Furin gene (fur) regulation in differentiating human megakaryoblastic Dami cells: involvement of the proximal GATA recognition motif in the P1 promoter and impact on the maturation of furin substrates

Abstract

The convertase furin is involved in the maturation of key growth/aggregation mediators synthesized by the platelet producers, megakaryocytes, but the regulation of furin in these cells remains unknown. Computer-assisted search of the furin promoter sequence revealed multiple potential binding motifs for GATA-1, suggesting that furin is expressed and regulated in these cells. Using megakaryoblastic Dami cells, we observed that fur mRNA expression increased gradually on phorbol 12-myristate 13-acetate-induced differentiation, reaching maximum levels (8.3-fold increase) at 10 days. Transient transfections with P1, P1A, or P1B fur-LUC-promoter constructs revealed that in Dami cells, the P1 promoter is the strongest and the most sensitive to forced expression of GATA-1. Coexpression of GATA-1 and its comodulator, Friend of GATA-1 (FOG-1), resulted in a cooperative increase in P1 activity. Deletion analysis indicated that important GATA-1-regulated sequences are located in the most proximal region of the P1 promoter. Further analysis revealed 2 potential GATA-binding motifs at positions ؊66 and ؉62. Point mutation of each of the 2 motifs indicated that the intactness of the first GATA site is required for full basal and GATA-1-stimulated promoter activity. Finally, the inhibition of furin activity through gene transfer of the inhibitor ␣1-AT-PDX led to a block in maturation of the furin substrates transforming growth factor-␤1 and platelet-derived growth factor. Taken together, these results indicate that the most proximal GATA element in the P1 promoter is needed for fur gene expression in megakaryoblastic cells. They also suggest that proper regulation of the fur gene in megakaryocytes has an impact on the activation of furin substrates involved in megakaryocyte maturation and platelet functions. IntroductionPlatelet generation implies a set of well-regulated processes that take place during megakaryocyte differentiation. These processes are mediated by bioactive proteins; among them are the growth/differentiation factors transforming growth factor-␤1 (⌻GF-␤1) 1,2 and platelet-derived growth factor (PDGF), 3 the key platelet adhesion molecules ␣ IIb 4,5 and von Willebrand factor (VWF), 6 and the cell-surface receptor Notch-1, 7 which are first synthesized as larger, inactive precursor molecules. Their activation occurs through limited endoproteolytic cleavage after a sequence of 2 or more basic residues (K or R). In the 1990s, 7 closely related mammalian subtilisin/kexin-like serine proteases with this cleavage specificity were discovered. They are grouped under the generic name proprotein convertases (PCs) and include PC1/PC3, PC2, PC4, PC5/PC6, PC7, PACE 4, and furin. 8 Among the convertases, furin represents the first and so far the best-characterized enzyme. The biologic importance of this convertase stems from the large number and variety of bioactive proteins and peptides that can be generated through its activity. These include key elements involved in normal and physiopathologic conditions, as exemplified b...

Notch1 Autoactivation via Transcriptional Regulation of Furin, Which Sustains Notch1 Signaling by Processing Notch1-Activating Proteases ADAM10 and Membrane Type 1 Matrix Metalloproteinase

“…The Hes1-reporter plasmid was a kind gift from Ryoichiro Kageyama (Kyoto University, Kyoto, Japan) (57). The human furin promoter-luciferase constructs pGL2-Basic and pGL2-SacI were generously provided by Claire M. Dubois (University of Sherbrooke, Sherbrooke, Quebec, Canada) (43). The mutations within the pGL2-SacI reporter were generated by PCR using primers 5=-CCTGTGACGTCA CAGCTCCTCATTCTGCGACAGTGG-3= and 5=-GAATGAGGAGCT GTGACGTCACAGGATGGTGGTTAG-3=, which replace the sequence 5=-TTCCCAC-3= with 5=-TCACAGC-3=.…”

“…Furin is a calcium-dependent serine protease that belongs to the family of proprotein convertases (PCs). The expression levels of furin differ for different cell types and degrees of cell differentiation (43)(44)(45)47). Furin has been reported to regulate tumor growth and malignant tumor phenotypes (48)(49)(50).…”

Potential role of APRIL as autocrine growth factor for megakaryocytopoiesis

“…10,11 Furin is involved in the maturation of growth factors and integrins synthesized by MKs and appears as a key enzyme for megakaryocytopoiesis and thrombocytopoiesis. 12,13 APRIL mRNA levels are reportedly low in normal tissues. In contrast, high mRNA levels have been detected in tumor cell lines and in several primary tumor tissues.…”

Hypoxia-enhanced Expression of the Proprotein Convertase Furin Is Mediated by Hypoxia-inducible Factor-1

“…The P1A and P1B promoters resemble housekeeping genes with multiple Sp-1 binding sites. On the other hand, the P1 promoter bears inducible gene features with the presence of TATA and CAAT elements in the proximal region and has been shown to be transactivated by C/EBP-␤, GATA-1, and Smads (22)(23)(24). Interestingly, computer-assisted analysis of the furin promoters uncovered the presence of putative hypoxia responsive elements (HREs) also found in many genes regulated by oxygen deprivation such as vascular endothelial growth factor, erythropoietin, and glucose transporter-1 (25)(26)(27)(28).…”