Role and regulation of the fibroblast growth factor axis in human thyroid follicular cells

Thyroidal levels of fibroblast growth factor-2 (FGF-2) and fibroblast growth factor receptor 1 (FGFR1) are elevated in human thyroid hyperplasia. To understand the significance of this, effects of FGFR1 activation on normal human thyrocyte growth and function in vitro and the regulation of FGF-2 and FGFR1 expression have been examined. FGF-2 stimulated cell growth, as measured by cell counting, and inhibited thyroid function as measured by 125I uptake. Sensitivity to FGF-2 disappeared after 7 days, although FGFR1 expression was maintained. Thyroid-stimulating hormone (TSH, 300 mU/l) increased FGFR1 mRNA expression within 4 h and protein expression by 8 h. Exogenous FGF-2 decreased FGFR1 protein. Endogenous FGF-2 levels were low (approximately 1-2 pg/microg protein), and TSH treatment decreased these by 50%. Protein kinase C (PKC) activation increased FGF-2 mRNA and FGF-2 secretion within 2 h. This effect was enhanced (4.4-fold) when cells were cultured in TSH. We conclude that TSH stimulates FGFR1 but not FGF-2 expression. PKC activation stimulates FGF-2 synthesis and secretion, and TSH synergizes with PKC activators. Increases in FGFR1 or FGF-2 or in both may contribute to goitrogenesis.     

Expression of fibroblast growth factor receptor 3 by fibroblast growth factor 2 in cultured chick embryo chondrocytes

Although fibroblast growth factor 2 (FGF2) and fibroblast growth factor receptor 3 (FGFR3) both inhibit longitudinal bone growth, little is known about the relationship between FGF2 and FGFR3. Accordingly, the current study examined the expression of FGFR3 mRNA after the administration of FGF2 using cultured chondrocytes from day 17 chick embryos to evaluate the relationship between FGF2 and FGFR3. The chondrocytes were isolated from the caudal one-third portion (LS) of sterna, peripheral regions (USP) and central core regions (USC) of the cephalic portion of the sterna, and lower portion of the proximal tibial growth plate (Ti) of day 17 chick embryo. The expression of FGFR1, FGFR3, and type II and X collagen mRNA in the chondrocytes from the LS, USP, USC, and Ti was determined. FGFR1 was not expressed in the LS and USP chondrocytes, yet strongly expressed in the USC and Ti chondrocytes. With a treatment of FGF2, the expression of FGFR1 slightly increased in the USC chondrocytes and was not related with the concentration of FGF2 in the Ti chondrocytes. FGFR3 was expressed in all the chondrocyte types, yet strongly increased in the LS, USC, USP, and Ti in that order according to the concentration of FGF2. For the LS and USP chondrocytes, the expression of FGFR3 with FGF2 increased in a 4-day culture, yet decreased in a 6-day culture, whereas for the USC chondrocytes, the expression of FGFR3 mRNA with FGF2 increased in a 2-day culture, yet decreased in a 4-day culture, suggesting that the hypertrophic chondrocytes were more numerous and sensitive compared to the proliferative chondrocytes. For all the chondrocyte types, FGF2 appeared to be up-regulated to FGFR3, as the expression of FGFR3 mRNA increased with a higher concentration of FGF2 until a peak level. In conclusion, FGF2 was found to up-regulate to FGFR3 until the peak level of FGFR3 mRNA expression, while in hypertrophic chondrocytes, FGFR3 appeared to cause the differentiaton of chondrocytes, resulting in the inhibition of longitudinal bone growth after the peak level of FGFR3 mRNA expression.     

FGFR4 and its novel splice form in myogenic cells: Interplay of glycosylation and tyrosine phosphorylation

The family of fibroblast growth factor receptors (FGFRs) is encoded by four distinct genes. FGFR1 and FGFR4 are both expressed during myogenesis, but whereas the function of FGFR1 in myoblast proliferation has been documented, the role of FGFR4 remains unknown. Here, we report on a new splice form of FGFR4 cloned from primary cultures of mouse satellite cells. This form, named FGFR4(-16), lacks the entire exon 16, resulting in a deletion within the FGFR kinase domain. Expression of FGFR4(-16) coincided with that of wild-type FGFR4 in all FGFR4-expressing tissues examined. Moreover, expression of both FGFR4 forms correlated with the onset of myogenic differentiation, as determined in mouse C2C12 cells and in the inducible myogenic system of 10T(1/2)-MyoD-ER cell line. Both endogenous and overexpressed forms of FGFR4 exhibited N-glycosylation. In contrast to FGFR1, induced homodimerization of FGFR4 proteins did not result in receptor tyrosine phosphorylation. Surprisingly, coexpression of FGFR4 forms and a chimeric FGFR1 protein resulted in FGFR4 tyrosine phosphorylation, raising the possibility that FGFR4 phosphorylation might be enabled by a heterologous tyrosine kinase activity. Collectively, the present study reveals novel characteristics of mouse FGFR4 gene products and delineates their expression pattern during myogenesis. Our findings suggest that FGFR4 functions in a distinctly different manner than the prototype FGFR during myogenic differentiation.     

From genotype to phenotype: the differential expression of FGF, FGFR, and TGFbeta genes characterizes human cranioskeletal development and reflects clinical presentation in FGFR syndromes.

Mutations in the fibroblast growth factor receptor (FGFR) genes 1, 2, and 3 are causal in a number of craniofacial dysostosis syndromes featuring craniosynostosis with basicranial and midfacial deformity. Great clinical variability is displayed in the pathologic phenotypes encountered. To investigate the influence of developmental genetics on clinical diversity in these syndromes, the expression of several genes implicated in their pathology was studied at sequential stages of normal human embryo-fetal cranial base and facial ossification (n = 6). At 8 weeks of gestation, FGFR1, FGFR2, and FGFR3 are equally expressed throughout the predifferentiated mesenchyme of the cranium, the endochondral skull base, and midfacial mesenchyme. Both clinically significant isoforms of FGFR2, IgIIIa/c and IgIIIa/b, are coexpressed in maxillary and basicranial ossification. By 10 to 13 weeks, FGFR1 and FGFR2 are broadly expressed in epithelia, osteogenic, and chondrogenic cell lineages. FGFR3, however, is maximally expressed in dental epithelia and proliferating chondrocytes of the skull base, but poorly expressed in the osteogenic tissues of the midface. FGF2 and FGF4, but not FGF7, and TGFbeta1 and TGFbeta3 are expressed throughout both osteogenic and chondrogenic tissues in early human craniofacial skeletogenesis. Maximal FGFR expression in the skull base proposes a pivotal role for syndromic growth dysplasia at this site. Paucity of FGFR3 expression in human midfacial development correlates with the relatively benign human mutant FGFR3 midfacial phenotypes. The regulation of FGFR expression in human craniofacial skeletogenesis against background excess ligand and selected cofactors may therefore play a profound role in the pathologic craniofacial development of children bearing FGFR mutations.     

Neuronal expression of fibroblast growth factor receptors in zebrafish

Fibroblast growth factor (FGF) signaling is important for a host of developmental processes such as proliferation, differentiation, tissue patterning, and morphogenesis. In vertebrates, FGFs signal through a family of four fibroblast growth factor receptors (FGFR 1-4), one of which is duplicated in zebrafish (FGFR1). Here we report the mRNA expression of the five known zebrafish fibroblast growth factor receptors at five developmental time points (24, 36, 48, 60, and 72 h postfertilization), focusing on expression within the central nervous system. We show that the receptors have distinct and dynamic expression in the developing zebrafish brain, eye, inner ear, lateral line, and pharynx. In many cases, the expression patterns are similar to those of homologous FGFRs in mouse, chicken, amphibians, and other teleosts.     

Neuronal expression of fibroblast growth factor receptors in zebrafish

Fibroblast growth factor (FGF) signaling is important for a host of developmental processes such as proliferation, differentiation, tissue patterning, and morphogenesis. In vertebrates, FGFs signal through a family of four fibroblast growth factor receptors (FGFR 1-4), one of which is duplicated in zebrafish (FGFR1). Here we report the mRNA expression of the five known zebrafish fibroblast growth factor receptors at five developmental time points (24, 36, 48, 60, and 72 h postfertilization), focusing on expression within the central nervous system. We show that the receptors have distinct and dynamic expression in the developing zebrafish brain, eye, inner ear, lateral line, and pharynx. In many cases, the expression patterns are similar to those of homologous FGFRs in mouse, chicken, amphibians, and other teleosts.     

Fibroblast growth factor receptor mediates fibroblast-dependent growth in EMMPRIN-depleted head and neck cancer tumor cells

Head and neck squamous cell carcinoma tumors (HNSCC) contain a dense fibrous stroma which is known to promote tumor growth, although the mechanism of stroma-mediated growth remains unclear. As dysplastic mucosal epithelium progresses to cancer, there is incremental overexpression of extracellular matrix metalloprotease inducer (EMMPRIN) which is associated with tumor growth and metastasis. Here, we present evidence that gain of EMMPRIN expression allows tumor growth to be less dependent on fibroblasts by modulating fibroblast growth factor receptor-2 (FGFR2) signaling. We show that silencing EMMPRIN in FaDu and SCC-5 HNSCC cell lines inhibits cell growth, but when EMMPRIN-silenced tumor cells were cocultured with fibroblasts or inoculated with fibroblasts into severe combined immunodeficient mice, the growth inhibition by silencing EMMPRIN was blunted by the presence of fibroblasts. Coculture experiments showed fibroblast-dependent tumor cell growth occurred via a paracrine signaling. Analysis of tumor gene expression revealed expression of FGFR2 was inversely related to EMMPRIN expression. To determine the role of FGFR2 signaling in EMMPRIN-silenced tumor cells, ligands and inhibitors of FGFR2 were assessed. Both FGF1 and FGF2 enhanced tumor growth in EMMPRIN-silenced cells compared with control vector-transfected cells, whereas inhibition of FGFR2 with blocking antibody or with a synthetic inhibitor (PD173074) inhibited tumor cell growth in fibroblast coculture, suggesting the importance of FGFR2 signaling in fibroblast-mediated tumor growth. Analysis of xenografted tumors revealed that EMMPRIN-silenced tumors had a larger stromal compartment compared with control. Taken together, these results suggest that EMMPRIN acquired during tumor progression promotes fibroblast-independent tumor growth.     

Involvement of fibroblast growth factor receptor 2 isoform switching in mammary oncogenesis

We identified the IIIb C2 epithelial cell-specific splice variant of fibroblast growth factor receptor 2 (FGFR2 IIIb C2) receptor tyrosine kinase in a screen for activated oncogenes expressed in T-47D human breast carcinoma cells. We found FGFR2 IIIb C2 expression in breast carcinoma cell lines and, additionally, expression of the mesenchymal-specific FGFR2 IIIc splice variant in invasive breast carcinomas. FGFR2 IIIc expression was associated with loss of epithelial markers and gain of mesenchymal markers. Although FGFR2 IIIb is expressed in epithelial cells, previous studies on FGFR2 IIIb transformation have focused on NIH 3T3 fibroblasts. Therefore, we compared the transforming activities of FGFR2 IIIb C2 in RIE-1 intestinal cells and several mammary epithelial cells. FGFR2 IIIb C2 caused growth transformation of epithelial cells but morphologic transformation of only NIH 3T3 cells. FGFR2 IIIb C2-transformed NIH 3T3, but not RIE-1 cells, showed persistent activation of Ras and increased cyclin D1 protein expression. NIH 3T3 but not RIE-1 cells express keratinocyte growth factor, a ligand for FGFR2 IIIb C2. Ectopic treatment with keratinocyte growth factor caused FGFR2 IIIb C2-dependent morphologic transformation of RIE-1 cells, as well as cyclin D1 up-regulation, indicating that both ligand-independent and stromal cell-derived, ligand-dependent mechanisms contribute to RIE-1 cell transformation. Our results support cell context distinct mechanisms of FGFR2 IIIb C2 transformation.     

Responsiveness of developing dental tissues to fibroblast growth factors: expression of splicing alternatives of FGFR1, -2, -3, and of FGFR4; and stimulation of cell proliferation by FGF-2, -4, -8, and -9

To elucidate the roles of fibroblast growth factors (FGF) in tooth development, we have analyzed the expression patterns of fibroblast growth factor receptors (FGFR) in mouse teeth by in situ hybridization and studied the effects of FGF-2, -4, -8, and -9 on cell proliferation in vitro by local application with beads on isolated dental mesenchymes. mRNAs of FGFR-1, -2, and -3 were localized by probes specific for the alternative splice variants IIIb and IIIc. The expression patterns of FGFR1, -2, and -3 were completely different, and the two splicing variants of FGFR1 and 2 exhibited different expression domains. FGFR4 was not expressed in the developing teeth. The IIIb splice forms of FGFR1 and -2 were expressed in the dental epithelium during morphogenesis. The IIIc splice form of FGFR1 was expressed both in epithelium and mesenchyme whereas FGFR2 IIIc was confined to the mesenchymal cells of the dental follicle. Both splice forms of FGFR3 were expressed in dental papilla mesenchyme. None of the FGF-receptors was detected in the primary enamel knot, the putative signaling center regulating tooth morphogenesis. This may explain the fact that enamel knot cells do not proliferate, although they express intensely mitogenic FGFs. Beads releasing FGF-2, -4, -8, or -9 proteins stimulated cell proliferation in cultured dental mesenchymes. These data, together with our earlier data on FGF expression [Kettunen and Thesleff (1998): Dev Dyn 211:256–268] suggest that FGF-8 and -9 mediate epithelial-mesenchymal interactions during tooth initiation. During advancing morphogenesis FGF-3, -4, and -9 may act both on mesenchyme and epithelium. Finally, the intense expression of FGFR1 in odontoblasts and ameloblasts, and FGFR2 IIIb in ameloblasts suggests that FGFs participate in regulation of their differentiation and/or secretory functions. Dev. Genet. 22:374–385, 1998. © 1998 Wiley-Liss, Inc.     

FGFR1-IIIb is a putative marker of pancreatic progenitor cells

The pancreas develops from buds that derive from the endodermal epithelium of the digestive tract. The progenitor cells that will give rise to the mature pancreatic cells reside within this epithelium. However, their exact identity remains unknown. In the present study, we searched for genes expressed by pancreatic progenitor cells. We focused our search on receptor tyrosine kinases. We found that fibroblast growth factor-IIIb (FGFR1-IIIb) expression is high in pancreatic epithelium enriched in progenitor cells. We next investigated FGFR1-IIIb expression throughout pancreatic development. At early stages of pancreas development, FGFR1-IIIb is expressed by pancreatic epithelial cells that resemble undifferentiated cells, while at later stages of development, FGFR1-IIIb expression decreases, concomitant with the expected decrease in the number of progenitor cells.     

Multiple Consequences of a Single Amino Acid Pathogenic RTK Mutation: The A391E Mutation in FGFR3

The A391E mutation in fibroblast growth factor receptor 3 (FGFR3) is the genetic cause for Crouzon syndrome with Acanthosis Nigricans. Here we investigate the effect of this mutation on FGFR3 activation in HEK 293 T cells over a wide range of fibroblast growth factor 1 concentrations using a physical-chemical approach that deconvolutes the effects of the mutation on dimerization, ligand binding, and efficiency of phosphorylation. It is believed that the mutation increases FGFR3 dimerization, and our results verify this. However, our results also demonstrate that the increase in dimerization is not the sole effect of the mutation, as the mutation also facilitates the phosphorylation of critical tyrosines in the activation loop of FGFR3. The activation of mutant FGFR3 is substantially increased due to a combination of these two effects. The low expression of the mutant, however, attenuates its signaling and may explain the mild phenotype in Crouzon syndrome with Acanthosis Nigricans. The results presented here provide new knowledge about the physical basis behind growth disorders and highlight the fact that a single RTK mutation may affect multiple steps in RTK activation.     

Differential signal transduction of alternatively spliced FGFR2 variants expressed in human mammary epithelial cells

Gene amplification and protein overexpression of fibroblast growth factor receptor 2 (FGFR2) characterize the SUM-52 breast cancer cell line developed in our laboratory. SUM-52 cells express nine distinct alternatively spliced isoforms of FGFR2. Among these isoforms are two otherwise identical FGFR2 variants that express either the C1 or C3 carboxyl terminus. FGFR2-C3 variants are not normally expressed by human mammary epithelial (HME) cells, and we have shown that overexpression of FGFR2-C3 in HME cells results in potent transformation. In particular, FGFR2-C3 expression leads to robust levels of constitutively tyrosine phosphorylated FRS2 in the absence of ligand stimulation. In contrast, overexpressed FGFR2-C1 requires constant stimulation with exogenous keratinocyte growth factor (KGF) to mimic the signaling capability of FGFR2-C3. However, activation of FRS2 that results from KGF-stimulated FGFR2-C1 signaling is transient and is associated with a mobility shift of FRS2 not observed when this signaling molecule is activated by the C3 isoform of FGFR2. Mutation of the only tyrosine phosphorylated site present in the C1 terminus and absent from C3, Tyr769, did not yield a receptor that rivaled the potent signaling of FGFR2-C3. We therefore conclude that aberrant expression of alternatively spliced isoforms of FGFR2 with the C3 carboxyl terminus in the SUM-52 breast cancer cells results in sustained activation of signal transduction leading to transformation.