Sphingosine kinase 1 is required for myristate-induced TNFα expression in intestinal epithelial cells

Journal Pre-proof Sphingosine kinase 1 is required for myristate-induced TNF␣ expression in intestinal epithelial cells Songhwa Choi, Justin M. Snider, Chris P. Cariello, Johana M. Lambert, Andrea K. Anderson, L. Ashley Cowart, Ashley J. Snider

PII:

S1098-8823(20)30016-2

DOI:

https://doi.org/10.1016/j.prostaglandins.2020.106423

Reference:

PRO 106423

To appear in:

Prostaglandins and Other Lipid Mediators

Received Date:

19 July 2019

Revised Date:

14 January 2020

Accepted Date:

27 January 2020

Please cite this article as: Choi S, Snider JM, Cariello CP, Lambert JM, Anderson AK, Cowart LA, Snider AJ, Sphingosine kinase 1 is required for myristate-induced TNF␣ expression in intestinal epithelial cells, Prostaglandins and Other Lipid Mediators (2020), doi: https://doi.org/10.1016/j.prostaglandins.2020.106423

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Title: Sphingosine kinase 1 is required for myristate-induced TNFα expression in intestinal epithelial cells Authors and Affiliations: Songhwa Choi1, Justin M. Snider1,2, Chris P. Cariello3, Johana M. Lambert4,5, Andrea K. Anderson4,5, L. Ashley Cowart4,6 ,Ashley J. Snider1,2,7*[email protected]

1Department

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of Medicine and Stony Brook Cancer Center, Stony Brook University, Stony Brook, NY 11794, USA 2Department

of Nutritional Sciences, College of Agriculture and Life Sciences, University of Arizona, Tucson, AZ 85721, USA of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY 11794,

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3Department

USA 4Department

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of Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, VA 23284, USA 5Department

Holmes McGuire Veterans’ Affairs Medical Center, Richmond, VA 23249, USA

7Northport

Veterans Affairs Medical Center, Northport, NY 11768, USA

*Corresponding Author:

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Ashley J. Snider

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6Hunter

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of Biochemistry & Molecular Biology, Medical University of South Carolina, Charleston, SC 29425, USA

1230 N. Cherry Avenue

BIO5 Institute, BSRL 372 Tucson, AZ, 85721

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Phone: +1-520-621-8093

Abstract:

Saturated fatty acids (SFA) have been known to trigger inflammatory signaling in metabolic tissues; however, the effects of specific SFAs in the intestinal epithelium have not been well studied. Several previous studies have implicated disruption in sphingolipid metabolism by oversupply of SFAs could affect the inflammatory process. Also, our previous studies have implicated sphingosine kinase 1 (SK1) and its product sphingosine-1-phosphate (S1P) as having key roles in the regulation of inflammatory processes in the intestinal epithelium. Therefore, to define the role for specific SFAs in inflammatory responses in intestinal epithelial cells, we

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examined myristate (C14:0) and palmitate (C16:0). Myristate, but not palmitate, significantly induced pro-inflammatory cytokine tumor necrosis factor α (TNFα), and it was SK1-dependent. Interestingly, myristate-induced TNFα expression was not suppressed by inhibition of S1P receptors (S1PRs), hinting at a potential novel intracellular target of S1P. Additionally, myristate regulated the expression of TNFα via JNK activation in an SK1-dependent manner, suggesting a novel S1PR-independent target as a mediator between SK1 and JNK in response to myristate. Lastly, a myristate-enriched milk fat-based diet (MFBD) increased expression of TNFα in colon tissues and elevated the S1P to sphingosine ratio, demonstrating the potential of myristateinvolved pathobiologies in intestinal tissues. Taken together our studies suggest that myristate regulates the expression of TNFα in the intestinal epithelium via regulation of SK1 and JNK.

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Keywords: Saturated fatty acid, myristate, intestinal inflammation, sphingosine kinase, MAPK

Introduction

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Saturated fatty acids (SFAs) from a high fat diet (HFD) have emerged as key molecules contributing to low-grade systemic inflammation. Excessive SFAs induce pro-inflammatory signaling in many cell types, including skeletal muscle cells, hepatocytes and adipose tissue macrophages, resulting in enhanced expression of cytokines such as tumor necrosis factor α (TNFα) and interleukin-6 (IL-6) (1-4). While SFA/HFD-induced inflammation has been studied extensively, most of these studies have focused on metabolic disorders such as insulin resistance caused by low-grade chronic inflammation. Correlations between HFD and colitis have been established; however, the specific fats and the mechanisms by which they alter or exacerbate colon inflammation in in vivo models is not clear (5-7). A systematic review evaluating the association between food intake and risk of inflammatory bowel disease (IBD) demonstrated that high intake of dietary fat is associated with increased risk of ulcerative colitis (8). These studies indicate that SFAs could be associated with inflammatory pathobiologies in intestinal tissues.

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Sphingolipids have been identified as bioactive lipids that regulate various functions in cells and tissues. SFAs are substrates for several sphingolipid enzymes including ceramide synthases (CerS) and serine palmitoyltransferases (SPT), and have been shown to alter sphingolipid metabolism [reviewed in (9)]. Specifically, ceramide has been shown to be increased by both HFD and SFAs (10-13). Sphingosine and sphingosine-1-phosphate (S1P) have also been shown to increase in plasma, liver and skeletal muscle, in vivo and in cultured cells, upon HFD or SFA administration (3, 4, 14-18). There are two enzymes that generate S1P, sphingosine kinase 1 and 2 (SK1 and SK2). Increases in S1P in response to HFD/SFA in skeletal muscle have been shown to primarily involve sphingosine kinase 1 (SK1), and not SK2 (3); however, liver from HFD-fed rats demonstrated increase of expression and activity of SK2, rather than SK1 (19). These studies suggest SK1 and SK2 could have distinct roles and regulation in different tissues in response to HFD. It has been well-documented that sphingolipids are key mediators in regulation of intestinal inflammatory responses. Specifically, we showed increased expression of SK1 in colon mucosa

from UC patients and protection from DSS-induced colitis in SK1 deficient mice (20). Moreover, pharmacologic inhibition of SK1 decreased colonic inflammation and suppressed recruitment of neutrophils in a DSS-induced colitis (21). Work by Liang et al. also, showed that loss of SK2 exacerbated DSS-induced colitis, partially via a compensatory increase in SK1 (22). Together these studies demonstrate a critical role for SK1/S1P in colon inflammation,

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S1P is present in low nanomolar concentrations in the cell and has high affinity for S1P receptors (S1PRs), giving significance as a signaling molecule. Although S1P is known to exert effects through S1PRs which are G protein-coupled receptors, intracellular S1P has also been reported to exert receptor-independent actions, such as regulation of NF-κB signaling through TNF receptor-associated factor 2 (TRAF2) (23) and enhanced gene transcription by direct binding to histone deacetylase 1/2 (HDAC1/2) (24). In addition, S1P can also mediate activation of thapsigargin-induced ER stress and further induce pro-inflammatory responses in keratinocytes by directly binding to the ER chaperone protein GRP94. This in turn promotes recruitment of TRAF2 to IRE1α and subsequent activation of NF-κB signaling (25). These studies suggest that S1P could regulate cellular functions through various receptor independent targets, as well as S1PRs.

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In this study, we examined the involvement of SK1 in SFA-induced inflammatory responses in IECs. Myristate significantly induced TNFα and COX2 expression in an SK1dependent manner. We further provide evidence that myristate-induced TNFα expression may be S1PR independent and due to activation of JNK signaling. Also, mice fed a milk-fat based diet (MFBD), which is high in myristate, exhibited increased TNFα expression in colon tissues. Together this study suggests that myristate-induced inflammatory responses in intestinal epithelium may be regulated in part by SK1, but not by S1PRs.

Reagents and materials

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Methods

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DMEM, penicillin-streptomycin, Pierce ECL substrate and BCA assay kit were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Fetal bovine serum (FBS) was from GE healthcare life sciences (Logan, UT, USA). Reagent grade (fatty acid-free) BSA was from Proliant Biologicals (Ankeny, IA, USA). Myristate, palmitate, myriocin and SP600125 were from Sigma Aldrich (St. Louis, MO, USA). VPC23019 was purchased from Tocris Bioscience (Bristol, United Kingdom). JTE-013 and ABC294640 were from Cayman Chemical (Ann Arbor, MI, USA). C17SPH and C17-DHS (internal standards for lipid measurement) were purchased by the Lipidomics Core Facility at Stony Brook University Medical Center from Avanti Polar Lipids (Alabaster, AL).

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Animals and diets

In all studies, C57BL/6 male and female mice (Jackson Laboratory, Bar Harbor, ME, USA) were given water and chow ad libitum. High fat diet (HFD, 60% of calories from fat, TD.09766) and an isocaloric low fat diet (16.8% of calories from fat, TD.08810) were purchased from Envigo, Inc. (Indianapolis, IN, USA). For the HFD studies, six 6-week-old male mice were placed in each group and maintained on the diets for a total of 18 weeks. At 18 weeks colons were isolated, snap-frozen, and homogenized for analysis. All animal procedures were approved by the Ralph H Johnson VA Medical Center and Medical University of South Carolina Institutional Animal Care and Use Committees (IACUC) and all studies conducted were based on NIH and the American Veterinary Medical Association guidelines.

Cell culture

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IEC6 rat intestinal epithelial cells and HCT116 human colorectal carcinoma cells were originally purchased from American Type Culture Collection (Manassas, VA, USA). DMEM medium was supplemented with 10% (v/v) FBS and 1% (v/v) penicillin-streptomycin antibiotics (growth media) for HCT116. 0.1 unit/ml bovine insulin (Sigma Aldrich) was also added to growth media for IEC6. Cells were kept in a humidified incubator at 37 °C with 5% CO2. Cells were seeded and treated as needed 24 h after plating. Cells were grown to a final confluence of ~80%. Prior to SFA treatment cells were serum-starved for 8 h. Pre-treatments with pharmacological inhibitors were carried out 1 h prior to the addition of SFAs unless otherwise indicated.

SFA treatment

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SFAs were prepared as described previously (12). Briefly, SFAs were dissolved in 100% ethanol to a concentration of 100mM, aliquoted and dried under nitrogen. Prior to cell treatment, SFAs were reconstituted to designated concentrations using DMEM supplemented with 2% fatty acidfree, low endotoxin-BSA. SFAs were conjugated to BSA via brief sonication, incubation at 55°C for 15 minutes, and were then cooled to 37°C. Prior to SFA treatment, cells were serum-starved for 8 h and then the media changed to media containing SFAs conjugated to BSA for the indicated times.

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Small interference RNA

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24 h after plating, cells were transfected with small interference RNA (siRNA) using Lipofectamine RNAiMAX (Thermo Fisher Scientific) according to the manufacturer’s protocol. 48 h posttransfection, media were changed, and cells were treated as indicated. siSK1 for rat (ID: s139426) and siSK1 for human (ID: s16957) Silencer Select siRNA (Thermo Fisher Scientific) were used in this study. AllStar Negative Control siRNA (Qiagen, Hilden, Germany) was used as a negative control.

Mass spectrometry for sphingolipid analysis

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For lipid extraction, cells were washed with ice-cold PBS, then directly lysed with 2 ml cell extraction mixture (2:3 70% isopropanol/ethyl acetate) followed by gentle scraping of the cells from the culture plate. Colon tissues were directly homogenized in cell extraction mixture using FastPrep-24 from MP Biomedicals (Santa Ana, CA, USA). The lysate and homogenate then were transferred to 15 ml Falcon tubes. The lipid samples were spiked with C17-SPH and C17-DHS (internal standards, 50pmol) and extracts were then analyzed by the Lipidomics Core Facility at Stony Brook University Medical Center, as described previously (26). Data were normalized by total lipid phosphate (Pi) present in the organic phase of the Bligh and Dyer extraction (27) detected by phosphomolybdate assay (28). Sphingolipid levels were expressed as pmol/nmol Pi.

RNA extraction and Quantitative real-time PCR

RNA extraction was performed using the PureLink RNA Mini Kit (Thermo Fisher Scientific) following the manufacturer’s protocol. One μg of RNA was then used for cDNA synthesis using the qScript cDNA SuperMix (Quantabio, Beverly, MA, USA) according to the manufacturer’s protocol.

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Real-time PCR was carried out using the Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). iTAQ master mix was purchased from Bio-Rad Laboratories (Hercules, CA, USA). The following TaqMan probes (Thermo Fisher Scientific) were used: rat TNFα (ID: Rn01525859_g1), rat COX2 (ID: Rn01483828_m1), rat SK1 (ID: Rn00682794_g1), human TNFα (ID: Hs01113624_g1), human SK1 (ID: Hs01116530_g1), human COX2 (Hs00153133_m1), mouse TNFα (ID: Mm00443260_g1), mouse SK1 (ID: Mm00448841_g1) and rat β-actin (ID: Rn00667869_m1), human RPLP0 (ID: Hs99999902_m1) and mouse β-actin (ID: Mm02619580_g1) were used as a housekeeping gene. Cycle threshold (Ct) values were obtained for each gene of interest and β-actin. ΔCt values were calculated, and the relative gene expression normalized to control samples was calculated from ΔΔCt values.

Immunoblot analysis

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Cells were washed with ice-cold PBS and then directly lysed in cold RIPA buffer containing protease inhibitor cocktail and phosphatase inhibitor cocktail (Sigma-Aldrich). Equal amounts of protein (20 μg) were boiled in 2x Laemmli Sample Buffer (Bio-Rad Laboratories) and separated by SDS-PAGE (4–15%, Tris-HCl) using the Bio-Rad Criterion system. Separated proteins were then transferred onto nitrocellulose membranes (Bio-Rad Laboratories) and blocked with 5% nonfat milk in PBS-0.05% Tween-20 for at 1 hour at room temperature. Anti-phospho-ERK, antiERK, anti-phospho-JNK, anti-JNK, anti-phospho-p38, anti-SK1 and anti-GAPDH were from Cell Signaling Technology (Danvers, MA, USA). Anti-COX2 was obtained from BD Biosciences (Franklin Lakes, NJ, USA). Horseradish peroxidase-labeled secondary antibodies were from Jackson ImmunoResearch (West Grove, PA, USA). Primary antibodies diluted 1:1000 or 1:5000 for GAPDH were then added to membranes and incubated at 4°C overnight. Membranes were washed 3 times with PBS-0.05% Tween-20 and then incubated with diluted 1:5000 horseradish peroxidase conjugated secondary antibodies for 1 hour at room temperature. Membranes were washed 3 times with PBS-0.05% Tween-20 and then incubated with Pierce ECL Substrate and exposed to X-ray films (Thermo Fisher Scientific) that were then processed and scanned.

Statistical analysis

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Statistical analysis was performed using GraphPad Prism (GraphPad Software). Data are presented as mean ± S.E.M. and were analyzed by two-tailed, unpaired Student’s t test or twoway analysis of variance with a Bonferroni post-test to correct for multiple comparisons, as appropriate. p<0.05 was considered statistically significant. Results

Myristate induces TNFα expression SFAs have been reported to trigger inflammatory responses, including TNFα expression, in various cell types and tissues. TNFα is a key pro-inflammatory cytokine which is involved in pathobiology of IBD. In this study, we examined the different effect of SFAs in TNFα expression in intestinal epithelial cells, using two common dietary FAs, myristate and palmitate. Cells were

treated with either vehicle (2% fatty acid-free, low endotoxin-BSA), 0.6mM myristate or palmitate for up to 24h. Interestingly, only myristate significantly increased TNFα expression with a maximal response at 16h (Figure 1A). Additionally, at 16h, TNFα expression increased in a dosedependent manner upon myristate treatment (Figure 1B). These data suggest that myristate, not palmitate, induces inflammatory response in IECs.

SK1 is required for myristate-induced S1P and TNFα

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The role of SKs and their products S1P and dihydro-S1P (dhS1P) in inflammatory responses has been studied in cells and in vivo. The role for S1P in inflammatory responses is controversial, yet several studies have reported that S1P functions as a pro-inflammatory mediator in various cell types (20, 22, 29). These studies suggest the potential involvement of SKs and (dh)S1P in myristate-induced TNFα. Therefore, we determined cellular S1P levels following myristate treatment. Myristate significantly and steadily increased S1P levels in IEC6 cells after treatment (Figure 2A); however, dhS1P levels were decreased (Figure 2B). These data hint that S1P, and not dhS1P, could be a play a role in the regulation of TNF upon myristate treatment.

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As there are 2 isoforms of SK capable of generating S1P and they are known to have different roles in the regulation of inflammation, we assessed the role for each isoform in myristate-induced TNFα expression. We initially adopted siRNA for SK1 and SK2 to answer this question. siRNA for SK1 decreased SK1 expression by approximately 70% (Figure 2C); however, siSK2 basally induced cell death (data not shown). Therefore, to examine the role for SK2 we utilized ABC294640, a pharmacological inhibitor for SK2. Interestingly, siSK1 abolished myristate-induced TNFα in IEC6 cells (Figure 2D), but ABC294640 failed to do so (Figure 2E). To substantiate these data, we utilized siRNA for SK1 in HCT116 colon cancer cells. Knockdown of SK1 also inhibited myristate-induced TNFα expression in these cells (Figure 2F&G). Also, the induction of S1P by myristate was suppressed by siSK1 (Figure 2H). These results suggest that SK1, not SK2, is required for myristate-induced TNFα expression and S1P generation.

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In addition to SKs, S1P lyase (SGPL1), the enzyme that irreversibly degrades S1P, can drastically alter cellular levels of S1P. To evaluate the effect of SGPL, and potentially augmented S1P in myristate-induced TNF we used siRNA for SGPL1. Unexpectedly, SGPL1 knockdown did not augment the effect of myristate-induced TNFα expression, despite a robust increase in basal intracellular S1P levels (Supplemental Figure 1A&B). These data suggest one of two possibilities; 1) myristate-induced TNFα is regulated by a specific pool of S1P, or 2) SK1, but not S1P, is required for myristate-induced TNFα expression.

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Although palmitate is the primary fatty acid to be utilized by SPT producing a d18sphingoid backbone, myristate can also be incorporated forming the d16-sphingoid backbone through de novo synthesis (30). Previous studies demonstrated that C16-dihydrosphingosine (dhSPH) has potent effects in cardiomyocytes (10), implicating that the inflammatory response by myristate may be due to C16-S1P instead of conventional C18-S1P. As expected, myristate significantly increased C16-S1P, but C16-dhS1P was not changed (Table 1). We utilized the SPT inhibitor, myriocin, as the d16-sphigoid backbone is generated only by de novo synthesis to determine the role of C16-S1P. Myriocin suppressed the generation of C16-S1P (Table 1); however, it did not inhibit myristate-induced TNFα expression (Supplemental Figure 2). These data suggest that C16-S1P from de novo synthesis may not be responsible for myristate-induced TNFα expression.

S1PR antagonists did not alter myristate-induced TNFα expression

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SK1-dependent TNFα expression requires JNK

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S1P generated by SKs is typically secreted into the extracellular space and exerts its function via S1PRs. To further define the potential role for S1P in myristate-induced TNFα, we administered exogenous S1P. Interestingly, treatment with exogenous 100nM S1P at 4, 8 and 16 hours did not appear to alter TNFα expression (Figure 3A), suggesting that myristate-induced S1P may not function via plasma membrane S1PRs. In order to determine if secreted S1P played a role in the TNFα response through its receptors, we examined the involvement of S1PRs in myristate-induced TNFα expression. While S1PRs have five isoforms, only 3 (S1PR1, S1PR2 and S1PR3) are predominantly expressed in epithelial cells (31). Therefore, we used VPC23019 to inhibit S1PR1 and S1PR3 and JTE-013 to antagonize S1PR2. Prior to performing the experiment with myristate, these antagonists were validated for efficacy. S1P has been shown to phosphorylate ERK through S1PRs; therefore, we examined ERK phosphorylation by S1P with and without pre-treatment with the antagonists. These antagonists suppressed the phosphorylation of ERK by S1P, demonstrating that they were effective in inhibiting S1PRs in IECs (Figure 3B). However, these antagonists did not disrupt myristate-induced TNFα expression (Figure 3C). These data suggest that myristate-induced TNFα is an S1PR-independent process, and possibly an S1P-independent process, although regulated in an SK1-dependent manner.

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NF-κB signaling is a well-known upstream regulator of TNFα expression. To identify the molecular mechanism by which myristate to induce TNFα, we analyzed the activation of NF-κB signaling upon myristate treatment. Unexpectedly, myristate did not induce translocation of p65 to nucleus in short term treatment or in long term treatment (Supplemental Figure 3). These results demonstrate that myristate-induction of TNFα does not involve NF-κB.

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MAPK signaling has also been shown to regulate of TNFα expression through AP-1 activation. Thus, we examined the regulation of MAPKs upon myristate treatment. JNK was significantly phosphorylated between 8 and 16 hours of myristate treatment, whereas phosphorylation of p38 and ERK with myristate were not different from vehicle (Figure 4A). To determine the involvement of JNK in myristate-induced TNFα expression, we utilized the JNK inhibitor, SP600125. SP600125 significantly suppressed expression of TNFα induced by myristate (Figures 4B). These data suggest the involvement of JNK in TNFα expression upon myristate treatment. Additionally, we explored the relationship between JNK and SK1, as both were required for the expression of TNFα upon myristate treatment. As shown in Figure 4C, phosphorylation of JNK upon myristate treatment was suppressed by knockdown of SK1. Phosphorylation of JNK in SK1 knockdown cells was decreased at the basal level in negative control siRNA treated cells, hinting that SK1 may be involved in basal regulation of JNK (Figure 4C). Similarly, SK1 knockdown also abolished myristate-induced JNK phosphorylation in HCT116 cells (Figure 4D). Together, these data suggest SK1 regulates TNFα expressing via JNK.

Myristate-induced COX2 is regulated by SK1 Cyclooxygenase 2 (COX2) is a key enzyme in inflammatory responses induced by proinflammatory cytokines, including TNFα (32). Therefore, we investigated the effect of myristate

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and involvement of SK1 in COX2 expression. Expression of COX2 mRNA was significantly augmented by myristate treatment with a maximal increase at 16h (Figure 5A). Indeed, at the 16h timepoint, myristate-induced COX2 expression was dose dependent (Figure 5B), demonstrating a similar response as that observed for TNFα expression with myristate (Figure 1). Next, we determined the involvement of SK1 with siRNA. Knockdown of SK1 notably abolished myristate-induced COX2 mRNA (Figure 5C), as well as protein expression (Figure 5D). COX2 expression was basally reduced in SK1 knockdown cells, similar to JNK phosphorylation (Figure 4C and 5D), suggesting that SK1 may basally suppress inflammatory responses in IECs. COX2 mRNA expression was significantly increased by myristate in HCT116 cells, and similar to IEC6 cells was suppressed by SK1 knockdown (Figure 5E). These data indicate that myristate may regulate inflammatory responses through SK1, including TNFα and COX2 expression.

Myristate-enriched HFD significantly increases TNFα expression in colon tissues

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To explore the effects of SFA on TNFα expression and regulation of SK1 in vivo in intestinal tissues, we maintained mice on a milk-fat based diet (MFBD), where C14:0 myristate is a major SFA in the diet (11), or an isocaloric low-fat control diet (CD) for 18 weeks. We then measured the expression of TNFα and SK1, as well as sphingolipid levels. TNFα mRNA expression was significantly increased in colon tissues from MFBD-fed mice (Figure 6A). mRNA expression of SK1 was not different between two groups (Figure 6B). However, relative S1P level to sphingosine was significantly increased in colon tissues from MFBD-fed mice (Figure 6C), hinting that SK activity may be enhanced in response to dietary myristate. Together these data demonstrate that a diet high in myristate (MFBD) results in increased TNFα expression, further suggesting a potential inflammatory role for SK1, protein or activity in intestinal tissues.

Discussion

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It has been shown that SFAs induce low level of inflammatory responses, and we and others have shown that SK1 is a key mediator in colon inflammation. Therefore, we set out to determine the relationship of SFA-induced inflammatory responses in intestinal epithelial cells and the potential involvement of SK1. In the present study, we show that myristate, but not palmitate, treatment of IECs resulted in expression of TNFα in an SK1-dependent manner. Interestingly, this did not require S1PRs. In an effort to elucidate the mechanism by which myristate induced TNFα expression in IECs, we demonstrated that JNK was activated by myristate in an SK1-dependent manner and required for TNFα expression. An additional downstream target and inflammatory signaling molecule, COX2, was also increased by myristate, in an SK1-dependent manner (summarized in Figure 7). In addition, our in vivo study with a myristate-enriched MFBD augmented TNFα expression and S1P levels, suggesting that myristate, and potentially myristate-mediated SK protein or activity, may be critical in the regulation of inflammatory responses upon HFD in colon tissues.

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Palmitate is well-known to induce pro-inflammatory responses in several cell types. We have reported that myristate, but not palmitate, enhanced expression of IL6 in IECs (12). In that work, we demonstrated that this increase in IL6 is dependent on myristate-specific regulation of C14-ceramide via CerS5/6. However, TNFα expression upon myristate treatment was not abolished in CerS5 or 6 knockdown cells (Supplement Figure 4). Our data therefore suggest that expression of TNFα and IL6 are regulated by separate mechanisms, and that SK1, and not CerS5/6 and C14-ceramide, are critical in TNFα regulation.

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Like IL6, TNFα induction was also a myristate specific response (Figure 1A); however, S1P levels were induced by both myristate and palmitate (Table 2), but unlike myristate, palmitate significantly increased dhS1P as well (Table 2). These data suggest that palmitate may promote de novo sphingolipid synthesis, while myristate likely results in S1P generation via the salvage pathway. Together our studies suggest that SK1, but not SK2, is required for myristate-induced TNFα induction and suggest the possibility that the SK1 protein and not S1P are required for this induction of TNFα.

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Many of the functions of S1P are attributed to its binding to one of five S1PRs. It has been shown that S1P generally exerts its function via these receptors in paracrine and/or autocrine manner. However, a few intracellular (S1PR-independent) targets of S1P have been suggested. In sphingolipid metabolism, S1P inhibits CerS2 by directly binding to a regulatory site of this enzyme, which may result in negative regulation of ceramide synthesis (33, 34). In addition, S1P directly binds to heat shock proteins (GRP94 and HSP90α) to increase ER stress responses and further activate innate immune responses (25). Our study showed myristate-induced TNFα expression was SK1 dependent, and S1PR independent, suggesting the involvement of additional S1P and/or SK1 targets. Moreover, recently Lee et al. reported that neuronal SK1 has acetyltransferase activity, which promotes the acetylation of COX2 (35). This study suggests that SK1 may regulate proteins directly independent of S1P. Our data showed that the expression of TNFα and COX2 were SK1 dependent (Figure 2&5), but it was not affected by cellular S1P levels with siRNA of SPGL1 (Supplemental Figure 1) or exogenous treatment of S1P (Figure 3A). These data suggest that SK1 may mediate inflammatory responses in an S1P and S1PR-independent manner in the intestinal epithelium, via a yet to be defined mechanism With respect to the role for SKs in inflammation, SK1 has been shown to play a more significant role in inflammation, as compared to SK2. Knockdown of SK1 alleviated inflammatory

responses with less systemic pro-inflammatory cytokines in collagen-induced arthritis model, but down regulation of SK2 with siRNA exacerbated the inflammatory conditions (36). Moreover, in colon inflammation, SK1 deletion protected mice from DSS-induced colitis with reduced expression of COX2 (20), while SK2 deletion significantly aggravated DSS-induced colitis and colitis-associated cancer (CAC) (22). In this study, to examine the role of SK2 in SFA-induced inflammatory response, we examined the siRNA of SK2 at first. However, knockdown of SK2 triggered cell death in IEC6 cells basally (data not shown). In addition, mRNA expression of SK2 was relatively and significantly higher than SK1 in these cells at basal level (Supplement Figure 5). These data suggest SK2 could be a more abundant isoform of SK in intestinal epithelial cells with potential cytoprotective or housekeeping functions, suggesting that SK1 is the more regulated by external stimuli and regulates stress-responses.

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In our previous studies, myristate significantly induced the pro-inflammatory cytokine IL6 through the IRE1α-XBP1 pathway in ER stress (12). Furthermore, IRE1α is known to activate JNK in response to ER stress (37), suggesting that myristate-induced TNFα may also be regulated by myristate-induced ER stress. However, unlike IL6, myristate-induced TNFα was not suppressed in IRE1α siRNA treated cells (Supplement Figure 6). Also, our preliminary data showed that myristate-induced phosphorylation of IRE1α was not abolished by SK1 knockdown (data not shown). Collectively these data demonstrate the regulation of TNFα upon myristate is likely separate from the processes involved in myristate-induced ER stress.

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Grant support

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In conclusion, this study demonstrates that myristate induced pro-inflammatory molecules, TNFα and COX2 expression, in intestinal epithelial cells. This inflammatory response required SK1 and JNK, but not S1PRs. Moreover, myristate-induced phosphorylation of JNK was inhibited by knockdown of SK1, which suggests SK1 as an upstream regulator of JNK. Together, these data suggest a myristate-specific inflammatory response in the intestinal epithelium, as well as the prospect of novel S1PR and/or S1P independent target(s) linking SK1 and JNK.

This work was supported by multiple grants and assistance programs: Veteran’s Administration Career Development Award (AJS); NCI P01 CA097132 (AJS); NIH R01 HL117233 and the Department of Veteran’s Affairs Merit grant I01BX000200 (LAC). The contents do not represent the views of the U.S. Department of Veterans Affairs or the United States Government.

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Author contributions

SC wrote the manuscript, performed conceptual and experimental design, as well as generation of data and analyses for cell culture and in vivo experiments. JMS performed lipid analyses from cell culture experiments. CC performed protein analyses from cell culture experiments. JML and AKA performed the in vivo HFD experiments and collected tissues for analysis. LAC contributed to experimental design, data analysis and manuscript editing. AJS conceived the original hypothesis, provided the necessary funding for materials and methods, and supervised the whole project.

Disclosures All authors declare that they have no conflicts of interest.

ACKNOWLEDGEMENTS.

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The authors would like to thank Lina M. Obeid for her expertise and suggestions for examining SKs; and Yusuf A. Hannun for his expert advice in sphingolipid biology. We also thank the Stony Brook University Lipidomics Shared Resource Core for lipid analysis.

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Figure legends

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Figure 1. Myristate elevates TNFα expression in intestinal epithelial cells. IEC6 cells were: A) treated with 0.6mM myristate (MYR), palmitate (PAL) or vehicle (VEH, 2% BSA) for the indicated times; B) treated with indicated concentration of MYR, PAL or VEH for 16h. mRNA expression TNFα was analyzed by qRT-PCR and normalized to β-actin. Data represent mean ± SEM, n=3, *p<0.05 and ***p<0.001 as compared to VEH treatment.

of ro -p re lP ur na Jo Figure 2. Myristate induces S1P and TNFα expression in an SK1-dependent manner. A&B) IEC6 cells were treated with 0.6mM MYR or VEH for indicated times. IEC6 cells were; C&D&H) transfected with 10nM AllStar negative control siRNA or SK1 siRNA or E) pre-treated with DMF

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(control) or 10μM ABC294640, and then treated with 0.6mM MYR or VEH for 16h. F&G) HCT116 cells were transfected with 10nM AllStar negative control siRNA or SK1 siRNA and then treated with 0.6mM MYR or VEH for 16h. A&B&H) Intracellular S1P and dhS1P levels were analyzed by ESI/MS/MS in the Stony Brook University Lipidomics Shared Resource Core and normalized to total lipid phosphate (Pi). C-G) mRNA expression SK1 or TNFα were analyzed by qRT-PCR and normalized to β-actin for IEC6 cells or RPLP0 for HCT116 cells. Data represent mean ± SEM, n=3, *p<0.05, **p<0.01 and ***p<0.001 as compared to VEH treatment; #p<0.05, ##p<0.01 and #p<0.05, ##p<0.01 and ###p<0.001 as compared to AllStar- or Control-MYR treatment.

of ro -p re lP ur na Jo Figure 3. S1PRs are not involved in myristate-induced TNFα expression. A) IEC6 cells were treated with 100nM S1P for the indicated times, and mRNA expression TNFα was analyzed by qRT-PCR and normalized to β-actin. B) IEC6 cells were pre-treated with DMSO, 10µM

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VPC23019 or 1µM JTE-013 for 30 min, and then treated with 100nM S1P for 5 min. Western blot analysis was performed to assess relative phosphorylation of ERK; representative blot shown for three experiments. C) IEC6 cells were pre-treated with DMSO, 10µM VPC23019 or 1µM JTE013 for 30 min, and then treated with 0.6mM MYR or VEH for 8h. mRNA expression TNFα was analyzed by qRT-PCR and normalized to β-actin. Data represent mean ± SEM, n=3, ****p<0.0001 as compared to DMSO-VEH treatment.

of ro -p re lP ur na Jo Figure 4. Myristate-induced TNFα expression in SK1- and JNK-dependent manner. IEC6 cells were: A) treated with 0.6mM MYR or VEH for indicated times; B) pre-treated with DMSO or 20µM SP600125 for 1 h, and then treated with 0.6mM MYR for 16h; C) transfected with 10nM AllStar

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negative control siRNA or SK1 siRNA, and then treated with 0.6mM MYR or VEH for 8h. D) HCT116 cells were transfected with 10nM AllStar negative control siRNA or SK1 siRNA, and then treated with 0.6mM MYR or VEH for 8h. A,C&D) Western blot analysis was performed to assess relative phosphorylation of MAPK; representative blot shown for three experiments. B) mRNA expression of TNFα was analyzed by qRT-PCR and normalized to β-actin. Data represent mean ± SEM, n=3, ****p<0.0001 as compared to DMSO VEH treatment; ###p<0.001 as compared to DMSO MYR treatment.

of ro -p re lP ur na Jo Figure 5. Myristate induces COX2 expression in and SK1-dependent manner. IEC6 cells were: A) treated with 0.6mM MYR or VEH for indicated times; B) treated with indicated concentrations of MYR for 16h; C&D) transfected with 10nM AllStar negative control siRNA or SK1 siRNA, and

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treated with 0.6mM MYR or VEH for 16h. E) HCT116 cells were transfected with 10nM AllStar negative control siRNA or SK1 siRNA, and treated with 0.6mM MYR or VEH for 16h. A-C&E) mRNA expression COX2 was analyzed by qRT-PCR and normalized to β-actin for IEC6 cells or RPLP0 for HCT116 cell. D) Western blot analysis was performed to assess relative COX2 expression; representative blot shown for three experiments (upper panel) and the expression of COX2 is quantified as OD ratio using ImageJ (lower panel). Data represent mean ± SEM, n=3, *p<0.05, **p<0.01, and ****p<0.0001 as compared to VEH treatment; ##p<0.01, ###p<0.001 and ####p<0.0001 as compared to AllStar MYR treatment.

of ro -p re lP ur na Jo Figure 6. HFD increases expression of TNFα mRNA in intestinal tissues. C57BL6 mice were fed a MBFD or an isocaloric CD for 18 weeks. A&B) mRNA expression levels of TNFα and SK1 in the colon were analyzed by qRT-PCR and normalized to β-actin. C) S1P and sphingosine level

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in the colon tissues was analyzed by ESI/MS/MS in the Stony Brook University Lipidomics Shared Resource Core and normalized to total lipid Pi, then relative levels of S1P:sphingosine were calculated. Data represent mean ± SEM, n≥9, **p<0.01 compared to CD.

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Figure 7. Proposed model for myristate-induced TNFα expression via SK1. Myristate treatment promotes S1P generation by SK1. Myristate-induced perturbation of SK1, and potentially S1P, activates JNK, which leads to TNFα expression. (Note: Red X indicates unlikely involvement of S1PRs in myristate-induced TNFα expression.)

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Table 1. C16-S1P levels following myristate treatment. C16-S1P

DMSO+VEH BDL

DMSO+MYR 1.07×10-3 ± 7.21×10-4

Myriocin+VEH BDL

Myriocin+MYR BDL

Unit: pmol/nmol of Pi BDL: Below detectable level

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IEC6 cells were pre-treated with DMSO or 100nM myriocin, and then treated with 0.6mM MYR or VEH for 16h. Intracellular C16-S1P level was analyzed by ESI/MS/MS in the Stony Brook University Lipidomics Shared Resource Core and normalized to total lipid phosphate (Pi). n=3.

Table 2. S1P and dhS1P levels upon SFA treatment. S1P dhS1P

VEH ± 1.07×10-2 -2 8.56×10 ± 1.83×10-2 1.28×10-1

MYR ± 2.00×10-2 b -2 6.53×10 ± 9.65×10-3 1.89×10-1

PAL ± 1.54×10-2 a -1 1.20×10 ± 6.77×10-3 a,c 1.77×10-1

Unit: pmol/nmol of Pi

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IEC6 cells were treated with 0.6mM MYR, PAL or VEH for 16h. Intracellular S1P and dhS1P levels were analyzed by ESI/MS/MS in the Stony Brook University Lipidomics Shared Resource Core and normalized to total lipid phosphate (Pi). n=3, a, p<0.05; b, p<0.01 as compared to VEH treatment and c, p<0.05 as compared to MYR treatment.