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A soluble fibroblast growth factor receptor is released from HL-60 promyelocytic leukemia cells: implications for
1Lawson Research Institute, St. Joseph's Health Centre, London, Ontario, Canada.
Abstract:
The biological activities of fibroblast growth factors (FGF) are mediated by specific cell membrane receptors (FGFR), which have three immunoglobulin-like IgG domains in the extracellular region. The carboxy-terminal segment of the third IgG domain of FGFR1 could be encoded by different exons, designated IIIa, IIIb, or IIIc. While exons IIIb or IIIc encode receptor forms with both intracellular and extracellular domains, the FGF receptor becomes potentially a secreted form lacking the intracellular domain and the transmembrane region when exon IIIa is expressed. Using reverse transcription polymerase chain reaction, we have found that mRNAs encoding the nucleotide sequences of FGFR1-IIIa and FGFR1-IIIc are expressed in HL-60 cells. FGFR1-IIIa fragment was synthesized by a glutathione S-transferase gene fusion system. The purified 33 kDa FGFR1-IIIa fragment fusion protein could bind [125I]-labelled FGF-2 in Western ligand blot analysis. Three species of proteins with the molecular weights of 82, 60, and 50 kDa were identified in serum-free, conditioned medium from HL-60 cells by Western blot using an antiserum against purified FGFR1-IIIa fragment fusion protein. Exposure to FGF-2 caused an increase in [3H]-thymidine incorporation into DNA of HL-60 cells and increased cell proliferation, but the addition of FGFR1-IIIa fragment fusion protein inhibited FGF-2-stimulated DNA synthesis and caused a dose-dependent inhibition of FGF-2-stimulated cell proliferation. The effects on DNA synthesis were partly reversed by antibody against the FGFR1-IIIa fragment. These results indicate that both cell membrane spanning and secreted FGF receptors are expressed in HL-60 cells, and that the actions of FGFs as paracrine growth factors could be modulated by secreted FGF receptor forms.
Insights
Secreted fibroblast growth factor receptor (FGFR) forms, including FGFR1-IIIa, are expressed in HL-60 cells. These secreted FGFRs can modulate fibroblast growth factor (FGF)-mediated cell proliferation and DNA synthesis.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Fibroblast growth factors (FGFs) exert biological activities via fibroblast growth factor receptors (FGFRs).
- FGFR1 exhibits alternative splicing, producing variants like FGFR1-IIIa, FGFR1-IIIb, and FGFR1-IIIc.
- The FGFR1-IIIa variant encodes a secreted form lacking intracellular and transmembrane domains.
Purpose of the Study:
- To investigate the expression of FGFR1 variants in HL-60 cells.
- To determine the functional role of secreted FGFR1-IIIa in modulating FGF-2 activity.
Main Methods:
- Reverse transcription polymerase chain reaction (RT-PCR) to detect mRNA expression.
- Glutathione S-transferase (GST) gene fusion system for protein fragment synthesis.
- Western ligand blot and Western blot analysis to detect protein binding and expression.
- Assays for [3H]-thymidine incorporation and cell proliferation to assess biological activity.
Main Results:
- Both FGFR1-IIIa and FGFR1-IIIc mRNAs are expressed in HL-60 cells.
- A 33 kDa FGFR1-IIIa fragment fusion protein binds FGF-2.
- Secreted FGFR1 proteins (82, 60, and 50 kDa) were detected in HL-60 cell conditioned medium.
- FGFR1-IIIa fragment inhibited FGF-2-stimulated DNA synthesis and proliferation in a dose-dependent manner.
Conclusions:
- HL-60 cells express both membrane-bound and secreted forms of FGF receptors.
- Secreted FGF receptor forms, such as FGFR1-IIIa, can act as modulators of FGF signaling.
- These findings suggest a regulatory mechanism for FGFs acting as paracrine growth factors.