Abstract

SH2-containing-5′inositol phosphatase-1 (SHIP), via removal of the 5′ phosphate group from PI(3,4,5) P3 (PIP3), influences signals downstream of cytokine/chemokine receptors that play a key role in megakaryocytopoiesis. Thombopoietin (TPO) influences megakaryocytes (MK) development by controlling their proliferation, differentiation and survival. SHIP phosphorylation, resulting from TPO receptor activation (c-mpl) influences MK cycling and proliferation. Stromal derived factor-1 (SDF-1/CXCL12) induces transendothelial MK migration facilitating platelet shedding. SHIP-deficient myeloid progenitors exhibit enhanced chemotaxis towards SDF-1/CXCL-12, indicating SHIP influences signaling downstream of its receptor (CXCR-4). In addition, colony-forming-unit-MK are decreased in SHIP−/− bone marrow (BM) and SHIP regulates PIP3 levels after thrombin or collagen platelet activation. To further explore SHIP effects on megakaryocytopoiesis, we measured MK compartment size, by flow cytometry, in mice with SHIP promoter/first exon deletion (SHIP−/−) and with inositol phosphatase deletion (SHIPΔIP/ΔIP) in order to confirm that the observed phenotype is highly penetrant. Lin− cKit+ CD41+ (n=5/strain), representing MK progenitors (MKP), were statistically significantly increased in BM (3-fold in SHIP−/− and SHIPΔIP/ΔIP), spleen (18-fold in SHIP−/−; 50.8-fold in SHIPΔIP/ΔIP) and peripheral blood (PB) (2.4-fold in SHIPΔIP/ΔIP) compared to their wild type (WT) littermates; not reaching statistical significance in SHIP−/− PB (1.6-fold). These findings suggest that SHIP may control MKP homeostasis. Lin− cKit- CD41+ (n=5), representing mature MK, were statistically significantly decreased in BM (2.6-fold in SHIP−/−; 2.2-fold in SHIPΔIP/ΔIP), increased in spleen (11-fold in SHIP−/−; 26.7-fold in SHIPΔIP/ΔIP) and PB (7.7-fold in SHIP−/−; 2.6-fold in SHIPΔIP/ΔIP); suggesting that SHIP may control MK redistribution. BM histopathology and Glycoprotein IIB/IIIa (CD41) immuno-staining showed that MK numbers were similar in SHIP−/− and WT, although morphologically we detected hypolobulated/micro (HM) MK in SHIP−/− and hyperlobulated (HL) MK in WT mice, consistent with MKP and MK flow cytometry phenotype respectively. Spleen histopathology and CD41 immuno-staining showed that the MK numbers were increased in SHIP−/−; cells with HM and HL morphology were present. These findings suggest that SHIP may control pathways that mediate MK localization and/or migration. Mean platelet numbers (0.95 x 106/μL SHIP−/−; 0.75 x 106/μL SHIPΔIP/ΔIP) were not significantly different compared to WT (1x 106/μL SHIP−/− and SHIPΔIP/ΔIP) although splenomegaly in SHIP−/− animals may prevent an increase in circulating platelets. In conclusion, SHIP regulates essential signaling pathways that control megakaryocytopoiesis in vivo. SHIP enzymatic activity could be targeted to increase MKP pool to enable this compartment to replenish platelets more rapidly following chemotherapy and radiation treatment.

Author notes

Corresponding author