International Journal of Molecular Sciences

Article Effects of Sphingosine-1-Phosphate on Cell Viability, Differentiation, and Expression of Adipocytes

Xiyuan Wu 1, Meena Kishore Sakharkar 1, Martin Wabitsch 2 and Jian Yang 1,*

1 Drug Discovery and Development Research Group, College of Pharmacy and Nutrition, University of Saskatchewan, 107 Wiggins Road, Saskatoon, SK S7N 5E5, Canada; [email protected] (X.W.); [email protected] (M.K.S.) 2 Department of Pediatrics and Adolescent Medicine, Ulm University Medical Center, Eythstr. 24, 89075 Ulm, Germany; [email protected] * Correspondence: [email protected]

 Received: 29 October 2020; Accepted: 4 December 2020; Published: 5 December 2020 

Abstract: Sphingosine-1-phosphate (S1P) is a highly potent sphingolipid metabolite, which controls numerous physiological and pathological process via its extracellular and intracellular functions. The breast is mainly composed of epithelial cells (mammary gland) and adipocytes (stroma). Adipocytes play an important role in regulating the normal functions of the breast. Compared to the vast amount studies on breast epithelial cells, the functions of S1P in breast adipocytes are much less known. Thus, in the current study, we used preadipocyte cell lines SGBS and mouse preadipocyte cell line 3T3-L1 as in vitro models to evaluate the effects of S1P on cell viability, differentiation, and gene expression in adipocytes. Our results showed that S1P increased cell viability in SGBS and 3T3-L1 preadipocytes but moderately reduced cell viability in differentiated SGBS and 3T3-L1 adipocytes. S1P was also shown to inhibit adipogenic differentiation of SGBS and 3T3-L1 at concentration higher than 1000 nM. Transcriptome analyses showed that S1P was more influential on gene expression in differentiated adipocytes. Furthermore, our network analysis in mature adipocytes showed that the upregulated DEGs (differentially expressed ) were related to regulation of lipolysis, PPAR (peroxisome proliferator-activated receptor) signaling, alcoholism, and toll-like receptor signaling, whereas the downregulated DEGs were overrepresented in cytokine–cytokine receptor interaction, focal adhesion, starch and sucrose metabolism, and nuclear receptors pathways. Together previous studies on the functions of S1P in breast epithelial cells, the current study implicated that S1P may play a critical role in modulating the bidirectional regulation of adipocyte-extracellular matrix-epithelial cell axis and maintaining the normal physiological functions of the breast.

Keywords: sphingosine-1-phosphate; preadipocyte; cell viability; adipocyte differentiation; gene expression; transcriptomics

1. Introduction Sphingosine-1-phosphate (S1P) is a highly potent sphingolipid metabolite which tightly controls various physiological and pathological processes via its extracellular and intracellular functions [1,2]. Its extracellular function follows an “inside-out” signaling model [3]. S1P is first synthesized from sphingosine by sphingosine kinases 1 (SphK1), which is located in the cytosol. Then, it is transported out of the cell to interact with a family of five G -coupled receptors (S1PR1–5) to regulate various cellular functions such as proliferation, apoptosis, differentiation, growth, migration, invasion, and angiogenesis [4–7]. However, the intracellular function of S1P is less understood. It is synthesized by sphingosine kinase 2 (SphK2) either inside the nucleus or in the perinuclear region. S1P then

Int. J. Mol. Sci. 2020, 21, 9284; doi:10.3390/ijms21239284 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 9284 2 of 16 epigenetically affects gene expression by inhibiting histone deacetylase HDAC1 and HDAC2 [8–10] and can induce cell apoptosis [11,12]. The breast is a highly specialized female exocrine organ responsible for producing milk to feed offspring. It is structurally divided into ductal epithelium and connective tissue stroma [13]. The ductal epithelium contains luminal, myoepithelial and basal cells and forms the mammary gland [14]. The stroma, which mainly contains adipocytes, plays an important role in regulating morphogenesis, development and homeostasis of the mammary gland [13,15,16]. S1P has been extensively studied for its regulatory role in normal physiology and pathogenesis of the mammary gland [17–20]. However, its function in breast adipocytes is far from being understood. Limited previous studies showed that S1P inhibits the differentiation of mouse preadipocyte 3T3-L1 cells at concentration higher than 0.5 µM[21]; and modulates adipocyte hypertrophy [22–24] and proinflammation response [25]. In addition, adipocytes constitute an abundant source of extracellular matrix (ECM) components in the breast [13,26], which play important roles in cell communication, adhesion, and homeostasis [27–29]. S1P can remodel the dynamic ECM and facilitate cell communication between adipocytes and ductal epithelial cells by altering the tight junction assembly [30]. A recent study showed that the level of S1P was about 10 times higher in the interstitial fluid (layer of fluid surrounding the mammary gland) than the mammary gland itself [31]. This implicates that S1P plays a critical role in regulating mammary ductal epithelium, stromal adipocyte tissue and their mutual communications. To get a better understanding of its functions in adipocytes, we evaluated the effects of S1P on cell viability, differentiation, and gene expression in both human pre- and differentiated adipocyte SGBS cells and mouse pre- and differentiated adipocyte 3T3-L1 cells. It was observed that S1P moderately increased cell viability in preadipocytes but decreased cell viability in differentiated adipocytes, and inhibited adipogenic differentiation at concentration higher than 1 µM. Compared to preadipocytes, the number of gene regulated by S1P was significantly increased in the differentiated adipocytes.

2. Results

2.1. Effect of S1P on Cell Viability of Pre- and Differentiated Adipocytes The effect of S1P on cell viability was evaluated against pre- and differentiated SGBS and 3T3-L1 adipocytes using the MTT assay (Figure1). For SGBS preadipocytes, cell viability was statistically significantly increased from 12% at 1000 nM to 28% at 5000 nM after 24 h of S1P treatment and by 10% (p < 0.05) at 2500 nM and 20% at 5000 nM (p < 0.05) after 48 h of S1P treatment. No statistically significant change in cell viability was observed for any S1P concentration at 72 h of treatment. For 3T3-L1 preadipocytes, cell viability was statistically significantly increased from 15% at 90 nM to 46% at 5000 nM at 24 h of S1P treatment, from 20% at 90 nM to 43% at 5000 nM at 48 h of S1P treatment, and by 21% at 1000 nM, 20% at 2500 nM and 27% at 5000 nM after 72 h of S1P treatment. In general, the effect of S1P on cell viability was stronger in 3T3-L1 preadipocytes and prolonged exposure to S1P weakened the effect. Cell viability was moderately decreased for both differentiated SGBS and 3T3-L1 adipocytes upon S1P treatment. For differentiated SGBS adipocytes, cell viability was statistically significantly decreased by 10–15% in the concentration of 500–5000 nM after 24 h of S1P treatment, and from 12% at 10 nM to 25% at 5000 nM at 48 h of S1P treatment. For differentiated 3T3-L1 adipocytes, cell viability was statistically significantly decreased from 8% at 90 nM to 21% at 5000 nM (except at 270 nM, p > 0.05) at 24 h of S1P treatment and 9% at 1000 nM to 18% at 5000 nM after 48 h of S1P treatment. No statistically significant change in cell viability was observed for both differentiated SGBS and 3T3-L1 adipocytes for 72 h of S1P treatment at any concentration. Similar to preadipocytes, prolonged treatment weakened the effect of S1P in differentiated adipocytes. The opposite effects on cell viability suggested that S1P elicited different functions between pre- and differentiated adipocytes. Int. J. Mol. Sci. 2020, 21, 9284 3 of 16 Int. J. Mol. Sci. 2020, 21, x 3 of 16

FigureFigure 1. Cell 1. Cell viability viability (% (% of of control) control) of preadipocyte preadipocyte SGBS SGBS cells cells(A), preadipocyte (A), preadipocyte 3T3-L1 cells 3T3-L1 (B), cells (B), diffdifferentiatederentiated adipocyteadipocyte SGBS cells cells (C (C) )and and differentiated differentiated adipocyte adipocyte 3T3-L1 3T3-L1 cells cells(D) under (D) under sphingosine-1-phosphatesphingosine-1-phosphate (S1P) (S1P) treatment treatment (10–5000 (10–5000 nm) nm) for fo 24r h,24 48h, h,48 andh, and 72 h,72 respectively.h, respectively. Cells Cells treated with methanoltreated with were methanol used as were the vehicleused as control.the vehicle Results control. were Results shown were in shown mean in SDmean and ± SD analyzed and by ± one-wayanalyzed ANOVA by one-way followed ANOVA by Dunnett’s followed post by Dunnett’s hoc test. post Data hoc were test. calculatedData were calculated from six from independent six independent experiments. A significant difference (*) was defined by p < 0.05 as compared to control experiments. A significant difference (*) was defined by p < 0.05 as compared to control under different under different time series (* 24 h, # 48 h, $ 72 h). time series (* 24 h, # 48 h, $ 72 h). Cell viability was moderately decreased for both differentiated SGBS and 3T3-L1 adipocytes 2.2. Effect of S1P on Adipocyte Differentiation upon S1P treatment. For differentiated SGBS adipocytes, cell viability was statistically significantly Todecreased examine by the 10–15% effect in ofthe S1Pconcentration on adipocyte of 500–5000 differentiation, nM after 24 h weof S1P treated treatment, the SGBSand from and 12% 3T3-L1 preadipocytesat 10 nM with to 25% S1P at (concentrations:5000 nM at 48 h of 100, S1P 500,treatment. 1000, For 2000 differentiated and 5000 nM) 3T3-L1 throughout adipocytes, the cell whole differentiationviability processwas statistically (14 days significantly for SGBS decreased and 10 days from for 8% 3T3-L1). at 90 nM Theto 21% lipid at 5000 content nM (except was much at 270 higher nM, p > 0.05) at 24 h of S1P treatment and 9% at 1000 nM to 18% at 5000 nM after 48 h of S1P treatment. in differentiated SGBS adipocytes than differentiated 3T3-L1 adipocytes (Figure2A) . Furthermore, No statistically significant change in cell viability was observed for both differentiated SGBS and 3T3- statisticallyL1 adipocytes significant for decrease72 h of S1P in treatment lipid content at any was concentration. observed inSimilar both dito ffpreadipocytes,erentiated adipocytes prolonged when S1P concentrationtreatment weakened was higher the effect than of 1000 S1P nM in differentiated (Figure2B,C). adipocytes. For di fferentiated SGBS adipocytes, the lipid content wasThe reduced opposite by effects 19% at on 1000 cell nM,viability 19% suggested at 2000 nM that and S1P 23%elicited at 5000different nM; functions whereas between for diff erentiatedpre- 3T3-L1and adipocytes, differentiated the adipocytes. lipid content was reduced by 32% at 1000 nM, 37% at 2000 nM and 44% at 5000 nM. Apparently, S1P exerted a much stronger inhibitory effect on differentiation in 3T3-L1 cells. 2.2. Effect of S1P on Adipocyte Differentiation Our currentInt. J. observation Mol. Sci. 2020, 21, isx consistent with previous studies [21]. 4 of 16 To examine the effect of S1P on adipocyte differentiation, we treated the SGBS and 3T3-L1 preadipocytes with S1P (concentrations: 100, 500, 1000, 2000 and 5000 nM) throughout the whole differentiation process (14 days for SGBS and 10 days for 3T3-L1). The lipid content was much higher in differentiated SGBS adipocytes than differentiated 3T3-L1 adipocytes (Figure 2A). Furthermore, statistically significant decrease in lipid content was observed in both differentiated adipocytes when S1P concentration was higher than 1000 nM (Figure 2B,C). For differentiated SGBS adipocytes, the lipid content was reduced by 19% at 1000 nM, 19% at 2000 nM and 23% at 5000 nM; whereas for differentiated 3T3-L1 adipocytes, the lipid content was reduced by 32% at 1000 nM, 37% at 2000 nM and 44% at 5000 nM. Apparently, S1P exerted a much stronger inhibitory effect on differentiation in 3T3-L1 cells. Our current observation is consistent with previous studies [21].

Figure 2. Changes in lipid accumulation (% of vehicle) in differentiated SGBS and 3T3-L1 adipocytes Figure 2. Changes in lipid accumulation (% of vehicle) in differentiated SGBS and 3T3-L1 adipocytes with S1P treatment throughout the whole differentiation process (14 days for SGBS and 10 days with S1P treatment throughout the whole differentiation process (14 days for SGBS and 10 days for for 3T3-L1).3T3-L1). SGBS SGBS and and 3T3-L1 3T3-L1 preadipocytespreadipocytes were were induced induced to differentiate to differentiate under underS1P treatment S1P treatment (concentration:(concentration: 100–5000 100–5000 nM). nM). Oil redOil red O stainingO staining waswas a appliedpplied to tovisualize visualize lipid droplets lipid droplets accumulated accumulated inside diffinsideerentiated differentiated adipocytes. adipocytes. Photos Photos were were taken taken under a alight light microscope microscope at 200× at magnification 200 magnification (A). The staining solution was extracted and quantified by absorbance of 492 nm, which represents× (A). The staining solution was extracted and quantified by absorbance of 492 nm, which represents the lipid content inside the differentiated SGBS adipocytes (B) and 3T3-L1 adipocytes (C). The results the lipid contentwere represented inside the as mean differentiated ± SD and analyzed SGBS adipocytesby one-way ANOVA (B) and followed 3T3-L1 by adipocytes Dunnett’s post (C). hoc The results were representedcomparisons. as meanData wereSD calculated and analyzed from three by independent one-way experiments. ANOVA followed A significant by difference Dunnett’s (*) post hoc ± comparisons.was defined Data wereby p < calculated0.05 as compared from with three vehicle independent control. experiments. A significant difference (*) was defined by p < 0.05 as compared with vehicle control. 2.3. Effect of S1P on Gene Expression in Pre- and Differentiated Adipocytes The gene expression affected by S1P treatment (concentration: 100 nM) was investigated by transcriptomics using the Affymetrix Clariom™ S Mouse/Human Array (Thermo Fisher Scientific). Differentially expressed genes (DEGs) were highlighted in green (fold change ≤ −1.5 and p < 0.05) and red (fold change ≥ 1.5 and p < 0.05) in the volcano plots (Figure 3). We identified 139 DEGs in SGBS preadipocytes, 178 DEGs in 3T3-L1 preadipocytes, 411 DEGs in differentiated SGBS adipocytes and 974 DEGs in differentiated 3T3-L1 adipocytes, respectively. The top 30 up- and downregulated DEGs are summarized in Table 1.

Figure 3. Volcano plots [fold change vs p-value (-log10)] displaying differentially expressed genes (DEGs) in pre- and differentiated adipocytes treated with 100 nM S1P compared to vehicle control.

Int. J. Mol. Sci. 2020, 21, x 4 of 16

Figure 2. Changes in lipid accumulation (% of vehicle) in differentiated SGBS and 3T3-L1 adipocytes with S1P treatment throughout the whole differentiation process (14 days for SGBS and 10 days for 3T3-L1). SGBS and 3T3-L1 preadipocytes were induced to differentiate under S1P treatment (concentration: 100–5000 nM). Oil red O staining was applied to visualize lipid droplets accumulated inside differentiated adipocytes. Photos were taken under a light microscope at 200× magnification (A). The staining solution was extracted and quantified by absorbance of 492 nm, which represents the lipid content inside the differentiated SGBS adipocytes (B) and 3T3-L1 adipocytes (C). The results were represented as mean ± SD and analyzed by one-way ANOVA followed by Dunnett’s post hoc comparisons. Data were calculated from three independent experiments. A significant difference (*) Int. J. Mol. Sci. 2020, 21, 9284 4 of 16 was defined by p < 0.05 as compared with vehicle control.

2.3. Effect Effect of S1P on Gene Expression in Pre- and DiDifferenfferentiatedtiated AdipocytesAdipocytes The gene expression aaffectedffected by S1PS1P treatmenttreatment (concentration:(concentration: 100 nM) was investigated by transcriptomics using using the the Affymetrix Affymetrix Clariom™ Clariom™ S Mouse/Human Mouse/Human Array (Thermo Fisher Fisher Scientific). Scientific). ≤ DifferentiallyDifferentially expressedexpressed genesgenes (DEGs)(DEGs) werewere highlighted highlighted in in green green (fold (fold change change −1.51.5 andand pp << 0.05) and ≤ − red (fold change ≥ 1.5 and p < 0.05)0.05) in in the the volcano volcano plots plots (Figure (Figure 33).). WeWe identifiedidentified 139139 DEGsDEGs inin SGBSSGBS ≥ preadipocytes, 178 DEGs in 3T3-L1 preadipocytes, 411 DEGs in di fferentiatedfferentiated SGBS adipocytes and 974 DEGs in differentiated differentiated 3T3-L1 adipocytes, respectively.respectively. The top 30 up- and downregulated DEGs are summarized in Table1 1..

Figure 3. VolcanoVolcano plots plots [fold change vs.vs pp-value-value (-log ( log10)])] displaying displaying differentially differentially expressed expressed genes − 10 (DEGs) in pre- and and differentiated differentiated adipocytes trea treatedted with 100 nM nM S1P S1P compared compared to to vehicle vehicle control. control. Genes with fold change 1.5 (green) and 1.5 (red) and p < 0.05 are highlighted in SGBS preadipocyte ≤ − ≥ (A), 3T3-L1 preadipocyte (B), differentiated SGBS adipocyte (C) and differentiated 3T3-L1 adipocyte (D). Int. J. Mol. Sci. 2020, 21, 9284 5 of 16

Table1. The top 30 up- and downregulated DEGs in pre- and differentiated SGBS and 3T3-L1 adipocytes.

Upregulated DEGs Downregulated DEGs Pre- Diff- Pre- Diff- Pre- Diff- Pre- Diff- SGBS SGBS 3T3-L1 3T3-L1 SGBS SGBS 3T3-L1 3T3-L1 PIK3C2B HAS1 Gm17332 Fstl1 FGF20 EMX2 Tmsb15b2 Stard9 CTPS2 CIDEC Ces2c Gm8281 OLFM3 ACAN Gm14092 Krtap16-3 ZNF506 TNFRSF19 Olfr1477 Mier2 FCER1A IFIT1 Gm13298 Folr1 UBE2D3 ZNF141 Sftpc Gm2237 MRAP SETD3 Wdr17 Usp26 TRABD2A GPRC5A Vmn1r46 Atg7 PHYHIPL IRAK1 Slc25a31 Mcoln2 OSBPL1A KLHL13 Tcp11l1 Lacc1 CEMIP FYCO1 Gm20823 Gad2 CISH THRSP Pou2f1 Depdc7 KLHDC8B DENND3 Olfr1436 Gm17482 BIRC3 ADAMTS9 Ssx9 Adipoq CDKAL1 NEO1 Vmn1r227 Gm17428 SCRIB CAT Olfr810 B3gnt9 TMEM170B ARID1A Olfr1323 Pygl CCDC54 SYN2 Gm14025 Trim13 FOXO1 STPG2 Olfr825 Ubash3b OR1B1 CLHC1 Gm3127 Slx4 SHISA4 PODN Ccdc152 Rnase9 PLSCR5 LIPE Olfr807 Lrrc9 DIRC3 HPS1 Iqcj Fam19a1 SPATA5 AGBL2 Lctl Eif6 PDE6A CDH22 Afp Chd7 SLC25A25 HACD3 Clec7a Rcor2 BRD9 TTLL12 Oas1a Gm11111 OR11H12 GK Ifna11 Esp36 FRY RASSF3 Gm20738 Vpreb2 LOC340074 NAT2 Atp8a1 Arhgap33 QRSL1 MICAL2 Gm10477 Gm2745 NDUFA8 PDE11A Sema3d Ensa FDXR RGS7 Olfr809 Col16a1 KSR1 CST2 5330417C22Rik Sh2b1 C17orf64 ATP11A Gm5662 Prlr API5L1 CD207 Olfr392 Pcdhb21 KIRREL3 LCTL Gm19668 Gm4399 CCDC160 SENP8 Klra23 Sema4g LY6G6C PITPNM2 Hsfy2 Gm4406 OR2F1 PDE1B Gm10037 D430020J02Rik MUC3A ADRBK1 Rhbdl3 Arhgef26 LONRF1 EPHA4 Olfr133 Saal1 DEFB104A MINK1 Tmprss11c Pnmal1 TRIM49D2 BCO1 4930402K13Rik Olfr1076 C9orf50 LTBP2 Lrit2 Defa3 POGZ HPD Pde9a Sssca1 TMEM129 MAN2A2 S100a14 Defa17 TACC3 LAIR1 Vmn1r4 Fbxw17 LAGE3 SCUBE3 Gm5538 Vmn2r110 PTK2 SLC38A4 Fpr-rs6 Zadh2 SLC2A7 GSK3A Mlana Lrrtm3 STK32A ADIPOQ 3425401B19Rik Abi3bp KRTAP1–5 MKL Sult2a2 LOC102642717 CCL20 LIPA Lama3 Fam180a RAB25 RFESD Olfr773 Gm10436 GMPR SMOX Pdk2 Mtor FLOT1 CCDC181 Car3 Olfr1258 C17orf47 NME8 Catsperd Foxm1 MYO1E RGS21 Elmod1 Defb25

2.4. (GO) Analysis We analyzed the GO terms for DEGs in pre- and differentiated SGBS and 3T3-L1 adipocytes using ConsensusPathDB [32]. The top 10 enriched GO terms (p < 0.05) in biological process, molecular function and cellular component are summarized in Figure4. It was obvious that the enriched GO terms were different between pre- and differentiated adipocytes and between SGBS and 3T3-L1. Int. J. Mol. Sci. 2020, 21, 9284 6 of 16 Int. J. Mol. Sci. 2020, 21, x 6 of 16

detection of chemical stimulus involved in sensory perception - signal transduction - neural nucleus development - cell morphogenesis - detection of stimulus involved in sensory perception - organ growth - synapse assembly - response to starvation - axon guidance - neuron projection guidance - detection of stimulus - G-protein coupled receptor signaling pathway - sensory perception of smell - negLog10_p_value sequestering of actin monomers - respiratory gaseous exchange - 6 chloride transport - 5 actin filament organization - 4 histamine secretion - 3 2 epithelial fluid transport - regulation of biological quality - regulation of molecular function - Count response to chemical - 40 cellular component organization - 80 cellular developmental process - 120 cellular response to stimulus - 160 anatomical structure development - multicellular organism development - cell communication - homophilic cell adhesion via plasma membrane adhesion molecules - negative regulation of transcription, DNA-templated - transcription from RNA polymerase II promoter - positive regulation of protein kinase B signaling - positive regulation of sequence-specific DNA binding transcription factor activity- glucose homeostasis - defense response to bacterium - positive regulation of I-kappaB kinase/NF-kappaB signaling - innate immune response - response to drug - Pre_3T3-L1 Pre_SGBS Diff_3T3-L1 Diff_SGBS A

lipid transporter activity - olfactory receptor activity - odorant binding - small molecule binding - ligase activity, forming carbon-nitrogen bonds - clathrin binding - activating transcription factor binding - bHLH transcription factor binding - ligase activity - sterol transporter activity - oxidoreductase activity, acting on NAD(P)H - G protein-coupled receptor binding - G-protein coupled receptor activity - negLog10_p_value transmembrane receptor activity - transmembrane signaling receptor activity - 6 signaling receptor activity - 5 signal transducer activity - 4 receptor activity - 3 2 molecular transducer activity - anion transmembrane transporter activity - extracellular matrix structural constituent - Count guanyl-nucleotide exchange factor activity - 50 oxidoreductase activity - 100 structural constituent of muscle - carbohydrate derivative binding - ion binding - cofactor binding - enzyme regulator activity - calcium ion binding - transcription regulatory region DNA binding - transcription factor activity, transcription factor binding - transcription factor binding - bitter taste receptor activity - chloride channel activity - heparin binding - serine-type endopeptidase activity -

Pre_3T3-L1 Pre_SGBS Diff_3T3-L1 Diff_SGBS

B

Figure 4. Cont.

Int. J. Mol. Sci. 2020, 21, 9284 7 of 16

Int. J. Mol. Sci. 2020, 21, x 7 of 16

integral component of membrane - plasma membrane - cell periphery - intrinsic component of membrane - GOneurotransmitterreceptor complex - ligase activity, forming carbon-nitrogen bonds - phosphatidylinositol 3-kinase complex - ion channel complex - transmembrane transporter complex - transporter complex - chemokine activity - membrane part - Count filamentous actin - 100 actin filament - 200 cell part - 300 cell - membrane - negLog10_p_value extracellular matrix - 4 endomembrane system - 3 extracellular matrix component - membrane region - 2 endoplasmic reticulum chaperone complex - basal part of cell - whole membrane - anchoring junction - contractile fiber part - cell-substrate junction - extracellular space - intermediate filament - extracellular region - protein-DNA complex - intermediate filament cytoskeleton - neuronal cell body -

Pre_3T3-L1 Pre_SGBS Diff_3T3-L1 Diff_SGBS

C

Figure 4. Top 10 enriched GO terms in biological process (A),), molecularmolecular functionfunction (B), andand cellularcellular component (C). For each cell type, the DEGDEG listlist waswas subjectedsubjected to GO analysis and the mostmost enrichedenriched GO terms were summed up. Size of the bubbles indica indicatestes counts of genes identified identified in each GO term, and colors ofof thethe bubblesbubbles showshow significancesignificance ((−loglog10 p-value)-value) of GO enriched fold changes comparedcompared − 10 to control.

2.5. Pathway Entichment Analysis 2.5. Pathway Entichment Analysis Up- and downregulated DEGs (|fold change| 1.5 and p < 0.05) were individually subjected to Up- and downregulated DEGs (|fold change| ≥≥ 1.5 and p < 0.05) were individually subjected to pathway enrichment using ConsensusPathDB. For the upregulated DEGs, the enriched pathways were pathway enrichment using ConsensusPathDB. For the upregulated DEGs, the enriched pathways inflammatory signaling and cytokine involved pathways in SGBS preadipocytes; cell cycle/mitosis were inflammatory signaling and cytokine involved pathways in SGBS preadipocytes; cell related pathways in 3T3-L1 preadipocytes; lipid metabolism-related pathways (e.g., PPAR signaling cycle/mitosis related pathways in 3T3-L1 preadipocytes; lipid metabolism-related pathways (e.g., pathway, metabolism of fatty acid, metabolism, and metabolism of lipids) in differentiated SGBS PPAR signaling pathway, metabolism of fatty acid, metabolism, and metabolism of lipids) in adipocytes; and G protein-coupled receptor signaling-related pathways (e.g., signaling by GPCR, differentiated SGBS adipocytes; and G protein-coupled receptor signaling-related pathways (e.g., GPCR ligand binding, and GPCR downstream signaling) in differentiated 3T3-L1 adipocytes, signaling by GPCR, GPCR ligand binding, and GPCR downstream signaling) in differentiated 3T3- respectively (Figure5A). For the downregulated DEGs, the enriched pathways were identified L1 adipocytes, respectively (Figure 5A). For the downregulated DEGs, the enriched pathways were to be metabolite regulation associated pathways in SGBS preadipocytes; GPCR signaling and olfactory identified to be metabolite regulation associated pathways in SGBS preadipocytes; GPCR signaling transduction pathways in 3T3-L1 preadipocytes; focal adhesion and axon guidance related pathways in and olfactory transduction pathways in 3T3-L1 preadipocytes; focal adhesion and axon guidance di erentiated SGBS adipocytes; and cell cycle and cell mitosis-related pathways (e.g., cell cycle, relatedff pathways in differentiated SGBS adipocytes; and cell cycle and cell mitosis-related pathways mitotic(e.g., cell and cycle, amplification mitotic andof amplification signal from of the signal kinetochores) from the kinetochores) in differentiated in differentiated 3T3-L1 adipocytes, 3T3-L1 respectivelyadipocytes, respectively (Figure5B). (Figure 5B).

Int. J. Mol. Sci. 2020, 21, 9284 8 of 16 Int. J. Mol. Sci. 2020, 21, x 8 of 16

G alpha (i) signalling events - Rheumatoid arthritis - Homo sapiens (human) - Chemokine receptors bind chemokines - Glucocorticoid Receptor Pathway - Leptin signaling pathway - Chemokine signaling pathway - Homo sapiens (human) - IL-17 signaling pathway - Homo sapiens (human) - JAK-STAT - TNF signaling pathway - Homo sapiens (human) - Cytokine-cytokine receptor interaction -Homo sapiens (human) - Signal Transduction - Nuclear Receptors - Platelet homeostasis - negLog10_p_value Amplification of signal from unattached kinetochores via a MAD2 inhibitory signal - 10.0 Amplification of signal from the kinetochores - Biological oxidations - 7.5 Cell Cycle, Mitotic - 5.0 Resolution of Sister Chromatid Cohesion - 2.5 Mitotic Spindle Checkpoint - RHO GTPases Activate Formins - Cell Cycle - Count Metabolism of lipids - 10 Fatty acid metabolism - 20 Regulation of lipolysis in adipocytes - Homo sapiens (human) - 30 AMPK signaling pathway -Homo sapiens (human) - 40 Metabolism - 50 PPAR signaling pathway -Homo sapiens (human) - Metabolism of amino acids and derivatives - Metabolism of steroids - Nuclear Receptors Meta-Pathway - Olfactory transduction - Mus musculus (mouse) - Signaling by GPCR - GPCR downstream signalling - GPCR ligand binding - GPCRs, Other - Non-odorant GPCRs - Class A/1 (Rhodopsin-like receptors) - Neuroactive ligand-receptor interaction - Mus musculus (mouse) - Pre_3T3-L1_up-regulated Pre_SGBS_up-regulated Diff_3T3-L1_up-regulated Diff_SGBS_upregulated A

B

FigureFigure 5. 5. PathwayPathway enrichment enrichment analysis analysis in pre- andand didifffferentiatederentiated SGBS SGBS and and 3T3-L1 3T3-L1 adipocytes. adipocytes. The The top top10 10 enriched enriched pathways pathways for for upregulated upregulated DEGs DEGs ( A(A)) andand downregulateddownregulated DEGs ( (BB)) DEGs DEGs are are plotted plotted separately.separately. Size Size of of the the bubbles bubbles indi indicatedcated the countcount ofof genesgenes identified identified in in each each pathway, pathway, and and color color of of the bubbles show significance ( log p-value) of enriched pathway compared to control. the bubbles show significance− (−log10 10 p-value) of enriched pathway compared to control. 3. Discussion 3. Discussion In the current study, we show the opposite effects of S1P on cell viability of adipocytes. S1P enhanced In the current study, we show the opposite effects of S1P on cell viability of adipocytes. S1P cell viability in human SGBS and mouse 3T3-L1 preadipocytes, but moderately decreased cell enhanced cell viability in human SGBS and mouse 3T3-L1 preadipocytes, but moderately decreased viability in differentiated SGBS and 3T3-L1 adipocytes. S1P also inhibited lipid accumulation during cell viability in differentiated SGBS and 3T3-L1 adipocytes. S1P also inhibited lipid accumulation differentiation in both adipocytes at concentration higher than 1000 nM. Our observation was in during differentiation in both adipocytes at concentration higher than 1000 nM. Our observation was line with previous studies that S1P enhanced cell proliferation and suppressed adipogenesis of in line with previous studies that S1P enhanced cell proliferation and suppressed adipogenesis of preadipocytes [21,33,34]. However, these previous studies were mainly carried out with mouse preadipocytes (e.g., 3T3-L1 and 3T3-F442A). The inhibition of adipogenesis was evaluated by the

Int. J. Mol. Sci. 2020, 21, 9284 9 of 16 preadipocytes [21,33,34]. However, these previous studies were mainly carried out with mouse preadipocytes (e.g., 3T3-L1 and 3T3-F442A). The inhibition of adipogenesis was evaluated by the downregulationInt. J. Mol. Sci. 2020 of, 21 adipocyte-specific, x transcriptional factors [34]. A high concentration of9 S1Pof 16 was shown to activate cAMP/PKA (cyclic AMP/protein kinase A), possibly via S1PR1/2, to promote lipolysis anddownregulation inhibit lipid accumulation of adipocyte-specific [35]. However, transcriptio thenal expressions factors [34]. of A genes high PRKACAconcentrationand ofPRKACB S1P waswere not changedshown to byactivate S1P treatment cAMP/PKA in pre-(cyclic and AM diP/proteinfferentiated kinase SGBS A), adipocytes. possibly via Similarly, S1PR1/2, to the promote expressions of mouselipolysis orthologs and inhibitPrkaca lipidand accumulationPrkacb were [35]. not However, altered bythe S1P expressions stimulus of in genes pre- andPRKACA differentiated and 3T3-L1PRKACB adipocytes. were not To changed explain by the S1P di fftreatmenterent eff ectsin pre- of S1Pand differentiated between pre- SGBS and diadipocytes.fferentiated Similarly, adipocytes, the expressions of mouse orthologs Prkaca and Prkacb were not altered by S1P stimulus in pre- and we identified genes with significantly different expression (p < 0.05) between differentiated and differentiated 3T3-L1 adipocytes. To explain the different effects of S1P between pre- and undifferentiated (i.e., pre-) SGBS and 3T3-L1 adipocytes. As shown in Figure6, the di fferentially differentiated adipocytes, we identified genes with significantly different expression (p < 0.05) expressedbetween genes differentiated were mainly and undifferentiated involved in the(i.e., PI3K-AKT pre-) SGBS pathwayand 3T3-L1 (e.g., adipocytes.AKT1-3 Asand shownPI3KR1-3 in ), sphingolipidFigure 6, the metabolism differentially and expressed S1P signaling genes pathway were mainly (e.g., involvedCERS1-6 in, SPHK1-2 the PI3K-AKTand S1PR1-5 pathway) and(e.g., other proteinAKT1-3 kinase and/phosphatase PI3KR1-3), sphingolipid signaling (e.g., metabolismROCK2 and, PLCB1-4 S1P signalingand PPP2Rs pathway). However, (e.g., CERS1-6 these, SPHK1- genes were regulated2 and S1PR1-5 differently) and between other protein SGBS kinase/pho and 3T3-L1sphatase adipocytes. signaling Specifically,(e.g., ROCK2, genesPLCB1-4SPHK1 and PPP2Rsand S1PR1). wereHowever, upregulated these ingenes both were diff regulatederentiated differently SGBS and between 3T3-L1 SGBS adipocytes and 3T3-L1 compared adipocytes. to Specifically, their respective preadipocytes,genes SPHK1 implicating and S1PR1 thatwere the upregulated SphK1-S1P-S1PR in both 1differensignalingtiated axis SGBS was and enhanced. 3T3-L1 adipocytes Because this signalingcompared axis to normally their respective promotes preadipocytes, cell proliferation, implicating other that the S1P-mediated SphK1-S1P-S1PR signaling1 signaling pathways axis was must be involvedenhanced. in Because decreasing this cellsignaling viability axis in normally the differentiated promotes SGBScell proliferation, and 3T3-L1 other adipocytes. S1P-mediated Moreover, signaling pathways must be involved in decreasing cell viability in the differentiated SGBS and 3T3- the expressions of S1PR2, S1PR3 and S1PR5 were downregulated in the differentiated SGBS adipocytes, L1 adipocytes. Moreover, the expressions of S1PR2, S1PR3 and S1PR5 were downregulated in the suggesting that S1P-S1PR2, S1P-S1PR3 and S1P-S1PR5 signaling pathways were likely to be impaired. differentiated SGBS adipocytes, suggesting that S1P-S1PR2, S1P-S1PR3 and S1P-S1PR5 signaling Furtherpathways studies were are likely warranted to be impaired. to identify Further which studies signaling are warranted pathway isto responsibleidentify which for signaling the reduced proliferationpathway is of responsible differentiated for the SGBS reduced and 3T3-L1proliferatio adipocytes.n of differentiated SGBS and 3T3-L1 adipocytes.

FigureFigure 6. Chord 6. Chord diagrams diagrams showing showing genes genes withwith si significantlygnificantly different different expressions expressions (p < ( p0.05)< 0.05) between between differentiateddifferentiated and and undi undifferentiatedfferentiated SGBS SGBS cellscells ( AA)) and and 3T3-L1 3T3-L1 cells cells (B) ( Bupon) upon 100 100nM nMS1P S1Ptreatment. treatment. TheThe up- up- and and downregulated downregulated genes genes are are shown shown inin magenta and and blue, blue, respectively. respectively.

OutOut of theof the DEGs DEGs identified identified from from transcriptomicstranscriptomics study, study, 9% 9% and and 17% 17% were were involved involved in human in human secretomesecretome in pre- in pre- and and diff erentiateddifferentiated SGBS SGBS adipocytes, adipocytes, respectively. respectively. Furthermore, Furthermore, about about 2% 2% of of the the DEGs DEGs were in the adipokine secretome in the differentiated SGBS adipocytes. This implicated that were in the adipokine secretome in the differentiated SGBS adipocytes. This implicated that S1P may S1P may play an important role in regulating the adipocyte secretory pattern, which, in turn, controls play an important role in regulating the adipocyte secretory pattern, which, in turn, controls the normal the normal functions of adipocytes and their communications with other types of cells. Taking into functionsconsideration of adipocytes that the and breast their is communications mainly composed with of otherepithelial types cells of cells.and adipocytes Taking into and consideration S1P is thataccumulated the breast is to mainly high concentration composed of in epithelial the interstitial cells fluid and [13,14,31], adipocytes the and current S1P isstudy accumulated suggests that to high concentrationS1P is highly in likely the interstitial to be crucial fluid for the [13 ,normal14,31], fu thenctions current of both study epithelial suggests cells that and S1P adipocytes, is highly as likely to bewell crucial as their for mutual the normal communications, functions of in both the breast. epithelial cells and adipocytes, as well as their mutual communications,Network analysis in the breast. was performed using Cytoscape [36] to investigate the biological relevance of theNetwork identified analysis pathways was performed on lipid metabolism using Cytoscape in mature [36] to adipocytes. investigate Multiple the biological functions relevance of the of the identifiedupregulated pathways DEGs on were lipid related metabolism to regulation in mature of lipolysis, adipocytes. PPAR Multiplesignaling, functions alcoholism, of and the upregulatedtoll-like receptor signaling (Figure 7A,C). The downregulated DEGs were overrepresented in cytokine–

Int. J. Mol. Sci. 2020, 21, 9284 10 of 16

DEGsInt. were J. Mol. related Sci. 2020, to 21 regulation, x of lipolysis, PPAR signaling, alcoholism, and toll-like receptor10 signaling of 16 (Figure7A,C). The downregulated DEGs were overrepresented in cytokine-cytokine receptor interaction, focalcytokine adhesion, receptor starch interaction, and sucrose focal metabolism, adhesion, st andarch nuclearand sucrose receptors metabolism, pathways and nuclear (Figure receptors7B,D). Thus, the functionalpathways (Figure role of 7B,D). S1P in Thus, lipid the metabolism functional role involved of S1P regulationin lipid metabolism of cytokines, involved receptors regulation signaling of cytokines, receptors signaling and focal adhesion. It is noteworthy that PPARs are ligand-activated and focal adhesion. It is noteworthy that PPARs are ligand-activated transcription factors. The PPAR transcription factors. The PPAR pathway, which is activated by fatty acids and their derivatives pathway, which is activated by fatty acids and their derivatives regulates numerous biological processes, regulates numerous biological processes, such as adipocyte differentiation, lipid/glucose metabolism, suchand as adipocyteenergy homeostasis. differentiation, lipid/glucose metabolism, and energy homeostasis.

FigureFigure 7. Pathway 7. Pathway network network analysis analysis based based onon up-up- and downregulated downregulated DEGs DEGs in differentiated in differentiated SGBS SGBS (A and(A andB, respectively) B, respectively) and and 3T3-L1 3T3-L1 (C (Cand andD D,, respectively) respectively) adipocytes. adipocytes. The The analysis analysis was was performed performed usingusing ClueGO. ClueGO. Enriched Enriched pathways pathways were were obtainedobtained from the the KEGG KEGG and and WikiPathway WikiPathway databases databases and and grouped based on shared genes (ranging from red circle: upregulated to blue circle: downregulated). grouped based on shared genes (ranging from red circle: upregulated to blue circle: downregulated). Size of the nodes indicates degree of significance, where only the most significant term/pathway is Size of the nodes indicates degree of significance, where only the most significant term/pathway is labeled as the name of the group. Function-related networks are grouped and may partially overlap. labeled as the name of the group. Function-related networks are grouped and may partially overlap.

We further mapped genes of the S1P-S1PR1 signaling pathway in the S1P-treated differentiated We further mapped genes of the S1P-S1PR1 signaling pathway in the S1P-treated differentiated SGBS and 3T3-L1 adipocytes. As shown in Figure 8, the regulation of S1P-S1PR1 signaling axis was SGBSsignificantly and 3T3-L1 different adipocytes. between As SGBS shown and in 3T3-L1 Figure cells.8, the A regulation unique feature of S1P-S1PR is that genes1 signaling AKT1, AKT2 axis was significantlyand AKT3 di werefferent downregulated between SGBS in differentiated and 3T3-L1 cells.SGBS Aadipocytes, unique feature whereas is thatthe ortholog genes AKT1, genes AKT2Akt1, and AKT3Akt2were and downregulated Akt3 were upregulated in differentiated in differentiated SGBS adipocytes, 3T3-L1 adipocytes. whereas AKTs theortholog are downstream genes Akt1, common Akt2 and Akt3targetswere upregulatedof multiple signaling in differentiated pathways 3T3-L1 other than adipocytes. S1P1-S1PR AKTs1, such are as downstream epidermal growth common factor targets of multiplereceptor signaling(EGFR) signaling pathways pathway other and than insulin S1P1-S1PR receptor1 (IR), such signaling as epidermal pathway, growth and play factor important receptor (EGFR)roles signaling in cell proliferation, pathway and migration insulin and receptor survival (IR) [37–39]. signaling Thus, pathway, the current and results play implicated important that roles in cell proliferation,regulation of S1P-S1PR migration1 and and crosstalk survival between [37–39]. S1P-S1PR Thus, the1 and current other results signaling implicated pathways that in mature regulation adipocytes might be species-specific. Further studies are required to identify the exact differences of of S1P-S1PR1 and crosstalk between S1P-S1PR1 and other signaling pathways in mature adipocytes S1P treatment between mouse and human adipocytes and investigate the transferability of mouse- might be species-specific. Further studies are required to identify the exact differences of S1P treatment based study results to human health research. between mouse and human adipocytes and investigate the transferability of mouse-based study results to human health research.

Int. J. Mol. Sci. 2020, 21, 9284 11 of 16 Int. J. Mol. Sci. 2020, 21, x 11 of 16

Figure 8. Mapping the S1P-S1PR signaling pathway in differentiated SGBS and 3T3-L1 adipocytes. Figure 8. Mapping the S1P-S1PR1 1 signaling pathway in differentiated SGBS and 3T3-L1 adipocytes.

WikiPathwayWikiPathway was was used used to to identify identify and and visualize visualize genesgenes involved in in the the S1P-S1PR S1P-S1PR1 signaling1 signaling axis axisin in S1P-treatedS1P-treated differentiated differentiated SGBS SGBS and and 3T3-L1 3T3-L1 adipocytes adipocytes compared compared toto thethe vehiclevehicle control. S1PR S1PR1 refers1 refers to sphingosine-1-phosphateto sphingosine-1-phosphate receptor receptor 1. 1.

4. Materials4. Materials and and Methods Methods

4.1. Materials4.1. Materials

Sphingosine-1-phosphateSphingosine-1-phosphate (S1P), (S1P), biotin,biotin, dD-pantothenic-pantothenic acid acid hemicalcium hemicalcium salt, salt, apo-transferrin apo-transferrin (human),(human), insulin, insulin, 3,30,5-triiodo- 3,3′,5-triiodo-l-thyronineL-thyronine sodium sodium salt (T3),salt rosiglitazone,(T3), rosiglitazone, dexamethasone, dexamethasone, 3-isobutyl-1- 3- methylxanthineisobutyl-1-methylxanthine (IBMX), hydrocortisone, (IBMX), hydrocortisone, fetal bovineserum fetal bovine (FBS), serum Oil red (FBS), O, and Oil 3-(4,5-dimethylthiazol- red O, and 3-(4,5- 2-yl)-2,5-diphenyltetrazoliumdimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were bromide purchased (MTT) fromwere purchased Sigma-Aldrich from Sigma-Aldrich Canada (Oakville, ON,Canada Canada). (Oakville,Trypsin-EDTA ON, Canada). (0.25%), Trypsin-EDTA phosphate-buffered (0.25%), salinephosphate-buffered (PBS) and penicillin-streptomycin saline (PBS) and penicillin-streptomycin were purchased from HyClone Laboratories Inc. (Logan, UT, USA). Triton were purchased from HyClone Laboratories Inc. (Logan, UT, USA). Triton X-100 (98%) and Dulbecco’s X-100 (98%) and Dulbecco’s phosphate-buffered saline (DPBS) was purchased from Thermo Fisher phosphate-buffered saline (DPBS) was purchased from Thermo Fisher Scientific (Burlington, ON, Canada). Scientific (Burlington, ON, Canada). Human preadipocyte cell line SGBS, which was derived from an Humaninfant preadipocyte with Simpson–Golabi–Beh cell line SGBS,mel which syndrome, was derived was established from an infant in coauthor with Simpson–Golabi–Behmel Martin Wabitsch’s syndromelaboratory., was Mouse established preadipocyte in coauthor cell line Martin 3T3-L1 Wabitsch’s was kindly laboratory. provided by MouseProfessor preadipocyte Jane Alcon, cell lineCollege 3T3-L1 of was Pharmacy kindly providedand Nutrition, by ProfessorUniversity Janeof Saskatchewan, Alcon, College Canada. of Pharmacy Dulbecco’s and modified Nutrition, UniversityEagle’s ofmedium Saskatchewan, (DMEM) Canada.and 1:1 mixture Dulbecco’s of Dulb modifiedecco’s modified Eagle’s mediumeagle’s medium (DMEM) with and ham’s 1:1 mixtureF12 of Dulbecco’smedium (DMEM-F12) modified eagle’s were mediumpurchased with from ham’s Gibco F12 (Thermo medium Fisher (DMEM-F12) Scientific, were Burlington, purchased ON, from GibcoCanada). (Thermo Fisher Scientific, Burlington, ON, Canada).

4.2. Cell4.2. Cell Culture Culture of Preadipocytes of Preadipocytes BothBoth cell cell lines lines were were cultured cultured underunder preadipocyte expansion expansion media media in T-75 in T-75cell culture cell culture flasks at flasks 37 °C with a humidified atmosphere of 5% CO2. Human preadipocyte cell line SGBS was cultured in at 37 ◦C with a humidified atmosphere of 5% CO2. Human preadipocyte cell line SGBS was DMEM-F12 supplemented with 10% FBS, 1% penicillin/streptomycin, and 10% 5 mM cultured in DMEM-F12 supplemented with 10% FBS, 1% penicillin/streptomycin, and 10% 5 mM pantothenate/biotin mixture (Pan/Bio; pantothenate:biotin = 1.7:3.3). Mouse preadipocyte cell line pantothenate/biotin mixture (Pan/Bio; pantothenate:biotin = 1.7:3.3). Mouse preadipocyte cell line 3T3-L1 was cultured in DMEM containing 10% FBS. The preadipocyte expansion media were 3T3-L1changed was cultured every two in to DMEM three days. containing 10% FBS. The preadipocyte expansion media were changed every two to three days. 4.3. Adipocyte Differentiation

After reaching 80–90% confluency (day 0), SGBS preadipocytes were washed with DPBS and then induced to differentiate in serum-free differentiation media (DMEM-F12 supplemented with 1% Int. J. Mol. Sci. 2020, 21, 9284 12 of 16 penicillin/streptomycin, 10% 5 mM Pan/Bio, 0.01 mg/mL human transferrin, 100 nM hydrocortisone, 0.2 nM T3, 25 nM dexamethasone, 250 µM IBMX, 2 µM rosiglitazone and 20 nM insulin). Four days after induction (day 4), the differentiation media were changed to postdifferentiation media by excluding IBMX, dexamethasone and rosiglitazone. The cells were cultured for 10 more days with media changed every four days. The SGBS cells were fully differentiated in about 8–12 days after induction as evidenced by changes in internal lipid droplet accumulation [40]. To differentiate 3T3-L1 preadipocytes, the cells were first cultured in preadipocyte expansion media for two days after reaching full confluence and before inducing differentiation (day 0). The cells were washed with DPBS and then induced to differentiate in differentiation media (DMEM containing 10% FBS, 1.0 µM dexamethasone, 0.5 mM IBMX and 1.0 µg/mL insulin) for another two days. On day 2, the media were replaced with the postdifferentiation media (DMEM with 10% FBS and 1.0 µg/mL insulin) and cultured for another six days. Cell culture media were changed every two to three days. Differentiation of 3T3-L1 cells were also evidenced by changes in internal lipid droplet accumulation.

4.4. Cell Viability Assay (MTT Assay) The effect of S1P on cell viability of pre- and differentiated SGBS and 3T3-L1 adipocytes were measured using the colorimetric MTT assay [41]. Briefly, for each adipocyte cell line, preadipocytes were seeded at 8 103 cells/well and differentiated adipocytes were seeded at 1 104 cells/well in × × 96 well plates. The seeding numbers were selected based on a pilot study. The cells were allowed to attach for 24 h before being treated with S1P (concentration: 10–5000 nM) for 24 h, 48 h, and 72 h, respectively. Methanol was used as a vehicle control. At the end of S1P treatment, the media were replaced with 100 µL MTT (0.5 mg/mL) dissolved in corresponding complete media and incubated for 4 h. Then, the media containing MTT were replaced with 150 µL DMSO to dissolve the formed formazans under constant shaking for 15 min on an orbital shaker. Absorbance was subsequently measured at 570 nm using a BioTek Synergy HK microplate reader (BioTek, Winooski, VT, USA). Cell viability was interpreted as the absorption ratio of treatment to control. The experiment was carried out in triplicate with six replicates in each test, and the data were shown in mean value SD as ± analyzed by GraphPad Prism 8 software (San Diego, CA, USA).

4.5. Oil Red O Staining Oil red O staining was used to measure lipid accumulation in differentiated SGBS and 3T3-L1 adipocytes. SGBS or 3T3-L1 preadipocytes were seeded at 2 104 cells/well in 24-well × plates and cultured with preadipocyte complete media before differentiation induction. On the induction day (day 0), media were replaced with differentiation media +/ S1P (concentration: − 100–5000 nM). After induction, the media were changed to postdifferentiation media +/ S1P for − SGBS on day 4 or 3T3-L1 on day 2. Media or media + S1P were changed every four days for SGBS or 2–3 days for 3T3-L1 throughout the whole adipogenic differentiation phase. On day 14 for SGBS or day 8 for 3T3-L1, lipid accumulation in differentiated adipocytes was measured using Oil red O staining. Briefly, cells were first washed with 1 PBS twice after removal of media and then fixed with × 4% paraformaldehyde in 1 PBS at room temperature for 30 min. The cells were stained with 0.2% Oil × red O working solution for 15 min after washing with 1 PBS twice. Stained cells were washed with × ddH2O until there was no pink color observed. Images were captured using an Olympus inverted phase microscope (Olympus Canada, Richmond Hill, ON, Canada) at 200 magnification after wells × were dried. Then, 2-propanol was used to solubilize the Oil red O dye, and the extraction was seeded in 96-well plates for lipid quantification by measuring absorbance at 492 nm using a BioTek Synergy HK microplate reader. Differentiation media + methanol was used as a vehicle control. Preadipocytes stained with Oil red O solution was used as a blank control. The results were presented as absorbance percentage normalized to control (100%) using the following formula:

(Atreat A )/(A A ) 100% (1) − blank control − blank × Int. J. Mol. Sci. 2020, 21, 9284 13 of 16

4.6. Transcriptome Analysis Pre- and differentiated SGBS and 3T3-L1 and SGBS adipocytes were treated with 100 nM S1P for 24 h before being collected by centrifugation. Treatment with methanol was used as a vehicle control. The collated cell pellets were sent to Applied Biosystems Microarray Research Services Laboratory (Thermo Fisher Scientific, Burlington, ON, Canada) for RNA extraction and transcriptome analysis. Extracted RNA samples were analyzed using the Affymetrix Clariom™ S Mouse/Human Array, which covers all known well-annotated genes. Raw data were normalized by Robust Multiple Averaging algorithms.

4.7. DEG Analysis For each adipocyte transcriptome analysis, gene expression values of the S1P-treated group were compared to methanol control group. Differentially expressed genes (DEGs) were defined as genes with |fold change| 1.5 and p < 0.05 controlled by a false discovery rate (FDR). DEGs of the differentiated ≥ SGBS adipocytes were filtered for secreted protein-coding genes based on the human secretome list from the Human Protein Atlas database [42], including those identified as released from adipocytes [43].

4.8. Gene Ontology (GO) and Pathway Enrichment Analyses GO and pathway enrichment analyses were performed on DEGs (|fold change| 1.5, p < 0.05, ≥ FDR controlled) identified from pre- and differentiated SGBS and 3T3-L1 adipocytes. Overrepresentation analysis (ORA) was performed using Consensus PathDB [32]. Each DEG list was input for defining overrepresented sets in two categories: GO-based sets and pathway-based sets. GO annotations were categorized into three GO terms: biological processes, molecular functions, and cellular components. Significant enrichment was calculated using hypergeometric testing with Benjamini–Hochberg FDR correction (cut-off of FDR < 0.05). Pathway enrichment analysis was only performed on DEGs from differentiated SGBS and 3T3-L1 adipocytes by employing the ORA tool in Consensus PathDB. Up- and downregulated DEGs were analyzed separately. We searched for pathways from three databases, KEGG [44,45], Reactome [46] and Wikipathway [47]. Minimal overlap with the input list of 2 and p-value cut-off at 0.05 were set as the default.

4.9. Statistical Analysis GraphPad Prism 8 was used to perform data analysis. Data for the MTT assay were calculated from three independent experiments with each experiment containing six replicates. Data for other analyses were calculated from three independent experiments with each experiment containing three replicates. Results were presented as mean SD. Data comparison was performed using the analysis of ± variance (ANOVA, one-way or two-way), followed by Dunnett’s or Tukey’s post hoc tests. Results were considered statistically significant for p < 0.05.

5. Conclusions The breast, which is mainly composed of epithelial cells and adipocytes, is a highly specialized female exocrine organ responsible for producing milk to feed offspring. Because S1P can accumulate up to 10 times higher in the interstitial fluid than the mammary gland itself, it may be essential for extracellular matrix remodeling and communications between epithelial cells and adipocytes. In this study, we showed that S1P exerted potent effects not only on adipocyte differentiation but also on cell viability and gene expression in both pre- and differentiated adipocytes. S1P-regulated pathways, such as lipolysis, PPAR signaling and cytokine–cytokine receptor interaction, will be investigated in our future studies to illustrate how S1P regulates the adipocyte-extracellular matrix-epithelial cell axis to maintain normal physiological functions of the breast and prevent development of breast cancer. Int. J. Mol. Sci. 2020, 21, 9284 14 of 16

Author Contributions: Conceptualization, J.Y.; methodology, M.K.S. and J.Y.; validation, X.W., M.K.S. and J.Y.; formal analysis, X.W., M.K.S. and J.Y.; investigation, X.W., M.K.S., M.W. and J.Y.; resources, M.W.; data curation, X.W. and J.Y.; writing—original draft preparation, X.W.; writing—review and editing, X.W., M.K.S., M.W. and J.Y.; visualization, X.W. and J.Y.; supervision, J.Y.; project administration, J.Y.; funding acquisition, J.Y. All authors have read and agreed to the published version of the manuscript. Funding: This research was supported by a President’s NSERC bridging grant (Number: 419247) from the University of Saskatchewan, Canada. M.K.S. would like to acknowledge the Natural Sciences and Engineering Research Council (Canada) for the award of a Discovery Grant (Number: 417652). Conflicts of Interest: The authors declare no conflict of interest.

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