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

Authors

  • Mi Ra Kim,

    1. Medical Research Center for Ischemic Tissue Regeneration, the Medical Research Institute, Department of Physiology, Busan, Republic of Korea
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  • Eun Su Jeon,

    1. Medical Research Center for Ischemic Tissue Regeneration, the Medical Research Institute, Department of Physiology, Busan, Republic of Korea
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  • Young Mi Kim,

    1. Medical Research Center for Ischemic Tissue Regeneration, the Medical Research Institute, Department of Physiology, Busan, Republic of Korea
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  • Jung Sub Lee,

    1. Department of Orthopaedic Surgery, School of Medicine, Pusan National University, Busan, Republic of Korea
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  • Jae Ho Kim

    Corresponding author
    1. Medical Research Center for Ischemic Tissue Regeneration, the Medical Research Institute, Department of Physiology, Busan, Republic of Korea
    • Department of Physiology, School of Medicine, Pusan National University, 1-Ga, Ami-Dong, Suh-Gu, Busan, 602-739, Republic of Korea
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    • Telephone: 82-51-240-7732; Fax: 82-51-246-6001


  • Author contributions: M.R.K.: conception and design, collection and assembly of data, data analysis and interpretation, manuscript writing; E.S.J. and Y.M.K.: collection and assembly of data, data analysis and interpretation; J.S.L.: provision of study material or patients, data analysis and interpretation; J.H.K.: conception and design, financial support, data analysis and interpretation, manuscript writing, final approval of manuscript; M. R. K. and E. S. J. contributed equally to this work.

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

  • §

    First published online in STEM CELLSExpress October 9, 2008.

Abstract

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 CELLS2009;27:191–199

INTRODUCTION

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 [1–4]. In vitro, MSCs differentiate to smooth muscle cells (SMCs) in response to transforming growth factor-β (TGF-β) [5, 6], and in vivo they are able to differentiate to SMCs and contribute to the remodeling of vasculature [7–9]. The phenotype of SMCs is characterized by expression of smooth muscle-specific contractile proteins such as α-smooth muscle actin (α-SMA), h1-calponin, smoothelin, and smooth muscle-myosin heavy chain (SM-MHC) [10], and the phenotypic expression of SMCs has been implicated in vascular development as well as in a variety of cardiovascular diseases including hypertension and atherosclerosis [10, 11]. In a previous study, we demonstrated that the expression levels of SMC-specific markers can be upregulated by treatment with sphingosylphosphorylcholine (SPC) and TGF-β1 in human adipose tissue-derived MSCs (hADSCs) [12], suggesting a potential role of MSCs as progenitor cells for SMCs. However, the molecular identities of extracellular factors that are involved in the differentiation of MSCs to SMCs are largely unknown.

Serum response factor (SRF) is a widely expressed transcription factor involved in orchestrating disparate programs of gene expression linked to muscle differentiation and cellular growth [13, 14]. SRF is required for expression of most SMC-specific markers via binding to highly conserved CArG cis-elements [CC(A/T)6GG] found in the promoters of most SMC-specific genes [14]. Myocardin, which is a member of the SAP domain family of nuclear proteins [15], functions as a SRF cofactor and plays a central role in the SRF-dependent expression of SMC-specific genes [16]. We demonstrated previously that both SRF and myocardin are involved in the smooth muscle differentiation of hADSCs induced by SPC and TGF-β [12].

Thromboxane A2 (TxA2) is a major prostanoid metabolite of arachidonic acid, which can be produced predominantly in platelets in response to a variety of physiological and pathological stimuli through isomerization of prostaglandin H2 by TxA2 synthase [17, 18]. TxA is a potent stimulator of platelet aggregation and smooth muscle contraction, and it plays a pivotal role in atherosclerotic vascular diseases, myocardial infarction, and bronchial asthma [19]. Its actions are primarily mediated by the TP receptor (TxA2 receptor), a G-protein-coupled receptor that triggers activation of phospholipase Cβ via Gq/11, leading to an increase in intracellular Ca2+ concentration ([Ca2+]i) [20] and consequent activation of the Ca2+/calmodulin (CaM)-dependent enzyme myosin light chain kinase (MLCK). U46619 elicits contraction of SMCs by inducing MLCK-dependent phosphorylation of the 20-kDa myosin light chain (MLC) [21, 22]. In addition to the elevation of [Ca2+]i, TxA2 stimulates the small GTPase RhoA via the G12/13 family of heterotrimeric G proteins and a guanine nucleotide-exchange factor [23]. Activated RhoA stimulates contraction and polymerization of actin filament in SMCs through the RhoA effector Rho kinase-dependent mechanism, resulting in sensitization of MLC phosphorylation to Ca2+ [24]. In addition to the stimulation of the contraction and actin polymerization, the RhoA-Rho kinase pathway plays a key role in SMC differentiation by regulating the integrity of the actin cytoskeleton [14, 25]. However, the functional roles of TxA2 in the differentiation of MSCs to SMCs have not been explored.

To determine whether TxA2 is involved in the differentiation of MSCs to contractile SMCs, we used the stable TxA2 analog U46619, because TxA2 itself is unstable. We demonstrate for the first time that U46619 induces differentiation of hADSCs to contractile smooth muscle-like cells through the CaM/MLCK- and RhoA-Rho kinase-dependent mechanisms.

MATERIALS AND METHODS

Materials

Phosphate-buffered saline (PBS), α-minimum essential medium (α-MEM), trypsin, fetal bovine serum, Lipofectamine 2000 reagent, fluo-4-AM, Alexa Fluor 488 goat anti-mouse antibody, and Alexa Fluor 568 phalloidin were purchased from Invitrogen (Carlsbad, CA, http://www.invitrogen.com). U46619, iloprost, prostaglandin F, prostaglandin D2, prostaglandin E2, leukotriene B4, and SQ29548 were purchased from Cayman Chemicals (Ann Arbor, MI, http://www.caymanchem.com). Anti-SM-MHC, anti-smoothelin, and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies, lysis buffer, and the Rho activation pull-down assay kit were purchased from Millipore (Temecula, CA, http://www.millipore.com). Anti-α-SMA and anti-h1-calponin antibodies, Ponceau S solution, Oil Red O, Alizarin Red S, W13, and ML-7 were from Sigma-Aldrich (St. Louis, MO, http://www.sigmaaldrich.com). Y27632 was purchased from EMD Biosciences (San Diego, CA, http://www.emdbiosciences.com). Anti-phospho-MLC20 (Ser-19) antibody was purchased from Cell Signaling Technology (Beverly, MA, http://www.cellsignal.com). Anti-RhoA antibody was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, http://www.scbt.com). Rat tail type I collagen was purchased from BD Biosciences (Bedford, MA, http://www.bdbiosciences.com). Culture plates were purchased from Nunc (Roskilde, Denmark, http://www.nuncbrand.com). Peroxidase-labeled secondary antibodies and the enhanced chemiluminescence Western blotting system were from Amersham Biosciences (Pittsburgh, PA, http://www4.gelifesciences.com). pEF-Bos-myc-Rho-kinase RB/PH (TT) was kindly provided by Dr. K. Kaibuchi (Nagoya University, Nagoya, Japan).

Cell Culture

Subcutaneous adipose tissue was obtained from elective surgical procedures with the patient's consent as approved by the institutional review board of Pusan National University Hospital. 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 salt solution buffer with 1% bovine serum albumin) for 60 minutes at 37°C with intermittent shaking. Details are available in the supporting information.

Western Blotting

Confluent, serum-starved hADSCs were treated with the appropriate conditions, washed with ice-cold PBS, and then lysed in lysis buffer (20 mM Tris-HCl, 1 mM ethylene glycol tetraacetic acid, 1 mM EDTA, 10 mM NaCl, 0.1 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, 30 mM sodium pyrophosphate, 25 mM β-glycerophosphate, and 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. 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 an enhanced chemiluminescence system.

Immunocytochemistry and Microscopy

Immunostaining and confocal microscopy were used to determine the subcellular distribution and organization of proteins. Cells were fixed in PBS containing 4% paraformaldehyde for 15 minutes, permeabilized with PBS containing 0.2% Triton X-100 for 10 minutes, and blocked with PBS containing 2% bovine serum albumin. For immunostaining, specimens were incubated with anti-α-SMA antibody for 2 h and Alexa Fluor 488-conjugated anti-mouse secondary antibody for 1 h. To stain F-actin filaments, the specimen was incubated with Alexa Fluor 543-conjugated phalloidin for 30 minutes followed by confocal microscopy. The images of the specimen were collected with a Leica TCL SP2 confocal microscope system (Leica Microsystems, Wetzler, Germany, http://www.leica-microsystems.com).

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 Moloney murine leukemia virus reverse transcriptase (Invitrogen) and 0.5 μg of oligo(dT) 15 primer (Promega, Madison, WI, http://www.promega.com). Details are available in the supporting information.

Transfection with Small Interference RNA

For small interference RNA (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 the manufacturer's instructions (Invitrogen). Details are available in the supporting information.

Measurement of [Ca2+]i

Spatially averaged photometric [Ca2+]i measurements from single cells were performed with the fluorescent Ca2+ indicator fluo-4-AM. Details are available in the supporting information.

RhoA Activation Assay

A Rho activation pull-down assay kit was used to measure the effect of U46619 on Rho activity in hADSCs. The cells were washed twice with α-MEM, incubated in serum-free α-MEM for 24 h, and then treated with U46619 for the indicated time before lysis of the cells. The activated Rho pull-down assay was performed according to the manufacturer's protocol. Protein was assayed before the pull-down assay to equalize total protein concentration of each treatment group.

Collagen Gel Lattice Contraction Assay

For measurement of contractility, cells were trypsinized from a monolayer culture by treatment with trypsin-EDTA, counted, and resuspended in α-MEM at a density of 1 × 106 cells/ml. The prepared cell suspension was added to collagen gel solution to achieve a final concentration of 3 mg of collagen/ml and 4 × 105 cells/ml, and then the cell collagen mixture was poured into 12-well culture plates. Plates were incubated for 1 h under standard culture conditions to polymerize the collagen cell lattices, and then the lattices were mechanically released from the bottom of the tissue culture dishes by gently pipetting medium at the lattice-dish interface to initiate collagen gel contraction. The extent of gel contraction of each cell population was analyzed by measuring the dimensions of the lattice before release and at specific times after release using a digital charge-coupled device camera. The area of gel lattices was determined by using Scion Image software (compliments of Scion Corporation, Frederick, MD, http://www.scioncorp.com). Relative lattice area was obtained by dividing the area at each time point by the initial area of the lattice.

Differentiation of hADSCs to Adipocytes or Osteoblasts

Differentiation of hADSCs to adipocytes or osteoblasts was induced by incubating the cells in adipogenic or osteogenic differentiation medium, respectively. Details are available in the supporting information.

Statistical Analysis

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

RESULTS

U46619 Induces Differentiation of hADSCs to Smooth Muscle-Like Cells

To explore whether TxA2 can induce differentiation of hADSCs to SMCs, hADSCs were treated with the indicated concentrations of U46619 for 4 days, and then the expression levels of α-SMA, an SMC marker, were determined by Western blotting. As shown in Figure 1A, U46619 dose dependently increased the expression of α-SMA with a maximal stimulation at 1 μM. The expression of α-SMA was observed after treatment of hADSCs with U46619 for 2 days and maximally increased on day 4 (Fig. 1B). U46619 stimulated the expression of not only α-SMA but also other SMC-specific markers such as h1-calponin, SM-MHC, and smoothelin (Fig. 1C). Because TxA2 is a prostanoid metabolite of arachidonic acid, we compared the effects of U46619 and other arachidonic acid metabolites on α-SMA expression. As shown in Figure 1D, iloprost (a stable analog of prostacyclin), prostaglandin F, prostaglandin D2, and prostaglandin E2 increased α-SMA expression, whereas the stimulatory effects of these prostanoid metabolites on α-SMA expression were less potent than those of U46619. In contrast to the prostanoid metabolite-stimulated α-SMA expression, leukotriene B4 had no significant impact on α-SMA expression. These results indicate that prostanoid metabolites derived from arachidonic acid stimulate differentiation of hADSCs to smooth muscle-like cells and that TxA2 is the most potent prostanoid metabolite stimulating smooth muscle differentiation.

Figure 1.

Effects of U46619 and arachidonic acid metabolites on differentiation of human adipose tissue-derived mesenchymal stem cells (hADSCs) to smooth muscle-like cells. (A): hADSCs were treated with the indicated concentrations of U46619 for 4 days. (B): hADSCs were treated with vehicle or 1 μM U46619 for the indicated time periods. (C): hADSCs were treated with vehicle or 1 μM U46619 for 4 days. (D): hADSCs were treated with 1 μM U46619, iloprost, PGF, PGD2, PGE2, and LTB4 for 4 days. The expression levels of α-SMA, calponin, SM-MHC, and smoothelin were determined by Western blotting. To ensure equal loading in each lane, the levels of GAPDH were determined by Western blotting. Representatives of three independent experiments are shown. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LTB4, leukotriene B4; PGD2, prostaglandin D2; PGE2, prostaglandin E2; PGF2α, prostaglandin F; α-SMA, α-smooth muscle actin; SM-MHC, smooth muscle-myosin heavy chain; w/o, without.

U46619 Induces α-SMA-Mediated Formation of Stress Fiber in hADSCs

To support the results that U46619 induced differentiation of hADSCs to smooth muscle-like cells, we next examined the effects of U46619 on intracellular distribution of α-SMA and actin filaments. As shown in Figure 2, treatment of hADSCs with 1 μM U46619 for 4 days increased the expression levels of α-SMA, which was localized in actin filaments. The increased assembly of actin filaments and thick fiber formation in response to U46619 was highly correlated with the increased expression of α-SMA, indicating that TxA2 induces differentiation of hADSCs to smooth muscle-like cells.

Figure 2.

Effects of U46619 on the formation of actin stress fibers and localization of α-SMA in human adipose tissue-derived mesenchymal stem cells (hADSCs). Serum-starved hADSCs were treated with vehicle or 1 μM U46619 for 4 days, and immunofluorescence staining was performed. α-SMA and F-actin were double-stained with anti-α-SMA and Alexa Fluor 568 phalloidin, and analyzed laser scanning confocal microscopy. Overlaid images of α-SMA (green) and F-actin (red) are shown. Representatives of three independent experiments are shown. Abbreviations: α-SMA, α-smooth muscle actin; w/o, without.

SRF and Myocardin Are Involved in U46619-Induced Differentiation of hADSCs to Smooth Muscle-Like Cells

Accumulating evidence suggests that myocardin plays a key role in the smooth muscle differentiation via the activation of SRF-dependent transcription of smooth muscle-specific genes [16]. Therefore, we determined the effects of U46619 on the mRNA levels of SRF and myocardin by RT-PCR. As shown in Figure 3A, U46619 treatment time dependently increased the mRNA levels of not only α-SMA but also of myocardin and SRF, indicative of an increased expression of myocardin and SRF during U46619-induced smooth muscle differentiation.

Figure 3.

Role of SRF and myocardin in U46619-induced α-SMA expression. (A): Serum-starved human adipose tissue-derived mesenchymal stem cells (hADSCs) were treated with vehicle or 1 μM U46619 for the indicated time periods, and the mRNA levels of SRF, myocardin, and GAPDH were determined by reverse transcription-polymerase chain reaction. (B): hADSCs were transfected with either control small interference RNA (siRNA) or si-SRF and then were treated with vehicle or 1 μM U46619 for 4 days. (C): hADSCs were transfected with either control siRNA or si-myocardin and then treated with vehicle or 1 μM U46619 for 4 days. The expression levels of α-SMA and GAPDH were determined by Western blotting. Representatives of three independent experiments are shown. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; si-myocardin, myocardin small interference RNA; si-SRF, serum response factor small interference RNA; α-SMA, α-smooth muscle actin; SRF, serum response factor.

We reported previously that siRNA-mediated depletion of endogenous SRF and myocardin abrogates the SPC-induced expression of α-SMA in hADSCs [12]. To assess the involvement of SRF and myocardin in the U46619-induced differentiation of hADSCs to smooth muscle-like cells, we examined the effects of siRNA-mediated knockdown of SRF and myocardin on the U46619-stimulated expression of α-SMA. As shown in Figure 3B and 3C, the U46619-stimulated expression of α-SMA was largely attenuated by the siRNA-based knockdown of either SRF or myocardin, which supports a crucial role of SRF and myocardin in U46619-induced SMC differentiation.

U46619 Induces Differentiation of hADSCs to Smooth Muscle-Like Cells Through TP Receptor-Mediated Elevation of [Ca2+]i

TxA2 induces a variety of cellular responses, including elevation of [Ca2+]i through activation of TP receptor [20]. Appropriately, we next explored whether U46619 increased [Ca2+]i through TP receptor-dependent mechanism in hADSCs. U46619 treatment transiently increased [Ca2+]i in hADSCs and preincubation of the cells with the TP receptor antagonist SQ29548 completely abrogated the U46619-induced elevation of [Ca2+]i (Fig. 4A and 4B). Furthermore, U46619-induced α-SMA expression was completely inhibited by pretreatment of the cells with SQ29548 (Fig. 4C). These results suggest that the stimulatory effects of U46619 on [Ca2+]i and α-SMA expression can be attributed exclusively to activation of TP receptor.

Figure 4.

Role of Ca2+/calmodulin in the U46619-induced α-SMA expression. Serum-starved human adipose tissue-derived mesenchymal stem cells (hADSCs) were loaded with 5 μM fluo-4-AM for 40 minutes at 37°C in the absence (A) or presence (B) of 10 μM SQ29548 and then exposed to 1 μM U46619. Ca2+-dependent fluorescence was measured every 1 second for indicated time periods, and fluorescence intensities of more than 10 different cells from time-lapse images were quantified over time. Results are expressed as a percentage of the control (0 seconds). Serum-starved hADSCs were treated with 1 μM U46619 in the absence or presence of 10 μM SQ29548 (C) or 7.5 μg/ml W13 (D) for 4 days. The expression levels of α-SMA and GAPDH were determined by Western blotting. Representatives of three independent experiments are shown. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; sec, second; α-SMA, α-smooth muscle actin; w/o, without.

TxA2-induced elevation of [Ca2+]i has been known to activate CaM in SMCs [20]. To assess whether Ca2+/CaM-dependent pathway is involved in the U46619-induced differentiation of hADSCs to smooth muscle-like cells, we examined the effect of the CaM-specific inhibitor W13 on the U46619-induced expression of α-SMA. As shown in Figure 4D, preincubation of hADSCs with W13 completely inhibited U46619-induced α-SMA expression, suggesting that TxA2-induced elevation of [Ca2+]i results in differentiation of hADSCs to smooth muscle-like cells through a CaM-dependent mechanism.

U46619 Induces Differentiation of hADSCs to Smooth Muscle-Like Cells Through a RhoA/Rho Kinase-Dependent Mechanism

RhoA plays a pivotal role in the expression of SMC-specific genes [26–28]. To explore the role of RhoA in the U46619-stimulated differentiation of hADSCs to smooth muscle-like cells, we determined the effect of U46619 on RhoA activity in hADSCs. As shown in Figure 5A, the amount of GTP-bound RhoA increased within 10 s in response to U46619 treatment. U46619-stimulated RhoA activation was abrogated by preincubation of the cells with SQ29548 (Fig. 5B), suggesting the involvement of TP receptor in the U46619-induced RhoA activation.

Figure 5.

Role of RhoA/Rho kinase-dependent pathway in the U46619-stimulated α-SMA expression. (A): Serum-starved human adipose tissue-derived mesenchymal stem cells (hADSCs) were treated with 1 μM U46619 for the indicated time periods. (B): Serum-starved hADSCs treated with 1 μM U46619 in the absence or presence of 10 μM SQ29548 for 5 minutes. GTP-bound RhoA was selectively precipitated by using glutathione S-transferase (GST)-Rho binding domain of Rhotekin (RBD) conjugated with glutathione-Sepharose 4B beads. The amounts of RhoA in the whole cell lysates or GST-RBD-bound RhoA (GTP-RhoA) were revealed after Western blotting using anti-RhoA antibody. (C): hADSCs were transfected with control DNA or dominant-negative mutant RhoAN19 (DN-RhoA), and then treated with 1 μM U46619 for 4 days. (D): Serum-starved hADSCs were treated with 1 μM U46619 in the absence or presence of 10 μM Y27632 for 4 days. (E): Serum-starved hADSCs were transfected with control DNA or DN-ROCK and then were treated with 1 μM U46619 for 4 days. The expression levels of α-SMA and GAPDH were determined by Western blotting with anti-α-SMA and anti-GAPDH antibodies, respectively. Representative data from three independent experiments are shown. Abbreviations: DN, dominant negative; DN-ROCK, dominant negative mutant of Rho kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; min, minutes; sec, second; α-SMA, α-smooth muscle actin.

To assess the involvement of RhoA in the U46619-induced smooth muscle differentiation of hADSCs, we determined the effect of a dominant-negative mutant of RhoA (RhoAN19) on U46619-stimulated α-SMA expression. As shown in Figure 5C, overexpression of RhoAN19 abrogated U46619-stimulated expression of α-SMA, indicating that TxA2 stimulates differentiation of hADSCs to smooth muscle-like cells through a RhoA-dependent mechanism.

Rho kinase has been implicated in the RhoA-induced cellular responses including cytoskeletal rearrangement and SMC differentiation [14, 25]. To clarify whether Rho kinase is involved in the U46619-induced smooth muscle differentiation of hADSCs, we examined the effect of the Rho kinase-specific inhibitor Y27632 on U46619-stimulated α-SMA expression. Pretreatment of hADSCs with Y27632 completely abrogated U46619-stimulated expression of α-SMA (Fig. 5D). Furthermore, transfection of hADSCs with the dominant-negative mutant of Rho kinase p160ROCK-RB/PH markedly attenuated U46619-stimulated expression of α-SMA (Fig. 5E). These results suggest that RhoA/Rho kinase-dependent pathway plays a key role in the U46619-induced differentiation of hADSCs to smooth muscle-like cells.

U46619 Stimulates Differentiation of hADSCs to Smooth Muscle-Like Cells Through a MLC-Dependent Mechanism

Regulatory light chains of myosin II (MLC20) plays a pivotal role in actin stress fiber formation and smooth muscle contraction [24]. To assess whether MLC20 could be activated in response to U46619 in hADSCs, we examined the effect of U46619 on the phosphorylation of MLC20 in hADSCs. Compared with the rapid activation of RhoA within 10 s after U46619 treatment (Fig. 5A), the phosphorylation levels of MLC20 were time dependently increased until 5 minutes (Fig. 6A), suggesting that phosphorylation of MLC20 occurred after RhoA activation. Both Ca2+/CaM/MLCK- and RhoA/Rho kinase-dependent pathways are involved in the phosphorylation of MLC20 and contraction of SMCs [24, 29–31]. Therefore, we next examined the involvement of the CaM/MLCK and the RhoA/Rho kinase pathways in U46619-stimulated phosphorylation of MLC20. As shown in Figure 6B, U46619-stimulated phosphorylation of MLC20 was markedly attenuated by pretreatment with the CaM inhibitor W13, the MLCK inhibitor ML-7, or Y27632, suggesting that both the CaM/MLCK and RhoA/Rho kinase pathways play a key role in U46619-stimulated MLC phosphorylation. In addition, we found that U46619-induced RhoA activation was not affected by W13 or ML-7 (data not shown), suggesting that the RhoA/Rho kinase pathway is independent of the CaM/MLCK pathway.

Figure 6.

Role of MLC-dependent pathway in the U46619-induced α-SMA expression. (A): Serum-starved human adipose tissue-derived mesenchymal stem cells (hADSCs) were treated with 1 μM U46619 for the indicated time periods. (B): Serum-starved hADSCs were pretreated with 10 μM Y27632, 10 μM ML-7, or 7.5 μg/ml W13 for 15 minutes and treated with 1 μM U46619 for 10 minutes. The phosphorylation levels of MLC20 were determined by Western blotting. To ensure equal loading in each lane, the levels of GAPDH were determined by Western blotting with anti-GAPDH antibody. (C): Serum-starved hADSCs were treated with 1 μM U46619 in the presence of indicated concentrations of ML-7 for 4 days. The expression levels of α-SMA and GAPDH were probed by Western blotting. Representative data from three independent experiments are shown. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; min, minutes; MLC, myosin light chain; p, phosphorylated; sec, seconds; α-SMA, α-smooth muscle actin; w/o, without.

To explore the involvement of MLCK in U46619-induced α-SMA expression, we examined the effect of ML-7 on the U46619-induced expression of α-SMA. As shown in Figure 6C, ML-7 dose dependently decreased U46619-stimulated expression of α-SMA, suggesting that MLCK plays a pivotal role in TxA2-stimulated α-SMA expression through phosphorylation of MLC20. Taken together, these results suggest that U46619 induces activation of MLC20 through CaM/MLCK- and RhoA/Rho kinase-dependent pathways and that activation of MLC20 is required for the U46619-induced differentiation of hADSCs to smooth muscle-like cells.

Contractility of the Smooth Muscle-Like Cells Differentiated from hADSCs

To assess whether the smooth muscle-like cells differentiated from hADSCs by U46619 treatment exhibit SMC-like contractile properties, we determined the contractility of the differentiated smooth muscle-like cells using a collagen gel lattice contraction assay. Differentiated smooth muscle-like cells and undifferentiated hADSCs were embedded in collagen lattices, and the dimension of the floating collagen lattices was determined at the indicated time periods. As shown in Figure 7A and 7B, the area of the collagen lattices embedded with the differentiated smooth muscle-like cells was time dependently decreased, whereas that of collagen lattices containing undifferentiated hADSCs was only slightly diminished. U46619 has been reported to elicit contraction of SMCs [21, 22]. To explore whether U46619 treatment could stimulate contraction of the differentiated smooth muscle-like cells, we examined the effect of U46619 on the contractility of collagen gel lattices embedded with the differentiated smooth muscle-like cells. Shrinkage of collagen gel lattices embedded with the differentiated smooth muscle-like cells was further stimulated by treatment of the collagen lattices with U46619 (supporting information Fig. 1). These results suggest that the smooth muscle-like cells differentiated from hADSCs by U46619 treatment exhibit smooth muscle-like contractile characteristics.

Figure 7.

Contractile activity of the smooth muscle-like cells differentiated from human adipose tissue-derived mesenchymal stem cells (hADSCs). (A): hADSCs were treated with vehicle or 1 μM U46619 for 4 days and then embedded in collagen gel lattices. The collagen gel lattices were incubated in serum-free medium, and the floating lattices were photographed at the indicated time by using a digital camera. (B): The area of gel lattices was determined by using Scion Image software and the relative lattice area was obtained by dividing the area at each time point by the initial area of the lattice. Data indicate mean ± SD (n = 4). *, p < .05; **, p < .01 by Student's t test. (C): hADSCs were treated with vehicle or 1 μM U46619 in the absence or presence of 10 μM ML-7 or 7.5 μg/ml W13 for 4 days and embedded in collagen gel lattices. The floating gel lattices were incubated in serum-free medium and photographed at the indicated time by using a digital camera. (D): The area of gel lattices was determined from (C) by using Scion Image software, and relative lattice area was obtained by dividing the area at each time point by the initial area of the lattice. Data indicate mean ± SD (n = 4). *, p < .05 by Student's t test. (E): hADSCs were treated with vehicle or 1 μM U46619 in the absence or presence of 10 μM Y27632 for 4 days and embedded in collagen gel lattices. The floating gel lattices were treated and analyzed as described above. (F): The area of gel lattices was determined by using Scion Image software and relative lattice area was obtained by dividing the area at each time point by the initial area of the lattice. Data indicate mean ± SD (n = 4). *, p < .05 by Student's t test. Abbreviations: h, hour; w/o, without.

To clarify whether the CaM/MLCK- and RhoA/Rho kinase-dependent mechanisms are involved in the increased contractility of the differentiated smooth muscle-like cells, hADSCs were exposed to U46619 in the absence or presence of W13, ML-7, or Y27632 for 4 days, and then the contractility of the cells was determined. As shown in Figure 7C and 7D, collagen gel lattices containing the cells, which were treated with U46619 in the presence of W13 or ML-7, were not shrunken. Furthermore, treatment of hADSCs with U46619 in the presence of Y27632 completely abrogated the increased contractility of the cells (Fig. 7E, 7F). Together with the results that the U46619-stimulated α-SMA expression was inhibited by preincubation with W13, ML-7, or Y27632, these results led us to conclude that CaM/MLCK- and RhoA/Rho kinase-dependent signaling pathways are responsible for the U46619-induced smooth muscle differentiation of hADSCs and increased contractility.

DISCUSSION

In the present study, we demonstrate for the first time that the stable TxA2 analog U46619 increases expression of several smooth muscle-specific contractile proteins, including α-SMA, calponin, SM-MHC, and smoothelin, which have been known as SMC markers [10]. Furthermore, the differentiated smooth muscle-like cells exhibited increased contractility, suggesting that U46619 induces differentiation of hADSCs to contractile smooth muscle-like cells. Accumulating evidence demonstrates that functional SMCs can be produced from various types of stem cells [32–34]. For example, multipotent adult progenitor cells isolated from bone marrow differentiate into functional SMCs in response to TGF-β1 [33]. In addition, contractile SMCs can be isolated from transgenic embryonic stem cell lines stably expressing a puromycin-resistance gene under the control of α-SMA or SM-MHC promoter [34]. Similar to the present study, we have reported that SPC also induces differentiation of hADSCs to smooth muscle-like cells [12]. Furthermore, MSCs isolated from lipoaspirate differentiate into contractile SMCs when cultured in a smooth muscle inductive medium containing fetal bovine serum and heparin [32]. hADSCs are highly useful for tissue engineering and regeneration strategies because they can be easily isolated from the adipose tissue of patients. Therefore, U46619-induced differentiation of hADSCs to SMCs provides a rapid and clinically relevant method for the engineering and regeneration of cardiovascular tissues.

In the present study, we demonstrate that U46619 treatment increases the expression of SRF and myocardin in hADSCs and that siRNA-mediated knockdown of SRF and myocardin attenuates the U46619-induced expression of α-SMA in hADSCs. It is well established that most SMC-specific differentiation marker genes are dependent on myocardin [35, 36] and that the onset of SRF expression correlates closely with SMC marker expression during development [13, 37]. TGF-β induces the expression of SRF, which in turn activates CArG-dependent smooth muscle differentiation [38, 39]. Furthermore, mouse embryos lacking myocardin die during the early stage of smooth muscle development and fail to express multiple smooth muscle marker genes in embryonic dorsal aorta and other vascular structures [40]. Therefore, these earlier results support the contention from the present study that both SRF and myocardin are required for U46619-induced differentiation of hADSCs to smooth muscle-like cells.

In the present study, we demonstrate that CaM/MLCK- and RhoA/Rho kinase-dependent pathways play a pivotal role in not only smooth muscle differentiation of hADSCs but also contractility, suggesting that both smooth muscle differentiation and contractility are regulated by the common signaling pathways. It has been documented that RhoA/Rho kinase-dependent pathway regulates both transcription of SMC-specific genes [26, 41] and SMC contraction [21, 22, 42, 43]. In addition, the Ca2+/CaM pathway plays a key role in SMC contraction through the stimulation of MLCK-mediated phosphorylation of MLC20 [31, 43]. The importance of Ca2+ in smooth muscle differentiation is supported by the recent observation that depolarization-induced Ca2+ influx through L-type voltage-gated Ca2+ channels in cultured aortic SMCs increases expression of SMC-specific genes [41]. The depolarization-induced SMC differentiation was abrogated by inhibition of Rho kinase by treatment with Y27632, suggesting that Rho kinase plays a role in the Ca2+-induced smooth muscle differentiation. These results support the idea that excitation signals, which induce contraction of SMCs, are also capable of regulating transcription of SMC-specific genes through excitation-transcription coupling [44]. In the present study, we demonstrated that the CaM inhibitor W13, MLCK inhibitor ML-7, and Rho kinase inhibitor Y27632 blocked U46619-induced MLC phosphorylation. These results suggest that TxA2 stimulates transcription of smooth muscle-specific genes and contractility by eliciting MLC-dependent actin polymerization, which may serve as a convergence point for the multiple signaling pathways that regulate differentiation of hADSCs to smooth muscle-like cells.

Vascular SMCs undergo phenotypic change during development, in vitro culture, and vascular diseases [45]. Unlike other muscle cells that are terminally differentiated, vascular SMCs spontaneously dedifferentiate from a “contractile” phenotype to a “synthetic” phenotype when isolated from the vessel and cultured in vitro [46]. Moreover, it has been documented that vascular SMCs are able to differentiate into a diverse range of mesenchymal lineages, including adipocytic and osteoblastic cell types [47, 48]. We found that the smooth muscle-like cells that differentiated from hADSCs by pretreatment with U46619 or TGF-β1 exhibited phenotypic dedifferentiation upon cultured in growth medium, as demonstrated by time-dependent decrease of α-SMA expression levels (supporting information Fig. 2). Furthermore, the differentiated smooth muscle-like cells were transdifferentiated into adipocytes or osteoblasts when they were cultured in adipogenic or osteogenic differentiation medium (supporting information Fig. 3). Taken together, these results suggest that the differentiated smooth muscle-like cells are not terminally differentiated cells but rather exhibit phenotypic plasticity that allows dedifferentiation and transdifferentiation depending on culture conditions.

MSCs distribute to all postnatal organ and perivascular niche compartments [49]. Several studies suggest that vascular pericytes or solitary SMCs, which are progenitors of SMCs involved in blood vessel formation, have phenotypic and physiological characteristics similar to MSCs [11, 50, 51]. Similar to MSCs, pericytes possess adipogenic, osteogenic, and chondrogenic differentiation potential [52, 53]. Furthermore, MSCs express pericyte markers and distribute in the perivascular location within adipose tissue [54]. Taken together, these results suggest that MSCs may play a pivotal role in the maintenance of vascular structure and generation of SMCs in adipose tissues and blood vessels, although physiological or pathological functions of TxA2-induced differentiation of hADSCs to smooth muscle-like cells have not been clarified. TxA2 is produced from macrophages and inflammatory cells and plays a pivotal role in vasoconstriction and atherosclerosis [17, 19], suggesting that TxA2 may be involved in the differentiation of perivascular MSCs to SMCs in vascular lesions. The functional implications of TxA2-induced differentiation of MSCs to smooth muscle-like cells within adipose and vascular tissues should be determined further.

ACKNOWLEDGMENTS

This work was supported by the Medical Research Center program of the Ministry of Science and Technology/Korea Science and Engineering Foundation (R13-2005-009) and the Korea Research Foundation grants (KRF-2007-521-C00225).

DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

The authors indicate no potential conflicts of interest.

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