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Keywords:

  • megakaryocyte differentiation;
  • G protein-coupled receptor 30;
  • G protein-coupled oestrogen receptor;
  • oestrogen receptor;
  • platelet formation

Megakaryocytes are bone-marrow precursor cells that differentiate to produce blood platelets via intermediate cytoplasmic extensions known as proplatelets (Patel et al, 2005). Recent advances in the understanding of megakaryocyte differentiation and platelet formation have been mostly obtained by biological studies of cultured cells. Although platelet formation from megakaryocytes has been actively studied, the molecular mechanisms of this process are still incompletely understood. Growing evidence has accrued that sex hormones may play a crucial role during megakaryopoiesis (Kostyak & Naik, 2007). Accordingly, it has been shown that high levels of oestrogens and conventional hormone replacement therapies increase the number of megakaryocytes in mice (Perry et al,2000) and in postmenopausal women (Bord et al, 2000). Nagata et al (2003) have also shown that oestradiol can be synthesised by murine megakaryocytes and that oestradiol positively affects proplatelet formation. In addition, Bord et al (2004) reported that oestrogens can promote megakaryocyte proliferation and maturation, thereby modulating the expression of the classical oestrogen receptors (ER)α and ERβ. We have previously demonstrated that oestrogens can potentiate platelet aggregation through a rapid and reversible ERβ-mediated signalling (Moro et al, 2005) and that membrane lipid rafts coordinate this pathway (Reineri et al, 2007).

Megakaryocytes and platelets are known to express ERβ (Khetawat et al, 2000). Genomic effects of oestrogens in megakaryocytes have been suggested to contribute to gender differences in platelet function. However, oestrogens can also induce rapid, non-genomic effects through interaction with classical ERs, localised on the plasma membrane, and through a member of the 7-transmembrane G protein-coupled receptor family, GPR30 (Cheskis et al, 2007), now known as G protein-coupled oestrogen receptor 1 (GPER). Despite the potential importance of GPER in megakaryocyte differentiation and maturation, its exact role in this process has not yet been determined. In this context, we sought to evaluate whether the expression of GPER could change during megakaryocytic differentiation.

CD34+ cells were isolated from human cord blood and cultured in StemSpan® Serum-Free Expansion Medium medium (Stem-Cell Technologies, Vancouver, BC, Canada) supplemented with 10 ng/ml thrombopoietin, interleukin (IL)6 and IL11 (all from PeproTech EC Ltd, London, UK) at 37°C in 5% CO2 to induce megakaryocyte differentiation.

After 13 days of culture cells were harvested, cytospun on glass coverslips, and stained with goat polyclonal anti-CD61 (Santa Cruz, Heidelberg, Germany) and secondary antibody conjugated with Alexa Fluor-488 (Invitrogen, Milan, Italy). Nuclear counterstaining was performed with Hoechst 33258. Specimens were mounted in Mowiol-488. Conventional fluorescence microscopy was performed through an Axioscope 2 Plus microscope (Carl Zeiss, Göttingen, Germany), using a 63/1·25 or a 100/1·30 Plan Neofluar oil-immersion objective. For each specimen, at least 100 cells were evaluated. The percentage of mature megakaryocytes positive for CD61 staining and with polyploid nuclei at the end of culture was 70 ± 15% (Fig 1).

image

Figure 1.  Representative immunofluorescence image of mature megakaryocytes derived from CD34+ cells, as revealed by staining for CD61 (scale bar = 20 μm).

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To evaluate GPER expression during megakaryocyte differentiation three independent experiments were performed, each using a different pool of three cord blood samples (a total of nine cord blood samples was used), to eliminate any biological variability.

GPER expression was assessed in cells on days 0 (CD34+), 3, 7 of differentiation, as well as in mature megakaryocytes isolated at day 13 by CD61 immunomagnetic beads technique (Miltenyi-Biotec, Bergisch Gladbach, Germany). Total RNA was extracted with the RNAqueous kit (Ambion Inc, Foster City, CA, USA) and reverse transcription reactions were performed using QuantiTect Reverse Transcription kit (Qiagen, Hilden, Germany). Real-time polymerase chain reaction was carried out on an ABI 7000 thermal cycler (Applied Biosystems, Foster City, CA 94404, USA) using the TaqMan chemistry. GAPDH was used as endogenous control, and CD34+ cells at day 0 of culture were used as calibrator. Each sample was analysed in triplicate and the results were consistent. GPER expression in CD34+ cells was arbitrarily set to 100. GPER expression decreased dramatically at day 3 (35·83 ± 13·25% with respect to calibrator; *P < 0·01). Moreover, GPER was no longer detectable both at day 7 and in mature megakaryocytes (CD61+) (Fig 2). The finding of a rapid decrease of GPER expression during the first stages of megakaryocytic differentiation leads to the conclusion that it is not involved in megakaryocyte maturation and proplatelet formation. However, we cannot firmly exclude that this receptor can eventually play a role in later stages of megakaryocytic differentiation. Future studies are also needed to evaluate the expression of GPER during haematopoietic stem cells differentiation in other cell lineages. Further research on the role of oestrogen-dependent signalling mediated by ERα and ERβ in megakaryocyte differentiation and platelet development is also warranted.

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Figure 2.  Differential expression of GPER during megakaryocyte differentiation. Results are expressed as percentage of GPER expression compared with expression levels in CD34+ (100%). Each sample was analysed in triplicate. We performed gene expression analysis using the comparative ΔΔCT method. Values are mean ± SEM of three independent experiments.

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Acknowledgements

  1. Top of page
  2. Acknowledgements
  3. Author contributions
  4. References

We thank Malara Alessandro, Badalucco Stefania and Pallotta Isabella (University of Pavia, Italy) for providing the necessary training regarding the isolation and culture of human cord blood CD34+ cells, and Drs Galli Licia and Perego Beatrice (University of Piemonte Orientale A. Avogadro) for umbilical cord blood collection.

This work was supported by grants from Ministero dell’Università e della Ricerca-PRIN 2007 (to FS), from Regione Piemonte -Ricerca Sanitaria Finalizzata 2006, 2007 and -Ricerca Scientifica Applicata 2004 and from Università del Piemonte Orientale-Ricerca Locale.

Author contributions

  1. Top of page
  2. Acknowledgements
  3. Author contributions
  4. References

CDV performed experiments, analysed data and wrote the paper. SB performed experiments and analysed data. A Balduini performed experiments and analysed data. AR performed experiments and analysed data. A Bagarotti performed experiments and analysed data. NS provided human cord blood samples. A Bertoni designed the project, analysed data and wrote the paper. FS financed the project and wrote the paper.

References

  1. Top of page
  2. Acknowledgements
  3. Author contributions
  4. References
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