Expression and function of fibroblast growth factor 10 and its receptor, fibroblast growth factor receptor 213, in bovine follicles

Some fibroblast growth factors (FGFs) affect ovarian follicle cell growth and/or differentiation. Whereas many FGFs activate several FGF receptors, FGF7 and FGF10 primarily activate only one, FGFR2B. As FGF7 is produced by bovine theca cells and acts on granulosa cells, we tested the hypothesis that FGF10 may also play a role in folliculogenesis in cattle. Reverse transcription-polymerase chain reaction demonstrated the presence of FGF10 mRNA in the oocytes and theca cells of the antral follicles, as well as in the preantral follicles. FGF10 protein was detected by immunohistochemistry in the oocytes of the preantral and antral follicles, and in the granulosa and theca cells of the antral follicles. FGF10 expression in theca cells changed during follicle development; mRNA abundance decreased with increasing follicular estradiol concentration in healthy follicles, and was lowest in highly atretic follicles. Culturing of granulosa cells in serum-free medium revealed FSH regulation of FGF10 receptor expression. The addition of FGF10 to cultured granulosa cells decreased the level of estradiol but did not alter cell proliferation. These data support a role for FGF10 in signaling to granulosa cells from theca cells and/or the oocyte.

Fibroblast growth factor-10 maintains the survival and promotes the growth of cultured goat preantral follicles

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

The fibroblast growth factor family: involvement in the regulation of folliculogenesis

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Expression of fibroblast growth factor receptors during development and regression of the bovine corpus luteum

There is evidence that fibroblast growth factors (FGFs) are involved in the regulation of growth and regression of the corpus luteum (CL). However, the expression pattern of most FGF receptors (FGFRs) during CL lifespan is still unknown. The objective of the present study was to determine the pattern of expression of 'B' and 'C' splice variants of FGFRs in the bovine CL. Bovine CL were collected from an abattoir and classed as corpora hemorrhagica (Stage I), developing (Stage II), developed (Stage III) or regressed (Stage IV) CL. Expression of FGFR mRNA was measured by semiquantitative reverse transcription-polymerase chain reaction and FGFR protein was localised by immunohistochemistry. Expression of mRNA encoding the 'B' and 'C' spliced forms of FGFR1 and FGFR2 was readily detectable in the bovine CL and was accompanied by protein localisation. FGFR1C and FGFR2C mRNA expression did not vary throughout CL lifespan, whereas FGFR1B was upregulated in the developed (Stage III) CL. FGFR3B, FGFR3C and FGFR4 expression was inconsistent in the bovine CL. The present data indicate that FGFR1 and FGFR2 splice variants are the main receptors for FGF action in the bovine CL.

Expression and Function of Fibroblast Growth Factor 18 in the Ovarian Follicle in Cattle

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Expression of fibroblast growth factor 10 and cognate receptors in the developing bovine ovary

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Potential Effects of Alendronate on Fibroblast Growth Factor 23 Levels and Effective Control of Hypercalciuria in an Adult with Jansen's Metaphyseal Chondrodysplasia

Context: Jansen's metaphyseal chondrodysplasia (JMC) is a rare autosomal dominant disorder caused by activating mutations in the PTH 1 receptor (PTH1R; PTH/PTHrP receptor), leading to chronic hypercalcemia and hypercalciuria. Hypophosphatemia is also a hallmark of JMC, and recently, increased fibroblast growth factor 23 (FGF23) levels have been reported in this syndrome. Hypercalcemia has been associated with increased cardiovascular risk; however, cardiovascular disease has not been extensively investigated in JMC patients. Objective: The aim of the study was to describe the long-term follow-up of a JMC patient with regard to the management of hypercalciuria, the evaluation of FGF23 levels under bisphosphonate treatment, and the investigation of cardiovascular repercussion of chronic hypercalcemia. Results: The diagnosis of JCM was confirmed by molecular analysis (p.H223R mutation in PTH1R). The patient was followed from 5 to 27 yr of age. Asymptomatic nephrolithiasis was diagnosed at 18 yr of age, prompting pharmacological management of hypercalciuria. Treatment with alendronate reduced hypercalciuria; however, normocalciuria was only obtained with the association of thiazide diuretic. Serum FGF23 levels, measured under alendronate treatment, were repeatedly within the normal range. Subclinical cardiovascular disease was investigated when the patient was 26 yr old, after 19 yr of sustained mild hypercalcemia; carotid and vertebral artery ultrasonography was normal, as well as coronary computed tomography angiography (calcium score = 0). Conclusion: The long-term follow-up of our JMC patient has provided insight on therapeutic strategies to control hypercalciuria, on the potential effects of alendronate on FGF23 levels, and on the lack of detectable cardiovascular disease at young adulthood after prolonged exposure to hypercalcemia. (J Clin Endocrinol Metab 97: 1098-1103, 2012)

The FGFRL1 receptor is shed from cell membranes, binds fibroblast growth factors (FGFs), and antagonizes FGF signaling in Xenopus embryos

FGFRL1 (fibroblast growth factor receptor like 1) is the fifth and most recently discovered member of the fibroblast growth factor receptor (FGFR) family. With up to 50% amino acid similarity, its extracellular domain closely resembles that of the four conventional FGFRs. Its intracellular domain, however, lacks the split tyrosine kinase domain needed for FGF-mediated signal transduction. During embryogenesis of the mouse, FGFRL1 is essential for the development of parts of the skeleton, the diaphragm muscle, the heart, and the metanephric kidney. Since its discovery, it has been hypothesized that FGFRL1 might act as a decoy receptor for FGF ligands. Here we present several lines of evidence that support this notion. We demonstrate that the FGFRL1 ectodomain is shed from the cell membrane of differentiating C2C12 myoblasts and from HEK293 cells by an as yet unidentified protease, which cuts the receptor in the membrane-proximal region. As determined by ligand dot blot analysis, cell-based binding assays, and surface plasmon resonance analysis, the soluble FGFRL1 ectodomain as well as the membrane-bound receptor are capable of binding to some FGF ligands with high affinity, including FGF2, FGF3, FGF4, FGF8, FGF10, and FGF22. We furthermore show that ectopic expression of FGFRL1 in Xenopus embryos antagonizes FGFR signaling during early development. Taken together, our data provide strong evidence that FGFRL1 is indeed a decoy receptor for FGFs.

Ablation of the tumor suppressor histidine triad nucleotide binding protein 1 is protective against hepatic ischemia/reperfusion injury

The identification of cellular pathways capable of limiting ischemia/reperfusion (I/R) injury remains a frontier in medicine, and its clinical relevance is urgent. Histidine triad nucleotide binding protein 1 (HINT1) is a tumor suppressor that influences apoptosis. Because apoptotic pathways are a feature of I/R injury, we asked whether Hint1 influences hepatic I/R injury. Hint1(-/-) and C57BL/6 mice were subjected to 70% liver ischemia followed by reperfusion for 3 or 24 hours or to a sham operation. The serum aminotransferase levels, histological lesions, apoptosis, reactive oxygen species, and expression of B cell lymphoma 2-associated X protein (Bax), heme oxygenase 1 (HO-1), interleukin-6 (IL-6), IL-10, tumor necrosis factor-a, Src, nuclear factor kappa B (p65/RelA), and c-Jun were quantified. The responses to toll-like receptor ligands and nicotinamide adenine dinucleotide phosphate oxidase activity in Kupffer cells were compared in Hint1(-/-) mice and C57BL/6 mice. After I/R, the levels of serum aminotransferases, parenchymal necrosis, and hepatocellular apoptosis were significantly lower in Hint1(-/-) mice versus control mice. Furthermore, Bax expression decreased more than 2-fold in Hint1(-/-) mice, and the increases in reactive oxygen species and HO-1 expression that were evident in wild-type mice after I/R were absent in Hint1(-/-) mice. The phosphorylation of Src and the nuclear translocation of p65 were increased in Hint1(-/-) mice, whereas the nuclear expression of phosphorylated c-Jun was decreased. The levels of the protective cytokines IL-6 and IL-10 were increased in Hint1(-/-) mice. These effects increased survival after I/R in mice lacking Hint1. Hint1(-/-) Kupffer cells were less activated than control cells after stimulation with lipopolysaccharides. CONCLUSION: The Hint1 protein influences the course of I/R injury, and its ablation in Kupffer cells may limit the extent of the injury.

Stromal derived growth factor alpha (SDF-1α) induces the differentiation of bone marrow progenitor cells (BMCs) into pericytes by regulating platelet derived growth factor B (PDGF-B) transcription

We previously demonstrated that bone marrow cells (BMCs) migrate to TC71 and A4573 Ewing’s sarcoma tumors where they can differentiate into endothelial cells (ECs) and pericytes and, participate in the tumor vascular development. This process of neo-vascularization, known as vasculogenesis, is essential for Ewing’s sarcoma growth with the soluble vascular endothelial growth factor, VEGF165, being the chemotactic factor for BMC migration to the tumor site. Inhibiting VEGF165 in TC71 tumors (TC/siVEGF7-1) inhibited BMC infiltration to the tumor site and tumor growth. Introducing the stromal-derived growth factor (SDF-1α) into the TC/siVEGF7-1 tumors partially restored vasculogenesis with infiltration of BMCs to a perivascular area where they differentiated into pericytes and rescued tumor growth. RNA collected from the SDF-1α-treated TC/siVEGF7-1 tumors also revealed an increase in platelet-derived growth factor B (PDGF-B) mRNA levels. PDGF-B expression is elevated in several cancer types and the role of PDGF-B and its receptor, PDGFR-β, has been extensively described in the process of pericyte maturation. However, the mechanisms by which PDGF-B expression is up-regulated during vascular remodeling and the process by which BMCs differentiate into pericytes during tumor vasculogenesis remain areas of investigation. In this study, we are the first to demonstrate that SDF-1α regulates the expression of PDGF-B via a transcriptional mechanism which involves binding of the ELK-1 transcription factor to the pdgf-b promoter. We are also first to validate the critical role of the SDF-1α/PDGF-B pathway in the differentiation of BMCs into pericytes both in vitro and in vivo. SDF-1α up-regulated PDGF-B expression in both TC/siVEGF7-1 and HEK293 cells. In contrast, down-regulating SDF-1α, down-regulated PDGF-B. We cloned the 2 kb pdgf-b promoter fragment into the pGL3 reporter vector and showed that SDF-1α induced pdgf-b promoter activity. We used chromatin immunoprecipitation (ChIP) and demonstrated that the ELK-1 transcription factor bound to the pdgf-b promoter in response to SDF-1α stimulation in both TC/siVEGF7-1 and HEK293 cells. We collected BMCs from the hind femurs of mice and cultured the cells in medium containing SDF-1α and PDGF-B and found that PDGFR-β+ BMCs differentiated into NG2 and desmin positive pericytes in vitro. In contrast, inhibiting SDF-1α and PDGF-B abolished this differentiation process. In vivo, we injected TC71 or A4573 tumor-bearing mice with the SDF-1α antagonist, AMD3100 and found that inhibiting SDF-1α signaling in the tumor microenvironment decreased the tumor microvessel density, decreased the tumor blood vessel perfusion and, increased tumor cell apoptosis. We then analyzed the effect of AMD3100 on vasculogenesis of Ewing’s sarcoma and found that BMCs migrated to the tumor site where they differentiated into ECs but, they did not form thick perivascular layers of NG2 and desmin positive pericytes. Finally, we stained the AMD3100-treated tumors for PDGF-B and showed that inhibiting SDF-1α signaling also inhibited PDGF-B expression. All together, these findings demonstrated that the SDF-1α/PDGF-B pathway plays a critical role in the formation of BM-derived pericytes during vasculogenesis of Ewing’s sarcoma tumors.

Fibroblast Growth Factor 23 Is an Independent and Specific Predictor of Mortality in Patients With Heart Failure and Reduced Ejection Fraction

BACKGROUND Strategies to improve risk prediction are of major importance in patients with heart failure (HF). Fibroblast growth factor 23 (FGF-23) is an endocrine regulator of phosphate and vitamin D homeostasis associated with an increased cardiovascular risk. We aimed to assess the prognostic effect of FGF-23 on mortality in HF patients with a particular focus on differences between patients with HF with preserved ejection fraction and patients with HF with reduced ejection fraction (HFrEF). METHODS AND RESULTS FGF-23 levels were measured in 980 patients with HF enrolled in the Ludwigshafen Risk and Cardiovascular Health (LURIC) study including 511 patients with HFrEF and 469 patients with HF with preserved ejection fraction and a median follow-up time of 8.6 years. FGF-23 was additionally measured in a second cohort comprising 320 patients with advanced HFrEF. FGF-23 was independently associated with mortality with an adjusted hazard ratio per 1-SD increase of 1.30 (95% confidence interval, 1.14-1.48; P<0.001) in patients with HFrEF, whereas no such association was found in patients with HF with preserved ejection fraction (for interaction, P=0.043). External validation confirmed the significant association with mortality with an adjusted hazard ratio per 1 SD of 1.23 (95% confidence interval, 1.02-1.60; P=0.027). FGF-23 demonstrated an increased discriminatory power for mortality in addition to N-terminal pro-B-type natriuretic peptide (C-statistic: 0.59 versus 0.63) and an improvement in net reclassification index (39.6%; P<0.001). CONCLUSIONS FGF-23 is independently associated with an increased risk of mortality in patients with HFrEF but not in those with HF with preserved ejection fraction, suggesting a different pathophysiologic role for both entities.