Glucocorticoid guides mobilization of bone marrow stem/progenitor cells via FPR and CXCR4 coupling

Background Our previous studies have proved the efficient exogenous repairing responses via bone marrow stem and progenitor cells (BMSPCs). However, the trafficking of endogenous bone marrow stem and progenitor cells to and from the bone marrow (BM) is a highly regulated process that remains to be elucidated. We aimed to study the relative importance of the hypothalamic-pituitary-adrenal (HPA) axis in the glucocorticoid-induced BMSPC mobilization. Methods The circulating mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs) were examined in Crh (+/+, −/−) mice after running stress or glucocorticoid mini-infusion. The MSCs and EPCs were investigated ex vivo after treatment with glucocorticoid and glucocorticoid receptor (GR) antagonist, RU486. The expression of chemotaxis receptors, N-formyl peptide receptor (FPR), and Cys-X-Cys receptor 4 (CXCR4) of MSCs and EPCs as well as their colocalization were investigated after treatment with glucocorticoid, glucocorticoid receptor (GR) antagonist (RU486), and FPR antagonist (Cyclosporin H). Results Forced running stress increased circulating MSCs and EPCs in mice, which was blunted when Crh was knocked out, and positively related to the levels of serum glucocorticoid. Prolonged glucocorticoid mini-infusion imitated the stress-induced increase in circulating MSCs and EPCs in Crh+/+ mice and rescued the impaired mobilization in circulating MSCs and EPCs in Crh−/− mice. Meanwhile, glucocorticoid promoted the chemotaxis of MSCs and EPCs ex vivo via GR, inhibited by RU486 (10 μM). Concurrently, glucocorticoid increased the expression of FPR of MSCs and EPCs, but inhibited their expression of CXCR4, followed by their changing colocalization in the cytoplasm. The GC-induced colocalization of FPR and CXCR4 was blunted by Cyclosporin H (1 μM). Conclusion Glucocorticoid-induced CXCR4-FPR responsiveness selectively guides the mobilization of BMSPCs, which is essential to functional tissue repair. Graphical abstract Schematic view of the role of glucocorticoid on the mobilization of bone marrow-derived stem/progenitor cells subsets in the present study. The HPA axis activation promotes the release of glucocorticoid, which regulates the directional migration of MSCs and EPCs mainly via GR. The possible mechanisms refer to the signal coupling of FPR and CXCR4. Their two-sided changes regulated by glucocorticoid are involved in the egress of MSCs and EPCs from BM, which is helpful for wound healing. MSCs, mesenchymal stem cells; EPCs, endothelial progenitor cells.

The synthetic chemoattractant Trp-Lys-Tyr-Met-Val-DMet activates neutrophils preferentially through the lipoxin A4 receptor

A D-methionine–containing peptide, Trp-Lys-Tyr-Met-Val-D-Met-NH2 (WKYMVm), featuring a unique receptor specificity was investigated with respect to its ability to activate neutrophil effector functions. The peptide was found to be more potent than the N-formylated peptide N-formyl-Met-Leu-Phe (fMLF) at inducing neutrophil chemotaxis, mobilization of neutrophil complement receptor 3 (CR3), and activation of the neutrophil NADPH-oxidase. The fact that binding of fML[3H]F was inhibited by both fMLF and WKYMVm suggests that N-formyl peptide receptor (FPR) is shared by these peptides. However, the neutrophil response induced by the WKYMVm peptide was insensitive to the fMLF antagonists, cyclosporin H, and Boc-FLFLF that specifically block the function of the FPR. These results suggest that even though WKYMVm may bind FPR the cells are activated preferentially through a receptor distinct from the FPR. Using transfected HL-60 cells expressing either the FPR or its neutrophil homologue FPRL1, also referred to as LXA4R because it has been shown to bind lipoxin A4, we show that WKYMVm is about 300-fold more active at mobilizing intracellular calcium through FPRL1 than through FPR. The WKYMVm activates FPRL1-expressing cells in a cyclosporin H-independent manner with an EC50 of around 75 pmol/L, whereas it activates FPR-expressing cells with an EC50 of around 25 nmol/L. The observation that exudated cells are primed in their response to WKYMVm suggests that FPRL1/LXA4R like FPR is stored in mobilizable organelles.

Synthesis and Crystal Structure Determination of Cyclosporin H

Acid-catalyzed degradation of cyclosporin A was studied in various solvents and products of reaction were monitored by HPLC. Identification of amino acids and their chirality were determined after hydrolysis and derivatization by GC-MS. Cyclosporin H was isolated as the principal product and its structure was determined by X-ray diffraction: Cyclosporin H- diethyl ether-water (1 : 0.5 : 1) crystallizes in the monoclinic space group I2 with a = 12.338(2) Å, b = 18.963(2) Å, c = 34.074(3) Å, β = 96.47(2)°, Z = 4, and V = 7 921.4(17) Å3.

Direct demonstration of a specific interaction between cyclophilin-D and the adenine nucleotide translocase confirms their role in the mitochondrial permeability transition

A fusion protein between cyclophilin-D (CyP-D) and glutathione S-transferase (GST) was shown to bind to purified liver inner mitochondrial membranes (IMMs) in a cyclosporin A (CsA)-sensitive manner. Binding was enhanced by diamide treatment of the IMMs. Immobilized GST–CyP-D avidly bound a single 30 kDa protein present in Triton X-100-solubilized IMMs; immunoblotting showed this to be the adenine nucleotide translocase (ANT). Binding was prevented by pretreatment of the CyP-D with CsA, but not with cyclosporin H. Purified ANT also bound specifically to GST–CyP-D, but porin did not, even in the presence of ANT.