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

SCL Assembles a Multifactorial Complex That Determines Glycophorin A Expression

SCL/TAL1 is a hematopoietic-specific transcription factor of the basic helix-loop-helix (bHLH) family that is essential for erythropoiesis. Here we identify the erythroid cell-specific glycophorin A gene (GPA) as a target of SCL in primary hematopoietic cells and show that SCL occupies the GPA locus in vivo. GPA promoter activation is dependent on the assembly of a multifactorial complex containing SCL as well as ubiquitous (E47, Sp1, and Ldb1) and tissue-specific (LMO2 and GATA-1) transcription factors. In addition, our observations suggest functional specialization within this complex, as SCL provides its HLH protein interaction motif, GATA-1 exerts a DNA-tethering function through its binding to a critical GATA element in the GPA promoter, and E47 requires its N-terminal moiety (most likely entailing a transactivation function). Finally, endogenous GPA expression is disrupted in hematopoietic cells through the dominant-inhibitory effect of a truncated form of E47 (E47-bHLH) on E-protein activity or of FOG (Friend of GATA) on GATA activity or when LMO2 or Ldb-1 protein levels are decreased. Together, these observations reveal the functional complementarities of transcription factors within the SCL complex and the essential role of SCL as a nucleation factor within a higher-order complex required to activate gene GPA expression

Differential Expression of the Tetraspanin CD9 in Normal and Leukemic Stem Cells

CD9 plays a crucial role in cellular growth, mobility, and signal transduction, as well as in hematological malignancy. In myeloid neoplasms, CD9 is involved in the altered interactions between leukemic and stromal cells. However, apart from its role in CD34+ progenitors and myeloid and megakaryocytic differentiation, its function in normal and leukemic pluripotent cells has not yet been determined. Very small embryonic-like stem cells (VSELs) are promising pluripotent stem cells found in adult tissues that can be developed for safe and efficient regenerative medicine. VSELs express different surface receptors of the highest importance in cell functioning, including CD9, and can be effectively mobilized after organ injury or in leukemic patients. In the present study, we observed that CD9 is among the most expressed receptors in VSELs under steady-state conditions; however, once the VSELs are expanded, CD9+ VSELs decrease and are more apoptotic. CD9– VSELs had no proliferative improvement in vitro compared to those that were CD9+. Interestingly, the addition of SDF-1 induced CD9 expression on the surface of VSELs, as observed by flow cytometry, and improved their migration. In addition, we observed, in the phenotypically identical VSELs present in the peripheral blood of patients with myeloproliferative neoplasms, compared to healthy subjects, a significantly higher number of CD9+ cells. However, in their hematopoietic stem cell (HSC) counterparts, the expression remained comparable. These results indicate that, likewise, in progenitors and mature cells, CD9 may play an important function in normal and malignant VSELs. This could explain the refractoriness observed by some groups of expanded stem cells to repairing efficiently damaged tissue when used as a source in cell therapies. Understanding the function of the CD9 receptor in normal and malignant CD34+ and VSELs, along with its relationship with the CXCR4/SDF-1 pathway, will enable advances in the field of adult pluripotent cell usage in regenerative medicine and in their role in leukemia

Molecular mechanisms of anthracyclines-mediated differentiation of human erythroleukemic cells.

International audienc

Molecular mechanisms of anthracyclines-mediated differentiation of human erythroleukemic cells.

International audienc

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

Schematic representation of the primary structure of proVEGF-C, its activating proteases and receptors R2 and R3.

<p>Upon synthesis and translocation into the Golgi network, proVEGF-C is processed at HSIIRR₂₁₄, dividing it into N-terminal (VEGF-C) and C-terminal polypeptides. The cleavage of proVEGF-C can also occurs <i>via</i> cell surface-attached convertases (PCs). Interaction of mature VEGF-C (wt) with the receptors VEGF/R3 and VEGF/R2 leads to Akt/ERK activation and functions. Mutation of the cleavage site of proVEGF-C (mut) prevents Akt/ERK activation and cell proliferation.</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