Dongwon Choi,1 Eunkyung Park,1 Eunson Jung,1 Young Jin Seong,1 Jaehyuk Yoo,1 Esak Lee,2 Mingu Hong,1 Sunju Lee,1 Hiroaki Ishida,3 James Burford,4 Janos Peti-Peterdi,4 Ralf H. Adams,5 Sonal Srikanth,6 Yousang Gwack,6 Christopher S. Chen,2 Hans J. Vogel,3 Chester J. Koh,7 Alex K. Wong,1 and Young-Kwon Hong1,*
1Division of Plastic and Reconstructive Surgery, Department of Surgery, Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, California, USA. 2Department of Biomedical Engineering and the Biological Design Center, Boston University; and Wyss Institute for Biologically Inspired Engineering, Harvard University; Boston, Massachusetts, USA. 3Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada. 4Department of Physiology and Biophysics, Zilkha Neurogenetic Institute, Keck School of Medicine, University of Southern California, Los Angeles, California, USA. 5Max Planck Institute for Molecular Biomedicine, Department of Tissue Morphogenesis, and University of Munster, Faculty of Medicine, Munster, Germany. 6Department of Physiology, David Geffen School of Medicine at UCLA, Los Angeles, California, USA. 7Division of Pediatric Urology, Texas Children’s Hospital, Baylor College of Medicine, Houston, Texas, USA.
*Address correspondence to: Young-Kwon Hong, Department of Surgery, University of Southern California, Norris Comprehensive Cancer Center, 1450 Biggy Street NRT6501, Los Angeles, California 90033, USA.
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 Kruppel-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.