Dysregulation of PSTPIP2 Due to Loss of GATA-1 Contributes to Aberrant Megakaryopoiesis

Abstract 854

GATA-1-deficiency causes dysregulation of multiple genes and leads to aberrant megakaryopoiesis. How the dysregulated genes may contribute to abnormal megakaryocyte development and differentiation is largely unknown. In this study, we focused on Pstpip2, a gene that was upregulated in GATA-1low and GATA-1s mutant megakaryocytes. A strong GATA-1 binding site in the intron 1 of Pstpip2 gene was previously identified by GATA-1 ChIP-Seq studies. These observations strongly suggest that Pstpip2 is a GATA-1 target gene. By using ChIP-PCR we showed that GATA-1 indeed bound to Pstpip2 intron 1 region in megakaryocytes. We also observed that Pstpip2 was expressed in myeloid cells and hematopoietic progenitors, but not lymphocytes. Noticeably, two cell lines with GATA-1 deficiencies, G1ME and CMK, which approximate the MEP and megakaryoblast stages, respectively, showed moderate expression of Pstpip2. Moreover, K562 cells displayed significant upregulation of PSTPIP2 and concomitant downregulation of GATA-1 during TPA-induced megakaryocytic differentiation. To study its function in megakaryocytic differentiation, we modulated PSTPIP2 expression by overexpression or shRNA knockdown. Ectopic expression of PSTPIP2 in K562 or CMK cells impaired TPA-induced megakaryocytic differentiation as evidenced by decreased CD41 expression. In contrast, downregulation of PSTPIP2 by shRNA promoted CD41 expression and increased polyploidy. These findings suggest that failure to suppress PSTPIP2 due to loss of GATA-1 may contribute to abnormal megakaryopoiesis.

To probe the mechanism by which PSTPIP2 inhibits megakaryocytic differentiation, we tested whether the interaction with its partner PEST-PTP or phosphorylation of key tyrosine residues in the protein by Src kinase is required. We created two mutants, W232A and Y323/333/F, which block its interaction with PEST-PTP or prevent tyrosine phosphorylation, respectively. Similar to WT PSTPIP2, both mutants retained the ability to inhibit megakaryocytic differentiation suggesting that inhibition of megakaryocytic differentiation does not depend on the PEST-PTP interaction or Src kinase phosphorylation. Separately, we have noted that PSTPIP2 also interacts with Fyn, Grb2, and Shc, suggesting that PSTPIP2 may affect signal transduction. Indeed, we found that ERK phosphorylation was downregulated by PSTPIP2. Since THPO/MPL signaling via JAK/STAT is the physiological pathway in megakaryocyte maturation rather than BCR-ABL signaling seen in K562 cells, we next analyzed the effect of PSTPIP2 on MPL signaling in G1ME cells. Consistent with our findings in K562 and CMK cells, both WT and mutant PSTPIP2 inhibited CD41 expression. Interestingly, ectopic expression of WT PSTPIP2 also caused cell cycle arrest at S phase in G1ME cells. PSTPIP2 also suppressed Erk and Shc phosphorylation. Considering the important role of MAPK/ERK signaling in megakaryocyte development, we believe that PSTPIP2 acts as a negative regulator of THPO-MPL signaling to suppress TPO-stimulated MAPK/ERK activation and subsequently inhibit megakaryopoiesis. Finally, we confirmed the function of PSTPIP2 in megakaryocyte differentiation using mouse bone marrow progenitor cells. Ectopic expression of PSTPIP2 significantly downregulated CD41 expression, reduced CFU-GM and CFU-Mk numbers, and reduced polyploidy, while knockdown of PSTPIP2 exhibited the opposite effects. Taken together, we have identified a new negative regulator of THPO-MPL signaling that is inhibited by GATA-1 in normal megakaryocyte development. Upregulation of PSTPIP2 may contribute to abnormal megakaryopoiesis observed in GATA-1-deficient megakaryocytes.