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Interaction of disintegrins with human neutrophils induces cytoskeleton reorganization, focal adhesion kinase activation, and extracellular-regulated kinase-2 nuclear translocation, interfering with the chemotactic function¹

ANA LUCIA J. COELHO^*, MARTA S. DE FREITAS^*, ANDREA MARIANO-OLIVEIRA^*, ANA L. OLIVEIRA-CARVALHO, RUSSOLINA B. ZINGALI and CHRISTINA BARJA-FIDALGO^*2

^* Departamento de Farmacologia, Instituto de Biologia, Universidade do Estado do Rio de Janeiro; and
Departamento de Bioquímica Médica, ICB/CCS, Universidade Federal do Rio de Janeiro, Rio do Janeiro, RJ, Brazil

²Correspondence: Departamento de Farmacologia, Instituto de Biologia, Universidade do Estado do Rio de Janeiro, Av. 28 de setembro 87, 5th Floor, Vila Isabel, Rio de Janeiro, 20551–030, RJ, Brazil. E-mail: barja-fidalgo{at}uerj.br

SPECIFIC AIMS

Disintegrins have often been described as passive blockers of integrins, inhibiting integrin-related functions like tumor cell metastasis, angiogenesis, and platelet aggregation. This report shows that jarastatin (JT) and two other RGD-monomeric disintegrins—kistrin (KR) and flavoridin (FL)—modulate human neutrophil (PMN) chemotaxis and chemokinesis by interfering with the activation of integrin-mediated signaling pathways.

PRINCIPAL FINDINGS

1. Disintegrins inhibited PMN chemotaxis in vitro and induced alterations in actin network dynamics
Incubation of PMN with JT, KR, or FL (0.1–10 µM) inhibited the chemotactic effect induced by N-formyl-methionyl-leucyl-phenylalanine peptide (fMLP; 0.1 µM) in Boyden chamber. Chemotaxis of PMN in vitro was partially reduced (10 µM=50%) by JT and completely inhibited by KR or FL (1 µM). In another set of experiments, PMN were allowed to migrate toward different concentrations of disintegrins (0.01–10 µM) placed into the bottom wells of the chamber. JT (10 µM), but not KR or FL, was shown to be chemotactic for PMN, an effect comparable to that of fMLP. On the other hand, KR but not JT or FL significantly enhanced (1 µM=142%) PMN locomotion when added to both sides of the chamber, showing a potent chemokinetic effect. In addition, interaction of PMN with JT or KR induced an expressive increase in actin polymerization to an extent similar to fMLP, used as positive control. In contrast, FL-stimulated PMN showed a significant decrease in F-actin content when compared with nonstimulated cells.

2. Involvement of tyrosine kinase signaling in the effect of disintegrins on PMN
To investigate the involvement of tyrosine kinase-mediated pathways, PMN were treated with genistein (80 µM), a tyrosine kinase inhibitor. Genistein did not alter the random chemotaxis or the basal contents of polymerized actin in PMN. In contrast, genistein partially inhibited the chemotactic effect of JT (40%) and reduced in 50% the increase in actin polymerization in PMN induced by JT or KR (1 µM). Similarly, positive effects of fMLP (0.1 µM) on both parameters were reduced in 40%. In agreement, JT, KR, and fMLP increased tyrosine phosphorylation in PMN. For comparison, cells were treated with two known PMN activators, fMLP (0.1 µM) and interleukin-8 (30 nM), which induced a significant increment in phosphotyrosine content. The effect of JT was similar to that induced by both stimulators, whereas the effect of KR was lower than JT.

3. Disintegrins induced focal adhesion kinase (FAK) activation in PMN
To determine whether disintegrins were able to induce FAK activation in PMN, the contents of tyrosine-phosphorylated FAK and FAK-associated with actin were both determined in cells stimulated with the peptides (Fig. 1 ). JT, KR, and FL (1 µM) increased FAK phosphorylation (Fig. 1A ) and association of FAK to actin cytoskeleton (Fig. 1B ), with JT being more potent inducer.

View larger version (47K): [in this window] [in a new window]   Figure 1. Disintegrins induce FAK activation in human neutrophils. The cellular content of phosphorylated FAK (A) and FAK associated with actin (B) was evaluated in PMN incubated with fMLP (0.1 µM), JT (1 µM), KR (1 µM), or FL (1 µM) for 5 min at 37°C. Cell lysates were immunoprecipitated with anti-FAK (A) or anti-actin (B) antibodies (lower inset) and then immunoblotted with anti-phosphotyrosine (A) or anti-FAK (B) antibodies (upper inset). Blots were analyzed by densitometry and the content of phosphorylated FAK (A: p-FAK) and FAK associated with actin (B: a-FAK) was expressed in arbitrary units. Results are representative of two similar experiments. *P < 0.05 in comparison with control cells.

4. Disintegrins induced Erk-2 activation in PMN
Erk-2, a mitogen-activated kinase (MAPK) of 42 KDa, is activated after its tyrosine phosphorylation, which allows its translocation to the nucleus. Figure 2 shows that Erk-2 was detected in nuclear extracts of nonstimulated cells 1 h after incubation with medium alone. The treatment of PMN with JT or KR (1 µM) for 1 h induced a significant increase on Erk-2 nuclear translocation, comparable to that caused by fMLP (0.1 µM). On the other hand, nuclear extracts from FL (1 µM) -treated PMN showed a reduced Erk-2 protein content when compared with nonstimulated cells, suggesting that this disintegrin may be inhibiting Erk-2 activation (Fig. 2) .

View larger version (38K): [in this window] [in a new window]   Figure 2. Effect of disintegrins on Erk-2 nuclear translocation in neutrophils. Cells were incubated with fMLP (0.1 µM), JT (1 µM), KR (1 µM), or FL (1 µM) for 1 h at 37°C. The nuclear content of Erk-2 was determined by anti-Erk-2 antibody immunoblotting (inset) and quantified by densitometry. Results are representative of three similar experiments. *P < 0.05 in comparison with control cells.

CONCLUSIONS

During the inflammatory process, the binding of integrin receptors to RGD-containing proteins from extracellular matrix stimulates PMN adhesion, chemotaxis, and phagocytosis. Here we present new data showing that the monomeric RGD-disintegrins JT, KR, and FL inhibited human PMN chemotaxis induced by fMLP. However, the data on the signaling pathways triggered by the interaction of the disintegrins with PMN suggested that JT, KR, and FL might act through distinct intracellular mechanisms to interfere with PMN functions. As demonstrated earlier, JT per se was able to induce PMN chemotaxis in vitro whereas KR and FL did not show any effect. This positive stimulation induced by JT is probably responsible for its only partial inhibitory effect on chemotaxis compared to the full inhibition seen with KR and FL. On the other hand, KR, but not JT or FL, showed a potent chemokinetic effect for PMN, which may be related to its inhibitory effect on PMN chemotaxis. The commitment of the actin network is a characteristic of integrin-mediated cell activation and may account for the anti-chemotactic effect of these disintegrins. Both JT and KR triggered a rapid and significant increase in actin polymerization whereas FL, which also inhibited chemotaxis, caused a decrease in F-actin content. These results suggest that disintegrins may interfere in integrin-mediated functions, acting differently on PMN actin dynamics.

Cytoskeleton reorganization triggered by integrin interactions is primarily associated with the activation of tyrosine kinases and the subsequent tyrosine phosphorylation of several proteins, resulting in the formation of focal adhesion complexes. The involvement of a tyrosine kinase activity in the effects of disintegrins in PMN was demonstrated by the inhibitory effect of genistein, a tyrosine kinase inhibitor, on the actin polymerization induced by JT and KR. In addition, we showed that JT and KR, but not FL, induced an increase in the content of tyrosine phosphorylated proteins. Recent works have addressed the effect of disintegrins, previously considered passive integrin-blocking agents, as activators of integrin-mediated signaling. Contortrostatin, a dimeric disintegrin, was shown to induce tyrosine phosphorylation of FAK and CAS in tumor cells, whereas monomeric disintegrins, echistatin, and FL had no effect. The authors impute the unique effect of contortrostatin to its homodimeric structure. By associating with activating kinases and adapter proteins, FAK, a focal adhesion kinase, serves as a scaffold on which signaling molecules mediated by integrin can modulate PMN responses. Our results showed that the monomeric disintegrins JT, KR, and FL induced tyrosine phosphorylation of FAK and its subsequent association with actin in PMN.

The trigger of several intracellular signaling pathways is often linked to FAK activation, including the activation of MAP kinases. Erk-2, a member of MAPK family, is activated by tyrosine phosphorylation and translocates to the nucleus affecting gene expression by directly phosphorylating transcription factors. The incubation of PMN with JT or KR (1 µM) for 1 h leads to Erk-2 activation, increasing the translocation of Erk-2 to the nucleus. In contrast, Erk-2 nuclear translocation was blocked in FL-stimulated cells, suggesting that this disintegrin inhibited Erk-2 activation. Although the involvement of MAPK pathway in PMN adhesion, degranulation, cytokine production, and apoptosis has been described, the precise role of Erk-2 in PMN is still unclear. Recently it was shown that contortrostatin induced Erk-2 activation in tumor cells, an effect that seems to be related to its negative effect on cell migration. Although the activation of Erk-2 by JT and KR could account for their inhibitory effect on PMN chemotaxis, the opposite effect of FL, which also inhibited chemotaxis, on Erk-2 nuclear translocation suggests that this pathway may be related to different effects of the disintegrins on PMN.

In conclusion, this work shows original findings on the effect of monomeric RGD-disintegrins on human neutrophil activation and function (Fig. 3 ). The data suggest that whereas JT and KR directly activate integrin-coupled signaling, FL, though able to activate FAK, acts as an inhibitor of integrin-mediated responses. Remarkably, JT and KR induced Erk-2 activation, an important signaling molecule, suggesting that by acting as regulators of integrin function, these peptides can modulate different responses in neutrophils.

View larger version (41K): [in this window] [in a new window]   Figure 3. Schematic diagram of the hypothesized effects of monomeric RGD-disintegrin on human neutrophil activation and functions. JT (A) and KR (B) trigger integrin-mediated signaling pathways leading to FAK and Erk-2 activation and changes on actin cytoskeleton. C) FL interacts with integrins on PMN, activates FAK and probably leads to the activation of negative effectors that inhibit integrin-mediated cell responses.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0812fje ; to cite this article, use FASEB J. (May 29, 2001) 10.1096/fj.00-0812fje

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