Fibroblast Growth Factor 6.

Fibroblast Growth Factor 6 (FGF6), also referred to as HST2 or HBGF6, is a member of the Fibroblast Growth Factor (FGF), the Heparin Binding Growth Factor (HBGF) and the Heparin Binding Secretory Transforming Gene (HST) families. The genomic and protein structure of FGF6 is highly conserved among varied species, as is its expression in muscle and muscle progenitor cells. Like other members of the FGF family, FGF6 regulates cell proliferation, differentiation, and migration. Specifically, it plays key roles in myogenesis and muscular regeneration, angiogenesis, along with iron transport and lipid metabolism. Similar to others from the FGF family, FGF6 also possesses oncogenic transforming activity, and as such is implicated in a variety of cancers.

LINC00265 promotes the viability, proliferation, and migration of bladder cancer cells via the miR-4677-3p/FGF6 axis.

BACKGROUND: Bladder cancer (BCa) is a common genitourinary malignancy with higher incidence in males. Long intergenic non-protein coding RNA 265 (LINC00265) is identified as an oncogene in many malignancies, while its role in BCa development remains unknown. PURPOSE: To explore the functions and mechanism of LINC00265 in BCa. RESEARCH DESIGN: Reverse transcription quantitative polymerase chain reaction was performed to examine LINC00265 expression in BCa cells. Cell counting kit-8 assays, colony formation assays, TdT-mediated dUTP Nick-End Labeling assays, and Transwell assays were conducted to examine BCa cell viability, proliferation, apoptosis, and migration. Luciferase reporter assays and RNA immunoprecipitation assays were carried out to explore the binding capacity between miR-4677-3p and messenger RNA fibroblast growth factor 6 (FGF6) (or LINC00265). Xenograft tumor model was established to explore the role of LINC00265 in vivo. RESULTS: LINC00265 was highly expressed in BCa cells. LINC00265 knockdown inhibited xenograft tumor growth and BCa cell viability, proliferation and migration while enhancing cell apoptosis. Moreover, LINC00265 interacted with miR-4677-3p to upregulate the expression of FGF6. FGF6 overexpression reversed the suppressive effect of LINC00265 knockdown on malignant phenotypes of BCa cells. CONCLUSIONS: LINC00265 promotes the viability, proliferation, and migration of BCa cells by binding with miR-4677-3p to upregulate FGF6 expression.

Skeletal muscle-targeted delivery of Fgf6 protects mice from diet-induced obesity and insulin resistance.

Obesity, a major health care issue, is characterized by metabolic abnormalities in multiple tissues, including the skeletal muscle. Although dysregulation of skeletal muscle metabolism can strongly influence the homeostasis of systemic energy, the underlying mechanism remains unclear. We found promoter hypermethylation and decreased gene expression of fibroblast growth factor 6 (FGF6) in the skeletal muscle of individuals with obesity using high-throughput sequencing. Reduced binding of the cyclic AMP responsive element binding protein-1 (CREB1) to the hypermethylated cyclic AMP response element, which is a regulatory element upstream of the transcription initiation site, partially contributed to the downregulation of FGF6 in patients with obesity. Overexpression of Fgf6 in mouse skeletal muscle stimulated protein synthesis, activating the mammalian target of rapamycin pathway, and prevented the increase in weight and the development of insulin resistance in high-fat diet-fed mice. Thus, our findings highlight the role played by Fgf6 in regulating skeletal muscle hypertrophy and whole-body metabolism, indicating its potential in strategies aimed at preventing and treating metabolic diseases.

FGF6 and FGF9 regulate UCP1 expression independent of brown adipogenesis.

Uncoupling protein-1 (UCP1) plays a central role in energy dissipation in brown adipose tissue (BAT). Using high-throughput library screening of secreted peptides, we identify two fibroblast growth factors (FGF), FGF6 and FGF9, as potent inducers of UCP1 expression in adipocytes and preadipocytes. Surprisingly, this occurs through a mechanism independent of adipogenesis and involves FGF receptor-3 (FGFR3), prostaglandin-E2 and interaction between estrogen receptor-related alpha, flightless-1 (FLII) and leucine-rich-repeat-(in FLII)-interacting-protein-1 as a regulatory complex for UCP1 transcription. Physiologically, FGF6/9 expression in adipose is upregulated by exercise and cold in mice, and FGF9/FGFR3 expression in human neck fat is significantly associated with UCP1 expression. Loss of FGF9 impairs BAT thermogenesis. In vivo administration of FGF9 increases UCP1 expression and thermogenic capacity. Thus, FGF6 and FGF9 are adipokines that can regulate UCP1 through a transcriptional network that is dissociated from brown adipogenesis, and act to modulate systemic energy metabolism.

FGF6 enhances muscle regeneration after nerve injury by relying on ERK1/2 mechanism.

BACKGROUND: Severe peripheral nerve injury leads to skeletal muscle atrophy and impaired limb function that is not sufficiently improved by existing treatments. Fibroblast growth factor 6 (FGF6) is involved in tissue regeneration and is dysregulated in denervated rat muscles. However, the way that FGF6 affects skeletal muscle repair after peripheral nerve injury has not been fully elucidated. METHODS: In this study, we investigated the role of FGF6 in the regeneration of denervated muscles using myoblast cells and an in vivo model of peripheral nerve injury. RESULTS: FGF6 promoted the viability and migration of C2C12 and primary myoblasts in a dose-dependent manner through FGFR1-mediated upregulation of cyclin D1. Low concentrations of FGF6 promoted myoblast differentiation through FGFR4-mediated activation of ERK1/2, which upregulated expression of MyHC, MyoD, and myogenin. FGFR-1, FGFR4, MyoD, and myogenin were not upregulated when FGF6 expression was inhibited in myoblasts by shRNA-mediated knockdown. Injection of FGF6 into denervated rat muscles enhanced the MyHC-IIb muscle fiber phenotype and prevented muscular atrophy. CONCLUSION: These findings indicate that FGF6 reduces skeletal muscle atrophy by relying on the ERK1/2 mechanism and enhances the conversion of slow muscle to fast muscle fibers, thereby promoting functional recovery of regenerated skeletal muscle after innervation.

Characterization and expression analysis of FGF6 (fibroblast growth factor 6) genes of grass carp (Ctenopharyngodon idellus) reveal their regulation on muscle growth.

The present study was conducted to investigate the regulative function of FGF6 in the muscle growth of grass carp (Ctenopharyngodon idellus) by the bioinformatics analysis and expression pattern analyses of FGF6 genes in different developmental stages and tissues, as well as the correlation analysis between muscle growth and FGF6 expression after fish were fed with different levels of dietary lotus leaf flavonoids (LLF) (0, 0.03%, 0.06%, 0.09%). Results showed that the FGF6a and FGF6b genes are two homologs of the FGF6 family, encoding 205 and 209 amino acids, respectively. Alignment of amino acid sequences and phylogenetic analysis demonstrated that FGF6a and FGF6b are highly conserved with other vertebrates. Quantitative RT-PCR analysis showed both FGF6a and FGF6b expressions were high in brain and muscle but low in other examined tissues. During embryonic development, FGF6a and FGF6b mRNA expressions could be detected as early as at fertilized egg stage and displayed the highest value at cleavage stage. Dietary LLF affected the gene expression of FGF6 in white muscle. The relative expression of FGF6a of 0.06% LLF group was significantly higher than that of 0.09% LLF group, while FGF6b expression of 0.06% LLF group was higher than those of other groups (P < 0.05). The muscle fiber diameter was significantly higher in 0.06% LLF group in comparison with other groups, while the fiber density in this group was lower (P < 0.05). Both FGF6a and FGF6b expressions were positively correlated with fiber diameter but negatively correlated with fiber density. These results collectively suggest that FGF6a and FGF6b play an important role in muscle growth regulation in grass carp.

A gene-based recessive diplotype exome scan discovers FGF6, a novel hepcidin-regulating iron-metabolism gene.

Standard analyses applied to genome-wide association data are well designed to detect additive effects of moderate strength. However, the power for standard genome-wide association study (GWAS) analyses to identify effects from recessive diplotypes is not typically high. We proposed and conducted a gene-based compound heterozygosity test to reveal additional genes underlying complex diseases. With this approach applied to iron overload, a strong association signal was identified between the fibroblast growth factor-encoding gene, FGF6, and hemochromatosis in the central Wisconsin population. Functional validation showed that fibroblast growth factor 6 protein (FGF-6) regulates iron homeostasis and induces transcriptional regulation of hepcidin. Moreover, specific identified FGF6 variants differentially impact iron metabolism. In addition, FGF6 downregulation correlated with iron-metabolism dysfunction in systemic sclerosis and cancer cells. Using the recessive diplotype approach revealed a novel susceptibility hemochromatosis gene and has extended our understanding of the mechanisms involved in iron metabolism.

Total substitution of dietary fish oil by vegetable oils stimulates muscle hypertrophic growth in Senegalese sole and the upregulation of fgf6.

The long term effects of fish oil (FO) substitution by increasing the levels of vegetable oils (VO), 0% (CTR), 50% (VO50) and 100% (VO100), in diets for Senegalese sole were evaluated in terms of skeletal muscle cellularity and expression of related genes. After 140 days of feeding, all fish had similar body weight and length. The inclusion of 50% VO did not result in differences in muscle cellularity, but dorsal muscle cross-sectional area and fast-twitch fibre diameter increased in fish fed total FO substitution, whilst fibre density was reduced (P < 0.05) in relation to CTR. The total number of fibres was similar in all treatments. FO substitution did not affect the transcript levels of myogenic genes (myf5, mrf4, myog, myod1, myod2), but resulted in a two-fold increase of fgf6 transcript levels compared to CTR (P < 0.05). The relative expression of igf-I was higher in VO100 than in VO50, but was similar to CTR. FO substitution resulted in cellularity changes related to the stimulation of muscle hypertrophic growth, but not hyperplastic growth, and associated with a nutritional modulation of fgf6 by dietary VO. This study indicates that 50% VO does not affect the muscle phenotype, but total FO substitution stimulates muscle hypertrophy.

ALK5-mediated transforming growth factor ß signaling in neural crest cells controls craniofacial muscle development via tissue-tissue interactions.

The development of the craniofacial muscles requires reciprocal interactions with surrounding craniofacial tissues that originate from cranial neural crest cells (CNCCs). However, the molecular mechanism involved in the tissue-tissue interactions between CNCCs and muscle progenitors during craniofacial muscle development is largely unknown. In the current study, we address how CNCCs regulate the development of the tongue and other craniofacial muscles using Wnt1-Cre; Alk5(fl/fl) mice, in which loss of Alk5 in CNCCs results in severely disrupted muscle formation. We found that Bmp4 is responsible for reduced proliferation of the myogenic progenitor cells in Wnt1-Cre; Alk5(fl/fl) mice during early myogenesis. In addition, Fgf4 and Fgf6 ligands were reduced in Wnt1-Cre; Alk5(fl/fl) mice and are critical for differentiation of the myogenic cells. Addition of Bmp4 or Fgf ligands rescues the proliferation and differentiation defects in the craniofacial muscles of Alk5 mutant mice in vitro. Taken together, our results indicate that CNCCs play critical roles in controlling craniofacial myogenic proliferation and differentiation through tissue-tissue interactions.

Preaxial polydactyly of the upper limb viewed as a spectrum of severity of embryonic events.

Preaxial polydactyly (PPD) is a common congenital abnormality and its classification varies among geneticists and hand surgeons. For example, the triphalangeal thumb, preaxial polysyndactyly, and the mirror hand deformity are considered as forms of PPD only in the genetics literature. Preaxial polydactyly is an error in the anteroposterior axis of the development of the upper limb. In this paper, the development of this axis is detailed and all molecular events that are known to lead to PPD are reviewed. Finally, based on the review, PPD is viewed as a spectrum of severity of embryonic events.

Cloning and expression analysis of Fgf5, 6 and 7 during early chick development.

FGFs with similar sequences can play different roles depending on the model organisms examined. Determining these roles requires knowledge of spatio-temporal Fgf gene expression patterns. In this study, we report the cloning of chick Fgf5, 6 and 7, and examine their gene expression patterns by whole mount in situ hybridization. We show that Fgf5's spatio-temporally restricted expression pattern indicates a potentially novel role during inner ear development. Fgf6 and Fgf7, although belonging to different subfamilies with diverged sequences, are expressed in similar patterns within the mesoderm. Alignment of protein sequences and phylogenetic analysis demonstrate that FGF5 and FGF6 are highly conserved between chick, human, mouse and zebrafish. FGF7 is similarly conserved except for the zebrafish, which has considerably diverged.

Pipeline for large-scale microdroplet bisulfite PCR-based sequencing allows the tracking of hepitype evolution in tumors.

Cytosine methylation provides an epigenetic level of cellular plasticity that is important for development, differentiation and cancerogenesis. We adopted microdroplet PCR to bisulfite treated target DNA in combination with second generation sequencing to simultaneously assess DNA sequence and methylation. We show measurement of methylation status in a wide range of target sequences (total 34 kb) with an average coverage of 95% (median 100%) and good correlation to the opposite strand (rho = 0.96) and to pyrosequencing (rho = 0.87). Data from lymphoma and colorectal cancer samples for SNRPN (imprinted gene), FGF6 (demethylated in the cancer samples) and HS3ST2 (methylated in the cancer samples) serve as a proof of principle showing the integration of SNP data and phased DNA-methylation information into "hepitypes" and thus the analysis of DNA methylation phylogeny in the somatic evolution of cancer.

Eccentric stimulation reveals an involvement of FGF6 in muscle resistance to mechanical stress.

The objective of this report was to analyse a potential role for FGF6 in muscle resistance to mechanical stress. Normal or regenerating muscles of FGF6 (-/-) mice versus wild-type mice were submitted to different protocols of damaging eccentric contractions (eccentric electrostimulation and intermittent downhill exercise). Then muscular structural properties were analysed by histological and immunochemistry techniques to evaluate the post-injury muscle recovery; their muscle contractile parameters (maximal tetanic force, kinetics properties and fatigue resistance) were assessed. The absence of FGF6 causes (1) a fast-to-slow myofibre type switch in adult control and regenerating Tibialis anterior (TA) muscle; (2) muscle weakness in regenerating muscles in animals submitted to eccentric exercise protocols due to aberrant extensive necrotic zones. These observations point out a crucial and unexpected role for FGF6 in muscle integrity and muscle protection against mechanical stress.

Regulation of osteoblast and osteoclast functions by FGF-6.

Fibroblast growth factor-6 (FGF-6) is known to be the key ligand for fibroblast growth factor receptor 4 (FGFR4) during muscle regeneration but its role in bone has yet to be verified. FGFR signaling is known to be important in the initiation and regulation of osteogenesis, so in this study the actions of FGF-6 on human osteoblasts and osteoclasts were investigated. Human primary osteoblasts (hOB) were used to study the effect of FGF-6 on proliferation (by ATP quantification), signal transduction (by ERK and AKT phosphorylation), differentiation (by alkaline phosphatase activity, APA), and mineralization (by calcein staining). To study FGF-6 activity on osteoclast differentiation, human bone marrow cells were used and tartrate-resistant acid phosphatase (TRAP) multinucleated cells together with actin filaments arrangements were quantified. Human primary mature osteoclasts were used to evaluate the effect of FGF-6 on osteoclast reabsorbing activity by reabsorbed pit measurements. FGF-6 >10(-9) M as FGF-2 10(-7) M induced hOB proliferation mediated by pERK together with a reduction in APA and reduced mineralization of the treated cells. Moreover FGF-6 increased the formation of TRAP-positive multinucleated cells in a dose-dependent manner (maximal effect at 10(-8) M). FGF-6-treated cells showed also a greater percentage of cells that formed typical osteoclast sealing zones. Mature osteoclasts cultured on dentine slice increased the area of reabsorption with a maximal effect of FGF-6 at 10(-12) M. FGF-6 may be considered a regulator of bone metabolism as shown by its activity on both osteoblasts and osteoclasts.

[The mRNA expression of P16(ink4a) and HST2 in benign prostatic hyperplasia tissues: a pilot study].

OBJECTIVE: To investigate the expressions of the aging gene P16(ink4a) and anti-aging gene HST2 in benign prostatic hyperplasia (BPH). METHODS: Twenty-three BPH and eighteen normal prostate specimens were collected and total RNA was extracted, followed by the reverse transcriptase polymerase chain reaction (RT-PCR). The expressions of P16(ink4a) was detected by semi-quantitative analysis in BPH and normal prostate tissues. RESULTS: P16(ink4a) mRNA, rather than HST2, was expressed in the BPH and normal prostate tissues. Semi-quantitative analysis showed that the P16(ink4a) mRNA expression in the normal prostate tissues (0.4868 +/- 0.545 was significantly higher than in the BPH tissues (0.2783 +/- 0.0268, with a statistical difference in between (P < 0. 05). CONCLUSION: P16(ink4a) might play an important role in the pathogenesis of BPH and is probably one of the factors of cell aging escape.

Sprouty gene expression is regulated by nerve and FGF6 during regeneration of mouse muscles.

Sprouty (Spry) proteins were identified as negative regulators of fibroblast growth factor (FGF) signaling in vertebrates and invertebrates. Given the importance of the FGFs in myogenesis, we performed cardiotoxin injury-induced regeneration experiments on soleus muscles of both, adult control and FGF6 ( - / - ) mutant mice and analyzed the accumulation of Spry (1, 2 and 4) transcripts using semi-quantitative and real-time RT-PCR assays and in situ hybridization. We also analyzed the effects of muscle denervation on the accumulation of Spry transcripts. The three Spry genes begin to be expressed as early as the first stages of muscle regeneration and are characterized by distinct expression patterns. Moreover, Spry gene expression was highly and differentially up-regulated, precociously by the lack of FGF6, and belatedly by muscle denervation strongly suggesting that the transient rise of Spry mRNA accumulation was associated to muscle differentiation. Rescue experiments supported the idea of a specific relationship between FGF6 and Spry 2, both being known for their particular involvement in myogenesis.

FGF6 in myogenesis.

Important functions in myogenesis have been proposed for FGF6, a member of the fibroblast growth factor family accumulating almost exclusively in the myogenic lineage. However, the analyses of Fgf6 (-/-) mutant mice gave contradictory results and the role of FGF6 during myogenesis remained largely unclear. Recent reports support the concept that FGF6 has a dual function in muscle regeneration, stimulating myoblast proliferation/migration and muscle differentiation/hypertrophy in a dose-dependent manner. The alternative use of distinct signaling pathways recruiting either FGFR1 or FGFR4 might explain the dual role of FGF6 in myogenesis. A role for FGF6 in the maintenance of a reserve pool of progenitor cells in the skeletal muscle has been also strongly suggested. The aim of this review is to summarize our knowledge on the involvement of FGF6 in myogenesis.

Global transcriptional characterization of SP and MP cells from the myogenic C2C12 cell line: effect of FGF6.

With the use of Hoechst staining techniques, we have previously shown that the C2C12 myogenic cell line contains a side population (SP) that is largely increased in the presence of fibroblast growth factor 6 (FGF6). Here, we compared transcriptional profiles from SP and main population (MP) cells from either C2C12 or FGF6-expressing C2C12. Expression profiles of SPs show that these cells are less differentiated than MPs and display some similarities to stem cells. Moreover, principal component analysis made it possible to distinguish specific contributions of either FGF6 or differentiation effects on gene expression profiles. This demonstrated that FGF6-expanded SPs were similar to parental C2C12-derived SPs. Conversely, FGF6-treated MPs differed from parental MPs and were more related to SP cells. These results show that FGF6 pushed committed myogenic cells toward a more immature phenotype resulting in the accumulation of cells with a SP phenotype. We propose that FGF6 conditioning could provide a way to expand the pool of immature cells by myoblast dedifferentiation.

Comparative genomics on mammalian Fgf6-Fgf23 locus.

CCND2-C12orf5-FGF23-FGF6 locus at human chromosome 12p13.32 and CCND1-ORAOV1-FGF19-FGF4 locus at human chromosome 11q13.3 are paralogous regions (paralogons) within the human genome. FGF23 is the causative factor for tumor-induced osteomalacia (TIO), a paraneoplastic disorder characterized by hypophosphatemia and skeletal undermineralization, and also for autosomal dominant hypophosphatemic rickets (ADHR). Here, rat Fgf6 and Fgf23 complete coding sequences were determined by using bioinformatics. Rat Fgf6 and Fgf23 genes, consisting of three exons, were located within AC103292.6 rat genome sequence. Rat Fgf6 and Fgf23 genes were clustered in tail-to-head manner with an interval of about 52 kb. Human FGF6 and FGF23 genes were clustered in tail-to-head manner with an interval of about 54 kb. Intergenic conserved region (IGCR) within the FGF6-FGF23 gene cluster was identified based on the evolutionary conservation. Human FGF6-FGF23 IGCR (nucleotide position 111648-112242 of AC008012.8 genome sequence) and rat Fgf6-Fgf23 IGCR (nucleotide position 156318-156894 of AC103292.6 genome sequence) showed 77.6% total nucleotide identity. CP2, E47, CREB and PAX4 binding sites were conserved among human FGF6, rat Fgf6, and mouse Fgf6 promoters. GATA and E47 binding sites were conserved among human FGF23, rat Fgf23, and mouse Fgf23 promoters. Because mouse Fgf23 mRNA was expressed in dendritic cells and activated spleen, tumor infiltrating dendritic cells are candidate sources of FGF23 secretion in TIO patients. This is the first report on comparative genomics analyses on human FGF6-FGF23 gene cluster and rodents Fgf6-Fgf23 gene cluster.