Zebrafish ProVEGF-C Expression, Proteolytic Processing and Inhibitory Effect of Unprocessed ProVEGF-C during Fin Regeneration

BACKGROUND: In zebrafish, vascular endothelial growth factor-C precursor (proVEGF-C) processing occurs within the dibasic motif HSIIRR(214) suggesting the involvement of one or more basic amino acid-specific proprotein convertases (PCs) in this process. In the present study, we examined zebrafish proVEGF-C expression and processing and the effect of unprocessed proVEGF-C on caudal fin regeneration. METHODOLOGY/PRINCIPAL FINDINGS: Cell transfection assays revealed that the cleavage of proVEGF-C, mainly mediated by the proprotein convertases Furin and PC5 and to a less degree by PACE4 and PC7, is abolished by PCs inhibitors or by mutation of its cleavage site (HSIIRR(214) into HSIISS(214)). In vitro, unprocessed proVEGF-C failed to activate its signaling proteins Akt and ERK and to induce cell proliferation. In vivo, following caudal fin amputation, the induction of VEGF-C, Furin and PC5 expression occurs as early as 2 days post-amputation (dpa) with a maximum levels at 4-7 dpa. Using immunofluorescence staining we localized high expression of VEGF-C and the convertases Furin and PC5 surrounding the apical growth zone of the regenerating fin. While expression of wild-type proVEGF-C in this area had no effect, unprocessed proVEGF-C inhibited fin regeneration. CONCLUSIONS/SIGNIFICANCES: Taken together, these data indicate that zebrafish fin regeneration is associated with up-regulation of VEGF-C and the convertases Furin and PC5 and highlight the inhibitory effect of unprocessed proVEGF-C on fin regeneration

Requirement of the PCs in VEGF-C-mediated Akt and ERK phosphorylation in ZF4 cells.

<p>Confluent ZF4 cells were serum starved for 24–48 h and then treated for 2 min at 37°C with media derived from LoVo cells transiently cotransfected with empty vectors and pSecTagB vector containing proVEGF-C construct (Control) or pSecTagB vector containing proVEGF-C cDNA and pIRES2-EGFP vector expressing full-length Furin, PACE4, PC5 or PC7 cDNAs. Equal amounts of cell lysates were subjected to Western blotting using an anti-phospho-Akt (A), or an anti-phospho-ERK (B). The anti-Akt and anti-ERK were used for data normalization. Results are representative of three experiments.</p

Expression of PC5 during fin regeneration.

<p>(A) Total RNA was isolated from fins (15-20 fins per time point) and analyzed by real-time PCR using specific primers for zebrafish PC5 or β-actin. Results are shown in the bar graph and are expressed as the ratio of the indicated transcripts relative to control (0 dpa). Results are shown as means ± S.E. of three experiments performed in triplicate. (B) Immunofluorescence analysis revealed that PC5 is expressed in all area of the regenerating fin and low signal was observed on the vessels (red signal, 25× objective).</p

Effect of proVEGF-C processing on ZF4 cells proliferation.

<p>(A) ZF4 cells were serum deprived overnight and then treated for 24 h with media derived from LoVo cells transiently cotransfected with empty vectors (Control) or empty vector and vector containing proVEGF-C construct or vector expressing proVEGF-C cDNA and vector expressing Furin cDNA. Cell proliferation was assessed using Cell Titer96 non-radioactive cell proliferation assay. Results are shown as means ± S.E. of three experiments performed in triplicate. (B) Total RNA derived from ZF4 cells was subjected to real-time PCR analysis using specific primers for the zebrafish VEGF-C receptors R2, R3 or β-actin. During PCR, the transcription of β-actin that was evaluated in each sample was used as endogenous control. Results are shown in the bar graph and are expressed as the ratio of the indicated transcripts relative to R2 transcript assigned to 100%. Results are shown as means ± S.E. of three experiments performed in duplicate.</p

PCs expression and activity in ZF4 cells.

<p>(A) Following total RNA extraction from 10⁴ x ZF4 cells, real-time PCR analysis was performed using specific primers for Furin, PC5 or β-actin zebrafish as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011438#s2" target="_blank">Material and Methods</a>. During PCR, the transcription of β-actin that was evaluated in each sample was used as endogenous control. Results are shown in the bar graph and are expressed as the percentage of the indicated transcripts relative to Furin transcript (100%). Data are shown as means ± S.E of three experiments performed in duplicate. (B) PCs activity in ZF4 cells was assessed by evaluating the cells protein extract ability to digest the universal PCs substrate, the fluorogenic peptide pERTKR-MCA at the indicated time periods. Digestion of pERTKR-MCA by recombinant Furin (2 unit/µl) is given for comparison. As can be seen, the PCs inhibitor peptidyl chloromethyl ketones (CMK) (10 µM) reduced dramatically the PCs activity in ZF4 cells and the activity of recombinant Furin. Results are representative of two experiments performed in triplicate and data are mean ± S.E. *p<0.005; **p<0.0001.</p

Uprocessed ProVEGF-C inhibits fin regeneration.

<p>(A) 24 h prior amputation of caudal fins (6 per group), empty vector (Control) or vector containing wild-type (wt) or mutant (mut) proVEGF-C constructs were injected in the fins and animals were allowed to regenerate at 28.5°C after fins amputation for 5 days. The microinjection of vector containing wild-type proVEGF-C had no effect on normal regeneration. In contrast, injection of the mutant proVEGF-C resulted in severe inhibition of fin regeneration. Results are representative of tree experiments. The corresponding percentages of regenerated area were deduced from the ratio of 100 x (fin surface regenerated in VEGF-Cwt)/(fin surface regenerated in Control) and 100 x (fin surface regenerated in VEGF-Cmut/fin surface regenerated in Control). (B) Total RNA derived from uncut fins was subjected to real-time PCR analysis using specific primers for the VEGF-C receptors R2, R3 or β-actin. During PCR, the transcription of β-actin that was evaluated in each sample was used as endogenous control. Results are shown in the bar graph and are expressed as the ratio of the indicated transcripts relative to R2 transcript assigned to 100%. Results are shown as means ± S.E. of three experiments performed in duplicate.</p

Processing of zebrafish proVEGF-C.

<p>(A) Processing of proVEGF-C was analyzed by Western blotting in zebrafish ZF4 cells transiently cotransfected with pSecTagB (Myc tag) vector containing the zebrafish proVEGF-C cDNA and the empty pIRES2-EGFP vector (Control) or pSecTagB expressing proVEGF-C and pIRES2-EGFP containing the PCs inhibitors; ppFurin or α1-PDX. Results of band intensities are shown in the bar graph that were deduced from the ratio of VEGF-C/(pro-VEGF-C + VEGF-C). Results are representative of three experiments. (B) The processing of proVEGF-C was analyzed by Western blotting using anti-Myc antibody on media derived from PCs-deficient LoVo cells transiently cotransfected with either pIRES2-EGFP vector alone and pSecTagB (Myc tag) containing the zebrafish VEGF-C cDNA (Control) or pIRES2-EGFP vector containing Furin, PACE4, PC5 or PC7 cDNA. The corresponding percentages of band intensities were deduced from the ratio of VEGF-C/(proVEGF-C+VEGF-C). Results are representative of three experiments.</p

Expression of Furin during fin regeneration.

<p>(A) Total RNA was isolated from fins (15–20 fins per time point) and analyzed by real-time PCR using specific primers for zebrafish Furin or β-actin. Results are shown in the bar graph and are expressed as the ratio of the indicated transcripts relative to control (0 dpa). Results are shown as means ± S.E. of three experiments performed in triplicate. (B) Immunofluorescence analysis revealed that Furin is mainly localized to the apical growth zone of the regenerating fin (red signal, 25× objective).</p

Primers used for Real-time PCR analysis.

<p>Primers used for Real-time PCR analysis.</p