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TAK1/MAP3K7, TAB2 and PKA-Cα Constitute a Novel Branch of TGFB/BMP Signaling in the Primary Cilium to Control Heart Development
Background: The heart is the first organ to form during human embryonic development in a process orchestrated spatially and temporarily by networks of morphogens, signaling pathways, transcription factors and chromatin modulators [1]. A significant part of these processes is linked to functions of both motile and primary cilia [2], the latter orchestrating canonical TGFB/BMP signaling in cardiomyogenesis [3]. Disturbing the developmental processes lead to congenital heart disease (CHD), the most common birth defect, a highly heterogenic spectrum of diseases with elusive etiology. TGFB Activated Kinase 1 (TAK1/MAP3K7), TAK1 Binding Protein 2 (TAB2) and cAMP-dependent protein kinase catalytic subunit alpha (PKA-Cα) all play major roles in non-canonical TGFB/BMP signaling, occurring when TAK1 autophosphorylates at Thr184,187 upon TAB2-binding [4] and when PKA-Cα phosphorylates TAK1 at Ser412 [5]. Rare genomic and intragenic variants in their respective genes result in rare multisystem disorders, such as frontometaphyseal dysplasia (FMD, OMIM #617137), Cardiospondylocarpofacial syndrome (CSCF, OMIM #157800) and cardioacrofacial dysplasia 1 (CAFD1, OMIM #601639), with clinical manifestations including CHD. Nonetheless, the spatiotemporal profile of TAK1, TAB2 and PKA-Cα in the context of cardiac development and disease remains largely unknown.
Aims: The current study was undertaken to advance our understanding of cardiac development and CHD by exploring the ciliary role of TAK1 and signaling partners in mediating molecular, cellular, and developmental processes orchestrating cardiogenesis.
Methods: Exome sequencing analysis; Generation and maintenance of tak1 and tab2 zebrafish mutant lines; zebrafish whole-mount immunostaining and proliferation assay; Alcian blue staining; Bulk-RNAseq on whole zebrafish hearts; Immunohistochemistry (IHC); immunocytochemistry; Generation and differentiation of mouse embryonic stem cell (mESC) gastruloids; Culture, stimulation assays and transfection of hTERT-RPE1 cells; P19Cl6 mouse teratoma stem cell maintainence, cardiomyocyte differentiation assay and inhibition assays; P19CL6 gene knock-out; immunofluorescence microscopy (IFM); SDS-PAGE and western blot analysis.
Results: We demonstrate a role for TAK1 in cardiac development, and that this role is dependent on its localization to the primary cilium. Homozygous zebrafish mutants for tak1 resulted in enlarged atrial chambers, limited trabeculation, tachycardia and electrical conduction defects, and with an increase in cell proliferation in both cardiac chambers. Bulk RNAseq of homozygous mutant hearts highlighted a significant decrease in expression of genes related to cardiomyocyte differentiation and extracellular matrix development. Specifically, we find that tak1-/- zebrafish recapitulate CSCF phenotypes including craniofacial defects and brachydactyly. IHC of zebrafish hearts and cardiomyogenic regions of mESC gastruloids unveiled that TAK1 predominantly localizes to the primary cilium. TGFB1 and BMP2 stimulation in RPE1 cells revealed that TAK1 is phosphorylated at the cilium at both Ser412 and Thr184,187 sites, and thus activated. Furthermore, patient mutated TAK1 failed to localize to the primary cilium of RPE1 cells. During cardiomyocytic differentiation of P19CL6 stem cells, we found that while TAK1 and PKA-Cα are stably localized to the primary cilium, TAB2 localization and TAK1 phosphorylation are upregulated in a differentiation-dependent manner. Knocking out Tak1 resulted in a delay of GATA4, α-actinin and Troponin-I expression, highlighting a crucial role for TAK1 in cardiomyogenesis. We were able to recapitulate this result using TAK1 inhibitors. Finally, we show that JNK1/2, a downstream target of TAK1 [6], was phosphorylated at the primary cilium in a differentiation-dependent manner, which is lost upon TAK1 inhibition.
Conclusions: We show that TAK1 plays a crucial role in cardiac development. Our work indicates TAK1 and its upstream regulators, TAB2 and PKA-Cα, undertake key roles at the primary cilium in mediating cardiomyocyte differentiation. We further suggest a model in which PKA-Cα-based crosstalk between non-canonical TGFB/BMP and Hedgehog signaling is key for cardiac development.
Funding: The study was supported by grants from the Danish Heart Foundation, Børnehjertefonden, Novo Nordisk Foundation, Carlsberg Foundation, European Commission Horizon 2020 research and innovation programme, Marie Skłodowska-Curie Innovative Training Networks (grant agreement No. 861329), Independent Research Fund Denmark (grant number 3103-00177B). The Novo Nordisk and Novozymes Scholarship Program, Læge Sophus Carl Emil Friis og hustru Olga Doris Friis’ Legat, Direktør Jacob Madsens og Hustru Olga Madsens Fond, Fonden til Lægevidenskabens Fremme, Helge Peetz og Verner Peetz og Hustru Vilma Peetz legat and Islamic Development Bank (IsDB).
Speakers
Oskar Kaaber Thomsen