Freishtat, Robert J. MD, MPH*†‡; Nagaraju, Kanneboyina DVM, PhD†‡; Jusko, William PhD§; Hoffman, Eric P. PhD†‡
Synthetic glucocorticoids (GCs), such as prednisone, are among the most widely prescribed drugs worldwide and are used to treat many acute and chronic inflammatory conditions.1 Glucocorticoids are very old drugs, yet they remain the standard of care for treatment of a variety of diseases including asthma, muscular dystrophy, autoimmune disorders, and arthritis. Other newer and more potent or targeted immunosuppressants have been tried in many of these disorders, but they have not shown the same efficacy as prednisone.2-4 Additionally, chronic use of prednisone is associated with significant adverse effects. It is clear that a greater understanding of the molecular action of GCs could lead to more optimized drugs and dosing regimens that improve efficacy and reduce adverse effects.
Glucocorticoids exert anti-inflammatory effects in large part through inhibition of nuclear factor-κB (NF-κB) and NF-κB downstream inflammatory targets.5 It is important to note that GCs have both short-term effects on protein-protein signaling occurring in minutes and longer-acting effects on messenger RNA transcription via GC receptor (GR) binding to target promoters (hours). The acute nontranscriptional activities via NF-κB seem responsible for most anti-inflammatory effects of GCs, whereas the receptor-mediated transcriptional activities account for most adverse effects.5
The current paradigm of GC efficacy is that efficacy is mediated through a direct anti-inflammatory role. Decreased inflammation in these disorders is thought to lead to decreased pathological tissue remodeling. However, this model has never been validated. In particular, improvements in inflammation have not been shown to improve the rate of lung function decline in asthma. Herein, we present an alternative paradigm, where GC efficacy is mediated through more successful tissue remodeling, with reduction in inflammation secondary to successful regeneration.
MECHANISMS OF GC ACTION
Molecular mechanistic studies of GCs in lung inflammation have focused on their impact on transcriptional targets of the hormone/receptor complex.6-8 However, it is widely recognized that the anti-inflammatory activities are not a direct consequence of GC/GR complex binding to gene targets, but rather GC effects on NF-κB protein signaling (either with or without receptor involvement). Importantly, GCs are known to have both transcriptional and signaling effects in the lung. They endogenously mediate physiological processes such as cortisol suppression, intracellular signaling, and trafficking.9
Many of the effects of GCs occur through binding and activation of the GR that is expressed in all tissues. The GR has many isoforms, each with different ligand and binding affinities and properties, tissue-specific effects, and nonreceptor (signaling) activities. The best characterized effect of GCs is their ability to bind soluble steroid hormone receptors and then move to the nucleus where the receptor complex directly binds to promoter elements and mediates gene transcription. In the case of GCs, there are well-studied steroid response cis-elements (sREs) in target gene promoters that are responsible for binding the ligand/receptor complex, with downstream modulation of gene transcription10,11 (Fig. 1). Ligand-activated GR dimers bind directly to sREs and directly or indirectly recruit molecules with histone acetyl transferase (HAT) activity, resulting in acetylation of lysines on H4 or H3, which opens up chromatin to facilitate gene transcription. Coactivators with HAT activity include the CREB binding protein (CBP) and the p300/CBP-associated factor (PCAF).12,13 These coactivators can also recruit other HATs.14 As an example, an in vivo temporal microarray study looking at the effects of a bolus of methylprednisolone in adrenalectomized rats showed approximately 200 genes differentially regulated at 2 hours.15
A second mechanism of GC action focuses on inflammatory genes that lack sRE cis-sites in their promoters but have functional NF-κB or activator protein 1 cis-sites that bind inflammatory mediator-induced transcription factors.16,17 Ligand-activated GR binds to these activated transcription factors, thereby blocking their binding to target cis-sites and up-regulation of target genes by GR-mediated trans-repression. Ligand-activated GR also binds to and inhibits coactivators with intrinsic HAT activity, resulting in inhibition of the HAT activity, recruitment of histone deacetylases, a decrease in histone acetylation, and a reduction in gene expression.
Additionally, GCs have a less characterized nontranscriptional (signaling) response. Prednisone has been shown to acutely activate the protein kinase C-dependent mitogen-activated protein kinase pathway via putative G-protein-coupled receptors in immune, brain, and lung cells.18,19 Other than the well-characterized translocation of the GR from the cytosol to the nucleus, a few proteins, such as annexin A1, 5-lipoxygenase, and S100A11, have been shown to translocate in response to GCs.18,20 Synthetic GCs have also been shown in multiple tissues to decrease membrane fluidity by altering the cholesterol/phospholipids ratio.21,22
THE CURRENT PARADIGM: INFLAMMATION LEADS TO REMODELING IN ASTHMA
Asthma is a complex, multifactorial disease comprising multiple different subtypes, rather than a single disease entity.23 Asthma is a chronic disease of the lower respiratory tract characterized by inflammation, airway hyperresponsiveness, and mucus obstruction in the airways. It reflects airway remodeling by goblet cell hyperplasia, mucus hypersecretion, smooth muscle hyperplasia, fibroblast proliferation, angiogenesis, and increased collagen deposition.24-27
Oral and inhaled GCs are the mainstay of acute and chronic asthma therapy. Glococorticoids, such as prednisone and fluticasone, are commonly used for their potent ability to modulate the inflammation that leads to acute exacerbations of asthma symptoms. For example, daily inhaled fluticasone reduces the frequency of acute asthma symptoms.28 As a result, depending on the severity and frequency of asthma symptoms, daily inhaled GCs are the standard of asthma care.29
Under the current paradigm, where asthmatic inflammation is assumed to lead to pathological tissue remodeling, it follows that anti-inflammatory treatment with GCs would diminish the typical accelerated loss of lung function over time in individuals with asthma.30 However, this is not the case. The Childhood Asthma Management Program studied the lung function of 5- to 12-year-olds with mild to moderate asthma being treated with budesonide or placebo. Although the budesonide group showed an initial improvement in lung function, this difference disappeared by the end of the 4-year study period.31 This finding has been confirmed repeatedly, most recently in an Inhaled Treatment as Regular Therapy in Early Asthma study.32 These investigators showed the same disappearance of lung function benefit from inhaled GCs (ie, budesonide) in children and adults after a 5-year study period. Furthermore, the bronchial reticular layer thickness in asthmatic patients treated with inhaled GCs is not diminished unless high doses are used.33
GLUCOCORTICOID REGULATION OF THE CELL CYCLE
Endogenous GC expression is tightly controlled by a system of regulatory loops known as the hypothalamic-pituitary-adrenal axis. Corticotropin-releasing hormone is secreted by the hypothalamus and stimulates secretion of adrenocorticotropic hormone (ACTH) by the anterior pituitary. The adrenocorticotropic hormone in turn stimulates the adrenal cortex to synthesize and secrete GCs, most notably cortisol and corticosterone. These secretions are pulsatile and occur as circadian rhythms entrained by the internal clock, the suprachiasmatic nucleus, and the light-dark cycle. This circadian rhythm of GCs further serves to reset the internal and peripheral clocks of the organism, including lung and lung-relevant cytokines such as IL-5.34 The hypothalamic-pituitary-adrenal axis is regulated by a negative feedback inhibition loop where GCs directly down-regulate secretion of corticotropin-releasing hormone and ACTH and by a positive feedback excitation loop where epinephrine and norepinephrine increase ACTH synthesis after being secreted in response to fear or stress. Glucocorticoids are highly expressed around the start of the organism's period of activity/waking (morning for diurnal and evening for nocturnal) and decrease over the course of this period.
The normal cell cycle consists of 2 mitotic stages separated by 2 "gap" stages where DNA is assessed for damage and can be repaired (Fig. 2). In peripheral tissues, such as lung, mitosis is regulated by a negative feedback loop among circadian oscillator gene products. Bmal1-Clock protein heterodimers enhance the expression of the Period genes, PER1, PER2, and PER3. These negatively feed back to inhibit the formation of Bmal1-Clock heterodimers.35 In addition, circadian oscillator gene expression is also regulated by endogenous GCs directly or indirectly through multiple signaling pathways.15,36-38 In particular, PER1 has a glucocorticoid response element in its promoter sequence,39-41 which increases PER1 transcription.42
Ironically, the circadian peak (ie, prewaking hours) in endogenous cortisol corresponds to an asthmatic clinical trough: minimum lung function43 and maximum inflammation.34,44,45 Interestingly, chronotherapeutic trials of exogenous GCs in nocturnal asthma have found improved lung function, symptoms, and inflammation with a single late-afternoon dose compared with the usual morning or even later-afternoon dosing.46-48 Knowledge is sparse regarding how circadian oscillators may contribute to asthma pathobiology. However, there is evidence of a GC-regulated circadian clock in the lung mediated by Clara cells.49 Furthermore, there is evidence of circadian variation in airway caliber, inflammation, and hyperreactivity.50
It should be noted that there is mounting evidence that many drug classes, including GCs, should be administered with respect to either the circadian rhythm of the disease's symptoms or of the endogenous analogues. Chronopharmacology and chronotherapy are currently being applied to diseases such as asthma, hypertension, rheumatoid arthritis, cancer, and rhinitis.50-52
Pharmacologic analogues of cortisol have been the standard treatment to generate and maintain a state of anti-inflammation in asthmatic patients for decades. As described, control of inflammation does not equate to restoration of normal lung remodeling. Studies need to be designed to test the hypothesis described herein that GC efficacy is mediated through more successful tissue remodeling, with reduction in inflammation secondary to successful regeneration. In vitro experiments of pulse GC dosing of primary human airway epithelial cells from normal and asthmatic donors can be a useful first step. Animal models of chronotherapeutic dosing of GCs in asthma can provide complementary data. Additionally, the relationships between GCs, circadian oscillator gene expression, and lung fibrosis/inflammation need to be studied. The results of these investigations would likely lead to optimized treatment regimens for asthma.
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