A transcriptomic approach to elucidate the physiological significance of human cytochrome P450 2S1 in bronchial epithelial cells
© Madanayake et al.; licensee BioMed Central Ltd. 2013
Received: 26 June 2013
Accepted: 21 November 2013
Published: 26 November 2013
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.
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.
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.
KeywordsCYP2S1 BEAS-2B Retinoic acid Arachidonic acid RNA Seq Orphan shRNA PGE2
Although the human genome was declared complete nearly a decade ago  and many of the proteins linked to these sequences have been identified, the challenge remains to characterize the functional significance of these proteins. It is particularly difficult to elucidate the metabolic activity of enzymes with no known substrate or critical function. Cytochrome P450s (CYPs) are heme-containing metabolic enzymes that typically catalyze the oxidation of endogenous and xenobiotic chemical substrates. Although these enzymes demonstrate a critical role in the metabolism of ~75% of all xenobiotic substrates, less than half of these enzymes have critical physiological functions [2–4]. A recent analysis of the human genome revealed approximately 57 distinct human CYPs . Roughly one-quarter of these CYPs are classified as orphans with little or no knowledge of their substrates and physiological significance. One of the most recently identified CYPs, Cytochrome P4502S1 (CYP2S1), was identified through a bioinformatics approach  and is among these orphan P450s.
CYP2S1 is likely to play an important role in regulating endogenous metabolism. CYP2S1 expression is sensitive to regulation by endogenous and exogenous chemicals. Retinoic acid significantly elevated CYP2S1 at the mRNA and protein level in a variety of human epithelial cells , including human lung cells . Oxygen deprivation within cultured cells also resulted in significant elevation of CYP2s1 mRNA within mouse hepatoma Hepa-1 cells ; however, a similar treatment in human monocytes did not alter expression . CYP2S1 expression was elevated in response to agonists of the arylhydrocarbon receptor, AhR, including dioxin and 3-methylchloranthrene (3-MC)  and components of cigarette smoke . Conversely, anti-inflammatory glucocorticoid agonist treatment of human lung cells at physiologically relevant concentrations significantly depleted CYP2S1 mRNA through epigenetic modulation of CYP2S1 . CYP2S1 was also elevated in chronic hyperproliferative diseases, including psoriasis  as well as multiple epithelial cancers [13–16]. It is likely that both transient and chronic changes in CYP2S1 expression alter metabolic activation of potential endogenous substrates and may reveal an important physiological role for CYP2S1.
Although it has been shown to effectively metabolize cancer therapeutics of the anthraquinone (AQ4N) [17, 18] and benzothiazole family (GW610 and SF203) , CYP2S1 is still considered an orphan with no known endogenous substrates. Candidate substrates have been identified and include the bioactive lipids all-trans retinoic acid (RA) [6, 20, 21] and metabolites of the arachidonic acid inflammatory cascade (in particular metabolites of the cyclooxygenase (prostaglandins) and lipoxygenase (HETES) pathways [9, 22]. CYP2S1-mediated metabolism of these lipids appears to require atypical metabolism (peroxide shunt pathway) and whether CYP2S1 contributes to the metabolism of these bioactive lipids is controversial . However, in vitro cellular assays in human and rodent cells appear to be consistent with metabolism of these endogenous substrates [9, 22, 23]. Previous work in our laboratory, examined the impact of CYP2S1 depletion on human bronchial epithelial (BEAS-2B) cells . Depletion of CYP2S1 in these cells led to enhanced cell proliferation and migration. Cell proliferation was, in part, attributed to modulation of arachidonic acid cascade, resulting in elevated levels of the inflammatory prostaglandin (PGE2) . The etiology of the change in migration, however, is still unclear.
Elucidating the physiological significance of an orphan cytochrome P450s is a complicated process hampered by difficulties in isolation and purification of CYPs as well as identifying possible substrates from a myriad of potential chemicals. Historically, a trial-and-error approach has been used to identify possible substrates. However, this approach is time and resource intensive and neglects chemicals outside of the chemical library. Recently, the Guengerich lab has successfully utilized advances in methodology and mass spectrometry analysis to identify endogenous substrates for a number of cytochrome P450s, including orphans [24, 25]. This approach is a significant advance in identification of novel endogenous substrates. However, it does not directly demonstrate the physiological impact of changes in P450 expression within human cells. To elucidate the physiological significance of alterations in P450 expression on biological pathways, we utilize next generation sequencing as a novel, unbiased approach to identify transcriptional changes in biological pathways within human cells. Specifically, we compare the transcriptomic profiles of two human bronchial epithelial (BEAS-2B) cell lines with differential CYP2S1 expression: CYP2S1 depleted (759) vs. control (SCRAM). Previous work in our lab demonstrated that CYP2S1 depleted cells enhanced cell proliferation and migration . We illustrate how pathway analysis of sequence-based differential expression results identified the molecular pathways perturbed in response to CYP2S1 depletion, provided insight into unexpected modes of action, and informed follow up experiments. Here we report how transcriptomic profiles are consistent with published phenotypes and proposed endogenous metabolism. Additionally, our results reveal novel changes in the mTOR signaling pathway, which has been linked to cell size . We pursued this phenotype experimentally and confirmed a significant increase in cellular diameter and volume in CYP2S1 depleted cells, suggesting a previously unknown role for CYP2S1-mediated metabolism in the regulation of cell growth.
Results and discussion
RNA-sequencing Analysis of CYP2S1 depleted human bronchial epithelial cells (BEAS-2B)
To identify specific differentially regulated genes between CYP2S1 depleted (759) and scrambled control (SCRAM) in human bronchial epithelial cells, we used the negative binomial test of significance as implemented in the Bioconductor package DESeq with significance defined as an adjusted p-value ≤0.05  (Figure 1C). CYP2S1 depletion resulted in the increased expression of 1159 genes while significantly reducing expression of 1326 genes. RNA-seq confirmed CYP2S1 depletion of approximately 3-fold in these samples. Quantitative PCR of CYP2S1 was performed (primers used as previously described  on each of the three biological replicates, and expressed relative to one of the most stable housekeeping genes identified in our study, b-actin (ACTB). The results demonstrate a high concordance in quantification of relative CYP2S1 expression between the two experimental methods (Figure 1D). A comprehensive list of differentially expressed genes is included in an additional file (Additional file 1).
DAVID analysis reveals biological pathways associated with previously published phenotypes
Statistically significant KEGG classifications of differentially expressed genes in CYP2S1 depleted cells
P value (EASE score)
TGF-beta signaling pathway
Nucleotide excision repair
mTOR signaling pathway
Glycine serine and threonine metabolism
Pathways in cancer
CYP2S1 depletion evokes changes in P450s that metabolize bioactive lipids
Cytochrome P450s altered in CYP2S1 depleted cells
Altered in CYP2S1 depleted BEAS-2B
Fold change & p value
Evidence for endogenous substrate metabolism
-3.2; p < 0.0001
-3.6; p < 0.05
Estrogen, bilirubin, melatonin, arachidonic acid. Reviewed in 
4.2; p ≤ 0.05
Saturated and unsaturated fatty acid hydroxylation including lauric acid (C12:0, myristic acid (C14:0); 11,14-eicosadienoic acid (C20:2); (C20:4) 
2.7; p ≤ 0.001
3.6; p < 0.05
Arachidonic acid epoxidation to epoxyeicosatrienoic acids (EETS). Reviewed in 
Pathway analysis for the synthesis and metabolism of proposed lipids substrates
CYP2S1-mediated metabolism of endogenous substrates remains controversial. However, there exists published biochemical and cellular evidence to suggest CYP2S1 mediated metabolism of the bioactive lipids including all-trans retinoic acid [6, 20, 21] as well as metabolic products of the arachidonic acid metabolism [9, 22, 23]. Transcriptional responses were evaluated for each pathway to determine whether physiological responses to CYP2S1 depletion were consistent with its proposed role in lipid metabolism.
Transcriptome analysis of retinol metabolism in CYP2S1 depleted cells
All-trans retinoic acid (RA) is transported to the cell as retinol (Vitamin A) and converted to retinal via oxidoreductases (ADH and SDR). Retinal is subsequently bioactivated to RA via retinal dehydrogenases (RALDHs) . Once formed, RA is inactivated via P450 oxidation to a variety of metabolites. The CYP26 family is the most effective at oxidizing RA [36–39]. Other CYPs’ (CYP1A, CYP2ABC, CYP3A, CYP4A) oxidation and conjugation of retinoic acid to multiple hydroxylated products as well as the glucuronide conjugate via UDP glucuronosyltransferase (UGT2B7) , respectively, is believed to inactivate RA. Heterologous expression of CYP2S1 has yielded contradictory results that either demonstrate [6, 20] or fail to show [17, 18] CYP2S1-mediated metabolism of RA. However, CYP2S1 expression within a cellular context in both chinese hamster ovary (CHO) cells and human keratinocytes (HaCaT) cells demonstrate that CYP2S1 contributes to retinoic acid metabolism in cells . To function, RA is shuttled to either cellular retinoic acid binding protein (CRABPII) or fatty acid binding proteins (FABP5) , depending on their expression levels within the cell. Once bound, RA is delivered by CRABP and FABP5 to retinoic acid receptor (RAR) or peroxisome proliferating receptor (PPAR), respectively, where it alters transcription of numerous downstream targets.
Transcriptome analysis of arachidonic acid metabolism in CYP2S1 depleted cells
CYP2S1 has been shown to metabolize products of the two major metabolic pathways of the arachidonic acid (AA) cascade, lipoxygenase and cyclooxygenase, in the presence of lipid peroxides . Fromel et al also demonstrated CYP2S1-mediated epoxidation of a variety of bioactive lipids including arachidonic acid to epoxyeicosatrienoic acid (EETs) in sf-9 cells expressing CYP2S1 . CYP2S1 depleted human BEAS-2B  and monocyte-derived macrophages  show increased levels of the inflammatory prostaglandin, PGE2. Conversely, over-expressing human CYP2S1 in rodent cells reduced synthesis of products of the cyclooxygenase pathway, PGE2 and PGD2 . Based on published data, as well changes in P450 expression (Table 2), we would have anticipated significant changes in arachidonic acid metabolism in CYP2S1 depleted cells (759) compared to controls (SCRAM). However, arachidonic acid metabolism was not identified in the KEGG or GO terms. In fact, the arachidonic acid GO term did not appear in our most stringent (p < 0.01) criteria, but it did show up as #175 when the p-value was relaxed (p < 0.05) (Additional file 4). It is possible that the inability to detect transcriptional changes within this pathway could suggest that either: i) subtle transcriptional changes, undetected as significantly different, in this pathway are sufficient to elicit significant changes in AA metabolism, or ii) pathway analysis relies on a priori knowledge of AA metabolism, which may not be sufficient to clearly discern this pathway. These data suggest that transcriptome analysis may underrepresent potentially important pathways altered in response to changes in CYP2S1 expression.
RNA-sequencing analysis reveals a novel phenotype in CYP2S1 depleted cells: differential regulation of cell size
To determine whether RNA-sequencing analysis could reveal novel functions or phenotypes associated with an orphan P450, we examined the literature for connections between the top KEGG pathways. Three of the top regulated pathways are involved in regulation of cell cycle, growth factor signaling, and mTOR signaling. A common connection between each of these is the regulation of cell size. In order to grow and divide, cells must double their cellular contents (Reviewed in ). CYP2S1 depleted BEAS-2B cells exhibit increased cell proliferation . Cell cycle control was listed as the most significant change in the KEGG classification. The growth factor TGFβ, exhibits crosstalk with the mTOR pathway . mTOR pathway is recognized as a central regulator in cell growth , and represents a potentially novel CYP2S1-regulated pathway.
Only one gene, eukaryotic initiation factor 4E binding protein 1 (eIF4EBP1; log2 fold = -1.29, p < 0.001) met the criteria of both statistical significance p < 0.05 and 2-fold decrease in expression. eIF4EBP1 interacts with the translation initation factor eIF4E inhibiting the assembly of translation complex [44, 45] The ratio of eIF4E/4E-BP is important for controlling translation of eIF4E-sensitive mRNA . In this experiment, eIF4EBP1’s significant downregulation would be consistent with an increase in cell size in CYP2S1 depleted cells, since increased protein synthesis is required for cell growth.
We propose that elevated PGE2 observed in response to CYP2S1-mediated modulation of the cyclooxygenase pathway , may stimulate the mTOR pathway to promote cell growth. Recently, PGE2 was shown to promote phosphorylation and activation of Akt through the EP4 receptor in prostate cancer cells . Furthermore, mitogen-stimulated activation of PI3K and Akt have been linked to the activation of the mammalian target of rapamycin complex I (mTORC1) . mTORC1 kinase activity, in turn, stimulates protein synthesis through the phosphorylation and inactivation of eIF4EB-P. Further experimentation is required to test whether CYP2S1-mediated changes in the cyclooxygenase pathway [22, 23], stimulating PGE2 synthesis, is the pathway linking CYP2S1-mediated metabolism to mTOR signaling and regulation of cell size in bronchial epithelial cells.
The data presented in this article represent a novel trancriptomic approach to identify the mode of action for alterations in orphan P450 expression. Although, a transcriptomic approach, alone, is not sufficient to identify endogenous substrates, it can provide clues into the biological significance of CYP2S1-mediated metabolism, and/or compensation for these metabolic shifts. Moreover, it can shed light on potentially novel important physiological pathways. Transcriptome findings of CYP2S1 depleted cells are consistent with previously published cellular phenotypes . The results also identified differential expression of genes involved in the mTOR pathway, a master regulator of cell size, which resulted in the identification of a novel, measurable phenotype (specifically, increase in cell size) in CYP2S1-depleted cells. Additionally, transcriptomic analysis was consistent with proposed endogenous lipid substrates as well as compensatory metabolic shifts in CYPs involved in lipid metabolism. Although this approach provides an excellent way of examining mode of action, it fails to identify specific chemical substrates and metabolic products. Conversely, a metabolomic approach identifies the metabolites but does not establish the physiological impact. Ultimately, a multi-omic approach integrating both transcriptomic and metabolomic analysis, may provide a powerful predictive approach to elucidate endogenous substrates as well as biological significance of orphan P450’s.
As indicated in our previous publication  we used the clones with the most significant difference in CYP2S1 expression for RNA-sequencing analysis (i.e. SCRAM#1 and 759#7). BEAS-2B CYP2S1 depleted cells (759 and 984) and scrambled controls (SCRAM) cells were grown in a six well plates to confluence and washed with PBS. RNA was isolated according to the manufactures protocol (Qiagen RNeasy) and eluted in 20ul of RNAse-free water. RNA quality was analyzed using the Bioanalyzer (Agilent 2100 Bioanalyzer with Agilent RNA 6000 Nano reagents) and samples with RNA integrity number (RIN) between 9 and 10 were sent to the National Center for Genomics Research (NCGR, Santa Fe, NM) for Illumina RNA sequencing or stored for qRT-PCR validation. In total, 3 biological replicates from each genotype (759#7, 984#1, and SCRAM#1) were assessed.
Library preparation and sequencing
Messenger RNA for 759 and SCRAM samples was isolated from total RNA samples with polyA selection, size selected and prepared into sequencing libraries with the TruSeq RNA sample preparation workflow from Illumina (San Diego, CA). Libraries were sequenced on the Illumina HiSeq 2000 platform, generating 141,896,712 1×50 nt single-end reads, averaging 11,824,726 reads per sample. Raw Illumina reads are available at the Sequence Read Archive at NCBI under the accession: SUB278513.
Read count based expression
For each sample, raw sequence reads were filtered and aligned to the human reference genome (GRCh37) with GSNAP . Subsequent management of the data was performed by Alpheus . Reads that aligned unambiguously to the human reference were binned based on annotated gene coordinates and summated to estimate expression level. Read count based expression estimates were evaluated for transcriptome-wide qualitative differences with JMP Genomics 6.0. Quantitative differential gene expression analysis was performed with the negative binomial test as implemented in the Bioconductor package DESeq . Genes were identified as differentially expressed if they had an adjusted (Benjamini-Hochberg False Discovery Rate (FDR) method for multiple testing correction) p-value of 0.05 or less.
Quantitative PCR analysis
cDNA was synthesized, using iScript reverse transcription supermix (BioRad, Hercules, CA), from 1 μg total RNA. Quantitative qRT-PCR was conducted using IQ sybr green supermix (BioRad) in accordance with manufacturers instructions, and performed using the BioRad CFX96. Primer efficiencies were calculated using standard curves obtained from plasmid-based amplicons. The housekeeping gene ACTB was selected based on consistency between samples, and samples were normalized to its expression. The qRT-PCR primers used as previously described .
Pathway and functional analysis of differentially expressed genes
To gain biological insight and contextualized differentially expressed genes (as described above), we further analyzed genes that were significantly altered with adjusted p-values of either <0.001 or <0.01. These subsets included 1579 and 2766 total genes, respectively. The open source database for annotation, visualization and integrated discovery (DAVID version 6.7) [28, 29] was employed to assess biological function of the selected genes. The functional annotation tool was used to cluster genes based on the degree of association. Functional annotation was set at high stringency and restricted to groups with an enrichment score greater than or equal to 1.3. Gene ontology (GO) terms and KEGG pathway were reported. Functional annotation tool is mainly provided the most relevant gene ontology terms (GO terms) associated with the gene list and we reported the mostly enriched GO term, which is GOTERM_BP_FAT. Additionally we looked into highly affected KEGG pathways with the gene list submitted.
Cell size determination
BEAS-2B cells were grown in 6 well plates to approximately 80% confluence, trypsinized, and cell diameter and volume were quantified using the Millipore scepter automated cell counter. Trypsinized cellular populations were gated between 10-24 μM to ensure that in-tact cells were identified. The final cell size represents the average cell size from a total of six biological replicates from each genotype.
PCR pathway array
The human mTOR RT profiler PCR pathway array (SABioscience Qiagen, Cat.no.330231 PAHS-098ZD) was used to validate mTOR regulation identified through RNA seq. RNA was isolated and analyzed as indicated above. cDNA was synthesized (Qiagen, RT first strand kit) and the PCR array was performed according to the manufactures protocol (Qiagen, Cat.no. 330231 PAHS-098ZD). PCR was run on the BIORAD CFX96 real-time cycler and data was analyzed using SABiosciences Excel based PCR array data analysis template (http://www.sabiosciences.com/pcrarraydataanalysis.php). The threshold cycle (CT) values, obtained through PCR, were exported to the SABiosciences Excel template. The ∆CT was calculated for each gene in relation to house keeping genes and ∆∆CT was calculated relative to the control sample (SCRAM). Based on the ∆∆CT values fold change (2(-∆∆CT)) was reported.
Cytochrome P450 2S1
Bronchial epithelial cells
- RNA seq:
Short Hairpin RNA
all trans Retinoic Acid
β - actin
Kyoto Encyclopedia of Genes and Genomes
Chinese Hamster Ovary cells
Cellular Retinoic Acid Binding Protein
Fatty Acid Binding Protein
Retinoic Acid Receptor
Peroxisome Proliferating Receptor
DEP domain containing mTOR-interacting protein
Eukaryotic Initiation Factor 4E binding protein 1
False Discovery Rate
Quantitative Real Time Polymerase Chain Reaction.
Funding for the RNA-sequencing and transcriptome analysis was provided through an NCGR NM-INBRE Sequencing Pilot Project Award (A.M.R). NCGR's work on this project was supported by the National Institute of General Medical Sciences (8P20GM103451-12). Regulation of CYP2S1 expression is funded by an NIH SCORE grant (1SC2CA179928-01) (A.M.R.).
The authors would also like to thank Immo Hansen and Jiannong Xu for critical reading of the manuscript, Patricia Gomez for technical assistance on cell size measurements, and Faye Schilkey (NCGR) for the support of the project.
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