The integrin alpha(v)beta8 mediates epithelial homeostasis through MT1-MMP-dependent activation of TGF-beta1

Abstract

Integrins, matrix metalloproteases (MMPs), and the cytokine TGF-beta have each been implicated in homeostatic cell behaviors such as cell growth and matrix remodeling. TGF-beta exists mainly in a latent state, and a major point of homeostatic control is the activation of TGF-beta. Because the latent domain of TGF-beta1 possesses an integrin binding motif (RGD), integrins have the potential to sequester latent TGF-beta (SLC) to the cell surface where TGF-beta activation could be locally controlled. Here, we show that SLC binds to alpha(v)beta8, an integrin expressed by normal epithelial and neuronal cells in vivo. This binding results in the membrane type 1 (MT1)-MMP-dependent release of active TGF-beta, which leads to autocrine and paracrine effects on cell growth and matrix production. These data elucidate a novel mechanism of cellular homeostasis achieved through the coordination of the activities of members of three major gene families involved in cell-matrix interactions.

Figure 1.

αvβ8 binds to LAP-β1 in an RGD- and cation-dependent fashion. (a) n-octylglucoside lysates from ¹²⁵I-cell surface-labeled β8-expressing HT1080 cells (1 ml) were passed over two identical LAP-β1–Sepharose columns (1 ml) and washed with 12 ml of wash buffer (shown in lanes 1, 2, and 3 are the 4th, 11th and 12th wash fractions). One column was eluted with 1-ml fractions containing 10 mM EDTA (fractions 4–8), and the other column was eluted with 1-ml fractions containing 1 mg/ml GRGESNK (lanes 1'–3') or 1 mg/ml GRGDSNK (lanes 4'–7'). Samples were resolved by 7.5% SDS-PAGE under nonreducing conditions and visualized by autoradiography. (b) EDTA and RGD elution fractions were immunoprecipitated with anti-β8 (14E5) and compared with anti-αv (L230) and anti-β8 (14E5) immunoprecipitations from cell lysates. The migration of the MW markers is shown on the left, and the expected migration of the αv (150 kD) and β8 (90 kD) subunits is shown on the right. Samples were resolved by 7.5% SDS-PAGE under nonreducing conditions and visualized by autoradiography. (c) ³⁵S metabolically labeled, (Translabel and ICN Biomedicals) truncated secreted αvβ8-AP fusion protein was applied to a 0.5-ml column of LAP-β1–Sepharose, washed sequentially with six fractions of wash buffer (lane 1, last wash fraction), and then eluted with 1 mg/ml GRGDSPK (lanes 2–6). On the left are the migrations of the MW markers, and on the right are the expected migrations of the truncated αv (140 kD) and the truncated β8-AP (130 kD) subunits. Samples were resolved by 10% SDS-PAGE under nonreducing conditions and visualized by autoradiography. (d) Supernatant containing secreted truncated αvβ8 with a COOH-terminal AP tag (AP-αvβ8) was applied to wells of a 96-well plate coated with either LAP-β1 (10 μg/ml) containing the RGD or the RGE binding motif or with VN (100 μg/ml) in the presence or absence of an anti-β8–blocking monoclonal antibody, 37E1. Specific binding was determined colorimetrically. An asterisk indicates increased binding of receptor to LAP (RGD) compared with antibody-treated or LAP (RGE) controls (p < 0.001). (e) Binding affinity of αvβ8 for LAP-β1 was determined using concentrated AP-αvβ8 and LAP-β1–Sepharose (1fM LAP-β1/bead). Receptor concentration was determined using purified placental AP (Applied Biosystems) as a standard. Dilutions of AP-β8 were incubated under equilibrium-binding conditions (overnight at 4°C) with 10 μl LAP-β1–Sepharose. Bound receptor was determined by luminescence using a CSPD substrate (Tropix; Applied Biosystems). (f) Adhesion of β8-expressing versus mock-transduced HT1080 cells to LAP-β1 (LAP) and SLC-coated wells of a 96-well plate. Cells (5 × 10⁴/well) were applied to each well, and after incubation for 1 h at 37°C unbound cells were removed by centrifugation. Absorbance (A ₅₉₅) after staining with Crystal violet is shown on the right. *p < 0.05; **p < 0.01.

Figure 2.

Cell surface expression of αvβ8 mediates activation of TGF-β. HT1080 (a), MvLu (b), SW480 (c), and H647 (d) cells either β8-transduced or mock-transduced were cocultured with TMLC reporter cells in the presence or absence of a neutralizing anti-β8 antibody (37E1) or pan–TGF-β neutralizing antibody (1D11). Relative luciferase units represent arbitrary units minus the TMLC background. Asterisks indicate increased luciferase activity of untreated β8-expressing cells compared with antibody-treated or mock controls. p < 0.001.

Figure 3.

The cytoplasmic domain of β8 is not required for cell adhesion to LAP-β1 or activation of TGF-β. (a) Construction of β8 subunit cytoplasmic truncation mutants. The full-length β8 (FL) subunit, a partial truncation mutant missing the COOH-terminal 11 amino acids (759), and a complete truncation mutant missing the complete β8 cytoplasmic domain (TM) were assembled by PCR mutagenesis and subcloned into retroviral vectors. (b) Immunoprecipitation analysis of surface-labeled SW480 cells, expressing FL, 759, TM, or retroviral backbone (mock) using an anti-β8 monoclonal antibody (37E1). The results demonstrate the presence and dimerization with the αv subunit on the cell surface and absence of the cytoplasmic domain in the TM construct. Biotinylated proteins were detected by Western blotting. Note that 37E1 is specific to αvβ8 because the two immunoprecipitated bands, corresponding to the αv subunit or the β8 subunit, were not seen in mock-transduced cells. Also, note that the TM construct was expressed at lower levels on the cell surface compared with 759 and FL. To determine the absence of the intracellular epitope in TM-expressing cells, cell lysates were immunoprecipitated with 37E1 and analyzed by Western blotting using a polyclonal anti-β8 antibody directed against the entire β8 cytoplasmic domain. In b (bottom), note that no signal for β8 is seen in the β8 immunoprecipitates of the truncation mutant (TM), indicating absence of the β8 cytoplasmic domain. (c) FACS^® of cytoplasmic deletion mutants (TM and 759) versus the wild type (FL) β8 subunit expressed in SW480 cells. Note the TM mutant is expressed at sixfold lower levels than the 759 or FL constructs. Histograms using arbitrary fluorescence units are shown. (d) Adhesion assays of SW480 cells expressing β8 truncation mutants demonstrate that the cytoplasmic domain of β8 is not required for adhesion to LAP-β1. Note that despite lower levels of surface expression of the TM construct, all constructs bound well to LAP-β1, whereas mock-transduced SW480 cells do not adhere to LAP-β1. (e-f) Demonstration that the β8 cytoplasmic domain is not required for activation of TGF-β. SW480 or HT1080 cells expressing the wild-type or truncation mutants were cocultured with TMLC reporter cells in the presence or absence of anti-β8 (37E1) or pan anti–TGF-β (1D11). Relative luciferase units are shown. Single and double asterisks indicate increased luciferase activity of untreated wild-type or mutant β8-expressing cells compared with antibody-treated or mock controls (*p < 0.01; **p < 0.001).

Figure 4.

Activation of TGF-β1 by αvβ8 is dependent on metalloprotease activity. (a) GM6001, a hydroxamated metalloprotease inhibitor, but not C1006, a nonhydroxamated control peptide, inhibits αvβ8-mediated but not αvβ6-mediated activation of TGF-β in SW480 cells. The asterisk indicates significant inhibition by GM6001 of β8-mediated activation compared with the other three groups (p < 0.01). (b) Only metalloprotease inhibitors inhibit αvβ8-mediated TGF-β activation. Inhibitors to aspartyl (pepstatin A), serine (PMSF, CK-23, aprotinin, and leupeptin), or cysteine (leupeptin and E64) proteases do not inhibit αvβ8-mediated TGF-β activation in SW480 cells. β8- and β6-mediated activation was determined by neutralization with 37E1 or 10D5 in a or 37E1 in b. The asterisk indicates significant inhibition by GM6001 compared with other inhibitors (p < 0.01).

Figure 5.

Active TGF-β is liberated into the supernatants of αvβ8-expressing cells. (a) Expression of β8 and β6 on the cell surface of β8-, β6-, and mock-transduced HT1080 cells using monoclonal antibodies specific for the β8 (14E5) or β6 (E7P6) integrin subunits. (b) Comparison of β8- and β6-mediated activation of TGF-β in cocultures with TMLC reporter cells. (c) Detection of active TGF-β liberated into the supernatant from β8-expressing but not β6- or mock-transduced HT1080 cells. Neutralizing antibodies to TGF-β were 1D11, or to β8 or β6 were 37E1 or 10D5, respectively. Relative luciferase units are shown in b and c. The asterisk indicates increased luciferase activity from supernatants of untreated β8-expressing cells compared with antibody-treated or mock controls (p < 0.01).

Figure 6.

MT1-MMP is deficient and can be reconstituted in the human lung carcinoma cell line H1264. (a) RT-PCR screening of tumor cell lines with MT1-MMP–specific primers demonstrates an absence of MT1-MMP in H1264 cells. A control amplification performed in parallel using β-actin primers is shown. (b) Western blotting confirms the expression of MT1-MMP and ΔMT1-MMP in transduced H1264 cells. Cell lysate from MT1-MMP or mock-transduced H1264 cells (40 μg protein) or cell supernatant from ΔMT1-MMP–transduced H1264 cells was resolved by 10% SDS page, and proteins were detected by Western blotting using an anti–MT1-MMP monoclonal antibody. The expected migration of the full-length form of MT1-MMP is 63 kD. Note that the secreted form (ΔMT1-MMP) migrates faster (54 kD), and the mock-infected cells express no MT1-MMP. (c) MT1-MMP, ΔMT1-MMP, or mock-transduced β8-overexpressing H1264 cells were plated onto 96-well dishes in serum-free medium containing recombinant pro–MMP-2. After an overnight incubation at 37°C in 5% CO₂, the supernatants were subjected to gelatin zymography (1 mg gelatin/ml, 10% SDS-PAGE). Pro-MMP2 migrates at 66 kD, and the fully activated form migrates at 59 kD. Note that only MT1-MMP–transduced H1264 cells activate MMP-2. MMP activity is shown as lucent bands against a dark Coomassie-stained background.

Figure 7.

αvβ8 mediates activation of TGF-β in β8-overexpressing H1264 cells reconstituted with MT1-MMP activity. (a) Flow cytometry of β8-transduced MT1-MMP, ΔMT1-MMP, or mock-transduced H1264 cells demonstrates equivalent levels of surface expression of β8 using an anti-β8 antibody (14E5). Histograms using arbitrary units are shown. (b) β8-overexpressing H1264 cells transduced with either MT1-MMP, ΔMT1-MMP, or the retroviral vector alone (mock) (1.6 × 10⁴) were cocultured with TMLC (1.6 × 10⁴) reporter cells in the presence or absence of inhibitors: anti-β8 (37E1), control peptide (C1006), GM6001, or the pan–TGF-β1 antibody (1D11). Asterisks indicate significantly different than untreated MT1-MMP–expressing cells. (c) The endogenous inhibitor TIMP-2 but not TIMP-1 inhibits αvβ8-mediated activation of TGF-β in H1264s cells. β8-overexpressing, MT1-MMP–expressing H1264s cells were cocultured with TMLC in the presence or absence of TIMP-1 (1 μg/ml), TIMP-2 (1 μg/ml), GM6001 (5 μM), anti-β8 (37E1), or pan-TGF-β1 (1D11). Relative luciferase units are shown (light units of cocultured cells in the presence or absence of inhibitors minus light units of TMLC cells alone) in b and c. Negative luciferase values were occasionally observed due to a small background of TGF-β activation by the TMLC cells. Single and double asterisks indicate treated cells compared with untreated cells (*p < 0.01; **p < 0.001).

Figure 8.

αvβ8 and MT1-MMP colocalize in substrate contacts. (a, top) Immunoprecipitation of MT1-MMP–GFP from ¹²⁵I cell surface-labeled MT1-MMP–GFP–expressing HT1080 cells. The catalytically active 90-kD MT1-MMP–GFP fusion protein (asterisk) was immunoprecipitated with anti-GFP antibodies from MT1-MMP–GFP–expressing HT1080 β8 cells but not mock-transduced HT1080 β8 cells. The 70-kD MT1-MMP–GFP band represents a catalytically inactive degradation product. (a, bottom) Gelatin zymography of supernatants from MT1-MMP–GFP–transduced or mock-transduced HT1080 β8 cells. The migration of Pro (Pro−) and active (Act.−) forms of MMP-2 are shown. (b–m) Confocal images of immunofluorescence microscopy. β8-expressing, MT1-MMP–GFP–expressing HT1080 cells (b–d and k–m); β8-expressing HT1080 cells (e–g); GFP-expressing HT1080 cells (h–j). Cells were allowed to attach 4 h to LAP-β1 (10 μg/ml coating concentration)-coated slides. After fixation and permeabilization, colocalization of β8 and GFP was determined using polyclonal anti-β8 and monoclonal anti-GFP antibodies. Pseudocolored confocal images of β8 (red) and GFP (green) staining taken in the plane of the substrate are shown. Bar, 7.5 μM.

Figure 9.

Cell surface-associated MT1-MMP cleaves and inactivates LAP-β1. (a) LAP-β1 is cleaved by incubation with β8-overexpressing MT1-MMP but not ΔMT1-MMP or mock-transduced H1264 cells. LAP-β1 (10 μg/ml) was incubated overnight with either no cells (lane 1), mock-transduced β8-overexpressing H1264 cells (lane 2), ΔMT1-MMP transduced, β8-overexpressing H1264 cells (lane 3), MT1-MMP transduced, β8-overexpressing H1264 cells (lane 4), 500 μg/ml of control peptide (C1006; lane 5), or 500 μg/ml hydroxymate inhibitor GM6001 (lane 6). 20 ng of the input LAP-β1 was resolved by 12.5% SDS-PAGE under reducing conditions. After immunoblotting with an anti-LAP antibody, the migration of the cleavage products calibrated to molecular weight standards (GIBCO BRL) is shown. Note that only LAP-β1 incubated in the presence of MT1-MMP is cleaved and that this cleavage is blocked by GM6001. (b) The TGF-β1 peptide GRRGDLATIH selectively inhibits αvβ8–LAP-β1 function. Adhesion assay of β8-overexpressing, MT1-MMP expressing H1264 cells (4 × 10⁴) to LAP-β1, VN, or fibronectin (FN) (10 μg/ml coating concentrations) in the presence or absence of 50 μg/ml of GRRGDLATIH. (c) LAP-β1 cleavage by MT1-MMP–transduced, β8-overexpressing H1264 cells is inhibited by GRRGDLATIH but not GRRGELATIH peptide (10 μg/ml). The degradation assay was performed and analyzed by immunoblotting as in a. No-cell control (lane 1); no-inhibitor control (lane 2); control GRRGELATIH peptide (lane 3); GRRGDLATIH peptide (lane 4). (d) LAP-β1 is inactivated by β8-overexpressing, MT1-MMP-expressing H1264 cells. LAP-β1 (5 μg) incubated overnight with β8-overexpressing, MT1-MMP-expressing or β8-overexpressing, mock-transduced H1264 cells was added to TMLC reporter cells in the presence of recombinant TGF-β1. As a control (white bars) TMLC reporter cells were incubated with only recombinant TGF-β and no LAP-β1. Relative luciferase units are shown. The asterisk indicates LAP-β1 incubated with mock control cells is not cleaved and decreases TGF-β activity compared with other groups (p < 0.05).

Figure 10.

The activation of TGF-β by αvβ8 is associated both with growth inhibition and with fibrogenesis in lung cancer tumor xenografts. (a) DNA synthesis is inhibited in αvβ8-expressing H647 cells, and the inhibition is reversed by TGF-β blocking antibodies (1D11). The asterisk indicates increased BrdU incorporation of antibody-treated β8-expressing cells compared with untreated β8-expressing cell (p < 0.05). (b) Histologic analysis of tumors grown in nude mice derived from either β8-expressing or mock-transduced H647 cells. Trichrome-stained sections highlighting dense collagen (green area between arrows) between islands of tumor cells (t) are shown. Bar, 75 μm. (c) Determination of active and SLC in β8-expressing and mock-transduced lung tumor xenografts. Relative luciferase units are shown. The asterisk indicates increased active TGF-β from β8-expressing tumors compared with mock-transduced tumors (*p < 0.01).