FGFR1 mRNAs Research Articles

Novel mineral regulatory pathways in ovine pregnancy: I. phosphate, klotho signaling, and sodium-dependent phosphate transporters.

Appropriate mineralization of the fetal skeleton requires an excess of phosphate in the fetus compared to the mother. However, mechanisms for placental phosphate transport are poorly understood. This study aimed to identify phosphate regulatory pathways in ovine endometria and placentae throughout gestation. Suffolk ewes were bred with fertile rams upon visual detection of estrus (Day 0). On Days 9, 12, 17, 30, 70, 90, 110, and 125 of pregnancy (n=3-14/Day), ewes were euthanized and hysterectomized. Phosphate abundance varied across gestational days in uterine flushings, allantoic fluid, and homogenized endometria and placentae (P<0.05). The expression of mRNAs for sodium-dependent phosphate transporters (SLC20A1 and SLC20A2) and klotho signaling mediators (FGF7, FGF21, FGF23, FGFR1-4, KL, KLB, ADAM10, and ADAM17) were quantified by qPCR. Day 17 conceptus tissue expressed SLC20A1, SLC20A2, KLB, FGF7, FGF21, FGF23, FGFR1, and FGFR2 mRNAs. Both sodium-dependent phosphate transporters and klotho signaling mediators were expressed in endometria and placentae throughout gestation. Gestational day influenced the expression of SLC20A1, ADAM10, ADAM17, FGF21, FGFR1, and FGFR3 mRNAs in both endometria and placentae (P<0.05). Gestational day influenced endometrial expression of FGF7 (P<0.001), and placental expression of FGF23 (P<0.05). Immunohistochemistry confirmed that both FGF23 and KL proteins were expressed in endometria and placentae throughout gestation. The observed spatiotemporal profile of KL-FGF signaling suggests a potential role in the establishment of pregnancy and regulation of fetal growth. This study provides a platform for further mechanistic investigation into the role for KL-FGF signaling in the regulation of phosphate transport at the ovine maternal-conceptus interface.

Functional conservation and divergence of duplicated fibroblast growth factor receptor 1 (fgfr1) genes in blunt snout bream (Megalobrama amblycephala)

Fgfr1 is a fibroblast growth factor receptor involved in regulating cell growth, proliferation, differentiation and migration. Here, we report the isolation and characterization of duplicated fgfr1 genes in blunt snout bream (Megalobrama amblycephala). Blunt snout bream fgfr1a and -1b cDNAs were found to share a relatively high sequence identity of 82%. During embryogenesis, both fgfr1a and -1b mRNAs were highly detected at zygotes but gradually decreased and then constantly expressed after 16hpf, besides a strong expression for the fgfr1b mRNA at 12hpf. Whole-mount in situ hybridization demonstrated that fgfr1a mRNA was transcribed at the eyes, mid-hindbrain boundary (MHB), brain, posterior somites and tailbud at 16hpf, while the fgfr1b mRNA was only detected at the eyes and posterior somites at the same period. At 28hpf embryos, both fgfr1a and -1b mRNAs were expressed in the eyes, brain, pharyngeal arches and tailbud, and in the eyes, brain, pharyngeal arches and notochord at 55hpf. In adult fish, fgfr1a mRNA was strongly expressed in the gill, gonad, brain and midgut, but examined relatively low in the skin and kidney. In contrast, the fgfr1b mRNA was highly detected in the brain and liver and quite low in the skin, gill and kidney. During starvation, both fgfr1a and -1b mRNAs were significantly up-regulated in the intestine and liver, but down-regulated in the brain. Moreover, duplicated fgfr1 mRNAs were differentially inhibited in tissues with exogenous recombinant hGH. Our results suggest that two fgfr1 genes play important roles in regulating growth and development in blunt snout bream.

Chronic Academic Stress Increases a Group of microRNAs in Peripheral Blood

MicroRNAs (miRNAs) play key roles in regulation of cellular processes in response to changes in environment. In this study, we examined alterations in miRNA profiles in peripheral blood from 25 male medical students two months and two days before the National Examination for Medical Practitioners. Blood obtained one month after the examination were used as baseline controls. Levels of seven miRNAs (miR-16, -20b, -26b, -29a, -126, -144 and -144*) were significantly elevated during the pre-examination period in association with significant down-regulation of their target mRNAs (WNT4, CCM2, MAK, and FGFR1 mRNAs) two days before the examination. State anxiety assessed two months before the examination was positively and negatively correlated with miR-16 and its target WNT4 mRNA levels, respectively. Fold changes in miR-16 levels from two days before to one month after the examination were inversely correlated with those in WNT4 mRNA levels over the same time points. We also confirmed the interaction between miR-16 and WNT4 3′UTR in HEK293T cells overexpressing FLAG-tagged WNT4 3′UTR and miR-16. Thus, a distinct group of miRNAs in periheral blood may participate in the integrated response to chronic academic stress in healthy young men.

The expression of fibroblast growth factor receptors during early bovine conceptus development and pharmacological analysis of their actions on trophoblast growth in vitro

The overall aim of this work was to examine the expression profiles for fibroblast growth factor receptors (FGFRs) and describe their biological importance during bovine pre- and peri-implantation conceptus development. FGFR1 and FGFR2 mRNAs were detected at 1-, 2-, 8-cell, morula and blastocyst stages whereas FGFR3 and FGFR4 mRNAs were detected after the 8-cell stage but not earlier. The abundance of FGFR1, FGFR3, and FGFR4 mRNAs increased at the morula and blastocyst stages. Immunofluorescence microscopy detected FGFR2 and FGFR4 exclusively in trophoblast cells whereas FGFR1 and FGFR3 were detected in both trophoblast cells and inner cell mass in blastocysts. Neither transcripts for FGF10 nor its receptor (FGFR2b) were temporally related to interferon τ (IFNT) transcript profile during peri- and postimplantation bovine conceptus development. A series of studies used a chemical inhibitor of FGFR kinase function (PD173074) to examine FGFR activation requirements during bovine embryo development. Exposing embryos to the inhibitor (1 μM) beginning on day 5 post-fertilization did not alter the percentage of embryos that developed into blastocysts or blastocyst cell numbers. The inhibitor did not alter the abundance of CDX2 mRNA but decreased (P<0.05) the relative abundance of IFNT mRNA in blastocysts. Exposing blastocysts to the inhibitor from days 8 to 11 post-fertilization reduced (P<0.05) the percentage of blastocysts that formed outgrowths after transfer to Matrigel-coated plates. In conclusion, each FGFR was detected in bovine embryos, and FGFR activation is needed to maximize IFNT expression and permit outgrowth formation.

BFGF siRNA expression plasmid inhibits growth of human LEC-B3 cells

Basic fibroblast growth factor (bFGF) has been thought to play an important role during the development of posterior capsule opacification (PCO) by promoting the growth of lens epithelial cells (LECs). In the present study, we sought to explore the inhibition effect on LEC-B3 cells growth by plasmid-based RNA interference (RNAi) targeting bFGF. It is a prospective study. LEC-B3 cells, an immortal human lens capsule epithelial cell line, were identified as human lens epithelium by immunohistochemical assay of alphaB-crystalline. Both bFGF and bFGF receptor 1 (FGFR1) mRNA expressions of LEC-B3 cells were determined by reverse transcription polymerase chain reaction (RT-PCR). Based on the identification of bFGF expression, plasmid-based RNA interference (RNAi) was used to inhibit bFGF mRNA expression of LEC-B3 cells by stable transfection. bFGF mRNA and protein levels were examined by real time PCR and western blot, respectively. Then the flow cytometry (FCM) was performed to evaluate proliferation cell nuclear antigen (PCNA) expression. PCNA level in bFGF-interfered LEC-B3 cells was compared to that of the control. P < 0.05 was regarded as statistically different. bFGF and FGFR1 mRNAs were abundantly expressed in LEC-B3 cells. bFGF mRNA and protein levels were both down-regulated by stable transfection of bFGF siRNA expression plasmid. PCNA expression was significantly decreased by inhibiting bFGF expression (t = -5.0011, P = 0.0005). bFGF and FGFR1 mRNA are extensively expressed in LEC-B3 cells. Plasmid-based RNAi targeting bFGF can lead to potent inhibition of LEC-B3 cells growth, which may play a part in dealing with the development of PCO.

Fibroblast growth factor-2 and its receptor expression in proliferating precursor cells of the subventricular zone in the adult rat brain

Several findings have suggested the existence in the subventricular zone (SVZ) from sagittal sections of adult rat brain of a trophic mechanism, mediated by fibroblast growth factor-2 (FGF-2) and its multiple high-affinity FGF receptors (FGFRs), regulating neurogenesis mainly by controlling precursor cell proliferation. However, no clear data are available on the expression of FGF-2 and FGFRs in proliferating precursor cells of the SVZ. To address these questions we examined FGF-2 mRNA and its FGFR mRNA expression in proliferating precursor cells of the SVZ by using a double labeling procedure, combining in situ hybridization for FGF-2 and its FGFR mRNA with immunohistochemistry for bromodeoxyuridine (BrdU), a marker for proliferating cells. The results showed that FGFR1 and FGFR2 mRNAs were expressed in BrdU-labeled proliferating precursor cells, whereas FGFR3 and FGF-2 mRNAs were not, suggesting that in the SVZ the proliferating precursor cells express FGFR1 or FGFR2 and they may respond to FGF-2 released by non-proliferating cells. The FGFR4 mRNA was not found expressed in the SVZ. In the future, by identifying the cell types expressing FGFRs, it will be possible to gain insight into the functional activity of FGF2 within the SVZ.

Changes in FGF and FGF receptor expression in low-frequency-stimulated rat muscles and rat satellite cell cultures

This study compares effects of chronic electrical stimulation on the expression levels of FGF-1, FGF-2 and their receptors (FGFR1, FGFR4) in rat tibialis anterior (TA) muscle of hypothyroid rat, as well as in satellite cell cultures derived from normal rat TA and soleus (SOL) muscles. In 5-day (5-d)-stimulated hypothyroid TA muscle, FGF-1 and FGF-2 mRNA levels were threefold elevated over control. FGFR1 and FGFR4 mRNAs were twofold and 1.5-fold elevated, respectively. In longer stimulated muscles, FGF-1 and FGFR4 mRNAs returned to basal levels, whereas FGF-2 mRNA remained elevated. FGFR1 mRNA decreased to control levels in 10-d stimulated muscles, but increased again after 20 days of stimulation. SOL- and TA-derived satellite cell cultures were stimulated for 5 days. At this time point, changes in myosin heavy chain isoforms were detectable consisting of increases in MHCI mRNA and decreases in MHCIIb and MHCIId mRNA. The comparison between 5-d-stimulated hypothyroid TA muscle and 5-d-stimulated TA- and SOL-derived satellite cell cultures revealed differences in the expression of FGF-1 and FGF-2, but similar expression levels of FGFR1 and FGFR4. Even though FGF-1 and FGF-2 mRNAs were elevated in the satellite cell cultures, their increases were less pronounced than in the stimulated hypothyroid muscle. Taking into consideration that skeletal muscle contains muscle fibres and various non-muscle tissues, e.g. blood vessels, these results suggest that the latter contribute to the observed increases in FGF-1 and FGF-2 expression in stimulated muscle.

Ciliary neurotrophic factor induces expression of the IGF type I receptor and FGF receptor 1 mRNAs in adult rat brain oligodendrocytes

Ciliary neurotrophic factor (CNTF) is produced and released in response to injury in the central nervous system (CNS). While CNTF initially was characterized as a trophic factor for neurons, more recent evidence supports roles for this factor in survival, proliferation, and maturation of oligodendrocyte lineage cells. Evidence is emerging to support the hypothesis that CNTF's actions may include enhancing other growth and trophic factors. Here we tested the hypothesis that CNTF can induce expression of receptors on oligodendrocytes for factors that are known to promote their generation, maturation, and survival. Specifically, we used an in vivo paradigm to test whether CNTF, when injected stereotactically into forebrain white matter of adult rats, could induce mRNA expression for the insulin-like growth factor (IGF) type I receptor (IGF-IR), fibroblast growth factor (FGF) receptor (FGFR)-1, FGFR3, and platelet-derived growth factor (PDGF) receptor-alpha (PDGFRα). We determined that CNTF injection increased expression of IGF-IR and FGFR1 mRNAs in adult white matter to 200–250% of control levels. Cellular analysis indicated that these receptor mRNAs were induced in interfascicular oligodendrocytes. In contrast, CNTF had no effect on levels of FGFR3 and PDGFRα mRNAs. These results suggest that CNTF enhances the sensitivity of oligodendrocytes to other mitogens and trophic factors via induction of their receptors. J. Neurosci. Res. 57:447–457, 1999. © 1999 Wiley-Liss, Inc.

Ciliary neurotrophic factor induces expression of the IGF type I receptor and FGF receptor 1 mRNAs in adult rat brain oligodendrocytes

Ciliary neurotrophic factor (CNTF) is produced and released in response to injury in the central nervous system (CNS). While CNTF initially was characterized as a trophic factor for neurons, more recent evidence supports roles for this factor in survival, proliferation, and maturation of oligodendrocyte lineage cells. Evidence is emerging to support the hypothesis that CNTF's actions may include enhancing other growth and trophic factors. Here we tested the hypothesis that CNTF can induce expression of receptors on oligodendrocytes for factors that are known to promote their generation, maturation, and survival. Specifically, we used an in vivo paradigm to test whether CNTF, when injected stereotactically into forebrain white matter of adult rats, could induce mRNA expression for the insulin-like growth factor (IGF) type I receptor (IGF-IR), fibroblast growth factor (FGF) receptor (FGFR)-1, FGFR3, and platelet-derived growth factor (PDGF) receptor-alpha (PDGFRalpha). We determined that CNTF injection increased expression of IGF-IR and FGFR1 mRNAs in adult white matter to 200-250% of control levels. Cellular analysis indicated that these receptor mRNAs were induced in interfascicular oligodendrocytes. In contrast, CNTF had no effect on levels of FGFR3 and PDGFRalpha mRNAs. These results suggest that CNTF enhances the sensitivity of oligodendrocytes to other mitogens and trophic factors via induction of their receptors.

Comparative localization of fibroblast growth factor receptor-1, -2, and -3 mRNAs in the rat brain: In situ hybridization analysis

The present study provides a detailed comparative description in the adult rat brain of areas that express mRNAs coding for the fibroblast growth factor subtype receptors 1-3 (FGFR1-3). One observation in this analysis was a widespread expression in the brain of all three FGFR mRNAs, according to the following rank order: FGFR1, diencephalon < telencephalon < mesencephalon and metencephalon < myelencephalon; FGFR2 and FGFR3, telencephalon < diencephalon < mesencephalon and metencephalon < myelencephalon. Another observation was an apparent cellular specificity in their basal expression. Thus, the FGFR1 mRNA was expressed mainly in large and weakly stained cells, whereas FGFR2 transcripts were expressed primarily in small and strongly stained cells and in cells of brain regions devoid of neuronal cells, such as the white matter. FGFR3 mRNA was always detected in small and strongly stained cells with scattered distribution and was not expressed in the white matter. However, FGFR2 mRNA was weakly expressed also in large cells localized in some nuclei of the lower brainstem, in the diagonal band, and in the septum. Furthermore, in the medial habenula and in the nuclei of the pons, there exists a high density of cells expressing both FGFR1 and FGFR2 (60-100%). With neurotoxic lesions involving 6-hydroxydopamine microinjections in the substantia nigra, reactive glial cells in the lesioned area and surrounding the cannula tract showed an increase in the expression of both FGFR1 and FGFR2 mRNAs, whereas no increased expression was found for FGFR3 mRNA. Taken together, these findings showed that these three FGF receptors exist in all subtypes of cells of each brain region. Their apparent cellular specificity suggests that these receptor subtypes can have a differential trophic role in the brain, reflecting the various biological activities shown by the ligands of the FGF family.

A comprehensive analysis of the distribution of FGF-2 and FGFR1 in the rat brain

We have examined the cellular distribution of both FGF-2 and FGFR1 immunoreactivity and their mRNAs throughout the normal adult rat brain in order to reconcile numerous disparate findings in the published literature. The results confirm a widespread distribution of FGF-2 and FGFR1 in the rat brain, and different regions express distinct patterns of FGF-2 and FGFR1 mRNA and protein: neuronal and non-neuronal cells show different subcellular distributions that vary according to the area where they are located. The intensity of the staining and hybridization also varies according to the loci examined and the cell type involved. Astrocytes contain the highest levels of FGF-2 and FGFR1 mRNAs, and characteristically, possess high levels of immunoreactive FGF-2 within the nucleus. Amongst non-neuronal cells, oligodendrocytes do not synthesize or contain significant levels of FGF-2 immunoreactivity however, they do express FGFR1 mRNA. In these cells, immunoreactive FGFR1 is mainly associated with the myelin sheaths of neuronal fibers. In ventricular systems, ependymal cells synthesize and contain immunoreactive FGFR1. In contrast, only cells lining the lateral wall of the IIIrd ventricle express FGF-2 mRNA. Subependymal cells contain high levels of both FGF-2 and FGFR1 immunoreactivity. Neurons express low levels of FGF-2 mRNA and immunoreactive FGF-2 is localized predominantly to the perikaryon. However, selected populations of neurons, such as CA2 field of the hippocampus, show high levels of FGF-2 mRNA, in which the nucleus is strongly immunopositive. Similarly, high levels of FGFR1 mRNA are localized to select populations of neurons (e.g. amygdala). FGFR1 immunoreactivity is mainly associated with myelinated fiber tracts (e.g. striatum), and some neurons show immunoreactivity in the perikaryon (e.g. hippocampus), the nucleus (e.g. mesencephalic trigeminal nucleus), or in axonal projections (e.g. hypothalamus). Remarkably, in many of the areas studied, FGF-2 and FGFR1 mRNA and/or their translated protein do not co-localize in neurons (e.g. neo-cortices) or even in the same regions of the brain (e.g. substantia nigra). In other instances, mRNAs for both FGF-2 and FGFR1 colocalize (e.g. supraoptic nucleus). The brain, in contrast to peripheral tissues, contains high levels of FGF-2 and actively expresses its gene under normal physiological conditions. The highly specific anatomical distribution of immunoreactive FGF-2 in neuronal and non-neuronal brain cells, supports the notion that it plays a multifunctional role in the CNS under normal physiology. By correlating the localization and the synthesis of FGF-2 and one of its high affinity receptors, FGFR1, in the CNS, it should be possible to obtain a better understanding of the roles of FGF-2 in normal and pathological conditions.

Acidic FGF and FGF receptors are specifically expressed in neurons of developing and adult rat dorsal root ganglia.

Employing complementary technical approaches, we have studied the expression of acidic fibroblast growth factor (aFGF) and FGF receptors in rat dorsal root ganglia. The results clearly showed that within spinal nerves aFGF and two high-affinity FGF receptors, FGFR-1 and FGFR-2, were prominently expressed in neurons, while expression in Schwann cells was undetectable. FGFR-3 and FGFR-4 were not expressed in dorsal root ganglia. Acidic FGF mRNA was detected in the majority of dorsal root ganglion neurons, including all size classes: FGFR-1 and FGFR-2 transcripts were only detected in subpopulations of mainly large and medium size neurons. In subcellular fractionation studies on dorsal root ganglion and spinal root tissue, aFGF was recovered in the soluble fraction and was thus not tightly associated with neuronal membranes. During development FGFR-1 and FGFR-2 mRNAs were found to be present at all stages examined (embryonic days 15-21 and postnatal days 1-120). Acidic FGF mRNA and protein were first detected at embryonic day 18, and their expression then increased progressively up to postnatal levels. In cultures of dorsal root ganglion neurons derived from day 15 embryos, aFGF expression was first detected 3 days after plating. The resulting neuron cultures continued to express aFGF in a Schwann cell-independent manner. In combination, these results indicate that aFGF expression in dorsal root ganglia is initiated and maintained in postmitotic neurons. Furthermore, the data suggest that the physiological function of aFGF in the peripheral nervous system is connected to processes specific to the mature sensory (and motor) system, such as the maintenance and survival of peripheral nerve neurons.

Differential expression of fibroblast growth factor receptors by human neurones, astrocytes and microglia.

Expression of fibroblast-growth factor receptors (FGFRs) was studied in human fetal neurones, astrocytes and microglia in culture. Northern blot analysis showed that neurones and microglia expressed the mRNAs for FGFR-1, FGFR-2, FGFR-3, FGFR-4 at different levels, whereas astrocytes expressed only FGFR-1 and FGFR-4 mRNAs. Immunocytochemical localization of FGFR-1 revealed that this receptor was predominantly localized on the axon hillock membrane in neurones, and was associated with the plasma membrane of ameboid, activated microglia and of glial-fibrillar acidic protein positive astrocytes. The expression of various members of the FGFR family in all the cell types investigated implicates FGFs in human brain development and functions.

Basic fibroblast growth factor (BFGF) and two of its receptors, FGFR1 and FGFR2: gene expression in the rat brain during postnatal development as determined by quantitative RT-PCR

Regional and temporal patterns of the expression of basic fibroblast growth factor (bFGF), and two of its high affinity receptors (FGFR1 and FGFR2), were examined in the male rat brain during early postnatal development; the reverse transcription-polymerase chain reaction (RT-PCR) was used to obtain mRNA measurements which were expressed relative to mRNA for GAPDH as a constant. In the rat cerebrum, the mRNAs for bFGF and for FGFR2 were relatively low in amount within the first postnatal week, but by 28 days, they were as high as in the 1-year-old rat cerebrum. In contrast, the expression of FGFR1 was biphasic: mRNA levels were higher at postnatal days 1 and 28 than at day 21. Quantitation of mRNA from microdissected regions of 28-day-old rat brain revealed that the expression of bFGF and of FGFR2 showed a marked variation between regions but the expression of FGFR1 appeared less variable between the regions that were analyzed. For all three genes the hippocampus appeared to have high relative amounts of mRNA. The temporal patterns of expression of bFGF, FGFR1 and FGFR2 also differed with brain region during early postnatal development. In the occipital cortex and inferior colliculus, the mRNAs for bFGF and FGFR2 both increased in amount during the first month, unlike that for FGFR1. However, in the cerebellum, the highest expression of bFGF and FGFR1 mRNAs occurred at postnatal day 1; FGFR2 expression apparently showed less change with age. The temporal changes in bFGF, FGFR1 and FGFR2 expression in different brain regions during early postnatal development suggest that receptor regulation may permit different physiological effects of bFGF according to brain region and developmental age.

Rat FGF receptor-4 mRNA in the brain is expressed preferentially in the medial habenular nucleus

The fibrobast growth factor (FGF) receptor family consists of four members, FGFR-1, FGFR-2, FGFR-3 and FGFR-4, that are closely related receptor tyrosine kinases. We examined the expression of rat FGFR-4 mRNA in the brain by in situ hybridization and compared it with that of the mRNAs for other FGF receptors. In contrast with FGFR-1, FGFR-2 and FGFR-3 mRNAs which are expressed widely in the brain, the FGFR-4 mRNA in the brain is expressed preferentially in the medial habenular nucleus neurons. The present finding indicates that FGFR-4 has a function specific to the medial habenular nucleus.

The structure and expression of the FGF receptor-1 mRNA isoforms in rat tissues

In this paper, we describe the structure of rat FGF receptor-1 mRNA isoforms and their expression in a variety of rat tissues. The rat FGFR-1 has the characteristics of FGFR-1 as well as mouse, human and chicken homologs. FGFR-1 mRNA was detectable in all the tissues examined by Northern analysis or polymerase chain reaction, indicating that FGFR-1 mRNA is widely expressed in rat tissues. The rat FGFR-1 mRNA has isoforms in both the extracellular and intracellular regions. The extracellular isoforms which have two or three immunoglobulin-like domains, are expressed almost equally in the tissues except for brain. However, the large form is a major form in the brain. Furthermore, in the brain, half of FGFR-1 mRNAs have the six nucleotides, which encode a potential serine-threonine kinase phosphorylation site in the intracellular juxtamembrane region, deleted. In contrast to the brain, the deletion isoform is a minor form in the other tissues. The tissue-specific expression of the isoforms indicates that they have different physiological functions. Although other isoforms of FGFR-1 mRNA in tumor cell lines have been reported, the isoforms were undetectable in all rat tissues examined, indicating the isoforms are products of abnormal alternative splicing in tumor cell lines.