Abstract
BackgroundAn increase in the frequency of circulating endothelial colony forming cells (ECFCs), the only subset of endothelial progenitor cells (EPCs) truly belonging to the endothelial phenotype, occurs in patients affected by primary myelofibrosis (PMF). Herein, they might contribute to the enhanced neovascularisation of fibrotic bone marrow and spleen. Store-operated Ca2+ entry (SOCE) activated by the depletion of the inositol-1,4,5-trisphosphate (InsP3)-sensitive Ca2+ store drives proliferation in ECFCs isolated from both healthy donors (N-ECFCs) and subjects suffering from renal cellular carcinoma (RCC-ECFCs). SOCE is up-regulated in RCC-ECFCs due to the over-expression of its underlying molecular components, namely Stim1, Orai1, and TRPC1.Methodology/Principal FindingsWe utilized Ca2+ imaging, real-time polymerase chain reaction, western blot analysis and functional assays to evaluate molecular structure and the functional role of SOCE in ECFCs derived from PMF patients (PMF-ECFCs). SOCE, induced by either pharmacological (i.e. cyclopiazonic acid or CPA) or physiological (i.e. ATP) stimulation, was significantly higher in PMF-ECFCs. ATP-induced SOCE was inhibited upon blockade of the phospholipase C/InsP3 signalling pathway with U73111 and 2-APB. The higher amplitude of SOCE was associated to the over-expression of the transcripts encoding for Stim2, Orai2–3, and TRPC1. Conversely, immunoblotting revealed that Stim2 levels remained constant as compared to N-ECFCs, while Stim1, Orai1, Orai3, TRPC1 and TRPC4 proteins were over-expressed in PMF-ECFCs. ATP-induced SOCE was inhibited by BTP-2 and low micromolar La3+ and Gd3+, while CPA-elicited SOCE was insensitive to Gd3+. Finally, BTP-2 and La3+ weakly blocked PMF-ECFC proliferation, while Gd3+ was ineffective.ConclusionsTwo distinct signalling pathways mediate SOCE in PMF-ECFCs; one is activated by passive store depletion and is Gd3+-resistant, while the other one is regulated by the InsP3-sensitive Ca2+ pool and is inhibited by Gd3+. Unlike N- and RCC-ECFCs, the InsP3-dependent SOCE does not drive PMF-ECFC proliferation.
Highlights
Primary myelofibrosis (PMF) is a Philadelphia chromosomenegative (Ph-neg) chronic myeloproliferative neoplasm (MPN) characterized by the following hallmarks: bone marrow (BM) fibrosis, myeloid metaplasia, splenomegaly, increased frequency of circulating CD34+ hematopoietic progenitor cells (HPCs), and a V617F mutation of the JAK2 gene in the hematopoietic lineage encountered in 63% of the patients [1,2]
Intracellular Ca2+ Release and Store-operated Ca2+ Entry are Abnormal in endothelial colony forming cells (ECFCs) Isolated from primary myelofibrosis (PMF) Patients The resting Ca2+ levels measured in PMF-ECFCs and ECFCs provided by healthy donors (N-ECFCs) were evaluated upon digital subtraction of the fluorescence background and were not statistically different (p,0.05), the average values of the Fura-2 ratio being 1.46360.015, n = 353, and 1.47060.022, n = 233, respectively
Cyclopiazonic acid (CPA) is a widely employed Sarco-Endoplasmic Reticulum Ca2+-ATPase (SERCA) inhibitor, which prevents the pump from counterbalancing the passive Ca2+ leak from the stores to the cytosol, thereby leading to a massive drop in the endoplasmic reticulum (ER) Ca2+ content which signals the Stromal interacting molecule 1 (Stim1)-mediated gating of store-operated Ca2+ channels on the plasma membrane
Summary
Primary myelofibrosis (PMF) is a Philadelphia chromosomenegative (Ph-neg) chronic myeloproliferative neoplasm (MPN) characterized by the following hallmarks: bone marrow (BM) fibrosis, myeloid metaplasia, splenomegaly, increased frequency of circulating CD34+ hematopoietic progenitor cells (HPCs), and a V617F mutation of the JAK2 gene in the hematopoietic lineage encountered in 63% of the patients [1,2]. An increase in the frequency of circulating endothelial colony forming cells (ECFCs), the only subset of endothelial progenitor cells (EPCs) truly belonging to the endothelial phenotype, occurs in patients affected by primary myelofibrosis (PMF) They might contribute to the enhanced neovascularisation of fibrotic bone marrow and spleen. SOCE is up-regulated in RCC-ECFCs due to the over-expression of its underlying molecular components, namely Stim, Orai, and TRPC1
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