Role of Dicer Enzyme in the Regulation of Store Operated Calcium Entry (SOCE) in CD4+ T Cells.
OBJECTIVE
Activation of T cell receptors (TCRs) in CD4+ T cells leads to a cascade of signalling reactions including increase of intracellular calcium (Ca2+) levels with subsequent Ca2+ dependent stimulation of gene expression, proliferation, cell motility and cytokine release. The increase of cytosolic Ca2+ results from intracellular Ca2+ release with subsequent activation of store-operated Ca2+ entry (SOCE). Previous studies suggested miRNAs are required for the development and functions of CD4+ T cells. An enzyme called Dicer is required during the process of manufacturing mature miRNAs from the precursor miRNAs. In this study, we explored whether loss of Dicer in CD4+ T cells affects SOCE and thus Ca2+ dependent regulation of cellular functions.


METHODS
We tested the expression of Orai1 by q-RT-PCR and flow cytometry. Further, we measured SOCE by an inverted phase-contrast microscope with the Incident-light fluorescence illumination system using Fura-2. Intracellular Ca2+ was also measured by flow cytometry using Ca2+ sensitive dye Fluo-4.


RESULTS
We found that in Dicer deficient (DicerΔ/Δ) mice Orai1 was downregulated at mRNA and protein level in CD4+ T cells. Further, SOCE was significantly smaller in DicerΔ/Δ CD4+ T cells than in CD4+ T cells isolated from wild-type (Dicerfl/fl) mice.


CONCLUSIONS
Our data suggest that miRNAs are required for adequate Ca2+ entry into CD4+ T cells and thus triggering of Ca2+ sensitive immune functions.
Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu city, China.
Shaqiu
S
Zhang
Tamer
T
Al-Maghout
Yuetao
Y
Zhou
Rosi
R
Bissinger
Abeer
A
Abousaab
Madhuri S
MS
Salker
Lisann
L
Pelzl
Bradley S
BS
Cobb
Anchun
A
Cheng
Yogesh
Y
Singh
Florian
F
Lang
D000814:Aniline Compounds / Q000378:metabolism; D000818:Animals; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D065096:Calcium Chelating Agents / Q000378:metabolism; D053487:DEAD-box RNA Helicases / Q000172:deficiency / Q000235:genetics*; D005456:Fluorescent Dyes / Q000378:metabolism; D016257:Fura-2 / Q000378:metabolism; D005786:Gene Expression Regulation; D017136:Ion Transport / Q000187:drug effects; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D000071740:ORAI1 Protein / Q000235:genetics* / Q000378:metabolism; D061251:Primary Cell Culture; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D043244:Ribonuclease III / Q000172:deficiency / Q000235:genetics*; D015398:Signal Transduction; D019284:Thapsigargin / Q000494:pharmacology; D014966:Xanthenes / Q000378:metabolism
D000814:Aniline Compounds; D065096:Calcium Chelating Agents; C409648:Fluo 4; D005456:Fluorescent Dyes; D035683:MicroRNAs; D000071740:ORAI1 Protein; C520430:Orai1 protein, mouse; D012333:RNA, Messenger; D014966:Xanthenes; D019284:Thapsigargin; C509781:Dicer1 protein, mouse; D043244:Ribonuclease III; D053487:DEAD-box RNA Helicases; D002118:Calcium; D016257:Fura-2