FGF2‐mediated upregulation of urokinase‐type plasminogen activator expression requires a MAP‐kinase dependent activation of poly(ADP‐ribose) polymerase

Abstract

Poly(ADP-ribosyl)ation is a post-translational modification of protein occurring in the nucleus by poly(ADP-ribose) polymerase enzyme activity. The main role of poly(ADP-ribose) polymerase system as "nick sensor" and DNA breaks repair is based on its activation via DNA strand breaks. Furthermore, poly(ADP-ribose) polymerase modifies the binding to DNA of several transcriptional factors by poly(ADP-ribosyl)ation, thereby regulating also transcriptional gene expression. We have analyzed whether poly(ADP-ribose) polymerase activity is involved in basic fibroblast growth factor (FGF2)-mediated upregulation of urokinase-type plasminogen activator (uPA) mRNA. We demonstrated that specific inhibition of poly(ADP-ribose) polymerase activity via 3-aminobenzamide (3ABA) or NAD+ deprivation prevents FGF2-mediated uPA mRNA over-expression and cell-associated plasminogen activator (PA) production in GM7373 endothelial cell line. We verified that FGF2 stimulates poly(ADP-ribose) polymerase activity by a DNA strand breaks-independent manner which involves a mitogen-activated protein kinases (MAPK)-dependent pathway, as confirmed by using PD98059 inhibitor and anisomycin stimulation. Poly(ADP-ribose) polymerase involved in this mechanism is mainly the 60 kDa molecular mass isoform, that presents an increase in serine phosphorylation in the presence of FGF2.

Low doses of 3-aminobenzamide, a poly(ADP-ribose) polymerase inhibitor, stimulate angiogenesis by regulating expression of urokinase type plasminogen activator and matrix metalloprotease 2

“…On the basis of the ratios between normalized values of uPA amplified products and of GAPDH products, we found that cells exposed to FGF2 for 24 h showed a 75% increase of uPA-mRNA (Figure 4b, left side; * p < 0.05 compared to control cells; n = 3). The addition of 3ABA inhibited FGF2-stimulated uPA gene expression (# p < 0.05 compared to FGF2 stimulated cells; n = 3), while it did not affect basal levels, confirming results previously obtained in transformed endothelial cells GM7373 [4]. No variation in uPAR mRNA levels was found in the presence of FGF2 nor in the presence of 3ABA, at least after 24 h of treatment (Figure 4b, right side).…”

“…Angiogenesis has been generally studied using a variety of approaches: both in vivo and in vitro models were developed to further explore individual steps and to better understand the molecular mechanisms involved in the angiogenic process. Previous studies from our laboratory have demonstrated that PARP inhibition could affect gene expression of the serine protease uPA by MAPK-dependent pathway, in transformed bovine endothelial cells [4,5]. Given the critical role of uPA and uPAR in angiogenesis and in many human diseases [20,21], it would be of great interest to evaluate the effect of PARP inhibition on some individual steps of angiogenesis which involve both uPA and uPAR gene expression and MMP-2 activity in a normal human cellular experimental system.…”

“…Recently, we found that, in transformed endothelial GM7373 cells, FGF2-dependent uPA upregulation is affected by poly(ADP-ribose) polymerase (PARP) activity stimulated by MAPK-dependent phosphorylation [4,5]. Poly(ADP-ribosyl)ation is a posttranslational modification of proteins involved in most cellular functions.…”

p38 and c-Jun N-Terminal Kinase Mitogen-Activated Protein Kinase Signaling Pathways Play Distinct Roles in the Response of Organogenesis-Stage Embryos to a Teratogen

“…Upstream MAPK kinase kinases (MAP3Ks) activate MAPK kinases, which in turn activate MAPKs by dual phosphorylation on threonine and tyrosine residues (Pearson et al, 2001). Downstream MAPK substrates include transcription factors or their components, such as activator protein 1 (AP-1) and p53 (Turjanski et al, 2007), molecules involved in the detection and response to DNA damage, such as poly(ADP-ribose) polymerase 1 and ␥H2AX (Caldini et al, 2005;Sluss and Davis, 2006), as well as a variety of pro-and antiapoptotic factors (Bogoyevitch and Kobe, 2006). Among the most widely studied MAPK families are extracellular signal-regulated kinases 1 and 2 (ERK1/2), p38 MAPKs (␣, ␤, ␥, and ␦), and c-Jun N-terminal kinases (JNKs; 1, 2, and 3) (Turjanski et al, 2007).…”

Modulation of urokinase plasminogen activator system by poly(ADP-ribose)polymerase-1 inhibition

“…Regulation of uPA and PARP system was shown to be interdependent in experiments done by Caldini and his group. uPA expression induced by FGF2 was increased by PARP activated through MAP kinase pathway and in other system PARP activity inhibition decreased uPA activity (Caldini et al 2005(Caldini et al , 2011. In A1235 cells PARP-1 inhibitor alone did not significantly change the growth rate and viability under our experimental conditions.…”