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Chemoattractant induces LFA-1 associated PI 3K activity and cell migration that are dependent on Fyn signaling

Giovanni Bernardini^*,1, Ji Yun Kim, Angela Gismondi^*, Eugene C. Butcher and Angela Santoni^*,

^* Department of Experimental Medicine and Pathology,
Istituto Pasteur-Fondazione Cenci Bolognetti, University "La Sapienza," Rome, Italy;
Department of Pathology, Stanford University School of Medicine, Stanford; Center for Molecular Biology and Medicine, Veteran Affairs Health Care System, Palo Alto, California, USA

¹Correspondence: Department of Experimental Medicine and Pathology University, "La Sapienza", Via Regina Elena 324, Rome 00161, Italy. E-mail: giovanni.bernardini{at}uniroma1.it

SPECIFIC AIMS

Lymphocyte function-associated antigen (LFA) -1 is a ß2 integrin family essential for leukocyte extravasation and migration into tissues. The ability of LFA-1 to interact with its ligands is dependent on its high-affinity/high-avidity state that can be reached after cell activation by a variety of stimuli including chemoattractants. Among a number of signaling intermediates, phosphatidylinositol 3kinase (PI 3K) and Src family protein tyrosine kinases (PTKs) contribute to chemoattractant-induced LFA-1 activation. The aim of our study was to identify the molecular mechanisms involved in regulating LFA-1 function by analyzing whether chemoattractant receptor triggering can induce the recruitment of Src and PI 3K family members in a complex with LFA-1 and to evaluate the relevance of Src/PI 3K cross-talk in chemoattractant-regulated integrin function.

PRINCIPAL FINDINGS

1. FMLP stimulation of TK1/FPR induces PTX-sensitive LFA-1-associated PI 3K activity
To investigate whether Src family PTKs and/or PI 3K can associate with LFA-1 on chemoattractant receptor stimulation, we used a mouse T cell lymphoma line, TK1, stably expressing the human N-formyl-Met-Leu-Phe (fMLP) receptor (fPR) as a model. In vitro PI 3K assays performed on LFA-1 immunoprecipitates from TK1/fPR cells stimulated with fMLP, CXCL-12 or left untreated showed that fMLP stimulation markedly increased LFA-1-associated PI 3K enzymatic activity that was detectable within 1 min and reached maximal levels at 3 min after stimulation and this event was sensitive to pertussis toxin (PTX) (Fig. 1 A, 1B ). Similar results were obtained with CXCL-12-stimulated normal mouse splenocytes.

View larger version (59K): [in this window] [in a new window]   Figure 1. FMLP induces PI 3K activity associated with LFA-1 and Fyn. TK1/fPR cells were serum deprived and 2.5 x 107 cells were stimulated with 1 µM fMLP, SDF-1 (50 nM) or for 3 min with vehicle (–) at 37°C under shaking, lysed, then immunoprecipitated. A) Anti-alphaL mAb (TIB213) immunoprecipitates were subjected to in vitro PI 3K kinase assay and radiolabeled PIP3 was resolved by TLC and detected by autoradiography. B) Cells were pretreated with PTX (200 ng/mL) or PP2 (10 µM) for 3 h and 40 min, respectively, or with DMSO alone (–), then stimulated and processed as above. IC represents immunoprecipitation with isotype control rat IgG2b. Bottom panels shows respectively alphaL (A) or p85 (B) loading controls. C) In vitro PI 3K assay was performed as above on Fyn immunoprecipitates obtained from cells pretreated or not with PTX (200 ng/mL) and PP2 (10 µM) before stimulation. D) Fyn kinase assay was performed on anti-Fyn immunoprecipitates from cells stimulated with fMLP. Sizes are indicated in kDas.

2. FMLP-induced LFA-1-associated PI 3K activity requires the Src tyrosine kinase Fyn
To investigate the mechanism of PI 3K activation upon fMLP stimulation, TK1/fPR cells were pretreated with the Src specific inhibitor PP2, and LFA-1 was immunoprecipitated from fMLP-stimulated cells. Inhibition of Src PTKs strongly impaired the LFA-1-associated PI 3K activity (Fig. 1B ). In an attempt to identify the Src kinase involved, we performed PI 3K assay on Fyn and Lck immunoprecipitates, two Src family PTKs abundantly expressed in this cell line. As shown in Fig. 1C , lipid kinase activity was rapidly up-regulated by fMLP stimulation (2- to 3-fold increase) in Fyn immunoprecipitates, but not in Lck immunoprecipitates (data not shown), suggesting that Src kinase-dependent activation of PI 3K may be specifically induced by Fyn PTK. As for LFA-1-associated PI 3K activity, preincubation of the cells with PTX and PP2 greatly reduced PI 3K activity associated with Fyn immunoprecipitates (Fig. 1C ). In addition, as shown in Fig. 1D , we could detect a rapid and persistent increase of Fyn autophosphorylation in TK1/FPR cells stimulated with fMLP as compared with cells treated with vehicle. These results support the hypothesis that Fyn catalytic activity is required for the regulation of PI 3K activation.

Since fPR engagement resulted in Fyn- and LFA-1-associated PI 3K activity with similar kinetics, we examined if Fyn association with LFA-1 was required to promote the integrin-associated PI 3K activity. Fyn was detected in alphaL immunoprecipitates from fMLP-stimulated cells, indicating that LFA-1 associates with a substantial amount of Fyn upon fMLP-stimulation with a kinetics consistent with that of the increased Fyn-associated PI 3K activity. It is important to note that transient overexpression of Fyn Ki (kinase inactive) and SH2mut (SH2 mutated) mutants in TK1/fPR cells resulted in impaired association of PI 3K activity with both Fyn and LFA-1, demonstrating an important role of Fyn in the activation of LFA-1-complexed PI 3K (data not shown). These results suggest that Fyn/PI 3K cross-talk may be relevant for the regulation of LFA-1 functions.

3. Role of Fyn in the regulation of LFA-1-dependent TK1/fPR migration in response to fMLP
To examine whether Fyn plays a role in chemoattractant-induced LFA-1-dependent cellular functions, we focused our studies on fMLP-induced chemotaxis on ICAM-1 substrate. Migration through both BSA- and ICAM-1-coated filters was maximal at 100 nM fMLP, but the number of migrated cells was strongly enhanced in the latter condition. Pretreatment of cells with alphaL but not ß1 blocking antibody reduced the level of migration on ICAM-1 coated surface to that of BSA-coated surface, which was unaffected by this treatment. These results indicate that TK1/FPR cell migration through ICAM-1 coated filters utilizes both LFA-1-dependent and -independent mechanisms. Moreover, fMLP-induced migration on both BSA and ICAM-1 requires PI 3K as the PI 3K specific inhibitor LY294002 reduces TK1/FPR cell migration (data not shown).

Interestingly, a significant decrease of fMLP-induced migration through ICAM-1 coated surface was observed in Fyn mutant-expressing cells. By contrast, when integrin-independent migration of TK1/FPR cells was assessed either by performing chemotaxis on ICAM-1 in the presence of LFA-1 blocking antibodies, or through BSA-coated filters, the overexpression of Fyn mutants did not significantly reduce chemotaxis (Fig. 2 A).

View larger version (20K): [in this window] [in a new window]   Figure 2. Fyn mutant-expressing TK1/fPR cells exhibit impaired chemotaxis on ICAM-1 substrate in response to fMLP. Chemotaxis through ICAM-1- (filled symbols) or BSA-coated filters (open symbols) of TK1/fPR cells cotransfected with GFP and empty vector, dominant negative Fyn Ki (A), or Fyn SH2 mutants (B) in response to different concentrations of fMLP. C) Chemotaxis through ICAM-1-coated filters of transiently transfected cells pretreated or not with anti-LFA-1 (TIB213) blocking antibody in response to fMLP (100 nM). (NC: no chemoattractant). Data are expressed as mean % of cell migration (±SE) evaluated on GFP+ cells from duplicate wells and represent results from 1 experiment of at least 3 independent experiments showing similar results. Student’s t test was performed to compare the migration of cells transfected with the empty vector with that of cells transfected with mutant forms of Fyn at individual concentrations of fMLP. *P < 0.05, **P < 0.01.

All together these data indicate that Fyn plays a crucial role in chemoattractant-induced migration on ICAM-1 via LFA-1, while it is dispensable for LFA-1-independent migration.

CONCLUSIONS AND SIGNIFICANCE

In this study we focused on fMLP/fPR pair to investigate the role of Src PTKs in chemoattractant-induced PI 3K activation and LFA-1-supported cell migration. In particular, we demonstrated that 1) PI 3K activity associates with LFA-1 following chemoattractant receptor activation; 2) Fyn PTK is rapidly recruited to the integrin receptor on chemoattractant signaling; 3) Fyn PTK regulates integrin-associated PI 3K activity on chemoattractant receptor triggering; 4) Proper regulation of Fyn activity is required to support integrin-dependent lymphocyte chemotaxis.

Three observations suggest that the lipid kinase activity we found associated with LFA-1 belongs to PI 3K(s) of the Class IA subfamily: 1) its activation depends on enzymatic activity and a functional SH2 domain of the Src PTK Fyn; 2) Class IA p85 regulatory subunit coimmunoprecipitates with LFA-1 (data not shown); 3) kinetics of phospholipid kinase activity in response to fMLP is compatible with the activation of PI 3K isoforms belonging to Class IA that has been demonstrated to occur within minutes after exposure to chemokines, differently from activation of Class IB, which occurs within seconds.

Our study demonstrates that chemoattractant-induced recruitment of Fyn to the integrin is required for LFA-1-associated PI 3K activation. The mechanisms of recruitment and activation of Fyn and PI 3K to LFA-1 are still under investigation. Direct interaction between Src and the cytoplasmic tail of the ß subunit of several integrins has recently been described, and it may be responsible for Fyn association with LFA-1. However, we can also speculate an indirect association of Fyn with LFA-1 and PI 3K, as our preliminary experiments indicate that Fyn-mediated regulation of PI 3K activity involves Pyk2, a GPCR-coupled PTK (Fig. 3 ).

View larger version (28K): [in this window] [in a new window]   Figure 3. Schematic representation of molecular events involved in Fyn-dependent migration induced by chemoattractant. In resting TK1/fPR cells, PI 3K is associated with LFA-1. Stimulation with chemoattractant induces a fast association of Fyn with the ß2 integrin leading to increased PI 3K activation, which requires the enzymatic activity of the Src kinase and is PTX sensitive. PI 3K activity associated with Fyn and LFA-1 may be the result of a direct association of PI 3K with Fyn (a) or may involve others intermediates such as Pyk2 (b).

Our study provides evidence that Fyn PTK is required for fMLP-promoted cell migration on ICAM-1, but not BSA, a function that also needs PI 3K activation. These results suggest that recruitment of Fyn and PI 3K to LFA-1 is involved in the regulation of chemoattractant-induced, integrin-supported migration. Induction of integrin-lateral mobility is required for leukocyte migration by promoting adhesive cluster formation at the leading edge and their movement toward the trailing edge of leukocytes. As recently described, PI 3K activation is crucial for the induction of LFA-1 lateral mobility. Thus, our data suggest that fMLP-induced activation of Fyn plays a role in promoting LFA-1 lateral mobility by regulating PI 3K.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3352fje;

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This Article Full Text (PDF) All Versions of this Article: 19/10/1305 04-3352fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bernardini, G. Articles by Santoni, A. Search for Related Content PubMed PubMed Citation Articles by Bernardini, G. Articles by Santoni, A.