Mesenchymal stem cells (MSCs) can differentiate into diverse cell types including adipogenic, osteogenic, chondrogenic and myogenic lineages. In the present study, we demonstrated for the first time that sphingosylphosphorylcholine (SPC) induces differentiation of human adipose-tissue-derived mesenchymal stem cells (hATSCs) to smooth-muscle-like cell types. SPC increased the expression levels of several smooth-muscle-specific genes, such as those for α-smooth-muscle actin (α-SMA), h1-calponin and SM22α, as effectively as transforming growth factor β (TGF-β1) and TGF-β3. SPC elicited delayed phosphorylation of Smad2 after 24 hours exposure, in contrast to rapid phosphorylation of Smad2 induced by TGF-β treatment for 10 minutes. Pretreatment of the cells with pertussis toxin or U0126, an MEK inhibitor, markedly attenuated the SPC-induced expression of β-SMA and delayed phosphorylation of Smad2, suggesting that the Gi/o-ERK pathway is involved in the increased expression of α-SMA through induction of delayed Smad2 activation. In addition, SPC increased secretion of TGF-β1 through an ERK-dependent pathway, and the SPC-induced expression of α-SMA and delayed phosphorylation of Smad2 were blocked by SB-431542, a TGF-β type I receptor kinase inhibitor, or anti-TGF-β1 neutralizing antibody. Silencing of Smad2 expression with small interfering RNA (siRNA) abrogated the SPC-induced expression of α-SMA. These results suggest that SPC-stimulated secretion of TGF-β1 plays a crucial role in SPC-induced smooth muscle cell (SMC) differentiation through a Smad2-dependent pathway. Both SPC and TGF-β increased the expression levels of serum-response factor (SRF) and myocardin, transcription factors involved in smooth muscle differentiation. siRNA-mediated depletion of SRF or myocardin abolished the α-SMA expression induced by SPC or TGF-β. These results suggest that SPC induces differentiation of hATSCs to smooth-muscle-like cell types through Gi/o-ERK-dependent autocrine secretion of TGF-β, which activates a Smad2-SRF/myocardin-dependent pathway.

STEM CELLS 2008;26: 789 -797 Disclosure of potential conflicts of interest is found at the end of this article.

S100A8 and S100A9 (S100A8/A9) are low-molecular weight members of the S100 family of calcium-binding proteins. Recent studies have reported S100A8/A9 promote tumorigenesis. We have previously reported that S100A8/A9 is mostly expressed in stromal cells and inflammatory cells between gastric tumor cells. However, the role of environmental S100A8/A9 in gastric cancer has not been defined. We observed in the present study the effect of S100A8/A9 on migration and invasion of gastric cancer cells. S100A8/ A9 treatment increased migration and invasionat lower concentrations that did not affect cell proliferation and cell viability. S100A8/A9 caused activation of p38 mitogenactivated protein kinase (MAPK) and nuclear factor-κB (NF-κB). The phosphorylation of p38 MAPK was not affected by the NF-κB inhibitor Bay whereas activation of NF-κB was blocked by p38 MAPK inhibitor SB203580, indicating that S100A8/A9-induced NF-κB activation is mediated by phosphorylation of p38 MAPK. S100A8/A9-induced cell migration and invasion was inhibited by SB203580 and Bay, suggesting that activation of p38 MAPK and NF-κB is involved in the S100A8/A9 induced cell migration and invasion. S100A8/A9 caused an increase in matrix metalloproteinase 2 (MMP2) and MMP12 expression, which were inhibited by SB203580 and Bay. S100A8/A9-induced cell migration and invasion was inhibited by MMP2 siRNA and MMP12 siRNA, indicating that MMP2 and MMP12 is related to the S100A8/A9 induced cell migration and invasion. Taken together, these results suggest that S100A8/A9 promotes cell migration and invasion through p38 MAPKdependent NF-κB activation leading to an increase of MMP2 and MMP12 in gastric cancer.

BackgroundEpithelial–mesenchymal transition (EMT) plays a significant role in tumor progression and invasion. Snail is a known regulator of EMT in various malignant tumors. This study investigated the role of Snail in gastric cancer.MethodsWe examined the effects of silenced or overexpressed Snail using lenti-viral constructs in gastric cancer cells. Immunohistochemical analysis of tissue microarrays from 314 patients with gastric adenocarcinoma (GC) was used to determine Snail’s clinicopathological and prognostic significance. Differential gene expression in 45 GC specimens with Snail overexpression was investigated using cDNA microarray analysis.ResultsSilencing of Snail by shRNA decreased invasion and migration in GC cell lines. Conversely, Snail overexpression increased invasion and migration of gastric cancer cells, in line with increased VEGF and MMP11. Snail overexpression (≥75% positive nuclear staining) was also significantly associated with tumor progression (P < 0.001), lymph node metastases (P = 0.002), lymphovascular invasion (P = 0.002), and perineural invasion (P = 0.002) in the 314 GC patients, and with shorter survival (P = 0.023). cDNA microarray analysis revealed 213 differentially expressed genes in GC tissues with Snail overexpression, including genes related to metastasis and invasion.ConclusionSnail significantly affects invasiveness/migratory ability of GCs, and may also be used as a predictive biomarker for prognosis or aggressiveness of GCs.

Sphingosylphosphorylcholine (SPC) has been implicated in a variety of cellular responses, including proliferation and differentiation. In this study, we demonstrate that D-erythro-SPC, but not L-threo-SPC, stereoselectively stimulated the proliferation of human adipose tissue-derived mesenchymal stem cells (hADSCs), with a maximal increase at 5 mM, and increased the intracellular concentration of Ca 21 ([Ca 21 ] i ) in hADSCs, which do not express known SPC receptors (i.e., OGR1, GPR4, G2A, and GPR12). The SPC-induced proliferation and increase in [Ca 21 ] i were sensitive to pertussis toxin (PTX) and the phospholipase C (PLC) inhibitor U73122, suggesting that PTX-sensitive G proteins, Gi or Go, and PLC are involved in SPC-induced proliferation. In addition, SPC treatment induced the phosphorylation of c-Jun and extracellular signal-regulated kinase, and SPC-induced proliferation was completely prevented by pretreatment with the c-Jun N-terminal kinase ( JNK)-specific inhibitor SP600125 but not with the MEK-specific inhibitor U0126. Furthermore, the SPC-induced proliferation and JNK activation were completely attenuated by overexpression of a dominant negative mutant of JNK2, and the SPC-induced activation of JNK was inhibited by pretreatment with PTX or U73122. Treatment of hADSCs with lysophosphatidic acid (LPA) receptor antagonist, Ki16425, had no impact on the SPC-induced increase in [Ca 21 ] i . However, SPC-induced proliferation was partially, but significantly, attenuated by pretreatment of the cells with Ki16425. These results indicate that SPC stimulates the proliferation of hADSCs through the Gi/Go-PLC-JNK pathway and that LPA receptors may be responsible in part for the SPC-induced prolifera-