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Dual function of focal adhesion kinase in regulating integrin-induced MMP-2 and MMP-9 release by human T lymphoid cells

Marta Segarra, Carme Vilardell, Kazue Matsumoto, Jordi Esparza, Ester Lozano, Carles Serra-Pages^*, Alvaro Urbano-Márquez, Kenneth M Yamada and Maria C Cid¹

Department of Internal Medicine and
^* Immunology, Hospital Clínic, University of Barcelona, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain; and
Craniofacial Developmental Biology and Regeneration Branch. National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda Maryland, USA

¹ Correspondence: Department of Internal Medicine. Hospital Clínic. Villarroel, 170. 08036 Barcelona, Spain. E-mail: mccid{at}clinic.ub.es

SPECIFIC AIMS

The aim of our study was to assess the role of focal adhesion kinase (FAK) in integrin-mediated gelatinase production by T lymphoid cells.

PRINCIPAL FINDINGS

1. FAK regulates integrin-dependent gelatinase (MMP-2 and MMP-9) expression and release by T lymphoid cells
Transient transfection of FAK wild-type cDNA into Jurkat T and primary T cells increased MMP-2 and MMP-9 production in response to fibronectin (FN), as assessed by gelatin zymography. FAK transfected cells showed enhanced MMP-2, MMP-9, and TIMP-1 mRNA in response to FN with no significant changes in MMP-14 and TIMP-2 expression.

However, both MMP-2 and MMP-9 appeared in the supernatant fluid long before an increase in gelatinase mRNA was detected by RT-PCR. Furthermore, blocking transcription with actinomycin D, which abrogated gelatinase mRNA induction by FN, did not inhibit the increase in gelatinase secretion triggered by FAK transfection. By contrast, blocking protein secretion with the inhibitors monensin and brefeldin A greatly reduced gelatinase production. Therefore, besides inducing MMP gene expression, FAK activation by integrin engagement has a prominent role in regulating gelatinase release.

To better understand the mechanisms through which FAK regulates gelatinase release, point mutants of FAK were transiently transfected into Jurkat cells. Point mutation of tyrosine 397 (FAK Y397F) has been shown to prevent recruitment of tyrosine kinases of the Src family, while mutation of lysine 454 (FAK K454R) abolishes FAK intrinsic kinase activity and mutation of tyrosine 925 (FAK Y925F) has been suggested to prevent Grb2 binding and the subsequent activation of the Ras/Raf-1/ERK signaling pathway. Mutation at the catalytic site did not decrease gelatinase production, indicating that intrinsic FAK kinase activity is not required for gelatinase production. Mutation of Y925 also did not reduce gelatinase release compared with FAK wild-type transfected cells.

In contrast, mutation at Y397 significantly decreased gelatinolytic signals, suggesting that recruitment of Src kinases by FAK into focal adhesions is necessary for gelatinase production.

2. Src and Src-FAK interaction are crucial for integrin-mediated gelatinase release by T lymphoid cell lines
Given that Lck is a major Src-type tyrosine kinase in cells of lymphoid lineage, we explored gelatinase induction by FN in J.CaM1.6 cells, a Jurkat-derived Lck-deficient T cell line. JCaM1.6 cells displayed dramatically reduced production of gelatinases in response to FN compared with Jurkat cells and it was restored by transfection with wild-type Lck.

The reduced gelatinase production in Y397F-transfected cells, combined with the requirement of Src tyrosine kinase activity in gelatinase induction, suggests that FAK/Src complex formation is crucial for integrin-mediated induction of gelatinases. In accord with this hypothesis, co-transfection of Lck and FAK wild-type into the Lck-deficient cell line J.CaM1.6 resulted in more strongly increased MMP-2 and MMP-9 production than transfection with either Lck or FAK individually.

3. Integrin-mediated stimulatory and inhibitory signals for gelatinase release are orchestrated by distinct domains of FAK: functional divergence between FRNK and FAT fragments
We previously showed that exposure to FN transduces stimulatory and inhibitory signals for MMP production by T lymphoid cells. We next explored whether these dual signals were coordinated by FAK by inducing overexpression of truncated forms. These consisted of the naturally occurring C-terminal domain FRNK, which lacks the kinase domain but retains binding sites for adaptor proteins and FAT, a shorter C-terminal truncated form that retains binding sites for paxillin and talin but lacks the proline-rich domains present in FRNK.

FRNK and FAT fragments had opposite effects on gelatinase release (Fig. 1 A). FRNK overexpression elicited a much lower release of gelatinases than FAK wild-type in response to FN. In contrast, FAT transfection produced an increase in both MMPs that was considerably higher even than FAK wild-type. The effects of FRNK and FAT were more prominent on gelatinase release than on gelatinase mRNA expression (Fig. 1C ). FRNK and FAT also had opposite effect on cell adhesion to FN: while FRNK decreased cell attachment, FAT increased it (Fig. 1D ). These findings suggest that these mutants may decrease cell motility through opposite mechanisms: by decreasing cell attachment or by inducing cell arrest.

View larger version (56K): [in this window] [in a new window]   Figure 1. Opposite effects of FAK truncated forms on gelatinase expression and release. A) Gelatin zymography of concentrated conditioned medium from Jurkat cells mock-transfected or transfected with FAK wild-type or FAK truncated forms FRNK and FAT, after exposure to soluble FN (left) or solid-phase FN (right). B) Western blot performed with lysates of transfected cells indicating the relative expression of FAK wild-type, FRNK fragment, and FAT subfragment. C) RT-PCR amplification of MMP-2, MMP-9 and ß2-µGlb using RNA obtained from the same cells as in panel B. D) Adhesion of transfected Jurkat cells to solid-phase FN and invasion through Matrigel-coated membranes.

We next tried to elucidate the molecular basis for the divergent effects of FRNK and FAT on gelatinase production. Compared with FRNK, FAT lacks two proline-rich domains with important functions. The proline-rich 1 domain is able to interact with SH3-containing pCAS (Crk-associated substrate) and the proline-rich 2 region is a binding site for GRAF (GTPase-activating protein for Rho associated with FAK). pCAS-mediated signals could be, then, stimulatory (through JNK) or predominantly inhibitory (through Ras/ERK or PI 3-kinase activation) for gelatinase secretion.

4. FRNK is able to mediate pCAS downstream signaling events regulating gelatinase release through mechanisms requiring Src-kinase activity
FRNK-recruited pCAS might be phosphorylated by other kinases and transmit downstream signaling events. It was recently shown that pCAS is able to directly recruit Src-type tyrosine kinases.

As a test of this hypothesis, Jurkat cells were transfected with FRNK mutated at prolines 712/715 (FRNK P712/715A), which is unable to recruit pCAS. As displayed in Fig. 2 A, this mutation prevented the inhibitory effect of FRNK on gelatinase release. Supporting a role for Src-type tyrosine kinases in phosphorylating FRNK-bound pCAS, FRNK transfection into the Lck-deficient J.CaM1.6 cell line not only failed to reduce MMP production but in fact resulted in an enhancement of MMP release even stronger than that achieved by FAT (Fig. 2B ). The suppressing effect of FRNK was reversed by inhibiting Src-type tyrosine kinase activity with PP2 (Fig. 2C ). The FRNK inhibitory effect, consequently, is dependent on Src-type tyrosine kinases.

View larger version (45K): [in this window] [in a new window]   Figure 2. FRNK inhibitory effect on gelatinase release requires Src activity and is associated with CasL phosphorylation and ERK activation. A) Gelatin zymography of conditioned medium obtained from Jurkat cells transiently transfected with the indicated FAK mutants and truncated forms after exposure to FN. B) Gelatin zymography of concentrated conditioned medium obtained from baseline JCaM1.6 cells, and J.CaM1.6 cells transiently transfected with FAK wild-type or truncated forms FRNK and FAT. C) Gelatin zymography of concentrated conditioned medium obtained from Jurkat cells mock transfected (–) and Jurkat cells transiently transfected with FRNK (+), untreated (–) or treated with Src-inhibitor PP2 (+) or MEK1 inhibitor PD98059 (+). D) Cell lysates obtained from Jurkat cells mock transfected (–) or transfected with FRNK (+), untreated (–) or treated with Src-inhibitor PP2 (+) and incubated with FN were subjected to SDS-PAGE and blotted onto nitrocellulose. Western blot was performed with a phosphotyrosine antibody, then the blot was stripped and reprobed with anti-pCAS antibody. E) Western blot analysis of cell lysates obtained from mock (–) or FRNK (+) transfected Jurkat cells, untreated (–) or treated with PP2, and exposed to FN. Blots were incubated with anti-phosphorylated ERK antibody, stripped and reprobed with anti-ERK antibody.

Further supporting the ability of FRNK to recruit pCAS and the ability of Src-type tyrosine kinases to phosphorylate it, FRNK overexpression did not interfere with phosphorylation of two pCAS family members present in lymphoid cells CasL and Sin (Fig. 2D ). However, both CasL and Sin phosphorylation were reduced by PP2 only in FRNK-transfected cells and not in the mock-transfected controls, supporting the idea that FRNK cooperates with Src-type tyrosine kinases to transmit downstream CasL/Sin signaling (Fig. 2D ). Consistent with this, FRNK-induced ERK phosphorylation was abrogated by the Src inhibitor PP2 (Fig. 2E ). As shown in Fig. 2C , the FRNK inhibitory effect on gelatinase release was reversed with the MEK1 inhibitor PD98059 indicating that FRNK transmits inhibitory signals for gelatinase release by a process requiring ERK activation.

5. FAT-increased gelatinase release requires Src-activity and is associated with reduced ERK activation
FAT subfragment lacks the predominantly inhibitory proline-rich regions but retains binding sites for important signaling and cytoskeleton proteins such as paxillin and talin. FAT enhanced gelatinase release was inhibited by the Src-family kinase inhibitor PP2 and was virtually unmodified by the JNK inhibitor SP600125, indicating that Src kinase activity but not JNK activity are necessary for FAT-enhanced gelatinase release.

CONCLUSIONS AND SIGNIFICANCE

Our findings indicate that, in T lymphoid cells, FAK regulates not only gelatinase production but also post-transcriptional gelatinase release in response to FN and that the scaffolding role of FAK rather than its kinase activity is the most relevant function in this process. FAK interaction with Src-type tyrosine kinases is essential in gelatinase release. As revealed by transient transfection of truncated forms, FAK is able to transduce both stimulatory and inhibitory signals for gelatinase release by T lymphoid cells through pathways leading to ERK1/2 inhibition and activation, respectively. Src-tyrosine kinase activity is required for both stimulatory and inhibitory functions. Inhibitory signals are mostly mediated by proline-rich regions, which also have an important role in regulating focal adhesion turnover. FAK emerges as an important mediator in coordinating stimulatory and inhibitory signals for gelatinase release. As depicted in Fig. 3 , this fine control may adapt pulse release of gelatinases to focal adhesion turnover.

View larger version (40K): [in this window] [in a new window]   Figure 3. Schematic model of FAK-mediated pulse gelatinase release coordinated to focal adhesion turnover. Cyclic activation/inhibition of ERK1/2 mediated by distinct domains of FAK is proposed as one of the involved pathways. A) Effective migration through the extracellular matrix requires focal adhesion turnover and pulse gelatinase release coordinated by entire FAK. B) The truncated form FRNK is unable to mediate this process and decreases MMP release as well as interaction with FN. C) The shorter fragment FAT freezes the cell in an adherent status with unrepressed gelatinase release.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3574fje;

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