Non-Canonical Wnt Signaling Regulates Hematopooetic Repopulation Through the Ryk Receptor

Abstract 852

Proper regulation of the balance between quiescence and proliferation is implicated in long-term maintenance of hematopoietic stem cell (HSC) function. Our lab has demonstrated that Wnt5a, a member of the Wnt family of ligands, induces HSC quiescence in vitro and enhances short and long-term hematopoietic repopulation (Nemeth, et al. 2007). To determine the mechanisms of Wnt5a signaling in HSCs we sought to identify potential Wnt5a receptors. The Ryk gene encodes a non-canonical Wnt receptor in lineage-negative, Sca-1+, c-kit+ (LSK) cells. Previous research using neural crest cells identified Ryk as a receptor for Wnt5a. We hypothesized that the ability of Wnt5a to enhance HSC quiescence and hematopoietic repopulation required the Ryk receptor. To test this hypothesis, we cultured C57BL/6 LSK cells in serum-free media with 25ng/ml SCF and Flt3, 250ng/ml recombinant Wnt5a (rWnt5a), and 1.0 μg/ml anti-Ryk neutralizing antibody (α-Ryk) for four days. We observed that compared to control treatment, rWnt5a significantly inhibited cellular expansion (control: 5.9 ± 0.1-fold; rWnt5a: 3.1 ± 0.3-fold; n = 4, p <0.01). The effect of Wnt5a on cellular expansion was blocked by the inhibition of Ryk with α-Ryk (α-Ryk + rWnt5a: 3.1 ± 0.3-fold; n = 4, p = 0.01 compared to rWnt5a alone). We confirmed these data using a genetic approach in which we transduced LSK cells with a lentiviral vector expressing a dominant-negative Ryk mutant; LSK cells transduced with this vector did not respond to rWnt5a treatment as measured by cell expansion. We measured the effects of rWnt5a and α-Ryk treatment on the cell cycle. rWnt5a treatment resulted in a modest decrease in the percentage of cells in the S/G2/M phases (control: 35.0 ± 1.9%; rWnt5a: 29.1 ± 0.5%, n=3, p < 0.01). Treatment with α-Ryk partially restored this response (α-Ryk + rWnt5a; 32.2 ± 2.0%, n=3, p = 0.06 compared to rWnt5a). We then tested the effects of rWnt5a and α-Ryk treatment on cell quiescence. As expected, rWnt5a treatment increased the percentage of cells in G0 compared to control (control: 11.3 ± 6.7%; rWnt5a: 26.1 ± 0.9%, n=3, p < 0.01). The effect of rWnt5a on quiescence was blocked by the addition of α-Ryk (α-Ryk + rWnt5a; 21.1 ± 1.4%, n=3, p < 0.05 compared to rWnt5a). To test the effects of inhibiting Ryk on Wnt5a-mediated enhancement of hematopoietic repopulation, we performed competitive bone marrow transplant assays. We cultured C57BL/6 LSK cells for 4 days and transplanted 1 × 10⁴ cells along with 5 × 10⁵ whole bone marrow cells from Ubc-GFP transgenic mice (C57BL/6 background) into lethally irradiated B6.SJL mice. At 4 and 16 weeks, we measured the contribution of cultured LSK cells to hematopoietic repopulation by determining the percentage of peripheral blood cells that were CD45.2+, GFP-. rWnt5a increased the contribution of cultured LSK cells to hematopoietic repopulation after 4 weeks (control: 5.3 ± 1.4%, n = 8, rWnt5a: 19.5 ± 7.1%, n = 6, p < 0.05) and 16 weeks (control: 4.5 ± 1.6% n = 8, rWnt5a: 32.0 ± 13.1%, n = 6, p = 0.01). Inhibiting Ryk blocked the effects of rWnt5a on reconstitution after 4 and 16 weeks (4 weeks: α-Ryk + rWnt5a; 5.8 ± 2.1% n = 6, p < 0.05 compared to rWnt5a; 16 weeks: α-Ryk + rWnt5a; 11.9 ± 7.1%, n = 6, p < 0.05 compared to rWnt5a). These data indicate that the effects of rWnt5a on hematopoietic cell proliferation and HSC engraftment are regulated by the Ryk receptor. To determine a mechanism by which Wnt5a induces quiescence and hematopoietic repopulation, we tested whether Wnt5a regulates production of reactive oxygen species (ROS). Excessive ROS production is linked to decreased HSC quiescence and reduced long-term function. We measured the level of ROS in our sorted LSK cells using the ROS probe dihydrodichlorofluorescein. We observed that treatment of LSK cells with rWnt5a resulted in a 1.6 ± 0.3-fold reduction in ROS levels compared to control (n = 3); this reduction was comparable to that observed using the proto-typical anti-oxidant N-acetyl cysteine. This reduction in ROS production was inhibited by the addition of α-Ryk (1.04 ± 0.1-fold compared to control, n = 3). Together, our data indicate that the Ryk receptor functionally interacts with Wnt5a to regulate hematopoietic stem and progenitor quiescence and function and that regulation of ROS production is a potential mechanism by which Wnt5a regulates HSC function.