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KLF2-induced actin shear fibers control both alignment to flow and JNK signaling in vascular endothelium

ABSTRACT: The shear stress-induced transcription factor Krüppel-like factor 2 (KLF2) confers anti-inflammatory properties to endothelial cells through inhibition of activator protein 1, presumably by interfering with MAPK cascades. To gain insight into the regulation of these cascades by KLF2, we used antibody arrays in combination with time-course mRNA micro-array analysis. No gross changes in MAPKs were detected, rather phosphorylation of actin cytoskeleton-associated proteins, including Focal Adhesion Kinase, was markedly repressed by KLF2. Furthermore, we demonstrate that KLF2-mediated inhibition of Jun NH2-terminal kinase (JNK) and its downstream targets ATF2/c-Jun is dependent on the cytoskeleton. Specifically, KLF2 directs the formation of typical short basal actin filaments, we term shear fibers, which are distinct from thrombin- or TNF-α-induced stress fibers. KLF2 is shown to be essential for shear stress-induced cell alignment, concomitant shear fiber assembly and inhibition of JNK signaling. These findings link the specific effects of shear-induced KLF2 on endothelial morphology to the suppression of JNK MAPK signaling in vascular homeostasis via novel actin shear fibers.

ORGANISM(S): Homo sapiens

PROVIDER: GSE19412 | GEO | 2009/12/11

SECONDARY ACCESSION(S): PRJNA121787

REPOSITORIES: GEO

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Project description:d9 and d12 Mks were either cultured statically or subjected to shear flow for 30 min; at d9, half the Mks were placed back in culture for 30 min (60 min time point) Megakaryocytes (Mks) are exposed to shear flow as they migrate from the bone marrow hematopoietic compartment into circulation thus releasing platelets and pro/preplatelets directly into the blood stream. Shear forces have been now established as promoting Mk maturation and platelet biogenesis. In order to understand the underlying mechanisms that modulate the response of Mks to shear forces, we carried out transcriptional analysis on immature and mature stem cell-derived Mks that were exposed to physiologically-relevant shear (2.5 dyn/cm2). In immature (d9) Mks, shear exposure upregulated genes related to growth and Mk maturation, while in mature (d12) Mks, it upregulated genes involved in apoptosis and intracellular transport. Following shear-flow exposure, 6 AP-1 transcripts (ATF4, JUNB, JUN, FOSB, FOS, and JUND) were upregulated at d9 and two AP-1 proteins (JunD and c-Fos) were upregulated both at d9 and d12. Our data show that MAPK signaling is linked to both the shear-stress response and AP-1 upregulation. JNK phosphorylation increased significantly following shear stimulation, while JNK inhibition reduced shear-induced JunD protein expression. Although p38 phosphorylation did not increase following shear flow, its inhibition reduced shear-induced JunD and c-Fos protein expression. JNK inhibition reduced fibrinogen binding of d9 and d12 platelet-like particle s (PLPs) and P-selectin expression at d12 PLPs, while p38 inhibition reduced fibrinogen binding of d12 PLPs. Here we show that mechanotransduction of shear forces in Mks results in JNK activation, AP-1 upregulation, and downstream transcriptional changes that promote maturation of immature Mks and platelet biogenesis in mature Mks. Two- and Three-condition experiment (flow vs. static culture condition, d9 vs. d12, and 30 min vs. 60 min at d9); Biological replicates: 3; Technical replicates: 1 (dye-swap)

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KLF2-induced actin shear fibers control both alignment to flow and JNK signaling in vascular endothelium

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