Expression of the High Molecular Weight Fibroblast Growth Factor-2 Isoform of 210 Amino Acids Is Associated with Modulation of Protein Kinases C δ and ε and ERK Activation

Abstract

The high molecular weight (HMW) fibroblast growth factor (FGF)-2 isoform of 210 amino acids initiated at a CUG start codon possesses a nuclear localization sequence and is not secreted. In contrast, the low molecular weight (LMW) isoform of 155 amino acids initiated at the AUG start codon can be secreted and activates the cell surface FGF receptors. The two isoforms possess different biological properties; however, little is known about the intracrine regulatory mechanisms involved in the biological effects of the HMW FGF-2 isoform. Using pancreatic cells stably transfected with cDNAs leading to the expression of either the HMW FGF-2 (A3 cells) or the LMW form (A5 cells), we provide evidence that the two FGF-2 isoforms differentially modulate PKC levels. The LMW FGF-2 up-regulated the PKC ⑀ levels by 1.6-fold; by contrast the HMW isoform down-regulated the level of this PKC isotype by about 3-fold and increased the amount of PKC ␦ by 1.7-fold. PKC mRNAs were also modified, suggesting that PKC expression was regulated at a pretranslational level. Additionally, expression of different levels of the HMW FGF-2 with an inducible expression system confirmed the role of this isoform on PKC ␦ and ⑀ expressions. Increased activation of ERK-1 and -2 was also observed in cells expressing the HMW FGF-2. By using different PKC inhibitors and a dominant negative PKC ␦, it was found that ERK activation was PKC ␦-dependent. These data indicate that expression of HMW FGF-2 can modify PKC levels by acting at the intracellular level and that the overexpression of PKC ␦ induces ERK-1/2 activation. The expression of a dominant negative FGFR1 did not reduce ERK-1/2 activation by the HMW FGF-2, suggesting that ERK activation does not require FGFR activity. The signaling cascade downstream of ERK might be involved in the known mitogenic effect exerted by this FGF-2 isoform.Basic FGF 1 (FGF-2) is a protein belonging to the family of heparin-binding growth factors and is produced by many cell types either normal or malignant (for review see Ref. 1). In tumors, it is one of the key factors regulating growth, blood supply, and invasiveness. These pleiotropic effects have been related to the binding of FGF-2 to high affinity FGF receptors or their splice variants and to low affinity binding sites (2). However, FGF-2 possesses some peculiar features supporting the involvement of other regulatory mechanisms in the final biological effects. Indeed, FGF-2 is synthesized from a single mRNA as five different isoforms with molecular masses of 18, 22, 22.5, 24, and 34 kDa through alternative translation at AUG and CUG start codons (3-5) and through internal ribosomal entries (6). The initiation of translation at the AUG codon gives rise to a peptide of 155 amino acids, and the initiation at the four CUG codons is responsible for the synthesis of the other isoforms that possess N-terminal extensions containing nuclear localization sequences (5, 7). Confocal and immunohistochemical analysis show that these nuclear localization sequence-containing iso...

Paracrine and autocrine effects of fibroblast growth factor-4 in endothelial cells

“…Experimental evidences point to di erent functions of FGF2 isoforms (Couderc et al, 1991;Bikfalvi et al, 1995;Esca t et al, 2000;Gaubert et al, 2001), possibly related to di erences in their subcellular localization and release. Indeed, HMW FGF2 isoforms are mostly recovered in the nucleus whereas M r 18 000 FGF2 is mostly cytosolic (Renko et al, 1990).…”

“…Nevertheless, FGF2 isoform(s) can be secreted in limited amounts by an alternative secretion pathway (Mignatti et al, 1992) and accumulate in the extracellular matrix, from where they are released by degrading enzymes (Ribatti et al, 1999a and references therein). Overexpression of the di erent FGF2 isoforms may have di erent biological consequences in transfected cells, suggesting that they may play di erent intracellular/extracellular functions (Couderc et al, 1991;Bikfalvi et al, 1995;Esca t et al, 2000;Gaubert et al, 2001). However, both M r 18 000 and M r 24 000 FGF2 isoforms show angiogenic activity in vitro and in vivo (Gualandris et al, 1994).…”

Dipeptidyl Peptidase Inhibits Malignant Phenotype of Prostate Cancer Cells by Blocking Basic Fibroblast Growth Factor Signaling Pathway

“…Several studies have linked bFGF to activation of the MAPK-ERK1/2 signaling pathway that plays a critical role in survival and migration of tumor cells (28,29). Indeed, bFGF-deficient endothelial cells lack ERK1/2 activation and loose their migratory potential (28).…”

“…Several studies have linked bFGF signaling to activation of the mitogen-activated protein kinase (MAPK)-extracellular signalregulated kinase (ERK)1/2 pathway that promotes cancer progression (28)(29)(30)(31). During cellular migration and angiogenesis, activation of ERK1/2 by bFGF increases the production of urokinase-type plasminogen activator (uPA), a serine protease that converts plasminogen to plasmin.…”

Nuclear FGF-2 facilitates cell survival in vitro and during establishment of metastases

“…The antiapoptotic function ascribed to FGF-2 in PC12 cells has been elucidated with PKC delta implicated in this event, which also requires the Ras/MAPK cascade signaling pathway (Wert and Palfrey, 2000). The high molecular weight FGF-2 isoform also regulates PKC delta and ERK signaling in pancreatic cells (Gaubert et al, 2001). Furthermore, in small-cell lung cancer cells, FGF-2 has been demonstrated to inhibit apoptosis via the control of inhibitor of apoptosis proteins (Pardo et al, 2002;Pardo et al, 2003).…”