Nuclear Localization Sequence of FGF1 Is Not Required for Its Intracellular Anti-Apoptotic Activity in Differentiated Cells.

Agata Lampart,Aleksandra Czyrek,Jacek Otlewski,Katarzyna Dominika Sluzalska,Malgorzata Zakrzewska,Antoni Wiedlocha,Aleksandra Szerszen

doi:10.3390/cells11030522

Abstract

Fibroblast growth factor 1 (FGF1) is considered primarily as a ligand for FGF surface receptors (FGFRs) through which it activates a number of cellular responses. In addition to its canonical mode of action, FGF1 can act intracellularly, before secretion or after internalization and translocation from the cell exterior. The role of FGF1 inside the cell is to provide additional protection against apoptosis and promote cell survival. The FGF1 protein contains a specific N-terminal nuclear localization sequence (NLS) that is essential for its efficient transport to the nucleus. Here, we investigated the role of this sequence in the anti-apoptotic response of FGF1. To this end, we produced recombinant FGF1 variants with mutated or deleted NLS and added them to apoptosis-induced cells in which FGFR1 was inactive, either as a result of chemical inhibition or kinase-dead mutation. After internalization, all FGF1 variants were able to protect the differentiated cells from serum starvation-induced apoptosis. To verify the results obtained for NLS mutants, we knocked down LRRC59, a protein that mediates the nuclear transport of FGF1. Upon LRRC59 silencing, we still observed a decrease in caspase 3/7 activity in cells treated exogenously with wild-type FGF1. In the next step, FGF1 variants with mutated or deleted NLS were expressed in U2OS cells, in which apoptosis was then induced by various factors (e.g., starvation, etoposide, staurosporine, anisomycin and actinomycin D). Experiments were performed in the presence of specific FGFR inhibitors to eliminate FGFR-induced signaling, potentially activated by FGF1 proteins released from damaged cells. Again, we found that the presence of NLS in FGF1 is not required for its anti-apoptotic activity. All NLS variants tested were able to act as wild type FGF1, increasing the cell viability and mitochondrial membrane potential and reducing the caspase 3/7 activity and PARP cleavage in cells undergoing apoptosis, both transiently and stably transfected. Our results indicate that the nuclear localization of FGF1 is not required for its intracellular anti-apoptotic activity in differentiated cells and suggest that the mechanism of the stress response differs according to the level of cell differentiation.

Highlights

Fibroblast growth factor 1 (FGF1) acts through specific interactions with surface FGF receptors (FGFRs) to activate intracellular signaling axes, such as PLCγ/PKC, PI3K/Akt, and Ras/MAPK, regulating cell growth, proliferation, survival, and playing a significant role in development [1,2,3]
We have recently shown that translocation of exogenous FGF1 protects cells from apoptosis through a pathway independent of FGF surface receptors (FGFRs) signaling [28]
We have recently reported that the translocation of exogenously administrated FGF1 protects various cell lines from apoptosis independently of FGFR activation [28]

Summary

Introduction

Fibroblast growth factor 1 (FGF1) acts through specific interactions with surface FGF receptors (FGFRs) to activate intracellular signaling axes, such as PLCγ/PKC, PI3K/Akt, and Ras/MAPK, regulating cell growth, proliferation, survival, and playing a significant role in development [1,2,3]. In addition to this canonical modus operandi, FGF1 has the unique ability to translocate across cell membranes, reaching the cytosol and nucleus [4,5,6,7,8]. Later studies have shown that deletion of this signal sequence results in loss of the protein stability, protease resistance and reduced affinity for FGFRs [11], which could explain it impaired biological activity.

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