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.

Oncostatin M (OSM) is a multifunctional cytokine of the interleukin-6 family and has been implicated in embryonic development, differentiation, inflammation, and regeneration of liver and bone. In the present study, we demonstrated that treatment of human adipose mesenchymal stem cells (hADSCs) with OSM-attenuated adipogenic differentiation, as indicated by decreased accumulation of intracellular lipid droplets and down-regulated expression of adipocytic markers, such as lipoprotein lipase and PPARgamma. However, OSM treatment stimulated osteogenic differentiation, as demonstrated by the increase in matrix mineralization and expression levels of osteogenic differentiation markers, including alkaline phosphatase, Runx2, and osteocalcin. OSM treatment induced activation of JAK2, JAK3, and ERK in hADSCs, and pre-treatment of hADSCs with the JAK2 inhibitor, AG490, significantly restored the OSM-induced inhibition of adipogenic differentiation. Whereas, the JAK3 inhibitor, WHI-P131, and the MEK inhibitor, U0126, had no effects on the anti-adipogenic activity of OSM. On the other hand, the pro-osteogenic activity of OSM was prevented by treatment of the cells with WHI-P131 or U0126, but not with AG490. These results indicate that distinct signaling pathways, including JAK2, JAK3, and MEK-ERK, play specific roles in the OSM-induced anti-adipogenic and pro-osteogenic differentiation of hADSCs.

Platelet-derived growth factor (PDGF) is a critical regulator of proliferation and migration for mesenchymal type cells. In this study, we examined the role of mitogen-activated protein (MAP) kinases in the PDGF-BB-induced proliferation and migration of human adipose tissue-derived mesenchymal stem cells (hATSCs). The PDGF-induced proliferation was prevented by a pretreatment with the c-Jun N-terminal kinase (JNK) inhibitor, SP600125. However, it was not prevented by a pretreatment with a p38 MAP kinase inhibitor, SB202190, and a specific inhibitor of the upstream kinase of extracellular signal-regulated kinase (ERK1/2), U0126. Treatment with PDGF induced the activation of JNK and ERK in hATSCs, and pretreatment with SP600125 specifically inhibited the PDGF-induced activation of JNK. Treatment with PDGF induced the cell cycle transition from the G0/G1 phase to the S phase, the elevated expression of cyclin D1, and the phosphorylation of Rb, which were prevented by a pretreatment with SP600125. In addition, the PDGF-induced migration of hATSCs was completely blocked by a pretreatment with SP600125, but not with U0126 and SB202190. These results suggest that JNK protein kinase plays a key role in the PDGF-induced proliferation and migration of mesenchymal stem cells.

Ageing is a natural process in living organisms throughout their lifetime, and most elderly people suffer from ageing-associated diseases. One suggested way to tackle such diseases is to rejuvenate stem cells, which also undergo ageing. Here we report that the thioredoxin-interacting protein (TXNIP)-p38 mitogen-activated protein kinase (p38) axis regulates the ageing of haematopoietic stem cells (HSCs), by causing a higher frequency of long-term HSCs, lineage skewing, a decrease in engraftment, an increase in reactive oxygen species and loss of Cdc42 polarity. TXNIP inhibits p38 activity via direct interaction in HSCs. Furthermore, cell-penetrating peptide (CPP)-conjugated peptide derived from the TXNIP-p38 interaction motif inhibits p38 activity via this docking interaction. This peptide dramatically rejuvenates aged HSCs in vitro and in vivo. Our findings suggest that the TXNIP-p38 axis acts as a regulatory mechanism in HSC ageing and indicate the potent therapeutic potential of using CPP-conjugated peptide to rejuvenate aged HSCs.

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-

The nuclear factor kappa B (NF-κB) pathway is pivotal in controlling survival and apoptosis of cancer cells. Macrophage migration inhibitory factor (MIF), a cytokine that regulates the immune response and tumorigenesis under inflammatory conditions, is upregulated in various tumors. However, the intracellular functions of MIF are unclear. In this study, we found that MIF directly interacted with thioredoxin-interacting protein (TXNIP), a tumor suppressor and known inhibitor of NF-κB activity, and MIF significantly induced NF-κB activation. MIF competed with TXNIP for NF-κB activation, and the intracellular MIF induced NF-κB target genes, including c-IAP2, Bcl-xL, ICAM-1, MMP2 and uPA, by inhibiting the interactions between TXNIP and HDACs or p65. Furthermore, we identified the interaction motifs between MIF and TXNIP via site-directed mutagenesis of their cysteine (Cys) residues. Cys and Cys of MIF and Cys and Cys of TXNIP were responsible for the interaction. MIF reversed the TXNIP-induced suppression of cell proliferation and migration. Overall, we suggest that MIF induces NF-κB activity by counter acting the inhibitory effect of TXNIP on the NF-κB pathway via direct interaction with TXNIP. These findings reveal a novel intracellular function of MIF in the progression of cancer.

Prostanoid metabolites are key mediators in inflammatory responses, and accumulating evidence suggests that mesenchymal stem cells (MSCs) can be recruited to injured or inflamed tissues. In the present study, we investigated whether prostanoid metabolites can regulate migration, proliferation, and differentiation potentials of MSCs. We demonstrated herein that the stable thromboxane A 2 (TxA 2 ) mimetic U46619 strongly stimulated migration and proliferation of human adipose tissue-derived MSCs (hADSCs). Furthermore, U46619 treatment increased expression of α-smooth muscle actin (α-SMA), a smooth muscle marker, in hADSCs, suggesting differentiation of hADSCs into smooth muscle-like cells. U46619 activated ERK and p38 MAPK, and pretreatment of the cells with the MEK inhibitor U0126 or the p38 MAPK inhibitor SB202190 abrogated the U46619-induced migration, proliferation, and α-SMA expression. These results suggest that TxA2 plays a key role in the migration, proliferation, and differentiation of hADSCs into smooth muscle-like cells through signaling mechanisms involving ERK and p38 MAPK.