FKBP52 is involved in the regulation of SOCE channels in the human platelets and MEG 01 cells

Abstract

Immunophilins are FK506-binding proteins that have been involved in the regulation of calcium homeostasis, either by modulating Ca(2+) channels located in the plasma membrane or in the rough endoplasmic reticulum (RE). We have investigated whether immunophilins would participate in the regulation of stored-operated Ca(2+) entry (SOCE) in human platelets and MEG 01. Both cell types were loaded with fura-2 for determining cytosolic calcium concentration changes ([Ca(2+)](c)), or stimulated and fixed to evaluate the protein interaction profile by performing immunoprecipitation and western blotting. We have found that incubation of platelets with FK506 increases Ca(2+) mobilization. Thapsigargin (TG)-evoked, Thr-evoked SOCE and TG-evoked Mn(2+) entry resulted in significant reduction by treatment of platelets with immunophilin antagonists. We confirmed by immunoprecipitation that immunophilins interact with transient receptor potential channel 1 (TRPC1) and Orai1 in human platelets. FK506 and rapamycin reduced the association between TRPC1 and Orai1 with FK506 binding protein (52) (FKBP52) in human platelets, and between TRPC1 and the type II IP(3)R, which association is known to be crucial for the maintenance of SOCE in human platelets. FKBP52 role in SOCE activation was confirmed by silencing FKBP52 using SiRNA FKBP52 in MEG 01 as demonstrated by single cell configuration imaging technique. TRPC1 silencing and depletion of cell of TRPC1 and FKBP52 simultaneously, impair activation of SOCE evoked by TG in MEG 01. Finally, in MEG 01 incubated with FK506 we observed a reduction in TRPC1/FKBP52 coupling, and similarly, FKBP52 silencing reduced the association between IP3R type II and TRPC1 during SOCE. All together, these results demonstrate that immunophilins participate in the regulation of SOCE in human platelets.

Pathophysiological Significance of Store-Operated Calcium Entry in Megakaryocyte Function: Opening New Paths for Understanding the Role of Calcium in Thrombopoiesis

“…Since that, Orai1 expression was observed in the human megakaryocytic cell line MEG-01 [172,173]. Borst et al demonstrated that Orai1 expression is modulated by the serum- and glucocorticoid-inducible kinase 1 (SGK1) [172], a kinase belonging to the AGC family of serine/threonine protein kinases [174,175], that can be regulated by a variety of different triggers, including hormones, thrombin, oxidative stress and growth factors [175].…”

Evaluation of the antiaggregant activity of ascorbyl phenolic esters with antioxidant properties

“…The reduction on Thr-evoked phospho-mTOR in Ser 2448 , which is targeted by Akt, was mostly evidenced in platelets preincubated with Gal Asc, Caf Asc, and Asc despite that the result were not significant regarding the values found in platelet non-treated with the esters. The impaired activation of mTOR upon Thr activation resulting from elevated basal activation levels might explain the reduction in calcium homeostasis and subsequent impaired aggregation in response to the physiological agonist, due to mTOR have been previously evidenced to participate in both signaling pathways in this cell type, as reported by our group and others [20,38,40]. In case of gallic acid, clinical trials using this phytochemical have reported inhibition of mTOR [70], which agree with the negative effect of Gal Asc over Akt-dependent mTOR phosphorylation evoked by Thr in our cell type.…”

Trpc1

“…While it has been shown that it participated in store-operated Ca 2+ entry pathways affecting platelet activation and thrombus formation (Rosado and Sage 2001;Lopez et al 2013;Jardin et al 2012;Rosado et al 2002), studies using platelets derived from the Trpc1 knockout mice could not show an effect on either store-operated Ca 2+ entry or platelet function, such as activation, aggregation, hemostasis, and thrombosis (Varga-Szabo et al 2008). …”