Fibroblast Growth Factor Receptor mRNA Research Articles

RETRACTED: Related transcriptional enhancer factor-1 induces fibroblast growth factor receptor-1 expression in endothelial cells

Fibroblast growth factor receptor-1 (FGFR-1) has been implicated in the process of cardiogenesis, although the underlying molecular mechanisms are poorly understood. In this study, we report the regulation of FGFR-1 expression by related transcriptional enhancer factor-1 (RTEF-1) in vitro (endothelial cells) and in vivo (RTEF-1 transgenic mice). FGFR-1 promoter activity, FGFR-1 mRNA and protein level were measured in bovine aortic endothelial cells (BAEC) in response to RTEF-1 and in endothelial cells isolated from livers in RTEF-1 transgenic mice. RTEF-1 stimulated FGFR-1 promoter activity in a dose-dependent manner. RTEF-1 enhanced FGFR-1 mRNA (4-fold) and protein expression (3.5-fold) whereas RTEF-1 siRNA decreased FGFR-1 protein expression (4-fold). FGFR-1 mRNA and protein expression were also increased in endothelial cells isolated from livers of RTEF-1 transgenic mice. Furthermore, RTEF-1 enhanced tubule formation whereas this was decreased by RTEF-1 knockdown. Moreover, increased relaxation of microvessels was found in RTEF-1 transgenic mice compared to wild-type mice. Our results indicate that RTEF-1 acts as a transcriptional stimulator of FGFR-1 in endothelial cells through its activation of the FGFR-1 promoter. RTEF-1 thus plays an important role in the regulation of FGFR-1 expression. These findings help further understand FGFR activity in angiogenesis and may lead to new therapeutic targets in ischemic vascular disorders.

Preserved expression of fibroblast growth factor (FGF)-2 and FGF receptor 1 in brain and spinal cord of amyotrophic lateral sclerosis patients

Impaired trophic support of motor neurons appears to be an important pathogenic factor in amyotrophic lateral sclerosis (ALS). We investigated the mRNA expression of the pluripotent fibroblast growth factor 2 (FGF-2) and its receptors in post mortem spinal cord of ALS and control patients. FGF-2 and FGF receptor (FGFR) 1 and 2 transcripts were first studied in the spinal cord using RT-PCR. The cellular distribution of FGF-2 and FGFR mRNA in the spinal cord, motor cortex and brain stem was then assessed by in situ hybridization histochemistry. RT-PCR revealed the presence of FGF-2 and FGF receptor 1 and 2 transcripts with no obvious differences between ALS and control spinal cord. Comparing mRNA expression in the motoneuron-containing ventral horn with the clinically and neuropathologically spared dorsal horn of ALS spinal cord displayed similar expression levels. At the cellular level, we found a prominent neuronal expression of FGF-2 and FGFR1. Interestingly, both morphologically intact and damaged motoneurons showed positive staining for FGF-2 and FGFR1 transcripts. The distribution of cells expressing FGF-2 and FGFR1 transcripts showed no differences between ALS and controls. Our data suggest that FGF-2 and FGFR1 expression is preserved in the motor system in end stage ALS.

Fas-activated serine/threonine phosphoprotein (FAST) is a regulator of alternative splicing

Fas-activated serine/threonine phosphoprotein (FAST) is a survival protein that is tethered to the outer mitochondrial membrane. In cells subjected to environmental stress, FAST moves to stress granules, where it interacts with TIA1 to modulate the process of stress-induced translational silencing. Both FAST and TIA1 are also found in the nucleus, where TIA1 promotes the inclusion of exons flanked by weak splice recognition sites such as exon IIIb of the fibroblast growth factor receptor 2 (FGFR2) mRNA. Two-hybrid interaction screens and biochemical analysis reveal that FAST binds to several alternative and constitutive splicing regulators, suggesting that FAST might participate in this process. The finding that FAST is concentrated at nuclear speckles also supports this contention. We show that FAST, like TIA1, promotes the inclusion of exon IIIb of the FGFR2 mRNA. Both FAST and TIA1 target a U-rich intronic sequence (IAS1) adjacent the 5' splice site of exon IIIb. However, unlike TIA1, FAST does not bind to the IAS1 sequence. Surprisingly, knockdown experiments reveal that FAST and TIA1 act independently of one another to promote the inclusion of exon IIIb. Mutational analysis reveals that FAST-mediated alternative splicing is separable from the survival effects of FAST. Our data reveal that nuclear FAST can regulate the splicing of FGFR2 transcripts.

Dynamic changes in the expression of growth factor receptors in the myocardium microvascular endothelium after murine myocardial infarction

After myocardial infarction, specific growth factors promote cardiac angiogenisis, leading to a therapeutic effect. Although this effect is mediated by specific receptors in the endothelium of the cardiac microvasculature, few studies have investigated dynamic changes in their expression. We explored this phenomenon in a murine model. We observed the mRNA expression of receptors by specific angiogenesis gene microarray at day 3 and day 7 after infarction. The vascular endothelial growth factor (VEGF) receptor Flk-1 was observed at the protein level at day 3 and day 7 by immunohistochemistry. The dynamic expression of fibroblast growth factor receptor-1 (FGFR-1) mRNA in the border zone and the noninfarcted zone at day 3, day 7, day 14, and day 42 was investigated by real-time PCR. Statistical significance was analyzed with SPSS 10.0 software using one-way analysis of variance (ANOVA). Three days after infarction, 9 receptors in the border zone and 7 receptors in the noninfarcted zone were down-regulated. Two receptors in the infarct edge and 5 receptors in the distant myocardium were up-regulated. However, at day 7, 11 receptors in the border zone were up-regulated, and only one was down-regulated. In the border zone, Flk-1 levels decreased at day 3 but increased significantly at day 7. Real-time PCR showed that FGFR-1 mRNA decreased markedly in the border zone at day 3 but increased afterward for at least 6 weeks. In the early stage (3 days) after infarction, the expression of receptors had decreased to some extent. However, at day 7, receptor expression was active and had moved from the distant noninfarcted zone to the border zone as a part of the acute repair process. Selecting the proper growth factors to target receptors with protective activity, and determining appropriate therapeutic timing may be important to the success of therapeutic angiogenesis.

Glypican-1 Is Frequently Overexpressed in Human Gliomas and Enhances FGF-2 Signaling in Glioma Cells

Signaling by fibroblast growth factor 2 (FGF-2), an autocrine stimulator of glioma growth, is regulated by heparan sulfate proteoglycans (HSPGs) via a ternary complex with FGF-2 and the FGF receptor (FGFR). To characterize glioma growth signaling, we examined whether altered HSPGs contribute to loss of growth control in gliomas. In a screen of five human glioma cell lines, U118 and U251 cell HSPGs activated FGF-2 signaling via FGFR1c. The direct comparison of U251 glioma cells with normal astrocyte HSPGs demonstrated that the glioma HSPGs had a significantly elevated ability to promote FGF-2-dependent mitogenic signaling via FGFR1c. This enhanced activity correlated with a higher level of overall sulfation, specifically the abundance of 2S- and 6S-containing disaccharides. Glioma cell expression of the cell-surface HSPG glypican-1 closely mirrored the FGF-2 coactivator activity. Furthermore, forced expression of glypican-1 in (glypican-1-deficient) U87 glioma cells enhanced their FGF-2 response. Immunohistochemical analysis revealed a highly significant overexpression of glypican-1 in human astrocytoma and oligodendroglioma samples compared with nonneoplastic gliosis. In summary, these observations suggest that altered HSPGs contribute to enhanced signaling of FGF-2 via FGFR1c in gliomas with glypican-1 playing a significant role in this mitogenic pathway.

Egr‐1 transcription activation exists in placental endothelium when vascular disease is present

To seek evidence of early vascular injury in the placental villous microcirculation in placental insufficiency identified by a high-resistance umbilical Doppler study by examining for expression of fibroblast growth factor receptor-1 (FGFR-1), its transcription factor, early growth response factor-1 (Egr-1) and plasma fibroblast growth factor-2 (FGF-2). Case-control study. University teaching hospital. Placentas and umbilical vein blood were collected at delivery from 12 women with normal pregnancy delivered at term and 14 with placental vascular disease defined by an abnormal umbilical artery Doppler study. Microvascular endothelial cells were isolated from fresh human placentas using collagenase digestion and Dynabeads coated with monoclonal antibody against CD31. RNA was extracted from the isolated endothelial cells. The messenger RNA (mRNA) expression of FGFR-1 and Egr-1 production were assessed by reverse transcription polymerase chain reaction and factored relative to 18S ribosomal RNA. To confirm that FGF-2 was playing a significant role in this microvascular endothelial cell injury in the placenta, we also measured the soluble fraction of FGF-2 in fetal plasma from same groups of pregnancies using an enzyme-linked immunosorbent assay. Microvascular endothelial cells expression of Egr-1mRNA, FGFR-1 mRNA and presence of soluble FGF-2 in fetal plasma. The soluble level of FGF-2 in the fetal placental circulation from pregnancy with placental vascular disease was increased when compared with normal pregnancy (median 10.15 pg/ml and interquartile range 5.34-21.83 pg/ml versus 4.46 pg/ml and 3.69-5.66 pg/ml; P < 0.05). Microvascular endothelial cells from the placental villi with placental vascular disease showed upregulation of both FGFR-1 mRNA expression (median 0.72 and interquartile range 0.40-1.64 versus 0.34 and 0.19-0.71; P<0.05) and Egr-1 expression (median 0.79 and interquartile range 0.27-1.86 versus 0.23 and 0.17-0.67; P<0.05) in comparison with normal pregnancy. Endothelial cells from the placental villi are upregulated for expression of Egr-1 transcription factor gene in placental vascular disease. The FGFR-1 activation and increase in FGF-2 in the fetal circulation are known to be very early features of the response of endothelium to injury. Egr-1 is a promoter of many key pathophysiologically relevant target genes, which influence the development of subsequent vascular lesions. This change may occur before the pathological features recognised on microscopy.

Ribozyme Cleavage Leads to Decreased Expression of Fibroblast Growth Factor Receptor 3 in Human Multiple Myeloma Cells, Which Is Associated with Apoptosis and Downregulation of Vascular Endothelial Growth Factor

The aim of this study was to investigate the fibroblast growth factor receptor 3 (FGFR3) mRNA cleavage by ribozymes targeting FGFR3, effect of growth inhibition and associated with mechanism on multiple myeloma (MM). We designated two ribozyme-expressing plasmids that target the FGFR3 genes, Rz52 and Rz32. In vitro catalytic activity of Rz52 and Rz32 in KMS11 cells decreased FGFR3 mRNA expression to 45% (p < 0.05) and 80% (p < 0.5), respectively, of that of the control. In vivo examination of the Rz52-transfected KMS11 clone showed that FGFR3 mRNA expression decreased to 20% (p < 0.05) of the control. In the Rz52-transfected H929 clone, FGFR3 mRNA decreased to 50% of the control. Protein expression of FGFR3 decreased to 70% of the parental KMS11 and H929 clones. DNA synthesis in the Rz52-transfected KMS11 clone decreased to 20% of that of the control, whereas the viability of cells decreased to 2% (p < 0.01) of that of the control. Ribozyme cleavage-associated increase in apoptosis of Rz52 KMS11 transfectants was twice that of the control. The inhibition of FGFR3 expression by ribozymes was associated with decreased vascular endothelial growth factor (VEGF) expression and upregulation of Flt-1 but not of the KDR receptor. Our data indicate that FGFR3 is an important cell survival and antiapoptotic factor for MM cells and that ribozyme-targeted downregulation of FGFR3 might be useful as a novel therapeutic intervention in MM characterized by t(4;14).

Fibroblast Growth Factor Receptor 3 Is Overexpressed in Urinary Tract Carcinomas and Modulates the Neoplastic Cell Growth

Fibroblast growth factor receptor 3 (FGFR3) mutations have been associated with achondroplastic syndromes and urinary bladder carcinomas. Here we describe changes in FGFR3 mRNA and protein expression in transitional carcinomas and determine the effect of monoclonal antibodies against FGFR3 in RT-112 cell line proliferation. We used microarray tools to evaluate FGFR3 mRNA expression in 22 urinary bladder carcinomas at different stages (noninvasive pTa, lamina propria invasive pT1, and muscular invasive pT2) and 7 nonneoplastic tissue controls. FGFR3 protein expression was evaluated by Western blotting in 15 different carcinomas and 3 nonneoplastic controls. Two hundred thirty-seven urinary bladder and renal pelvis carcinomas and 21 negative controls were tested on tissue microarrays by immunohistochemistry. The effect on cell proliferation in the RT-112 bladder cancer cell line of monoclonal antibodies against FGFR3 was also evaluated. Overexpression of FGFR3 mRNA was found in pTa and pT1 stage carcinomas (fold change >8) and in pT2 carcinomas (fold change >4). Nonneoplastic urinary bladder samples do not express FGFR3 protein. However, 83% of pTa, 100% of pT1, and 50% of pT2 carcinomas expressed FGFR3 as determined by Western blotting. By immunohistochemistry, FGFR3 was positive in 71.4% of pTa, 72% of pT1, and 49.2% of pT2 cases as well as 61.5% of upper urinary tract carcinomas. Proliferation of the RT-112 cell line was inhibited with monoclonal antibodies against FGFR3. FGFR3 seems to play an important role in transitional cell carcinoma development. Our results suggest that FGFR3 antagonists could be developed as possible therapeutics for treatment of urinary tract carcinoma.

Basic fibroblast growth factor and fibroblast growth factor receptor-1 in the human hippocampal formation

Basic fibroblast growth factor (bFGF) is an important mitogen and neurotrophic factor that binds and signals through the high-affinity receptor, fibroblast growth factor receptor 1 (FGFR1). However, only a limited amount of information is available concerning the molecular forms and anatomical distribution of fibroblast growth factors (FGFs) in the normal human brain. We found multiple bFGF and FGFR1 mRNA transcripts which vary in expression pattern across human brain regions. Using in situ hybridization and immunohistochemistry, we localized bFGF and FGFR1 mRNA and protein to cells in the normal adult human hippocampus and caudal entorhinal cortex (ERC). The majority of pyramidal neurons contained FGFR1 mRNA and protein in the mesial temporal lobe, with neurons in the CA2/CA3 region demonstrating the highest levels of FGFR1 mRNA. In contrast to FGFR1, bFGF mRNA expression was detected at very low levels in a small fraction of the neurons in the human hippocampus and caudal ERC. While bFGF mRNA may be expressed at low levels in neurons, bFGF-immunopositive cells with astrocytic features were detected throughout the mesial temporal lobe in rats, monkeys and humans. bFGF immunoreactive processes are found traversing the dentate gyrus, and bFGF immunoreactive cells are found in the neurogenic subgranular zone in all three mammalian species studied. The anatomical distribution of these two FGF family members suggests that bFGF is endogenously positioned to be involved in ongoing neurogenesis in the adult hippocampus, and that FGF trophic signaling to differentiated neurons could involve the release of astrocytic bFGF acting on neuronal FGFR1 in the normal adult human hippocampus.

Over-expression of fibroblast growth factor receptor 3 in human hepatocellular carcinoma

To describe the significant over-expression of fibroblast growth factor receptor 3 (FGFR3), which is a signal transduction and cell proliferation related gene in hepatocellular carcinoma (HCC). Following DNA microarray, Northern blot and quantitative real-time PCR were employed to confirm FGFR3 expression difference in HCC tissues and surrounding non-neoplastic liver tissue. FGFR3 expression levels were further determined by immunohistochemical study in 43 cases of HCC. Northern blot results showed the significant over-expression of FGFR3 in HCC tissues, which was consistent with that from DNA microarray. Quantitative real-time PCR demonstrated that the mean ratio of FGFR3 mRNA to glyceraldehyde-3-phosphate dehydrogenase (GADPH) mRNA in HCC tissue was 0.250, whereas the ratio in non-neoplastic liver tissue was 0.014. Statistical analyses of 43 cases of HCC revealed that HCC scored higher than the matched non-neoplastic liver tissues. Examination of clinicopathological features revealed a strong correlation of over-expression of FGFR3 with poor tumor differentiation and high nuclear grade. Over-expression of FGFR3 may play an important role in liver carcinogenesis. FGFR3 may be an ideal candidate as a molecular marker in the diagnosis of HCC and a potential therapeutic target.

Expression of growth-factor Receptors in Rat Socket coagulum-derived Cells

The purpose of this study was to determine if periodontal ligament (PDL) fibroblasts are capable of expressing growth factor receptors during their differentiation. PDL cells, obtained from 2-day-old coagulum in the tooth socket of Sprague-Dawley rats after tooth extraction, were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and antibiotics. Confluent cells were grown in the presence of 50 μg/ml ascorbic acid (AA), 10mM β-glycerophosphate (βGP), and 5 µM dexamethasone (Dex) for up to 3 weeks. Morphological changes in PDL cells during the 3-week cultures in the presence of AA, βGP, and Dex were investigated under a phase-contrast microscope. The messenger ribonucleic acid (mRNA) expression pattern of growth factors, including platelet-derived growth factor receptor-alpha and beta (PDGF-Rα and PDGFRβ), insulin-like growth factor type 1 and 2 receptors (IGF-1R and IGF-2R), and fibroblast growth factor receptor-4 (FGF-R4), were investigated in PDL cells, using Northern blot analysis. Based on the appearance of PDL cell cultures under the phase-contrast microscope, 4 morphologically distinct stages were identified: the confluent stage, the multilayer stage, the nodule stage and the mineralization stage. In the confluent stage (stage Ⅰ), PDL cells had a fibroblast-like or polygonal shape and formed a monolayer. On the 7(superscript th) day after treatment with AA, βGP, and Dex (stage Ⅱ), cell proliferation occurred in focal areas generating multilayered cells with a fibroblastic shape. After the 14(superscript th) day (stage Ⅲ) the multilayered cells formed round nodules by depositing a matrix between the cell layers. Heavy mineralization of the matrix was first seen on the 21st day after mineralized medium treatment and was characterized by the presence of refringent material covering the nodules, referred to as the mineralization stage (stage Ⅳ). By Northern blot analysis, it was determined that PDGF-Rα was weakly expressed in the confluent stage and then increased with the progression of PDL cell differentiation, while the level of PDGF-Rβ transcripts remained high throughout the 4 stages. IGF-1R gene activities were also high throughout the four stages. In contrast, expressions of IGF-2R and FGF-R4 mRNA were undetectable throughout differentiation. These results indicate that growth factor receptors are differentially expressed by PDL cells, which may be involved in the regulation of growth and differentiation of PDL cells.

Expression pattern of fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) system members in bovine corpus luteum endothelial cells during treatment with FGF-2, VEGF or oestradiol.

The development of the corpus luteum (CL) is accompanied by very active angiogenesis. We hypothesize that during this process endothelial cells (EC) are under the control of several angiogenic factors and steroids. The aim of this study was to examine the expression of the angiogenic growth factor systems - fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) - in EC derived from the bovine CL. Endothelial cells were cultured in serum-free medium and treated for 24 h with different concentrations of oestradiol (range from 10(-13) to 10(-5) mol/l), VEGF or FGF-2 (1, 10 and 100 ng/ml, respectively) and compared with untreated controls. Cells were harvested, total RNA extracted and subjected to semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Treatment with oestradiol or FGF-2 stimulated the expression of FGF-2, but VEGF treatment showed no effect on the FGF-2 expression. FGF-2 or VEGF treatment resulted in an up-regulation of the FGF receptor (FGFR) mRNA. However, no FGF-1 expression was detected in EC. For the VEGF system, treatment with FGF-2, VEGF or oestradiol did not affect VEGF expression. However, the presence of FGF-2 in the medium up-regulated the expression of both VEGF receptors (VEGFR-1 and VEGFR-2), whereas oestradiol or VEGF treatment showed no effect on the expression of these receptors. Our results reveal that functional angiogenic growth factor systems were expressed in vitro in bovine EC derived from the CL. This suggests that the angiogenic FGF and VEGF system members were regulated by FGF or VEGF, but not by oestradiol-17beta.

Fibroblast growth factor receptor-3 is expressed in undifferentiated intestinal epithelial cells during murine crypt morphogenesis.

Prior studies have demonstrated that fibroblast growth factor receptor-3 (FGFR-3) regulates proliferation of undifferentiated intestinal epithelial cells in vitro. However, the function(s) of FGFR-3-mediated signaling during intestinal development and epithelial differentiation in vivo remain unknown. The goal of this study was to define the temporal, regional, and cell-specific patterns of FGFR-3 expression and its ligands during normal intestinal ontogeny and epithelial regeneration. Both the IIIb and IIIc isoforms of FGFR-3 mRNA, which result from differential splicing of the FGFR-3 primary transcript, were detected in mouse small intestine as early as embryonic day 16. FGFR-3 levels peaked in the small intestine from 7 to 21 days after birth and decreased thereafter to reach the low levels observed in adult mice. FGFR-3 IIIb and IIIc mRNA levels were highest in the duodenum and proximal jejunum with lower levels of both seen in the distal jejunum, ileum, and colon. FGFR-3 was expressed in a subset of proliferating undifferentiated crypt epithelial cells located in the intervillous epithelium and in the lower half of nascently forming crypts but not in differentiated epithelial cell types. FGFR-3 IIIb was the dominant isoform expressed in both small intestinal and colonic crypts. Expression of FGF1, FGF2, and FGF9, known ligands of FGFR-3, paralleled patterns of FGFR-3 expression during gut development. These data suggest that signaling through FGFR-3 plays a role in regulating morphogenic events involved in formation of intestinal crypts and/or the fate of epithelial stem cells.

A novel ginseng saponin metabolite induces apoptosis and down-regulates fibroblast growth factor receptor 3 in myeloma cells

Ginseng (the root of Panax ginseng C.A. Meyer, Araliaceae) has been used as a crude drug taken orally for preventive and therapeutic purposes in Asian countries as a traditional medicine. In the current study, we have investigated the antitumor effect of a novel ginseng protopanaxadiol saponin bacterial metabolic derivative, 20-O-beta-D-glucopyranosyl-20(S)-protopanaxadiol (IH-901), in eight human myeloma cell lines. IH-901 inhibited the proliferation of all myeloma cell lines examined. Despite the fibroblast growth factor receptor 3 (FGFR3) overexpression due to a chromosomal translocation t(4;14)(q16.3;q32.3) in KMS-11 myeloma cells, IH-901 induced apoptosis in a dose- and time-dependent way in this cell line. Treatment of KMS-11 with IH-901 resulted in the formation of internucleosomal DNA fragments, poly (ADP-ribose) polymerase cleavage, and the activation of caspase-3. IH-901 also caused the down-regulation of FGFR3 mRNA and protein expression and inhibited ERK activity in KMS-11 cells. Our results demonstrate that IH-901 induces apoptosis and inhibits FGFR3 expression and signaling in KMS-11 cells, suggesting candidacy for the chemoprevention and the treatment of myeloma.

Expression of fibroblast growth factor 2 and its receptor during skeletal muscle development from turkeys with different growth rates

Fibroblast growth factor 2 (FGF2) is a key regulator of muscle cell proliferation and differentiation. To address how FGF2 and fibroblast growth factor receptor 1 (FGFR1) gene expression influences skeletal muscle development and growth, pectoralis major muscle was isolated at embryonic days (ED) 14, 16, 18, 20, 22, and 24, and at 1-, 8-, 12-, and 16-week posthatch from a turkey line (F) selected only for increased 16-week body weight and its genetic control line (RBC2). The mRNA levels of FGF2 and FGFR1 were measured by semi-quantitative reverse transcription polymerase chain reaction. Compared to the RBC2 line males, the F line males had higher FGF2 mRNA levels at ED 14 and 16, and higher FGFR1 mRNA levels at ED 18, but down-regulated FGF2 and FGFR1 gene expression at ED 22. Although no FGF2 mRNA was detected in posthatch muscle tissue, the F line turkeys had more FGFR1 gene expression at 8-, 12-, and 16-week posthatch than the RBC2 line turkeys. During myogenic satellite cell proliferation, the F line cells had higher FGF2 and FGFR1 mRNA levels than the RBC2 line cells. The satellite cell responsiveness to FGF2 treatment was evaluated by the ability of the cells to proliferate. The male satellite cells were more responsive to FGF2 stimulation than the female cells in both lines. These results suggest that the F line turkeys have increased FGF2 signaling that may affect muscle cell proliferation and differentiation, which may also lead to an enhancement in muscle development and growth rate.

Expression of fibroblast growth factor receptor-3 (FGFR3), signal transducer and activator of transcription-1, and cyclin-dependent kinase inhibitor p21 during endochondral ossification: differential role of FGFR3 in skeletal development and fracture repair.

Increasing evidence suggests that fibroblast growth factor receptor-3 (FGFR3) is a negative regulator of endochondral bone growth; however, its role during skeletal repair is unknown. Using a rat model of closed femoral fracture healing, we analyzed the spatial and temporal expression of FGFR3. To assess a possible role for FGFR3 during healing, we also analyzed the spatial and temporal expression of signal transducer and activator of transcription-1 (STAT1) and cyclin-dependent kinase inhibitor p21, important mediators of FGFR3 signaling. Before these experiments, we studied the spatial expression of FGFR3 during skeletal development in mouse embryos. At 16.5 and 19.5 d post coitum, FGFR3 mRNA was strongly expressed in resting and proliferating chondrocytes but weakly in hypertrophic chondrocytes and not in osteoblasts. In contrast, during fracture repair, it was strongly expressed in prehypertrophic chondrocytes, and the expression level reached a maximum on d 14. Immunoreactivity for STAT1 was detected in the cytoplasm of chondrocytes on d 4 and 7 and both in the cytoplasm and nucleus of hypertrophic chondrocytes on d 14. Furthermore, FGFR3, STAT1, and p21 exhibited a similar temporal expression profile, suggesting that FGFR3-mediated STAT1-p21 signaling plays a role in fracture repair. These results indicate a differential role of FGFR3 in skeletal development and fracture repair.

Thyroid hormone activates fibroblast growth factor receptor-1 in bone.

Thyroid hormone (T3) and the T3 receptor (TR) alpha gene are essential for bone development whereas adult hyperthyroidism increases the risk of osteoporotic fracture. We isolated fibroblast growth factor receptor-1 (FGFR1) as a T3-target gene in osteoblasts by subtraction hybridization. FGFR1 mRNA was induced 2- to 3-fold in osteoblasts treated with T3 for 6-48 h, and FGFR1 protein was stimulated 2- to 4-fold. Induction of FGFR1 was independent of mRNA half-life and abolished by actinomycin D and cycloheximide, indicating the involvement of an intermediary protein. Fibroblast growth factor 2 (FGF2) stimulated MAPK in osteoblasts, and pretreatment with T3 for 6 h induced a more rapid response to FGF that was increased in magnitude by 2- to 3-fold. Similarly, T3 enhanced FGF2-activated autophosphorylation of FGFR1, but did not modify FGF2-induced phosphorylation of the docking protein FRS2. These effects were abolished by the FGFR-selective inhibitors PD166866 and PD161570. In situ hybridization analyses of TRalpha-knockout mice, which have impaired ossification and skeletal mineralization, revealed reduced FGFR1 mRNA expression in osteoblasts and osteocytes, whereas T3 failed to stimulate FGFR1 mRNA or enhance FGF2-activated MAPK signaling in TRalpha-null osteoblasts. These findings implicate FGFR1 signaling in T3-dependent bone development and the pathogenesis of skeletal disorders resulting from thyroid disease.

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.

Down-regulation of (IIIb) and (IIIc) isoforms of fibroblast growth factor receptor 2 (FGFR2) is associated with malignant progression in human prostate.

Fibroblast growth factors (FGFs) and their receptors (FGFRs) have a critical function in the cellular stroma/epithelium interaction for the development and homeostasis of human prostate. Imbalance in expression of these factors is associated with malignancy in several cancers. To quantify the expression of fibroblast growth factor receptor isoforms FGFR2(IIIb), FGFR2(IIIc), FGFR1(IIIc), and fibroblast growth factors FGF2 and FGF7 in normal and tumoral human prostate tissues, and human prostatic epithelial cell lines, we used quantitative real-time polymerase chain reaction. The expression of FGFR2(IIIb) mRNA is down-regulated in 60% of the tumors studied (P < 0.0001). Furthermore, FGFR2(IIIb) is significantly reduced in androgen-independent tumors (AI) compared with androgen-responsive tumors (AD) (P = 0.02). A significant reduction in FGFR2(IIIc) expression is also observed in 80% of tumors (P = 0.001). However, unlike FGFR2(IIIb), the down-regulation of FGFR2(IIIc) is not related to the androgen-independent status (P = 0.09). On the other hand, neither FGFR1(IIIc) nor FGF2 and FGF7 have shown any significant variation in expression between normal and cancerous specimens. These findings propose that decreased expression of not only FGFR2(IIIb) but also FGFR2(IIIc) isoforms may be a critical step in prostate cancer progression and furthermore suggest that FGFR2(IIIb) expression could be used as a marker for prostate cancer evolution from androgen-dependent to androgen-independent status.

Concomitant expression of heparin-binding epidermal growth factor-like growth factor mRNA and basic fibroblast growth factor mRNA in myocardial infarction in rats.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is mitogenic and chemotactic for many cell types. HB-EGF is induced in pathological states which require cell mitogenesis and proliferation, including angiogenesis, and has been reported to interact functionally with basic fibroblast growth factor (bFGF). To test our hypothesis that HB-EGF mRNA expression is increased in myocardial infarction, we used Northern hybridization in rats to investigate the expression of HB-EGF and EGF receptor mRNAs expression in the infarct zone compared to the expression of bFGF and FGF receptor mRNAs. We also performed in situ hybridization to identify the cells responsible for HB-EGF mRNA production. HB-EGF mRNA rapidly increased after ligation (mean +/- SE, 5.6+/-0.23-fold increase at 6 hours compared to the preligation heart levels) and reached a maximum level (9.1+/-0.42-fold increase) around 12 hours. HB-EGF mRNA then gradually decreased on day 1 (5.8+/-1.0-fold increase), day 2 (3.2+/-0.94-fold increase) and day 3 (1.9+/-0.33-fold increase) after ligation. Parallel changes in bFGF mRNA expression were observed (6, 12 hours, days 1, 2 and 3; 3.6+/-0.42-, 5.3+/-0.12-, 2.3+/-0.12-, 1.7+/-0.03- and 0.95+/-0.03-fold increase, respectively). EGF receptor (ErbB-1) mRNA was gradually increased on day 2 (2.4+/-0.53-fold increase), day 7(4.0+/-0.61-fold increase) and day 14 (7.0+/-0.61-fold increase). Similarly, FGF receptor (FGF receptor-1) mRNA was gradually increased (days 2,7 and 14; 1.3+/-0.13-, 1.5+/-0.17- and 2.3+/-0.15-fold increase, respectively). Reperfusion after a 2-hour ligation (too late to salvage myocytes) enhanced HB-EGF (12 hours, 16.8+/-1.8-fold increase) and bFGF (12 hours, 10.4+/-1.1-fold increase) mRNA expression. The cells responsible for the increased production of HB-EGF mRNA were shown by in situ hybridization to be surviving myocytes located in the infarct peripheral zone around infarct necrotizing tissue. In conclusion, our results demonstrated a rapid increase in HB-EGF mRNA expression concomitant with an increase in bFGF mRNA expression, suggesting that HB-EGF and bFGF might play some role in the course of pathological changes in the infarct in the early inflammatory phase. Reperfusion at times too late to salvage myocytes accelerated sequential changes in the expression of both HB-EGF and bFGF mRNAs.