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Neoplasia. Feb 2012; 14(2): 95–107.
PMCID: PMC3306255

Membrane-Bound and Exosomal Metastasis-Associated C4.4A Promotes Migration by Associating with the α6β4 Integrin and MT1-MMP1,2


Metastasis-associated C4.4A, which becomes upregulated during wound healing and, in some tumors, during tumor progression, is known to be frequently associated with hypoxia. With the function of C4.4A still unknown, we explored the impact of hypoxia on C4.4A expression and functional activity. Metastatic rat and human tumor lines upregulate C4.4A expression when cultured in the presence of CoCl2. Although hypoxia-inducible factor 1α (HIF-1α) becomes upregulated concomitantly, HIF-1α did not induce C4.4A transcription. Instead, hypoxia-induced C4.4A up-regulation promoted in vivo and in vitro wound healing, where increased migration on the C4.4A ligands laminin-111 and -332 was observed after a transient period of pronounced binding. Increased migration was accompanied by C4.4A associating with α6β4, MT1-MMP1, and TACE and by laminin fragmentation. Hypoxia also promoted the release of C4.4A in exosomes and TACE-mediated C4.4A shedding. The association of C4.4A with α6β4 and MT1-MMP1 was maintained in exosomes and exosomal α6β4- and MT1-MMP1-associated C4.4A but not shed C4.4A sufficient for laminin degradation. Hypoxia-induced recruitment of α6β4 toward raft-located C4.4A, MT1-MMP, and TACE allows for a shift from adhesion to motility, which is supported by laminin degradation. These findings provide the first explanation for the C4.4A contribution to wound healing and metastasis.


C4.4A is a glycosyl-phosphatidyl-inositol-anchored molecule and belongs, like the urokinase-type plasminogen activator receptor (uPAR), to the Ly6 family [1–3]. C4.4A shares with uPAR three-finger protein domains, characterized by three to six S-S bridges, which guarantee maintenance of domain structure by stabilizing the hydrophobic nucleus of the protein [4,5]. uPAR has three and C4.4A two strongly hydrophobic three-finger protein domain [6]. C4.4A has 5 to 6 N-glycosylation sites close to the second TFP domain and 15 O-glycosylation sites in a Ser/Thr-rich region at the C-terminus [7]. C4.4A associates with laminins (LN) 111 and 332 (formerly LN1 and LN5) and galectin-3 [8].

C4.4A expression is restricted to the basal and suprabasal layers of squamous epithelium in nontransformed tissue [1,2,7,9,10], where it becomes upregulated during wound healing [7,10]. High C4.4A expression has been seen in several types of carcinoma like mammary, renal cell, colorectal [11–13] and most pronounced non-small cell lung cancer [14]. Its expression in other types of cancer, like esophageal cancer becomes regulated during tumor progression [15,16]. C4.4A transcription requires C/EBPβ and is enhanced by JunD or c-Jun [17], which fits to upregulated expression during wound healing [18–22].

We here aimed to shed light on the molecular mechanism of C4.4A activities, which remain elusive, despite C4.4A being consistently associated with tumor progression and wound healing [3]. These two activities of C4.4A are frequently associated with hypoxia [23,24], which initiates a gene transcription program frequently through hypoxia-inducible factor (HIF) [25,26]. Stabilized HIF-1α undergoes nuclear translocation and associates with transcriptional coactivators [27,28]. Because the C4.4A promoter contains three potential HIF-1α response elements (HREs), we first evaluated whether hypoxia-induced HIF-1α may contribute to C4.4A transcription and whether hypoxia influences C4.4A activity in wound healing and tumor cell migration. Under hypoxia, C4.4A forms a complex with α6β4 and MMP14 (formerly MT1-MMP), which promotes motility possibly through focalized LN332 degradation.

Materials and Methods

Tumor Lines

The rat tumor lines were BSp73ASML (ASML, C4.4A+, α6β4+, metastasizing), BSp73AS (AS, C4.4-, α6β4-, nonmetastasizing) [29], and BSp73AS1B1 (AS1B1, C4.4A cDNA-transfected AS clone, C4.4A+, α6β4-). Thecoding sequenceof the C4.4A cDNA has been cloned into the pcDNA3 vector with a CMV promoter to drive C4.4A transcription [1]; Progressor (Prog) (C4.4A+ α6β4+) [30], 804G (LN332 secreting) [31], and the human A431 (LN332 secreting) [32] were maintained in RPMI/10% fetal calf serum (FCS). The human pancreatic cancer lines Capan-2 (metastasizing) [33], Colo357 (metastasizing) [34], 8.18 (weakly metastasizing) (Tumor Bank, German Cancer Research Center, Heidelberg, Germany; personal observations), and BxPC3 (nonmetastasizing) [35] were maintained in RPMI/10% FCS/10 mM Na-pyruvate. Confluent cultures were trypsinized and split. Where indicated, cells were treated with 100 to 200 µM CoCl2 for 6 to 24 hours or maintained at 1% O2 for 6 to 12 hours.

Antibodies, Matrix Proteins, and Inhibitors

Antibodies, matrix proteins, and inhibitors are listed in Table W1.

Vesicle Depletion and Exosome Preparation

Cells were cultured (48 hours) in serum-free medium. Cleared supernatants (2 x 10 minutes at 500g, 1 x 20 minutes at 2000g, 1 x 30 minutes at 10,000g) were centrifuged (90 minutes at 100,000g) and washed (phosphate-buffered saline, 90 minutes at 100,000g). The supernatant was collected as vesicle-depleted fraction. The pellet (crude exosomes) was suspended in 2.5 M sucrose, overlaid by a continuous sucrose gradient (0.25–2 M), and centrifuged (15 hours at 150,000g).

Flow Cytometry

Flow cytometry followed routine procedures. For intracellular staining, cells were fixed and permeabilized. For chloramphenicol acetyltransferase (CAT) assay standardization, cells were cotransfected with the EGFP-C1 plasmid to evaluate transfection efficacy. Cells were analyzed in a FACScan using the Cell Quest analysis program.

Reverse Transcription-Polymerase Chain Reaction

Total RNA preparation followed standard procedures. Primers are listed in Table W2.

C4.4A Promoter Constructs, Mutations, Transfection, and CAT Assay

A 588-bp and a 1.9-kbp C4.4A promoter constructs have been used [17]. Three CGTG HRE sequences of C4.4A at -673 bp (HRE1), -1183 bp (HRE2), and -1557 bp (HRE3) are shown in Table W3. C4.4A sequences were inserted in promoterless pBLCAT3-Basis (pBLCATB3), which contains the CAT gene [36]. Tk promoter-containing pBLCAT2 served as control [37]. Transfection (3 µg of pBLCAT3-C4.4A, 1 µg of EGFP-C1 or 1.75 µg of pBLCAT3-C4.4A, 1.25 µg of p(HA) HIF-1α 401Δ603 [HIF-1α with oxygen-dependent degradation domain deletion], 1 µg of EGFP-C1) was done as described [17]. For the CAT assay [38], lysates were standardized for protein content and transfection efficacy. Thin-layer chromatography was quantitated using BAS-1800II PhosphorImager (Fuji, Dusseldorf, Germany).

Immunoprecipitation, SDS-PAGE, and Western blot

Cell lysates (60 minutes, 4°C, HEPES buffer, 1% Brij96, protease inhibitor cocktail) were centrifuged (13,000g for 10 minutes at 4°C), incubated with antibody (overnight), and precipitated with ProteinG Sepharose (1 hour at 4°C). Washed immune complexes were dissolved in Laemmli buffer. Precipitates/lysates were resolved on 10% SDS-PAGE. Proteins were transferred to nitrocellulose membranes (30 V for 12 hours at 4°C); membranes were blocked, blotted with primary and HRP-conjugated secondary antibodies (1 hour at room temperature), and developed with the ECL kit or were stained with Coomassie blue.

Immunofluorescence and Immunohistochemistry

Cells seeded on bovine serum albumin (BSA)-, LN111-, LN332-, or fibronectin (FN)-coated cover slides were fixed; permeabilized; blocked; incubated with primary antibody (60 minutes at 4°C); fluorochrome-conjugated secondary antibody (60 minutes at 4°C); blocked, incubated with a second, dye-labeled primary antibody (60 minutes at 4°C); and washed. Where indicated, cells were removed by EDTA. Cover slides were mounted in Elvanol (Sigma Aldrich, Steinheim, Germany). Shock-frozen skin sections (7 µm) were exposed to primary antibody, biotinylated secondary antibody, and alkaline phosphatase-conjugated avidin-biotin complex solutions. Sections were counter stained with hematoxylin and eosin. Digitized images were generated using a Leica DMRBE microscope (Leica, Wetzlar, Germany), a SPOT CCD camera, and Software SPOT2.1.2 (Sterling Heights, MI).

Adhesion and Migration Assays

Adhesion to coated 96-well plates was determined after 30 and 240 minutes (37°C). Nonadherent cells were removed by washing. Migration was evaluated in Boyden chambers seeding cells in the upper chamber (RPMI/1% BSA) with/without CoCl2 and/or protease inhibitors. The lower chamber, separated by an 8-µm pore size polycarbonate membrane, contained RPMI/1% BSA or LN332 (804G supernatant). In both assays, cells were stained with crystal violet, measuring OD595nm after lysis. Adhesion/migration is presented as percentage input cells. For in vitro wound healing, a subconfluent monolayer was scratched. Wound closure (light microscopy) is presented as percentage reduction of the freshly wounded area.

Rats and Treatment

A 1-cm-diameter full-thickness skin area was excised from the shaved back of 8-week-old BDX rats. At the time of excision, after 4 and 7 days, rats received 100 µg of control IgG or C4.4 in 100 µl of phosphate-buffered saline, perilesionally. Sterile gauze covering the wound was fixed with a whole-body bandage. A 1.5-cm-diameter area, including the wound, excised immediately, after 1, 3, 7, and 10 days, was shock frozen. Animal experiments were government approved (Baden-Wuerttemberg).


Values represent the mean ± SD of triplicates and/or three repetitions. P < .05 (Student's t test) was considered statistically significant.


Hypoxia-Induced C4.4A Up-regulation

The impact of hypoxia on C4.4A expression was evaluated in two metastatic C4.4A+ rat tumor lines Prog and ASML, in AS1B1 cells (C4.4A cDNA-transfected AS cells) [1,29], and in human pancreatic tumor lines.

Cells were cultured in the presence of CoCl2, which mimics hypoxic conditions [39]. High C4.4A expression in ASML and Prog cells was increased after 6 hours of 200 µM CoCl2 treatment and increased further during a 12-hour culture period. Culturing in 1% O2 exerted similar effects (data not shown). AS1B1 cells showed a minor insignificant increase only after 12 hours of CoCl2 treatment. AS cells remained C4.4A- (Figure 1, A and B). Two human pancreatic cancer cell lines, Colo357 and Capan-2, which express C4.4A at a medium to low level, responded to CoCl2 treatment with a dose- and time-dependent increase in C4.4A expression, where a subfraction showed a very high expression after 200 µM CoCl2 treatment. 8.18 cells, which do not express C4.4A, revealed very weak expression in approximately 15% of cells after 12 hours of 200 µM CoCl2 treatment. BxPC3 cells remained C4.4A- (Figure 1, C and and1D).1D). Thus, hypoxia can contribute to C4.4A protein up-regulation.

Figure 1
Hypoxia-induced C4.4A up-regulation: (A, B) ASML, Prog, AS, and AS1B1 cells and (C, D) the human pancreatic cancer lines Capan-2, Colo357, BxPC3, and 8.18 were cultured in the presence of titrated amounts of CoCl2 for 6 to 24 hours. (A, C) Expression ...

HIF-1α Does Not Promote C4.4A Transcription

Under hypoxia, stabilized HIF-1α acts as a transcription factor. Under normoxia, AS, AS1B1, and ASML cells express HIF-1α at a low level. In ASML cells, HIF-1α showed a strong, dose-dependent increase after 12 hours of CoCl2 treatment. Up-regulation was weak after 6 hours and declined beyond 12 hours. An increase in HIF-1α was also seen in CoCl2-treated Capan-2 and BxPC3 cells (Figure 2A). Notably, the latter do not express C4.4A after CoCl2 treatment. This finding suggests that HIF-1α, if at all, may only act as a cotranscription factor for C4.4A.

Figure 2
HIF-1α does not promote C4.4A transcription: (A) ASML, Capan-2, and BxPC3 cells were cultured in the presence of 200 µM CoCl2 for 6 to 24 hours. HIF-1α expression was evaluated by WB; actin served as control. (B, C) The -1- to ...

The C4.4A promoter containing three HREs at -673, -1183, and -1557 bp (Table W3), we controlled for HIF-1α cotranscription factor activity in hypoxia. However, when the C4.4A promoter from -588 (no HRE) or from -1957 to -1 bp were inserted into promoterless pBLCAT3, CAT activity of transfected ASML cells, cultured under hypoxia, was not significantly changed (Figure 2B). Furthermore, only a slight increase in CAT activity was observed on cotransfection with pCDNA3-HIF-1α, irrespective of whether the promoter construct contained (1957 bp) or did not contain (588 bp) the three HRE (Figure 2C).

Thus, hypoxia-induced C4.4A protein up-regulation does, at least, not predominantly proceed at the transcription level.

Hypoxia-Induced C4.4A Up-regulation, LN332 Adhesion, Keratinocyte Migration, and Tumor Cell Motility

Keratinocytes express C4.4A and C4.4A is engaged in wound repair [3,7,8]. Controlling for C4.4A expression and the impact of anti-C4.4A (C4.4) on wound repair after full-thickness skin excision revealed a strong C4.4A expression at the leading front of migrating keratinocytes, and wound closure was strongly delayed in C4.4-treated rats (Figure 3).

Figure 3
Keratinocyte migration during wound healing and C4.4A expression: The back skin of BDX rats was wounded by a 1-cm-diameter full-thickness skin excision. Rats received at days 0, 4, and 7 after wounding a perilesional injection of 100 µg of control ...

With wound repair frequently being associated with hypoxia, we next asked whether hypoxia-induced C4.4A up-regulation affects adhesion or migration on LN332, which, in humans and rats, is a major C4.4A ligand [1,8,10].

ASML, Prog, AS, and AS1B1 cells adhere better to LN332 than BSA. In addition, after 30 minutes, significantly more CoCl2-treated than untreated ASML, Prog, and AS1B1, but not AS cells, adhere to LN332 (Figure 4A). We also evaluated binding to collagen I, III, and IV as well as to FN and vitronectin. Binding did not differ between AS and AS1B1 cells (data not shown), which is in line with the results of pull-down assays that revealed LN332 and (weakly) LN111, but not other matrix, proteins to bind to recombinant C4.4A [8]. Because the increase in binding was more pronounced in ASML and Prog, which both, and distinct to AS, express α6β4, we next asked for the contribution of C4.4A versus α6β4 [40] to LN332 adhesion. C4.4 andB5.5(anti-α6β4) inhibited LN332 binding of untreated and CoCl2-treated ASML and Prog cells with equal efficacy, and no additive inhibitory effect was seen in the presence of both antibodies (data not shown). Binding of CoCl2-treated ASML and Prog cells, but not of AS and AS1B1 cells to BSA, was also slightly inhibited by C4.4 and B5.5, which may be a consequence of both these lines, distinct to AS cells, secreting LN332 [44] (unpublished observations). Binding of AS1B1 cells was only inhibited by C4.4 (Figure 4B).

Figure 4
The impact of hypoxia and upregulated C4.4A expression on LN332 adhesion. (A, B) Untreated and CoCl2-treated ASML, Prog, AS, and AS1B1 cells were seeded on BSA- or 804G supernatant (LN332)-coated plates and cultured for 30 minutes with or without CoCl ...

Because inhibition by C4.4 and B5.5 was stronger in CoCl2-treated cells, we speculated that stress could support joint C4.4A-α6β4 activities. C4.4A and α6β4 did not colocalize in ASML and hardly in Prog cells, when seeded on BSA-coated plates. Weak colocalization was seen in the presence of CoCl2 or on LN332-coated plates. Instead, C4.4A and α6β4 strongly colocalized in CoCl2-treated ASML and Prog cells seeded on LN332 (Figure 4C) and hypoxia strengthened C4.4A-α6β4 coimmunoprecipitation (Figure 4D).

C4.4A-mediated LN332 binding is transient and could be restored in the presence of a protease inhibitor [1]. This also accounts for CoCl2-treated ASML and Prog cells. After 4 hours, adhesion to LN332 did not consistently exceed adhesion to BSA and the adhesion supporting effect of CoCl2 treatment was weak or abolished. Adhesion of CoCl2-treated ASML and Prog cells was partly restored in the presence of broad serine protease (aprotinin) and MMP (MMP-Inh.II) inhibitors. Instead, in C4.4A-, α6β4- AS cells' adhesion to LN332 was stronger after 4 hours than after 30 minutes. Adhesion was not influenced by CoCl2 and remained unaltered in the presence of MMP-Inh.II. In AS1B1 cells, slightly reduced adhesion to LN332 was at a borderline significant level restored by MMP-Inh.II only in CoCl2-treated cells (Figure 5A). This indicated an essential contribution of C4.4A to reduced LN332 adhesion, which becomes strengthened by α6β4.

Figure 5
The impact of protease inhibitors on adhesion to and migration on LN332: (A) Untreated and CoCl2-treated ASML and Prog cells were seeded on BSA- or 804G supernatant (LN332)-coated plates. Where indicated, cultures contained CoCl2 and/or aprotinin or MMP-Inh.II. ...

As LN332 adhesion decreased with time, hypoxia enhanced migration of ASML and Prog cells on LN332 as revealed by in vitro wound healing. Both C4.4 and B5.5 inhibited wound closure of ASML and Prog cells on LN332-coated plates in the presence or absence of CoCl2 (Figure 5B). In line with the MMP-Inh.II-restored adhesiveness to LN332, MMP-Inh.II interfered with migration. MMP-Inh.II exerted no or a minor effect on wound healing of Prog and ASML cells on BSA but significantly inhibited wound healing on LN332 in the presence of CoCl2 (Figure 5C).

Hypoxia-induced increased migratory activity also accounted for transwell migration of ASML and Prog cells, which was promoted by CoCl2 treatment and inhibited by C4.4 and B5.5 (Figure 5D). Transwell migration toward LN332 was inhibited by MMP-Inh.II, although the effect was weak for Prog cells (Figure 5E).

Taken together, hypoxia supports an association of the two LN332 receptors C4.4A and α6β4. Both molecules promote short-term LN332 adhesion but lasting migration. An MMP inhibitor restores adhesiveness and interferes with migration. Thus, hypoxia might be accompanied by increased protease activity accounting for LN332 degradation, where LN332 degradation products can exert chemotactic activity [41–43]. Alternatively, hypoxia might contribute to C4.4A shedding or release.

C4.4A Cooperation with Proteases

ASML [44] and Prog cells secrete LN332. C4.4A poorly colocalized with LN332 on BSA-coated plates under normoxic conditions. Colocalization was promoted when CoCl2-treated cells were seeded on LN332-coated plates (Figure 6A). Because the experimental setting did not allow to judge on LN332 degradation, ASML and Prog cells were seeded in the presence or absence of CoCl2 on uncoated plates, removing cells after 2 days of culture by EDTA and staining for LN111, LN332, and FN. In CoCl2-treated cultures, the rim staining for LN332 was stronger and more focalized, but the area below the cell body was free of LN332. Instead, FN remained deposited below the cell body independent of CoCl2 treatment. LN111 deposition was reduced but not abolished. Staining of cell-free areas confirmed poor LN332, high FN, and intermediate LN111 recovery (Figure 6B).

Figure 6
Cooperation of C4.4A and MMP14 in LN332 degradation. (A) ASML and Prog cells cultured on 804G supernatant (LN332)-coated cover slides with or without CoCl2 were fixed and stained with anti-LNγ2/anti-mouse-Cy2 and C4.4-TxR. Single fluorescence ...

These findings argued for LN332 and (partial) LN111 fragmentation. To control the hypothesis, LN111 and LN332 (804G supernatant) were cocultured for 24 hours with ASML cells in the presence of CoCl2 with/without MMP-Inh.II. Cells were removed, and the LNs were separated by SDS-PAGE. Gels were stained with Coomassie blue or proteins were transferred and blotted with anti-LN111 or anti-LNγ2. Coomassie blue staining provided evidence for LN111 and LN332 fragmentation, which was reduced when cultures contained MMP-Inh.II. Western blot (WB) confirmed LNγ2 degradation, and a weaker LN111 band was recovered when LN111 was cocultured with CoCl2-treated ASML cells but not when cultures contained MMP-Inh.II (Figure 6C).

Thus, C4.4A might promote motility through associated proteases involved in LN degradation. According to the inhibitory activity of MMP-Inh.II, MMP14 was considered a possible candidate. Weak colocalization of MMP14 with C4.4A and α6β4 in resting cells became strong when CoCl2-treated cells were grown on LN332-coated slides (Figure 7A), which corresponded to an increase in coimmunoprecipitation of C4.4A and α6β4 with MMP14 in CoCl2-treated ASML cells. C4.4A and more pronounced α6β4 also coimmunoprecipitated TACE, but not uPA, uPAR, MMP2, and MMP9. Although the C4.4A and α6β4 association with TACE was CoCl2 treatment independent, TACE clustering was more pronounced in CoCl2-treated ASML cells (Figure 7, B–D).

Figure 7
The C4.4A association with proteases, C4.4A shedding, and exosome inclusion in hypoxia. (A) ASML and Prog, grown on BSA- or 804G supernatant (LN332)-coated plates in the presence or absence of CoCl2, were stained with anti-MMP14/anti-rabbit-Cy2 and C4.4-TxR ...

The association of C4.4A with TACE, possibly through α6β4, and the fact that C4.4A has a highly sensitive cleavage site [45] and is delivered in exosomes [8] prompted us to evaluate whether CoCl2 treatment supports C4.4A cleavage and/or exosomal release. C4.4A is recovered in exosomes, a higher amount being recovered from CoCl2-treated cells. Shed C4.4A is only recovered in the supernatant of CoCl2-treated ASML cells. C4.4A shedding is largely abolished in the presence of the TACE inhibitor (TAPI). However, TAPI did not influence the delivery in exosomes. Exosomal C4.4A and α6β4 also coimmunoprecipitate with MMP14 and TACE (Figure 7E).

These findings raised the question whether exosomal or shed C4.4A would contribute to LN degradation. When exosomes and supernatant from CoCl2-treated ASML cells were coincubated with LN111 and LN332, exosomes, but not shed C4.4A, partly degraded LN111 and LN332. LN degradation by exosomes was also inhibited by MMP-Inh.II (Figure 7F). The experiment was repeated with hLN332, which confirmed partial degradation by Colo357-derived exosomes and inhibition of degradation by MMP-Inh.II. Blot analysis with anti-LNα3, -β3, and -γ2 provided evidence for partial degradation of all three LN332 chains (Figure 7G).

Taken together, hypoxia strengthens C4.4A expression and promotes migration on LN111 and LN332. Migration is accompanied by α6β4 and MMP14 recruitment toward C4.4A and focalized LN5 fragmentation. Owing to the high recovery of C4.4A- and α6β4-associated MMP14 in exosomes, the process of focalized LN332 degradation can also proceed in the surrounding tissue.


The function of metastasis-associated C4.4A, with very restricted expression in normal tissue [1,2,7,9,10], remains elusive [3]. We here report that hypoxia-induced C4.4A up-regulation promotes transition from a sessile toward a mobile phenotype through associating with α6β4 and proteases, which supports focalized matrix degradation. The process is strengthened by the release of C4.4A, α6β4, and MMP14 into exosomes.

Regulation of C4.4A Expression in Hypoxia

Low oxygen and, as a surrogate, CoCl2 treatment [39] are accompanied by HIF-1α transport to the nucleus, where it acts as a transcription factor for multiple genes [26,28]. Because hypoxia is accompanied by upregulated C4.4A expression and the C4.4A promoter contains three HREs, we considered the possibility that HIF-1α may contribute to C4.4A transcription. However, cotransfection with HIF-1α did not show an HIF-1α contribution to C4.4A transcription. C/EBPβ, essentially required for C4.4A transcription [17], has also been associated with metastasis-associated gene transcription [46], gene expression in differentiating keratinocytes, and wound repair [47,48]. However, although a high CAT activity was observed after cotransfection with C/EBPβ, we did not observe a further increase by CoCl2 treatment (data not shown). Thus, high C4.4A expression under hypoxia may be a consequence of protein stabilization. The finding that CoCl2 treatment did not induce expression in C4.4A-negative tumor lines is in line with this interpretation.

Cooperation of C4.4A with α6β4 and MMP14

C4.4A is strongly expressed in migrating keratinocytes during wound repair where a direct contribution to keratinocyte migration has been demonstrated by C4.4, significantly inhibiting wound healing. In vitro, CoCl2 treatment promoted migration of C4.4A+ tumor cells on LN332, a major C4.4A ligand [8], after a transient increase in LN332 adhesion.

The engagement of C4.4A in the shift toward motility on LN332 could rely on hypoxia-induced associations of C4.4A with α6β4 and MMP14. On LN332, but not on BSA, C4.4A colocalizes and associates with α6β4, with focalized colocalization and association being stronger under hypoxia. ASML, Prog, and AS/AS1B1 also express α3β1 and α6β1. However, neither of these integrins coimmunoprecipitated C4.4A in resting cells or in stimulated cells. Thus, it is the association between C4.4A and α6β4 that has bearing on adhesion/migration of ASML and Prog cells. LN332 is a major ligand for α6β4 [40,49], and one could have speculated that C4.4A only acts as an accessory molecule. This has been excluded because recombinant C4.4A binds LN332 [8] and AS1B1 cells, which do not express α6β4 [1], also showing pronounced LN332 binding. However, the effect of C4.4A by itself as demonstrated in AS1B1 cells on LN332 binding and motility (data not shown) is much weaker than in cells, where C4.4A associates with α6β4 in hypoxia (ASML, Prog). In line with this finding, adhesion and migration on LN332 became equally well inhibited by C4.4 and anti-α6β4.

Pronounced adhesion of ASML and Prog cells to LN332 is transient. It is known that α6β4 contributes to tumor cell motility [49–54] that can be initiated through binding of α6β4 to LN332. Furthermore, α6β4 becomes recruited toward rafts in stimulatory conditions [55,56]. This is accompanied by phosphorylation of the β4 cytoplasmic domain [57–59], recruitment of Shc, and activation of downstream signaling molecules [60] like rac with a central role in directional cell migration [61,62]. Taking this into account, it becomes likely that the joint activity of C4.4A with α6β4 accounts for the shift from adhesion to motility. Whether C4.4A actively recruits α6β4 or whether α6β4 and C4.4A come into vicinity through their joint ligand(s) remains to be explored.

Finally, proteolytic processing of LN332 also can support cell migration [63]. The rat γ2 chain can be cleaved by MMP2 and MMP14 [42]. One of the γ2 fragments interacts with and activates the epidermal growth factor receptor [64]. Different LN332 fragments from the γ2 and the α3 chain also account for the deposition into the matrix underlying cultured cells [65,66]. MMP14 and hepsin can also cleave the β3 chain [43,67], where hepsin cleavage promotes motility on LN332 [67]; the α3 chain can be cleaved by several proteases including plasmin [68]. Three observations are in line with hypoxia-induced C4.4A up-regulation and the association with α6β4 accounting for the pronounced migration due to LN332 fragmentation: 1) broad serine or MMP inhibitors promoted LN332 adhesion, 2) LN332 deposits were not recovered in CoCl2-treated cultures, and 3) an MMP inhibitor-reduced migration on LN332. Because aprotinin partially restored adhesion to LN332, but MMP-Inh.II exerted stronger effects on cell migration, we tested several proteases for associating with C4.4A and α6β4. Both LN332 ligands do not associate with uPAR, uPA, MMP2, or MMP9 but associate with TACE and more strongly in hypoxia with MMP14. In addition, rat and human LN332 becomes degraded by CoCl2-treated C4.4A+ and α6β4+ tumor cells, and a broad MMP inhibitor interferes with degradation. Whether additional proteases like hepsin may also associate with C4.4A and contribute to LN332 degradation [67] remains to be explored.

Thus, the engagement of C4.4A in tissue remodeling, wound repair, and tumor progression may well rely on its hypoxia-induced association with α6β4 and MMP14.

Hypoxia and C4.4A Release

C4.4A shedding and exosomal release [11] are both stimulated by hypoxia. The increase in soluble C4.4A could be linked to TACE that cleaves C4.4A [69]. Using SILAC (stable isotope labeling by amino acids in cell culture), the authors demonstrate in a human mammary carcinoma line that C4.4A is a new substrate for both ADAM10 and ADAM17/TACE and speculate that cleavage of C4.4A by ADAM10 and TACE may contribute to tumor progression [69]. Our findings of coimmunoprecipitation of C4.4A and α6β4 with TACE, the increased recovery of C4.4A in vesicle-free culture supernatant, and the inhibition of C4.4A release by TAPI are fully in line with the described findings and support the authors' interpretation that the C4.4A-TACE association may contribute to metastasizing tumor cell motility. The increase in exosomal C4.4A corresponds to a pronounced exosome release under stress [70]. In line with the internalization of membrane microdomains, such that internalized protein complexes remain intact [71], exosomes collected from CoCl2-treated tumor cells contained C4.4A, α6β4, and MMP14, where, for the latter, clathrin- and caveolin-dependent internalization has been described [72]. Importantly, the exosomal α6β4- and MMP14-associated C4.4A, but not soluble C4.4A, supports LN332 degradation. This confirms that not C4.4A by itself but a complex of C4.4A and α6β4 with proteases is required for LN fragmentation. Exosomal proteins are functionally active [73], which also accounts for MMP14 [74]. Accordingly, the activity of the C4.4A- and α6β4-associated MMP14 is not restricted to the cell membrane but can additionally prepare the surrounding of migrating epithelial and tumor cells, thus extending the operational range of C4.4A.


As summarized in Figure 8, hypoxia-induced C4.4A up-regulation is accompanied, after a transient increase in LN adhesion, by pronounced migration, where migration on LN depends on the association of C4.4A with α6β4 under hypoxia. C4.4A- and α6β4-associated MMP14 could contribute to the shift toward migration by facilitating focalized LN degradation. Importantly, hypoxia also strengthens the release of exosomes, which express C4.4A- and α6β4-associated MMP14. Exosomes thus expand the range of functional activity of C4.4A in wound healing, tissue remodeling, and tumor cell spread.

Figure 8
Schematic presentation of C4.4A, α6β4, and protease cooperation in LN migration. (A) C4.4A, MMP14, and TACE are raft located molecules. In resting cells, α6β4 is located outside rafts. Both C4.4A and α6β ...

Supplementary Material

Supplementary Figures and Tables:


The authors thank M. Li-Weber (Department of Tumor Immunology, German Cancer Research Center, Heidelberg, Germany) for the HIF-1α 401Δ603 plasmid and C. Niesik and F. Stefani for help with animal experiments and immunohistology.


α6β4 integrin
serine protease inhibitor
AS cells transfected with C4.4A cDNA
HIF response element
formerly LN1
formerly LN5
membrane type 1 matrix metalloproteinase/MT1-MMP
TACE inhibitor
Western blot


1This work was supported by the Deutsche Krebshilfe (10-1821-Zö3 to M.Z.). H. Ngora is a PhD grant recipient of the Deutscher Akademischer Austausch Dienst (German Academic Exchange Service). The authors declare no conflict of interest.

2This article refers to supplementary materials, which are designated by Tables W1 to W3 and are available online at www.neoplasia.com.


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