The NAMPT Promoter Is Regulated by Mechanical Stress, Signal Transducer and Activator of Transcription 5, and Acute Respiratory Distress Syndrome–Associated Genetic Variants

Abstract

Increased nicotinamide phosphoribosyltransferase (NAMPT) transcription is mechanistically linked to ventilator-induced inflammatory lung injury (VILI), with VILI severity attenuated by reduced NAMPT bioavailability. The molecular mechanisms of NAMPT promoter regulation in response to excessive mechanical stress remain poorly understood. The objective of this study was to define the contribution of specific transcription factors, acute respiratory distress syndrome (ARDS)-associated single nucleotide polymorphisms (SNPs), and promoter demethylation to NAMPT transcriptional regulation in response to mechanical stress. In vivo NAMPT protein expression levels were examined in mice exposed to high tidal volume mechanical ventilation. In vitro NAMPT expression levels were examined in human pulmonary artery endothelial cells exposed to 5 or 18% cyclic stretch (CS), with NAMPT promoter activity assessed using NAMPT promoter luciferase reporter constructs with a series of nested deletions. In vitro NAMPT transcriptional regulation was further characterized by measuring luciferase activity, DNA demethylation, and chromatin immunoprecipitation. VILI-challenged mice exhibited significantly increased NAMPT expression in bronchoalveolar lavage leukocytes and in lung endothelium. A mechanical stress-inducible region (MSIR) was identified in the NAMPT promoter from 22,428 to 22,128 bp. This MSIR regulates NAMPT promoter activity, mRNA expression, and signal transducer and activator of transcription 5 (STAT5) binding, which is significantly increased by 18% CS. In addition, NAMPT promoter activity was increased by pharmacologic promoter demethylation and inhibited by STAT5 silencing. ARDS-associated NAMPT promoter SNPs rs59744560 (2948G/T) and rs7789066 (22,422A/G) each significantly elevated NAMPT promoter activity in response to 18% CS in a STAT5-dependent manner. Our results show that NAMPT is a key novel ARDS therapeutic target and candidate gene with genetic/epigenetic transcriptional regulation in response to excessive mechanical stress.Keywords: acute respiratory distress syndrome; cyclic stretch; nicotinamide phosphoribosyltransferase; B cell colony-enhancing factor; signal transducer and activator of transcription 5 Clinical RelevanceNicotinamide phosphoribosyltransferase (NAMPT)/pre-B cell colony-enhancing factor is a key novel molecular marker and therapeutic target for acute lung injury. This study promotes the interpretation of the genetic and epigenetic regulation of NAMPT in response to excessive mechanical stress.Acute respiratory distress syndrome (ARDS) is characterized by severe hypoxemia and a persistently high mortality rate (z 30%) (1, 2). Mechanical ventilation is a life-saving intervention in critically ill patients with respiratory failure due to ARDS; however, excessive mechanical ventilation contributes directly to inflammatory lung injury, a process known

Endotoxin‐ and Mechanical Stress–Induced Epigenetic Changes in the Regulation of the Nicotinamide Phosphoribosyltransferase Promoter

“…Preliminary analysis showed dose-dependent increases in NAMPT expression upon pharmacological inhibition of DNA methyltransferases. 8 As a complementary approach, in vitro methylation of the NAMPT promoter luciferase constructs by CpG methyltransferase also showed decreased expression (data not shown). Using bisulfite se- quencing of DNA from human lung endothelium, we extended our studies to investigate changes in methylation occurring in the NAMPT promoter at single-base resolution and demonstrated decreases in DNA methylation occurring in response to excessive mechanical stress and endotoxin challenges.…”

“…Our earlier publication has described the intricate involvement of signal transducer and activator of transcription 5 (STAT5) in the regulation of NAMPT in response to mechanical stress. 8 Because HDAC activity is required for transcription of STAT5-responsive genes, 19 inhibition of HDAC activity may lead to decreased binding of STAT5 transcription factors to the NAMPT promoter, thus leading to the attenuated responses. In contrast to the observations of HDAC inhibition attenuating NAMPT gene and protein expression, silencing of Sirt-1, a class III HDAC, resulted in significantly increased NAMPT gene and protein expression, suggesting distinct mechanisms of action between class I and II HDACs versus class III HDACs.…”

“…7 Although our earlier studies have also elucidated specific transcription factors that bind and augment NAMPT promoter, no studies to date have evaluated the epigenetic regulation of NAMPT expression with excessive mechanical stress. 8 Epigenetics has emerged as a critical novel regulator of gene transcription. The major epigenetic mechanisms, including DNA methylation, histone modifications, and noncoding RNA-mediated posttranscriptional regulation of messenger RNA (mRNA), have been implicated in a multitude of physiological processes, such as cancer, cellular differentiation, inflammation, [9][10][11] and VILI.…”

Sp1‐Mediated Nonmuscle Myosin Light Chain Kinase Expression and Enhanced Activity in Vascular Endothelial Growth Factor–Induced Vascular Permeability

“…Luciferase promoter assays were performed as described elsewhere. 11 mRNA extraction, complementary DNA (cDNA) synthesis, quantitative polymerase chain reaction (qPCR), and chromatin immunoprecipitation (ChIP) analysis mRNA was isolated from lung ECs using RNA isolation kits (Promega). cDNA was synthesized using the High-Capacity cDNA Re-verse Transcription Kit (Applied Biosystems, Foster City, CA).…”

“…In addition to these epidemiologic studies, race and sex differences in ARDS deaths in the United States over the past several decades have clearly demonstrated an increase in incidence and mortality due to sepsis and acute lung injury in African Americans and Latinos compared with non-Latino whites, 3,4 highlighting the contribution of genetic components in disease susceptibility and severity. [5][6][7][8][9][10][11][12] Increased lung vascular permeability is a major pathobiologic feature of ARDS and drives morbidity and mortality. At a mechanistic level, increased gaps between lung endothelium are directly related to the extent of lung fluid imbalance and lung edema.…”