Published online before print November 13, 2008, doi: 10.1161/CIRCRESAHA.108.180752
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© 2008 American Heart Association, Inc.
Molecular Medicine |
MEKK3 Initiates Transforming Growth Factor β2–Dependent Epithelial-to-Mesenchymal Transition During Endocardial Cushion Morphogenesis
From the Department of Pharmacology and Toxicology (M.V.S., D.M.B., P.P., E.R., R.R.V., T.D.C.), Department of Molecular and Cellular Biology (T.D.C.), Steele Children’s Research Center (T.D.C.), and BIO5 Institute (R.R.V., T.D.C.), University of Arizona, Tucson.
Correspondence to Todd D. Camenisch, PhD, University of Arizona, Department of Pharmacology and Toxicology, College of Pharmacy, 1703 E Mabel St, Tucson, AZ 85721. E-mail camenisch{at}pharmacy.arizona.edu
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Abstract |
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Congenital heart defects occur at a rate of 5% and are the most prevalent birth defects. A better understanding of the complex signaling networks regulating heart development is necessary to improve repair strategies for congenital heart defects. The mitogen-activated protein 3 kinase (MEKK3) is important to early embryogenesis, but developmental processes affected by MEKK3 during heart morphogenesis have not been fully examined. We identify MEKK3 as a critical signaling molecule during endocardial cushion development. We report the detection of MEKK3 transcripts to embryonic hearts before, during, and after cardiac cushion cells have executed epithelial-to-mesenchymal transition (EMT). MEKK3 is observed to endocardial cells of the cardiac cushions with a diminishing gradient of expression into the cushions. These observations suggest that MEKK3 may function during production of cushion mesenchyme as required for valvular development and septation of the heart. We used a kinase inactive form of MEKK3 (MEKK3KI) in an in vitro assay that recapitulates in vivo EMT and show that MEKK3KI attenuates mesenchyme formation. Conversely, constitutively active MEKK3 (ca-MEKK3) triggers mesenchyme production in ventricular endocardium, a tissue that does not normally undergo EMT. MEKK3-driven mesenchyme production is further substantiated by increased expression of EMT-relevant genes, including TGFβ2, Has2, and periostin. Furthermore, we show that MEKK3 stimulates EMT via a TGFβ2-dependent mechanism. Thus, the activity of MEKK3 is sufficient for developmental EMT in the heart. This knowledge provides a basis to understand how MEKK3 integrates signaling cascades activating endocardial cushion EMT.
Key Words: epithelial-to-mesenchymal transition • MEKK3 • TGFβ2 • endocardial cushions • heart
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Introduction |
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The formation of endocardial cushions is an important step in valvuloseptal development. After the heart has undergone rightward looping, extracellular matrix deposits in the atrioventricular canal (AVC) and outflow tract form the initial endocardial cushions.1 A vital event in endocardial cushion morphogenesis is epithelial-to-mesenchymal transition (EMT), where specific cells in the endocardium surrounding the cardiac cushions are activated to delaminate from the endocardial layer, transform into mesenchyme, and migrate into the underlying extracellular matrix.2 This newly formed tissue will be remodeled to form valves and septa, creating the partitioned 4-chamber heart.
Approximately 5% of children born have a congenital heart defect (CHD).3 CHDs include valvular and septal defects that cause insufficient blood flow through the heart. These patients often require surgery during infancy, and such defects can cause complications later in life. Although there is increased knowledge about genetic factors that are responsible for specific CHDs, little is known how these genetic errors translate into CHD phenotypes. Misexpression or improper coding of effector proteins may lead to defective extracellular and/or intracellular signaling, resulting in altered cellular processes critical to heart development. Marfan’s syndrome is an example of disrupted signaling during heart formation. A mutation in the fibrillin-1 gene, an extracellular matrix protein that regulates transforming growth factor (TGF)β, results in myxomatous valves.4 There are also a variety of Marfan-like diseases in which mutations in the TGFβ receptors are responsible for valvular defects,5 further suggesting that TGFβ signaling must be well regulated during valvulogenesis. Another example is Noonan syndrome, a disorder caused by dysregulated signal transduction by an overactive tyrosine phosphatase, Shp2.6 Transgenic mice with a common Shp2 mutation, Q79R, found in patients with Noonan syndrome are prone to pulmonary valve stenosis attributable to elevated cell proliferation and reduced apoptosis during endocardial cushion development.7,8 Wild-type Shp2 activity is involved in mitogen-activated protein kinase (MAPK) signaling downstream of receptor tyrosine kinases,9 such as epidermal growth factor receptor, which regulate heart valve formation.10 Regulation of MAPK pathways, including Ras/Raf/MEK/extracellular signal-regulated kinase (ERK), is affected by TGFβ,11 but this has yet to be examined in endocardial cushion development.
Among signaling factors important for cardiac cushion EMT are bone morphogenetic protein (BMP)2 and TGF-β2. BMP2 is vital for development of endocardial cushions as conditionally removing BMP2 from AVC myocardium causes abnormal segmentation of AV myocardium and failure to form endocardial cushions.12 TGFβ2 is also important in activating endocardial cushion EMT and regulating cushion morphogenesis.13–15 Mice deficient for TGFβ2 exhibit cardiac defects, including dilated aortic walls, overriding tricuspid valves, myxomatous valves, and outflow tract malformations.15,16 Although TGFβ family growth factors are important to cardiac cushion development, the regulation and action of these factors is not completely understood.
MAPK cascades often begin with ligand-mediated activation of a receptor. Those involved in developmental processes include receptor tyrosine kinases, G protein–coupled receptors, and TGFβ/BMP receptors among others. The activated receptor recruits intracellular effectors that mediate specific signaling pathways. For example, epidermal growth factor receptor tyrosine kinase activation leads to recruitment of effectors, such as Grb2 and Sos, which lead to activation of the small GTPase Ras. Consequently, a MAP3K, like Raf, will be activated by Ras, so that it phosphorylates and activates downstream MAPKK (MEK). The activated MAPKK will then phosphorylate a MAPK (p38, ERK, ERK5, or JNK), which translocates into the nucleus to mediate the activities of transcription factors.9,17 These cascades are responsible for control of cell proliferation, differentiation, apoptosis, or migration. MAPKs, such as ERK, have been implicated during endocardial cushion development,7,8,18 but their roles are still being defined. Regulators of ERK MAPK, including Shp2, Ras, and Raf, are also implicated in proper heart valve development as mutations in these genes affect cardiac cushion EMT and cause valvular defects.7,19–21
We have previously shown that the MAPKKK MEKK4 is necessary but not sufficient for cardiac cushion EMT during heart development.22 The present work elucidates the role of MEKK3 with different functions than MEKK4 during cardiac cushion morphogenesis. Our results show that MEKK3 is both necessary and sufficient for EMT in the embryonic heart, further supported by previous results that demonstrate loss of MEKK3 is embryonic lethal.23 Finally, MEKK3 functions via a TGFβ2-dependent mechanism to produce valve mesenchyme.
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Materials and Methods |
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Immunodetection on Embryo Sections
Mouse embryos were collected from stages embryonic day (E)9.5 to E12.5 and processed as described previously.22 Rabbit polyclonal antibody24 and goat anti–rabbit-Alexa594 secondary antibody (Molecular Probes, Invitrogen, Carlsbad, Calif) was used to detect MEKK3 documenting fluorescence with a Leica DMLB fluorescence microscope (Leica, Bannockburn, Ill) with Image ProPlus software (Media Cybernetics, Bethesda, Md). For vimentin detection, an anti-vimentin antibody (Santa Cruz Biotechnology, Santa Cruz, Calif)22 was used with an anti–goat-Alexa594 secondary antibody (Molecular Probes). Nuclear staining was accomplished with Hoechst dye (Molecular Probes). Tissue sections and cells treated with only secondary antibody were used to control for background staining (data not shown).
RT-PCR and Real-time RT-PCR
RNA was isolated from embryonic hearts at stages E9, E9.5, E10.5, and E14.5 with TRIzol (Invitrogen). Reverse transcription reactions were accomplished using a reverse-transcription reaction kit (Fermentas, Glen Burnie, Md). Primers for PCR are described in the expanded Materials and Methods section in the online data supplement, available at http://circres.ahajournals.org.
Real-time PCR was performed using a Roche 480 LightCycler (Roche, Indianapolis, Ind). Real-time RT-PCR was performed in triplicate, and statistics are included as SDs.
Endocardial Cushion and Ventricular Explants
Embryos were removed from pregnant female mice at E9.5 (Sigma, St Louis, Mo). Hearts were dissected from embryos at E9.5, and microdissection of the AVC cushions and ventricles was performed in Tyrode’s buffer.14,19,25 AVCs and ventricles were dissected and placed in adenoviral-transfection mixes as described previously.22 Four explants were placed per type I collagen gel and hydrated overnight with 0.1% ITS in Opti-MEM (Gibco-BRL) according to published methods.14,22 Additional transfection mix was placed on each explant for 3 hours and replaced by complete media (1% bovine growth serum [Hyclone, Logan, Utah]; 1% anti-mycotic/anti-biotic, 1x medium 199 [Gibco-BRL]; 0.01% ITS [Gibco-BRL]). AVC explants were allowed to incubate at 37°C for 48 hours, whereas ventricular explants incubated at 37°C for 72 hours. Cultures were used for RNA isolation or fixed with 2% paraformaldehyde for detection of vimentin and cell enumeration. For TGFβ2 neutralizing experiments, anti-TGFβ2 and IgG-isotype control (R&D systems, Minneapolis, Minn) were used at a concentration of 250 pg/mL in media.26 Other neutralization treatments are described in the expanded Materials and Methods section (online data supplement). Some cultures were provided with 5-bromodeoxyuridine (BrdUrd) as detailed in the expanded Materials and Methods section (online data supplement). Statistics for experiments were calculated using the Student’s 2-sample unpaired t test and SD.
Preparation of Adenoviral-Mediated Transfection Mixture for Explants
Adenoviral-mediated transfections were accomplished as previously described.19,22 Plasmids for mammalian expression (pCMV) containing wt-MEKK3, the kinase domain of MEKK3 (ca-MEKK3), or MEKK3KI (K391M) (1 µg per explant) were added with adenovirus that expresses green fluorescent protein (Ad5-GFP, 108 pfu) (Iowa Gene Vector Transfer Core) in Opti-MEM (Gibco-BRL). Details are listed in the online data supplement.
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Results |
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MEKK3 Expression in the Embryonic Heart
MEKK3 transcripts are detected in the rudimentary heart at stages E9, E9.5, E10.5, and E14.5, stages before, during, and following cardiac cushion EMT (Figure I, A, in the online data supplement).14 To localize MEKK3 protein in the embryonic heart, we performed immunofluorescence staining on embryo sections from E9.5 to E12.5 with an anti-MEKK3 antibody. MEKK3 is detected to the myocardium at all stages examined. At E9.5 to E10.5, MEKK3 is also detected to the endocardium lining the AVC cushions and a diminished gradient of expression in mesenchymal cells within the cushion matrix (Figure 1A, 1B, 1D, and 1E). Additionally, little MEKK3 is detected in the ventricular endocardium, which does not normally undergo transition to mesenchyme (Figure 1C and 1F). This immunolocalization places MEKK3 in the appropriate area of the heart when EMT occurs. At later stages E11.5 (supplemental Figure I, B and C) and E12.5 (supplemental Figure I, D and E), MEKK3 is localized to the endocardium, although moderately diminished at E12.5. This prolonged expression of MEKK3 beyond the EMT period suggests other roles for MEKK3 in endocardial cell function.
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MEKK3 Is Necessary for Cardiac Cushion EMT
In vivo EMT is recapitulated in vitro via the endocardial cushion explant assay.2 In this assay, endocardial cushions of the AVC are microdissected from E9.5 embryos and explanted onto collagen gels.14 The endocardial cells migrate onto the gel, where they undergo EMT and invade into the collagen matrix. Mesenchyme formation and invasion is scored to determine the extent of EMT. Mesenchymal cells are distinguished as individual, elongated cells with filopodia, whereas epithelial cells have cell–cell contacts with adjacent cells and have a rectangular or rounded morphology.2,19 In addition, immunofluorescent detection of vimentin indicates mesenchymal cells, because its expression is upregulated in cells of this phenotype.27 We used this assay to determine whether the kinase activity of MEKK3 is required for EMT and production of AVC cushion mesenchyme. Kinase inactive MEKK3 (MEKK3KI), which acts as a dominant negative, was used in the AVC endocardial cushion explant assay. MEKK3KI was created by mutating the active site lysine at position 391 to methionine (K391M).24 Naïve and Ad5-GFP–infected cultures were used as controls and displayed normal amounts of mesenchyme production (Figure 2A, 2B, and 2D). Addition of wild-type MEKK3 (wt-MEKK3) to explant cultures did not have an effect on AVC cushion EMT (Figure 2D). In contrast, MEKK3KI causes a 3-fold reduction in mesenchyme formation compared to control explant cultures (Figure 2C and 2D). Active caspase-3 detection in AVC explants with MEKK3KI shows a significant 2-fold increase in apoptosis compared to controls after 24 hours (Figure 3A through 3C; P=0.0077). This suggests that MEKK3 kinase activity is necessary for developmental EMT during endocardial cushion morphogenesis and functions in endothelial cell survival.
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MEKK3 Is Sufficient to Activate EMT
Ventricular endocardium does not undergo EMT because the myocardium of the ventricle is not competent to promote transformation of AVC endocardium, suggesting the myocardium underlying the AVC is regionally restricted for promoting mesenchyme formation.2 Therefore, explants of endocardium derived from the ventricle are used to screen candidate factors for sufficiency to induce EMT. For example, Alk2, a TGFβ/BMP receptor, has been determined sufficient for EMT in this manner.28 We established ventricular endocardial explants to determine whether the catalytic activity of MEKK3 can drive EMT. A constitutively active form of MEKK3 (ca-MEKK3) was created by truncating the N-terminal region, leaving the fully active kinase domain.29 Naïve and Ad5-GFP transfected ventricular endocardium exhibited little to no mesenchyme formation (Figure 4A and 4D). Explants treated with constitutively active Alk2 (ca-Alk2), which has been shown to activate EMT in this system,28 served as a positive control, driving mesenchyme outgrowth (Figure 4B and 4D). ca-MEKK3 also induced EMT in explanted ventricular endocardium to levels comparable to those with ca-Alk2 (Figure 4B through 4D). Mesenchymal cells were confirmed by vimentin detection in cells of ca-MEKK3 and Alk2 control cultures (Figure 4B and 4C). In addition, cell proliferation was examined by BrdUrd incorporation into ventricular explant cultures (Figure 5). The number of proliferating cells is significantly increased in ca-MEKK3 cultures (P=0.0037), and increased proliferation is observed primarily within endothelial cells (P=0.0007) (Figure 5D and 5E), suggesting that ca-MEKK3 is primarily involved in endothelial cell proliferation. These data demonstrate that ca-MEKK3 is sufficient for the production of cardiac mesenchyme, partly by increasing the population of endocardial cells available for EMT.
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MEKK3 Triggers Expression of Factors That Promote EMT
A TGFβ2/TGFβ type III receptor (TβRIII) signaling cascade is critical for the EMT program.30 Additionally, molecules including BMP2, Has2, and Snail2 (Slug) are implicated in this process.12,19,31 Importantly, periostin is a marker of mature cushion mesenchyme.32,33 Therefore, real-time RT-PCR was performed on RNA samples prepared from ventricular explants with or without ca-MEKK3 to examine the expression of these EMT-related genes. MEKK3 mRNA is increased in ventricular explants provided with ca-MEKK3, which along with fluorescence detection of GFP (supplemental Figure II), demonstrates transfection of the cultures (Figure 6). TβRIII is expressed 64 times more in the ca-MEKK3 samples compared to controls (Figure 6). TβRIII is required to mediate TGFβ2-induced EMT.30,34 Because TGFβ2 is necessary in promoting EMT in endocardial cushions,14,26 we detected a substantial increase in TGFβ2 message in ca-MEKK3 samples but not in controls (Figure 6). Furthermore, expression of Snail2 (Slug), a transcription factor upregulated by TGFβ2 signaling,31 is also induced by ca-MEKK3–expressing ventricular cultures (Figure 6). BMP2 is also necessary for mesenchyme production from the AVC cushion endocardium and is expressed in AVC cushion myocardium but not in the ventricular myocardium.12,35 BMP2 expression is not observed in control ventricular explant cultures, but ca-MEKK3 induces expression of BMP2 in this system (Figure 6). TGFβ1 and TGFβ3 are also induced by ca-MEKK3 as compared to control samples (Figure 6). Examining expression of genes that are normally activated by cells undergoing EMT,19 we detected induced expression of hyaluronan synthase 2 (Has2) compared to controls, further supporting a role for MEKK3 in mesenchyme production (Figure 6). Periostin expression confirms the mature mesenchymal phenotype of cells that invade into the extracellular matrix.32,36 Expression of periostin is significantly increased in ca-MEKK3 cultures compared to controls (Figure 6), which coincides with the increase in mesenchymal cells observed in Figure 4. These data suggest that ca-MEKK3 in ventricular explants cultures is inducing expression of genes necessary for the onset of EMT and eventually genes that are indicative of mesenchyme production.
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-, synthase 1). Genes examined were MEKK3, TβRIII, TGFβ1, TGFβ3, TGFβ2, BMP2, Snail2, Has2, and Periostin. Ad-GFP control (C) cultures (open bars) vs ca-MEKK3 (M) cultures (filled bars). Primers to the MEKK3 kinase domain were used to check transfection of ca-MEKK3. Normalized expression was calculated using mean control value for the gene analyzed.
ca-MEKK3 Induces Production of TGFβ2
The observation that MEKK3 activity increases expression of TGFβ2, TβRIII, and Slug suggests that it may be upstream of this key TGFβ growth factor cascade as required for EMT. Therefore, we examined explant supernatants for the presence of TGFβ2 to determine whether there is an increase in TGFβ2 that coincides with elevated gene expression. A significant 4-fold increase in TGFβ2 is detected in ca-MEKK3 explant cultures compared to GFP controls (Figure 7A). Conversely, a 4.4-fold decrease in TGFβ2 is detected in endocardial cushion explants treated with kinase inactive MEKK3 (MEKK3KI) (supplemental Figure III). Thus, MEKK3 increases production of TGFβ2 message and protein. Next, we examined phosphorylated Smad2 (p-Smad2) as a functional target of TGFβ signaling in ventricular explants cultures with or without ca-MEKK3. Detection of filamentous actin (F-actin, green) shows maintained cell–cell junctions between ventricular endocardial cells in controls (Figure 7B), while these junctions are dismantled in the presence of ca-MEKK3, indicating initiation of EMT (Figure 7C). Although low levels are detected in control ventricular endocardium, we observe increased p-Smad2 in cultures with ca-MEKK3 (Figure 7, compare D and E). Collectively, these observations support that MEKK3 induces EMT through TGFβ2.
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Neutralization of TGFβ2 Inhibits ca-MEKK3-induced EMT
Our observations strongly suggest MEKK3 stimulates TGFβ2 production coincident with EMT. To determine whether TGFβ2 is a central factor mediating MEKK3-induced EMT, ca-MEKK3–expressing ventricular explant cultures were cultured with or without neutralizing anti-TGFβ2 antibody. As expected, ca-MEKK3 is able to induce mesenchyme production in explants treated with control IgG (Figure 8C) and those with ca-MEKK3 alone (Figure 8, compare A and B). In contrast, addition of anti-TGFβ2 dramatically inhibits the ability of ca-MEKK3 to stimulate mesenchyme formation (Figure 8D and 8E). Because increases in expression were also observed for BMP2, TGFβ1, and TGFβ3, it was necessary to examine whether these factors have a significant contribution to ca-MEKK3-induced mesenchyme production. Effects of neutralizing each of these factors in cultures of ventricular endocardium with ca-MEKK3 were assessed for EMT. We used the BMP antagonist noggin and antibodies against TGFβ3 and TGFβ1 in these experiments. Decreases in EMT are observed with noggin and anti-TGFβ3 treatment in ca-MEKK3–expressing ventricular explants; however, mesenchyme production in each was significantly higher than with anti-TGFβ2 treatment (Figure 8 and supplemental Figure III, C and D). Blockade of TGFβ1 has no significant effect on ca-MEKK3–induced EMT (Figure 8E and supplemental Figure III, E). Thus, neutralizing TGFβ2 induced by ca-MEKK3 blocks EMT restoring the normal phenotype of the ventricular endocardium, which does not normally undergo EMT. Blocking activities of BMP2, TGFβ3, or TGFβ1 does not fully restore the normal phenotype (supplemental Figure IV). Furthermore, TGFβ2 is elevated even with blockade of BMP2, TGFβ3, or TGFβ1 compared with control cultures (supplemental Figure V). The dramatic reduction in EMT when TGFβ2 is neutralized also suggests that the other TGFβ factors do not compensate for TGFβ2 loss in this system. Collectively, these data show that the production of endocardial-derived mesenchyme by active MEKK3 involves a TGFβ2-dependent mechanism.
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Discussion |
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MEKK3 knockout mice exhibit myocardial and endocardial defects, which are responsible, in part, for their death at E10.5.37 Although not a primary focus of Yang et al,37 the authors show an acellular AVC cushion in the MEKK3-deficient embryos. These defects are consistent with localization of MEKK3 to the myocardium and AVC cushion endocardium in our studies. Importantly, we detected very little MEKK3 in ventricular endocardium, which is a region in the heart that does not execute developmental EMT.2 This restricted detection of MEKK3 to the cushion endocardium is consistent with expression patterns of other proteins necessary and sufficient for cardiac cushion EMT. One example is Alk2, a TGFβ/BMP receptor, which is sufficient to activate mesenchyme production during valvulogenesis.28 Hence, localization of MEKK3 to the AVC cushion endocardium implies that it is also involved in activation of endocardium to become mesenchyme. Moreover, MEKK3 kinase activity is necessary for the transition from endocardium to mesenchyme, because endocardial cushion explants failed to display normal levels of mesenchymal cells with MEKK3KI. We observe endocardial migration onto the gel, but a majority of cells do not become vimentin-positive mesenchymal cells in the presence of MEKK3KI (see supplemental Figure II and Figure 2). There is also an increase in apoptosis observed in AVC explant cultures provided with MEKK3KI, which is consistent with MEKK3 being involved in endothelial cell survival in MEKK3-deficient mice.38 Remarkably, ca-MEKK3 is sufficient to induce EMT from ventricular endocardium, similar to that with ca-Alk2.22,28 Additionally, an increase in endothelial cell proliferation is observed in ca-MEKK3–expressing ventricular endocardium, consistent with decreased endothelial cell proliferation in the MEKK3-deficient embryonic heart.38 Together, these data demonstrate that MEKK3 is necessary and sufficient for mesenchyme production, and this is partially attributable to increased proliferation and survival of endocardial cells available for the EMT program.
Our studies show that TGFβ2 is necessary for ca-MEKK3-driven EMT in ventricular endocardium. This growth factor is especially important for mesenchyme production in the murine heart.14 TGFβ2 involvement in endocardial cushion morphogenesis is supported by a TGFβ2 knockout mouse model that exhibits heart defects including valve and outflow tract malformations.15,16 This phenotype also demonstrates that TGFβ2 has developmental functions which are not compensated for by the other TGFβs. In addition, MEKK3 is upstream of the MAPKs p38 and JNK, which are important in activating ATF2, a transcription factor that stimulates TGFβ2 expression.39 Normally ventricular endocardium expresses little TβRIII, a receptor necessary for TGFβ2-induced EMT.30 In our studies, TβRIII and TGFβ2 are both upregulated in response to ca-MEKK3, demonstrating a link between the MAP3K, MEKK3, and a defined EMT-activating pathway. Mesenchyme production by ca-MEKK3 in ventricular endocardium was ablated by neutralizing TGFβ2, demonstrating that MEKK3 is upstream of TGFβ2-mediated EMT. Consistent with this, we detected an increase in Snail231 during ca-MEKK3 conditions, suggesting intact TGFβ2 activity during MEKK3 initiated EMT. We detected a number of additional genes upregulated as a result of MEKK3 activity including Has2, which is a synthetase that produces hyaluronan, a vital extracellular molecule required for cardiac EMT.19 Another gene upregulated by MEKK3 is TGFβ3, which becomes expressed in cushion mesenchyme and contributes to formation of fibrous structures in the developing heart.23 In addition, BMP2 message is induced by ca-MEKK3 but not in control cultures. Alk3, a BMP receptor, in the myocardium is needed for TGFβ2 production and cushion morphogenesis, so MEKK3 may act upstream of BMP2 signaling for TGFβ2-dependent EMT.40 BMP2 also promotes cell migration and tissue maturation during endocardial cushion morphogenesis, demonstrating its additional role in post-EMT events.41 Interestingly, BMP2 is shown to act synergistically with TGFβ3 to promote cardiac cushion EMT in chicken.42 Because we show that neutralizing BMP2 or TGFβ3 in ca-MEKK3–expressing ventricular explants does not decrease mesenchyme production to the extent by blocking TGFβ2, it is likely that these molecules either enhance TGFβ2-induced EMT or play a role in the invasion step of EMT. TGFβ1 expression increases with ca-MEKK3 in ventricular explants; however, there is no significant decrease in MEKK3-driven mesenchyme production after blockade of TGFβ1. Embryos from female TGFβ1 knockout mice demonstrate in utero mortality with cardiac defects, whereas females that are heterozygous or wild-type for TGFβ1 are able to transfer TGFβ1 to the developing embryo for normal cardiac development.43 Letterio et al also indicate that TGFβ1 is important to endocardial cell survival.43 This may explain why fewer cells are observed with neutralizing TGFβ1 than other groups (data not shown), although the percentage of cells undergoing EMT is relatively equal to ca-MEKK3–expressing ventricles. Periostin is expressed by mesenchymal cells within the endocardial cushions,36 and its overexpression causes transition of cells to mesenchyme.44 Periostin expression is significantly increased in samples with ca-MEKK3 versus controls, which supports the ability of MEKK3 to produce mature mesenchyme. Collectively, our studies show that MEKK3 mediates EMT and production of cushion mesenchyme via a TGFβ2-dependent mechanism.
This is the first time that MEKK3 has been implicated in developmental EMT and functionally linked to early events in heart valve formation. We observe MEKK3 is mediating pathways, specifically through TGFβ2, that are activating EMT. As such, active MEKK3 is sufficient for mesenchyme production. Deciphering upstream mediators of MEKK3 will be important to connect factors that establish an activated endothelium with the ability to execute EMT. In summary, we present MEKK3 as a critical signaling effector in the pathways that mediate endocardial cushion EMT.
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Acknowledgments |
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We thank S. Lalani, B. Eaker, J. Sollome, and A. Fritz for technical assistance. We acknowledge Dr J. V. Barnett (Vanderbilt University) for generously providing ca-Alk2 adenovirus, Dr R. Runyan (University of Arizona) for providing blocking antibodies, and Dr J. Schroeder for advice on the manuscript.
Sources of Funding
This work was supported by NIH grants HLBI087736 (to M.V.S.), HLBI077493 (to T.D.C), and AG019710 (to R.R.V.). We also thank PANDA (People Acting Now Discover Answers) for support through the Steele Children’s Research Center (to T.D.C.).
Disclosures
None.
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Footnotes |
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Original received September 6, 2007; resubmission received June 4, 2008; revised resubmission received October 30, 2008; accepted October 30, 2008.
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References |
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