Shear stimulation of FOXC1 and FOXC2 differentially regulates cytoskeletal activity during lymphatic valve maturation
Abstract
Mutations in the transcription factor FOXC2 are predominately associated with lymphedema. Herein, we demonstrate a key role for related factor FOXC1, in addition to FOXC2, in regulating cytoskeletal activity in lymphatic valves. FOXC1 is induced by laminar, but not oscillatory, shear and inducible, endothelial-specific deletion impaired postnatal lymphatic valve maturation in mice. However, deletion of Foxc2 induced valve degeneration, which is exacerbated in Foxc1; Foxc2 mutants. FOXC1 knockdown (KD) in human lymphatic endothelial cells increased focal adhesions and actin stress fibers whereas FOXC2-KD increased focal adherens and disrupted cell junctions, mediated by increased ROCK activation. ROCK inhibition rescued cytoskeletal or junctional integrity changes induced by inactivation of FOXC1 and FOXC2 in vitro and vivo respectively, but only ameliorated valve degeneration in Foxc2 mutants. These results identify both FOXC1 and FOXC2 as mediators of mechanotransduction in the postnatal lymphatic vasculature and posit cytoskeletal signaling as a therapeutic target in lymphatic pathologies.
Data availability
All data generated or analysed during this study are included in the manuscript and supporting files.
Article and author information
Author details
Funding
National Institutes of Health (R01HL126920,R01HL144129)
- Tsutomu Kume
National Institutes of Health (5T32HL094293)
- Pieter R Norden
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: All procedures and animal studies were approved by Northwestern University's institutional animal care and use committee (IACUC) (Protocol: IS00008794) or by the Animal Ethics Committee of Vaud, Switzerland.
Copyright
© 2020, Norden et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 2,898
- views
-
- 330
- downloads
-
- 49
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Further reading
-
- Cell Biology
The excessive cosolute densities in the intracellular fluid create a physicochemical condition called macromolecular crowding (MMC). Intracellular MMC entropically maintains the biochemical thermodynamic equilibria by favouring associative reactions while hindering transport processes. Rapid cell volume shrinkage during extracellular hypertonicity elevates the MMC and disrupts the equilibria, potentially ushering cell death. Consequently, cells actively counter the hypertonic stress through regulatory volume increase (RVI) and restore the MMC homeostasis. Here, we establish fluorescence anisotropy of EGFP as a reliable tool for studying cellular MMC and explore the spatiotemporal dynamics of MMC during cell volume instabilities under multiple conditions. Our studies reveal that the actin cytoskeleton enforces spatially varying MMC levels inside adhered cells. Within cell populations, MMC is uncorrelated with nuclear DNA content but anti-correlated with the cell spread area. Although different cell lines have statistically similar MMC distributions, their responses to extracellular hypertonicity vary. The intensity of the extracellular hypertonicity determines a cell's ability for RVI, which correlates with Nuclear Factor Kappa Beta (NFkB) activation. Pharmacological inhibition and knockdown experiments reveal that Tumour Necrosis Factor Receptor 1 (TNFR1) initiates the hypertonicity induced NFkB signalling and RVI. At severe hypertonicities, the elevated MMC amplifies cytoplasmic microviscosity and hinders Receptor Interacting Protein Kinase 1 (RIPK1) recruitment at the TNFR1 complex, incapacitating the TNFR1-NFkB signalling and consequently, RVI. Together, our studies unveil the involvement of TNFR1-NFkB signalling in modulating RVI and demonstrate the pivotal role of MMC in determining cellular osmoadaptability.
-
- Cell Biology
- Immunology and Inflammation
Arpin was discovered as an inhibitor of the Arp2/3 complex localized at the lamellipodial tip of fibroblasts, where it regulated migration steering. Recently, we showed that arpin stabilizes the epithelial barrier in an Arp2/3-dependent manner. However, the expression and functions of arpin in endothelial cells (EC) have not yet been described. Arpin mRNA and protein are expressed in EC and downregulated by pro-inflammatory cytokines. Arpin depletion in Human Umbilical Vein Endothelial Cells causes the formation of actomyosin stress fibers leading to increased permeability in an Arp2/3-independent manner. Instead, inhibitors of ROCK1 and ZIPK, kinases involved in the generation of stress fibers, normalize the loss-of-arpin effects on actin filaments and permeability. Arpin-deficient mice are viable but show a characteristic vascular phenotype in the lung including edema, microhemorrhage, and vascular congestion, increased F-actin levels, and vascular permeability. Our data show that, apart from being an Arp2/3 inhibitor, arpin is also a regulator of actomyosin contractility and endothelial barrier integrity.