MiR-221 Influences Effector Functions and Actin Cytoskeleton in Mast Cells

Abstract

Mast cells have essential effector and immunoregulatory functions in IgE-associated allergic disorders and certain innate and adaptive immune responses, but the role of miRNAs in regulating mast cell functions is almost completely unexplored. To examine the role of the activation-induced miRNA miR-221 in mouse mast cells, we developed robust lentiviral systems for miRNA overexpression and depletion. While miR-221 favored mast cell adhesion and migration towards SCF or antigen in trans-well migration assays, as well as cytokine production and degranulation in response to IgE-antigen complexes, neither miR-221 overexpression, nor its ablation, interfered with mast cell differentiation. Transcriptional profiling of miR-221-overexpressing mast cells revealed modulation of many transcripts, including several associated with the cytoskeleton; indeed, miR-221 overexpression was associated with reproducible increases in cortical actin in mast cells, and with altered cellular shape and cell cycle in murine fibroblasts. Our bioinformatics analysis showed that this effect was likely mediated by the composite effect of miR-221 on many primary and secondary targets in resting cells. Indeed, miR-221-induced cellular alterations could not be recapitulated by knockdown of one of the major targets of miR-221. We propose a model in which miR-221 has two different roles in mast cells: in resting cells, basal levels of miR-221 contribute to the regulation of the cell cycle and cytoskeleton, a general mechanism probably common to other miR-221-expressing cell types, such as fibroblasts. Vice versa, upon induction in response to mast cell stimulation, miR-221 effects are mast cell-specific and activation-dependent, contributing to the regulation of degranulation, cytokine production and cell adherence. Our studies provide new insights into the roles of miR-221 in mast cell biology, and identify novel mechanisms that may contribute to mast cell-related pathological conditions, such as asthma, allergy and mastocytosis.

Two Functionally Distinct Subsets of Mast Cells Discriminated By IL-2–Independent CD25 Activities

“…To investigate this possibility further, we reconstituted CD25 expression in CD25KO mast cells by lentiviral transduction. As we previously reported (15,33), efficiency of BMMC transduction was routinely 40-50%, as assessed by expression of surface CD25 (Fig. 4D).…”

Dnmt3a restrains mast cell inflammatory responses

“…We therefore assessed mast cell responses to physiological stimulation with IgE and antigen. Interestingly, upon acute stimulation, mast cells lacking Dnmt3a showed significantly increased ability to release the content of their cytoplasmic granules, as assessed both by annexin V binding [which occurs at sites of secretory granule fusion with the plasma membrane (19)(20)(21)] and release of β-hexosaminidase (an enzyme normally stored in cytoplasmic granules) in the culture supernatant ( Fig. 1 D and E).…”

miR-146a and NF-κB1 Regulate Mast Cell Survival and T Lymphocyte Differentiation

“…Next, we evaluated whether the impaired Th2 responses observed in these mice could be partially explained by an altered ability of mast cells to perform their effector functions (degranulation, cytokine production) in response to acute stimulation. The extent of mast cell degranulation was assessed both in vitro, by measuring release of ␤-hexosaminidase from cytoplasmic granules upon stimulation (18), and in vivo, by transferring mast cells into mast cell-deficient recipient mice (Kit W-sh/W-sh ) and performing passive cutaneous anaphylaxis (PCA) experiments. Even in the absence of p50, mast cells degranulated normally in response to IgE crosslinking or PMA and ionomycin stimulation, both in vitro and in vivo ( Fig.…”

“…Mast cell degranulation was assessed as described previously (18). Briefly, 5 ϫ 10 4 cells were resuspended in 50 l Opti-MEM-1% FBS and stimulated for 1 h with either PMA and ionomycin or IgE-antigen complexes.…”