Fibroblast growth factor-2 stimulates endothelial nitric oxide synthase expression and inhibits apoptosis by a nitric oxide-dependent pathway in Nb2 lymphoma cells.
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.
Murphy
P R
PR
Departments of Physiology and Biophysics, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7.
Limoges
M
M
Dodd
F
F
Boudreau
R T
RT
Too
C K
CK
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001120:Arginine / Q000494:pharmacology; D002455:Cell Division / Q000187:drug effects; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000502:physiology*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects / Q000502:physiology*; D008223:Lymphoma; D009569:Nitric Oxide / Q000502:physiology*; D019001:Nitric Oxide Synthase / Q000235:genetics*; D052250:Nitric Oxide Synthase Type III; D011388:Prolactin / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014158:Transcription, Genetic / Q000187:drug effects; D014407:Tumor Cells, Cultured
D012333:RNA, Messenger; D016222:Fibroblast Growth Factor 2; D009569:Nitric Oxide; D011388:Prolactin; D001120:Arginine; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; C496316:Nos3 protein, rat