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¹ Department of Biochemistry and Molecular Biology, Faculty of Medicine, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan
² KAN Research Institute Inc., Kobe MI R&D Center, Kobe 650-0047, Japan
³ Department of Biochemistry and Molecular Biology, Faculty of Medicine, Division of Molecular and Cellular Biology, Graduate School of Medicine, Kobe University, Kobe, 650-0017, Japan

	Abstract

Top Abstract Introduction Results Discussion Experimental procedures References

 
It was previously shown that platelet-derived growth factor (PDGF) receptor physically and functionally interacts with integrin _vβ₃, effectively inducing cell movement. We previously showed that Necl-5, originally identified as a poliovirus receptor, interacts with integrin _vβ₃ and enhances its clustering and the formation of focal complexes at the leading edges of moving cells, resulting in an enhancement of cell movement. We showed here that Necl-5 additionally interacts with PDGF receptor in NIH3T3 cells and regulates the interaction between PDGF receptor and integrin _vβ₃, effectively inducing directional cell movement. PDGF receptor co-localized with Necl-5 and integrin _vβ₃ at peripheral ruffles over lamellipodia, which were formed at the leading edges of moving cells in response to PDGF, but not at the focal complexes under these ruffles, whereas Necl-5 and integrin _vβ₃ co-localized at these focal complexes. The clustering of these three molecules at peripheral ruffles required the activation of integrin _vβ₃ by vitronectin and the PDGF-induced activation of the small G protein Rac and subsequent re-organization of the actin cytoskeleton. These results indicate a key role of Necl-5 in directional cell movement by physically and functionally interacting with both integrin _vβ₃ and PDGF receptor.

	Introduction

Top Abstract Introduction Results Discussion Experimental procedures References

 
Cells move toward chemotactic substances (chemoattractants), such as growth factors, chemokines and cytokines (Gumbiner 1996; Lauffenburger & Horwitz 1996). These substances first bind to their cell surface receptors, which then transduce signals for cell movement. It was previously believed that the receptors for chemoattractants localize at leading edges of moving cells, but their localizations and regulatory mechanisms remained undetermined. It was reported that T cells respond to chemokines, such as SDF1, and that chemokine receptors, such as CXCR4, localize at leading edges (Shimonaka et al. 2003). Contrary to T cells, it was reported that, in amoeba Dictiostellium discoideum, a cell surface receptor for cyclic AMP, a chemoattractant for this amoeba, is uniformly distributed along the plasma membrane, but that its downstream signaling molecules, such as PTEN and PI3-kinase, are unevenly distributed: PI3-kinase localizes at leading edges whereas PTEN localizes at rear edges (Comer & Parent 2002; Weiner 2002). Thus, the localization of each receptor varies and the mechanisms underlying their localizations remain unknown. Fibroblasts respond to platelet-derived growth factor (PDGF) as a chemoattractant, and polarize in the direction of higher concentrations of PDGF (Ronnstrand & Heldin 2001). However, the localization and behavior of PDGF receptor remain unknown.

At leading edges, special structures necessary for cell movement are dynamically formed: these structures include protrusions, such as filopodia and lamellipodia, peripheral ruffles, focal complexes and focal adhesions (Hall 1998; Rottner et al. 1999; Zaidel-Bar et al. 2004). These structures are formed by re-organization of the actin cytoskeleton, which is regulated by the actions of the Rho family small G proteins: lamellipodia and ruffles are formed by the action of Rac; filopodia are formed by the action of Cdc42; and focal complexes are formed by the action of Rac and Cdc42 (Rottner et al. 1999). The formation of these structures is inhibited by the action of Rho. Focal complexes are transformed to focal adhesions by inactivation of Cdc42 and Rac and activation of Rho (Rottner et al. 1999; Ballestrem et al. 2001). The activities of these small G proteins are cooperatively regulated by receptors and integrins.

Integrins are cell-matrix adhesion molecules that are essential for cell movement in cooperation with cell surface receptors for chemoattractants (van der Flier & Sonnenberg 2001; Comoglio et al. 2003). Integrins form cell-matrix junctions called focal complexes and focal adhesions (Cram & Schwarzbauer 2004). Focal complexes are smaller in size than focal adhesions, and are formed at contact sites between protrusions and extracellular matrix (ECM) proteins at leading edges (Rottner et al. 1999; Zaidel-Bar et al. 2004). Focal adhesions are formed at sites to the rear of focal complexes. Ruffles randomly attach to matrix and some of them form new focal complexes; pre-existing focal complexes are transformed to focal adhesions. Integrins have at least two forms: conformations with either low or high affinity for their ECM binding partners (van der Flier & Sonnenberg 2001; Takagi et al. 2002; Cram & Schwarzbauer 2004). When talin binds to the low-affinity form, this integrin is converted to the high-affinity form (Cram & Schwarzbauer 2004). Upon binding to ECM proteins, integrins transduce signals inside cells that then cause the re-organization of the actin cytoskeleton, eventually resulting in the clustering of integrins and the formation of focal complexes and focal adhesions (Cram & Schwarzbauer 2004). Of the many integrins, integrin _vβ₃ forms focal complexes and this integrin is often up-regulated in cancer cells (Ballestrem et al. 2001; Guo & Giancotti 2004).

It was previously shown that PDGF receptor physically interacts with integrin _vβ₃ (Schneller et al. 1997; Woodard et al. 1998) and that this interaction synergistically enhances cell movement and proliferation (Woodard et al. 1998). However, the regulatory and molecular mechanisms of this interaction remain unknown.

We recently found that nectin-like molecule (Necl)-5 forms a complex with integrin _vβ₃ and enhances its clustering and subsequent formation of focal complexes at the leading edges of moving NIH3T3 cells in response to PDGF (Minami et al. 2007). This action of Necl-5 requires both the activation of PDGF receptor by PDGF and the activation of integrin _vβ₃ by vitronectin, an ECM protein that binds to integrin _vβ₃ (Cheresh 1987). Necl-5 was originally identified as a poliovirus receptor (PVR)/CD155 in humans (Mendelsohn et al. 1989; Koike et al. 1990) and as the product of a gene, Tage4, which is over-expressed in colon carcinomas in rodents (Chadeneau et al. 1994). PVR/CD155 was also shown to be over-expressed in many human cancer cells (Gromeier et al. 2000; Masson et al. 2001; Bottino et al. 2003). This molecule, with four nomenclatures, was re-named Necl-5 (Takai et al. 2003). However, it remains unknown how the Necl-5-integrin complex is clustered at leading edges or how cells determine the direction of movement in response to PDGF.

In the present study, we attempted to address these issues and found that Necl-5 interacts not only with integrin _vβ₃ but also with PDGF receptor and that the Necl-5-PDGF receptor complex also localizes at peripheral ruffles over lamellipodia at the leading edges of NIH3T3 cells, which were formed in response to PDGF in a vitronectin-dependent manner. We discuss the role of this interaction in directional movement. In this study, we studied concerning PDGF receptor β and it is simply referred to as PDGF receptor.

	Results

Top Abstract Introduction Results Discussion Experimental procedures References

 
Co-localization of PDGF receptor with Necl-5 and integrin _Vβ₃ at peripheral ruffles over lamellipodia of leading edges

We previously showed that when NIH3T3 cells are sparsely plated on µ-slide VI flow dishes pre-coated with vitronectin, starved of serum and directionally stimulated by PDGF, most cells become polarized and form protrusive lamellipodia, peripheral ruffles over the lamellipodia, focal complexes under the ruffles and focal adhesions at leading edges in the direction of higher concentrations of PDGF (Minami et al. 2007). The formation of these leading edge structures requires the activation of PDGF receptor by PDGF, the activation of integrin _Vβ₃ by vitronectin, and the expression of Necl-5. Necl-5 physically interacts with integrin _Vβ₃ and the Necl-5-integrin _Vβ₃ complex localizes at peripheral ruffles over the lamellipodia of leading edges and the focal complexes under the ruffles, but not at the focal adhesions. Consistent with these observations, the immunofluorescence signals for Necl-5 and integrin β₃ were concentrated and co-localized at peripheral ruffles over the lamellipodia (Fig. 1Aa). In the basal sections of the cells, the signals for Necl-5 and integrin β₃ were observed at the focal complexes under the peripheral ruffles (Fig. 1Aa; fuzzy dot-like structures and line-like focal complexes (FXs)). The signal for integrin β₃, but not that for Necl-5, was observed at the focal adhesions at sites to the rear of leading edges. Essentially the same results were obtained when cells were stained for integrin _V instead of integrin β₃ (data not shown). Indeed, unless otherwise specified, the same results were essentially obtained for both integrin _V and integrin β₃ in the experiments that follow. The signal for PDGF receptor was faintly detected at the peripheral ruffles and co-localized with those for Necl-5 and integrin β₃ in the middle sections of the cells. In the basal sections of the cells, the signal for PDGF receptor was detected as an indistinct signal under the peripheral ruffles. By co-localization analysis, PDGF receptor loosely co-localized with Necl-5, but weakly co-localized with integrin β₃. Thus, PDGF receptor might not be concentrated at focal complexes.

View larger version (92K): [in this window] [in a new window]   Figure 1  Co-localization of PDGF receptor with Necl-5 and integrin Vβ3 at peripheral ruffles over lamellipodia of leading edges. (A–C) Immunofluorescence images of PDGF-stimulated NIH3T3 cells cultured on vitronectin-coated µ-slide dishes. Cells were double or triple stained with the anti-Necl-5, the anti-PDGF receptor β and the anti-integrin β3 mAbs. (A) Wild-type NIH3T3 cells; (B) Necl-5-NIH3T3 cells; (C) wild-type NIH3T3 cells transfected with an siRNA vector; (Ca) Necl-5-knockdown-NIH3T3 cells; (Cb) control-NIH3T3 cells. (Aa, Ba, Ca, Cb) in the presence of PDGF; (Ab, Bb) in the absence of PDGF. Arrowheads, leading edges; FXs, focal complexes; FA, focal adhesion; bars, 10 µm. Inset boxes show the areas of high magnification images. The high magnification images are indicated as intensity ratio images. Analysis of the co-localization of the two indicated proteins and generation of the intensity ratio images were performed using IMAGEJ 1.34S software with an RG2B co-localization plug-in. The results shown are representative of three independent experiments.

In NIH3T3 cells in which Necl-5 was knocked down by an siRNA vector (Necl-5-knockdown-NIH3T3 cells), definite leading edges were not formed, the signal for Necl-5 was negligible (Fig. 1Ca and b), and the signal for integrin b₃ was not concentrated at any region, as described previously (Minami et al. 2007) (data not shown). The signal for PDGF receptor was not concentrated at any peripheral region, although dot-like signals for PDGF receptor were detected inside cells (Fig. 1Ca and Cb). These signals might represent endocytosed PDGF receptor. When NIH3T3 or Necl-5-NIH3T3 cells were similarly cultured on dishes coated with laminin, an ECM protein that does not bind to integrin _Vβ₃ (Cheresh 1987), instead of vitronectin-coated ones, or were cultured on vitronectin-coated dishes but not stimulated by PDGF, they did not show polarized structures with protrusions, ruffles or focal complexes (Fig. 1Ab and Bb and data not shown) (Minami et al. 2007). None of the signal for PDGF receptor, Necl-5, or integrin β₃ was concentrated at any peripheral region. These results are consistent with our earlier observations (Minami et al. 2007) and indicate that the formation of the leading edge structures and the localization of PDGF receptor as well as Necl-5 and integrin _Vβ₃ at peripheral ruffles require the activation of PDGF receptor by PDGF, the activation of integrin _Vβ₃ by vitronectin, and the expression of Necl-5.

Physical interaction of Necl-5 with PDGF receptor

We then examined whether Necl-5 physically interacts with PDGF receptor using a co-immunoprecipitation assay. We co-expressed HA-tagged PDGF receptor (PDGF receptor-HA) and either of FLAG-tagged Necl-5 (FLAG-Necl-5) or FLAG alone as a control, in HEK293 cells, and cultured the cells in suspension in the presence or absence of PDGF and AG1296. We used HEK293 cells because the expression of integrin _Vβ₃ was not significantly detected by Western blotting in this cell line (data not shown). The suspension culture enabled the detection of a possible cis-interaction between Necl-5 and PDGF receptor on the plasma membrane, rather than trans-interaction. The presence of PDGF predominantly made PDGF receptor the tyrosine phosphorylated form, whereas the presence of AG1296 made it the non-phosphorylated form (data not shown). AG1296 is an inhibitor of the kinase activity of PDGF receptor (Kovalenko et al. 1997). Because over-expression of PDGF receptor induces autophosphorylation of this protein to some degree even in the absence of PDGF (data not shown), this inhibitor was added to keep PDGF receptor from being auto-phosphorylated. When FLAG-Necl-5 was immunoprecipitated from cell lysates using the anti-FLAG monoclonal antibody (mAb), PDGF receptor-HA was co-immunoprecipitated with FLAG-Necl-5 irrespective of the presence or absence of PDGF and AG1296 (Fig. 2A). The amount of co-immunoprecipitated PDGF receptor-HA in the presence or absence of PDGF and AG1296 was not significantly different. PDGF receptor-HA was not co-immunoprecipitated with FLAG from cells expressing FLAG as a control, irrespective of the presence or absence of PDGF and AG1296. These results indicate that Necl-5 physically interacts in cis with both the tyrosine-phosphorylated and non-phosphorylated forms of PDGF receptor to similar extents.

View larger version (46K): [in this window] [in a new window]   Figure 2  Physical interaction of Necl-5 with PDGF receptor. (A) Physical interaction of Necl-5 with PDGF receptor. HEK293 cells were transfected with various combinations of indicated vectors and cultured in suspension under the indicated conditions. FLAG-tagged Necl-5, Necl-5-CP, or Necl-5-EC was immunoprecipitated using the anti-FLAG mAb and samples were assessed by Western blotting using the anti-FLAG pAb mAb and the anti-HA mAb. (B) Physical interaction between Necl-5, PDGF receptor and integrin Vβ3 at an endogenous level. NIH3T3 cells were cultured on vitronectin-coated dishes and starved of serum. Cells were treated with DTSSP and then subjected to a co-immunoprecipitation assay using the anti-integrin V pAb. Samples were assessed by Western blotting using the anti-integrin V mAb, the anti-Necl-5 mAb and the anti-PDGF receptor pAb. (C) Physical interaction between PDGF receptor and integrin Vβ3. EGFP-tagged integrin β3 was immunoprecipitated using the anti-GFP pAb and samples were assessed by Western blotting using the anti-GFP pAb, the anti-integrin V mAb and the anti-HA mAb. The results shown are representative of three independent experiments.

We then confirmed that endogenous Necl-5 physically interacts not only with endogenous integrin _Vβ₃ but also with endogenous PDGF receptor. NIH3T3 cells were cultured on vitronectin-coated dishes, treated with the chemical cross-linker DTSSP, and then endogenous integrin _V was immunoprecipitated from cell lysates. DTSSP was used as we previously showed an interaction between endogenous Necl-5 and endogenous integrin _Vβ₃ using this reagent in NIH3T3 cells (Minami et al. 2007). Necl-5 and PDGF receptor were co-immunoprecipitated with integrin _V from cell lysates (Fig. 2B). Taken together, these findings suggest that Necl-5 physically interacts with both integrin _Vβ₃ and PDGF receptor at peripheral ruffles over lamellipodia of leading edges of NIH3T3 cells, which are formed in response to PDGF in a vitronectin-dependent manner.

It was previously shown by a co-immunoprecipitation assay using endogenous proteins from NIH3T3 and rat microvascular endothelial cells that PDGF receptor physically interacts with integrin _Vβ₃ (Schneller et al. 1997; Woodard et al. 1998), but it remains unknown whether this interaction is direct or mediated by Necl-5, because we previously showed that Necl-5 physically interacts with integrin _Vβ₃ (Minami et al. 2007) and we showed here that Necl-5 physically interacts with PDGF receptor. In addition, it was shown that integrin _Vβ₃ has two forms: low- and high-affinity forms (van der Flier & Sonnenberg 2001; Takagi et al. 2002; Cram & Schwarzbauer 2004). We previously showed that Necl-5 physically interacts with both forms to similar extents (Minami et al. 2007), but it remains unknown with which form of integrin _Vβ₃ PDGF receptor interacts. We next examined these two issues. We expressed PDGF receptor-HA, and either EGFP-tagged integrin β₃ (integrin β₃-EGFP) and integrin _V, or EGFP alone as a control, in HEK293 cells, and cultured the cells in suspension in the presence or absence of PDGF and AG1296. Mn²⁺ and cyclo-RGDfV, an integrin-binding peptide, were also added to the medium to make predominantly the high-affinity form of integrin _Vβ₃ (Takagi et al. 2002). Alternatively, we co-expressed PDGF receptor-HA, and either EGFP-tagged integrin β₃^T329C/A347C (integrin β₃^T329C/A347C-EGFP) and integrin _V, or EGFP alone as a control, in HEK293 cells, and cultured the cells in suspension in the presence or absence of PDGF and AG1296. Ca²⁺ and Mg²⁺ were added to the medium to make predominantly the low-affinity form of integrin _Vβ₃. Integrin β₃^T329C/A347C does not make the high-affinity form with integrin _V (Luo et al. 2004). We used HEK293 cells because the expression of Necl-5 was not significantly detected by Western blotting in this cell line (data not shown). When integrin β₃-EGFP was immunoprecipitated from cell lysates using the anti-GFP polyclonal antibody (pAb), integrin _V and PDGF receptor-HA were co-immunoprecipitated with it, irrespective of the presence or absence of PDGF and AG1296 (Fig. 2C). Essentially the same results were obtained when integrin β₃^T329C/A347C-EGFP was immunoprecipitated. None of these molecules was co-immunoprecipitated with EGFP from cells expressing EGFP as a control. These results are consistent with earlier observations (Schneller et al. 1997; Woodard et al. 1998) and indicate that PDGF receptor physically interacts with both the low- and high-affinity forms of integrin _Vβ₃ to similar extents and that these interactions are not mediated by Necl-5.

Taken together, the present and previous results indicate that Necl-5, PDGF receptor and integrin _Vβ₃ can form any combinations of binary complex.

Actin cytoskeleton-dependent localization of PDGF receptor at peripheral ruffles

It was shown that the actin cytoskeleton is necessary for the clustering of integrin in general (Cram & Schwarzbauer 2004). Because PDGF is necessary for the formation of leading edge structures, including lamellipodia, peripheral ruffles over the lamellipodia, focal complexes under the ruffles, and focal adhesions, and the localization of PDGF receptor, Necl-5, and integrin _Vβ₃ at their specific sites, we examined whether the localization of PDGF receptor as well as Necl-5 and integrin _Vβ₃ at the peripheral ruffles is dependent on the actin cytoskeleton. When NIH3T3 cells were sparsely plated on µ-slide VI flow dishes pre-coated with vitronectin, starved of serum and directionally stimulated by PDGF in the presence of cytochalasin D, cells did not form protrusions, such as lamellipodia and ruffles (Fig. 3A). The immunofluorescence signal for actin stress fibers was lost, but the signal for peripheral actin remained and that for actin patches appeared inside the cells. The signal for integrin β₃ was faintly detected at focal adhesions. Focal complexes were not observed at any region. The signal for Necl-5 was not concentrated at any peripheral region. The signal for PDGF receptor was also not concentrated at any peripheral region, but was detected as internalized dot-like structures inside the cells. Essentially the same results were obtained when this experiment was performed using Necl-5-NIH3T3 cells, except for a decrease in the intensity of the internalized dot-like signals for PDGF receptor (Fig. 3B). These results indicate that the localization of PDGF receptor as well as Necl-5 and integrin _Vβ₃ at peripheral ruffles is dependent on the actin cytoskeleton.

View larger version (33K): [in this window] [in a new window]   Figure 3  Actin cytoskeleton-dependent localization of PDGF receptor at peripheral ruffles. (A and B) Immunofluorescence images of PDGF-stimulated NIH3T3 cells cultured on vitronectin-coated µ-slide dishes in the presence of 1 µM cytochalasin D. Cells were triple stained with various combinations of the anti-Necl-5, the anti-integrin β3, the anti-PDGF receptor β and the anti-actin mAbs. (A) Wild-type NIH3T3 cells; (B) Necl-5-NIH3T3 cells. Bars, 10 µm. The results shown are representative of three independent experiments.

We previously showed that the PDGF-induced activation of Rac is involved in the formation of lamellipodia and peripheral ruffles, the Necl-5-dependent clustering of integrin _Vβ₃, and the formation of focal complexes at the leading edges of moving NIH3T3 cells (Minami et al. 2007). We examined whether the PDGF-induced activation of Rac is involved in the localization of PDGF receptor as well as Necl-5 and integrin _Vβ₃ at peripheral ruffles. When NIH3T3 cells expressing a dominant-negative mutant of Rac1 (Rac1DN) were sparsely plated on µ-slide VI flow dishes pre-coated with vitronectin, starved of serum and directionally stimulated by PDGF, the extents of protrusions, ruffles, and focal complexes were significantly reduced compared with those in NIH3T3 cells (Fig. 4A and see Fig. 1Aa). Moreover, the immunofluorescence signal for Necl-5 or integrin β₃ was not concentrated at any peripheral region; consistent with our earlier observations (Minami et al. 2007). The signal for PDGF receptor was not concentrated at any peripheral region. Essentially the same results were obtained when this experiment was performed using Necl-5-NIH3T3 cells (Fig. 4B). These results indicate that the PDGF-induced activation of Rac is involved in the localization of PDGF receptor as well as Necl-5 and integrin _Vβ₃ at peripheral ruffles.

View larger version (21K): [in this window] [in a new window]   Figure 4  Rac-dependent localization of PDGF receptor at peripheral ruffles. (A and B) Immunofluorescence images of PDGF-stimulated NIH3T3 cells expressing Rac1DN, cultured on vitronectin-coated µ-slide dishes. Cells were transfected with myc-tagged Rac1DN and triple stained with various combinations of the anti-Necl-5, the anti-integrin β3, the anti-PDGF receptor β and the anti-myc mAbs. (A) Wild-type NIH3T3 cells; (B) Necl-5-NIH3T3 cells. Bars, 10 µm. The results shown are representative of three independent experiments.

Regulation of the interaction between PDGF receptor and integrin _Vβ₃ by Necl-5 and PDGF

We finally studied the role of the interaction between Necl-5 and PDGF receptor. We first examined whether this interaction affects the interaction between PDGF receptor and integrin _Vβ₃. Necl-5-NIH3T3 cells were cultured on vitronectin-coated dishes in the presence or absence of PDGF, treated with DTSSP, and then endogenous integrin _V was immunoprecipitated from cell lysates. Necl-5 and PDGF receptor were co-immunoprecipitated with integrin _V from cell lysates, irrespective of the presence or absence of PDGF (Fig. 5A). The amount of Necl-5 co-immunoprecipitated with integrin _V was not affected by PDGF, but the amount of PDGF receptor co-immunoprecipitated with integrin _V was 10%–20% reduced by PDGF. When wild-type NIH3T3 cells were similarly subjected to the same co-immunoprecipitation assay, Necl-5 and PDGF receptor were co-immunoprecipitated with integrin _V from cell lysates, irrespective of the presence or absence of PDGF (Fig. 5B, see also Fig. 2B). The amount of Necl-5 co-immunoprecipitated with integrin _V was not affected by PDGF, but the amount of PDGF receptor co-immunoprecipitated with integrin _V was 50%–60% reduced by PDGF. When Necl-5-knockdown-NIH3T3 cells were similarly subjected to the same co-immunoprecipitation assay, PDGF receptor, but not Necl-5, was co-immunoprecipitated with integrin _V, irrespective of the presence or absence of PDGF (Fig. 5C). The amount of PDGF receptor co-immunoprecipitated with integrin _V was about 90% reduced by PDGF. These results are consistent with those of the immunofluorescence microscopic examination shown in Fig. 1 and indicate that Necl-5 and PDGF regulate the interaction between PDGF receptor and integrin _Vβ₃: PDGF reduces the interaction and Necl-5 blocks this reduction.

View larger version (61K): [in this window] [in a new window]   Figure 5  Regulation of the interaction between PDGF receptor and integrin Vβ3 by Necl-5 and PDGF. NIH3T3 cells were cultured on vitronectin-coated dishes, starved of serum and cultured in the absence or presence of PDGF. Cells were treated with DTSSP and then subjected to a co-immunoprecipitation assay using the anti-integrin V pAb. Samples were assessed by Western blotting using the anti-integrin V mAb, the anti-Necl-5 mAb and the anti-PDGF receptor pAb. (A) Necl-5-NIH3T3 cells, (B) NIH3T3 cells, (C) control-NIH3T3 and Necl-5-knockdown-NIH3T3 cells. Asterisks, the relative level of the amounts of co-immunoprecipitated PDGF receptor in the absence or presence of PDGF. Intensities were normalized to 1.0 for respective cells cultured in the absence of PDGF. The results shown are representative of three independent experiments.

Enhancement of cell movement by Necl-5 and PDGF

We previously showed that Necl-5 enhances the movement of L and NIH3T3 cells in the presence of serum containing various growth factors and ECM proteins (Ikeda et al. 2004; Fujito et al. 2005). Using a Boyden chamber assay, we then examined whether over-expression or knockdown of Necl-5 affects directional cell movement. Necl-5-NIH3T3 and Necl-5-knockdown-NIH3T3 cells were cultured on vitronectin-coated cell culture inserts and stimulated by PDGF. Necl-5-NIH3T3 cells moved more rapidly than wild-type NIH3T3 cells, whereas Necl-5-knockdown-NIH3T3 cells moved more slowly than control-NIH3T3 cells (Fig. 6A and B). Movement of these three types of cells was dependent on vitronectin and they did not move on BSA- or laminin-coated cell culture inserts (data not shown) (Woodard et al. 1998; Ikeda et al. 2004). These results are consistent with our earlier observations (Ikeda et al. 2004; Fujito et al. 2005) and indicate that the expression level of Necl-5 regulates PDGF-induced directional cell movement.

View larger version (24K): [in this window] [in a new window]   Figure 6  Enhancement of cell movement by Necl-5 and PDGF. For measurement of cell movement with a Boyden chamber assay, NIH3T3 cells were starved of serum and incubated on cell culture inserts coated with vitronectin in the presence of PDGF in the bottom well for 16 h. (A) Wild-type NIH3T3 and Necl-5-NIH3T3 cells. The cells were stained by crystalviolet and the migrated cells were counted. (B) Control- and Necl-5-knockdown-NIH3T3 cells. Wild-type NIH3T3 cells were transfected with an siRNA vector, pBS-EGFP-H1-Necl-5 or pBS-EGFP-H1-control and their transfection efficiencies were determined to be about 20%. The EGFP-positive migrated cells were counted. The number of control EGFP-positive migrated cells was consistent with that of control migrated cells in (A), considering the transfection efficiency. *P < 0.001. The results shown are the means ± SE of three independent experiments.

	Discussion

Top Abstract Introduction Results Discussion Experimental procedures References

It has been shown that PDGF receptor physically interacts with integrin _Vβ₃ (Schneller et al. 1997; Woodard et al. 1998). We previously showed that Necl-5 physically interacts with integrin _Vβ₃ (Minami et al. 2007). In the present study, we confirmed these earlier observations and further showed that Necl-5 physically interacts with PDGF receptor through their extracellular regions. The cytoplasmic region of Necl-5 was not necessary for this interaction, but it may regulate the interaction, because the amount of PDGF receptor-HA co-immunoprecipitated with FLAG-Necl-5-CP (FLAG-Necl-5 with the cytoplasmic region deleted) was about 3 times as much as that co-immunoprecipitated with the full-length FLAG-Necl-5. There are theoretically cis- and trans-interactions between Necl-5 and PDGF receptor; however, the results of the immunoprecipitation assay using suspended HEK293 cells support an interaction in cis. Thus, Necl-5 and PDGF receptor on the plasma membrane of the same cell physically interact with each other in cis.

PDGF receptor and integrin _Vβ₃ each have two forms: tyrosine-phosphorylated and non-phosphorylated forms and low- and high-affinity forms, respectively (Heldin et al. 1998; Takagi et al. 2002; Cram & Schwarzbauer 2004). Which forms of PDGF receptor and integrin interact with each other was not known, but we showed here that PDGF receptor and integrin _Vβ₃ have potencies to interact with any forms to similar extents. In addition, we showed here that Necl-5 interacts equally with both forms of PDGF receptor. PDGF receptor, Necl-5 and integrin _Vβ₃ all co-localize at peripheral ruffles over lamellipodia of leading edges of NIH3T3 cells. These three molecules might form a ternary complex there, but it is practically difficult to obtain definitive evidence for this conclusion because each of Necl-5, PDGF receptor, and integrin _Vβ₃ can form any combinations of binary complex. Thus, at present we conclude that each of Necl-5, PDGF receptor and integrin _Vβ₃ forms at least each binary complex and localizes at peripheral ruffles of lamellipodia of leading edges.

It was previously shown that cell movement requires the synergistic activity of PDGF- and integrin-induced signaling (Woodard et al. 1998). PDGF-induced signaling requires integrin _Vβ₃ activation by binding to its ECM proteins (Woodard et al. 1998). The binding of talin to integrin increases the affinity of integrin for its specific ECM protein, and the binding of integrin to its specific ECM protein transduces signals inside cells leading to the re-organization of the actin cytoskeleton, which is necessary for integrin clustering (Cram & Schwarzbauer 2004). These inside-out and outside-in signaling pathways from the growth factor receptor and integrin bring about the formation of focal complexes and focal adhesions. Consistently, we previously showed that PDGF-induced, Necl-5- and integrin _Vβ₃-dependent formation of leading edge structures requires the PDGF-induced activation of Rac and subsequent re-organization of the actin cytoskeleton (Minami et al. 2007). In the present study, we confirmed these earlier observations and showed that the actin cytoskeleton and Rac activation are further necessary for the localization of PDGF receptor at peripheral ruffles over lamellipodia of leading edges of NIH3T3 cells. In addition, we showed here that Necl-5 and PDGF regulate the interaction between PDGF receptor and integrin _Vβ₃: PDGF reduces the interaction and Necl-5 blocks this reduction. Thus, Necl-5 plays a key role in this interaction. Taken together, these findings suggest that when PDGF reaches and binds to its receptor and induces Rac activation at specific regions; Rac activated in this way induces re-organization of the actin cytoskeleton, which induces formation of leading edge structures, formation of complexes of Necl-5, PDGF receptor, and integrin _Vβ₃, and concentration of these molecules at specific sites. The clustered complexes then enhance PDGF-induced Rac activation in a positive-feedback manner. Such a feedback amplification mechanism might efficiently form leading edge structures in the direction of higher concentrations of PDGF.

The present result that activated PDGF receptor is highly concentrated at peripheral ruffles over lamellipodia of leading edges, but not at focal complexes, suggests that PDGF-induced signaling, at least the PDGF-induced activation of Rac, is transduced from the peripheral ruffles, but not from the focal complexes, the focal adhesions, or other areas. Receptors including PDGF receptor undergo internalization by endocytosis upon binding of their specific ligands (Heldin et al. 1998). After PDGF receptor is down-regulated from the cell surface of the peripheral ruffles, PDGF-induced signaling is supposed to be diminished. When such ruffles attach to ECM proteins, they form new protrusions and focal complexes. This interpretation is consistent with the fact that PDGF receptor was not concentrated at focal complexes where Necl-5 and integrin _Vβ₃ were concentrated. Thereafter, Necl-5, PDGF receptor and integrin _Vβ₃, which remain diffusely on the plasma membrane, are recruited to peripheral ruffles to cluster in response to PDGF in a manner dependent on the activation of integrin _Vβ₃ and transduce the next cycle of signals. Thus, the dynamic clustering of Necl-5, PDGF receptor, and integrin _Vβ₃, the formation of peripheral ruffles, the dynamic clustering of Necl-5 and integrin _Vβ₃, and the formation of focal complexes may determine the direction of cell movement toward higher concentrations of PDGF. However, further studies are necessary to elucidate the detailed mechanisms underlying these dynamic events, which are essential for efficient directional cell movement.

	Experimental procedures

Top Abstract Introduction Results Discussion Experimental procedures References

 
Constructions

pCAGIPuro-FLAG-Necl-5, pCAGIZeo-FLAG-Necl-5-CP and pCAGIZeo-FLAG-Necl-5-EC were prepared as described previously (Ikeda et al. 2003, 2004). A small interfering RNA (siRNA) vector against Necl-5 (pBS-EGFP-H1-Necl-5) and a control siRNA vector against luciferase (pBS-EGFP-H1-control) were prepared as described previously (Fujito et al. 2005; Kajita et al. 2007). pBS-H1 vector was a gift from Dr H. Shibuya (Tokyo Medical and Dental University, Tokyo, Japan). Expression vectors were kindly provided as follows: wild-type human integrin _V and human integrin β₃ (pcDNA3-_V and -β₃) were from Dr J. C. Norman (University of Leicester, Leicester, UK), and a dominant-negative mutant of human integrin β₃ (pcDNA3.1-Myc-His(+)-β₃^T329C/A347C) was from Dr J. Takagi (Osaka University, Suita, Japan). To obtain EGFP-tagged integrin β₃^T329C/A347C (pEGFP-N3-integrin β₃^T329C/A347C), a fragment of integrin β₃^T329C/A347C from pcDNA3.1-Myc-His(+)-β₃^T329C/A347C was cloned into pEGFP-N3 vector (Clontech, Palo Alto, CA). To obtain EGFP-tagged wild-type integrin β₃ (pEGFP-N3-integrin β₃), a fragment of the extracellular region of integrin β₃^T329C/A347C in pEGFP-N3-integrin β₃^T329C/A347C was replaced with the same region of wild-type integrin β₃ from pcDNA3-β₃. To obtain HA-tagged PDGF receptor β (pcDNA3.1Hyg-PDGFRβ-HA), a fragment of PDGF receptor β, the stop codon of which had been deleted by PCR cloning from human PDGF receptor β cDNA, and an oligonucleotide encoding a HA-tag and a stop codon, were cloned into pcDNA3.1Hyg (Invitrogen, Carlsbad, CA). Human PDGF receptor β cDNA was a gift from Dr H. Matsui (Kobe University, Kobe, Japan). pEFBOS-myc-Rac1DN was prepared as described previously (Komuro et al. 1996).

Cell culture, transfection and siRNA experiments

NIH3T3 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% calf serum. HEK293 cells were maintained in DMEM supplemented with 10% fetal calf serum. Necl-5-NIH3T3 cells (NIH3T3 cells stably expressing FLAG-tagged Necl-5) were prepared as described previously (Kajita et al. 2007). For transient expression experiments, cells were transfected with various expression vectors using LipofectAMINE 2000 reagent (Invitrogen). Necl-5-knockdown-NIH3T3 cells (NIH3T3 cells in which Necl-5 was knocked down) were prepared as described previously (Fujito et al. 2005; Kajita et al. 2007). Briefly, NIH3T3 cells were transfected with pBS-EGFP-H1-Necl-5. As a control, pBS-EGFP-H1-control was used. For the co-immunoprecipitation assay with Necl-5-knocked down NIH3T3 cells, a double-stranded 25 nt RNA duplex, stealth RNAi, to Necl-5 (5'-AGCGCCAG GGCAAUAUGCUU-CUAAU-3') and a control stealth RNAi duplex were purchased from Invitrogen and NIH3T3 cells were transfected with stealth RNAi duplex using LipofectAMINE RNAiMAX reagent (Invitrogen).

Antibodies and reagents

A rat mAb against the extracellular region of Necl-5 (mAb-i, 1A8–8) was prepared as described previously (Ikeda et al. 2003). Rabbit anti-PDGF receptor β mAb (Y92) was purchased from Abcam. Hamster anti-integrin _V and β₃ mAbs (H9.2B8 and 2C9.G2, respectively; for immunostaining) were purchased from BD Biosciences (Pharmingen, NJ). The following mouse mAbs were purchased from commercial sources: anti-integrin _V (for Western blotting; BD Biosciences), anti-FLAG M2 (for immunoprecipitation and Western blotting; Sigma, St Louis, MO), anti-actin (Chemicon, Temecula, CA) and anti-HA mAbs (Babco, Berekely, CA). The following rabbit pAbs were purchased from commercial sources: anti-PDGF receptor β (Upstate Biotechnology, Lake Placid, NY), anti-FLAG (for Western blotting; Sigma) and anti-GFP pAbs (for immunoprecipitation and Western blotting; MBL, Woburn, MA). Hybridoma cells (9E10) expressing a mouse anti-Myc mAb were purchased from the American Type Culture Collection. Fatty acid-free BSA, laminin, ConA, cyclo-RGDfV and cytochalasin D were purchased from Sigma. AG1296 was purchased from Calbiochem (SanDiego, CA). HRP-conjugated secondary Abs were purchased from GE Healthcare (Little Chalfont, UK). Fluorophore (FITC, Cy3 and Cy5)-conjugated secondary Abs were purchased from Jackson ImmunoResearch (West Grove, PA). Human recombinant PDGF-BB was purchased from PeproTech (London, UK). Vitronectin was purified from human plasma (Kohjinbio, Saitama, Japan) as described previously (Yatohgo et al. 1988). The chemical cross-linker 3,3'-dithiobis [sulfosuccinimidylpropionate] (DTSSP), which is membrane-insoluble and has a disulfide bond, was purchased from Pierce (Rockford, IL).

Directional stimulation by PDGF

A concentration gradient of PDGF was generated as described previously (Minami et al. 2007). Briefly, a µ-Slide VI flow (uncoated; Ibidi) was used. The µ-Slide VI flow has six parallel channels, which were coated with 5 µg/mL vitronectin. Cells were seeded at a density of 5 x 10³ cells/cm², cultured for 16 h, and starved of serum by incubating in DMEM containing 0.5% BSA for 1 h. A concentration gradient of PDGF was generated using DMEM containing 0.5% BSA and 30 ng/mL PDGF, according to manufacturer's protocol. For the experiments using cytochalasin D, cells were treated with 1 mM cytochalasin D during serum starvation and PDGF stimulation. After 30 min of PDGF stimulation, cells were fixed with acetone : methanol (1:1) at –20 °C. Alternatively, cells were fixed with 4% paraformaldehyde/PBS and then incubated with 100% MetOH at –20 °C. Cells were incubated with 1% BSA in PBS, and then with 20% BlockAce (Osaka, Japan) in PBS, prior to immunofluorescence microscopy. Samples were analyzed using digital eclipse C1si-ready (Nikon, Tokyo, Japan) confocal laser-scanning microscope systems.

Co-immunoprecipitation assay using HEK293 cells

HEK293 cells were co-transfected with various combinations of plasmids, cultured for 24 h, detached with 0.05% trypsin and 0.53 mM EDTA, and treated with a trypsin inhibitor (Sigma). To make predominantly the high-affinity form of integrin _Vβ₃, cells were cultured in suspension with Ca²⁺-free DMEM (Invitrogen) containing 0.5% BSA, 1 mM MnCl₂, 50 µg/mL cyclo-RGDfV and/or 30 ng/mL PDGF or 50 µM AG1296 for 30 min, collected by centrifugation, washed with Wash buffer (20 mM Tris–HCl at pH 8.0, 150 mM NaCl and 1 mM Na₃VO₄) and lysed with Buffer A (20 mM Tris–HCl at pH 8.0, 150 mM NaCl, 1 mM MnCl₂, 10% glycerol, 1% Nonidet P-40, 10 mM NaF, 1 mM Na₃VO₄, 10 µg/mL leupeptin, 2 µg/mL aprotinin and 10 µM APMSF). To make predominantly the low-affinity form of integrin _Vβ₃, cells were cultured in suspension with DMEM containing 0.5% BSA and/or 30 ng/mL PDGF or 50 µM AG1296 for 30 min, collected by centrifugation, washed with Wash buffer and lysed with Buffer B (20 mM Tris–HCl at pH 8.0, 150 mM NaCl, 1 mM CaCl₂, 1 mM MgCl₂, 10% glycerol, 1% Nonidet P-40, 10 mM NaF, 1 mM Na₃VO₄, 10 µg/mL leupeptin, 2 µg/mL aprotinin and 10 µm APMSF). The lysates were rotated for 30 min and subjected to centrifugation at 12 000 g for 20 min. The supernatant was pre-cleared with protein G-Sepharose 4 Fast Flow beads (GE Healthcare) at 4 °C for 1 h, incubated with the anti-FLAG mAb at 4 °C for 4 h and then incubated with protein G-Sepharose beads at 4 °C for 2 h. After the beads were extensively washed with Buffer A or B, bound proteins were eluted by boiling the beads in SDS Sample Buffer (60 mM Tris–HCl, pH 6.7, 3% SDS, 2% 2-mercaptoethanol and 5% glycerol) for 5 min and subjected to SDS-PAGE followed by Western blotting.

Co-immunoprecipitation assay using NIH3T3 cells

NIH3T3, Necl-5-NIH3T3 and Necl-5-knockdown-NIH3T3 cells were plated at a density of 5 x 10³ cells per square centimeter on vitronectin-coated dishes, cultured for 16 h, starved of serum with DMEM containing 0.5% BSA for 1 h, and then stimulated by DMEM containing 0.5% BSA and/or 3 ng/mL PDGF. After 30 min, cells were washed with ice-cold PBS and incubated with 2 mM DTSSP in PBS at 4 °C for 2 h. To quench the cross-linking reaction, 1 M Tris–HCl (pH 7.5) was added at a final concentration of 20 mM. Cells were washed with PBS and lysed with Buffer B. The lysates were rotated for 30 min and subjected to centrifugation at 12 000 g for 20 min. The supernatant was pre-cleared with protein A-Sepharose (GE Healthcare) at 4 °C for 1 h, incubated with the anti-integrin _V pAb or rabbit serum, as a control, at 4 °C for 16 h, and then incubated with protein A-Sepharose beads at 4 °C for 4 h. After the beads were extensively washed with Buffer B, bound proteins were eluted by boiling the beads in SDS Sample Buffer for 5 min and subjected to SDS-PAGE followed by Western blotting.

Boyden chamber assay

Boyden chamber assay was performed as described previously (Ikeda et al. 2004). For the assay using NIH3T3 and Necl-5-NIH3T3 cells, PET membrane cell culture inserts (8.0-µm pores, BD Falcon) were used. For the assay using Necl-5-knockdown-NIH3T3 cells, NIH3T3 cells were transfected with pBS-EGFP-H1-Necl-5 or pBS-EGFP-H1-control and HTS fluoroBlock cell culture inserts (8.0-µm pores, BD Falcon) were used. Cell culture inserts were coated with 3 µg/mL of vitronectin and then blocked with 1% BSA/PBS. Cells that had been serum starved with DMEM containing 0.5% BSA for 18 h were detached with 0.05% trypsin and 0.53 mM EDTA and then treated with a trypsin inhibitor. Cells were then re-suspended in DMEM containing 0.5% BSA and seeded at a density of 4 x 10⁴ cells per insert. Cells were incubated at 37 °C for 14 h in the presence of 30 ng/mL PDGF-BB. PDGF-BB was added only to the bottom well in order to generate a concentration gradient. After incubation, the inserts were washed with PBS and the cells were fixed using 3.7% formaldehyde/PBS. Migrated cells were observed by Crystal Violet staining (for NIH3T3 and Necl-5-NIH3T3 cells) or EGFP fluorescence (for control-NIH3T3 cells and Necl-5-knockdown-NIH3T3 cells). The number of migrated cells in five randomly chosen fields per insert was counted by microscopic examination.

	Acknowledgements

 
We thank Drs J. Takagi, J. C. Norman, H. Matsui and H. Shibuya for their generous gifts of reagents. This work was supported by grants-in-aid for Scientific Research and for Cancer Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (2005, 2006, 2007).

	Footnotes

 
Communicated by: Eisuke Nishida

^* Correspondence: E-mail: ytakai{at}med.kobe-u.ac.jp

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Received: 10 November 2007
Accepted: 29 November 2007

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