Fibroblast Growth Factor-2 Stimulates Endothelial Nitric Oxide Synthase Expression and Inhibits Apoptosis by a Nitric Oxide-Dependent Pathway in Nb2 Lymphoma Cells¹

Abstract

We recently reported that the rat Nb2 T lymphoma cells expressed messenger RNAs (mRNAs) encoding both fibroblast growth factor-2 (FGF-2) and the FGF receptor, suggesting possible paracrine and/or autocrine roles for FGF-2 in lymphoma cell function. We have also shown that the Nb2 cells expressed endothelial nitric oxide synthase (eNOS) and produced low levels of nitric oxide (NO) that inhibited apoptosis of PRL-deprived cells via a PRL-independent, bcl-2-mediated pathway. In this study the effects of PRL and FGF-2 on Nb2 cell survival and NO production were further investigated. The percentages of nonapoptotic cells in PRL-treated vs. PRL-deprived cultures after 6 days were 95% and 53%, respectively. Addition of FGF-2 to PRL-deprived Nb2 cells did not stimulate cell proliferation, but the onset of apoptosis was significantly inhibited, such that more than 85% of the cells remained nonapoptotic after 6 days. The steady state levels of bcl-2 and bag-1 mRNAs were low in PRL-deprived Nb2 cells, but were markedly increased by PRL or FGF-2. bcl-2 expression was induced within 1 h of PRL or FGF-2 addition and continued to increase to a level 20- to 25-fold above the control level within 24 h. bag-1 expression also increased within 1 h after the addition of PRL or FGF-2, was maximal within 8 h, and declined slowly thereafter. The levels of eNOS mRNAs were low but detectable in growth-arrested Nb2 cells, and PRL further down-regulated eNOS mRNA levels over the next 24 h. In contrast, FGF-2 significantly increased eNOS mRNA levels within 2 h to reach a peak 10-fold induction by 12 h. FGF-2 stimulation of eNOS mRNA was accompanied by a 2- to 3.5-fold increase in cellular levels of the eNOS protein and a 2.5-fold increase in serine-phosphorylated eNOS. However, the ratio of serine-phosphorylated eNOS vs. total cellular eNOS was unchanged, indicating that FGF-2 did not affect the serine phosphorylation status of eNOS. Nb2 cells produced low basal levels of NO, which increased with increasing L-arginine concentrations. PRL did not further increase NO release in the presence of L-arginine (0.1 or 1 mM), but FGF-2 significantly (P: </= 0.05) increased NO release in the presence of 0.1 and 1 mM L-arginine. Furthermore, coincubation of aminoguanidine (NOS inhibitor) with FGF-2 completely abrogated the protective effect of FGF-2 on bcl-2 and bag-1 mRNA levels in PRL-deprived Nb2 cells. In summary, FGF-2 inhibited apoptosis of PRL-deprived Nb2 cells. This antiapoptotic action of FGF-2 appears to be mediated by stimulation of eNOS expression, increased levels of cellular NO, and stimulation of expression of the antiapoptotic genes bcl-2 and bag-1.

Insulin Dynamically Regulates Calmodulin Gene Expression by Sequential O-Glycosylation and Phosphorylation of Sp1 and Its Subcellular Compartmentalization in Liver Cells

“…The phosphoserine-specific antibody has a high titer against phosphoserine/bovine serum albumin, and we were able to use it routinely at a 1:1000 dilution for Western blots. The anti-phosphoserine antibody has been used by many investigators, and its specificity is well documented in the literature (17,18). Protein A-Sepharose, SYPRO, Bio-Rad protein assay reagent, and kaleidoscope protein molecular weight markers were obtained from Bio-Rad.…”

Fibroblast Growth Factor Type 2 (FGF2) Administration Attenuated the Clinical Manifestations of Preeclampsia in a Murine Model Induced by L-NAME

“…FGF stimulation was shown to be necessary for the maintenance of VEGF receptor 2 (VEGFR2) levels and, in its absence, VEGFR2 expression rapidly declined, leading to reduced production of NO, impaired angiogenesis and arteriogenesis and eventually, loss of vascular integrity (Murakami et al, 2011). FGF type 2 (FGF2 or FGFb), one of the most studied FGF family members; it induces endothelium-derived de novo synthesis of vasodilators, including endothelial NO, and is a very potent inducer of angiogenesis (Parsons-Wingerter et al, 2000;Murphy et al, 2001). Besides its mitogenic effect on vascular smooth muscle and endothelial cells in vitro and in vivo, previous studies suggest that FGF2 might be involved in the endothelial feedback loop activated by prohypertensive peptides and to play a role in the vascular remodeling that appears in hypertension (Ozkan et al, 2008).…”