Anthony N. Gerber

Kruppel-like factor 15 regulates skeletal muscle lipid flux and exercise adaptation

The ability of skeletal muscle to enhance lipid utilization during exercise is a form of metabolic plasticity essential for survival. Conversely, metabolic inflexibility in muscle can cause organ dysfunction and disease. Although the transcription factor Kruppel-like factor 15 (KLF15) is an important regulator of glucose and amino acid metabolism, its endogenous role in lipid homeostasis and muscle physiology is unknown. Here we demonstrate that KLF15 is essential for skeletal muscle lipid utilization and physiologic performance. KLF15 directly regulates a broad transcriptional program spanning all major segments of the lipid-flux pathway in muscle. Consequently, Klf15-deficient mice have abnormal lipid and energy flux, excessive reliance on carbohydrate fuels, exaggerated muscle fatigue, and impaired endurance exercise capacity. Elucidation of this heretofore unrecognized role for KLF15 now implicates this factor as a central component of the transcriptional circuitry that coordinates physiologic flux of all three basic cellular nutrients: glucose, amino acids, and lipids.

The glucocorticoid receptor and KLF15 regulate gene expression dynamics and integrate signals through feed-forward circuitry

ABSTRACT The glucocorticoid receptor (GR) regulates adaptive transcriptional programs that alter metabolism in response to stress. Network properties that allow GR to tune gene expression to match specific physiologic demands are poorly understood. We analyzed the transcriptional consequences of GR activation in murine lungs deficient for KLF15, a transcriptional regulator of amino acid metabolism that is induced by glucocorticoids and fasting. Approximately 7% of glucocorticoid-regulated genes had altered expression in Klf15-knockdown (Klf15−/−) mice. KLF15 formed coherent and incoherent feed-forward circuits with GR that correlated with the expression dynamics of the glucocorticoid response. Coherent feed-forward gene regulation by GR and KLF15 was characterized by combinatorial activation of linked GR-KLF15 regulatory elements by both factors and increased GR occupancy, while expression of KLF15 reduced GR occupancy at the incoherent target, MT2A. Serum deprivation, which increased KLF15 expression in a GR-independent manner in vitro, enhanced glucocorticoid-mediated induction of feed-forward targets of GR and KLF15, such as the loci for the amino acid-metabolizing enzymes proline dehydrogenase and alpha-aminoadipic semialdehyde synthase. Our results establish feed-forward architecture as an organizational principle for the GR network and provide a novel mechanism through which GR integrates signals and regulates expression dynamics.

Expression Profiling Identifies Klf15 as a Glucocorticoid Target That Regulates Airway Hyperresponsiveness

Glucocorticoids (GCs), which activate GC receptor (GR) signaling and thus modulate gene expression, are widely used to treat asthma. GCs exert their therapeutic effects in part through modulating airway smooth muscle (ASM) structure and function. However, the effects of genes that are regulated by GCs on airway function are not fully understood. We therefore used transcription profiling to study the effects of a potent GC, dexamethasone, on human ASM (HASM) gene expression at 4 and 24 hours. After 24 hours of dexamethasone treatment, nearly 7,500 genes had statistically distinguishable changes in expression; quantitative PCR validation of a 40-gene subset of putative GR-regulated genes in 6 HASM cell lines suggested that the early transcriptional targets of GR signaling are similar in independent HASM lines. Gene ontology analysis implicated GR targets in controlling multiple aspects of ASM function. One GR-regulated gene, the transcription factor, Kruppel-like factor 15 (Klf15), was already known to modulate vascular smooth and cardiac muscle function, but had no known role in the lung. We therefore analyzed the pulmonary phenotype of Klf15(-/-) mice after ovalbumin sensitization and challenge. We found diminished airway responses to acetylcholine in ovalbumin-challenged Klf15(-/-) mice without a significant change in the induction of asthmatic inflammation. In cultured cells, overexpression of Klf15 reduced proliferation of HASM cells, whereas apoptosis in Klf15(-/-) murine ASM cells was increased. Together, these results further characterize the GR-regulated gene network in ASM and establish a novel role for the GR target, Klf15, in modulating airway function.

Glucocorticoids enhance muscle endurance and ameliorate Duchenne muscular dystrophy through a defined metabolic program

Significance Classic physiological studies have documented the endurance-promoting effects of glucocorticoid (GC) hormones on skeletal muscle. Pharmacologic GC therapy also improves muscle function in patients with Duchenne muscular dystrophy (DMD), a genetic muscle-wasting disease. Despite these well-established physiological and clinical observations, the molecular basis underlying the beneficial effects of GCs in skeletal muscle remains obscure. This study shows that physiological effects of GCs on muscle endurance and their therapeutic effect in DMD are mediated, in part, via activation of a potent metabolic gene called Kruppel-like factor 15 (KLF15). Importantly, KLF15 does not drive GC-mediated muscle wasting. These data shed light on the poorly understood ergogenic properties of GCs, findings that may inform steroid-sparing therapies for DMD and other muscle diseases. Classic physiology studies dating to the 1930s demonstrate that moderate or transient glucocorticoid (GC) exposure improves muscle performance. The ergogenic properties of GCs are further evidenced by their surreptitious use as doping agents by endurance athletes and poorly understood efficacy in Duchenne muscular dystrophy (DMD), a genetic muscle-wasting disease. A defined molecular basis underlying these performance-enhancing properties of GCs in skeletal muscle remains obscure. Here, we demonstrate that ergogenic effects of GCs are mediated by direct induction of the metabolic transcription factor KLF15, defining a downstream pathway distinct from that resulting in GC-related muscle atrophy. Furthermore, we establish that KLF15 deficiency exacerbates dystrophic severity and muscle GC–KLF15 signaling mediates salutary therapeutic effects in the mdx mouse model of DMD. Thus, although glucocorticoid receptor (GR)-mediated transactivation is often associated with muscle atrophy and other adverse effects of pharmacologic GC administration, our data define a distinct GR-induced gene regulatory pathway that contributes to therapeutic effects of GCs in DMD through proergogenic metabolic programming.

Cistrome-based Cooperation Between Airway Epithelial Glucocorticoid Receptor and NF-κB Orchestrates Anti-inflammatory Effects

Antagonism of pro-inflammatory transcription factors by monomeric glucocorticoid receptor (GR) has long been viewed as central to glucocorticoid (GC) efficacy. However, the mechanisms and targets through which GCs exert therapeutic effects in diseases such as asthma remain incompletely understood. We previously defined a surprising cooperative interaction between GR and NF-κB that enhanced expression of A20 (TNFAIP3), a potent inhibitor of NF-κB. Here we extend this observation to establish that A20 is required for maximal cytokine repression by GCs. To ascertain the global extent of GR and NF-κB cooperation, we determined genome-wide occupancy of GR, the p65 subunit of NF-κB, and RNA polymerase II in airway epithelial cells treated with dexamethasone, TNF, or both using chromatin immunoprecipitation followed by deep sequencing. We found that GR recruits p65 to dimeric GR binding sites across the genome and discovered additional regulatory elements in which GR-p65 cooperation augments gene expression. GR targets regulated by this mechanism include key anti-inflammatory and injury response genes such as SERPINA1, which encodes α1 antitrypsin, and FOXP4, an inhibitor of mucus production. Although dexamethasone treatment reduced RNA polymerase II occupancy of TNF targets such as IL8 and TNFAIP2, we were unable to correlate specific binding sequences for GR or occupancy patterns with repressive effects on transcription. Our results suggest that cooperative anti-inflammatory gene regulation by GR and p65 contributes to GC efficacy, whereas tethering interactions between GR and p65 are not universally required for GC-based gene repression.

Response Element Composition Governs Correlations Between Binding Site Affinity and Transcription in Glucocorticoid Receptor Feed-forward Loops

Background: Feed-forward loops are utilized in glucocorticoid signaling and can bestow temporal control to gene regulation. Results: Cooperation with KLF15 enhances low affinity glucocorticoid receptor binding site activity in coherent feed-forward loops controlling amino acid catabolism. Conclusion: Feed-forward response element composition contributes to temporal diversity of transcriptional regulation by glucocorticoids. Significance: Cooperative feed-forward regulatory control may underpin glucocorticoid-induced metabolic side effects. Combinatorial gene regulation through feed-forward loops (FFLs) can bestow specificity and temporal control to client gene expression; however, characteristics of binding sites that mediate these effects are not established. We previously showed that the glucocorticoid receptor (GR) and KLF15 form coherent FFLs that cooperatively induce targets such as the amino acid-metabolizing enzymes AASS and PRODH and incoherent FFLs exemplified by repression of MT2A by KLF15. Here, we demonstrate that GR and KLF15 physically interact and identify low affinity GR binding sites within glucocorticoid response elements (GREs) for PRODH and AASS that contribute to combinatorial regulation with KLF15. We used deep sequencing and electrophoretic mobility shift assays to derive in vitro GR binding affinities across sequence space. We applied these data to show that AASS GRE activity correlated (r2 = 0.73) with predicted GR binding affinities across a 50-fold affinity range in transfection assays; however, the slope of the linear relationship more than doubled when KLF15 was expressed. Whereas activity of the MT2A GRE was even more strongly (r2 = 0.89) correlated with GR binding site affinity, the slope of the linear relationship was sharply reduced by KLF15, consistent with incoherent FFL logic. Thus, GRE architecture and co-regulator expression together determine the functional parameters that relate GR binding site affinity to hormone-induced transcriptional responses. Utilization of specific affinity response functions and GR binding sites by FFLs may contribute to the diversity of gene expression patterns within GR-regulated transcriptomes.

Regulation of MUC5B Expression in Idiopathic Pulmonary Fibrosis

&NA; The gain‐of‐function mucin 5B (MUC5B) promoter variant, rs35705950, confers the largest risk, genetic or otherwise, for the development of idiopathic pulmonary fibrosis; however, the mechanisms underlying the regulation of MUC5B expression have yet to be elucidated. Here, we identify a critical regulatory domain that contains the MUC5B promoter variant and has a highly conserved forkhead box protein A2 (FOXA2) binding motif. This region is differentially methylated in association with idiopathic pulmonary fibrosis, MUC5B expression, and rs35705950. In addition, we show that this locus binds FOXA2 dynamically, and that binding of FOXA2 is necessary for enhanced expression of MUC5B. In aggregate, our findings identify novel targets to regulate the expression of MUC5B.

Feed-forward transcriptional programming by nuclear receptors: Regulatory principles and therapeutic implications

Nuclear receptors (NRs) are widely targeted to treat a range of human diseases. Feed-forward loops are an ancient mechanism through which single cell organisms organize transcriptional programming and modulate gene expression dynamics, but they have not been systematically studied as a regulatory paradigm for NR-mediated transcriptional responses. Here, we provide an overview of the basic properties of feed-forward loops as predicted by mathematical models and validated experimentally in single cell organisms. We review existing evidence implicating feed-forward loops as important in controlling clinically relevant transcriptional responses to estrogens, progestins, and glucocorticoids, among other NR ligands. We propose that feed-forward transcriptional circuits are a major mechanism through which NRs integrate signals, exert temporal control over gene regulation, and compartmentalize client transcriptomes into discrete subunits. Implications for the design and function of novel selective NR ligands are discussed.

Glucocorticoids and the Lung

The lung is a major clinical target of glucocorticoid-based therapeutics, and GR signaling has broad effects on respiratory physiology and inflammation. During lung development, expression of GR in the mesenchyme is required for normal terminal alveolar epithelial differentiation. Prenatal administration of exogenous glucocorticoids (GCs) to prevent neonatal respiratory distress syndrome, however, promotes alveolar maturation and accelerates surfactant expression in a manner consistent with direct effects on the developing alveolar epithelium. Likewise, cell autonomous effects of GCs in regulating gene expression and phenotype of the airway epithelium and airway smooth muscle have been demonstrated to control important therapeutic effects of GCs in treating asthma and chronic obstructive pulmonary disease. Here, mechanisms and consequences of GR signaling in the developing lung and in treating obstructive lung disease are reviewed, with a focus on direct effects of GR signaling on alveolar differentiation, surfactant expression, and airway epithelial and smooth muscle pathophysiology.

Glucocorticoid and TNF signaling converge at A20 (TNFAIP3) to repress airway smooth muscle cytokine expression

Airway smooth muscle is a major target tissue for glucocorticoid (GC)-based asthma therapies, however, molecular mechanisms through which the GC receptor (GR) exerts therapeutic effects in this key airway cell type have not been fully elucidated. We previously identified the nuclear factor-κB (NF-κB) inhibitor, A20 (TNFAIP3), as a mediator of cytokine repression by glucocorticoids (GCs) in airway epithelial cells and defined cooperative regulation of anti-inflammatory genes by GR and NF-κB as a key mechanistic underpinning of airway epithelial GR function. Here, we expand on these findings to determine whether a similar mechanism is operational in human airway smooth muscle (HASM). Using HASM cells derived from normal and fatal asthma samples as an in vitro model, we demonstrate that GCs spare or augment TNF-mediated induction of A20 (TNFAIP3), TNIP1, and NFKBIA, all implicated in negative feedback control of NF-κB-driven inflammatory processes. We applied chromatin immunoprecipitation and reporter analysis to show that GR and NF-κB directly regulate A20 expression in HASM through cooperative induction of an intronic enhancer. Using overexpression, we show for the first time that A20 and its interacting partner, TNIP1, repress TNF signaling in HASM cells. Moreover, we applied small interfering RNA-based gene knockdown to demonstrate that A20 is required for maximal cytokine repression by GCs in HASM. Taken together, our data suggest that inductive regulation of A20 by GR and NF-κB contributes to cytokine repression in HASM.