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A Transcriptomic Approach to Elucidate the Physiological Significance Of Madanayake et al. BMC Genomics 2013, 14:833 http://www.biomedcentral.com/1471-2164/14/833 RESEARCH ARTICLE Open Access A transcriptomic approach to elucidate the physiological significance of human cytochrome P450 2S1 in bronchial epithelial cells Thushara W Madanayake1, Ingrid E Lindquist2, Nicholas P Devitt2, Joann Mudge2 and Aaron M Rowland1* Abstract Background: Cytochrome P450 2S1 (CYP2S1) is an orphan P450 with an unknown biological function. Data from our laboratory and others suggest that CYP2S1 may have an important physiological role in modulating the synthesis and metabolism of bioactive lipids including prostaglandins and retinoids. CYP2S1 expression is elevated in multiple epithelial-derived cancers as well as in the chronic hyperproliferative disease psoriasis. Whether CYP2S1 expression in proliferative disease is protective, detrimental, or neutral to disease progression remains to be determined. Two human bronchial epithelial cells (BEAS-2B) were constructed to represent chronic depletion of CYP2S1 using short-hairpin RNA (shRNA) silencing directed toward the 3’UTR (759) and exon 3 (984) of the CYP2S1 gene and compared with a non-targeting shRNA control (SCRAM). Both CYP2S1 mRNA and protein were depleted by approximately 75% in stable cell lines derived from both targeted shRNA constructs (759 and 984). To elucidate the biological significance of CYP2S1, we analyzed transcriptome alterations in response to CYP2S1 depletion in human lung cells. Results: RNA-sequencing (RNA-seq) analysis was performed to compare the transcriptome of the control (SCRAM) and the CYP2S1-depleted (759) BEAS-2B cell lines. Transcriptomes of the replicates from the two cell lines were found to be distinct populations as determined using Principal Component Analysis and hierarchical clustering. Approximately 1000 genes were differentially expressed in response to CYP2S1 depletion. Consistent with our previous phenotypes, DAVID analysis revealed altered regulation in key pathways implicated in cell proliferation and migration. Transcriptomic profiles were also consistent with the metabolism of proposed endogenous substrates. Pathway analysis also revealed significant expression changes within mTOR signaling, a critical pathway in cell growth. To determine whether these changes manifest as altered cell size, cell diameter and volume were calculated, revealing that CYP2S1 depletion promotes cell growth in BEAS-2B cells. Conclusions: These data suggest that pathway analysis of sequence-based gene expression is a powerful method to identify pathways and phenotypic alterations in response to changes in orphan enzyme expression. Our results suggest a novel role for CYP2S1-mediated metabolism in modulating BEAS-2B cell size. These findings warrant further studies on CYP2S1 regulated pathways to elucidate potential substrates of CYP2S1. Keywords: CYP2S1, BEAS-2B, Retinoic acid, Arachidonic acid, RNA Seq, Orphan, shRNA, PGE2 * Correspondence: [email protected] 1Department of Chemistry and Biochemistry, New Mexico State University, Las Cruces, NM 88003, USA Full list of author information is available at the end of the article © 2013 Madanayake et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Madanayake et al. BMC Genomics 2013, 14:833 Page 2 of 13 http://www.biomedcentral.com/1471-2164/14/833 Background (peroxide shunt pathway) and whether CYP2S1 contrib- Although the human genome was declared complete nearly utes to the metabolism of these bioactive lipids is contro- a decade ago [1] and many of the proteins linked to these versial [18]. However, in vitro cellular assays in human sequences have been identified, the challenge remains to and rodent cells appear to be consistent with metabolism characterize the functional significance of these proteins. It of these endogenous substrates [9,22,23]. Previous work in is particularly difficult to elucidate the metabolic activity of our laboratory, examined the impact of CYP2S1 depletion enzymes with no known substrate or critical function. Cyto- on human bronchial epithelial (BEAS-2B) cells [23]. De- chrome P450s (CYPs) are heme-containing metabolic en- pletion of CYP2S1 in these cells led to enhanced cell pro- zymes that typically catalyze the oxidation of endogenous liferation and migration. Cell proliferation was, in part, and xenobiotic chemical substrates. Although these enzymes attributed to modulation of arachidonic acid cascade, demonstrate a critical role in the metabolism of ~75% of all resulting in elevated levels of the inflammatory prostaglan- xenobiotic substrates, less than half of these enzymes have din (PGE2) [23]. The etiology of the change in migration, critical physiological functions [2-4]. A recent analysis of the however, is still unclear. human genome revealed approximately 57 distinct human Elucidating the physiological significance of an CYPs [4]. Roughly one-quarter of these CYPs are classified orphan cytochrome P450s is a complicated process as orphans with little or no knowledge of their substrates hampered by difficulties in isolation and purification of and physiological significance. One of the most recently CYPs as well as identifying possible substrates from a identified CYPs, Cytochrome P4502S1 (CYP2S1), was iden- myriad of potential chemicals. Historically, a trial-and- tified through a bioinformatics approach [5] and is among error approach has been used to identify possible these orphan P450s. substrates. However, this approach is time and resource CYP2S1 is likely to play an important role in regulating intensive and neglects chemicals outside of the chem- endogenous metabolism. CYP2S1 expression is sensitive to ical library. Recently, the Guengerich lab has success- regulation by endogenous and exogenous chemicals. Retin- fully utilized advances in methodology and mass oic acid significantly elevated CYP2S1 at the mRNA and spectrometry analysis to identify endogenous sub- protein level in a variety of human epithelial cells [6], in- strates for a number of cytochrome P450s, including cluding human lung cells [7]. Oxygen deprivation within orphans [24,25]. This approach is a significant advance cultured cells also resulted in significant elevation of in identification of novel endogenous substrates. How- CYP2s1 mRNA within mouse hepatoma Hepa-1 cells [8]; ever, it does not directly demonstrate the physiological however, a similar treatment in human monocytes did not impactofchangesinP450expressionwithinhuman alter expression [9]. CYP2S1 expression was elevated in re- cells. To elucidate the physiological significance of al- sponse to agonists of the arylhydrocarbon receptor, AhR, terationsinP450expressiononbiologicalpathways,we including dioxin and 3-methylchloranthrene (3-MC) [10] utilize next generation sequencing as a novel, unbiased and components of cigarette smoke [11]. Conversely, anti- approach to identify transcriptional changes in bio- inflammatory glucocorticoid agonist treatment of human logical pathways within human cells. Specifically, we lung cells at physiologically relevant concentrations signifi- compare the transcriptomic profiles of two human cantly depleted CYP2S1 mRNA through epigenetic modu- bronchial epithelial (BEAS-2B) cell lines with differen- lation of CYP2S1 [12]. CYP2S1 was also elevated in chronic tial CYP2S1 expression: CYP2S1 depleted (759) vs. hyperproliferative diseases, including psoriasis [6] as well as control (SCRAM). Previous work in our lab demon- multiple epithelial cancers [13-16]. It is likely that both strated that CYP2S1 depleted cells enhanced cell prolif- transient and chronic changes in CYP2S1 expression alter eration and migration [23]. We illustrate how pathway metabolic activation of potential endogenous substrates analysis of sequence-based differential expression and may reveal an important physiological role for results identified the molecular pathways perturbed in CYP2S1. response to CYP2S1 depletion, provided insight into Although it has been shown to effectively metabolize unexpected modes of action, and informed follow up cancer therapeutics of the anthraquinone (AQ4N) [17,18] experiments. Here we report how transcriptomic and benzothiazole family (GW610 and SF203) [19], profiles are consistent with published phenotypes and CYP2S1 is still considered an orphan with no known en- proposed endogenous metabolism. Additionally, our dogenous substrates. Candidate substrates have been iden- results reveal novel changes in the mTOR signaling tified and include the bioactive lipids all-trans retinoic pathway, which has been linked to cell size [26]. We acid (RA) [6,20,21] and metabolites of the arachidonic pursued this phenotype experimentally and confirmed acid inflammatory cascade (in particular metabolites of a significant increase in cellular diameter and volume the cyclooxygenase (prostaglandins) and lipoxygenase in CYP2S1 depleted cells, suggesting a previously un- (HETES) pathways [9,22]. CYP2S1-mediated metabolism known role for CYP2S1-mediated metabolism in the of these lipids appears to require atypical metabolism regulation of cell growth. Madanayake et al. BMC Genomics 2013, 14:833 Page 3 of 13 http://www.biomedcentral.com/1471-2164/14/833 Results and discussion DAVID analysis reveals biological pathways associated RNA-sequencing Analysis
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