Hae Young Song

Sphingosylphosphorylcholine induces differentiation of human mesenchymal stem cells into smooth-muscle-like cells through a TGF-β-dependent mechanism

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.

Cancer‐Derived Lysophosphatidic Acid Stimulates Differentiation of Human Mesenchymal Stem Cells to Myofibroblast‐Like Cells

Lysophosphatidic acid (LPA) is enriched in ascites of ovarian cancer patients and is involved in growth and invasion of ovarian cancer cells. Accumulating evidence suggests cancer‐associated myofibroblasts play a pivotal role in tumorigenesis through secreting stromal cell‐derived factor‐1 (SDF‐1). In the present study, we demonstrate that LPA induces expression of α‐smooth muscle actin (α‐SMA), a marker for myofibroblasts, in human adipose tissue‐derived mesenchymal stem cells (hADSCs). The LPA‐induced expression of α‐SMA was completely abrogated by pretreatment of the cells with Ki16425, an antagonist of LPA receptors, or by silencing LPA1 or LPA2 isoform expression with small interference RNA (siRNA). LPA elicited phosphorylation of Smad2/3, and siRNA‐mediated depletion of endogenous Smad2/3 or adenoviral expression of Smad7, an inhibitory Smad, abrogated the LPA induced expression of α‐SMA and phosphorylation of Smad2/3. LPA‐induced secretion of transforming growth factor (TGF)‐β1 in hADSCs, and pretreatment of the cells with SB431542, a TGF‐β type I receptor kinase inhibitor, or anti‐TGF‐β1 neutralizing antibody inhibited the LPA‐induced expression of α‐SMA and phosphorylation of Smad2. Furthermore, ascites from ovarian cancer patients or conditioned medium from ovarian cancer cells induced expression of α‐SMA and phosphorylation of Smad2, and pretreatment of the cells with Ki16425 or SB431542 abrogated the expression of α‐SMA and phosphorylation of Smad2. In addition, LPA increased the expression of SDF‐1 in hADSCs, and pretreatment of the cells with Ki16425 or SB431562 attenuated the LPA‐stimulated expression of SDF‐1. These results suggest that cancer‐derived LPA stimulates differentiation of hADSCs to myofibroblast‐like cells and increases SDF‐1 expression through activating autocrine TGF‐β1‐Smad signaling pathway.

Role of c-Jun N-terminal kinase in the PDGF-induced proliferation and migration of human adipose tissue-derived mesenchymal stem cells

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.

Oncostatin M promotes osteogenesis and suppresses adipogenic differentiation of human adipose tissue-derived mesenchymal stem cells

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 PPARγ. 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. J. Cell. Biochem. 101:1238–1251, 2007.

Sphingosylphosphorylcholine induces proliferation of human adipose tissue-derived mesenchymal stem cells via activation of JNK

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 μM, and increased the intracellular concentration of Ca2+ ([Ca2+]i) in hADSCs, which do not express known SPC receptors (i.e., OGR1, GPR4, G2A, and GPR12). The SPC-induced proliferation and increase in [Ca2+]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 [Ca2+]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 proliferation.

Lysophosphatidic acid mediates migration of human mesenchymal stem cells stimulated by synovial fluid of patients with rheumatoid arthritis

Migration of mesenchymal stem cells plays a key role in regeneration of injured tissues. Rheumatoid arthritis (RA) is a chronic inflammatory disease and synovial fluid (SF) reportedly contains a variety of chemotactic factors. This study was undertaken to investigate the role of SF in migration of human bone marrow-derived mesenchymal stem cells (hBMSCs) and the molecular mechanism of SF-induced cell migration. SF from RA patients greatly stimulated migration of hBMSCs and the SF-induced migration was completely abrogated by pretreatment of the cells with the lysophosphatidic acid (LPA) receptor antagonist Ki16425 and by small interfering RNA- or lentiviral small hairpin RNA-mediated silencing of endogenous LPA(1)/Edg2. Moreover, SF from RA patients contains higher concentrations of LPA and an LPA-producing enzyme autotoxin than normal SF. In addition, SF from RA patients increased the intracellular concentration of calcium through a Ki16425-sensitive mechanism and pretreatment of the cells with the calmodulin inhibitor W7 or calmodulin-dependent protein kinase II inhibitor KN93 abrogated the SF-induced cell migration. These results suggest that LPA-LPA(1) plays a key role in the migration of hBMSCs induced by SF from RA patients through LPA(1)-dependent activation of calmodulin-dependent protein kinase II.

Platelet-Activating Factor Receptor Mediates Oxidized Low Density Lipoprotein-Induced Migration of Bone Marrow-Derived Mesenchymal Stem Cells

Background: Oxidized low density lipoprotein (oxLDL) is involved in the development of vascular diseases. Platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glyero-3-phosphorylcholine) is a key component of oxLDL. Methods: In the present study, we evaluate whether oxLDL can regulate migration of human bone-marrow derived stem cells (hBMSCs) and characterize the role of PAF in the oxLDL-induced migration. Results and Conclusions: oxLDL stimulated chemotaxis of hBMSCs in vitro. Treatment of the cells with BN52021, a specific antagonist of PAF receptor (PAF-R), completely blocked the cell migration induced by PAF, but not platelet-derived growth factor (PDGF-BB). Using PAF-R-specific small interfering RNA, it was demonstrated that silencing of endogenous PAF-R expression significantly attenuated cell migration induced by PAF, but not PDGF-BB, suggesting the specific involvement of PAF-R in the oxLDL-induced cell migration. In addition, PAF-induced migration of hBMSCs was abrogated by pretreating cells with mitogen-activated protein kinase (MAPK) inhibitors, including the MEK inhibitor U0126, the p38 MAPK inhibitor SB202190, and the JNK inhibitor SP600125. Moreover, adenoviral overexpression of a dominant negative mutant of p38 MAPK blocked PAF-stimulated migration. Taken together, these results suggest that PAF plays a pivotal role in the oxLDL-induced recruitment of hBMSCs through mechanisms involving PAF-R-dependent activation of MAPKs.

Oxidized phosphatidylcholine induces migration of bone marrow-derived mesenchymal stem cells through Krüppel-like factor 4-dependent mechanism

Platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine) and PAF-like oxidized phospholipids including 1-palmitoyl-2-oxovaleroyl-sn-glycero-3-phosphorylcholine (POVPC) are generated upon LDL oxidation. The aim of this study was to evaluate the question of whether POVPC can regulate migration of human bone marrow-derived stem cells (hBMSCs) and to characterize signaling mechanisms involved in the POVPC-induced cell migration. POVPC treatment resulted in dose- and time-dependent increase of hBMSCs migration. Treatment of cells with BN52021, a specific antagonist of PAF receptor, completely blocked cell migration induced by not only PAF but also POVPC. Silencing of endogenous PAF receptor expression using PAF receptor-specific small interfering RNA resulted in significant attenuation of cell migration induced by PAF or POVPC. Both PAF and POVPC induced expression of Krüppel-like factor 4 (KLF4) in hBMSCs. POVPC- or PAF-induced cell migration was abrogated by small interfering RNA-mediated depletion of endogenous KLF4. These results suggest that PAF receptor plays a pivotal role in POVPC-induced migration of human BMSCs through PAF receptor-mediated expression of KLF4.

Sphingosine-1-Phosphate-Induced Migration and Differentiation of Human Mesenchymal Stem Cells to Smooth Muscle Cells

Migration and differentiation of mesenchymal stem cells are crucial for tissue regeneration in response to injury. Sphingosine-1-phosphate (S1P) is a bioactive lipid that regulates a variety of biological processes, including proliferation, survival, differentiation and motility. In the present study, we determined the role of S1P in migration and differentiation of human bone marrow-derived mesenchymal stem cells (BMSCs). S1P stimulated migration of BMSCs in a dose- and time-dependent manner, and pre-incubation of the cells with pertussis toxin completely abrogated S1P-induced migration, suggesting involvement of Gi-coupled receptors in S1P-induced cell migration. S1P elicited elevation of intracellular concentration of Ca 2+ ([Ca 2+ ]i) and pretreatment with VPC23019, an antagonist of S1P₁/S1P₃, blocked S1P-induced migration and increase of [Ca 2+ ]i. Small interfering RNA-mediated knockdown of endogenous S1P1 attenuated S1P-induced migration of BMSCs. Furthermore, S1P treatment induced expression of α-smooth muscle actin (α-SMA), a smooth muscle marker, and pretreatment with VPC23019 abrogated S1P-induced α-SMA expression. S1P induced phosphorylation of p38 mitogen-activated protein kinase (MAPK), and pretreatment of cells with SB202190, an inhibitor of p38 MAPK, or adenoviral overexpression of a dominant-negative mutant of the p38 MAPK blocked S1P-induced cell migration and α-SMA expression. Taken together, these results suggest that S1P stimulates migration and smooth muscle differentiation of BMSCs through an S1P1-p38 MAPK-dependent mechanism.