Fibroblast growth factor-16 is a growth factor for embryonic brown adipocytes

In rat embryos, fibroblast growth factor (FGF)-16 is predominantly expressed in brown adipose tissue. To elucidate the role of FGF-16, we examined the expression of FGF-16 mRNA in rat embryonic brown adipose tissue at different developmental stages by Northern blotting analysis and in situ hybridization. FGF-16 mRNA was expressed abundantly in brown adipose tissue during embryonic day 17. 5, embryonic days 17.5-19.5, and thereafter at lower levels into the neonatal period. The expression profile of FGF-16 mRNA well corresponds to the proliferative profile of embryonic brown adipose tissue reported. We also examined the mitogenic activity of recombinant rat FGF-16 for primary brown adipocytes prepared from rat embryonic brown adipose tissue. FGF-16 showed significant mitogenic activity for primary brown adipocytes. The mitogenic activity was found to be exerted by binding and activating FGF receptor-4 in the brown adipose tissue. As a great induction of proliferation of rat brown adipose tissue during cold acclimation was reported, we also examined the expression of FGF-16 mRNA in the brown adipose tissue during cold acclimation by Northern blotting analysis. The expression of FGF-16 mRNA was not increased, but rather decreased. The expression profile of FGF-16 mRNA and the mitogenic activity of FGF-16 reported here indicate that FGF-16 is a unique growth factor involved in proliferation of embryonic brown adipose tissue.     

Fibroblast growth factor is an inhibitor of chondrocyte terminal differentiation

The effects of basic fibroblast growth factor (bFGF) on terminal differentiation of chondrocytes and cartilage-matrix calcification were investigated. Rabbit growth-plate chondrocytes maintained as a pelleted mass in a centrifuge tube produced an abundant proteoglycan matrix during the matrix-maturation stage, yielding a cartilage-like tissue. Thereafter, they terminally differentiated to hypertrophic chondrocytes which produced high levels of alkaline phosphatase. These cells induced extensive calcification of the matrix in the absence of additional phosphate (Kato, Y., Iwamoto, M., Koike, T., Suzuki, F., and Takano, Y. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 9552-9556). Addition of bFGF to the chondrocyte cultures abolished the increases in alkaline phosphatase activity, 45Ca deposition, and the calcium content. These effects were dose-dependent, reversible, and observed in the presence of cytosine arabinoside, an inhibitor of DNA synthesis. The inhibitory effects could be observed only when chondrocytes were exposed to bFGF in a transition period between the matrix-maturation and hypertrophic stages. As chondrocytes differentiated to hypertrophic cells, bFGF became less effective in inhibiting the expression of the mineralization-related phenotypes. The present study also shows that although the rate of [35S]sulfate incorporation into large, chondroitin sulfate proteoglycan in the cell-matrix fraction is very high during the matrix-maturation stage, it abruptly decreases by 90% after terminal differentiation. Furthermore, the terminal differentiation-associated decrease in proteoglycan synthesis was delayed by bFGF. These results provide evidence that bFGF inhibits terminal differentiation of chondrocytes and calcification.     

Fibroblast growth factor-2-induced signaling through lipid raft-associated fibroblast growth factor receptor substrate 2 (FRS2)

The plasma membrane is not homogeneous but contains specific subcompartments characterized by their unique lipid and protein composition. Based on their enrichment in various signaling molecules, these membrane microdomains are recognized to be sites of localized signal transduction for a number of extracellular stimuli. We have previously shown that fibroblast growth factor-2 (FGF2) induced a specific signaling response within a lipid raft membrane microdomain in human neuroblastoma cells characterized by the tyrosine phosphorylation of a p80 phosphoprotein. Herein, we show that this protein is the signaling adaptor FRS2 and that it is localized exclusively to lipid rafts in vitro and in vivo. We have examined how the tyrosine phosphorylation and serine-threonine phosphorylation of FRS2 within lipid rafts affect the response of cells to FGF2 signaling. Our data suggest that activation of protein kinase C, Src family kinases, and MEK1/2 are involved in regulating serine-threonine phosphorylation of FRS2, which can indirectly affect FRS2 phosphotyrosine levels. We also show that Grb2 is recruited to lipid rafts during signaling events and that activation of MEK1/2 by different mechanisms within lipid rafts may lead to different cellular responses. This work suggests that compartmentalized signaling within lipid rafts may provide a level of specificity for growth factor signaling.     

Epidermal growth factor receptor promotes tumor progression and contributes to gemcitabine resistance in osteosarcoma

Osteosarcoma(OS)is the most common type of primary malignant tumors that originate in the bone.Resistance to chemotherapy confers a poor prognosis on OS patient...     

Fibroblast growth factor-2 immunoreactivity is present in the central and peripheral auditory pathways of adult rats

Recent findings have pointed out the role of neurotrophic factors in the survival and maintenance of neurons of the auditory system. Basic fibroblast growth factor (bFGF, FGF-2) is a potent neurotrophic molecule whose actions can be seen in the central and peripheral nervous systems. In the present study, FGF-2 immunoreactivity was analyzed in the auditory pathways of the adult rat, employing a well-characterized polyclonal antibody against FGF-2. In the cochlea, FGF-2 immunoreactivity was observed in the inner and outer hair cells of the organ of Corti, spiral ganglion neurons, spiral limbus, and stria vascularis. Stereological methods employing optical fractionator revealed the presence of 84.5, 15, and 0.5% of spiral ganglion neurons possessing FGF-2 immunoreactivity of strong, moderate, and weak intensity, respectively. In the central auditory pathways, FGF-2 immunoreactivity was found in the cytoplasm of the neurons of the cochlear nuclei, trapezoid body nuclei, medial geniculate nucleus, and inferior colliculus. The two-color immunoperoxidase method showed FGF-2 immunoreactivity in the nuclei of astrocytes throughout the central auditory pathway. Computer-assisted microdensitometric image analysis revealed higher levels of specific mean gray values of FGF-2 immunoreactivity in the trapezoid body and ventral cochlear nucleus and also in the spiral ganglion and inner hair cells. Sections incubated with FGF-2 antibody preabsorbed with human recombinant FGF-2 showed no immunoreaction in the majority of the studied regions, exhibiting only a slight immunoreactive product in the hair cells of the organ of Corti. Furthermore, no changes in immunoreactivity were observed in sections incubated with FGF-2 antiserum preincubated with human recombinant acidic FGF (FGF-1). The findings suggest that FGF-2 may exert paracrine and autocrine actions on neurons of the central and peripheral auditory systems and may be of importance in the mechanism of hearing diseases.     

ATP depletion is an important factor in DHEA-induced growth inhibition and apoptosis in BV-2 cells

Dehydroepiandrosterone (DHEA), a major steroid secreted by the adrenal gland, is known to have antiproliferative effects but the mechanism is unclear. We recently reported that DHEA induces growth inhibition and apoptosis in BV-2 cells and these effects are inversely associated with glucose concentrations in the medium. Here, we further showed that incubation of BV-2 cells with DHEA under glucose deprivation (G0) led to dose- and time-dependent decrease in cellular ATP levels. The decrease in ATP preceded growth inhibition and apoptosis induced by DHEA and all these effects of DHEA (i.e., loss of ATP, antiproliferation and apoptosis) were prevented by glucose added at 4.5 mg/ml (G4.5) during incubation. In addition, two ATP-depleting agents, iodoacetic acid (IAA) and 2,4-dinitrophenylhydrazine (2,4-DNP), potentiated the antiproliferative and apoptotic effects of DHEA. We also determined whether decrease in nucleic acid synthesis (due to glucose-6-phosphate dehydrogenase inhibition by DHEA) contributes to DHEA-induced antiproliferation and apoptosis. Using a mixture of deoxyribonucleotides (DN) and ribonucleotides (RN), we showed that DNRN had little or no effect on DHEA-induced growth inhibition and apoptosis. We also showed that mevalonic acid (MVA) did not affect DHEA-induced antiproliferation and apoptotic effects, indicating that protein isoprenylation and membrane association are not affected by DHEA in BV-2 cells. Taken together, the present results demonstrate that depletion of ATP by DHEA plays an important role in DHEA-induced antiproliferation and apoptosis of BV-2 cells.     

Fibroblast growth factor-2 and vascular endothelial growth factor expression in the ileocecal region of quail (Coturnix coturnix japonica)

Abstract

We undertook this study to immunolocalize in quail vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF-2) in the ileocecal region, which is a significant entry point for intestinal immunity. Diffuse cytoplasmic reaction for FGF-2 and VEGF was observed in the epithelial cells of the distal ileum and proximal cecal mucosa. VEGF immunoreactive cells, which give strong intracytoplasmic immunoreaction, were observed in the lamina propria of both intestinal parts. FGF-2 immunoreactive cells were seen in the lamina propria and germinative centers of lymph follicles in the cecum mucosa. Expressions of FGF-2 and VEGF in healthy quail intestines indicate that these factors have physiological roles in quail.     

Production of an insulin-like growth factor by osteosarcoma

To test the possibility that osteosarcoma cells produce their own growth factors, we measured levels of insulin and somatomedin C (SMC), an insulin-like growth factor, in culture media of two cell lines derived from patients with that disease. SMC but not insulin levels increased three- to ten-fold over a period of 7 days paralleling the increases in cell number. Production of SMC was inhibited by cycloheximide.     

Fibroblast growth factor-2 regulates expression of osteopontin in periodontal ligament cells

Osteopontin is a protein found in the bone-related matrix and plays multiple regulatory roles in mineralizing and non-mineralizing tissue. In osteogenic cell-lines, the expression of osteopontin increases with the progression of differentiation, but both the expression and function of osteopontin vary with the cell type and its activation state. In this study, we examined the expression of osteopontin by clones established from mouse periodontal ligament, in response to inorganic phosphate and fibroblast growth factor (FGF)-2, which can induce periodontal tissue regeneration. The involvement of inorganic phosphate in the expression of osteopontin during the course of cell differentiation of a clone MPDL22 was confirmed by addition of foscarnet, an inorganic phosphate transport inhibitor. Although FGF-2 decreased the mRNA expression of almost every bone-related protein in MPDL22, FGF-2 upregulated the expression of osteopontin in MPDL22 at both mRNA and protein levels. Interestingly, FGF-2 enhanced the concentration of osteopontin in the culture supernatant of MPDL22, whereas inorganic phosphate did not. The FGF-2-induced osteopontin in the culture supernatant seems to be involved in cell survival activity. An immunohistochemical study showed that the FGF-2-induced osteopontin was mainly present in perinuclear matrices while the inorganic phosphate-induced osteopontin was associated with extracellular matrices in addition to perinuclear matrices. The present results indicated that FGF-2 induces unique expression of osteopontin, which may play a role different from the other bone-related proteins during the process of periodontal tissue regeneration by FGF-2.     

Fibroblast growth factor-2 in hyperplastic pituitaries of D2R knockout female mice

Dopamine D2 receptor (D2R) knockout (KO) female mice develop chronic hyperprolactinemia and pituitary hyperplasia. Our objective was to study the expression of the mitogen fibroblast growth factor (FGF2) and its receptor, FGFR1, comparatively in pituitaries from KO and wild-type (WT) female mice. We also evaluated FGF2 subcellular localization and FGF2 effects on pituitary function. FGF2-induced prolactin release showed a similar response pattern in both genotypes, even though basal and FGF2-stimulated release was higher in KO. FGF2 stimulated pituitary cellular proliferation (MTS assay and [(3)H]thymidine incorporation), with no differences between genotypes. FGF2 concentration (measured by ELISA) in whole pituitaries or cultured cells was lower in KO (P < 0.00001 and 0.00014). Immunofluorescence histochemistry showed less FGF2 in pituitaries from KO females and revealed a distinct FGF2 localization pattern between genotypes, being predominantly nuclear in KO and cytosolic in WT pituitaries. Finally, FGF2 could not be detected in the conditioned media from pituitary cultures of both genotypes. FGFR1 levels (Western blot and immunohistochemistry) were higher in pituitaries of KO. Basal concentration of phosphorylated ERKs was lower in KO cells (P = 0.018). However, when stimulated with FGF2, a significantly higher increment of ERK phosphorylation was evidenced in KO cells (P < or = 0.02). We conclude that disruption of the D2R caused an overall decrease in pituitary FGF2 levels, with an increased distribution in the nucleus, and increased FGFR1 levels. These results are important in the search for reliable prognostic indicators for patients with pituitary dopamine-resistant prolactinomas, which will make tumor-specific therapy possible.     

Fibroblast growth factor-2 is a downstream mediator of phosphatidylinositol 3-kinase-Akt signaling in 14,15-epoxyeicosatrienoic acid-induced angiogenesis

To determine the efficacy of cytochrome P450 2C9 metabolites of arachidonic acid, viz. 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids (EETs), in inducing angiogenesis, we have studied their effects on human dermal microvascular endothelial cell (HDMVEC) tube formation and migration. All four EETs stimulated HDMVEC tube formation and migration in a dose-dependent manner. Because 14,15-EET was found to be slightly more efficacious than 5,6-, 8,9-, and 11,12-EETs in stimulating HDMVEC tube formation and migration, we next focused on elucidation of the signaling mechanisms underlying its angiogenic activity. 14,15-EET stimulated Akt and S6K1 phosphorylation in Src- and phosphatidylinositol 3-kinase (PI3K)-dependent manner in HDMVECs. Inhibition of Src and PI3K-Akt-mTOR signaling by both pharmacological and dominant-negative mutant approaches suppressed 14,15-EET-induced HDMVEC tube formation and migration in vitro and Matrigel plug angiogenesis in vivo. In addition, 14,15-EET induced the expression of fibroblast growth factor-2 (FGF-2) in Src- and PI3K-Akt-dependent and mTOR-independent manner in HDMVECs. Neutralizing anti-FGF-2 antibodies completely suppressed 14,15-EET-induced HDMVEC tube formation and migration in vitro and Matrigel plug angiogenesis in vivo. Together, these results show for the first time that Src and PI3K-Akt signaling via targeting in parallel with FGF-2 expression and mTOR-S6K1 activation plays an indispensable role in 14,15-EET-induced angiogenesis.     

Expression of a transmembrane phosphotyrosine phosphatase inhibits cellular response to platelet-derived growth factor and insulin-like growth factor-1.

Tyrosine phosphorylation is a mechanism of signal transduction shared by many growth factor receptors and oncogene products. Phosphotyrosine phosphatases (PTPases) potentially modulate or counter-regulate these signaling pathways. To test this hypothesis, the transmembrane PTPase CD45 (leukocyte common antigen) was expressed in the murine cell line C127. Hormone-dependent autophosphorylation of the platelet-derived growth factor (PDGF) and insulin-like growth factor-1 (IGF-1) receptors was markedly reduced in cells expressing the transmembrane PTPase. Tyrosine phosphorylation of other PDGF-dependent phosphoproteins (160, 140, and 55 kDa) and IGF-1-dependent phosphoproteins (145 kDa) was similarly decreased. Interestingly, the pattern of growth factor-independent tyrosine phosphorylations was comparable in cells expressing the PTPase and control cells. This suggests a selectivity or accessibility of the PTPase limited to a subset of cellular phosphotyrosyl proteins. The maximum mitogenic response to PDGF and IGF-1 in cells expressing the PTPase was decreased by 67 and 71%, respectively. These results demonstrate that a transmembrane PTPase can both affect the tyrosine phosphorylation state of growth factor receptors and modulate proximal and distal cellular responses to the growth factors.     

Fibroblast growth factor-2 during postnatal development of the tracheal basement membrane zone

Thickening of the basement membrane zone (BMZ) is a characteristic of several airway diseases; however, very little is known about how this process occurs. The purpose of this study was to define development of the BMZ in the trachea of growing rhesus monkeys at 1, 2, 3, and 6 mo of age. We measured immunoreactivity of collagen types I, III, and V to detect structural changes in the developing BMZ. To detect more dynamic, functional components of the epithelial-mesenchymal trophic unit, we evaluated the distribution of perlecan, fibroblast growth factor-2 (FGF-2), and fibroblast growth factor receptor-1 (FGFR-1). One-month-old monkeys had a mean collagen BMZ width of 1.5 +/- 0.7 microm that increased to 4.4 +/- 0.4 microm in 6-mo-old monkeys. Perlecan was localized in the BMZ of the epithelium at all ages. FGF-2 was strongly expressed in basal cells at 1-3 mo. At 6 mo, FGF-2 was expressed throughout the BMZ and weakly in basal cells. FGFR-1 immunoreactivity was expressed by basal cells and cilia and weakly in the nuclei of columnar cells at all time points. These data indicate that development of the BMZ is a postnatal event in the rhesus monkey that involves FGF-2.     

Fibroblast growth factor receptor 1 is principally responsible for fibroblast growth factor 2-induced catabolic activities in human articular chondrocytes

Introduction  

Cartilage degeneration driven by catabolic stimuli is a critical pathophysiological process in osteoarthritis (OA). We have defined fibroblast growth factor 2 (FGF-2) as a degenerative mediator in adult human articular chondrocytes. Biological effects mediated by FGF-2 include inhibition of proteoglycan production, up-regulation of matrix metalloproteinase-13 (MMP-13), and stimulation of other catabolic factors. In this study, we identified the specific receptor responsible for the catabolic functions of FGF-2, and established a pathophysiological connection between the FGF-2 receptor and OA.     

Fibroblast growth factor 2 is more dynamic than vascular endothelial growth factor A during the follicle-luteal transition in the cow

Luteal inadequacy is a major cause of infertility in a number of species. During the early luteal phase, progesterone production requires the rapid growth of the corpus luteum (CL), which is in turn dependent on angiogenesis. In the present study, we examined the temporal changes in vascular endothelial growth factor A (VEGFA), fibroblast growth factor 2 (FGF2) and secreted protein, acidic, cysteine-rich (osteonectin) (SPARC) during the follicular-luteal transition and CL development in the cow. Luteal VEGFA concentrations increased as the CL developed but were lower in the regressing CL. Conversely, luteal FGF2 concentrations were highest immediately postovulation in the collapsed follicle and declined as the CL developed. Furthermore, three FGF2 isoforms were present in the collapsed follicle, but only one isoform was detected in older CL. Interestingly, FGF2 concentrations increased in the regressing CL. Western blot analysis for SPARC showed the presence of two isoforms, which were constitutively expressed throughout CL development. Further studies investigated the regulation of FGF2 by LH, which showed that FGF2 concentrations in preovulatory follicular fluid were higher in those animals that had experienced an LH surge. Moreover, LH stimulated FGF2 production in dispersed luteal cells. Conversely, the LH surge had no effect on follicular fluid VEGFA concentrations. In conclusion, FGF2 was more dynamic than VEGFA and SPARC during the follicular-luteal transition, which suggests that FGF2 plays a key role in the initiation of angiogenesis at this time. Furthermore, it is likely that this is stimulated by the LH surge. The results also suggest that VEGFA and SPARC have a more constitutive, but essential, role in the development of the CL vasculature.     

Transforming growth factor beta is an important immunomodulatory protein for human B lymphocytes

The growth and differentiation of B cells to immunoglobulin (Ig)-secreting cells is regulated by a variety of soluble factors. This study presents data that support a role for transforming growth factor (TGF)-beta in this regulatory process. B lymphocytes were shown to have high-affinity receptors for TGF-beta that were increased fivefold to sixfold after in vitro activation. The addition of picogram quantities of TGF-beta to B cell cultures suppressed factor-dependent, interleukin 2 (IL 2) B cell proliferation and markedly suppressed factor-dependent (IL 2 or B cell differentiation factor) B cell Ig secretion. In contrast, the constitutive IgG production by an Epstein Barr virus-transformed B cell line was not modified by the presence of TGF-beta in culture. This cell line was found to lack high-affinity TGF-beta receptors. The degree of inhibition of B cell proliferation observed in in vitro cultures was found to be dependent not only on the concentration of TGF-beta added but also on the concentration of the growth stimulatory substance (IL 2) present. By increasing the IL 2 concentrations in culture, the inhibition of proliferation induced by TGF-beta could be partially overcome. In contrast, the inhibition of Ig secretion induced by TGF-beta could not be overcome by a higher concentration of stimulatory factor, demonstrating that the suppression of B cell differentiation by TGF-beta is not due solely to its effects on proliferation. Furthermore, it was demonstrated that B lymphocytes secrete TGF-beta. Unactivated tonsillar B cells had detectable amounts of TGF-beta mRNA on Northern blot analysis, and B cell activation with Staphylococcus aureus Cowan (SAC) resulted in a twofold to threefold increase in TGF-beta mRNA. Supernatants conditioned by unactivated B cells had small amounts of TGF-beta, SAC activation of the B cells resulted in a sixfold to sevenfold increase in the amount of TGF-beta present in the supernatants. Thus, B lymphocytes synthesize and secrete TGF-beta and express receptors for TGF-beta. The addition of exogenous TGF-beta to cultures of stimulated B cells inhibits subsequent proliferation and Ig secretion. TGF-beta may function as an autocrine growth inhibitor that limits B lymphocyte proliferation and ultimate differentiation.