The data identify a new cellular and molecular mechanism underlying BAV. The availability of an animal model for the most frequent human BAV opens the way for the elucidation of BAV pathogenesis and the development of much needed therapies.
We investigated the role of phospholipase D (PLD) and its product phosphatidic acid (PA) in myogenic differentiation of cultured L6 rat skeletal myoblasts. Arginine-vasopressin (AVP), a differentiation inducer, rapidly activated PLD in a Rho-dependent way, as shown by almost total suppression of activation by C3 exotoxin pretreatment. Addition of 1-butanol, which selectively inhibits PA production by PLD, markedly decreased AVP-induced myogenesis. Conversely, myogenesis was potentiated by PLD1b isoform overexpression but not by PLD2 overexpression, establishing that PLD1 is involved in this process. The expression of the PLD isoforms was differentially regulated during differentiation. AVP stimulation of myoblasts induced the rapid formation of stress fiber-like actin structures (SFLSs). 1-Butanol selectively inhibited this response, whereas PLD1b overexpression induced SFLS formation, showing that it was PLD dependent. Endogenous PLD1 was located at the level of SFLSs, and by means of an intracellularly expressed fluorescent probe, PA was shown to be accumulated along these structures in response to AVP. In addition, AVP induced a PLD-dependent neosynthesis of phosphatidylinositol 4,5-bisphosphate (PIP2), which also was accumulated along actin fibers. These data support the hypothesis that PLD participates in myogenesis through PA- and PIP2-dependent actin fiber formation.
Angiotensin II (AII), a potent vasoactive hormone, acts on numerous organs via G-protein-coupled receptors and elicits cell-specific responses. At the level of the heart, AII stimulation alters gene transcription and leads to cardiomyocyte hypertrophy. Numerous intracellular signaling pathways are activated in this process; however, which of these directly link receptor activation to transcriptional regulation remains undefined. We used the atrial natriuretic factor (ANF) gene (NPPA) as a marker to elucidate the signaling cascades involved in AII transcriptional responses. We show that ANF transcription is activated directly by the AII type 1 receptor and precedes the development of myocyte hypertrophy. This response maps to STAT and GATA binding sites, and the two elements transcriptionally cooperate to mediate signaling through the JAK-STAT and protein kinase C (PKC)-GATA-4 pathways. PKC phosphorylation enhances GATA-4 DNA binding activity, and STAT-1 functionally and physically interacts with GATA-4 to synergistically activate AII and other growth factor-inducible promoters. Moreover, GATA factors are able to recruit STAT proteins to target promoters via GATA binding sites, which are sufficient to support synergy. Thus, STAT proteins can act as growth factor-inducible coactivators of tissue-specific transcription factors. Interactions between STAT and GATA proteins may provide a general paradigm for understanding cell specificity of cytokine and growth factor signaling.
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