indicating that estrogens play a partial role in antagonizing the anti-inflammatory effects of glucocorticoids. These data also indicate that pathways independent of estrogen signaling are likely playing a role in the sexually dimorphic actions of glucocorticoids.

Further evidence supporting the sexually dimorphic actions of glucocorticoids come from studies in which dexamethasone exerts differential regulation of the drug-metabolizing proteins. In particular, Cyp3a4 has been shown to display differential expression patterns between males and females, a phenomenon that has classically been thought to be driven by the sexually dimorphic actions of GH. It was recently shown that GR signaling was also involved in the GH-mediated sexual dimorphic regulation of Cyp3a4,⁴⁰ further supporting the case that glucocorticoids exert their actions in a sexually dimorphic manner.

While the direct mechanism by which glucocorticoids exert their actions in a sexually dimorphic manner is still not known, there are several possible answers to this question. First, an interplay between glucocorticoids and ERs and ARs cannot be ruled out. In fact, there are many reports indicating GRs and ERs can interact and modulate downstream signaling. For example, estrogens have been shown to antagonize the glucocorticoid induction of the glucocorticoid-induced leucine zipper (GILZ) gene,²⁴ an important gene for anti-inflammatory actions of glucocorticoids. ER antagonism of GR signaling could potentially be a mechanism underlying the less efficient response of females to corticosteroid therapy compared to men.

Another potential mechanism could be the differential expression of gender-specific coactivators and transcription factors. For example, there are several coregulatory molecules, such as prohibitin 2 and mediator complex subunit 12, that are enriched in females, while males express higher levels of other coregulators, such as TATA box–binding protein.³⁵ Moreover, glucocorticoid treatment increases the list of gender-specific coregulators, further implicating the role of gender-specific coregulators in modulating the glucocorticoid effect in males versus females.³⁵

Conclusion

There are many factors that differentiate physiology between males and females. These factors have classically been attributed to differences in the chromosomes we possess, particularly the allosomes, as well as the sex hormones that we produce. It is now appreciated that these factors do not contribute to disease alone and that other factors independent of sex hormones and chromosomes, such as glucocorticoids, insulin, and growth hormone, contribute to the sexual dimorphism observed in both physiology and pathophysiological states.

Historically, women have been excluded from clinical trials, dramatically limiting our knowledge on how sexual dimorphism in gene expression can affect disease progression and treatment efficacy.⁴¹ This has led to the majority of current therapies, with synthetic glucocorticoids being no exception, being prescribed equally among males and females even though many different diseases are known to display a sexually dimorphic pattern in etiology and progression. From the work of Duma et al. it can now be appreciated that glucocorticoids exert their anti-inflammatory effects more potently in males compared to females, suggesting synthetic glucocorticoids may not be the most efficacious treatment paradigm for chronic inflammatory diseases in women. If these findings are confirmed in humans, it may require rethinking of how synthetic glucocorticoids are prescribed. Hopefully, future studies will define the molecular basis underlying the sexually dimorphic actions of glucocorticoids, which will help us design more personalized treatment strategies in which gender is taken into account.