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(Circulation Research. 2008;103:635.)
© 2008 American Heart Association, Inc.

Cellular Biology

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

Eun Su Jeon^*, Won Sun Park^*, Mi Jeong Lee, Young Mi Kim, Jin Han^#, Jae Ho Kim^#

From the Medical Research Center for Ischemic Tissue Regeneration (E.S.J., M.J.L., Y.M.K., J.H.K.), the Medical Research Institute, Department of Physiology, College of Medicine, Pusan National University; and National Research Laboratory for Mitochondrial Signaling (W.S.P., J.H.), FIRST Mitochondria Research Group, Department of Physiology and Biophysics, College of Medicine, Cardiovascular and Metabolic Disease Center, Inje University, Busan, Republic of Korea.

Correspondence to Jae Ho Kim, PhD, Pusan National University College of Medicine, Department of Physiology, 1-Ga, Ami-Dong, Suh-Gu, Busan 602-739, Republic of Korea. E-mail jhkimst{at}pusan.ac.kr

	Abstract

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

Key Words: sphingosylphosphorylcholine • mesenchymal stem cells • smooth muscle • differentiation • Rho kinase

	Introduction

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Smooth muscle cells (SMCs) play important roles in angiogenesis, blood vessel maintenance, and blood pressure regulation. SMCs exhibit a contractile phenotype characterized by high expression of specific contractile proteins including -smooth muscle actin (-SMA), calponin, SM22, smoothelin, h-caldesmon, and smooth muscle-myosin heavy chain (SM-MHC).^1,2 The phenotypic SMC expression pattern has been implicated not only in vascular development but also in cardiovascular diseases like hypertension and atherosclerosis.^2,3 A variety of ion channels regulate SMC contractility; among them are the voltage-dependent L-type Ca²⁺ channels and the big conductance Ca²⁺-activated K⁺ (BK_Ca) channels.^4,5 Although SMC differentiation is an essential component of vascular development, little is known about the specific factors and molecular mechanisms that designate stem cells to differentiate into functional SMCs that express a SMC-specific set of contractile proteins and ion channels.

An increasing body of evidence demonstrates that transcription of smooth muscle-specific genes is mediated by serum response factor (SRF) and myocardin family of SRF cofactors, ie, myocardin, myocardin-related transcription factor (MRTF)-A (or MKL1), and MRTF-B (or MKL2).^2,6,7 SRF regulates SMC-specific transcription by binding to the CArG boxes found in the promoters of nearly all of SMC marker genes. Myocardin, in turn, constitutively activates SRF-dependent transcription.^8–11 In unstimulated cells, MRTF-A/B are sequestered in the cytoplasm through direct interaction with G-actin, but RhoA/Rho kinase–mediated actin polymerization depletes the G-actin pool, which frees MRTF from G-actin to enter the nucleus where it can stimulate SRF-dependent transcription of SMC-specific genes.¹² Therefore, these results suggest that the RhoA/Rho kinase pathway plays a key role in SMC differentiation by regulating the integrity of the cytoskeleton and the cellular locale of MRTF.^13,14

Mesenchymal stem cells (MSCs) can be isolated from a variety of tissues, such as bone marrow, periosteum, trabecular bone, synovium, skeletal muscle, deciduous teeth, and adipose tissues, and they exhibit self-renewal and long-term viability, as well as the potential to differentiate along adipogenic, osteogenic, chondrogenic, or myogenic lineages.^15–18 In vitro, MSCs differentiate into SMCs in response to transforming growth factor (TGF)-β,^19,20 mechanical stress,²¹ and direct contact with vascular endothelial cells.²² Moreover, adoptively transferred MSCs differentiate into SMCs and contribute to the remodeling of vasculature in vivo.^23–25 The ability of MSCs to generate SMCs makes them an ideal model to study de novo generation of SMCs, and it renders them useful in other applications as well, including tissue engineering. It has been reported that human adipose tissue–derived MSCs (hADSCs) have similar expression profiles and differentiation potential to human bone marrow–derived MSCs.²⁶ Therefore, hADSCs are highly useful for the study of de novo generation of SMCs and vascular tissue engineering for clinical application.

Sphingosylphosphorylcholine (SPC) is a sphingolipid generated by N-deacylation of sphingomyelin, among the most abundant lipids in the cell membrane. SPC enhances Ca²⁺ sensitization, and it thereby induces contraction of a variety of smooth muscle tissues, including gastrointestinal smooth muscle, mesenteric microvessels, and coronary and cerebral arteries.^27,28 In a previous study, we demonstrated that SPC induced differentiation of hADSCs into smooth muscle–like cells and that small interference siRNA-mediated depletion of endogenous SRF and myocardin attenuated the SPC-induced expression of SMC markers.²⁹

These observations suggest that SRF and myocardin are an essential components of SPC-induced differentiation of hADSCs into SMCs, but no known studies have addressed whether the differentiated SMCs exhibit smooth muscle–like contractile responses or whether the RhoA/Rho kinase/MRTF pathway is involved in this SPC-induced differentiation process. In the present study, we demonstrated for the first time that SPC induced differentiation of MSCs to contractile SMCs by activating the RhoA/Rho kinase/MRTF-dependent pathway.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
hADSCs were isolated from human subcutaneous adipose tissue and cultured in growth medium (-minimum essential medium supplemented with 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin). The collagen gel lattice contraction assay was performed as previously described³⁰ with a slight modification. The voltage-dependent Ca²⁺ and BK_Ca currents were measured with patch-clamp techniques. Spatially averaged photometric [Ca²⁺] measurements from single cells were determined by administering the fluorescent Ca²⁺ indicator fluo-4-acetoxymethyl ester, and measuring the resulting fluorescence with a laser scanning confocal microscope. A commercial RhoA activation assay kit was used to measure the RhoA activity. Protein expression and phosphorylation levels were determined by Western blotting. Immunostaining and confocal microscopy were used to determine the subcellular distribution and organization of proteins.

An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
SMCs Differentiated From hADSCs by SPC Treatment Exhibited Increased Contractility
Treating of hADSCs with 2 µmol/L SPC for 4 days elicited their differentiation into SMCs, as demonstrated by their increased expression of the SMC-specific transcription factor myocardin and the following SMC-specific contractile proteins: -SMA, calponin, h-caldesmon, smoothelin, and SM-MHC (Figure I in the online data supplement). In humans, 2 isoforms have been described: smoothelin-A (59 kDa), which is predominantly expressed in visceral SMC, and smoothelin-B (100 kDa), which is found in vascular SMC (van Eys et al, 1997). In the present study, an 100-kDa protein band was recognized by Western blotting with anti-smoothelin antibody, suggesting SPC-induced expression of smoothelin-B in hADSCs. To explore whether SMCs differentiated from hADSCs (hADSC-SMCs) as a result of SPC treatment were capable of contracting and generating force, we measured the contractile ability of the cells by using collagen lattice contraction assay. In this assay, SMCs that are embedded in attached collagen matrices generate isometric tension, and releasing the collagen lattice from the culture dish causes mechanical unloading and contraction as the actin cytoskeleton of the embedded cells becomes free to contract the deformable collagen lattice.³¹ We reconstituted either hADSC-SMCs or undifferentiated hADSCs (where indicated) into these collagen lattices, and we allowed the embedded cells to shrink the floating lattices by incubating them with serum-free medium at 37°C for the indicated time periods. As shown in Figure 1A and 1B, incorporation of hADSC-SMCs time-dependently reduced the size of the collagen lattices, indicating basal contractile tone. However, the collagen lattices containing undifferentiated hADSCs contracted to a lesser extent than those containing hADSC-SMCs.

View larger version (19K): [in this window] [in a new window]   Figure 1. Smooth muscle–like contractile responses of hADSC-SMCs. A, hADSCs and hADSC-SMCs were embedded in collagen gel lattices, and gel contraction was photographed at the indicated time by using a digital camera. B, The area of the gel lattices was determined with 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 represent means±SD (n=4). *P<0.05 vs control (0 hour) by 2-way ANOVA and Scheffe’s post hoc test. C, The collagen lattices were exposed to 60 mmol/L KCl or 1 µmol/L carbachol for 24 hours, and the area of gel lattices was determined. Data represent means±SD (n=5). *P<0.05 by 2-way ANOVA and Scheffe’s post hoc test.

Because membrane depolarization and contractile agonists regulate SMC contractility,³² we evaluated whether these factors affects the contractility of hADSC-SMCs by measuring the effects of KCl and carbachol on the contraction of collagen lattices containing hADSC-SMCs. As shown in Figure 1C, exposure of the collagen lattices containing hADSC-SMCs to either 60 mmol/L KCl or 1 µmol/L carbachol enhanced their shrinkage. However, collagen gel lattices harboring undifferentiated hADSCs did not contract in response to KCl or carbachol. These results suggest that hADSC-SMCs exhibited contractility in response to membrane depolarization or contractile stimuli.

L-Type Ca²⁺ Channels Play a Role in hADSC-SMCs Contraction
Contraction of SMCs stems from membrane depolarization and an increase in Ca²⁺ influx mediated by voltage-gated L-type Ca²⁺ channels.³³ To explore whether L-type Ca²⁺ channels were involved in the contractility of hADSC-SMCs, we examined their expression level of Cav_1.2, a smooth muscle–specific L-type Ca²⁺ channel isoform.³⁴ As shown in Figure 2A, treating hADSCs with 2 µmol/L SPC for 4 days markedly increased their Cav_1.2 expression levels. To functionally correlate this expression of L-type Ca²⁺ channel to a Ca²⁺ current, we recorded the Ba²⁺-sensitive and voltage-dependent Ca²⁺ currents in the hADSC-SMCs using Bay K8644, an L-type Ca²⁺ channel opener, in perforated patch-clamp assays. As shown in Figure 2B and 2C, the hADSC-SMCs exhibited Ba²⁺-sensitive inward current in the presence of Bay K8644, in contrast to the lack of a significant current in undifferentiated hADSCs, suggesting that L-type Ca²⁺ channels are highly expressed in hADSC-SMCs.

View larger version (35K): [in this window] [in a new window]   Figure 2. Role of voltage-dependent L-type Ca2+ channels in the contractility of hADSC-SMCs. A, The expression levels of Cav1.2 and tubulin in hADSC-SMCs or undifferentiated hADSCs were determined by Western blotting. B, The Ba2+-sensitive and voltage-dependent L-type Ca2+ current traces were determined in undifferentiated hADSCs (left) or hADSC-SMCs (right) in the presence of 10 µmol/L Bay K8644. Representative data from 6 different experiments are shown. C, Current density of Ca2+ current in hADSC-SMCs and undifferentiated hADSCs. Data represent means±SD (n=6). *P<0.01 by Student’s t test. D, Collagen gel lattices embedded with hADSC-SMCs or hADSCs were exposed to serum-free medium containing 10 µmol/L Bay K8644 or a vehicle (w/o), and gel contraction was photographed at the indicated time using a digital camera. The relative lattice area was obtained by dividing the area at each time point by the initial area of the lattice. Data represent means±SD (n=4). *P<0.05 vs respective controls (0 hour), #P<0.05; 2-way ANOVA and Scheffe’s post hoc test. E, Collagen gel lattices embedded with hADSC-SMCs or hADSCs were exposed to serum-free medium containing 10 µmol/L Bay K8644 or a vehicle in the absence or presence of 10 µmol/L nifedipine for 24 hours, and gel contraction was photographed using a digital camera. The relative lattice area was obtained by dividing the area at 24 hours by the initial area of the lattice. Data represent means±SD (n=5). *P<0.05 vs respective controls, #P<0.05; 2-way ANOVA and Scheffe’s post hoc test.

To explore whether L-type Ca²⁺ channel–mediated Ca²⁺ influx is involved in the contractile response of the hADSC-SMCs, we determined the effect of Bay K8644 on the contraction of collagen lattices embedded with hADSC-SMCs or undifferentiated hADSCs. As shown in Figure 2D, Bay K8644 stimulated contraction of the collagen lattices containing hADSC-SMCs, whereas it had no significant impact on the contraction of the collagen lattices containing undifferentiated hADSCs. Furthermore, treating the collagen lattices with nifedipine (an L-type Ca²⁺ channel blocker) during the contraction assay completely abrogated the basal and Bay K8644–stimulated contractile responses (Figure 2E). Taken together, these results suggest that SPC increased the expression of L-type Ca²⁺ channels and that these channels are responsible for the contractile response induced by membrane depolarization.

L-Type Ca²⁺ Channels Were Involved in the Membrane Depolarization–Induced Contractility of hADSCs
We next explored whether the L-type Ca²⁺ channel–mediated Ca²⁺ influx was involved in the membrane depolarization-induced contractility of hADSC-SMCs. As shown in supplemental Figure IIA, treating the cells with 60 mmol/L KCl increased their intracellular Ca²⁺ concentration, but it had no effect on undifferentiated hADSCs. Moreover, pretreating the hADSC-SMCs with 10 µmol/L nifedipine completely abrogated not only the KCl-induced elevation of intracellular Ca²⁺ concentration but also hADSC-SMC contractility (supplemental Figure IIB). These results suggest that membrane depolarization stimulated hADSC-SMC contractility by enhancing Ca²⁺ influx through their L-type Ca²⁺ channels.

Ca²⁺-Activated K⁺ Channel Were Involved in the hADSC-SMC Contractile Response
BK_Ca channels reportedly play a pivotal role in vessel relaxation by inducing membrane hyperpolarization, which leads to closure of the L-type Ca²⁺ channels and removal of Ca²⁺ from the cytoplasm.³⁵ To explore the involvement of BK_Ca channels in the contractile response of hADSC-SMCs, we examined their BK_Ca channel expression level by Western blotting. As shown in Figure 3A, BK_Ca channel expression was higher in hADSC-SMCs than in undifferentiated hADSCs. To further confirm that these BK_Ca channels were functional in hADSC-SMCs, we measured BK_Ca channel activity in a whole cell configuration. Figure 3B shows that depolarizing voltage steps from -80 to 60 mV generated BK_Ca currents, identified as such because they were sensitive to iberiotoxin, a specific BK_Ca inhibitor. The amplitude of the outward currents in the hADSC-SMCs was substantial throughout the entire voltage range, in contrast to the constitutively low BK_Ca current in undifferentiated hADSCs (Figure 3B and 3C). Together with the increased expression of BK_Ca in the hADSC-SMCs, these results suggest that functional BK_Ca channels are upregulated in hADSC-SMCs.

View larger version (14K): [in this window] [in a new window]   Figure 3. Role of BKCa channels in the contractility of hADSC-SMCs. A, The expression levels of BKCa1.1 and tubulin in hADSC-SMCs or undifferentiated hADSCs were determined by Western blotting. B, BKCa currents were recorded in a whole cell configuration in response to voltage steps (from –80 to +60 mV for 600 ms) for hADSCs and hADSC-SMCs. C, Current–voltage (I–V) relationship of the BKCa currents in hADSCs () and hADSC-SMCs () (n=6). D, Collagen gel lattices embedded with undifferentiated hADSCs (hADSC) or hADSC-SMCs (SMC) were exposed to serum-free medium containing 100 nmol/L iberiotoxin and/or 10 µmol/L Bay K8644 for 12 hours, and the relative lattice area was obtained by dividing the area at 12 hours by the initial area of the lattice. Data represent means±SD (n=4). *P<0.05 by 2-way ANOVA and Scheffe’s post hoc test.

To delineate the involvement of BK_Ca in SMC contraction, we determined the effect of iberiotoxin on the contractile ability of the hADSC-SMCs. As shown in Figure 3D, iberiotoxin potentiated the Bay K8644-induced contraction of collagen lattices containing hADSC-SMCs. Therefore, it is likely that pharmacological inhibition of BK_Ca amplified the contraction induced by L-type Ca²⁺ channel opening, because it prevented membrane potential repolarization.

SPC Induced SMC Differentiation of hADSCs Through a RhoA/Rho Kinase–Dependent Mechanism
Accumulating evidence suggests that RhoA-mediated rearrangement of the actin cytoskeleton plays a pivotal role in SMC differentiation.¹⁴ To explore whether SPC activated RhoA in hADSCs, we treated the cells with SPC and measured the resulting GTP-loaded RhoA levels with a RhoA activation assay. As shown in Figure 4A, we could detect GTP-RhoA as early as 2 minutes following SPC treatment, after which GTP-RhoA levels were maximally increased at 5 minutes. To elucidate whether RhoA activation participated in the SPC-induced differentiation of hADSCs into SMCs, we examined the effect of a dominant-negative RhoA mutant (RhoA^N19) on expression of SMC-specific markers in response to SPC. As shown in Figure 4B, ectopic overexpression of RhoA^N19 inhibited the SPC-induced expression of -SMA, suggesting that RhoA plays a key role in the SPC-induced differentiation of hADSCs into SMCs.

View larger version (21K): [in this window] [in a new window]   Figure 4. Role of RhoA/Rho kinase–dependent pathway in the SPC-induced differentiation of hADSCs to SMCs. A, Serum-starved hADSCs were treated with 2 µmol/L D-erythro-SPC for the indicated time periods. The amounts of RhoA in the whole cell lysates (Total RhoA) or the GTP-bound RhoA precipitated from the lysates were revealed by Western blotting using anti-RhoA antibody. B, hADSCs were transfected with control DNA or dominant-negative mutant RhoAN19 (DN-RhoA-HA) and treated with 2 µmol/L D-erythro-SPC for 4 days. C, Serum-starved hADSCs were pretreated with 10 µmol/L Y27632 or a vehicle and treated with 2 µmol/L D-erythro-SPC for 4 days. D, Serum-starved hADSCs were transfected with an adenovirus bearing RB/PH (TT) and treated with 2 µmol/L D-erythro-SPC for 4 days. The expression levels of -SMA, calponin, SM-MHC, smoothelin, h-caldesmon, myocardin, HA-tagged DN-RhoA, and tubulin were determined by Western blot analyses. An anti-myocardin antibody (Abcam Inc) was used at a dilution of 1:1000 for Western blotting.

To explore whether RhoA operated through one of its major downstream targets, Rho kinase, to mediate the SPC-induced differentiation of hADSCs to SMCs, we examined whether we could inhibit this differentiation with Y27632, a Rho kinase–specific inhibitor. As shown in Figure 4C, pretreating cells with Y27632 abrogated their SPC-induced expression of the SMC-specific markers, including -SMA, calponin, SM-MHC, smoothelin, h-caldesmon, and myocardin. To confirm these results, we examined the effect of RB/PH (TT), a dominant-negative mutant of Rho kinase,³⁶ on the SPC-induced -SMA expression in hADSCs. As shown in Figure 4D, overexpression of the RB/PH (TT) abrogated the SPC-stimulated expression of -SMA, suggesting a key role of Rho kinase in the SPC-induced differentiation of hADSCs to SMCs.

To support the notion that Rho kinase is involved in SPC-induced differentiation of hADSCs to SMCs, we next examined whether the SPC-induced expression and intracellular localization of -SMA was influenced by Y27632. As shown in supplemental Figure III, treating hADSCs with 2 µmol/L SPC for 4 days increased their expression of -SMA, which localized to F-actin filaments. The increased assembly of actin filaments and thick fiber formation in response to SPC correlated strongly with the increased -SMA expression, whereas pretreating the cells with Y27632 completely blocked -SMA expression and actin polymerization induced by SPC. These results suggest that Rho kinase is crucial for SPC-induced expression of -SMA and actin polymerization in hADSCs.

Rho Kinase Was Essential for SPC-Induced Contractility
To explore whether Rho kinase–mediated expression of contractile proteins is responsible for the increased contractility, we treated hADSCs with SPC in the absence or presence of Y27632 for 4 days, after which we examined their contractility. Consistent with the result that Y27632 abrogated the expression of -SMA in hADSCs, pretreatment with Y27632 also completely abrogated SPC-induced contractility (supplemental Figure IV). Therefore, these results suggest that Rho kinase stimulates expression of contractile proteins in SPC-differentiated hADSC-SMCs to promote their increased contractility.

MRTF-A Is Critical for SPC-Induced Differentiation of hADSCs to SMCs
Recent reports suggest that the transcription factor SRF and its cofactors (myocardin, MRTF-A, and MRTF-B) regulate expression of smooth muscle-specific genes.^2,6,7 We previously demonstrated that SPC treatment increases expression levels of SRF and myocardin and that siRNA-mediated depletion of SRF or myocardin abrogated the SPC-induced expression of -SMA.²⁹ To explore the effects of SPC on the expression of MRTF isoforms, we measured the mRNA levels of myocardin and MRTF isoforms after SPC treatment. As shown in Figure 5A and 5B, SPC treatment significantly increased the expression levels of myocardin and MRTF-A, but not of MRTF-B, suggesting that myocardin and MRTF-A may be associated with the SPC-induced differentiation of hADSCs to SMCs.

View larger version (24K): [in this window] [in a new window]   Figure 5. Role of MRTF-A in the SPC-induced differentiation of hADSCs into SMCs. A, Serum-starved hADSCs were treated with vehicles or 2 µmol/L D-erythro-SPC for 12 hours, and the mRNA levels of myocardin, MRTF-A, MRTF-B, and GAPDH were determined by RT-PCR. B, The relative mRNA levels of myocardin, MRTF-A, and MRTF-B were determined by real-time RT-PCR and normalized to those of GAPDH. Data represent means±SD (n=4). *P<0.05 vs control (w/o); 2-way ANOVA and Scheffe’s post hoc test. C, hADSCs were transfected with control siRNA or si-MRTF-A. The mRNA levels of MRTF-A and GAPDH were determined by RT-PCR. D, The siRNA-transfected cells were treated with vehicles or 2 µmol/L SPC for 4 days. The expression levels of -SMA, calponin, SM-MHC, smoothelin, h-caldesmon, myocardin, and tubulin were determined by Western blotting.

To clarify whether MRTF-A is involved in this differentiation process, we examined the effects of siRNA-mediated MRTF-A depletion on SPC-induced expression of SMC-specific markers. Transfection of hADSCs with an MRTF-specific siRNA attenuated endogenous MRTF-A expression (Figure 5C), which significantly inhibited SPC-induced expression of -SMA, calponin, SM-MHC, h-caldesmon, smoothelin, and myocardin (Figure 5D). These results suggest that MRTF-A plays a key role in the SPC-induced differentiation of hADSCs to SMCs.

According to previous studies, MRTF localizes to the cytoplasm in unstimulated cells and RhoA/Rho kinase–mediated actin polymerization elicits translocation of MRTF to the nucleus, where it promotes transcription of SMC-specific genes.^12,37 To explore whether SPC induced translocation of MRTF-A through a Rho kinase-dependent mechanism, we determined the intracellular localization of FLAG-tagged MRTF-A in response to SPC treatment. As shown in Figure 6, MRTF-A was largely cytoplasmic in serum-starved hADSCs, but SPC treatment led to translocation of MRTF-A to the nucleus. This translocation was inhibitable by pretreating the cells with Y27632. These results suggest that SPC induced differentiation of hADSCs to SMCs through Rho kinase-dependent nuclear translocation of MRTF-A.

View larger version (11K): [in this window] [in a new window]   Figure 6. Role of Rho kinase in the SPC-induced nuclear translocation of MRTF-A. hADSCs were transfected with p3xFlag-CMV-7.1 plasmid bearing MRTF-A, serum-starved for 48 hours, and treated with 2 µmol/L D-erythro-SPC in the absence or presence of 10 µmol/L Y27632 for 24 hours. FLAG-tagged MRTF-A and the nuclei were visualized by immunocytochemistry with anti-FLAG antibody and staining with Hoechst 33258 dye, respectively, and analyzed with a Leica TCS-SP2 laser scanning confocal microscope. The bottom images show overlaid images corresponding to FLAG-MRTF-A (green) and nucleus (blue). Scale bar=50 µm.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
SMCs express ion channels that are responsible for their contractile activity. Specifically, the contractile process involves voltage-dependent Ca²⁺ channel–mediated Ca²⁺ influx, Ca²⁺/calmodulin-dependent contraction, and K⁺ channel–mediated repolarization of membrane potential.^4,5 In the present study, we demonstrated for the first time that SPC induced differentiation of hADSCs to functional SMCs. First, hADSC-SMCs expressed SMC-specific contractile proteins and ion channels, including L-type Ca²⁺ and BK_Ca channels. Second, hADSC-SMCs exhibited contractility in response to the L-type Ca²⁺ channel opener Bay K8644 and membrane depolarization induced by KCl, and the L-type Ca²⁺ channel blocker nifedipine blocked the contractile responses. Third, the BK_Ca channel blocker iberiotoxin potentiated the Bay K8644–induced contractile activity of hADSC-SMCs, presumably because it inhibited repolarization. In support of the present study, it was recently reported that TGF-β1 induced differentiation of bone marrow–derived multipotent adult progenitor cells into functional SMCs. These cells expressed functional L-type Ca²⁺ channels and exhibited a contractile reaction in response to KCl, the L-type Ca²⁺ channel opener FPL64176, serotonin, and endothelin-1.³⁸ In addition, functional SMCs could be isolated from transgenic embryonic stem cell lines that stably expressed a puromycin-resistance gene under the control of either -SMA or SM-MHC promoter.³⁹ These SMCs displayed agonist-induced Ca²⁺ transients and expressed functional Ca²⁺ channels; moreover, they generated a contractile force within collagen gels in response to endothelin-1 or membrane depolarization. hADSCs have a clinical advantage as progenitors of functional SMCs because large amounts of hADSCs can easily be isolated from patient adipose tissues. Therefore, these results led us to suggest that SPC-induced differentiation of hADSCs into functional SMCs would be highly useful for vascular tissue engineering.

In the present study, we demonstrated that blocking the RhoA/Rho kinase pathway, either by overexpressing dominant-negative RhoA (DN-RhoA) or Rho kinase (DN-Rho kinase) constructs or by pretreating cells with Y27632, completely abrogated the SPC-induced expression of SMC-specific markers. Consistent with these results, an increasing body of evidence demonstrates a key role for RhoA-dependent signaling in the expression of smooth muscle–specific genes in a variety of cell types: Inhibition of RhoA by C3 exoenzyme or DN-RhoA or pharmacological inhibition of Rho kinase with Y27632 abrogated TGF-β1–induced expression of -SMA in Monc-1 neural crest stem cells.⁴⁰ Sphingosine-1-phosphate increased RhoA activity and -SMA expression in SMCs, and inhibition of Rho kinase by Y27632 attenuated this sphingosine-1-phosphate–stimulated -SMA expression.⁴¹ Furthermore, inhibiting Rho kinase activity with Y27632 abrogated the renal fibrosis–associated increase in -SMA expression⁴² and TGF-β1–induced -SMA expression in gingival fibroblasts.⁴³ These results support the notion that the RhoA/Rho kinase signaling pathway plays a key role in the SPC-induced differentiation of hADSCs to SMCs.

In the present study, we demonstrated that SPC treatment promoted MRTF-A expression and that knockdown of endogenous MRTF-A expression abrogated the SPC-induced differentiation of hADSCs to SMCs. Furthermore, SPC induced translocation of MRTF-A from the cytosol to the nucleus through a Rho kinase–dependent mechanism. Activation of RhoA has been reported to induce actin polymerization through the ROCK/LIM kinase (LIMK)/cofilin pathway, which stabilizes F-actin and mDia1 and promotes the assembly of monomeric G-actin into F-actin filaments.^44,45 Actin polymerization resulted in the translocation of MRTF-A from the cytoplasm to the nucleus.^12,46,47 Pharmacological inhibition of Rho kinase with Y27632 abrogated nuclear translocation of MRTF-A induced by sphingosine-1-phosphate or mechanical force.^48,49 Therefore, it is likely that MRTF-A is a key factor in transducing Rho/actin signaling from the cytoplasm to the nucleus and in activating transcription of SMC-specific genes in response to SPC treatment. In addition, we demonstrated that either knockdown of endogenous MRTF-A or pharmacological inhibition of Rho kinase attenuated the SPC-induced expression of myocardin. Taken together with the previous report that myocardin plays a key role in the SPC-induced expression of SMC-specific markers in hADSCs,²⁹ these results suggest that Rho kinase-dependent activation of MRTF-A appears to act upstream of the myocardin pathway during SMC differentiation of hADSCs.

The physiological and pathophysiological significance of MSCs in cardiovascular development and diseases is still unclear, attributable to a lack of precise knowledge regarding their anatomic distribution within tissues. However, several lines of evidence 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.¹⁸ Similar to MSCs, pericytes possess adipogenic, osteogenic, and chondrogenic differentiation potential.^50,51 The present study, together with these earlier studies, suggest that MSCs may be SMC progenitors within local tissue and that SPC may promote angiogenesis and vasculogenesis by inducing differentiation of MSCs to SMCs.

	Acknowledgments

 
We thank Mi Ra Kim and Kyung Hye Kim for technical assistance and Chi Dae Kim for helpful comments and suggestions.

Sources of Funding

This work was supported by the MRC program of MOST/KOSEF (R13-2005-009) and Korea Science and Engineering Foundation grant R0A-2007-000-20085-0.

Disclosures

None.

	Footnotes

 
*Both authors contributed equally to this work.

^#Both authors contributed equally to this work.

Original received January 4, 2008; resubmission received June 6, 2008; revised resubmission received July 3, 2008; accepted July 24, 2008.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Shanahan CM, Weissberg PL, Metcalfe JC. Isolation of gene markers of differentiated and proliferating vascular smooth muscle cells. Circ Res. 1993; 73: 193–204.[Abstract]

2. 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]

3. Liu C, Nath KA, Katusic ZS, Caplice NM. Smooth muscle progenitor cells in vascular disease. Trends Cardiovasc Med. 2004; 14: 288–293.[CrossRef][Medline] [Order article via Infotrieve]

4. Ledoux J, Werner ME, Brayden JE, Nelson MT. Calcium-activated potassium channels and the regulation of vascular tone. Physiology (Bethesda). 2006; 21: 69–78.[CrossRef][Medline] [Order article via Infotrieve]

5. Marks AR. Calcium channels expressed in vascular smooth muscle. Circulation. 1992; 86 (suppl III): III-61–III-67.[Medline] [Order article via Infotrieve]

6. Parmacek MS. Myocardin-related transcription factors: critical coactivators regulating cardiovascular development and adaptation. Circ Res. 2007; 100: 633–644.[Abstract/Free Full Text]

7. Pipes GC, Creemers EE, Olson EN. The myocardin family of transcriptional coactivators: versatile regulators of cell growth, migration, and myogenesis. Genes Dev. 2006; 20: 1545–1556.[Abstract/Free Full Text]

8. Du KL, Ip HS, Li J, Chen M, Dandre F, Yu W, Lu MM, Owens GK, Parmacek MS. Myocardin is a critical serum response factor cofactor in the transcriptional program regulating smooth muscle cell differentiation. Mol Cell Biol. 2003; 23: 2425–2437.[Abstract/Free Full Text]

9. Yoshida T, Sinha S, Dandre F, Wamhoff BR, Hoofnagle MH, Kremer BE, Wang DZ, Olson EN, Owens GK. Myocardin is a key regulator of CArG-dependent transcription of multiple smooth muscle marker genes. Circ Res. 2003; 92: 856–864.[Abstract/Free Full Text]

10. Chen J, Kitchen CM, Streb JW, Miano JM. Myocardin: a component of a molecular switch for smooth muscle differentiation. J Mol Cell Cardiol. 2002; 34: 1345–1356.[CrossRef][Medline] [Order article via Infotrieve]

11. Wang DZ, Li S, Hockemeyer D, Sutherland L, Wang Z, Schratt G, Richardson JA, Nordheim A, Olson EN. Potentiation of serum response factor activity by a family of myocardin-related transcription factors. Proc Natl Acad Sci U S A. 2002; 99: 14855–14860.[Abstract/Free Full Text]

12. Miralles F, Posern G, Zaromytidou AI, Treisman R. Actin dynamics control SRF activity by regulation of its coactivator MAL. Cell. 2003; 113: 329–342.[CrossRef][Medline] [Order article via Infotrieve]

13. Miano JM. Serum response factor: toggling between disparate programs of gene expression. J Mol Cell Cardiol. 2003; 35: 577–593.[CrossRef][Medline] [Order article via Infotrieve]

14. Cen B, Selvaraj A, Prywes R. Myocardin/MKL family of SRF coactivators: key regulators of immediate early and muscle specific gene expression. J Cell Biochem. 2004; 93: 74–82.[CrossRef][Medline] [Order article via Infotrieve]

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

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

17. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR. Multilineage potential of adult human mesenchymal stem cells. Science. 1999; 284: 143–147.[Abstract/Free Full Text]

18. Short B, Brouard N, Occhiodoro-Scott T, Ramakrishnan A, Simmons PJ. Mesenchymal stem cells. Arch Med Res. 2003; 34: 565–571.[CrossRef][Medline] [Order article via Infotrieve]

19. 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] [Order article via Infotrieve]

20. Wang D, Park JS, Chu JS, Krakowski A, Luo K, Chen DJ, Li S. 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]

21. Kobayashi N, Yasu T, Ueba H, Sata M, Hashimoto S, Kuroki M, Saito M, Kawakami M. Mechanical stress promotes the expression of smooth muscle-like properties in marrow stromal cells. Exp Hematol. 2004; 32: 1238–1245.[CrossRef][Medline] [Order article via Infotrieve]

22. Ball SG, Shuttleworth AC, Kielty CM. Direct cell contact influences bone marrow mesenchymal stem cell fate. Int J Biochem Cell Biol. 2004; 36: 714–727.[CrossRef][Medline] [Order article via Infotrieve]

23. Davani S, Marandin A, Mersin N, Royer B, Kantelip B, Herve P, Etievent JP, Kantelip JP. Mesenchymal progenitor cells differentiate into an endothelial phenotype, enhance vascular density, and improve heart function in a rat cellular cardiomyoplasty model. Circulation. 2003; 108 (suppl II): II-253–II-258.[Medline] [Order article via Infotrieve]

24. Yoon YS, Wecker A, Heyd L, Park JS, Tkebuchava T, Kusano K, Hanley A, Scadova H, Qin G, Cha DH, Johnson KL, Aikawa R, Asahara T, Losordo DW. Clonally expanded novel multipotent stem cells from human bone marrow regenerate myocardium after myocardial infarction. J Clin Invest. 2005; 115: 326–338.[CrossRef][Medline] [Order article via Infotrieve]

25. Gojo S, Gojo N, Takeda Y, Mori T, Abe H, Kyo S, Hata J, Umezawa A. In vivo cardiovasculogenesis by direct injection of isolated adult mesenchymal stem cells. Exp Cell Res. 2003; 288: 51–59.[CrossRef][Medline] [Order article via Infotrieve]

26. Lee RH, Kim B, Choi I, Kim H, Choi HS, Suh K, Bae YC, Jung JS. Characterization and expression analysis of mesenchymal stem cells from human bone marrow and adipose tissue. Cell Physiol Biochem. 2004; 14: 311–324.[CrossRef][Medline] [Order article via Infotrieve]

27. Bitar KN, Yamada H. Modulation of smooth muscle contraction by sphingosylphosphorylcholine. Am J Physiol. 1995; 269: G370–G377.[Medline] [Order article via Infotrieve]

28. Bischoff A, Czyborra P, Fetscher C, Meyer Zu HD, Jakobs KH, Michel MC. Sphingosine-1-phosphate and sphingosylphosphorylcholine constrict renal and mesenteric microvessels in vitro. Br J Pharmacol. 2000; 130: 1871–1877.[CrossRef][Medline] [Order article via Infotrieve]

29. Jeon ES, Moon HJ, Lee MJ, Song HY, Kim YM, Bae YC, Jung JS, Kim JH. 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]

30. Bell E, Ivarsson B, Merrill C. Production of a tissue-like structure by contraction of collagen lattices by human fibroblasts of different proliferative potential in vitro. Proc Natl Acad Sci U S A. 1979; 76: 1274–1278.[Abstract/Free Full Text]

31. Grinnell F. Fibroblast-collagen-matrix contraction: growth-factor signalling and mechanical loading. Trends Cell Biol. 2000; 10: 362–365.[CrossRef][Medline] [Order article via Infotrieve]

32. Kuriyama H, Kitamura K, Itoh T, Inoue R. Physiological features of visceral smooth muscle cells, with special reference to receptors and ion channels. Physiol Rev. 1998; 78: 811–920.[Abstract/Free Full Text]

33. Abernethy DR, Soldatov NM. Structure-functional diversity of human L-type Ca2+ channel: perspectives for new pharmacological targets. J Pharmacol Exp Ther. 2002; 300: 724–728.[Abstract/Free Full Text]

34. Moosmang S, Lenhardt P, Haider N, Hofmann F, Wegener JW. Mouse models to study L-type calcium channel function. Pharmacol Ther. 2005; 106: 347–355.[CrossRef][Medline] [Order article via Infotrieve]

35. Nelson MT, Quayle JM. Physiological roles and properties of potassium channels in arterial smooth muscle. Am J Physiol. 1995; 268: C799–C822.[Medline] [Order article via Infotrieve]

36. Amano M, Chihara K, Nakamura N, Kaneko T, Matsuura Y, Kaibuchi K. The COOH terminus of Rho-kinase negatively regulates rho-kinase activity. J Biol Chem. 1999; 274: 32418–32424.[Abstract/Free Full Text]

37. Posern G, Treisman R. Actin’ together: serum response factor, its cofactors and the link to signal transduction. Trends Cell Biol. 2006; 16: 588–596.[CrossRef][Medline] [Order article via Infotrieve]

38. Ross JJ, Hong Z, Willenbring B, Zeng L, Isenberg B, Lee EH, Reyes M, Keirstead SA, Weir EK, Tranquillo RT, Verfaillie CM. Cytokine-induced differentiation of multipotent adult progenitor cells into functional smooth muscle cells. J Clin Invest. 2006; 116: 3139–3149.[CrossRef][Medline] [Order article via Infotrieve]

39. Sinha S, Wamhoff BR, Hoofnagle MH, Thomas J, Neppl RL, Deering T, Helmke BP, Bowles DK, Somlyo AV, Owens GK. Assessment of contractility of purified smooth muscle cells derived from embryonic stem cells. Stem Cells. 2006; 24: 1678–1688.[CrossRef][Medline] [Order article via Infotrieve]

40. Chen S, Crawford M, Day RM, Briones VR, Leader JE, Jose PA, Lechleider RJ. RhoA modulates Smad signaling during transforming growth factor-beta-induced smooth muscle differentiation. J Biol Chem. 2006; 281: 1765–1770.[Abstract/Free Full Text]

41. Lockman K, Hinson JS, Medlin MD, Morris D, Taylor JM, Mack CP. Sphingosine 1-phosphate stimulates smooth muscle cell differentiation and proliferation by activating separate serum response factor co-factors. J Biol Chem. 2004; 279: 42422–42430.[Abstract/Free Full Text]

42. Nagatoya K, Moriyama T, Kawada N, Takeji M, Oseto S, Murozono T, Ando A, Imai E, Hori M. Y-27632 prevents tubulointerstitial fibrosis in mouse kidneys with unilateral ureteral obstruction. Kidney Int. 2002; 61: 1684–1695.[CrossRef][Medline] [Order article via Infotrieve]

43. Smith PC, Caceres M, Martinez J. Induction of the myofibroblastic phenotype in human gingival fibroblasts by transforming growth factor-beta1: role of RhoA-ROCK and c-Jun N-terminal kinase signaling pathways. J Periodontal Res. 2006; 41: 418–425.[CrossRef][Medline] [Order article via Infotrieve]

44. Copeland JW, Treisman R. The diaphanous-related formin mDia1 controls serum response factor activity through its effects on actin polymerization. Mol Biol Cell. 2002; 13: 4088–4099.[Abstract/Free Full Text]

45. Sotiropoulos A, Gineitis D, Copeland J, Treisman R. Signal-regulated activation of serum response factor is mediated by changes in actin dynamics. Cell. 1999; 98: 159–169.[CrossRef][Medline] [Order article via Infotrieve]

46. Du KL, Chen M, Li J, Lepore JJ, Mericko P, Parmacek MS. Megakaryoblastic leukemia factor-1 transduces cytoskeletal signals and induces smooth muscle cell differentiation from undifferentiated embryonic stem cells. J Biol Chem. 2004; 279: 17578–17586.[Abstract/Free Full Text]

47. Cen B, Selvaraj A, Burgess RC, Hitzler JK, Ma Z, Morris SW, Prywes R. Megakaryoblastic leukemia 1, a potent transcriptional coactivator for serum response factor (SRF), is required for serum induction of SRF target genes. Mol Cell Biol. 2003; 23: 6597–6608.[Abstract/Free Full Text]

48. Hinson JS, Medlin MD, Lockman K, Taylor JM, Mack CP. Smooth muscle cell-specific transcription is regulated by nuclear localization of the myocardin-related transcription factors. Am J Physiol Heart Circ Physiol. 2007; 292: H1170–H1180.[Abstract/Free Full Text]

49. Zhao XH, Laschinger C, Arora P, Szaszi K, Kapus A, McCulloch CA. Force activates smooth muscle alpha-actin promoter activity through the Rho signaling pathway. J Cell Sci. 2007; 120: 1801–1809.[Abstract/Free Full Text]

50. Farrington-Rock C, Crofts NJ, Doherty MJ, Ashton BA, Griffin-Jones C, Canfield AE. Chondrogenic and adipogenic potential of microvascular pericytes. Circulation. 2004; 110: 2226–2232.[Abstract/Free Full Text]

51. Schor AM, Canfield AE, Sutton AB, Arciniegas E, Allen TD. Pericyte differentiation. Clin Orthop Relat Res. 1995; 81–91.

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