Mumin Md Abdul Æ Anu Alice Thomas
Received: 12 March 2009 / Accepted: 8 July 2009 / Published online: 25 July 2009 Ó to the authors 2009
Abstract Basic fibroblastgrowthfactor (bFGF), a pro- tein, plays a key role in wound healing and blood vessel regeneration. However, bFGF is easily degraded in bio- logic systems. Mesoporous silica nanoparticles (MSNs) with well-tailored porous structure have been used for hosting guest molecules for drug delivery. Here, we report an in situ route to load bFGF in MSNs for a prolonged release. The average diameter (d) of bFGF-loaded MSNs is 57 ± 8 nm produced by a water-in-oil microemulsion method. The in vitro releasing profile of bFGF from MSNs in phosphate buffer saline has been monitored for 20 days through a colorimetric enzyme linked immunosorbent assay. The loading efficiency of bFGF in MSNs is esti- mated at 72.5 ± 3%. In addition, the cytotoxicity test indicates that the MSNs are not toxic, even at a concen- tration of 50 lg/mL. It is expected that the in situ loading method makes the MSNs a new delivery system to deliver protein drugs, e.g. growth factors, to help blood vessel regeneration and potentiate greater angiogenesis.
In multivariate regression analysis the relationship between LVMI and cFGF23 remained significant after stepwise adjustment for haemoglobin and GFR (Table 2). After adjustment for GFR, LVEDD and LVESD no longer correlated with FGF23.
To determine if cFGF23 could induce cardiac hypertrophy, we expressed the cDNA sequence encoding amino acids 178 to 251 of FGF23 in Chinese hamster ovary cells and produced a purified protein using the Myc-tag of the protein. We cultivated adult rat ventricular myocytes (ARVMs) for 24hrs in the presence of the purified cFGF23. As shown in figure 1B cFGF23 significantly increased in a concentration dependent manner the cell surface area of ARVMs. This effect was similar to that observed with FGF2, a fibroblastgrowthfactor known to induce cardiac hypertrophy. ARVMs, did not express αKlotho mRNA but expressed various types of FGFR in western blot experiments (data not shown). To determine if FGFR could mediate cFGF23-induced cardiomyocyte hypertrophy, ARVMs were incubated with cFGF23 or FGF2 alone or in the presence of a FGFR inhibitor (PD166866 from Sigma-Aldrich). The FGFR inhibitor fully prevented both cFGF23 and FGF2 induced hypertrophy (Figure 1B). Treatment of ARVMs with cFGF23 also resulted in a significant increase in the beta myosin heavy chain mRNA expression, a marker of cardiac hypertrophy (online supplementary figure S2).
Edited by Michael H. Wigler, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, and approved March 10, 1998 (received for review November 18, 1997)
ABSTRACT Chromosome 8p11–12 is the site of a recurrent breakpoint in a myeloproliferative disorder that involves lym- phoid (T- or B-cell), myeloid hyperplasia and eosinophilia, and evolves toward acute leukemia. This multilineage involvement suggests the malignant transformation of a primitive hemato- poietic stem cell. In this disorder, the 8p11–12 region is associ- ated with three different partners 6q27, 9q33, and 13q12. We describe here the molecular characterization of the t(8;13) trans- location that involves the FGFR1 gene from 8p12, encoding a tyrosine kinase receptor for members of the fibroblastgrowthfactor family, and a gene from 13q12, tentatively named FIM (Fused In Myeloproliferative disorders). FIM is related to
Germany, 6 International Collaborative Center on GrowthFactor Research, Life Science Institute, Wenzhou
University-Wenzhou Medical University, Wenzhou, China
Members of the PEA3 transcription factors are emerging as bone fide targets for fibroblastgrowthfactor (FGF) signaling. Among them, ETV4 and ETV5 appear to mediate FGF10 signaling during early embryonic lung development. In this paper, recently obtained Tg(Etv4-GFP) and Etv5 CreERT 2−RFP fluorescent reporter lines were generally characterized during early embryonic development and in the context of FGF10 signaling, in particular. We found that both Tg(Etv4-GFP) and Etv5 CreERT 2−RFP were primarily expressed in the epithelium of the lung during embryonic development. However, the expression of Etv5 CreERT 2−RFP was much higher than that of Tg(Etv4-GFP), and continued to increase during development, whereas Tg(Etv4-GFP) decreased. The expression patterns of the surrogate fluorescent protein GFP and RFP for ETV4 and ETV5, respectively, agreed with known regions of FGF10 signaling in various developing organs, including the lung, where ETV4-GFP was seen primarily in the distal epithelium and to a lesser extent in the surrounding mesenchyme. As expected, ETV5-RFP was restricted to the lung epithelium, showing a decreasing expression pattern from distal buds to proximal conducting airways. FGF10 inhibition experiments confirmed that both
The 484 nucleotides (nts)-long alternatively translated region (ATR) of the human fibroblastgrowthfactor 2 (FGF-2) mRNA contains four CUG and one AUG translation initiation codons. While the 5 end proximal CUG codon is initiated by a cap-dependent translation process, the other four initiation codons are initiated by a mechanism of internal entry of ribosomes. We undertook here a detailed analysis of the cis -acting elements defining the FGF-2 internal ribosome entry site (IRES). A thorough deletion analysis study within the 5 ATR led us to define a 176 nts-long region as being necessary and sufficient for IRES function at four codons present in a downstream 308 nts-long RNA segment. Unexpectedly, a single IRES module is therefore responsible for translation initiation at four distantly localized codons. The determination of the FGF-2 5 ATR RNA secondary structure by enzymatic and chemical probing experiments showed that the FGF-2 IRES contained two stem-loop regions and a G-quartet motif that constitute novel structural determinants of IRES function. MESH Keywords Alternative Splicing ; Base Sequence ; Cell Line ; Codon, Initiator ; genetics ; DNA, Complementary ; genetics ; FibroblastGrowthFactor 2 ; genetics ; Gene Expression ; Humans ; Molecular Sequence Data ; Nucleic Acid Conformation ; Peptide Chain Initiation, Translational ; RNA, Messenger ; chemistry ; genetics ; Ribosomes ; metabolism ; Sequence Deletion ; Transfection
Keywords: FGFR3; Periosteal derived cells; Osteogenic differentia- tion; Bone formation
The importance of the FibroblastGrowthFactor (FGF) receptor 3 (FGFR3) signalling pathway in endochondral bone development, and more specifically in the regulation of chondrocyte proliferation and differentiation in the epiphyseal growth plate, was discovered when an activating mutation in FGFR3 was found to cause achondroplasia, the common form of human dwarfism . Even though the mechanism of action of these mutations is not fully understood, pathways involved in FGFR3 signalling such as the STAT and Mitogen Activating Protein Kinase (MAPK) pathways have been suggested to play a role [2-4]. Inhibition of chondrocyte proliferation is caused by induced phosphorylation of STAT-1, which after translocation into the nucleus induces transcription of cell cycle inhibitors such as p21 . In addition, the MAPK-pathway has been suggested to play a role due to phosphorylation of Erk1/2 and p38 [6-8]. The latter may cause premature and enhanced osteoblast differentiation of cells carrying activating mutations in FGFR3. This was confirmed in a study by Su et al., where enhanced phosphorylation of Erk-1/2 was suggested to be responsible for elevated cell proliferation together with impaired bone matrix mineralisation . Phosphorylation of p38 in the same study was correlated to promotion of osteogenic differentiation but reduction in proliferation and cell induced mineralisation.
plates and incubated at 37 °C in 95% humidity for 3 h. These micromasses were allowed to deposit ECM and to develop cell-to-cell, cell-to-matrix, and cell-to-plastic interactions in a three-dimensional environment for the course of 28 days. To induce chondrocyte differenti- ation, cells were cultured with 500 μl of chemically defined chondrogenic medium [ 26 ] consisting of LG- DMEM (Gibco, UK) supplemented with 100 μM ascorbate-2-phosphate, 100 nM dexamethasone, 40 μg/ ml proline, and insulin-transferrin-selenium (ITS) + premix universal culture supplement (BD Biosciences, Bedford, MA), including 6.25 mg/ml insulin, 6.25 mg/ ml transferrin, 6.25 mg/ml selenious acid, 1.25 mg/ml bovine serum albumin (BSA), and 5.35 mg/ml linoleic acid. This basal chondrogenic medium (BCM) was supple- mented with 10 ng/ml transforming growthfactor (TGF)- β1, 100 ng/ml bone morphogenetic protein (BMP)2, 100 ng/ml growth differentiation factor (GDF)5, 0.2 ng/ml fibroblastgrowthfactor (FGF)2, and 1 ng/ml BMP6, as described elsewhere [ 23 ]. Rho kinase inhibitor Y27632 [ 27 ] (20 μM) was used to minimize cytoskeletal tension and prevent cell detachment and the formation of a nec- rotic center. This medium is referred as “GF cocktail”. Culture medium was refreshed every 2 days. Negative control aggregates (CTRL) were cultured in BCM with no supplementation of GFs. For osteochondral defect im- plantation, positive control aggregates of hPDCs were cul- tured in BCM supplemented with 20 μM Rho kinase inhibitor Y27632 and 10 ng/ml of TGF-β1 for 28 days (TGF-β1 aggregates).
Five fibroblastgrowthfactor 2 (FGF-2) isoforms are synthesized from human FGF-2 mRNA by a process of alternative initiation of translation. The regulation of FGF-2 isoform expression by the mRNA 5823-nucleotide- long 3 ⴕ-untranslated region containing eight alternative polyadenylation sites was examined. Because previous studies had shown that FGF-2 expression was regulated in primary cells but not in transformed cells, primary human skin fibroblasts were used in this study. Using an approach of cell transfection with synthetic reporter mRNAs, a novel translational enhancer (3 ⴕ-TE) was iden- tified in the 1370-nucleotide mRNA segment located up- stream from the eighth poly(A) site. Deletion mutagen- esis showed that the 3 ⴕ-TE was composed of two domains with additive effects. The 3 ⴕ-TE exhibited the unique feature of modulating the use of FGF-2 alternative ini- tiation codons, which favored the relative expression of CUG-initiated isoforms. Interestingly, the use of an al- ternative polydenylation site removing the 3 ⴕ-TE was detected in skin fibroblasts in response to heat shock and cell density variations. At high cell densities, 3 ⴕ-TE removal was correlated with a loss of CUG-initiated FGF-2 expression. These data show that the FGF-2 mRNA 3 ⴕ-untranslated region is able to modulate FGF-2 isoform expression by the coupled processes of transla- tion activation and alternative polyadenylation.
Received January 23, 2009; Revised June 9, 2009; Accepted June 11, 2009
Fibroblastgrowthfactor 1 (FGF1) is involved in muscle development and regeneration. The FGF1 gene contains four tissue-specific promoters allow- ing synthesis of four transcripts with distinct leader regions. Two of these transcripts contain internal ribosome entry sites (IRESs), which are RNA ele- ments allowing mRNA translation to occur in condi- tions of blockade of the classical cap-dependent mechanism. Here, we investigated the function and the regulation of FGF1 during muscle differentiation and regeneration. Our data show that FGF1 protein expression is induced in differentiating myoblasts and regenerating mouse muscle, whereas siRNA knock-down demonstrated FGF1 requirement for myoblast differentiation. FGF1 induction occurred at both transcriptional and translational levels, involving specific activation of both promoter A and IRES A, whereas global cap-dependent transla- tion was inhibited. Furthermore, we identified, in the FGF1 promoter A distal region, a cis-acting ele- ment able to activate the IRES A-driven translation. These data revealed a mechanism of molecular coupling of mRNA transcription and translation, involving a unique process of IRES activation by a promoter element. The crucial role of FGF1 in
Key Words. Bone engineering • Mesenchymal stem cells • Hypoxia • Intramembranous ossi ﬁcation • Calvaria
The craniofacial area is prone to trauma or pathologies often resulting in large bone damages. One potential treatment option is the grafting of a tissue-engineered construct seeded with adult mesenchymal stem cells (MSCs). The dental pulp appears as a relevant source of MSCs, as dental pulp stem cells display strong osteogenic properties and are ef ﬁcient at bone formation and repair. Fibroblastgrowthfactor-2 (FGF-2) and/or hypoxia primings were shown to boost the angiogenesis potential of dental pulp stem cells from human exfoliated deciduous teeth (SHED). Based on these ﬁndings, we hypothesized here that these primings would also improve bone for- mation in the context of craniofacial bone repair. We found that both hypoxic and FGF-2 prim- ings enhanced SHED proliferation and osteogenic differentiation into plastically compressed collagen hydrogels, with a much stronger effect observed with the FGF-2 priming. After implan- tation in immunode ﬁcient mice, the tissue-engineered constructs seeded with FGF-2 primed SHED mediated faster intramembranous bone formation into critical size calvarial defects than the other groups (no priming and hypoxia priming). The results of this study highlight the interest of FGF-2 priming in tissue engineering for craniofacial bone repair. STEM CELLS TRANSLATIONAL MEDICINE 2019;8:844–857
Basic fibroblastgrowthfactor (bFGF) exerts a mitogenic effect on cortical neuroblasts, whereas neurotrophin 3 (NT3) promotes differentiation in these cells. Here we provide evidence that both the mitogenic effect of bFGF and the differentiation-promoting effect of NT3 are linked with modifications of cell cycle kinetics in mouse cortical precursor cells. We adapted an in vitro assay, which makes it possible to evaluate (1) the speed of progression of the cortical precursors through the cell cycle, (2) the duration of individual phases of the cell cycle, (3) the proportion of proliferative versus differentiative divisions, and (4) the influence on neuroglial differentiation. Contrary to what has been claimed previously, bFGF promotes proliferation via a change in cell cycle kinetics by simultaneously decreasing G1 duration and increasing the proportion of proliferative divisions. In contrast, NT3 lengthens G1 and promotes differentiative divisions. We investigated the molecular foundations of these effects and show that bFGF downregulates p27kip1 and upregulates cyclin D2 expression. This contrasts with NT3, which upregulates p27kip1 and downregulates cyclin D2 expression. Neither bFGF nor NT3 influences the proportion of glia or neurons in short to medium term cultures. The data point to links between the length of the G1 phase and the type of division of cortical precursors: differentiative divisions are correlated with long G1 durations, whereas proliferative divisions correlate with short G1 durations. The present results suggest that concerted mechanisms control the progressive increase in the cell cycle duration and proportion of differentiative divisions that is observed as corticogenesis proceeds.
MASON, R.J. (1985). Stimulation of DNA synthesis in cultured rat alveolar type II cells. Exp. Lung Res. 8, 53–66.
LESLIE, C.C., MCCORMICK-SHANNON, K., and MASON, R.J. (1990). Heparin-binding growth factors stimulate DNA synthesis in rat alveolar type II cells. Am. J. Respir. Cell Mol. Biol. 2, 99–106. LESLIE, C.C., MCCORMICK-SHANNON, K., SHANNON, J.M., GARRICK, B., DAMM, D., ABRAHAM, J.A., and MASON, R.J. (1997). Heparin-binding EGF-like growthfactor is a mitogen for rat alveolar type II cells. Am. J. Respir. Cell Mol. Biol. 16, 379–387. PANOS, R.J., RUBIN, J.S., CSAKY, K.G., AARONSON, S.A., and MA- SON, R.J. (1993). Keratinocyte growthfactor and hepatocyte growthfactor/scatter factor are heparin-binding growth factors for alveolar type II cells in fibroblast-conditioned medium. J. Clin. Invest. 92, 969–977. PANOS, R.J., BAK, P.M., SIMONET, W.S., RUBIN, J.S., and SMITH, L.J. (1995). Intratracheal instillation of keratinocyte growthfactor decreases hyperoxia-induced mortality in rats. J. Clin. Invest. 96, 2026–2033.
but not hnRNP C1/C2, mRNAs are loaded on polysomes in a growth-dependent manner (40). As a consequence, the intracellular levels of hnRNP A1 would increase very rapidly after a proliferation stimulus. Indeed, the level of hnRNP A1 increases after transformation of rat embryo fibroblasts (41), consistent with previous reports of increased hnRNP A1 expression in transformed and rapidly proliferating cells (42,43). In addition to the regulation of hnRNP A1 levels, its activity is regulated by its subcellular localization. The expression of a permanently active mutant of zeta-PKC promotes the cytoplasmic accumulation of hnRNP A1 in proliferating cells (44). Furthermore, the subcellular localization of hnRNP A1 can be modulated by the MKK3/6-p38 pathway in response to stress such as osmotic shock and irradiation with UV-C light (45). Since our study highlights a novel function of hnRNP A1 in translation, the deregulation of hnRNP A1 level or activity may have important consequences for the translational regulation of several genes.
FibroblastGrowth factors (FGFs) are growth factors which have diverse biological activities including broad mitogenic and cell survival activities. FGFs constitute a large family of 22 distinct polypeptide growth factors varying in size from 17 to 34 kDa and have between 13 to 71% sequence homology. More specifically, FGF8 and FGF18 are both important modulator of granulosa cell functions. FGF8 and FGF18 are homologous factors which possess similar sequence homology, but we hypothesized they may interact to FGFRs differently leading to distinct effects, particularly on granulosa cell growth and induce proliferation following a short period of exposition. This study was performed to investigate the effects of FGF8 and FGF18 on ovine granulosa cells proteome. Ovine ovaries were obtained from adult sheep’s irrespective of stage of estrous cycle and were cultured using a standard protocol. Granulosa cells were harvested from follicles then, seeded and cultivated. After, they were exposed to 10 ng/mL of FGF8 or FGF18. Cell proteins were extracted, cysteine bonds were reduced and acetylated and proteins were digested with trypsin. Tryptic peptides were analyzed using a bottom-up proteomic approach, mass spectrometry and a label-free quantitation method. The results obtained revealed following treatment with FGF8 or FGF18 for 30 minutes, an important shift toward upregulation for the entire granulosa cell proteome was measured. Additionally, several proteins, including ATF1, STAT3, MAPK1, MAPK3, MAPK14, PLCG1, PLCG2, PKCA, PIK3CA, RAF1, GAB1 and BAG2 were significantly upregulated (> 1.5-fold; p < 0.01). Results are suggesting the activation of the MAPK/ERK pathway as expected. However, it is important to note that ATF1 and STAT3 are important transcription factors involved in cell growth, proliferation and survival and consequently can hamper or rescue the normal ovine reproductive system function. Both are strongly interacting with the MAPK/ERK pathway. They are directly involved in the growth, proliferation and mechanisms of cell survival. This very significant increase of STAT3 and ATF1 could have important consequences on the viability of the oocyte.
Figure 8. Schematic representation of the HSPG in the pericellular matrix. In the crowded pericellular matrix (macromolecular concentration of ,400 mg/mL), proteins and proteoglycans interact with each other. Only HSPG proteoglycans (in black and grey), the FGF2 (in green) and few other HS partners (in blue), are represented here for clarity. The HS-binding partners, which may be membrane-associated or ‘‘soluble’’ in the matrix, include growth factors, cytokines, chemokines, enzymes, matrix proteins, and numerous cell-surface receptors. Proteoglycan core proteins (black, inserted in the membrane) are shown with their HS chains (dark grey lines), which are between 40 to 160 nm long. Along these chains, dark grey rectangles represent strong binding sites for FGF2, and light grey rectangles represent weaker binding sites for FGF2. These binding sites form non-random networks of heterogeneously distributed binding sites within which the FGF2 moves by translocating from one site to another (a). A path of the FGF2 is shown by green arrows and some of its successive positions marked as a green circle. This motion of the FGF2 is independent of the motion of the protein core of the HSPG itself (b). Movement of the HS chains (c) to which the FGF2 is attached may also contribute to the motion of the FGF2 within the pericellular matrix. Note that the many endogenous binding partners of HS chains and HSPG core proteins may, in some conditions, severely restrict the diffusion of the protein core and of the HS chains (asterisks).
2.1.2 Granulosa cells
GC are important for oocyte maturation as they provide nutrients that support further development. As follicles grow and the antrum cavity is formed, the GC separates into two anatomically and phenotypically different subtypes: the cumulus granulosa cells (CGC), which are in direct contact with the oocyte, have a high rate of proliferation, low steroidogenic capacity, low LH receptor (LHCGR) expression and high levels of insulin growthfactor I (IGF-1); and the mural granulosa cells (MGC) which have a primarily endocrine function and support follicle growth, and which undergo terminal differentiation to luteal cells after ovulation. The interaction between oocytes and CGC is complex; CGC express characteristics distinct from the MGC that are acquired under the influence of the oocyte and which promote cell differentiation and development of the GC (Albertini et al., 2001). The oocyte achieves this by secreting labile paracrine signaling factors, and perturbation of this signaling results in the production of an oocyte unable to undergo normal maturation (Yeo et al., 2009). It is possible that MGC are antagonist or insufficient for supporting the last stages of oocyte maturation (Eppig et al., 1997).
The transient nuclear accumulation of FGF-2 䡠RSK2 com-
plexes associated with the maintenance of a high level of RSK2 activity toward the Ser-10 phosphorylation of histone H3 could represent a mechanism of direct signaling to chromatin through histone modification, which may cause a sustained chromatin remodeling (34) and/or facilitate transcription of genes related to cellular response induced by FGF-2 (35–37). Taken together, all of these findings strengthen the concept that a growing number of growth factors elicit their mitogenic response in a bifunctional manner by activating cell surface receptors and by directly associating with nuclear targets.