Chiarelli R et al. (2022), Vanadium Modulates Proteolytic Activities and M...

ECB-ART-51148

Int J Mol Sci 2022 Nov 17;2322:. doi: 10.3390/ijms232214238.

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Vanadium Modulates Proteolytic Activities and MMP-14-Like Levels during Paracentrotus lividus Embryogenesis.

Chiarelli R , Martino C , Scudiero R , Geraci F .

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The increasing industrial use of vanadium (V), as well as its recent medical use in various pathologies has intensified its environmental release, making it an emerging pollutant. The sea urchin embryo has long been used to study the effects induced by metals, including V. In this study we used an integrated approach that correlates the biological effects on embryo development with proteolytic activities of gelatinases that could better reflect any metal-induced imbalances. V-exposure caused morphological/morphometric aberrations, mainly concerning the correct distribution of embryonic cells, the development of the skeleton, and the embryo volume. Moreover, V induced a concentration change in all the gelatinases expressed during embryo development and a reduction in their total proteolytic activity. The presence of three MMP-like gelatinases (MMP-2, -9, and -14) was also demonstrated and their levels depended on V-concentration. In particular, the MMP-14-like protein modified its expression level during embryo development in a time- and dose-dependent manner. This enzyme also showed a specific localization on filopodia, suggesting that primary mesenchyme cells (PMCs) could be responsible for its synthesis. In conclusion, these results indicate that an integrated study among morphology/morphometry, proteolytic activity, and MMP-14 expression constitutes an important response profile to V-action.

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	Figure 1. Morphological and morphometric effects induced by V during embryonic development. Images representative of embryos from 12 h to 48 h of development/treatment. (A1,B1,C1,D1,E1,F1,G1,H1,I1) Control embryos; (A2,B2,C2,D2,E2,F2,G2,H2,I2) 1 mM V-treated embryos; (A3,B3,C3,D3,E3,F3,G3,H3,I3) 500 µM V-treated embryos. Control and V-treated embryos were observed at different development/treatment intervals: (A1–A3) 12 h; (B1–B3) 15 h; (C1–C3) 18 h; (D1–D3) 21 h; (E1–E3) 24 h; (F1–F3) 30 h; (G1–G3) 36 h; (H1–H3) 42 h; and (I1–I3) 48 h. Scale bar = 45 µm. Images are representative of three independent experiments. The line graph shows data related to the volumetric analysis. Experiments were performed in triplicate and data are expressed as means ± standard deviation (n = 3 ± SD). Statistical analysis was performed using t-test with * p ≤ 0.05.
	Figure 2. Amount of V and Ca incorporated in embryos after 48 h of development/treatment. Embryos were cultured under normal growth conditions and with V 1 mM or 500 µM. (A) V and (B) Ca content. Values reported in the pie charts show the ionic concentrations detected for V (µg/g weight embryos) and Ca (mg/g weight embryos). Experiments were performed in triplicate and data are expressed as means ± standard deviation (n = 3 ± SD). Statistical analysis was performed using t-test with p ≤ 0.01; * p ≤ 0.0005.
	Figure 3. Metal-related enzymatic activities analysed by gelatin substrate gel zymography. (A) Zymograms showing bands in lysates of control and V-treated (1 mM, 500 µM) embryos from 12 h to 48 h of development/treatment. (B) Line graph reporting the modulation over time of the total proteolytic activity for each development stage and V-concentration. (C) Pie chart showing the distribution of the total proteolytic activity for the entire time of development/treatments. (D) Zymogram showing gelatinase bands in lysates of control and V-treated (1 mM; 500 µM; 100 µM; 50 µM; 1 µM; 500 nM; 100 nM) embryos at 36 h of development. (E) Histogram showing the total proteolytic activity of control and V-treated (1 mM; 500 µM; 100 µM; 50 µM; 1 µM; 500 nM; 100 nM) embryos at 36 h of development. Experiments were performed in triplicate and data are expressed as means ± standard deviation (n = 3 ± SD). The band intensity of panels A and D was measured by Quantity One software. Statistical analysis was performed using t-test with * p ≤ 0.05; p ≤ 0.01; * p ≤ 0.0005.
	Figure 4. Single proteolytic activities. Line graph reporting the modulation over time, from 12 h to 48 h of each proteolytic activity, in controls and V-treated (1 mM, 500 µM) embryos. According to their molecular weight, the identified proteases are as follows: (A1,A2) 309 kDa; (B1,B2) 255 kDa; (C1,C2) 177 kDa; (D1,D2) 79 kDa; (E1,E2) 59 kDa; (F1,F2) 34 kDa; (G1,G2) 30 kDa; (H1,H2) 25 kDa; and (I1,I2) 22 kDa. The pie charts display the value of each gelatinase activity related to the whole development/treatment time. Experiments were performed in triplicate and graph data are expressed as means ± standard deviation (n = 3 ± SD). Statistical analysis was performed using t-test with * p ≤ 0.05; p ≤ 0.01; * p ≤ 0.0005.
	Figure 5. Immunoblotting detection and quantitative analysis for MMPs. (A–C) Total lysates of control and V-treated embryos (1 mM, 500 μM) after 36 h of development/treatment, immunoreacted with anti- (A) MMP-2, (B) MMP-9, and (C) MMP-14 antibodies. Histograms showed the densitometric analysis of the obtained bands. (D) Total lysates of control and V-treated embryos (1 mM, 500 μM) after 12, 15, 18, 21, 24, 30, 36, 42, and 48 h of development/treatment immunoreacted with anti-MMP-14 antibodies. Histograms showed the densitometric analysis of the bands. (E) Total lysates of control and V-treated (1 mM; 500 µM; 100 µM; 50 µM; 1 µM; 500 nM; 100 nM) embryos after 36 h of development/treatment immunoreacted with anti-MMP-14 antibodies. Histograms showed the densitometric analysis of the bands. Actin was used as a loading control. Relative protein expression, reported as arbitrary units, was calculated as the band density ratio to that of actin. Experiments were performed in triplicate and data are expressed as means ± standard deviation (n = 3 ± SD). Statistical analysis was performed using t-test with * p ≤ 0.05; p ≤ 0.01; * p ≤ 0.0005.
	Figure 6. Immunolocalization analysis of MMP-14-like protease in whole mount embryos. Shown are representative confocal microscopy images of equatorial optical sections of embryos at 36 h of development/treatment. (A1,B1,C1) MMP-14-like protein detection; (A2,B2,C2,D2) nuclei counterstaining with propidium iodide (PI); (A3,B3,C3,D3) merge of green (MMP-14) and red (PI) signals. (A1–A3) Control embryo; (D1) negative control with only the secondary antibody; (B1–B3) 1 mM V-treated embryo; (C1–C3) 500 µM V-treated embryo. (A4,B4,C4) Higher magnification of a section of (A3) control, (B3) 1 mM V-treated, and (C3) 500 µM V-treated embryo, respectively. White arrows indicate the localization of MMP-14-like in filopodia. Scale bar = 45 µm.

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	Figure 1. Morphological and morphometric effects induced by V during embryonic development. Images representative of embryos from 12 h to 48 h of development/treatment. (A1,B1,C1,D1,E1,F1,G1,H1,I1) Control embryos; (A2,B2,C2,D2,E2,F2,G2,H2,I2) 1 mM V-treated embryos; (A3,B3,C3,D3,E3,F3,G3,H3,I3) 500 µM V-treated embryos. Control and V-treated embryos were observed at different development/treatment intervals: (A1–A3) 12 h; (B1–B3) 15 h; (C1–C3) 18 h; (D1–D3) 21 h; (E1–E3) 24 h; (F1–F3) 30 h; (G1–G3) 36 h; (H1–H3) 42 h; and (I1–I3) 48 h. Scale bar = 45 µm. Images are representative of three independent experiments. The line graph shows data related to the volumetric analysis. Experiments were performed in triplicate and data are expressed as means ± standard deviation (n = 3 ± SD). Statistical analysis was performed using t-test with * p ≤ 0.05.
	Figure 2. Amount of V and Ca incorporated in embryos after 48 h of development/treatment. Embryos were cultured under normal growth conditions and with V 1 mM or 500 µM. (A) V and (B) Ca content. Values reported in the pie charts show the ionic concentrations detected for V (µg/g weight embryos) and Ca (mg/g weight embryos). Experiments were performed in triplicate and data are expressed as means ± standard deviation (n = 3 ± SD). Statistical analysis was performed using t-test with p ≤ 0.01; * p ≤ 0.0005.
	Figure 3. Metal-related enzymatic activities analysed by gelatin substrate gel zymography. (A) Zymograms showing bands in lysates of control and V-treated (1 mM, 500 µM) embryos from 12 h to 48 h of development/treatment. (B) Line graph reporting the modulation over time of the total proteolytic activity for each development stage and V-concentration. (C) Pie chart showing the distribution of the total proteolytic activity for the entire time of development/treatments. (D) Zymogram showing gelatinase bands in lysates of control and V-treated (1 mM; 500 µM; 100 µM; 50 µM; 1 µM; 500 nM; 100 nM) embryos at 36 h of development. (E) Histogram showing the total proteolytic activity of control and V-treated (1 mM; 500 µM; 100 µM; 50 µM; 1 µM; 500 nM; 100 nM) embryos at 36 h of development. Experiments were performed in triplicate and data are expressed as means ± standard deviation (n = 3 ± SD). The band intensity of panels A and D was measured by Quantity One software. Statistical analysis was performed using t-test with * p ≤ 0.05; p ≤ 0.01; * p ≤ 0.0005.
	Figure 4. Single proteolytic activities. Line graph reporting the modulation over time, from 12 h to 48 h of each proteolytic activity, in controls and V-treated (1 mM, 500 µM) embryos. According to their molecular weight, the identified proteases are as follows: (A1,A2) 309 kDa; (B1,B2) 255 kDa; (C1,C2) 177 kDa; (D1,D2) 79 kDa; (E1,E2) 59 kDa; (F1,F2) 34 kDa; (G1,G2) 30 kDa; (H1,H2) 25 kDa; and (I1,I2) 22 kDa. The pie charts display the value of each gelatinase activity related to the whole development/treatment time. Experiments were performed in triplicate and graph data are expressed as means ± standard deviation (n = 3 ± SD). Statistical analysis was performed using t-test with * p ≤ 0.05; p ≤ 0.01; * p ≤ 0.0005.
	Figure 5. Immunoblotting detection and quantitative analysis for MMPs. (A–C) Total lysates of control and V-treated embryos (1 mM, 500 μM) after 36 h of development/treatment, immunoreacted with anti- (A) MMP-2, (B) MMP-9, and (C) MMP-14 antibodies. Histograms showed the densitometric analysis of the obtained bands. (D) Total lysates of control and V-treated embryos (1 mM, 500 μM) after 12, 15, 18, 21, 24, 30, 36, 42, and 48 h of development/treatment immunoreacted with anti-MMP-14 antibodies. Histograms showed the densitometric analysis of the bands. (E) Total lysates of control and V-treated (1 mM; 500 µM; 100 µM; 50 µM; 1 µM; 500 nM; 100 nM) embryos after 36 h of development/treatment immunoreacted with anti-MMP-14 antibodies. Histograms showed the densitometric analysis of the bands. Actin was used as a loading control. Relative protein expression, reported as arbitrary units, was calculated as the band density ratio to that of actin. Experiments were performed in triplicate and data are expressed as means ± standard deviation (n = 3 ± SD). Statistical analysis was performed using t-test with * p ≤ 0.05; p ≤ 0.01; * p ≤ 0.0005.
	Figure 6. Immunolocalization analysis of MMP-14-like protease in whole mount embryos. Shown are representative confocal microscopy images of equatorial optical sections of embryos at 36 h of development/treatment. (A1,B1,C1) MMP-14-like protein detection; (A2,B2,C2,D2) nuclei counterstaining with propidium iodide (PI); (A3,B3,C3,D3) merge of green (MMP-14) and red (PI) signals. (A1–A3) Control embryo; (D1) negative control with only the secondary antibody; (B1–B3) 1 mM V-treated embryo; (C1–C3) 500 µM V-treated embryo. (A4,B4,C4) Higher magnification of a section of (A3) control, (B3) 1 mM V-treated, and (C3) 500 µM V-treated embryo, respectively. White arrows indicate the localization of MMP-14-like in filopodia. Scale bar = 45 µm.