Eun Su Jeon

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.

Tumor Necrosis Factor-α-Activated Human Adipose Tissue–Derived Mesenchymal Stem Cells Accelerate Cutaneous Wound Healing through Paracrine Mechanisms

Human adipose tissue-derived mesenchymal stem cells (ASCs) stimulate regeneration of injured tissues by secretion of various cytokines and chemokines. Wound healing is mediated by multiple steps including inflammation, epithelialization, neoangiogenesis, and proliferation. To explore the paracrine functions of ASCs on regeneration of injured tissues, cells were treated with tumor necrosis factor-α (TNF-α), a key inflammatory cytokine, and the effects of TNF-α-conditioned medium (CM) on tissue regeneration were determined using a rat excisional wound model. We demonstrated that TNF-α CM accelerated wound closure, angiogenesis, proliferation, and infiltration of immune cells into the cutaneous wound in vivo. To assess the role of proinflammatory cytokines IL-6 and IL-8, which are included in TNF-α CM, IL-6 and IL-8 were depleted from TNF-α CM using immunoprecipitation. Depletion of IL-6 or IL-8 largely attenuated TNF-α CM-stimulated wound closure, angiogenesis, proliferation, and infiltration of immune cells. These results suggest that TNF-α-activated ASCs accelerate cutaneous wound healing through paracrine mechanisms involving IL-6 and IL-8.

Angiotensin II-induced differentiation of adipose tissue-derived mesenchymal stem cells to smooth muscle-like cells

Angiotensin II (Ang II) is involved in the development of cardiovascular disease and vascular remodeling. In this study, we demonstrate that treatment of human adipose tissue-derived mesenchymal stem cells (hADSCs) with Ang II increased the expression of smooth muscle-specific genes, including alpha-smooth muscle actin (alpha-SMA), calponin, h-caldesmon, and smooth muscle myosin heavy chain (SM-MHC), and also elicited the secretion of transforming growth factor-beta1 (TGF-beta1) and delayed phosphorylation of Smad2. The Ang II-induced expression of alpha-SMA and delayed phosphorylation of Smad2 were blocked by pretreatment of the cells with a TGF-beta type I receptor kinase inhibitor, SB-431542, small interference RNA-mediated depletion of endogenous Smad2, and adenoviral expression of Smad7. Furthermore, the Ang II-induced TGF-beta1 secretion, alpha-SMA expression, and delayed phosphorylation of Smad2 in hADSCs were abrogated by the MEK inhibitor U0126, suggesting a pivotal role of MEK/ERK pathway in the Ang II-induced activation of TGF-beta1-Smad2 signaling pathway. The smooth muscle-like cells which were differentiated from hADSCs by Ang II treatment exhibited contraction in response to 60mM KCl. These results suggest that Ang II induces differentiation of hADSCs to contractile smooth muscle-like cells through ERK-dependent activation of the autocrine TGF-beta1-Smad2 crosstalk pathway.

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.

A Rho Kinase/Myocardin-Related Transcription Factor-A–Dependent Mechanism Underlies the Sphingosylphosphorylcholine-Induced Differentiation of Mesenchymal Stem Cells Into Contractile Smooth Muscle Cells

Sphingosylphosphorylcholine (SPC) induces differentiation of human adipose tissue–derived mesenchymal stem cells (hADSCs) to smooth muscle cells (SMCs). In the present study, we characterized contractile and ion channel properties of SMCs differentiated from hADSCs (hADSC-SMCs) as a result of SPC treatment, and we investigated the molecular mechanisms involved in the SPC-induced differentiation. Using in vitro collagen gel lattice contraction and whole cell patch clamp, we showed that the hADSC-SMCs expressed functional L-type voltage-gated Ca2+ channels and contractile activities in response to KCl, carbachol, and the L-type Ca2+ channel opener Bay K8644, whereas the L-type Ca2+ channel blocker nifedipine abrogated the contractility of hADSC-SMCs. Furthermore, hADSC-SMCs expressed functional big conductance Ca2+-activated K+ (BKCa) channels, and the BKCa channel blocker iberiotoxin potentiated the Bay K8644-stimulated contractility of the hADSC-SMCs, indicating that these cells exhibited SMC-like contractile characteristics. SPC activated RhoA in hADSCs and pretreatment with the Rho kinase inhibitor Y27632 or by overexpression of dominant-negative mutants of RhoA or Rho kinase completely abrogated the SPC-induced differentiation of hADSCs into SMCs. SPC also increased the expression levels of myocardin-related transcription factor (MRTF)-A, a transcription factor involved in smooth muscle differentiation, in hADSCs. Small interference RNA–mediated depletion of endogenous MRTF-A abolished the SPC-induced differentiation of hADSCs into SMCs. Furthermore, SPC promoted nuclear translocation of MRTF-A, and pharmacological inhibition of Rho kinase blocked this effect. These results suggest that SPC induced differentiation of hADSCs into contractile SMCs through a mechanism involving RhoA/Rho kinase–dependent nuclear translocation of MRTF-A.

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.

Thromboxane A2 Induces Differentiation of Human Mesenchymal Stem Cells to Smooth Muscle-Like Cells†‡§

Thromboxane A2 (TxA2) is involved in smooth muscle contraction and atherosclerotic vascular diseases. Accumulating evidence suggests a pivotal role for mesenchymal stem cells (MSCs) in vascular remodeling. In the present study, we demonstrate for the first time that the TxA2 mimetic U46619 induces differentiation of human adipose tissue‐derived MSCs (hADSCs) to smooth muscle‐like cells, as demonstrated by increased expression of smooth muscle‐specific contractile proteins such as α‐smooth muscle actin (α‐SMA), calponin, smoothelin, and smooth muscle‐myosin heavy chain. Using an in vitro collagen gel lattice contraction assay, we showed that U46619‐induced expression of the contractile proteins was associated with increased contractility of the cells. U46619 increased the intracellular Ca2+ concentration in hADSCs and pretreatment of the cells with the thromboxane receptor antagonist SQ29548 or the calmodulin (CaM) inhibitor W13 abrogated the U46619‐induced α‐SMA expression and contractility, suggesting a pivotal role of Ca2+/CaM in the U46619‐stimulated smooth muscle differentiation of hADSCs. In addition, U46619 elicited activation of RhoA in hADSCs, and pretreatment of the cells with the Rho kinase‐specific inhibitor Y27632 or overexpression of the dominant‐negative mutants of RhoA and Rho kinase blocked U46619‐stimulated α‐SMA expression and contractility. Furthermore, U46619 induced phosphorylation of myosin light chain (MLC) through CaM/MLC kinase‐ and Rho kinase‐dependent pathways, and the MLC kinase inhibitor ML‐7 abrogated U46619‐induced α‐SMA expression and contractility. These results suggest that U46619 induces differentiation of hADSCs to contractile smooth muscle‐like cells through CaM/MLCK‐ and RhoA‐Rho kinase‐dependent actin polymerization. STEM CELLS 2009;27:191–199

Ovarian cancer-derived lysophosphatidic acid stimulates secretion of VEGF and stromal cell-derived factor-1α from human mesenchymal stem cells

Lysophosphatidic acid (LPA) stimulates growth and invasion of ovarian cancer cells and tumor angiogenesis. Cancer-derived LPA induces differentiation of human adipose tissue-derived mesenchymal stem cells (hASCs) to α-smooth muscle actin (α-SMA)-positive cancer-associated fibroblasts. Presently, we explored whether cancer-derived LPA regulates secretion of pro-angiogenic factors from hASCs. Conditioned medium (CM) from the OVCAR-3 and SKOV3 ovarian cancer cell lines stimulated secretion angiogenic factors such as stromal-derived factor-1α (SDF-1α) and VEGF from hASCs. Pretreatment with the LPA receptor inhibitor Ki16425 or short hairpin RNA lentiviral silencing of the LPA1 receptor abrogated the cancer CM-stimulated expression of α-SMA, SDF-1, and VEGF from hASCs. LPA induced expression of myocardin and myocardin-related transcription factor-A, transcription factors involved in smooth muscle differentiation, in hASCs. siRNA-mediated depletion of endogenous myocardin and MRTF-A abrogated the expression of α-SMA, but not SDF-1 and VEGF. LPA activated RhoA in hASCs and pretreatment with the Rho kinase inhibitor Y27632 completely abrogated the LPA-induced expression of α-SMA, SDF-1, and VEGF in hASCs. Moreover, LPA-induced α-SMA expression was abrogated by treatment with the ERK inhibitor U0126 or the phosphoinositide-3-kinase inhibitor LY294002, but not the PLC inhibitor U73122. LPA-induced VEGF secretion was inhibited by LY294002, whereas LPA-induced SDF-1 secretion was markedly attenuated by U0126, U73122, and LY294002. These results suggest that cancer-secreted LPA induces differentiation of hASCs to cancer-associated fibroblasts through multiple signaling pathways involving Rho kinase, ERK, PLC, and phosphoinositide-3-kinase.