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

Marjolein Meinders, Divine I Kulu, Harmen J G van de Werken, Mark Hoogenboezem, Hans Janssen, Rutger W W Brouwer, Wilfred F J van Ijcken, Erik-Jan Rijkers, Jeroen A A Demmers, Imme Krüger, Timo K van den Berg, Guntram Suske, Laura Gutiérrez, Sjaak Philipsen

Research output: Contribution to journalArticleAcademicpeer-review

Abstract

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.

Original languageEnglish
Pages (from-to)1957-67
Number of pages11
JournalBlood
Volume125
Issue number12
DOIs
Publication statusPublished - 19 Mar 2015

Cite this

Meinders, M., Kulu, D. I., van de Werken, H. J. G., Hoogenboezem, M., Janssen, H., Brouwer, R. W. W., ... Philipsen, S. (2015). Sp1/Sp3 transcription factors regulate hallmarks of megakaryocyte maturation and platelet formation and function. Blood, 125(12), 1957-67. https://doi.org/10.1182/blood-2014-08-593343
Meinders, Marjolein ; Kulu, Divine I ; van de Werken, Harmen J G ; Hoogenboezem, Mark ; Janssen, Hans ; Brouwer, Rutger W W ; van Ijcken, Wilfred F J ; Rijkers, Erik-Jan ; Demmers, Jeroen A A ; Krüger, Imme ; van den Berg, Timo K ; Suske, Guntram ; Gutiérrez, Laura ; Philipsen, Sjaak. / Sp1/Sp3 transcription factors regulate hallmarks of megakaryocyte maturation and platelet formation and function. In: Blood. 2015 ; Vol. 125, No. 12. pp. 1957-67.
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abstract = "Sp1 and Sp3 belong to the specificity proteins (Sp)/Kr{\"u}ppel-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.",
keywords = "Animals, Azepines/chemistry, Blood Platelets/cytology, Bone Marrow/metabolism, Flow Cytometry, Lectins, C-Type/metabolism, Megakaryocytes/cytology, Mice, Mice, Knockout, Naphthalenes/chemistry, Platelet Aggregation, Platelet Membrane Glycoproteins/metabolism, Proteome, Signal Transduction, Sp1 Transcription Factor/genetics, Sp3 Transcription Factor/genetics, Spleen/metabolism, Thrombocytopenia/metabolism, Transcription Factors/metabolism",
author = "Marjolein Meinders and Kulu, {Divine I} and {van de Werken}, {Harmen J G} and Mark Hoogenboezem and Hans Janssen and Brouwer, {Rutger W W} and {van Ijcken}, {Wilfred F J} and Erik-Jan Rijkers and Demmers, {Jeroen A A} and Imme Kr{\"u}ger and {van den Berg}, {Timo K} and Guntram Suske and Laura Guti{\'e}rrez and Sjaak Philipsen",
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Meinders, M, Kulu, DI, van de Werken, HJG, Hoogenboezem, M, Janssen, H, Brouwer, RWW, van Ijcken, WFJ, Rijkers, E-J, Demmers, JAA, Krüger, I, van den Berg, TK, Suske, G, Gutiérrez, L & Philipsen, S 2015, 'Sp1/Sp3 transcription factors regulate hallmarks of megakaryocyte maturation and platelet formation and function' Blood, vol. 125, no. 12, pp. 1957-67. https://doi.org/10.1182/blood-2014-08-593343

Sp1/Sp3 transcription factors regulate hallmarks of megakaryocyte maturation and platelet formation and function. / Meinders, Marjolein; Kulu, Divine I; van de Werken, Harmen J G; Hoogenboezem, Mark; Janssen, Hans; Brouwer, Rutger W W; van Ijcken, Wilfred F J; Rijkers, Erik-Jan; Demmers, Jeroen A A; Krüger, Imme; van den Berg, Timo K; Suske, Guntram; Gutiérrez, Laura; Philipsen, Sjaak.

In: Blood, Vol. 125, No. 12, 19.03.2015, p. 1957-67.

Research output: Contribution to journalArticleAcademicpeer-review

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T1 - Sp1/Sp3 transcription factors regulate hallmarks of megakaryocyte maturation and platelet formation and function

AU - Meinders, Marjolein

AU - Kulu, Divine I

AU - van de Werken, Harmen J G

AU - Hoogenboezem, Mark

AU - Janssen, Hans

AU - Brouwer, Rutger W W

AU - van Ijcken, Wilfred F J

AU - Rijkers, Erik-Jan

AU - Demmers, Jeroen A A

AU - Krüger, Imme

AU - van den Berg, Timo K

AU - Suske, Guntram

AU - Gutiérrez, Laura

AU - Philipsen, Sjaak

N1 - © 2015 by The American Society of Hematology.

PY - 2015/3/19

Y1 - 2015/3/19

N2 - 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.

AB - 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.

KW - Animals

KW - Azepines/chemistry

KW - Blood Platelets/cytology

KW - Bone Marrow/metabolism

KW - Flow Cytometry

KW - Lectins, C-Type/metabolism

KW - Megakaryocytes/cytology

KW - Mice

KW - Mice, Knockout

KW - Naphthalenes/chemistry

KW - Platelet Aggregation

KW - Platelet Membrane Glycoproteins/metabolism

KW - Proteome

KW - Signal Transduction

KW - Sp1 Transcription Factor/genetics

KW - Sp3 Transcription Factor/genetics

KW - Spleen/metabolism

KW - Thrombocytopenia/metabolism

KW - Transcription Factors/metabolism

U2 - 10.1182/blood-2014-08-593343

DO - 10.1182/blood-2014-08-593343

M3 - Article

VL - 125

SP - 1957

EP - 1967

JO - Blood

JF - Blood

SN - 0006-4971

IS - 12

ER -

Meinders M, Kulu DI, van de Werken HJG, Hoogenboezem M, Janssen H, Brouwer RWW et al. Sp1/Sp3 transcription factors regulate hallmarks of megakaryocyte maturation and platelet formation and function. Blood. 2015 Mar 19;125(12):1957-67. https://doi.org/10.1182/blood-2014-08-593343