Differential Effects of Posttranslational Modifications of CXCL8/Interleukin-8 on CXCR1 and CXCR2 Internalization and Signaling Properties

CXCL8 or interleukin (IL)-8 directs neutrophil migration and activation through interaction with CXCR1 and CXCR2 that belong to the family of G protein-coupled receptors (GPCRs). Naturally occurring posttranslational modifications of the NH2-terminal region of CXCL8 affect its biological activities, but the underlying molecular mechanisms are only partially understood. Here, we studied the implications of site-specific citrullination and truncation for the signaling potency of CXCL8. Native CXCL8(1-77), citrullinated [Cit5]CXCL8(1-77) and the major natural isoform CXCL8(6-77) were chemically synthesized and tested in internalization assays using human neutrophils. Citrullinated and truncated isoforms showed a moderately enhanced capacity to induce internalization of CXCR1 and CXCR2. Moreover, CXCL8-mediated activation of Gαi-dependent signaling through CXCR1 and CXCR2 was increased upon modification to [Cit5]CXCL8(1-77) or CXCL8(6-77). All CXCL8 variants promoted recruitment of β-arrestins 1 and 2 to CXCR1 and CXCR2. Compared to CXCL8(1-77), CXCL8(6-77) showed an enhanced potency to recruit β-arrestin 2 to both receptors, while for [Cit5]CXCL8(1-77) only the capacity to induce β-arrestin 2 recruitment to CXCR2 was increased. Both modifications had no biasing effect, i.e., did not alter the preference of CXCL8 to activate either Gαi-protein or β-arrestin-dependent signaling through its receptors. Our results support the concept that specific chemokine activities are fine-tuned by posttranslational modifications.

Oxysterols and EBI2 promote osteoclast precursor migration to bone surfaces and regulate bone mass homeostasis

Bone surfaces attract hematopoietic and nonhematopoietic cells, such as osteoclasts (OCs) and osteoblasts (OBs), and are targeted by bone metastatic cancers. However, the mechanisms guiding cells toward bone surfaces are essentially unknown. Here, we show that the Gαi protein–coupled receptor (GPCR) EBI2 is expressed in mouse monocyte/OC precursors (OCPs) and its oxysterol ligand 7α,25-dihydroxycholesterol (7α,25-OHC) is secreted abundantly by OBs. Using in vitro time-lapse microscopy and intravital two-photon microscopy, we show that EBI2 enhances the development of large OCs by promoting OCP motility, thus facilitating cell–cell interactions and fusion in vitro and in vivo. EBI2 is also necessary and sufficient for guiding OCPs toward bone surfaces. Interestingly, OCPs also secrete 7α,25-OHC, which promotes autocrine EBI2 signaling and reduces OCP migration toward bone surfaces in vivo. Defective EBI2 signaling led to increased bone mass in male mice and protected female mice from age- and estrogen deficiency–induced osteoporosis. This study identifies a novel pathway involved in OCP homing to the bone surface that may have significant therapeutic potential.

Recombinant DEK Regulates Hematopoietic Stem and Progenitor Cells in a CXCR2- GαI Protein-Dependent Manner

DEK, a non-histone nuclear phosphoprotein involved in heterochromatin remodeling, is released from cells and can regulate hematopoiesis (Broxmeyer et al., 2012, Stem Cells Dev., 21: 1449; 2013, Stem Cells, 31: 1447). Marrow from DEK-/- mice manifest increased hematopoietic progenitor cell (HPC) numbers and cycling status and decreased long-term and secondary hematopoietic stem cells (HSC) engrafting capability. Moreover, recombinant mouse (rmu) DEK inhibited HPC colony formation in vitro. We now show that rmuDEK is myelosuppressive in vitro in an S-phase specific manner and reversibly decreases numbers and cycling status of CFU-GM, BFU-E, and CFU-GEMM in vivo, with DEK-/- mice being much more sensitive than control mice to this suppression. In addition, rmuDEK treatment of WT mice in vivo reversibly reduces the phenotypic number of ST-HSC, MPP, CMP, GMP, and MEP in the marrow. In vivo administration of rmuDEK to DEK-/- mice greatly enhanced the number of phenotypic LT-HSC and functional HSC in these mice (competitive HSC repopulation in lethally irradiated mice). DEK-/- mice are also more sensitive to 5-FU than control mice resulting in slower hematopoietic cell recovery, possibly due to the enhanced cycling status of HPC in DEK-/- mice. To determine the biological role of extracellular DEK, we utilized a reagent that could inactivate its function. This inactivating agent, but not its control, neutralized the inhibitory effect of rmuDEK. In addition, treating marrow cells in vitro with truncated rmuDEK by pretreating the DEK with the enzyme DPP4 (which DEK has targeted truncation sites for) also blocked the inhibitory effects of DEK suggesting that DEK must be in its full length form in order to perform its function Upon our discovery that the DEK protein has a Glu-Leu-Arg (ELR) motif, similar to that of CXC chemokines such as IL-8, we hypothesized that DEK may manifest at least some of its actions through CXCR2, known to bind and mediate the actions of IL-8 and MIP-2. In order to examine if this is indeed the case we first confirmed expression of CXCR2 on the surface of HSC and HPC. To determine whether rmuDEK’s inhibitory function is mediated through the CXCR2 receptor, a neutralizing monoclonal antibody against CXCR2 was utilized. Marrow treated in vitro with rmuDEK, rhIL-8, or rmuMIP-2 inhibited colony formation; however pretreating marrow cells with the neutralizing CXCR2 antibody blocked the inhibitory effect of these proteins. Marrow treated with rmuMIP-1α (a chemokine that does not bind to CXCR2) also inhibited colony formation; however neutralizing CXCR2 antibody had no effect on the ability of MIP-1α to inhibit colony formation. Neutralizing CXCR4, a chemokine receptor that binds SDF-1 but not IL-8, MIP-2 or MIP-1α, had no effect on the inhibition of colony formation. DEK inhibition of CFU-GM colony formation is dependent on Gαi-protein-coupled receptor signaling as determined through the use of pertussis toxin. This is a unique mechanism since IL-8 and MIP-1α had been previously reported by us to be insensitive to the inhibitory effects of pertussis toxin. As extracellular DEK can remodel chromatin in non-hematopoietic cells in vitro (Kappes et al., 2011, Genes Dev., 25: 673; Saha et al., 2013, PNAS, 110: 6847), we next assessed the effects of DEK on the heterchromatin marker H3K9Me3 in the nucleus of purified mouse Lineage negative, Sca-1 positive, c-kit positive (LSK) marrow cells by imaging flow cytometry. RmuDEK enhanced the presence of H3K9Me3 in the nucleus of DEK-/- LSK marrow cells indicating that rmuDEK can be internalized by LSK cells and mediate heterchromatin formation. These results add to emerging evidence that DEK, a unique nuclear protein, plays a role in regulating hematopoiesis through a CXCR2/Gαi protein signaling pathway. Its modulation may be of practical use. Disclosures No relevant conflicts of interest to declare.

Regulation of Estradiol and Progesterone Production by CRH-R1 and -R2 Is through Divergent Signaling Pathways in Cultured Human Placental Trophoblasts

CRH and its related peptides urocortins (UCN) have been identified in placenta and implicated to play pivotal roles in the regulation of pregnancy and parturition in humans. The objectives of present study were to investigate the effects of endogenous CRH and its related peptides in the regulation of steroid production in placenta. Placental trophoblasts were isolated from term placenta tissues and cultured for 72 h. Estradiol (E2) and progesterone (P4) contents in culture media were determined by radioimmunoassay. Treatment of cultured trophoblasts with CRH or UCNI antibody showed decreased E2, whereas increased P4 production. Treatment of cells with CRH receptor type 1 antagonist antalarmin or CRH receptor type 2 (CRH-R2) antagonist astressin-2b also decreased E2 but increased P4 production. Knockdown of CRH receptor type 1 or CRH-R2 cells showed a decrease in E2 production and an increase in P4 production. In CRH-R2 knockdown cells, CRH stimulated GTP-bound Gαs protein and phosphorylated phospholipase C-β3. Adenylyl cyclase and protein kinase A inhibitors blocked CRH-induced increased E2 production but not decreased P4 production. PLC inhibitor U73122 and protein kinase C inhibitor chelerythrine blocked the effects of CRH on E2 and P4 production in CRH-R2 knockdown cells. UCNIII, the specific CRH-R2 agonist, stimulated GTP-bound Gαi protein and phosphorylated phospholipase C-β3 expression. Both U73122 and chelerythrine blocked UCNIII-induced increased E2 production and decreased P4 production. We suggest that CRH and its related peptides might be involved in changes in the progesterone to estrogen ratio during human pregnancy.