TIMP-1 Induces an EMT-Like Phenotypic Conversion in MDCK Cells Independent of Its MMP-Inhibitory Domain

Matrix metalloproteinases (MMPs) and their endogenous inhibitors (TIMPs) regulate epithelial-mesenchymal transition (EMT) critical for the development of epithelial organs as well as cancer cell invasion. TIMP-1 is frequently overexpressed in several types of human cancers and serves as a prognostic marker. The present study investigates the roles of TIMP-1 on the EMT process and formation of the lumen-like structure in a 3D Matrigel culture of MDCK cells. We show that TIMP-1 overexpression effectively prevents cell polarization and acinar-like structure formation. TIMP-1 induces expression of the developmental EMT transcription factors such as SLUG, TWIST, ZEB1 and ZEB2, leading to downregulation of epithelial marker and upregulation of mesenchymal markers. Importantly, TIMP-1′s ability to induce the EMT-like process is independent of its MMP-inhibitory domain. To our surprise, TIMP-1 induces migratory and invasive properties in MDCK cells. Here, we present a novel finding that TIMP-1 signaling upregulates MT1-MMP and MMP-2 expression, and potentiates MT1-MMP activation of pro-MMP-2, contributing to tumor cell invasion. In spite of the fact that TIMP-1, as opposed to TIMP-2, does not interact with and inhibit MT1-MMP, TIMP-1 may act as a key regulator of MT1-MMP/MMP-2 axis. Collectively, our findings suggest a model in which TIMP-1 functions as a signaling molecule and also as an endogenous inhibitor of MMPs. This concept represents a paradigm shift in the current view of TIMP-1/MT1-MMP interactions and functions during cancer development/progression

Knockdown of MT1-MMP abrogated MMP-2 activation in the MDCK-T1, T1D cells.

<p>MDCK-T1 (A) and MDCK-T1D (B) cells in the absence or presence of control or MT1-MMP siRNAs were untreated (-) or treated (+) with ConA for 18 hr. The protein levels of MT1-MMP in cell lysates and MMP-2 in conditioned media were determined by immunoblot analysis. Conditioned media were analyzed by gelatin zymography. P, pro-MMP-2: I, intermediate form of MMP-2: A, active MMP-2.</p

TIMP-1 regulates EMT markers and induces motility of MDCK cells, independent of its MMP-inhibitory domain. A.

<p>RT-PCR analysis (left panel) of epithelial marker E-cadherin and mesenchymal markers N-cadherin, Fibronectin, and Vimentin. Immunoblot analysis (right panel) of E-cadherin (after long and short exposures) and vimentin in MDCK-Neo, -T1, and –T1D cells. <b>B.</b> RT-PCR analysis of EMT transcriptional factors SNAIL, SLUG, TWIST, ZEB1 and ZEB2. <b>C.</b> A scratch migration assay was performed in MDCK-Neo, -T1 and T1D cells for 18 h.</p

TIMP-1 induces cell survival signaling in MDCK cells, independent of its MMP-inhibitory domain. A.

<p>Schematic representation of the T1 and T1D proteins. The 12 cysteine (C) residues are linked to form six disulfide bonds; the 23 amino acids-long secretion signal sequence (amino acid 1–23) is indicated. A TIMP-1 mutant encoding the C-terminal and partial N-terminal (amino acid 66–184) regions of human T1 is indicated as TID. <b>B.</b> (Left Panel) RT-PCR analysis of TIMP-1 mRNA in MDCK-Neo, MDCK-T1 and MDCK-T1D cells using primers common to human and canine TIMP-1 mRNA. <b>B.</b> (Right Panel) Immunoblot analysis of TIMP-1 in cell lysates or conditioned media (CM) using anti-TIMP-1 Ab that recognizes the N-terminal domain of TIMP-1 (top panel) or anti-FLAG antibody (middle panel). <b>C.</b> Immunoblot analysis of Erk and Akt using MDCK-Neo, -T1, and –T1D cell lysates. <b>D-E.</b> DEVDase activity in MDCK-Neo, MDCK-T1, and MDCK-T1D cells upon treatment with 0.5 uM staurosporine for 4 hr (D) and serum-free culturing for 48 hr (E). DEVDase activity was normalized per microgram protein and each bar represents the mean ± s.d. of triplicates in three independent experiments. <b>F-G</b>. Apoptotic cells were quantified by FACS after F2N12S labeling and SYTOX AADvanced™ dead cell stain. (F) Representative FACS data without or with 0.5 µM staurosporine for 16 hr. (Upper left-dead cells; Lower left-apoptotic cells; Lower right-live cells). (G) Bar graph represents the mean percentages of apoptotic cells ± s.d. in the three independent experiment. Asterisk (*) indicates a <i>P</i> value <0.001 using a Paired T-test.</p

TIMP-1 signaling disrupts MDCK cell polarization and acini formation in a 3D Matrigel culture.

<p>Phalloidin staining (red) and blue-fluorescent nuclear staining with DAPI in MDCK-Neo (<b>A</b>), -T1 (<b>B</b>), and –T1D (<b>C</b>) cells in a 3D Matrigel culture for indicated periods. <b>D.</b> Percentage of polarized acini examined in cross sections through the middle of developing acini of MDCK-Neo at 12 days, and T1, T1D at 23 days. (More than 40 spheroids were analyzed for each condition from three independent experiments and the means <u>+</u> s.d. were shown). <b>E-F</b>. At the indicated time points, MDCK-Neo, T1, T1D cell proliferation was assessed by trypan blue exclusion assay (E) or MTT assay (F). (Shown are means <u>+</u> s.d of three independent experiments).</p

Identification of CD63 as a tissue inhibitor of metalloproteinase-1 interacting cell surface protein

This study identified CD63, a member of the tetraspanin family, as a TIMP-1 interacting protein by yeast two-hybrid screening. Immunoprecipitation and confocal microscopic analysis confirmed CD63 interactions with TIMP-1, integrin β1, and their co-localizations on the cell surface of human breast epithelial MCF10A cells. TIMP-1 expression correlated with the level of active integrin β1 on the cell surface independent of cell adhesion. While MCF10A cells within a three-dimensional (3D) matrigel matrix form polarized acinar-like structures, TIMP-1 overexpression disrupted breast epithelial cell polarization and inhibited caspase-mediated apoptosis in centrally located cells, necessary for the formation and maintenance of the hollow acinar-like structures. Small hairpin RNA (shRNA)-mediated CD63 downregulation effectively reduced TIMP-1 binding to the cell surface, TIMP-1 co-localization with integrin β1, and consequently reversed TIMP-1-mediated integrin β1 activation, cell survival signaling and apoptosis inhibition. CD63 downregulation also restored polarization and apoptosis of TIMP-1 overexpressing MCF10A cells within a 3D-matrigel matrix. Taken together, the present study identified CD63 as a cell surface binding partner for TIMP-1, regulating cell survival and polarization via TIMP-1 modulation of tetraspanin/integrin signaling complex