Stem Cells
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

First published online December 6, 2007
Stem Cells Vol. 26 No. 3 March 2008, pp. 789 -797
doi:10.1634/stemcells.2007-0742; www.StemCells.com
© 2008 AlphaMed Press

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
2007-0742v1
26/3/789    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Reprints/Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jeon, E. S.
Right arrow Articles by Kim, J. H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jeon, E. S.
Right arrow Articles by Kim, J. H.

THE STEM CELL NICHE

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

Eun Su Jeona, Hyun Jung Moona, Mi Jeong Leea, Hae Young Songa, Young Mi Kima, Mong Chob, Dong-Soo Suhc, Man-Soo Yoonc, Chulhun L. Changd, Jin Sup Junga, Jae Ho Kima

aMedical Research Center for Ischemic Tissue Regeneration of Pusan National University and the Medical Research Institute, Busan, Republic of Korea;
bDepartment of Internal Medicine,
cDepartment of Obstetrics and Gynecology, and
dDepartment of Laboratory Medicine, College of Medicine, Pusan National University, Busan, Republic of Korea

Key Words. Lysophosphatidic acid • Mesenchymal stem cells • Myofibroblasts • Stromal cell-derived factor-1; {alpha}-smooth muscle actin

Correspondence: Correspondence: Jae Ho Kim, Ph.D., Department of Physiology, School of Medicine, Pusan National University, 1-Ga, Ami-Dong, Suh-Gu, Busan, 602-739, Republic of Korea. Telephone: 82-51-240-7732; Fax: 82-51-246-6001. e-mail: jhkimst{at}pusan.ac.kr

Received on September 4, 2007; accepted for publication on November 26, 2007.

First published online in STEM CELLS EXPRESS  December 6, 2007.

   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
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 {alpha}-smooth muscle actin ({alpha}-SMA), a marker for myofibroblasts, in human adipose tissue-derived mesenchymal stem cells (hADSCs). The LPA-induced expression of {alpha}-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 {alpha}-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 {alpha}-SMA and phosphorylation of Smad2. Furthermore, ascites from ovarian cancer patients or conditioned medium from ovarian cancer cells induced expression of {alpha}-SMA and phosphorylation of Smad2, and pretreatment of the cells with Ki16425 or SB431542 abrogated the expression of {alpha}-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.

Disclosure of potential conflicts of interest is found at the end of this article.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Tumors are composed of neoplastic cells and non-neoplastic stromal cell components, including fibroblasts, myofibroblasts, endothelial cells, pericytes, and inflammatory cells [1]. Myofibroblasts (also called carcinoma-associated fibroblasts or cancer stroma) have been shown to play important roles during cancer progression [1–3] and exhibit smooth muscle cell (SMC)-like features, including contractile stress fibers and de novo expression of SMC markers, including {alpha}-smooth muscle actin ({alpha}-SMA) and calponin [4–6]. They stimulate tumorigenesis, angiogenesis, and invasion in a variety of solid tumors, including prostate, breast, and ovarian carcinomas [1, 7–10]. In addition, it has been reported that myofibroblasts express stromal cell-derived factor-1 (SDF-1)/CXCL12, a homeostatic chemokine that signals through the cognate G protein-coupled receptor, CXCR4, that is expressed in endothelial progenitor cells and a variety of human carcinoma cells [3, 11]. Myofibroblast-derived SDF-1 plays a key role in the progression of breast cancer by enhancing growth of tumor cells and by recruiting endothelial progenitor cells that are required for tumor angiogenesis [10]. CXCR4 is essentially required for metastatic spread to organs where SDF-1 is expressed, such as the bone marrow, and tumor-cell survival and growth are favored [11]. These observations suggest that SDF-1 derived from myofibroblasts plays a pivotal role in the tumorigenesis and metastasis. However, the molecular identity of extracellular factors and the molecular mechanisms involved in the enhanced expression of SDF-1 in myofibroblasts are largely unknown.

Lysophosphatidic acid (LPA) is a small bioactive phospholipid produced by activated platelets, mesothelial cells, fibroblasts, adipocytes, and some cancer cells [12–14]. LPA is involved in a variety of physiological and pathophysiological responses including wound healing, production of angiogenic factors, chemotaxis, neointima formation, tumor cell invasion, metastasis, and cell cycle progression [13, 15]. In addition, LPA has been known to stimulate contraction of myofibroblasts isolated from patients with Dupuytren's disease and induce migration of human hepatic myofibroblasts [16, 17]. The biological functions of LPA have been shown to be mediated through several G protein-coupled receptors, that is, LPA1/EDG-2, LPA2/EDG-4, LPA3/EDG-7, LPA4/GPR23, and LPA5/GPR92 [18]. Increasing body of evidence suggests that LPA is relevant to the tumorigenesis and metastasis of ovarian cancer; LPA levels have been reported to be elevated in the blood and ascites of patients with ovarian cancer [19–21], and LPA activates adhesion, migration, and invasion of several ovarian cancer cell lines [22, 23]. Moreover, small interference RNA (siRNA)-mediated silencing of LPA1 expression or pharmacological inhibition of LPA1 blocked bone metastasis of ovarian cancer cells in vivo [24], suggesting that LPA plays crucial roles in tumorigenesis and metastasis of ovarian cancer by activating LPA receptors.

Mesenchymal stem cells (MSCs) have a self-renewal capacity, long-term viability, and differentiation potential toward diverse cell types, such as adipogenic, osteogenic, chondrogenic, and myogenic lineages [25–28]. This suggests potential clinical applications of MSCs for regenerative medicine. However, MSCs could also play an adverse effect that favors tumor growth: Tumor cells mixed with MSCs, when transplanted subcutaneously, exhibited elevated capability of proliferation, rich angiogenesis in tumor tissues, and high metastatic activity [29]. MSCs constitute a large proportion of non-neoplastic stromal cells within the tumor microenvironment and constitutively secrete the chemokine SDF-1 [11]. These results indicate a pivotal role of MSCs played in the tumorigenesis and tumor angiogenesis by secreting SDF-1.

It has been documented that transforming growth factor (TGF)-β1 from cancer cells is involved in the differentiation of stromal cell types to myofibroblasts [1, 30, 31], and that TGF-β1 stimulates expression of {alpha}-SMA, which is a marker gene of myofibroblasts, in bone marrow-derived MSCs in vitro [32, 33]. Furthermore, in an earlier study we demonstrated that the expression level of {alpha}-SMA in human adipose tissue-derived mesenchymal stem cells (hADSCs) was up-regulated by sphingosylphosphorylcholine [34], which has also been known to be elevated in ascites from ovarian cancer patients [19]. SPC up-regulates secretion of TGF-β1, and the autocrine signaling pathway involving TGF-β1-Smad2 is required for the SPC-stimulated expression of {alpha}-SMA [34], suggesting that MSCs may act as progenitors for myofibroblasts that are closely associated with tumorigenesis. However, to our knowledge, it has not yet been studied whether the malignant effusion from patients with ovarian cancer induces differentiation of MSCs to myofibroblasts and whether LPA, SPC, and/or TGF-β1 are involved in the malignant effusion-induced myofibroblast differentiation.

In this study, we demonstrated for the first time that LPA, but not SPC, in the malignant ascites from ovarian cancer patients induces differentiation of MSCs to myofibroblast-like cells, as evidenced by enhanced expression of {alpha}-SMA and SDF-1, and that the autocrine TGF-β1-Smad signaling loop plays a pivotal role in the LPA-induced expression of {alpha}-SMA and SDF-1.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Materials
{alpha}-Minimum essential medium, phosphate-buffered saline (PBS), trypsin, fetal bovine serum, and Lipofectamine 2000 reagent were purchased from Invitrogen (Carlsbad, CA, http://www.invitrogen.com). Smad3 inhibitor (SIS3) was purchased from EMD Biosciences, Inc. (San Diego, http://www.emdbiosciences.com). Human recombinant TGF-β1, anti-TGF-β1 neutralizing antibody (Catalog number: AF-101-NA), and enzyme-linked immunosorbent assay (ELISA) kits for human TGF-β1 (Catalog number: DY240) and human SDF-1 (Catalog number: DY350) were purchased from R&D systems, Inc. (Minneapolis, http://www.rndsystems.com). Anti-phospho-Smad2 (Ser465/467), anti-phospho-Smad3/Smad1, anti-Smad2, and anti-Smad3 antibodies were obtained from Cell Signaling Technology (Beverly, MA, http://www.cellsignal.com). Peroxidase-labeled secondary antibodies were from Amersham Biosciences. Alexa Fluor 488 goat anti-mouse antibody and Alexa Fluor 568 phalloidin were from Molecular Probes, Inc. (Eugene, OR, http://probes.invitrogen.com). Anti-smooth muscle myosin heavy chain (SM-MHC) antibody was purchased from Millipore (Temecula, CA, http://www.millipore.com). One-Oleoyl-sn-glycero-3-phosphate (1-oleoyl-LPA), SB431542, Ki16425, anti-{alpha}-SMA, anti-calponin, and anti-tubulin antibodies were from Sigma-Aldrich (St. Louis, http://www.sigmaaldrich.com).

Cell Culture
Subcutaneous adipose tissue was obtained from elective surgeries with patient's consent as approved by the Institution Review Board. For isolation of hADSCs, adipose tissues were washed at least three times with sterile PBS and treated with an equal volume of collagenase type I suspension (1g/l of Hanks' balanced saline solution [HBSS] buffer with 1% bovine serum albumin) for 60 minutes at 37°C with intermittent shaking. Details are available in supplemental online data.

Western Blotting
Serum-starved hADSCs were treated with appropriate conditions, washed with ice-cold phosphate-buffered saline (PBS), and then lysed in lysis buffer (20 mM Tris-HCl, 1 mM EGTA, 1 mM EDTA, 10 mM NaCl, 0.1 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, 30 mM sodium pyrophosphate, 25 mM β-glycerol phosphate, 1% Triton X-100, pH 7.4). Lysates were resolved by SDS-polyacrylamide gel electrophoresis, transferred onto a nitrocellulose membrane, and then stained with 0.1% Ponceau S solution (Sigma-Aldrich). After blocking with 5% nonfat milk, the membranes were immunoblotted with various antibodies, and the bound antibodies were visualized with horseradish peroxidase-conjugated secondary antibodies using the enhanced chemiluminescence Western blotting system (ECL, Amersham Biosciences, Pittsburgh, http://www4.gelifesciences.com).

Isolation of Aorta and Mesenteric Artery from Rat
Wistar male rats were anesthetized by intraperitoneal injection of sodium pentobarbital (30 mg/kg). The descending thoracic aorta and superior mesenteric artery were quickly excised, and homogenized in lysis buffer (20 mM Tris-HCl, 1 mM EGTA, 1 mM EDTA, 10 mM NaCl, 0.1 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, 30 mM sodium pyrophosphate, 25 mM β-glycerol phosphate, 1% Triton X-100, pH 7.4).

Ascites from Patients with Ovarian Cancer or Liver Cirrhosis
Ascites were obtained from four patients with stage III ovarian cancer (range, 48–73 years) and four patients with liver cirrhosis (range, 43–77 years) with patient's consent as approved by the Institution Review Board. Approximately 10 ml of aspirations from ascites were collected, immediately centrifuged at 1,000 x g for 20 minutes to remove cells, and the supernatant was stored at –70°C until use.

Reverse Transcription-Polymerase Chain Reaction Analysis
Cells were treated as indicated, and total cellular RNA was extracted by the Trizol method (Invitrogen). For reverse transcription-polymerase chain reaction (RT-PCR) analysis, aliquots of 2 µg each of RNA were subjected to cDNA synthesis with 200 U of M-MLV reverse transcriptase (Invitrogen) and 0.5 µg of oligo (dT) 15 primer (Promega, Madison, WI, http://www.promega.com). Details are available in supplemental online data.

Transfection with Small Interference RNA
For siRNA experiments, hADSCs were plated on 60-mm dishes at 70% confluence, and they were then transfected with appropriate siRNAs by using the Lipofectamine 2000 reagent according to manufacturer's instructions (Invitrogen). Details are available in supplemental online data.

Adenoviral Infection
Recombinant adenoviruses, expressing Smad7 or LacZ, were kindly provided by Dr. Kohei Miyazono (The Cancer Institute, Tokyo University, Tokyo, Japan) and were used individually at a multiplicity of infection of 50, as previously described [35].

Immunocytochemistry and Microscopy
Immunostaining and confocal microscopy were used to determine the subcellular distribution and organization of proteins. Details are available in supplemental online data.

Preparation of Conditioned Medium
Human ovarian adenocarcinoma cell line OVCAR-3 (ATCC No. HTB-161) were purchased from American Type Culture Collection (Manassas, VA, http://www.atcc.org). OVCAR-3 cells were plated on 100-mm cell culture dishes and cultured in growth medium until reaching confluence. The cells were briefly rinsed twice with PBS, and then incubated with 10 ml of fresh, serum-free {alpha}-minimum essential medium for 48 hours before collecting media. The conditioned medium (CM) was centrifuged at 2000 rpm for 10 minutes to remove cell debris, filtered using 0.45-µm Millipore Ultrafree centrifugal filters (Millipore, Temecula, CA, http://www.millipore.com), and stored at –70°C for subsequent use.

Enzyme-Linked Immunosorbent Assay
Commercially available sandwich ELISA kits were used to evaluate the secretion of TGF-β1 or SDF-1{alpha} in the CM derived from hADSCs. Details are available in supplemental online data.

Statistical Analysis
The results of multiple observations are presented as mean ± SD. Statistical significance was assessed by using Student's unpaired t-test where indicated in the figure legends.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
LPA Induces the Expression of {alpha}-SMA in hADSCs by Activating LPA Receptors
To explore whether LPA can stimulate differentiation of hADSCs to myofibroblast-like cells, we examined the effect of LPA on the expression of {alpha}-SMA, which is considered as the most reliable marker of differentiated myofibroblasts. Thus, hADSCs were treated with serum-free medium containing different concentrations of LPA for 4 days, and the expression levels of {alpha}-SMA were determined by Western blotting. As shown in Figure 1A, the expression level of {alpha}-SMA in hADSCs was dose-dependently increased by LPA treatment with a maximal increase at 5 µM LPA. The LPA-induced expression of {alpha}-SMA occurred after the exposure of hADSCs to LPA for 2 days and the expression level of {alpha}-SMA was maximally increased on day 4 (Fig. 1B).


Figure 1
View larger version (22K):
[in this window]
[in a new window]

  		Figure 1. Lysophosphatidic acid stimulates expression level of {alpha}-smooth muscle actin ({alpha}-SMA) in human adipose tissue-derived mesenchymal stem cells (hADSCs). (A): Serum-starved hADSCs were treated with indicated concentrations of 1-Oleoyl-sn-glycero-3-phosphate (1-oleoyl-LPA) for 4 days. (B): Serum-starved hADSCs were treated with vehicle or 5 µM 1-oleoyl-LPA for indicated time periods. (C): Serum-starved hADSCs were pretreated with vehicle or 10 µM Ki16425 for 15 minutes, and challenged with either 5 µM 1-oleoyl-LPA, 2 µM sphingosylphosphorylcholine, or 2 ng/ml transforming growth factor (TGF)-β1 for 4 days. The expression levels of {alpha}-SMA and tubulin were determined by Western blot analysis with anti-{alpha}-SMA and anti-tubulin antibodies, respectively. (D): The expression and distribution of {alpha}-SMA (green channel) and F-actin (red channel) were determined by double-staining with anti-{alpha}-SMA and Alexa Fluor 568 phalloidin and analyzed by a fluorescence microscopy. Scale bar = 100 µm. Representative data from three independent experiments are shown. Abbreviations: {alpha}-SMA, {alpha}-smooth muscle actin; LPA, lysophosphatidic acid; SPC, sphingosylphosphorylcholine; w/o, without.

 
Myofibroblasts have been reported to express several SMC-specific contractile proteins, including {alpha}-SMA and calponin, and they can be distinguished from fully differentiated SMCs by their general lack of SM-MHC [6, 30, 36]. To further support the notion that LPA induces differentiation of hADSC to myofibroblasts, we determined the expression levels of the SMC markers in response to LPA. As shown in supplemental online Fig. 1, LPA increased the expression levels of {alpha}-SMA and calponin in hADSCs, whereas the expression of SM-MHC was not detected in the LPA-treated cells, in contrast to evident expression of SM-MHC in rat aorta and mesenteric artery. These results suggest that LPA induces differentiation of hADSCs to myofibroblast-like cells, but not to fully differentiated SMCs.

To assess the involvement of LPA receptors in the LPA-induced {alpha}-SMA expression, we examined the effect of Ki16425, an antagonist specific to LPA receptors, on the {alpha}-SMA expression induced by LPA. As shown in Figure 1C, 10 µM Ki16425 completely inhibited the LPA-stimulated {alpha}-SMA expression, suggesting LPA receptors are involved in the LPA-induced {alpha}-SMA expression. We recently reported that both SPC and TGF-β1 stimulate the expression of {alpha}-SMA in hADSCs [34]. Therefore, to ascertain whether Ki16425 specifically inhibits the LPA-stimulated {alpha}-SMA expression, we examined the effect of Ki16425 on the {alpha}-SMA expression induced by SPC or TGF-β1. In contrast to its potent inhibitory effect on the LPA-stimulated {alpha}-SMA expression, Ki16425 had no significant impact on the expression of {alpha}-SMA increased by SPC or TGF-β1 (Fig. 1C), suggesting that Ki16425 selectively attenuates the LPA-stimulated {alpha}-SMA expression in hADSCs.

To further confirm the result above, we examined actin organization by double staining for {alpha}-SMA and F-actin filaments. As shown in Figure 1D, treatment of hADSCs with 5 µM LPA for 4 days increased the expression levels of {alpha}-SMA, which is localized in F-actin filaments. The increased assembly of actin filaments and thick fiber in response to LPA was highly correlated with increased expression of {alpha}-SMA, whereas pretreatment of the cells with Ki16425 completely blocked the expression of {alpha}-SMA and actin polymerization induced by LPA.

In a previous study, we reported that three isoforms of LPA receptors, including LPA1, LPA2, and LPA3, are expressed in hADSCs [37]. Therefore, in the present study, we determined the involvement of the LPA receptors in the LPA-induced {alpha}-SMA expression by using siRNA-mediated depletion of each LPA receptor. The endogenous expression of LPA1, LPA2, or LPA3 was specifically down-regulated by transfection of siRNA specific for each LPA receptor isoform (Fig. 2A). Depletion of endogenous LPA1 or LPA2 completely attenuated the LPA-induced expression of {alpha}-SMA in hADSCs, whereas knock down of LPA3 receptor partially inhibited the LPA-stimulated {alpha}-SMA expression (Fig. 2B and C). These results indicate that LPA1 and LPA2 play a crucial role in the expression of {alpha}-SMA in hADSCs induced by LPA.


Figure 2
View larger version (29K):
[in this window]
[in a new window]

  		Figure 2. Role of lysophosphatidic acid receptors in the LPA-induced expression of {alpha}-smooth muscle actin. (A): Human adipose tissue-derived mesenchymal stem cells (hADSCs) were transfected with control small interference RNA (siRNA) or siRNAs specific for LPA1, LPA2, or LPA3 respectively. The mRNA levels of LPA1, LPA2, LPA3, and GAPDH were determined by semi-quantitative RT-PCR. (B): The siRNA-transfected hADSCs were treated with vehicles or 5 µM 1-Oleoyl-sn-glycero-3-phosphate (1-oleoyl-LPA) for 4 days. The expression levels of {alpha}-SMA and tubulin were determined by Western blot analysis. Representative data from three independent experiments are shown. (C): The densities of {alpha}-SMA and tubulin were quantified from the experiments of the panel (B), and the expression levels of {alpha}-SMA were normalized to tubulin levels in the samples. The data are presented as a percentage of control (mock-treated cells). Data represent mean ± SD. (n = 3). These data were analyzed by using the Student's t-test and considered to be significantly different when p < .05 (*). Abbreviations: {alpha}-SMA, {alpha}-smooth muscle actin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LPA, lysophosphatidic acid.

 
LPA-Induced {alpha}-SMA Expression Is Mediated by Smad-Dependent Pathway
TGF-β induces {alpha}-SMA expression through oligomerization of type I and type II TGF-β receptor kinases and subsequent phosphorylation of cytoplasmic signaling molecules, Smads [38–40]. We have recently demonstrated that TGF-β1 rapidly induced phosphorylation of Smad2 within 10 minutes, in contrast to delayed phosphorylation of Smad2 in response to SPC [34]. To assess whether LPA can induce phosphorylation of Smads, we examined the effects of LPA on the phosphorylation of Smad2 and Smad3. LPA treatment maximally elicited phosphorylation of Smad2 and Smad3 after exposure of hADSCs for 48 hours, and the LPA-induced phosphorylation of Smad2 and Smad3 was sustained by day 4, indicating delayed phosphorylation of Smad2 and Smad3 in response to LPA (Fig. 3A). The LPA-induced late phosphorylation of Smad2 and Smad3 was completely abolished by pretreatment of the cells with Ki16425 (Fig. 3B), suggesting that LPA receptors are involved in the delayed activation of Smad2 and Smad3 in response to LPA.


Figure 3
View larger version (65K):
[in this window]
[in a new window]

  		Figure 3. Role of Smad2 and Smad3 in the lysophosphatidic acid-induced expression of {alpha}-smooth muscle actin in human adipose tissue-derived mesenchymal stem cells (hADSCs). (A): Serum-starved hADSCs were treated with vehicles or 5 µM 1-Oleoyl-sn-glycero-3-phosphate (1-oleoyl-LPA) for the indicated time. (B): Serum-starved hADSCs were pretreated with vehicles or 10 µM Ki16425 for 15 minutes and exposed to vehicles or 5 µM 1-oleoyl-LPA for 4 days. (C): Serum-starved hADSCs were transfected with control small interference RNA (siRNA) (Control), Smad2 siRNA (si-Smad2), or Smad3 siRNA (si-Smad3), and then challenged with 5 µM 1-oleoyl-LPA for 4 days. (D): Serum-starved hADSCs were pretreated with vehicles or 10 µM Smad3 inhibitor (SIS3) for 15 minutes and exposed to vehicles or 5 µM 1-oleoyl-LPA for 4 days. (E): hADSCs were infected with adenoviruses carrying β-galactosidase (Control) or FLAG-tagged Smad7 (FLAG-Smad7), and then treated with vehicles or 5 µM 1-oleoyl-LPA for 4 days. The expression levels of {alpha}-SMA, tubulin, FLAG-tagged Smad7, Smad2, and Smad3 were determined by Western blotting with anti-{alpha}-SMA, anti-tubulin, anti-FLAG, and anti-Smad2 and anti-Smad3 antibodies, respectively. The phosphorylation levels of Smad2 and Smad3 were determined by Western blotting with anti-p-Smad2 and anti-p-Smad3 antibodies, respectively. Representative data from three independent experiments are shown. Abbreviations: {alpha}-SMA, {alpha}-smooth muscle actin; LPA, lysophosphatidic acid; p-Smad, phosphorylated Smad; si-Smad2, Smad2 siRNA; si-Smad3, Smad3 siRNA.

 
In order to clarify whether Smad2 and Smad3 play a crucial role in the {alpha}-SMA expression induced by LPA, we examined the effects of siRNA-mediated knockdown of Smad2 or Smad3 on the LPA-stimulated expression of {alpha}-SMA. Transfection of hADSCs with Smad2 siRNA down-regulated the expression level of Smad2, and completely abrogated the late phosphorylation of Smad2 and {alpha}-SMA expression induced by LPA, without significant impact on the phosphorylation and expression level of Smad3 (Fig. 3C). Furthermore, Smad3 siRNA inhibited the LPA-induced expression of {alpha}-SMA and late phosphorylation of Smad3. To confirm the involvement of Smad3 in the LPA-induced expression of {alpha}-SMA, we examined the effect of SIS3, a specific inhibitor of Smad3 [41], on the LPA-stimulated {alpha}-SMA expression and Smad phosphorylation. SIS3 inhibited the LPA-induced phosphorylation of Smad3, but not of Smad2, suggesting that SIS3 selectively inhibits Smad3-dependent pathway. Consistent with the inhibition of LPA-induced {alpha}-SMA expression by siRNA-mediated depletion of Smad3, the LPA induction of {alpha}-SMA was markedly attenuated by pretreatment of hADSCs with SIS3 (Fig. 3D).

To further confirm the involvement of Smad2 and Smad3 in the LPA induction of {alpha}-SMA, we examined the effect of Smad7, an inhibitory Smad isoform, on the phosphorylation levels of Smad2 and Smad3 and expression level of {alpha}-SMA increased by LPA. Adenoviral overexpression of Smad7 attenuated the LPA-induced phosphorylation of Smad2 and Smad3 (Fig. 3E). Moreover, Smad7 expression completely abrogated the LPA-induced expression of {alpha}-SMA. These results suggest that activation of Smad2 and Smad3 play a pivotal role in the LPA-induced expression of {alpha}-SMA in hADSCs.

LPA-Induced {alpha}-SMA Expression is Dependent on Autocrine TGF-β1-Dependent Pathway
To explore whether the LPA-induced late activation of Smad and expression of {alpha}-SMA are mediated by increased release of TGF-β1, we determined the secreted level of TGF-β1 by using ELISA. As shown in Figure 4A, LPA time-dependently stimulated the secretion of TGF-β1 in hADSCs with a maximal stimulation at 48 hours. Inhibition of TGF-β type I receptor by SB431542, a selective inhibitor of TGF-β type I receptor kinase activity, completely abrogated the {alpha}-SMA expression and delayed phosphorylation of Smad2 in response to LPA or TGF-β1 (Fig. 4B). To assess whether autocrine release of TGF-β1 is involved in the LPA-induced {alpha}-SMA expression, we determined the effects of anti-TGF-β1 neutralizing antibody on the LPA-induced increase of {alpha}-SMA. As shown in Figure 4B, {alpha}-SMA expression and late phosphorylation of Smad2 in response to LPA or TGF-β1 were blocked by pre-incubation of the cells with the anti-TGF-β1 neutralizing antibody. These results clearly suggest that LPA induces expression of {alpha}-SMA through activating TGF-β1-Smad pathway in hADSCs.


Figure 4
View larger version (27K):
[in this window]
[in a new window]

  		Figure 4. Role of transforming growth factor (TGF)-β-dependent pathway in the lysophosphatidic acid (LPA)-induced expression of {alpha}-smooth muscle actin ({alpha}-SMA) and the delayed activation of Smad2. (A): Human adipose tissue-derived mesenchymal stem cells (hADSCs) were exposed to serum-free medium containing either vehicles or 5 µM 1-oleoyl-LPA for the indicated time periods and the conditioned medium was then subjected to ELISA for determination of the protein levels of TGF-β1. Data represent mean ± SD. (n = 3). * indicates p < .05 versus control (0 minute). (B): Serum-starved hADSCs were pretreated with vehicles, 10 µM SB431542, or 0.2 µg/ml anti-TGF-β1 neutralizing antibody, and then treated with 5 µM 1-Oleoyl-sn-glycero-3-phosphate (1-oleoyl-LPA) or 2 ng/ml TGF-β1 for 4 days. The expression levels of {alpha}-SMA, tubulin, and Smad2 were determined by Western blotting. The phosphorylation level of Smad2 was determined by Western blotting with anti-p-Smad2 antibody. Representative data from three independent experiments are shown. Abbreviations: {alpha}-SMA, {alpha}-smooth muscle actin; h, hour; LPA, lysophosphatidic acid; min, minutes; p-Smad2, phosphorylated Smad2; TGF-β1, transforming growth factor-β1; w/o, without.

 
Malignant Ascites Induce the Expression of {alpha}-SMA in hADSCs through LPA-TGF-β1-Dependent Crosstalk Pathway
To explore whether ascites from ovarian cancer patients can induce differentiation of hADSCs to myofibroblasts, we determined the expression level of {alpha}-SMA after exposure of hADSCs to different concentrations of ascites for 4 days. As shown in Figure 5A, malignant ascites derived from four different patients with ovarian cancer dose-dependently increased the expression level of {alpha}-SMA with a maximal stimulation at 5%. In contrast, nonmalignant ascites from four different patients with liver cirrhosis had no significant impact on the expression level of {alpha}-SMA, suggesting that malignant ascites from ovarian cancer patients specifically increase the expression level of {alpha}-SMA in hADSCs.


Figure 5
View larger version (51K):
[in this window]
[in a new window]

  		Figure 5. Malignant ascites from ovarian cancer patients induce the expression of {alpha}-smooth muscle actin and delayed phosphorylation of Smad2 through lysophosphatidic acid (LPA)-transforming growth factor (TGF)-β-dependent mechanism. (A): Serum-starved human adipose tissue-derived mesenchymal stem cells (hADSCs) were treated for 4 days with indicated concentrations of ascites from patients with ovarian cancer (four cases) or liver cirrhosis (four cases). (B): Serum-starved hADSCs were pretreated with vehicles or 10 µM Ki16425 for 15 minutes, and exposed to 1% malignant ascites from an ovarian cancer patient (Case 1) for 4 days. (C): Serum-starved hADSCs were pretreated with vehicles or 10 µM SB431542, and then exposed to 1% ovarian cancer ascites (Case 1) for 4 days. The expression levels of {alpha}-SMA, tubulin, and Smad2 were determined by Western blotting. The phosphorylation level of Smad2 was determined by Western blotting with anti-p-Smad2 antibody. Representative data from three independent experiments are shown. Abbreviations: {alpha}-SMA, {alpha}-smooth muscle actin; p-Smad2, phosphorylated Smad2.

 
To assess whether the malignant ascites-induced expression of {alpha}-SMA is mediated by LPA, we examined the effects of Ki16425 on the malignant ascites-induced {alpha}-SMA expression. Figure 5B shows that Ki16425 markedly inhibited the {alpha}-SMA expression increased by the malignant ascites, implying that LPA is involved in the malignant ascites-induced {alpha}-SMA expression.

To assess whether TGF-β1-Smad-dependent pathway is involved in the malignant ascites-induced expression of {alpha}-SMA, we next examined the effects of SB431542 on the {alpha}-SMA expression increased by the malignant ascites. As shown in Figure 5C, SB431542 completely abrogated the malignant ascites-induced expression of {alpha}-SMA and delayed phosphorylation of Smad2. These results suggest that LPA in malignant ascites stimulates the expression of {alpha}-SMA through autocrine TGF-β1-Smad signaling loop.

Ovarian Cancer Cells Stimulate the Expression of {alpha}-SMA in hADSCs through LPA-TGF-β1-Dependent Crosstalk Pathway
An increasing body of evidence suggests that ovarian cancer cells produce LPA through mechanisms involving phospholipase D and phospholipase A2 [42–44]. To assess whether ovarian cancer cells promote the expression of {alpha}-SMA in hADSCs through producing LPA, we examined the effects of CM, which was derived from OVCAR-3 human ovarian cancer cells, on the expression level of {alpha}-SMA and phosphorylation of Smad2 in hADSCs. As shown in Figure 6A, CM from OVCAR-3 cells dose-dependently increased the expression level of {alpha}-SMA and delayed phosphorylation of Smad2 in hADSCs. The OVCAR-3 CM-induced expression of {alpha}-SMA and delayed phosphorylation of Smad2 were markedly abrogated by pharmacological inhibition of LPA receptor with Ki16425 (Fig. 6B). Furthermore, the TGF-β type I receptor inhibitor, SB431542, completely attenuated the delayed phosphorylation of Smad2 and {alpha}-SMA expression in response to OVCAR-3 CM (Fig. 6C).


Figure 6
View larger version (48K):
[in this window]
[in a new window]

  		Figure 6. Conditioned medium from OVCAR-3 cells induces the expression of {alpha}-smooth muscle actin ({alpha}-SMA) and delayed phosphorylation of Smad2. (A): Serum-starved human adipose tissue-derived mesenchymal stem cells (hADSCs) were exposed to indicated concentrations of conditioned medium (CM) from OVCAR-3 for 4 days. (B): Serum-starved hADSCs were pretreated with 10 µM Ki16425 or vehicles for 15 minutes, and exposed to 50% OVCAR-3 CM for 4 days. (C): Serum-starved hADSCs were pretreated with 10 µM SB431542 or vehicles for 15 minutes, and exposed to 50% OVCAR-3 CM for 4 days. The expression levels of {alpha}-SMA, tubulin, and Smad2 were determined by Western blotting. The phosphorylation levels of Smad2 were determined by Western blotting with anti-p-Smad2 antibody. Representative data from three independent experiments are shown. Abbreviations: {alpha}-SMA, {alpha}-smooth muscle actin; CM, condition medium; p-Smad2, phosphorylated Smad2.

 
To further support the involvement of autocrine TGF-β1-dependent pathway in the {alpha}-SMA expression induced by malignant ascites and OVCAR-3 CM, we measured the concentrations of TGF-β1 present in the malignant ascites and OVCAR-3 CM by using ELISA analysis. As shown in supplemental online Fig. 2A, TGF-β1 could be detected in the ascites derived from three different patients with ovarian cancer, but not in OVCAR-3 CM, whereas exposure of hADSCs to the ascites and OVCAR-3 CM greatly up-regulated secretion of TGF-β1 from hADSCs; this suggests that the autocrine secretion of TGF-β1 from hADSCs is responsible for the {alpha}-SMA expression induced by the malignant ascites and OVCAR-3 CM. We next examined the effect of anti-TGF-β1 neutralizing antibody on the expression of {alpha}-SMA and calponin induced by the malignant ascites and OVCAR-3 CM. Pretreatment of hADSCs with anti-TGF-β1 neutralizing antibody completely abrogated the expression of {alpha}-SMA and calponin induced by the malignant ascites or OVCAR-3 CM (supplemental online Fig. 2B). These results suggest that LPA secreted from ovarian cancer cells induces differentiation of hADSCs to myofibroblast-like cells through stimulating the autocrine TGF-β1-dependent pathway.

LPA Stimulates Production of SDF-1 from hADSCs through TGF-β1-Smad-Dependent Pathway
Cancer-associated myofibroblasts play a key role in angiogenesis during tumorigenesis by producing SDF-1 [3, 45]. To explore whether myofibroblast-like cells which were differentiated from hADSCs in response to LPA can produce SDF-1, we examined the effects of LPA on the mRNA level of SDF-1 by using RT-PCR analysis. The mRNA levels of SDF-1 in hADSCs were dose-dependently up-regulated by LPA treatment (Fig. 7A). Consistent with these results, LPA dose-dependently increased the level of SDF-1 protein in the CM from hADSCs, further supporting the result that LPA induces production of SDF-1 in hADSCs (Fig. 7B). Exposure of hADSCs to 5 µM LPA significantly increased the mRNA level of SDF-1 at 12 hours and maximally at 24 hours (Fig. 7C). Not only LPA but also TGF-β1 increased the expression level of SDF-1 in hADSCs, suggesting a possibility that LPA stimulates SDF-1 production in hADSCs through TGF-β1-dependent mechanism.


Figure 7
View larger version (39K):
[in this window]
[in a new window]

  		Figure 7. Lysophosphatidic acid in the malignant ascites or OVCAR3 conditioned medium (CM) induces the expression of stromal cell-derived factor-1 through activation of autocrine transforming growth factor-β1-dependent signaling pathway. (A): Serum-starved human adipose tissue-derived mesenchymal stem cells (hADSCs) were treated with indicated concentrations of 1-Oleoyl-sn-glycero-3-phosphate (1-oleoyl-LPA) for 24 hours. The mRNA levels of SDF-1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were determined by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). (B): hADSCs were treated with indicated concentrations of 1-oleoyl-LPA for 48 hours. The conditioned medium was subjected to ELISA, for determination of the levels of SDF-1 protein. Data represent mean ± SD. (n = 3). * indicates p < .05 and ** indicates p < .01 versus control (0 µM). (C): Serum-starved hADSCs were exposed to vehicles, 5 µM 1-oleoyl-LPA, or 2 ng/ml TGF-β1 for the indicated time periods. (D): Serum-starved hADSCs were pretreated with vehicles, 10 µM Ki16425, or 10 µM SB431542 for 15 minutes, and then exposed to 5 µM 1-oleoyl-LPA, 1% ovarian cancer ascites (Case 1), 50% OVCAR-3 CM, or 2 ng/ml TGF-β1 for 24 hours. The mRNA levels of SDF-1 and GAPDH were determined by semi-quantitative RT-PCR. (E): Serum-starved hADSCs were pretreated with vehicles, 10 µM Ki16425, or 10 µM SB431542 for 15 minutes, and then exposed to 5 µM 1-oleoyl-LPA or 2 ng/ml TGF-β1 for 48 hours. The CM was subjected to ELISA for determination of the protein levels of SDF-1. Data represent mean ± SD. (n = 3). * indicates p < .05. Abbreviations: {alpha}-SMA, {alpha}-smooth muscle actin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LPA, lysophosphatidic acid; SDF-1, stromal cell-derived factor-1; TGF-β1, transforming growth factor-β1.

 
To explore whether malignant ascites and OVCAR-3 CM increase the expression of SDF-1 through the LPA-dependent activation of TGF-β1-Smad pathway, we determined the effect of malignant ascites or OVCAR-3 CM on SDF-1 expression in the absence or presence of Ki16425 or SB431542. As shown in Figure 7D, LPA as well as malignant ascites and OVCAR-3 CM up-regulated the levels of SDF-1 mRNA in hADSCs. Consistently, malignant ascites derived from three patients with ovarian cancer and OVCAR-3 CM induced secretion of SDF-1 from hADSCs (supplemental online Fig. 3). The increased expression of SDF-1 in response to LPA, malignant ascites, or OVCAR-3 CM was abrogated by pretreatment of the cells with Ki16425 or SB431542. However, TGF-β1-stimulated expression of SDF-1 was attenuated by pretreatment of the cells with SB431542, but not with Ki16425 (Fig. 7D). These results suggest that LPA is responsible for the increased expression of SDF-1 induced by malignant ascites or OVCAR-3 CM through activation of TGF-β1-dependent pathway.

To further confirm these results, we determined the effects of LPA and TGF-β1 on the secretion of SDF-1 protein in the absence or presence of Ki16425 or SB431542 by using ELISA. Exposure of the cells to either LPA or TGF-β1 significantly increased the secretion of SDF-1 (Fig. 7E). The increased secretion of SDF-1 in response to LPA was completely inhibited by pretreatment of the cells with Ki16425 or SB431542, whereas TGF-β1-induced production of SDF-1 was abrogated by SB431542, but not by Ki16425, implying that autocrine TGF-β1-Smad-signaling pathway is necessary for the LPA-induced expression of SDF-1. The stimulatory effect of LPA on SDF-1 production was more potent than that of TGF-β1, in contrast to the less potent effect of LPA on the expression of {alpha}-SMA than TGF-β1 (Fig. 4B). Therefore, these results suggest that activation of the autocrine TGF-β1-Smad signaling loop is not sufficient for the LPA-induced production of SDF-1, and that not only TGF-β1-dependent pathway but also TGF-β1-independent mechanism may be required for the LPA-induced expression of SDF-1.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 Acknowledgments
 References
 
In this study, we showed that LPA stimulated the expression of {alpha}-SMA and calponin in hADSCs, whereas the expression of SM-MHC was not induced by LPA treatment. In contrast to the shared expression of {alpha}-SMA and calponin in myofibroblasts and SMCs, SM-MHC has been considered as the best discriminating marker identified to date for the SMCs [6], suggesting that LPA induces differentiation of hADSCs to myofibroblasts, but not to fully differentiated SMCs. LPA has been reported to promote the expression of {alpha}-SMA and calponin in neural progenitor cells isolated from the embryonic cerebral cortex [46]. In contrast, LPA treatment decreased the expression of calponin and caldesmon in vascular SMCs isolated from rat aorta [47]. Intriguingly, it has recently been reported that MCAT elements within {alpha}-SMA promoter are required for {alpha}-SMA transcription in myofibroblasts, but not in adult SMCs, suggesting that adult SMCs and myofibroblasts use distinct transcriptional mechanisms for {alpha}-SMA expression [48]. Therefore, it is likely that the effect of LPA on the expression of these SMC markers varies depending on the context of cells, and the differential effects of LPA on the {alpha}-SMA expression may be due to the different transcriptional mechanisms. It is still unclear, however, whether myofibroblasts and SMCs are distinctly different cell types. The transcriptional mechanism involved in the LPA-induced expression of SMC markers in hADSCs should be further clarified.

In the present study, we showed that LPA stimulated the expression of {alpha}-SMA and delayed activation of Smad2 and Smad3 through the autocrine activation of TGF-β receptor. The LPA-induced expression of {alpha}-SMA and delayed phosphorylation of Smad2 and Smad3 were completely blocked by pharmacological inhibition of TGF-β receptor or pretreatment with anti-TGF-β1 neutralizing antibody. Furthermore, LPA stimulated the production of TGF-β1, followed by activation of Smad2/3. These results suggest that the autocrine TGF-β1-Smad signaling loop plays a key role in the LPA-induced expression of {alpha}-SMA. Furthermore, TGF-β has been suggested to promote tumorigenesis by inducing angiogenesis, suppressing apoptotic activity, or enhancing cell motility as tumor progresses [49, 50]. Therefore, the LPA-induced activation of TGF-β-Smad autocrine pathways seems to provide a new signaling mechanism for the LPA-induced tumorigenesis.

The present study demonstrated for the first time that malignant ascites from ovarian cancer patients induced the expression of {alpha}-SMA, whereas ascites from liver cirrhosis patients had no significant impact on the expression of {alpha}-SMA; this suggests that the malignant ascites specifically induce differentiation of MSCs to myofibroblast-like cells. It has been reported that LPA and SPC are abundant in malignant ascites from patients with ovarian cancer, compared to ascites from patients with nonmalignant liver diseases [19]. We reported previously that SPC induced the expression of {alpha}-SMA through G-coupled receptor-dependent pathways in hADSCs [34]. It is therefore plausible to suggest that SPC plays a key role in the malignant ascites-stimulated expression of {alpha}-SMA. However, the involvement of SPC in the malignant ascite-induced expression of {alpha}-SMA can be excluded, because Ki16425, the LPA receptor-selective antagonist, completely attenuated the expression of {alpha}-SMA in response to LPA or the malignant ascites, but not SPC. Furthermore, siRNA-mediated depletion of endogenous LPA receptors markedly abrogated the LPA-induced expression of {alpha}-SMA. These results suggest that LPA, but not SPC, plays a pivotal role in the differentiation of hADSCs to myofibroblast-like cells induced by malignant ascites.

The importance of reactive tumor microenvironment is increasingly appreciated. LPA has been shown to be an essential microenvironmental factor in ovarian cancer, and high levels of LPA in the ascitic fluid and sera of patients have been correlated with poor prognosis of the disease [20, 51]. High levels of LPA up to 80 µM, which exceeds the levels required to stimulate the expression of {alpha}-SMA in MSCs, have been reported in the ascitic fluid of ovarian cancer patients [19, 20, 51]. Moreover, LPA has been shown to be produced by ovarian cancer cells [42–44, 52]. In the present study, we demonstrated that CM from OVCAR-3 ovarian cancer cells induced the expression of {alpha}-SMA in hADSCs. Furthermore, the increased expression of {alpha}-SMA in response to OVCAR-3 CM was blocked by pharmacological inhibition of LPA receptors. These result led us to suggest that cancer-derived LPA promotes differentiation of MSCs toward myofibroblast-like cells in tumor microenvironment. High concentration of LPA has been reported in malignant ascites derived from patients with pancreatic cancer [53] as well as ovarian cancer, therefore, it remains to be determined whether ascites derived from other malignant tumors can also induce differentiation of hADSCs to myofibroblasts.

Recent studies indicate that stromal cells in the primary tumor are an important source of SDF-1. In myofibroblasts isolated from breast cancer specimens, SDF-1 expression was significantly up-regulated compared to fibroblasts obtained from normal breast tissue [10, 45]. Myofibroblasts-derived SDF-1 not only increases carcinoma cell growth directly through the CXCR4 receptor displayed on tumor cells, but also serves to recruit endothelial progenitor cells into tumors, thereby stimulating neoangiogenesis [3]. Moreover, SDF-1 produced by human prostatic carcinoma-associated fibroblasts promotes malignancy of benign human prostatic epithelial cells [54]. In the present study, we demonstrated for the first time that LPA in the malignant ascites or the CM from ovarian cancer cells promotes the expression and secretion of SDF-1 in hADSCs through TGF-β1-dependent mechanism. In support of the present study, it has been reported that CM from C85 colorectal cancer cells promote the production of SDF-1 in rat MSCs [55]. Taken together with these results, the present study provides evidences that LPA derived from cancer cells is responsible for the enhanced expression of SDF-1 in carcinoma-associated myofibroblasts. Further studies are needed to clarify whether the LPA-induced differentiation of MSCs to myofibroblast-like cells is involved in the tumor growth and angiogenesis in vivo.


   DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
References
 
This work was supported by the MRC program of MOST/KOSEF (R13-2005-009) and a grant from the National R&D Program for Cancer Control, Ministry of Health & Welfare, Republic of Korea (00000033). We are grateful to Dr. Kohei Miyazono for providing us the adenoviruses expressing Smad7.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 

  1. De Wever O, Mareel M. Role of tissue stroma in cancer cell invasion. J Pathol 2003;200:429–447.[CrossRef][Medline]

  2. Desmoulière A, Guyot C, Gabbiani G. The stroma reaction myofibroblast: a key player in the control of tumor cell behavior. Int J Dev Biol 2004;48:509–517.[CrossRef][Medline]

  3. Orimo A, Weinberg RA. Stromal fibroblasts in cancer: a novel tumor-promoting cell type. Cell Cycle 2006;5:1597–1601.[Medline]

  4. Darby I, Skalli O, Gabbiani G. Alpha-smooth muscle actin is transiently expressed by myofibroblasts during experimental wound healing. Lab Invest 1990;63:21–29.[Medline]

  5. Tomasek JJ, Gabbiani G, Hinz B et al. Myofibroblasts and mechano-regulation of connective tissue remodelling. Nat Rev Mol Cell Biol 2002;3:349–363.[CrossRef][Medline]

  6. Owens GK, Kumar MS, Wamhoff BR. Molecular regulation of vascular smooth muscle cell differentiation in development and disease. Physiol Rev 2004;84:767–801.[Abstract/Free Full Text]

  7. Sappino AP, Skalli O, Jackson B et al. Smooth-muscle differentiation in stromal cells of malignant and non-malignant breast tissues. Int J Cancer 1988;41:707–712.[Medline]

  8. Ganss R. Tumor stroma fosters neovascularization by recruitment of progenitor cells into the tumor bed. J Cell Mol Med 2006;10:857–865.[CrossRef][Medline]

  9. Olumi AF, Grossfeld GD, Hayward SW et al. Carcinoma-associated fibroblasts direct tumor progression of initiated human prostatic epithelium. Cancer Res 1999;59:5002–5011.[Abstract/Free Full Text]

  10. Orimo A, Gupta PB, Sgroi DC et al. Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion. Cell 2005;121:335–348.[CrossRef][Medline]

  11. Burger JA, Kipps TJ. CXCR4: a key receptor in the crosstalk between tumor cells and their microenvironment. Blood 2006;107:1761–1767.[Abstract/Free Full Text]

  12. Aoki J. Mechanisms of lysophosphatidic acid production. Semin Cell Dev Biol 2004;15:477–489.[CrossRef][Medline]

  13. Mills GB, Moolenaar WH. The emerging role of lysophosphatidic acid in cancer. Nat Rev Cancer 2003;3:582–591.[CrossRef][Medline]

  14. Gaits F, Fourcade O, Le Balle F et al. Lysophosphatidic acid as a phospholipid mediator: pathways of synthesis. FEBS Lett 1997;410:54–58.[CrossRef][Medline]

  15. Birgbauer E, Chun J. New developments in the biological functions of lysophospholipids. Cell Mol Life Sci 2006;63:2695–2701.[CrossRef][Medline]

  16. Parizi M, Howard EW, Tomasek JJ. Regulation of LPA-promoted myofibroblast contraction: role of Rho, myosin light chain kinase, and myosin light chain phosphatase. Exp Cell Res 2000;254:210–220.[CrossRef][Medline]

  17. Tangkijvanich P, Melton AC, Chitapanarux T et al. Platelet-derived growth factor-BB and lysophosphatidic acid distinctly regulate hepatic myofibroblast migration through focal adhesion kinase. Exp Cell Res 2002;281:140–147.[CrossRef][Medline]

  18. Murph M, Tanaka T, Liu S et al. Of spiders and crabs: the emergence of lysophospholipids and their metabolic pathways as targets for therapy in cancer. Clin Cancer Res 2006;12:6598–6602.[Abstract/Free Full Text]

  19. Xiao YJ, Schwartz B, Washington M et al. Electrospray ionization mass spectrometry analysis of lysophospholipids in human ascitic fluids: comparison of the lysophospholipid contents in malignant vs nonmalignant ascitic fluids. Anal Biochem 2001;290:302–313.[CrossRef][Medline]

  20. Xu Y, Shen Z, Wiper DW et al. Lysophosphatidic acid as a potential biomarker for ovarian and other gynecologic cancers. JAMA 1998;280:719–723.[Abstract/Free Full Text]

  21. Westermann AM, Havik E, Postma FR et al. Malignant effusions contain lysophosphatidic acid (LPA)-like activity. Ann Oncol 1998;9:437–442.[Abstract/Free Full Text]

  22. Bian D, Su S, Mahanivong C et al. Lysophosphatidic acid stimulates ovarian cancer cell migration via a Ras-MEK kinase 1 pathway. Cancer Res 2004;64:4209–4217.[Abstract/Free Full Text]

  23. Xu Y, Fang XJ, Casey G et al. Lysophospholipids activate ovarian and breast cancer cells. Biochem J 1995;309Pt 3:933–940.[Medline]

  24. Boucharaba A, Serre CM, Guglielmi J et al. The type 1 lysophosphatidic acid receptor is a target for therapy in bone metastases. Proc Natl Acad Sci U S A 2006;103:9643–9648.[Abstract/Free Full Text]

  25. Barry FP, Murphy JM. Mesenchymal stem cells: clinical applications and biological characterization. Int J Biochem Cell Biol 2004;36:568–584.[CrossRef][Medline]

  26. Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science 1997;276:71–74.[Abstract/Free Full Text]

  27. Pittenger MF, Mackay AM, Beck SC et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999;284:143–147.[Abstract/Free Full Text]

  28. Short B, Brouard N, Occhiodoro-Scott T et al. Mesenchymal stem cells. Arch Med Res 2003;34:565–571.[CrossRef][Medline]

  29. Zhu W, Xu W, Jiang R et al. Mesenchymal stem cells derived from bone marrow favor tumor cell growth in vivo. Exp Mol Pathol 2006;80:267–274.[CrossRef][Medline]

  30. Hinz B. Formation and function of the myofibroblast during tissue repair. J Invest Dermatol 2007;127:526–537.[CrossRef][Medline]

  31. Li H, Fan X, Houghton J. Tumor microenvironment: The role of the tumor stroma in cancer. J Cell Biochem 2007;101:805–815.[CrossRef][Medline]

  32. Kinner B, Zaleskas JM, Spector M. Regulation of smooth muscle actin expression and contraction in adult human mesenchymal stem cells. Exp Cell Res 2002;278:72–83.[CrossRef][Medline]

  33. Wang D, Park JS, Chu JS et al. Proteomic profiling of bone marrow mesenchymal stem cells upon transforming growth factor beta1 stimulation. J Biol Chem 2004;279:43725–43734.[Abstract/Free Full Text]

  34. Jeon ES, Moon HJ, Lee MJ et al. Sphingosylphosphorylcholine induces differentiation of human mesenchymal stem cells into smooth-muscle-like cells through a TGF-beta-dependent mechanism. J Cell Sci 2006;119:4994–5005.[Abstract/Free Full Text]

  35. Fujii M, Takeda K, Imamura T et al. Roles of bone morphogenetic protein type I receptors and Smad proteins in osteoblast and chondroblast differentiation. Mol Biol Cell 1999;10:3801–3813.[Abstract/Free Full Text]

  36. Gabbiani G. The myofibroblast in wound healing and fibrocontractive diseases. J Pathol 2003;200:500–503.[CrossRef][Medline]

  37. Jeon ES, Song HY, Kim MR et al. Sphingosylphosphorylcholine induces proliferation of human adipose tissue-derived mesenchymal stem cells via activation of JNK. J Lipid Res 2006;47:653–664.[Abstract/Free Full Text]

  38. Hu B, Wu Z, Phan SH. Smad3 mediates transforming growth factor-beta-induced alpha-smooth muscle actin expression. Am J Respir Cell Mol Biol 2003;29:397–404.[Abstract/Free Full Text]

  39. Qiu P, Ritchie RP, Fu Z et al. Myocardin enhances Smad3-mediated transforming growth factor-beta1 signaling in a CArG box-independent manner: Smad-binding element is an important cis element for SM22alpha transcription in vivo. Circ Res 2005;97:983–991.[Abstract/Free Full Text]

  40. Sinha S, Hoofnagle MH, Kingston PA et al. Transforming growth factor-beta1 signaling contributes to development of smooth muscle cells from embryonic stem cells. Am J Physiol Cell Physiol 2004;287:C1560–C1568.[Abstract/Free Full Text]

  41. Jinnin M, Ihn H, Tamaki K. Characterization of SIS3, a novel specific inhibitor of Smad3, and its effect on transforming growth factor-beta1-induced extracellular matrix expression. Mol Pharmacol 2006;69:597–607.[Abstract/Free Full Text]

  42. Luquain C, Singh A, Wang L et al. Role of phospholipase D in agonist-stimulated lysophosphatidic acid synthesis by ovarian cancer cells. J Lipid Res 2003;44:1963–1975.[Abstract/Free Full Text]

  43. Eder AM, Sasagawa T, Mao M et al. Constitutive and lysophosphatidic acid (LPA)-induced LPA production: role of phospholipase D and phospholipase A2. Clin Cancer Res 2000;6:2482–2491.[Abstract/Free Full Text]

  44. Shen Z, Belinson J, Morton RE et al. Phorbol 12-myristate 13-acetate stimulates lysophosphatidic acid secretion from ovarian and cervical cancer cells but not from breast or leukemia cells. Gynecol Oncol 1998;71:364–368.[CrossRef][Medline]

  45. Allinen M, Beroukhim R, Cai L et al. Molecular characterization of the tumor microenvironment in breast cancer. Cancer Cell 2004;6:17–32.[CrossRef][Medline]

  46. Morishita R, Nagata K, Ito H et al. Expression of smooth muscle cell-specific proteins in neural progenitor cells induced by agonists of G protein-coupled receptors and transforming growth factor-beta. J Neurochem 2007;101:1031–1040.[CrossRef][Medline]

  47. Hayashi K, Takahashi M, Nishida W et al. Phenotypic modulation of vascular smooth muscle cells induced by unsaturated lysophosphatidic acids. Circ Res 2001;89:251–258.[Abstract/Free Full Text]

  48. Gan Q, Yoshida T, Li J et al. Smooth muscle cells and myofibroblasts use Distinct transcriptional mechanisms for smooth muscle alpha-actin expression. Circ Res 2007;101:883–892.[Abstract/Free Full Text]

  49. Bierie B, Moses HL. TGF-beta and cancer. Cytokine Growth Factor Rev 2006;17:29–40.[CrossRef][Medline]

  50. Jakowlew SB. Transforming growth factor-beta in cancer and metastasis. Cancer Metastasis Rev 2006;25:435–457.[CrossRef][Medline]

  51. Xu Y, Gaudette DC, Boynton JD et al. Characterization of an ovarian cancer activating factor in ascites from ovarian cancer patients. Clin Cancer Res 1995;1:1223–1232.[Abstract]

  52. Sengupta S, Xiao YJ, Xu Y. A novel laminin-induced LPA autocrine loop in the migration of ovarian cancer cells. FASEB J 2003;17:1570–1572.[Abstract/Free Full Text]

  53. Yamada T, Sato K, Komachi M et al. Lysophosphatidic acid (LPA) in malignant ascites stimulates motility of human pancreatic cancer cells through LPA1. J Biol Chem 2004;279:6595–6605.[Abstract/Free Full Text]

  54. Ao M, Franco OE, Park D et al. Cross-talk between Paracrine-Acting Cytokine and Chemokine Pathways Promotes Malignancy in Benign Human Prostatic Epithelium. Cancer Res 2007;67:4244–4253.[Abstract/Free Full Text]

  55. Menon LG, Picinich S, Koneru R et al. Differential gene expression associated with migration of mesenchymal stem cells to conditioned medium from tumor cells or bone marrow cells. STEM CELLS 2007;25:520–528.[Abstract/Free Full Text]





This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
2007-0742v1
26/3/789    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Reprints/Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jeon, E. S.
Right arrow Articles by Kim, J. H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jeon, E. S.
Right arrow Articles by Kim, J. H.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
STEM CELLS THE ONCOLOGIST CME ALPHAMED PRESS JOURNALS