Laminar flow downregulates Notch activity to promote lymphatic sprouting
Dongwon Choi, … , Alex K. Wong, Young-Kwon Hong
Dongwon Choi, … , Alex K. Wong, Young-Kwon Hong
Published March 6, 2017
Citation Information: J Clin Invest. 2017;127(4):1225-1240. https://doi.org/10.1172/JCI87442.
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Research Article Vascular biology

Laminar flow downregulates Notch activity to promote lymphatic sprouting

Abstract

The major function of the lymphatic system is to drain interstitial fluid from tissue. Functional drainage causes increased fluid flow that triggers lymphatic expansion, which is conceptually similar to hypoxia-triggered angiogenesis. Here, we have identified a mechanotransduction pathway that translates laminar flow–induced shear stress to activation of lymphatic sprouting. While low-rate laminar flow commonly induces the classic shear stress responses in blood endothelial cells and lymphatic endothelial cells (LECs), only LECs display reduced Notch activity and increased sprouting capacity. In response to flow, the plasma membrane calcium channel ORAI1 mediates calcium influx in LECs and activates calmodulin to facilitate a physical interaction between Krüppel-like factor 2 (KLF2), the major regulator of shear responses, and PROX1, the master regulator of lymphatic development. The PROX1/KLF2 complex upregulates the expression of DTX1 and DTX3L. DTX1 and DTX3L, functioning as a heterodimeric Notch E3 ligase, concertedly downregulate NOTCH1 activity and enhance lymphatic sprouting. Notably, overexpression of the calcium reporter GCaMP3 unexpectedly inhibited lymphatic sprouting, presumably by disturbing calcium signaling. Endothelial-specific knockouts of Orai1 and Klf2 also markedly impaired lymphatic sprouting. Moreover, Dtx3l loss of function led to defective lymphatic sprouting, while Dtx3l gain of function rescued impaired sprouting in Orai1 KO embryos. Together, the data reveal a molecular mechanism underlying laminar flow–induced lymphatic sprouting.

Authors

Dongwon Choi, Eunkyung Park, Eunson Jung, Young Jin Seong, Jaehyuk Yoo, Esak Lee, Mingu Hong, Sunju Lee, Hiroaki Ishida, James Burford, Janos Peti-Peterdi, Ralf H. Adams, Sonal Srikanth, Yousang Gwack, Christopher S. Chen, Hans J. Vogel, Chester J. Koh, Alex K. Wong, Young-Kwon Hong

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Figure 5

KLF2 forms a complex with PROX1 and CaM, and is required for lymphatic sprouting.

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KLF2 forms a complex with PROX1 and CaM, and is required for lymphatic s...
(A) FLAG-KLF2 and/or Myc-PROX1 were expressed in HEK293 cells, and co-IP assays were performed using anti-FLAG (top panel) or anti-Myc antibody (bottom panel). (B) Co-IP assays were performed against FLAG-KLF2 and Myc-PROX1 in HEK293 cells in the presence of DMSO (CTR), Bapta-AM (Bapta, 10 μM), ionomycin (Iono, 1 μM), or Bapta-AM (10 μM)/ionomycin (1 μM). (C) CaM promotes PROX1/KLF2 interaction. FLAG-KLF2 and Myc-PROX1 were expressed in HEK293 cells with CaM (HA-CaM) or not (Empty). Co-IP was performed using IgG or anti-FLAG antibody. (D) Serial Co-IP assays showing a protein complex formation among KLF2, PROX1, and CaM. These 3 proteins were expressed in HEK293 cells and first immunoprecipitated using FLAG-antibody beads (1st Co-IP) and then using anti-HA antibody (2nd Co-IP) for final immunodetection of Myc-PROX1. See details in Supplemental Information. (E) LECs were cultured under static or laminar flow (2 dyn/cm2), and co-IP assays were performed using IgG or anti-PROX1 antibody, followed by immunoblotting against KLF2 or PROX1. (F) Effect of calcium chelation on PROX1/KLF2 complex formation. (Pre-Bapta) LECs were pretreated with Bapta-AM (10 μM) for 10 minutes and exposed to laminar flow (2 dyn/cm2) for 24 hours before co-IP assay. (Post-Bapta) LECs were exposed to laminar flow for 0.5 hours, treated with Bapta-AM (10 μM), and subjected to laminar flow (2 dyn/cm2) for an additional 0.5 hours before co-IP assay. (G) qRT-PCR analyses showing that ORAI1 inhibition by SKF-96365 (SKF, 3 μM) reduced the flow-induced KLF2 upregulation in LECs. (H) Western blot assays showing protein levels of KLF2, NICD1, and β-actin in LECs, which were transfected with scrambled siRNA (siCTR) or KLF2 siRNA (siKLF2) and exposed to laminar flow (2 dyne/cm2). A vertical line marks spliced lanes. (I and J) Lymphatic and blood vessels of the back skins of control (CTR) embryos [Cdh5(PAC)-CreERT2 Klf2+/+ Prox1-EGFP] and Klf2ECKO embryos [Cdh5(PAC)-CreERT2 Klf2fl/fl Prox1-EGFP] were visualized by lymphatic-specific EGFP signal and CD31 immunostaining, respectively, at E15.5. Equivalent anatomic locations were chosen for i and iii, and for ii and iv. Scale bars: 100 μm. (J) Vascular analyses were performed and graphed. Data are expressed as SEM and SD of a representative of 3 independent experiments. *P < 0.05; #P < 0.01; §P < 0.001.