1Department of Molecular Physiology and Biological Physics. University of Virginia, Charlottesville, Virginia 22908; and 2Department of Medicine, University of Manchester, Manchester M13 9PT, United Kingdom
Submitted 5 May 2004 ; accepted in final form 7 August 2004
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ABSTRACT |
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embryoid body; Smad
In vivo, 50% of TGF-
1-null mice die in utero at days 10.511.5 with defects in the yolk sac vasculature, including decreased vessel wall integrity and reduced contact between endothelial and mesenchymal cells (10). A similar phenotype has been reported in the TGF-
type II receptor-null mouse (25). Null mutations in other TGF-
signaling pathway components such as Alk-1 (24, 33), endoglin (5, 20), and Smad5 (6) also lead to intrauterine death at midgestation because of fragile and dilated or hemorrhagic vessels. In these knockout models, fetal death, likely due to effects on endothelial stability and tube formation leading to an abnormal vasculature, occurs before significant recruitment or maturation of vascular SMCs. Nevertheless, some of these studies have reported loss of early SMC coating around the nascent dorsal aorta in null animals (20, 24), suggesting that the TGF-
signaling pathway may be required for SMC differentiation. Alternatively, the reported reduction in SMC number may simply be due to nonspecific effects, because the null embryos show significant growth retardation, edema, and necrosis by the time SMCs are first detected. Moreover, if TGF-
signaling is indeed a requirement for SMC development, it is not possible to distinguish the primary effects of the null mutation on SMCs from those secondary to the defective endothelial network or to effects mediated through other cell types.
Meanwhile, there is compelling evidence that TGF-1 coordinately upregulates a variety of SMC differentiation marker genes including smooth muscle
-actin (SM
A), smooth muscle myosin heavy chain (SMMHC), and h1-calponin in cultured SMCs derived from mature blood vessels (3, 14). Because SMC differentiation is characterized by the upregulation of these and other SM-specific genes, it has been postulated that TGF-
1 may promote SMC development from pluripotential precursors. This hypothesis was initially investigated in vitro, with primary cultures of neural crest cells (32) and more recently with neural crest-derived Monc-1 cells (8). These studies provided evidence that addition of TGF-
1 induced these cells to express several SMC markers, including SM
A and calponin. However, although the cells were able to respond to exogenous growth factor by expressing SMC differentiation marker genes, no loss-of-function studies were done to determine whether endogenous TGF-
1 signaling was a component of the normal developmental pathway of SMCs derived from the neural crest. Additional studies on mouse embryonic 10T1/2 cells (15) provided evidence that exogenous TGF-
1 induced SM
A expression whereas antibody-mediated inhibition of TGF-
reduced expression of this marker when the cells were cocultured with bovine endothelial cells (ECs). However, SM
A is expressed by many cell types other than SMCs (26), and evidence that TGF-
1-treated 10T1/2 cells also expressed the much more definitive SMC marker SMMHC was equivocal because the studies relied on use of an antibody that we have found shows very low sensitivity for murine SMMHC, leading to problems of cross-reactivity with nonmuscle myosin heavy chains including embryonic SM myosin heavy chain [SMemb or nonmuscle myosin heavy chain B (NMMHC-B)]. Indeed, we reported (35) that TGF-
does not induce expression of either SMMHC or the potent SMC/cardiac myocyte-specific transcriptional activator myocardin in 10T1/2 cells. Thus the 10T1/2 system may be a model of myofibroblast activation rather than true SMC development.
In summary, there is a lack of direct evidence that TGF- signaling contributes to differentiation of SMCs during embryonic development. Moreover, previous studies have relied on model systems that may not recapitulate normal developmental control processes in that they used cells already predetermined to particular cell fates rather than pluripotential embryonic stem cells (ESCs). Finally, of critical importance, cells derived in most of these experiments have not been shown to possess any contractile activitya hallmark of mature SMCs. Although a recent study examining TGF-
1-stimulated Monc-1 cells did document a change in the morphology of some cells in response to carbachol (8), the extent and nature of this change have not been fully investigated. Indeed, a limitation of many in vitro models of SMC development is that contractile ability of the resultant cells has not been clearly established, and it is conceivable that in these systems only a subset of the developmental programs required for the formation of SMC lineage are activated.
The focus of the present studies was to directly investigate the role of TGF- in SMC development with an ESC-embryoid body (EB) model of early SMC lineage development previously described by Drab and colleagues (11) wherein cells develop full contractile capabilities. This system is particularly intriguing because it is derived from aggregates of differentiating ESCs, which recapitulate many of the events of early embryonic development including development of mesoderm, the visceral yolk sac, and blood islands. The ESC-EB model also recreates the multiple cell types and the heterotypic cell-cell and cell-matrix interactions that occur in the developing embryo and thus may represent a more "physiological" system in which to study SMC development. Of interest, results of the present studies show that SM-specific gene expression was downregulated by a soluble truncated TGF-
type II receptor, an anti-TGF-
1 antibody, or small interfering (si)RNAs directed against Smad2 or Smad3, indicating that endogenous TGF-
1 signaling through Smad2 and Smad3 plays an important role in the development of SMCs from totipotential ESCs.
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MATERIALS AND METHODS |
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RNA extraction and RT-PCR.
RNA was extracted from ESC-EBs at a variety of time points with TRIzol (GIBCO), and the manufacturer's instructions were followed. Reverse transcription was carried out on 1 µg of total RNA. Quantitative real-time PCR was performed with Taqman chemistry probes and an iCycler (Bio-Rad). Sequences for all primers and probes (Integrated DNA Technologies, Coralville, IA) were as follows: SMA forward CGCTGTCAGGAACCCTGAGA, reverse CGAAGCCGGCCTTACAGA, probe CAGCACAGCCCTGGTGTGCGAC; SMMHC forward TGGACACCATGTCAGGGAAA, reverse ATGGACACAAGTGCTAAGCAGTCT, probe AGAACACTAAACGACAGCAGAGCCCAGC; 18S forward CGGCTACCACATCCAAGGAA, reverse AGCTGGAATTACCGCGGC, probe TGCTGGCACCAGACTTGCCCTC; glyceraldehyde 3-phosphate dehydrogenase (GAPDH) forward ggctcatgaccacagtccat, reverse gcctgcttcaccaccttct, probe cctggagaaacctgccaagtatgatgac; platelet endothelial cell adhesion molecule-1 [PECAM-1; amplification assessed by SYBR Green (Molecular Probes, Eugene, OR) incorporation with no probe] forward cttgagcctcaccaagctct, reverse actctcgcaatccaggaatc. Conventional PCR was carried out with the following primers for semiquantitative assessment of the SM1 and SM2 isoforms of SMMHC: SMMHC forward AGGAAACACCAAGGTCAAGCA, reverse CCCTGACATAGTGTCCAACTG (36 cycles); SM22
forward tccagtccacaaacgaccaagc, reverse gaattgagccacctgttccatctg (25 cycles); myocardin forward AAACCAGGCCCCCTCCC, reverse CGGATTCGAAGCTGTTGTCTT, 34 cycles; smoothelin-B forward tcagaggcttctccaacactaagag, reverse ttggctctcgatttggggttggttg (27 cycles); GAPDH forward TCTTCACCACCATGGAGAAGG, reverse GTTGTCATGGATGACCTTGGCC (22 cycles).
Immunocytochemistry.
EBs were fixed with 4% paraformaldehyde for 10 min and then washed with phosphate-buffered saline, air dried, and stored at 80°C until required for immunocytochemistry. Specimens were then rehydrated, pretreated with 0.6% H2O2 in methanol and 0.5% Triton X-100, and blocked with 3% BSA-3% normal horse, sheep, or goat serum (Jackson, West Grove, PA). Primary antibodies were anti-chicken SMMHC (12), a rabbit anti-serum used at 1:2,000 dilution; anti-SMA (clone 1A4; Sigma), a monoclonal antibody used at 1:5,000 dilution; and anti-PECAM-1 (Santa Cruz Biotechnology, Santa Cruz, CA), a goat polyclonal antibody used at 1:500 dilution. Negative controls were performed with appropriate nonimmune serum or pooled immunoglobulins (Igs; Jackson). Detection was performed either with a fluorescence-labeled secondary antibody (Jackson) or with the avidin-biotin complex (ABC) Elite detection kit (Vector Laboratories, Burlingame, CA) and diaminobenzidine as the substrate.
Generation of adenoviruses and inhibition of TGF- signaling.
Recombinant adenoviruses Ad5-RIIs and Ad5-lacZ were generated as previously described (18). Ad5-RIIs contains a truncated human TGF-
type II receptor cDNA under the transcriptional control of the major immediate-early human cytomegalovirus (CMV) enhancer/promoter (MIEhCMV 700 to +50). Ad5-lacZ contains the cDNA for Escherichia coli
-galactosidase under control of MIEhCMV. Stocks were purified by cesium chloride gradient centrifugation and titered by serial dilution end point assay. Viruses were added to ESC-EB culture medium at a concentration of 3 x 106 pfu/ml on days 10, 13, 17, 21, and 25. Specific inhibition of TGF-
1 was achieved by supplementing ESC-EB medium daily from day 10 to day 28 with an isoform-specific antibody (R&D, Minneapolis, MN) or a nonimmune IgG control at 0.5 µg/ml. All experiments were carried out at least three times. ESC-EBs in all experiments were harvested for RNA on day 28.
siRNA generation, transfection, and reporter expression.
Plasmids expressing siRNAs to Smad2, Smad3, a scrambled control, and an empty vector control were generated as previously described (35). Plasmids encoding the siRNAs were cotransfected into differentiating ESC-EBs along with a SMA or SMMHC promoter-luciferase reporter construct and a CMV-Renilla luciferase construct (Promega, Madison, WI) to normalize for transfection efficiency. FuGENE 6 (Roche) was used for transfection in accordance with the manufacturer's protocol. Cell lysates were harvested with Reporter lysis buffer (Promega) and a freeze-thaw step. Luciferase activity was measured with a luciferase assay system (Promega) and corrected for Renilla luciferase activity as detected with 270 nM coelenterazine (Biosynth, Naperville, IL). All experiments were carried out in triplicate and repeated at least three times.
Smad immunoblotting. COS-7 cells were cotransfected with an expression vector for Smad2 or Smad3 and a plasmid expressing siRNAs to Smad2, Smad3, a scrambled control, or an empty vector control. Cell lysate was harvested 72 h after transfection with a modified radioimmunoprecipitation buffer (150 mM NaCl, 50 mM Tris·HCl pH 8.0, 1% NP-40, 0.5% deoxycholate, and 2 mM EDTA) with complete EDTA-free protease inhibitor cocktail (Roche). Protein concentration was measured with a bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL), and 10 µg of protein was denatured and run on a 10% SDS polyacrylamide gel. The protein was blotted onto a polyvinylidene difluoride (PVDF) membrane and blocked with 4% nonfat milk. Primary antibodies directed against Smad2 or Smad3 (Upstate, Waltham, MA) were applied at 1:1,000 dilution, and an anti-rabbit-horseradish peroxidase secondary was used at 1:3,000. The blot was visualized with an enhanced chemiluminescence kit (Amersham Biosciences, Piscataway, NJ) according to the manufacturer's guidelines.
Statistical analysis. Quantitative data on mRNA expression levels were compared with unpaired t-tests. Luciferase activity in multiple groups was compared with ANOVA. A probability value of P < 0.05 was selected as significant for all tests.
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RESULTS |
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Plating of ESC-EBs on a gelatin-coated surface and treatment with atRA and DBcAMP led to an extensive outgrowth from the central cellular aggregate (Fig. 1A). The ESC-EBs were harvested and fixed at day 15 and day 28. Multiple cell types were detected throughout the outgrowth with immunocytochemistry, including SMCs (Fig. 1, B and C) and ECs (Fig. 1D). No overt vascular structures were seen, but the ECs formed a primitive, poorly organized vascular plexus at day 15. Immunocytochemical analyses showed numerous SMA-positive cells at both time points, whereas numerous SMMHC-positive cells were detected at day 28. Negative controls carried out by substituting nonimmune serum or pooled Igs for primary antibodies did not result in a signal (data not shown). SMCs were distributed sparsely throughout much of the ESC-EB outgrowth and occasionally as dense aggregates (Fig. 2C). The SM2 isoform, found predominantly in postnatal SM-containing tissues (1, 4, 36), was detected with RT-PCR on RNA extracted from the whole ESC-EBs at day 28 (Fig. 1E). Thus the presence of this isoform of SMMHC is indicative of the development of mature contractile SMCs within the ESC-EBs. In addition, the expression of several other markers of SMCSM22
, myocardin, and smoothelin-Bwas detected by RT-PCR (Fig. 1F) and supported the conclusion that genuine SMCs developed in the ESC-EBs.
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Differential upregulation of SMCs and endothelial markers in ESC-EBs.
Next, it was important to determine the time course of increase in SM-selective gene expression more accurately to carry out loss-of-function interventions at appropriate time points. Because immunocytochemistry and conventional RT-PCR are poorly quantitative, we used real-time RT-PCR to accurately quantify SM- and EC-selective gene expression over a range of time points during development of SMCs within the ESC-EB system. SMC markers were expressed at very low levels during early development in the ESC-EBs and increased significantly from day 10 onward (Fig. 3). Interestingly, the greatest increase in SMA occurred between day 10 and day 15 (Fig. 3A), whereas SMMHC expression increased later, between day 15 and day 28 (Fig. 3B). This difference in temporal expression pattern between SM
A and SMMHC is reminiscent of the time course of expression seen in vivo in the developing embryo.
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Endogenous TGF-1 signaling is required for SMC-specific gene expression in ESC-EBs.
To directly test the role of endogenous TGF-
signaling in differentiation of SMCs within the ESC-EB system, cells were infected with Ad5-RIIs, a recombinant adenovirus that expresses a soluble TGF-
type II receptor that has been shown to efficiently inhibit TGF-
signaling in vivo and in vitro (18). Effects on expression of SMC marker genes were measured with real-time RT-PCR in the Ad5-RIIs treatment group vs. a control group treated with Ad5-LacZ, a control virus expressing
-galactosidase, at equivalent multiplicity of infection. Inhibition of TGF-
signaling downregulated SM
A and SMMHC mRNA expression in the ESC-EBs (Fig. 4). These results suggested that endogenous TGF-
signaling was required for SMC-specific gene expression during the development of SMCs from ESCs. A limitation of this experiment was that the soluble TGF-
type II receptor does not distinguish effectively between the TGF-
1 and TGF-
3 isoforms. However, because data on whole animal knockout models implicate the TGF-
1 isoform as potentially required for SMC development, we used an anti-TGF-
1 antibody to specifically inhibit this isoform in the ESC-EBs. Treatment of developing ESC-EBs with the anti-TGF-
1 antibody also reduced SMC-specific gene expression compared with a nonimmune IgG control (Fig. 5). Thus SMC development in this system appears to be mediated, at least in part, by TGF-
1 isoform signaling through the type II receptor.
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The efficacy and specificity of the siRNA expression constructs were tested by coexpression of Smad2 or Smad3 with either the matching Smad siRNA expression vector or a variety of control constructs in COS-7 cells. Immunoblotting studies showed that the siRNA directed against Smad2 was highly effective and almost completely abolished Smad2 protein expression in cells cotransfected with a Smad2 expression vector (Fig. 7A). Moreover, no significant effect was seen on Smad3 expression (Fig. 7B), confirming the specific nature of the Smad2-targeted siRNA. The siRNA expression construct directed against Smad3 was also effective and significantly downregulated but did not completely abrogate expression of the target gene (Fig. 7B). Interestingly, Smad3-targeted siRNAs also appeared to have a minor effect on Smad2 expression (Fig. 7A). The gene-specific nature of suppression induced by our siRNA expression plasmids was further confirmed by comparing results with a scrambled control siRNA or an empty vector control. These control constructs had no effect on Smad expression.
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DISCUSSION |
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In this study, a surprising and significant finding was that expression of the SMA promoter was dependent on both Smad2 and Smad3 signaling whereas expression of the more SMC-selective promoter, SMMHC, was only Smad2 dependent. This may be due to the differential signaling requirements of these two SMC markers during development. Indeed, it has been shown that Smad2 and Smad3 have widely differing roles in TGF-
1 stimulation of embryonic fibroblasts (27). However, another possibility is that the SM
A expression detected in the present study was from a variety of cell types in addition to SMCs because other cells, which also develop in the ESC-EBs, are known to express this gene during development (26). Interestingly, Smad2 activation has been reported in response to TGF-
1 in cultured SMCs (17). In addition, although a role for Smad2 in myofibroblast activation cannot be totally excluded (19), Smad3 has been identified by several groups as a key effector molecule in TGF-
1-dependent activation of SM-specific genes in embryonic fibroblasts (7, 28). Accordingly, it is possible that the specific Smad2 dependence displayed by the SMMHC promoter, which is restricted to SMCs during development (22), is more representative of the signaling requirements in developing SMCs, whereas the dependence of the SM
A promoter on both Smad2 and Smad3 reflects signals from SMCs plus myofibroblasts or other muscle types. Further data supporting this conclusion come from knockout experiments in which mice that are null for Smad3 are born with a normal vasculature (9, 34). Together, the results suggest that Smad3 may not be required for SMC development/gene expression but rather for myofibroblast activation.
In this study, as in other studies on ESC-EBs (13), EC development was noted in a network that resembled a loose, early vascular plexus. However, no mature blood vessels were found to develop, and a consistent association between ECs and SMCs was not found. A variety of studies have suggested that proximity or contact of ECs is required for SMC precursors to develop into SMCs and that heterotypic cell-cell interactions are required to allow SMCs to mature and stabilize the endothelial network (16). In this study, although there was some overlap in the distribution of ECs and early SMCs, there was not a consistent association between these cell types, and in many cases there appeared to be significant numbers of SMCs in the absence of closely associated ECs. However, heterotypic cell-cell interactions were not examined in great detail, and it is possible that, given the nature of the ESC-EB, where multiple cell layers are stacked on top of each other, a small number of ECs may have been interspersed within the SMC clusters. Additionally, EC-SMC interactions may be transient in this system, rendering these associations difficult to document. Indeed, it is thought that hemodynamic forces are required to maintain endothelial stability in the vasculature (31), and it is possible that in their absence EC-SMC interactions are not stabilized or that ECs are lost through apoptosis. Consistent with this hypothesis, we found that PECAM-1 expression in the ESC-EB peaks at day 10 and then decreases steadily (Fig. 3C). Despite these uncertainties, the key issue is that the ESC-EBs create an environment in which mature, contractile SMCs develop, and that disruption of TGF-1 signaling within this system inhibited SMC differentiation. An additional inherent limitation of the published TGF-
1 mouse knockout studies was that it was unclear whether the effects on SMCs were primary or secondary to loss of TGF-
1 signaling in ECs. In the present study, we attempted to minimize perturbation of EC development and to selectively target SMC development by delaying inhibition of growth factor function until EC gene expression had peaked. However, it is not possible to be certain that EC or indeed other cell functions were not affected in any way and that the effects documented are indeed direct effects of TGF-
1 on SMCs. Thus, although it is clear that endogenous TGF-
1 signaling through at least Smad2 is required in the ESC-EBs for SMC development, further studies in which inhibition of TGF-
1 signaling is restricted to SMCs are required to more clearly answer questions on cellular specificity. Transgenic mouse systems have been developed in which cre-recombinase is expressed solely in SMCs (30) and could be used to inactivate components of the TGF-
signaling pathway in a SM-specific manner. This type of study would clarify whether activation of the TGF-
signaling pathway specifically in SMCs is required for expression of SMC markers. However, because promoter-enhancers capable of directing DNA-modifying enzymes in a SMC-restricted manner are only expressed once the cell has already started differentiating, this system will only have utility for examining the late stages of differentiation-maturation and not the initial induction of SMC lineage in multipotential cells. To investigate earlier stages of the developmental process, interventions would need to be targeted to SMC progenitors. However, because these cells by definition do not express any SMC markers, it has not been possible to identify or specifically target this presumptive cell population to date.
The development of mature contractile SMCs from pluripotential precursors may be considered as a series of steps involving first a commitment to the SMC lineage, then differentiation into early immature SMCs, and finally maturation into the mature contractile phenotype (26). Which of these steps are regulated by TGF-1? In previous studies (8, 15, 32) TGF-
1 promoted the development of an early SM-like cell from neural crest cells or 10T1/2 cells, suggesting a role for the growth factor in both the commitment and early differentiation steps. However, it remains unclear whether TGF-
1 signaling is required for commitment and early differentiation of SMCs in vivo in the embryo. In the present study, TGF-
1 signaling was disrupted from day 10 onward, a time at which SM
A expression had begun to increase but SMMHC expression remained low (Fig. 3, A and B). Thus it is likely that commitment to the SMC lineage had already occurred and the major effect of our loss-of-function interventions was on the maturation process and possibly on early differentiation.
The ESC-EB system recapitulates many of the events of early embryonic development. Multiple cell types are obtained, allowing for the heterotypic cell-cell and cell-matrix interactions that are characteristic of key developmental events in vivo. The generation of multiple cell types is a strength of this system in that it recreates some of the cell-cell interactions and environmental cues present in the developing embryo. However, there are also significant limitations of the ESC-EB system in that such multiplicity of cell types renders results derived from this system more difficult to interpret because information from the cell type of interest is measured on a background of other cell types. Nevertheless, the present studies emphasize the power of the ESC-EB model for dissecting out developmental mechanisms that will be greatly enhanced by the use of transgenic and/or null ESC lines for such experiments in the future.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Supplemental material for this article is available online at http://ajpcell.physiology.org/cgi/content/full/00221.2004/DC1.
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