Calcium signalling: my first love!
I spent five years in the lab of Prof. Franco Tanzi and Prof. Francesco Moccia at the University of Pavia and I gained a deep understanding of the molecular mechanisms that generates different calcium signals.
During my master and my PhD I studied the calcium signals induced by the Vascular Endothelial Growth Factor (VEGF) in endothelial progenitor cells (EPCs). EPCs are mobilised from the bone marrow to the site of tissue regeneration and sustain neovascularization after acute vascular injury and upon the angiogenic switch in solid tumours. Therefore, they represent a suitable tool for cell-based therapy in regenerative medicine and provide a novel promising target in the fight against cancer. Notably, VEGF is the most potent stimulus for EPC proliferation and incorporation into neovessels, but very little was known about the signalling pathways it activates in these cells.
Firstly, we proved that VEGF induces calcium oscillations that require the interplay between InsP3-dependent calcium release and store-operated calcium entry (SOCE) and promote EPC growth and tubulogenesis by engaging NF-κB (Dragoni et al., Stem Cells 2011).
Unlike EPCs extracted from peripheral blood (PB-EPCs), EPCs derived from umbilical cord blood (UCB-ECPs) express the canonical transient receptor potential channel 3 (TRPC3) that mediates diacylglycerol-dependent calcium entry. For this reason, the calcium response to VEGF in UCB-EPCs is shaped by a different calcium machinery as compared with PB-ECFCs, and TRPC3 stands out as a promising target in EPC-based therapy (Dragoni et al., Stem Cells and Dev 2013).
Additionally, we discovered that PB-EPCs possess a functional TRPV4 channel before their engraftment into nascent vessels (Dragoni et al., J Cell Physiol 2015).
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This is a graphical abstract of my first paper.
VEGF binds VEGFR2, leading to the receptor autophosphorylation and consequent activation of PLC.
PLC cleaves PIP2 to DAG and InsP3.
InsP3 binds to its receptor on the ER, leading to its opening and the depletion of the ER calcium stores.
The store depletion causes the activation of the calcium sensor Stim1 which activates Orai1 on the plasma membrane, leading to calcium entry (SOCE). The interplay between calcium release and calcium entry generates oscillations that lead to CaMK activation, and IKBa phosphorylation. IKBa detaches from NFkB that can now translocate into the nucleus and activate the transcription of the genes responsible for cell proliferation and tubulogenesis.
I then investigated if EPCs extracted from patients suffering from different types of malignancies express a functioning calcium machinery and how they respond to VEGF. We observed that two distinct signalling pathways mediate SOCE in EPCs derived from primary myelofibrosis patients (PMF-ECFC); one is activated by passive store depletion and is gadolinium-resistant, while the other one is regulated by the InsP3-sensitive calcium pool and is inhibited by gadolinium. Moreover, VEGF-induced calcium oscillations modestly stimulate PMF-ECFC growth and in vitro angiogenesis. These results may explain the modest effect of anti-VEGF therapies in PMF patients (Dragoni et al., PLoS One 2014; Dragoni et al., Exp Hematol 2015).
Finally, we had the opportunity to study proper cancer cells and we discovered that a functional SOCE is expressed but does not control proliferation of metastatic renal cellular carcinoma cells isolated from patients resistant to multikinase inhibitors (Dragoni et al., Biomed Res Int 2014).