CXC chemokine receptor 4 signaling upon co-activation with stromal cell-derived factor-1α and ubiquitin

Abstract

Recently, we reported that extracellular ubiquitin functions as another agonist of CXC chemokine receptor (CXCR)4. Whereas the cognate CXCR4 ligand, stromal cell-derived factor (SDF)-1α, is also a CXCR7 agonist, ubiquitin does not bind to CXCR7. Because both ligands are present in the extracellular environment, co-activation of CXCR4 appears to be physiologically relevant. CXCR4 mediated effects of ubiquitin, however, are not well understood and consequences of co-activation of CXCR4 with both ligands are unknown. Utilizing proximity ligation assays and flow cytometry, we detected CXCR4, but not CXCR7, on the cell surface of THP-1 cells, which suggests that confounding effects of CXCR7 are unlikely. Time course and magnitude of reduction of cell surface CXCR4 expression were comparable after stimulation of THP-1 cells with both ligands. SDF-1α was more efficacious than ubiquitin to mobilize Ca2+. Co-stimulation of THP-1 cells with both ligands resulted in synergistic effects on Ca2+ fluxes at suboptimal ligand concentrations. Homologous desensitization of Ca2+ fluxes was detectable with both ligands. SDF-1α pre-stimulation desensitized ubiquitin induced Ca2+ fluxes, but not vice versa. Effects of SDF-1α and ubiquitin on cAMP levels, Akt and ERK1/2 phosphorylation and chemotactic responses were additive. The chemotactic activities of ubiquitin and SDF-1α were sensitive to AMD3100, pertussis toxin, U73122, LY94002 and U0126. These data suggest that CXCR4 activation with SDF-1α and ubiquitin results in partially synergistic effects on cellular signaling events and in differential effects on receptor desensitization. The ligand ratio that is present in the extracellular environment may contribute to the regulation of CXCR4 mediated functions.

Chemokine (C-X-C Motif) Receptor 4 and Atypical Chemokine Receptor 3 Regulate Vascular α1-Adrenergic Receptor Function

“…After systemic administration, the volume of distribution of AMD3100 is small, its half-life in rats is approximately 60 min and binding of AMD3100 to CXCR4, unlike binding of CXCL12 and ubiquitin to CXCR4, does not result in ligand-induced receptor internalization (14,43,54). Thus, in our experiments, withdrawal of blood removed large proportions of AMD3100 from the systemic circulation and subsequent fluid resuscitation further reduced the remaining drug concentrations.…”

A prototypic modified risk tobacco product exhibits reduced effects on chemotaxis and transendothelial migration of monocytes compared with a reference cigarette

“…Consistent with this finding, gremlin-1 has been shown to inhibit macrophage migration inhibitory factor (MIF)-dependent monocyte migration and adhesion to activated endothelial cells (Muller et al, 2014). MIF has been identified as a noncognate ligand of CXCR4 (Bernhagen et al, 2007), the classical chemokine receptor for CXCL12 (Tripathi et al, 2014). Furthermore, MIF elicits monocyte arrest in inflamed and atherosclerotic arteries (Bernhagen et al, 2007).…”

“…The expression of CXCL12 in human atherosclerotic plaques (Abi-Younes et al, 2000), the Abbreviations: 7-AAD, 7-aminoactinomycin D; BSA, bovine serum albumin; CIM, cell invasion/migration; CO, carbon monoxide; CS, cigarette smoke; CXCL12, C-X-C motif ligand 12; DNPH, dinitrophenylhydrazine; E-plate, electrical impedance plate; fMLP, N-formylmethionyl-leucyl-phenylalanine; GM-CSF, granulocyte-macrophage colony-stimulating factor; HCAECs, human coronary arterial endothelial cells; HGF, hepatocyte growth factor; ICAM1, intercellular adhesion molecule-1; IL-8, interleukin-8; LC-MS, liquid chromatography-electrospray ionization tandem mass spectrometry; MCP-1, monocyte chemoattractant protein-1; NO, nitrogen monoxide; pMRTP, prototypic modified risk tobacco product; RTCA-DP, real-time cell analyzer dualplate; SELE, E-selectin; TEM, transendothelial migration; THP-1, human monocytic cell line; TNF-α, tumor necrosis factor-α; VCAM1, vascular cell adhesion molecule. association between CXCL12 levels and coronary artery disease (Damas et al, 2002), and the potent chemotactic effects of CXCL12 on peripheral blood mononuclear cells (Liu et al, 2006) prompted us to investigate the effects of extracts from the reference cigarette 3R4F and a prototypic modified risk tobacco product (pMRTP) on CXCL12-dependent transendothelial migration (TEM) of THP-1 cells, a monocytic cell line that expresses CXCR4 (Tripathi et al, 2014).…”

Heteromerization of chemokine (C-X-C motif) receptor 4 with α1A/B-adrenergic receptors controls α1-adrenergic receptor function

“…We sought to evaluate whether heteromeric complexes between α 1 -AR and CXCR4 are expressed on the cell surface of VSMC. Thus, we used proximity ligation assays (PLA) to visualize individual receptors and receptorreceptor interactions (39)(40)(41). PLA have been previously used to observe individual proteins and interactions between individual endogenous proteins at a single-molecule resolution (39).…”

“…Duolink proximity ligation assays were performed as described previously (40,41). In brief, VSMC and A7r5 cells were grown and fixed on eight-well tissue culture slides.…”