Thrombopoietin Enhances Expression of Vascular Endothelial Growth Factor (VEGF) in Primitive Hematopoietic Cells through Hif-1α.

The development of hematopoietic stem cells (HSCs) is orchestrated by numerous hematopoietic cytokines, growth factors and chemokines. For the most part these proteins are secreted from bone marrow microenviromental cells, including fibroblasts, endothelial cells and stromal cells, or are displayed on their cell surfaces. HSCs have also been shown to produce numerous cytokines and growth factors. Interestingly, secreted cytokines have also been reported to induce the production of additional cytokines from neighboring cells, suggesting that cytokines drive networks of other cytokines to support hematopoietic cell development. Vascular endothelial growth factor (VEGF), a major regulator of angiogenesis and vasculogenesis, also plays an important role in HSC development, where it acts in an intracellular autocrine fashion to promote cell survival. The secretion of VEGF from endothelial or smooth muscle cells is regulated by extracellular stimuli, inflammatory cytokines and hypoxia. In contrast, it is not clear whether synthesis of VEGF in HSCs is regulated by extracellular signals. Because several early acting cytokines, including TPO, affect VEGF-A expression in hematopoietic cell lines, we hypothesized that TPO could be a regulator of a HSC VEGF autocrine loop. We found that TPO induces VEGF transcripts in primitive marrow derived sca-1⁺/c-kit⁺/Gr-1⁻ hematopoietic cells, and that VEGF transcripts are reduced in these cells when derived from Tpo−/− mice. Additional studies determined that TPO induces VEGF expression by increasing the nuclear levels of its primary transcription factor, both in the TPO-dependent primitive hematopoietic cell line UT-7/TPO and in purified sca-1⁺/c-kit⁺/Gr-1⁻marrow cells. Elevation of HIF-1α by TPO is achieved by two different mechanisms; augmented protein synthesis and enhanced stabilization. The latter mechanism is dependent on heat shock protein 90 (Hsp90), as inhibition of Hsp90 function by the specific inhibitor geldanamycin inhibited the TPO-dependent stabilization of HIF-1α. In additional studies we also established that VEGF expression was important for the favorable TPO effect on primitive hematopoietic cells, as blockade of the VEGF receptor with a specific inhibitor, SU5416 (0.1mM), substantially blunted TPO induced growth of sorted single sca-1⁺/c-kit⁺/Gr-1⁻marrow cells in serum-free cultures. Importantly, this inhibitor does not affect TPO induced phosphorylation of Jak2, ERK and AKT in UT-7/TPO cells even at a 100-fold higher concentration. Along with our previous findings that TPO affects Hox transcription factors that regulate HSC proliferation, these data contribute to our growing understanding of the mechanisms by which a hormone can influence HSC development.