Science.gov

Sample records for acidic fibroblast growth

  1. Structure of rat acidic fibroblast growth factor at 1.4 Å resolution

    SciTech Connect

    Kulahin, Nikolaj; Kochoyan, Arthur; Berezin, Vladimir; Bock, Elisabeth; Gajhede, Michael

    2007-02-01

    The structure of rat acidic fibroblast growth factor was determined and compared with those of human, bovine and newt origin. The rat and human structures were found to be very similar. Fibroblast growth factors (FGFs) constitute a family of 22 structurally related heparin-binding polypeptides that are involved in the regulation of cell growth, survival, differentiation and migration. Here, a 1.4 Å resolution X-ray structure of rat FGF1 is presented. Two molecules are present in the asymmetric unit of the crystal and they coordinate a total of five sulfate ions. The structures of human, bovine and newt FGF1 have been published previously. Human and rat FGF1 are found to have very similar structures.

  2. The effect of pantothenic acid deficiency on keratinocyte proliferation and the synthesis of keratinocyte growth factor and collagen in fibroblasts.

    PubMed

    Kobayashi, Daisaku; Kusama, Miho; Onda, Masaaki; Nakahata, Norimichi

    2011-01-01

    It has been reported that pantothenic acid (vitamin B5) and panthenol, an alcohol derivative of pantothenic acid, have beneficial moisturizing effects on the skin. However, few studies have investigated the mechanism of action of pantothenic acid on skin tissues. We tried to clarify the role of pantothenic acid on skin function by using keratinocytes and fibroblasts. The depletion of pantothenic acid from the culture medium suppressed keratinocyte proliferation and promoted differentiation. Moreover, pantothenic acid depletion decreased the synthesis of keratinocyte growth factor and procollagen 4a2 in fibroblasts. These results suggest that pantothenic acid is essential for maintaining keratinocyte proliferation and differentiation. PMID:21258175

  3. Uptake and Intracellular Transport of Acidic Fibroblast Growth Factor: Evidence for Free and Cytoskeleton-anchored Fibroblast Growth Factor Receptors

    PubMed Central

    Citores, Lucía; Wesche, Jørgen; Kolpakova, Elona; Olsnes, Sjur

    1999-01-01

    Endocytic uptake and intracellular transport of acidic FGF was studied in cells transfected with FGF receptor 4 (FGFR4). Acidification of the cytosol to block endocytic uptake from coated pits did not inhibit endocytosis of the growth factor in COS cells transfected with FGFR4, indicating that it is to a large extent taken up by an alternative endocytic pathway. Fractionation of the cells demonstrated that part of the growth factor receptor was present in a low-density, caveolin-containing fraction, but we were unable to demonstrate binding to caveolin in immunoprecipitation studies. Upon treatment of the cells with acidic FGF, the activated receptor, together with the growth factor, moved to a juxtanuclear compartment, which was identified as the recycling endosome compartment. When the cells were lysed with Triton X-100, 3-([3-chloramidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate, or 2-octyl glucoside, almost all surface-exposed and endocytosed FGFR4 was solubilized, but only a minor fraction of the total FGFR4 in the cells was found in the soluble fraction. The data indicate that the major part of FGFR4 is anchored to detergent-insoluble structures, presumably cytoskeletal elements associated with the recycling endosome compartment. PMID:10564275

  4. Large-scale production of bioactive recombinant human acidic fibroblast growth factor in transgenic silkworm cocoons.

    PubMed

    Wang, Feng; Wang, Riyuan; Wang, Yuancheng; Zhao, Ping; Xia, Qingyou

    2015-01-01

    With an increasing clinical demand for functional therapeutic proteins every year, there is an increasing requirement for the massive production of bioactive recombinant human acidic fibroblast growth factor (r-haFGF). In this present study, we delicately explore a strategy for the mass production of r-haFGF protein with biological activity in the transgenic silkworm cocoons. The sequence-optimized haFGF was inserted into an enhanced sericin-1 expression system to generate the original transgenic silkworm strain, which was then further crossed with a PIG jumpstarter strain to achieve the remobilization of the expression cassette to a "safe harbor" locus in the genome for the efficient expression of r-haFGF. In consequence, the expression of r-haFGF protein in the mutant line achieved a 5.6-fold increase compared to the original strain. The high content of r-haFGF facilitated its purification and large-scald yields. Furthermore, the r-haFGF protein bioactively promoted the growth, proliferation and migration of NIH/3T3 cells, suggesting the r-haFGF protein possessed native mitogenic activity and the potential for wound healing. These results show that the silk gland of silkworm could be an efficient bioreactor strategy for recombinant production of bioactive haFGF in silkworm cocoons. PMID:26567460

  5. Large-scale production of bioactive recombinant human acidic fibroblast growth factor in transgenic silkworm cocoons

    NASA Astrophysics Data System (ADS)

    Wang, Feng; Wang, Riyuan; Wang, Yuancheng; Zhao, Ping; Xia, Qingyou

    2015-11-01

    With an increasing clinical demand for functional therapeutic proteins every year, there is an increasing requirement for the massive production of bioactive recombinant human acidic fibroblast growth factor (r-haFGF). In this present study, we delicately explore a strategy for the mass production of r-haFGF protein with biological activity in the transgenic silkworm cocoons. The sequence-optimized haFGF was inserted into an enhanced sericin-1 expression system to generate the original transgenic silkworm strain, which was then further crossed with a PIG jumpstarter strain to achieve the remobilization of the expression cassette to a “safe harbor” locus in the genome for the efficient expression of r-haFGF. In consequence, the expression of r-haFGF protein in the mutant line achieved a 5.6-fold increase compared to the original strain. The high content of r-haFGF facilitated its purification and large-scald yields. Furthermore, the r-haFGF protein bioactively promoted the growth, proliferation and migration of NIH/3T3 cells, suggesting the r-haFGF protein possessed native mitogenic activity and the potential for wound healing. These results show that the silk gland of silkworm could be an efficient bioreactor strategy for recombinant production of bioactive haFGF in silkworm cocoons.

  6. Large-scale production of bioactive recombinant human acidic fibroblast growth factor in transgenic silkworm cocoons

    PubMed Central

    Wang, Feng; Wang, Riyuan; Wang, Yuancheng; Zhao, Ping; Xia, Qingyou

    2015-01-01

    With an increasing clinical demand for functional therapeutic proteins every year, there is an increasing requirement for the massive production of bioactive recombinant human acidic fibroblast growth factor (r-haFGF). In this present study, we delicately explore a strategy for the mass production of r-haFGF protein with biological activity in the transgenic silkworm cocoons. The sequence-optimized haFGF was inserted into an enhanced sericin-1 expression system to generate the original transgenic silkworm strain, which was then further crossed with a PIG jumpstarter strain to achieve the remobilization of the expression cassette to a “safe harbor” locus in the genome for the efficient expression of r-haFGF. In consequence, the expression of r-haFGF protein in the mutant line achieved a 5.6-fold increase compared to the original strain. The high content of r-haFGF facilitated its purification and large-scald yields. Furthermore, the r-haFGF protein bioactively promoted the growth, proliferation and migration of NIH/3T3 cells, suggesting the r-haFGF protein possessed native mitogenic activity and the potential for wound healing. These results show that the silk gland of silkworm could be an efficient bioreactor strategy for recombinant production of bioactive haFGF in silkworm cocoons. PMID:26567460

  7. Ferulic Acid Exerts Anti-Angiogenic and Anti-Tumor Activity by Targeting Fibroblast Growth Factor Receptor 1-Mediated Angiogenesis

    PubMed Central

    Yang, Guang-Wei; Jiang, Jin-Song; Lu, Wei-Qin

    2015-01-01

    Most anti-angiogenic therapies currently being evaluated target the vascular endothelial growth factor (VEGF) pathway; however, the tumor vasculature can acquire resistance to VEGF-targeted therapy by shifting to other angiogenesis mechanisms. Therefore, other therapeutic agents that block non-VEGF angiogenic pathways need to be evaluated. Here, we identified ferulic acid as a novel fibroblast growth factor receptor 1 (FGFR1) inhibitor and a novel agent with potential anti-angiogenic and anti-cancer activities. Ferulic acid demonstrated inhibition of endothelial cell proliferation, migration and tube formation in response to basic fibroblast growth factor 1 (FGF1). In ex vivo and in vivo angiogenesis assays, ferulic acid suppressed FGF1-induced microvessel sprouting of rat aortic rings and angiogenesis. To understand the underlying molecular basis, we examined the effects of ferulic acid on different molecular components and found that ferulic acid suppressed FGF1-triggered activation of FGFR1 and phosphatidyl inositol 3-kinase (PI3K)-protein kinase B (Akt) signaling. Moreover, ferulic acid directly inhibited proliferation and blocked the PI3K-Akt pathway in melanoma cell. In vivo, using a melanoma xenograft model, ferulic acid showed growth-inhibitory activity associated with inhibition of angiogenesis. Taken together, our results indicate that ferulic acid targets the FGFR1-mediated PI3K-Akt signaling pathway, leading to the suppression of melanoma growth and angiogenesis. PMID:26473837

  8. Stimulation of proliferation of a human osteosarcoma cell line by exogenous acidic fibroblast growth factor requires both activation of receptor tyrosine kinase and growth factor internalization.

    PubMed Central

    Wiedłocha, A; Falnes, P O; Rapak, A; Muñoz, R; Klingenberg, O; Olsnes, S

    1996-01-01

    U2OS Dr1 cells, originating from a human osteosarcoma, are resistant to the intracellular action of diphtheria toxin but contain toxin receptors on their surfaces. These cells do not have detectable amounts of fibroblast growth factor receptors. When these cells were transfected with fibroblast growth factor receptor 4, the addition of acidic fibroblast growth factor to the medium induced tyrosine phosphorylation, DNA synthesis, and cell proliferation. A considerable fraction of the cell-associated growth factor was found in the nuclear fraction. When the growth factor was fused to the diphtheria toxin A fragment, it was still bound to the growth factor receptor and induced tyrosine phosphorylation but did not induce DNA synthesis or cell proliferation, nor was any fusion protein recovered in the nuclear fraction. On the other hand, when the fusion protein was associated with the diphtheria toxin B fragment to allow translocation to the cytosol by the toxin pathway, the fusion protein was targeted to the nucleus and stimulated both DNA synthesis and cell proliferation. In untransfected cells containing toxin receptors but not fibroblast growth factor receptors, the fusion protein was translocated to the cytosol and targeted to the nucleus, but in this case, it stimulated only DNA synthesis. These data indicate that the following two signals are required to stimulate cell proliferation in transfected U2OS Dr1 cells: the tyrosine kinase signal from the activated fibroblast growth factor receptor and translocation of the growth factor into the cell. PMID:8524304

  9. Acidic Fibroblast Growth Factor Promotes Endothelial Progenitor Cells Function via Akt/FOXO3a Pathway

    PubMed Central

    Wang, Yuqiang; Cao, Qing; Sang, Tiantian; Liu, Fang; Chen, Shuyan

    2015-01-01

    Acidic fibroblast growth factor (FGF1) has been suggested to enhance the functional activities of endothelial progenitor cells (EPCs). The Forkhead homeobox type O transcription factors (FOXOs), a key substrate of the survival kinase Akt, play important roles in regulation of various cellular processes. We previously have shown that FOXO3a is the main subtype of FOXOs expressed in EPCs. Here, we aim to determine whether FGF1 promotes EPC function through Akt/FOXO3a pathway. Human peripheral blood derived EPCs were transduced with adenoviral vectors either expressing a non-phosphorylable, constitutively active triple mutant of FOXO3a (Ad-TM-FOXO3a) or a GFP control (Ad-GFP). FGF1 treatment improved functional activities of Ad-GFP transduced EPCs, including cell viability, proliferation, antiapoptosis, migration and tube formation, whereas these beneficial effects disappeared by Akt inhibitor pretreatment. Moreover, EPC function was declined by Ad-TM-FOXO3a transduction and failed to be attenuated even with FGF1 treatment. FGF1 upregulated phosphorylation levels of Akt and FOXO3a in Ad-GFP transduced EPCs, which were repressed by Akt inhibitor pretreatment. However, FGF1 failed to recover Ad-TM-FOXO3a transduced EPCs from dysfunction. These data indicate that FGF1 promoting EPC function is at least in part mediated through Akt/FOXO3a pathway. Our study may provide novel ideas for enhancing EPC angiogenic ability and optimizing EPC transplantation therapy in the future. PMID:26061278

  10. Zoledronic acid suppresses transforming growth factor-β-induced fibrogenesis by human gingival fibroblasts

    PubMed Central

    KOMATSU, YUKO; IBI, MIHO; CHOSA, NAOYUKI; KYAKUMOTO, SEIKO; KAMO, MASAHARU; SHIBATA, TOSHIYUKI; SUGIYAMA, YOSHIKI; ISHISAKI, AKIRA

    2016-01-01

    Bisphosphonates (BPs) are analogues of pyro-phosphate that are known to prevent bone resorption by inhibiting osteoclast activity. Nitrogen-containing BPs, such as zoledronic acid (ZA), are widely used in the treatment of osteoporosis and bone metastasis. However, despite having benefits, ZA has been reported to induce BP-related osteonecrosis of the jaw (BRONJ) in cancer patients. The molecular pathological mechanisms responsible for the development of BRONJ, including necrotic bone exposure after tooth extraction, remain to be elucidated. In this study, we examined the effects of ZA on the transforming growth factor-β (TGF-β)-induced myofibroblast (MF) differentiation of human gingival fibroblasts (hGFs) and the migratory activity of hGFs, which are important for wound closure by fibrous tissue formation. The ZA maximum concentration in serum (Cmax) was found to be approximately 1.47 µM, which clinically, is found after the intravenous administration of 4 mg ZA, and ZA at this dose is considered appropriate for the treatment of cancer bone metastasis or bone diseases, such as Erdheim-Chester disease. At Cmax, ZA significantly suppressed i) the TGF-β-induced promotion of cell viability, ii) the TGF-β-induced expression of MF markers such as α-smooth muscle actin (α-SMA) and type I collagen, iii) the TGF-β-induced migratory activity of hGFs and iv) the expression level of TGF-β type I receptor on the surfaces of hGFs, as well as the TGF-β-induced phosphorylation of Smad2/3. Thus, ZA suppresses TGF-β-induced fibrous tissue formation by hGFs, possibly through the inhibition of Smad-dependent signal transduction. Our findings partly elucidate the molecular mechanisms underlying BRONJ and may prove to be beneficial to the identification of drug targets for the treatment of this symptom at the molecular level. PMID:27176567

  11. Optimization of acidic fibroblast growth factor (FGF-1) and its delivery through a modified degradable fibrin scaffold

    NASA Astrophysics Data System (ADS)

    Pandit, Abhay Smashikant

    The aim of this investigation was to develop a degradable fibrin wound dressing that can deliver an optimized dose of acidic fibroblast growth factor (FGF-1). This aim led to three distinct phases of study. In the first phase, a structurally modified fibrin degradable scaffold was developed and tested in a rabbit ear ulcer model. A significant increase in the angiogenic and fibroblastic response with a corresponding decrease in healing time was seen in the modified fibrin-treated ulcers as compared with untreated ulcers and ulcers treated with non-modified fibrin systems. In the second phase of the study, a biochemical factor, FGF-1, was added to this scaffold. An optimal dose of 8 mug of FGF-1 was determined to be required to initiate a desired wound-healing response in a rabbit ear ulcer model, based on an enhanced angiogenic and fibroblastic response and an increased epithelialization rate. The objective of the last phase was to investigate the efficacy of a modified scaffold as a vehicle for FGF-1. In vivo testing was conducted in a full-thickness defect model in a rabbit. Improvements were seen in the angiogenic and fibroblastic responses in the FGF-1/modified fibrin treatment group and, hence, FGF-1/modified fibrin was the preferred treatment. In conclusion, the modified fibrin/FGF-1 matrix served as a suitable vehicle for the growth factor, providing a desired healing response and a desirable release rate and, thus, was determined to be an effective scaffold.

  12. Zoledronic acid suppresses transforming growth factor-β-induced fibrogenesis by human gingival fibroblasts.

    PubMed

    Komatsu, Yuko; Ibi, Miho; Chosa, Naoyuki; Kyakumoto, Seiko; Kamo, Masaharu; Shibata, Toshiyuki; Sugiyama, Yoshiki; Ishisaki, Akira

    2016-07-01

    Bisphosphonates (BPs) are analogues of pyrophosphate that are known to prevent bone resorption by inhibiting osteoclast activity. Nitrogen-containing BPs, such as zoledronic acid (ZA), are widely used in the treatment of osteoporosis and bone metastasis. However, despite having benefits, ZA has been reported to induce BP-related osteonecrosis of the jaw (BRONJ) in cancer patients. The molecular pathological mechanisms responsible for the development of BRONJ, including necrotic bone exposure after tooth extraction, remain to be elucidated. In this study, we examined the effects of ZA on the transforming growth factor-β (TGF‑β)-induced myofibroblast (MF) differentiation of human gingival fibroblasts (hGFs) and the migratory activity of hGFs, which are important for wound closure by fibrous tissue formation. The ZA maximum concentration in serum (Cmax) was found to be approximately 1.47 µM, which clinically, is found after the intravenous administration of 4 mg ZA, and ZA at this dose is considered appropriate for the treatment of cancer bone metastasis or bone diseases, such as Erdheim-Chester disease. At Cmax, ZA significantly suppressed i) the TGF‑β-induced promotion of cell viability, ii) the TGF‑β-induced expression of MF markers such as α-smooth muscle actin (α-SMA) and type I collagen, iii) the TGF‑β-induced migratory activity of hGFs and iv) the expression level of TGF‑β type I receptor on the surfaces of hGFs, as well as the TGF‑β-induced phosphorylation of Smad2/3. Thus, ZA suppresses TGF‑β-induced fibrous tissue formation by hGFs, possibly through the inhibition of Smad‑dependent signal transduction. Our findings partly elucidate the molecular mechanisms underlying BRONJ and may prove to be beneficial to the identification of drug targets for the treatment of this symptom at the molecular level. PMID:27176567

  13. TAT-Mediated Acidic Fibroblast Growth Factor Delivery to the Dermis Improves Wound Healing of Deep Skin Tissue in Rat

    PubMed Central

    Tang, Lu; Zheng, Lulu; Jin, Zi; Yu, Bingjie; Wang, Zhitao; Lin, Peng; Yu, Weidan; Li, Haiyan; Li, Xiaokun; Wang, Xiaojie

    2015-01-01

    Background The definition of deep tissue injury was derived from multiple clinical cases as “A purple or maroon localized area of discolored intact skin or blood-filled blister due to damage of underlying soft tissue from pressure and/or shear”. Acidic fibroblast growth factor (aFGF) significantly improves wound healing under diabetic conditions. However, to date, the therapeutic application of aFGF has been limited, due to its low delivery efficiency and short half-life. Methodology/Principal Findings Using an animal model of magnet-induced pressure ulcers, transactivator of transcription protein (TAT)-aFGF was evaluated for transdermal delivery and wound healing. Immunohistochemistry and Western blotting were also performed to determine the expression of transforming growth factor (TGF)-β1, α-smooth muscle actin (α-SMA), CD68, proliferating cell nuclear antigen (PCNA) and TGF-β-receptor II (TGF- βRII) in cultured human dermal fibroblasts. We found that that mice treated with TAT-aFGF had higher accumulation of aFGF in both dermis and subcutaneous tissues compared with mice treated with aFGF alone. In the remodeling phase, TAT-aFGF treatment decreased the expression of α-SMA to normal levels, thereby facilitating normal wound healing processes and abrogating hypertrophic scarring. In human dermal fibroblasts, TAT-aFGF reversed the suppressive effect of TNF-α on α-SMA expression and restored TGF-βRII and TGF-β1 expression. Conclusions/Significance Our results demonstrate that TAT-aFGF has a favorable therapeutic effect on the healing of subcutaneous deep tissue injury. PMID:26271041

  14. Circulating intestinal fibroblast growth factor 19 has a pronounced diurnal variation and modulates hepatic bile acid synthesis in man.

    PubMed

    Lundåsen, T; Gälman, C; Angelin, B; Rudling, M

    2006-12-01

    Bile acids (BAs) traversing the enterohepatic circulation exert several important metabolic effects. Their hepatic synthesis, controlled by the enzyme cholesterol 7alpha-hydroxylase (CYP7A1), has a unique diurnal variation in man. Here we provide evidence that the transintestinal flux of BAs regulates serum levels of intestinal fibroblast growth factor 19 (FGF19) that in turn modulate BA production in human liver. Basal FGF19 levels varied by 10-fold in normal subjects, and were reduced following treatment with a BA-binding resin and increased upon feeding the BA chenodeoxycholic acid. Serum FGF19 levels exhibited a pronounced diurnal rhythm with peaks occurring 90-120 min after the postprandial rise in serum BAs. The FGF19 peaks in turn preceded the declining phase of BA synthesis. The diurnal rhythm of serum FGF19 was abolished upon fasting. We conclude that, in humans, circulating FGF19 has a diurnal rhythm controlled by the transintestinal BA flux, and that FGF19 modulates hepatic BA synthesis. Through its systemic effects, circulating FGF19 may also mediate other known BA-dependent effects on lipid and carbohydrate metabolism. PMID:17116003

  15. Investigation of the structural stability of the human acidic fibroblast growth factor by hydrogen-deuterium exchange.

    PubMed

    Chi, Ya-Hui; Kumar, Thallampuranam Krishnaswamy S; Kathir, Karuppanan Muthusamy; Lin, Dong-Hai; Zhu, Guang; Chiu, Ing-Ming; Yu, Chin

    2002-12-24

    The conformational stability of the human acidic fibroblast growth factor (hFGF-1) is investigated using amide proton exchange and temperature-dependent chemical shifts, monitored by two-dimensional NMR spectroscopy. The change in free energy of unfolding (DeltaG(u)) of hFGF-1 is estimated to be 5.00 +/- 0.09 kcal.mol(-)(1). Amide proton-exchange rates of 74 residues (in hFGF-1) have been unambiguously measured, and the exchange process occurs predominately according to the conditions of the EX2 limit. The exchange rates of the fast-exchanging amide protons exposed to the solvent have been measured using the clean SEA-HSQC technique. The amide proton protection factor and temperature coefficient estimates show reasonably good correlation. Residues in beta-strands II and VI appear to constitute the stability core of the protein. Among the 12 beta-strands constituting the beta-barrel architecture of hFGF-1, beta-strand XI, located in the heparin binding domain, exhibits the lowest average protection factor value. Amide protons involved in the putative folding nucleation site in hFGF-1, identified by quench-flow NMR studies, do not represent the slow-exchanging core. Residues in portions of hFGF-1 experiencing high conformational flexibility mostly correspond to those involved in receptor recognition and binding. PMID:12484774

  16. Increased acidic fibroblast growth factor concentrations in the serum and cerebrospinal fluid of patients with Alzheimer's disease.

    PubMed

    Mashayekhi, Farhad; Hadavi, Mahvash; Vaziri, Hamid Reza; Naji, Mohammad

    2010-03-01

    Acidic fibroblast growth factor (aFGF), also called FGF-1, which influences the proliferation and differentiation of various cell types in vitro, was originally isolated from neural tissue. It is released from the ependymal cells of the cerebral third ventricle into the cerebrospinal fluid (CSF). FGF-1 promotes the survival of neurons. Reactive astrocytes express FGF-1 in the brain of patients with Alzheimer's disease (AD). By comparing the CSF proteome of patients with AD and normal controls it might be possible to identify proteins that have a role in AD. Because CSF is in contact with the extracellular space of the brain, modifications in the brain biochemistry could be reflected in the CSF. The aim of this study was to determine concentrations of serum and CSF FGF-1 in patients with AD. This study consisted of 64 CSF samples, from patients with AD (n=32) and those without (normal controls) (n=32). The level of CSF and serum FGF-1 in patients with AD was higher than in patients without AD. We conclude that FGF-1 is a constant component of human serum and CSF and that FGF-1 may be involved in the pathophysiology of AD. PMID:20079650

  17. Heparin-binding growth factor type 1 (acidic fibroblast growth factor): a potential biphasic autocrine and paracrine regulator of hepatocyte regeneration.

    PubMed Central

    Kan, M; Huang, J S; Mansson, P E; Yasumitsu, H; Carr, B; McKeehan, W L

    1989-01-01

    Heparin-binding growth factor type 1 (HBGF-1; sometimes termed acidic fibroblast growth factor) is potentially an important factor in liver regeneration. HBGF-1 alone (half-maximal effect at 60 pM) stimulated hepatocyte DNA synthesis and bound to a high-affinity receptor (Kd = 62 pM; 5000 per cell). Epidermal growth factor (EGF) neutralized or masked the mitogenic effect of HBGF-1 concurrent with appearance of low-affinity HBGF-1 binding sites. HBGF-1 reduced the inhibitory effect of transforming growth factor type beta (TGF-beta) on the EGF stimulus. Nanomolar levels of HBGF-1 decreased the EGF stimulus. An increase in hepatic HBGF-1 gene expression after partial hepatectomy precedes increases in expression of the EGF homolog, TGF-alpha, and nonparenchymal-cell-derived TGF-beta in the regenerating liver. Expression of HBGF-1 mRNA occurs in both hepatocytes and nonparenchymal cells and persists for 7 days in liver tissue after partial hepatectomy. HBGF-1 acting through a high-affinity receptor is a candidate for the early autocrine stimulus that drives hepatocyte DNA synthesis prior to or concurrent with the EGF/TGF-alpha stimulus. It may allow hepatocyte proliferation to proceed in the presence of low levels of TGF-beta. An EGF/TGF-alpha-dependent change in HBGF-1 receptor phenotype and increasing levels of nonparenchymal-cell-derived HBGF-1 and TGF-beta may serve to limit hepatocyte proliferation. Images PMID:2477840

  18. The pro-fibrotic properties of transforming growth factor on human fibroblasts are counteracted by caffeic acid by inhibiting myofibroblast formation and collagen synthesis.

    PubMed

    Mia, Masum M; Bank, Ruud A

    2016-03-01

    Fibrosis is a chronic disorder affecting many organs. A universal process in fibrosis is the formation of myofibroblasts and the subsequent collagen deposition by these cells. Transforming growth factor beta1 (TGFβ1) plays a major role in the formation of myofibroblasts, e.g. by activating fibroblasts. Currently, no treatments are available to circumvent fibrosis. Caffeic acid phenethyl ester (CAPE) shows a broad spectrum of biological activities, including anti-fibrotic properties in vivo in mice and rats. However, little is known about the direct effects of CAPE on fibroblasts. We have tested whether CAPE is able to suppress myofibroblast formation and collagen formation of human dermal and lung fibroblasts exposed to TGFβ1, and found that this was indeed the case. In fact, the formation of myofibroblasts by TGFβ1 and subsequent collagen formation was completely abolished by CAPE. The same was observed for fibronectin and tenascin C. The lack of myofibroblast formation is likely due to the suppression of GLI1 and GLI2 expression by CAPE because of diminished nuclear SMAD2/3 levels. Post-treatment with CAPE after myofibroblast formation even resulted in a partial reversal of myofibroblasts into fibroblasts and/or reduction in collagen formation. Major discrepancies were seen between mRNA levels of collagen type I and cells stained positive for collagen, underlining the need for protein data in fibrosis studies to make reliable conclusions. PMID:26453399

  19. Heparin modulation of the neurotropic effects of acidic and basic fibroblast growth factors and nerve growth factor on PC12 cells

    SciTech Connect

    Neufeld, G.; Gospodarowicz, D.; Dodge, L.; Fujii, D.K.

    1987-04-01

    Nerve growth factor (NGF) and acidic or basic fibroblast growth factor (aFGF and bFGF, respectively) induce neurite outgrowth from the rat pheochromocytoma cell line, PC12. The neurites induced by these three factors are stable for up to a month in cell culture in the continued presence of any of the above growth factors. bFGF (ED50 = 30 pg/ml) is 800 fold more potent in stimulating neurite outgrowth than aFGF (ED50 = 25 ng/ml) and 260 fold more potent than NGF (ED50 = 8 ng/ml). While the neurotropic activities of aFGF and NGF are potentiated by heparin, that of bFGF is both partially inhibited or stimulated, depending upon the concentration of bFGF. Radioreceptor binding experiments show that aFGF and bFGF bind to a common binding site on the PC12 cell surface. Affinity labeling studies demonstrate a single receptor with an apparent molecular weight of 145,000 daltons, which corresponds to the high molecular weight receptor identified in BHK-21 cells. NGF does not appear to compete with aFGF or bFGF for binding to the receptor. Heparin blocked the binding of bFGF to the receptor but had only a small inhibitory effect on the binding of aFGF to the receptor. Thus, it appears that heparin inhibition of the neurotropic effects of bFGF occurs, at least in part, by impairing the interaction of bFGF with the receptor, while having little effect on that of aFGF. The stimulatory effects of heparin on the neurotropic activity of aFGF, bFGF, and NGF may occur through a site not associated with the respective cellular receptor for the growth factors.

  20. A natural kinase-deficient variant of fibroblast growth factor receptor 1.

    PubMed

    Wang, L Y; Edenson, S P; Yu, Y L; Senderowicz, L; Turck, C W

    1996-08-01

    A fibroblast growth factor receptor 1 variant missing 37 amino acids from the carboxy-terminal tyrosine kinase catalytic domain was discovered in human lung fibroblasts and several other human cell lines. The receptor variant binds specifically to acidic fibroblast growth factor but has no tyrosine kinase activity. It was found that cellular transfectants expressing the fibroblast growth factor receptor 1 variant are mitogenically inactive and ligand binding to the receptor causes neither receptor autophosphorylation nor phospholipase C-gamma transphosphorylation. The fibroblast growth factor receptor 1 variant therefore represents an inactive receptor for acidic fibroblast growth factor. Since both kinase and kinase-deficient receptor forms are expressed in cells, it is conceivable that the kinase-deficient receptor plays an important role in regulating cellular responses elicited by acidic fibroblast growth factor stimulation. PMID:8756477

  1. Secreted or nonsecreted forms of acidic fibroblast growth factor produced by transfected epithelial cells influence cell morphology, motility, and invasive potential.

    PubMed Central

    Jouanneau, J; Gavrilovic, J; Caruelle, D; Jaye, M; Moens, G; Caruelle, J P; Thiery, J P

    1991-01-01

    Addition of exogenous acidic fibroblast growth factor (aFGF) to NBT-II epithelial carcinoma cells results in fibroblastic transformation and cell motility. We have generated aFGF-producing NBT-II cells by transfection with recombinant expression vectors containing human aFGF cDNA, or the human aFGF cDNA coupled to a signal peptide (SP) sequence. The effects of the nonsecreted and the secreted 16-kDa growth factor on the morphology, motility, and cell invasive potential (gelatinase activity) were compared. aFGF coupled to a SP was actively secreted out of the producing cells. The secretion of aFGF was not necessary for induction of gelatinase activity, as this was observed in NBT-II cells producing aFGF with or without SP. Production of aFGF, whether secreted or not secreted, resulted in increased in vitro motility of most isolated clones; however, there was no correlation between aFGF level and motility rate. The data suggest that expression of aFGF in NBT-II cells induces metastatic potential through an autocrine or intracrine mechanism. Images PMID:1707175

  2. Hepatocyte FRS2α is essential for the endocrine fibroblast growth factor to limit the amplitude of bile acid production induced by prandial activity.

    PubMed

    Wang, C; Yang, C; Chang, J Y F; You, P; Li, Y; Jin, C; Luo, Y; Li, X; McKeehan, W L; Wang, F

    2014-01-01

    In addition to being positively regulated by prandial activity, bile acid production is also negatively controlled by the endocrine fibroblast growth factor 19 (FGF19) or the mouse ortholog FGF15 from the ileum that represses hepatic cholesterol 7 α-hydroxylase (Cyp7a1) expression through activating FGF receptor four (FGFR4). However, how these two regulatory mechanisms interplay to control bile acid homeostasis in the body and the downstream pathways by which FGFR4 regulates Cyp7a1 expression are not fully understood. Here we report that hepatocyte FGFR substrate 2α (FRS2α), a scaffold protein essential for canonical FGFRs to activate the ERK and AKT pathways, was required for the regulation of bile acid production by the FGF15/19-FGFR4 signaling axis. This occurred through limiting the extent of increases in Cyp7a1 expression induced by prandial activity. Excess FGFR4 kinase activity reduced the amplitude of the increase whereas a lack of FGFR4 augmented the increase of Cyp7a1 expression in the liver. Ablation of Frs2α alleles in hepatocytes abrogated the regulation of Cyp7a1 expression by FGFR4. Together, the results demonstrate that FRS2α-mediated pathways are essential for the FGF15/FGF19-FGFR4 signaling axis to control bile acid homeostasis. PMID:25056539

  3. Fibroblast Growth Factor 21 Limits Lipotoxicity by Promoting Hepatic Fatty Acid Activation in Mice on Methionine and Choline-deficient Diets

    PubMed Central

    Fisher, ffolliott M.; Chui, Patricia C.; Nasser, Imad A.; Popov, Yury; Cunniff, Jeremy C.; Lundasen, Thomas; Kharitonenkov, Alexei; Schuppan, Detlef; Flier, Jeffrey S.; Maratos-Flier, Eleftheria

    2014-01-01

    Background & Aims Nonalcoholic fatty liver disease (NAFLD) is a common consequence of human and rodent obesity. Disruptions in lipid metabolism lead to accumulation of triglycerides and fatty acids, which can promote inflammation and fibrosis and lead to nonalcoholic steatohepatitis (NASH). Circulating levels of fibroblast growth factor (FGF)21 increase in patients with NAFLD or NASH, so we assessed the role of FGF21 in the progression of murine fatty liver disease, independent of obesity, caused by methionine and choline deficiency. Methods C57BL/6 wild-type and FGF21-knockout (FGF21-KO) mice were placed on methionine- and choline-deficient (MCD), high-fat, or control diets for 8–16 weeks. Mice were weighed; serum and liver tissues were collected and analyzed for histology, levels of malondialdehyde and liver enzymes, gene expression, and lipid content. Results The MCD diet increased hepatic levels of FGF21 mRNA more than 50-fold and serum levels 16-fold, compared with the control diet. FGF21-KO mice had more severe steatosis, fibrosis, inflammation, and peroxidative damage than wild-type C57BL/6 mice. FGF21-KO mice had reduced hepatic fatty acid activation and β oxidation, resulting in increased levels of free fatty acid. FGF21-KO mice given continuous subcutaneous infusions of FGF21 for 4 weeks while on MCD diets had reduced steatosis and peroxidative damage, compared with mice not receiving FGF21. The expression of genes that regulate inflammation and fibrosis were reduced in FGF21-KO mice given FGF21, similar to those of wild-type mice. Conclusions FGF21 regulates fatty acid activation and oxidation in livers of mice. In the absence of FGF21, accumulation of inactivated fatty acids results in lipotoxic damage and increased steatosis. PMID:25083607

  4. Upregulation of hepatic bile acid synthesis via fibroblast growth factor 19 is defective in gallstone disease but functional in overweight individuals

    PubMed Central

    Harsch, Simone; Matysik, Silke; Lütjohann, Dieter; Schmitz, Gerd; Stange, Eduard F

    2014-01-01

    Background: Fibroblast growth factor 19 (FGF19) is an enteric hormone regulating bile acid de novo synthesis by sensing ileal bile acid flux. However, the role of FGF19 in cholelithiasis has not yet been elucidated and therefore is investigated in the present study. Methods: Total mRNA and protein were isolated from ileal biopsies and used for tissue expression analysis. FGF19, 7α-hydroxycholesterol (7α-OH-Chol), 27-hydroxycholesterol (27-OH-Chol), and different bile acids were determined in the blood samples. Results: FGF19 serum levels did not differ between gallstone carriers and controls but were significantly decreased in the overweight individuals (−32%, p = 0.0002), irrespective of gallstone status (normalweight to overweight controls −29%, p = 0.0017; normalweight to overweight gallstone carriers −44%, p = 0.0338), and correlated inversely with bodyweight (p < 0.0001, ρ = −0.3317). Compared to non-overweight controls, apical sodium-dependent bile acid transporter expression was significantly diminished in the non-overweight gallstone carriers (−42%, PmRNA = 0.0393; −52%, pprotein = 0.0169) as well as in the overweight controls (−24%, PmRNA = 0.0148; −43%, pprotein = 0.0017). FGF19 expression varied widely and was similar in all groups. A significant negative correlation was noted between 7α-OH-Chol, 27-OH-Chol, and FGF19 serum levels (p < 0.01; ρ7α-OH-Chol = −0.2155; ρ27-OH-Chol = −0.2144) in obesity. Conclusion: Upregulation of hepatic bile acid synthesis via FGF 19 is defective in gallstone disease but functional in overweight individuals. PMID:25360305

  5. Abrogation of fibroblast activation protein enzymatic activity attenuates tumor growth.

    PubMed

    Cheng, Jonathan D; Valianou, Matthildi; Canutescu, Adrian A; Jaffe, Eileen K; Lee, Hyung-Ok; Wang, Hao; Lai, Jack H; Bachovchin, William W; Weiner, Louis M

    2005-03-01

    Tumor-associated fibroblasts are functionally and phenotypically distinct from normal fibroblasts that are not in the tumor microenvironment. Fibroblast activation protein is a 95 kDa cell surface glycoprotein expressed by tumor stromal fibroblasts, and has been shown to have dipeptidyl peptidase and collagenase activity. Site-directed mutagenesis at the catalytic site of fibroblast activation protein, Ser624 --> Ala624, resulted in an approximately 100,000-fold loss of fibroblast activation protein dipeptidyl peptidase (DPP) activity. HEK293 cells transfected with wild-type fibroblast activation protein, enzymatic mutant (S624A) fibroblast activation protein, or vector alone, were inoculated subcutaneously into immunodeficient mouse to assess the contribution of fibroblast activation protein enzymatic activity to tumor growth. Overexpression of wild-type fibroblast activation protein showed growth potentiation and enhanced tumorigenicity compared with both fibroblast activation protein S624A and vector-transfected HEK293 xenografts. HEK293 cells transfected with fibroblast activation protein S624A showed tumor growth rates and tumorigenicity potential similar only to vector-transfected HEK293. In vivo assessment of fibroblast activation protein DPP activity of these tumors showed enhanced enzymatic activity of wild-type fibroblast activation protein, with only baseline levels of fibroblast activation protein DPP activity in either fibroblast activation protein S624A or vector-only xenografts. These results indicate that the enzymatic activity of fibroblast activation protein is necessary for fibroblast activation protein-driven tumor growth in the HEK293 xenograft model system. This establishes the proof-of-principle that the enzymatic activity of fibroblast activation protein plays an important role in the promotion of tumor growth, and provides an attractive target for therapeutics designed to alter fibroblast activation protein-induced tumor growth by targeting

  6. Transforming growth factor-beta 1 and fibroblast growth factors in rat growth plate.

    PubMed

    Jingushi, S; Scully, S P; Joyce, M E; Sugioka, Y; Bolander, M E

    1995-09-01

    Chondrocytes in the growth plate progress in an orderly fashion from resting through proliferating to hypertrophic cells. In the region of hypertrophic chondrocytes, the cartilage is invaded by capillary loops and endochondral ossification is initiated. It is currently believed that growth factors may regulate the proliferation and maturation of chondrocytes and the synthesis of extracellular matrix in the growth plate. The ordered sequence of proliferation and differentiation observed in the growth plate provides a unique opportunity to study the role of acidic fibroblast growth factor, basic fibroblast growth factor, and transforming growth factor-beta 1 in the regulation of these processes. In this study, expression of the mRNA of these growth factors was examined using total RNA extracted from the physis and epiphysis of rat tibias. Transforming growth factor-beta 1 mRNA was detected by Northern hybridization. Expression of the genes encoding acidic and basic fibroblast growth factors was demonstrated by polymerase chain reaction amplification. In addition, using polyclonal antibodies against these growth factors, we localized them by immunohistochemical analysis. Strong intracellular staining with a predominantly nuclear pattern was observed in chondrocytes from the proliferating and upper hypertrophic zones. In contrast, chondrocytes in the resting zone stained only faintly for the presence of these growth factors. Some chondrocytes in the resting zone adjacent to the proliferating zone stained with these antibodies, and the antibodies also stained cells in the zone of Ranvier, which regulates latitudinal bone growth. Lastly, the location of transforming growth factor-beta 1 was examined further with use of a polyclonal antipeptide antibody specific for its extracellular epitope.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7472755

  7. Human recombinant endopeptidase PHEX has a strict S1' specificity for acidic residues and cleaves peptides derived from fibroblast growth factor-23 and matrix extracellular phosphoglycoprotein.

    PubMed Central

    Campos, Marcelo; Couture, Constance; Hirata, Izaura Y; Juliano, Maria A; Loisel, Thomas P; Crine, Philippe; Juliano, Luiz; Boileau, Guy; Carmona, Adriana K

    2003-01-01

    The PHEX gene (phosphate-regulating gene with homologies to endopeptidases on the X chromosome) encodes a protein (PHEX) with structural homologies to members of the M13 family of zinc metallo-endopeptidases. Mutations in the PHEX gene are responsible for X-linked hypophosphataemia in humans. However, the mechanism by which loss of PHEX function results in the disease phenotype, and the endogenous PHEX substrate(s) remain unknown. In order to study PHEX substrate specificity, combinatorial fluorescent-quenched peptide libraries containing o -aminobenzoic acid (Abz) and 2,4-dinitrophenyl (Dnp) as the donor-acceptor pair were synthesized and tested as PHEX substrates. PHEX showed a strict requirement for acidic amino acid residues (aspartate or glutamate) in S(1)' subsite, with a strong preference for aspartate. Subsites S(2)', S(1) and S(2) exhibited less defined specificity requirements, but the presence of leucine, proline or glycine in P(2)', or valine, isoleucine or histidine in P(1) precluded hydrolysis of the substrate by the enzyme. The peptide Abz-GFSDYK(Dnp)-OH, which contains the most favourable residues in the P(2) to P(2)' positions, was hydrolysed by PHEX at the N-terminus of aspartate with a k(cat)/ K(m) of 167 mM(-1) x s(-1). In addition, using quenched fluorescence peptides derived from fibroblast growth factor-23 and matrix extracellular phosphoglycoprotein sequences flanked by Abz and N -(2,4-dinitrophenyl)ethylenediamine, we showed that these physiologically relevant proteins are potential PHEX substrates. Finally, our results clearly indicate that PHEX does not have neprilysin-like substrate specificity. PMID:12678920

  8. Mapping the response of human fibroblast growth factor 21 (FGF21) promoter to serum availability and lipoic acid in HepG2 hepatoma cells.

    PubMed

    Xia, Mengna; Erickson, Anjeza; Yi, Xiaohua; Moreau, Régis

    2016-03-01

    The hormone-like polypeptide, fibroblast growth factor 21 (FGF21), is a major modulator of lipid and glucose metabolism and an exploratory treatment strategy for obesity related metabolic disorders. The costs of recombinant FGF21 and mode of delivery by injection are important constraints to its wide therapeutic use. The stimulation of endogenous FGF21 production through diet is being explored as an alternative approach. To that end, we examined the mechanism(s) by which serum manipulation and lipoic acid (a dietary activator of FGF21) induce FGF21 in human hepatocellular carcinoma HepG2 cells. Serum withdrawal markedly induced FGF21 mRNA levels (88 fold) and FGF21 secreted in the media (19 fold). Lipoic acid induced FGF21 mRNA 7 fold above DMSO-treated control cells and FGF21 secretion 3 fold. These effects were several-fold greater than those of PPARα agonist, Wy14643, which failed to induce FGF21 above and beyond the induction seen with serum withdrawal. The use of transcription inhibitor, actinomycin D, revealed that de novo mRNA synthesis drives FGF21 secretion in response to serum starvation. Four previously unrecognized loci in FGF21 promoter were nucleosome depleted and enriched in acetylated histone H3 revealing their role as transcriptional enhancers and putative transcription factor binding sites. FGF21 did not accumulate to a significant degree in induced HepG2 cells, which secreted FGF21 time dependently in media. We conclude that lipoic acid cell signaling connects with the transcriptional upregulation of FGF21 and it may prove to be a safe and affordable means to stimulate FGF21 production. PMID:26691139

  9. Abscisic acid ameliorates the systemic sclerosis fibroblast phenotype in vitro

    SciTech Connect

    Bruzzone, Santina; Battaglia, Florinda; Mannino, Elena; Parodi, Alessia; Fruscione, Floriana; Basile, Giovanna; Salis, Annalisa; Sturla, Laura; Negrini, Simone; Kalli, Francesca; Stringara, Silvia; Filaci, Gilberto; and others

    2012-05-25

    Highlights: Black-Right-Pointing-Pointer ABA is an endogenous hormone in humans, regulating different cell responses. Black-Right-Pointing-Pointer ABA reverts some of the functions altered in SSc fibroblasts to a normal phenotype. Black-Right-Pointing-Pointer UV-B irradiation increases ABA content in SSc cultures. Black-Right-Pointing-Pointer SSc fibroblasts could benefit from exposure to ABA and/or to UV-B. -- Abstract: The phytohormone abscisic acid (ABA) has been recently identified as an endogenous hormone in humans, regulating different cell functions, including inflammatory processes, insulin release and glucose uptake. Systemic sclerosis (SSc) is a chronic inflammatory disease resulting in fibrosis of skin and internal organs. In this study, we investigated the effect of exogenous ABA on fibroblasts obtained from healthy subjects and from SSc patients. Migration of control fibroblasts induced by ABA was comparable to that induced by transforming growth factor-{beta} (TGF-{beta}). Conversely, migration toward ABA, but not toward TGF-{beta}, was impaired in SSc fibroblasts. In addition, ABA increased cell proliferation in fibroblasts from SSc patients, but not from healthy subjects. Most importantly, presence of ABA significantly decreased collagen deposition by SSc fibroblasts, at the same time increasing matrix metalloproteinase-1 activity and decreasing the expression level of tissue inhibitor of metalloproteinase (TIMP-1). Thus, exogenously added ABA appeared to revert some of the functions altered in SSc fibroblasts to a normal phenotype. Interestingly, ABA levels in plasma from SSc patients were found to be significantly lower than in healthy subjects. UV-B irradiation induced an almost 3-fold increase in ABA content in SSc cultures. Altogether, these results suggest that the fibrotic skin lesions in SSc patients could benefit from exposure to high(er) ABA levels.

  10. c-Fos mediates repression of the apical sodium-dependent bile acid transporter by fibroblast growth factor-19 in mice

    PubMed Central

    Ghosh, Ayantika; Chen, Frank; Banerjee, Swati; Xu, Ming

    2013-01-01

    Fibroblast growth factor-19 (FGF-19), a bile acid-responsive enterokine, is secreted by the ileum and regulates a variety of metabolic processes. These studies examined the signal transduction pathways operant in FGF-19-mediated repression of the apical sodium-dependent bile acid transporter (ASBT). Responses to FGF-19 were assessed in Caco-2 and CT-26 cells and in mice where c-fos was conditionally silenced in the intestine by a cre-lox strategy. FGF-19 treatment of Caco-2 cells or wild-type mice led to a significant reduction in ASBT protein expression and enhanced phosphorylation of extracellular signaling kinase 1/2 (ERK1/2), c-Fos, and c-Jun. FGF-19 treatment of Caco-2 cells led to a reduction in activity of the human ASBT promoter and this repression could be blocked by treatment with a mitogen-activated protein kinase/ERK kinase (MEK1/2) inhibitor or by silencing jun kinase 1, jun kinase 2, c-fos, or c-jun. Site directed mutagenesis of a c-fos binding element in the ASBT promoter blocked FGF-19-mediated repression in luciferase reporter constructs. ASBT promoter activity was repressed by FGF-19 in CT-26 cells and this repression could be reduced by MEK1/2 inhibition or silencing c-fos. FGF-19-mediated repression of ASBT protein expression was abrogated in mice where c-fos was conditionally silenced in the intestine. In contrast, ASBT was repressed in the c-Fos expressing gallbladders of the same mice. The studies demonstrate that FGF-19 represses the expression of ASBT in the ileum and gallbladder via a signal transduction pathway involving MEK1/2, ERK1/2, JNK1, JNK2, and c-Fos. PMID:24309182

  11. Relevance of charge balance and hyaluronic acid on alginate-chitosan sponge microstructure and its influence on fibroblast growth.

    PubMed

    Orellana, Sandra L; Giacaman, Annesi; Pavicic, Francisca; Vidal, Alejandra; Moreno-Villoslada, Ignacio; Concha, Miguel

    2016-10-01

    The study of biomaterials by electrical charge scaling to explore the role of net charge on biocompatibility and suitability for tissue regeneration has been limited as has the search for products that could improve this first-rate variable. In the present study, we prepared sponges composed of chitosan/alginate (CS/ALG) with or without hyaluronic acid (HA) by mixing polymer stock solutions of different net electric charge ratios (n(+/) n(-) ), and then lyophilizing them to obtain porous materials. The electric charge ratios n(+/) n(-) studied were 0.3, 0.8, 1.0, and 2.5 for CS/ALG and 0.3, 1.0, 1.9, and 3.7 for CS/ALG/HA sponges. Under these conditions a role for net electric charge balance over sponge microstructure rearrangement, protection to dissolution, cellular proliferation, and cell-cell interactions was apparent, effects that were enhanced by copolymer modification with HA. Mass balance, electric charge, and specific products that influence both such as HA, have a potential in biomaterials for wound healing. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2537-2543, 2016. PMID:27238940

  12. The Prevention of Diabetic Cardiomyopathy by Non-Mitogenic Acidic Fibroblast Growth Factor Is Probably Mediated by the Suppression of Oxidative Stress and Damage

    PubMed Central

    Zhang, Chi; Zhang, Linbo; Chen, Shali; Feng, Biao; Lu, Xuemian; Bai, Yang; Liang, Guang; Tan, Yi; Shao, Minglong; Skibba, Melissa; Jin, Litai; Li, Xiaokun; Chakrabarti, Subrata; Cai, Lu

    2013-01-01

    Background Emerging evidence showed the beneficial effect of acidic fibroblast growth factor (aFGF) on heart diseases. The present study investigated whether non-mitogenic aFGF (nm-aFGF) can prevent diabetic cardiomyopathy and the underlying mechanisms, if any. Methodology/Principal Findings Type 1 diabetes was induced in mice by multiple intraperitoneal injections of low-dose streptozotocin. Hyperglycemic and age-matched control mice were treated with or without nm-aFGF at 10 µg/kg daily for 1 and 6 months. Blood pressure and cardiac function were assessed. Cardiac H9c2 cell, human microvascular endothelial cells, and rat cardiomyocytes were exposed to high glucose (25 mM) for mimicking an in vitro diabetic condition for mechanistic studies. Oxidative stress, DNA damage, cardiac hypertrophy and fibrosis were assessed by real-time qPCR, immunofluorescent staining, Western blotting, and pathological examination. Nm-aFGF significantly prevented diabetes-induced hypertension and cardiac dysfunction at 6 months. Mechanistic studies demonstrated that nm-aFGF showed the similar preventive effect as the native aFGF on high glucose-induced oxidative stress (increase generation of reactive oxygen species) and damage (cellular DNA oxidation), cell hypertrophy, and fibrotic response (increased mRNA expression of fibronectin) in three kinds of cells. These in vitro findings were recaptured by examining the heart of the diabetic mice with and without nm-aFGF. Conclusions These results suggest that nm-aFGF can prevent diabetic cardiomyopathy, probably through attenuation of cardiac oxidative stress, hypertrophy, and fibrosis. PMID:24349248

  13. S100A13-C2A binary complex structure-a key component in the acidic fibroblast growth factor for the non-classical pathway

    SciTech Connect

    Mohan, Sepuru K.; Rani, Sandhya G.; Kumar, Sriramoju M.; Yu Chin

    2009-03-13

    Fibroblast growth factors (FGFs) are key regulators of cell proliferation, differentiation, tumor-induced angiogenesis and migration. FGFs are essential for early embryonic development, organ formation and angiogenesis. They play important roles in tumor formation, inflammation, wound healing and restenosis. The biological effects of FGFs are mediated through the activation of the four transmembrane phosphotyrosine kinase receptors (FGFRs) in the presence of heparin sulfate proteoglycans (HSPGs) and therefore require the release of FGFs into the extracellular space. However, FGF-1 lacks the signal peptide required for the releasing of these proteins through the classical endoplasmic reticulum (ER)-Golgi secretary pathway. Maciag et al. demonstrated that FGF-1 is exported through a non-classical release pathway involving the formation of a specific multiprotein complex [M. Landriscina, R. Soldi, C. Bagala, I. Micucci, S. Bellum, F. Tarantini, I. Prudovsky, T. Maciag, S100A13 participates in the release of fibroblast growth factor 1 in response to heat shock in vitro, J. Biol. Chem. 276 (2001) 22544-22552; C.M. Carreira, T.M. LaVallee, F. Tarantini, A. Jackson, J.T. Lathrop, B. Hampton, W.H. Burgess, T. Maciag, S100A13 is involved in the regulation of fibroblast growth factor-1 and p40 synaptotagmin-1 release in vitro, J. Biol. Chem. 273 (1998) 22224-22231; T.M. LaValle, F. Tarantini, S. Gamble, C.M. Carreira, A. Jackson, T. Maciag, Synaptotagmin-1 is required for fibroblast growth factor-1 release, J. Biol. Chem. 273 (1998) 22217-22223; C. Bagala, V. Kolev, A. Mandinova, R. Soldi, C. Mouta, I. Graziani, I, Prudovsky, T. Maciag, The alternative translation of synaptotagmin 1 mediates the non-classical release of FGF1, Biochem. Biophys. Res. Commun. 310 (2003) 1041-1047]. The protein constituents of this complex include FGF-1, S100A13 (a Ca{sup 2+}-binding protein), and the p40 form of synaptotagmin 1 (Syt1). To understand the molecular events in the FGF-1 releasing

  14. In vitro stress effect on degradation and drug release behaviors of basic fibroblast growth factor – poly(lactic-co-glycolic-acid) microsphere

    PubMed Central

    Xiong, Yan; Yu, Zeping; Lang, Yun; Hu, Juanyu; Li, Hong; Yan, Yonggang; Tu, Chongqi; Yang, Tianfu; Song, Yueming; Duan, Hong; Pei, Fuxing

    2016-01-01

    Objective To study the degradation and basic fibroblast growth factor (bFGF) release activity of bFGF – poly(lactic-co-glycolic-acid) microsphere (bFGF-PLGA MS) under stress in vitro, including the static pressure and shearing force-simulating mechanical environment of the joint cavity. Method First, bFGF-PLGA MSs were created. Meanwhile, two self-made experimental instruments (static pressure and shearing force loading instruments) were initially explored to provide stress-simulating mechanical environment of the joint cavity. Then, bFGF-PLGA MSs were loaded into the two instruments respectively, to study microsphere degradation and drug release experiments. In the static pressure loading experiment, normal atmospheric pressure loading (approximately 0.1 MPa), 0.35 MPa, and 4.0 MPa pressure loading and shaking flask oscillation groups were designed to study bFGF-PLGA MS degradation and bFGF release. In the shearing force loading experiment, a pulsating pump was used to give the experimental group an output of 1,000 mL/min and the control group an output of 10 mL/min to carry out bFGF-PLGA MS degradation and drug release experiments. Changes of bFGF-PLGA MSs, including microsphere morphology, quality, weight-average molecular weight of polymer, and microsphere degradation and bFGF release, were analyzed respectively. Results In the static pressure loading experiment, bFGF-PLGA MSs at different pressure were stable initially. The trend of molecular weight change, quality loss, and bFGF release was consistent. Meanwhile, microsphere degradation and bFGF release rates in the 4.0 MPa pressure loading group were faster than those in the normal and 0.35 MPa pressure loading groups. It was the fastest in the shaking flask group, showing a statistically significant difference (P<0.0001). In the shearing force loading experiment, there were no distinctive differences in the rates of microsphere degradation and bFGF release between experimental and control group. Meanwhile

  15. Identification of Amino Acid Residues in Fibroblast Growth Factor 14 (FGF14) Required for Structure-Function Interactions with Voltage-gated Sodium Channel Nav1.6.

    PubMed

    Ali, Syed R; Singh, Aditya K; Laezza, Fernanda

    2016-05-20

    The voltage-gated Na(+) (Nav) channel provides the basis for electrical excitability in the brain. This channel is regulated by a number of accessory proteins including fibroblast growth factor 14 (FGF14), a member of the intracellular FGF family. In addition to forming homodimers, FGF14 binds directly to the Nav1.6 channel C-tail, regulating channel gating and expression, properties that are required for intrinsic excitability in neurons. Seeking amino acid residues with unique roles at the protein-protein interaction interface (PPI) of FGF14·Nav1.6, we engineered model-guided mutations of FGF14 and validated their impact on the FGF14·Nav1.6 complex and the FGF14:FGF14 dimer formation using a luciferase assay. Divergence was found in the β-9 sheet of FGF14 where an alanine (Ala) mutation of Val-160 impaired binding to Nav1.6 but had no effect on FGF14:FGF14 dimer formation. Additional analysis revealed also a key role of residues Lys-74/Ile-76 at the N-terminal of FGF14 in the FGF14·Nav1.6 complex and FGF14:FGF14 dimer formation. Using whole-cell patch clamp electrophysiology, we demonstrated that either the FGF14(V160A) or the FGF14(K74A/I76A) mutation was sufficient to abolish the FGF14-dependent regulation of peak transient Na(+) currents and the voltage-dependent activation and steady-state inactivation of Nav1.6; but only V160A with a concomitant alanine mutation at Tyr-158 could impede FGF14-dependent modulation of the channel fast inactivation. Intrinsic fluorescence spectroscopy of purified proteins confirmed a stronger binding reduction of FGF14(V160A) to the Nav1.6 C-tail compared with FGF14(K74A/I76A) Altogether these studies indicate that the β-9 sheet and the N terminus of FGF14 are well positioned targets for drug development of PPI-based allosteric modulators of Nav channels. PMID:26994141

  16. Fibroblast growth factor 23 and bone mineralisation

    PubMed Central

    Guo, Yu-Chen; Yuan, Quan

    2015-01-01

    Fibroblast growth factor 23 (FGF23) is a hormone that is mainly secreted by osteocytes and osteoblasts in bone. The critical role of FGF23 in mineral ion homeostasis was first identified in human genetic and acquired rachitic diseases and has been further characterised in animal models. Recent studies have revealed that the levels of FGF23 increase significantly at the very early stages of chronic kidney disease (CKD) and may play a critical role in mineral ion disorders and bone metabolism in these patients. Our recent publications have also shown that FGF23 and its cofactor, Klotho, may play an independent role in directly regulating bone mineralisation instead of producing a systematic effect. In this review, we will discuss the new role of FGF23 in bone mineralisation and the pathophysiology of CKD-related bone disorders. PMID:25655009

  17. The Fibroblast Growth Factor signaling pathway

    PubMed Central

    Ornitz, David M; Itoh, Nobuyuki

    2015-01-01

    The signaling component of the mammalian Fibroblast Growth Factor (FGF) family is comprised of eighteen secreted proteins that interact with four signaling tyrosine kinase FGF receptors (FGFRs). Interaction of FGF ligands with their signaling receptors is regulated by protein or proteoglycan cofactors and by extracellular binding proteins. Activated FGFRs phosphorylate specific tyrosine residues that mediate interaction with cytosolic adaptor proteins and the RAS-MAPK, PI3K-AKT, PLCγ, and STAT intracellular signaling pathways. Four structurally related intracellular non-signaling FGFs interact with and regulate the family of voltage gated sodium channels. Members of the FGF family function in the earliest stages of embryonic development and during organogenesis to maintain progenitor cells and mediate their growth, differentiation, survival, and patterning. FGFs also have roles in adult tissues where they mediate metabolic functions, tissue repair, and regeneration, often by reactivating developmental signaling pathways. Consistent with the presence of FGFs in almost all tissues and organs, aberrant activity of the pathway is associated with developmental defects that disrupt organogenesis, impair the response to injury, and result in metabolic disorders, and cancer. © 2015 Wiley Periodicals, Inc. PMID:25772309

  18. Cultured human foreskin fibroblasts produce a factor that stimulates their growth with properties similar to basic fibroblast growth factor

    SciTech Connect

    Story, M.T. )

    1989-05-01

    To determine if fibroblasts could be a source of fibroblast growth factor (FGF) in tissue, cells were initiated in culture from newborn human foreskin. Fibroblast cell lysates promoted radiolabeled thymidine uptake by cultured quiescent fibroblasts. Seventy-nine percent of the growth-promoting activity of lysates was recovered from heparin-Sepharose. The heparin-binding growth factor reacted on immunoblots with antiserum to human placenta-derived basic FGF and competed with iodinated basic FGF for binding to antiserum to (1-24)bFGF synthetic peptide. To confirm that fibroblasts were the source of the growth factor, cell lysates were prepared from cells incubated with radiolabeled methionine. Heparin affinity purified material was immunoprecipitated with basic FGF antiserum and electrophoresed. Radiolabeled material was detected on gel autoradiographs in the same molecular weight region as authentic iodinated basic FGF. The findings are consistant with the notion that cultured fibroblasts express basic FGF. As these cells also respond to the mitogen, it is possible that the regulation of their growth is under autocrine control. Fibroblasts may be an important source of the growth factor in tissue.

  19. Fibroblast Growth Factor Signaling in Metabolic Regulation

    PubMed Central

    Nies, Vera J. M.; Sancar, Gencer; Liu, Weilin; van Zutphen, Tim; Struik, Dicky; Yu, Ruth T.; Atkins, Annette R.; Evans, Ronald M.; Jonker, Johan W.; Downes, Michael Robert

    2016-01-01

    The prevalence of obesity is a growing health problem. Obesity is strongly associated with several comorbidities, such as non-alcoholic fatty liver disease, certain cancers, insulin resistance, and type 2 diabetes, which all reduce life expectancy and life quality. Several drugs have been put forward in order to treat these diseases, but many of them have detrimental side effects. The unexpected role of the family of fibroblast growth factors in the regulation of energy metabolism provides new approaches to the treatment of metabolic diseases and offers a valuable tool to gain more insight into metabolic regulation. The known beneficial effects of FGF19 and FGF21 on metabolism, together with recently discovered similar effects of FGF1 suggest that FGFs and their derivatives carry great potential as novel therapeutics to treat metabolic conditions. To facilitate the development of new therapies with improved targeting and minimal side effects, a better understanding of the molecular mechanism of action of FGFs is needed. In this review, we will discuss what is currently known about the physiological roles of FGF signaling in tissues important for metabolic homeostasis. In addition, we will discuss current concepts regarding their pharmacological properties and effector tissues in the context of metabolic disease. Also, the recent progress in the development of FGF variants will be reviewed. Our goal is to provide a comprehensive overview of the current concepts and consensuses regarding FGF signaling in metabolic health and disease and to provide starting points for the development of FGF-based therapies against metabolic conditions. PMID:26834701

  20. Fibroblast Growth Factor 23 in Long-Duration Spaceflight

    NASA Technical Reports Server (NTRS)

    Bokhari, R.; Zwart, S. R.; Fields, E.; Heer, M.; Sibonga, J.; Smith, S. M.

    2015-01-01

    Many nutritional factors influence bone, from the basics of calcium and vitamin D, to factors which influence bone through acid/base balance, including protein, sodium, and more. Fibroblast growth factor 23 (FGF23) is a recently identified factor, secreted from osteocytes, which is involved in classic (albeit complex) feedback loops controlling phosphorus homeostasis through both vitamin D and parathyroid hormone (PTH) (1, 2). As osteocytes are gravity sensing cells, it is important to determine if there are changes in FGF23 during spaceflight. In extreme cases, such as chronic kidney disease, FGF23 levels are highly elevated. FGF23 imbalances, secondary to dietary influences, may contribute to skeletal demineralization and kidney stone risk during spaceflight.

  1. Copper binding affinity of the C2B domain of synaptotagmin-1 and its potential role in the nonclassical secretion of Acidic Fibroblast Growth Factor

    PubMed Central

    Jayanthi, Srinivas; Kathir, Karuppanan Muthusamy; Rajalingam, Dakshinamurthy; Furr, Mercede; Daily, Anna; Thurman, Ryan; Rutherford, Lindsay; Chandrashekar, Reena; Adams, Paul; Prudovsky, Igor; Suresh Kumar, Thallapuranam Krishnaswamy

    2014-01-01

    Fibroblast growth factor 1 (FGF1) is a heparin-binding proangiogenic protein. FGF1 lacks the conventional N-terminal signal peptide required for secretion through the endoplasmic reticulum (ER) -Golgi secretory pathway. FGF1 is released through a Cu2+ - mediated nonclassical secretion pathway. The secretion of FGF1 involves the formation of a Cu2+- mediated multiprotein release complex (MRC) including FGF1, S100A13 (a calcium-binding protein) and p40 synaptotagmin (Syt1). It is believed that binding of Cu2+ to the C2B domain is important for the release of FGF1 in to the extracellular medium. In this study, using a variety of biophysical studies, Cu2+ and lipid interactions of the C2B domain of Syt1were characterized. Isothermal titration calorimetry (ITC) experiments reveal that C2B domain binds to Cu2+ in a biphasic manner involving an initial endothermic and a subsequent exothermic phase. Fluorescence energy transfer experiments using Tb3+ show that there are two Cu2+- binding pockets on the C2B domain, and one of these is also a Ca2+- binding site. Lipid-binding studies using ITC demonstrate that the C2B domain preferentially binds to small unilamellar vesicles of phosphatidyl serine (PS). Results of the differential scanning calorimetry and limited trypsin digestion experiments suggest that C2B domain is marginally destabilized upon binding to PS vesicles. These results, for the first time, suggest that the main role of the C2B domain of Syt1 is to serve as an anchor for the FGF1 MRC on the membrane bilayer. In addition, binding of the C2B domain to the lipid bilayer is shown to significantly decrease the binding affinity of the protein to Cu2+. The study provides valuable insights on the sequence of structural events that occur in the nonclassical secretion of FGF1. PMID:25224745

  2. Fibroblast growth factor 15 deficiency impairs liver regeneration in mice

    PubMed Central

    Kong, Bo; Huang, Jiansheng; Zhu, Yan; Li, Guodong; Williams, Jessica; Shen, Steven; Aleksunes, Lauren M.; Richardson, Jason R.; Apte, Udayan; Rudnick, David A.

    2014-01-01

    Fibroblast growth factor (FGF) 15 (human homolog, FGF19) is an endocrine FGF highly expressed in the small intestine of mice. Emerging evidence suggests that FGF15 is critical for regulating hepatic functions; however, the role of FGF15 in liver regeneration is unclear. This study assessed whether liver regeneration is altered in FGF15 knockout (KO) mice following 2/3 partial hepatectomy (PHx). The results showed that FGF15 KO mice had marked mortality, with the survival rate influenced by genetic background. Compared with wild-type mice, the KO mice displayed extensive liver necrosis and marked elevation of serum bile acids and bilirubin. Furthermore, hepatocyte proliferation was reduced in the KO mice because of impaired cell cycle progression. After PHx, the KO mice had weaker activation of signaling pathways that are important for liver regeneration, including signal transducer and activator of transcription 3, nuclear factor-κB, and mitogen-activated protein kinase. Examination of the KO mice at early time points after PHx revealed a reduced and/or delayed induction of immediate-early response genes, including growth-control transcription factors that are critical for liver regeneration. In conclusion, the results suggest that FGF15 deficiency severely impairs liver regeneration in mice after PHx. The underlying mechanism is likely the result of disrupted bile acid homeostasis and impaired priming of hepatocyte proliferation. PMID:24699334

  3. Autophagy is required for IL-2-mediated fibroblast growth

    SciTech Connect

    Kang, Rui; Tang, Daolin; Lotze, Michael T.; Zeh III, Herbert J.

    2013-02-15

    Autophagy is an evolutionarily conserved pathway responsible for delivery of cytoplasmic material into the lysosomal degradation pathway to enable vesicular exocytosis. Interleukin (IL)-2 is produced by T-cells and its activity is important for immunoregulation. Fibroblasts are an immune competent cell type, playing a critical role in wound healing, chronic inflammation, and tumor development. Although autophagy plays an important role in each of these processes, whether it regulates IL-2 activity in fibroblasts is unknown. Here, we show that autophagy is required for IL-2-induced cell growth in fibroblasts. IL-2 significantly induced autophagy in mouse embryonic fibroblasts (MEFs) and primary lung fibroblasts. Autophagy inhibitors (e.g., 3-methylamphetamine and bafilomycin A1) or knockdown of ATG5 and beclin 1 blocked clinical grade IL-2-induced autophagy. Moreover, IL-2 induced HMGB1 cytoplasmic translocation in MEFs and promoted interaction between HMGB1 and beclin1, which is required for autophagy induction. Pharmacological and genetic inhibition of autophagy inhibited IL-2-induced cell proliferation and enhanced IL-2-induced apoptosis. These findings suggest that autophagy is an important pro-survival regulator for IL-2-induced cell growth in fibroblasts.

  4. Retinoic acid stimulation of human dermal fibroblast proliferation is dependent on suboptimal extracellular Ca2+ concentration

    SciTech Connect

    Varani, J.; Shayevitz, J.; Perry, D.; Mitra, R.S.; Nickoloff, B.J.; Voorhees, J.J. )

    1990-06-01

    Human dermal fibroblasts failed to proliferate when cultured in medium containing 0.15 mmol/l (millimolar) Ca2+ (keratinocyte growth medium (KGM)) but did when the external Ca2+ concentration was raised to 1.4 mmol/l. All-trans retinoic acid (retinoic acid) stimulated proliferation in KGM but did not further stimulate growth in Ca2(+)-supplemented KGM. The ability of retinoic acid to stimulate proliferation was inhibited in KGM prepared without Ca2+ or prepared with 0.03 mmol/l Ca2+ and in KGM treated with 1 mmol/l ethylene-glycol-bis-(beta-aminoethyl ether)N,N'-tetra acetic acid. Using 45Ca2+ to measure Ca2+ influx and efflux, it was found that retinoic acid minimally increased Ca2+ uptake into fibroblasts. In contrast, retinoic acid treatment of fibroblasts that had been pre-equilibrated for 1 day with 45Ca2+ inhibited release of intracellular Ca2+ into the extracellular fluid. Retinoic acid also stimulated 35S-methionine incorporation into trichloroacetic acid-precipitable material but in contrast to its effect on proliferation, stimulation of 35S-methionine incorporation occurred in both high-Ca2+ and low-Ca2+ medium. These data indicate that retinoic acid stimulation of proliferation, but not protein synthesis, is dependent on the concentration of Ca2+ in the extracellular environment.

  5. Fibroblast Growth Factor-2 Alters the Nature of Extinction

    ERIC Educational Resources Information Center

    Graham, Bronwyn M.; Richardson, Rick

    2011-01-01

    These experiments examined the effects of the NMDA-receptor (NMDAr) antagonist MK801 on reacquisition and re-extinction of a conditioned fear that had been previously extinguished before injection of fibroblast growth factor-2 (FGF2) or vehicle. Recent findings have shown that relearning and re-extinction, unlike initial learning and extinction,…

  6. Functional restoration using basic fibroblast growth factor (bFGF) infusion in Kainic acid induced cognitive dysfunction in rat: neurobehavioural and neurochemical studies.

    PubMed

    Srivastava, Nishi; Seth, Kavita; Srivastava, Nalini; Khanna, Vinay K; Agrawal, Ashok Kumar

    2008-07-01

    Neurogenesis occurs in dentate gyrus of adult hippocampus under the influence of various mitogenic factors. Growth factors besides instigating the proliferation of neuronal progenitor cells (NPCs) in dentate gyrus, also supports their differentiation to cholinergic neurons. In the present study, an attempt has been made to investigate the neurotrophic effect of bFGF in Kainic acid (KA) induced cognitive dysfunction in rats. Stereotaxic lesioning using (KA) was performed in hippocampal CA3 region of rat's brain. Four-weeks post lesioning rats were assessed for impairment in learning and memory using Y maze followed by bFGF infusion in dentate gyrus region. The recovery was evaluated after bFGF infusion using neurochemical, neurobehavioural and immunohistochemical approaches and compared with lesioned group. Significant impairment in learning and memory (P < 0.01) observed in lesioned animals, four weeks post lesioning exhibited significant restoration (P < 0.001) following bFGF infusion twice at one and four week post lesion. The bFGF infused animals exhibited recovery in hippocampus cholinergic (76%)/ dopaminergic (46%) receptor binding and enhanced Choline acetyltransferase (ChAT) immunoreactivity in CA3 region. The results suggest restorative potential of bFGF in cognitive dysfunctions, possibly due to mitogenic effect on dentate gyrus neurogenic area leading to generation and migration of newer cholinergic neurons. PMID:17955369

  7. Cyclic phosphatidic acid and lysophosphatidic acid induce hyaluronic acid synthesis via CREB transcription factor regulation in human skin fibroblasts.

    PubMed

    Maeda-Sano, Katsura; Gotoh, Mari; Morohoshi, Toshiro; Someya, Takao; Murofushi, Hiromu; Murakami-Murofushi, Kimiko

    2014-09-01

    Cyclic phosphatidic acid (cPA) is a naturally occurring phospholipid mediator and an analog of the growth factor-like phospholipid lysophosphatidic acid (LPA). cPA has a unique cyclic phosphate ring at the sn-2 and sn-3 positions of its glycerol backbone. We showed before that a metabolically stabilized cPA derivative, 2-carba-cPA, relieved osteoarthritis pathogenesis in vivo and induced hyaluronic acid synthesis in human osteoarthritis synoviocytes in vitro. This study focused on hyaluronic acid synthesis in human fibroblasts, which retain moisture and maintain health in the dermis. We investigated the effects of cPA and LPA on hyaluronic acid synthesis in human fibroblasts (NB1RGB cells). Using particle exclusion and enzyme-linked immunosorbent assays, we found that both cPA and LPA dose-dependently induced hyaluronic acid synthesis. We revealed that the expression of hyaluronan synthase 2 messenger RNA and protein is up-regulated by cPA and LPA treatment time dependently. We then characterized the signaling pathways up-regulating hyaluronic acid synthesis mediated by cPA and LPA in NB1RGB cells. Pharmacological inhibition and reporter gene assays revealed that the activation of the LPA receptor LPAR1, Gi/o protein, phosphatidylinositol-3 kinase (PI3K), extracellular-signal-regulated kinase (ERK), and cyclic adenosine monophosphate response element-binding protein (CREB) but not nuclear factor κB induced hyaluronic acid synthesis by the treatment with cPA and LPA in NB1RGB cells. These results demonstrate for the first time that cPA and LPA induce hyaluronic acid synthesis in human skin fibroblasts mainly through the activation of LPAR1-Gi/o followed by the PI3K, ERK, and CREB signaling pathway. PMID:24845645

  8. Extracellular depolymerization of hyaluronic acid in cultured human skin fibroblasts

    SciTech Connect

    Nakamura, T.; Takagaki, K.; Kubo, K.; Morikawa, A.; Tamura, S.; Endo, M. )

    1990-10-15

    The chain length of ({sup 3}H)hyaluronic acid synthesized by cultivating human skin fibroblasts in the presence of ({sup 3}H)glucosamine was investigated. ({sup 3}H)Hyaluronic acid obtained from the matrix fraction was excluded from a Sepharose CL-2B column irrespective of the incubation period, whereas that from the medium was depolymerized into a constant chain length (Mr = 40,000). The reducing and non-reducing terminals of the depolymerized hyaluronic acid were N-acetylglucosamine and glucuronic acid, respectively. Prolonged incubation produced no oligosaccharides as shown by examination of hyaluronidase digests, suggesting the presence of a novel endo-beta-N-acetylglucosaminidase in cultured human skin fibroblasts.

  9. Monomethylarsonous acid inhibited endogenous cholesterol biosynthesis in human skin fibroblasts.

    PubMed

    Guo, Lei; Xiao, Yongsheng; Wang, Yinsheng

    2014-05-15

    Human exposure to arsenic in drinking water is a widespread public health concern, and such exposure is known to be associated with many human diseases. The detailed molecular mechanisms about how arsenic species contribute to the adverse human health effects, however, remain incompletely understood. Monomethylarsonous acid [MMA(III)] is a highly toxic and stable metabolite of inorganic arsenic. To exploit the mechanisms through which MMA(III) exerts its cytotoxic effect, we adopted a quantitative proteomic approach, by coupling stable isotope labeling by amino acids in cell culture (SILAC) with LC-MS/MS analysis, to examine the variation in the entire proteome of GM00637 human skin fibroblasts following acute MMA(III) exposure. Among the ~6500 unique proteins quantified, ~300 displayed significant changes in expression after exposure with 2 μM MMA(III) for 24 h. Subsequent analysis revealed the perturbation of de novo cholesterol biosynthesis, selenoprotein synthesis and Nrf2 pathways evoked by MMA(III) exposure. Particularly, MMA(III) treatment resulted in considerable down-regulation of several enzymes involved in cholesterol biosynthesis. In addition, real-time PCR analysis showed reduced mRNA levels of select genes in this pathway. Furthermore, MMA(III) exposure contributed to a distinct decline in cellular cholesterol content and significant growth inhibition of multiple cell lines, both of which could be restored by supplementation of cholesterol to the culture media. Collectively, the present study demonstrated that the cytotoxicity of MMA(III) may arise, at least in part, from the down-regulation of cholesterol biosynthesis enzymes and the resultant decrease of cellular cholesterol content. PMID:24625837

  10. Monomethylarsonous acid inhibited endogenous cholesterol biosynthesis in human skin fibroblasts

    SciTech Connect

    Guo, Lei; Xiao, Yongsheng; Wang, Yinsheng

    2014-05-15

    Human exposure to arsenic in drinking water is a widespread public health concern, and such exposure is known to be associated with many human diseases. The detailed molecular mechanisms about how arsenic species contribute to the adverse human health effects, however, remain incompletely understood. Monomethylarsonous acid [MMA(III)] is a highly toxic and stable metabolite of inorganic arsenic. To exploit the mechanisms through which MMA(III) exerts its cytotoxic effect, we adopted a quantitative proteomic approach, by coupling stable isotope labeling by amino acids in cell culture (SILAC) with LC-MS/MS analysis, to examine the variation in the entire proteome of GM00637 human skin fibroblasts following acute MMA(III) exposure. Among the ∼ 6500 unique proteins quantified, ∼ 300 displayed significant changes in expression after exposure with 2 μM MMA(III) for 24 h. Subsequent analysis revealed the perturbation of de novo cholesterol biosynthesis, selenoprotein synthesis and Nrf2 pathways evoked by MMA(III) exposure. Particularly, MMA(III) treatment resulted in considerable down-regulation of several enzymes involved in cholesterol biosynthesis. In addition, real-time PCR analysis showed reduced mRNA levels of select genes in this pathway. Furthermore, MMA(III) exposure contributed to a distinct decline in cellular cholesterol content and significant growth inhibition of multiple cell lines, both of which could be restored by supplementation of cholesterol to the culture media. Collectively, the present study demonstrated that the cytotoxicity of MMA(III) may arise, at least in part, from the down-regulation of cholesterol biosynthesis enzymes and the resultant decrease of cellular cholesterol content. - Highlights: • MMA(III)-induced perturbation of the entire proteome of GM00637 cells is studied. • Quantitative proteomic approach revealed alterations of multiple cellular pathways. • MMA(III) inhibits de novo cholesterol biosynthesis. • MMA

  11. An expandable, inducible hemangioblast state regulated by fibroblast growth factor.

    PubMed

    Vereide, David T; Vickerman, Vernella; Swanson, Scott A; Chu, Li-Fang; McIntosh, Brian E; Thomson, James A

    2014-12-01

    During development, the hematopoietic and vascular lineages are thought to descend from common mesodermal progenitors called hemangioblasts. Here we identify six transcription factors, Gata2, Lmo2, Mycn, Pitx2, Sox17, and Tal1, that "trap" murine cells in a proliferative state and endow them with a hemangioblast potential. These "expandable" hemangioblasts (eHBs) are capable, once released from the control of the ectopic factors, to give rise to functional endothelial cells, multilineage hematopoietic cells, and smooth muscle cells. The eHBs can be derived from embryonic stem cells, from fetal liver cells, or poorly from fibroblasts. The eHBs reveal a central role for fibroblast growth factor, which not only promotes their expansion, but also facilitates their ability to give rise to endothelial cells and leukocytes, but not erythrocytes. This study serves as a demonstration that ephemeral progenitor states can be harnessed in vitro, enabling the creation of tractable progenitor cell lines. PMID:25458896

  12. Asymmetrical Macromolecular Complex Formation of Lysophosphatidic Acid Receptor 2 (LPA2) Mediates Gradient Sensing in Fibroblasts*

    PubMed Central

    Ren, Aixia; Moon, Changsuk; Zhang, Weiqiang; Sinha, Chandrima; Yarlagadda, Sunitha; Arora, Kavisha; Wang, Xusheng; Yue, Junming; Parthasarathi, Kaushik; Heil-Chapdelaine, Rick; Tigyi, Gabor; Naren, Anjaparavanda P.

    2014-01-01

    Chemotactic migration of fibroblasts toward growth factors relies on their capacity to sense minute extracellular gradients and respond to spatially confined receptor-mediated signals. Currently, mechanisms underlying the gradient sensing of fibroblasts remain poorly understood. Using single-particle tracking methodology, we determined that a lysophosphatidic acid (LPA) gradient induces a spatiotemporally restricted decrease in the mobility of LPA receptor 2 (LPA2) on chemotactic fibroblasts. The onset of decreased LPA2 mobility correlates to the spatial recruitment and coupling to LPA2-interacting proteins that anchor the complex to the cytoskeleton. These localized PDZ motif-mediated macromolecular complexes of LPA2 trigger a Ca2+ puff gradient that governs gradient sensing and directional migration in response to LPA. Disruption of the PDZ motif-mediated assembly of the macromolecular complex of LPA2 disorganizes the gradient of Ca2+ puffs, disrupts gradient sensing, and reduces the directional migration of fibroblasts toward LPA. Our findings illustrate that the asymmetric macromolecular complex formation of chemoattractant receptors mediates gradient sensing and provides a new mechanistic basis for models to describe gradient sensing of fibroblasts. PMID:25542932

  13. Asymmetrical macromolecular complex formation of lysophosphatidic acid receptor 2 (LPA2) mediates gradient sensing in fibroblasts.

    PubMed

    Ren, Aixia; Moon, Changsuk; Zhang, Weiqiang; Sinha, Chandrima; Yarlagadda, Sunitha; Arora, Kavisha; Wang, Xusheng; Yue, Junming; Parthasarathi, Kaushik; Heil-Chapdelaine, Rick; Tigyi, Gabor; Naren, Anjaparavanda P

    2014-12-26

    Chemotactic migration of fibroblasts toward growth factors relies on their capacity to sense minute extracellular gradients and respond to spatially confined receptor-mediated signals. Currently, mechanisms underlying the gradient sensing of fibroblasts remain poorly understood. Using single-particle tracking methodology, we determined that a lysophosphatidic acid (LPA) gradient induces a spatiotemporally restricted decrease in the mobility of LPA receptor 2 (LPA2) on chemotactic fibroblasts. The onset of decreased LPA2 mobility correlates to the spatial recruitment and coupling to LPA2-interacting proteins that anchor the complex to the cytoskeleton. These localized PDZ motif-mediated macromolecular complexes of LPA2 trigger a Ca(2+) puff gradient that governs gradient sensing and directional migration in response to LPA. Disruption of the PDZ motif-mediated assembly of the macromolecular complex of LPA2 disorganizes the gradient of Ca(2+) puffs, disrupts gradient sensing, and reduces the directional migration of fibroblasts toward LPA. Our findings illustrate that the asymmetric macromolecular complex formation of chemoattractant receptors mediates gradient sensing and provides a new mechanistic basis for models to describe gradient sensing of fibroblasts. PMID:25542932

  14. Role of fibroblast growth factors in organ regeneration and repair.

    PubMed

    El Agha, Elie; Kosanovic, Djuro; Schermuly, Ralph T; Bellusci, Saverio

    2016-05-01

    In its broad sense, regeneration refers to the renewal of lost cells, tissues or organs as part of the normal life cycle (skin, hair, endometrium etc.) or as part of an adaptive mechanism that organisms have developed throughout evolution. For example, worms, starfish and amphibians have developed remarkable regenerative capabilities allowing them to voluntarily shed body parts, in a process called autotomy, only to replace the lost parts afterwards. The bizarre myth of the fireproof homicidal salamander that can survive fire and poison apple trees has persisted until the 20th century. Salamanders possess one of the most robust regenerative machineries in vertebrates and attempting to draw lessons from limb regeneration in these animals and extrapolate the knowledge to mammals is a never-ending endeavor. Fibroblast growth factors are potent morphogens and mitogens that are highly conserved among the animal kingdom. These growth factors play key roles in organogenesis during embryonic development as well as homeostatic balance during postnatal life. In this review, we provide a summary about the current knowledge regarding the involvement of fibroblast growth factor signaling in organ regeneration and repair. We also shed light on the use of these growth factors in previous and current clinical trials in a wide array of human diseases. PMID:26459973

  15. Reduced growth factor requirement of keloid-derived fibroblasts may account for tumor growth

    SciTech Connect

    Russell, S.B.; Trupin, K.M.; Rodriguez-Eaton, S.; Russell, J.D.; Trupin, J.S.

    1988-01-01

    Keloids are benign dermal tumors that form during an abnormal wound-healing process is genetically susceptible individuals. Although growth of normal and keloid cells did not differ in medium containing 10% (vol/vol) fetal bovine serum, keloid culture grew to significantly higher densities than normal cells in medium containing 5% (vol/vol) fetal bovine serum, keloid cultures grew to significantly higher densities than normal cells in medium containing 5% (vol/vol) plasma or 1% fetal bovine serum. Conditioned medium from keloid cultures did not stimulate growth of normal cells in plasma nor did it contain detectable platelet-derived growth factor or epidermal growth factor. Keloid fibroblasts responded differently than normal adult fibroblasts to transforming growth factor ..beta... Whereas transforming growth factor ..beta.. reduced growth stimulation by epidermal growth factor in cells from normal adult skin or scars, it enhanced the activity of epidermal growth factor in cells from normal adult skin or scars, it enhanced the activity of epidermal growth factor in cells from keloids. Normal and keloid fibroblasts also responded differently to hydrocortisone: growth was stimulated in normal adult cells and unaffected or inhibited in keloid cells. Fetal fibroblasts resembled keloid cells in their ability to grow in plasma and in their response to hydrocortisone. The ability of keloid fibroblasts to grow to higher cell densities in low-serum medium than cells from normal adult skin or from normal early or mature scars suggests that a reduced dependence on serum growth factors may account for their prolonged growth in vivo. Similarities between keloid and fetal cells suggest that keloids may result from the untimely expression of growth-control mechanism that is developmentally regulated.

  16. Keratinocyte growth factor and hepatocyte growth factor/scatter factor are heparin-binding growth factors for alveolar type II cells in fibroblast-conditioned medium.

    PubMed Central

    Panos, R J; Rubin, J S; Csaky, K G; Aaronson, S A; Mason, R J

    1993-01-01

    Epithelial-mesenchymal interactions mediate aspects of normal lung growth and development and are important in the restoration of normal alveolar architecture after lung injury. To determine if fibroblasts are a source of soluble growth factors for alveolar type II cells, we investigated the effect of fibroblast-conditioned medium (CM) on alveolar type II cell DNA synthesis. Serum-free CM from confluent adult human lung fibroblasts was concentrated fivefold by lyophilization. Type II cells were isolated from adult rats by elastase dissociation and incubated with [3H]thymidine and varying dilutions of concentrated CM and serum from day 1 to 3 of culture. Stimulation of type II cell DNA synthesis by fibroblast-CM was maximal after 48 h of conditioning and required the presence of serum. The activity of the CM was eliminated by boiling and by treatment with trypsin, pepsin, or dithiothreitol and was additive with saturating concentrations of acidic fibroblast growth factor, epidermal growth factor, and insulin. The growth factor activity bound to heparin-Sepharose and was eluted with 0.6 and 1.0 M NaCl. Neutralizing antibody studies demonstrated that the primary mitogens isolated in the 0.6 and 1.0 M NaCl fractions were keratinocyte growth factor (KGF, fibroblast growth factor 7) and hepatocyte growth factor/scatter factor (HGF/SF), respectively. HGF/SF was demonstrated in the crude CM and KGF was detected in the 0.6 M NaCl eluent by immunoblotting. Northern blot analysis confirmed that the lung fibroblasts expressed both KGF and HGF/SF transcripts. Human recombinant KGF and HGF/SF induced a concentration- and serum-dependent increase in rat alveolar type II cell DNA synthesis. We conclude that adult human lung fibroblasts produce at least two soluble heparin-binding growth factors, KGF and HGF/SF, which promote DNA synthesis and proliferation of rat alveolar type II cells in primary culture. KGF and HGF/SF may be important stimuli for alveolar type II cell

  17. Fibroblast Growth Factor 21 Analogs for Treating Metabolic Disorders

    PubMed Central

    Zhang, Jun; Li, Yang

    2015-01-01

    Fibroblast growth factor (FGF) 21 is a member of the endocrine FGF subfamily. FGF21 expression is induced under different disease conditions, such as type 2 diabetes, obesity, chronic kidney diseases, and cardiovascular diseases, and it has a broad spectrum of functions in regulating various metabolic parameters. Many different approaches have been pursued targeting FGF21 and its receptors to develop therapeutics for treating type 2 diabetes and other aspects of metabolic conditions. In this article, we summarize some of these key approaches and highlight the potential challenges in the development of these agents. PMID:26594197

  18. Dyskeratosis Congenita Dermal Fibroblasts are Defective in Supporting the Clonogenic Growth of Epidermal Keratinocytes

    PubMed Central

    Buckingham, Erin M.; Goldman, Frederick D.; Klingelhutz, Aloysius J.

    2012-01-01

    Telomere shortening is associated with cellular senescence and aging. Dyskeratosis congenita (DC) is a premature aging syndrome caused by mutations in genes for telomerase components or telomere proteins. DC patients have very short telomeres and exhibit aging-associated pathologies including epidermal abnormalities and bone marrow failure. Here, we show that DC skin fibroblasts are defective in their ability to support the clonogenic growth of epidermal keratinocytes. Conditioned media transfer experiments demonstrated that this defect was largely due to lack of a factor or factors secreted from the DC fibroblasts. Compared to early passage normal fibroblasts, DC fibroblasts express significantly lower transcript levels of several genes that code for secreted proteins, including Insulin-like Growth Factor 1 (IGF1) and Hepatocyte Growth Factor (HGF). Aged normal fibroblasts with short telomeres also had reduced levels of IGF1 and HGF, similar to early passage DC fibroblasts. Knockdown of IGF1 or HGF in normal fibroblasts caused a reduction in the capacity of conditioned media from these fibroblasts to support keratinocyte clonogenic growth. Surprisingly, reconstitution of telomerase in DC fibroblasts did not significantly increase transcript levels of IGF1 or HGF or substantially increase the ability of the fibroblasts to support keratinocyte growth, indicating that the gene expression defect is not readily reversible. Our results suggest that telomere shortening in dermal fibroblasts leads to reduction in expression of genes such as IGF1 and HGF and that this may cause a defect in supporting normal epidermal proliferation. PMID:23251848

  19. Fibroblast growth factor receptors, developmental corruption and malignant disease.

    PubMed

    Kelleher, Fergal C; O'Sullivan, Hazel; Smyth, Elizabeth; McDermott, Ray; Viterbo, Antonella

    2013-10-01

    Fibroblast growth factors (FGF) are a family of ligands that bind to four different types of cell surface receptor entitled, FGFR1, FGFR2, FGFR3 and FGFR4. These receptors differ in their ligand binding affinity and tissue distribution. The prototypical receptor structure is that of an extracellular region comprising three immunoglobulin (Ig)-like domains, a hydrophobic transmembrane segment and a split intracellular tyrosine kinase domain. Alternative gene splicing affecting the extracellular third Ig loop also creates different receptor isoforms entitled FGFRIIIb and FGFRIIIc. Somatic fibroblast growth factor receptor (FGFR) mutations are implicated in different types of cancer and germline FGFR mutations occur in developmental syndromes particularly those in which craniosynostosis is a feature. The mutations found in both conditions are often identical. Many somatic FGFR mutations in cancer are gain-of-function mutations of established preclinical oncogenic potential. Gene amplification can also occur with 19-22% of squamous cell lung cancers for example having amplification of FGFR1. Ontologic comparators can be informative such as aberrant spermatogenesis being implicated in both spermatocytic seminomas and Apert syndrome. The former arises from somatic FGFR3 mutations and Apert syndrome arises from germline FGFR2 mutations. Finally, therapeutics directed at inhibiting the FGF/FGFR interaction are a promising subject for clinical trials. PMID:23880303

  20. Design and characteristics of cytotoxic fibroblast growth factor 1 conjugate for fibroblast growth factor receptor-targeted cancer therapy

    PubMed Central

    Szlachcic, Anna; Zakrzewska, Malgorzata; Lobocki, Michal; Jakimowicz, Piotr; Otlewski, Jacek

    2016-01-01

    Fibroblast growth factor receptors (FGFRs) are attractive candidate cancer therapy targets as they are overexpressed in multiple types of tumors, such as breast, prostate, bladder, and lung cancer. In this study, a natural ligand of FGFR, an engineered variant of fibroblast growth factor 1 (FGF1V), was conjugated to a potent cytotoxic drug, monomethyl auristatin E (MMAE), and used as a targeting agent for cancer cells overexpressing FGFRs, similar to antibodies in antibody–drug conjugates. The FGF1V–valine–citrulline–MMAE conjugate showed a favorable stability profile, bound FGFRs on the cell surface specifically, and efficiently released the drug (MMAE) upon cleavage by the lysosomal protease cathepsin B. Importantly, the conjugate showed a prominent cytotoxic effect toward cell lines expressing FGFR. FGF1V–vcMMAE was highly cytotoxic at concentrations even an order of magnitude lower than those found for free MMAE. This effect was FGFR-specific as cells lacking FGFR did not show any increased mortality. PMID:27563235

  1. Design and characteristics of cytotoxic fibroblast growth factor 1 conjugate for fibroblast growth factor receptor-targeted cancer therapy.

    PubMed

    Szlachcic, Anna; Zakrzewska, Malgorzata; Lobocki, Michal; Jakimowicz, Piotr; Otlewski, Jacek

    2016-01-01

    Fibroblast growth factor receptors (FGFRs) are attractive candidate cancer therapy targets as they are overexpressed in multiple types of tumors, such as breast, prostate, bladder, and lung cancer. In this study, a natural ligand of FGFR, an engineered variant of fibroblast growth factor 1 (FGF1V), was conjugated to a potent cytotoxic drug, monomethyl auristatin E (MMAE), and used as a targeting agent for cancer cells overexpressing FGFRs, similar to antibodies in antibody-drug conjugates. The FGF1V-valine-citrulline-MMAE conjugate showed a favorable stability profile, bound FGFRs on the cell surface specifically, and efficiently released the drug (MMAE) upon cleavage by the lysosomal protease cathepsin B. Importantly, the conjugate showed a prominent cytotoxic effect toward cell lines expressing FGFR. FGF1V-vcMMAE was highly cytotoxic at concentrations even an order of magnitude lower than those found for free MMAE. This effect was FGFR-specific as cells lacking FGFR did not show any increased mortality. PMID:27563235

  2. Inhibitory activities of omega-3 Fatty acids and traditional african remedies on keloid fibroblasts.

    PubMed

    Olaitan, Peter B; Chen, I-Ping; Norris, James E C; Feinn, Richard; Oluwatosin, Odunayo M; Reichenberger, Ernst J

    2011-04-01

    Keloids develop when scar tissue responds to skin trauma with proliferative fibrous growths that extend beyond the boundaries of the original wound and progress for several months or years. Keloids most frequently occur in individuals of indigenous sub-Saharan African origin. The etiology for keloids is still unknown and treatment can be problematic as patients respond differently to various treatment modalities. Keloids have a high rate of recurrence following surgical excision. Some West African patients claim to have had successful outcomes with traditional African remedies-boa constrictor oil (BCO) and shea butter-leading the authors to investigate their effects on cultured fibroblasts. The effects of emulsions of BCO, fish oil, isolated omega-3 fatty acids, and shea butter were tested in comparison to triamcinolone regarding inhibition of cell growth in keloid and control fibroblast cultures. In a series of controlled studies, it was observed that fish oil and BCO were more effective than triamcinolone, and that cis-5, 8, 11, 14, 17-eicosapentaenoic acid was more effective than -linolenic acid. While cell counts in control cultures continuously decreased over a period of 5 days, cell counts in keloid cultures consistently declined between day 1 and day 3, and then increased between day 3 and day 5 for all tested reagents except for fish oil. These results suggest that oils rich in omega-3 fatty acids may be effective in reducing actively proliferating keloid fibroblasts. Additional studies are warranted to investigate whether oils rich in omega-3 fatty acids offer effective and affordable treatment for some keloid patients, especially in the developing world. PMID:24489452

  3. Construction of a yeast artificial chromosome contig encompassing the human acidic fibroblast growth factor (FGF1) gene: Toward the cloning of the ANLL/MDS tumor-suppressor gene

    SciTech Connect

    Chiu, Ing-Ming; Gilmore, E.C.; Liu, Yang; Payson, R.A. )

    1994-02-01

    The region surrounding the human acidic fibroblast growth factor (FGF1) locus on chromosome 5q31 is of particular interest since it represents a critical region consistently lost in acute nonlymphocytic leukemia (ANLL) or myelodysplastic syndrome (MDS) patients who have a demonstrable deletion of the distal portion of the long arm of chromosome 5. It is proposed that an ANLL/MDS leukemia suppressor gene resides on 5q31. The authors have previously shown that the gene is most likely localized between FGF1 and PDGFRB/CSF1R loci. The region has also been linked to at least four other genetic diseases, Treacher Collins syndrome, diastrophic dysplasia, limb-girdle muscular dystrophy, and an autosomal dominant deafness, by linkage analysis. Here, they describe yeast artificial chromosomes (YAC) spanning 450 kb around the FGF1 gene. Six YAC clones were isolated from a human YAC library and their restriction enzyme maps were determined. The overlap of the clones with each other and with FGF1 cosmid and phage clones was characterized. Three of the YAC clones were found to contain the entire FGF1 gene, which spans more than 100 kb. Proximal and distal ends of several of these YAC clones were isolated for further overlap cloning. The proximal ends of both Y2 and Y4 were localized to previously isolated FGF1 DNA by sequence analysis. The distal ends of these two clones also hybridized to a human-hamster hybrid containing chromosome 5 as the only human genetic material. These results suggest that these YAC clones represent colinear DNA around the FGF1 locus. None of the YAC clones were found to contain the CD 14 and GRL genes, the closest known proximal and distal markers (relative to the centromere) to the FGF1 gene, respectively. This contig is useful for the overlap cloning of the 5q31 region and for reverse genetic strategies for the isolation of disease genes in the region. 46 refs., 7 figs., 5 tabs.

  4. Role of the fibroblast growth factor receptor axis in cholangiocarcinoma.

    PubMed

    Ang, Celina

    2015-07-01

    Advanced cholangiocarcinoma (CCA) is a highly lethal disease with limited therapeutic options beyond cytotoxic chemotherapy. Molecular profiling of CCA has provided insights into the pathogenesis of this disease and identified potential therapeutic targets. The fibroblast growth factor receptor (FGFR) axis is important for maintaining tissue homeostasis. Aberrations in FGFR activity have been implicated in the development and progression of CCA and other malignancies, which has generated significant interest in exploring FGFR's therapeutic potential. FGFR2 fusion events are present in up to 17% of intrahepatic CCAs and appear to predict sensitivity to FGFR inhibitors even after progression on chemotherapy. These observations have led to a clinical trial evaluating FGFR inhibition in patients with CCA enriched for FGFR alterations. This review summarizes current knowledge about the role of the FGFR pathway in cholangiocarcinogenesis and ongoing work in developing FGFR-directed therapies as an antineoplastic strategy for CCA. PMID:25678238

  5. Fibroblast Growth Factors: Biology, Function, and Application for Tissue Regeneration

    PubMed Central

    Yun, Ye-Rang; Won, Jong Eun; Jeon, Eunyi; Lee, Sujin; Kang, Wonmo; Jo, Hyejin; Jang, Jun-Hyeog; Shin, Ueon Sang; Kim, Hae-Won

    2010-01-01

    Fibroblast growth factors (FGFs) that signal through FGF receptors (FGFRs) regulate a broad spectrum of biological functions, including cellular proliferation, survival, migration, and differentiation. The FGF signal pathways are the RAS/MAP kinase pathway, PI3 kinase/AKT pathway, and PLCγ pathway, among which the RAS/MAP kinase pathway is known to be predominant. Several studies have recently implicated the in vitro biological functions of FGFs for tissue regeneration. However, to obtain optimal outcomes in vivo, it is important to enhance the half-life of FGFs and their biological stability. Future applications of FGFs are expected when the biological functions of FGFs are potentiated through the appropriate use of delivery systems and scaffolds. This review will introduce the biology and cellular functions of FGFs and deal with the biomaterials based delivery systems and their current applications for the regeneration of tissues, including skin, blood vessel, muscle, adipose, tendon/ligament, cartilage, bone, tooth, and nerve tissues. PMID:21350642

  6. Biology of FGFRL1, the fifth fibroblast growth factor receptor.

    PubMed

    Trueb, Beat

    2011-03-01

    FGFRL1 (fibroblast growth factor receptor like 1) is the most recently discovered member of the FGFR family. It contains three extracellular Ig-like domains similar to the classical FGFRs, but it lacks the protein tyrosine kinase domain and instead contains a short intracellular tail with a peculiar histidine-rich motif. The gene for FGFRL1 is found in all metazoans from sea anemone to mammals. FGFRL1 binds to FGF ligands and heparin with high affinity. It exerts a negative effect on cell proliferation, but a positive effect on cell differentiation. Mice with a targeted deletion of the Fgfrl1 gene die perinatally due to alterations in their diaphragm. These mice also show bilateral kidney agenesis, suggesting an essential role for Fgfrl1 in kidney development. A human patient with a frameshift mutation exhibits craniosynostosis, arguing for an additional role of FGFRL1 during bone formation. FGFRL1 contributes to the complexity of the FGF signaling system. PMID:21080029

  7. Fibroblast growth factor signaling in skeletal development and disease

    PubMed Central

    Ornitz, David M.; Marie, Pierre J.

    2015-01-01

    Fibroblast growth factor (FGF) signaling pathways are essential regulators of vertebrate skeletal development. FGF signaling regulates development of the limb bud and formation of the mesenchymal condensation and has key roles in regulating chondrogenesis, osteogenesis, and bone and mineral homeostasis. This review updates our review on FGFs in skeletal development published in Genes & Development in 2002, examines progress made on understanding the functions of the FGF signaling pathway during critical stages of skeletogenesis, and explores the mechanisms by which mutations in FGF signaling molecules cause skeletal malformations in humans. Links between FGF signaling pathways and other interacting pathways that are critical for skeletal development and could be exploited to treat genetic diseases and repair bone are also explored. PMID:26220993

  8. Recombinant basic fibroblast growth factor accelerates wound healing.

    PubMed

    McGee, G S; Davidson, J M; Buckley, A; Sommer, A; Woodward, S C; Aquino, A M; Barbour, R; Demetriou, A A

    1988-07-01

    Basic fibroblast growth factor (bFGF) stimulates extracellular matrix metabolism, growth, and movement of mesodermally derived cells. We have previously shown that collagen content in polyvinyl alcohol sponges increased after bFGF treatment. We hypothesized that bFGF-treated incisional wounds would heal more rapidly. After intraperitoneal pentobarbital anesthesia, male, 200- to 250-g, Sprague-Dawley rats (n = 27) each underwent two sets of paired, transverse, dorsal incisions closed with steel sutures. On Day 3 postwounding, 0.4 ml of bFGF (recombinant, 400 ng. Synergen) or normal saline was injected into one of each paired incisions. Animals were killed with ether on postwounding Days 5, 6, and 7 and their dorsal pelts were excised. Fresh or formalin-fixed wound strips were subjected to tensile strength measurements using a tensiometer. Breaking energy was calculated. Wound collagen content (hydroxyproline) was measured in wound-edge samples following hydrolysis using high-performance liquid chromatography. There was an overall significant increase in fresh wound tensile strength (13.7 +/- 1.06 vs 19.1 +/- 1.99 g/mm, P less than 0.01) and wound breaking energy (476 +/- 47 vs 747 +/- 76 mm2, P less than 0.001) in bFGF-treated incisions. There was an increase in wound collagen content which was not statistically significant and there was no difference in fixed incisional tensile strength. Histologic examination showed better organization and maturation in bFGF wounds. Recombinant bFGF accelerates normal rat wound healing. This may be due to earlier accumulation of collagen and fibroblasts and/or to greater collagen crosslinking in bFGF-treated wounds. PMID:3392988

  9. Fibroblast growth factor (FGF) signaling in development and skeletal diseases

    PubMed Central

    Teven, Chad M.; Farina, Evan M.; Rivas, Jane; Reid, Russell R.

    2014-01-01

    Fibroblast growth factors (FGF) and their receptors serve many functions in both the developing and adult organism. Humans contain 18 FGF ligands and four FGF receptors (FGFR). FGF ligands are polypeptide growth factors that regulate several developmental processes including cellular proliferation, differentiation, and migration, morphogenesis, and patterning. FGF-FGFR signaling is also critical to the developing axial and craniofacial skeleton. In particular, the signaling cascade has been implicated in intramembranous ossification of cranial bones as well as cranial suture homeostasis. In the adult, FGFs and FGFRs are crucial for tissue repair. FGF signaling generally follows one of three transduction pathways: RAS/MAP kinase, PI3/AKT, or PLCγ. Each pathway likely regulates specific cellular behaviors. Inappropriate expression of FGF and improper activation of FGFRs are associated with various pathologic conditions, unregulated cell growth, and tumorigenesis. Additionally, aberrant signaling has been implicated in many skeletal abnormalities including achondroplasia and craniosynostosis. The biology and mechanisms of the FGF family have been the subject of significant research over the past 30 years. Recently, work has focused on the therapeutic targeting and potential of FGF ligands and their associated receptors. The majority of FGF-related therapy is aimed at age-related disorders. Increased understanding of FGF signaling and biology may reveal additional therapeutic roles, both in utero and postnatally. This review discusses the role of FGF signaling in general physiologic and pathologic embryogenesis and further explores it within the context of skeletal development. PMID:25679016

  10. A p53 growth arrest protects fibroblasts from anticancer agents.

    PubMed

    McCormack, E S; Bruskin, A M; Borzillo, G V

    1997-01-01

    Reversible inhibitors of the cell cycle such as the TGF-betas have been exploited to protect dividing cells from exposure to anticancer drugs and radiation. Here, rat embryo fibroblast (REF) lines expressing different p53 mutations were used to test whether the p53 growth arrest could also chemoprotect cells from high doses of anticancer drugs. Whereas the doubling times of the different REF lines at 37 degrees C were similar, cells bearing temperature-sensitive mutations (mouse 135V or human 143A) were growth arrested at 31 degrees C. Temperature-dependent p53 activity was associated with increased levels of MDM2 and p21/WAF1, and the induction of an integrated p53-responsive luciferase gene. The REF lines exhibited similar sensitivities to common anticancer drugs when grown at 37 degrees C. However, when exposed to the same agents following transient incubation at 31 degrees C, the p53-arrested cells exhibited a marked survival advantage as shown by colony-forming assays. Chemoprotection was not universal, in that colony formation was not enhanced significantly after treatment with cisplatin or 5-fluorouracil, two drugs which can cause cellular damage throughout the cell cycle. Like other negative growth regulators, an activated p53 checkpoint may mediate the survival of cells exposed to drugs that target DNA synthesis or mitosis. PMID:9351895

  11. Fibroblast Growth Factor-23 in Bed Rest and Spaceflight

    NASA Technical Reports Server (NTRS)

    Bokhari, R.; Zwart, S. R; Fields, E.; Heer, M.; Sibonga, J.; Smith, S. M.

    2014-01-01

    Many nutritional factors influence bone, from the basics of calcium and vitamin D, to factors which influence bone through acid/base balance, including protein, sodium, and more. Fibroblast growth factor 23 (FGF23) is a recently identified factor, secreted from osteocytes, which is involved in classic (albeit complex) feedback loops controlling phosphorus homeostasis through both vitamin D and parathyroid hormone (PTH) (1, 2). As osteocytes are gravity sensing cells, it is important to determine if there are changes in FGF23 during spaceflight. In extreme cases, such as chronic kidney disease, FGF23 levels are highly elevated. FGF23 imbalances, secondary to dietary influences, may contribute to skeletal demineralization and kidney stone risk during spaceflight. Presented with an imbalanced dietary phosphorus to calcium ratio, increased secretion of FGF23 will inhibit renal phosphorus reabsorption, resulting in increased excretion and reduced circulating phosphorus. Increased intake and excretion of phosphorus is associated with increased kidney stone risk in both the terrestrial and microgravity environments. Highly processed foods and carbonated beverages are associated with higher phosphorus content. Ideally, the dietary calcium to phosphorus ratio should be at minimum 1:1. Nutritional requirements for spaceflight suggest that this ratio not be less than 0.67 (3), while the International Space Station (ISS) menu provides 1020 mg Ca and 1856 mg P, for a ratio of 0.55 (3). Subjects in NASA's bed rest studies, by design, have consumed intake ratios much closer to 1.0 (4). FGF23 also has an inhibitory influence on PTH secretion and 1(alpha)-hydroxylase, both of which are required for activating vitamin D with the conversion of 25-hydroxyvitamin D to 1,25-dihydroxyvitamin D. Decreased 1,25-dihydroxyvitamin D will result in decreased intestinal phosphorus absorption, and increased urinary phosphorus excretion (via decreased renal reabsorption). Should a decrease in 1

  12. Fibroblast growth factor is an inhibitor of chondrocyte terminal differentiation

    SciTech Connect

    Kato, Y.; Iwamoto, M. )

    1990-04-05

    The effects of basic fibroblast growth factor (bFGF) on terminal differentiation of chondrocytes and cartilage-matrix calcification were investigated. Rabbit growth-plate chondrocytes maintained as a pelleted mass in a centrifuge tube produced an abundant proteoglycan matrix during the matrix-maturation stage, yielding a cartilage-like tissue. Thereafter, they terminally differentiated to hypertrophic chondrocytes which produced high levels of alkaline phosphatase. These cells induced extensive calcification of the matrix in the absence of additional phosphate. Addition of bFGF to the chondrocyte cultures abolished the increases in alkaline phosphatase activity, {sup 45}Ca deposition, and the calcium content. These effects were dose-dependent, reversible, and observed in the presence of cytosine arabinoside, an inhibitor of DNA synthesis. The inhibitory effects could be observed only when chondrocytes were exposed to bFGF in a transition period between the matrix-maturation and hypertrophic stages. As chondrocytes differentiated to hypertrophic cells, bFGF became less effective in inhibiting the expression of the mineralization-related phenotypes. The present study also shows that although the rate of ({sup 35}S)sulfate incorporation into large, chondroitin sulfate proteoglycan in the cell-matrix fraction is very high during the matrix-maturation stage, it abruptly decreases by 90% after terminal differentiation. Furthermore, the terminal differentiation-associated decrease in proteoglycan synthesis was delayed by bFGF. These results provide evidence that bFGF inhibits terminal differentiation of chondrocytes and calcification.

  13. Binding, internalization, and degradation of basic fibroblast growth factor in human microvascular endothelial cells

    SciTech Connect

    Bikfalvi, A.; Dupuy, E.; Inyang, A.L.; Tobelem, G. ); Fayein, N.; Courtois, Y. ); Leseche, G. )

    1989-03-01

    The binding, internalization, and degradation of basic fibroblast growth factor (bFGF) in human omental microvascular endothelial cells (HOME cells) were investigated. Binding studies of bFGF in human endothelial cells have not yet been reported. Basic FGF bound to HOME cells. The number of low-affinity binding sites was found to be variable. Washing the cells with 2 M phosphate-buffered saline removed completely {sup 125}I-bFGF bound to low-affinity binding sites but decreased also the high-affinity binding. The majority of the surface-bound {sup 125}I-bFGF was removed by washing the cells with acetic acid buffer at pH 3. At this temperature, degradation of the internalized ligand was followed after 1 hour by the appearance of three major bands of 15,000 10,000, and 8,000 Da and was inhibited by chloroquine. These results demonstrated two classes of binding sites for bFGF in HOME cells; the number of high-affinity binding sites being larger than the number reported for bovine capillary endothelial cells. The intracellular processing of bFGF in HOME cells seems to be different from that of heparin binding growth factor-1 in murine lung capillary endothelial cells and of eye-derived growth factor-1 in Chinese hamster fibroblasts.

  14. Induction of fibroblast apolipoprotein E expression during apoptosis, starvation-induced growth arrest and mitosis.

    PubMed Central

    Quinn, Carmel M; Kågedal, Katarina; Terman, Alexei; Stroikin, Uri; Brunk, Ulf T; Jessup, Wendy; Garner, Brett

    2004-01-01

    Apolipoprotein E (apoE) mediates the hepatic clearance of plasma lipoproteins, facilitates cholesterol efflux from macrophages and aids neuronal lipid transport. ApoE is expressed at high levels in hepatocytes, macrophages and astrocytes. In the present study, we identify nuclear and cytosolic pools of apoE in human fibroblasts. Fibroblast apoE mRNA and protein levels were up-regulated during staurosporine-induced apoptosis and this was correlated with increased caspase-3 activity and apoptotic morphological alterations. Because the transcription of apoE and specific pro-apoptotic genes is regulated by the nuclear receptor LXR (liver X receptor) alpha, we analysed LXRalpha mRNA expression by quantitative real-time PCR and found it to be increased before apoE mRNA induction. The expression of ABCA1 (ATP-binding cassette transporter A1) mRNA, which is also regulated by LXRalpha, was increased in parallel with apoE mRNA, indicating that LXRalpha probably promotes apoE and ABCA1 transcription during apoptosis. Fibroblast apoE levels were increased under conditions of serum-starvation-induced growth arrest and hyperoxia-induced senescence. In both cases, an increased nuclear apoE level was observed, particularly in cells that accumulated lipofuscin. Nuclear apoE was translocated to the cytosol when mitotic nuclear disassembly occurred and this was associated with an increase in total cellular apoE levels. ApoE amino acid sequence analysis indicated several potential sites for phosphorylation. In vivo studies, using 32P-labelling and immunoprecipitation, revealed that fibroblast apoE can be phosphorylated. These studies reveal novel associations and potential roles for apoE in fundamental cellular processes. PMID:14656220

  15. Effect of transforming growth factor beta on synthesis of glycosaminoglycans by human lung fibroblasts

    SciTech Connect

    Dubaybo, B.A.; Thet, L.A. )

    1990-09-01

    The processes of lung growth, injury, and repair are characterized by alterations in fibroblast synthesis and interstitial distribution of extracellular matrix components. Transforming growth factor beta (TGF-beta), which is postulated to play a role in modulating lung repair, alters the distribution of several matrix components such as collagen and fibronectin. We studied the effect of TGF-beta on the synthesis and distribution of the various glycosaminoglycans (GAGs) and whether these effects may explain its role in lung repair. Human diploid lung fibroblasts (IMR-90) were exposed to various concentrations of TGF-beta (0-5 nM) for variable periods of time (0-18 h). Newly synthesized GAGs were labeled with either (3H)glucosamine or (35S)sulfate. Individual GAGs were separated by size exclusion chromatography after serial enzymatic and chemical digestions and quantitated using scintillation counting. There was a dose-dependent increase in total GAG synthesis with maximal levels detected after 6 h of exposure. This increase was noted in all individual GAG types measured and was observed in both the cell associated GAGs (cell-matrix fraction) as well as the GAGs released into the medium (medium fraction). In the cell-matrix fraction, TGF-beta increased the proportion of heparan sulfate that was membrane bound as well as the proportion of dermatan sulfate in the intracellular compartment. In the medium fraction, TGF-beta increased the proportion of hyaluronic acid, chondroitin sulfate and dermatan sulfate released. We conclude that the role of TGF-beta in lung growth and repair may be related to increased synthesis of GAGs by human lung fibroblasts as well as alterations in the distribution of individual GAGs.

  16. The suppression of fibroblast growth factor 2/fibroblast growth factor 4-dependent tumour angiogenesis and growth by the anti-growth factor activity of dextran derivative (CMDB7).

    PubMed Central

    Bagheri-Yarmand, R.; Kourbali, Y.; Mabilat, C.; Morère, J. F.; Martin, A.; Lu, H.; Soria, C.; Jozefonvicz, J.; Crépin, M.

    1998-01-01

    Our previous studies showed that carboxymethyl benzylamide dextran (CMDB7) blocks basic fibroblast growth factor (FGF-2)-dependent cell proliferation of a human breast epithelial line (HBL100), suggesting its potential role as a potent antiangiogenic substance. The derived cell line (HH9), which was transformed with the hst/FGF4 gene, has been shown to be highly proliferative in vitro and to induce angiogenic tumours in nude mice. We show here that CMDB7 inhibits the mitogenic activities of the conditioned media from HBL 100 and HH9 cells in a dose-dependent manner. When HH9 cells were injected s.c. into nude mice, CMDB7 treatment (300 mg kg(-1) week(-1)) suppressed the tumour take and the tumour growth by about 50% and 80% respectively. Immunohistochemical analysis showed a highly significant decrease, by more than threefold, in the endothelial density of viable tumour regions, together with a significant increase in the necrosis area. This antiangiogenic activity of CMDB7 was further demonstrated by direct inhibition of calf pulmonary artery (CPAE) and human umbilical vein (HUVEC) endothelial cell proliferation and migration in vitro. In addition, we showed that CMDB7 inhibits specifically the mitogenic effects of the growth factors that bind to heparin such as FGF-2, FGF-4, platelet-derived growth factor (PDGF-BB) and transforming growth factor (TGF-beta1), but not those of epidermal growth factor (EGF) and insulin-like growth factor (IGF-1). These results demonstrate that CMDB7 inhibits FGF-2/FGF-4-dependent tumour growth and angiogenesis, most likely by disrupting the autocrine and paracrine effects of growth factors released from the tumour cells. Images Figure 4 PMID:9662260

  17. The Use of Fibroblast Growth Factor 23 Testing in Patients with Kidney Disease

    PubMed Central

    2014-01-01

    The emergence of fibroblast growth factor 23 as a potentially modifiable risk factor in CKD has led to growing interest in its measurement as a tool to assess patient risk and target therapy. This review discusses the analytical and clinical challenges faced in translating fibroblast growth factor 23 testing into routine practice. As for other bone mineral markers, agreement between commercial fibroblast growth factor 23 assays is poor, mainly because of differences in calibration, but also, these differences reflect the variable detection of hormone fragments. Direct comparison of readout from different assays is consequently limited and likely hampers setting uniform fibroblast growth factor 23–directed targets. Efforts are needed to standardize assay output to enhance clinical use. Fibroblast growth factor 23 is robustly associated with cardiovascular and renal outcomes in patients with CKD and adds value to risk assessments based on conventional risk factors. Compared with most other mineral markers, fibroblast growth factor 23 shows better intraindividual temporal stability, with minimal diurnal and week-to-week variability, but substantial interindividual variation, maximizing discriminative power for risk stratification. Conventional therapeutic interventions for the CKD–mineral bone disorder, such as dietary phosphate restriction and use of oral phosphate binders or calcimimetics, are associated with variable efficacy at modulating circulating fibroblast growth factor 23 concentrations, like they are for other mineral metabolites. Dual therapy with dietary phosphate restriction and noncalcium-based binder use achieves the most consistent fibroblast growth factor 23–lowering effect and seems best monitored using an intact assay. Additional studies are needed to evaluate whether strategies aimed at reducing levels or antagonizing its action have beneficial effects on clinical outcomes in CKD patients. Moreover, a better understanding of the mechanisms

  18. X-Ray structure and biophysical properties of rabbit fibroblast growth factor 1

    SciTech Connect

    Lee, Jihun; Blaber, Sachiko I.; Irsigler, Andre; Aspinwall, Eric; Blaber, Michael

    2010-01-14

    The rabbit is an important and de facto animal model in the study of ischemic disease and angiogenic therapy. Additionally, fibroblast growth factor 1 (FGF-1) is emerging as one of the most important growth factors for novel pro-angiogenic and pro-arteriogenic therapy. However, despite its significance, the fundamental biophysical properties of rabbit FGF-1, including its X-ray structure, have never been reported. Here, the cloning, crystallization, X-ray structure and determination of the biophysical properties of rabbit FGF-1 are described. The X-ray structure shows that the amino-acid differences between human and rabbit FGF-1 are solvent-exposed and therefore potentially immunogenic, while the biophysical studies identify differences in thermostability and receptor-binding affinity that distinguish rabbit FGF-1 from human FGF-1.

  19. The influence of different nanostructured scaffolds on fibroblast growth

    NASA Astrophysics Data System (ADS)

    Chung, I.-Cheng; Li, Ching-Wen; Wang, Gou-Jen

    2013-08-01

    Skin serves as a protective barrier, modulating body temperature and waste discharge. It is therefore desirable to be able to repair any damage that occurs to the skin as soon as possible. In this study, we demonstrate a relatively easy and cost-effective method for the fabrication of nanostructured scaffolds, to shorten the time taken for a wound to heal. Various scaffolds consisting of nanohemisphere arrays of poly(lactic-co-glycolic acid) (PLGA), polylactide and chitosan were fabricated by casting using a nickel (Ni) replica mold. The Ni replica mold is electroformed using the highly ordered nanohemisphere array of the barrier-layer surface of an anodic aluminum oxide membrane as the template. Mouse fibroblast cells (L929s) were cultured on the nanostructured polymer scaffolds to investigate the effect of these different nanohemisphere arrays on cell proliferation. The concentration of collagen type I on each scaffold was then measured through enzyme-linked immunosorbent assay to find the most effective scaffold for shortening the wound-healing process. The experimental data indicate that the proliferation of L929 is superior when a nanostructured PLGA scaffold with a feature size of 118 nm is utilized.

  20. Fibroblast growth factors as tissue repair and regeneration therapeutics.

    PubMed

    Nunes, Quentin M; Li, Yong; Sun, Changye; Kinnunen, Tarja K; Fernig, David G

    2016-01-01

    Cell communication is central to the integration of cell function required for the development and homeostasis of multicellular animals. Proteins are an important currency of cell communication, acting locally (auto-, juxta-, or paracrine) or systemically (endocrine). The fibroblast growth factor (FGF) family contributes to the regulation of virtually all aspects of development and organogenesis, and after birth to tissue maintenance, as well as particular aspects of organism physiology. In the West, oncology has been the focus of translation of FGF research, whereas in China and to an extent Japan a major focus has been to use FGFs in repair and regeneration settings. These differences have their roots in research history and aims. The Chinese drive into biotechnology and the delivery of engineered clinical grade FGFs by a major Chinese research group were important enablers in this respect. The Chinese language clinical literature is not widely accessible. To put this into context, we provide the essential molecular and functional background to the FGF communication system covering FGF ligands, the heparan sulfate and Klotho co-receptors and FGF receptor (FGFR) tyrosine kinases. We then summarise a selection of clinical reports that demonstrate the efficacy of engineered recombinant FGF ligands in treating a wide range of conditions that require tissue repair/regeneration. Alongside, the functional reasons why application of exogenous FGF ligands does not lead to cancers are described. Together, this highlights that the FGF ligands represent a major opportunity for clinical translation that has been largely overlooked in the West. PMID:26793421

  1. Fibroblast growth factors as tissue repair and regeneration therapeutics

    PubMed Central

    Kinnunen, Tarja K.

    2016-01-01

    Cell communication is central to the integration of cell function required for the development and homeostasis of multicellular animals. Proteins are an important currency of cell communication, acting locally (auto-, juxta-, or paracrine) or systemically (endocrine). The fibroblast growth factor (FGF) family contributes to the regulation of virtually all aspects of development and organogenesis, and after birth to tissue maintenance, as well as particular aspects of organism physiology. In the West, oncology has been the focus of translation of FGF research, whereas in China and to an extent Japan a major focus has been to use FGFs in repair and regeneration settings. These differences have their roots in research history and aims. The Chinese drive into biotechnology and the delivery of engineered clinical grade FGFs by a major Chinese research group were important enablers in this respect. The Chinese language clinical literature is not widely accessible. To put this into context, we provide the essential molecular and functional background to the FGF communication system covering FGF ligands, the heparan sulfate and Klotho co-receptors and FGF receptor (FGFR) tyrosine kinases. We then summarise a selection of clinical reports that demonstrate the efficacy of engineered recombinant FGF ligands in treating a wide range of conditions that require tissue repair/regeneration. Alongside, the functional reasons why application of exogenous FGF ligands does not lead to cancers are described. Together, this highlights that the FGF ligands represent a major opportunity for clinical translation that has been largely overlooked in the West. PMID:26793421

  2. Experimental Myocardial Infarction Upregulates Circulating Fibroblast Growth Factor‐23

    PubMed Central

    Andrukhova, Olena; Slavic, Svetlana; Odörfer, Kathrin I; Erben, Reinhold G

    2015-01-01

    ABSTRACT Myocardial infarction (MI) is a major cause of death worldwide. Epidemiological studies have linked vitamin D deficiency to MI incidence. Because fibroblast growth factor‐23 (FGF23) is a master regulator of vitamin D hormone production and has been shown to be associated with cardiac hypertrophy per se, we explored the hypothesis that FGF23 may be a previously unrecognized pathophysiological factor causally linked to progression of cardiac dysfunction post‐MI. Here, we show that circulating intact Fgf23 was profoundly elevated, whereas serum vitamin D hormone levels were suppressed, after induction of experimental MI in rat and mouse models, independent of changes in serum soluble Klotho or serum parathyroid hormone. Both skeletal and cardiac expression of Fgf23 was increased after MI. Although the molecular link between the cardiac lesion and circulating Fgf23 concentrations remains to be identified, our study has uncovered a novel heart–bone–kidney axis that may have important clinical implications and may inaugurate the new field of cardio‐osteology. © 2015 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research (ASBMR). PMID:25858796

  3. Fibroblast growth factors, old kids on the new block.

    PubMed

    Li, Xiaokun; Wang, Cong; Xiao, Jian; McKeehan, Wallace L; Wang, Fen

    2016-05-01

    The fibroblast growth factors (FGFs) are a family of cell intrinsic regulatory peptides that control a broad spectrum of cellular activities. The family includes canonic FGFs that elicit their activities by activating the FGF receptor (FGFR) tyrosine kinase and non-canonic members that elicit their activities intracellularly and via FGFR-independent mechanisms. The FGF signaling axis is highly complex due to the existence of multiple isoforms of both ligands and receptors, as well as cofactors that include the chemically heterogeneous heparan sulfate (HS) cofactors, and in the case of endocrine FGFs, the Klotho coreceptors. Resident FGF signaling controls embryonic development, maintains tissue homeostasis, promotes wound healing and tissue regeneration, and regulates functions of multiple organs. However, ectopic or aberrant FGF signaling is a culprit for various diseases, including congenital birth defects, metabolic disorder, and cancer. The molecular mechanisms by which the specificity of FGF signaling is achieved remain incompletely understood. Since its application as a druggable target has been gradually recognized by pharmaceutical companies and translational researchers, understanding the determinants of FGF signaling specificity has become even more important in order to get into the position to selectively suppress a particular pathway without affecting others to minimize side effects. PMID:26768548

  4. Hyaluronic acid abrogates ethanol-dependent inhibition of collagen biosynthesis in cultured human fibroblasts

    PubMed Central

    Donejko, Magdalena; Przylipiak, Andrzej; Rysiak, Edyta; Miltyk, Wojciech; Galicka, Elżbieta; Przylipiak, Jerzy; Zaręba, Ilona; Surazynski, Arkadiusz

    2015-01-01

    Introduction The aim of the study was to evaluate the effect of ethanol on collagen biosynthesis in cultured human skin fibroblasts, and the role of hyaluronic acid (HA) in this process. Regarding the mechanism of ethanol action on human skin fibroblasts we investigated: expression of β1 integrin and insulin-like growth factor 1 receptor (IGF-IR), signaling pathway protein expression: mitogen-activated protein kinases (MAPKs), protein kinase B (Akt), nuclear factor kappa B (NF-κB) transcription factor, cytotoxicity assay and apoptosis, metalloproteinase activity, as well as the influence of HA on these processes. Materials and methods Collagen biosynthesis, activity of prolidase, DNA biosynthesis, and cytotoxicity were measured in confluent human skin fibroblast cultures that have been treated with 25, 50, and 100 mM ethanol and with ethanol and 500 µg/mL HA. Western blot analysis and zymography were performed to evaluate expression of collagen type I, β1 integrin receptor, IGF-IR, NF-κB protein, phospho-Akt protein, kinase MAPK, caspase 9 activity, and matrix metalloproteinases (MMP-9 and MMP-2). Results Ethanol in a dose-dependent manner lead to the impairment of collagen biosynthesis in fibroblast cultures through decreasing prolidase activity and expression of β1 integrin and IGF-IR. This was accompanied by an increased cytotoxicity, apoptosis and lowered expression of the signaling pathway proteins induced by β1 integrin and IGF-IR, that is, MAPK (ERK1/2) kinases. The lowered amount of synthesized collagen and prolidase activity disturbance may also be due to the activation of NF-κB transcription factor, which inhibits collagen gene expression. It suggests that the decrease in fibroblast collagen production may be caused by the disturbance in its biosynthesis but not degradation. The application of HA has a protective effect on disturbances caused by the examined substances. It seems that regulatory mechanism of ethanol-induced collagen aberration take

  5. Improved cellular response of ion modified poly(lactic acid-co-glycolic acid) substrates for mouse fibroblast cells.

    PubMed

    Adhikari, Ananta Raj; Geranpayeh, Tanya; Chu, Wei Kan; Otteson, Deborah C

    2016-03-01

    In this report, the effects of argon (Ar) ion irradiation on poly(lactic acid-co-glycolic acid) (PLGA) substrates on biocompatibility were studied. PLGA scaffold substrates were prepared by spin coating glass surfaces with PLGA dissolved in anhydrous chloroform. Previously, we showed that surface modifications of PLGA films using ion irradiation modulate the inherent hydrophobicity of PLGA surface. Here we show that with increasing ion dose (1×10(12) to 1×10(14) ions/cm(2)), hydrophobicity and surface roughness decreased. Biocompatibility for NIH3T3 mouse fibroblast cells was increased by argon irradiation of PLGA substrates. On unirradiated PLGA films, fibroblasts had a longer doubling time and cell densities were 52% lower than controls after 48 h in vitro. Argon irradiated PLGA substrates supported growth rates similar to control. Despite differences in cell cycle kinetics, there was no detectible cytotoxicity observed on any substrate. This demonstrates that argon ion irradiation can be used to tune the surface microstructure and generate substrates that are more compatible for the cell growth and proliferation. PMID:26706518

  6. Neonatal fibroblast growth factor treatment enhances cocaine sensitization.

    PubMed

    Clinton, Sarah M; Turner, Cortney A; Flagel, Shelly B; Simpson, Danielle N; Watson, Stanley J; Akil, Huda

    2012-11-01

    Growth factors are critical in neurodevelopment and neuroplasticity, and recent studies point to their involvement in addiction. We previously reported increased levels of basic fibroblast growth factor (FGF2) in high novelty/drug-seeking rats (bred high responders, bHR) compared to low novelty/drug-seeking rats(bred low responders, bLRs). The present study asked whether an early life manipulation of the FGF system(a single FGF2 injection on postnatal day 2) can impact cocaine sensitization and associated neurobiological markers in adult bHR/bLR animals. Neonatal FGF2- and vehicle-treated bHR/bLR rats were sensitized to cocaine(7 daily injections, 15 mg/kg/day, i.p.) in adulthood. Neonatal FGF2 markedly increased bLRs' typically low psychomotor sensitization to cocaine (day 7 locomotor response to cocaine), but had little effect on bHRs' cocaine sensitization. Gene expression studies examined dopaminergic molecules as well as FGF2 and the FGFR1 receptor in cocaine naïve animals, to investigate possible neurobiological alterations induced by neonatal FGF2 exposure that may influence behavioral response to cocaine. bLRs showed decreased tyrosine hydroxylase in the ventral tegmental area (VTA), decreased D1 and increased D2 receptor expression in the nucleus accumbens core, as well as decreased FGF2 in the VTA, substantia nigra, accumbens core, and caudate putamen compared to bHRs. Neonatal FGF2 selectively increased D1 receptor and FGF2 mRNA in the accumbens core of bLRs, which may contribute to their heightened cocaine sensitization. Our results suggest increased FGF2 in the mesodopaminergic circuit (as in baseline bHRs and neonatal FGF2-exposed bLRs vs. baseline bLRs) enhances an individual's susceptibility to cocaine sensitization and may increase vulnerability to drug seeking and addiction. PMID:22819969

  7. Neonatal Fibroblast Growth Factor Treatment Enhances Cocaine Sensitization

    PubMed Central

    Clinton, Sarah M.; Turner, Cortney A.; Flagel, Shelly B.; Simpson, Danielle N.; Watson, Stanley J.; Akil, Huda

    2012-01-01

    Growth factors are critical in neurodevelopment and neuroplasticity, and recent studies point to their involvement in addiction. We previously reported increased levels of basic fibroblast growth factor (FGF2) in high novelty/drug-seeking rats (bred High Responders, bHR) compared to low novelty/drug-seeking rats (bred Low Responders, bLRs). The present study asked whether an early life manipulation of the FGF system (a single FGF2 injection on postnatal day 2) can impact cocaine sensitization and associated neurobiological markers in adult bHR/bLR animals. Neonatal FGF2- and vehicle-treated bHR/bLR rats were sensitized to cocaine (7 daily injections, 15 mg/kg/day, i.p.) in adulthood. Neonatal FGF2 markedly increased bLRs’ typically low psychomotor sensitization to cocaine (day 7 locomotor response to cocaine), but had little effect on bHRs’ cocaine sensitization. Gene expression studies examined dopaminergic molecules as well as FGF2 and the FGFR1 receptor in cocaine naïve animals, to investigate possible neurobiological alterations induced by neonatal FGF2 exposure that may influence behavioral response to cocaine. bLRs showed decreased tyrosine hydroxylase in the ventral tegmental area (VTA), decreased D1 and increased D2 receptor expression in the nucleus accumbens core, as well as decreased FGF2 in the VTA, substantia nigra, accumbens core, and caudate putamen compared to bHRs. Neonatal FGF2 selectively increased D1 receptor and FGF2 mRNA in the accumbens core of bLRs, which may contribute to their heightened cocaine sensitization. Our results suggest increased FGF2 in the mesodopaminergic circuit (as in baseline bHRs and neonatal FGF2-exposed bLRs vs. baseline bLRs) enhances an individual’s susceptibility to cocaine sensitization and may increase vulnerability to drug seeking and addiction. PMID:22819969

  8. General pharmacology of recombinant human basic fibroblast growth factor.

    PubMed

    Okumura, M; Yajima, M; Nishimura, T; Ikeda, H; Nishimori, T

    1996-07-01

    General pharmacological effects of recombinant human basic fibroblast growth factor (bFGF) were investigated. 1. Central nervous system: Basic FGF produced almost no effect on the general symptoms and behaviors of mice. Basic FGF did not influence the spontaneous motor activity, hexobarbital-induced anesthesia, electroshock seizure threshold, pentylenetetrazole-induced seizure in mice and normal body temperature and spinal reflex in rats up to a dose of 1 mg/kg (s.c., i.v.). As regards pain sensation, it inhibited the acetic acid-induced writhing at 1 mg/kg (s.c.). No abnormal waves were observed in spontaneous EEG of the rabbit up to 1 mg/kg (i.v.) of bFGF, but at 0.1 mg/kg it had a slight effect on the ratio of EEG levels and at 1 mg/kg induced an increase in rest period, disappearance in the period of fast wave sleep and a decrease in the period of deep sleep. 2. Somatic nervous system: Basic FGF did not influence the corneal reflex, twitch response of the skin and diaphragm-phrenic nerve preparations. 3. Autonomic nervous system and smooth muscle: Basic FGF showed little effects on the spontaneous movement of the isolated ileum, contraction induced by various agonists in isolated ileum, resting tension and noradrenaline(NA)-induced contraction of the aorta, resting tension and histamine-induced contraction of isolated trachea, spontaneous movement and 5-HT-induced contraction of isolated strips of stomach fundus, NA-induced contraction of isolated vas deferens of the rat up to the concentration of 10(-4) g/ml. Basic FGF augmented the tone of the isolated non-pregnant uterus at the concentrations of 10(-5) g/ml and above and inhibited or tended to inhibit the contractile tension of non-pregnant or pregnant uterus at 10(-4) g/ml, but it did not influence the spontaneous movement of the uterus, either the non-pregnant or pregnant, under in situ conditions even at a dose of 1 mg/kg (i.v.). Basic FGF did not influence the pupil size. 4. Respiratory and circulatory

  9. Imatinib mesylate inhibits platelet derived growth factor stimulated proliferation of rheumatoid synovial fibroblasts

    SciTech Connect

    Sandler, Charlotta; Joutsiniemi, Saima; Lindstedt, Ken A.; Juutilainen, Timo; Kovanen, Petri T.; Eklund, Kari K. . E-mail: kari.eklund@hus.fi

    2006-08-18

    Synovial fibroblast is the key cell type in the growth of the pathological synovial tissue in arthritis. Here, we show that platelet-derived growth factor (PDGF) is a potent mitogen for synovial fibroblasts isolated from patients with rheumatoid arthritis. Inhibition of PDGF-receptor signalling by imatinib mesylate (1 {mu}M) completely abrogated the PDGF-stimulated proliferation and inhibited approximately 70% of serum-stimulated proliferation of synovial fibroblasts. Similar extent of inhibition was observed when PDGF was neutralized with anti-PDGF antibodies, suggesting that imatinib mesylate does not inhibit pathways other than those mediated by PDGF-receptors. No signs of apoptosis were detected in synovial fibroblasts cultured in the presence of imatinib. These results suggest that imatinib mesylate specifically inhibits PDGF-stimulated proliferation of synovial fibroblasts, and that inhibition of PDGF-receptors could represent a feasible target for novel antirheumatic therapies.

  10. Pentadecapeptide BPC 157 enhances the growth hormone receptor expression in tendon fibroblasts.

    PubMed

    Chang, Chung-Hsun; Tsai, Wen-Chung; Hsu, Ya-Hui; Pang, Jong-Hwei Su

    2014-01-01

    BPC 157, a pentadecapeptide derived from human gastric juice, has been demonstrated to promote the healing of different tissues, including skin, muscle, bone, ligament and tendon in many animal studies. However, the underlying mechanism has not been fully clarified. The present study aimed to explore the effect of BPC 157 on tendon fibroblasts isolated from Achilles tendon of male Sprague-Dawley rat. From the result of cDNA microarray analysis, growth hormone receptor was revealed as one of the most abundantly up-regulated genes in tendon fibroblasts by BPC 157. BPC 157 dose- and time-dependently increased the expression of growth hormone receptor in tendon fibroblasts at both the mRNA and protein levels as measured by RT/real-time PCR and Western blot, respectively. The addition of growth hormone to BPC 157-treated tendon fibroblasts dose- and time-dependently increased the cell proliferation as determined by MTT assay and PCNA expression by RT/real-time PCR. Janus kinase 2, the downstream signal pathway of growth hormone receptor, was activated time-dependently by stimulating the BPC 157-treated tendon fibroblasts with growth hormone. In conclusion, the BPC 157-induced increase of growth hormone receptor in tendon fibroblasts may potentiate the proliferation-promoting effect of growth hormone and contribute to the healing of tendon. PMID:25415472

  11. Expression of fibroblast growth factor 23 by canine soft tissue sarcomas.

    PubMed

    Hardcastle, M R; Dittmer, K E

    2016-09-01

    Tumour-induced osteomalacia (TIO) is a rare paraneoplastic syndrome of humans. Some mesenchymal tumours (often resembling haemangiopericytomas) express molecules that normally regulate phosphorus metabolism; most frequently, fibroblast growth factor 23. Patients develop renal phosphate wasting and inappropriately low serum concentrations of 1, 25 (OH)2 vitamin D3 , leading to osteomalacia. Surgical removal of the tumour is curative. The authors examined expression of canine fibroblast growth factor 23 in 49 soft tissue sarcomas, and control tissues from normal adult dogs. RNA extracted from bone or formalin-fixed, paraffin-embedded tissues was analysed by end point and quantitative reverse transcriptase-polymerase chain reaction. Fibroblast growth factor 23 expression was detected in bone, lung, kidney, lymph node and thymus. Fifteen of 49 sarcomas (31%) expressed fibroblast growth factor 23, three of these had high relative expression and some features resembling phosphatonin-expressing mesenchymal tumours of humans. Further work is required to determine whether TIO may occur in dogs. PMID:24923416

  12. Lactic Acid Bacteria Convert Human Fibroblasts to Multipotent Cells

    PubMed Central

    Ohta, Kunimasa; Kawano, Rie; Ito, Naofumi

    2012-01-01

    The human gastrointestinal tract is colonized by a vast community of symbionts and commensals. Lactic acid bacteria (LAB) form a group of related, low-GC-content, gram-positive bacteria that are considered to offer a number of probiotic benefits to general health. While the role of LAB in gastrointestinal microecology has been the subject of extensive study, little is known about how commensal prokaryotic organisms directly influence eukaryotic cells. Here, we demonstrate the generation of multipotential cells from adult human dermal fibroblast cells by incorporating LAB. LAB-incorporated cell clusters are similar to embryoid bodies derived from embryonic stem cells and can differentiate into endodermal, mesodermal, and ectodermal cells in vivo and in vitro. LAB-incorporated cell clusters express a set of genes associated with multipotency, and microarray analysis indicates a remarkable increase of NANOG, a multipotency marker, and a notable decrease in HOX gene expression in LAB-incorporated cells. During the cell culture, the LAB-incorporated cell clusters stop cell division and start to express early senescence markers without cell death. Thus, LAB-incorporated cell clusters have potentially wide-ranging implications for cell generation, reprogramming, and cell-based therapy. PMID:23300571

  13. Retinoic Acid, GABA-ergic, and TGF-β Signaling Systems Are Involved in Human Cleft Palate Fibroblast Phenotype

    PubMed Central

    Baroni, Tiziano; Bellucci, Catia; Lilli, Cinzia; Pezzetti, Furio; Carinci, Francesco; Becchetti, Ennio; Carinci, Paolo; Stabellini, Giordano; Calvitti, Mario; Lumare, Eleonora; Bodo, Maria

    2006-01-01

    During embryogenesis, a complex interplay between extracellular matrix (ECM) molecules, regulatory molecules, and growth factors mediates morphogenetic processes involved in palatogenesis. Transforming growth factor-β (TGF-β), retinoic acid (RA), and γ-aminobutyric acid (GABA)ergic signaling systems are also potentially involved. Using [3H]glucosamine and [35S]methionine incorporation, anion exchange chromatography, semiquantitative radioactive RT-PCR, and a TGF-β binding assay, we aimed to verify the presence of phenotypic differences between primary cultures of secondary palate (SP) fibroblasts from 2-year-old subjects with familial nonsyndromic cleft lip and/or palate (CLP-SP fibroblasts) and age-matched normal SP (N-SP) fibroblasts. The effects of RA—which, at pharmacologic doses, induces cleft palate in newborns of many species—were also studied. We found an altered ECM production in CLP-SP fibroblasts that synthesized and secreted more glycosaminoglycans (GAGs) and fibronectin (FN) compared with N-SP cells. In CLP-SP cells, TGF-β3 mRNA expression and TGF-β receptor number were higher and RA receptor-α (RARA) gene expression was increased. Moreover, we demonstrated for the first time that GABA receptor (GABRB3) mRNA expression was upregulated in human CLP-SP fibroblasts. In N-SP and CLP-SP fibroblasts, RA decreased GAG and FN secretion and increased TGF-β3 mRNA expression but reduced the number of TGF-β receptors. TGF-β receptor type I mRNA expression was decreased, TGF-β receptor type II was increased, and TGF-β receptor type III was not affected. RA treatment increased RARA gene expression in both cell populations but upregulated GABRB3 mRNA expression only in N-SP cells. These results show that CLP-SP fibroblasts compared with N-SP fibroblasts exhibit an abnormal phenotype in vitro and respond differently to RA treatment, and suggest that altered crosstalk between RA, GABAergic, and TGF-β signaling systems could be involved in human cleft

  14. Action of fibroblast growth factor-2 on the intervertebral disc

    PubMed Central

    Li, Xin; An, Howard S; Ellman, Michael; Phillips, Frank; Thonar, Eugene J; Park, Daniel K; Udayakumar, Ranjith K; Im, Hee-Jeong

    2008-01-01

    Introduction Fibroblast growth factor 2 (FGF2) is a growth factor that is immediately released after cartilage injury and plays a pivotal role in cartilage homeostasis. In human adult articular cartilage, FGF2 mediates anti-anabolic and potentially catabolic effects via the suppression of proteoglycan (PG) production along with the upregulation of matrix-degrading enzyme activity. The aim of the present study was to determine the biological effects of FGF2 in spine disc cells and to elucidate the complex biochemical pathways utilized by FGF2 in bovine intervertebral disc (IVD) cells in an attempt to further understand the pathophysiologic processes involved in disc degeneration. Methods We studied the effect of FGF2 on IVD tissue homeostasis by assessing MMP-13 expression (potent matrix-degrading enzyme), PG accumulation, and PG synthesis in the bovine spine IVD, as well as evaluating whether FGF2 counteracts known anabolic factors such as BMP7. To understand the molecular mechanisms by which FGF2 antagonizes BMP7 activity, we also investigated the signaling pathways utilized by FGF2 in bovine disc tissue. Results The primary receptor expressed in bovine nucleus pulposus cartilage is FGFR1, and this receptor is upregulated in degenerative human IVD tissue compared with normal IVD tissue. Stimulation of bovine nucleus pulposus cells cultured in monolayer with FGF2 augmented the production of MMP-13 at the transcriptional and translational level in a dose-dependent manner. Stimulation of bovine nucleus pulposus cells cultured in alginate beads for 21 days with FGF2 resulted in a dose-dependent decrease in PG accumulation, due at least in part to the inhibition of PG synthesis. Further studies demonstrate that FGF2 (10 ng/ml) antagonizes BMP7-mediated acceleration of PG production in bovine nucleus pulposus cells via the upregulation of noggin, an inhibitor of the transforming growth factor beta/bone morphogenetic protein signaling pathway. Chemical inhibitor studies

  15. Immunohistochemical localization of basic fibroblast growth factor in bovine ovarian follicles.

    PubMed

    van Wezel, I L; Umapathysivam, K; Tilley, W D; Rodgers, R J

    1995-12-29

    Basic fibroblast growth factor (bFGF, FGF2) controls cell proliferation and differentiation in many organs and tissues. In the ovary, cells proliferate and differentiate during folliculogenesis and during formation of the corpus luteum. While previous studies have inferred a role for bFGF in these processes, the precise contribution of bFGF to follicular activation or recruitment has not been established. For this reason, bFGF was immunolocalized in bovine follicles, using anti-bFGF immunoglobulin specific for the 1-24-amino acid terminus of the 18-kDa peptide. Basic FGF was immunolocalized to the cytoplasm of oocytes from bovine primordial and primary follicles. Strong immunostaining was also observed in corpora lutea, the ovarian surface epithelium, and smooth muscle cells surrounding blood vessels, while substantial levels of immunostaining were also present in cells of the theca interna. In most of the healthy antral follicles examined, the three or so layers of granulosa cells which were closest to the basement membrane were also stained, with greatest levels of staining at the most basal region of each cell. Atretic antral follicles had significant and uniform levels of immunostaining throughout the theca interna and the membrana granulosa. Immunostaining as described above was reduced to background levels when the primary specific immunoglobulin was preabsorbed with a 350 molar excess of peptide comprising the NH2-terminal 24 amino acids of bFGF. Based upon our previous observations and those reported here, we propose that basic fibroblast growth factor is synthesized by immature oocytes, especially those from primordial and primary follicles, and that bFGF has a potential role in activating follicle growth via stimulation of granulosa cell proliferation and follicular basement membrane synthesis. PMID:8824888

  16. Cytokine regulation of human lung fibroblast hyaluronan (hyaluronic acid) production. Evidence for cytokine-regulated hyaluronan (hyaluronic acid) degradation and human lung fibroblast-derived hyaluronidase.

    PubMed Central

    Sampson, P M; Rochester, C L; Freundlich, B; Elias, J A

    1992-01-01

    We characterized the mechanisms by which recombinant (r) tumor necrosis factor (TNF), IFN-gamma, and IL-1, alone and in combination, regulate human lung fibroblast hyaluronic acid (HA) production. Each cytokine stimulated fibroblast HA production. The combination of rTNF and rIFN-gamma resulted in a synergistic increase in the production of high molecular weight HA. This was due to a synergistic increase in hyaluronate synthetase activity and a simultaneous decrease in HA degradation. In contrast, when rTNF and rIL-1 were combined, an additive increase in low molecular weight HA was noted. This was due to a synergistic increase in hyaluronate synthetase activity and a simultaneous increase in HA degradation. Human lung fibroblasts contained a hyaluronidase that, at pH 3.7, depolymerized high molecular weight HA to 10-40 kD end products of digestion. However, hyaluronidase activity did not correlate with fibroblast HA degradation. Instead, HA degradation correlated with fibroblast-HA binding, which was increased by rIL-1 plus rTNF and decreased by rIFN-gamma plus rTNF. Recombinant IL-1 and rTNF weakly stimulated and rIL-1 and rTNF in combination further augmented the levels of CD44 mRNA in lung fibroblasts. In contrast, rIFN-gamma did not significantly alter the levels of CD44 mRNA in unstimulated or rTNF stimulated cells. These studies demonstrate that rIL-1, rTNF, and rIFN-gamma have complex effects on biosynthesis and degradation which alter the quantity and molecular weight of the HA produced by lung fibroblasts. They also show that fibroblast HA degradation is mediated by a previously unrecognized lysosomal-type hyaluronidase whose function may be regulated by altering fibroblast-HA binding. Lastly, they suggest that the CD44 HA receptor may be involved in this process. Images PMID:1401082

  17. Hepatic Aryl Hydrocarbon Receptor Attenuates Fibroblast Growth Factor 21 Expression.

    PubMed

    Girer, Nathaniel G; Murray, Iain A; Omiecinski, Curtis J; Perdew, Gary H

    2016-07-15

    The Aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor involved in many physiological processes. Several studies indicate that AHR is also involved in energy homeostasis. Fibroblast growth factor 21 (FGF21) is an important regulator of the fasting and feeding responses. When administered to various genetic and diet-induced mouse models of obesity, FGF21 can attenuate obesity-associated morbidities. Here, we explore the role of AHR in hepatic Fgf21 expression through the use of a conditional, hepatocyte-targeted AHR knock-out mouse model (Cre(Alb)Ahr(Fx/Fx)). Compared with the congenic parental strain (Ahr(Fx/Fx)), non-fasted Cre(Alb)Ahr(Fx/Fx) mice exhibit a 4-fold increase in hepatic Fgf21 expression, as well as elevated expression of the FGF21-target gene Igfbp1 Furthermore, in vivo agonist activation of AHR reduces hepatic Fgf21 expression during a fast. The Fgf21 promoter contains several putative dioxin response elements (DREs). Using EMSA, we demonstrate that the AHR-ARNT heterodimer binds to a specific DRE that overlaps binding sequences for peroxisome proliferator-activated receptor α (PPARα), carbohydrate response element-binding protein (ChREBP), and cAMP response element-binding protein, hepatocyte specific (CREBH). In addition, we reveal that agonist-activated AHR impairs PPARα-, ChREBP-, and CREBH-mediated promoter activity in Hepa-1 cells. Accordingly, agonist treatment in Hepa-1 cells ablates potent ER stress-driven Fgf21 expression, and pre-treatment with AHR antagonist blocks this effect. Finally, we show that pre-treatment of primary human hepatocytes with AHR agonist diminishes PPARα-, glucose-, and ER stress-driven induction of FGF21 expression, indicating the effect is not mouse-specific. Together, our data show that AHR contributes to hepatic energy homeostasis, partly through the regulation of FGF21 expression and signaling. PMID:27226639

  18. Fibroblast Growth Factor 23 in Patients Undergoing Peritoneal Dialysis

    PubMed Central

    Isakova, Tamara; Xie, Huiliang; Barchi-Chung, Allison; Vargas, Gabriela; Sowden, Nicole; Houston, Jessica; Wahl, Patricia; Lundquist, Andrew; Epstein, Michael; Smith, Kelsey; Contreras, Gabriel; Ortega, Luis; Lenz, Oliver; Briones, Patricia; Egbert, Phyllis; Ikizler, T. Alp; Jueppner, Harald

    2011-01-01

    Summary Background and objectives Fibroblast growth factor 23 (FGF23) is an independent risk factor for mortality in patients with ESRD. Before FGF23 testing can be integrated into clinical practice of ESRD, further understanding of its determinants is needed. Design, setting, participants, & measurements In a study of 67 adults undergoing peritoneal dialysis, we tested the hypothesis that longer dialysis vintage and lower residual renal function and renal phosphate clearance are associated with higher FGF23. We also compared the monthly variability of FGF23 versus parathyroid hormone (PTH) and serum phosphate. Results In unadjusted analyses, FGF23 correlated with serum phosphate (r = 0.66, P < 0.001), residual renal function (r = −0.37, P = 0.002), dialysis vintage (r = 0.31, P = 0.01), and renal phosphate clearance (r = −0.38, P = 0.008). In adjusted analyses, absence of residual renal function and greater dialysis vintage associated with higher FGF23, independent of demographics, laboratory values, peritoneal dialysis modality and adequacy, and treatment with vitamin D analogs and phosphate binders. Urinary and dialysate FGF23 clearances were minimal. In three serial monthly measurements, within-subject variability accounted for only 10% of total FGF23 variability compared with 50% for PTH and 60% for serum phosphate. Conclusions Increased serum phosphate, loss of residual renal function, longer dialysis vintage, and lower renal phosphate clearance are associated with elevated FGF23 levels in ESRD patients undergoing peritoneal dialysis. FGF23 may be a more stable marker of phosphate metabolism in ESRD than PTH or serum phosphate. PMID:21903990

  19. Acute exercise increases fibroblast growth factor 21 in metabolic organs and circulation.

    PubMed

    Tanimura, Yuko; Aoi, Wataru; Takanami, Yoshikazu; Kawai, Yukari; Mizushima, Katsura; Naito, Yuji; Yoshikawa, Toshikazu

    2016-06-01

    Fibroblast growth factor 21, a metabolic regulator, plays roles in lipolysis and glucose uptake in adipose tissues and skeletal muscles. Its expression in skeletal muscle is upregulated upon activation of the phosphatidylinositol 3-kinase/Akt signaling pathway, which is induced by exercise and muscle contraction. We examined the increase of fibroblast growth factor 21 after acute exercise in metabolic organs, especially skeletal muscles and circulation. Participants exercised on bicycle ergometers for 60 min at 75% of their V˙O2max. Venous blood samples were taken before exercise and immediately after exercise. In an animal study, male ICR mice were divided into sedentary and exercise groups. Mice in the exercise group performed treadmill exercises at 30 m min(-1) for 60 min. Shortly thereafter, blood, liver, and skeletal muscle samples were taken from mice. Acute exercise induced the increase of serum fibroblast growth factor 21 in both humans and mice, and increased fibroblast growth factor 21 expression in the skeletal muscles and the liver of mice. Acute exercise activated Akt in mice skeletal muscle. Acute exercise increases fibroblast growth factor 21 concentrations in both serum and metabolic organs. Moreover, results show that acute exercise increased the expression of fibroblast growth factor 21 in skeletal muscle, accompanied by the phosphorylation of Akt in mice. PMID:27335433

  20. Phosphorylation-regulated Nucleocytoplasmic Trafficking of Internalized Fibroblast Growth Factor-1

    PubMed Central

    Więdłocha, Antoni; Nilsen, Trine; Wesche, Jørgen; Sørensen, Vigdis; Małecki, Jędrzej; Marcinkowska, Ewa; Olsnes, Sjur

    2005-01-01

    Fibroblast growth factor-1 (FGF-1), which stimulates cell growth, differentiation, and migration, is capable of crossing cellular membranes to reach the cytosol and the nucleus in cells containing specific FGF receptors. The cell entry process can be monitored by phosphorylation of the translocated FGF-1. We present evidence that phosphorylation of FGF-1 occurs in the nucleus by protein kinase C (PKC)δ. The phosphorylated FGF-1 is subsequently exported to the cytosol. A mutant growth factor where serine at the phosphorylation site is exchanged with glutamic acid, to mimic phosphorylated FGF-1, is constitutively transported to the cytosol, whereas a mutant containing alanine at this site remains in the nucleus. The export can be blocked by leptomycin B, indicating active and receptor-mediated nuclear export of FGF-1. Thapsigargin, but not leptomycin B, prevents the appearance of active PKCδ in the nucleus, and FGF-1 is in this case phosphorylated in the cytosol. Leptomycin B increases the amount of phosphorylated FGF-1 in the cells by preventing dephosphorylation of the growth factor, which seems to occur more rapidly in the cytoplasm than in the nucleus. The nucleocytoplasmic trafficking of the phosphorylated growth factor is likely to play a role in the activity of internalized FGF-1. PMID:15574884

  1. Irradiated fibroblast-induced bystander effects on invasive growth of squamous cell carcinoma under cancer-stromal cell interaction.

    PubMed

    Kamochi, Noriyuki; Nakashima, Masahiro; Aoki, Shigehisa; Uchihashi, Kazuyoshi; Sugihara, Hajime; Toda, Shuji; Kudo, Sho

    2008-12-01

    The irradiated fibroblast-induced response of non-irradiated neighboring cells is called 'radiation-induced bystander effect', but it is unclear in non-irradiated human squamous cell carcinoma (SCC) cells. The present study shows that irradiated fibroblasts promoted the invasive growth of T3M-1 SCC cells, but not their apoptosis, more greatly than non-irradiated fibroblasts, using collagen gel invasion assay, immunohistochemistry and Western blot. The number of irradiated fibroblasts decreased to about 30% of that of non-irradiated fibroblasts, but irradiated fibroblasts increased the growth marker ki-67 display of SCC cells more greatly than non-irradiated fibroblasts. Irradiated fibroblasts did not affect the apoptosis marker ss-DNA expression of SCC cells. Irradiated fibroblasts enhanced the display of the following growth-, invasion- and motility-related molecules in SCC cells more greatly than non-irradiated fibroblasts: c-Met, Ras, mitogen-activated protein kinase (MAPK) cascade (Raf-1, MEK-1 and ERK-1/2), matrix metalloproteinase-1 and -9, laminin 5 and filamin A. Irradiated fibroblasts, but not non-irradiated ones, formed irradiation-induced foci (IRIF) of the genomic instability marker p53-binding protein 1 (53BP1) and expressed transforming growth factor-beta1 (TGF- beta1). Irradiated fibroblasts in turn enabled SCC cells to enhance 53BP1 IRIF formation more extensively than non-irradiated fibroblasts. Finally, effects of irradiated fibroblasts on growth and apoptosis of another HEp-2 SCC cell type were similar to those of T3M-1. These results suggest that irradiated fibroblasts promotes invasion and growth of SCC cells by enhancement of invasive growth-related molecules above through TGF- beta1-mediated bystander mechanism, in which irradiated fibroblast-induced genomic instability of SCC cells may be involved. PMID:19018771

  2. Oleic, Linoleic and Linolenic Acids Increase ROS Production by Fibroblasts via NADPH Oxidase Activation

    PubMed Central

    Hatanaka, Elaine; Dermargos, Alexandre; Hirata, Aparecida Emiko; Vinolo, Marco Aurélio Ramirez; Carpinelli, Angelo Rafael; Newsholme, Philip; Armelin, Hugo Aguirre; Curi, Rui

    2013-01-01

    The effect of oleic, linoleic and γ-linolenic acids on ROS production by 3T3 Swiss and Rat 1 fibroblasts was investigated. Using lucigenin-amplified chemiluminescence, a dose-dependent increase in extracellular superoxide levels was observed during the treatment of fibroblasts with oleic, linoleic and γ-linolenic acids. ROS production was dependent on the addition of β-NADH or NADPH to the medium. Diphenyleneiodonium inhibited the effect of oleic, linoleic and γ-linolenic acids on fibroblast superoxide release by 79%, 92% and 82%, respectively. Increased levels of p47phox phosphorylation due to fatty acid treatment were detected by Western blotting analyses of fibroblast proteins. Increased p47phox mRNA expression was observed using real-time PCR. The rank order for the fatty acid stimulation of the fibroblast oxidative burst was as follows: γ-linolenic > linoleic > oleic. In conclusion, oleic, linoleic and γ-linolenic acids stimulated ROS production via activation of the NADPH oxidase enzyme complex in fibroblasts. PMID:23579616

  3. Oleic, linoleic and linolenic acids increase ros production by fibroblasts via NADPH oxidase activation.

    PubMed

    Hatanaka, Elaine; Dermargos, Alexandre; Hirata, Aparecida Emiko; Vinolo, Marco Aurélio Ramirez; Carpinelli, Angelo Rafael; Newsholme, Philip; Armelin, Hugo Aguirre; Curi, Rui

    2013-01-01

    The effect of oleic, linoleic and γ-linolenic acids on ROS production by 3T3 Swiss and Rat 1 fibroblasts was investigated. Using lucigenin-amplified chemiluminescence, a dose-dependent increase in extracellular superoxide levels was observed during the treatment of fibroblasts with oleic, linoleic and γ-linolenic acids. ROS production was dependent on the addition of β-NADH or NADPH to the medium. Diphenyleneiodonium inhibited the effect of oleic, linoleic and γ-linolenic acids on fibroblast superoxide release by 79%, 92% and 82%, respectively. Increased levels of p47 (phox) phosphorylation due to fatty acid treatment were detected by Western blotting analyses of fibroblast proteins. Increased p47 (phox) mRNA expression was observed using real-time PCR. The rank order for the fatty acid stimulation of the fibroblast oxidative burst was as follows: γ-linolenic > linoleic > oleic. In conclusion, oleic, linoleic and γ-linolenic acids stimulated ROS production via activation of the NADPH oxidase enzyme complex in fibroblasts. PMID:23579616

  4. Tracking and Increasing Viability of Topically Injected Fibroblasts Suspended in Hyaluronic Acid Filler.

    PubMed

    You, Hi-Jin; Namgoong, Sik; Rhee, Sung-Mi; Han, Seung-Kyu

    2016-03-01

    A new injectable tissue-engineered soft tissue consisting of a mixture of hyaluronic acid (HA) filler and cultured human fibroblasts have been developed by the authors. To establish this method as a standard treatment, a further study was required to determine whether the injected fibroblasts could stay at the injected place or move to other sites. In addition, effective strategies were needed to increase viability of the injected fibroblasts. The purpose of this study was to track the injected fibroblasts and to determine the effect of adding prostaglandin E1 (PGE1) or vitamin C on the viability of fibroblasts.Human fibroblasts labeled with fluorescence dye were suspended in HA filler and injected into 4 sites on the back of nude mice. The injected bioimplants consisted of one of the 4 followings: HA filler without cells (HA group), fibroblasts suspended in HA filler (HA + FB group), PGE1-supplemented fibroblasts in HA filler (HA + FB + PGE1 group), and vitamin C-supplemented fibroblasts in HA filler (HA + FB + VC group). At 4 weeks after injection, locations and intensities of the fluorescence signals were evaluated using a live imaging system.The fluorescence signals of the fibroblast-containing groups were visible only at the injected sites without dispersing to other sites. The HA +FB + PGE1 group showed a significantly higher fluorescence signal than the HA + FB and the HA + FB +VC groups (P < 0.05, each). There was no statistical difference between the HA + FB and HA + FB +VC groups (P = 0.69).The results of the current study collectively suggest that injected fibroblasts suspended in HA filler stay at the injected place without moving to other sites. In addition, PGE1 treatment may increase the remaining rhodamine B isothiocynanate dye at the injected site of the human dermal fibroblasts. PMID:26854786

  5. Rapid increase in fibroblast growth factor 21 in protein malnutrition and its impact on growth and lipid metabolism.

    PubMed

    Ozaki, Yori; Saito, Kenji; Nakazawa, Kyoko; Konishi, Morichika; Itoh, Nobuyuki; Hakuno, Fumihiko; Takahashi, Shin-Ichiro; Kato, Hisanori; Takenaka, Asako

    2015-11-14

    Protein malnutrition promotes hepatic steatosis, decreases insulin-like growth factor (IGF)-I production and retards growth. To identify new molecules involved in such changes, we conducted DNA microarray analysis on liver samples from rats fed an isoenergetic low-protein diet for 8 h. We identified the fibroblast growth factor 21 gene (Fgf21) as one of the most strongly up-regulated genes under conditions of acute protein malnutrition (P<0·05, false-discovery rate<0·001). In addition, amino acid deprivation increased Fgf21 mRNA levels in rat liver-derived RL-34 cells (P<0·01). These results suggested that amino acid limitation directly increases Fgf21 expression. FGF21 is a polypeptide hormone that regulates glucose and lipid metabolism. FGF21 also promotes a growth hormone-resistance state and suppresses IGF-I in transgenic mice. Therefore, to determine further whether Fgf21 up-regulation causes hepatic steatosis and growth retardation after IGF-I decrease in protein malnutrition, we fed an isoenergetic low-protein diet to Fgf21-knockout (KO) mice. Fgf21-KO did not rescue growth retardation and reduced plasma IGF-I concentration in these mice. Fgf21-KO mice showed greater epididymal white adipose tissue weight and increased hepatic TAG and cholesterol levels under protein malnutrition conditions (P<0·05). Overall, the results showed that protein deprivation directly increased Fgf21 expression. However, growth retardation and decreased IGF-I were not mediated by increased FGF21 expression in protein malnutrition. Furthermore, FGF21 up-regulation rather appears to have a protective effect against obesity and hepatic steatosis in protein-malnourished animals. PMID:26330054

  6. Hydroxyeicosatetraenoic acid metabolism in cultured human skin fibroblasts. Evidence for peroxisomal beta-oxidation.

    PubMed Central

    Gordon, J A; Figard, P H; Spector, A A

    1990-01-01

    To determine whether the peroxisome is responsible for hydroxyeicosatetraenoic acid (HETE) oxidation, 12- and 15-HETE oxidation was measured in normal and peroxisomal deficient skin fibroblasts from patients with Zellweger's (cerebrohepatorenal) syndrome. When incubated for 1 h with normal fibroblasts, reverse phase HPLC indicated that 24% of the 12-HETE radioactivity was converted to one major polar metabolite. Chemical derivatization followed by reverse phase HPLC and TLC indicated that this metabolite is 8-hydroxyhexadecatrienoic acid [16:3(8-OH)]. Similarly, 33% of the added 15-HETE was also converted to a more polar metabolite. Neither 12- nor 15-HETE were converted to any metabolites by the peroxisomal deficient (Zellweger) cells. No defect in HETE oxidation was found in other human fibroblast cell lines with diverse metabolic abnormalities. Zellweger fibroblasts accumulated increased amounts of 12-HETE, compared with normal fibroblasts. As in the normal cells, most of the 12-HETE incorporated into Zellweger fibroblasts was present in the choline and ethanolamine phosphoglycerides. Protein synthesis, lysosomal acid lipase activity, and mitochondrial butyrate oxidation were not impaired in the Zellweger fibroblasts. Since the Zellweger cells do not convert 12- and 15-HETE to oxidative metabolites, peroxisomes appear to be the cellular organelle responsible for HETE oxidation. Images PMID:2318972

  7. Blocking Fibroblast Growth Factor Receptor Signaling Inhibits Tumor Growth, Lymphangiogenesis, and Metastasis

    PubMed Central

    Larrieu-Lahargue, Frédéric; Welm, Alana L.; Bouchecareilh, Marion; Alitalo, Kari; Li, Dean Y.; Bikfalvi, Andreas; Auguste, Patrick

    2012-01-01

    Fibroblast Growth Factor receptor (FGFR) activity plays crucial roles in tumor growth and patient survival. However, FGF (Fibroblast Growth Factor) signaling as a target for cancer therapy has been under-investigated compared to other receptor tyrosine kinases. Here, we studied the effect of FGFR signaling inhibition on tumor growth, metastasis and lymphangiogenesis by expressing a dominant negative FGFR (FGFR-2DN) in an orthotopic mouse mammary 66c14 carcinoma model. We show that FGFR-2DN-expressing 66c14 cells proliferate in vitro slower than controls. 66c14 tumor outgrowth and lung metastatic foci are reduced in mice implanted with FGFR-2DN-expressing cells, which also exhibited better overall survival. We found 66c14 cells in the lumen of tumor lymphatic vessels and in lymph nodes. FGFR-2DN-expressing tumors exhibited a decrease in VEGFR-3 (Vascular Endothelial Growth Factor Receptor-3) or podoplanin-positive lymphatic vessels, an increase in isolated intratumoral lymphatic endothelial cells and a reduction in VEGF-C (Vascular Endothelial Growth Factor-C) mRNA expression. FGFs may act in an autocrine manner as the inhibition of FGFR signaling in tumor cells suppresses VEGF-C expression in a COX-2 (cyclooxygenase-2) or HIF1-α (hypoxia-inducible factor-1 α) independent manner. FGFs may also act in a paracrine manner on tumor lymphatics by inducing expression of pro-lymphangiogenic molecules such as VEGFR-3, integrin α9, prox1 and netrin-1. Finally, in vitro lymphangiogenesis is impeded in the presence of FGFR-2DN 66c14 cells. These data confirm that both FGF and VEGF signaling are necessary for the maintenance of vascular morphogenesis and provide evidence that targeting FGFR signaling may be an interesting approach to inhibit tumor lymphangiogenesis and metastatic spread. PMID:22761819

  8. NMR characterization of copper and lipid interactions of the C2B domain of synaptotagmin I—relevance to the non-classical secretion of the human acidic fibroblast growth factor (hFGF-1)

    PubMed Central

    Kathir, Karuppanan Muthusamy; Gao, Li; Rajalingam, Dakshinamurthy; Daily, Anna E.; Brixey, Sherri; Liu, Huimin; Davis, Dan; Adams, Paul; Prudovsky, Igor; Kumar, Thallapuranam Krishnaswamy Suresh

    2013-01-01

    Human fibroblast growth factor (hFGF-1) is a ~17 kDa heparin binding cytokine. It lacks the conventional hydrophobic N-terminal signal sequence and is secreted through non-classical secretion routes. Under stress, hFGF-1 is released as a multiprotein complex consisting of hFGF-1, S100A13 (a calcium binding protein), and p40 synaptotagmin (Syt1). Copper (Cu2+) is shown to be required for the formation of the multiprotein hFGF-1 release complex (Landriscina et al.,2001; Di Serio et al., 2008). Syt1, containing the lipid binding C2B domain, is believed to play an important role in the eventual export of the hFGF-1 across the lipid bilayer. In this study, we characterize Cu2+ and lipid interactions of the C2B domain of Syt1 using multidimensional NMR spectroscopy. The results highlight how Cu2+ appears to stabilize the protein bound to pS vesicles. Cu2+ and lipid binding interface mapped using 2D 1H–15N heteronuclear single quantum coherence experiments reveal that residues in β-strand I contributes to the unique Cu2+ binding site in the C2B domain. In the absence of metal ions, residues located in Loop II and β-strand IV contribute to binding to unilamelar pS vesicles. In the presence of Cu2+, additional residues located in Loops I and III appear to stabilize the protein-lipid interactions. The results of this study provide valuable information towards understanding the molecular mechanism of the Cu2+-induced non-classical secretion of hFGF-1. PMID:19835837

  9. Photodynamic therapy inhibit Fibroblast Growth Factor-10 induced keratinocyte differentiation and proliferation through ROS in Fibroblast Growth Factor Receptor-2b pathway

    PubMed Central

    Gozali, Maya Valeska; Yi, Fei; Zhang, Jia-an; Liu, Juan; Wu, Hong-jin; Xu, Yang; Luo, Dan; Zhou, Bing-rong

    2016-01-01

    5-aminolevulinic acid-photodynamic therapy (ALA-PDT) is known to be effective in several skin diseases such as acne, actinic keratoses, condyloma acuminata. However, some detailed mechanisms of ALA-PDT to treat these skin diseases still remain elusive. In this study, we aimed to investigate mechanism of ALA-PDT in in-vitro and in-vivo models. For in vitro, we use human keratinocyte cell line (HaCaT) cells. CCK-8 was used to detect cell proliferation activity, immunofluorescence and western blotting method to detect the content of keratin (K)1, K6, K16, protein kinase C (PKC), fibroblast growth factor receptor-2b (FGFR2b) protein, ELISA and RT-PCR to detect expression of interleukin (IL) 1α in the cell supernatant, and detect reactive oxygen species (ROS). For in vivo, we use 20 rabbits to induce hyperkeratosis acne model in their ear. Dermatoscope was used to see follicle hyperkeratosis and skin biopsy to analyze histology and immunohistochemical of PKC, FGFR2b, K1, K6 and K16. Results from this study suggest that ROS stimulated by ALA-PDT lead to inhibition of FGFR2b pathway in PKC downstream to cause reduction of IL1α expression, and eventually, keratinocytes differentiation and proliferation. Our data thus reveal a treatment mechanism of ALA-PDT underlying hyperkeratosis related dermatoses. PMID:27273653

  10. Photodynamic therapy inhibit Fibroblast Growth Factor-10 induced keratinocyte differentiation and proliferation through ROS in Fibroblast Growth Factor Receptor-2b pathway.

    PubMed

    Gozali, Maya Valeska; Yi, Fei; Zhang, Jia-An; Liu, Juan; Wu, Hong-Jin; Xu, Yang; Luo, Dan; Zhou, Bing-Rong

    2016-01-01

    5-aminolevulinic acid-photodynamic therapy (ALA-PDT) is known to be effective in several skin diseases such as acne, actinic keratoses, condyloma acuminata. However, some detailed mechanisms of ALA-PDT to treat these skin diseases still remain elusive. In this study, we aimed to investigate mechanism of ALA-PDT in in-vitro and in-vivo models. For in vitro, we use human keratinocyte cell line (HaCaT) cells. CCK-8 was used to detect cell proliferation activity, immunofluorescence and western blotting method to detect the content of keratin (K)1, K6, K16, protein kinase C (PKC), fibroblast growth factor receptor-2b (FGFR2b) protein, ELISA and RT-PCR to detect expression of interleukin (IL) 1α in the cell supernatant, and detect reactive oxygen species (ROS). For in vivo, we use 20 rabbits to induce hyperkeratosis acne model in their ear. Dermatoscope was used to see follicle hyperkeratosis and skin biopsy to analyze histology and immunohistochemical of PKC, FGFR2b, K1, K6 and K16. Results from this study suggest that ROS stimulated by ALA-PDT lead to inhibition of FGFR2b pathway in PKC downstream to cause reduction of IL1α expression, and eventually, keratinocytes differentiation and proliferation. Our data thus reveal a treatment mechanism of ALA-PDT underlying hyperkeratosis related dermatoses. PMID:27273653

  11. Fibroblast Growth Factors and Vascular Endothelial Growth Factor Promote Cardiac Reprogramming under Defined Conditions

    PubMed Central

    Yamakawa, Hiroyuki; Muraoka, Naoto; Miyamoto, Kazutaka; Sadahiro, Taketaro; Isomi, Mari; Haginiwa, Sho; Kojima, Hidenori; Umei, Tomohiko; Akiyama, Mizuha; Kuishi, Yuki; Kurokawa, Junko; Furukawa, Tetsushi; Fukuda, Keiichi; Ieda, Masaki

    2015-01-01

    Summary Fibroblasts can be directly reprogrammed into cardiomyocyte-like cells (iCMs) by overexpression of cardiac transcription factors, including Gata4, Mef2c, and Tbx5; however, this process is inefficient under serum-based culture conditions, in which conversion of partially reprogrammed cells into fully reprogrammed functional iCMs has been a major hurdle. Here, we report that a combination of fibroblast growth factor (FGF) 2, FGF10, and vascular endothelial growth factor (VEGF), termed FFV, promoted cardiac reprogramming under defined serum-free conditions, increasing spontaneously beating iCMs by 100-fold compared with those under conventional serum-based conditions. Mechanistically, FFV activated multiple cardiac transcriptional regulators and converted partially reprogrammed cells into functional iCMs through the p38 mitogen-activated protein kinase and phosphoinositol 3-kinase/AKT pathways. Moreover, FFV enabled cardiac reprogramming with only Mef2c and Tbx5 through the induction of cardiac reprogramming factors, including Gata4. Thus, defined culture conditions promoted the quality of cardiac reprogramming, and this finding provides new insight into the mechanism of cardiac reprogramming. PMID:26626177

  12. miR-100 Inhibits the Growth and Migration of Burn-Denatured Fibroblasts

    PubMed Central

    Ying, Jianghui; Liu, Yunfeng; Yang, Ruijin; Zhang, Yong; Xu, Jianjun

    2016-01-01

    Background Burn-denatured dermis is able to regain the function and shape of normal dermis; however, the potential mechanisms are still vague. The aim of this study was to investigate roles of miR-100 involved in the growth and migration of burn-denatured fibroblasts. Material/Methods Quantitative real-time polymerase chain reaction(qRT-PCR) was used to assess the expression of miR-100. Transient transfection of miR-100 mimics and inhibitor was used to up-regulate or down-regulate the expression of miR-100. Cell proliferation and colony formation assay were used to test the cell growth, and wound healing assay and transwell migration assay were used to evaluate the cell migration. Results miR-100 expression was notably downregulated in the burn-denatured fibroblasts compared to normal controls. Functionally, transfection of miR-100 inhibitors improved the growth and migration abilities of burn-denatured fibroblasts. In contrast, upregulation of miR-100 inhibits the growth and migration of burn-denatured fibroblasts. Conclusions Based on these observations, we concluded that miR-100 can inhibit the growth and migration of burn-denatured fibroblasts. PMID:26928010

  13. Potentiation of fibroblast growth by nodular sclerosing Hodgkin's disease cell cultures.

    PubMed

    Newcom, S R; O'Rourke, L

    1982-07-01

    Cell cultures were established from 8 lymph nodes replaced by nodular sclerosing Hodgkin's disease. Serum-containing and serum-free conditioned media from these cultures potentiated fibroblast growth and were found to be consistently more potent than fibroblast growth factor, 100 ng/ml, every other day. Both a proliferative response and transformation-like growth were observed using BALB/c 3T3 cells, human diploid fibroblasts, and human embryonic fibroblasts as target cells. The Hodgkin's disease growth factor(s) was not produced by fibroblasts or lymphocytes in the Hodgkin's cultures and was most potent when the Hodgkin's cultures had been enriched with Hodgkin's giant cells. Removal of normal macrophages decreased the proliferative activity but did not eliminate it or nonadherent growth of 3T3 cells in agar. Control cultures of 6 nonmalignant lymph nodes, a Lennert's lymphoma, a mixed cellularity Hodgkin's disease lymph node, and a malignant histiocytosis cell line suggested that among lymph node disorders, this feature may be relatively specific for nodular sclerosing Hodgkin's disease. PMID:6211204

  14. Hypoxia induces expression of connective tissue growth factor in scleroderma skin fibroblasts

    PubMed Central

    Hong, K-H; Yoo, S-A; Kang, S-S; Choi, J-J; Kim, W-U; Cho, C-S

    2006-01-01

    Connective tissue growth factor (CTGF) plays a role in the fibrotic process of systemic sclerosis (SSc). Because hypoxia is associated with fibrosis in several profibrogenic conditions, we investigated whether CTGF expression in SSc fibroblasts is regulated by hypoxia. Dermal fibroblasts from patients with SSc and healthy controls were cultured in the presence of hypoxia or cobalt chloride (CoCl2), a chemical inducer of hypoxia-inducible factor (HIF)-1α. Expression of CTGF was evaluated by Northern and Western blot analyses. Dermal fibroblasts exposed to hypoxia (1% O2) or CoCl2 (1–100 µM) enhanced expression of CTGF mRNA. Skin fibroblasts transfected with HIF-1α showed the increased levels of CTGF protein and mRNA, as well as nuclear staining of HIF-1α, which was enhanced further by treatment of CoCl2. Simultaneous treatment of CoCl2 and transforming growth factor (TGF)-β additively increased CTGF mRNA in dermal fibroblasts. Interferon-γ inhibited the TGF-β-induced CTGF mRNA expression dose-dependently in dermal fibroblasts, but they failed to hamper the CoCl2-induced CTGF mRNA expression. In addition, CoCl2 treatment increased nuclear factor (NF)-κB binding activity for CTGF mRNA, while decreasing IκBα expression in dermal fibroblasts. Our data suggest that hypoxia, caused possibly by microvascular alterations, up-regulates CTGF expression through the activation of HIF-1α in dermal fibroblasts of SSc patients, and thereby contributes to the progression of skin fibrosis. PMID:17034590

  15. Fibroblast growth factor receptor 1 is principally responsible for fibroblast growth factor 2-induced catabolic activities in human articular chondrocytes

    PubMed Central

    2011-01-01

    Introduction Cartilage degeneration driven by catabolic stimuli is a critical pathophysiological process in osteoarthritis (OA). We have defined fibroblast growth factor 2 (FGF-2) as a degenerative mediator in adult human articular chondrocytes. Biological effects mediated by FGF-2 include inhibition of proteoglycan production, up-regulation of matrix metalloproteinase-13 (MMP-13), and stimulation of other catabolic factors. In this study, we identified the specific receptor responsible for the catabolic functions of FGF-2, and established a pathophysiological connection between the FGF-2 receptor and OA. Methods Primary human articular chondrocytes were cultured in monolayer (24 hours) or alginate beads (21 days), and stimulated with FGF-2 or FGF18, in the presence or absence of FGFR1 (FGF receptor 1) inhibitor. Proteoglycan accumulation and chondrocyte proliferation were assessed by dimethylmethylene blue (DMMB) assay and DNA assay, respectively. Expression of FGFRs (FGFR1 to FGFR4) was assessed by flow cytometry, immunoblotting, and quantitative real-time PCR (qPCR). The distinctive roles of FGFR1 and FGFR3 after stimulation with FGF-2 were evaluated using either pharmacological inhibitors or FGFR small interfering RNA (siRNA). Luciferase reporter gene assays were used to quantify the effects of FGF-2 and FGFR1 inhibitor on MMP-13 promoter activity. Results Chondrocyte proliferation was significantly enhanced in the presence of FGF-2 stimulation, which was inhibited by the pharmacological inhibitor of FGFR1. Proteoglycan accumulation was reduced by 50% in the presence of FGF-2, and this reduction was successfully rescued by FGFR1 inhibitor. FGFR1 inhibitors also fully reversed the up-regulation of MMP-13 expression and promoter activity stimulated by FGF-2. Blockade of FGFR1 signaling by either chemical inhibitors or siRNA targeting FGFR1 rather than FGFR3 abrogated the up-regulation of matrix metalloproteinases 13 (MMP-13) and a disintegrin and

  16. Growth factor modulation of fibroblast proliferation, differentiation, and invasion: implications for tissue valve engineering.

    PubMed

    Narine, Kishan; De Wever, Olivier; Van Valckenborgh, Dillis; Francois, Katrien; Bracke, Marc; DeSmet, Stefaan; Mareel, Marc; Van Nooten, Guido

    2006-10-01

    We have previously shown that transforming growth factor-beta1 (TGF-beta1) stimulates transdifferentiation of fibroblasts into smooth muscle alpha-actin (alpha-SMA) positive myofibroblasts. However, TGF-beta, as such, is unsuitable for effective population of a heart valve matrix, because it dose-dependently inhibits growth of fibroblasts. The aim of this study was to investigate combinations of other growth factors with TGF-beta to stimulate the proliferation of suitably differentiated cells and to enhance their invasion into aortic valve matrices. Human dermal mesenchymal cells (hDMC1.1) were treated with combinations of growth factors to stimulate these cells to trans-differentiate into myofibroblasts, to proliferate, and to invade. Growth factors were chosen after expression of their respective receptors was confirmed in hDMC1.1 using reverse transcriptase polymerase chain reaction. We combined TGF-beta with several growth factors such as insulin-like growth factor (IGF-1, IGF-2), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and platelet-derived growth factor (PDGF-AA, PDGF-BB, and PDGFAB). Nuclear Ki67 staining, MTT assay, and cell counting revealed that only EGF and bFGF were capable of overcoming TGF-beta-induced growth inhibition. However, bFGF but not EGF inhibited TGF-beta-induced alpha-SMA expression, as evidenced by immuno-cytochemistry and Western blotting. A growth factor cocktail (TGF-beta, EGF, bFGF) has been established that maintains TGF-beta-induced trans-differentiation but overcomes TGF-beta-induced growth inhibition while stimulating fibroblast proliferation and invasion. PMID:17518640

  17. Ketoprofen S(+) enantiomer inhibits prostaglandin production and cell growth in 3T6 fibroblast cultures.

    PubMed

    Sánchez, T; Moreno, J J

    1999-04-01

    The ketoprofen S(+) enantiomer inhibits with great stereoselectivity both prostaglandin H synthase isoenzymes. Thus, the biological effects of ketoprofen on inflammation are due almost entirely to the S(+) isomer. Here, we report that the S(+) enantiomer, at doses that inhibit prostaglandin synthesis, is effective in reducing DNA synthesis and 3T6 fibroblast growth. Our data suggest that prostaglandins are involved in the control of 3T6 fibroblast growth and that the effect of the ketoprofen S(+) enantiomer on 3T6 proliferation is correlated with its effects on prostaglandin H synthase and prostaglandin production. PMID:10323281

  18. Fibroblast growth factor receptor 4 (FGFR4) deficiency improves insulin resistance and glucose metabolism under diet-induced obesity conditions.

    PubMed

    Ge, Hongfei; Zhang, Jun; Gong, Yan; Gupte, Jamila; Ye, Jay; Weiszmann, Jennifer; Samayoa, Kim; Coberly, Suzanne; Gardner, Jonitha; Wang, Huilan; Corbin, Tim; Chui, Danny; Baribault, Helene; Li, Yang

    2014-10-31

    The role of fibroblast growth factor receptor 4 (FGFR4) in regulating bile acid synthesis has been well defined; however, its reported role on glucose and energy metabolism remains unresolved. Here, we show that FGFR4 deficiency in mice leads to improvement in glucose metabolism, insulin sensitivity, and reduction in body weight under high fat conditions. Mechanism of action studies in FGFR4-deficient mice suggest that the effects are mediated in part by increased plasma levels of adiponectin and the endocrine FGF factors FGF21 and FGF15, the latter of which increase in response to an elevated bile acid pool. Direct actions of increased bile acids on bile acid receptors, and other potential indirect mechanisms, may also contribute to the observed metabolic changes. The results described herein suggest that FGFR4 antagonists alone, or in combination with other agents, could serve as a novel treatment for diabetes. PMID:25204652

  19. Histological Effect of Basic Fibroblast Growth Factor on Chronic Vocal Fold Scarring in a Rat Model

    PubMed Central

    Tateya, Ichiro; Tateya, Tomoko; Sohn, Jin-Ho; Bless, Diane M.

    2016-01-01

    Objectives Vocal fold scarring is one of the most challenging laryngeal disorders to treat and there are currently no consistently effective treatments available. Our previous studies have shown the therapeutic potential of basic fibroblast growth factor (bFGF) for vocal fold scarring. However, the histological effects of bFGF on scarred vocal fold have not been elucidated. The aim of this study was to examine the histological effects of bFGF on chronic vocal fold scarring. Methods Sprague-Dawley rats were divided into phosphate buffered saline (sham) and bFGF groups. Unilateral vocal fold stripping was performed and the drug was injected into the scarred vocal fold for each group 2 months postoperatively. Injections were performed weekly for 4 weeks. Two months after the last injection, larynges were harvested and histologically analyzed. Results A significant increase of hyaluronic acid was observed in the vocal fold of the bFGF group compared with that of the sham group. However, there was no remarkable change in collagen expression nor in vocal fold contraction. Conclusion Significant increase of hyaluronic acid by local bFGF injection was thought to contribute to the therapeutic effects on chronic vocal fold scarring. PMID:26976028

  20. Genetic deletion of fibroblast growth factor 14 recapitulates phenotypic alterations underlying cognitive impairment associated with schizophrenia.

    PubMed

    Alshammari, T K; Alshammari, M A; Nenov, M N; Hoxha, E; Cambiaghi, M; Marcinno, A; James, T F; Singh, P; Labate, D; Li, J; Meltzer, H Y; Sacchetti, B; Tempia, F; Laezza, F

    2016-01-01

    Cognitive processing is highly dependent on the functional integrity of gamma-amino-butyric acid (GABA) interneurons in the brain. These cells regulate excitability and synaptic plasticity of principal neurons balancing the excitatory/inhibitory tone of cortical networks. Reduced function of parvalbumin (PV) interneurons and disruption of GABAergic synapses in the cortical circuitry result in desynchronized network activity associated with cognitive impairment across many psychiatric disorders, including schizophrenia. However, the mechanisms underlying these complex phenotypes are still poorly understood. Here we show that in animal models, genetic deletion of fibroblast growth factor 14 (Fgf14), a regulator of neuronal excitability and synaptic transmission, leads to loss of PV interneurons in the CA1 hippocampal region, a critical area for cognitive function. Strikingly, this cellular phenotype associates with decreased expression of glutamic acid decarboxylase 67 (GAD67) and vesicular GABA transporter (VGAT) and also coincides with disrupted CA1 inhibitory circuitry, reduced in vivo gamma frequency oscillations and impaired working memory. Bioinformatics analysis of schizophrenia transcriptomics revealed functional co-clustering of FGF14 and genes enriched within the GABAergic pathway along with correlatively decreased expression of FGF14, PVALB, GAD67 and VGAT in the disease context. These results indicate that Fgf14(-/-) mice recapitulate salient molecular, cellular, functional and behavioral features associated with human cognitive impairment, and FGF14 loss of function might be associated with the biology of complex brain disorders such as schizophrenia. PMID:27163207

  1. Fibroblast growth factor 21 deficiency exacerbates chronic alcohol-induced hepatic steatosis and injury

    PubMed Central

    Liu, Yanlong; Zhao, Cuiqing; Xiao, Jian; Liu, Liming; Zhang, Min; Wang, Cuiling; Wu, Guicheng; Zheng, Ming-Hua; Xu, Lan-Man; Chen, Yong-Ping; Mohammadi, Moosa; Chen, Shao-Yu; Cave, Matthew; McClain, Craig; Li, Xiaokun; Feng, Wenke

    2016-01-01

    Fibroblast growth factor 21 (FGF21) is a hepatokine that regulates glucose and lipid metabolism in the liver. We sought to determine the role of FGF21 in hepatic steatosis in mice exposed to chronic alcohol treatment and to discern underlying mechanisms. Male FGF21 knockout (FGF21 KO) and control (WT) mice were divided into groups that were fed either the Lieber DeCarli diet containing 5% alcohol or an isocaloric (control) diet for 4 weeks. One group of WT mice exposed to alcohol received recombinant human FGF21 (rhFGF21) in the last 5 days. Liver steatosis and inflammation were assessed. Primary mouse hepatocytes and AML-12 cells were incubated with metformin or rhFGF21. Hepatic genes and the products involved in in situ lipogenesis and fatty acid β-oxidation were analyzed. Alcohol exposure increased circulating levels and hepatic expression of FGF21. FGF21 depletion exacerbated alcohol-induced hepatic steatosis and liver injury, which was associated with increased activation of genes involved in lipogenesis mediated by SREBP1c and decreased expression of genes involved in fatty acid β-oxidation mediated by PGC1α. rhFGF21 administration reduced alcohol-induced hepatic steatosis and inflammation in WT mice. These results reveal that alcohol-induced FGF21 expression is a hepatic adaptive response to lipid dysregulation. Targeting FGF21 signaling could be a novel treatment approach for alcoholic steatohepatitis. PMID:27498701

  2. Fibroblast growth factor 21 deficiency exacerbates chronic alcohol-induced hepatic steatosis and injury.

    PubMed

    Liu, Yanlong; Zhao, Cuiqing; Xiao, Jian; Liu, Liming; Zhang, Min; Wang, Cuiling; Wu, Guicheng; Zheng, Ming-Hua; Xu, Lan-Man; Chen, Yong-Ping; Mohammadi, Moosa; Chen, Shao-Yu; Cave, Matthew; McClain, Craig; Li, Xiaokun; Feng, Wenke

    2016-01-01

    Fibroblast growth factor 21 (FGF21) is a hepatokine that regulates glucose and lipid metabolism in the liver. We sought to determine the role of FGF21 in hepatic steatosis in mice exposed to chronic alcohol treatment and to discern underlying mechanisms. Male FGF21 knockout (FGF21 KO) and control (WT) mice were divided into groups that were fed either the Lieber DeCarli diet containing 5% alcohol or an isocaloric (control) diet for 4 weeks. One group of WT mice exposed to alcohol received recombinant human FGF21 (rhFGF21) in the last 5 days. Liver steatosis and inflammation were assessed. Primary mouse hepatocytes and AML-12 cells were incubated with metformin or rhFGF21. Hepatic genes and the products involved in in situ lipogenesis and fatty acid β-oxidation were analyzed. Alcohol exposure increased circulating levels and hepatic expression of FGF21. FGF21 depletion exacerbated alcohol-induced hepatic steatosis and liver injury, which was associated with increased activation of genes involved in lipogenesis mediated by SREBP1c and decreased expression of genes involved in fatty acid β-oxidation mediated by PGC1α. rhFGF21 administration reduced alcohol-induced hepatic steatosis and inflammation in WT mice. These results reveal that alcohol-induced FGF21 expression is a hepatic adaptive response to lipid dysregulation. Targeting FGF21 signaling could be a novel treatment approach for alcoholic steatohepatitis. PMID:27498701

  3. Fibroblast growth factor (Fgf) signaling pathway regulates liver homeostasis in zebrafish.

    PubMed

    Tsai, Su-Mei; Liu, Da-Wei; Wang, Wen-Pin

    2013-04-01

    In mammals, fibroblast growth factor (FGF) signaling controls liver specification and regulates the metabolism of lipids, cholesterol, and bile acids. FGF signaling also promotes hepatocyte proliferation, and helps detoxify hepatotoxin during liver regeneration after partial hepatectomy. However, the function of Fgf in zebrafish liver is not yet well understood, specifically for postnatal homeostasis. The current study analyzed the expression of fgf receptors (fgfrs) in the liver of zebrafish. We then investigated the function of Fgf signaling in the zebrafish liver by expressing a dominant-negative Fgf receptor in hepatocytes (lfabp:dnfgfr1-egfp, lf:dnfr). Histological analysis showed that our genetic intervention resulted in a small liver size with defected medial expansion of developing livers in transgenic (Tg) larvae. Morphologically, the liver lobe of lf:dnfr adult fish was shorter than that of control. Ballooning degeneration of hepatocytes was observed in fish as young as 3 months. Further examination revealed the development of hepatic steatosis and cholestasis. In adult Tg fish, we unexpectedly observed increased liver-to-body-weight ratios, with higher percentages of proliferating hepatocytes. Considering all these findings, we concluded that as in mammals, in adult zebrafish the metabolism of lipid and bile acids in the liver are regulated by Fgf signaling. Disruption of the Fgf signal-mediated metabolism might indirectly affect hepatocyte proliferation. PMID:22820869

  4. A heparin-mimicking polymer conjugate stabilizes basic fibroblast growth factor

    NASA Astrophysics Data System (ADS)

    Nguyen, Thi H.; Kim, Sung-Hye; Decker, Caitlin G.; Wong, Darice Y.; Loo, Joseph A.; Maynard, Heather D.

    2013-03-01

    Basic fibroblast growth factor (bFGF) is a protein that plays a crucial role in diverse cellular functions, from wound healing to bone regeneration. However, a major obstacle to the widespread application of bFGF is its inherent instability during storage and delivery. Here, we describe the stabilization of bFGF by covalent conjugation with a heparin-mimicking polymer, a copolymer consisting of styrene sulfonate units and methyl methacrylate units bearing poly(ethylene glycol) side chains. The bFGF conjugate of this polymer retained bioactivity after synthesis and was stable to a variety of environmentally and therapeutically relevant stressors—such as heat, mild and harsh acidic conditions, storage and proteolytic degradation—unlike native bFGF. Following the application of stress, the conjugate was also significantly more active than the control conjugate system in which the styrene sulfonate units were omitted from the polymer structure. This research has important implications for the clinical use of bFGF and for the stabilization of heparin-binding growth factors in general.

  5. Intracellular modification of /sup 125/I-labeled epidermal growth factor by normal human foreskin fibroblasts

    SciTech Connect

    Schaudies, R.P.; Savage, C.R. Jr.

    1986-02-01

    Intracellular processing of /sup 125/I-labeled epidermal growth factor (EGF) in normal human foreskin fibroblasts was examined after incubation with saturating concentrations of (/sup 125/I)EGF. This report describes the column chromatographic separation of multiple processed forms of EGF generated by human foreskin fibroblasts and their structural characterization. More than 95% of the cell-bound (/sup 125/I)EGF was converted into multiple forms, which were separated into four distinct peaks of radioactivity using columns of Bio-Gel P-150 equilibrated with 0.2% sodium dodecyl sulfate. These were designated peaks 1-4. Cellular generation of these four peaks was dependent on culture conditions. Differences in absolute and relative amounts of peaks 1-4 were observed as a function of time of incubation at 37 C. In addition, chromatographic profiles of cell-associated /sup 125/I varied in relation to cell density. The radioactivity in peak 1 comigrated with /sup 125/I-labeled native EGF on nondenaturing polyacrylamide gels (pH 9.5), whereas peaks 2 and 3 exhibited more rapid electrophoretic mobilities. Electrophoretic mobilities of the radioactivity in peaks 2 and 3 were indistinguishable from those of chemically prepared derivatives of (/sup 125/I)EGF which were lacking either one or six amino acid residues from the carboxyterminus, respectively. The EGF receptor bound the radioactive material in peak 2 with an affinity equal to or greater than that of EGF; however, the radioactivity in peak 3 was bound to a much lesser extent. The radiolabel in both peaks 2 and 3 was greater than 95% precipitable by antiserum to native EGF. The labeled material in peak 4 was composed of (/sup 125/I)monoiodotyrosine, /sup 125/I-, and an unidentified peptide. None of the radiolabeled compounds in peak 4 interacted with the EGF receptor or with antiserum to native EGF.

  6. Fibroblast growth factor receptor 3 effects on proliferation and telomerase activity in sheep growth plate chondrocytes

    PubMed Central

    2012-01-01

    Background Fibroblast growth factor receptor 3 (FGFR3) inhibits growth-plate chondrocyte proliferation and limits bone elongation. Gain-of-function FGFR3 mutations cause dwarfism, reduced telomerase activity and shorter telomeres in growth plate chondroyctes suggesting that FGFR3 reduces proliferative capacity, inhibits telomerase, and enhances senescence. Thyroid hormone (T3) plays a role in cellular maturation of growth plate chondrocytes and a known target of T3 is FGFR3. The present study addressed whether reduced FGFR3 expression enhanced telomerase activity, mRNA expression of telomerase reverse transcriptase (TERT) and RNA component of telomerase (TR), and chondrocyte proliferation, and whether the stimulation of FGFR3 by T3 evoked the opposite response. Results Sheep growth-plate proliferative zone chondrocytes were cultured and transfected with siRNA to reduce FGFR3 expression; FGFR3 siRNA reduced chondrocyte FGFR3 mRNA and protein resulting in greater proliferation and increased TERT mRNA expression and telomerase activity (p < 0.05). Chondrocytes treated with T3 significantly enhanced FGFR3 mRNA and protein expression and reduced telomerase activity (p < 0.05); TERT and TR were not significantly reduced. The action of T3 at the growth plate may be partially mediated through the FGFR3 pathway. Conclusions The results suggest that FGFR3 inhibits chondrocyte proliferation by down-regulating TERT expression and reducing telomerase activity indicating an important role for telomerase in sustaining chondrocyte proliferative capacity during bone elongation. PMID:23216972

  7. Alteration of Connective Tissue Growth Factor (CTGF) Expression in Orbital Fibroblasts from Patients with Graves’ Ophthalmopathy

    PubMed Central

    Chang, Pei-Chen; Wei, Yau-Huei

    2015-01-01

    Graves’ ophthalmopathy (GO) is a disfiguring and sometimes blinding disease, which is characterized by inflammation and swelling of orbital tissues, with fibrosis and adipogenesis being predominant features. The aim of this study is to investigate whether the expression levels of fibrosis-related genes, especially that of connective tissue growth factor (CTGF), are altered in orbital fibroblasts of patients with GO. The role of oxidative stress in the regulation of CTGF expression in GO orbital fibroblasts is also examined. By a SYBR Green-based real time quantitative PCR (RT-QPCR), we demonstrated that the mRNA expression levels of fibronectin, apolipoprotein J, and CTGF in cultured orbital fibroblasts from patients with GO were significantly higher than those of age-matched normal controls (p = 0.007, 0.037, and 0.002, respectively). In addition, the protein expression levels of fibronectin, apolipoprotein J, and CTGF analyzed by Western blot were also significantly higher in GO orbital fibroblasts (p = 0.046, 0.032, and 0.008, respectively) as compared with the control. Furthermore, after treatment of orbital fibroblasts with a sub-lethal dose of hydrogen peroxide (200 μM H2O2), we found that the H2O2-induced increase of CTGF expression was more pronounced in the GO orbital fibroblasts as compared with those in normal controls (20% vs. 7%, p = 0.007). Importantly, pre-incubation with antioxidants including N-acetylcysteine (NAC) and vitamin C, respectively, resulted in significant attenuation of the induction of CTGF in GO orbital fibroblasts in response to H2O2 (p = 0.004 and 0.015, respectively). Taken together, we suggest that oxidative stress plays a role in the alteration of the expression of CTGF in GO orbital fibroblasts that may contribute to the pathogenesis and progression of GO. Antioxidants may be used in combination with the therapeutic agents for effective treatment of GO. PMID:26599235

  8. The Role of Vascular Endothelial Growth Factors and Fibroblast Growth Factors in Angiogenesis during Otitis Media

    PubMed Central

    Husseman, Jacob; Palacios, Sean D.; Rivkin, Alexander Z.; Oehl, Heinz; Ryan, Allen F.

    2012-01-01

    The middle ear response to otitis media includes transformation and hyperplasia of the mucosal epithelium and subepithelial connective tissue. Significant neovascularization is also noted, which occurs both to support the hypertrophied mucosa and to mediate the increased trafficking of leukocytes. We investigated the role of two known potent angiogenic growth factor families, the fibroblast growth factors (FGFs) and vascular endothelial growth factors (VEGFs), in middle ear mucosal angiogenesis. DNA microarrays were used to evaluate the expression of FGFs and VEGFs, as well as their receptors and unique signaling proteins, in the middle ears of mice undergoing a complete course of acute bacterial otitis media. In addition, a member of each family was introduced to the middle ear submucosal compartment of the normal middle ears of guinea pigs, by a continuous-release osmotic minipump system over 1 week. During the course of bacterial otitis media, a significant regulation of a number of genes important for angiogenesis was identified. Histologic evaluation of middle ear mucosa following micropump infusion of both FGF1 and VEGF-A showed significant angiogenesis at the site of infusion in comparison to control saline infusion. These results support a role for FGFs and VEGFs in the neovascularization of the middle ear mucosa during otitis media, and offer a potential avenue for therapeutic intervention. PMID:22104377

  9. The effect of valinomycin in fibroblasts from patients with fatty acid oxidation disorders

    SciTech Connect

    Ndukwe Erlingsson, Uzochi Chimdinma; Iacobazzi, Francesco; Liu, Aiping; Ardon, Orly; Pasquali, Marzia; Longo, Nicola

    2013-08-09

    Highlights: •Valinomycin can cause mitochondrial stress and stimulate fatty acid oxidation. •Cells with VLCAD deficiency fail to increase fatty acid oxidation in response to valinomycin. •Response to valinomycin can help in the diagnosis of VLCAD deficiency. -- Abstract: Disorders of the carnitine cycle and of the beta oxidation spiral impair the ability to obtain energy from fats at time of fasting and stress. This can result in hypoketotic hypoglycemia, cardiomyopathy, cardiac arrhythmia and other chronic medical problems. The in vitro study of fibroblasts from patients with these conditions is impaired by their limited oxidative capacity. Here we evaluate the capacity of valinomycin, a potassium ionophore that increases mitochondrial respiration, to increase the oxidation of fatty acids in cells from patients with inherited fatty acid oxidation defects. The addition of valinomycin to fibroblasts decreased the accumulation of the lipophilic cation tetraphenylphosphonium (TPP{sup +}) at low concentrations due to the dissipation of the mitochondrial membrane potential. At higher doses, valinomycin increased TPP{sup +} accumulation due to the increased potassium permeability of the plasma membrane and subsequent cellular hyperpolarization. The incubation of normal fibroblasts with valinomycin increased [{sup 14}C]-palmitate oxidation (measured as [{sup 14}C]O{sub 2} release) in a dose-dependent manner. By contrast, valinomycin failed to increase palmitate oxidation in fibroblasts from patients with very long chain acyl CoA dehydrogenase (VLCAD) deficiency. This was not observed in fibroblasts from patients heterozygous for this condition. These results indicate that valinomycin can increase fatty acid oxidation in normal fibroblasts and could be useful to differentiate heterozygotes from patients affected with VLCAD deficiency.

  10. Autofluorescence Imaging of Living Pancreatic Islets Reveals Fibroblast Growth Factor-21 (FGF21)-Induced Metabolism

    PubMed Central

    Sun, Mark Y.; Yoo, Eunjong; Green, Brenda J.; Altamentova, Svetlana M.; Kilkenny, Dawn M.; Rocheleau, Jonathan V.

    2012-01-01

    Fibroblast growth factor-21 (FGF21) has therapeutic potential for metabolic syndrome due to positive effects on fatty acid metabolism in liver and white adipose tissue. FGF21 also improves pancreatic islet survival in excess palmitate; however, much less is known about FGF21-induced metabolism in this tissue. We first confirmed FGF21-dependent activity in islets by identifying expression of the cognate coreceptor Klothoβ, and by measuring a ligand-stimulated decrease in acetyl-CoA carboxylase expression. To further reveal the effect of FGF21 on metabolism, we employed a unique combination of two-photon and confocal autofluorescence imaging of the NAD(P)H and mitochondrial NADH responses while holding living islets stationary in a microfluidic device. These responses were further correlated to mitochondrial membrane potential and insulin secretion. Glucose-stimulated responses were relatively unchanged by FGF21. In contrast, responses to glucose in the presence of palmitate were significantly reduced compared to controls showing diminished NAD(P)H, mitochondrial NADH, mitochondrial membrane potential, and insulin secretion. Consistent with the glucose-stimulated responses being smaller due to continued fatty acid oxidation, mitochondrial membrane potential was increased in FGF21-treated islets by using the fatty acid transport inhibitor etomoxir. Citrate-stimulated NADPH responses were also significantly larger in FGF21-treated islets suggesting preference for citrate cycling rather than acetyl-CoA carboxylase-dependent fatty acid synthesis. Overall, these data show a reduction in palmitate-induced potentiation of glucose-stimulated metabolism and insulin secretion in FGF21-treated islets, and establish the use of autofluorescence imaging and microfluidic devices to investigate cell metabolism in a limited amount of living tissue. PMID:23283237

  11. Individual Differences in the Expression of Conditioned Fear Are Associated with Endogenous Fibroblast Growth Factor 2

    ERIC Educational Resources Information Center

    Graham, Bronwyn M.; Richardson, Rick

    2016-01-01

    These experiments examined the relationship between the neurotrophic factor fibroblast growth factor 2 (FGF2) and individual differences in the expression of conditioned fear. Experiments 1 and 2 demonstrated that rats naturally expressing low levels of contextual or cued fear have higher levels of hippocampal FGF2 relative to rats that express…

  12. Problem-Based Test: The Effect of Fibroblast Growth Factor on Gene Expression

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2011-01-01

    This paper shows the results of an experiment in which the effects of fibroblast growth factor (FGF), actinomycin D (Act D; an inhibitor of transcription), and cycloheximide (CHX; an inhibitor of translation) were studied on the expression of two genes: a gene called "Fnk" and the gene coding for glyceraldehyde-3-phosphate dehydrogenase (GAPDH).…

  13. Platelet-derived growth factor-D promotes fibrogenesis of cardiac fibroblasts.

    PubMed

    Zhao, Tieqiang; Zhao, Wenyuan; Chen, Yuanjian; Li, Victoria S; Meng, Weixin; Sun, Yao

    2013-06-15

    Platelet-derived growth factor (PDGF)-D is a newly recognized member of the PDGF family with its role just now being understood. Our previous study shows that PDGF-D and its receptors (PDGFR-β) are significantly increased in the infarcted heart, where PDGFR-β is primarily expressed by fibroblasts, indicating the involvement of PDGF-D in the development of cardiac fibrosis. In continuing with these findings, the current study explored the molecular basis of PDGF-D on fibrogenesis. Rat cardiac fibroblasts were isolated and treated with PDGF-D (200 ng/ml medium). The potential regulation of PDGF-D on fibroblast growth, phenotype change, collagen turnover, and the transforming growth factor (TGF)-β pathway were explored. We found: 1) PDGF-D significantly elevated cardiac fibroblast proliferation, myofibroblast (myoFb) differentiation, and type I collagen secretion; 2) matrix metalloproteinase (MMP)-1, MMP-2, and MMP-9 protein levels were significantly elevated in PDGF-D-treated cells, which were coincident with increased expressions of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2; 3) PDGF-D significantly enhanced TGF-β1 synthesis, which was eliminated by TGF-β blockade with small-interfering RNA (siRNA); 4) the stimulatory role of PDGF-D on fibroblast proliferation and collagen synthesis was abolished by TGF-β blockade; and 5) TGF-β siRNA treatment significantly suppressed PDGF-D synthesis in fibroblasts. These observations indicate that PDGF-D promotes fibrogenesis through multiple mechanisms. Coelevations of TIMPs and MMPs counterbalance collagen degradation. The profibrogenic role of PDGF-D is mediated through activation of the TGF-β1 pathway. TGF-β1 exerts positive feedback on PDGF-D synthesis. These findings suggest the potential therapeutic effect of PDGFR blockade on interstitial fibrosis in the infarcted heart. PMID:23585135

  14. Optimal Viscosity and Particle Shape of Hyaluronic Acid Filler as a Scaffold for Human Fibroblasts.

    PubMed

    Kim, Deok-Yeol; Namgoong, Sik; Han, Seung-Kyu; Won, Chang-Hoon; Jeong, Seong-Ho; Dhong, Eun-Sang; Kim, Woo-Kyung

    2015-07-01

    The authors previously reported that cultured human fibroblasts suspended in a hyaluronic acid filler can produce human dermal matrices with extended in vivo stability in animal and clinical studies. The present study was undertaken to determine the optimal viscosity and particle shape of hyaluronic acid filler as a scaffold for cultured human dermal fibroblasts to enhance the maximal viability of injected cells. The fibroblasts were suspended in either 1 of 3 hyaluronic acid viscosities at 2 different particle shapes. The viscosities used in this study were low (600,000-800,000 centipoises), moderate (2,000,000-4,000,000 centipoises), and high (8,000,000-12,000,000 centipoises). The particle shape was evaluated by testing round and irregular shapes. The fibroblast mixed bioimplants were injected into the back of individual athymic nude mice. The levels of type I collagen were measured using fluorescent-activated cell sorting (FACS) and immunohistochemical staining at 16 weeks after the injections. Results of FACS demonstrated that the mean cell ratio with human collagens in the moderate viscosity group was greater than those of control, low, and high viscosity groups. An immunohistochemical study showed similar results. The moderate viscosity group demonstrated the highest positive staining of human collagens. However, there were no significant differences between groups of irregular and round shape particles. A hyaluronic acid bioimplant with moderate viscosity is superior to that with low or high viscosity in the viability for human fibroblasts. However, the particle shape does not influence the viability of the fibroblasts. PMID:26163839

  15. Mutations in fibroblast growth factor receptors: Phenotypic consequences during eukaryotic development

    SciTech Connect

    Park, W.J.; Bellus, G.A.; Jabs, E.W.

    1995-10-01

    Recently, a tremendous amount of excitement and interest has been generated by the rapid succession of discoveries in the human fibroblast growth factor receptor (FGFR) field. In less than a year, mutations in three FGFRs (FGFR1-FGFR3) have been associated with three skeletal dysplasias and four craniosynostotic syndromes. FGFRs are members of the receptor tyrosine kinase family that bind fibroblast growth factors (FGFs). The FGF family consists of structurally related polypeptides that play a key role in numerous aspects of embryogenesis, growth, and homeostasis. FGFs have a potent growth stimulatory and/or differentiation-inducing effect on cells such as those derived from the early-embryonic mesoderm or ectoderm. In addition to mitogenesis and differentiation, FGFs also stimulate chemotaxis, cell survival, and angiogenesis. FGFs mediate cellular responses on binding to and activation of FGFRs. 45 refs., 2 figs., 1 tab.

  16. Enhanced effect of fibroblast growth factor-2-containing dalteparin/protamine nanoparticles on hair growth

    PubMed Central

    Takabayashi, Yuki; Nambu, Masaki; Ishihara, Masayuki; Kuwabara, Masahiro; Fukuda, Koichi; Nakamura, Shingo; Hattori, Hidemi; Kiyosawa, Tomoharu

    2016-01-01

    Purpose Although treatments for alopecia are in high demand, not all treatments are safe and reliable. Dalteparin/protamine nanoparticles (D/P NPs) can effectively carry growth factors (GFs) such as fibroblast GF (FGF)-2. The purpose of this study was to identify the effects of FGF-2-containing D/P NPs (FGF-2&D/P NPs) on hair growth. Patients and methods In this study, the participants were 12 volunteers with thin hair. One milliliter of FGF-2 (100 ng/mL) and D/P NPs (56 μg/mL) was applied and massaged on the skin of the scalp by the participants twice a day. They were evaluated for 6 months. Participants were photographed using a digital camera for general observation and a hair diagnosis system for measuring hair diameter. Results The mean diameter of the hairs was significantly higher following the application of FGF-2&D/P NPs for 6 months. Objective improvements in thin hair were observed in two cases. Nine participants experienced greater bounce and hair resilience. Conclusion The transdermal application of FGF-2&D/P NPs to the scalp can be used as a new treatment for alopecia. PMID:27274299

  17. Nicotine Stimulates Nerve Growth Factor in Lung Fibroblasts through an NFκB-Dependent Mechanism

    PubMed Central

    Wongtrakool, Cherry; Grooms, Kora; Bijli, Kaiser M.; Crothers, Kristina; Fitzpatrick, Anne M.; Hart, C. Michael

    2014-01-01

    Rationale Airway hyperresponsiveness (AHR) is classically found in asthma, and persistent AHR is associated with poor asthma control. Although airway smooth muscle (ASM) cells play a critical pathophysiologic role in AHR, the paracrine contributions of surrounding cells such as fibroblasts to the contractile phenotype of ASM cells have not been examined fully. This study addresses the hypothesis that nicotine promotes a contractile ASM cell phenotype by stimulating fibroblasts to increase nerve growth factor (NGF) secretion into the environment. Methods Primary lung fibroblasts isolated from wild type and α7 nicotinic acetylcholine receptor (α7 nAChR) deficient mice were treated with nicotine (50 µg/ml) in vitro for 72 hours. NGF levels were measured in culture media and in bronchoalveolar lavage (BAL) fluid from asthmatic, smoking and non-smoking subjects by ELISA. The role of the NFκB pathway in nicotine-induced NGF expression was investigated by measuring NFκB nuclear translocation, transcriptional activity, chromatin immunoprecipitation assays, and si-p65 NFκB knockdown. The ability of nicotine to stimulate a fibroblast-mediated, contractile ASM cell phenotype was confirmed by examining expression of contractile proteins in ASM cells cultured with fibroblast-conditioned media or BAL fluid. Results NGF levels were elevated in the bronchoalveolar lavage fluid of nicotine-exposed mice, current smokers, and asthmatic children. Nicotine increased NGF secretion in lung fibroblasts in vitro in a dose-dependent manner and stimulated NFκB nuclear translocation, p65 binding to the NGF promoter, and NFκB transcriptional activity. These responses were attenuated in α7 nAChR deficient fibroblasts and in wild type fibroblasts following NFκB inhibition. Nicotine-treated, fibroblast-conditioned media increased expression of contractile proteins in ASM cells. Conclusion Nicotine stimulates NGF release by lung fibroblasts through α7 nAChR and NFκB dependent pathways

  18. Comparison of human dermal fibroblasts (HDFs) growth rate in culture media supplemented with or without basic fibroblast growth factor (bFGF).

    PubMed

    Abdian, Narges; Ghasemi-Dehkordi, Payam; Hashemzadeh-Chaleshtori, Morteza; Ganji-Arjenaki, Mahbobe; Doosti, Abbas; Amiri, Beheshteh

    2015-12-01

    Basic fibroblast growth factor (bFGF or FGF-2) is a member of the FGF family secreted by different kinds of cells like HDFs and it is an important nutritional factor for cell growth and differentiation. The HDFs release bFGF in culture media at very low. The present study aims to investigate the HDFs growth rate in culture media supplemented either with or without bFGF. In brief, HDFs were isolated from human foreskin sample and were cultured in vitro in media containing bFGF and lack of this factor. The cells growth rate was calculated by trypan blue. The karyotyping was performed using G-banding to investigate the chromosomal abnormality of HDFs in both groups. Total RNA of each groups were extracted and cDNA samples were synthesized then, real-time Q-PCR was used to measure the expression level of p27kip1 and cyclin D1 genes normalized to internal control gene (GAPDH). The karyotype analysis showed that HDFs cultured in media or without bFGF had normal karyotype (46 chromosomes, XY) and chromosomal abnormalities were not observed. The cell growth rates in both groups were normal with proliferated exponentially but the slope of growth curve in HDFs cultured in media containing bFGF was increased. Karyotyp test showed that bFGF does not affect on cytogenetic stability of cells. The survey of p27kip1 and cyclin D1 genes by real-time Q-PCR showed that the expression level of these genes were up-regulated when adding bFGF in culture media (p < 0.05). The findings of the present study demonstrate that appropriate supplementation of culture media with growth factor like bFGF could enhance the proliferation and differentiation capacity of cells and improve cells growth rate. Similarly, fibroblast growth factors did not induce any chromosomal abnormality in cells. Furthermore, in HDFs cultured in bFGF supplemented media, the p27kip1 and cyclin D1 genes were up-regulated and suggesting an important role for bFGF in cell-cycle regulation and progression and fibroblast

  19. Fibroblast growth factor 7 inhibits cholesterol 7{alpha}-hydroxylase gene expression in hepatocytes

    SciTech Connect

    Sun, Zhichao; Yu, Xuemei; Wu, Weibin; Jia, Dongwei; Chen, Yinle; Ji, Lingling; Liu, Xijun; Peng, Xiaomin; Li, Yintao; Yang, Lili; Ruan, Yuanyuan; Gu, Jianxin; Ren, Shifang; Zhang, Songwen

    2012-07-13

    Highlights: Black-Right-Pointing-Pointer FGF7 strongly and rapidly down-regulates the expression of CYP7A1 in hepatocytes. Black-Right-Pointing-Pointer FGF7 suppresses the expression of CYP7A1 via FGFR2 and downstream JNK activation. Black-Right-Pointing-Pointer Blocking FGF7 abrogates HSC-induced inhibition of CYP7A1 expression in hepatocytes. -- Abstract: Cholesterol 7{alpha}-hydroxylase (CYP7A1) is the initial and rate-limiting enzyme for bile acid synthesis. Transcription of the CYP7A1 gene is regulated by bile acids, nuclear receptors and cytokines. Fibroblast growth factor 7 (FGF7) secreted from activated hepatic stellate cells (HSC) during chronic liver fibrosis regulates hepatocyte survival and liver regeneration. In the carbon tetrachloride (CCl{sub 4})-induced fibrotic mouse liver, we demonstrated that the expression of CYP7A1 was largely decreased while the expression of FGF7 was significantly increased. We further demonstrated that FGF7 inhibited CYP7A1 gene expression in hepatocytes. Knockdown study by short interfering RNA, kinase inhibition and phosphorylation assays revealed that the suppression of CYP7A1 expression by FGF7 was mediated by FGFR2 and its downstream JNK signaling cascade. The FGF7 neutralizing antibody restored CYP7A1 expression in Hep3B cells treated with conditioned medium from HSC. In summary, the data suggest that FGF7 is a novel regulator of CYP7A1 expression in hepatocytes and may prevent hepatocytes from accumulating toxic bile acids during liver injury and fibrosis.

  20. Accelerated fracture healing in transgenic mice overexpressing an anabolic isoform of fibroblast growth factor 2.

    PubMed

    Hurley, Marja M; Adams, Douglas J; Wang, Liping; Jiang, Xi; Burt, Patience Meo; Du, Erxia; Xiao, Liping

    2016-03-01

    The effect of targeted expression of an anabolic isoform of basic fibroblast growth factor (FGF2) in osteoblastic lineage on tibial fracture healing was assessed in mice. Closed fracture of the tibiae was performed in Col3.6-18 kDaFgf2-IRES-GFPsaph mice in which a 3.6 kb fragment of type I collagen promoter (Col3.6) drives the expression of only the 18 kD isoform of FGF2 (18 kDaFgf2/LMW) with green fluorescent protein-sapphire (GFPsaph) as well as Vector mice (Col3.6-IRES-GFPsaph, Vector) that did not harbor the FGF2 transgene. Radiographic, micro-CT, DEXA, and histologic analysis of fracture healing of tibiae harvested at 3, 10 and 20 days showed a smaller fracture callus but accelerated fracture healing in LMWTg compared with Vector mice. At post fracture day 3, FGF receptor 3 and Sox 9 mRNA were significantly increased in LMWTg compared with Vector. Accelerated fracture healing was associated with higher FGF receptor 1, platelet derived growth factors B, C, and D, type X collagen, vascular endothelial cell growth factor, matrix metalloproteinase 9, tartrate resistant acid phosphatase, cathepsin K, runt-related transcription factor-2, Osterix and Osteocalcin and lower Sox9, and type II collagen expression at 10 days post fracture. We postulate that overexpression of LMW FGF2 accelerated the fracture healing process due to its effects on factors that are important in chondrocyte and osteoblast differentiation and vascular invasion. PMID:26252425

  1. Effects of Activin in Embryoid Bodies Expressing Fibroblast Growth Factor 5.

    PubMed

    Shirouzu, Yasumasa; Yanai, Goichi; Yang, Kai-Chiang; Sumi, Shoichiro

    2016-06-01

    Nodal/activin signaling is indispensable for embryonic development. We examined what activin does to the embryoid bodies (EBs) produced from mouse embryonic stem cells (mESCs) expressing an epiblast marker. The EBs were produced by culturing mESCs by the hanging drop method for 24 hours. The resulting EBs were transferred onto gelatin-coated dishes and allowed to further differentiate. The 24-hour EBs showed a stronger expression of fibroblast growth factor (FGF)5 and Brachyury (specific to the epiblast) in comparison with mESCs. Treating the transferred EBs with activin A maintained transcript levels of FGF5 and Oct4, while inhibiting definitive endoderm differentiation. The activin A treatment reversed the endoderm differentiation induced by retinoic acid (RA), while the inhibition of nodal/activin signaling promoted RA-induced endoderm differentiation. Inhibition of nodal/activin signaling in EBs, including epiblast-like cells, promotes differentiation into the endoderm, facilitating the transition from the pluripotent state to specification of the endoderm. PMID:27253628

  2. Heparin binding preference and structures in the fibroblast growth factor family parallel their evolutionary diversification

    PubMed Central

    Jiang, Chao; Wilkinson, Mark C.

    2016-01-01

    The interaction of a large number of extracellular proteins with heparan sulfate (HS) regulates their transport and effector functions, but the degree of molecular specificity underlying protein–polysaccharide binding is still debated. The 15 paracrine fibroblast growth factors (FGFs) are one of the paradigms for this interaction. Here, we measure the binding preferences of six FGFs (FGF3, FGF4, FGF6, FGF10, FGF17, FGF20) for a library of modified heparins, representing structures in HS, and model glycosaminoglycans, using differential scanning fluorimetry. This is complemented by the identification of the lysine residues in the primary and secondary binding sites of the FGFs by a selective labelling approach. Pooling these data with previous sets provides good coverage of the FGF phylogenetic tree, deduced from amino acid sequence alignment. This demonstrates that the selectivity of the FGFs for binding structures in sulfated polysaccharides and the pattern of secondary binding sites on the surface of FGFs follow the phylogenetic relationship of the FGFs, and so are likely to be the result of the natural selection pressures that led to the expansion of the FGF family in the course of the evolution of more complex animal body plans. PMID:27030175

  3. Minireview: Roles of Fibroblast Growth Factors 19 and 21 in Metabolic Regulation and Chronic Diseases.

    PubMed

    Zhang, Fangfang; Yu, Lechu; Lin, Xiufei; Cheng, Peng; He, Luqing; Li, Xiaokun; Lu, Xuemian; Tan, Yi; Yang, Hong; Cai, Lu; Zhang, Chi

    2015-10-01

    Fibroblast growth factor (FGF)19 and FGF21 are hormones that regulate metabolic processes particularly during feeding or starvation, thus ultimately influencing energy production. FGF19 is secreted by the intestines during feeding and negatively regulates bile acid synthesis and secretion, whereas FGF21 is produced in the liver during fasting and plays a crucial role in regulating glucose and lipid metabolism, as well as maintaining energy homeostasis. FGF19 and FGF21 are regarded as late-acting hormones because their functions are only used after insulin and glucagon have completed their actions. Although FGF19 and FGF21 are activated under different conditions, they show extensively functional overlap in terms of improving glucose tolerance, insulin sensitivity, weight loss, and lipid, and energy metabolism, particularly in pathological conditions such as diabetes, obesity, metabolic syndrome, and cardiovascular and renal diseases. Most patients with these metabolic diseases exhibit reduced serum FGF19 levels, which might contribute to its etiology. In addition, the simultaneous increase in serum FGF21 levels is likely a compensatory response to reduced FGF19 levels, and the 2 proteins concertedly maintain metabolic homeostasis. Here, we review the physiological and pharmacological cross talk between FGF19 and FGF21 in relation to the regulation of endocrine metabolism and various chronic diseases. PMID:26308386

  4. Fibroblast Growth Factor 21 Suppresses Adipogenesis in Pig Intramuscular Fat Cells

    PubMed Central

    Wang, Yongliang; Liu, Xinyi; Hou, Liming; Wu, Wangjun; Zhao, Shuhong; Xiong, Yuanzhu

    2015-01-01

    Fibroblast growth factor 21 (FGF21) plays an important role in the treatment of disease associated with muscle insulin resistance which is characterized by various factors, such as intramuscular triglyceride (IMT) content. Studies have also shown that FGF21 inhibits triglyceride synthesis in vivo. However, the precise mechanism whereby FGF21 regulates triglyceride metabolism in intramuscular fat (IMF), which may influence the muscle insulin sensitivity, is not clearly understood. In order to understand the role of FGF21 in IMF deposition, we performed FGF21 overexpression in IMF cells by stable transfection. Our results showed that FGF21 inhibited the key adipogenesis gene mRNA expression of peroxisome proliferator-activated receptor gamma (PPARG), CCAAT/enhancer-binding protein (CEBP) family by reducing lysine-specific demethylase 1 (LSD1) expression which led to significant decline in lipid accumulation, and the result was confirmed by Western blot. Moreover, triggered by FGF21, parts of the adipokines—fatty acid-binding protein 4 (FABP4), glucose transporter 4 (GLUT4), adiponectin (ADIPOQ), and perilipin (PLIN1)—were also down-regulated. Furthermore, FGF21 gene expression was suppressed by transcription factor CEBP beta (CEBPB) which contributed strongly to triglyceride synthesis. Taken together, our study is the first to experimentally demonstrate FGF21 emerging as an efficient blockade of adipogenesis in IMF, thus also providing a new understanding of the mechanism whereby FGF21 improves insulin sensitivity. PMID:26703591

  5. Fibroblast Growth Factor 8 Deficiency Compromises the Functional Response of the Serotonergic System to Stress

    PubMed Central

    Brooks, Leah R.; Pals, Heide L.; Enix, Courtney L.; Woolaver, Rachel A.; Paul, Evan D.; Lowry, Christopher A.; Tsai, Pei-San

    2014-01-01

    Functionally heterogeneous populations of serotonergic neurons, located within the dorsal raphe nucleus (DR), play a role in stress-related behaviors and neuropsychiatric illnesses such as anxiety and depression. Abnormal development of these neurons may permanently alter their structure and connections, making the organism more susceptible to anxiety-related disorders. A factor that critically regulates the development of serotonergic neurons is fibroblast growth factor 8 (Fgf8). In this study, we used acute restraint stress followed by behavioral testing to examine whether Fgf8 signaling during development is important for establishing functional stress- and anxiety-related DR neurocircuits in adulthood. Wild-type and heterozygous male mice globally hypomorphic for Fgf8 were exposed to acute restraint stress and then tested for anxiety-like behavior on the elevated plus-maze. Further, we measured c-Fos immunostaining as a marker of serotonergic neuronal activation and tissue 5-hydroxyindoleacetic acid concentrations as a marker of serotonin functional output. Results showed that Fgf8 hypomorphs exhibited 1) an exaggerated response of DR anxiety-promoting circuits and 2) a blunted response of a DR panic-inhibiting circuit to stress, effects that together were associated with increased baseline anxiety-like behavior. Overall, our results provide a neural substrate upon which Fgf8 deficiency could affect stress response and support the hypothesis that developmental disruptions of serotonergic neurons affect their postnatal functional integrity. PMID:24992493

  6. Fibroblast Growth Factor 21 Suppresses Adipogenesis in Pig Intramuscular Fat Cells.

    PubMed

    Wang, Yongliang; Liu, Xinyi; Hou, Liming; Wu, Wangjun; Zhao, Shuhong; Xiong, Yuanzhu

    2016-01-01

    Fibroblast growth factor 21 (FGF21) plays an important role in the treatment of disease associated with muscle insulin resistance which is characterized by various factors, such as intramuscular triglyceride (IMT) content. Studies have also shown that FGF21 inhibits triglyceride synthesis in vivo. However, the precise mechanism whereby FGF21 regulates triglyceride metabolism in intramuscular fat (IMF), which may influence the muscle insulin sensitivity, is not clearly understood. In order to understand the role of FGF21 in IMF deposition, we performed FGF21 overexpression in IMF cells by stable transfection. Our results showed that FGF21 inhibited the key adipogenesis gene mRNA expression of peroxisome proliferator-activated receptor gamma (PPARG), CCAAT/enhancer-binding protein (CEBP) family by reducing lysine-specific demethylase 1 (LSD1) expression which led to significant decline in lipid accumulation, and the result was confirmed by Western blot. Moreover, triggered by FGF21, parts of the adipokines--fatty acid-binding protein 4 (FABP4), glucose transporter 4 (GLUT4), adiponectin (ADIPOQ), and perilipin (PLIN1)--were also down-regulated. Furthermore, FGF21 gene expression was suppressed by transcription factor CEBP beta (CEBPB) which contributed strongly to triglyceride synthesis. Taken together, our study is the first to experimentally demonstrate FGF21 emerging as an efficient blockade of adipogenesis in IMF, thus also providing a new understanding of the mechanism whereby FGF21 improves insulin sensitivity. PMID:26703591

  7. Hyperbaric oxygen attenuates cell growth in skin fibroblasts cultured in a high-glucose medium.

    PubMed

    Lin, Hen-I; Chu, Shi-Jye; Perng, Wann-Cherng; Wu, Chin-Pyng; Lin, Zuei-Yin; Huang, Kun-Lun

    2008-01-01

    Hyperglycemia and hypoxia synergistically retard diabetic wound healing. We investigated the direct effect of hyperbaric and normobaric hyperoxia on skin fibroblasts cultured in a high-glucose medium. Detroit 551 human dermal fibroblasts cultured in Dulbecco's modified Eagle's medium containing d-glucose had reduced cell survival compared with cells grown in normal glucose medium; survival was 27.5+/-3.8% lower in 25 mM glucose and 30.6+/-3.7% lower in 50 mM glucose. Cell survival decreased because of inhibition of cell proliferation and enhanced cell death. Daily hyperbaric oxygen therapy at 2.5 atmosphere absolute for 90 minutes on 3 consecutive days reduced cell proliferation and increased cell death in normal cultured fibroblasts. Hyperbaric oxygen therapy and high-glucose medium had a synergistic effect and reduced survival by 37.6+/-4.4% (25 mM glucose) and 39.6+/-5.1% (50 mM glucose). The effects of hyperbaric oxygen and high-glucose medium were associated with overproduction of reactive oxygen species. Our results suggest that direct exposure of skin fibroblasts to hyperbaric oxygen affects cell growth and superimposes the toxic effect of high glucose. This cytotoxicity may be related to the production of reactive oxygen species in the fibroblasts. PMID:18638270

  8. Neurotensin Decreases the Proinflammatory Status of Human Skin Fibroblasts and Increases Epidermal Growth Factor Expression

    PubMed Central

    Miguel Neves, Bruno; Cruz, Maria Teresa; Carvalho, Eugénia

    2014-01-01

    Fibroblasts colonization into injured areas during wound healing (WH) is responsible for skin remodelling and is also involved in the modulation of inflammation, as fibroblasts are immunologically active. Herein, we aimed to determine neurotensin effect on the immunomodulatory profile of fibroblasts, both in homeostatic and inflammatory conditions. Neurotensin mediated responses occurred through NTR1 or NTR3 receptors, while under inflammatory conditions NTR1 expression increase seemed to modulate neurotensin responses. Among different immunomodulatory genes, CCL11, IL-8, and IL-6 were the most expressed genes, while CCL4 and EGF were the less expressed genes. After neurotensin exposure, IL-8 mRNA expression was increased while CCL11 was decreased, suggesting a proinflammatory upregulation and chemoattractant ability downregulation of fibroblasts. Under inflammatory conditions, gene expression was significantly increased. After neurotensin exposure, CCL4 and IL-6 mRNA expression were decreased while CCL11 was increased, suggesting again a decrease in the chemoattractant capacity of fibroblasts and in their proinflammatory status. Furthermore, the expression of EGF, a crucial growth factor for skin cells proliferation and WH, was increased in all conditions. Overall, neurotensin, released by nerve fibers or skin cells, may be involved in the decrease of the chemotaxis and the proinflammatory status in the proliferation and remodelling phases of WH. PMID:25180119

  9. Endogenous insulin-like growth factor (IGF) binding proteins cause IGF-1 resistance in cultured fibroblasts from a patient with short stature.

    PubMed Central

    Tollefsen, S E; Heath-Monnig, E; Cascieri, M A; Bayne, M L; Daughaday, W H

    1991-01-01

    The ED50 of insulin-like growth factor (IGF)-I-stimulated alpha-aminoisobutyric acid (AIB) uptake (mean +/- SD) in cultured fibroblasts from a child with short stature that we have reported (1.40 +/- 0.24 nM), is significantly higher than the ED50 of IGF-I-stimulated AIB uptake in fibroblasts from 11 normal subjects (0.42 +/- 0.12 nM) and from 127 short children (0.35 +/- 0.11 nM). Similarly, the ED50 of IGF-I-stimulated thymidine incorporation in fibroblasts from this child is 2.8 times higher than that in fibroblasts from four normal subjects. To minimize potential modulation of IGF-I action by endogenous IGF binding proteins in these assays, fibroblast responsiveness to [Q3,A4,Y15,L16]IGF-I, an IGF-I variant that has a 600-fold reduced affinity for serum IGF binding proteins, has been examined. The biological activity of this variant is comparable in the patient's and normal fibroblasts, suggesting that the resistance to IGF-I action cannot be attributed to a defective IGF-I receptor. To investigate directly the possibility that IGF-I sensitivity in the patient's fibroblasts is reduced by endogenous IGF binding proteins (IGFBP), binding proteins that are secreted into AIB assay buffer during a 3-h collection and that are cell-associated at the end of the collection have been analyzed. Ligand blot analysis of conditioned AIB assay buffer demonstrates that fibroblasts from the patient secrete 1.3-2.2 times more of Mr 46,400/42,900, 32,000, and 26,800 binding proteins than normal fibroblasts. The major difference between fibroblasts from the patient and from normal subjects is a striking 10-fold increase in the amount of a cell surface Mr 32,000 binding protein in the patient's fibroblasts. The Mr 32,000 binding protein is similar in size to IGFB-1 and different from IGFBP-2 and IGFBP-3, but it does not cross-react with an antibody against IGFBP-1. We conclude that the resistance to IGF-I action in the patient's fibroblasts is caused by an abnormal production and

  10. Intercellular transport of lysosomal acid lipase mediates lipoprotein cholesteryl ester metabolism in a human vascular endothelial cell-fibroblast coculture system.

    PubMed Central

    Sando, G N; Ma, G P; Lindsley, K A; Wei, Y P

    1990-01-01

    We present results from studies of human cell culture models to support the premise that the extracellular transport of lysosomal acid lipase has a function in lipoprotein cholesteryl ester metabolism in vascular tissue. Vascular endothelial cells secreted a higher fraction of cellular acid lipase than did smooth muscle cells and fibroblasts. Acid lipase and lysosomal beta-hexosaminidase were secreted at approximately the same rate from the apical and basolateral surface of an endothelial cell monolayer. Stimulation of secretion with NH4Cl did not affect the polarity. We tested for the ability of secreted endothelial lipase to interact with connective tissue cells and influence lipoprotein cholesterol metabolism in a coculture system in which endothelial cells on a micropore filter were suspended above a monolayer of acid lipase-deficient (Wolman disease) fibroblasts. After 5-7 d, acid lipase activity in the fibroblasts reached 10%-20% of the level in normal cells; cholesteryl esters that had accumulated from growth in serum were cleared. Addition of mannose 6-phosphate to the coculture medium blocked acid lipase uptake and cholesterol clearance, indicating that lipase released from endothelial cells was packaged into fibroblast lysosomes by a phosphomannosyl receptor-mediated pathway. Supplementation of the coculture medium with serum was not required for lipase uptake and cholesteryl ester hydrolysis by the fibroblasts, but was necessary for cholesterol clearance. Results from our coculture model suggest that acid lipase may be transported from intact endothelium to cells in the lumen or the wall of a blood vessel. We postulate that delivery of acid hydrolases and lipoproteins to a common endocytic compartment may occur and have an impact on cellular lipoprotein processing. PMID:2150334

  11. ATP differentially upregulates fibroblast growth factor 2 and transforming growth factor α in neonatal and adult mice: effect on neuroproliferation.

    PubMed

    Jia, C; Cussen, A R; Hegg, C C

    2011-03-17

    Multiple neurotrophic factors play a role in proliferation, differentiation and survival in the olfactory epithelium (OE); however, the signaling cascade has not been fully elucidated. We tested the hypotheses that ATP induces the synthesis and secretion of two neurotrophic factors, fibroblast growth factor 2 (FGF2) and transforming growth factor alpha (TGFα), and that these neurotrophic factors have a role in inducing proliferation. Protein levels of FGF2 and TGFα were increased 20 h post-intranasal instillation of ATP compared to vehicle control in adult Swiss Webster mice. Pre-intranasal treatment with purinergic receptor antagonist pyridoxal-phosphate-6-azophenyl-20,40-disulfonic acid (PPADS) significantly blocked this ATP-induced increase, indicating that upregulation of FGF2 and TGFα expression is mediated by purinergic receptor activation. However, in neonatal mouse, intranasal instillation of ATP significantly increased the protein levels of FGF2, but not TGFα. Likewise, ATP evoked the secretion of FGF2, but not TGFα, from neonatal mouse olfactory epithelial slices and PPADS significantly blocked ATP-evoked FGF2 release. To determine the role of FGF2 and TGFα in inducing proliferation, 5-bromo-2-deoxyuridine (BrdU) incorporation was examined in adult olfactory epithelium. Intranasal treatment with FGF receptor inhibitor PD173074 or epidermal growth factor receptor inhibitor AG1478 following ATP instillation significantly blocked ATP-induced BrdU incorporation. Collectively, these data demonstrate that ATP induces proliferation in adult mouse olfactory epithelium by promoting FGF2 and TGFα synthesis and activation of their receptors. These data suggest that different mechanisms regulate neurogenesis in neonatal and adult OE, and FGF2 and TGFα may have different roles throughout development. PMID:21187124

  12. Autocrine and paracrine actions of intestinal fibroblast-derived insulin-like growth factors.

    PubMed

    Simmons, J G; Pucilowska, J B; Lund, P K

    1999-04-01

    Paracrine and autocrine actions of the insulin-like growth factors (IGFs) are inferred by local expression within the bowel. CCD-18Co cells, IEC-6 cells, and immunoneutralization were used to analyze whether IGFs have direct autocrine or paracrine effects on proliferation of cultured intestinal fibroblasts and epithelial cells. Growth factor expression was analyzed by ribonuclease protection assay and RT-PCR. Extracellular matrix (ECM) was analyzed for effects on cell proliferation. CCD-18Co cells express IGF-II mRNAs and low levels of IGF-I mRNA. Conditioned medium from CCD-18Co cells (CCD-CM) stimulated proliferation of IEC-6 and CCD-18Co cells. Neutralization of IGF immunoreactivity in CCD-CM reduced but did not abolish this effect. RT-PCR and immunoneutralization demonstrated that other growth factors contribute to mitogenic activity of CCD-CM. Preincubation of CCD-CM with ECM prepared from IEC-6 or CCD-18Co cells reduced its mitogenic activity. ECM from CCD-18Co cells enhanced growth factor-dependent proliferation of IEC-6 cells. IEC-6 cell ECM inhibited IGF-I action on CCD-18Co cells. We conclude that IGF-II is a potent autocrine mitogen for intestinal fibroblasts. IGF-II interacts with other fibroblast-derived growth factors and ECM to stimulate proliferation of intestinal epithelial cells in a paracrine manner. PMID:10198323

  13. Effects of growth factors on the proliferation of human keratinocytes and fibroblasts in vitro.

    PubMed

    Kim, D S; Korting, H C; Schäfer-Korting, M

    1998-01-01

    Growth/differentiation factor-5 (GDF-5) is a new member of the transforming growth factor-beta (TGF-beta) superfamily of multifunctional peptide growth factors that appear to mediate many key events in cell growth and development. The effects of GDF-5 and other growth factors (epidermal growth factor, EGF; TGF-beta 1) on the proliferation of human keratinocytes and fibroblasts compared with desoximetasone and calcipotriol have been investigated. The proliferation rate was determined by a hemocytometer, MTT assay and the incorporation of [3H]-thymidine. Moreover, cell cycle analyses were performed and the influence on interleukin-1 alpha (IL-1 alpha) production in keratinocytes was measured by enzyme-linked immunosorbent assay (ELISA) because of its pronounced proinflammatory effect. In keratinocytes, GDF-5 stimulated cell proliferation to a minor extent. The drug already proved to be effective at very low concentrations (0.1 ng/ml). Growth stimulatory effects with EGF have been observed only in keratinocyte basal medium (KBM), but not in keratinocyte growth medium (KGM). TGF-beta 1 markedly inhibited the proliferation of keratinocytes at concentrations > 1 ng/ml. Calcipotriol and desoximetasone also showed a dose-dependent cell growth inhibition in epidermal cell cultures. IL-1 alpha synthesis was greatly suppressed by calcipotriol 10(-8)-10(-6) M. EGF at 10 ng/ml, in contrast, strongly stimulated IL-1 alpha production. Neither GDF-5 nor TGF-beta 1 had a significant effect on IL-1 alpha production in keratinocyte monolayer cultures. In fibroblasts, GDF-5 induced very weak antiproliferative effects. Calcipotriol and desoximetasone also inhibited cell growth in fibroblast cultures whereas proliferation and DNA synthesis were strongly stimulated by 1 ng/ml EGF. There was, however, a contradiction between TGF-beta 1 results on fibroblasts. Whereas TGF-beta 1 increased proliferation in cell number determination and in the thymidine incorporation assay, MTT assays showed

  14. Biostimulatory effects of low-level laser therapy on epithelial cells and gingival fibroblasts treated with zoledronic acid

    NASA Astrophysics Data System (ADS)

    Basso, F. G.; Pansani, T. N.; Turrioni, A. P. S.; Kurachi, C.; Bagnato, V. S.; Hebling, J.; de Souza Costa, C. A.

    2013-05-01

    Low-level laser therapy (LLLT) has been considered as an adjuvant treatment for bisphosphonate-related osteonecrosis, presenting positive clinical outcomes. However, there are no data regarding the effect of LLLT on oral tissue cells exposed to bisphosphonates. This study aimed to evaluate the effects of LLLT on epithelial cells and gingival fibroblasts exposed to a nitrogen-containing bisphosphonate—zoledronic acid (ZA). Cells were seeded in wells of 24-well plates, incubated for 48 h and then exposed to ZA at 5 μM for an additional 48 h. LLLT was performed with a diode laser prototype—LaserTABLE (InGaAsP—780 nm ± 3 nm, 25 mW), at selected energy doses of 0.5, 1.5, 3, 5, and 7 J cm-2 in three irradiation sessions, every 24 h. Cell metabolism, total protein production, gene expression of vascular endothelial growth factor (VEGF) and collagen type I (Col-I), and cell morphology were evaluated 24 h after the last irradiation. Data were statistically analyzed by Kruskal-Wallis and Mann-Whitney tests at 5% significance. Selected LLLT parameters increased the functions of epithelial cells and gingival fibroblasts treated with ZA. Gene expression of VEGF and Col-I was also increased. Specific parameters of LLLT biostimulated fibroblasts and epithelial cells treated with ZA. Analysis of these in vitro data may explain the positive in vivo effects of LLLT applied to osteonecrosis lesions.

  15. Profiling of anti-fibrotic signaling by hepatocyte growth factor in renal fibroblasts

    SciTech Connect

    Schievenbusch, Stephanie; Strack, Ingo; Scheffler, Melanie; Wennhold, Kerstin; Maurer, Julia; Nischt, Roswitha; Dienes, Hans Peter; Odenthal, Margarete

    2009-07-17

    Hepatocyte growth factor (HGF) is a multifunctional growth factor affecting cell proliferation and differentiation. Due to its mitogenic potential, HGF plays an important role in tubular repair and regeneration after acute renal injury. However, recent reports have shown that HGF also acts as an anti-inflammatory and anti-fibrotic factor, affecting various cell types such as renal fibroblasts and triggering tubulointerstitial fibrosis of the kidney. The present study provides evidence that HGF stimulation of renal fibroblasts results in the activation of both the Erk1/2 and the Akt pathways. As previously shown, Erk1/2 phosphorylation results in Smad-linker phosphorylation, thereby antagonizing cellular signals induced by TGF{beta}. By siRNA mediated silencing of the Erk1/2-Smad linkage, however, we now demonstrate that Akt signaling acts as an auxiliary pathway responsible for the anti-fibrotic effects of HGF. In order to define the anti-fibrotic function of HGF we performed comprehensive expression profiling of HGF-stimulated renal fibroblasts by microarray hybridization. Functional cluster analyses and quantitative PCR assays indicate that the HGF-stimulated pathways transfer the anti-fibrotic effects in renal interstitial fibroblasts by reducing expression of extracellular matrix proteins, various chemokines, and members of the CCN family.

  16. Stereoconfiguration of bisphosphatidic and semilysobisphosphatidic acids from cultured hamster fibroblasts (BHK cells).

    PubMed

    Somerharju, P; Brotherus, J; Kahma, K; Renkonen, O

    1977-04-26

    Monolayers of hamster fibroblasts (BHK cells) were incubated in Eagle's minimal essential medium under conditions where an increase in the levels of all cellular bisphosphatidic acids takes place. Bisphosphatidic acid and semilysobisphosphatidic were isolated from these cells and subjected to strong alkaline hydrolysis. Stereochemical analysis of the hydrolysis products revealed that the majority of the molecules of both lipids are derivatives of sn-1-glycerophospho-sn-1'-glycerol, the structure previously found to be the "backbone" of lysobisphosphatidic acid, (bis(monoacylglycerol)phosphate) from BHK cells and other sources. This finding suggests a close metabolic relationship between the three bisphosphatidic acid derivatives of BHK cells. PMID:857898

  17. Characterization of the molecular defect in infantile and adult acid alpha-glucosidase deficiency fibroblasts.

    PubMed Central

    Beratis, N G; LaBadie, G U; Hirschhorn, K

    1978-01-01

    Different clinical expressions of acid alpha-glucosidase deficiency have been described. The present study was undertaken to investigate the basic metabolic defect in the infantile and adult forms of the disease. Acid alpha-glucosidase (EC 3.2.1.20) was purified from normal and from adult acid alpha-glucosidase deficiency fibroblasts. The pH optimum; Michaelis constant; electrophoretic mobility in starch; thermal denaturation at pH 4.0 and 7.0; and inhibition by turanose, alpha-methylglucoside and trehalose were the same in purified enzyme from normal and mutant cells. Placental acid alpha-glucosidase was purified to, or near, homogeneity. Monospecific antibodies raised against the enzyme in each of three enzyme peaks obtained from the last purification step were found to cross-react with the enzyme of all three peaks, and with purified, normal fibroblast enzyme. Cross-reacting material (CRM) also was identified in fibroblast lysates from normal subjects and from both forms of acid alpha-glucosidase deficiency. The amount of CRM in the adult form appeared to be significantly less than in normal cells or cells from the infantile form. Enzyme activity was demonstrated in the immune complexes of the normal and adult acid alpha-glucosidase deficiency fibroblasts, but not of the infantile form. Competition for antibody binding sites was observed between normal and both types of mutant enzymes. The findings indicate that this case of infantile acid alpha-glucosidase deficiency is the result of a structural gene mutation which causes the synthesis of a catalytically inactive (CRM-positive) enzyme protein. It appears that in the adult form, the mutation causes a reduction in the amount of the enzyme protein present in the cells. Images PMID:34626

  18. Intravitreous transplantation of encapsulated fibroblasts secreting the human fibroblast growth factor 2 delays photoreceptor cell degeneration in Royal College of Surgeons rats

    PubMed Central

    Uteza, Yves; Rouillot, Jean-Sébastien; Kobetz, Alexandra; Marchant, Dominique; Pecqueur, Sèverine; Arnaud, Emmanuelle; Prats, Hervé; Honiger, Jiri; Dufier, Jean-Louis; Abitbol, Marc; Neuner-Jehle, Martin

    1999-01-01

    We developed an experimental approach with genetically engineered and encapsulated mouse NIH 3T3 fibroblasts to delay the progressive degeneration of photoreceptor cells in dark-eyed Royal College of Surgeons rats. These xenogeneic fibroblasts can survive in 1.5-mm-long microcapsules made of the biocompatible polymer AN69 for at least 90 days under in vitro and in vivo conditions because of their stable transfection with the gene for the 18-kDa form of the human basic fibroblast growth factor (hFGF-2). Furthermore, when transferred surgically into the vitreous cavity of 21-day-old Royal College of Surgeons rats, the microencapsulated hFGF-2-secreting fibroblasts provoked a local delay of photoreceptor cell degeneration, as seen at 45 days and 90 days after transplantation. This effect was limited to 2.08 mm2 (45 days) and 0.95 mm2 (90 days) of the retinal surface. In both untreated eyes and control globes with encapsulated hFGF-2-deficient fibroblasts, the rescued area (of at most 0.08 mm2) was significantly smaller at both time points. Although, in a few ocular globes, surgical trauma induced a reorganization of the retinal cytoarchitecture, neither microcapsule rejection nor hFGF-2-mediated tumor formation were detected in any treated eyes. These findings indicate that encapsulated fibroblasts secreting hFGF-2 or perhaps other agents can be applied as potential therapeutic tools to treat retinal dystrophies. PMID:10077648

  19. Genome-wide identification, phylogeny, and expression of fibroblast growth genes in common carp.

    PubMed

    Jiang, Likun; Zhang, Songhao; Dong, Chuanju; Chen, Baohua; Feng, Jingyan; Peng, Wenzhu; Mahboob, Shahid; Al-Ghanim, Khalid A; Xu, Peng

    2016-03-10

    Fibroblast growth factors (FGFs) are a large family of polypeptide growth factors, which are found in organisms ranging from nematodes to humans. In vertebrates, a number of FGFs have been shown to play important roles in developing embryos and adult organisms. Among the vertebrate species, FGFs are highly conserved in both gene structure and amino-acid sequence. However, studies on teleost FGFs are mainly limited to model species, hence we investigated FGFs in the common carp genome. We identified 35 FGFs in the common carp genome. Phylogenetic analysis revealed that most of the FGFs are highly conserved, though recent gene duplication and gene losses do exist. By examining the copy number of FGFs in several vertebrate genomes, we found that eight FGFs in common carp have undergone gene duplications, including FGF6a, FGF6b, FGF7, FGF8b, FGF10a, FGF11b, FGF13a, and FGF18b. The expression patterns of all FGFs were examined in various tissues, including the blood, brain, gill, heart, intestine, muscle, skin, spleen and kidney, showing that most of the FGFs were ubiquitously expressed, indicating their critical role in common carp. To some extent, examination of gene families with detailed phylogenetic or orthology analysis verified the authenticity and accuracy of assembly and annotation of the recently published common carp whole genome sequences. Gene families are also considered as a unique source for evolutionary studies. Moreover, the whole set of common carp FGF gene family provides an important genomic resource for future biochemical, physiological, and phylogenetic studies on FGFs in teleosts. PMID:26691502

  20. Single-domain antibodies that compete with the natural ligand fibroblast growth factor block the internalization of the fibroblast growth factor receptor 1

    SciTech Connect

    Veggiani, Gianluca; Ossolengo, Giuseppe; Aliprandi, Marisa; Cavallaro, Ugo; Marco, Ario de

    2011-05-20

    Highlights: {yields} Recombinant antibodies for FGFR1 were isolated from a llama naive library in VHH format. {yields} These antibodies compete with the natural ligand FGF-2 for the same epitope on FGFR1. {yields} The antibody competition inhibits the FGF-2-dependent internalization of FGFR1. -- Abstract: Single-domain antibodies in VHH format specific for fibroblast growth factor receptor 1 (FGFR1) were isolated from a phage-display llama naive library. In particular, phage elution in the presence of the natural receptor ligand fibroblast growth factor (FGF) allowed for the identification of recombinant antibodies that compete with FGF for the same region on the receptor surface. These antibodies posses a relatively low affinity for FGFR1 and were never identified when unspecific elution conditions favoring highly affine binders were applied to panning procedures. Two populations of competitive antibodies were identified that labeled specifically the receptor-expressing cells in immunofluorescence and recognize distinct epitopes. Antibodies from both populations effectively prevented FGF-dependent internalization and nuclear accumulation of the receptor in cultured cells. This achievement indicates that these antibodies have a capacity to modulate the receptor physiology and, therefore, constitute powerful reagents for basic research and a potential lead for therapeutic applications.

  1. A new syndrome with ethylmalonic aciduria and normal fatty acid oxidation in fibroblasts.

    PubMed

    Burlina, A B; Dionisi-Vici, C; Bennett, M J; Gibson, K M; Servidei, S; Bertini, E; Hale, D E; Schmidt-Sommerfeld, E; Sabetta, G; Zacchello, F

    1994-01-01

    We describe four Italian male infants with a novel clinical phenotype characterized by orthostatic acrocyanosis, relapsing petechiae, chronic diarrhea, progressive pyramidal signs, mental retardation, and brain magnetic resonance imaging abnormalities. The first symptoms appeared after the termination of breast-feeding and introduction of formula feeding. Marked persistent 2-ethylmalonic aciduria was associated with abnormal excretion of C4-C5(n-butyryl-, isobutyryl-, isovaleryl-, and 2-methylbutyryl-)acylglycines and acylcarnitines and with intermittent lactic acidosis. Short- and branched-chain plasma acylcarnitine levels were also elevated. 2-Ethylmalonic aciduria is generally regarded as being indicative of a defect in fatty acid oxidation. Extensive studies of cultured fibroblasts failed to reveal such a defect. The observation of intermittent urinary excretion of 2-ethylhydracrylic acid pointed to involvement of the isoleucine R pathway in ethylmalonate biosynthesis. This hypothesis was tentatively corroborated by the biochemical responses to an oral isoleucine challenge in two patients. However, fibroblast studies showed normal oxidation rates of (14C)isoleucine (ul), indicating that this is not a defect of isoleucine oxidation expressed in skin fibroblasts. In one of two patients tested, cytochrome c oxidase activity was partially reduced (45%) in cultured fibroblasts. This unique clinical and biochemical phenotype identifies a new metabolic encephalopathy of yet undetermined cause. PMID:8283379

  2. Fibroblast growth factor receptor inhibitors as a cancer treatment: from a biologic rationale to medical perspectives.

    PubMed

    Dieci, Maria Vittoria; Arnedos, Monica; Andre, Fabrice; Soria, Jean Charles

    2013-03-01

    The fibroblast growth factor/fibroblast growth factor receptor (FGF/FGFR) signaling pathway plays a fundamental role in many physiologic processes, including embryogenesis, adult tissue homeostasis, and wound healing, by orchestrating angiogenesis. Ligand-independent and ligand-dependent activation have been implicated in a broad range of human malignancies and promote cancer progression in tumors driven by FGF/FGFR oncogenic mutations or amplifications, tumor neoangiogenesis, and targeted treatment resistance, thereby supporting a strong rationale for anti-FGF/FGFR agent development. Efforts are being pursued to develop selective approaches for use against this pathway by optimizing the management of emerging, class-specific toxicity profiles and correctly designing clinical trials to address these different issues. PMID:23418312

  3. Role of fibroblast growth factor (FGF) signaling in the neuroendocrine control of human reproduction.

    PubMed

    Miraoui, Hichem; Dwyer, Andrew; Pitteloud, Nelly

    2011-10-22

    Fibroblast growth factor (FGF) signaling is critical for a broad range of developmental processes. In 2003, Fibroblast growth factor receptor 1 (FGFR1) was discovered as a novel locus causing both forms of isolate GnRH Deficiency, Kallmann syndrome [KS with anosmia] and normosmic idiopathic hypogonadotropic hypogonadism [nIHH] eventually accounting for approximately 10% of gonadotropin-releasing hormone (GnRH) deficiency cases. Such cases are characterized by a broad spectrum of reproductive phenotypes from severe congenital forms of GnRH deficiency to reversal of HH. Additionally, the variable expressivity of both reproductive and non-reproductive phenotypes among patients and family members harboring the identical FGFR1 mutations has pointed to a more complex, oligogenic model for GnRH deficiency. Further, reversal of HH in patients carrying FGFR1 mutations suggests potential gene-environment interactions in human GnRH deficiency disorders. PMID:21664428

  4. Fibroblast growth factors preserve blood-brain barrier integrity through RhoA inhibition after intracerebral hemorrhage in mice.

    PubMed

    Huang, Bin; Krafft, Paul R; Ma, Qingyi; Rolland, William B; Caner, Basak; Lekic, Tim; Manaenko, Anatol; Le, Mai; Tang, Jiping; Zhang, John H

    2012-04-01

    Fibroblast growth factors (FGFs) maintain and promote vascular integrity; however whether FGFs protect the blood-brain barrier (BBB) after intracerebral hemorrhage (ICH) remains unexplored. In this present study, we hypothesized that exogenous FGF administration attenuates brain injury after ICH, specifically by preserving endothelial adherens junctions, therefore reducing vasogenic brain edema and attenuating neurofunctional deficits in mice subjected to experimental ICH. Acid fibroblast growth factor (FGF1) or basic fibroblast growth factor (FGF2) was administered intracerebroventricularly (ICV) at 0.5 h after intrastriatal injection of bacterial collagenase (cICH) or autologous whole blood (bICH). Fibroblast growth factor receptor (FGFR) inhibitor PD173074 and phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 were additionally administered with FGF2. The selective Rho-associated coiled-coil forming protein serine/threonine kinase (ROCK) inhibitor Y27632 was independently administered at 0.5 h after cICH. Brain water content and neurofunctional deficits were evaluated at 24 and 72h after ICH induction. Evans blue extravasation as well as Western blot analysis for the quantification of activated FGFR, Akt, Ras-related C3 botulinum toxin substrate 1 (Rac1), Ras homolog gene family member A (RhoA) and adherens junction proteins (p120-catenin, β-catenin and VE-cadherin) were conducted at 72 h post-cICH. FGF treatment reduced perihematomal brain edema and improved neurofunctional deficits at 72 h after experimental ICH (p<0.05, compared to vehicle); however, FGFR and PI3K inhibition reversed these neuroprotective effects. Exogenous FGF2 increased activated FGFR, Akt, and Rac1 but reduced activated RhoA protein expression at 72 h after cICH (p<0.05, compared to vehicle), which was reversed by FGFR and PI3K inhibition. Y27632 treatment reduced brain injury at 72 h after cICH (p<0.05, compared to vehicle) and increased the expression of catenins (p120-catenin,

  5. Upregulation of fibroblast growth factor 1 in the synovial membranes of patients with late stage osteoarthritis.

    PubMed

    Li, R; Wang, B; He, C Q; Yang, Y Q; Guo, H; Chen, Y; Du, T H

    2015-01-01

    Osteoarthritis (OA) is a degenerative disease of the systemic joint that involves multiple cytokines and growth factors. Fibroblast growth factor 1 (FGF-1) is increased in patients with rheumatic arthritis. The aim of this study was to determine whether the expression and secretion of FGF-1 differed in synovial tissue from patients with late stage OA from that in normal tissues. We selected eight patients with late stage OA and eight healthy donors for this study. An enzyme-linked immunosorbent assay was used to determine the amount of FGF-1 in the synovial fluid and in the culture medium of synovial fibroblasts. Real time quantitative polymerase chain reaction (qPCR) analysis was performed to examine the expression levels of FGF-1 and FGF receptor 2 (FGFR2) in synovial and cartilage tissues. We detected FGF-1 in the synovial fluid from all eight donors, as well as in the culture medium of synovial fibroblasts. Synovial fluid from patients with OA and culture medium of OA synovial fibroblasts contained significantly more FGF-1 than those from controls. FGF-1 expression was also lower in the synovial membranes of normal donors than in those of OA patients. FGFR2 expression was also higher in OA cartilage than in normal cartilage. Overall, these results demonstrated that FGF-1 synthesis and secretion by synovial fibroblasts were significantly increased in OA. FGFR2 expression was also shown to be upregulated in patients with OA. These findings suggest that increased FGF-1 signaling correlates with an OA pathological condition. PMID:26400350

  6. Sustained calcium influx activated by basic fibroblast growth factor in Balb-c 3T3 fibroblasts.

    PubMed Central

    Munaron, L; Distasi, C; Carabelli, V; Baccino, F M; Bonelli, G; Lovisolo, D

    1995-01-01

    1. We have investigated the ionic events elicited in Balb-c 3T3 fibroblasts by basic fibroblast growth factor (bFGF), a peptide that binds to membrane receptors with tyrosine kinase activity and has a mitogenic action on many cell types. The peptide (0.2-100 ng ml-1) caused the appearance of an inward current, as observed in whole-cell patch-clamp experiments at a holding potential of -50 mV, that could last for tens of minutes and had a peak density of 4.6 +/- 2.6 pA pF-1. The reversal potential was 18.8 +/- 16.7 mV. 2. The current was reversibly abolished by removal of bFGF from the external bath. Inhibition of low-affinity FGF receptors had no effect on the activation of the inward current; it was completely abolished when cells were pre-incubated with tyrphostin or 5'-methylthioadenosine (MTA), two inhibitors of the tyrosine kinase activity of the high-affinity FGF receptors. The inward current was not activated by the emptying of internal calcium stores, as tested with 200 nM thapsigargin. 3. Values of peak current density comparable to control ones were obtained when either all Na+ ions or all Ca2+ ions were removed from the external solution; when both ions were completely removed, no inward current could be observed. The inward current was not affected by 2 microM nifedipine, and was reversibly blocked by the imidazole derivative SK&F 96365-A. 4. Measurements of free intracellular calcium concentration ([Ca2+]i) with the dye fura-2 showed that bFGF elicited sustained increases in [Ca2+]i that were completely dependent on external calcium and on the presence of the agonist and could last more than 1 h. 5. Single channel currents (conductance 7.9 pS) in response to bFGF stimulation could be recorded in the cell-attached configuration with 100 mM CaCl2 in the pipette. When the resting potential was brought near to 0 mV by external perfusion in a high-K+ solution, Vrev was about 0 mV. 6. We conclude that in Balb-c 3T3 cells bFGF induces an inward current that

  7. Sustained calcium influx activated by basic fibroblast growth factor in Balb-c 3T3 fibroblasts.

    PubMed

    Munaron, L; Distasi, C; Carabelli, V; Baccino, F M; Bonelli, G; Lovisolo, D

    1995-05-01

    1. We have investigated the ionic events elicited in Balb-c 3T3 fibroblasts by basic fibroblast growth factor (bFGF), a peptide that binds to membrane receptors with tyrosine kinase activity and has a mitogenic action on many cell types. The peptide (0.2-100 ng ml-1) caused the appearance of an inward current, as observed in whole-cell patch-clamp experiments at a holding potential of -50 mV, that could last for tens of minutes and had a peak density of 4.6 +/- 2.6 pA pF-1. The reversal potential was 18.8 +/- 16.7 mV. 2. The current was reversibly abolished by removal of bFGF from the external bath. Inhibition of low-affinity FGF receptors had no effect on the activation of the inward current; it was completely abolished when cells were pre-incubated with tyrphostin or 5'-methylthioadenosine (MTA), two inhibitors of the tyrosine kinase activity of the high-affinity FGF receptors. The inward current was not activated by the emptying of internal calcium stores, as tested with 200 nM thapsigargin. 3. Values of peak current density comparable to control ones were obtained when either all Na+ ions or all Ca2+ ions were removed from the external solution; when both ions were completely removed, no inward current could be observed. The inward current was not affected by 2 microM nifedipine, and was reversibly blocked by the imidazole derivative SK&F 96365-A. 4. Measurements of free intracellular calcium concentration ([Ca2+]i) with the dye fura-2 showed that bFGF elicited sustained increases in [Ca2+]i that were completely dependent on external calcium and on the presence of the agonist and could last more than 1 h. 5. Single channel currents (conductance 7.9 pS) in response to bFGF stimulation could be recorded in the cell-attached configuration with 100 mM CaCl2 in the pipette. When the resting potential was brought near to 0 mV by external perfusion in a high-K+ solution, Vrev was about 0 mV. 6. We conclude that in Balb-c 3T3 cells bFGF induces an inward current that

  8. Expression and function of fibroblast growth factor (FGF) 9 in hepatic stellate cells and its role in toxic liver injury

    SciTech Connect

    Antoine, Marianne; Wirz, Werner; Tag, Carmen G.; Gressner, Axel M.; Marvituna, Meltem; Wycislo, Mathias; Hellerbrand, Claus; Kiefer, Paul . E-mail: paul.kiefer@klinik.uni-regensburg.de

    2007-09-21

    Hepatic injury and regeneration of the liver are associated with activation of hepatic stellate cells (HSC). Fibroblast growth factors (FGFs) and their receptors are important regulators of repair in various tissues. HSC express FGFR3IIIc as well as FGFGR4 and different spliced FGFR1IIIc and FGFR2IIIc isoforms which differ in the presence or absence of the acid box and of the first Ig-like domain. Expression of FGF9, known to be capable to activate the HSC FGFR2/3-isoforms, was increased in HSC in liver slice cultures after exposition to carbon tetrachloride, as an acute liver injury model. FGF9 significantly stimulated 3-H thymidine incorporation of hepatocytes, but failed to induce DNA synthesis in HSC despite the fact that FGF9 induced a sustained activation of extracellular signal-related kinases (ERK) 1/2. FGF9 induced an increased phosphorylation of Tyr436 of the fibroblast growth factor receptor substrate (FRS) 2, while phosphorylation of Tyr196 which is required for efficient Grb2 recruitment remained unchanged. Our findings suggest that HSC FGF9 provide a paracrine mitogenic signal to hepatocytes during acute liver injury, while the autocrine FGF9 signaling appears to be not sufficient to induce cell proliferation.

  9. Cardiac fibroblasts are predisposed to convert into myocyte phenotype: Specific effect of transforming growth factor. beta

    SciTech Connect

    Eghbali, M.; Tomek, R.; Woods, C.; Bhambi, B. )

    1991-02-01

    Cardiac fibroblasts are mainly responsible for the synthesis of major extracellular matrix proteins in the heart, including fibrillar collagen types I and III and fibronectin. In this report we show that these cells, when stimulated by transforming growth factor {beta}{sub 1} (TGF-{beta}{sub 1}), acquire certain myocyte-specific properties. Cultured cardiac fibroblasts from adult rabbit heart were treated with TGF-{beta}{sub 1}, (10-15 ng/ml) for different periods of time. Northern hybridization analysis of total RNA showed that cells treated with TGF-{beta}{sub 1} became stained with a monoclonal antibody to muscle-specific actin. After treatment of quiescent cells with TGF-{beta}{sub 1}, cell proliferation (as measured by ({sup 3}H)thymidine incorporation) was moderately increased. Cultured cardiac fibroblasts at the subconfluent stage, when exposed to TGF-{beta}{sub 1} in the presence of 10% fetal bovine serum, gave rise to a second generation of slowly growing cells that expressed muscle-specific actin filaments. The findings demonstrate that cardiac fibroblasts can be made to differentiate into cells that display many characteristics of cardiac myocytes. TGF-{beta}{sub 1} seems to be a specific inducer of such conversion.

  10. Osteogenic Responses in Fibroblasts Activated by Elastin Degradation Products and Transforming Growth Factor-β1

    PubMed Central

    Simionescu, Agneta; Simionescu, Dan T.; Vyavahare, Narendra R.

    2007-01-01

    Our objective was to establish the role of fibroblasts in medial vascular calcification, a pathological process known to be associated with elastin degradation and remodeling. Rat dermal fibroblasts were treated in vitro with elastin degradation products and transforming growth factor (TGF)-β1, factors usually present in deteriorated matrix environments. Cellular changes were monitored at the gene and protein level by reverse transcriptase-polymerase chain reaction, enzyme-linked immunosorbent assay, immunofluorescence, and von Kossa staining for calcium deposits. By 21 days, multicellular calcified nodules were formed in the presence of elastin degradation products and TGF-β1 separately and to a significantly greater extent when used together. Before mineralization, cells expressed α-smooth muscle actin and large amounts of collagen type I and matrix metalloproteinase-2, characteristic features of myofibroblasts, key elements in tissue remodeling and repair. Stimulated cells expressed increased levels of core-binding factor α1, osteocalcin, alkaline phosphatase, and osteoprotegerin, representative bone-regulating proteins. For most proteins analyzed, TGF-β1 synergistically amplified responses of fibroblasts to elastin degradation products. In conclusion, elastin degradation products and TGF-β1 promote myofibroblastic and osteogenic differentiation in fibroblasts. These results support the idea that elastin-related calcification involves dynamic remodeling events and suggest the possibility of a defective tissue repair process. PMID:17591959

  11. Calcium-Alginate Hydrogel-Encapsulated Fibroblasts Provide Sustained Release of Vascular Endothelial Growth Factor

    PubMed Central

    Hunt, Nicola C.; Shelton, Richard M.; Henderson, Deborah J.

    2013-01-01

    Vascularization of engineered or damaged tissues is essential to maintain cell viability and proper tissue function. Revascularization of the left ventricle (LV) of the heart after myocardial infarction is particularly important, since hypoxia can give rise to chronic heart failure due to inappropriate remodeling of the LV after death of cardiomyocytes (CMs). Fibroblasts can express vascular endothelial growth factor (VEGF), which plays a major role in angiogenesis and also acts as a chemoattractant and survival factor for CMs and cardiac progenitors. In this in vitro model study, mouse NIH 3T3 fibroblasts encapsulated in 2% w/v Ca-alginate were shown to remain viable for 150 days. Semiquantitative reverse transcription–polymerase chain reaction and immunohistochemistry demonstrated that over 21 days of encapsulation, fibroblasts continued to express VEGF, while enzyme-linked immunosorbent assay showed that there was sustained release of VEGF from the Ca-alginate during this period. The scaffold degraded gradually over the 21 days, without reduction in volume. Cells released from the Ca-alginate at 7 and 21 days as a result of scaffold degradation were shown to retain viability, to adhere to fibronectin in a normal manner, and continue to express VEGF, demonstrating their potential to further contribute to maintenance of cardiac function after scaffold degradation. This model in vitro study therefore demonstrates that fibroblasts encapsulated in Ca-alginate provide sustained release of VEGF. PMID:23082964

  12. Insulin-like growth factor I receptor expression and function in fibroblasts from two patients with deletion of the distal long arm of chromosome 15.

    PubMed

    Siebler, T; Lopaczynski, W; Terry, C L; Casella, S J; Munson, P; De Leon, D D; Phang, L; Blakemore, K J; McEvoy, R C; Kelley, R I

    1995-12-01

    Most patients with deletion of the distal long arm of chromosome 15 have intrauterine growth retardation and postnatal growth deficiency in addition to developmental abnormalities. It has been proposed that the absence of one copy of the insulin-like growth factor I (IGF-I) receptor gene may play a role in the growth deficiency seen in this syndrome. To address this question we examined IGF-I receptor expression and function in fibroblasts from two patients with deletion of the distal long arm of chromosome 15 (15q26.1-->qter). Quantitative Southern blot analysis of the IGF-I receptor gene was performed on HindIII digests of fibroblast DNA. Radioactivity in the 1.7-kilobase receptor fragment in the two patients was 55% and 51% of the values in controls, consistent with the absence of one copy of the IGF-I receptor gene. IGF-I receptor messenger ribonucleic acid levels were quantitated by a solution hybridization/nuclease protection assay. Receptor messenger ribonucleic acid levels in the two patients were 45% and 52% of the values in controls. Northern blotting demonstrated normal size IGF-I receptor transcripts and affinity crosslinking of [125I]IGF-I to Triton X-100-solubilized fibroblasts demonstrated a normal size receptor in the patients. Analysis of placental membranes prepared from one patient revealed no difference in [125I]IGF-I binding. In the patients' fibroblasts, however, binding of [125I]long [R3]-IGF-I to the IGF-I receptor was significantly reduced, as assessed by the amount of radioactivity competed by the monoclonal antibody alpha IR-3 or insulin and Scatchard analysis of binding data. To assess IGF-I receptor function, stimulation of [alpha-1-14C]-methylaminoisobutyric acid transport and stimulation of [methyl-3H]thymidine incorporation into DNA by a full range of IGF-I concentrations was examined in patient and control fibroblasts. There was a significant decrease in the maximal response to IGF-I in both assays for one of the two patients when

  13. Free bone graft reconstruction of irradiated facial tissue: Experimental effects of basic fibroblast growth factor stimulation

    SciTech Connect

    Eppley, B.L.; Connolly, D.T.; Winkelmann, T.; Sadove, A.M.; Heuvelman, D.; Feder, J. )

    1991-07-01

    A study was undertaken to evaluate the potential utility of basic fibroblast growth factor in the induction of angiogenesis and osseous healing in bone previously exposed to high doses of irradiation. Thirty New Zealand rabbits were evaluated by introducing basic fibroblast growth factor into irradiated mandibular resection sites either prior to or simultaneous with reconstruction by corticocancellous autografts harvested from the ilium. The fate of the free bone grafts was then evaluated at 90 days postoperatively by microangiographic, histologic, and fluorochrome bone-labeling techniques. Sequestration, necrosis, and failure to heal to recipient osseous margins was observed both clinically and histologically in all nontreated irradiated graft sites as well as those receiving simultaneous angiogenic stimulation at the time of graft placement. No fluorescent activity was seen in these graft groups. In the recipient sites pretreated with basic fibroblast growth factor prior to placement of the graft, healing and reestablishment of mandibular contour occurred in nearly 50 percent of the animals. Active bone formation was evident at cortical margins adjacent to the recipient sites but was absent in the more central cancellous regions of the grafts.

  14. Lysophosphatidic acid receptor-5 negatively regulates cellular responses in mouse fibroblast 3T3 cells

    SciTech Connect

    Dong, Yan; Hirane, Miku; Araki, Mutsumi; Fukushima, Nobuyuki; Tsujiuchi, Toshifumi

    2014-04-04

    Highlights: • LPA{sub 5} inhibits the cell growth and motile activities of 3T3 cells. • LPA{sub 5} suppresses the cell motile activities stimulated by hydrogen peroxide in 3T3 cells. • Enhancement of LPA{sub 5} on the cell motile activities inhibited by LPA{sub 1} in 3T3 cells. • The expression and activation of Mmp-9 were inhibited by LPA{sub 5} in 3T3 cells. • LPA signaling via LPA{sub 5} acts as a negative regulator of cellular responses in 3T3 cells. - Abstract: Lysophosphatidic acid (LPA) signaling via G protein-coupled LPA receptors (LPA{sub 1}–LPA{sub 6}) mediates a variety of biological functions, including cell migration. Recently, we have reported that LPA{sub 1} inhibited the cell motile activities of mouse fibroblast 3T3 cells. In the present study, to evaluate a role of LPA{sub 5} in cellular responses, Lpar5 knockdown (3T3-L5) cells were generated from 3T3 cells. In cell proliferation assays, LPA markedly stimulated the cell proliferation activities of 3T3-L5 cells, compared with control cells. In cell motility assays with Cell Culture Inserts, the cell motile activities of 3T3-L5 cells were significantly higher than those of control cells. The activity levels of matrix metalloproteinases (MMPs) were measured by gelatin zymography. 3T3-L5 cells stimulated the activation of Mmp-2, correlating with the expression levels of Mmp-2 gene. Moreover, to assess the co-effects of LPA{sub 1} and LPA{sub 5} on cell motile activities, Lpar5 knockdown (3T3a1-L5) cells were also established from Lpar1 over-expressing (3T3a1) cells. 3T3a1-L5 cells increased the cell motile activities of 3T3a1 cells, while the cell motile activities of 3T3a1 cells were significantly lower than those of control cells. These results suggest that LPA{sub 5} may act as a negative regulator of cellular responses in mouse fibroblast 3T3 cells, similar to the case for LPA{sub 1}.

  15. Transforming growth factor-β inhibits IQ motif containing guanosine triphosphatase activating protein 1 expression in lung fibroblasts via the nuclear factor-κB signaling pathway.

    PubMed

    Zong, Chuanyue; Zhang, Xianlong; Xie, Ying; Cheng, Jiawen

    2015-07-01

    IQ motif containing guanosine triphosphatase activating protein 1 (IQGAP1) is associated with idiopathic pulmonary fibrogenesis (IPF); however, characterization of the expression of IQGAP1 in lung fibroblasts has remained elusive. The present study therefore evaluated IQGAP1 expression in mouse and human lung fibroblasts under fibrotic conditions via western blot analysis. It was revealed that IQGAP1 expression levels were significantly decreased in lung fibroblasts isolated from bleomycin-challenged mice than in those of control mice. Transforming growth factor-β (TGF-β) induced differentiation, as well as decreased expression of IQGAP1 in WI-38 cells human lung fibroblasts. Furthermore, inhibition of nuclear factor (NF)-κB activation restored the TGF-β-induced inhibition of IQGAP1 expression in WI-38 cells. In lysophosphatidic acid (LPA)-challenged WI-38 cells, the expression of IQGAP1 was also decreased, while neutralized anti-TGF-β antibody treatment restored the LPA-induced inhibition of IQGAP1 expression. These data indicated that TGF-β inhibited IQGAP1 expression in lung fibroblasts via the NF-κB signaling pathway, presenting a potential novel therapeutic target for the treatment of IPF. PMID:25684348

  16. Fibroblast Growth Factor 21-Null Mice Do Not Exhibit an Impaired Response to Fasting

    PubMed Central

    Antonellis, Patrick Joseph; Hayes, Meghan Patricia; Adams, Andrew Charles

    2016-01-01

    Fibroblast growth factor 21 (FGF21) is a pleotropic metabolic regulator, expression of which is elevated during fasting. To this end, the precise role played by FGF21 in the biology of fasting has been the subject of several recent studies, which have demonstrated contributions to the regulation of both lipid and carbohydrate metabolism. In the present study, we compared wild-type (WT) and FGF21-null (FGF21KO) mice, demonstrating that, despite the significant induction of FGF21 during fasting in the WT animals, our strain of FGF21-null mice exhibits only limited impairments in their adaptation to nutrient deprivation. Specifically, fasted FGF21KO mice display a mild attenuation of gluconeogenic transcriptional induction in the liver accompanied by partially blunted glucose production in response to a pyruvate challenge. Furthermore, FGF21KO mice displayed only minor impairments in lipid metabolism in the fasted state, limited to accumulation of hepatic triglycerides and a reduction in expression of genes associated with fatty acid oxidation. To address the possibility of compensation to germline deletion of FGF21, we further interrogated the role of endogenous FGF21 via acute pharmacological blockade of FGF21 signaling. At the transcriptional level, we show that FGF21 signaling is required for full induction of gluconeogenic and oxidative genes in the liver. However, corroborating our findings in FGF21KO mice, pharmacological blockade of the FGF21 axis did not profoundly disrupt the physiological response to fasting. Taken as a whole, these data demonstrate that, while FGF21 is partially required for appropriate gene expression during the fed to fasted transition, its absence does not significantly impact the downstream physiology of the fasted state. PMID:27445980

  17. Enhanced in vitro refolding of fibroblast growth factor 15 with the assistance of SUMO fusion partner.

    PubMed

    Kong, Bo; Guo, Grace L

    2011-01-01

    Fibroblast growth factor 15 (Fgf15) is the mouse orthologue of human FGF19. Fgf15 is highly expressed in the ileum and functions as an endocrine signal to regulate liver function, including bile acid synthesis, hepatocyte proliferation and insulin sensitivity. In order to fully understand the function of Fgf15, methods are needed to produce pure Fgf15 protein in the prokaryotic system. However, when expressed in Escherichia coli (E. coli), the recombinant Fgf15 protein was insoluble and found only in inclusion bodies. In the current study, we report a method to produce recombinant Fgf15 protein in E. coli through the use of small ubiquitin-related modifier (SUMO) fusion tag. Even though the SUMO has been shown to strongly improve protein solubility and expression levels, our studies suggest that the SUMO does not improve Fgf15 protein solubility. Instead, proper refolding of Fgf15 protein was achieved when Fgf15 was expressed as a partner protein of the fusion tag SUMO, followed by in vitro dialysis refolding. After refolding, the N-terminal SUMO tag was cleaved from the recombinant Fgf15 fusion protein by ScUlp1 (Ubiquitin-Like Protein-Specific Protease 1 from S. cerevisiae). With or without the SUMO tag, the refolded Fgf15 protein was biologically active, as revealed by its ability to reduce hepatic Cyp7a1 mRNA levels in mice. In addition, recombinant Fgf15 protein suppressed Cyp7a1 mRNA levels in a dose-dependent manner. In summary, we have developed a successful method to express functional Fgf15 protein in prokaryotic cells. PMID:21655243

  18. Fibroblast growth factor 21-deficient mice demonstrate impaired adaptation to ketosis.

    PubMed

    Badman, Michael K; Koester, Anja; Flier, Jeffrey S; Kharitonenkov, Alexei; Maratos-Flier, Eleftheria

    2009-11-01

    Fibroblast growth factor 21 (FGF21) is a key metabolic regulator. Expressed primarily in liver and adipose tissue, FGF21 is induced via peroxisome proliferator-activated receptor (PPAR) pathways during states requiring increased fatty acid oxidation including fasting and consumption of a ketogenic diet. To test the hypothesis that FGF21 is a physiological regulator that plays a role in lipid oxidation, we generated mice with targeted disruption of the Fgf21 locus (FGF21 knockout). Mice lacking FGF21 had mild weight gain and slightly impaired glucose homeostasis, indicating a role in long-term energy homeostasis. Furthermore, FGF21KO mice tolerated a 24-h fast, indicating that FGF21 is not essential in the early stages of starvation. In contrast to wild-type animals in which feeding KD leads to dramatic weight loss, FGF21KO mice fed KD gained weight, developed hepatosteatosis, and showed marked impairments in ketogenesis and glucose control. This confirms the physiological importance of FGF21 in the adaptation to KD feeding. At a molecular level, these effects were accompanied by lower levels of expression of PGC1alpha and PGC1beta in FGF21KO mice, strongly implicating these key transcriptional regulators in the action of FGF21. Furthermore, within the liver, the maturation of the lipogenic transcription factor sterol regulatory element-binding protein-1c was increased in FGF21KO mice, implicating posttranscriptional events in the maladaptation of FGF21KO mice to KD. These data reinforce the role of FGF21 is a critical regulator of long-term energy balance and metabolism. Mice lacking FGF21 cannot respond appropriately to a ketogenic diet, resulting in an impaired ability to mobilize and utilize lipids. PMID:19819944

  19. Intranasal nanoparticles of basic fibroblast growth factor for brain delivery to treat Alzheimer's disease.

    PubMed

    Zhang, Chi; Chen, Jie; Feng, Chengcheng; Shao, Xiayan; Liu, Qingfeng; Zhang, Qizhi; Pang, Zhiqing; Jiang, Xinguo

    2014-01-30

    Disabilities caused by neurodegeneration have become one of the main causes of mortality in elderly population, with drug distribution to the brain remaining one of the most difficult challenges in the treatment of the central nervous system (CNS) diseases due to the existence of blood-brain barrier. Lectins modified polyethylene glycol-polylactide-polyglycolide (PEG-PLGA) nanoparticles could enhance the drug delivery to the brain following intranasal administration. In this study, basic fibroblast growth factor (bFGF) was entrapped in nanoparticles conjugated with Solanum tuberosum lectin (STL), which selectively binds to N-acetylglucosamine on the nasal epithelial membrane for its brain delivery. The resulting nanoparticles had uniform particle size and negative zeta potential. The brain distribution of the formulations following intranasal administration was assessed using radioisotopic tracing method. The areas under the concentration-time curve of (125)I-bFGF in the olfactory bulb, cerebrum, and cerebellum of rats following nasal application of STL modified nanoparticles (STL-bFGF-NP) were 1.79-5.17 folds of that of rats with intravenous administration, and 0.61-2.21 and 0.19-1.07 folds higher compared with intranasal solution and unmodified nanoparticles, respectively. Neuroprotective effect was evaluated using Mirror water maze task in rats with intracerebroventricular injection of β-amyloid25-35 and ibotenic acid. The spatial learning and memory of Alzheimer's disease (AD) rats in STL-bFGF-NP group were significantly improved compared with AD model group, and were also better than other preparations. The results were consistent with the value of choline acetyltransferase activity of rat hippocampus as well as the histological observations of rat hippocampal region. The histopathology assays also confirmed the in vivo safety of STL-bFGF-NP. These results clearly indicated that STL-NP was a promising drug delivery system for peptide and protein drugs such as

  20. Lepidopteran Ortholog of Drosophila Breathless Is a Receptor for the Baculovirus Fibroblast Growth Factor

    PubMed Central

    Katsuma, Susumu; Daimon, Takaaki; Mita, Kazuei; Shimada, Toru

    2006-01-01

    The Bombyx mori nucleopolyhedrovirus (BmNPV) encodes a gene homologous to the mammalian fibroblast growth factor (FGF) family. We report the cloning of B. mori and Spodoptera frugiperda orthologous genes (Bmbtl and Sfbtl, respectively) of Drosophila melanogaster breathless (btl) encoding a receptor for Branchless/FGF and show that these genes encode the receptor for a baculovirus-encoded FGF (vFGF). Sequence analysis showed that BmBtl is composed of 856 amino acid residues, which potentially encodes a 97.3-kDa polypeptide and shares structural features and sequence similarities with the FGF receptor family. Reverse transcription-PCR experiments showed that Bmbtl was abundantly expressed in the trachea and midgut in B. mori larvae, with moderate expression observed in the hemocytes and the B. mori cultured cell line BmN. We generated Sf-9 cells that stably expressed His-tagged BmBtl. Western blot analysis revealed that BmBtl was an ∼110-kDa protein. Immunoprecipitation experiments showed that BmNPV vFGF markedly phosphorylated BmBtl in Sf-9 cells. In addition, we found that BmBtl overexpression enhanced the migration activity for BmNPV vFGF. Furthermore, we generated Sf-9 cells in which Sfbtl was knocked down by transfection with double-strand RNA-expressing plasmids. In these cells, cell motility triggered by vFGF was markedly reduced. These results strongly suggest that the Btl orthologs, BmBtl and SfBtl, are the receptors for vFGF, which mediate vFGF-induced host cell chemotaxis. PMID:16699027

  1. Fibroblast growth factor-23 increases mouse PGE2 production in vivo and in vitro

    PubMed Central

    Syal, Ashu; Schiavi, Susan; Chakravarty, Sumana; Dwarakanath, Vangipuram; Quigley, Raymond; Baum, Michel

    2014-01-01

    Fibroblast growth factor-23 (FGF-23) has been implicated in the renal phosphate wasting in X-linked hypophosphatemia, tumor-induced osteomalacia, and autosomal dominant hypophosphatemic rickets. Recently, we demonstrated that Hyp mice have greater urinary PGE2 levels compared with C57/B6 mice and that indomethacin administration in vivo and in vitro ameliorates the phosphate transport defect in Hyp mice. To determine further whether altered prostaglandin metabolism plays a role in the renal phosphate transport defect in Hyp mice, we incubated renal proximal tubules with arachidonic acid. We find that PGE2 production was higher in Hyp mice than in C57/B6 mice. Incubation of C57/B6 mouse renal proximal tubules with FGF-23R176Q, an active mutant form of FGR23, increased tubular PGE2 production, an effect that was inhibited by 50 μM PD-98059 and 10 μM SB-203580, inhibitors of the MAP kinase pathway. C57/B6 mice injected with FGF-23R176Q had a ~10-fold increase in PGE2 excretion 24 h after intraperitoneal injection of FGF-23R176Q compared with vehicle-treated controls. Finally, we show that PGE2 inhibited both phosphate and volume absorption in mouse proximal convoluted tubules perfused in vitro and reduced brush-border membrane vesicle NaPi-2a protein abundance from renal cortex incubated in vitro with PGE2. In conclusion, FGF-23 increases urinary and renal tubular PGE2 production via the MAP kinase pathway and PGE2 inhibits proximal tubule phosphate transport. PMID:16144964

  2. Fibroblast growth factor-23 increases mouse PGE2 production in vivo and in vitro.

    PubMed

    Syal, Ashu; Schiavi, Susan; Chakravarty, Sumana; Dwarakanath, Vangipuram; Quigley, Raymond; Baum, Michel

    2006-02-01

    Fibroblast growth factor-23 (FGF-23) has been implicated in the renal phosphate wasting in X-linked hypophosphatemia, tumor-induced osteomalacia, and autosomal dominant hypophosphatemic rickets. Recently, we demonstrated that Hyp mice have greater urinary PGE2 levels compared with C57/B6 mice and that indomethacin administration in vivo and in vitro ameliorates the phosphate transport defect in Hyp mice. To determine further whether altered prostaglandin metabolism plays a role in the renal phosphate transport defect in Hyp mice, we incubated renal proximal tubules with arachidonic acid. We find that PGE2 production was higher in Hyp mice than in C57/B6 mice. Incubation of C57/B6 mouse renal proximal tubules with FGF-23R176Q, an active mutant form of FGR23, increased tubular PGE2 production, an effect that was inhibited by 50 microM PD-98059 and 10 microM SB-203580, inhibitors of the MAP kinase pathway. C57/B6 mice injected with FGF-23R176Q had a approximately 10-fold increase in PGE2 excretion 24 h after intraperitoneal injection of FGF-23R176Q compared with vehicle-treated controls. Finally, we show that PGE2 inhibited both phosphate and volume absorption in mouse proximal convoluted tubules perfused in vitro and reduced brush-border membrane vesicle NaPi-2a protein abundance from renal cortex incubated in vitro with PGE2. In conclusion, FGF-23 increases urinary and renal tubular PGE2 production via the MAP kinase pathway and PGE2 inhibits proximal tubule phosphate transport. PMID:16144964

  3. The growth regulatory fibroblast IK channel is the prominent electrophysiological feature of rat prostatic cancer cells.

    PubMed

    Rane, S G

    2000-03-16

    Physiological effectors for mitogenic cell growth control remain to be determined for mammalian tumor cells, particularly those derived from prostatic tissue. One such effector for mitogenic Ras/MAPK signaling in fibroblasts is an intermediate-conductance, calcium-activated potassium channel (FIK). In this study patch-clamp electrophysiology was used to show that both AT2.1 and MatLyLu rat prostate cancer cell lines express high levels of a current identified as FIK, based on the following criteria: activation by elevation of intracellular calcium, voltage independence, potassium selectivity, and block by charybdotoxin (ChTX) and the Stichodactyla helianthus potassium channel neurotoxin (StK). FIK current densities in AT2.1 and MatLyLu cells were comparable to the high levels seen in fibroblasts transfected with oncogenic Ras or Raf, suggesting hyperactivity of the Ras/MAPK pathway in prostatic cancer cells. Voltage-gated sodium current was present in most MatLyLu cells but absent from AT2.1 cells, and all AT2.1 cells had voltage-gated potassium currents. Thus, FIK is the main electrophysiological feature of rat prostatic cancer cells as it is for mitogenically active fibroblasts, suggesting it may play a similar growth regulatory role in both. PMID:10708575

  4. Correction of Hypertension by Normalization of Endothelial Levels of Fibroblast Growth Factor and Nitric Oxide Synthase in Spontaneously Hypertensive Rats

    NASA Astrophysics Data System (ADS)

    Cuevas, Pedro; Garcia-Calvo, Margarita; Carceller, Fernando; Reimers, Diana; Zazo, Mercedes; Cuevas, Begona; Munoz-Willery, Isabel; Martinez-Coso, Victoria; Lamas, Santiago; Gimenez-Gallego, Guillermo

    1996-10-01

    Acidic and basic fibroblast growth factors (FGFs) share a wide range of diverse biological activities. To date, low levels of FGF have not been correlated with a pathophysiologic state. We report that blood vessels of spontaneously hypertensive rats are shown to be associated with a marked decrement in endothelial basic FGF content. This decrement correlates both with hypertension and with a decrease in the endothelial content of nitric oxide synthase. restoration of FGF to physiological levels in the vascular wall, either by systemic administration or by in vivo gene transfer, significantly augmented the number of endothelial cells with positive immunostaining for nitric oxide synthase, corrected hypertension, and ameliorated endothelial-dependent responses to vasoconstrictors. These results suggest an important role for FGFs in blood pressure homeostasis and open new avenues for the understanding of the etiology and treatment of hypertension.

  5. Common nucleotide sequence of structural gene encoding fibroblast growth factor 4 in eight cattle derived from three breeds.

    PubMed

    Sato, Sho; Takahashi, Toshikiyo; Nishinomiya, Hiroshi; Katoh, Makiko; Itoh, Ryu; Yokoo, Masaki; Yokoo, Mari; Iha, Momoe; Mori, Yuki; Kasuga, Kano; Kojima, Ikuo; Kobayashi, Masayuki

    2012-03-01

    Fibroblast growth factor 4 (FGF4) is considered as a crucial gene for the proper development of bovine embryos. However, the complete nucleotide sequences of the structural genes encoding FGF4 in identified breeds are still unknown. In the present study, direct sequencing of PCR products derived from genomic DNA samples obtained from three Japanese Black, two Japanese Shorthorn and three Holstein cattle, revealed that the nucleotide sequences of the structural gene encoding FGF4 matched completely among these eight cattle. On the other hand, differences in the nucleotide sequences, leading to substitutions, insertions or deletions of amino acid residues were detected when compared with the already reported sequence from unidentified breeds. We cannot rule out a possibility that the structural gene elucidated in the present study is widely distributed in cattle. To the best of our knowledge, this is the first determination of the complete nucleotide sequence of the structural gene encoding bovine FGF4 in identified breeds. PMID:22435631

  6. Fibroblast and endothelial outgrowth from human Tenon's explants: inhibition of fibroblast growth by 5HT receptor antagonism.

    PubMed

    Mullaney, P; Curren, B; Collum, L; Kilfeather, S

    1991-11-01

    Tenon's layer capsule is a major source of fibroplasia associated with post-traumatic wound healing in the external eye and failure of filtration surgery for glaucoma (trabeculectomy). We have investigated the cell types involved in outgrowth from human Tenon's layer explants in culture. Outgrowth from explants maintained in fetal bovine serum (5%) and fibroblast conditioned medium (25%) consisted of endothelial cells exhibiting factor VIII antibody staining and were located to sectioned capillary ends. In media supplemented with human serum (10%), fibroblast outgrowth was observed from the entire perimeter of explants. The mitogenic stimulus of human serum on fibroblasts, monitored by 3H-thymidine uptake, was dose-dependent and accompanied by a 200% increase in inositol phosphate production. 5HT induced a significant increase in fibroblast proliferation and 5HT receptor antagonism with methysergide (20 microM) reduced serum-induced mitogenesis by 30%. These findings support the role of fibroblasts in failure of filtration surgery for glaucoma and suggest a role for 5HT in serum-derived Tenon's fibroplasia. PMID:1811281

  7. Basic fibroblast growth factor binds to subendothelial extracellular matrix and is released by heparitinase and heparin-like molecules

    SciTech Connect

    Bashkin, P.; Doctrow, S.; Klagsbrun, M.; Svahn, C.M.; Folkman, J.; Vlodavsky, I. )

    1989-02-21

    Basic fibroblast growth factor (bFGF) exhibits specific binding to the extracellular matrix (ECM) produced by cultured endothelial cells. Binding was saturable as a function both of time and of concentration of {sup 125}I-bFGF. Scatchard analysis of FGF binding revealed the presence of about 1.5 x 10{sup 12} binding sites/mm{sup 2} ECM with an apparent k{sub D} of 610 nM. FGF binds to heparan sulfate (HS) in ECM as evidenced by (i) inhibition of binding in the presence of heparin or HS at 0.1-1 {mu}g/mL, but not by chondroitin sulfate, keratan sulfate, or hyaluronic acid at 10 {mu}g/mL, (ii) lack of binding to ECM pretreated with heparitinase, but not with chondroitinase ABC, and (iii) rapid release of up to 90% of ECM-bound FGF by exposure to heparin, HS, or heparitinase, but not to chondroitin sulfate, keratan sulfate, hyaluronic acid, or chondroitinase ABC. Oligosaccharides derived from depolymerized heparin, and as small as the tetrasaccharide, released the ECM-bound FGF, but there was little or no release of FGF by modified nonanticoagulant heparins such as totally desulfated heparin, N-desulfated heparin, and N-acetylated heparin. FGF released from ECM was biologically active, as indicated by its stimulation of cell proliferation and DNA synthesis in vascular endothelial cells and 3T3 fibroblasts. Similar results were obtained in studies on release of endogenous FGF-like mitogenic activity from Descement's membranes of bovine corneas. It is suggested that ECM storage and release of bFGF provide a novel mechanism for regulation of capillary blood vessel growth. Whereas ECM-bound FGF may be prevented from acting on endothelial cells, its displacement by heparin-like molecules and/or HS-degrading enzymes may elicit a neovascular response.

  8. Medium-chain fatty acids undergo elongation before {beta}-oxidation in fibroblasts

    SciTech Connect

    Jones, Patricia M. . E-mail: Patti.Jones@childrens.com; Butt, Yasmeen; Messmer, Bette; Boriak, Richard; Bennett, Michael J.

    2006-07-21

    Although mitochondrial fatty acid {beta}-oxidation (FAO) is considered to be well understood, further elucidation of the pathway continues through evaluation of patients with FAO defects. The FAO pathway can be examined by measuring the 3-hydroxy-fatty acid (3-OHFA) intermediates. We present a unique finding in the study of this pathway: the addition of medium-chain fatty acids to the culture media of fibroblasts results in generation of 3-OHFAs which are two carbons longer than the precursor substrate. Cultured skin fibroblasts from normal and LCHAD-deficient individuals were grown in media supplemented with various chain-length fatty acids. The cell-free medium was analyzed for 3-OHFAs by stable-isotope dilution gas-chromatography/mass-spectrometry. Our finding suggests that a novel carbon chain-length elongation process precedes the oxidation of medium-chain fatty acids. This previously undescribed metabolic step may have important implications for the metabolism of medium-chain triglycerides, components in the dietary treatment of a number of disorders.

  9. Differential regulation of Na/H antiporter by acid in renal epithelial cells and fibroblasts.

    PubMed Central

    Moe, O W; Miller, R T; Horie, S; Cano, A; Preisig, P A; Alpern, R J

    1991-01-01

    Increased Na/H antiporter activity has been demonstrated after in vivo chronic metabolic acidosis as well as in vitro acid preincubation of cultured rabbit renal tubule cells. To study the underlying molecular mechanisms of this adaptive increase in Na/H antiporter activity, the present studies examined the effect of low pH media on Na/H antiporter activity and mRNA abundance in cultured renal tubule cells. Na/H antiporter activity was increased by 60% in a mouse renal cortical tubule cell line (MCT), and by 90% in an opossum kidney cell line (OKP) after 24 h of preincubation in acid (low [HCO3]) media. The ethylisopropylamiloride sensitivity of the Na/H antiporters were different in these two cell lines (MCT IC50 = 65 nM; OKP IC50 = 4.5 microM). In MCT cells, Na/H antiporter mRNA abundance measured by RNA blots increased by two- to fivefold after 24 h in low [HCO3] media. Na/H antiporter mRNA abundance was also increased in MCT cells with high CO2 preincubation as well as in rat renal cortex with in vivo chronic acid feeding. In contrast to renal epithelia, acid preincubation of NIH 3T3 fibroblasts led to suppression of Na/H antiporter activity. RNA blots of 3T3 fibroblasts revealed the same size Na/H antiporter transcript as in MCT cells. However, Na/H antiporter mRNA levels were suppressed by acid preincubation. These studies demonstrate differential regulation of Na/H antiporter activity and mRNA abundance in renal epithelial cells and fibroblasts in response to an acidotic environment. Images PMID:1658050

  10. Role of Arachidonic Acid in Promoting Hair Growth

    PubMed Central

    Munkhbayar, Semchin; Jang, Sunhyae; Cho, A-Ri; Choi, Soon-Jin; Shin, Chang Yup; Eun, Hee Chul; Kim, Kyu Han

    2016-01-01

    Background Arachidonic acid (AA) is an omega-6 polyunsaturated fatty acid present in all mammalian cell membranes, and involved in the regulation of many cellular processes, including cell survival, angiogenesis, and mitogenesis. The dermal papilla, composed of specialized fibroblasts located in the bulb of the hair follicle, contributes to the control of hair growth and the hair cycle. Objective This study investigated the effect of AA on hair growth by using in vivo and in vitro models. Methods The effect of AA on human dermal papilla cells (hDPCs) and hair shaft elongation was evaluated by MTT assay and hair follicle organ culture, respectively. The expression of various growth and survival factors in hDPCs were investigated by western blot or immunohistochemistry. The ability of AA to induce and prolong anagen phase in C57BL/6 mice was analyzed. Results AA was found to enhance the viability of hDPCs and promote the expression of several factors responsible for hair growth, including fibroblast growth factor-7 (FGF-7) and FGF-10. Western blotting identified the role of AA in the phosphorylation of various transcription factors (ERK, CREB, and AKT) and increased expression of Bcl-2 in hDPCs. In addition, AA significantly promoted hair shaft elongation, with increased proliferation of matrix keratinocytes, during ex vivo hair follicle culture. It was also found to promote hair growth by induction and prolongation of anagen phase in telogen-stage C57BL/6 mice. Conclusion This study concludes that AA plays a role in promoting hair growth by increasing the expression of growth factors in hDPCs and enhancing follicle proliferation and survival. PMID:26848219

  11. Carbachol stimulates a different phospholipid metabolism than nerve growth factor and basic fibroblast growth factor in PC12 cells.

    PubMed Central

    Pessin, M S; Altin, J G; Jarpe, M; Tansley, F; Bradshaw, R A; Raben, D M

    1991-01-01

    We have examined 1,2-diglycerides (DGs) generated in PC12 cells in response to the muscarinic agonist carbachol and compared them with those generated in response to the differentiation factors nerve growth factor and basic fibroblast growth factor. Whereas carbachol stimulates a greater release of inositol phosphates, all three agonists generate similar levels of DGs. In this report, we have analyzed the molecular species of PC12 DGs generated in response to these three agonists. Additionally, we have analyzed the molecular species of PC12 phospholipids. The data indicate that 1) after 1 min of either nerve growth factor or basic fibroblast growth factor stimulation, DGs arise primarily from phosphoinositide hydrolysis; 2) in contrast, after 1 min of carbachol stimulation, DG are generated equally by both phosphoinositide and phosphatidylcholine hydrolysis; and 3) after 15 min of stimulation by any of these agonists, DGs are generated largely by phosphatidylcholine hydrolysis, with a smaller component arising from the phosphoinositides. These results suggest that at least part of the mechanism by which PC12 cells distinguish between different agonists is via alterations in phospholipid sources and kinetics of DG generation. PMID:1892912

  12. Epidermal growth factor (EGF) antagonizes transforming growth factor (TGF)-beta1-induced collagen lattice contraction by human skin fibroblasts.

    PubMed

    Park, J S; Kim, J Y; Cho, J Y; Kang, J S; Yu, Y H

    2000-12-01

    Wound contraction plays an important role in healing, but in extreme conditions, it may lead to excessive scar formation and pathological wound contracture. To date, the key regulator of excessive contracture is known to be transforming growth factor-beta (TGF-beta1). In this study, we have evaluated epidermal growth factor (EGF) antagonism in fibroblast-populated collagen lattice (FPCL) gel contraction, which has been generally used as an in vitro model thought to mimic wound contraction in vivo. As expected, TGF-beta1 treatment enhanced normal fibroblast-induced collagen gel contraction in a dose-dependent manner. In contrast, EGF did not affect normal gel formation, but significantly antagonized TGF-beta1-induced gel formation (p<0.05 at 100 ng/ml), whereas the other growth factor, platelet-derived growth factor (PDGF), did not altered either normal or TGF-beta1-induced gel contractions. Similarly, EGF treatment, but not PDGF, also significantly suppressed TGF-beta1 release that was autologously elicited by TGF-beta1 treatment (p<0.01 at 100 ng/ml). Therefore, the results suggest that EGF may negatively regulate the role of TGF-beta1 through attenuating autologous release of TGF-beta1. PMID:11145189

  13. Dynamic Regulation of Platelet-Derived Growth Factor Receptor α Expression in Alveolar Fibroblasts during Realveolarization

    PubMed Central

    Chen, Leiling; Acciani, Thomas; Le Cras, Tim; Lutzko, Carolyn

    2012-01-01

    Although the importance of platelet-derived growth factor receptor (PDGFR)-α signaling during normal alveogenesis is known, it is unclear whether this signaling pathway can regulate realveolarization in the adult lung. During alveolar development, PDGFR-α–expressing cells induce α smooth muscle actin (α-SMA) and differentiate to interstitial myofibroblasts. Fibroblast growth factor (FGF) signaling regulates myofibroblast differentiation during alveolarization, whereas peroxisome proliferator-activated receptor (PPAR)-γ activation antagonizes myofibroblast differentiation in lung fibrosis. Using left lung pneumonectomy, the roles of FGF and PPAR-γ signaling in differentiation of myofibroblasts from PDGFR-α–positive precursors during compensatory lung growth were assessed. FGF receptor (FGFR) signaling was inhibited by conditionally activating a soluble dominant-negative FGFR2 transgene. PPAR-γ signaling was activated by administration of rosiglitazone. Changes in α-SMA and PDGFR-α protein expression were assessed in PDGFR-α–green fluorescent protein (GFP) reporter mice using immunohistochemistry, flow cytometry, and real-time PCR. Immunohistochemistry and flow cytometry demonstrated that the cell ratio and expression levels of PDGFR-α–GFP changed dynamically during alveolar regeneration and that α-SMA expression was induced in a subset of PDGFR-α–GFP cells. Expression of a dominant-negative FGFR2 and administration of rosiglitazone inhibited induction of α-SMA in PDGFR-α–positive fibroblasts and formation of new septae. Changes in gene expression of epithelial and mesenchymal signaling molecules were assessed after left lobe pneumonectomy, and results demonstrated that inhibition of FGFR2 signaling and increase in PPAR-γ signaling altered the expression of Shh, FGF, Wnt, and Bmp4, genes that are also important for epithelial–mesenchymal crosstalk during early lung development. Our data demonstrate for the first time that a comparable

  14. Newborn human skin fibroblasts senesce in vitro without acquiring adult growth factor requirements

    SciTech Connect

    Wharton, W.

    1984-01-01

    Cultures of human fibroblasts were prepared from chest skin obtained either from newborns (less than 3 months old) or adults (more than 35 years old) and maintained in vitro until they senesced. Adult cells grew logarithmically in medium supplemented with whole blood serum but not with platelet-poor plasma. Early passage cells obtained from newborns grew equally well in either plasma- or serum-supplemented medium. The difference in growth factor requirements between adult and newborn cells persisted through the lifespan of the cells; i.e., newborn cells did not develop adult hormonal requirements when maintained in culture. Thus, in vitro cellular aging can be distinguished from some types of differentiation.

  15. Inhibition of fibroblast growth factor receptor 3-dependent lung adenocarcinoma with a human monoclonal antibody.

    PubMed

    Yin, Yongjun; Ren, Xiaodi; Smith, Craig; Guo, Qianxu; Malabunga, Maria; Guernah, Ilhem; Zhang, Yiwei; Shen, Juqun; Sun, Haijun; Chehab, Nabil; Loizos, Nick; Ludwig, Dale L; Ornitz, David M

    2016-05-01

    Activating mutations in fibroblast growth factor receptor 3 (FGFR3) have been identified in multiple types of human cancer and in congenital birth defects. In human lung cancer, fibroblast growth factor 9 (FGF9), a high-affinity ligand for FGFR3, is overexpressed in 10% of primary resected non-small cell lung cancer (NSCLC) specimens. Furthermore, in a mouse model where FGF9 can be induced in lung epithelial cells, epithelial proliferation and ensuing tumorigenesis is dependent on FGFR3. To develop new customized therapies for cancers that are dependent on FGFR3 activation, we have used this mouse model to evaluate a human monoclonal antibody (D11) with specificity for the extracellular ligand-binding domain of FGFR3, that recognizes both human and mouse forms of the receptor. Here, we show that D11 effectively inhibits signaling through FGFR3 in vitro, inhibits the growth of FGFR3-dependent FGF9-induced lung adenocarcinoma in mice, and reduces tumor-associated morbidity. Given the potency of FGF9 in this mouse model and the absolute requirement for signaling through FGFR3, this study validates the D11 antibody as a potentially useful and effective reagent for treating human cancers or other pathologies that are dependent on activation of FGFR3. PMID:27056048

  16. Genomic organization of the mouse fibroblast growth factor receptor 3 (Fgfr3) gene

    SciTech Connect

    Perez-Castro, A.V.; Wilson, J.; Altherr, M.R.

    1995-11-20

    The fibroblast growth factor receptor 3 (Fgfr3) protein is a tyrosine kinase receptor involved in the signal transduction of various fibroblast growth factors. Recent studies suggest its important role in normal development. In humans, mutation in Fgfr3 is responsible for growth disorders such as achondroplasia, hypoachondroplasia, and thanatophoric dysplasia. Here, we report the complete genomic organization of the mouse Fgfr3 gene. The murine gene spans approximately 15 kb and consists of 19 exons and 18 introns. One major and one minor transcription initiation site were identified. Position +1 is located 614 nucleotides upstream from the ATG initiation codon. The translation initiation and termination sites are located in exons 2 and 19, respectively. Five Sp1 sites, two AP2 sites, one Zeste site, and one Krox 24 site were observed in the 5{prime}-flanking region. The Fgfr3 promoter appears to be contained within a CpG island and, as is common in genes having multiple Sp1-binding sites, lacks a TATA box. 35 refs., 3 figs., 1 tab.

  17. Inhibition of fibroblast growth factor receptor 3-dependent lung adenocarcinoma with a human monoclonal antibody

    PubMed Central

    Yin, Yongjun; Ren, Xiaodi; Smith, Craig; Guo, Qianxu; Malabunga, Maria; Guernah, Ilhem; Zhang, Yiwei; Shen, Juqun; Sun, Haijun; Chehab, Nabil; Loizos, Nick; Ludwig, Dale L.; Ornitz, David M.

    2016-01-01

    ABSTRACT Activating mutations in fibroblast growth factor receptor 3 (FGFR3) have been identified in multiple types of human cancer and in congenital birth defects. In human lung cancer, fibroblast growth factor 9 (FGF9), a high-affinity ligand for FGFR3, is overexpressed in 10% of primary resected non-small cell lung cancer (NSCLC) specimens. Furthermore, in a mouse model where FGF9 can be induced in lung epithelial cells, epithelial proliferation and ensuing tumorigenesis is dependent on FGFR3. To develop new customized therapies for cancers that are dependent on FGFR3 activation, we have used this mouse model to evaluate a human monoclonal antibody (D11) with specificity for the extracellular ligand-binding domain of FGFR3, that recognizes both human and mouse forms of the receptor. Here, we show that D11 effectively inhibits signaling through FGFR3 in vitro, inhibits the growth of FGFR3-dependent FGF9-induced lung adenocarcinoma in mice, and reduces tumor-associated morbidity. Given the potency of FGF9 in this mouse model and the absolute requirement for signaling through FGFR3, this study validates the D11 antibody as a potentially useful and effective reagent for treating human cancers or other pathologies that are dependent on activation of FGFR3. PMID:27056048

  18. A basic fibroblast growth factor analog for protection and mitigation against acute radiation syndromes.

    PubMed

    Casey-Sawicki, Kate; Zhang, Mei; Kim, Sunghee; Zhang, Amy; Zhang, Steven B; Zhang, Zhenhuan; Singh, Ravi; Yang, Shanmin; Swarts, Steven; Vidyasagar, Sadasivan; Zhang, Lurong; Zhang, Aiguo; Okunieff, Paul

    2014-06-01

    The effects of fibroblast growth factors and their potential as broad-spectrum agents to treat and mitigate radiation injury have been studied extensively over the past two decades. This report shows that a peptide mimetic of basic fibroblast growth factor (FGF-P) protects and mitigates against acute radiation syndromes. FGF-P attenuates both sepsis and bleeding in a radiation-induced bone marrow syndrome model and reduces the severity of gastrointestinal and cutaneous syndromes; it should also mitigate combined injuries. FGF-2 and FGF-P induce little or no deleterious inflammation or vascular leakage, which distinguishes them from most other growth factors, angiogenic factors, and cytokines. Although recombinant FGFs have proven safe in several ongoing clinical trials, they are expensive to synthesize, can only be produced in limited quantity, and have limited shelf life. FGF-P mimics the advantageous features of FGF-2 without these disadvantages. This paper shows that FGF-P not only has the potential to be a potent yet safe broad-spectrum medical countermeasure that mitigates acute radiotoxicity but also holds promise for thermal burns, ischemic wound healing, tissue engineering, and stem-cell regeneration. PMID:24776903

  19. Changes in expression and secretion patterns of fibroblast growth factor 8 and Sonic Hedgehog signaling pathway molecules during murine neural stem/progenitor cell differentiation in vitro☆

    PubMed Central

    Lu, Jiang; Lu, Kehuan; Li, Dongsheng

    2012-01-01

    In the present study, we investigated the dynamic expression of fibroblast growth factor 8 and Sonic Hedgehog signaling pathway related factors in the process of in vitro hippocampal neural stem/progenitor cell differentiation from embryonic Sprague-Dawley rats or embryonic Kunming species mice, using fluorescent quantitative reverse transcription-PCR and western blot analyses. Results demonstrated that the dynamic expression of fibroblast growth factor 8 was similar to fibroblast growth factor receptor 1 expression but not to other fibroblast growth factor receptors. Enzyme-linked immunosorbent assay demonstrated that fibroblast growth factor 8 and Sonic Hedgehog signaling pathway protein factors were secreted by neural cells into the intercellular niche. Our experimental findings indicate that fibroblast growth factor 8 and Sonic Hedgehog expression may be related to the differentiation of neural stem/progenitor cells. PMID:25624789

  20. Diversity of Interstitial Lung Fibroblasts Is Regulated by Platelet-Derived Growth Factor Receptor α Kinase Activity.

    PubMed

    Green, Jenna; Endale, Mehari; Auer, Herbert; Perl, Anne-Karina T

    2016-04-01

    Epithelial-mesenchymal cell interactions and factors that control normal lung development are key players in lung injury, repair, and fibrosis. A number of studies have investigated the roles and sources of epithelial progenitors during lung regeneration; such information, however, is limited in lung fibroblasts. Thus, understanding the origin, phenotype, and roles of fibroblast progenitors in lung development, repair, and regeneration helps address these limitations. Using a combination of platelet-derived growth factor receptor α-green fluorescent protein (PDGFRα-GFP) reporter mice, microarray, real-time polymerase chain reaction, flow cytometry, and immunofluorescence, we characterized two distinct interstitial resident fibroblasts, myo- and matrix fibroblasts, and identified a role for PDGFRα kinase activity in regulating their activation during lung regeneration. Transcriptional profiling of the two populations revealed a myo- and matrix fibroblast gene signature. Differences in proliferation, smooth muscle actin induction, and lipid content in the two subpopulations of PDGFRα-expressing fibroblasts during alveolar regeneration were observed. Although CD140α(+)CD29(+) cells behaved as myofibroblasts, CD140α(+)CD34(+) appeared as matrix and/or lipofibroblasts. Gain or loss of PDGFRα kinase activity using the inhibitor nilotinib and a dominant-active PDGFRα-D842V mutation revealed that PDGFRα was important for matrix fibroblast differentiation. We demonstrated that PDGFRα signaling promotes alveolar septation by regulating fibroblast activation and matrix fibroblast differentiation, whereas myofibroblast differentiation was largely PDGFRα independent. These studies provide evidence for the phenotypic and functional diversity as well as the extent of specificity of interstitial resident fibroblasts differentiation during regeneration after partial pneumonectomy. PMID:26414960

  1. Growth hormone promoted tyrosyl phosphorylation of growth hormone receptors in murine 3T3-F442A fibroblasts and adipocytes

    SciTech Connect

    Foster, C.M.; Shafer, J.A.; Rozsa, F.W.; Wang, X.; Lewis, S.D.; Renken, D.A.; Natale, J.E.; Schwartz, J.; Carter-Su, C.

    1988-01-12

    Because many growth factor receptors are ligand-activated tyrosine protein kinases, the possibility that growth hormone (GH), a hormone implicated in human growth, promotes tyrosyl phosphorylation of its receptor was investigated. /sup 125/I-Labeled human GH was covalently cross-linked to receptors in intact 3T3-F442A fibroblasts, a cell line which differentiates into adipocytes in response to GH. The cross-linked cells were solubilized and passed over a column of phosphotyrosyl binding antibody immobilized on protein A-Sepharose. Immunoadsorbed proteins were eluted with a hapten (p-nitrophenyl phosphate) and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The eluate from the antibody column contained in M/sub r/ 134,000 /sup 125/I-GH-receptor complex. A similar result was obtained when the adipocyte form of 3T3-F442A cells was used in place of fibroblast form. O-Phosphotyrosine prevented /sup 125/I-GH-receptor complexes from binding to the antibody column, whereas O-phosphoserine and O-phosphothreonine did not. In studies of GH-promoted phosphorylation in 3T3-F442A fibroblasts labeled metabolically with (/sup 32/P)P/sub i/, GH was shown to stimulate formation of a /sup 32/P-labeled protein which bound to immobilized phosphotyrosyl binding antibodies. The molecular weight of 114,000 obtained for this protein is similar to that expected for non-cross-linked GH receptor. These observations provide strong evidence that binding of GH to its receptor stimulates phosphorylation of tyrosyl residues in the GH receptor.

  2. In situ detection of basic fibroblast growth factor by highly specific antibodies.

    PubMed Central

    Schulze-Osthoff, K.; Risau, W.; Vollmer, E.; Sorg, C.

    1990-01-01

    Basic fibroblast growth factor (bFGF) is thought to be of major importance for fibrosis and angiogenesis. Despite intensive studies dealing with the biochemistry and multiple biologic effects of bFGF, the cellular distribution is virtually unknown. Therefore, using the indirect immunoperoxidase technique, we examined the effect of bFGF on a large pattern of normal, inflammatory, and tumorous human tissues. Staining was performed on cryostat sections with a highly specific affinity-purified antiserum. In normal tissues, especially those of the thymus and placenta, mainly dendritic cells contained the growth factor. High levels of bFGF were also detected in basal cells and gland epithelial cells of skin biopsies. A conspicuous expression was observed in chronic inflammatory tissues corresponding to a generally pronounced proliferation of fibroblasts and endothelial cells in these situations. Tumors revealed a very heterogenous staining pattern. In some lesions, bFGF was predominantly present in infiltrating and endothelial cells. In several, neoplasms tumor cells exhibited an intensive staining. In some, especially vascular tumors, bFGF could not be detected. From the staining results it is concluded that angiogenesis is not simply controlled by the presence of bFGF but is mediated by a balance of several angiogenic inducers and inhibitors. Images Figure 1 Figure 2 PMID:1695484

  3. Connective tissue growth factor/CCN2-null mouse embryonic fibroblasts retain intact transforming growth factor-{beta} responsiveness

    SciTech Connect

    Mori, Yasuji; Hinchcliff, Monique; Wu, Minghua; Warner-Blankenship, Matthew; Lyons, Karen M.

    2008-03-10

    Background: The matricellular protein connective tissue growth factor (CCN2) has been implicated in pathological fibrosis, but its physiologic role remains elusive. In vitro, transforming growth factor-{beta} (TGF-{beta}) induces CCN2 expression in mesenchymal cells. Because CCN2 can enhance profibrotic responses elicited by TGF-{beta}, it has been proposed that CCN2 functions as an essential downstream signaling mediator for TGF-{beta}. To explore this notion, we characterized TGF-{beta}-induced activation of fibroblasts from CCN2-null (CCN2{sup -/-}) mouse embryos. Methods: The regulation of CCN2 expression was examined in vivo in a model of fibrosis induced by bleomycin. Cellular TGF-{beta} signal transduction and regulation of collagen gene expression were examined in CCN2{sup -/-} MEFs by immunohistochemistry, Northern, Western and RT-PCR analysis, immunocytochemistry and transient transfection assays. Results: Bleomycin-induced skin fibrosis in the mouse was associated with substantial CCN2 up-regulation in lesional fibroblasts. Whereas in vitro proliferation rate of CCN2{sup -/-} MEFs was markedly reduced compared to wild type MEFs, TGF-{beta}-induced activation of the Smad pathways, including Smad2 phosphorylation, Smad2/3 and Smad4 nuclear accumulation and Smad-dependent transcriptional responses, were unaffected by loss of CCN2. The stimulation of COL1A2 and fibronectin mRNA expression and promoter activity, and of corresponding protein levels, showed comparable time and dose-response in wild type and CCN2{sup -/-} MEFs, whereas stimulation of alpha smooth muscle actin and myofibroblast transdifferentiation showed subtle impairment in MEFs lacking CCN2. Conclusion: Whereas endogenous CCN2 plays a role in regulation of proliferation and TGF-{beta}-induced myofibroblast transdifferentiation, it appears to be dispensable for Smad-dependent stimulation of collagen and extracellular matrix synthesis in murine embryonic fibroblasts.

  4. Fibroblast growth factor-2 stimulates adipogenic differentiation of human adipose-derived stem cells

    SciTech Connect

    Kakudo, Natsuko . E-mail: kakudon@takii.kmu.ac.jp; Shimotsuma, Ayuko; Kusumoto, Kenji

    2007-07-27

    Adipose-derived stem cells (ASCs) have demonstrated a capacity for differentiating into a variety of lineages, including bone, cartilage, or fat, depending on the inducing stimuli and specific growth and factors. It is acknowledged that fibroblast growth factor-2 (FGF-2) promotes chondrogenic and inhibits osteogenic differentiation of ASCs, but thorough investigations of its effects on adipogenic differentiation are lacking. In this study, we demonstrate at the cellular and molecular levels the effect of FGF-2 on adipogenic differentiation of ASCs, as induced by an adipogenic hormonal cocktail consisting of 3-isobutyl-1-methylxanthine (IBMX), dexamethasone, insulin, and indomethacin. FGF-2 significantly enhances the adipogenic differentiation of human ASCs. Furthermore, in cultures receiving FGF-2 before adipogenic induction, mRNA expression of peroxisome proliferator-activated receptor {gamma}2 (PPAR{gamma}2), a key transcription factor in adipogenesis, was upregulated. The results of FGF-2 supplementation suggest the potential applications of FGF-2 and ASCs in adipose tissue regeneration.

  5. Sustained activation of fibroblast transforming growth factor-beta/Smad signaling in a murine model of scleroderma.

    PubMed

    Takagawa, Shinsuke; Lakos, Gabriella; Mori, Yasuji; Yamamoto, Toshiyuki; Nishioka, Kiyoshi; Varga, John

    2003-07-01

    Transforming growth factor-beta is responsible for triggering a cascade of events leading to fibrosis in scleroderma. The Smads are intracellular signal transducers recently shown to mediate fibroblast activation and other profibrotic responses elicited by transforming growth factor-betain vitro. To understand better the involvement of Smads in the pathogenesis of fibrosis, we examined Smad expression and activation in situ in a murine model of scleroderma. Bleomycin injections induced striking dermal infiltration with macrophages by 3 d, and progressive fibrosis by 2 wk. Infiltrating macrophages and resident fibroblasts expressed Smad3, the positive mediator for transforming growth factor-beta responses. Importantly, in bleomycin-injected skin, fibroblasts showed predominantly nuclear localization of Smad3 and intense staining for phospho-Smad2/3. Furthermore, phosphorylated Smad2/3 in fibroblasts was detected even after the resolution of inflammation. Expression of Smad7, the endogenous inhibitor of transforming growth factor-beta/Smad signaling, was strongly induced in dermal cells by transforming growth factor-beta, but not by bleomycin injections. Collectively, these results indicate that bleomycin-induced murine scleroderma is associated with rapid and sustained induction of transforming growth factor-beta/Smad signaling in resident dermal fibroblasts. Despite apparent activation of the intracellular transforming growth factor-beta signaling pathway in the lesional dermis, the expression of transforming growth factor-beta-inducible Smad7 was not upregulated. In light of the critical function of Smad7 as an endogenous inhibitor of Smad signaling that restricts the duration and magnitude of transforming growth factor-beta responses, and as a mediator of apoptosis, relative Smad7 deficiency observed in the present studies may account for sustained activation of transforming growth factor-beta/Smad signaling in lesional tissues. These findings raise the

  6. Prostaglandin E2 regulates angiogenesis via activation of fibroblast growth factor receptor-1.

    PubMed

    Finetti, Federica; Solito, Raffaella; Morbidelli, Lucia; Giachetti, Antonio; Ziche, Marina; Donnini, Sandra

    2008-01-25

    Prostaglandin E(2) (PGE(2)) behaves as a mitogen in epithelial tumor cells as well as in many other cell types. We investigated the actions of PGE(2) on microvascular endothelial cells (capillary venular endothelial cells) with the purpose of delineating the signaling pathway leading to the acquisition of the angiogenic phenotype and to new vessel formation. PGE(2) (100 nM) produced activation of the fibroblast growth factor receptor 1 (FGFR-1), as measured by its phosphorylation, but not of vascular endothelial growth factor receptor 2. PGE(2) stimulated the EP3 subtype receptor, as deduced by abrogation of EP3 Galpha(i) subunit activity through pertussis toxin. Consistent with this result, in human umbilical venular endothelial cells missing the EP3 receptor, PGE(2) did not phosphorylate FGFR-1. Upon binding to its receptor, PGE(2) initiated an autocrine/paracrine signaling cascade involving the intracellular activation of c-Src, activation of matrix metalloproteinase (predominantly MMP2), which in turn caused the mobilization of membrane-anchored fibroblast growth factor-2 (FGF-2). In fact, in cells unable to release FGF-2 the transfection with both FGFR-1 and EP3 did not result in FGFR-1 phosphorylation in response to PGE(2). Relevance for the FGF2-FGFR-1 system was highlighted by confocal analysis, showing receptor internalization after cell exposure to the prostanoid. ERK1/2 appeared to be the distal signal involved, its phosphorylation being sensitive to either cSrc inhibitor or FGFR-1 blocker. Finally, PGE(2) stimulated cell migration and capillary formation in aortic rings, which were severely reduced by inhibitors of signaling molecules or by receptor antagonist. In conclusion, this study provides evidence for the involvement of FGFR-1 through FGF2 in eliciting PGE(2) angiogenic responses. This signaling pattern is similar to the autocrine-paracrine mechanism which operates in endothelial cells to support neovascular growth. PMID:18042549

  7. Acid phosphatase activity and intracellular collagen degradation by fibroblasts in vitro.

    PubMed

    Yajima, T

    1986-01-01

    Human gingival fibroblasts were cultured with collagen fibrils. The precise process of collagen phagocytosis and the relationship between acid phosphatase activity and intracellular degradation of collagen were investigated by cytochemical methods at the ultrastructural level. The collagen fibrils were first engulfed at one end by cellular processes, or the cell membrane wrapped itself around the middle of the fibrils. Collagen phagocytosis induced acid phosphatase activity in the fibroblast Golgi-endoplasmic reticulum-lysosome system. By application of the tracer lanthanum, deposits were observed in the intercellular spaces and along the fibrils being phagocytosed. At this stage, primary lysosomes were seen in close proximity to the collagen being engulfed, but no signs of fusion were observed. When the fibrils had been interiorized in whole or in part, they ultimately became enclosed within phagosomes, and no tracer was observed along the interiorized portion of the fibrils. Primary lysosomes then fused with these collagen-containing phagosomes to form phagolysosomes. Collagen degradation occurred within these bodies even though the end of a fibril might have protruded outside of the cell. These results suggest that selective and controlled phagocytosis of collagen and intracellular digestion of it may play a central role in the physiological remodeling and metabolic breakdown of the collagen of connective tissues. PMID:3742560

  8. Improved regeneration potential of fibroblasts using ascorbic acid-blended nanofibrous scaffolds.

    PubMed

    Sridhar, Sreepathy; Venugopal, Jayarama Reddy; Ramakrishna, Seeram

    2015-11-01

    Two-dimensional scaffolds, three-dimensional scaffolds, and dermal substitutes are extensively used for biomedical applications in skin tissue regeneration. Not much explored synthetic polymers, like poly(l-lactic acid)-co-poly-(ε-caprolactone) (PLACL), natural polymers, like silk fibroin (SF), and active inducing agents, such as ascorbic acid (AA) and tetracycline hydrochloride (TCH), represent a favorable matrix for fabricating dermal substitutes to engineer artificial skin for wound repair. The profligate nature of residing skin cells near the wound site is a paramount to survival and also regulating stem cells and other cellular networks and mechanical forces. PLACL/SF/TCH/AA nanofibrous scaffolds were fabricated by electrospinning and characterized for fiber morphology, membrane porosity, wettability, and significant subchains using Fourier transform infrared spectroscopy for culturing human-derived dermal fibroblasts. The PLACL, PLACL/SF, PLACL/SF/TCH, and PLACL/SF/TCH/AA scaffolds obtained diameters between 250 and 340 nm. The secretion of collagen by the laboratory-grown fibroblasts over the AA-blended scaffolds was found to be significantly higher compared with that of other scaffolds. The obtained results positively prove that introduction of naturally secreting compounds (AA) by the cells into the nanofibrous scaffolds will favor cell's microenvironment and eventually leads to complete tissue regeneration. PMID:25903719

  9. D1398G Variant of MET Is Associated with Impaired Signaling of Hepatocyte Growth Factor in Alveolar Epithelial Cells and Lung Fibroblasts.

    PubMed

    Atanelishvili, Ilia; Shirai, Yuichiro; Akter, Tanjina; Noguchi, Atsushi; Ash, Kurt T; Misra, Suniti; Ghatak, Sibnath; Silver, Richard M; Bogatkevich, Galina S

    2016-01-01

    Pulmonary fibrosis represents the terminal stage of a diverse group of lung diseases including scleroderma associated interstitial lung disease. The molecular mechanisms underlying the pathogenesis of lung fibrosis are not well understood and there is a great need for more effective treatment for this lethal disease. We recently discovered a small fragment of hepatocyte growth factor (HGF) receptor MET as a peptide designated "M10," with strong antifibrotic properties. Furthermore, we showed that aspartic acid at position 1398 of MET is essential for M10 generation. The current study was undertaken to investigate the D1398G variant of MET in which aspartic acid at position 1398 was mutated to glycine resulting in loss of M10. We demonstrate that lung fibroblasts, A549, and primary alveolar epithelial cells (AEC) expressing D1398G MET exhibit reduced auto-phosphorylation on tyrosine residues and reduced activation of Ras and MAPK. HGF treatment of scleroderma lung fibroblasts as well as HGF treatment of TGFβ-treated normal lung fibroblasts transfected with wild type MET is associated with decreased collagen, connective tissue growth factor (CTGF, CCN2) and smooth muscle α-actin (SMA). However, HGF has no such effects in cells transfected with MET D1398G. Cisplatin- and FasL-induced apoptosis is significantly reduced in AEC transfected with MET wild type, but not in AEC transfected with MET D1398G. We conclude that the D1398G variant of MET is associated with compromised phosphorylation and impaired HGF signaling in lung fibroblasts and AEC, two cell types implicated in the pathogenesis of pulmonary fibrosis associated with scleroderma. Ongoing studies will explore the frequency of this variant and its relationship to pulmonary outcomes in scleroderma patients. PMID:27584154

  10. Covalent Targeting of Fibroblast Growth Factor Receptor Inhibits Metastatic Breast Cancer.

    PubMed

    Brown, Wells S; Tan, Li; Smith, Andrew; Gray, Nathanael S; Wendt, Michael K

    2016-09-01

    Therapeutic targeting of late-stage breast cancer is limited by an inadequate understanding of how tumor cell signaling evolves during metastatic progression and by the currently available small molecule inhibitors capable of targeting these processes. Herein, we demonstrate that both β3 integrin and fibroblast growth factor receptor-1 (FGFR1) are part of an epithelial-mesenchymal transition (EMT) program that is required to facilitate metastatic outgrowth in response to fibroblast growth factor-2 (FGF2). Mechanistically, β3 integrin physically disrupts an interaction between FGFR1 and E-cadherin, leading to a dramatic redistribution of FGFR1 subcellular localization, enhanced FGF2 signaling and increased three-dimensional (3D) outgrowth of metastatic breast cancer cells. This ability of β3 integrin to drive FGFR signaling requires the enzymatic activity of focal adhesion kinase (FAK). Consistent with these mechanistic data, we demonstrate that FGFR, β3 integrin, and FAK constitute a molecular signature capable of predicting decreased survival of patients with the basal-like subtype of breast cancer. Importantly, covalent targeting of a conserved cysteine in the P-loop of FGFR1-4 with our newly developed small molecule, FIIN-4, more effectively blocks 3D metastatic outgrowth as compared with currently available FGFR inhibitors. In vivo application of FIIN-4 potently inhibited the growth of metastatic, patient-derived breast cancer xenografts and murine-derived metastases growing within the pulmonary microenvironment. Overall, the current studies demonstrate that FGFR1 works in concert with other EMT effector molecules to drive aberrant downstream signaling, and that these events can be effectively targeted using our novel therapeutics for the treatment of the most aggressive forms of breast cancer. Mol Cancer Ther; 15(9); 2096-106. ©2016 AACR. PMID:27371729

  11. Fibroblast growth factor receptors as novel therapeutic targets in SNF5-deleted malignant rhabdoid tumors.

    PubMed

    Wöhrle, Simon; Weiss, Andreas; Ito, Moriko; Kauffmann, Audrey; Murakami, Masato; Jagani, Zainab; Thuery, Anne; Bauer-Probst, Beatrice; Reimann, Flavia; Stamm, Christelle; Pornon, Astrid; Romanet, Vincent; Guagnano, Vito; Brümmendorf, Thomas; Sellers, William R; Hofmann, Francesco; Roberts, Charles W M; Graus Porta, Diana

    2013-01-01

    Malignant rhabdoid tumors (MRTs) are aggressive pediatric cancers arising in brain, kidney and soft tissues, which are characterized by loss of the tumor suppressor SNF5/SMARCB1. MRTs are poorly responsive to chemotherapy and thus a high unmet clinical need exists for novel therapies for MRT patients. SNF5 is a core subunit of the SWI/SNF chromatin remodeling complex which affects gene expression by nucleosome remodeling. Here, we report that loss of SNF5 function correlates with increased expression of fibroblast growth factor receptors (FGFRs) in MRT cell lines and primary tumors and that re-expression of SNF5 in MRT cells causes a marked repression of FGFR expression. Conversely, siRNA-mediated impairment of SWI/SNF function leads to elevated levels of FGFR2 in human fibroblasts. In vivo, treatment with NVP-BGJ398, a selective FGFR inhibitor, blocks progression of a murine MRT model. Hence, we identify FGFR signaling as an aberrantly activated oncogenic pathway in MRTs and propose pharmacological inhibition of FGFRs as a potential novel clinical therapy for MRTs. PMID:24204904

  12. Fibroblast Growth Factor Receptors as Novel Therapeutic Targets in SNF5-Deleted Malignant Rhabdoid Tumors

    PubMed Central

    Wöhrle, Simon; Jagani, Zainab; Thuery, Anne; Bauer-Probst, Beatrice; Reimann, Flavia; Stamm, Christelle; Pornon, Astrid; Romanet, Vincent; Guagnano, Vito; Brümmendorf, Thomas; Sellers, William R.; Hofmann, Francesco; Roberts, Charles W. M.; Graus Porta, Diana

    2013-01-01

    Malignant rhabdoid tumors (MRTs) are aggressive pediatric cancers arising in brain, kidney and soft tissues, which are characterized by loss of the tumor suppressor SNF5/SMARCB1. MRTs are poorly responsive to chemotherapy and thus a high unmet clinical need exists for novel therapies for MRT patients. SNF5 is a core subunit of the SWI/SNF chromatin remodeling complex which affects gene expression by nucleosome remodeling. Here, we report that loss of SNF5 function correlates with increased expression of fibroblast growth factor receptors (FGFRs) in MRT cell lines and primary tumors and that re-expression of SNF5 in MRT cells causes a marked repression of FGFR expression. Conversely, siRNA-mediated impairment of SWI/SNF function leads to elevated levels of FGFR2 in human fibroblasts. In vivo, treatment with NVP-BGJ398, a selective FGFR inhibitor, blocks progression of a murine MRT model. Hence, we identify FGFR signaling as an aberrantly activated oncogenic pathway in MRTs and propose pharmacological inhibition of FGFRs as a potential novel clinical therapy for MRTs. PMID:24204904

  13. Fibroblast growth factor receptor 3 protein is overexpressed in oral and oropharyngeal squamous cell carcinoma.

    PubMed

    Koole, Koos; van Kempen, Pauline M W; Swartz, Justin E; Peeters, Ton; van Diest, Paul J; Koole, Ron; van Es, Robert J J; Willems, Stefan M

    2016-02-01

    Fibroblast growth factor receptor 3 (FGFR3) is a member of the fibroblast growth factor receptor tyrosine kinase family. It has been identified as a promising therapeutic target in multiple types of cancer. We have investigated FGFR3 protein expression and FGFR3 gene copy-numbers in a single well-documented cohort of oral and oropharyngeal squamous cell carcinoma. Tissue microarray sets containing 452 formalin-fixed paraffin-embedded tissues were immunohistochemically stained with an anti-FGFR3 antibody and hybridized with a FGFR3 fluorescence in situ hybridization probe. FGFR3 protein expression was correlated with clinicopathological and survival data, which were retrieved from electronic medical records. FGFR3 mRNA data of 522 head and neck squamous cell carcinoma (HNSCC) were retrieved from The Cancer Genome Atlas (TCGA). Fibroblast growth factor receptor 3 (FGFR3) protein was overexpressed in 48% (89/185) of oral and 59% (124/211) of oropharyngeal squamous cell carcinoma. Overexpression of FGFR3 protein was not related to overall survival or disease-free survival in oral (HR[hazard ratio]: 0.94; 95% CI: 0.64-1.39; P = 0.77, HR: 0.94; 95% CI: 0.65-1.36; P = 0.75) and oropharyngeal squamous cell carcinoma (HR: 1.21; 95% CI: 0.81-1.80; P = 0.36, HR: 0.42; 95% CI: 0.79-1.77; P = 0.42). FGFR3 mRNA was upregulated in 3% (18/522) of HNSCC from the TCGA. The FGFR3 gene was gained in 0.6% (1/179) of oral squamous cell carcinoma but no amplification was found in oral and oropharyngeal squamous cell carcinoma. In conclusion, FGFR3 protein is frequently overexpressed in oral and oropharyngeal squamous cell carcinoma. Therefore, it may serve as a potential therapeutic target for FGFR3-directed therapies in oral and oropharyngeal squamous cell carcinoma. PMID:26711175

  14. Vitamin D inhibition of pro-fibrotic effects of transforming growth factor β1 in lung fibroblasts and epithelial cells

    PubMed Central

    Ramirez, Allan M.; Wongtrakool, Cherry; Welch, Teresa; Steinmeyer, Andreas; Zügel, Ulrich; Roman, Jesse

    2010-01-01

    The mechanisms that control fibroproliferation and matrix deposition in lung fibrosis remain unclear. We speculate that vitamin D deficiency may contribute to pulmonary fibrosis since vitamin D deficiency has been implicated in several diseases. First, we confirmed the presence of vitamin D receptors (VDR) in cultured NIH/3T3 and lung fibroblasts. Fibroblasts transfected with a vitamin D response element – reporter construct and exposed to the active vitamin D metabolite, 1,25(OH)2D3, showed increased promoter activity indicating VDR functionality in these cells. Testing the effects of 1,25(OH)2D3 on fibroblasts treated with transforming growth factor β1 (TGFβ1), considered a driver of many fibrotic disorders, we found that 1,25(OH)2D3 inhibited TGFβ1-induced fibroblast proliferation in a dose-dependent fashion. 1,25(OH)2D3 also inhibited TGFβ1 stimulation of α-smooth muscle actin expression and polymerization and prevented the upregulation of fibronectin and collagen in TGFβ1-treated fibroblasts. Finally, we examined how 1,25(OH)2D3 affects epithelial-mesenchymal transformation of lung epithelial cells upon exposure to TGFβ1. We showed that the TGFβ1-induced upregulation of mesenchymal cell markers and abnormal expression of epithelial cell markers were blunted by 1,25(OH)2D3. These observations suggest that under TGFβ1 stimulation, 1,25(OH)2D3 inhibits the profibrotic phenotype of lung fibroblasts and epithelial cells. PMID:19931390

  15. The effect of ursolic and oleanolic acids on human skin fibroblast cells.

    PubMed

    Wójciak-Kosior, Magdalena; Paduch, Roman; Matysik-Woźniak, Anna; Niedziela, Piotr; Donica, Helena

    2011-01-01

    In this article, we look at how ursolic and oleanolic acids can be used for the purpose of quality control of natural products used in dermatocosmetology as well as of various other therapeutic preparations. Ursolic acid (UA) and oleanolic acid (OA) are pentacyclic triterpenes and they are constituents of many medicinal herbs. In this study, we analyzed the cytotoxic and anti-proliferative activity of OA and UA against normal human skin fibroblasts (HSF). Additionally, the scavenging activity of free radicals of both acids was analyzed. The sensitivity of cells to OA and UA activity was determined using a standard spectrophotometric (MTT) assay. The free radical scavenging activity of OA and UA was measured using the DPPH• test. The F-actin cytoskeletal proteins organization was analyzed using TRITC-phalloidine fluorescent staining. The cytotoxic activity of the analyzed acids was determined using Neutral Red (NR) uptake assay. Of the two isomeric compounds, UA showed a higher cytotoxic activity against HSF cells than did OA. Our investigations showed that OA, in view of its non-toxic nature, may be used as a supplementary factor for dermal preparations. PMID:22252762

  16. Effects of epidermal growth factor and keratinocyte growth factor on the growth of oropharyngeal keratinocytes in coculture with autologous fibroblasts in a three-dimensional matrix.

    PubMed

    Blaimauer, Karin; Watzinger, Elisabeth; Erovic, Boban M; Martinek, Helga; Jagersberger, Tamara; Thurnher, Dietmar

    2006-01-01

    Tissue engineering of oropharyngeal mucosa is rendered complex by the fact that oropharyngeal keratinocytes are difficult to culture in the long term and do not grow well after several subcultivations. Three populations of oropharyngeal keratinocytes were isolated by a method based on different levels of beta(1)-integrin expression. In particular, keratinocytes were isolated between cell fractions that adhere rapidly on collagen-IV-coated culture dishes (RAC-IV) and populations that are less adherent (RAC-IV-D). The total fraction of both subpopulations served as a control (RAC-IV-T). The epidermal growth factor (EGF) and the keratinocyte growth factor (KGF) were examined with regard to their effects on the growth of the three populations. Growth curves of all three cell fractions grown with or without EGF were generated, and different concentrations of EGF and KGF were tested. EGF did not change any growth characteristics of the cells, with the exception of the speed of growth. Best growth was achieved with a physiologic EGF concentration of 0.15-1.5 ng/ml and a KGF concentration of 15 ng/ml. Finally, we cocultured oropharyngeal keratinocytes and their autologous fibroblasts in a three-dimensional matrix using Matrigeltrade mark. Oropharyngeal keratinocytes grown in coculture formed larger colonies than keratinocytes grown without fibroblasts. In conclusion, we were able to optimize the supplement of EGF and KGF in standard medium for the long-term culture of primary oropharyngeal keratinocytes. The use of Matrigel as a scaffold for three-dimensional cocultures of oropharyngeal keratinocytes and fibroblasts might signify a step forward in the development of a transplantable mucosa construct. PMID:16804300

  17. mTOR inhibition improves fibroblast growth factor receptor targeting in hepatocellular carcinoma

    PubMed Central

    Scheller, T; Hellerbrand, C; Moser, C; Schmidt, K; Kroemer, A; Brunner, S M; Schlitt, H J; Geissler, E K; Lang, S A

    2015-01-01

    Background: Systemic therapy has proven only marginal effects in hepatocellular carcinoma (HCC) so far. The aim of this study was to evaluate the effect of targeting fibroblast growth factor receptor (FGFR) on tumour and stromal cells in HCC models. Methods: Human and murine HCC cells, endothelial cells (ECs), vascular smooth muscle cells (VSMCs), hepatic stellate cells (HSCs), human HCC samples, FGFR inhibitor BGJ398 and mammalian target of rapamycin (mTOR) inhibitor rapamycin were used. Effects on growth, motility, signalling and angiogenic markers were determined. In vivo subcutaneous and syngeneic orthotopic tumour models were used. Results: In tumour cells and ECs, targeting FGFR showed significant inhibitory effects on signalling and motility. Minor effects of FGFR inhibition were observed on VSMCs and HSCs, which were significantly enhanced by combining FGFR and mTOR blockade. In vivo daily (5 mg kg−1) treatment with BGJ398 led to a significant growth inhibition in subcutaneous tumour models, but only a combination of FGFR and mTOR blockade impaired tumour growth in the orthotopic model. This was paralleled by reduced tumour cell proliferation, vascularisation, pericytes and increased apoptosis. Conclusions: Targeting FGFR with BGJ398 affects tumour cells and ECs, whereas only a combination with mTOR inhibition impairs recruitment of VSMCs and HSCs. Therefore, this study provides evidence for combined FGFR/mTOR inhibition in HCC. PMID:25688743

  18. Selection and characterization of a human neutralizing antibody to human fibroblast growth factor-2

    SciTech Connect

    Tao, Jun; Xiang, Jun-Jian; Li, Dan; Deng, Ning; Wang, Hong; Gong, Yi-Ping

    2010-04-09

    Compelling evidences suggest that fibroblast growth factor-2 (FGF-2) plays important roles in tumor growth, angiogenesis and metastasis. Molecules blocking the FGF-2 signaling have been proposed as anticancer agents. Through screening of a human scFv phage display library, we have isolated several human single-chain Fv fragments (scFvs) that bind to human FGF-2. After expression and purification in bacteria, one scFv, named 1A2, binds to FGF-2 with a high affinity and specificity, and completes with FGF-2 binding to its receptor. This 1A2 scFv was then cloned into the pIgG1 vector and expressed in 293T cells. The purified hIgG1-1A2 antibody showed a high binding affinity of 8 x 10{sup -9} M to rhFGF-2. In a set of vitro assays, it inhibited various biological activities of FGF-2 such as the proliferation, migration and tube formation of human umbilical vein endothelial cells. More importantly, hIgG1-1A2 antibody also efficiently blocked the growth while inducing apoptosis of glioma cells. For the first time, we generated a human anti-FGF-2 antibody with proven in vitro anti-tumor activity. It may therefore present a new therapeutic candidate for the treatment of cancers that are dependent on FGF-2 signaling for growth and survival.

  19. Distinct Roles for Fibroblast Growth Factor Signaling in Cerebellar Development and Medulloblastoma

    PubMed Central

    Emmenegger, Brian A.; Hwang, Eugene I.; Moore, Colin; Markant, Shirley L.; Brun, Sonja N.; Dutton, John W.; Read, Tracy-Ann; Fogarty, Marie P.; Singh, Alok R.; Durden, Donald L.; Yang, Chaofeng; McKeehan, Wallace L.; Wechsler-Reya, Robert J.

    2013-01-01

    Cerebellar granule neurons are the most abundant neurons in the brain, and a critical element of the circuitry that controls motor coordination and learning. In addition, granule neuron precursors (GNPs) are thought to represent cells of origin for medulloblastoma, the most common malignant brain tumor in children. Thus, understanding the signals that control the growth and differentiation of these cells has important implications for neurobiology and neuro-oncology. Our previous studies have shown that proliferation of GNPs is regulated by Sonic hedgehog (Shh), and that aberrant activation of the Shh pathway can lead to medulloblastoma. Moreover, we have demonstrated that Shh-dependent proliferation of GNPs and medulloblastoma cells can be blocked by basic fibroblast growth factor (bFGF). But while the mitogenic effects of Shh signaling have been confirmed in vivo, the inhibitory effects of bFGF have primarily been studied in culture. Here we demonstrate that mice lacking FGF signaling in GNPs exhibit no discernable changes in GNP proliferation or differentiation. In contrast, activation of FGF signaling has a potent effect on tumor growth: treatment of medulloblastoma cells with bFGF prevents them from forming tumors following transplantation, and inoculation of tumor-bearing mice with bFGF markedly inhibits tumor growth in vivo. These results suggest that activators of FGF signaling may be useful for targeting medulloblastoma and other Shh-dependent tumors. PMID:23045271

  20. Rat embryo fibroblast cells expressing human papillomavirus 1a genes exhibit altered growth properties and tumorigenicity.

    PubMed Central

    Green, M; Brackmann, K H; Loewenstein, P M

    1986-01-01

    Human papillomavirus 1a (HPV1a) induces benign tumors (papillomas or warts) in humans under natural conditions of infection but has not been found to replicate significantly in cell culture or in experimental animals. To establish model systems to study the oncogenic properties and expression of HPV genes, we established cell lines by cotransfecting the 3Y1 rat fibroblast cell line with HPV1a DNA constructs containing an intact early gene region and the Tn5 neomycin resistance gene. Most cell lines selected for expression of the neomycin resistance gene by treatment with the antibiotic G-418 contained viral DNA in a high-molecular-weight form. The growth characteristics of several cell lines containing high copy numbers of HPV1a DNA were studied further. They were shown to differ from the parental cell line and from G-418-resistant cell lines that did not incorporate viral DNA in the following properties: morphological alteration, increased cell density at confluence, growth in 0.5% serum, efficient anchorage-independent growth in soft agar, and rapid formation of tumors in nude mice. Those cell lines that possessed altered growth properties and tumorigenicity were found to express abundant quantities of polyadenylated virus-specific RNA species in the cytoplasm. Images PMID:3023676

  1. Distinct roles for mammalian target of rapamycin complexes in the fibroblast response to transforming growth factor-beta.

    PubMed

    Rahimi, Rod A; Andrianifahanana, Mahefatiana; Wilkes, Mark C; Edens, Maryanne; Kottom, Theodore J; Blenis, John; Leof, Edward B

    2009-01-01

    Transforming growth factor-beta (TGF-beta) promotes a multitude of diverse biological processes, including growth arrest of epithelial cells and proliferation of fibroblasts. Although the TGF-beta signaling pathways that promote inhibition of epithelial cell growth are well characterized, less is known about the mechanisms mediating the positive response to this growth factor. Given that TGF-beta has been shown to promote fibrotic diseases and desmoplasia, identifying the fibroblast-specific TGF-beta signaling pathways is critical. Here, we investigate the role of mammalian target of rapamycin (mTOR), a known effector of phosphatidylinositol 3-kinase (PI3K) and promoter of cell growth, in the fibroblast response to TGF-beta. We show that TGF-beta activates mTOR complex 1 (mTORC1) in fibroblasts but not epithelial cells via a PI3K-Akt-TSC2-dependent pathway. Rapamycin, the pharmacologic inhibitor of mTOR, prevents TGF-beta-mediated anchorage-independent growth without affecting TGF-beta transcriptional responses or extracellular matrix protein induction. In addition to mTORC1, we also examined the role of mTORC2 in TGF-beta action. mTORC2 promotes TGF-beta-induced morphologic transformation and is required for TGF-beta-induced Akt S473 phosphorylation but not mTORC1 activation. Interestingly, both mTOR complexes are necessary for TGF-beta-mediated growth in soft agar. These results define distinct and overlapping roles for mTORC1 and mTORC2 in the fibroblast response to TGF-beta and suggest that inhibitors of mTOR signaling may be useful in treating fibrotic processes, such as desmoplasia. PMID:19117990

  2. The effect of platelet-derived growth factor on cell division and glycosaminoglycan synthesis by human skin and scar fibroblasts.

    PubMed

    Savage, K; Siebert, E; Swann, D

    1987-07-01

    The effect of platelet-derived growth factor (PDGF) on cell division and glycosaminoglycan (GAG) synthesis by fibroblasts isolated from skin and scar was measured. We found that PDGF stimulates cell division more efficiently in normal skin fibroblasts than in scar fibroblasts and decreases GAG synthesis in skin and scar fibroblasts. Using a 4-h pulse label with [3H]thymidine ([3H]Thd) following a 20-h incubation of confluent monolayer cultures with 0-5 units PDGF/ml Dulbecco's modified Eagle's medium, we found a concentration-dependent increase in [3H]Thd incorporation. After incubation of fibroblasts with [3H]glucosamine and 35SO4 in the presence or absence of PDGF, labeled constituents were isolated from the extracellular, pericellular, and cellular fractions by pronase digestion and column chromatography on Sepharose CL4B or DEAE-cellulose and analyzed by cellulose acetate electrophoresis. The presence of PDGF decreased the total amount of 35S incorporated into macromolecules by skin and scar fibroblasts and resulted in an altered distribution of labeled GAGs. Dermal fibroblasts exposed to PDGF for 24 h incorporated a greater percentage of radiolabeled 35S into dermatan sulfate prime (DS') and less into dermatan sulfate (DS) in the extracellular fractions and a greater percentage of 35S into heparan sulfate (HS) in the pericellular fractions than did parallel cultures grown in the absence of PDGF. It is thought than PDGF may have an effect on scar formation by increasing the fibroblast population in the wound tissue and by affecting the total amount and types of matrix components synthesized. PMID:3598205

  3. Silk fibroin sponges with cell growth-promoting activity induced by genetically fused basic fibroblast growth factor.

    PubMed

    Kambe, Yusuke; Kojima, Katsura; Tamada, Yasushi; Tomita, Naohide; Kameda, Tsunenori

    2016-01-01

    Transgenic silkworm technology has enabled the biological properties of silk fibroin protein to be altered by fusion to recombinant bioactive proteins. However, few studies have reported the fabrication of genetically modified fibroin proteins into three-dimensional spongy structures to serve as scaffolds for tissue engineering. We generated a transgenic silkworm strain that produces fibroin fused to basic fibroblast growth factor (bFGF) and processed the fibroin into a spongy structure using a simple freeze/thaw method. NIH3T3 mouse embryonic fibroblasts grown on bFGF-fused fibroin sponges proliferated and spread out well, showing half the population doubling time of cells cultured on wild-type fibroin sponges. Furthermore, the number of primary rabbit articular chondrocytes growing on bFGF-fused fibroin sponges was around five-times higher than that of the wild-type control at 3-days post cell-seeding. As the physical properties of wild-type and bFGF-fused fibroin sponges were almost identical, it is suggested that bFGF fused to fibroin retained its biological activity, even after the bFGF-fused fibroin was fabricated into the spongy structure. The bFGF-fused fibroin sponge has the potential for widespread application in the field of tissue engineering, and the method of fabricating this structure could be applicable to other recombinant bioactive fibroin proteins. PMID:26190702

  4. The role of mutations and overexpression of the fibroblast growth factor receptor-3 in bladder cancer.

    PubMed

    Wang, Q Y; Zhao, Y; Zhang, R

    2015-12-01

    Bladder cancer (BC) is the seventh most common cancer worldwide. Throughout the last decade, several studies demonstrated that the fibroblast growth factor (FGF) signalling is altered in a significant proportion of patients with BC. FGF receptor (FGFR) 3 may thus serve as a promising biomarker for BC. Mutations of this gene are prevalent in BC (e.g., found in 74% of non-invasive papillary tumours), suggesting that FGFR3 status is an important event in BC. The aim of this review was to overview the outcomes of different mutations in FGFR3 receptor in the context of BC. We first described FGFR3 receptor and continue with mutations of FGFR3 gene, including activating mutations and overexpression of this gene. Finally, we addressed the clinical relevance of mutated FGFR3 gene. PMID:26542242

  5. BMP-9 enhances fibroblast growth factor 21 expression and suppresses obesity.

    PubMed

    Kim, Sooho; Choe, Senyon; Lee, Dong Kun

    2016-07-01

    Although BMP-9 has been reported to induce browning of white adipose tissues (WATs) and suppress high fat diet-induced obesity, detailed molecular mechanism needs to be further elucidated. We report here that administration of MB109, a recombinant derivative of human BMP-9, into obese mice enhanced gene expression of fibroblast growth factor 21 (FGF21), a metabolic regulator, and alleviates a spectrum of pathological symptoms due to high fat diet-induced obesity. In addition, periodical injection of MB109 (500μg/kg/week) reduced an amount of lipid droplets in the liver, serum levels of alanine aminotransferase (ALT), and total cholesterol. These results indicate that MB109 is also effective to treat obesity-mediated non-alcoholic fatty liver disease (NAFLD). PMID:27085971

  6. Use of fibroblast growth factor 2 for expansion of chondrocytes and tissue engineering

    NASA Technical Reports Server (NTRS)

    Martin, Ivan (Inventor); Freed, Lisa E. (Inventor); Langer, Robert (Inventor); Vunjak-Novakovic, Gordana (Inventor)

    2003-01-01

    The present invention provides an improved method for expanding cells for use in tissue engineering. In particular the method provides specific biochemical factors to supplement cell culture medium during the expansion process in order to reproduce events occurring during embryonic development with the goal of regenerating tissue equivalents that resemble natural tissues both structurally and functionally. These specific biochemical factors improve proliferation of the cells and are capable of de-differentiation mature cells isolated from tissue so that the differentiation potential of the cells is preserved. The bioactive molecules also maintain the responsiveness of the cells to other bioactive molecules. Specifically, the invention provides methods for expanding chondrocytes in the presence of fibroblast growth factor 2 for use in regeneration of cartilage tissue.

  7. Fibroblast Growth Factor 21 As an Emerging Therapeutic Target for Type 2 Diabetes Mellitus.

    PubMed

    So, Wing Yan; Leung, Po Sing

    2016-07-01

    Fibroblast growth factor (FGF) 21 is a distinctive member of the FGF family that functions as an endocrine factor. It is expressed predominantly in the liver, but is also found in adipose tissue and the pancreas. Pharmacological studies have shown that FGF21 normalizes glucose and lipid homeostasis, thereby preventing the development of metabolic disorders, such as obesity and diabetes. Despite growing evidence for the therapeutic potential of FGF21, paradoxical increases of FGF21 in different disease conditions point to the existence of FGF21 resistance. In this review, we give a critical appraisal of recent advances in the understanding of the regulation of FGF21 production under various physiological conditions, its antidiabetic actions, and the clinical implications. We also discuss recent preclinical and clinical trials using engineered FGF21 analogs in the management of diabetes, as well as the potential side effects of FGF21 therapy. PMID:27031294

  8. Carbon nanoparticles downregulate expression of basic fibroblast growth factor in the heart during embryogenesis

    PubMed Central

    Wierzbicki, Mateusz; Sawosz, Ewa; Grodzik, Marta; Hotowy, Anna; Prasek, Marta; Jaworski, Sławomir; Sawosz, Filip; Chwalibog, André

    2013-01-01

    Carbon nanoparticles, with their high biocompatibility and low toxicity, have recently been considered for biomedical applications, including antiangiogenic therapy. Critical to normal development and tumor formation, angiogenesis is the process of forming capillary blood vessels from preexisting vessels. In the present study, we evaluated the effects of diamond and graphite nanoparticles on the development of chicken embryos, as well as vascularization of the chorioallantoic membrane and heart at the morphological and molecular level. Nanoparticles did not affect either body/heart weight or serum indices of the embryos’ health. However, vascularization of the heart and the density of branched vessels were significantly reduced after treatment with diamond nanoparticles and, to a lesser extent, graphite nanoparticles. Application of nanoparticles significantly downregulated gene and protein expression of the proangiogenic basic fibroblast growth factor, indicating that both diamond and graphite nanoparticles inhibit angiogenesis. PMID:24039425

  9. Alternative Splicing of Fibroblast Growth Factor Receptor IgIII Loops in Cancer

    PubMed Central

    Holzmann, Klaus; Grunt, Thomas; Heinzle, Christine; Sampl, Sandra; Steinhoff, Heinrich; Reichmann, Nicole; Kleiter, Miriam; Hauck, Marlene; Marian, Brigitte

    2012-01-01

    Alternative splicing of the IgIII loop of fibroblast growth factor receptors (FGFRs) 1–3 produces b- and c-variants of the receptors with distinctly different biological impact based on their distinct ligand-binding spectrum. Tissue-specific expression of these splice variants regulates interactions in embryonic development, tissue maintenance and repair, and cancer. Alterations in FGFR2 splicing are involved in epithelial mesenchymal transition that produces invasive, metastatic features during tumor progression. Recent research has elucidated regulatory factors that determine the splice choice both on the level of exogenous signaling events and on the RNA-protein interaction level. Moreover, methodology has been developed that will enable the in depth analysis of splicing events during tumorigenesis and provide further insight on the role of FGFR 1–3 IIIb and IIIc in the pathophysiology of various malignancies. This paper aims to summarize expression patterns in various tumor types and outlines possibilities for further analysis and application. PMID:22203889

  10. Stable Expression of Basic Fibroblast Growth Factor in Chloroplasts of Tobacco.

    PubMed

    Wang, Yun-Peng; Wei, Zheng-Yi; Zhong, Xiao-Fang; Lin, Chun-Jing; Cai, Yu-Hong; Ma, Jian; Zhang, Yu-Ying; Liu, Yan-Zhi; Xing, Shao-Chen

    2016-01-01

    Basic fibroblast growth factor (bFGF) is a multifunctional factor in acceleration of cell proliferation, differentiation and transference, and therefore widely used in clinical applications. In this study, expression vector pWX-Nt03 harboring a codon-optimized bFGF gene was constructed and introduced into the tobacco chloroplasts by particle bombardment. After four rounds of selection, bFGF was proved to integrate into the chloroplast genome of regenerated plants and two of four transgenic plants were confirmed to be homoplastomic by PCR and Southern hybridization. ELISA assay indicated that bFGF represented approximately 0.1% of total soluble protein in the leaves of transplastomic tobacco plants. This is the first report of bFGF expression via chloroplast transformation in model plant, providing an additional option for the production of chloroplast-produced therapeutic proteins. PMID:26703590

  11. Stable Expression of Basic Fibroblast Growth Factor in Chloroplasts of Tobacco

    PubMed Central

    Wang, Yun-Peng; Wei, Zheng-Yi; Zhong, Xiao-Fang; Lin, Chun-Jing; Cai, Yu-Hong; Ma, Jian; Zhang, Yu-Ying; Liu, Yan-Zhi; Xing, Shao-Chen

    2015-01-01

    Basic fibroblast growth factor (bFGF) is a multifunctional factor in acceleration of cell proliferation, differentiation and transference, and therefore widely used in clinical applications. In this study, expression vector pWX-Nt03 harboring a codon-optimized bFGF gene was constructed and introduced into the tobacco chloroplasts by particle bombardment. After four rounds of selection, bFGF was proved to integrate into the chloroplast genome of regenerated plants and two of four transgenic plants were confirmed to be homoplastomic by PCR and Southern hybridization. ELISA assay indicated that bFGF represented approximately 0.1% of total soluble protein in the leaves of transplastomic tobacco plants. This is the first report of bFGF expression via chloroplast transformation in model plant, providing an additional option for the production of chloroplast-produced therapeutic proteins. PMID:26703590

  12. Fibroblast growth factor receptor signaling as therapeutic targets in gastric cancer

    PubMed Central

    Yashiro, Masakazu; Matsuoka, Tasuku

    2016-01-01

    Fibroblast growth factor receptors (FGFRs) regulate a variety of cellular functions, from embryogenesis to adult tissue homeostasis. FGFR signaling also plays significant roles in the proliferation, invasion, and survival of several types of tumor cells. FGFR-induced alterations, including gene amplification, chromosomal translocation, and mutations, have been shown to be associated with the tumor initiation and progression of gastric cancer, especially in diffuse-type cancers. Therefore, the FGFR signaling pathway might be one of the therapeutic targets in gastric cancer. This review aims to provide an overview of the role of FGFR signaling in tumorigenesis, tumor progression, proliferation, and chemoresistance. We also discuss the accumulating evidence that demonstrates the effectiveness of using clinical therapeutic agents to inhibit FGFR signaling for the treatment of gastric cancer. PMID:26937130

  13. Apocynin Attenuates Cardiac Injury in Type 4 Cardiorenal Syndrome via Suppressing Cardiac Fibroblast Growth Factor-2 With Oxidative Stress Inhibition

    PubMed Central

    Liu, Yang; Liu, Yu; Liu, Xun; Chen, Jie; Zhang, Kun; Huang, Feifei; Wang, Jing-Feng; Tang, Wanchun; Huang, Hui

    2015-01-01

    Background Type 4 cardiorenal syndrome (CRS) refers to the cardiac injury induced by chronic kidney disease. We aimed to assess oxidative stress and cardiac injury in patients with type 4 CRS, determine whether the antioxidant apocynin attenuated cardiac injury in rats with type 4 CRS, and explore potential mechanisms. Methods and Results A cross-sectional study was conducted among patients with type 4 CRS (n=17) and controls (n=16). Compared with controls, patients with type 4 CRS showed elevated oxidative stress, which was significantly correlated with cardiac hypertrophy and decreased ejection fraction. In vivo study, male Sprague-Dawley rats underwent 5/6 subtotal nephrectomy and sham surgery, followed with apocynin or vehicle treatment for 8 weeks. Eight weeks after surgery, the 5/6 subtotal nephrectomy rats mimicked type 4 CRS, showing increased serum creatinine, cardiac hypertrophy and fibrosis, and decreased ejection fraction compared with sham-operated animals. Cardiac malondialdehyde, NADPH oxidase activity, fibroblast growth factor-2, and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation increased significantly in the 5/6 subtotal nephrectomy rats. These changes were significantly attenuated by apocynin. In vitro study showed that apocynin reduced angiotensin II–induced NADPH oxidase–dependent oxidative stress, upregulation of fibroblast growth factor-2 and fibrosis biomarkers, and ERK1/2 phosphorylation in cardiac fibroblasts. Importantly, the ERK1/2 inhibitor U0126 reduced the upregulation of fibroblast growth factor-2 and fibrosis biomarkers in angiotensin II–treated fibroblasts. Conclusions Oxidative stress is a candidate mediator for type 4 CRS. Apocynin attenuated cardiac injury in type 4 CRS rats via inhibiting NADPH oxidase–dependent oxidative stress-activated ERK1/2 pathway and subsequent fibroblast growth factor-2 upregulation. Our study added evidence to the beneficial effect of apocynin in type 4 CRS. PMID:26109504

  14. Evidence for the involvement of fibroblast growth factor 10 in lipofibroblast formation during embryonic lung development.

    PubMed

    Al Alam, Denise; El Agha, Elie; Sakurai, Reiko; Kheirollahi, Vahid; Moiseenko, Alena; Danopoulos, Soula; Shrestha, Amit; Schmoldt, Carole; Quantius, Jennifer; Herold, Susanne; Chao, Cho-Ming; Tiozzo, Caterina; De Langhe, Stijn; Plikus, Maksim V; Thornton, Matthew; Grubbs, Brendan; Minoo, Parviz; Rehan, Virender K; Bellusci, Saverio

    2015-12-01

    Lipid-containing alveolar interstitial fibroblasts (lipofibroblasts) are increasingly recognized as an important component of the epithelial stem cell niche in the rodent lung. Although lipofibroblasts were initially believed merely to assist type 2 alveolar epithelial cells in surfactant production during neonatal life, recent evidence suggests that these cells are indispensable for survival and growth of epithelial stem cells during adulthood. Despite increasing interest in lipofibroblast biology, little is known about their cellular origin or the molecular pathways controlling their formation during embryonic development. Here, we show that a population of lipid-droplet-containing stromal cells emerges in the developing mouse lung between E15.5 and E16.5. This is accompanied by significant upregulation, in the lung mesenchyme, of peroxisome proliferator-activated receptor gamma (master switch of lipogenesis), adipose differentiation-related protein (marker of mature lipofibroblasts) and fibroblast growth factor 10 (previously shown to identify a subpopulation of lipofibroblast progenitors). We also demonstrate that although only a subpopulation of total embryonic lipofibroblasts derives from Fgf10(+) progenitor cells, in vivo knockdown of Fgfr2b ligand activity and reduction in Fgf10 expression lead to global reduction in the expression levels of lipofibroblast markers at E18.5. Constitutive Fgfr1b knockouts and mutants with conditional partial inactivation of Fgfr2b in the lung mesenchyme reveal the involvement of both receptors in lipofibroblast formation and suggest a possible compensation between the two receptors. We also provide data from human fetal lungs to demonstrate the relevance of our discoveries to humans. Our results reveal an essential role for Fgf10 signaling in the formation of lipofibroblasts during late lung development. PMID:26511927

  15. [Improvement of yield and purity of human fibroblast growth factor-21].

    PubMed

    Yu, Dan; Ye, Xianlong; Ren, Guiping; Xu, Pengfei; Li, Shujie; Niu, Zeshan; Li, Deshan

    2014-04-01

    Fibroblast growth factor -21 (FGF-21) is a recently discovered metabolic regulation factor, regulating glucose and lipid metabolism and increasing insulin sensitivity. FGF-21 is expected to be a potential anti-diabetic drug. Expression of FGF-21 as inclusion bodies has advantages for high yield and purity, but the bioactivity of the protein is almost totally lost after denature and renature. That is why FGF-21 is currently expressed in soluble form. As a result, the yield is considerably low. In this study, we used SUMO vector to express SUMO-human FGF-21 (SUMO-hFGF-21) in form of inclusion body. We optimized the culture conditions to increase the yield of the bioactive human fibroblast growth factor-21. We applied the hollow fiber membrane filtration column to enrich the bacteria, wash, denature and renature inclusion bodies. After affinity and gel filtration chromatography, we examined the hypoglycemic activity of FGF-21 by the glucose uptake assay in HepG2 cells. We also detected the blood glucose concentration of type 2 diabetic db/db model mice after short or long-term treatment. The results show that the yield of ihFGF-21 was 4 times higher than that of shFGF-21. The yield was 20 mg/L for ihFGF-21 vs. 6 mg/L for shFGF-21. The purity of ihFGF-21 was above 95%, while shFGF-21 was 90%. Compared with the traditional method of extracting inclusion bodies, the production cycle was about three times shortened by application of hollow fiber membrane filtration column technology, but the bioactivity did not significantly differ. This method provides an efficient and cost-effective strategy to the pilot and industrial production of hFGF-21. PMID:25195255

  16. Cell Type-Dependent Nonspecific Fibroblast Growth Factor Signaling in Apert Syndrome.

    PubMed

    Yeh, Erika; Atique, Rodrigo; Fanganiello, Roberto Dalto; Sunaga, Daniele Yumi; Ishiy, Felipe Augusto André; Passos-Bueno, Maria Rita

    2016-08-15

    Apert Syndrome (AS) is one of the most severe forms of craniosynostosis. It is caused by gain-of-function mutations in the receptor fibroblast growth factor receptor 2 (FGFR2), which leads to ligand-receptor promiscuity. Here, we aimed to better understand the behavior of mesenchymal stem cells (MSCs) and of fibroblastoid cells, cellular populations that are part of the suture complex, when stimulated with different fibroblast growth factors (FGFs). We also aimed to verify whether FGFR2 specificity loss due to AS mutations would change their signaling behavior. We tested this hypothesis through cell proliferation and differentiation assays and through gene expression profiling. We found that FGF19 and FGF10 increase proliferation of fibroblastoid cells harboring the FGFR2 p.S252W mutation, but not of mutant MSCs. FGF19 and FGF10 were associated with different expression profiles in p.S252W cells. Further, in accordance to our gene expression microarray data, FGF19 decreases bone differentiation rate of mutant fibroblastoid cells and increases bone differentiation rate of MSCs. This effect in osteogenesis appears to be mediated by BMP signaling. The present data indicate that non-natural FGFR2 ligands, such as FGF10 and FGF19, are important factors in the pathophysiology of AS. Further research is needed to determine the role of modulation of MSC proliferation or use of FGF19 or anti-BMP2 as inhibitors of osteogenesis in AS subjects' cells, and whether these findings can be used in the clinical management of AS. PMID:27339175

  17. Lactobacillus sakei lipoteichoic acid inhibits MMP-1 induced by UVA in normal dermal fibroblasts of human.

    PubMed

    You, Ga-Eun; Jung, Bong-Jun; Kim, Hye-Rim; Kim, Han-Geun; Kim, Tae-Rahk; Chung, Dae-Kyun

    2013-10-28

    Human skin is continuously exposed to ultraviolet (UV)-induced photoaging. UVA increases the activity of MMP-1 in dermal fibroblasts through mitogen-activated protein kinase (MAPK), p38, signaling. The irradiation of keratinocytes by UVA results in the secretion of the inflammatory cytokine, tumor necrosis factor-α (TNF-α), and the stimulation of MMP-1 in normal human dermal fibroblasts (NHDFs). Lipoteichoic acid (LTA) is a component of the cell wall of gram-positive Lactobacillus spp. of bacteria. LTA is well known as an anti-inflammation molecule. LTA of the bacterium Lactobacillus plantarum has an anti-photoaging effect, but the potential anti-photoaging effect of the other bacteria has not been examined to date. The current study showed that L. sakei LTA (sLTA) has an immune modulating effect in human monocyte cells. Our object was whether inhibitory effects of sLTA on MMP-1 are caused from reducing the MAPK signal in NHDFs. It inhibits MMP-1 and MAPK signaling induced by UVA in NHDFs. We also confirmed effects of sLTA suppressing TNF-α inducing MMP-1 in NHDFs. PMID:23851272

  18. Fibroblasts behavior after N-acetylcysteine and amino acids exposure: extracellular matrix gene expression.

    PubMed

    Avantaggiato, Anna; Palmieri, Annalisa; Bertuzzi, Gianluigi; Carinci, Francesco

    2014-06-01

    Reactive oxygen species (ROS) are chemically reactive molecules with impaired electrons that make them unstable and able to react easily with a great variety of molecules. The main targets of ROS are DNA, proteins, and membrane phospholipids. In the skin, ROS are able to affect the production of collagen and elastin, the main components of the extracellular matrix (ECM). This action contributes to the skin's aging. N-Acetylcysteine (NAC) is an acetylated cysteine residue with excellent anti-oxidant activity that boosts glutathione (GSH) levels. This study evaluates the effect of a solution of NAC and amino acids, which is used in aesthetic medicine as an intra-dermal injective treatment, on fibroblast behavior. To this aim, the expression levels of some ECM-related genes (HAS1, HYAL1 ELN, ELANE, MMP2, MMP3, MMP13, COL1A1, COL3A1) were analyzed on cultured dermal fibroblasts using real-time reverse transcription polymerase chain reaction (RT-PCR). All but two collagen genes were up-regulated after 24 hr of treatment. PMID:24438160

  19. Retinoic acid treatment of fibroblasts causes a rapid decrease in ( sup 3 H)inositol uptake

    SciTech Connect

    Sinha, R.; Creek, K.E.; Silverman-Jones, C.; de Luca, L.M. )

    1989-04-01

    NIH 3T3 fibroblasts treated with all-trans-retinoic acid (RA) showed a dramatic decrease in the uptake of ({sup 3}H)inositol compared to solvent-treated controls. The onset of RA-induced inhibition of ({sup 3}H)inositol uptake was rapid with a 10-15% decrease occurring after 2-3 h of RA exposure and 60-70% reduction after 16 h of RA treatment. A progressive dose-dependent decrease in inositol uptake was found as the concentration of RA increased from 10{sup {minus}8} to 10{sup {minus}5} M and the effect was fully reversible within 48 h after RA removal. RA inhibition of inositol uptake was also observed in 3T3-Swiss and Balb/3T3 cells but not in two virally transformed 3T3 cell lines. Phlorizin, amiloride, and monensin inhibited inositol uptake by 66, 74, and 58%, respectively, and this inhibition was additive when the cells were treated with RA as well as these inhibitors. A decreased incorporation of ({sup 3}H)inositol into polyphosphoinositides was also observed in RA-treated cells but not to the same extent as for ({sup 3}H)inositol uptake. In conclusion, RA treatment of 3T3 fibroblasts decreases the uptake of ({sup 3}H)inositol by up to 70% within 8 to 10 h at near physiological concentrations in a reversible and specific manner.

  20. Science of Hyaluronic Acid Beyond Filling: Fibroblasts and Their Response to the Extracellular Matrix.

    PubMed

    Landau, Marina; Fagien, Steven

    2015-11-01

    Loss of viscoelasticity is one of the primarily signs of skin aging, followed by appearance of visible wrinkles. Hyaluronic acid (HA)-based fillers are widely used to fill wrinkles and compensate for volume loss. Recent clinical observations demonstrate persistence of the filling effect longer than the biological availability of the filler. Stimulation of new collagen by cross-linked HA and up-regulation of elastin have been suggested as possible explanation to this observation and have been supported experimentally. Cross-linked HA substitutes for fragmented collagen in restoring extracellular matrix required for normal activity of fibroblasts, such as collagen and elastin production. To restore extracellular matrix efficiently, serial monthly treatments are required. Boosting of facial and nonfacial skin through fibroblast activation is a new indication for HA-based products. Injectable HA has also been recently registered in Europe as agents specific for the improvement of skin quality (Restylane Skinboosters). Further explanation of the possible mechanisms supported by long-term clinical examples is presented herein. PMID:26441098

  1. Asiatic Acid Isolated From Centella Asiatica Inhibits TGF-β1-induced Collagen Expression in Human Keloid Fibroblasts via PPAR-γ Activation

    PubMed Central

    Bian, Difei; Zhang, Jizhou; Wu, Xin; Dou, Yannong; Yang, Yan; Tan, Qian; Xia, Yufeng; Gong, Zhunan; Dai, Yue

    2013-01-01

    Keloids are fibroproliferative disorders characterized by exuberant extracellular matrix deposition and transforming growth factor (TGF)-β/Smad pathway plays a pivotal role in keloid pathogenesis. Centella asiatica extract has been applied in scar management for ages. As one of its major components, asiatic acid (AA) has been recently reported to inhibit liver fibrosis by blocking TGF-β/Smad pathway. However, its effect on keloid remains unknown. In order to investigate the effects of AA on cell proliferation, invasion and collagen synthesis, normal and keloid fibroblasts were exposed to TGF-β1 with or without AA. Relevant experiments including 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, 5-ethynyl-2-deoxyuridine (EdU) incorporation assay, Transwell invasion assay, enzyme-linked immunosorbent assay, Western blot, quantitative polymerase chain reaction and RNA interference assay were conducted. As a result, keloid fibroblasts showed higher responsiveness to TGF-β1 stimulation than normal fibroblasts in terms of invasion and collagen synthesis. AA could suppress TGF-β1-induced expression of collagen type I, inhibit Smad 2/3 phosphorylation and plasminogen activator inhibitor-1 (PAI-1) expression, while elevate Smad 7 protein level. Noteworthy, the effects of AA on keloid fibroblasts could be abrogated by PPAR-γ antagonist GW9662 and by silencing of PPAR-γ. The present study demonstrated that AA inhibited TGF-β1-induced collagen and PAI-1 expression in keloid fibroblasts through PPAR-γ activation, which suggested that AA was one of the active constituents of C. asiatica responsible for keloid management, and could be included in the arsenal for combating against keloid. PMID:24250248

  2. Vitamin D3 supplementation increases fibroblast growth factor-23 in HIV-infected youth treated with tenofovir disoproxil fumarate

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tenofovir (TDF) is associated with phosphaturia and elevated 1,25 dihydroxy vitamin D (1,25-OH(2)D). Fibroblast growth factor-23 causes phosphaturia and increases in response to elevated 1,25-OH(2)D. Vitamin D binding proetin (VDBP) binds to 1,25-OH(2)D, decreasing biologic activity, and is elevated...

  3. Vitamin D Supplementation increases fibroblast growth factor-23 in HIV-infected youth treated with tenofovir disoproxil fumarate

    Technology Transfer Automated Retrieval System (TEKTRAN)

    BACKGROUND: Tenofovir (TDF) is associated with phosphaturia and elevated 1,25 dihydroxy vitamin D (1,25-OH(2)D). Fibroblast growth factor 23 (FGF23) causes phosphaturia and increases in response to elevated 1,25-OH(2)D. Vitamin D binding protein (VDBP) binds to 1,25-OH(2)D, decreasing its biologic...

  4. All-trans retinoic acid and rapamycin normalize Hutchinson Gilford progeria fibroblast phenotype

    PubMed Central

    Pellegrini, Camilla; Columbaro, Marta; Capanni, Cristina; D'Apice, Maria Rosaria; Cavallo, Carola; Murdocca, Michela; Lattanzi, Giovanna; Squarzoni, Stefano

    2015-01-01

    Hutchinson Gilford progeria syndrome is a fatal disorder characterized by accelerated aging, bone resorption and atherosclerosis, caused by a LMNA mutation which produces progerin, a mutant lamin A precursor. Progeria cells display progerin and prelamin A nuclear accumulation, altered histone methylation pattern, heterochromatin loss, increased DNA damage and cell cycle alterations. Since the LMNA promoter contains a retinoic acid responsive element, we investigated if all-trans retinoic acid administration could lower progerin levels in cultured fibroblasts. We also evaluated the effect of associating rapamycin, which induces autophagic degradation of progerin and prelamin A. We demonstrate that all-trans retinoic acid acts synergistically with low-dosage rapamycin reducing progerin and prelamin A, via transcriptional downregulation associated with protein degradation, and increasing the lamin A to progerin ratio. These effects rescue cell dynamics and cellular proliferation through recovery of DNA damage response factor PARP1 and chromatin-associated nuclear envelope proteins LAP2α and BAF. The combined all-trans retinoic acid-rapamycin treatment is dramatically efficient, highly reproducible, represents a promising new approach in Hutchinson-Gilford Progeria therapy and deserves investigation in ageing-associated disorders. PMID:26359359

  5. All-trans retinoic acid and rapamycin normalize Hutchinson Gilford progeria fibroblast phenotype.

    PubMed

    Pellegrini, Camilla; Columbaro, Marta; Capanni, Cristina; D'Apice, Maria Rosaria; Cavallo, Carola; Murdocca, Michela; Lattanzi, Giovanna; Squarzoni, Stefano

    2015-10-01

    Hutchinson Gilford progeria syndrome is a fatal disorder characterized by accelerated aging, bone resorption and atherosclerosis, caused by a LMNA mutation which produces progerin, a mutant lamin A precursor. Progeria cells display progerin and prelamin A nuclear accumulation, altered histone methylation pattern, heterochromatin loss, increased DNA damage and cell cycle alterations. Since the LMNA promoter contains a retinoic acid responsive element, we investigated if all-trans retinoic acid administration could lower progerin levels in cultured fibroblasts. We also evaluated the effect of associating rapamycin, which induces autophagic degradation of progerin and prelamin A. We demonstrate that all-trans retinoic acid acts synergistically with low-dosage rapamycin reducing progerin and prelamin A, via transcriptional downregulation associated with protein degradation, and increasing the lamin A to progerin ratio. These effects rescue cell dynamics and cellular proliferation through recovery of DNA damage response factor PARP1 and chromatin-associated nuclear envelope proteins LAP2α and BAF. The combined all-trans retinoic acid-rapamycin treatment is dramatically efficient, highly reproducible, represents a promising new approach in Hutchinson-Gilford Progeria therapy and deserves investigation in ageing-associated disorders. PMID:26359359

  6. Pharmacological inhibition of fibroblast growth factor (FGF) receptor signaling ameliorates FGF23-mediated hypophosphatemic rickets.

    PubMed

    Wöhrle, Simon; Henninger, Christine; Bonny, Olivier; Thuery, Anne; Beluch, Noemie; Hynes, Nancy E; Guagnano, Vito; Sellers, William R; Hofmann, Francesco; Kneissel, Michaela; Graus Porta, Diana

    2013-04-01

    Fibroblast growth factor 23 (FGF23) is a circulating factor secreted by osteocytes that is essential for phosphate homeostasis. In kidney proximal tubular cells FGF23 inhibits phosphate reabsorption and leads to decreased synthesis and enhanced catabolism of 1,25-dihydroxyvitamin D3 (1,25[OH]2 D3 ). Excess levels of FGF23 cause renal phosphate wasting and suppression of circulating 1,25(OH)2 D3 levels and are associated with several hereditary hypophosphatemic disorders with skeletal abnormalities, including X-linked hypophosphatemic rickets (XLH) and autosomal recessive hypophosphatemic rickets (ARHR). Currently, therapeutic approaches to these diseases are limited to treatment with activated vitamin D analogues and phosphate supplementation, often merely resulting in partial correction of the skeletal aberrations. In this study, we evaluate the use of FGFR inhibitors for the treatment of FGF23-mediated hypophosphatemic disorders using NVP-BGJ398, a novel selective, pan-specific FGFR inhibitor currently in Phase I clinical trials for cancer therapy. In two different hypophosphatemic mouse models, Hyp and Dmp1-null mice, resembling the human diseases XLH and ARHR, we find that pharmacological inhibition of FGFRs efficiently abrogates aberrant FGF23 signaling and normalizes the hypophosphatemic and hypocalcemic conditions of these mice. Correspondingly, long-term FGFR inhibition in Hyp mice leads to enhanced bone growth, increased mineralization, and reorganization of the disturbed growth plate structure. We therefore propose NVP-BGJ398 treatment as a novel approach for the therapy of FGF23-mediated hypophosphatemic diseases. PMID:23129509

  7. Fibroblast growth factor-2 promotes keratan sulfate proteoglycan expression by keratocytes in vitro

    NASA Technical Reports Server (NTRS)

    Long, C. J.; Roth, M. R.; Tasheva, E. S.; Funderburgh, M.; Smit, R.; Conrad, G. W.; Funderburgh, J. L.

    2000-01-01

    Keratocytes of the corneal stroma produce a specialized extracellular matrix responsible for corneal transparency. Corneal keratan sulfate proteoglycans (KSPG) are unique products of keratocytes that are down-regulated in corneal wounds and in vitro. This study used cultures of primary bovine keratocytes to define factors affecting KSPG expression in vitro. KSPG metabolically labeled with [(35)S]sulfate decreased during the initial 2-4 days of culture in quiescent cultures with low serum concentrations (0.1%). Addition of fetal bovine serum, fibroblast growth factor-2 (FGF-2), transforming growth factor beta, or platelet derived growth factor all stimulated cell division, but only FGF-2 stimulated KSPG secretion. Combined with serum, FGF-2 also prevented serum-induced KSPG down-regulation. KSPG secretion was lost during serial subculture with or without FGF-2. Expression of KSPG core proteins (lumican, mimecan, and keratocan) was stimulated by FGF-2, and steady state mRNA pools for these proteins, particularly keratocan, were significantly increased by FGF-2 treatment. KSPG expression therefore is supported by exogenous FGF-2 and eliminated by subculture of the cells in presence of serum. FGF-2 stimulates KSPG core protein expression primarily through an increase in mRNA pools.

  8. Fibroblast growth factor receptor signaling in hereditary and neoplastic disease: biologic and clinical implications.

    PubMed

    Helsten, Teresa; Schwaederle, Maria; Kurzrock, Razelle

    2015-09-01

    Fibroblast growth factors (FGFs) and their receptors (FGFRs) are transmembrane growth factor receptors with wide tissue distribution. FGF/FGFR signaling is involved in neoplastic behavior and also development, differentiation, growth, and survival. FGFR germline mutations (activating) can cause skeletal disorders, primarily dwarfism (generally mutations in FGFR3), and craniofacial malformation syndromes (usually mutations in FGFR1 and FGFR2); intriguingly, some of these activating FGFR mutations are also seen in human cancers. FGF/FGFR aberrations reported in cancers are mainly thought to be gain-of-function changes, and several cancers have high frequencies of FGFR alterations, including breast, bladder, or squamous cell carcinomas (lung and head and neck). FGF ligand aberrations (predominantly gene amplifications) are also frequently seen in cancers, in contrast to hereditary syndromes. There are several pharmacologic agents that have been or are being developed for inhibition of FGFR/FGF signaling. These include both highly selective inhibitors as well as multi-kinase inhibitors. Of note, only four agents (ponatinib, pazopanib, regorafenib, and recently lenvatinib) are FDA-approved for use in cancer, although the approval was not based on their activity against FGFR. Perturbations in the FGFR/FGF signaling are present in both inherited and malignant diseases. The development of potent inhibitors targeting FGF/FGFR may provide new tools against disorders caused by FGF/FGFR alterations. PMID:26224133

  9. Role of fibroblast growth factor type 1 and 2 in carbon tetrachloride-induced hepatic injury and fibrogenesis.

    PubMed

    Yu, Chundong; Wang, Fen; Jin, Chengliu; Huang, Xinqiang; Miller, David L; Basilico, Claudio; McKeehan, Wallace L

    2003-10-01

    Genomic ablation of hepatocyte-specific fibroblast growth factor receptor (FGFR)4 in mice revealed a role of FGF signaling in cholesterol and bile acid metabolism and hepatolobular restoration in response to injury without effect on liver development or hepatocyte proliferation. Although the potential role of all 23 FGF polypeptides in the liver is still unclear, the most widely studied prototypes, FGF1 and FGF2, are present and have been implicated in liver cell growth and function in vitro. To determine whether FGF1 and FGF2 play a role in response to injury and fibrosis, we examined the impact of both acute and chronic exposure to carbon tetrachloride (CCl(4)) in the livers of FGF1- and FGF2-deficient mice. After acute CCl(4) exposure, FGF1(-/-)FGF2(-/-) mice exhibited an accelerated release of serum alanine aminotransferase similar to FGFR4 deficiency, but no effect on overall hepatolobular restoration or bile acid metabolism. FGF1(-/-)FGF2(-/-) mice exhibited a normal increase in alpha-smooth muscle actin and desmin associated with activation and migration of hepatic stellate cells to damage, but a reduced level of hepatic stellate cell-derived matrix collagen alpha1(I) synthesis. Liver fibrosis resulting from chronic CCl(4) exposure was markedly decreased in the livers of FGF1/FGF2-deficient mice. These results suggest an agonist role for FGF1 and FGF2 in specifically insult-induced liver matrix deposition and hepatic fibrogenesis and a potential target for the prevention of hepatic fibrosis. PMID:14507672

  10. Ca2+- and PKC-dependent stimulation of PGE2 synthesis by deoxycholic acid in human colonic fibroblasts.

    PubMed

    Zhu, Yingting; Hua, Ping; Rafiq, Shazia; Waffner, Eric J; Duffey, Michael E; Lance, Peter

    2002-09-01

    We investigated prostanoid biogenesis by human colonic fibroblasts (CCD-18Co cells and nine primary fibroblast cultures) exposed to a primary (cholic, CA) or a secondary (deoxycholic, DCA) bile acid. Basal PGE2 levels in CCD-18Co cultures and fibroblast strains initiated from normal and adenocarcinomatous colon, respectively, were 1.7 +/- 0.3, 4.0 +/- 2.0, and 15.0 +/- 4.8 ng/mg protein. Peak levels 24 h after exposure to DCA (300 microM) rose, respectively, seven-, six- and sevenfold, but CA elicited no such responses. Increases in PGE2 synthesis were preceded by sequential increases in PGH synthase-2 mRNA and protein expression and were fully prevented by a nonselective (indomethacin) or a selective (celecoxib) nonsteroidal anti-inflammatory drug. DCA, but not CA, caused abrupt, transient increases in fibroblast intracellular Ca2+ concentration ([Ca2+]i) approximately 1 min after exposure. Increased [Ca2+]i was required for DCA-mediated induction of PGE2 synthesis, and protein kinase C was a further essential component of this signaling pathway. Colonic fibroblasts may be a major target for prostanoid biogenesis induced by fecal bile acids and, potentially, other noxious actions of these agents. PMID:12181161