Multimerization of monocyte chemoattractant protein-1 is not required for glycosaminoglycan-dependent transendothelial chemotaxis

Chemokines interact with specific G-protein-coupled cell-surface receptors and with glycosaminoglycans (GAGs), such as heparan sulphate. Although chemokines often form multimers in solution, this process may be enhanced following interaction with GAGs on the cell surface, or within the extracellular matrix. However, the significance of multimerization for chemokine function remains controversial. In the present study, a fusion protein was prepared between the prototypical human CC chemokine, monocyte chemoattractant protein-1 (MCP-1; also known as CCL-2) and a large secreted placental alkaline phosphatase (SEAP) moiety. This fusion protein (MCP-1–SEAP) remained monomeric under conditions that promote oligomerization of the native chemokine. Radioligand binding showed that both native MCP-1 and MCP-1–SEAP competed for the same site on the surface of HEK-293 cells expressing the CCR2b chemokine receptor. The interaction between either chemokine species and endothelial cell surface GAGs was antagonized by the addition of the heparan sulphate-like molecule, heparin. Both MCP-1 and MCP-1–SEAP induced a Ca2+-flux in the THP-1 monocytic cell line, and were equally effective at promoting transendothelial chemotaxis of mononuclear immune cells, with maximal migration being produced by treatment with 12nM of either species. In each case this chemotactic response was almost completely antagonized by the addition of heparin. The importance of interaction between either native MCP-1 or MCP-1–SEAP and cell-surface GAGs for transcellular migration was demonstrated by the almost complete absence of leucocyte chemotaxis across monolayers of GAG-deficient mutant cells. In summary, this study shows that multimerization is neither necessary for, nor potentiates, the biological activity of MCP-1. However, the results do clearly demonstrate the importance of the interaction between MCP-1 and cell-surface heparan sulphate for transmonolayer leucocyte chemotaxis.

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Stimulation of FcγR receptors induces monocyte chemoattractant protein-1 in the human monocytic cell line THP-1 by a mechanism involving IκB-α degradation and formation of p50/p65 NF-κB/Rel complexes

International Immunology ◽

10.1093/intimm/12.4.547 ◽

2000 ◽

Vol 12 (4) ◽

pp. 547-554 ◽

Cited By ~ 19

Author(s):

Andrés Alonso ◽

Yolanda Bayón ◽

Marta Renedo ◽

Mariano Sánchez Crespo

Keyword(s):

Cell Line ◽

Monocytic Cell ◽

Monocytic Cell Line ◽

Monocyte Chemoattractant Protein 1 ◽

Monocyte Chemoattractant ◽

Monocyte Chemoattractant Protein ◽

Human Monocytic Cell Line ◽

Human Monocytic Cell ◽

Stimulation Of

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Monocyte chemoattractant protein 1 causes differential signalling mediated by proline-rich tyrosine kinase 2 in THP-1 cells

Biochemical Journal ◽

10.1042/bj3550751 ◽

2001 ◽

Vol 355 (3) ◽

pp. 751-756 ◽

Cited By ~ 16

Author(s):

Masahide YAMASAKI ◽

Hidenori ARAI ◽

Noboru ASHIDA ◽

Kenji ISHII ◽

Toru KITA

Keyword(s):

Tyrosine Kinase ◽

Tyrosine Phosphorylation ◽

Tyrosine Kinases ◽

Signalling Pathways ◽

Monocytic Cell ◽

Monocytic Cell Line ◽

Monocyte Chemoattractant Protein 1 ◽

Monocyte Chemoattractant ◽

Monocyte Chemoattractant Protein ◽

Tyrosine Kinase 2

Monocyte chemoattractant protein 1 (MCP-1) has a crucial role in atherogenesis and inflammation. However, MCP-1-mediated signalling pathways in monocytes have not been fully elucidated. In the present study we investigated the role of tyrosine kinases such as proline-rich tyrosine kinase 2 (Pyk2) in MCP-1-mediated signal transduction in the monocytic cell line THP-1. Pyk2 was tyrosine phosphorylated very quickly after stimulation with MCP-1. We found that Lyn, Shc and paxillin were also tyrosine phosphorylated by MCP-1. We examined the association of these molecules by immunoprecipitation and immunoblot analysis. The association of Pyk2 with Lyn was dependent on stimulation with MCP-1 and on tyrosine phosphorylation of Pyk2. Phosphorylation of p38 was also dependent on tyrosine phosphorylation of Pyk2. However, the association of Pyk2 with paxillin and Grb2 was not affected by stimulation with MCP-1. Phosphorylation of ERK (extracellular-signal-regulated protein kinase) was not affected by overexpression of kinase-negative Pyk2. Our results indicate that Pyk2 forms a complex with paxillin, Grb2 and Lyn in THP-1 cells. However, Pyk2 is not always involved in MCP-1-mediated signalling pathways.

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Homocysteine induces monocyte chemoattractant protein-1 expression by activating NF-κB in THP-1 macrophages

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.2001.280.6.h2840 ◽

2001 ◽

Vol 280 (6) ◽

pp. H2840-H2847 ◽

Cited By ~ 65

Author(s):

Guoping Wang ◽

Yaw L. Siow ◽

Karmin O

Keyword(s):

Underlying Mechanism ◽

Inhibitory Protein ◽

Monocytic Cell ◽

Monocyte Chemoattractant Protein 1 ◽

Arterial Walls ◽

Monocyte Chemoattractant ◽

Monocyte Chemoattractant Protein ◽

Monocyte Migration ◽

Human Monocytic Cell ◽

Monocyte Chemotaxis

Homocysteinemia is an independent risk factor for cardiovascular disorders. The recruitment of monocytes is an important event in atherogenesis. Monocyte chemoattractant protein-1 (MCP-1) is a potent chemokine that stimulates monocyte migration into the intima of arterial walls. The objective of the present study was to investigate the effect of homocysteine on MCP-1 expression in macrophages and the underlying mechanism of such effect. Human monocytic cell (THP-1)-derived macrophages were incubated with homocysteine. By nuclease protection assay and ELISA, homocysteine (0.05–0.2 mM) was shown to significantly enhance the expression of MCP-1 mRNA (up to 2.6-fold) and protein (up to 4.8-fold) in these cells. Homocysteine-induced MCP-1 expression resulted in increased monocyte chemotaxis. The increase in MCP-1 expression was associated with activation of nuclear factor (NF)-κB due to increased phosphorylation of the inhibitory protein (IκB-α) as well as reduced expression of IκB-α mRNA in homocysteine-treated cells. In conclusion, our results demonstrate that homocysteine, at pathological concentration, stimulates MCP-1 expression in THP-1 macrophages via NF-κB activation.

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