Ceramide Synthases Expression and Role of Ceramide Synthase-2 in the Lung: Insight from Human Lung Cells and Mouse Models

et al. (2013) Ceramide Synthases Expression and Role of Ceramide Synthase-2 in the Lung: Insight from Human Lung Cells and Mouse Models. PLoS ONE 8(5): e62968. doi:10.1371/journal.pone.0062968 Ceramide Synthases Expression and Role of Ceramide Synthase-2 in the Lung: Insight from Human Lung Cells and Mouse Models Irina Petrache 0 Krzysztof Kamocki 0 Christophe Poirier 0 Yael Pewzner-Jung 0 Elad L. Laviad 0 Kelly S. Schweitzer 0 Mary Van Demark 0 Matthew J. Justice 0 Walter C. Hubbard 0 Anthony H. Futerman 0 Hong Wei Chu, National Jewish Health, United States of America 0 1 Division of Pulmonary and Critical Care Medicine, Department of Medicine, Indianapolis, Indiana, United States of America, 2 Richard L. Roudebush Veteran Affairs Medical Center, Indianapolis, Indiana, United States of America, 3 Department of Biological Chemistry, Weizmann Institute of Science , Rehovot , Israel , 4 Department of Clinical Pharmacology, Johns Hopkins University , Baltimore, Maryland , United States of America Increases in ceramide levels have been implicated in the pathogenesis of both acute or chronic lung injury models. However, the role of individual ceramide species, or of the enzymes that are responsible for their synthesis, in lung health and disease has not been clarified. We now show that C24- and C16-ceramides are the most abundant lung ceramide species, paralleled by high expression of their synthetic enzymes, ceramide synthase 2 (CerS2) and CerS5, respectively. Furthermore, the ceramide species synthesis in the lung is homeostatically regulated, since mice lacking very long acyl chain C24-ceramides due to genetic deficiency of CerS2 displayed a ten-fold increase in C16-ceramides and C16-dihydroceramides along with elevation of acid sphingomyelinase and CerS5 activities. Despite relatively preserved total lung ceramide levels, inhibition of de novo sphingolipid synthesis at the level of CerS2 was associated with significant airflow obstruction, airway inflammation, and increased lung volumes. Our results suggest that ceramide species homeostasis is crucial for lung health and that CerS2 dysfunction may predispose to inflammatory airway and airspace diseases. - Funding: This work was supported by the NIH R01HL077328 (IP), the Reba and John Smith and English Chairs of Respiratory Diseases (IP) and a Bi-national Scientific Foundation research grant (2009242) (IP and AHF). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Ceramide, a signaling sphingolipid involved in cell differentiation and apoptosis, has received great attention recently due to reports of abnormal ceramide accumulation in prevalent lung diseases such as acute lung injury, cystic fibrosis, or chronic obstructive pulmonary disease (COPD). Furthermore, the de novo pathway of ceramide synthesis has been implicated in asthma. Ceramide, which consists of multiple molecular species distinguished by fatty acyl chain length, saturation, and a-hydroxylation, is synthesized by a family of ceramide synthases (CerS). Six CerS exist, each using defined acyl chains for synthesis of dihydroceramides (DHCer) and ceramides. Thus, CerS1 uses mostly C18-CoA, CerS2 uses C22 to C24-CoAs, CerS3 uses C26 and higher acyl CoAs [1], CerS4 uses C18- and C20-CoAs, and CerS5 and CerS6 use mostly C16-CoA [2] (Fig. 1). These CerS have defined tissue distribution [3]. For instance, lung epithelial cells exhibit high levels of CerS5 expression, but little is known about CerS role in the lung, in general. To date, the role of specific ceramides in lung function has not been addressed. The goal of our study was to investigate the CerS expression profile and the role of CerS2 in the lung. The pathways by which ceramides are synthesized intracellular include sphingomyelin hydrolysis performed by acid or neutral sphingomyelinases, and de novo synthesis, which requires serine palmitoyl transferase (SPT) activation, itself regulated by ORMDL proteins [4], followed by CerS activation to generate dihydroceramide, which is then desaturated to ceramide. The metabolism of ceramide either by deacylation to sphingosine or by glycosylation to glycosylated ceramides can itself be harnessed in a recycling fashion to re-synthesize or deglycosylate to ceramides, respectively [5] (Fig. 1). Although there might be acyl-chain type preference in the action of several of ceramide generating enzymes, CerS are primarily responsible for ceramide species-specificity. Understanding the role of specific CerS in lung biology is important, given the increasing appreciation of ceramide species-specific cellular function [68] and the potential need for selective targeting of only deleterious ceramide species. Recently, several groups, including ours, used molecular approaches to individually inhibit the expression of CerS, in order to understand their function in vivo, in various organs. We created a CerS2-null mouse which is unable to synthesize very long acyl chain (VLC) ceramides. These mice are characterized by liver pathology and deficient myelin maintenance in the brain [9]. The impact of loss of any CerS, including CerS2 on the murine lung pathology or function has not yet been described. We hypothesized that because of the central role of ceramides in sphingolipid metabolism, and the importance of ceramide and its various metabolites in cell maintenance and Figure 1. Ceramide metabolic pathways. Ceramide can be synthesized via the de novo pathway regulated by serine palmitoyl transferase (SPT), ceramide synthases (CerS; isoforms and their preferred substrates described in tabular format), and desaturases (DEGS); via sphingomyelinase pathway regulated by sphingomyelinases (SMases); or via the recycling pathway. doi:10.1371/journal.pone.0062968.g001 immune regulation, mice deficient in CerS2 will exhibit abnormal lung pathophysiology. Understanding the impact of CerS2 on lung biology will be useful in understating the regulation of sphinoglipid metabolism in the lung in general, and in the future design of therapies that target various ceramide species and metabolites. In the current study, we determined the expression of ceramide species and CerS in the lung and in principal alveolar cells, and examined the importance of CerS2 in lung function. We demonstrate that CerS2 is essential for proper lung sphingolipid homeostasis and airway function. Materials and Methods Chemicals and Reagents All chemicals and reagents were from Sigma-Aldrich (St. Louis, MO), unless otherwise stated. Cell Culture Beas2B cells, a transformed human bronchial cell line, were a kind gift from Dr. Augustine Choi, Harvard University and were originally purchased from American Type Culture Collection (ATCC, Manassas, VA). They were used from passages 512. Primary human small airway epithelial cells (SAEC) and human lung microv (...truncated)