Processing of laminin-5 and its functional consequences: role of plasmin and tissue-type plasminogen activator

Abstract

The laminin-5 component of the extracellular matrices of certain cultured cells such as the rat epithelial cell line 804G and the human breast epithelial cell MCF-10A is capable of nucleating assembly of cell- matrix adhesive devices called hemidesmosomes when other cells are plated upon them. These matrices also impede cell motility. In contrast, cells plated onto the laminin-5-rich matrices of pp126 epithelial cells fail to assemble hemidesmosomes and are motile. To understand these contradictory phenomena, we have compared the forms of heterotrimeric laminin-5 secreted by 804G and MCF-10A cells with those secreted by pp126 cells, using a panel of laminin-5 subunit-specific antibodies. The alpha3 subunit of laminin-5 secreted by pp126 cells migrates at 190 kD, whereas that secreted by 804G and MCF-10A cells migrates at 160 kD. The pp126 cell 190-kD alpha3 chain of laminin-5 can be specifically proteolyzed by plasmin to a 160-kD species at enzyme concentrations that do not apparently effect the laminin-5 beta and gamma chains. After plasmin treatment, pp126 cell laminin-5 not only impedes cell motility but also becomes competent to nucleate assembly of hemidesmosomes. The possibility that plasmin may play an important role in processing laminin-5 subunits is supported by immunofluorescence analyses that demonstrate colocalization of laminin-5 and plasminogen in the extracellular matrix of MCF-10A and pp126 cells. Whereas tissue-type plasminogen activator (tPA), which converts plasminogen to plasmin, codistributes with laminin-5 in MCF-10A matrix, tPA is not present in pp126 extracellular matrix. Treatment of pp126 laminin-5-rich extracellular matrix with exogenous tPA results in proteolysis of the laminin-5 alpha3 chain from 190 to 160 kD. In addition, plasminogen and tPA bind laminin-5 in vitro. In summary, we provide evidence that laminin-5 is a multifunctional protein that can act under certain circumstances as a motility and at other times as an adhesive factor. In cells in culture, this functional conversion appears dependent upon and is regulated by tPA and plasminogen.

Figure 1

The extracellular matrices of MCF-10A, 804G, and pp126 cells (lanes 1–3) were prepared according to Gospodarowicz (1984). Approximately 15 μg of each sample was run on the lane of a 6% SDS-PAGE gel that was subsequently stained with Coomassie brilliant blue. The α3, β3, and γ2 chains of laminin-5 are indicated (see Fig. 2 for the corresponding immunoblots). Note that in MCF-10A and 804G cell matrices the 160-kD form of the α3 chain and 155-kD form of the γ2 chain migrate at about the same molecular weight. In addition, the 105-kD form of the γ2 chain is stained poorly by Coomassie brilliant blue. Molecular weight standards are indicated to the left.

Figure 2

The laminin-5 subunit compositions of the extracellular matrices (ECM) of MCF-10A, 804G, pp126, SCC12, and NHEK cells are shown (lanes 1–6). Approximately 10 μg of matrix protein was run on each lane of a 6% gel, transferred to nitrocellulose, and then processed for immunoblotting using laminin-5 subunit– specific antibody preparations. (a) The laminin-5 β3 chain was identified using the monoclonal antibody clone 17. Clone 17 antibody recognizes a protein of 145 kD in all of the human matrices but shows no reactivity with 804G cell matrix (compare lane 2 with lanes 1, 3, 5, and 6). (b) Antiserum J20, against the laminin-5 γ2 chain, recognizes 155-kD polypeptides in matrix preparations derived from MCF-10A, 804G, pp126, and NHEK cells (lanes 1, 2, 3, and 6), as well as a 105-kD species in the matrices of MCF-10A, 804G, SCC12, and NHEK cells (lanes 1, 2, 5, and 6). (c) The laminin-5 α3 chain is identified using the mouse monoclonal antibody 10B5, which recognizes a polypeptide migrating at 160 kD in 804G and MCF-10A cell matrix (lanes 1 and 2), and a protein of 190 kD in the matrices of pp126, NHEK, and SCC12 cells (lanes 3, 5, and 6).Approximately 50 μg of the matrix of pp126 cells was treated for 90 min with a 1-ml PBS solution containing plasmin (+Pm) at a concentration of 1 μg/ml and then the treated matrix preparation was probed with the β3, γ2, and α3 subunit-specific antibodies (lane 4 in each blot). The mobility of the β3 and γ2 subunits are unaffected by such treatment (compare b, lanes 3 and 4), whereas the α3 subunit migrates at 160 kD, compared with 190 kD in the untreated matrix (compare c, lanes 3 and 4). Molecular weight standards are indicated to the left.

Figure 2

The laminin-5 subunit compositions of the extracellular matrices (ECM) of MCF-10A, 804G, pp126, SCC12, and NHEK cells are shown (lanes 1–6). Approximately 10 μg of matrix protein was run on each lane of a 6% gel, transferred to nitrocellulose, and then processed for immunoblotting using laminin-5 subunit– specific antibody preparations. (a) The laminin-5 β3 chain was identified using the monoclonal antibody clone 17. Clone 17 antibody recognizes a protein of 145 kD in all of the human matrices but shows no reactivity with 804G cell matrix (compare lane 2 with lanes 1, 3, 5, and 6). (b) Antiserum J20, against the laminin-5 γ2 chain, recognizes 155-kD polypeptides in matrix preparations derived from MCF-10A, 804G, pp126, and NHEK cells (lanes 1, 2, 3, and 6), as well as a 105-kD species in the matrices of MCF-10A, 804G, SCC12, and NHEK cells (lanes 1, 2, 5, and 6). (c) The laminin-5 α3 chain is identified using the mouse monoclonal antibody 10B5, which recognizes a polypeptide migrating at 160 kD in 804G and MCF-10A cell matrix (lanes 1 and 2), and a protein of 190 kD in the matrices of pp126, NHEK, and SCC12 cells (lanes 3, 5, and 6).Approximately 50 μg of the matrix of pp126 cells was treated for 90 min with a 1-ml PBS solution containing plasmin (+Pm) at a concentration of 1 μg/ml and then the treated matrix preparation was probed with the β3, γ2, and α3 subunit-specific antibodies (lane 4 in each blot). The mobility of the β3 and γ2 subunits are unaffected by such treatment (compare b, lanes 3 and 4), whereas the α3 subunit migrates at 160 kD, compared with 190 kD in the untreated matrix (compare c, lanes 3 and 4). Molecular weight standards are indicated to the left.

Figure 3

Approximately 10 μg of either MCF-10A (lanes 1 and 3) or pp126 cell (lanes 2 and 4) ECM was run on lanes of a 6% gel, transferred to nitrocellulose, and then processed for immunoblotting using either a mouse serum (Cta3) raised against residues 1561–1713 at the COOH terminus of the α3 subunit of laminin-5 (lanes 1 and 2) or the α3 subunit monoclonal antibody 10B5 (lanes 3 and 4). Antiserum Cta3 recognizes a 190-kD protein in pp126 ECM (lane 2), but does not show reactivity with any polypeptide in MCF-10A ECM (lane 1). The 190-kD species in pp126 ECM is also recognized by 10B5 antibodies (lane 4). However, unlike the Cta3 serum, 10B5 antibodies recognize a 160-kD protein in MCF-10A ECM (lane 3). Molecular weight markers are indicated at the left of the immunoblots.

Figure 4

SCC12 cells were plated onto pp126 ECM, pp126 ECM that had been treated with plasmin (Pm) (1 μg/ml for 90 min), MCF-10A ECM, affinity-purified pp126 laminin-5, affinity-purified pp126 laminin-5 treated with plasmin (Pm) (1 μg/ml for 90 min), or pp126 ECM treated with 10 μg/ml tPA overnight. After 1 h the motility of the SCC12 cells was assayed by video microscopy and then quantitated as the average of the total displacements of each cell over a 2-h period. For each substrate, we performed at least three trials in which a total of at least ninety cells were evaluated. Note that SCC12 cells show similar motility on pp126 ECM and affinity-purified pp126 laminin-5. They show less motility on plasmin-treated pp126 ECM, plasmin-modified purified pp126 laminin-5, MCF-10A ECM, and pp126 ECM treated with tPA. Error bars indicate standard deviations.

Figure 5

Approximately 10 μg of MCF-10A and pp126 ECM and 5 μg of laminin-1 were processed for SDS-PAGE and then separated polypeptides were transferred to nitrocellulose. The nitrocellulose sheet was then subjected to immunoblotting using a monoclonal antibody against the β1 subunit of laminin. This antibody recognizes a 200-kD protein in laminin-1 but no obvious polypeptides in MCF-10A or pp126 ECM.

Figure 6

Radiolabeled laminin-5 was captured by the laminin-5 γ2 chain antibody GB3 from the conditioned medium of pp126 cells. One sample was left untreated, whereas the other was digested with 1 μg/ml plasmin for 90 min. Approximately 2 μg of both preparations were then processed for SDS-PAGE and then the gel dried and was exposed to film. Note that the untreated material consists of three prominent polypeptides of 190-, 145- and 105-kD molecular weight (the α, β and γ chains of laminin-5, respectively), whereas in the plasmin-modified material there are major polypeptides of 160 (processed α3 subunit, indicated as α3*), 145, and 100 kD. Note that there is a minor amount of the 190-kD unprocessed α subunit in the plasmin-modified preparation. Molecular weights are indicated to the left of the gel profiles.

Figure 7

SCC12 cells were maintained for 24 h on plasmin-modified (1 μg/ml for 90 min) pp126 matrix (a), plasmin-modified purified pp126 laminin-5 (b), and plasmin-modified pp126- derived laminin-5 that had been incubated for 30 min at 37°C with 50 μg/ml of the laminin-5 function-inhibiting antibody 1947 before addition of the cells (c). The SCC12 cells were then processed for electron microscopy and cross-sections of the cells were prepared. In a and b, several hemidesmosomes in the SCC12 cells are observed at sites of cell– matrix association (arrows). Each has a triangular, trilayered plaque that is associated with intermediate filaments (curved arrows in a and b). c, the SCC12 cell has assembled no obvious hemidesmosomes along regions of cell–matrix interaction. The arrow indicates one hemidesmosome-like structure that is not associated with the substrate. This structure may even be a half-desmosome. a and c are at the same magnification. Together, these results provide evidence that plasmin-modified pp126 cell laminin-5 is capable of inducing the assembly of hemidesmosomes. Bars: (a and c) 500 nm; (b) 200 nm.

Figure 8

MCF-10A cells (a–c and g–i) and pp126 cells (d–f and j–l) were processed for indirect double-label immunofluorescence microscopy using antibodies against laminin-5 (a, d, g, and j) in combination with either an antibody against plasminogen (b and e) or tPA (h and k). Note that there is colocalization of plasminogen and laminin-5 staining patterns in a and b as well as d and e. In g and h, tPA shows codistribution with laminin-5. In k, tPA localizes to cell bodies but does not colocalize with laminin-5 in j. c, f, i, and l are phase images. Bar, 25 μm.

Figure 9

Approximately 10 μg of untreated MCF-10A cell matrix (lane 1), untreated pp126 cell matrix (lane 2), or pp126 cell matrix treated for 16 h with tPA (∼50 μg of matrix incubated for 16 h with 1 ml of tPA at a concentration of either 5 or 10 μg/ml) (lanes 3 and 4) were subjected to SDS-PAGE on 6% gels, transferred to nitrocellulose, and then processed for immunoblotting using the laminin-5 α3 subunit antibody 10B5. In untreated pp126 cell matrix, the α3 subunit shows a molecular weight of 190 kD (lane 2) (refer to Fig. 2). The α3 chain from tPA-treated pp126 matrices comigrates with the α3 chain of MCF-10A matrix at 160 kD (lanes 1, 3, and 4). Note the small amount of residual 190-kD polypeptide in the 5 μg/ml tPA-treated pp126 matrix (lane 3). Molecular weight standards are shown to the left.

Figure 10

Approximately 1 ng of laminin-5 (LN-5), fibronectin (FN), and bovine serum albumin (BSA) were dotted onto nitrocellulose (left). After blocking, the membranes were incubated with 20 μg/ml of tPA, plasminogen (Pg), or uPA overnight at 4°C (top). Bound protein was then detected with the appropriate antibody. Note that tPA binds both fibronectin and laminin-5, whereas plasminogen binds only laminin-5. uPA binds both BSA and fibronectin and also shows some binding to laminin-5.