Sp1/Sp3 transcription factors regulate hallmarks of megakaryocyte maturation and platelet formation and function

Abstract

Key Points• Megakaryocyte-specific Sp1/ Sp3 double-knockout mice display thrombocytopenia, platelet dysfunction, and defects in megakaryocyte maturation.• Selective Mylk inhibition by ML7 affects proplatelet formation and stabilization and ITAM receptor-mediated platelet aggregation.Sp1 and Sp3 belong to the specificity proteins (Sp)/Krüppel-like transcription factor family. They are closely related, ubiquitously expressed, and recognize G-rich DNA motifs. They are thought to regulate generic processes such as cell-cycle and growth control, metabolic pathways, and apoptosis. Ablation of Sp1 or Sp3 in mice is lethal, and combined haploinsufficiency results in hematopoietic defects during the fetal stages. Here, we show that in adult mice, conditional pan-hematopoietic (Mx1-Cre) ablation of either Sp1 or Sp3 has minimal impact on hematopoiesis, whereas the simultaneous loss of Sp1 and Sp3 results in severe macrothrombocytopenia. This occurs in a cell-autonomous manner as shown by megakaryocyte-specific (Pf4-Cre) double-knockout mice. We employed flow cytometry, cell culture, and electron microscopy and show that although megakaryocyte numbers are normal in bone marrow and spleen, they display a less compact demarcation membrane system and a striking inability to form proplatelets. Through megakaryocyte transcriptomics and platelet proteomics, we identified several cytoskeleton-related proteins and downstream effector kinases, including Mylk, that were downregulated upon Sp1/Sp3 depletion, providing an explanation for the observed defects in megakaryopoiesis. Supporting this notion, selective Mylk inhibition by ML7 affected proplatelet formation and stabilization and resulted in defective ITAM receptor-mediated platelet aggregation. (Blood. 2015; 125(12):1957-1967 IntroductionPlatelets are the blood cells responsible for maintaining the body hemostasis and, in humans, ;10 11 platelets are produced by megakaryocytes daily.1 Megakaryopoiesis is the process whereby hematopoietic progenitor cells differentiate into mature megakaryocytes, which produce platelets in a process known as thrombopoiesis. 2,3 This takes place at sites of primary hematopoiesis, ie, the bone marrow and, in mice, also in the spleen. 4 The hormone thrombopoietin (TPO), produced by the liver, regulates platelet production. TPO levels in plasma are inversely proportional to the megakaryocyte/ platelet mass. 5 Megakaryocyte differentiation is characterized by highly specialized cellular changes including endomitosis (leading to polyploid cells), accumulation of a and dense granules, and development of a demarcation membrane system (DMS), which is required as a membrane reservoir to produce large numbers of platelets.2,3 Mature megakaryocytes migrate toward the proximity of blood vessels through which they protrude cytoplasmic microtubulerich extensions into the blood stream, known as proplatelets, from which platelets are shed.6 Coordinated interactions between the membrane, cytoskeletal machinery and intracellular signaling are essential for prop...

Gfi1b controls integrin signaling-dependent cytoskeleton dynamics and organization in megakaryocytes

“…In control mice, most of these cells were polyploid (>4N) with a 16N population being predominant (~60% of all megakaryocytes) followed by the 8N and 32N populations, each representing 10–20%, and a very low number of 64N cells (Figures 4D and Online Supplementary Figure S2A ), which is in agreement with previous reports. 18,19 Megakaryocytes from Pf4 -Cre Gfi1b fl/fl mice were also polyploid, but had a different distribution: the 32N subset was not affected by Gfi1b loss, the 16N megakaryocytes were notably decreased and the 2N, 4N and 8N fractions were slightly increased compared to controls (Figure 5D and Online Supplementary Figure S2A ). Gating these subsets into a rainbow plot based on their ploidy showed that the increase in smaller megakaryo cytes in the Gfi1b knockout mice was primarily due to an increase in cells of the 2N and 4N subsets (Figure 4E).…”

Integrated genome-wide analysis of expression quantitative trait loci aids interpretation of genomic association studies

“…For example, Clusters 10 and 51 target an over-abundance of genes with promoter regions containing motifs specific to transcription factors SP1 and NRF1 . Indeed, transcription factor SP1 has recently been shown to regulate platelet formation in mouse [25]. Changes in activities of these transcription factors may mediate the effect of genetic variants in these clusters on platelet formation and dynamics.…”

miR-29c contribute to glioma cells temozolomide sensitivity by targeting O6-methylguanine-DNA methyltransferases indirectly

“…Previous research has shown that the family member miR-29b functions as an oncogene by inhibiting tumor suppressors such as phosphatase and tensin homolog (PTEN) [30]. In this report, we provide evidence that miR-29c regulate MGMT expression indirectly via targeting of transcription factor Specificity Protein 1 (Sp1), a universal zinc finger transcription factor that in expressed in nearly all cells and tissues [31, 32]. We show that the SP1 expression increasing the response to TMZ in glioma cell lines.…”