Pleiotrophin, a multifunctional cytokine and growth factor, induces leukocyte responses through the integrin Mac-1

Di Shen,Nataly P Podolnikova,Valentin P Yakubenko,Christopher L Ardell,Arnat Balabiyev,Tatiana P Ugarova,Xu Wang

doi:10.1074/jbc.m116.773713

Di Shen, Nataly P Podolnikova + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m116.773713

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Abstract

Pleiotrophin (PTN) is a multifunctional, cationic, glycosaminoglycan-binding cytokine and growth factor involved in numerous physiological and pathological processes, including tissue repair and inflammation-related diseases. PTN has been shown to promote leukocyte responses by inducing their migration and expression of inflammatory cytokines. However, the mechanisms through which PTN mediates these responses remain unclear. Here, we identified the integrin Mac-1 (αMβ2, CD11b/CD18) as the receptor mediating macrophage adhesion and migration to PTN. We also found that expression of Mac-1 on the surface of human embryonic kidney (HEK) 293 cells induced their adhesion and migration to PTN. Accordingly, PTN promoted Mac-1-dependent cell spreading and initiated intracellular signaling manifested in phosphorylation of Erk1/2. While binding to PTN, Mac-1 on Mac-1-expressing HEK293 cells appears to cooperate with cell-surface proteoglycans because both anti-Mac-1 function-blocking mAb and heparin were required to block adhesion. Moreover, biolayer interferometry and NMR indicated a direct interaction between the αMI domain, the major ligand-binding region of Mac-1, and PTN. Using peptide libraries, we found that in PTN the αMI domain bound sequences enriched in basic and hydrophobic residues, indicating that PTN conforms to the general principle of ligand-recognition specificity of the αMI domain toward cationic proteins/peptides. Finally, using recombinant PTN-derived fragments, we show that PTN contains two distinct Mac-1-binding sites in each of its constitutive domains. Collectively, these results identify PTN as a ligand for the integrin Mac-1 on the surface of leukocytes and suggest that this interaction may play a role in inflammatory responses.

Highlights

Pleiotrophin (PTN) is a multifunctional, cationic, glycosaminoglycan-binding cytokine and growth factor involved in numerous physiological and pathological processes, including tissue repair and inflammation-related diseases
Because PTN has high affinity for glycosaminoglycans and heparan sulfate proteoglycan (HSPG) N-syndecan binds PTN, we examined the possibility that HSPGs on wild-type HEK293 cells mediate adhesion to PTN
When Mac-1 HEK293 cells were treated with both mAb 44a and heparin, cell adhesion was inhibited by Ͼ95%

Summary

Results

To assess whether PTN can bind Mac-1, we initially examined the ability of Mac-1 HEK293 to adhere to immobilized PTN. Treatment of control HEK293 cells with PTN did not induce phosphorylation of Erk1/2 (Fig. 4A) These data suggest that in this system initiation of intracellular signaling by PTN requires both Mac-1 and HSPGs. To further characterize the Mac-1–PTN interactions and determine domains of Mac-1 responsible for PTN binding, we analyzed the binding parameters of the interaction between the ␣MI domain and PTN. To investigate the contribution of each domain to Mac-1 binding, we prepared truncated PTN fragments corresponding to the NTD (residues 1–57), CTD (residues 58 –114), and PTN-short (residues 1–114) with the C-terminal tail deleted (Fig. 8A) and examined their ability to support cell adhesion and induce MAPK activation (Fig. 8, B–F). The data showed that removing the C-terminal tail of PTN did not reduce significantly the ability of PTN-short to support adhesion of Mac-1 HEK293 cells compared with intact PTN The maximal level of adhesion mediated by these frag-

37 PTN 44a

Discussion

47. AAAAAAAAA 4247 30

Experimental procedures