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SLP76 interactome in resting and activated primary mast cells

ABSTRACT: We used a knock-in (KI) mouse expressing SLP-76 with a C-terminal One-Strep-Tag (OST). Such a system allowed the replacement in the genome of SLP-76 by its tagged version and its expression in physiological amounts. Then, by combining high affinity purification and label-free quantitative proteomics, we report here the first description of the SLP76 interactome in resting and activated primary cultured mast cells derived from mice expressing an OST at the C-terminus of SLP-76 (Slp76OST/OST mice).

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: Hesse Anne-Marie

PROVIDER: PXD000052 | Pride | 2013-08-28

REPOSITORIES: Pride

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Proteomic analysis of the SH2 domain-containing leukocyte protein of 76 kDa (SLP76) interactome in resting and activated primary mast cells [corrected].

Bounab Yacine Y Hesse Anne-Marie- AM Iannascoli Bruno B Grieco Luca L Couté Yohann Y Niarakis Anna A Roncagalli Romain R Lie Eunkyung E Lam Kong-Peng KP Demangel Caroline C Thieffry Denis D Garin Jérôme J Malissen Bernard B Daëron Marc M

Molecular & cellular proteomics : MCP 20130702 10

We report the first proteomic analysis of the SLP76 interactome in resting and activated primary mouse mast cells. This was made possible by a novel genetic approach used for the first time here. It consists in generating knock-in mice that express signaling molecules bearing a C-terminal tag that has a high affinity for a streptavidin analog. Tagged molecules can be used as molecular baits to affinity-purify the molecular complex in which they are engaged, which can then be studied by mass spec ...[more]

PMID: 23820730

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Project description:Investigations of teriparatide (rPTH) as a potential treatment for critical defects have demonstrated the predicted anabolic effects on bone formation, and significant non-anabolic effects on healing via undefined mechanisms. Specifically, studies in murine models of structural allograft healing demonstrated that rPTH treatment increased angiogenesis (vessels <30μm), and decreased arteriogenesis (>30 μm) and mast cell numbers, which lead to decreased fibrosis and accelerated healing. To better understand these non-anabolic effects, we interrogated osteogenesis, vasculogenesis and mast cell accumulation in mice randomized to placebo (saline), rPTH (20µg/kg/2days), or the mast cell inhibitor sodium cromolyn (SC) (24µg/kg/2days), via longitudinal micro-CT and multiphoton laser scanning microscopy (MPLSM), in a critical calvaria defect model. Micro-CT demonstrated that SC significantly increased defect window closure and new bone volume versus placebo (p<0.05), although these effects were not as great as rPTH. Interestingly, both rPTH and SC has similar inhibitory effects on arteriogenesis versus placebo (p<0.05) without affecting total vascular volume. MPLSM time course studies in untreated mice revealed that large numbers of mast cells were detected 1 day post-op (43 +/- 17), peaked at 6 days (76 +/- 6), and were still present in the critical defect at the end of the experiment on day 30 (20 +/- 12). In contrast, angiogenesis was not observed until day 4, and functional vessels were first observed on 6 days, demonstrating that mast cell accumulation precedes vasculogenesis. To confirm a direct role of mast cells on osteogenesis and vasculogenesis, we demonstrated that specific diphtheria toxin-α deletion in Mcpt5-Cre-iDTR mice results in similar affects as SC treatment in WT mice. Collectively, these findings demonstrate that mast cells inhibit bone defect healing by stimulating arteriogenesis associated with fibrotic scaring, and that an efficacious non-anabolic effect of rPTH therapy on bone repair is suppression of arteriogenesis and fibrosis secondary to mast cell inhibition.And then, we need to figure out how rPTH works on mast cells. So we used the co-culture of osteoblast and mast cells, and perform RNAseq, by which we aim to screen out the factors in mast cells that are potential mediators for rPTH effects in bone formation, blood vessel formation, and fibrosis formation.

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