Mouse C2C12 cells, which constitutively express S1PR1, S1PR2, and S1PR3 , were used as positive controls (data not shown).
First, we analysed by reverse transcription and real-time PCR the expression of the five S1P receptor (S1PR) subtypes and we found that BM-MSCs expressed three of five S1PRs, S1PR1, S1PR2, and S1PR3 (Figures 1(a) and 1(b)), whereas in this experimental conditions, S1PR4 and S1PR5 subtypes were not detectable.
As shown in (Figures 1(a) and 1(b)), in high-density cell culture conditions, the real-time PCR analysis indicated that S1PR1 displayed a marked increase, S1PR3 a slight one, whereas S1PR2 did not change, suggesting a prevalent role of S1PR1 in the high-density pool of BM-MSCs.
However, our preliminary results indicate that S1PR2 antagonist is not effective in regulating MMP-2 activity in BM-MSCs, whereas a specific antagonist of S1PR3 is not commercially available.
FTY720-phosphate functions as a noncompetitive inhibitor of various S1PRs [30,31], such as S1PR1, S1PR3
, S1PR4, and S1PR5, but not S1PR2, receptors [27, 32, 33].
In breast cancer cell lines, however, the use of tamoxifen and medroxyprogesterone result in the downregulation of S1PR3
and stimulation of S1PR2 with activation of autophagy of the breast cancer cells towards death, again demonstrating the detrimental role of S1P pathway activation in cancer .
Circulating myeloid cells such as monocytes respond to S1P mainly through S1PR3
 or S1PR5 .
Adding to the concept of tumor as wounds that do not heal, it was reported that LCN2 from macrophages exposed to apoptotic cells and thus, linked to S1P signaling via S1PR3
, supported the proliferation and healing of renal epithelium, once inflammatory conditions were terminated .
S1PR1, S1PR2, and S1PR3 specifically contribute to SIP-induced cell motility .
S1PR1 agonist (SEW2871), S1PR1 antagonist (W146), S1PR2 antagonist (JTE-013), and S1PR3 antagonist (CAY10444) were from Cayman Chemical (Ann Arbor, MI, USA).
Where indicated, cells were transfected with S1PR1-3 siRNA 48 h prior to S1P stimulation (1 [micro]M, 5 min) or pretreated for 1 h with W146 (S1PR1 antagonist), JTE-013 (S1PR2 antagonist) or CAY10444 (S1PR3 antagonist).
Human Bone Marrow-Derived Stem Cells Express S1PR1, S1PR2, and S1PR3. In line with previous studies, human BMSCs were confirmed to express CD44, CD105, CD166, CD73, and lack expression of CD14, CD45, and CD34 (Figure 1(a)).