Enhanced Expression of Stim, Orai, and TRPC Transcripts and Proteins in Endothelial Progenitor Cells Isolated from Patients with Primary Myelofibrosis

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.

MANAGEMENT OF ENDOCRINE DISEASE: Klinefelter syndrome, cardiovascular system, and thromboembolic disease: review of literature and clinical perspectives

“…Congruent with this observation, Ru et al (39) showed that in KS subjects testosterone levels were not correlated with the number of EPCs (Table 1). Given the growing interest of the scientific community in the study of EPCs (40,41,42,43,44), further studies are needed to explain the relationship between EPCs and KS.…”

Bradykinin‐mediated Ca2+ signalling regulates cell growth and mobility in human cardiac c‐Kit+ progenitor cells

“…This implies that TRPC3 and TRPC4 are not involved in mediating bradykinin‐induced Ca 2+ influx. La 3+ is a well‐known TRP channel blocker; however, a recent study reported that a low concentration of La 3+ at 10 μmol L −1 can selectively block SOCE channel 23. We therefore used 10 μmol L −1 La 3+ to test whether SOCE channel is involved in mediating the bradykinin‐induced Ca 2+ influx.…”

Dysregulation of VEGF-induced proangiogenic Ca2+ oscillations in primary myelofibrosis-derived endothelial colony-forming cells

“…Cell culture, solutions, Ca 2þ measurements, and proliferation assays All technical details of ECFC isolation and cultivation, measurement of intracellular Ca 2þ dynamics, and proliferation assays have been described elsewhere [9,17,22] and are detailed in the Supplementary Methods (online only, available at www.exphem. org).…”

“…More recently, we found that the Ca 2þ toolkit undergoes a dramatic rearrangement also in EPCs derived from PMF patients bearing the JAK2 mutation [17]. These cells exhibited a reduction in InsP 3 -depedent Ca 2þ release and two pharmacologically distinguishable types of SOCCs: one is stimulated by passive ER depletion, is inhibited by BTP-2 and La 3þ , and is resistant to Gd 3þ , whereas the other is tuned by the InsP 3 -sensitive Ca 2þ pool and inhibited by BTP-2, La 3þ , and Gd 3þ [17]. Unlike EPCs derived from peripheral blood of healthy donors and from umbilical cord blood (UCB) [15,18], the pharmacologic blockade of store-operated Ca 2þ entry (SOCE) did not block PMF-derived EPC proliferation when the cells were grown in an endothelial growth medium enriched with growth factors, including VEGF [17].…”

“…For instance, a lower ER Ca 2þ concentration and the downregulation of InsP 3 Rs prevent VEGF from triggering Ca 2þ oscillations in RCC-derived EPCs [3,9,10]. More recently, we found that the Ca 2þ toolkit undergoes a dramatic rearrangement also in EPCs derived from PMF patients bearing the JAK2 mutation [17]. These cells exhibited a reduction in InsP 3 -depedent Ca 2þ release and two pharmacologically distinguishable types of SOCCs: one is stimulated by passive ER depletion, is inhibited by BTP-2 and La 3þ , and is resistant to Gd 3þ , whereas the other is tuned by the InsP 3 -sensitive Ca 2þ pool and inhibited by BTP-2, La 3þ , and Gd 3þ [17].…”