Journal: International Journal of Molecular Sciences

Article Title: Extracellular Matrix Remodeling by Fibroblast-MMP14 Regulates Melanoma Growth

doi: 10.3390/ijms222212276

Figure Lengend Snippet: B16F1 cell proliferation and migration on MMP14 Sf−/− and MMP14 Sf+/+ fibroblast matrix. ( a ) Schematic representation of fibroblast culture to obtain their matrix (picture of Coomassie-stained matrix after fibroblast removal) and seeding B16F1 cells on it. ( b ) BrdU incorporation measurement of B16F1 cells cultured on fibroblast matrix. ( c ) Colony outgrowth assay showing melanoma migration on fibroblast matrix (left) and quantification (right). Experiments were repeated three times. mWT (MMP14 Sf+/+ matrix, control matrix) n = 3; mSF −/− (MMP14 Sf−/− matrix) n = 3; continuous line, cell front after 0 h; dashed line, cell front after indicated period of time; * p < 0.05; *** p < 0.001; scale, 100 µm.

Article Snippet: Recombinant human MMP14 100 ng/µL (918-MPN, R&D Systems, Wiesbaden, Germany) was activated using 0.1 µg/mL trypsin (15090046, Thermo Fisher Scientific, Darmstadt, Germany) in 50 mM Tris, 0.15 M NaCl, 10 mM CaCl 2 , 5 µM ZnCl 2 , 0.05% Brij-35, pH 7.5 for 1 h at 37 °C.

Techniques: Migration, Staining, BrdU Incorporation Assay, Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: Extracellular Matrix Remodeling by Fibroblast-MMP14 Regulates Melanoma Growth

doi: 10.3390/ijms222212276

Figure Lengend Snippet: Analysis of MMP14 Sf−/− and MMP14 Sf+/+ fibroblast matrices. ( a ) Mass Spectrometric and ( b ) immunoblot analysis of fibroblast matrix and conditioned medium (8% SDS-PAGE). ( c ) Transcriptional analysis of fibroblast lysates for collagens VI (COL6A1) and XIV (COL14A1). ( d ) In vitro processing of recombinant collagen XIV (Coll XIV) and human MMP14 visualized on immunoblot and silver staining (4–12% Bis-Tris gradient gel). Collagen XIV fragments generated by MMP14 are indicated with black arrows. MMP14 Sf+/+ (WT), n = 3; MMP14 Sf−/− (SF −/− ), n = 3; pro-MMP14, inactive form of MMP14; * p < 0.05.

Article Snippet: Recombinant human MMP14 100 ng/µL (918-MPN, R&D Systems, Wiesbaden, Germany) was activated using 0.1 µg/mL trypsin (15090046, Thermo Fisher Scientific, Darmstadt, Germany) in 50 mM Tris, 0.15 M NaCl, 10 mM CaCl 2 , 5 µM ZnCl 2 , 0.05% Brij-35, pH 7.5 for 1 h at 37 °C.

Techniques: Western Blot, SDS Page, In Vitro, Recombinant, Silver Staining, Generated

Journal: International Journal of Molecular Sciences

Article Title: Extracellular Matrix Remodeling by Fibroblast-MMP14 Regulates Melanoma Growth

doi: 10.3390/ijms222212276

Figure Lengend Snippet: Melanoma cells proliferation on collagen XIV coatings. BrdU incorporation analysis of B16F1 cells cultured on ( a ) collagen XIV (Coll XIV) (1 to 10 µg/mL) and ( b ) on serum (FCS) alone and in combination with collagen XIV (1 to 10 µg/mL). ( c ) Proliferation was measured in cells cultured on matrix from control fibroblasts (mWT) alone or with the addition of collagen XIV (1 to 10 µg/mL) and MMP14 Sf−/− (SF −/− ) fibroblasts. Experiments were repeated three times. mWT (MMP14 Sf+/+ matrix, control matrix), n = 3; mSF −/− (MMP14 Sf−/− matrix), n = 3; * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Recombinant human MMP14 100 ng/µL (918-MPN, R&D Systems, Wiesbaden, Germany) was activated using 0.1 µg/mL trypsin (15090046, Thermo Fisher Scientific, Darmstadt, Germany) in 50 mM Tris, 0.15 M NaCl, 10 mM CaCl 2 , 5 µM ZnCl 2 , 0.05% Brij-35, pH 7.5 for 1 h at 37 °C.

Techniques: BrdU Incorporation Assay, Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: Extracellular Matrix Remodeling by Fibroblast-MMP14 Regulates Melanoma Growth

doi: 10.3390/ijms222212276

Figure Lengend Snippet: B16F1 cell migration on MMP14 Sf+/+ fibroblast matrix enriched with collagen XIV. Colony outgrowth assay on MMP14 Sf+/+ fibroblast matrix (mWT) containing 10 µg/mL collagen XIV (Coll XIV). The FCS-coated surface was used as a control. Below is the quantification in the percentage of the migrated area. Experiments were repeated three times. mWT (MMP14 Sf+/+ matrix), n = 3; FCS, n = 3; a continuous line marks the cell border at time 0 h (this time point is shown in the insert); dashed line, cells border after indicated period of time; * p < 0.05; scale: 100 µm.

Article Snippet: Recombinant human MMP14 100 ng/µL (918-MPN, R&D Systems, Wiesbaden, Germany) was activated using 0.1 µg/mL trypsin (15090046, Thermo Fisher Scientific, Darmstadt, Germany) in 50 mM Tris, 0.15 M NaCl, 10 mM CaCl 2 , 5 µM ZnCl 2 , 0.05% Brij-35, pH 7.5 for 1 h at 37 °C.

Techniques: Migration

Journal: International Journal of Molecular Sciences

Article Title: Extracellular Matrix Remodeling by Fibroblast-MMP14 Regulates Melanoma Growth

doi: 10.3390/ijms222212276

Figure Lengend Snippet: Analysis of B16F1 cell adhesion on collagen XIV coating and fibroblast matrix. ( a ) Melanoma adhesion assay on collagen XIV (Coll XIV) coatings (1 µg/mL; 10 µg/mL), ( b ) wildtype fibroblast matrix (mWT), in combination with 10 µg/mL collagen XIV (mWT + Coll XIV) and MMP14 Sf−/− matrix (mSF −/− ). Fibronectin (FN) was used as positive control. Experiments were repeated three times. mWT (MMP14 Sf+/+ matrix, control matrix), n = 3, mWT + Coll XIV, n = 3; mSF −/− (MMP14 Sf−/− matrix), n = 3; * p < 0.05; **** p < 0.0001; scale, 100 µm.

Article Snippet: Recombinant human MMP14 100 ng/µL (918-MPN, R&D Systems, Wiesbaden, Germany) was activated using 0.1 µg/mL trypsin (15090046, Thermo Fisher Scientific, Darmstadt, Germany) in 50 mM Tris, 0.15 M NaCl, 10 mM CaCl 2 , 5 µM ZnCl 2 , 0.05% Brij-35, pH 7.5 for 1 h at 37 °C.

Techniques: Cell Adhesion Assay, Positive Control

Journal: International Journal of Molecular Sciences

Article Title: Extracellular Matrix Remodeling by Fibroblast-MMP14 Regulates Melanoma Growth

doi: 10.3390/ijms222212276

Figure Lengend Snippet: Collagen XIV expression in mouse melanoma. Immunofluorescence staining of collagen XIV (red) and DAPI (blue) in melanoma from MMP14 Sf+/+ and MMP14 Sf−/− mice. The graph represents quantified signal intensity per area measured within peritumoral tissue (100 µm radius from the tumor). MMP14 Sf+/+ (WT), n = 6; MMP14 Sf−/− (SF −/− ), n = 5; t, tumor; e, epidermis. ** p < 0.01; scale, 100 µm.

Article Snippet: Recombinant human MMP14 100 ng/µL (918-MPN, R&D Systems, Wiesbaden, Germany) was activated using 0.1 µg/mL trypsin (15090046, Thermo Fisher Scientific, Darmstadt, Germany) in 50 mM Tris, 0.15 M NaCl, 10 mM CaCl 2 , 5 µM ZnCl 2 , 0.05% Brij-35, pH 7.5 for 1 h at 37 °C.

Techniques: Expressing, Immunofluorescence, Staining

Journal: International Journal of Molecular Sciences

Article Title: Extracellular Matrix Remodeling by Fibroblast-MMP14 Regulates Melanoma Growth

doi: 10.3390/ijms222212276

Figure Lengend Snippet: Analysis of collagen XIV and MMP14 expression in human tissue samples. Immunofluorescence detection of collagen XIV (red) and immunohistochemical staining for MMP14 in human nevi and melanoma. The nuclei are in blue (DAPI). Average collagen XIV signal intensity per area is shown on the left. Yellow arrowheads, nests of melanocytic nevi; white/black arrowheads, cutaneous melanoma nests; e, epidermis; n, nevus; t, tumor; s, stroma; scale, 100 µm.

Article Snippet: Recombinant human MMP14 100 ng/µL (918-MPN, R&D Systems, Wiesbaden, Germany) was activated using 0.1 µg/mL trypsin (15090046, Thermo Fisher Scientific, Darmstadt, Germany) in 50 mM Tris, 0.15 M NaCl, 10 mM CaCl 2 , 5 µM ZnCl 2 , 0.05% Brij-35, pH 7.5 for 1 h at 37 °C.

Techniques: Expressing, Immunofluorescence, Immunohistochemical staining, Staining

Journal: Cells

Article Title: Bivalent Inhibitor with Selectivity for Trimeric MMP-9 Amplifies Neutrophil Chemotaxis and Enables Functional Studies on MMP-9 Proteoforms

doi: 10.3390/cells9071634

Figure Lengend Snippet: Inhibitory capacity of monovalent and bivalent carboxylate inhibitors 5 and 7 on proteases with importance for acute inflammation.

Article Snippet: MMP-14/MT1-MMP (NS0 cell-derived) was also purchased from R & D systems (Minneapolis, MN, USA) and activated by incubation with recombinant human furin (cat. No. 1503-SE-010, R & D systems, Minneapolis, MO, USA) in activation buffer (50 mM Tris, 1 mM CaCl 2 , 0.5% Brij-35, pH 9) for 1.5 h and activity was tested in assay buffer (50 mM Tris, 3 mM CaCl 2 , 1 μM ZnCl 2 , pH 8.5).

Techniques: Inhibition

Journal: bioRxiv

Article Title: MMP14 cleaves PTH1R in the chondrocyte derived osteoblast lineage, curbing signaling intensity for proper bone anabolism

doi: 10.1101/2022.08.17.504252

Figure Lengend Snippet: (A), Schematic diagram showing the isolation of osteochondrogenic cells from mouse tibia for single-cell transcriptomics (scRNAseq). (B), Scatter-plot showing the 3420 single cells, 3110 of which belong to the chondro-osteogenic clusters, as identified in the P6 sample by dimension reduction method UMAP. HC: hypertrophic chondrocytes. Obs: osteoblasts. RBC: red blood cells. (C), Upper panel: heatmap showing the expression patterns of P6 signature genes in the preHC, HC and immature Ob clusters, when comparing in a one-vs-all manner among the three. Representative signature genes of interest are listed by the side. Lower panel: signal track indicating the level of tdTomato expression inferred from the WPRE signal. (D), Representative immunostaining showing the spatial distribution of MMP14 and X-gal stained (blue) in hematoxylin stained sections from C10Cre;RLacZ tibae. HZ, hypertrophic zone; TB, trabecular region; CB, cortical region. Black arrows in enlarged figures indicate LacZ+;MMP14+ cells. Red arrows indicate Mmp14+;LacZ-cells. Dotted line draws chondro-osseous junction. Scale bar 200μm,50μm(magnified picture).(E-H), Scatter-plot showing the integration of the mature and immature osteoblasts of the P6 (E) and the P56 (F) mice. (H-J), (I), Piecharts showing the percentages of mature osteoblasts in the both samples. (J), A panel of scatter-plots showing the expression pattern of a list of selected markers, representing the different osteoblast differentiation states. See also , Extended Table S1&2.

Article Snippet: The peptides and recombinant proteins were incubated with recombinant MMP14 (BML-SE259-0010, Enzo Lifesciences) for 16 hours and the mixture was analyzed using western blotting or MALDI-TOF mass spectrometry (Centre for Proteomics, The University of Hong Kong).

Techniques: Isolation, Single-cell Transcriptomics, Expressing, Immunostaining, Staining

Journal: bioRxiv

Article Title: MMP14 cleaves PTH1R in the chondrocyte derived osteoblast lineage, curbing signaling intensity for proper bone anabolism

doi: 10.1101/2022.08.17.504252

Figure Lengend Snippet: (A), Cells in cortical bone and groove of ranvier at P10. Yellow arrow indicates Megakaryocytes. Green arrow indicate endosteal bone lining cells. Black arrow indicates perichondral cells. (B), Bright field and fluorescence images of radius, ulna and scapula of C10Cre;RYFP;Mmp14 +/+ and C10Cre;RYFP;Mmp14 -/- mice at P3. Green fluorescence label HC-derived cells. Red arrows mark chondro-osseous junction. Yellow arrow marks trabecular region. HZ represents hypertrophic zone. (C), Immunofluorescence staining of RFP and Edu-labeled cells counterstained with DAPI in C10Cre;RtdT;Mmp14+/+ and C10Cre;RtdT;Mmp14 -/- tibia at P3. For quantification, the trabecular bone is divided into four zones, each 0.1 mm in thickness. The number of RFP+(red), RFP+Edu+(red and green) cells in each region were counted. Data are presented as means ± SEM ** p<0.01, * p<0.05, unpaired t test. (D), Immunostaining of Endomucin (Endo) labeling blood vessels below chondro-osseous junction comparing wild-type (yellow bar)and Mmp14-/-(green bar) mice at P3. The vascular density and number of Ecmn cells are presented (n=9). Data are presented as means ± SEM ** p<0.01, * p<0.05, unpaired t test. HZ, hypertrophic zone. TB, trabecular bone.

Article Snippet: The peptides and recombinant proteins were incubated with recombinant MMP14 (BML-SE259-0010, Enzo Lifesciences) for 16 hours and the mixture was analyzed using western blotting or MALDI-TOF mass spectrometry (Centre for Proteomics, The University of Hong Kong).

Techniques: Fluorescence, Derivative Assay, Immunofluorescence, Staining, Labeling, Immunostaining

Journal: bioRxiv

Article Title: MMP14 cleaves PTH1R in the chondrocyte derived osteoblast lineage, curbing signaling intensity for proper bone anabolism

doi: 10.1101/2022.08.17.504252

Figure Lengend Snippet: (A), H&E staining and in situ hybridization of Col10a1 , Col1a1 , Mmp13 , Mmp14 in Mmp14 ΔHC and control mice at P10. White signals marks mRNA expression of corresponding genes. HZ, hypertrophic zone; TB, trabecular region. (B), Edu labeling assay in Mmp14 ΔHC and control mice. HC-descendants, nucleus and proliferating cells were labeled with RFP(red), DAPI(blue) and Edu(green). (C), Tartrate-resistant acid phosphatase (TRAP) staining comparing Mmp14 ΔHC and control mice. Black arrows mark TRAP+ cells HZ, Hypertrophic Zone. TB, Trabecular bone. Counting of TRAP+ cells suggests the number of osteoclasts are not significantly affected in the mutants(n=6 for control, n=4 for Mmp14 ΔHC mutants). (D), Immunofluorescence staining of RFP, Perilipin, GFP and DAPI labeling tdTomato(Red), adipocytes(white), osteoblasts(green) and nucleus at P10. Quantitation of RFP+PER+, RFP-PER+, and PER+ cells at distal tibia comparing Mmp14 ΔHC mutants and control at P10. Unpaired t-test are used for PER+ cells. Paired t-test is used for PER+RFP+ cells. Quantitation is shown in (n=5) (p<0.05) Unpaired t-test.

Article Snippet: The peptides and recombinant proteins were incubated with recombinant MMP14 (BML-SE259-0010, Enzo Lifesciences) for 16 hours and the mixture was analyzed using western blotting or MALDI-TOF mass spectrometry (Centre for Proteomics, The University of Hong Kong).

Techniques: Staining, In Situ Hybridization, Expressing, Labeling, Immunofluorescence, Quantitation Assay

Journal: bioRxiv

Article Title: MMP14 cleaves PTH1R in the chondrocyte derived osteoblast lineage, curbing signaling intensity for proper bone anabolism

doi: 10.1101/2022.08.17.504252

Figure Lengend Snippet: ( A ), Violin plots showing the expression levels of Pth1r and Mmp14 in the HC-derived and non-HC-derived osteoblasts. n.s.: not significant. (B) , HEK293T cells expressing HA-tagged human PTH1R were transfected with either WT(MT1-FL) or E/A catalytic inactive mutant MMP14 (MT1 EA). β-actin is used as a loading control. Image representative of three independent experiments. ( C ), HEK293T cells expressing mouse Pth1r(without HA-tag) were transfected with either WT(MT1-FL) or E/A catalytic inactive mutant MMP14 (MT1-EA). Mouse Pth1r was deglycosylated, detected with anti-PTH1R antibody and β-actin is used as a internal loading control. Images are representative of 3 independent experiments. ( D ), His-tagged rPTH1R-ECD was incubated with recombinant catalytic domain of MMP14 at two enzyme/substrate ratios (rPTH1R-ECD only, 1:50, 1:10, and 1:10 with EDTA). The protein mixture were deglycosylated(left panel). Western blotting analyses used specific antibody indicated. Data are representative of three independent experiments. ( E ), Representative western blot trabecular bone protein lysate Mmp14+/+(left lane) and Mmp14-/-(right lane) mice at P14 (n=2). ( F ), Synthetic peptide from extracellular domain of PTH1R 55-67 was incubated with recombinant MMP14. The presence of 770Da suggests a fragment of VLQRPAS.

Article Snippet: The peptides and recombinant proteins were incubated with recombinant MMP14 (BML-SE259-0010, Enzo Lifesciences) for 16 hours and the mixture was analyzed using western blotting or MALDI-TOF mass spectrometry (Centre for Proteomics, The University of Hong Kong).

Techniques: Expressing, Derivative Assay, Transfection, Mutagenesis, Incubation, Recombinant, Western Blot

Journal: bioRxiv

Article Title: MMP14 cleaves PTH1R in the chondrocyte derived osteoblast lineage, curbing signaling intensity for proper bone anabolism

doi: 10.1101/2022.08.17.504252

Figure Lengend Snippet: (A), Western blot of HEK293T cells transfected with human PTH1R-HA, MT1-FL, MT1-EA, ADAM10, ADAM15 and ADAM17. (B), Co-immunoprecipitation of MMP14 with PTH1R in HEK293T cells transfected with MT1-FL and PTH1R-HA. PTH1R-HA was immunoprecipitated with anti-HA antibody in G-agarose. (C), Cell lysate from HEK293T cells transfected with PTH1R-HA, MMP14-Flag were immunoprecipitated with anti-Flag-tag antibody and analyzed by western blot. (D), Cleaved fragment of PTH1R were observed in cultured trabecular bone osteoblasts analyzed by western blot from Mmp14+/+(left lane) and Mmp14-/-(right lane) mice at P14.

Article Snippet: The peptides and recombinant proteins were incubated with recombinant MMP14 (BML-SE259-0010, Enzo Lifesciences) for 16 hours and the mixture was analyzed using western blotting or MALDI-TOF mass spectrometry (Centre for Proteomics, The University of Hong Kong).

Techniques: Western Blot, Transfection, Immunoprecipitation, FLAG-tag, Cell Culture

Journal: bioRxiv

Article Title: MMP14 cleaves PTH1R in the chondrocyte derived osteoblast lineage, curbing signaling intensity for proper bone anabolism

doi: 10.1101/2022.08.17.504252

Figure Lengend Snippet: ( A ), MMP14 can inhibit PTH signaling in HEK293 cells. HEK293 cells stably expressing PTH1R-HA were transfected with empty vector(EV), MMP14(MT1-FL) and catalytic inactive MMP14(MT1-EA). Cells were challenged with 1×10-7M PTH in vitro for 0,30 and 60 minutes. PTH1R, MMP14, phospho-creb(p-creb), total-creb(t-creb), phosphor-Erk, total-Erk and loading control β -actin were analyzed by western blotting. ( B ), Relative level of p-creb/t-creb and p-Erk/t-Erk were quantitated and measured using ImageJ (n=6 for p-Creb/t-Creb and n=5 for p-Erk/t-Erk). One-way ANOVA was used to determine statistical significance. Data are presented as means ± SEM. ** p<0.01, * p<0.05. ( C ), HEK 293 cells expressing PTH1R were challenged with PTH for 30,60 and 180 minutes and the level of cAMP with MT1-FL,MT1-EA or empty vector were measured(n=5). One-way ANOVA was used to determine statistical significance. ( D ), Trabecular osteoblasts extracted from Mmp14+/+(red bar) and Mmp14-/-(blue bar) mice were challenged with 1×10-7M PTH( – ). Cell lysate were analyzed by western blotting for p-Creb and t-Creb level (left). Right, quantification of relative level of p-Creb/t-Creb in trabecular osteoblasts extracted from Mmp14+/+ and Mmp14-/-mice (n=5). Data are presented as means ± SEM. ** p<0.01, * p<0.05, unpaired t test. ( E ), Schematic diagram showing MMP14 cleaves PTH1R at amino acid 61. Orange stripe indicates mutation sites. Seven-pass transmembrane domains are in green. PTH1R transduces signals via G-protein complex α,β,γ.

Article Snippet: The peptides and recombinant proteins were incubated with recombinant MMP14 (BML-SE259-0010, Enzo Lifesciences) for 16 hours and the mixture was analyzed using western blotting or MALDI-TOF mass spectrometry (Centre for Proteomics, The University of Hong Kong).

Techniques: Stable Transfection, Expressing, Transfection, Plasmid Preparation, In Vitro, Western Blot, Mutagenesis

Journal: bioRxiv

Article Title: MMP14 cleaves PTH1R in the chondrocyte derived osteoblast lineage, curbing signaling intensity for proper bone anabolism

doi: 10.1101/2022.08.17.504252

Figure Lengend Snippet: (A), Relative level of PTH1R in HEK293T cells co-transfected with empty vector, MMP14 and MMP14-EA. ( B ), Immunofluorescence staining of MMP14 in P56 mice treated with vehicle(left) and PTH( – ).

Article Snippet: The peptides and recombinant proteins were incubated with recombinant MMP14 (BML-SE259-0010, Enzo Lifesciences) for 16 hours and the mixture was analyzed using western blotting or MALDI-TOF mass spectrometry (Centre for Proteomics, The University of Hong Kong).

Techniques: Transfection, Plasmid Preparation, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: MMP14 cleaves PTH1R in the chondrocyte derived osteoblast lineage, curbing signaling intensity for proper bone anabolism

doi: 10.1101/2022.08.17.504252

Figure Lengend Snippet: Upon PTH binding, PTH1R generates cAMP signals to activate cAMP response element binding protein (Creb). Activation of PTH1R causes upregulation of MMP14. MMP14 in turn cleaves PTH1R to inhibit its signaling activity. In the chondrocyte lineage, pre-hypertrophic chondrocytes (pre-HC) differentiate to HCs, translocate to the subchondral space and subsequently to HC-pre-osteoblasts contributing to trabecular, endosteal and endocortical bone and can respond to PTH treatment. Together, MMP14 moderates the HC derivatives’ response to PTH ensuring a controlled supply of osteoblast precursors and thereby bone anabolism. Obs, osteoblasts, PHZ, Hypertrophic zone. HZ, Hypertrophic zone. TB, Trabecular bone.

Article Snippet: The peptides and recombinant proteins were incubated with recombinant MMP14 (BML-SE259-0010, Enzo Lifesciences) for 16 hours and the mixture was analyzed using western blotting or MALDI-TOF mass spectrometry (Centre for Proteomics, The University of Hong Kong).

Techniques: Binding Assay, Activation Assay, Activity Assay