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ScientificWorldJournal
2014 Jan 30;2014:467907. doi: 10.1155/2014/467907.
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Functional characterization of tissue inhibitor of metalloproteinase-1 (TIMP-1) N- and C-terminal domains during Xenopus laevis development.
Nieuwesteeg MA, Willson JA, Cepeda M, Fox MA, Damjanovski S.
???displayArticle.abstract??? Extracellular matrix (ECM) remodeling is essential for facilitating developmental processes. ECM remodeling, accomplished by matrix metalloproteinases (MMPs), is regulated by endogenous tissue inhibitors of metalloproteinases (TIMPs). While the TIMP N-terminal domain is involved in inhibition of MMP activity, the C-terminal domain exhibits cell-signaling activity, which is TIMP and cell type dependent. We have previously examined the distinct roles of the Xenopus laevis TIMP-2 and -3 C-terminal domains during development and here examined the unique roles of TIMP-1 N- and C-terminal domains in early X. laevis embryos. mRNA microinjection was used to overexpress full-length TIMP-1 or its individual N- or C-terminal domains in embryos. Full-length and C-terminal TIMP-1 resulted in increased lethality compared to N-terminal TIMP-1. Overexpression of C-terminal TIMP-1 resulted in significant decreases in mRNA levels of proteolytic genes including TIMP-2, RECK, MMP-2, and MMP-9, corresponding to decreases in MMP-2 and -9 protein levels, as well as decreased MMP-2 and MMP-9 activities. These trends were not observed with the N-terminus. Our research suggests that the individual domains of TIMP-1 are capable of playing distinct roles in regulating the ECM proteolytic network during development and that the unique functions of these domains are moderated in the endogenous full-length TIMP-1 molecule.
Figure 1. Evolutionary conservation of TIMP-1 N- and C-terminal domains. Sequence analysis comparing amino acid sequence identity of X. laevis TIMP-1 N- and C-terminal domains versus known (at the time of publication) vertebrate species. Light purple boxes represent species whose N-terminal domains were more highly conserved with X. laevis TIMP-1 than their C-terminal domains. Light pink boxes represent species whose C-terminal domains were more highly conserved with X. laevis TIMP-1 than their N-terminal domains. Blue boxes represent species with equal conservation with both X. laevis TIMP-1 N- and C-terminal domains.
Figure 2. Confirmation of full-length, N-terminal, or C-terminal TIMP-1 constructs overexpressed in X. laevis embryos. 4 ng of mRNA coding for full-length (T1FL), N-terminal (T1N), or C-terminal (T1C) TIMP-1 constructs was microinjected into X. laevis embryos at the 1-cell stage. (a) Overexpression of TIMP-1 mRNA as shown by RT-PCR analysis. RNA was isolated from stage 15 embryos. Control (uninjected) embryos expressed TIMP-1 at very low levels. Primers specific to each construct were used to confirm mRNA levels of T1FL (678 bp), T1N (453 bp), and T1C (252 bp). RT-PCR of EF1α was used as a loading control. (b) Increased levels of TIMP-1 constructs as shown by Western blot analysis. All constructs are HA tagged. Protein was isolated from stage 30 embryos. Anti-HA antibodies were used to confirm expression of each construct at the protein level (T1FL = 26 KDa, T1N = 18 KDa, and T1C = 12 KDa). No HA was detected in control uninjected embryos. β-actin was used as protein loading control.
Figure 3. Phenotypic effects of overexpression of full-length, N-terminal, or C-terminal TIMP-1 constructs. Following injection, photographs were taken of representative embryos at stage 30. (a) Control (uninjected) embryos were phenotypically normal. Microinjection of T1FL (b) and T1N (c) constructs both resulted in normal anterior (head) development but truncated posterior axes. (d) Microinjection of T1C constructs resulted in lack of head structures, other head defects, and neural tube closure failure. Panel (a) magnification is lower than in (b), (c), and (d). Embryos are 1 mm in diameter.
Figure 4. Overexpression of all three TIMP-1 constructs leads to abnormal development and death. Following injection of mRNA constructs at the 1-cell stage, embryos were scored for a normal phenotype at stages 15 and 30. Dead and abnormal embryos were counted as containing morphological defects. The graph illustrates the percentage of normal embryos following injection of GFP mRNA (GFP), or full-length (T1FL), N-terminal (T1N), or C-terminal (T1C) TIMP-1 mRNA constructs at the given stage. Control embryos are uninjected. Results are based on 3 independent sets of experiments; bars indicate standard error.
Figure 5. Effect of overexpression of full-length, N-terminal, or C-terminal TIMP-1 on mRNA levels of MMP inhibitors. Semiquantitative RT-PCR analysis was used to measure changes in mRNA levels of (a) TIMP-2, (b) TIMP-3, and (c) RECK at stage 30, following microinjection of 4 ng of TIMP-1 full-length (T1FL), N-terminal (T1N), or C-terminal (T1C) constructs into X. laevis embryos at the 1-cell stage. In each case mRNA levels were measured relative to Ef1α. The results are presented as mean ± standard error from 3 independent experiments. *P < 0.05 and **P < 0.01, all versus control (CONT; uninjected) embryos, as analyzed by one-way ANOVA and Dunnett's post hoc test.
Figure 6. Effect of overexpression of full-length, N-terminal, or C-terminal TIMP-1 on mRNA levels of MMPs. Semiquantitative RT-PCR analysis was used to measure changes in mRNA levels of (a) MMP-2, (b) MMP-9, and (c) MT1-MMP at stage 30, following microinjection of 4 ng of TIMP-1 full-length (T1FL), N-terminal (T1N), or C-terminal (T1C) constructs into X. laevis embryos at the 1-cell stage. In each case mRNA levels were measured relative to Ef1α. The results are presented as mean ± standard error from 3 independent experiments. *P < 0.05 and **P < 0.01, all versus control (CONT; uninjected) embryos, as analyzed by one-way ANOVA and Dunnett's post hoc test.
Figure 7. Zymography demonstrated altered levels of active MMP-2 and MMP-9 following overexpression of TIMP-1 constructs. 4 ng of mRNA coding for full-length (T1FL), N-terminal (T1N), or C-terminal (T1C) constructs was injected into X. laevis embryos at the 1-cell stage and protein was isolated from stage 30 embryos. Gelatin zymography was used to measure changes in (a) MMP-2 and (b) MMP-9 activity. Zymogram (top) is representative of one experiment where the bright brand represents the active forms (63 and 84 kDa) of MMP-2 and MMP-9, respectively. Graphs represent quantification of above zymogram, and data is presented as mean ± standard error from 3 independent experiments. *P < 0.05 and **P < 0.01, all versus control (CONT; uninjected) embryos, as analyzed by one-way ANOVA and Dunnett's post hoc test.
Figure 8. Reverse zymography demonstrating full-length and N-terminal, but not C-terminal, TIMP-1 can directly inhibit MMP activity. 4 ng of mRNA coding for full-length (T1FL), N-terminal (T1N), or C-terminal (T1C) TIMP-1 constructs was injected into X. laevis embryos at the 1-cell stage and protein was isolated from stage 30 embryos. MMP-inhibitory activity is represented by dark bands. White arrows indicate the location of the expected inhibitory band of the T1FL (26 KDa) and T1N (18 KDa) constructs, respectively. Black arrow represents position of T1C (12 KDa) construct, which cannot inhibit MMP activity as suggested by the light band.
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