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A PKC-dependent recruitment of MMP-2 controls semaphorin-3A growth-promoting effect in cortical dendrites.

Gonthier B, Koncina E, Satkauskas S, Perraut M, Roussel G, Aunis D, Kapfhammer JP, Bagnard D - PLoS ONE (2009)

Bottom Line: The in situ analysis of MMP-2 expression and activity is consistent with a functional growth assay demonstrating in vitro that the pharmacological inhibition of MMP-2 reduces the growth of cortical dendrites in response to Sema3A.Hence, our results suggest that the selective recruitment and activation of MMP-2 in response to Sema3A requires a PKC alpha dependent mechanism.Altogether, we provide a second set of data supporting MMPs as effectors of the growth-promoting effects of semaphorins, and we identify the potential signalling pathway involved.

View Article: PubMed Central - PubMed

Affiliation: INSERM U575, Physiopathologie du Système Nerveux, Strasbourg, France.

ABSTRACT
There is increasing evidence for a crucial role of proteases and metalloproteinases during axon growth and guidance. In this context, we recently described a functional link between the chemoattractive Sema3C and Matrix metalloproteinase 3 (MMP3). Here, we provide data demonstrating the involvement of MMP-2 to trigger the growth-promoting effect of Sema3A in cortical dendrites. The in situ analysis of MMP-2 expression and activity is consistent with a functional growth assay demonstrating in vitro that the pharmacological inhibition of MMP-2 reduces the growth of cortical dendrites in response to Sema3A. Hence, our results suggest that the selective recruitment and activation of MMP-2 in response to Sema3A requires a PKC alpha dependent mechanism. Altogether, we provide a second set of data supporting MMPs as effectors of the growth-promoting effects of semaphorins, and we identify the potential signalling pathway involved.

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Sema3A-dependent expression and activity of MMP-2.(A) Western Blotting analysis of MMP-2 and MMP-9 expression. The expression of MMP-2 increases in the presence of Sema3A, while no modification occurs for MMP-9. (B) Determination of MMP-2 and MMP-9 enzymatic activity by ELISA assay. MMP-2 activity increases in the presence of Sema3A without modification MMP-9 activity. (* p<0.01 student's t test/control, ns not significant).
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pone-0005099-g005: Sema3A-dependent expression and activity of MMP-2.(A) Western Blotting analysis of MMP-2 and MMP-9 expression. The expression of MMP-2 increases in the presence of Sema3A, while no modification occurs for MMP-9. (B) Determination of MMP-2 and MMP-9 enzymatic activity by ELISA assay. MMP-2 activity increases in the presence of Sema3A without modification MMP-9 activity. (* p<0.01 student's t test/control, ns not significant).

Mentions: To characterize further the involvement of gelatinases in the growth promoting effect of Sema3A, we performed western blot analysis (4 independent experiments) and enzymatic activity assays (3 independent experiments including triplicates of each experimental condition). As depicted in Figure 5A, cortical neurons in culture express both MMP-2 and MMP-9 consistently with RT-PCR and in situ observations. The addition of Sema3A in the culture medium induced 49% increase of MMP-2 expression while MMP-9 expression remained unchanged. Thus, Sema3A triggers a specific induction of MMP-2 expression. Using an Elisa-based activity assay, we also found that the induction of MMP-2 expression was associated with an increased enzymatic activity (+23%) in culture media while MMP-9 activity was not changed (Figure 5B). These results demonstrate that Sema3A signalling triggers a specific induction of MMP-2 expression and activity. To demonstrate the specificity of the link between MMP-2 and Sema3A we monitored MMP-2 activity in the presence of a function-blocking antibody directed against Neuropilin-1, the major binding receptor of Sema3A. To this end, cortical neurons were grown in control conditions or in the presence of 100 ng/ml Sema3A, with or without 1 μg/ml anti-NRP1. As expected from previous studies, the selective blocking of NRP1 suppressed the Sema3A-dependent growth promotion of cortical dendrites (Figure 6A). Consistently, we found that blocking NRP1 suppressed Sema3A-induced augmentation of MMP-2 activity (Figure 6B).


A PKC-dependent recruitment of MMP-2 controls semaphorin-3A growth-promoting effect in cortical dendrites.

Gonthier B, Koncina E, Satkauskas S, Perraut M, Roussel G, Aunis D, Kapfhammer JP, Bagnard D - PLoS ONE (2009)

Sema3A-dependent expression and activity of MMP-2.(A) Western Blotting analysis of MMP-2 and MMP-9 expression. The expression of MMP-2 increases in the presence of Sema3A, while no modification occurs for MMP-9. (B) Determination of MMP-2 and MMP-9 enzymatic activity by ELISA assay. MMP-2 activity increases in the presence of Sema3A without modification MMP-9 activity. (* p<0.01 student's t test/control, ns not significant).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2663036&req=5

pone-0005099-g005: Sema3A-dependent expression and activity of MMP-2.(A) Western Blotting analysis of MMP-2 and MMP-9 expression. The expression of MMP-2 increases in the presence of Sema3A, while no modification occurs for MMP-9. (B) Determination of MMP-2 and MMP-9 enzymatic activity by ELISA assay. MMP-2 activity increases in the presence of Sema3A without modification MMP-9 activity. (* p<0.01 student's t test/control, ns not significant).
Mentions: To characterize further the involvement of gelatinases in the growth promoting effect of Sema3A, we performed western blot analysis (4 independent experiments) and enzymatic activity assays (3 independent experiments including triplicates of each experimental condition). As depicted in Figure 5A, cortical neurons in culture express both MMP-2 and MMP-9 consistently with RT-PCR and in situ observations. The addition of Sema3A in the culture medium induced 49% increase of MMP-2 expression while MMP-9 expression remained unchanged. Thus, Sema3A triggers a specific induction of MMP-2 expression. Using an Elisa-based activity assay, we also found that the induction of MMP-2 expression was associated with an increased enzymatic activity (+23%) in culture media while MMP-9 activity was not changed (Figure 5B). These results demonstrate that Sema3A signalling triggers a specific induction of MMP-2 expression and activity. To demonstrate the specificity of the link between MMP-2 and Sema3A we monitored MMP-2 activity in the presence of a function-blocking antibody directed against Neuropilin-1, the major binding receptor of Sema3A. To this end, cortical neurons were grown in control conditions or in the presence of 100 ng/ml Sema3A, with or without 1 μg/ml anti-NRP1. As expected from previous studies, the selective blocking of NRP1 suppressed the Sema3A-dependent growth promotion of cortical dendrites (Figure 6A). Consistently, we found that blocking NRP1 suppressed Sema3A-induced augmentation of MMP-2 activity (Figure 6B).

Bottom Line: The in situ analysis of MMP-2 expression and activity is consistent with a functional growth assay demonstrating in vitro that the pharmacological inhibition of MMP-2 reduces the growth of cortical dendrites in response to Sema3A.Hence, our results suggest that the selective recruitment and activation of MMP-2 in response to Sema3A requires a PKC alpha dependent mechanism.Altogether, we provide a second set of data supporting MMPs as effectors of the growth-promoting effects of semaphorins, and we identify the potential signalling pathway involved.

View Article: PubMed Central - PubMed

Affiliation: INSERM U575, Physiopathologie du Système Nerveux, Strasbourg, France.

ABSTRACT
There is increasing evidence for a crucial role of proteases and metalloproteinases during axon growth and guidance. In this context, we recently described a functional link between the chemoattractive Sema3C and Matrix metalloproteinase 3 (MMP3). Here, we provide data demonstrating the involvement of MMP-2 to trigger the growth-promoting effect of Sema3A in cortical dendrites. The in situ analysis of MMP-2 expression and activity is consistent with a functional growth assay demonstrating in vitro that the pharmacological inhibition of MMP-2 reduces the growth of cortical dendrites in response to Sema3A. Hence, our results suggest that the selective recruitment and activation of MMP-2 in response to Sema3A requires a PKC alpha dependent mechanism. Altogether, we provide a second set of data supporting MMPs as effectors of the growth-promoting effects of semaphorins, and we identify the potential signalling pathway involved.

Show MeSH