Alternative Splicing of Fibroblast Growth Factor Receptor 3 Produces a Secreted Isoform That Inhibits Fibroblast Growth Factor–Induced Proliferation and Is Repressed in Urothelial Carcinoma Cell Lines

Abstract

Fibroblast growth factor receptors (FGFRs) are a family of receptor tyrosine kinases that play key roles in proliferation, differentiation, and tumorigenesis. FGFR3 was identified as the major family member expressed in both normal human urothelium and cultured normal human urothelial (NHU) cells and was expressed as the IIIb isoform. We also identified a splice variant, FGFR3 #8-10, lacking exons encoding the COOH-terminal half of immunoglobulin-like domain III and the transmembrane domain. Previous reports have assumed that this is a cancer-specific splice variant. We showed that FGFR3 #8-10 is a normal transcript in NHU cells and is translated, N-glycosylated, and secreted. Primary urothelium expressed high levels of FGFR3 transcripts. In culture, levels were reduced in actively proliferating cells but increased at confluence and as cells approached senescence. Cells overexpressing FGFR3 IIIb showed FGF1-induced proliferation, which was inhibited by the addition of FGFR3 #8-10. In bladder tumor cell lines derived from aggressive carcinomas, there were significant alterations in the relative expression of isoforms including an overall decrease in the proportion of FGFR3 #8-10 and predominant expression of FGFR3 IIIc in some cases. In summary, alternative splicing of FGFR3 IIIb in NHU cells represents a normal mechanism to generate a transcript that regulates proliferation and in bladder cancer, the ratio of FGFR3 isoforms is significantly altered. (Cancer Res 2005; 65(22): 10441-9)

Altered Splicing of FGFR1 Is Associated with High Tumor Grade and Stage and Leads to Increased Sensitivity to FGF1 in Bladder Cancer

“…18 Individual isoform products were cloned into pGEM T-easy, sequenced, and subcloned into pFB-HYG. 24 FGFR1 and shRNA constructs 18 were transfected into Phoenix A packaging cells (ATCC, Manassas, VA), using siPORT XP-1 transfection agent (Ambion, Warrington, UK). After 48 hours, medium was harvested, 0.4 m filtered, and mixed in equal amounts with fresh medium containing 8 g/ml polybrene (Sigma, Poole, UK).…”

“…Real-time RT-PCR analysis was performed using SYBR Green I as reporter and ROX as reference dye (Perkin-Elmer, Seer Green, UK), using SDHA as an internal control, as described previously. 24 FGFR1 isoform levels were analyzed using the following primers: FGFR1␣F, 5Ј-ctctaactgcagaacttgggatgt-3Ј; FGFR1␣R, 5Ј-ccagggctgggcttgtt-3Ј; FGFR1␤F, 5Ј-gaccttgcctgaacaagatgctc-3Ј; FGFR1␤R, 5Ј-gcactgcatgcaatttcttttcc-3Ј.…”

An essential role for FGF receptor signaling in lens development

“…FGFR1 and FGFR2 additionally exist in a IIIa isoform which introduces a stop codon prior to the transmembrane domain resulting in a secreted extracellular domain [37,38]. Secreted isoforms of FGFR3 [39] and FGFR4 [32] have also been described . It is thought that these secreted isoforms of FGFRs may negatively regulate FGFR signaling.…”

Fibroblast growth factor receptor 1 and cytokeratin 20 expressions and their relation to prognostic variables in bladder cancer

“…cDNA synthesis, and quantitative real-time PCR mRNA was reverse transcribed and cDNA was synthesized with a reverse transcription kit (Reverse Transcriptase Master Mix, Roche Diagnostics) according to the manufacturer's instructions. FGFR1 and CK20 expressions were detected by RT-PCR as previously described with modifications (Tomlinson et al, 2005;Pu et al, 2008). Hypoxanthine phosphoribosyl transferase (HPRT) was used as a reference gene.…”