Asthmatic Serum And Cytokines Induced Down-regulation Of FKBP12.6 Expression In Airway Smooth Muscle Cell And Its Role In Stress Induced Ca²⁺ Release

FKBP12.6 is a member of FKBPs immunophilins family, its molecular weight is 12.6kDa. Ryanodine receptors is a sarcoplasmic reticulum calcium release channel, which have three subtypes, including RyR1, RyR2 and RyR3. RyR2 is expressed on cardiac myocytes and airway smooth muscle cells which plays a key role in the calcium release of sarcoplasmic reticulum. FKBP12.6 can bind to RyR2 specifically , and stabilize RyR2. It was reported that binding of FKBP12.6 to RyR2 can stabilize cardiomyocytes from sarcoplasmic reticulum calcium release, which is the target for treatment of heart failure. Does the interaction of FKBP12.6 and RyR2 play a key role in airway smooth musle cells calcium release ? We hypothesized that the expression level of FKBP12.6 in airway smooth muscle cells related to the release of calcium from sarcoplasmic reticulum.Objective:To investigate FKBP12.6 expression level on sarcoplasmic reticulum calcium release in asthmatic airway smooth muscle cells.Methods:1. The expression of FKBP12.6 in asthmatic serum or cytokines treated bronchial smooth muscle cells are detected by Real-time PCR and western blot.2. Use immunoflorescence and confocal laser microscopy to detect the co-localization of FKBP12.6 and RyR2. The dissociation of FKBP12.6 from RyR2 in asthmatic serum or cytokines treated bronchial smooth muscle cells are detected by immunoprecipitation. 3. After transfected with FKBP12.6 RNA interference and FKBP12.6 overexpression virus vector, bronchial smooth muscle cells are labeled with calcium fluorescent indicator Fura-2, then monitor the dynamic changes of cytosolic free calcium concentration ([Ca2+]i) in response to 10μM bradykinin by calcium fluorescence imaging system.Results:1. Asthmatic serum or cytokines treatment induced down-regulation of FKBP12.6 mRNA and proteins expression in contrast to control (p<0.05);2. The binding of FKBP12.6 RyR2 decreased significantly in Asthmatic serum treated airway smooth muscle cells.3. The co-localization of FKBP12.6 and RyR2 decreased dramatically asthmatic serum or cytokines treated airway smooth muscle cells by immunoflorescence.4. After transfected with FKBP12.6 interfering RNA virus, the duration of 10-5mol/L bradykinin induced calcium increase then return to baseline is longer than control; after transfected with FKBP12.6 overexpression virus vector, the magnitude of intracellular calcium increase was siginficantly lower than that of control, and the duration is 1/3 of control.Conclusion:The results showed that asthmatic serum or cytokines IL-5, IL-13, TNF-αtreatment induced down-regulation of FKBP12.6 in airway smooth muscle cells of rats, lead to reduced binding of FKBP12.6, and the RyR2 calcium release channel become more unstable, which maybe contribute to asthmatic inflammation induced airway smooth muscle hyperresponsiveness. Increase the expression of FKBP12.6 could stabilize RyR2 channel, increase the binding of FKBP12.6 to RyR2, which can reduce the calcium release on sarcoplasmic reticulum.