Abstract
The chemokine receptor CXCR4 plays a fundamental role in homeostasis and pathology by orchestrating recruitment and positioning of immune cells, under the guidance of a CXCL12 gradient. The ability of chemokines to form heterocomplexes, enhancing their function, represents an additional level of regulation on their cognate receptors. In particular, the multi-faceted activity of the heterocomplex formed between CXCL12 and the alarmin HMGB1 is emerging as an unexpected player able to modulate a variety of cell responses, spanning from tissue regeneration to chronic inflammation. Nowadays, little is known on the selective signaling pathways activated when CXCR4 is triggered by the CXCL12/HMGB1 heterocomplex. In the present work, we demonstrate that this heterocomplex acts as a CXCR4 balanced agonist, activating both G protein and β-arrestins-mediated signaling pathways to sustain chemotaxis. We generated β-arrestins knock out HeLa cells by CRISPR/Cas9 technology and show that the CXCL12/HMGB1 heterocomplex-mediated actin polymerization is primarily β-arrestin1 dependent, while chemotaxis requires both β-arrestin1 and β-arrestin2. Triggering of CXCR4 with the CXCL12/HMGB1 heterocomplex leads to an unexpected receptor retention on the cell surface, which depends on β-arrestin2. In conclusion, the CXCL12/HMGB1 heterocomplex engages the β-arrestin proteins differently from CXCL12, promoting a prompt availability of CXCR4 on the cell surface, and enhancing directional cell migration. These data unveil the signaling induced by the CXCL12/HMGB1 heterocomplex in view of identifying biased CXCR4 antagonists or agonists targeting the variety of functions it exerts.
Highlights
Chemokines are a family of chemoattractant cytokines that orchestrate the recruitment and the positioning of immune cells via G protein-coupled receptors (GPCRs), under homeostatic, or pathological conditions
Cell migration induced by the CXCL12/HMGB1 heterocomplex was accompanied by a significant increase in the accumulated distance, compared to cells stimulated with a sub-optimal CXCL12 dose (Figure 1E)
The actin polymerization induced by both the heterocomplex and the optimal concentration of CXCL12 was significantly decreased in β-arrestins KO compared to wt HeLa cells, suggesting a contribution of both β-arrestins in this process (Figures 3E,F). These results indicate that the CXCL12/HMGB1 heterocomplex-mediated actin polymerization depends on both β-arrestin isoforms, with a prominent role exerted by β-arrestin1
Summary
Chemokines are a family of chemoattractant cytokines that orchestrate the recruitment and the positioning of immune cells via G protein-coupled receptors (GPCRs), under homeostatic, or pathological conditions. The chemokine CXCL12 can form a heterocomplex with the alarmin HMGB1 [6, 7], which enhances the recruitment of human and mouse cells to inflammatory and regenerative sites via the chemokine receptor CXCR4 [6, 8,9,10]. Chemokine receptor signaling controlling cell migration is mostly dependent on heterotrimeric Gi protein activation, while receptor desensitization and endocytosis is linked to the activity of β-arrestins. This process has been demonstrated to be far more complex since β-arrestins can directly mediate chemokine receptor signaling, sustaining important cellular responses, including cytoskeleton remodeling, and chemotaxis [11, 12]. Depending on the tissue context, cell type, and chemokine receptor engaged, a given agonist could activate one or both pathways, defining a “biased” or “balanced” signaling [13,14,15]
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