Involvement of TGFβ1 in autocrine regulation of proplatelet formation in healthy subjects and patients with primary myelofibrosis

Abstract

Megakaryocytes release platelets into the bloodstream by elongating proplatelets. In this study, we showed that human megakaryocytes constitutively release Transforming Growth Factor β1 and express its receptors. Importantly, Transforming Growth Factor β1 downstream signaling, through SMAD2/3 phosphorylation, was shown to be active in megakaryocytes extending proplatelets, indicating a type of autocrine stimulation on megakaryocyte development. Furthermore, inactivation of Transforming Growth Factor β1 signaling, by the receptor inhibitors SB431542 and Stemolecule ALK5 inhibitor, determined a significant decrease in proplatelet formation. Recent studies indicated a crucial role of Transforming Growth Factor β1 in the pathogenesis of primary myelofibrosis. We demonstrated that primary myelofibrosis-derived megakaryocytes expressed increased levels of bioactive Transforming Growth Factor β1; however, higher levels of released Transforming Growth Factor β1 did not lead to enhanced activation of downstream pathways. Overall, these data propose Transforming Growth Factor β1 as a new element in the autocrine regulation of proplatelet formation in vitro. Despite the increase in Transforming Growth Factor β1 this mechanism seems to be preserved in primary myelofibrosis.

Figures

Figure 1.

Autocrine TGFβ1 impact on human megakaryocyte maturation and platelet production. Megakaryocytes were derived from human umbilical cord blood progenitor cells as described in the Online Supplementary Appendix. RNA was extracted from CD61+ megakaryocytes and qRT-PCR of the TGFβ1 expression was performed at day 7, 10 and 13 of megakaryocyte differentiation. mRNA levels were normalized to expression levels at Day 7 of culture (bars represent mean±SD, n=3 different experiments, *P<0.05, ANOVA and Bonferroni t-test as post-hoc test) (A). TGFβ1 was constitutively released into the conditioned medium during megakaryocyte differentiation in culture. Total (B) and bioactive TGFβ1 (C) levels in culture supernatants were determined by biological assay and data normalized to cell numbers (means±SD, n=3 separate experiments, *P<0.05, ANOVA and Bonferroni's t-test as post-hoc test). TGFβ1 receptors were expressed by human megakaryocytes in culture. Megakaryocytes were lysed and subjected to Western blot analysis. TβRI and TβRII receptors were detected in human megakaryocytes at day 7, 10 and 13 of culture. The membrane was reprobed with anti–b-actin to ensure equal loading (representative of 5 different experiments) (D). Western blot analysis of pSMAD (E), pAkt and PTEN (F) in human megakaryocytes revealed that the signaling involving pSMAD2/3, pAkt and PTEN was activated in mature megakaryocytes. Samples were also probed with anti-SMAD2/3, anti-Akt and anti–β-actin antibodies to ensure equal loading (representative of 5 different experiments). Mature megakaryocytes seeded in presence or absence of the TβRI inhibitors, SB431542 (10 μM) and specific ALK5 inhibitor (10 μM), or the Akt inhibitor, AKTI-1/2 (10 μM), showed a significant reduction of proplatelet formation (ppf) relative to not treated controls (bars represent means±SD, n=3 separate experiments, *P<0,05) (G). Western blot analysis of megakaryocytes treated (+) or not (–) with SB431542 (10 μM) (H) or ALK5 specific inhibitor (10 μM) (I) at Day 13 of culture. Decrease of SMAD2/3 phosphorylation determined by TβRI inhibition affected PTEN expression and subsequent Akt activation. The membranes were reprobed with anti–β-actin to ensure equal loading (representative of 5 different experiments). Mature megakaryocytes seeded in presence or absence of human recombinant TGFβ1 (1 ng/mL) showed a significant increase of proplatelet formation (ppf) relative to untreated controls (bars represent means±SD, n=5 separate experiments, *P<0.05) (L).

Figure 2.

Autocrine TGFβ1 signaling in PMF derived megakaryocytes. Megakaryocytes were derived from peripheral blood progenitors of patients with primary myelofibrosis (PMF) and healthy donor controls (CTRL), as described in the Online Supplementary Appendix. Mature megakaryocytes from PMF, showed a significant reduction of proplatelet formation (ppf) relative to CTRL (bars represent means±SD, n=11 separate experiments, *P<0.05) (A). Total (B) and bioactive TGFβ1 (C) levels in culture supernatants were determined by biological assay and data normalized to cell number (means±SD, n=11 separate experiments). PMF derived megakaryocytes showed a significant increase in both total and bioactive TGF secretion as compared to CTRL. Megakaryocytes were lysed and subjected to Western blot analysis. TGFβ1 receptors were equally expressed in CTRL and PMF mature megakaryocytes in vitro. The membrane was reprobed with anti–b-actin to ensure equal loading (representative of 3 different experiments) (D). Western blot analysis of pSMAD, pAkt and PTEN revealed a similar activation in CTRL and PMF derived megakaryocytes, despite the increased levels of TGFβ1 observed in PMF culture medium (representative of 3 different experiments) (E). PMF derived megakaryocytes seeded in presence (+) or absence (–) of the TβRI inhibitor, SB431542 (10 μM), showed a significant reduction of SMAD2/3 and Akt phosphorylation and conversely an increase in PTEN expression. The membranes were reprobed with anti–β-actin to ensure equal loading (representative of 3 different experiments) (F).