Fibroblast Growth Factor Activity Research Articles

Ret rescues mitochondrial morphology and muscle degeneration of Drosophila Pink1 mutants

Parkinson's disease (PD)-associated Pink1 and Parkin proteins are believed to function in a common pathway controlling mitochondrial clearance and trafficking. Glial cell line-derived neurotrophic factor (GDNF) and its signaling receptor Ret are neuroprotective in toxin-based animal models of PD. However, the mechanism by which GDNF/Ret protects cells from degenerating remains unclear. We investigated whether the Drosophila homolog of Ret can rescue Pink1 and park mutant phenotypes. We report that a signaling active version of Ret (RetMEN2B) rescues muscle degeneration, disintegration of mitochondria and ATP content of Pink1 mutants. Interestingly, corresponding phenotypes of park mutants were not rescued, suggesting that the phenotypes of Pink1 and park mutants have partially different origins. In human neuroblastoma cells, GDNF treatment rescues morphological defects of PINK1 knockdown, without inducing mitophagy or Parkin recruitment. GDNF also rescues bioenergetic deficits of PINK knockdown cells. Furthermore, overexpression of RetMEN2B significantly improves electron transport chain complex I function in Pink1 mutant Drosophila. These results provide a novel mechanism underlying Ret-mediated cell protection in a situation relevant for human PD.

Effects of combinations of BMP-2 with FGF-2 and/or VEGF on HUVECs angiogenesis in vitro and CAM angiogenesis in vivo

Angiogenesis, a complex biologic process, is regulated by a large number of angiogenic factors, including vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2). Whether Bone morphogenetic proteins-2 (BMP-2), the osteoinductive factor, could significantly reinforce the effect of VEGF and FGF-2 on angiogenesis has not been studied in detail. To study the positive effects of multiple growth factors on angiogenesis, HUVECs were treated with BMP-2, VEGF, or FGF-2 singly and in binary and ternary combinations. This study further investigates the optimal timing of the ternary combination of BMP-2, VEGF and FGF-2 for angiogenesis in the chorioallantoic membrane (FGF-2 CAM). Results of single applications of BMP-2, VEGF, or FGF-2 suggested that HUVECs angiogenesis could be promoted in a dose-dependent manner and that the optimal concentration of BMP, VEGF and FGF-2 was 10, 50 and 1 ng/mL, respectively. These results indicated that the angiogenic activity of VEGF and FGF-2 was amplified by combining with BMP-2. The ternary combination of BMP-2, VEGF and FGF-2 exhibited a positive and synergistic effect on HUVECs angiogenesis, with the lower concentrations of each factor (1 ng/mL of BMP-2, 25 ng/mL of VEGF and 0.1 ng/mL of FGF-2) being sufficient to show synergistic promotion. When VEGF and FGF-2 were added in the initial activation stage and BMP-2 was added in the maturation stage, both HUVECs angiogenesis in vitro and CAM angiogenesis in vivo could be enhanced more effectively. These results could provide a basis for the controlled release systems capable of delivering multiple factors sequentially to promote angiogenesis in tissue engineering.

The role of FGF2 in spinal cord trauma and regeneration research

The role of <scp>FGF</scp>2 in spinal cord trauma and regeneration research

Expression of a Secreted Fibroblast Growth Factor Binding Protein-1 (FGFBP1) in Angioproliferative Kaposi Sarcoma.

ObjectiveKaposi’s sarcoma (KS) is an angioproliferative disease frequently seen in patients with the acquired immunodeficiency syndrome (AIDS). Previous studies suggest that the HIV-1 protein Tat and Fibroblast Growth Factor 2 (FGF-2) have synergistic angiogenic effects in AIDS-KS tumors. However, the mechanisms by which FGF-2 is released and activated in KS tumors are not clearly defined. We carried out this study to determine whether an FGF-binding protein (FGFBP1 or BP1) that enhances the angiogenic activity of FGF-2 is expressed in AIDS-KS tumors, and to define whether BP1, FGF-2, and HIV-Tat protein-protein interactions could play a potential clinically role in the pathogenesis of AIDS-KS.MethodsBP1 was localized in AIDS-KS lesions by immunohistochemistry and in situ hybridization studies. The binding of radiolabeled FGF-2 to His-tagged BP1 or the FGF-receptor 1 was assessed in the presence and absence of HIV-Tat and other viral proteins. Mice carrying tetracycline-regulated BP1 transgene mice were used to determine whether activation of BP1 during wound healing induces KS-like lesions.ResultsBP1 expression was detected in AIDS-KS tumor keratinocytes, spindle cells, and infiltrating mononuclear cells. In addition, HIV-Tat competed for the binding of FGF-2 to immobilized BP1, but does not affect the interactions of FGF-2 with its high affinity receptor (FGFR-1). In contrast, two other HIV-proteins, Nef and gp120, did not affect the binding of FGF-2 to BP1 or to FGFR-1. Finally, up-regulation of BP1 expression in tetracycline-regulated –conditional BP1 transgenic mice subjected to skin wounds, induced KS-like skin lesions.ConclusionTaking into consideration the results of previous studies showing that both HIV-Tat and BP1 enhance the mitogenic and angiogenic activity of locally-stored FGF-2, both in vitro and in vivo, our findings suggest a novel mechanism by which the release and activity of FGFs can be modulated in AIDS-KS tumors by HIV-Tat as well as BP1.

Diagnostic Modalities for FGF23-Producing Tumors in Patients with Tumor-Induced Osteomalacia.

Fibroblast growth factor 23 (FGF23) is a hormone that is produced by osteocytes and regulates phosphate and vitamin D metabolism through binding to the Klotho-FGF receptor complex. Excessive actions of FGF23 cause several kinds of hypophosphatemic rickets/osteomalacia. Tumor-induced rickets/osteomalacia (TIO) is a paraneoplastic syndrome caused by overproduction of FGF23 from the responsible tumors. Because TIO is cured by complete resection of the causative tumors, it is of great clinical importance to locate these tumors. Several imaging methods including skeletal survey by magnetic resonance imaging and octreotide scintigraphy have been used to identify the tumors that cause TIO. However, none of these imaging studies indicate that the detected tumors are actually producing FGF23. Recently, systemic venous sampling was conducted for locating FGF23-producing tumor in suspected patients with TIO and demonstrated that this test might be beneficial to a subset of patient. Further studies with more patients are necessary to establish the clinical utility of venous sampling in patients with TIO.

Increasing dietary phosphorus intake from food additives: potential for negative impact on bone health.

It is important to consider whether habitual high phosphorus intake adversely affects bone health, because phosphorus intake has been increasing, whereas calcium intake has been decreasing in dietary patterns. A higher total habitual dietary phosphorus intake has been associated with higher serum parathyroid hormone (PTH) and lower serum calcium concentrations in healthy individuals. Higher serum PTH concentrations have been shown in those who consume foods with phosphorus additives. These findings suggest that long-term dietary phosphorus loads and long-term hyperphosphatemia may have important negative effects on bone health. In contrast, PTH concentrations did not increase as a result of high dietary phosphorus intake when phosphorus was provided with adequate amounts of calcium. Intake of foods with a ratio of calcium to phosphorus close to that found in dairy products led to positive effects on bone health. Several randomized controlled trials have shown positive relations between dairy intake and bone mineral density. In our loading test with a low-calcium, high-phosphorus lunch provided to healthy young men, serum PTH concentrations showed peaks at 1 and 6 h, and serum fibroblast growth factor 23 (FGF23) concentrations increased significantly at 8 h after the meal. In contrast, the high-calcium, high-phosphorus meal suppressed the second PTH and FGF23 elevations until 8 h after the meal. This implies that adequate dietary calcium intake is needed to overcome the interfering effects of high phosphorus intake on PTH and FGF23 secretion. FGF23 acts on the parathyroid gland to decrease PTH mRNA and PTH secretion in rats with normal kidney function. However, increased serum FGF23 is an early alteration of mineral metabolism in chronic kidney disease, causing secondary hyperthyroidism, and implying resistance of the parathyroid gland to the action of FGF23 in chronic kidney disease. These findings suggest that long-term high-phosphorus diets may impair bone health mediated by FGF23 resistance both in chronic kidney disease patients and in the healthy population.

Liver-Enriched Transcription Factor CREBH Interacts With Peroxisome Proliferator-Activated Receptor α to Regulate Metabolic Hormone FGF21

Lipid metabolism is tightly regulated by nuclear receptors, transcription factors, and cellular enzymes. In this study, we demonstrated that the liver-enriched transcription factor CREBH (cAMP-responsive element binding protein, hepatocyte specific) and peroxisome proliferator-activated receptor α (PPARα) function as binary transcriptional activators to regulate lipid metabolism by activating fibroblast growth factor 21 (FGF21), a hepatic hormone that regulates whole-body energy homeostasis. Gain- and loss-of-function studies indicated that CREBH regulates triglyceride and fatty acid metabolism in animals under fasting or on an atherogenic high-fat (AHF) diet. CREBH and PPARα act as interactive trans-activators that regulate each other for their expression. Activated CREBH protein interacts with PPARα to form a functional complex upon fasting or the AHF diet, and both factors are required for induction of the metabolic hormone FGF21. The CREBH-PPARα complex was found to bind to integrated CRE-PPAR-responsive element-binding motifs in the FGF21 gene promoter. Whereas CREBH and PPARα function in synergy to activate FGF21 gene expression, PPARα relies on CREBH to exert its trans-activation effect on FGF21. Supporting the key role of CREBH in regulating FGF21, infusion of recombinant FGF21 protein can reverse hypertriglyceridemia and hypoketonemia and partially rescue nonalcoholic steatohepatitis developed in the CREBH-null mice after the AHF diet. Our study demonstrated a transcriptional regulatory axis of CREBH-PPARα-FGF21 in maintaining lipid homeostasis under metabolic stress. The functional relationship between CREBH and PPARα in regulating FGF21 may represent an important transcriptional coactivation mechanism that orchestrates the processes of energy supply upon metabolic alteration.

Fibroblast Growth Factor Receptor 1 Signaling in Adult Cardiomyocytes Increases Contractility and Results in a Hypertrophic Cardiomyopathy

Fibroblast growth factors (FGFs) and their receptors are highly conserved signaling molecules that have been implicated in postnatal cardiac remodeling. However, it is not known whether cardiomyocyte-expressed FGF receptors are necessary or sufficient for ventricular remodeling in the adult heart. To determine whether cardiomyocytes were competent to respond to an activated FGF receptor, and to determine if this signal would result in the development of hypertrophy, we engineered a doxycycline (DOX)-inducible, cardiomyocyte-specific, constitutively active FGF receptor mouse model (αMHC-rtTA, TRE-caFgfr1-myc). Echocardiographic and hemodynamic analysis indicated that acute expression of caFGFR1 rapidly and directly increased cardiac contractility, while chronic expression resulted in significant hypertrophy with preservation of systolic function. Subsequent histologic analysis showed increased cardiomyocyte cross-sectional area and regions of myocyte disarray and fibrosis, classic features of hypertrophic cardiomyopathy (HCM). Analysis of downstream pathways revealed a lack of clear activation of classical FGF-mediated signaling pathways, but did demonstrate a reduction in Serca2 expression and troponin I phosphorylation. Isolated ventricular myocytes showed enhanced contractility and reduced relaxation, an effect that was partially reversed by inhibition of actin-myosin interactions. We conclude that adult cardiomyocytes are competent to transduce FGF signaling and that FGF signaling is sufficient to promote increased cardiomyocyte contractility in vitro and in vivo through enhanced intrinsic actin-myosin interactions. Long-term, FGFR overexpression results in HCM with a dynamic outflow tract obstruction, and may serve as a unique model of HCM.

Biochemical and Functional Characterization of the Klotho-VS Polymorphism Implicated in Aging and Disease Risk

Klotho (KL) is an age-regulating protein named after the Greek goddess who spins the thread of life. Mice deficient in KL are normal throughout development, but rapidly degenerate and display a variety of aging-associated abnormalities that eventually lead to decreased life expectancy. While multiple genetic association studies have identified KL polymorphisms linked with changes in disease risk, there is a paucity of concrete mechanistic data to explain how these amino acid substitutions alter KL protein function. The KLVS polymorphism is suggested to lead to changes in protein trafficking although the mechanism is unclear. Our studies have sought to further investigate the functional differences in the KLVS variant that result in increased risk of many age-related diseases. Our findings suggest that the F352V and C370S substitutions lead to alterations in processing as seen by differences in shedding and half-life. Their co-expression in KLVS results in a phenotype resembling wild-type, but despite this intragenic complementation there are still changes in homodimerization and interactions with FGFR1c. Taken together, these studies suggest that KLVS leads to altered homodimerization that indirectly leads to changes in processing and FGFR1c interactions. These findings help elucidate the functional differences that result from the VS polymorphism, which will help clarify how alterations in KL function can lead to human disease and affect cognition and lifespan.

Phosphate wasting and fibroblast growth factor-23

The purpose of this study is to review the regulation of phosphate and recent progress in fibroblast growth factor-23 (FGF-23), a key phosphate regulatory hormone. Phosphate is required for mineralization of bone, muscle strength and a host of biologic functions. Phosphate is sensed by bone that responds with secretion of FGF-23. The major action of FGF-23 is to stimulate phosphaturia. Feedback loops between FGF-23, 1,25-dihydroxyvitamin D3 and parathyroid hormone maintain phosphate homeostasis. Information about FGF-23 has accumulated from studies in patients with oncogenic osteomalacia and inherited disorders of phosphate wasting rickets that explains the pathophysiology. Exciting new discoveries have highlighted FGF-23 as an independent risk factor for cardiovascular disease in patients with chronic kidney disease. The phosphate sensor triggering FGF-23 production remains to be identified. Derangements in FGF-23 production, half-life or downstream response are responsible for several disorders of phosphate wasting, rickets and oncogenic osteomalacia. Very high levels of FGF-23 in renal failure are an independent risk for cardiovascular disease.

Regulation of renal phosphate transport by FGF23 is mediated by FGFR1 and FGFR4.

Fibroblast growth factor 23 (FGF23) is a bone-derived hormone that acts on the proximal tubule to decrease phosphate reabsorption and serum levels of 1,25-dihydroxyvitamin D₃ [1,25(OH)₂ Vitamin D₃]. Abnormal FGF23 metabolism has been implicated in several debilitating hypophosphatemic and hyperphosphatemic disorders. The renal receptors responsible for the phosphaturic actions of FGF23 have not been elucidated. There are four fibroblast growth factor receptors (FGFR); 1-4 with "b" and "c" isoforms for receptors 1, 2, and 3. FGFR1, 3, and 4 are expressed in the mouse proximal tubule, and deletion of any one receptor did not affect serum phosphate levels, suggesting that more than one receptor is involved in mediating the phosphaturic actions of FGF23. To determine the receptors responsible for the phosphaturic actions of FGF23, we studied Fgfr1 (kidney conditional) and Fgfr4 (global) double mutant mice (Fgfr1⁻/⁻/Fgfr4⁻/⁻). Fgfr1⁻/⁻/Fgfr4⁻/⁻ mice have higher FGF23 levels than their wild-type counterparts (108.1 ± 7.3 vs. 4,953.6 ± 675.0 pg/ml; P < 0.001). Despite the elevated FGF23 levels, Fgfr1⁻/⁻/Fgfr4⁻/⁻ mice have elevated serum phosphorus levels, increased brush-border membrane vesicle (BBMV) phosphate transport, and increased Na-P(i) cotransporter 2c (NaPi-2c) protein expression compared with wild-type mice. These data are consistent with FGFR1 and FGFR4 being the critical receptors for the phosphaturic actions of FGF23.

FGF2 Promotes Resistance To Quizartinib In Vitro, and FGF2 Increases In The Marrow Of Patients Prior To Resistance

BackgroundPatients with acute myeloid leukemia and internal tandem duplication of fms-like tyrosine kinase receptor-3 (FLT3+ AML) make up about 25% of newly diagnosed AML and have a poor prognosis. As a tyrosine kinase, FLT3 is an attractive therapeutic target, however the majority of patients develop resistance. Although FLT3 inhibitors initially induce a rapid decline of circulating leukemia cells, the decline of blasts in the bone marrow occurs more slowly suggesting that leukemia cells within the bone marrow microenvironment are less sensitive to the effects of FLT3 kinase inhibition. We tested multiple cytokines, growth factors and extracellular matrix proteins from the microenvironment to determine which proteins were most protective of FLT3+ AML cell lines in vitro. ResultsWe exposed the FLT3+ AML cell line, MOLM-14, to the FLT3 inhibitor quizartinib (AC220) and found that two of the most protective proteins in our screen were fibroblast growth factor 2 (FGF2) and, consistent with previous reports, FLT3 ligand (FL). FGF2 is expressed in bone marrow stromal cells and promotes resistance to kinase-inhibitors in other malignancies, including chronic myeloid leukemia (Traer et al, submitted). FGF2-mediated resistance to quizartinib could be overcome by addition of the specific FGF receptor (FGFR) inhibitor PD173074. To identify the FGFR responsible for resistance, we targeted FGFR1-4 with siRNAs and evaluated the protective effects of FGF2 in the presence of quizartinib. Only siRNA targeting FGFR1 was able to attenuate the protective effects of FGF2. FGF2 activation of FGFR1 led to downstream activation of the MAPK pathway, circumventing the effects of quizartinib on FLT3.Since patients develop resistance over several months, we tested the protective effects of FGF2 on MOLM-14 cells in extended cultures. After 8 weeks, all cultures supplemented with FGF2 eventually resumed growth (n=4). In contrast, only 2 of 4 long-term MOLM-14 cells cultured in quizartinib alone resumed growth after 12 weeks. We sequenced the FLT3 kinase domain and found no mutations in 3 of 4 FGF2-supplemented cultures. However, one FGF2-supplemented culture developed a kinase domain mutation after >3 months suggesting continued clonal evolution. In contrast, the resistant cultures grown in quizartinib alone both developed resistance through mutation of the FLT3 kinase domain, providing evidence that the microenvironment affects the kinetics and mechanism of resistance.We then examined FGF2 in the bone marrow in response to quizartinib, using biopsies from FLT3+ AML patients on the phase II clinical trial. By immunohistochemistry, FGF2 expression was only modestly increased in patients prior to initiation of quizartinib (31.4% compared to 23.2% of normal marrow samples, p=0.19). However, FGF2 increased significantly during treatment with quizartinib (51%, p=0.0013) and remained elevated until disease relapse. Thus, residual leukemia cells exist in an environment with relatively increased FGF2 and this population eventually develops overt kinase resistance, similar to our in vitro model. As previous reports have shown that FL also increases during therapy with FLT3 inhibitors, we suspect that FL and FGF2 may work cooperatively to promote resistance in the microenvironment. Since FLT3 inhibitors that also target FGFR, such as ponatinib, are currently in clinical trials it will be interesting to compare patterns of resistance in these patients. ConclusionsOur data supports a model in which paracrine FGF2 from bone marrow stroma promotes survival of residual leukemia cells, eventually leading to quizartinib resistance and disease relapse. In vitro resistance occurs via FGF2 ligand-induced activation of the FGFR1/MAPK pathway, and can be overcome by concomitant inhibition of FLT3 and FGFR, at least initially, however continued clonal evolution can lead to development of kinase domain mutations. Overall, our results illustrate the importance of ligand-induced resistance and the need to develop rational inhibitor combinations, particularly in diseases like FLT3+ AML that routinely develop resistance to kinase inhibitors. Disclosures:Kovacsovics:Ambit: Investigator on clinical trial for Ambit. Dr. Kovacsovics received no research support from Ambit. Other. Tyner:InCyte Corporation: Research Funding. Druker:Ambit Biosciences: Consultancy.

Carbon nanotubes functionalized with fibroblast growth factor accelerate proliferation of bone marrow-derived stromal cells and bone formation

Multi-walled carbon nanotubes (MWCNTs) were functionalized with fibroblast growth factor (FGF) and the advantages of their use as scaffolds for bone augmentation were evaluated in vitro and in vivo. The activity of FGF was assessed by measuring the effect on the proliferation of rat bone marrow stromal cells (RBMSCs). The presence of FGF enhanced the proliferation of RBMSCs and the FGF covalently conjugated to the nanotubes (FGF–CNT) showed the same effect as FGF alone. In addition, FGF–CNT coated sponges were implanted between the parietal bone and the periosteum of rats and the formation of new bone was investigated. At day 14 after implantation, a larger amount of newly formed bone was clearly observed in most pores of FGF–CNT coated sponges. These findings indicated that MWCNTs accelerated new bone formation in response to FGF, as well as the integration of particles into new bone during its formation. Scaffolds coated with FGF–CNT could be considered as promising novel substituting materials for bone regeneration in future tissue engineering applications.

Fibroblast growth factor-2 expression in the preimplantation equine conceptus and endometrium of pregnant and cyclic mares

Uterine-derived growth factors and cytokines play essential roles in regulating preimplantation conceptus development. In several species, fibroblast growth factor-2 (FGF2) promotes embryogenesis, trophoblast cell migration, and adhesion. This study investigated mRNA expression for FGF2, its receptors (FGFR1-4), the activating factor FGF binding protein (FGF-BP) in equine endometrium and trophectoderm during early pregnancy and the estrous cycle, and localized FGF2 protein in both endometrium and conceptus tissues. FGF2, FGFRs1-4, and FGFBP mRNAs were expressed in endometrium throughout the estrous cycle and early pregnancy, and in days 14 to 28 conceptus membranes. FGF2 transcription was higher during estrus than on days 7 or 14 of diestrus, suggesting estrogen dependency. Endometrial expression of FGF2 mRNA and protein increased as pregnancy progressed from days 21 and day 28; FGF2 protein was localized predominantly in the luminal and glandular epithelium. FGF2 mRNA was detectable in trophectoderm from as early as day 14, and transcription and translation increased in day 21 and 28 allantochorion. FGF2 protein was localized mainly in the trophectoderm up to day 21 but was present in both trophectoderm and endoderm of day 28 allantochorion. FGFR1 mRNA was down-regulated in the endometrium at day 7 of diestrus but increased again by day 14. Gene expression for all of the FGFR2 splice variants, including FGFR2IIIc, was up-regulated during estrus. During early pregnancy, endometrial FGFR1 expression decreased, whereas FGFR2IIIc expression did not change. Conceptus mRNA expression for all FGFRs increased as pregnancy progressed. FGFBP expression remained unchanged in endometrium, but increased in the conceptus between days 14 and 28, suggesting a role in regulating FGF2 activity in the developing conceptus. We conclude that during weeks 3 and 4 of pregnancy, the equine endometrial epithelium produces FGF2, which may play a role in trophoblast development and adhesion.

Fibroblast Growth Factor 2 Causes G2/M Cell Cycle Arrest in Ras-Driven Tumor Cells through a Src-Dependent Pathway

We recently reported that paracrine Fibroblast Growth Factor 2 (FGF2) triggers senescence in Ras-driven Y1 and 3T3Ras mouse malignant cell lines. Here, we show that although FGF2 activates mitogenic pathways in these Ras-dependent malignant cells, it can block cell proliferation and cause a G2/M arrest. These cytostatic effects of FGF2 are inhibited by PD173074, an FGF receptor (FGFR) inhibitor. To determine which downstream pathways are induced by FGF2, we tested specific inhibitors targeting mitogen-activated protein kinase (MEK), phosphatidylinositol 3 kinase (PI3K) and protein kinase C (PKC). We show that these classical mitogenic pathways do not mediate the cytostatic activity of FGF2. On the other hand, the inhibition of Src family kinases rescued Ras-dependent malignant cells from the G2/M irreversible arrest induced by FGF2. Taken together, these data indicate a growth factor-sensitive point in G2/M that likely involves FGFR/Ras/Src pathway activation in a MEK, PI3K and PKC independent manner.

Growth and Regression in Bovine Corpora Lutea: Regulation by Local Survival and Death Pathways

The bovine corpus luteum (CL) is a transient gland with a life span of only 18 days in the cyclic cow. Mechanisms controlling CL development and secretory function may involve factors produced both within and outside this gland. Although luteinizing hormone (LH) surge is the main trigger of ovulation and granulosa cells luteinization, many locally produced agents such as arachidonic acid (AA) metabolites, growth factors and cytokines were shown to complement gonadotropins action in the process of CL development. Bovine CL is a highly vascular gland, where the very rapid angiogenesis rate (until Day 5 of the cycle) results in the development of a capillary network, endowing this gland with one of the highest blood flow rate per unit mass in the body. Angiogenesis in the developing CL is later followed by either controlled regression of the microvascular tree in the non-fertile cycle or maintenance and stabilization of the blood vessels, as seen during pregnancy. Different luteal cell types (both steroidogenic and accessory luteal cells: immune cells, endothelial cells, pericytes and fibroblasts) are involved in the pro- and/or anti-angiogenic responses. The balance between pro- and anti-angiogenic responses to the main luteolysin - prostaglandin F2α (PGF2α) could be decisive in whether or not PGF2α induces CL regression. Fibroblast growth factor-2 (FGF2) may be one of the factors that modulate the angiogenic response to PGF2α. Manipulation of local production and action of FGF2 will provide new tools for reproductive management of dairy cattle. Luteolysis is characterized by a rapid decrease in progesterone production, followed by structural regression. Factors like endothelin-1, cytokines (tumour necrosis factorα, interferons) and nitric oxide were all shown to play critical roles in functional and structural regression of the CL by inhibiting steroidogenesis and inducting apoptosis.

Heparin Modulates the Mitogenic Activity of Fibroblast Growth Factor by Inducing Dimerization of its Receptor. A 3D View by Using NMR

In vitro mitogenesis assays have shown that sulfated glycosaminoglycans (GAGs; heparin and heparan sulfate) cause an enhancement of the mitogenic activity of fibroblast growth factors (FGFs). Herein, we report that the simultaneous presence of FGF and the GAG is not an essential requisite for this event to take place. Indeed, preincubation with heparin (just before FGF addition) of cells lacking heparan sulfate produced an enhancing effect equivalent to that observed when the GAG and the protein are simultaneously added. A first structural characterization of this effect by analytical ultracentrifugation of a soluble preparation of the heparin-binding domain of fibroblast growth factor receptor 2 (FGFR2) and a low molecular weight (3 kDa) heparin showed that the GAG induces dimerization of FGFR2. To derive a high resolution structural picture of this molecular recognition process, the interactions of a soluble heparin-binding domain of FGFR2 with two different homogeneous, synthetic, and mitogenically active sulfated GAGs were analyzed by NMR spectroscopy. These studies, assisted by docking protocols and molecular dynamics simulations, have demonstrated that the interactions of these GAGs with the soluble heparin-binding domain of FGFR induces formation of an FGFR dimer; its architecture is equivalent to that in one of the two distinct crystallographic structures of FGFR in complex with both heparin and FGF1. This preformation of the FGFR dimer (with similar topology to that of the signaling complex) should favor incorporation of the FGF component to form the final assemblage of the signaling complex, without major entropy penalty. This cascade of events is probably at the heart of the observed activating effect of heparin in FGF-driven mitogenesis.

GDNF-independent ureteric budding: role of PI3K-independent activation of AKT and FOSB/JUN/AP-1 signaling

SummaryA significant fraction of mice deficient in either glial cell-derived neurotrophic factor (GDNF) or its co-receptors (Gfrα1, Ret), undergoes ureteric bud (UB) outgrowth leading to the formation of a rudimentary kidney. Previous studies using the isolated Wolffian duct (WD) culture indicate that activation of fibroblast growth factor (FGF) receptor signaling, together with suppression of BMP/Activin signaling, is critical for GDNF-independent WD budding (Maeshima et al., 2007). By expression analysis of embryonic kidney from Ret(−/−) mice, we found the upregulation of several FGFs, including FGF7. To examine the intracellular pathways, we then analyzed GDNF-dependent and GDNF-independent budding in the isolated WD culture. In both conditions, Akt activation was found to be important; however, whereas this occurred through PI3-kinase in GDNF-dependent budding, in the case of GDNF-independent budding, Akt activation was apparently via a PI3-kinase independent mechanism. Jnk signaling and the AP-1 transcription factor complex were also implicated in GDNF-independent budding. FosB, a binding partner of c-Jun in the formation of AP-1, was the most highly upregulated gene in the ret knockout kidney (in which budding had still occurred), and we found that its siRNA-mediated knockdown in isolated WDs also blocked GDNF-independent budding. Taken together with the finding that inhibition of Jnk signaling does not block Akt activation/phosphorylation in GDNF-independent budding, the data support necessary roles for both FosB/Jun/AP-1 signaling and PI3-kinase-independent activation of Akt in GDNF-independent budding. A model is proposed for signaling events that involve Akt and JNK working to regulate GDNF-independent WD budding.

Neonatal Iron Deficiency Causes Abnormal Phosphate Metabolism by Elevating FGF23 in Normal and ADHR Mice

Fibroblast growth factor 23 (FGF23) gain of function mutations can lead to autosomal dominant hypophosphatemic rickets (ADHR) disease onset at birth, or delayed onset following puberty or pregnancy. We previously demonstrated that the combination of iron deficiency and a knock-in R176Q FGF23 mutation in mature mice induced FGF23 expression and hypophosphatemia that paralleled the late-onset ADHR phenotype. Because anemia in pregnancy and in premature infants is common, the goal of this study was to test whether iron deficiency alters phosphate handling in neonatal life. Wild-type (WT) and ADHR female breeder mice were provided control or iron-deficient diets during pregnancy and nursing. Iron-deficient breeders were also made iron replete. Iron-deficient WT and ADHR pups were hypophosphatemic, with ADHR pups having significantly lower serum phosphate (p < 0.01) and widened growth plates. Both genotypes increased bone FGF23 mRNA (>50 fold; p < 0.01). WT and ADHR pups receiving low iron had elevated intact serum FGF23; ADHR mice were affected to a greater degree (p < 0.01). Iron-deficient mice also showed increased Cyp24a1 and reduced Cyp27b1, and low serum 1,25-dihydroxyvitamin D (1,25D). Iron repletion normalized most abnormalities. Because iron deficiency can induce tissue hypoxia, oxygen deprivation was tested as a regulator of FGF23, and was shown to stimulate FGF23 mRNA in vitro and serum C-terminal FGF23 in normal rats in vivo. These studies demonstrate that FGF23 is modulated by iron status in young WT and ADHR mice and that hypoxia independently controls FGF23 expression in situations of normal iron. Therefore, disturbed iron and oxygen metabolism in neonatal life may have important effects on skeletal function and structure through FGF23 activity on phosphate regulation.

Decrease of FGF Receptor (FGFR) and Interstitial Fibrosis in the Kidney of Streptozotocin-induced Diabetic Rats

Fibrosis is the final disorder of end-stage renal disease. Activation of fibroblast growth factor (FGF) 23-klotho axis could suppress renal fibrosis in mice. Also, a marked decrease of klotho expression was observed in the kidney of streptozotocin-induced diabetic rats (STZ rats). However, relation of FGF in renal fibrosis remained unclear. This study was aimed to screen the effect of hyperglycemia on FGF receptor (FGFR) and fibrosis in kidney of rats with diabetic nephropathy and investigate this potential mechanism in cultured Madin-Darby Canine Kidney (MDCK) epithelial cells. STZ rats were used to treat with insulin or phloridzin at the dose sufficient to correct hyperglycemia for understanding the changes of renal dysfunction. The cultured MDCK cells were also used to treat with high glucose, hydrogen peroxide, or tiron in addition to transfection of siRNA to silence the klotho. Both insulin and phloridzin reversed fibrosis and FGFR expressions in kidney of STZ rats. It was confirmed in high glucose-exposed MDCK cells. However, klotho failed to modify the level of FGFR in MDCK cells. Meanwhile, FGFR was restored by tiron in MDCK cells and in diabetic rats without changing blood glucose. In conclusion, interstitial fibrosis and decreased FGFR expression are observed in the kidney of diabetic rats. This change is reversed by tiron without the correction of blood glucose. Also, klotho has no effect on expression of FGFR. Thus, decrease of oxidative stress is useful for the recovery of FGFR expression and improvement of renal fibrosis in type-1 like diabetic rats.