Splicing Reporter Mice Revealed the Evolutionally Conserved Switching Mechanism of Tissue-Specific Alternative Exon Selection

Since alternative splicing of pre-mRNAs is essential for generating tissue-specific diversity in proteome, elucidating its regulatory mechanism is indispensable to understand developmental process or tissue-specific functions. We have been focusing on tissue-specific regulation of mutually exclusive selection of alternative exons because this implies the typical molecular mechanism of alternative splicing regulation and also can be good examples to elicit general rule of “splice code”. So far, mutually exclusive splicing regulation has been explained by the outcome from the balance of multiple regulators that enhance or repress either of alternative exons discretely. However, this “balance” model is open to questions of how to ensure the selection of only one appropriate exon out of several candidates and how to switch them. To answer these questions, we generated an original bichromatic fluorescent splicing reporter system for mammals using fibroblast growth factor-receptor 2 (FGFR2) gene as model. By using this splicing reporter, we demonstrated that FGFR2 gene is regulated by the “switch-like” mechanism, in which key regulators modify the ordered splice-site recognition of two mutually exclusive exons, eventually ensure single exon selection and their distinct switching. Also this finding elucidated the evolutionally conserved “splice code,” in which combination of tissue-specific and broadly expressed RNA binding proteins regulate alternative splicing of specific gene in a tissue-specific manner. These findings provide the significant cue to understand how a number of spliced genes are regulated in various tissue-specific manners by a limited number of regulators, eventually to understand developmental process or tissue-specific functions

Exogenous Addition of a C-Xylopyranoside Derivative Stimulates Keratinocyte Dermatan Sulfate Synthesis and Promotes Migration

As C-Xyloside has been suggested to be an initiator of glycosaminoglycan (GAG) synthesis, and GAGs such as Dermatan sulfate (DS) are potent enhancers of fibroblast growth factor (FGF) - 10 action, we investigated if a C-Xylopyranoside derivative, (C-β-D-xylopyranoside-2-hydroxy-propane, C-Xyloside), could promote DS production by cultured normal human keratinocytes, how this occurs and if C-Xyloside could also stimulate FGF-dependent cell migration and proliferation. C-Xyloside-treated keratinocytes greatly increased secretion of total sulfated GAGs. Majority of the induced GAG was chondroitin sulfate/dermatan sulfate (CS/DS) of which the major secreted GAG was DS. Cells lacking xylosyltransferase enzymatic activity demonstrated that C-Xyloside was able to stimulate GAG synthesis without addition to core proteins. Consistent with the observed increase in DS, keratinocytes treated with C-Xyloside showed enhanced migration in response to FGF-10 and secreted into their culture media GAGs that promoted FGF-10-dependent cellular proliferation. These results indicate that C-Xyloside may enhance epithelial repair by serving as an initiator of DS synthesis

The synthetic inhibitor of Fibroblast Growth Factor Receptor PD166866 controls negatively the growth of tumor cells in culture

<p>Abstract</p> <p>Background</p> <p>Many experimental data evidence that over-expression of various growth factors cause disorders in cell proliferation. The role of the Fibroblast Growth Factors (FGF) in growth control is indisputable: in particular, FGF1 and its tyrosine kinase receptor (FGFR1) act through a very complex network of mechanisms and pathways. In this work we have evaluated the antiproliferative activity effect of PD166866, a synthetic molecule inhibiting the tyrosin kinase action of FGFR1.</p> <p>Methods</p> <p>Cells were routinely grown in Dulbecco Modified Eagle's medium supplemented with newborn serum and a penicillin-streptomycin mixture.</p> <p>Cell viability was evaluated by Mosmann assay and by trypan blue staining. DNA damage was assessed by <it>in situ </it>fluorescent staining with Terminal Deoxynucleotidyl Transferase dUTP nick end labeling (TUNEL assay).</p> <p>Assessment of oxidative stress at membrane level was measured by quantitative analysis of the intra-cellular formation of malonyl-dialdheyde (MDA) deriving from the decomposition of poly-unsaturated fatty acids.</p> <p>The expression of Poly-ADP-Ribose-Polymerase (PARP), consequent to DNA fragmentation, was evidenced by immuno-histochemistry utilizing an antibody directed against an N-terminal fragment of the enzyme.</p> <p>Results</p> <p>The bioactivity of the drug was investigated on Hela cells. Cytoxicity was assessed by the Mosmann assay and by vital staining with trypan blue. The target of the molecule is most likely the cell membrane as shown by the significant increase of the intracellular concentration of malonyl-dihaldheyde. The increase of this compound, as a consequence of the treatment with PD166866, is suggestive of membrane lipoperoxidation. The TUNEL assay gave a qualitative, though clear, indication of DNA damage. Furthermore we demonstrate intracellular accumulation of poly-ADP-ribose polymerase I. This enzyme is a sensor of nicks on the DNA strands and this supports the idea that treatment with the drug induces cell death.</p> <p>Conclusions</p> <p>Data presented in this work show that PD166866 has clear antiproliferative effects. The negative control of cell proliferation may be exerted through the activation of the apoptotic pathway. The results of experiments addressing this specific point, such as: evaluation of DNA damage, lipoperoxidation of the cell membrane and increase of expression of PARP, an enzyme directly involved in DNA repair. Results suggest that cells exposed to PD16866 undergo apoptosis. However, concomitant modes of cell death cannot be ruled out. The possible use of this drug for therapeutic purposes is discussed.</p

Identification of Mechanosensitive Genes during Embryonic Bone Formation

Although it is known that mechanical forces are needed for normal bone development, the current understanding of how biophysical stimuli are interpreted by and integrated with genetic regulatory mechanisms is limited. Mechanical forces are thought to be mediated in cells by “mechanosensitive” genes, but it is a challenge to demonstrate that the genetic regulation of the biological system is dependant on particular mechanical forces in vivo. We propose a new means of selecting candidate mechanosensitive genes by comparing in vivo gene expression patterns with patterns of biophysical stimuli, computed using finite element analysis. In this study, finite element analyses of the avian embryonic limb were performed using anatomically realistic rudiment and muscle morphologies, and patterns of biophysical stimuli were compared with the expression patterns of four candidate mechanosensitive genes integral to bone development. The expression patterns of two genes, Collagen X (ColX) and Indian hedgehog (Ihh), were shown to colocalise with biophysical stimuli induced by embryonic muscle contractions, identifying them as potentially being involved in the mechanoregulation of bone formation. An altered mechanical environment was induced in the embryonic chick, where a neuromuscular blocking agent was administered in ovo to modify skeletal muscle contractions. Finite element analyses predicted dramatic changes in levels and patterns of biophysical stimuli, and a number of immobilised specimens exhibited differences in ColX and Ihh expression. The results obtained indicate that computationally derived patterns of biophysical stimuli can be used to inform a directed search for genes that may play a mechanoregulatory role in particular in vivo events or processes. Furthermore, the experimental data demonstrate that ColX and Ihh are involved in mechanoregulatory pathways and may be key mediators in translating information from the mechanical environment to the molecular regulation of bone formation in the embryo

Fibroblast growth factor 19 expression correlates with tumor progression and poorer prognosis of hepatocellular carcinoma

<p>Abstract</p> <p>Background</p> <p>Although fibroblast growth factor 19 (FGF19) can promote liver carcinogenesis in mice, its involvement in human hepatocellular carcinoma (HCC) has not been well investigated. FGF19, a member of the FGF family, has unique specificity for its receptor FGFR4. This study aimed to clarify the involvement of FGF19 in the development of HCC.</p> <p>Methods</p> <p>We investigated human FGF19 and FGFR4 expression in 40 hepatocellular carcinoma specimens using quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) analysis and immunohistochemistry. Moreover, we examined the expression and the distribution of FGF19 and FGFR4 in 5 hepatocellular carcinoma cell lines (HepG2, HuH7, HLE, HLF, and JHH7) using RT-PCR and immunohistochemistry. To test the role of the FGF19/FGFR4 system in tumor progression, we used recombinant FGF19 protein and small interfering RNA (siRNA) of <it>FGF19 </it>and <it>FGFR4 </it>to regulate their concentrations.</p> <p>Results</p> <p>We found that FGF19 was significantly overexpressed in HCCs as compared with corresponding noncancerous liver tissue (<it>P </it>< 0.05). Univariate and multivariate analyses revealed that the tumor <it>FGF19 </it>mRNA expression was an independent prognostic factor for overall and disease-free survival. Moreover, we found that the FGF19 recombinant protein could increase the proliferation (<it>P </it>< 0.01, <it>n </it>= 12) and invasion (<it>P </it>< 0.01, <it>n </it>= 6) capabilities of human hepatocellular carcinoma cell lines and inhibited their apoptosis (<it>P </it>< 0.01, <it>n </it>= 12). Inversely, decreasing <it>FGF19 </it>and <it>FGFR4 </it>expression by siRNA significantly inhibited proliferation and increased apoptosis in JHH7 cells (<it>P </it>< 0.01, <it>n </it>= 12). The postoperative serum FGF19 levels in HCC patients was significantly lower than the preoperative levels (<it>P </it>< 0.01, <it>n </it>= 29).</p> <p>Conclusions</p> <p>FGF19 is critically involved in the development of HCCs. Targeting FGF19 inhibition is an attractive potential therapeutic strategy for HCC.</p

FGF Signaling Pathway in the Developing Chick Lung: Expression and Inhibition Studies

Background: Fibroblast growth factors (FGF) are essential key players during embryonic development. Through their specific cognate receptors (FGFR) they activate intracellular cascades, finely regulated by modulators such as Sprouty. Several FGF ligands (FGF1, 2, 7, 9, 10 and 18) signaling through the four known FGFRs, have been implicated in lung morphogenesis. Although much is known about mammalian lung, so far, the avian model has not been explored for lung studies. Methodology/Principal Findings: In this study we provide the first description of fgf10, fgfr1-4 and spry2 expression patterns in early stages of chick lung development by in situ hybridization and observe that they are expressed similarly to their mammalian counterparts. Furthermore, aiming to determine a role for FGF signaling in chick lung development, in vitro FGFR inhibition studies were performed. Lung explants treated with an FGF receptor antagonist (SU5402) presented an impairment of secondary branch formation after 48 h of culture; moreover, abnormal lung growth with a cystic appearance of secondary bronchi and reduction of the mesenchymal tissue was observed. Branching and morphometric analysis of lung explants confirmed that FGFR inhibition impaired branching morphogenesis and induced a significant reduction of the mesenchyme. Conclusions/Significance: This work demonstrates that FGFRs are essential for the epithelial-mesenchymal interactions tha

Targeting FGFR4 Inhibits Hepatocellular Carcinoma in Preclinical Mouse Models

The fibroblast growth factor (FGF)-FGF receptor (FGFR) signaling system plays critical roles in a variety of normal developmental and physiological processes. It is also well documented that dysregulation of FGF-FGFR signaling may have important roles in tumor development and progression. The FGFR4–FGF19 signaling axis has been implicated in the development of hepatocellular carcinomas (HCCs) in mice, and potentially in humans. In this study, we demonstrate that FGFR4 is required for hepatocarcinogenesis; the progeny of FGF19 transgenic mice, which have previously been shown to develop HCCs, bred with FGFR4 knockout mice fail to develop liver tumors. To further test the importance of FGFR4 in HCC, we developed a blocking anti-FGFR4 monoclonal antibody (LD1). LD1 inhibited: 1) FGF1 and FGF19 binding to FGFR4, 2) FGFR4–mediated signaling, colony formation, and proliferation in vitro, and 3) tumor growth in a preclinical model of liver cancer in vivo. Finally, we show that FGFR4 expression is elevated in several types of cancer, including liver cancer, as compared to normal tissues. These findings suggest a modulatory role for FGFR4 in the development and progression of hepatocellular carcinoma and that FGFR4 may be an important and novel therapeutic target in treating this disease

Clathrin- and Dynamin-Independent Endocytosis of FGFR3 – Implications for Signalling

Endocytosis of tyrosine kinase receptors can influence both the duration and the specificity of the signal emitted. We have investigated the mechanisms of internalization of fibroblast growth factor receptor 3 (FGFR3) and compared it to that of FGFR1 which is internalized predominantly through clathrin-mediated endocytosis. Interestingly, we observed that FGFR3 was internalized at a slower rate than FGFR1 indicating that it may use a different endocytic mechanism than FGFR1. Indeed, after depletion of cells for clathrin, internalization of FGFR3 was only partly inhibited while endocytosis of FGFR1 was almost completely abolished. Similarly, expression of dominant negative mutants of dynamin resulted in partial inhibition of the endocytosis of FGFR3 whereas internalization of FGFR1 was blocked. Interfering with proposed regulators of clathrin-independent endocytosis such as Arf6, flotillin 1 and 2 and Cdc42 did not affect the endocytosis of FGFR1 or FGFR3. Furthermore, depletion of clathrin decreased the degradation of FGFR1 resulting in sustained signalling. In the case of FGFR3, both the degradation and the signalling were only slightly affected by clathrin depletion. The data indicate that clathrin-mediated endocytosis is required for efficient internalization and downregulation of FGFR1 while FGFR3, however, is internalized by both clathrin-dependent and clathrin-independent mechanisms

The FGFR4-G388R Polymorphism Promotes Mitochondrial STAT3 Serine Phosphorylation to Facilitate Pituitary Growth Hormone Cell Tumorigenesis

Pituitary tumors are common intracranial neoplasms, yet few germline abnormalities have been implicated in their pathogenesis. Here we show that a single nucleotide germline polymorphism (SNP) substituting an arginine (R) for glycine (G) in the FGFR4 transmembrane domain can alter pituitary cell growth and hormone production. Compared with FGFR4-G388 mammosomatotroph cells that support prolactin (PRL) production, FGFR4-R388 cells express predominantly growth hormone (GH). Growth promoting effects of FGFR4-R388 as evidenced by enhanced colony formation was ascribed to Src activation and mitochondrial serine phosphorylation of STAT3 (pS-STAT3). In contrast, diminished pY-STAT3 mediated by FGFR4-R388 relieved GH inhibition leading to hormone excess. Using a knock-in mouse model, we demonstrate the ability of FGFR4-R385 to promote GH pituitary tumorigenesis. In patients with acromegaly, pituitary tumor size correlated with hormone excess in the presence of the FGFR4-R388 but not the FGFR4-G388 allele. Our findings establish a new role for the FGFR4-G388R polymorphism in pituitary oncogenesis, providing a rationale for targeting Src and STAT3 in the personalized treatment of associated disorders