action of transforming growth factor b (TGFb) involves the regulation of sphingosine kinase (SphK)1, critically implicated in S1P formation [12]. In order to completely utilize the therapeutic potential of these cells, it is important to understand their intrinsic properties and the role of the microenvironment in modulating their behaviour and function. To this end, to fully individuate the potential biological action of the pleiotropic cue S1P, we established transcriptional profiles of human mesoangioblasts treated with the bioactive sphingolipid. Presented results demonstrate that S1P promotes differentiation of human mesoangioblasts towards SM cells by transcriptional up-regulation of GATA6 and LMCD1. Moreover, we present evidence that TGFb-induced differentiation of mesoangio- blasts into SM relies on GATA6 and LMCD1 and that the induction of these two transcription factors depends on SphK/S1P axis. Methods Materials Biochemicals, cell culture reagents, Dulbecco’s modified Eagle’s medium (DMEM), fetal calf serum (FCS), protease inhibitor cocktail, bovine serum albumin (BSA), Tetramethylrhodamine B isothiocyanate (TRITC)-phalloidin conjugate were purchased from Sigma (St. Louis, MO, USA). RNeasy Micro Kit was from Qiagen, (Valencia, CA, USA). TGFb1 and basic fibroblast growth factor were purchased from PeproTech (London, UK). SKI-2 [2- (p-hydroxyanilino)-4-(p11chlorophenyl)thiazole] and D-erythro-S1P were from Calbiochem (San Diego, CA, USA). Enhanced chemiluminescence (ECL) reagents were obtained from GE Healthcare Europe GmbH (Milan, Italy). Coomassie Blue reagent was from Bio-Rad (Hercules, CA, USA). Secondary antibodies conjugated to horseradish peroxidase, anti-laminin, anti-calponin 1 (CNN1), anti-GATA6, anti-tropomyosin 1 (TPM1) antibodies, and non fat dry milk were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Goat anti-transgelin (TAGLN) antibodies was from Everest Biotech (Oxfordshire, OX, UK). Fluorescein-conjugated horse anti-mouse and anti-rabbit secondary antibodies were obtained from Vector (Burlingame, CA, USA). High Capacity cDNA Reverse Transcription Kit and all reagents and probes required to perform Real-Time PCR were from Applied Biosystems Inc. (Foster City, CA, USA). Two short interfering RNA (siRNA) duplexes corresponding to two DNA target sequences of human GATA6 (59- GACAGAACGUGAUU- CUCGUdTdT-39 and 59- GCUCAAGUAUUCGGGUCAA- dTdT-39), human LMCD1 (59-GCCAUUACUGCGAGAGU- CUdTdT-39 and 59-GGUCUACUCGGACAGGGCAdTdT-39), one siRNA duplex corresponding to scrambled siRNA (59- UUCUCCGAACGUGUCACGUdTdT-39), Mission siRNA du- plex corresponding to DNA target sequences of human SphK1 (SASI Hs01_00169403) and Mission siRNA duplex corresponding to DNA target sequences of human SphK2 (SASI Hs01_00158544) were purchased from Sigma-Proligo (The Woodlands, TX, USA). Lipofectamine RNAiMAXTM were purchased from Life Technol- ogies (Carlsbad, CA, USA). MatrigelTM basement membrane matrix was purchased from BD Biosciences (Bedford, MA, USA). The specific S1P1/3 antagonist, VPC23019 [13] and the S1P2 antagonist JTE23019 [14] were obtained from Tocris Cookson Ltd. (Bristol, UK). The ABCC1 inhibitor MK571 [15] was from Enzo Lifesciences, (Farmingdale, NY, USA). Cell culture Human mesoangioblasts were routinely grown as previously described [16]. S1P Differentiates Mesoangioblasts For SM differentiation experiments, routine culture medium on 90% confluent cells was changed to DMEM and incubated with 0.11 mmol/L TGFb or 1 mmol/L S1P. In some experiments cells were treated with 10 mmol/L SKI-2, 1 mmol/L VPC23019 and 1 mmol/L JTE013 45 min before agonist challenge. Short interfering RNA administration To silence SphK1, SphK2, GATA6 or LMCD1, mesoangio- blasts were transfected with siRNA using a non specific siRNA as control. Cells grown into 6-well dishes (80,000 cells/well) in Mega Cell Medium supplemented with 5% heat inactivated FCS, were transfected with the 21-nucleotide duplexes using RNAiMAXTM Reagent according to manufacturer’s instructions as previously demonstrated [17]. Briefly, RNAiMAXTM Reagent was incubated with siRNA (200 nM) at room temperature for 20 min and successively the lipid/RNA complexes were added with gentle agitation to the cells. After 48 h from the beginning of transfection cells were used for the experiments. To evaluate the specific knock- down of target genes, quantitative Real-Time PCR was per- formed. cDNA microarray Agilent whole human genome oligonucleotide microarrays (44 K) were used to examine alterations in gene expression of cells after S1P treatment (Agilent Technologies, Santa Clara, CA). For the hybridization, 2 mg of total RNA were used. Labeling reaction, hybridization and washing of the array were performed following the protocols provided by Agilent according to the Two color microarray-based gene expression analysis. Arrays were scanned by using a 4000B Scanner (Axon Instruments, Union City, CA, USA). Each hybridization produced a 16-bit image, which was processed using the Agilent Feature Extraction software (v8.1). In order to identify differentially expressed genes, the data obtained from Feature Extraction were analyzed by ‘‘Significance Analysis of Microarray’’ (SAM) algorithm [18] using the R package siggenes. All the statistical analyses were performed by using the statistical language R [19]. Immunostaining and fluorescence microscopy Cells were seeded on Lab-Tek II Chamber slides (Nalge Nunc International, Naperville, IL, USA), challenged with 1 mmol/L S1P or 0.11 mmol/L TGFb for 48 h and processed as previously described [20]. Images were obtained using a Leica SP5 laser scanning confocal microscope with 406 and 636 objectives. Western blot analysis Total cell lysates were prepared and subjected to SDS-PAGE electrophoresis and Western analysis as previously described [21]. Quantitative Real-Time RT-PCR Total RNA (2 mg), extracted with RNeasy Micro Kit from human mesoangioblasts, was reverse-transcribed using High Capacity cDNA Reverse Transcription Kit following the manu- facturer’s protocols. The quantification of target gene mRNAs was performed by Real-Time PCR employing TaqMan Gene Expression Assays. Simultaneous amplification of the target sequences (TPM1, Hs01576435_m1, CNN1, Hs00154543_m1, GATA6, Hs00232018_m1, TRPS1, Hs00232645_m1, LMCD1, Hs00205871_m1, Gene Expression assays, Applied Biosystems, Foster City, CA) together with the housekeeping gene, 18S rRNA, was carried out essentially as previously described [11]. The 22DDCT method was applied as a comparative method of PLoS ONE | www.plosone.org 2 May 2011 | Volume 6 | Issue 5 | e20389