Phosphatidylinositol transfer proteins regulate megakaryocyte TGF-β1 secretion and hematopoiesis in mice

We hypothesized that megakaryocyte (MK) phosphoinositide signaling mediated by phosphatidylinositol transfer proteins (PITPs) contributes to hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) regulation. Conditional knockout mice lacking PITPs specifically in MKs and platelets (pitp alpha(-/-) and pitp alpha(-/-)/beta(-/-)) bone marrow (BM) manifested decreased numbers of HSCs, MK-erythrocyte progenitors, and cycling HPCs. Further, pitp alpha(-/-)/beta(-/-) BM had significantly reduced engrafting capability in competitive transplantation and limiting dilution analysis. Conditioned media (CM) from cultured pitp alpha(-/-) and pitp alpha(-/-)/beta(-/-) BM MKs contained higher levels of transforming growth factor beta 1 (TGF-beta 1) and interleukin-4 (IL-4), among other myelosuppressive cytokines, than wildtype BM MKs. Correspondingly, BM flush fluid from pitp alpha(-/-) and pitp alpha(-/-)/beta(-/-) mice had higher concentrations of TGF-beta 1. CM from pitp alpha(-/-) and pitp alpha(-/-)/beta(-/-) MKs significantly suppressed HPC colony formation, which was completely extinguished in vitro by neutralizing anti-TGF-beta antibody, and treatment of pitp alpha(-/-)/beta(-/-) mice in vivo with anti-TGF-beta antibodies completely reverted their defects in BM HSC and HPC numbers. TGF-beta and IL-4 synergized to inhibit HPC colony formation in vitro. Electron microscopy analysis of pitp alpha(-/-)/beta(-/-) MKs revealed ultrastructural defects with depleted alpha-granules and large, misshaped multivesicular bodies. Von Willebrand factor and thrombospondin-1, like TGF-beta, are stored in MK alpha-granules and were also elevated in CM of cultured pitp alpha(-/-)/beta(-/-) MKs. Altogether, these data show that ablating PITPs in MKs indirectly dysregulates hematopoiesis in the BM by disrupting alpha-granule physiology and secretion of TGF-beta 1.