Msx2 and Necdin Combined Activities Are Required for Smooth Muscle Differentiation in Mesoangioblast Stem Cells
Little is known about the molecular mechanism underlying specification and differentiation of smooth muscle (SM), and this is, at least in part, because of the few cellular systems available to study the acquisition of a SM phenotype in vitro. Mesoangioblasts are vessel-derived stem cells that can be induced to differentiate into different cell types of the mesoderm, including SM. We performed a DNA microarray analysis of a mesoangioblast clone that spontaneously expresses an immature SM phenotype and compared it with a sister clone mainly composed of undifferentiated progenitor cells. This study allowed us to define a gene expression profile for “stem” cells versus smooth muscle cells (SMCs) in the absence of differentiation inducers such as transforming growth factor β. Two transcription factors, msx2 and necdin, are expressed at least 100 times more in SMCs than in stem cells, are coexpressed in all SMCs and tissues, are induced by transforming growth factor β, and, when coexpressed, induce a number of SM markers in mesoangioblast, fibroblast, and endothelial cell lines. Conversely, their downregulation through RNA interference results in a decreased expression of SM markers. These data support the hypothesis that Msx2 and necdin act as master genes regulating SM differentiation in at least a subset of SMCs.
Smooth muscle cells (SMCs) regulate a variety of functions, such as arterial tone, airway resistance, and gastrointestinal and genitourinary tract contractility; alterations of vascular smooth muscle cells (VSMCs) contribute to occlusive arteriosclerosis, which often leads to heart and brain stroke, the major causes of death in developed countries.1,2 Still, little is known about the developmental control of SMC specification and differentiation. For example, vascular development requires the correct spatial and temporal expression of specific sets of genes in at least 2 different progenitor cells, that is, the angioblasts that form a primary vascular network and the pericytes that surround the network and differentiate into VSMCs. SMCs may also derive from angioblast transdifferentiation and from migrating neural crest cells. This complex embryological origin suggests a similar complexity in its molecular control. Indeed, until now, no early specific marker or master gene has been demonstrated to play a role comparable to that of MyoD in skeletal muscle development.
Insights have been recently provided by work on the transcription factor modulator recognition factor 2 (Mrf2), which has been shown to be expressed at the onset of SM differentiation and to be a candidate regulator of SMC differentiation and proliferation.3,4
Moreover, myocardin, a transcriptional coactivator of serum response factor (SRF), has been shown to play a crucial role in smooth myogenesis, as shown by severe reduction of SM differentiation in the myocardin null embryo.5–7
We recently identified and characterized a class of vessel-associated stem cells called mesoangioblasts, which in the fetal stage of development are distributed to tissue anlagen through angiogenesis and through the circulation. They contribute to perinatal tissue growth and may represent the ancestors of postnatal stem cells;8–10 they also contribute to repair of cardiac and skeletal muscle.11,12
Here we show that 1 of the mesoangioblast clones isolated, although maintaining stem cell features such as pluripotency and self-renewal ability, also displays phenotypic traits of definitive SMCs. This led us to compare this cell line, through microarray analysis, with a sister clone (mainly composed of undifferentiated cells) to search for differentially expressed genes that may control the process of SM differentiation. Here we report that 2 such genes, msx2 and necdin, are likely candidates as regulators of SM differentiation.
Mesoangioblast cell clones were derived from the dorsal aorta of C57/Bl6 9.5 dpc embryos and grown as described.9
Cell Proliferation Assay
D16 and D351 cells were seeded in microtiter plates (tissue culture grade 96 wells) at an initial density of 3×103 cells/well in 100 μL of complete medium. The relative increase in cell number was measured after 4 hours, 24 hours, 48 hours, and 72 hours using the Cell Proliferation Kit II (XTT) assay (Roche).
Immunofluorescence on cells and cryostat sections was performed as reported.9 The antibodies used in this study and their working dilution were as follows: α smooth muscle actin (αSMA) monoclonal antibody (mAb) (1:350) (Sigma), SM22 mAb (1:300),13 calponin mAb (1:500) (Sigma), SM-MyoHC 1 e 2 mAb (1:400),14 rabbit anti-sarcomeric myosin (1:300), and MF20 mAb (1:2),9 NC243.15
Biotin-Labeled cRNA Transcription and GeneChip Hybridization
RNA (5 μg), isolated from quasi-confluent D16 and D351 culture, was converted into double-stranded cDNA by reverse transcription using the cDNA synthesis SuperScript Choice System kit (Invitrogen). Labeled cRNA was generated from the cDNA sample by in vitro transcription (Enzo bio array HY RNA transcript labeling kit; Enzo) supplemented with biotin-11-CTP and biotin-16-UTP. Fifteen micrograms of biotinylated cRNA were fragmented, assessed by gel electrophoresis, and placed in a hybridization cocktail containing 4 biotinylated hybridization controls (BioB, BioC, BioD, and Cre). Samples were hybridized to an identical lot of Affymetrix MGU74Av2, MGU74Bv2, and MGU74Cv2 GeneChip arrays for 16 hours. GeneChips were washed and stained according to the Eukaryotic GE WS2 protocol (Affymetrix).
Microarray Data Analysis
The images from the scanned chips were processed using the Affymetrix Microarray Analysis Suite 5.0 (MAS 5.0; see the online data supplement available at http://circres.ahajournals.org).
Constructs and Transfections
Necdin ORF and 3′ probes were isolated by polymerase chain reaction (PCR) on 129 genomic DNA using the following primers, respectively, and using Pfu Taq polymerase (Promega): NECF1, AAAGATCTGTCCTGCTCTGATCCGAAGG; NECR2, AAGAATTTCGTATGGGTCAGAAACTATCAGTG (necdin ORF, 1020 bp); NECF2, CAAGGATCCACTGATAGTTTCTGACCCATAC; and NECR1, AAGAATTCGCCAGTTGAAGTCATATGGAG (necdin 3′ probe, 400 bp). Necdin 3′ probe fragment was cloned in pBluescript KS- (Stratagene); necdin ORF and Msx2 ORF (a gift from David Sassoon, Brookdale Department of Molecular, Cell and Developmental Biology, Mount Sinai Medical School, New York) were cloned in pCDNA3 (Invitrogen) and in pIRES2EGFP (Clontech).
D16 cells were transiently transfected with the necdin and msx2 expression vectors, alone or in combination, using the lipofectamin reagent (Invitrogen). Twenty-four hours after transfection, the serum concentration was lowered to facilitate differentiation, and after an additional 48 hours, cells were processed for immunofluorescence, or total RNA was extracted for RT-PCR analysis. Transfection efficiency (≈50%) of pCDNA3-based expression vectors was determined by cotransfection with pCMV-LacZ. D16 was also stably transfected with the same expression vectors. A pool of neomicin-resistant cells for each transfection combination were grown at low serum concentration for 4 days and processed for immunofluorescence.
In Situ Hybridization
In situ hybridization whole mount and on mouse cryostat sections was performed according to procedures described,16,17 using the necdin 3′ probe.
Reverse Transcription—Polymerase Chain Reaction
RNA (1 μg) collected from various cell types and dissected embryos was converted into double-stranded cDNA by reverse transcription using the cDNA synthesis Thermoscript RT-PCR System kit (Invitrogen). cDNA was then amplified using specific primers (see the online data supplement).
Small interfering RNA (siRNA)–expressing vectors, psiUc/necdin and psiUc/msx2, were obtained by cloning in the BglII and XhoI sites of the psiUx vector.17a DNA fragments were derived by annealing the following oligos: necdin F, 5′GATCTCAACAACCGTATGCCCATGACATTTGTGTAGTGTCATGGGCATACGGTTGTTTGACTTTCTGGAGTTTCAAAAGTAGAC-3′; necdin R, 5′TCGAGTCTACTTTTGAAACTCCAGAAAGTCAAACAACCGTATGCCCATG- ACACTACACAAATGTCATGGGCATACGGTTGTTGA-3′; Msx2 F, 5′GATCTCAACAGTACCTGTCCATAGCAGTTTGTGTAGCTGCTATGGACAGGTACTGTTTGACTTTCTGGAGTTTCAAAAGTAGAC-3′; Msx2 R, 5′TCGAGTCTACTTTTGAAACTCCAG-AAAGTCAAACAGTACCTGTCCATAGCAGCTACACAAAC-TGCTATGGACAGGTACTGTTGA-3′.
The selected target sequences on necdin and msx2 were taken from National Center for Biotechnology Information Database accession numbers NM_010882 (nucleotides 621 to 641) and NM_013601 (nucleotides 857 to 877), respectively.
For Northern Blot analysis on 10 μg of total RNA, we used the following oligos: α necdin, 5′-ACAACCGTATGCCCATGACA-3′; α msx2, 5′-ACAGTACCTGTCCATAGCAG-3′.
Characterization of a Mesoangioblast Cell Clone Displaying Features of SMCs
We isolated mesoangioblast cell clones from the dorsal aorta of a C57BL6 9.5 dpc embryo. These clones (called clones D) express the endothelial markers characteristic of mesoangioblast cells, including Sca-1, CD34, and Thy-1.9
The large majority of mesoangioblast cell clones contain a variable percentage of cells (ranging from 5% to 30%) that express the SM marker αSMA. When treated with transforming growth factor (TGF) β1, cells in all clones differentiate into αSMA-positive SMCs, although with percentages that vary from clone to clone. However, one clone, D351, contains 80% to 90% of αSMA-positive cells, even in the absence of TGFβ (Figure S1A through S1D in the online data supplement). D351 cells appear morphologically different, the cells being more flattened and extended compared with another clone, D16, which contains very few αSMA-positive cells (Figure 1A and 1B).
D351 cells proliferate at a lower rate than D16 cells (Figure 1C), are more resistant to apoptosis induced by serum starvation (Figure S1E through S1L in the online data supplement), and express SMC-specific markers such as SM22, calponin1, and SM-specific myosin (SM-MyoHC), in addition to αSMA (Figure 1D). Under the same conditions, D16 does not express SM22, calponin, or SM-MyHC1 and -2.
Nevertheless, D351 cells were able to differentiate in vitro into a variety of cell types, such as skeletal muscle, cardiac muscle, and osteocytes (Figure S2A through S2F in the online data supplement), with similar efficiency as D16 cells. The occurrence of transdifferentiation from SM to skeletal muscle or bone was documented by detection of a SM marker (calponin) in the same cytoplasm of cells that were also expressing sarcomeric myosin or alkaline phosphatase, respectively (Figure S2G through S2L in the online data supplement).
These observations led us to conclude that D351 cells still retain, at least in vitro, the pluripotency characteristic of mesoangioblasts, even if they have acquired a more defined SMC phenotype.
Phenotypic Analysis of Mesoangioblast Cell Clones by DNA Microarray
We then defined the molecular phenotype of D16 and D351 cells by DNA microarray technique to provide a thorough definition of the gene expression profile with respect to “stemness,” embryological origin, and presence of lineage-specific markers in the absence of differentiation inducers. Fluorescent-labeled cRNA from D16 and D351 cells was hybridized to the Affymetrix Murine Genome U74v2 set, which consists of 3 GeneChip probe arrays (U74Av2, U74Bv2, U74Cv2), interrogating ≈36 000 mouse genes and expressed sequence tag clusters from the UniGene database (Build 74). As shown in the scatter plot in Figure 1E and in the online data supplement, D16 and D351 express a similar number of sequences (12 696 in D16 and 14 162 in D351), with a slight increase in complexity in D351 (+11.5%). Most sequences are expressed in both D16 and D351 (11516). In this category, 6648 sequences are not changed in the 2 cell populations, whereas 2204 show an increased expression in D16 and 2664 in D351. The number of sequences that can be considered specifically expressed in either cell population, on the basis of a significant change probability value, is 499 in D16 and 1038 in D351. The complete set of data from the hybridization experiment has been submitted to the Gene Expression Omnibus repository18 and is available online at the site http://www.ncbi.nlm.nih.gov/geo (accession numbers: GSM8510, GSM8511, GSM8512, GSM8513, GSM8514, GSM8515, GSM8516, GSM8517, GSM8518, GSE552, GSE554, GSE555, and GSE563) (see also information in the online data supplement).
Identification of SM-Differentiation Cofactors
The microarray revealed the presence of differentially expressed transcription factor encoding messengers in the D351 pool that may represent putative transcriptional master genes for SM differentiation or genes involved in maintaining a differentiated state.
Among the transcription factor expressed exclusively in D351 (listed in Table S2 in the online data supplement), we decided to focus on the 2 most highly expressed transcription factors and whose role has never been studied in the context of SM development: necdin, a MAGE protein19,20 and msx2, a homeodomain-containing protein.21,22
To investigate whether these genes could indeed initiate or facilitate SM development and differentiation, we first analyzed their expression pattern during embryogenesis. Msx2 is already known to be expressed in the neural crest cells,23 progenitors for different types of SMCs, and also in the SM layer of many embryonic vessels24 (B. Robert, personal communication).
To gain information on the embryonic expression of necdin outside the central nervous system,25–27 we performed in situ hybridization and immunofluorescence on cryostat sections of E10.5 and E16.5 embryos. (E indicates embryonic day.)
This analysis showed that in E10.5 embryos, necdin is expressed in the neural tube (Figure 2A), in the myotomes (Figure 2B), ) and in mesenchymal condensation between the branchial arches pouch and the branchial arteries, 1 site of origin for vascular SMCs28 (arrow in Figure 2A). At E16.5, expression is found in brain, spinal chord, cranial nerves and dorsal root ganglion (data not shown), and in major skeletal muscle masses in the limbs (Figure 2E). In the abdomen, expression is found also in the external muscular layer outlining the gut (Figure 2C and 2D, arrows), and staining is also present in cells associate with some vessels in the limbs (Figure 2F, arrow). The localization of necdin in the SM layer in the gut, small vessels, and in the dorsal aorta is confirmed by immunostaining using an antibody specific for necdin15 that largely overlaps with αSMA-positive cells (Figure S2G through S2J in the online data supplement).
Expression of Msx2 and Necdin in SMCs and Tissues
To test a possible role of msx2 and necdin in differentiation of SM, we investigated whether these genes are upregulated on TGFβ-induced SM differentiation of mesoangioblast cells. We also tested their expression in other cell types, before and after TGFβ treatment, including fibroblasts (3T3), embryonic fibroblasts (10T1/2), endothelial cells (H5V), and SMCs (rat SMC [RSMC]), as well as in embryonic aorta and gut at different developmental stages. Untreated D16 cells do not express detectable levels of msx2 and necdin, which are induced by TGFβ after 18 hours, 36 hours, and 72 hours; this correlates with the expression of SM myosin (SM-MyoHC) and SM22. Interestingly, msx2 and necdin are coexpressed only in SMCs, both in cell lines and tissues. We also tested the expression of other transcription factors, which have been suggested to play a role in SM differentiation including myocardin, myocardin-related transcription factor (MRTFA), serum response factor (SRF), and Mrf2α4–6,29,30 (Figure 3A). Mrf2α, SRF, and MRTFA are expressed by both D351 and D16, before and after TGFβ stimulation, but also in fibroblast, endothelial, and SMC lines at variable but consistent levels. As expected, expression of myocardin is restricted to SM cell lines and tissues, but no myocardin transcripts were observed in mesoangioblast cells in any condition (Figure 3A).
Msx2 and Necdin Are Necessary and Sufficient to Activate SM Gene Expression in Different Cell Types
Msx2 and necdin were then overexpressed, individually or in combination, in D16 cells (which do not express either), in 10T1/2 cells that express only necdin, and in H5V cells (which express only msx2). Expression of either gene alone in D16 cells modestly increased the percentage of D16 cells expressing αSMA (Figure 3B), but did not induce expression of other SM markers; however, the combined expression of the 2 genes resulted in the induction of the SM-specific genes SM22, calponin, and SM-MyoHC (Figure 3B and 3C). In addition, the proliferation rate of D16 stably transfected with both genes, but did with a single one, appeared to be reduced (not shown). Expression of SM-specific myosin was also induced in 10T1/2 cells transfected with msx2 and at lower levels in H5V cells transfected with necdin, although expression of calponin was not detected in these conditions. The same was also observed when both necdin and msx2 were overexpressed in 10T1/2 and H5V (not shown).
To verify whether expression of SM markers was because of a direct transcriptional activation of the SMC genes by msx2 and necdin, we tested the ability of these 2 factors, alone or in combination, to transactivate SMC promoters, in D16 and 10T1/2 cells. We observed that msx2 and necdin, either alone or together, are incapable of activating the expression of the Luciferase reporters driven by 4 different SMC promoters, α-SMA, SM22, calponin, and γ-SMA (Figure S3A in the online data supplement), thus suggesting that their action is likely indirect.
Finally, we wanted to ascertain if loss of expression of msx2 and necdin in D351 cells could affect their ability to express a SM phenotype. We transfected D351 with vectors expressing siRNA specific for necdin and msx2, alone or in combination. Forty-eight hours after transfection, we detected expression of the siRNA in the cells (Figure 4A). After 72 hours, we observed a reduction in the level of necdin and msx2 transcripts (Figure 4B), accompanied by a decreased expression of αSMA, SM22, calponin, and SM-MyoHC (Figure 4C through 4E), which appeared to be greater in cells expressing both siRNAs. We observed similar results when we transfected both siRNA in RSMCs (Figure 4F and 4G and data not shown).
Origin of SM
Tracing the lineage of SM differentiation has been hampered by the lack of early markers, and, consequently, little knowledge is available on the origin of SMCs, apart from the observation that they arise from both the splanchnic mesoderm and the neural crest; similarly, little is known about the developmental program that leads to their differentiation.1,31–33 Because SMCs readily de-differentiate in vitro and show the same plasticity in vivo, it is conceivable that extrinsic positional cues, likely emanating from adjacent epithelia, play an important part in maintaining and, likewise, in inducing the differentiated SM phenotype. Moreover, different adjacent epithelia may induce subtle phenotypic differences in the associated SMCs. At least in the case of vascular development, SM may also derive from transdifferentiation of endothelial cells and, as mentioned above, from neural crest cells in specific districts of the vascular tree. In this context, it is interesting to note that mesoangioblasts, originally derived from angioblastic cells, at low frequency undergo differentiation into αSMA positive cells, suggesting that they mimic in vitro a phenomenon naturally occurring in vivo.10
Phenotypic Features of Mesoangioblast-Derived SMCs Revealed by Microarray Analysis
Both mesoangioblast cell clones D351 and D16 originate from the embryonic dorsal aorta and share a number of markers and similar transdifferentiation ability, but differ in the spontaneous expression of αSMA and other SMC markers.
Microarray analysis of transcripts of the 2 cell clones confirmed the SM phenotype of D351 cells, which express a number of SM-specific genes. Despite the expression of differentiated markers, D351 cells express some of the stem-cell–specific markers present in D16 cells, such as Sca-1 and Thy-1, but not others, such as CD34, indicating that D351 may represent an intermediate progenitor state toward terminally differentiated SM. This is also suggested by the maintained ability to differentiate into other mesoderm cell types, such as cardiac and skeletal muscle as well as bone.
Transcription Factors Selectively Expressed in Mesoangioblast-Derived SM
Among several transcription factors highly expressed in D351 cells, we focused on msx2 and necdin, whose role had never been associated with SM development and differentiation. Msx2 is expressed in the neural crest, and it is known to play different roles in osteogenesis, chondrogenesis, or in patterning cardiac neural crest.21,23,34 In addition, recent evidence indicates that msx2 is also expressed in the SM layer of many embryonic vessels24 (and B. Robert, personal communication).
Necdin has mainly been implicated in neuronal differentiation and has been associated with the Prader–Willi syndrome.19,20,35 In addition, we found that necdin is expressed in the mesenchyme of the interbranchial arch region, where SMCs that will surround the branchial arteries originate;28 later on, it is expressed in the external muscular layer surrounding the gut and many vessels.
The undifferentiated mesoangioblast stem cell clone D16 does not express SM markers, but is competent for SM differentiation on TGFβ1 induction. A first response to TGFβ1 stimulation is indeed the simultaneous upregulation of necdin and msx2. These results indicate that, at least in vitro, these 2 genes are early targets of TGFβ1. Interestingly, msx2 and necdin are coexpressed only in SMCs or SM-fated cells, such as the mesoangioblast D351 line, whereas non-SMCs, such as 10T1/2 or H5V, express either one or the other. When overexpressed in D16 cells, in the absence of other growth factors, necdin and msx2 together (but not alone) are necessary and sufficient to initiate the differentiation program and to induce the appearance of several SMC markers, thus mimicking the action of TGFβ stimulation. A similar effect was also seen in the embryonic fibroblast line 10T1/2, transfected only with msx2, and in the endothelial cell line H5V, transfected only with necdin, thus “complementing” the already expressed cofactor. In this situation, however, only some SMC markers are induced, such as SM-specific myosin, but not others, such as calponin. This suggests that for the complete induction of the SM differentiation pathway, other factors are needed in endothelial and fibroblast cells.
Conversely, when both necdin and msx2 are downregulated by RNA interference in both D351 and RSMCs, decreased expression of SM markers is observed, suggesting that necdin and msx2 also play a role in the maintenance of the SM-differentiated state.
Interestingly, it has been suggested that both necdin and msx2 have a role as transcriptional corepressors or direct transcriptional repressors.36–39 In particular, necdin has been shown to facilitate cell cycle exit and to promote survival by preventing p53-mediated apoptosis.40–42 Msx2 appears to play roles in cell cycle control and has a proapoptotic effect in vivo, particularly in osteogenesis.43–45 Intriguingly, just recently, it has been shown that necdin and msx2 form a stable complex via MAGE-D1 (NRAGE, Dlxin-1) (K. Yoshikawa, H. Taniura, I. Nishimura, and K. Yoshikawa, manuscript in preparation).
It has been independently reported that myocardin (a serum response cofactor), MRTFA, and Mrf2α and -β (A-T rich interaction domain transcription factors) play a central role in SM differentiation. Myocardin is expressed in visceral and vascular SMC during development,5 and in cotransfection assays of rat aortic cells or 10T1/2 it activates several SMC-specific genes in a TGFβ-independent way.6,29 The 2 myocardin-related transcription factors, MRTFA and -B, expressed in numerous embryonic and adult tissues, have been shown to interact with SRF and to stimulate its transcriptional activity.30 More recently, it has been demonstrated that myocardin mutant mice show a severe deficiency in SMC development.7 D16 and D351 mesoangioblasts do express, constitutively, MRF2α and MRTFA, as well as SRF, whereas they do not express myocardin, before or after TGFβ-induced SMCs differentiation. Thus, it appears that TGFβ-mediated mesoangioblast differentiation into SMC is myocardin independent; on the other hand, necdin and msx2 may cooperate with MRFTA or Mrf2α in the course of this process.
The scenario is also complicated by some of these proteins acting mainly as transcriptional activators, whereas others, including msx2 and necdin, act as repressors. Thus, a role for these 2 proteins in the process may be to repress transcription of another repressor of differentiation or, alternatively, to switch from repressors to activators, depending on their molecular partners, such as in the case of Tcf/Groucho ve Tcf/b catenin molecular complexes.46 Indeed, transient transfection of msx2 and necdin does not activate transcription of smooth promoter–directed reporter genes, but causes accumulation of the relative protein product in the cytoplasm. Whichever the underlying molecular mechanism, msx2 and necdin are necessary and sufficient to activate the SM program and, thus, act as SM master genes at least in a subset of progenitors. They act independently of myocardin (which is not expressed by mesoangioblasts nor induced by TGFβ), indicating that the latter gene may play a crucial role in SM differentiation, but is not absolutely necessary for this process, at least in a subset of SMCs. This fact may explain why ablation of the myocardin gene results in a severe, but not complete, disappearance of SM.
Clearly, more experiments and deeper analysis of null mice are required to definitively clarify the role of these factors in SM differentiation. Currently, the simplest interpretation of the different data is that the embryological and phenotypic heterogeneity of SMCs may easily relate to the existence of different molecular entry points into the differentiation program, each mediated by different combinations of transcription factors, possibly activated at different times and in different places during embryonic, fetal, and postnatal development.
This work was supported by grants from Telethon, the European Community, Duchenne Parent Project Italia/Compagnia di San Paolo, Fondazione Istituto Pasteur-Cenci Bolognetti (FIRB), Consiglio Nazionale delle Ricerche (CNR), and the Italian Ministries of Health and of Education, University and Research (MIUR). We are grateful to Anna Innocenzi for her help with histology and immunofluorescence and to members of the laboratory for their critical reading of the manuscript. We are also indebted to Saverio Sartore for antibodies and to David Sassoon for constructs.
Original received January 28, 2004; revision received May 3, 2004; accepted May 7, 2004.
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