3-Mercaptopyruvate sulfurtransferase disruption in dermal fibroblasts facilitates adipogenic trans-differentiation

Journal Pre-proof 3-Mercaptopyruvate sulfurtransferase disruption in dermal fibroblasts facilitates adipogenic trans-differentiation Elena A. Ostrakhovitch, Shin Akakura, Reiko Sanokawa-Akakura, Siamak Tabibzadeh PII:

S0014-4827(19)30557-9

DOI:

https://doi.org/10.1016/j.yexcr.2019.111683

Reference:

YEXCR 111683

To appear in:

Experimental Cell Research

Received Date: 1 April 2019 Revised Date:

13 October 2019

Accepted Date: 17 October 2019

Please cite this article as: E.A. Ostrakhovitch, S. Akakura, R. Sanokawa-Akakura, S. Tabibzadeh, 3-Mercaptopyruvate sulfurtransferase disruption in dermal fibroblasts facilitates adipogenic transdifferentiation, Experimental Cell Research (2019), doi: https://doi.org/10.1016/j.yexcr.2019.111683. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc.

3-Mercaptopyruvate sulfurtransferase disruption in dermal fibroblasts facilitates adipogenic transdifferentiation

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Elena A. Ostrakhovitch , Shin Akakura , Reiko Sanokawa-Akakura , Siamak Tabibzadeh

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Frontiers in Bioscience Research Institute in Aging and Cancer, 16471 Scientific Way, Irvine, CA 92618

*Correspondence: [email protected]

AUTHOR’S CONTRIBUTIONS

Conceptualization: EA and ST. Methodology: EA, and ST. Investigation: EA, SA, RA. Resources: ST, Writing: ST and EA. Funding acquisition: ST

Key Words: Transsulfuration, 3-Mercaptopyruvate sulfurtransferase, de-differentiation, Adipogenesis Running title: MPST safeguards fibroblasts differentiated state

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Abstract

Transitioning from a differentiated state to a higher-order of plasticity, by partial rather than full reactivation of pluripotency genes, might be a better approach in regenerative medicine. Hydrogen sulfide plays a crucial role in the maintenance and differentiation of mesenchymal stem cells (MSC) that have the potential to differentiate to a diverse group of mesenchymally derived cells. It was shown that these cells show a heavy reliance on cystathionineβ-synthase (CBS)-derived hydrogen sulfide (H2S) during differentiation. We have found that expression and activity of 3-mercaptopyruvate sulfurtransferase (MPST), one of three enzymes that hat regulates H2S biosynthesis, is significantly lower in MSC as compared with lineage-restricted dermal fibroblasts. Here, we tested the hypothesis that suppression of MPST in dermal fibroblasts might induce plasticity-related changes and broaden the transdifferentiation potency. Inactivation of MPST with phenylpyruvate (PP) or by siRNA silencing led to diminished H2S production associated with increased production of reactive oxygen species (ROS) and lactic acid. Accumulation of α-ketoglutarate (α-KG), a key metabolite required for the expression of ten-eleven translocation hydroxylase (TET), was associated with stimulated transcription of pluripotency related genes including OCT4, KLF4, SOX2, and NANOG. Suppression of TET1 gene and inhibition of glycolysis with glucose analog, 2-desoxy-d-glucose, or hexokinase II inhibitor significantly reduced expression of pluripotency genes following MPST inactivation or knockdown. MPST disruption promoted the conversion of fibroblasts into adipocytes as evidenced by a significant increase in expression of adipocyte-specific genes, PPARγ, and UCP1, and intracellular accumulation of oil Red-O positive fat droplets. Inhibition of glycolysis inhibited these changes. Under induced differentiation conditions, fibroblasts with disrupted MPST show the potency to differentiate to white adipogenic lineage. Thus, MPST inactivation or silencing enhances the plasticity of dermal fibroblasts in a TET1 and glycolysis dependent manner and promotes adipogenic transdifferentiation.

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Abbreviations HDF: human dermal fibroblast cells, MSCs: mesenchymal stem cells, iPSC: induced pluripotent cells, OXPHOS, oxidative phosphorylation, PP: Phenylpyruvate, CBS: cystathionine-beta-synthase, CSE: cystathionine gamma-lyase, MPST: mercaptopyruvate sulfurtransferase, TET1: Ten-eleven translocation methylcytosine dioxygenase 1, OCT4A: POU domain class 5, transcription factor 1, SOX2: SRY (sex-determining region Y)-box 2, NANOG: Homeobox Transcription Factor Nanog, KLF4: Kruppel like factor 4, cMYC: MYC Proto-Oncogene, LDHA: lactate dehydrogenase A, 3BrP: 3-Bromopyruvate , α-ketoglutarate: αKG, UCP1: uncoupling protein 1, UCP2: uncoupling protein 2, ADRB1: adrenoceptor beta 1, PPARG: peroxisome proliferator activated receptor gamma, PPARGC1A: PPARgamma Coactivator 1alpha, ADIPOQ: adiponectin, Asc-1: Solute Carrier Family 7 (Neutral Amino Acid Transporter Light Chain, Asc System), SLC1A3 (ASCT2): Solute Carrier Family 1 Member 3, SLC1A5: Solute Carrier Family 1 Member 5, SLC3A2: Solute Carrier Family 3 Member 2

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INTRODUCTION

Differentiation restricts or locks the plasticity of cells in a differentiated state. Thus, differentiation silences pluripotency genes and leads to the expression of a set of genes that locks the cell identity [1]. The notion that differentiation is terminally fixed, stable and cannot be reversed was changed when it was shown that, by the introduction of OKSM factors (OCT4A, KLF4, SOX2, cMYC), the differentiation properties could be altered [2, 3]. The plasticity of differentiated cells can be induced by various cell injuries, stress, and pharmacological interventions or conditioning. The ability to overcome the stress or injury, which is an inevitable part of life, requires plasticity in differentiated cells to enable their adaptation and survival. Dedifferentiation step is required before the induction of trans-differentiation. It was shown that brief pluripotency-inducing transcriptional reprogramming could promote cell fate changes in somatic cells [4]. Reliance on aerobic glycolysis appears to reset the epigenetic landscape that controls cell fate and physiology both in iPS and cancer cells [5, 6]. Energy metabolism shifts from glycolysis to oxidative phosphorylation (OXPHOS) as differentiation progresses, and the reverse occurs with the reprogramming path in fibroblasts [7]. The progressive resetting of metabolism and metabolite levels parallel progressive global epigenetic modifications and gene expression during reprogramming [8]. Transcriptome and proteome analysis has revealed that during dedifferentiation of somatic cells, the shift in bioenergetics, from primary reliance on OXPHOS to glycolysis, precedes and facilitates nuclear reprogramming [9, 10]. During the first 3-4 days of mouse embryonic fibroblast (MEF) reprogramming, consistent with the increase in glycolysis, these cells acidify their environment and only transiently change their oxygen consumption [11, 12]. Successfully reprogrammed cells exhibit a 2-fold increase in extracellular acidification [9, 13, 14]. The extent of the metabolic changes is lower in partially reprogrammed cells [15, 16]. Stimulation of glycolytic activity enhances, whereas inhibition of glycolysis reduces the efficiency of nuclear reprogramming of fibroblasts [9, 13, 14]. Thus, the glycolytic switch is required during trans-differentiation from one lineage to another lineage [17]. The trans-sulfuration and its upstream and downstream pathways are critically important in an array of diverse metabolic functions that are required for homeostasis. Hydrogen sulfide (H2S), a primary product of this pathway, is formed by at least three enzymes; namely cystathionine beta-synthase (CBS), cystathionine-γ-lyase (CTH or CSE), 3-mercaptopyruvate sulfurtransferase (MPST or 3MST). Intramitochondrial H2S produced by MPST acts as a stimulator of oxidative phosphorylation (OXPHOS) thereby increasing mitochondrial ATP production [18, 19]. In mesenchymal stem cells (MSCs), the expression of CBS and CSE was shown to correlate with osteogenic differentiation potential [20, 21]. Furthermore, CBS but not CSE is required for the chondrogenic differentiation of MSC [20].

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Fibroblasts share many characteristics with MSCs and show a similar pattern of gene expression and set of cell markers, yet, they show only a limited trans-differentiation potential [22, 23]. Fibroblasts and adipocytes can be derived from mesenchymal stem cells, suggesting that fibroblasts can transform into adipocytes under certain conditions. It was recently demonstrated that fibroblasts from human skeletal muscle can undergo adipogenic differentiation and facilitate the formation of fatty deposits in muscle [24]. Here, we tested whether MPST might influence the trans-differentiation potential of fibroblasts. To this end, we examined whether MPST inactivation or knockdown induces a metabolic switch that modifies the differentiation potential of human fibroblasts. The results reported here show that down-regulation or inactivation of MPST is consistent with a higher level of cellular plasticity as evidenced by the expression of pluripotency markers and enhanced plasticity of such cells to trans-differentiate into adipocytes.

MATERIALS AND METHODS

Materials Human dermal fibroblasts (HDF) were obtained from ATCC (Manassas, VA). Mesenchymal stem cells (MSCs), isolated from a 25-year-old male donor, were purchased from ReachBio Research lab (Seattle, WA). Chemicals were from Sigma-Aldrich Company (St Louis, MO) or Fisher Scientific (Pittsburgh, PA). siRNAs to CBS, TET1 and MPST were purchased from Santa Cruz Biotechnology (Paso Robles, CA) and IDT (San Diego, CA). Sequences of the siRNA and primers used for gene amplifications or silencing are shown in Tables 1 and 2.

Cell culture and viability assay Human dermal fibroblasts were maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented 5

with 10% fetal bovine serum (FBS) (Sigma-Aldrich Company, St Louis, MO). For experiments, cells (5×10 ) were grown in αMEM supplemented with knockout serum replacement (Gibco, Gaithersburg, MD) and incubated in o

humidified atmosphere of 5% CO2 at 37 C. Fibroblasts were used in passages 2 to 12 and were cultured in the absence or presence of specified chemicals for indicated number of days.

MSCs (ReachBio Research lab, Seattle, WA) were maintained in MesenPRO RS basal medium (Gibco-Life technologies, Gaithersburg, MD) with MesenPRO RS™ growth supplement. The MSCs were cultured at 37°C in 5% CO2 and low oxygen tension (5% O2) to sustain the multipotential phenotype of MSCs [25]. MSCs were used in passages 1 to 3.

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XF (extracellular flux) bioenergetic analysis Oxygen consumption rates (OCR) and pH measurements were performed using the XF24 Extracellular Flux analyzer (Seahorse Bioscience, North Billerica, MA) as described by the manufacturer and the bioenergetic assays were performed using the protocol provided in the XF24 manual (Wu et al., 2007). The concentrations of oligomycin, FCCP and antimycin A/Rotenone were optimized using Mito stress test kit (Seahorse Biosciences, Billerica, MA). Briefly, cells were plated at 30,000 cells/well in XF24 polystyrene cell culture plates (Seahorse Bioscience, North Billerica). Cells were incubated for 24 hours in a humidified 37°C incubator with 10% CO 2 (αMEM medium). Prior to performing an assay, growth medium in the wells of XF cell plates were exchanged with the appropriate assay medium (XF Base Medium supplemented with pyruvate) to achieve a minimum of 1/1000 dilution of growth medium. 600 µL of the assay medium was added to cells in each XF assay. While sensor cartridges were calibrated, cell plates were incubated at 37°C in a 37°C/non-CO

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incubator for 60 minutes prior to the start of an assay. All

compounds were prepared at appropriate concentrations in desired assay medium and adjusted to pH 7.4 before use. The compounds were administered in a total volume of 75 µL for each injection port. In a typical experiment, 3 baseline measurements were recorded prior to the addition of any compound, and 3 response measurements were recorded after the addition of each compound. Basal OCR and ECAR were measured, as well as the changes in oxygen consumption caused by the addition of the metabolic inhibitors described above. OCR was reported as absolute rates (pmoles/min for OCR) or expressed as a percentage of the baseline oxygen consumption. Level of OCR and ECAR was normalized to the protein content. Three independent experiments were performed in quintuplicate.

Transfection and RNA interference 5

For transfection, 2-5x10 cells were cultured in DMEM medium without serum or antibiotics. Then, K2 transfection reagent (Biontex, Germany) complexed with targeting (siRNA) and non-targeting scrambled (Scr) control RNAs (Cruz Biotechnology, Paso Robles, CA) were added to cell cultures. Each oligonucleotide was dissolved in 100 µM Duplex Buffer (100 mM Potassium Acetate, 30 mM HEPES, pH 7.5) and mixed in equal molar amounts, at a final concentration of 10 µM per oligonucleotide. Oligonucleotides were annealed at 94°C for 2 minutes and then cooled to room temperature for 2 hours. After 48 hours of transfection, the medium was replaced with αMEM medium supplemented with knockout serum replacement medium and cells were treated for 3 additional days prior to being harvested for analysis.

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Cell protein extraction and Western blotting Proteins from cells were extracted in a lysis buffer consisting of 50 mM Tris–HCl, pH7.5, 150 mM NaCl, 1% NP-40, 2 mM EDTA, 1 mM PMSF, 1 mM Na3VO4, 50 mM NaF, and protease inhibitor cocktail. Total protein content was quantified by Bio-Rad protein assay system based on Bradford dye-binding method (Bio-Rad Lab, Hercules, CA). Lysates with equal amounts of total protein were separated in 8, 10 or 12% polyacrylamide gels by SDS-PAGE electrophoresis under reducing conditions. Proteins were transferred to PVDF membrane and blots were probed with antibodies as detailed in the text and were detected by ECL enhanced chemiluminescence (Amersham ECL Plus Western Blotting Detection Reagents, GE Healthcare Life Sciences, Pittsburgh, PA) using C-Digital Imager (Li-COR, Lincoln, NE). Densitometric analysis of blots was performed using ImageJ analysis software using β-actin as a loading control.

RNA extraction, cDNA synthesis and quantitative real-time PCR Total RNA was extracted from cultured cells with TRI reagent (Sigma-Aldrich, St Louis, MO) following the manufacture’s instruction. Total RNA (1 µg) was reverse transcribed in a final volume of 20 µL using Thermo Scientific (Gaithersburg, MD) cDNA synthesis kit. cDNA was used in 10 µL reactions for quantitative real time PCR. The reaction conditions for qPCR were as follows: initial denaturation step at 95°C for 10 min, 40 cyc les of 95°C for 15 sec, 60°C for 45 sec, followed by melting curve analysis. Real-time quantitative PCR reaction mixture consisted of 5 µl iTaq Universal SYBR Green supermix (Bio-Rad, Hercules, CA), 1 µl upstream primer, 1 µl downstream primer, 1 µl cDNA and 1 µl ddH2O reactions. qPCR was performed using LightCycler 96 system (Roche Diagnostics, Indianapolis, IN). Relative expression level for each gene was normalized to that of β-ACTIN as a housekeeping −∆∆Ct

gene. The data obtained from qPCR were analyzed by relative quantification using 2

method as described

previously [26, 27]. Data were processed using Roche Real-Time PCR Analysis Software LightCycler 96 SW1.1 (Roche molecular diagnostics, Pleasanton, CA).

Biochemical analysis H2S levels were quantified by using Free Radical Analyzer (TBR4100 and ISO-H2S-2, World Precision 6

Instruments, Sarasota, FL) in accord with the manufacturer’s instruction. Briefly, 1×10 viable cells in PBS, were o

incubated at 37 C for 1 hr. Cells were then centrifuged and the supernatants were analyzed. Prior to the measurements, the sensor was polarized and calibrated by adding four aliquots of the Na2S stock solution at the final concentrations of 0.25, 0.5, 1 and 2 µM.

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Lactic acid was quantified by a spectrophotometric method using 1% p-phenylphenol as described previously [28]. Intracellular content of α-ketoglutarate (αKG) was quantified by a kit in accordance with the manufacturer’s instruction (Sigma-Aldrich, St Louis, MO). 2-

Sulfate (SO4 ) level in the medium was analyzed according to the method described by Wrobel et al and Golterman et al [29, 30]. We used changes in sulfate level as indicator of H2S catabolism [31]. However, we have to point out that cysteinesulfinate dependent oxidation of cysteine is another source of sulfate [32]. Activity of MPST in cell lysate was assessed as described previously [33, 34]. Cells were washed with PBS, lysed and total protein content was quantified by Bio-Rad protein assay system based on Bradford dye-binding method (Bio-Rad Lab, Hercules, CA). 10 µl of cell lysate was used for analysis of MPST activity. Briefly, the reaction mixture containing: 100 µl of 0.12M phosphate buffer (pH = 8.0), 10 µl of 1M Na2SO3, 30 µl of 0.1M DTT, 30 µl of distilled water, 10 µl of cell lysate and 20 µl of 0.1M 3-mercaptopyruvate was incubated at 37°C for 15 min. In blank samples, 3-mercaptopyruvate was omitted. Subsequently, 50 µl of 1.2M HClO4 were added and the mixture was centrifuged at 3 000 x g for 10 min. After centrifugation, 67 µl of the supernatant were collected and supplemented with 800 µl of 0.12M phosphate buffer (pH 8.0) and 67 µl of 0.1M N-Ethylmaleimide (NEM) and 33 µl of NADH. NEM was present to block the residual 3MP which is also a substrate of LDH [33]. Then, 1.5 µl of LDH were added and absorbance at a wavelength λ = 340 nm was measured 2 min after LDH addition. The difference between initial absorbance and its value after LDH addition corresponds to the amount of the pyruvate formed. The activity of the enzyme was expressed as µmoles of product formed during 1 min per mg protein. The enzyme activity was expressed as nmoles of pyruvate per 1 mg of protein produced during 1 min of incubation at 37°C.

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NAD /NADH levels were measured using NAD+/NADH assay kit (Bio Assay Systems, Hayward CA) following manufacturer's instruction. Measurement of intracellular ROS generation using DCFDA. The intracellular ROS generation was measured using 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA). Cells were loaded with 20 µM 2′,7′◦ dichlorodihydrofluorescein diacetate (DCFH-DA) for 30 min at 37 C. Fluorescence intensity was analyzed with excitation at 495 nm and emission at 520 nm using Infinite M Nano plate reader (Tecan, Switzerland). Arbitrary fluorescence values were normalization to protein level.

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Differentiation protocol Adipogenic differentiation For adipogenic differentiation, DMEM:HF12 (1:1) was supplemented with 10% FBS, 100 nM dexamethasone (Sigma-Aldrich, St Louis, MO), 10 µg/ml insulin ( (Sigma-Aldrich, St Louis, MO), 250 µM isobutyl methylxanthine (IBMX) (Cayman Chemical, Ann Arbor, MI) and 200 µM indomethacin (Sigma-Aldrich, St Louis, MO) and 1% ITS media supplement solution that included of 10 µg/ml insulin, 5.5 µg/ml transferrin, and 5 ng/ml selenium (Lonza, Walkersville, MD). Medium was changed every 4-5 days. Negative control cells for adipogenic differentiation o

received DMEM supplemented with 10% FBS. Cells were maintained at 37 C and 5% CO2. After 25 days of differentiation, cells were stained with Oil Red.

Histochemical staining Cells were fixed in 4% paraformaldehyde for 5 minutes at room temperature, and then they were washed with PBS/0.1% Tween-20 for 5 minutes. Alkaline phosphatase was visualized in cells via histochemical detection with 4-nitroblue tetrazolium (NBT) as a substrate and 5-bromo-4-chloro-8-indolilphosphate (BCIP) as a coupler and then equilibrated with NTMT solution (100 mM Tris pH 9.5, 100 mM NaCl, 50 mM MgCl2, 0.1% Tween-20). Color reaction was developed by incubating cells in NBT/BCIP solution and after 40 minutes, reaction was stopped by washing cells in PBS. For other histochemical staining, monolayer cells, cultured in differentiation medium or corresponding control DMEM medium were fixed in 10% neutral buffered formalin for 30 minutes at room temperature. Intracellular fat deposits were stained with Oil Red O. Oil red O (0.5% in isopropanol) was diluted with distilled water (3:2) and then filtered through a 0.45 µm filter. Cells fixed in 10% w/v formaldehyde were incubated with Oil Red solution for 30 min at room temperature and then cells were washed with water. The stained fat droplets were visualized and photographed with a light microscope (Olympus, Model IX50). Lipid density was assessed by extracting the Oil Red O with isopropanol and measuring the absorbance at 510 nm using SpectaMAX Plus plate reader (Molecular Devices, San Jose, California).

Statistical analysis All experiments were performed, at least in triplicates, and were repeated at least three times. The results are presented as means ± SEM derived from at least three experiments. Statistical analysis was performed using two-group comparisons by means of Student's t-test. Error bars in figures represent the Standard Deviation of the mean. Statistical significance is shown as *: p≤0.05, **: p≤0.005, ***: p ≤0.0005.

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RESULTS

TET1, CBS, CSE, and MPST are differentially expressed in human dermal fibroblasts (HDFs) and mesenchymal stem cells (MSCs) HDFs and MSCs share many properties including a spindle morphology, a set of common cell surface markers, as well as proliferation and differentiation potential. However, expression of pluripotency markers such as OCT4 and SSEA-4 which is important to stem cell identity and degree of differentiation potential sets the MSCs apart from fibroblasts. Adult multipotent stem cells including mesenchymal stem cells express Oct4, a transcription factor required for stem cell maintenance, but they do not express other transcription factors such as Nanog and Sox2 [35]. Consistent with previous reports, real-time quantitative PCR showed that OCT4A but not SOX2 and NANOG were significantly expressed in MSCs, while, as expected for differentiated cells, OCT4A, SOX2, and NANOG were not expressed in dermal fibroblasts (Figure 1A) [36, 37]. Fibroblasts and mesenchymal stem cells (MSCs) also differed in respect to the expression of Ten Eleven Translocation (TET) genes, which encode a family of hydroxymethylase enzymes, involved in regulating DNA methylation dynamics and which promote the acquisition of a naïve pluripotent state [38]. TET1 transcripts were expressed at a high copy number in MSCs while they were expressed at a low level in fibroblasts (Figure 1B). We next compared the expression level of major trans-sulfuration enzymes, CBS, CSE and MPST that drive production of H2S, which modulate diverse metabolic functions, in fibroblasts and MSCs [18, 19]. As assessed by qPCR, CBS was highly expressed in MSCs, while its expression was significantly lower in fibroblasts (Figure 1C). CSE expression remained essentially unchanged, while MPST expression was almost one order of magnitude lower in MSCs as compared to HDFs (Figure 1C). The MPST activity was also significantly lower in MSCs as compared to that in fibroblasts (Figure 1D). MPST silencing leads to metabolic changes and upregulates TET1 and pluripotency genes Since expression and activity of MPST were low in multipotent MSCs and high in lineage-restricted fibroblasts, we tested whether MPST is involved in programs that regulate differentiation and plasticity. To test the impact of MPST, MPST was knocked down using a short interfering RNA (siRNA). Successful silencing of MPST was confirmed by the suppression of its gene and protein expression, activity, a decrease in the production of H2S and 2-

amount of sulfate (SO4 ), the products of H2S oxidation, released to medium. (Figure 2A-E). The silencing of MPST

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in dermal fibroblasts resulted in the up-regulation of CBS but did not affect CSE (Figure 2A-B). Diminished production of H2S was responsible for the accumulation of reactive oxygen species (ROS) and oxidation of NADH (Figure 2F-G). MPST plays an essential role in cellular metabolism, mitochondrial function, and ATP production. For the biogenic role of MPST, analysis of lactate showed that MPST silencing led to a significant increase in lactic acid production and elevated extracellular acidification rate (ECAR), which is an indicator of the level of glycolysis (Figure 3A-B). However, there was no difference between the oxygen consumption rate (OCR) of control and MPST knockdown indicating that while MPST knockdown increases lactic acidosis, it does not affect OXPHOS (Figure 3C, Supplementary Figure 1). To further support the notion that MPST silencing impacts the aerobic glycolysis, we incubated cells with the 3-bromopyruvate (3BrP) that inhibits the activity of Hexokinase II by alkylation [39]. Cell viability and the formation of lactic acid were significantly suppressed following the addition of 3BrP in cells treated with MPST siRNA (Figure 3D-E). Treatment with a donor of H2S, NaHS, rescued the effect of MPST silencing on the production of lactic acid (Figure 3F). These data suggest that the silencing of MPST results in a metabolic shift towards glycolysis and that depletion of hydrogen sulfide as a result of disruption of MPST and consequent accumulation of ROS responsible for the metabolic shift. An increase in aerobic glycolysis can be achieved by limiting the entry of pyruvate into mitochondria either by inactivation of pyruvate dehydrogenase (PDH) complex or by the expression mitochondrial uncoupling proteins, UCP1 and UCP2. Uncoupling protein 1 (UCP1) is a transporter for H+ and fatty acid anions and plays an important role in adrenergic thermogenesis in brown-fat cells through uncoupling of OXPHOS from heat generation by mitochondria [40]. UCP2 regulates ATP and reactive oxygen species production by affecting the mitochondrial respiratory chain and affects metabolic functions by limiting mitochondrial glucose oxidation [41, 42]. To decipher whether MPST regulates these uncoupling proteins, we examined the expression of UCP1 and UCP2 in cells with MPST knockdown. Whereas the expression of UCP1 and UCP2 was repressed in fibroblasts, MPST knockdown resulted in their up-regulation (Figure 4A-B) [43]. The metabolic changes in fibroblasts, whose MPST was knocked down, led to a marked increase in αketoglutarate (α-KG) concentration (Figure 4C). It has been shown that the metabolite, α-KG, is required for the demethylase activity of TETs and for maintaining OCT4 gene expression and OCT4 reactivation [38]. Concomitant with the high levels of α-KG, the expression of TET1 as well as OCT4A, KLF4, SOX2, C-MYC and NANOG, was increased in fibroblasts with MPST knockdown (Figure 4D-E). The induction of SOX2 expression was highest (>8 fold), whereas KLF4 and C-MYC expression levels were less influenced (<2 fold). The up-regulation of SOX2 was confirmed by Western blotting (Figure 4E). The treatment of fibroblasts with 3BrP prevented the expression of pluripotency genes suggesting that MPST acts as a direct link between gaining metabolic acidosis and gene

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expressions (Figure 4F). Previously, it was shown that lactate, the end product of glycolysis, is a strong enhancer of UCP1 expression [44]. The expression of UCP1 in cells with MPST knockdown was blocked by inhibitor treatment. These data indicate that the ability of cells with silenced MPST to express UCP1 depended on lactic acid availability. Treatment with the donor of H2S, NaHS, diminished the effect of MPST silencing on the expression of SOX2 (Supplementary Figure 2).

Inactivation of MPST by phenylpyruvate leads to metabolic changes, accumulation of intracellular α-KG, and upregulation of pluripotency genes Since the activity of MPST was low in MSCs and high in HDF, we tested the hypothesis that reliance on MPST in differentiated cells might be responsible for the low differentiation potential of fibroblasts. To this end, we tested the effect of a known inhibitor of MPST, sodium phenylpyruvate (PP) which, when administered at 30 mM, reduces the activity of purified MPST to 11% of control values [45]. The treatment of fibroblasts with 1 mM PP for 6 days led to a significant reduction in its activity (Figure 5A). Despite an increase in CBS, the inactivation of MPST led to a decrease in the production of H2S and the release of sulfate by fibroblasts (Figure 5B-C). Cells treated with PP had the increased level of ROS production (Figure 5D). These results are consistent with results from the MPST knockdown model. Inactivation of MPST with PP shifted bioenergetics and enhanced the reliance of cells on glycolysis as evident by a pronounced increase in extracellular lactic acid (Figure 5E). Lactic acidosis in fibroblasts, which were treated with PP, was accompanied by the up-regulation of UCP1 and UCP2 (Figure 5F). Consistent with results obtained from MPST knockdown, in-activation of MPST with PP resulted in an increase in the level of α-ketoglutarate and expression level of TET1 both at RNA and protein level (Figure 6A-C). Coordinately, the expression of a core pluripotency genes OCT4A, KLF4, SOX2, and NANOG was up-regulated in cells that were treated for 6 days with 1 mM PP (Figure 6C). We further demonstrated that glycolysis is required for PP-induced increase in pluripotency gene markers (Figure 6D). To inhibit glycolysis, we used 2-Deoxy-D-glucose (2DG), a synthetic glucose analog. The treatment of fibroblasts with 200 µM of 2DG prevented the increase of OCT4 and NANOG induced by MPST inactivation with PP (Figure 6D). The induction of pluripotency gene markers was TET1 dependent as siRNA targeting of TET1 expression (Figure 6E) inhibited the PP induced expression of pluripotency gene markers (Figure 6F). Inactivation of MPST promotes adipogenic trans-differentiation of fibroblasts It was shown that OCT4 and SOX2 overexpression significantly promote the ability of MSCs to transdifferentiate into adipocytes and that SOX2 favors adipogenesis in MSCs via the upregulation of the inducer of adipogenesis, peroxisome proliferator-activated receptor-γ, PPARγ [46, 47]. Thus, we considered that the increase in

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expression of pluripotency genes, OCT4, and SOX2 in fibroblasts with MPST knockdown or PP treatment might similarly lead to an increase in the potency of fibroblasts to differentiate to an adipocyte lineage. MPST knockdown or Inactivation of MPST for 6 days by treatment with 1 mM PP promoted adipogenic differentiation as evidenced by an increase in accumulation of Oil Red O positive intracellular fat droplets and reactivation of UCP1 (Figure 7A-C). As was mentioned earlier, the increased level of lactate can induce the expression of mitochondrial UCP1. To test whether the production of lactic acid may be responsible for UCP1 expression, cells were treated with 2DG. The treatment of fibroblasts with 2DG prevented the UCP1 and adipocyte-specific transcription factor, PPARγ, the expression that could be induced by PP (Figure 7C). Since UCP1 is regulated in adipocytes via adrenergic signaling mechanism, we treated cells for 6 days jointly with PP and with 5 µM isoproterenol, a β-adrenergic agonist. This treatment further enhanced the expression of UCP1 and β-adrenergic receptor genes, ADRB1 and ADRB3, by about 10-fold over the control values and led to the intracellular accumulation of oil red O positive lipid droplets (Figure 7DF). UCP1 and ADRB3, are highly expressed in the inner membrane of mitochondria in brown adipose tissue and, under certain circumstances, in white adipose tissue [48]. Inactivation or silencing of MPST facilitates induced adipogenic trans-differentiation of fibroblasts Treatment of fibroblasts with PP followed by treatment with an adipogenic inducing medium containing IBMX, dexamethasone, indomethacin, and insulin for 25 days [49], increased expression of PPARγ, ADIPONECTIN (ADIPOQ), LEPTIN, and ASC-1 which are enriched in white adipose tissue (Figure 8A-B). Although MPST inactivation triggered the expression of UCP1, which is predominantly expressed in the inner membrane of mitochondria in brown adipose tissue, this expression was completely suppressed following induced adipogenesis (Figure 8A). Fibroblasts treated with PP and then subjected to adipogenic differentiation exhibited high storage of intra-cellular fat deposits (Figure 8B-C). Since PP might have off-target effects, we considered differentiating the modified fibroblasts with MPST knockdown due to its specificity. The silencing of MPST promoted adipogenic trans-differentiation (Figure 9A-C). Adipogenesis forced by MPST silencing was accompanied by morphological changes, typical of adipocytes including accumulation of intracellular lipid droplets as evidenced by Oil Red O staining (Figure 9A-B). Several key adipogenic genes such as PPARγ, PPARγC1, ADIPOQ, LEPTIN, and ASC-1 were significantly up-regulated (Figure 9C). The silencing of MPST triggered the expression of UCP1, which is predominantly expressed in the inner membrane of mitochondria in brown adipose tissue and under certain circumstances in white adipose tissue. However, this expression was prominent in DMEM but not in adipogenic inducing medium.

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DISCUSSION Shifts from relying on OXPHOS to glycolysis in differentiated cells change a number of parameters including cell cycle, biomass, and redox state and generates metabolites that influence cell fate. The data presented here reveal that suppression of the high level of MPST in fibroblasts modifies the metabolism in fibroblasts and supports up to the 2.5-fold increase in lactic acid production. Inhibition of the activity of MPST using PP, the α-Keto acids that, in a concentration-dependent manner, inhibits MPST, also increases extracellular acidification by lactic acid [45, 50]. PP was shown to have an inhibitory effect on the transport of pyruvate, pyruvate oxidation and oxygen consumption [51-54]. Fibroblasts with disrupted MPST show transition to glycolysis and acidification of their environment by excessive lactic acid production. The elevated concentration of extracellular lactate, as a result of suppression of MPST activity, might limit mitochondrial pyruvate since PP was shown to restrain the transport of pyruvate to mitochondria and to reduce oxygen consumption and pyruvate oxidation [51-55]. Increasing the level of ROS might be responsible for the induction of glycolysis since treatment with H2S donor, NaHS, prevents the accumulation of lactic acid. Wu et al reported that the energy metabolism shifts to anaerobic glycolysis as an adaptive response to oxidative stress in the primary cultures of skin fibroblasts [56]. The authors showed that hexokinase type II (HK II), lactate dehydrogenase (LDH) and glucose transporter 1 (GLUT1) were up-regulated in skin fibroblasts exposed to hydrogen peroxide. During oxidative stress, oxidatively modified proteins essentially associate with the tricarboxylic acid cycle and the mitochondrial function (α-ketoglutarate dehydrogenase (α-KGDH), succinate dehydrogenase, pyruvate dehydrogenase complex). Brand and colleagues had shown that ROS activate mitochondrial uncoupling proteins, UCP1 and UCP2, via modification of cysteine residues [57]. On the other hand, the extracellular acidification by MPST inactivation or knockdown might also be related to the changes in mitochondrial uncoupling proteins, which are known to redirect glucose-derived carbon from mitochondrial oxidation into pentose phosphate shunt which leads to acidification of the environment [43, 58]. The overexpression of UCP1 increases glucose uptake and lactate output without changes in oxygen consumption [59]. High levels of UCP2 decreases pyruvate oxidation and decouples glycolysis from OXPHOS by shunting pyruvate out of the mitochondria [43]. It has been suggested that UCPs block the usage of pyruvate by the TCA cycle by re-routing the mitochondrial function to fatty acid and glutamate oxidation [60]. Thus, suppression of the activity of MPST by PP might affect the level of mitochondrial pyruvate as a principle cause for the elevated concentration of extracellular lactate. Since extracellular acidification and increased lactate production is a hallmark of metabolic reprogramming, it has the potential to alter the fate of cells [58, 61]. Glycolytic pathway gene expression and DNA methylation patterns also change during reprogramming. During somatic cell reprogramming in murine cells, up-regulation of glycolytic genes including GLUT1, HXKE, and LDHA precedes the expression of pluripotency markers (OCT4,

14

NANOG, and SOX2) [16]. We showed that the fibroblasts with MPST knockdown express pluripotency markers due to an increase in glycolysis since extra-cellular acidification and reactivation of pluripotency markers by these cells could be inhibited by the inhibitors of glycolysis including treatment with 3-BP or 2-DG. α-Ketoglutarate is one of the intermediary metabolites of the TCA cycle and is also a glutaminolysis end product. Glutamine enters into the cell and is sequentially converted to glutamate and then to α-KG. Fibroblasts with silenced or inactivated MPST showed a significant increase in intracellular α-KG. The finding that expression of glutamine transporters and, therefore, uptake of glutamine were diminished in MPST knockdown cells (Supplementary Fig. 3) suggests that the overall accumulation of α-KG was unlikely due to increased glutaminolysis. Increasing the level of ROS leads to oxidative α-KGDH inhibition and accumulation of α-KG [62]. Since the accumulation of ROS was detected in cells with silenced or inactivated MPST, it is likely that the accumulation of αKG in these cells results from oxidative α-KGDH inhibition. Accumulation of α-KG may lead to α-KGDH-dependent oxidative stress which in turn may profoundly affect cell redox state and metabolism. Whether an increase in glycolysis is a consequence of KGDH inhibition or is part of an overall increase in glucose metabolism may be worth elucidating. α-KG is a required co-factor and activator for histone and DNA demethylation by α-KG-dependent hydroxylases such as members of TET family (TET1, TET2, and TET3) and α-KG-dependent demethylases such as mjC-domain-containing histone demethylases (JHDMs) [63, 64]. TET1 regulates the balance between the glycolysis and oxidative metabolic pathways and has been shown to substitute OCT4 in the OSKM cocktail for the generation of iPS cells [14, 65, 66]. DNA demethylation of promoter regions of pluripotency genes including NANOG, SOX2, and OCT4 by TET1 occurs very early during reprogramming of fibroblasts [67]. Silencing or inactivation of MPST results in a glycolytic shift and a coordinate re-activation of OCT4 and SOX2, the core transcription factor circuitry that is normally silenced in fibroblasts and which can activate the enhancers that regulate the expression of other pluripotency genes, including NANOG [68-70]. Cells with disrupted MPST, fail to activate the pluripotency program and, for this reason, they are more reminiscent of partially reprogrammed iPS cells that are in a transient stage of reprogramming [71-73]. Our results show that the expression of pluripotency genes can be effectively blocked by TET1 siRNA knockdown in cells undergoing MPST inactivation or knockdown which shows that the induction of pluripotency genes is TET1 dependent. The expression of OCT4 and SOX2 in fibroblasts with MPST inactivation or knockdown might favor their adipogenic potential. SOX2 overexpression in mouse embryonic stem cells promotes mesoderm differentiation and adipogenesis [74], while its depletion prevents adipogenic differentiation [46]. Also, overexpression of OCT4 alone or with SOX2, improved adipogenesis in MSCs [37, 47]. Fibroblasts, which overexpress UCP1 through MPST

15

inactivation or knockdown undergo rapid lipogenesis as evidenced by intracellular accumulation of Oil Red positive fat droplets. A similar increase in UCP proteins, mitochondrial uncoupling and a gradual increase in cellular lipids is also seen on day 4 of cellular reprogramming [75, 76]. One of the key genes that regulate lipogenesis in cells with MPST inactivation or knockdown, PPARγ, has been shown to regulate UCP1 gene expression by binding to PPARγbinding sites in UCP1 gene promoter, leading to the transformation of fibroblasts into adipocytes [77]. Moreover, PPARγ is known to be demethylated by TET proteins during adipogenesis [78]. Although fibroblasts share phenotypes that resemble those of mesenchymal stem cells, they lack differentiation and colony-forming potential. However, fibroblasts show some degree of phenotype plasticity that can be controlled. For example, it was shown that fibroblasts have the potential to differentiate into various mesodermal lineages [79, 80]. The disruption of the fibroblastic transcriptional regulatory network, that includes PPRX1, OSR1, TWIST2, and LHX9, was shown to facilitate adipogenic trans-differentiation [81]. Takeda and co-authors recently reported a method for direct conversion of human fibroblasts to brown adipocyte using inhibitors for GSK3, MEK, and p53, and for TGF-beta and BMP signaling [82]. Here, we show that silencing or inactivation of a single protein, MPST, can promote de-differentiation in fibroblasts and prime these cells for adipogenic trans-differentiation. There are two main types of adipose tissues, white adipose tissue (WAT) which lacks UCP1 and brown adipose tissue (BAT), which is rich in mitochondria that contain UCP1. Brown adipocytes show increased uptake of glucose, express several glycolytic enzymes and release high amounts of lactate [83, 84]. Suppression of glycolytic flux in brown adipocytes by knockdown of hexokinase II (HK II) decreases glucose uptake and reduces thermogenesis that cannot be rescued by supplementation with pyruvate [85]. Similarly, inhibition of glycolysis suppressed the expression of UCP1 in cells with inactivated MPST. In brown and brite (brown-like-in-white) fat cells, UCP1 expression is regulated by β-adrenergic receptors (ADRBs) [86-88]. Treatment with isoproterenol leads to the up-regulation of UCP1 and consequently leads to the disruption of the coupling efficiency of the electron transport chain, enhancing glycolysis [89]. β-Adrenergic activation leads to the induction of several glycolytic enzymes including HK1 and HKII in cultured brown adipocytes [83]. Here, we show that inactivation of MPST in fibroblast cells causes reactivation of β-adrenergic receptors, increases UCP1, which is otherwise not normally expressed in fibroblasts, and concomitantly enhances glycolysis. It was shown that the increased level of lactate resulted in increased expression of mitochondrial uncoupling protein 1 (UCP1) in muscle cells [90]. An elevation in ROS generation was shown to associate with mitochondrial proliferation [91-93]. ROS is also the underlying trigger of UCP1 induction. It was reported that H2O2 treatment induced UCP1 expression in brown-fat fibroblasts even when PGC-1α was ablated, thus separating UCP1 induction from mitochondrial biogenesis [94].

16

Fibroblasts treated with an inhibitor of MPST become responsive to β-adrenergic agonist, isoproterenol, as evidenced by enhanced adipogenesis and expression of UCP1 and ADRB1 and 3. In response to small molecules, including insulin, dexamethasone, and isobutylmethylxanthine (IBMX), MSCs differentiate into white adipocytes [95, 96]. Fibroblasts thath undergo MPST inactivation or silencing become sensitive to the conditions that are found to promote differentiation in MSCs to WAT. These cells while silence their UPC1, highly express adipocyte-specific transcription factors, PPARγ, and other key adipogenic genes such as PPARGC1, ADIPOQ, LEPTIN, ASC-1. In summary, the results presented here, demonstrate that, by optimizing the combination and dosage of small molecules regulating MPST activity, it is possible to induce sufficient plasticity in fibroblasts to unlock their adipogenic potential.

AUTHOR CONTRIBUTIONS Conceptualization: EA and ST. Methodology: EA, and ST. Investigation: EA, SA, RA. Resources: ST, Writing: ST and EA. Funding acquisition: ST

DECLARATIONS OF INTEREST

The authors do not declare any conflict of interest.

REFERENCES

[1] [2] [3] [4]

[5] [6] [7] [8]

M.J. Boland, K.L. Nazor, J.F. Loring, Epigenetic regulation of pluripotency and differentiation, Circ Res 115 (2014) 311-324. J.B. Gurdon, Adult frogs derived from the nuclei of single somatic cells, Dev Biol 4 (1962) 256-273. K. Takahashi, S. Yamanaka, Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors, Cell 126 (2006) 663-676. O. Bar-Nur, C. Verheul, A.G. Sommer, J. Brumbaugh, B.A. Schwarz, I. Lipchina, A.J. Huebner, G. Mostoslavsky, K. Hochedlinger, Lineage conversion induced by pluripotency factors involves transient passage through an iPSC stage, Nat Biotechnol 33 (2015) 761-768. C.V. Dang, Links between metabolism and cancer, Genes Dev 26 (2012) 877-890. P.S. Ward, C.B. Thompson, Metabolic reprogramming: a cancer hallmark even warburg did not anticipate, Cancer Cell 21 (2012) 297-308. J. Mathieu, H. Ruohola-Baker, Metabolic remodeling during the loss and acquisition of pluripotency, Development 144 (2017) 541-551. M.H. Chin, M.J. Mason, W. Xie, S. Volinia, M. Singer, C. Peterson, G. Ambartsumyan, O. Aimiuwu, L. Richter, J. Zhang, I. Khvorostov, V. Ott, M. Grunstein, N. Lavon, N. Benvenisty, C.M. Croce, A.T. Clark, T. Baxter, A.D. Pyle, M.A. Teitell, M. Pelegrini, K. 17

[9]

[10]

[11]

[12]

[13] [14]

[15]

[16]

[17] [18]

[19]

[20]

Plath, W.E. Lowry, Induced pluripotent stem cells and embryonic stem cells are distinguished by gene expression signatures, Cell Stem Cell 5 (2009) 111-123. C.D. Folmes, T.J. Nelson, A. Martinez-Fernandez, D.K. Arrell, J.Z. Lindor, P.P. Dzeja, Y. Ikeda, C. Perez-Terzic, A. Terzic, Somatic oxidative bioenergetics transitions into pluripotency-dependent glycolysis to facilitate nuclear reprogramming, Cell Metab 14 (2011) 264-271. J. Zhang, E. Nuebel, G.Q. Daley, C.M. Koehler, M.A. Teitell, Metabolic regulation in pluripotent stem cells during reprogramming and self-renewal, Cell Stem Cell 11 (2012) 589-595. Y.S. Kida, T. Kawamura, Z. Wei, T. Sogo, S. Jacinto, A. Shigeno, H. Kushige, E. Yoshihara, C. Liddle, J.R. Ecker, R.T. Yu, A.R. Atkins, M. Downes, R.M. Evans, ERRs Mediate a Metabolic Switch Required for Somatic Cell Reprogramming to Pluripotency, Cell Stem Cell 16 (2015) 547-555. K.E. Hawkins, S. Joy, J.M. Delhove, V.N. Kotiadis, E. Fernandez, L.M. Fitzpatrick, J.R. Whiteford, P.J. King, J.P. Bolanos, M.R. Duchen, S.N. Waddington, T.R. McKay, NRF2 Orchestrates the Metabolic Shift during Induced Pluripotent Stem Cell Reprogramming, Cell Rep 14 (2016) 1883-1891. Y. Yoshida, K. Takahashi, K. Okita, T. Ichisaka, S. Yamanaka, Hypoxia enhances the generation of induced pluripotent stem cells, Cell Stem Cell 5 (2009) 237-241. M.A. Esteban, T. Wang, B. Qin, J. Yang, D. Qin, J. Cai, W. Li, Z. Weng, J. Chen, S. Ni, K. Chen, Y. Li, X. Liu, J. Xu, S. Zhang, F. Li, W. He, K. Labuda, Y. Song, A. Peterbauer, S. Wolbank, H. Redl, M. Zhong, D. Cai, L. Zeng, D. Pei, Vitamin C enhances the generation of mouse and human induced pluripotent stem cells, Cell Stem Cell 6 (2010) 71-79. S.J. Park, S.A. Lee, N. Prasain, D. Bae, H. Kang, T. Ha, J.S. Kim, K.S. Hong, C. Mantel, S.H. Moon, H.E. Broxmeyer, M.R. Lee, Metabolome Profiling of Partial and Fully Reprogrammed Induced Pluripotent Stem Cells, Stem Cells Dev 26 (2017) 734-742. A.D. Panopoulos, O. Yanes, S. Ruiz, Y.S. Kida, D. Diep, R. Tautenhahn, A. Herrerias, E.M. Batchelder, N. Plongthongkum, M. Lutz, W.T. Berggren, K. Zhang, R.M. Evans, G. Siuzdak, J.C. Izpisua Belmonte, The metabolome of induced pluripotent stem cells reveals metabolic changes occurring in somatic cell reprogramming, Cell Res 22 (2012) 168-177. L. Lai, E. Reineke, D.J. Hamilton, J.P. Cooke, Glycolytic Switch Is Required for Transdifferentiation to Endothelial Lineage, Circulation 139 (2019) 119-133. M. Fu, W. Zhang, L. Wu, G. Yang, H. Li, R. Wang, Hydrogen sulfide (H2S) metabolism in mitochondria and its regulatory role in energy production, Proc Natl Acad Sci U S A 109 (2012) 2943-2948. C. Szabo, C. Ransy, K. Modis, M. Andriamihaja, B. Murghes, C. Coletta, G. Olah, K. Yanagi, F. Bouillaud, Regulation of mitochondrial bioenergetic function by hydrogen sulfide. Part I. Biochemical and physiological mechanisms, Br J Pharmacol 171 (2014) 2099-2122. L. Gambari, G. Lisignoli, E. Gabusi, C. Manferdini, F. Paolella, A. Piacentini, F. Grassi, Distinctive expression pattern of cystathionine-beta-synthase and cystathionine-gammalyase identifies mesenchymal stromal cells transition to mineralizing osteoblasts, J Cell Physiol 232 (2017) 3574-3585. 18

[21]

[22]

[23]

[24]

[25] [26] [27] [28] [29]

[30] [31] [32] [33] [34]

[35]

[36]

Y. Liu, R. Yang, X. Liu, Y. Zhou, C. Qu, T. Kikuiri, S. Wang, E. Zandi, J. Du, I.S. Ambudkar, S. Shi, Hydrogen sulfide maintains mesenchymal stem cell function and bone homeostasis via regulation of Ca(2+) channel sulfhydration, Cell Stem Cell 15 (2014) 6678. D.T. Covas, R.A. Panepucci, A.M. Fontes, W.A. Silva, Jr., M.D. Orellana, M.C. Freitas, L. Neder, A.R. Santos, L.C. Peres, M.C. Jamur, M.A. Zago, Multipotent mesenchymal stromal cells obtained from diverse human tissues share functional properties and geneexpression profile with CD146+ perivascular cells and fibroblasts, Exp Hematol 36 (2008) 642-654. R.A. Denu, S. Nemcek, D.D. Bloom, A.D. Goodrich, J. Kim, D.F. Mosher, P. Hematti, Fibroblasts and Mesenchymal Stromal/Stem Cells Are Phenotypically Indistinguishable, Acta Haematol 136 (2016) 85-97. C.C. Agley, A.M. Rowlerson, C.P. Velloso, N.R. Lazarus, S.D. Harridge, Human skeletal muscle fibroblasts, but not myogenic cells, readily undergo adipogenic differentiation, J Cell Sci 126 (2013) 5610-5625. A. Mohyeldin, T. Garzon-Muvdi, A. Quinones-Hinojosa, Oxygen in stem cell biology: a critical component of the stem cell niche, Cell Stem Cell 7 (2010) 150-161. K.J. Livak, T.D. Schmittgen, Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method, Methods 25 (2001) 402-408. E.C. Jensen, Real-time reverse transcription polymerase chain reaction to measure mRNA: use, limitations, and presentation of results, Anat Rec (Hoboken) 295 (2012) 1-3. K.A. Taylor, A simple colorimetric assay for muramic acid and lactic acid. , Applied Biochemistry and Biotechnology 56 (1996) 49-58. M. Wrobel, P. Sura, Z. Srebro, Sulfurtransferases and the content of cysteine, glutathione and sulfane sulfur in tissues of the frog Rana temporaria, Comp Biochem Physiol B Biochem Mol Biol 125 (2000) 211-217. H.L.D.G.B.-W. GoltermanI, I.M, Colorimetric determination of sulphate in freshwater with a chromate reagent Hydrobiologia 228 (1992) 111-115. R.O. Beauchamp, Jr., J.S. Bus, J.A. Popp, C.J. Boreiko, D.A. Andjelkovich, A critical review of the literature on hydrogen sulfide toxicity, Crit Rev Toxicol 13 (1984) 25-97. M.H. Stipanuk, I. Ueki, Dealing with methionine/homocysteine sulfur: cysteine metabolism to taurine and inorganic sulfur, J Inherit Metab Dis 34 (2011) 17-32. W.N. Valentine, J.K. Frankenfeld, 3-Mercaptopyruvate sulfurtransferase (EC 2.8.1.2): a simple assay adapted to human blood cells, Clin Chim Acta 51 (1974) 205-210. M. Wrobel, H. Jurkowska, L. Sliwa, Z. Srebro, Sulfurtransferases and cyanide detoxification in mouse liver, kidney, and brain, Toxicol Mech Methods 14 (2004) 331337. L.A. Boyer, T.I. Lee, M.F. Cole, S.E. Johnstone, S.S. Levine, J.P. Zucker, M.G. Guenther, R.M. Kumar, H.L. Murray, R.G. Jenner, D.K. Gifford, D.A. Melton, R. Jaenisch, R.A. Young, Core transcriptional regulatory circuitry in human embryonic stem cells, Cell 122 (2005) 947-956. C. Moriscot, F. de Fraipont, M.J. Richard, M. Marchand, P. Savatier, D. Bosco, M. Favrot, P.Y. Benhamou, Human bone marrow mesenchymal stem cells can express insulin and key transcription factors of the endocrine pancreas developmental pathway upon genetic and/or microenvironmental manipulation in vitro, Stem Cells 23 (2005) 594-603. 19

[37] [38]

[39]

[40] [41]

[42]

[43]

[44]

[45] [46]

[47]

[48] [49]

[50] [51]

T.M. Liu, Y.N. Wu, X.M. Guo, J.H. Hui, E.H. Lee, B. Lim, Effects of ectopic Nanog and Oct4 overexpression on mesenchymal stem cells, Stem Cells Dev 18 (2009) 1013-1022. G. Marsboom, G.F. Zhang, N. Pohl-Avila, Y. Zhang, Y. Yuan, H. Kang, B. Hao, H. Brunengraber, A.B. Malik, J. Rehman, Glutamine Metabolism Regulates the Pluripotency Transcription Factor OCT4, Cell Rep 16 (2016) 323-332. Z. Chen, H. Zhang, W. Lu, P. Huang, Role of mitochondria-associated hexokinase II in cancer cell death induced by 3-bromopyruvate, Biochimica et biophysica acta 1787 (2009) 553-560. A.M. Bertholet, Y. Kirichok, UCP1: A transporter for H(+) and fatty acid anions, Biochimie 134 (2017) 28-34. A. Vozza, G. Parisi, F. De Leonardis, F.M. Lasorsa, A. Castegna, D. Amorese, R. Marmo, V.M. Calcagnile, L. Palmieri, D. Ricquier, E. Paradies, P. Scarcia, F. Palmieri, F. Bouillaud, G. Fiermonte, UCP2 transports C4 metabolites out of mitochondria, regulating glucose and glutamine oxidation, Proc Natl Acad Sci U S A 111 (2014) 960-965. J. Su, J. Liu, X.Y. Yan, Y. Zhang, J.J. Zhang, L.C. Zhang, L.K. Sun, Cytoprotective Effect of the UCP2-SIRT3 Signaling Pathway by Decreasing Mitochondrial Oxidative Stress on Cerebral Ischemia-Reperfusion Injury, Int J Mol Sci 18 (2017). J. Zhang, I. Khvorostov, J.S. Hong, Y. Oktay, L. Vergnes, E. Nuebel, P.N. Wahjudi, K. Setoguchi, G. Wang, A. Do, H.J. Jung, J.M. McCaffery, I.J. Kurland, K. Reue, W.N. Lee, C.M. Koehler, M.A. Teitell, UCP2 regulates energy metabolism and differentiation potential of human pluripotent stem cells, EMBO J 30 (2011) 4860-4873. A. Carriere, Y. Jeanson, S. Berger-Muller, M. Andre, V. Chenouard, E. Arnaud, C. Barreau, R. Walther, A. Galinier, B. Wdziekonski, P. Villageois, K. Louche, P. Collas, C. Moro, C. Dani, F. Villarroya, L. Casteilla, Browning of white adipose cells by intermediate metabolites: an adaptive mechanism to alleviate redox pressure, Diabetes 63 (2014) 3253-3265. D.W. Porter, S.I. Baskin, The effect of three alpha-keto acids on 3-mercaptopyruvate sulfurtransferase activity, J Biochem Toxicol 11 (1996) 45-50. E. Seo, U. Basu-Roy, P.H. Gunaratne, C. Coarfa, D.S. Lim, C. Basilico, A. Mansukhani, SOX2 regulates YAP1 to maintain stemness and determine cell fate in the osteo-adipo lineage, Cell reports 3 (2013) 2075-2087. S.M. Han, S.H. Han, Y.R. Coh, G. Jang, J. Chan Ra, S.K. Kang, H.W. Lee, H.Y. Youn, Enhanced proliferation and differentiation of Oct4- and Sox2-overexpressing human adipose tissue mesenchymal stem cells, Experimental & molecular medicine 46 (2014) e101. Y.H. Lee, S.N. Kim, H.J. Kwon, J.G. Granneman, Metabolic heterogeneity of activated beige/brite adipocytes in inguinal adipose tissue, Scientific reports 7 (2017) 39794. M.A. Scott, V.T. Nguyen, B. Levi, A.W. James, Current methods of adipogenic differentiation of mesenchymal stem cells, Stem cells and development 20 (2011) 17931804. D.A. Wing, S.I. Baskin, Modifiers of mercaptopyruvate sulfurtransferase catalyzed conversion of cyanide to thiocyanate in vitro, J Biochem Toxicol 7 (1992) 65-72. A.P. Halestrap, M.D. Brand, R.M. Denton, Inhibition of mitochondrial pyruvate transport by phenylpyruvate and alpha-ketoisocaproate, Biochim Biophys Acta 367 (1974) 102108. 20

[52] [53] [54] [55] [56]

[57]

[58]

[59]

[60]

[61] [62] [63]

[64] [65]

[66]

[67]

J. Swierczynski, Z. Aleksandrowicz, M. Zydowo, Inhibition of pyruvate oxidation by skeletal muscle mitochondria by phenylpyruvate, Acta Biochim Pol 23 (1976) 85-92. R.K. Howell, M. Lee, Influence of alpha-ketoacids on the respiration of brain in vitro, Proc Soc Exp Biol Med 113 (1963) 660-663. B.B. Gallagher, The effect of phenylpyruvate on oxidative-phosphorylation in brain mitochondria, J Neurochem 16 (1969) 1071-1076. T. Itoh, Effects of sodium phenylpyruvate on amino acid formation in brain, Can J Biochem 43 (1965) 835-840. S.B. Wu, Y.H. Wei, AMPK-mediated increase of glycolysis as an adaptive response to oxidative stress in human cells: implication of the cell survival in mitochondrial diseases, Biochim Biophys Acta 1822 (2012) 233-247. K.S. Echtay, D. Roussel, J. St-Pierre, M.B. Jekabsons, S. Cadenas, J.A. Stuart, J.A. Harper, S.J. Roebuck, A. Morrison, S. Pickering, J.C. Clapham, M.D. Brand, Superoxide activates mitochondrial uncoupling proteins, Nature 415 (2002) 96-99. W. Zhou, M. Choi, D. Margineantu, L. Margaretha, J. Hesson, C. Cavanaugh, C.A. Blau, M.S. Horwitz, D. Hockenbery, C. Ware, H. Ruohola-Baker, HIF1alpha induced switch from bivalent to exclusively glycolytic metabolism during ESC-to-EpiSC/hESC transition, EMBO J 31 (2012) 2103-2116. Y. Si, S. Palani, A. Jayaraman, K. Lee, Effects of forced uncoupling protein 1 expression in 3T3-L1 cells on mitochondrial function and lipid metabolism, J Lipid Res 48 (2007) 826-836. I. Samudio, M. Fiegl, M. Andreeff, Mitochondrial uncoupling and the Warburg effect: molecular basis for the reprogramming of cancer cell metabolism, Cancer Res 69 (2009) 2163-2166. M.G. Vander Heiden, L.C. Cantley, C.B. Thompson, Understanding the Warburg effect: the metabolic requirements of cell proliferation, Science 324 (2009) 1029-1033. L. Tretter, V. Adam-Vizi, Alpha-ketoglutarate dehydrogenase: a target and generator of oxidative stress, Philos Trans R Soc Lond B Biol Sci 360 (2005) 2335-2345. W. Xu, H. Yang, Y. Liu, Y. Yang, P. Wang, S.H. Kim, S. Ito, C. Yang, P. Wang, M.T. Xiao, L.X. Liu, W.Q. Jiang, J. Liu, J.Y. Zhang, B. Wang, S. Frye, Y. Zhang, Y.H. Xu, Q.Y. Lei, K.L. Guan, S.M. Zhao, Y. Xiong, Oncometabolite 2-hydroxyglutarate is a competitive inhibitor of alpha-ketoglutarate-dependent dioxygenases, Cancer Cell 19 (2011) 17-30. W.G. Kaelin, Jr., S.L. McKnight, Influence of metabolism on epigenetics and disease, Cell 153 (2013) 56-69. Y. Gao, J. Chen, K. Li, T. Wu, B. Huang, W. Liu, X. Kou, Y. Zhang, H. Huang, Y. Jiang, C. Yao, X. Liu, Z. Lu, Z. Xu, L. Kang, J. Chen, H. Wang, T. Cai, S. Gao, Replacement of Oct4 by Tet1 during iPSC induction reveals an important role of DNA methylation and hydroxymethylation in reprogramming, Cell Stem Cell 12 (2013) 453-469. R. Khoueiry, A. Sohni, B. Thienpont, X. Luo, J.V. Velde, M. Bartoccetti, B. Boeckx, A. Zwijsen, A. Rao, D. Lambrechts, K.P. Koh, Lineage-specific functions of TET1 in the postimplantation mouse embryo, Nat Genet 49 (2017) 1061-1072. N. Bhutani, J.J. Brady, M. Damian, A. Sacco, S.Y. Corbel, H.M. Blau, Reprogramming towards pluripotency requires AID-dependent DNA demethylation, Nature 463 (2010) 1042-1047. 21

[68] [69]

[70]

[71]

[72]

[73]

[74] [75]

[76]

[77]

[78]

[79]

[80]

[81]

[82]

L. Chen, G.Q. Daley, Molecular basis of pluripotency, Hum Mol Genet 17 (2008) R2327. W.E. Lowry, L. Richter, R. Yachechko, A.D. Pyle, J. Tchieu, R. Sridharan, A.T. Clark, K. Plath, Generation of human induced pluripotent stem cells from dermal fibroblasts, Proc Natl Acad Sci U S A 105 (2008) 2883-2888. J.L. Chew, Y.H. Loh, W. Zhang, X. Chen, W.L. Tam, L.S. Yeap, P. Li, Y.S. Ang, B. Lim, P. Robson, H.H. Ng, Reciprocal transcriptional regulation of Pou5f1 and Sox2 via the Oct4/Sox2 complex in embryonic stem cells, Molecular and cellular biology 25 (2005) 6031-6046. A. Meissner, T.S. Mikkelsen, H. Gu, M. Wernig, J. Hanna, A. Sivachenko, X. Zhang, B.E. Bernstein, C. Nusbaum, D.B. Jaffe, A. Gnirke, R. Jaenisch, E.S. Lander, Genomescale DNA methylation maps of pluripotent and differentiated cells, Nature 454 (2008) 766-770. R. Sridharan, J. Tchieu, M.J. Mason, R. Yachechko, E. Kuoy, S. Horvath, Q. Zhou, K. Plath, Role of the murine reprogramming factors in the induction of pluripotency, Cell 136 (2009) 364-377. T.S. Mikkelsen, J. Hanna, X. Zhang, M. Ku, M. Wernig, P. Schorderet, B.E. Bernstein, R. Jaenisch, E.S. Lander, A. Meissner, Dissecting direct reprogramming through integrative genomic analysis, Nature 454 (2008) 49-55. J.L. Kopp, B.D. Ormsbee, M. Desler, A. Rizzino, Small increases in the level of Sox2 trigger the differentiation of mouse embryonic stem cells, Stem cells 26 (2008) 903-911. A. Vazquez-Martin, B. Corominas-Faja, S. Cufi, L. Vellon, C. Oliveras-Ferraros, O.J. Menendez, J. Joven, R. Lupu, J.A. Menendez, The mitochondrial H(+)-ATP synthase and the lipogenic switch: new core components of metabolic reprogramming in induced pluripotent stem (iPS) cells, Cell Cycle 12 (2013) 207-218. L. Wang, T. Zhang, L. Wang, Y. Cai, X. Zhong, X. He, L. Hu, S. Tian, M. Wu, L. Hui, H. Zhang, P. Gao, Fatty acid synthesis is critical for stem cell pluripotency via promoting mitochondrial fission, EMBO J 36 (2017) 1330-1347. J.H. Chen, K.J. Goh, N. Rocha, M.P. Groeneveld, M. Minic, T.G. Barrett, D. Savage, R.K. Semple, Evaluation of human dermal fibroblasts directly reprogrammed to adipocyte-like cells as a metabolic disease model, Dis Model Mech 10 (2017) 1411-1420. K. Fujiki, A. Shinoda, F. Kano, R. Sato, K. Shirahige, M. Murata, PPARgamma-induced PARylation promotes local DNA demethylation by production of 5hydroxymethylcytosine, Nat Commun 4 (2013) 2262. J.P. Junker, P. Sommar, M. Skog, H. Johnson, G. Kratz, Adipogenic, chondrogenic and osteogenic differentiation of clonally derived human dermal fibroblasts, Cells Tissues Organs 191 (2010) 105-118. K. Lorenz, M. Sicker, E. Schmelzer, T. Rupf, J. Salvetter, M. Schulz-Siegmund, A. Bader, Multilineage differentiation potential of human dermal skin-derived fibroblasts, Exp Dermatol 17 (2008) 925-932. Y. Tomaru, R. Hasegawa, T. Suzuki, T. Sato, A. Kubosaki, M. Suzuki, H. Kawaji, A.R. Forrest, Y. Hayashizaki, F. Consortium, J.W. Shin, H. Suzuki, A transient disruption of fibroblastic transcriptional regulatory network facilitates trans-differentiation, Nucleic Acids Res 42 (2014) 8905-8913. Y. Takeda, Y. Harada, T. Yoshikawa, P. Dai, Direct conversion of human fibroblasts to brown adipocytes by small chemical compounds, Sci Rep 7 (2017) 4304. 22

[83]

[84]

[85]

[86]

[87] [88] [89]

[90]

[91]

[92]

[93]

[94]

[95]

[96]

A.L. Basse, M.S. Isidor, S. Winther, N.B. Skjoldborg, M. Murholm, E.S. Andersen, S.B. Pedersen, C. Wolfrum, B. Quistorff, J.B. Hansen, Regulation of glycolysis in brown adipocytes by HIF-1alpha, Scientific reports 7 (2017) 4052. C. Petersen, M.D. Nielsen, E.S. Andersen, A.L. Basse, M.S. Isidor, L.K. Markussen, B.M. Viuff, I.H. Lambert, J.B. Hansen, S.F. Pedersen, MCT1 and MCT4 Expression and Lactate Flux Activity Increase During White and Brown Adipogenesis and Impact Adipocyte Metabolism, Scientific reports 7 (2017) 13101. S. Winther, M.S. Isidor, A.L. Basse, N. Skjoldborg, A. Cheung, B. Quistorff, J.B. Hansen, Restricting glycolysis impairs brown adipocyte glucose and oxygen consumption, Am J Physiol Endocrinol Metab 314 (2018) E214-E223. E.M. Rohlfs, K.W. Daniel, R.T. Premont, L.P. Kozak, S. Collins, Regulation of the uncoupling protein gene (Ucp) by beta 1, beta 2, and beta 3-adrenergic receptor subtypes in immortalized brown adipose cell lines, The Journal of biological chemistry 270 (1995) 10723-10732. B. Cannon, J. Nedergaard, Brown adipose tissue: function and physiological significance, Physiol Rev 84 (2004) 277-359. Y. Jiang, D.C. Berry, J.M. Graff, Distinct cellular and molecular mechanisms for beta3 adrenergic receptor-induced beige adipocyte formation, Elife 6 (2017). C.N. Miller, J.Y. Yang, E. England, A. Yin, C.A. Baile, S. Rayalam, Isoproterenol Increases Uncoupling, Glycolysis, and Markers of Beiging in Mature 3T3-L1 Adipocytes, PLoS One 10 (2015) e0138344. N. Kim, M. Nam, M.S. Kang, J.O. Lee, Y.W. Lee, G.S. Hwang, H.S. Kim, Piperine regulates UCP1 through the AMPK pathway by generating intracellular lactate production in muscle cells, Sci Rep 7 (2017) 41066. G. Barja de Quiroga, M. Lopez-Torres, R. Perez-Campo, M. Abelenda, M. Paz Nava, M.L. Puerta, Effect of cold acclimation on GSH, antioxidant enzymes and lipid peroxidation in brown adipose tissue, Biochem J 277 ( Pt 1) (1991) 289-292. R.J. Mailloux, C.N. Adjeitey, J.Y. Xuan, M.E. Harper, Crucial yet divergent roles of mitochondrial redox state in skeletal muscle vs. brown adipose tissue energetics, FASEB J 26 (2012) 363-375. E.T. Chouchani, L. Kazak, M.P. Jedrychowski, G.Z. Lu, B.K. Erickson, J. Szpyt, K.A. Pierce, D. Laznik-Bogoslavski, R. Vetrivelan, C.B. Clish, A.J. Robinson, S.P. Gygi, B.M. Spiegelman, Mitochondrial ROS regulate thermogenic energy expenditure and sulfenylation of UCP1, Nature 532 (2016) 112-116. J. St-Pierre, S. Drori, M. Uldry, J.M. Silvaggi, J. Rhee, S. Jager, C. Handschin, K. Zheng, J. Lin, W. Yang, D.K. Simon, R. Bachoo, B.M. Spiegelman, Suppression of reactive oxygen species and neurodegeneration by the PGC-1 transcriptional coactivators, Cell 127 (2006) 397-408. L. Janderova, M. McNeil, A.N. Murrell, R.L. Mynatt, S.R. Smith, Human mesenchymal stem cells as an in vitro model for human adipogenesis, Obesity research 11 (2003) 6574. D.L. Morganstein, P. Wu, M.R. Mane, N.M. Fisk, R. White, M.G. Parker, Human fetal mesenchymal stem cells differentiate into brown and white adipocytes: a role for ERRalpha in human UCP1 expression, Cell research 20 (2010) 434-444.

23

Table 1. Real-time qPCR primers Gene

Forward primer (5’->3’)

Tm

Reverse primer (5’->3’)

Tm

β-ACTIN

TTG CCG ACA GGA TGC AGA AGG A

61

AGG TGG ACA GCG AGG CCA GGA T

65

c-MYC

AAA CAC AAA CTT GAA CAG CTA C

51.8

ATT TGA GGC AGT TTA CAT TAT GG

51.4

OCT4A

CTCCTGGAGGGCCAGGAATC

59.8

CCACATCGGCCTGTGTATAT

54.4

KLF4

ACC AGG CAC TAC CGT AAA CAC A

58.5

GGT CCG ACC TGG AAA ATG CT

57.7

SOX2

CCCAGCAGACTTCACATGT

55

CCTCCCATTTCCCTCGTTTT

55.1

NANOG

TGAACCTCAGCTACAAACAG

52.3

TGGTGGTAGGAAGAGTAAAG

51

TET1

CAGAACCTAAACCACCCGTG

64.2

TGCTTCGTAGCGCCATTGTAA

67.5

UCP1

CAATCACCGCTGTGGTAAAAAC

54.9

GTAGAGGCCGATCCTGAGAGA

57.5

UCP2

GGAGGTGGTCGGAGATACCAA

58.3

ACAATGGCATTACGAGCAACAT

55.4

CBS

TCA AGA GCA ACG ATG AGG AG

54.4

ATG TAG TTC CGC ACT GAG TC

54.7

CSE

AGAAGGTGATTGACATTGAAGG

61.9

CAATAGGAGATGGAACTGCTC

60.3

MPST

CGC CGT GTC ACT GCT TGA T

58.3

CAC CTGGAAGCGCCGGGATT

62.9

ADRB1

AAGGAACATCAGCAAGCCAC

55

GTGAACTCGAAGCCCACAAT

55.5

ADRB3

CTGCCAGTTCTCACCTTCTGA

57.9

AACTCGTCCCCAAATTCACCC

57.9

ADIPOQ

GGCTTTCCGGGAATCCAAGG

58.6

TGGGGATAGTAACGTAAGTCTCC

55.4

PPARG

TCCATGCTGTTATGGGTGAA

54

GGGAGTGGTCTTCCATTACG

55

PPARGC1A

CCAAGTCGTTCACATCTAGTTCA

54.6

TCTGAGTCTGTATGGAGTGACAT

54.9

LEPTIN

CACACGCAGTCAGTCTCCTC

57.6

AGGTTCTCCAGGTCGTTGG

56.8

ASC-1

GTGGCGCTCAAGAAGGAGAT

57.3

CCTTGGGCGAGATGAAGAT

54.5

Primers were purchased from IDT (San Diego, CA).

Table 2. siRNA sequences Target

Sense primer (5’->3’)

Antisense primer (5’->3’)

MPST si RNA (h) pool 1

GCGCCGCUUUCUUCGACAU

AUGUCGAAGAAAGCGGCGC

MPST si RNA (h) pool 2

GAGAAGAGCCCUGAGGAG

CCGCCUUCAUCAAGACCUA

MPST si RNA (h) pool 3

CCACCCACGUCGUGAUCUA

AGAAAGUGGACCUGUCUAA

TET1 si RNA (h)

Santa Cruz Biotech sc-90457

Santa Cruz Biotech sc-90457

24

FIGURE LEGENDS Figure 1. Differential gene signatures of fibroblasts and MSCs. A-C, The expression of OCT4A, TET1 and CBS, CSE, and MPST was quantified by qPCR in human dermal fibroblasts (HDF) and MSC cells and normalized against β-ACTIN. D, The activity of MPST was quantified in HDF and MSC cells. Experiments were performed in triplicates in three independent experiments. Error bars represent the Standard Deviation of the mean. Statistical significance is shown as ** (p ≤0.005), and *** (p ≤0.0005) compared to HDF.

Figure. 2. MPST depletion results in redox imbalance. Cells were transfected with Src or MPST si RNA for 48 h and cultured in αMEM/SR for 3 days. A, Quantitative PCR analysis of CBS, CSE and MPST. The gene expression was normalized to Scr si control. B, Representative Western blots and relative fold changes of CBS, CSE and MPST normalized to β-Actin. C, Analysis of MPST activity. D, Quantitation of H2S production. E, Level of sulfate in the medium. F, Analysis of ROS production. Relative mean +

fluorescence intensity of DCF are shown after normalization to protein level. G, Levels of NADH and NAD . Data were collected from 5-10 independent experiments and presented as the mean ± SD. Statistical significance is shown as * (p≤0.05), ** (p ≤0.005), and *** (p ≤0.0005) compared to Scr si control.

Figure. 3. Metabolic changes in response to MPST silencing. A, Knockdown of MPST resulted in increased lactate level. Results are shown relative to Scr si control. Cellular bioenergetics was measured using Seahorse XF 24 Analyzer. B, Extracellular acidification rate (ECAR), as indicator of increased glycolytic rate. C, OCR and ECAR measurements. D-E, Cells were transfected with Scr control and MPST siRNA for 48 h. Cells were cultured in αMEM/SR for additional 3 days without and with 3BrP and then cell viability, as a measure of metabolic activity, (D) and lactic acid production (E) were analyzed. Results were expressed as a percentage of data obtained from treated cells to the untreated Scr si controls. F, Assessment of lactic acid production in control and MPST knockdown cells treated without and with 100 µM NaHS. Data were collected from at least three independent experiments and presented as the mean ± SD. Statistical significance is shown as * (p≤0.05), ** (p ≤0.005), and *** (p ≤0.0005) compared to Scr si control.

25

Figure. 4. MPST silencing increases expression of uncoupling proteins and reactivates genes associated with stem cell identity. Cells were transfected with Src or MPST si RNA for 48 h and cultured in αMEM/SR for 3 days and cells were assessed. A, Quantitative PCR of UCP1 and UCP2. B, Representative Western blotting and relative fold changes of UCP1. C, Quantitation of αKG. D, Quantitative PCR analysis of TET1 and pluripotency markers. E. Representative Western blotting and relative fold changes of TET1 and SOX2. Data were collected from 3-5 independent experiments and presented as the mean ± SD. Statistical significance is shown as *** (p ≤0.0005) compared to Scr si control. Fibroblasts with MPST knockdown are highly dependent on glycolysis for the expression of pluripotency genes F, Cells were treated for 6 days with Scr control and MPST siRNA and simultaneously treated without and with 3BrP. The expression of CBS, UCP1, OCT4, NANOG, and SOX2 expression was analyzed by quantitative PCR analysis. Data were collected from three independent experiments. Error bars represent the Standard Deviation of the mean. Statistical significance is shown as * and

#

(p≤0.05), ** and

##

(p ≤0.005), and *** (p ≤0.0005) compared to Scr si

#

control (*) and to Scr si control treated with 3BrP ( ).

Figure 5. Metabolic changes in response to inactivation of MPST with phenylpyruvate. Fibroblasts were treated for 6 days with 1 mM PP and then they were subjected to: A, Analysis of MPST activity. B, H2S released into the culture medium was measured as described in Materials and Methods. C, Level of sulfate in the medium. D, Analysis of ROS production. Relative mean fluorescence intensity of DCF are shown after normalization to protein level. E, Analysis of lactic acid production. F, Quantitative PCR analysis of UCP1 and UCP2 gene expression. The data were pooled from three independent experiments. Error bars represent the Standard Deviation of the mean. Statistical significance is shown as * (p≤0.05) and *** (p ≤0.0005) compared to untreated control.

Figure 6. Reactivation of pluripotency markers following MPST inactivation is glycolysis and TET1 dependent. Fibroblasts were treated for six days without and with 1 mM PP. A, Quantitative analysis of αKG production. B, Western blotting of TET1 and SOX2. C, Quantitative PCR analysis of TET1 and pluripotency markers.

26

D, Cells were treated without and with 200 µM of 2-deoxy-D-glucose (2DG) and then cells were assessed by quantitative PCR for analysis of OCT4 and NANOG. Statistical significance is shown as *** (p ≤0.0005) compared to control and as ### (p ≤0.0005) compared to PP treated cells. Fibroblasts were treated for six days without and with 1 mM PP in presence or absence of Src control and TET1 siRNA. E, TET1 knockdown was sufficient to prevent TET1 induction by Western Blotting. Then, these cells were subjected to quantitative PCR analysis. F, Quantitative PCR analysis of TET1 and pluripotency markers. Data were collected from four independent experiments and presented as the mean ± SD. Statistical significance is shown as ** (p ≤0.005), ***and ### (p ≤0.0005) compared to Scr si control (*) and to Scr si control treated with PP (#).

Figure 7. Downregulation of MPST facilitates adipogenesis. A, HDF were transfected with Src or MPST si RNA for 48 h and after 3 days of culture in αMEM/SR and then lipid droplets were visualized with oil red O. Scale bars, 100 µM. B, Fibroblasts were treated for 6 days without and with 1 mM PP and then lipid droplets were visualized with Oil Red O. Scale bars, 100 µM. C, Cells were treated without and with 200 µM of 2-deoxy-D-glucose (2DG) and then cells were assessed by quantitative PCR for analysis of PPARG and UCP1. D, HDF were transfected with Src or MPST si RNA for 48 h and cultured in αMEM/SR for 6 days in the absence or presence of 5 µM isoproterenol. Intracellular lipid deposits were visualized with oil red O staining. Scale bars, 100 µM. E, Cells were treated for 6 days without and with 1 mM PP in the presence of 5 µM isoproterenol in αMEM/SR. Intracellular lipid deposits were visualized with Oil Red O staining. Scale bars, 100 µM. F, Quantitative PCR analysis of the expression of UCP1, ADRB1, and ADRB3. Data were collected from three independent experiments and presented as the mean ± SD. Statistical significance is shown as ***and ### (p ≤0.0005) compared to control (*) and to control treated with PP (#).

Figure 8. Treatment of fibroblasts with PP facilitates adipogenesis under induced adipogenic conditions. HDF cells were treated for 6 days in the presence of 1 mM phenypyruvate (PP) and then were cultured for an additional 25 days in an adipogenic inducing medium that contained IBMX, dexamethasone, indomethacin and insulin. Cells were then assessed for: A, Quantitative PCR analysis of expression of adipogenic markers in cells maintained in DMEM/10%FBS or an adipogenic medium. Data were normalized to the level of untreated with PP control in DMEM/10%FBS. B, Staining of the lipid droplets with Oil Red O. C, Spectrophotometric quantification of optical density of oil Red O at 510 nm.

27

Data were collected from four independent experiments. Error bars represent the Standard Deviation of the mean. Statistical significance is shown as *** and

###

(p ≤0.0005) compared to untreated with PP control maintained in #

DMEM/10%FBS (*) and to untreated with PP cells maintained in adipogenic inducing medium ( ).

Figure 9. Effects of MPST silencing on induced adipogenic differentiation. HDF cells were treated with Scr or MPST siRNA for six days and then they were subjected to adipogenic differentiation in the absence of adipogenic inducers such as IBMX, dexamethasone, indomethacin and insulin for 25 days. A, The lipid droplets were visualized with Oil Red O. Scale bars, 100 µM. B, Spectrophotometric quantification of optical density of oil red O at 510 nm in stained cells was carried to assess the intracellular lipid load. C. Quantitative analysis of UCP1, PPARG, PPARGC1, ADIPOQ, LEPTIN and ASC-1. Data were collected from three independent experiments. Error bars represent the Standard Deviation of the mean. Statistical significance is shown as *** and

###

(p ≤0.0005) compared to untreated with PP control maintained in DMEM/10%FBS (*) and to untreated #

with PP cells maintained in adipogenic inducing medium ( ).

28

Table 1. Real-time qPCR primers Gene

Forward primer (5’->3’)

Tm

Reverse primer (5’->3’)

Tm

β-ACTIN

TTG CCG ACA GGA TGC AGA AGG A

61

AGG TGG ACA GCG AGG CCA GGA T

65

c-MYC

AAA CAC AAA CTT GAA CAG CTA C

51.8

ATT TGA GGC AGT TTA CAT TAT GG

51.4

OCT4A

CTCCTGGAGGGCCAGGAATC

59.8

CCACATCGGCCTGTGTATAT

54.4

KLF4

ACC AGG CAC TAC CGT AAA CAC A

58.5

GGT CCG ACC TGG AAA ATG CT

57.7

SOX2

CCCAGCAGACTTCACATGT

55

CCTCCCATTTCCCTCGTTTT

55.1

NANOG

TGAACCTCAGCTACAAACAG

52.3

TGGTGGTAGGAAGAGTAAAG

51

TET1

CAGAACCTAAACCACCCGTG

64.2

TGCTTCGTAGCGCCATTGTAA

67.5

UCP1

CAATCACCGCTGTGGTAAAAAC

54.9

GTAGAGGCCGATCCTGAGAGA

57.5

UCP2

GGAGGTGGTCGGAGATACCAA

58.3

ACAATGGCATTACGAGCAACAT

55.4

CBS

TCA AGA GCA ACG ATG AGG AG

54.4

ATG TAG TTC CGC ACT GAG TC

54.7

CSE

AGAAGGTGATTGACATTGAAGG

61.9

CAATAGGAGATGGAACTGCTC

60.3

MPST

CGC CGT GTC ACT GCT TGA T

58.3

CAC CTGGAAGCGCCGGGATT

62.9

ADRB1

AAGGAACATCAGCAAGCCAC

55

GTGAACTCGAAGCCCACAAT

55.5

ADRB3

CTGCCAGTTCTCACCTTCTGA

57.9

AACTCGTCCCCAAATTCACCC

57.9

ADIPOQ

GGCTTTCCGGGAATCCAAGG

58.6

TGGGGATAGTAACGTAAGTCTCC

55.4

PPARG

TCCATGCTGTTATGGGTGAA

54

GGGAGTGGTCTTCCATTACG

55

PPARGC1A

CCAAGTCGTTCACATCTAGTTCA

54.6

TCTGAGTCTGTATGGAGTGACAT

54.9

LEPTIN

CACACGCAGTCAGTCTCCTC

57.6

AGGTTCTCCAGGTCGTTGG

56.8

ASC-1

GTGGCGCTCAAGAAGGAGAT

57.3

CCTTGGGCGAGATGAAGAT

54.5

Primers were purchased from IDT (San Diego, CA).

Table 2. siRNA sequences Target

Sense primer (5’->3’)

Antisense primer (5’->3’)

MPST si RNA (h) pool 1

GCGCCGCUUUCUUCGACAU

AUGUCGAAGAAAGCGGCGC

MPST si RNA (h) pool 2

GAGAAGAGCCCUGAGGAG

CCGCCUUCAUCAAGACCUA

MPST si RNA (h) pool 3

CCACCCACGUCGUGAUCUA

AGAAAGUGGACCUGUCUAA

TET1 si RNA (h)

Santa Cruz Biotech sc-90457

Santa Cruz Biotech sc-90457

C

OCT4 expression, relative to β-Actin

Gene expression, relative to β-Actin

0.00E+00

5.00E-04

1.00E-03

1.50E-03

2.00E-03

2.50E-03

3.00E-03

3.50E-03

4.00E-03

4.50E-03

0.00E+00

1.00E-03

2.00E-03

3.00E-03

4.00E-03

5.00E-03

6.00E-03

7.00E-03

HDF

CBS CSE MPST

HDF MSCs

***

MSCs

***

B

TET1 expression, relative to β-Actin

A

D

0.00E+00

5.00E-03

1.00E-02

1.50E-02

2.00E-02

2.50E-02

3.00E-02

3.50E-02

MPST activity, nmoles pyruvate/min/mg protein

0

50

100

150

200

250

300

350 ***

HDF MSCs

HDF MSCs

Figure 1

Gene expression, relative to Scr si Control

50

100

150

200

250

300

0 SiRNA

H2S, nM

D

0

0.5

1

1.5

2

2.5

3

Scr

CBS

***

MPST

***

CSE

E

MPST

***

0.05

0.1

0.15

0.2

0.25

0.3

0.35

Scr

MPST

***

F

SiRNA Scr MPST

β-Actin

MPST

CSE

CBS

B

0 SiRNA

SO42-, mM/106 cells

A

**

SiRNA

0

2000

4000

6000

8000

10000

12000

Scr

CBS

14000

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

MPST

CSE

MPST

***

G

C

0

20

40

60

80

100

120

0 SiRNA

0.05

0.1

0.15

0.2 *

Scr

Scr MPST

SiRNA

0.25

NADH/NAD

+

Relative protein level (normalized to β-Actin) Relative DCF fluorescence intensity

MPST activity, Relative to Scr si Control

MPST

***

Figure 2

D

0

50

100

150

200

250

SiRNA

0

20

40

60

80

100

120

SiRNA

Lactic acid, % of Scr si Control

Viability, % of scrambled Control

Scr

Scr

**

MPST

MPST

***

B

0

0.5

1

1.5

2

2.5

3

E

0

SiRNA

50

100

150

200

250

SiRNA

ECAR (mpH/min/µg protein)

Lactic acid, % of scrambled Control

MPST

*** ###

MPST

***

Scr

Scr

*

C

F

OCR pmol/min/µg protein

A

0

200

0

2

4

6

8

10

12

NaHS

0

50

100

150

Lactic acid, % of scrambled Control

2

-

***

+

ECAR, mpH/min/µg protein

1

Scr siRNA

3

MPST siRNA

Figure 3

E

Gene expression, relative to Scr si Control

***

Scr

UCP1

-Actin SiRNA

SOX2

TET1

0

2

4

6

8

10

12

MPST

UCP2

***

B

2

0

0.5

1

UCP1

0

0.2

0.4

0.6

0.8

1

1.2

1.4

*

TET1

*

SOX2

***

SiRNA Scr MPST

β-Actin

Relative protein level (normalized to β-Actin)

1.5

Relative protein level (normalized to β-Actin)

1.6

F

C

Gene expression, relative to scrambled Control

A

αKG, nmole/mg protein 0

2

4

6

8

10

12

14

16

Scr

CBS

SiRNA

0

0.05

0.1

0.15

0.2

0.25

UCP1

MPST

***

1

2

4

8

Scr si

***

OCT4A

***

OCT4A NANOG SOX2

D Gene expression, relative to Scr si Control

CBS

UCP1

C-MYC

***

MPST si

TET1

***

SOX2

*** #

NANOG

***

OCT4A NANOG

***

SOX2

*** ##

KLF4

***

***

Figure 4

A

B

300

***

1

0.25

***

0.8

SO42-, mM/106 cells

250 200 H2S, nM

MPST activity, Relative to Control

1.2

C

0.6 0.4 0.2

150 100 50

0

0

PP

-

PP

+

D

-

0.2 0.15 0.1 0.05 0 PP

+

E

***

-

F

6000 5000 4000 3000 2000 1000 0 PP

‐

+

Lactic acid, Relative to Control

ROS, relative intensity normalized to protein level

*

Gene expression, relative to β-Actin

7.00E-03

7000

+

3.5 ***

3 2.5 2 1.5 1 0.5 0 PP

-

+

6.00E-03

UCP1 UCP2

***

5.00E-03 4.00E-03 3.00E-03 2.00E-03 1.00E-03

*

0.00E+00

PP

-

+

Figure 5

A

D

αKG, nmole/mg protein

0.3

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

PP

0

0.05

0.1

0.15

0.2

0.25

Gene expression, relative to Control

***

+

OCT4

PBS

‐

***

2DG

###

2DG

###

E

β-Actin PP

0

0.2

0.4

0.6

0.8

1

1.2

1.4

TET1

Relative TET1 level (normalized to β-Actin)

NANOG

PBS

***

B

SiRNA

β-Actin

TET1

-

Scr

+

**

TET1

C

F

Gene expression, relative to β-Actin Gene expression, relative to Control 0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

***

TET1 si OCT4

Scr si

###

OCT4A KLF4

***

Scr si

SOX2

***

**

KLF4

TET1 si

###

TET1 si SOX2

Scr si

NANOG TET1

***

TET1 si NANOG

Scr si

###

Figure 6

D

A

MPST si

Scr si

MPST si

Scr si

Isoproterenol

E

B

+

-

PP

+

-

PP

Isoproterenol

0

1

2

3

4

5

6

7

8

9

Gene expression, relative to β‐Actin Isoproterenol 5 µM

PP 1 mM

0.00E+00

1.00E-04

2.00E-04

3.00E-04

*** *** 4.00E-04

5.00E-04

6.00E-04

7.00E-04

F

C

Gene expression, relative to Control

‐

‐

ADRB3

ADRB1

UCP1

PPARG

PBS

***

+ ‐

+ 

###

2DG UCP1

*** *** ***

PBS

‐

2DG

###

***

+

+

#########

Figure 7

B

Gene expression, relative to Control in DMEM/10%FBS

A

PPARG

PP 1  mM

Adipogenic  medium 

DMEM 

1

2

4

8

16

32

64

128

‐

ADPOQ

DMEM

LEPTIN

+

ASC‐1

UCP1

***

PPARG

###

C

LEPTIN

ADIPOQ

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

###

ASC‐1

***

UCP1

DMEM  Adipogenic  medium 

Adipogenic medium

###

###

Optical density of Oil red at 510 nm

Figure 8

C

A

1

10

100

1000

***

Scr

***

***

***

MPST

DMEM

UCP1 PPARG PPARGC1 ADIPOQ LEPTIN ASC-1 UCP1

***

SiRNA

Adipogenic medium

DMEM

*** *** *** ≠

0

0.1

0.2

0.3

0.4

0.5

0.6 *** ***

***

***

DMEM Adipogenic medium

MPST si

Scr si

Adipogenic medium

PPARGPPARGC1 ADIPOQ LEPTIN ASC-1

≠

B Optical density of Oil red at 510 nm

Gene expression, relative to Scr si Control in DMEM/10%FBS

Figure 9

Conflict of interest disclosure

The authors declare that they have no conflict of interest. The authors certify that they have NO affiliations with or involvement in any organization or entity with any financial interest or nonfinancial interest in the materials discussed in this manuscript