- Open Access
Akt promotes Endocardial-Mesenchyme Transition
© Meadows et al; licensee BioMed Central Ltd. 2009
- Received: 14 May 2009
- Accepted: 21 September 2009
- Published: 21 September 2009
Endothelial to mesenchyme transition (EndMT) can be observed during the formation of endocardial cushions from the endocardium, the endothelial lining of the atrioventricular canal (AVC), of the developing heart at embryonic day 9.5 (E9.5). Many regulators of the process have been identified; however, the mechanisms driving the initial commitment decision of endothelial cells to EndMT have been difficult to separate from processes required for mesenchymal proliferation and migration. We have several lines of evidence that suggest a central role for Akt signaling in committing endothelial cells to enter EndMT. Akt1 mRNA was restricted to the endocardium of endocardial cushions while they were forming. The PI3K/Akt signaling pathway is necessary for mesenchyme outgrowth, as sprouting was inhibited in AVC explant cultures treated with the PI3K inhibitor LY294002. Furthermore, endothelial marker, VE-cadherin, was downregulated and mesenchyme markers, N-cadherin and Snail, were induced in response to expression of a constitutively active form of Akt1 (myrAkt1) in endothelial cells. Finally, we isolated the function of Akt1 signaling in the commitment to the transition using a transgenic model where myrAkt1 was pulsed only in endocardial cells and turned off after EndMT initiation. In this way, we determined that increased Akt signaling in the endocardium drives EndMT and discounted its other functions in cushion mesenchymal cells.
- Endocardial Cushion
- PI3K Inhibitor LY294002
- Endocardial Cell
- Akt1 mRNA
- lacZ Staining
Prior to EndMT, the heart is a tube consisting of an inner endocardium and an outer myocardium separated by a thin layer of extracellular matrix called the cardiac jelly. On E9.5, signals from the myocardium and cardiac jelly induce a subset of endothelial cells in the endocardium to transform into mesenchyme cells. They migrate into the cardiac jelly and proliferate, eventually remodeling the cardiac cushions into heart valve leaflets and septa for a partitioned heart [1–4]. Several coordinated signals in the endocardium and myocardium that modulate EndMT in the AVC have been characterized. Developmental defects in cardiac tissues of TGFβ2 knockout mice, resulting in perinatal mortality, have been attributed to problems with epithelial-mesenchymal transition which underscores that increased TGFβ2 expression between E8.5 and E9.5 is necessary for EndMT to occur [5, 6]. This induction is facilitated by endocardial activation of Notch which also stimulates Snail transcriptional repression of vascular endothelial (VE)-cadherin [7, 8]. VE-cadherin becomes delocalized from cell junctions, facilitating the formation of the sprouting phenotype characteristic of mesenchyme cells . VE-cadherin downregulation is a requisite for EndMT and, like EMT in epithelial cells, coincident N-cadherin upregulation marks the mesenchymal state. By E10.5, VEGF localization in the myocardium causes the cessation of EndMT [10, 11]. It is unclear whether Akt mediates any of these signals, however, PI3K-Akt signaling has been established as a component of EndMT in a number of systems.
In mammary epithelial cells and tumors isolated from mouse mammary glands, Akt is activated in response to EMT induction [12, 13]. The inhibition of PI3K-Akt signaling in metastatic breast tumor cells reduces EMT and transcriptional responses promoted by TGFβ . Moreover, expression of myrAkt in squamous carcinoma cells is sufficient to drive EMT including the relocation of epithelial (E)-cadherin from cell junctions to cytoplasmic granules and the induction of mesenchyme markers, N-cadherin and vimentin . Also, the importance of the Akt signaling pathway in EndMT is underscored by a number of downstream Akt pathways. Akt pathway targets including βCatenin, Notch, and Snail are essential for EndMT and regulate the formation of endocardial cushion [7, 8, 16]. Recently, PI3K signaling was determined to be necessary for AVC mesenchyme outgrowth, as pharmacological inhibition of Akt using an allosteric inhibitor (EMD Akt inhibitor XI) stunted mesenchyme outgrowth in cardiac cushion explants . It is not clear from this pharmacological inhibition, which can target endocardial, myocardial and mesenchyme cells, whether initiation of EndMT is prevented or if there is a subsequent inhibition on the migration and proliferation of mesenchyme cells since the assay relies on counting the cells that have invaded the collagen gel. We have investigated the hypothesis that Akt signaling in endocardial cells initiates EndMT in the developing heart cushions.
In Situ Hybridization
In situ hybridization was performed on E10.5 CD-1 embryos as described in . Specific riboprobes designed to the 3' untranslated regions (3'UTRs) of Akt1 sense, 5' agactctgatcatcatccctgggt 3', and antisense, 5' actctcgctgatccacatcctgag 3' were produced using the T3 transcription reaction kit and Digoxigenin (DIG)-labeling (Roche).
Timed matings between VE-cadherin:tTA and either TET:myrAkt1 mice or VE-cadherin:tTA and TET:lacZ were used to generate embryos expressing VE-cadherin:tTA, TET:myrAkt1, VE-cadherin:tTA/TET:myrAkt1, TET:lacZ, and VE-cadherin:tTA/TET:lacZ. E11.5 embryos were fixed in 3.7% paraformaldehyde, embedded in OCT, and sectioned. MyrAkt embryos were stained with phospho-Akt1 (Upstate) to confirm transgene expression and localization. LacZ embryos were stained with x-gal (Specialty Media) to confirm LacZ localization and were counterstained with eosin.
Ex Vivo AVC Assay
The AVC explants were cultured as previously described in . Relative differences in mesenchyme outgrowth were scored after 48 hours in culture (1 = monolayer growth, 2 = mesenchyme sprouts, 3 = mesenchyme sprouts with extensive migration). Inhibitor used: 10 μM LY294002 (Sigma). Antibody: Cy3 conjugated α SMA (Sigma).
Isolation of primary mouse endothelial cells, Immunofluorescence, Western Blot and RT-PCR
Primary endothelial cells were isolated from the hearts or lungs of wildtype or VE-cadherin:tTA/TET:myrAkt1 mice as described previously . VE-cadherin:tTA/TET:myrAkt1 cells were cultured in the presence of 2 μg/ml TET (Fischer) to suppress myrAkt expression. Immunofluorescent staining and western blot analysis were performed according to standard protocols. Antibodies: N-cadherin and VE-cadherin (BD-Biosciences), Snail (Abcam), Tubulin (Calbiochem). RT-PCR analysis was performed as described previously . Primers: VE-cadherin sense, 5' ggccctggacagactgca 3' and antisense, 5' ttcgtggaggagctgatc 3'. GAPDH sense, 5' ggcaaattcaacggcacagt 3' and antisense, 5' aagatggtgatgggcttccc 3'.
Since the tTA is turned off when the ve-cadherin promoter is downregulated in EndMT, we interpret these data to demonstrate that myrAkt expression in advance of EndMT is sufficient to drive increased EndMT in the endocardium. Together, these data establish a role for Akt1 as a novel signaling component driving the endocardial commitment to EndMT in the developing heart. Interestingly, deletion of only Akt1 does not impair mesenchyme transition, as embryos from Akt1 null mice develop normally. It is likely Akt3 compensates for the absence of Akt1 when it is disrupted, as it is upregulated in endothelial cells isolated from Akt1 knockout endothelial cells . Furthermore, while disruption of no single Akt gene is lethal, Akt1/Akt3 null embryos die between E11 and E12, and have vascular defects and hemorrhages, implicating an endothelial defect. Furthermore, a requirement for Akt1 and Akt3 in heart function was demonstrated by the observation that postnatal day 3 (P3) hearts from Akt1-/- Akt3+/- mice are reduced in size and have enlarged atria and ventricles indicative of a failing cardiovascular system . These mice were reported to have septation defects and thickened valves, which implicates defects in endocardial cushion development and maturation. Together, these data suggest that Akt1 can drive EndMT, and there can be functional redundancy in the Akt isoforms in the absence of Akt1.
Recent studies indicating that endocardium undergoes EndMT following heart injury and leads to fibrosis open the possibility that Akt1 signaling may also participate in pathological processes in adult hearts . Further study is required to investigate this possibility and to fully elucidate the signaling cascade driving Akt-mediated EndMT. In addition, the unique contribution Akt has in the myocardial and mesenchyme cell populations during endocardial cushion formation need to be determined. Our study defines for the first time a novel role for Akt1 at the earliest stages of endocardial commitment to becoming a mesenchymal phenotype.
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