Scientists at the Oklahoma Medical Research Foundation have broken new ground in understanding how the lymphatic system works, potentially opening the door for future therapies. The lymphatic system is a network of vessels and lymph nodes that spans the entire body. 

Wnt/β-catenin signaling is necessary for lymphatic vascular development. Oscillatory shear stress (OSS) enhances Wnt/β-catenin signaling in cultured lymphatic endothelial cells (LECs) to induce expression of the lymphedema-associated transcription factors GATA2 and FOXC2.

However, the mechanisms by which OSS regulates Wnt/β-catenin signaling and GATA2 and FOXC2 expression are unknown. We show that OSS activates autocrine Wnt/β-catenin signaling in LECs in vitro.

Tissue-specific deletion of Wntless, which is required for the secretion of Wnt ligands, reveals that LECs and vascular smooth muscle cells are complementary sources of lymphatic endothelial cells that regulate lymphatic vascular development in vivo.

Further, the LEC master transcription factor PROX1 forms a complex with β-catenin and the TCF/LEF transcription factor TCF7L1 to enhance Wnt/β-catenin signaling and promote FOXC2 andGATA2 expression in LECs.

Thus, the work defines Wnt sources, reveals that PROX1 directs cell fate by acting as a Wnt signaling component, and dissects the mechanisms of PROX1 and Wnt synergy.

OSS Activates Autocrine Wnt/β-Catenin Signaling in LECsIn Vitro

Wnt/β-catenin signaling is activated upon the interaction of canonical Wnt ligands with Frizzled receptors and LRP5 and 6 (LRP5/6) co-receptors. Wnt ligands induce the phosphorylation of LRP5/6.

The researchers observed increased phosphorylation of LRP6 in primary human LECs (HLECs) exposed to OSS, suggesting that Wnt ligands are involved in OSS-mediated activation of Wnt/β-catenin signaling.

To evaluate whether Wnt ligands are important for OSS-mediated FOXC2 and GATA2 expression we exposed HLECs to OSS in the presence of recombinant (r) DKK1, which inhibits the interaction between LRP5/6 and Wnt ligands.

The expression of FOXC2GATA2, and the positive control AXIN2 was reduced in OSS-exposed HLECs cultured in the presence of rDKK1 relative to controls, indicating that Wnt ligands are necessary for the OSS-mediated enhancement of FOXC2 and GATA2 expression.

In the intestinal epithelium, Paneth cells are considered the source of Wnt ligands that regulate the differentiation and proliferation of epithelial stem cells in ex vivo organoids.  

However, abolishing Wnt secretion from Paneth cells does not significantly affect intestinal epithelial homeostasis in vivo. Stromal cells that are located near the intestinal crypts produce Wnt ligands and compensate for the loss of Paneth cell-derived Wnt ligands.

Similarly, although autocrine Wnt/β-catenin signaling is sufficient for activating the expression of FOXC2 and GATA2 in vitro, Wnt ligands from vascular smooth muscle cells play a complementary role in vivo.