Cloning and high level expression of a synthetic gene for human basic fibroblast growth factor.

A gene encoding human basic fibroblast growth factor has been chemically synthesized, cloned and expressed in Escherichia coli as a biologically active protein. The 465 bp gene was assembled by enzymatic ligation of 6 pairs of oligonucleotides and cloned in the expression vector pLCII downstream from the strong PL promoter. This promoter directed the synthesis of a fusion protein between a 31 amino acids fragment of the lambda phage cII protein and bFGF. A four amino acid recognition sequence for the site-specific protease fXa was introduced in the plasmid construct and this allowed cleavage of the fusion protein at the boundary between cII and bFGF. bFGF was purified close to homogeneity using a Heparin-Sepharose column and Mono S cation exchange chromatography. The use of the pLCII expression system resulted in the accumulation of 20 to 25 mg of purified bFGF per l of bacterial culture. The recombinant bFGF was mitogenic for mouse 3T3 fibroblasts and the dose-response curve was similar to the one for native bFGF.     

Differential gene expression of human keratinocyte HaCaT cells induced by fibroblast growth factor 10 treatment

Fibroblast growth factor 10 (FGF10) has multiple biological activities involved in angiogenesis, mitogenesis, cellular differentiation, development, and tissue injury repair. Our previous studies revealed that treatment of FGF10 remarkably stimulated HaCaT cell proliferation and abbreviated cell apoptosis. However, the molecular mechanisms remain largely unknown. The aim of this study was to investigate FGF10-induced modifications in gene expression in the HaCaT cells by using the cDNA microarray technique. The microarray data showed that FGF10 modified the expression of 2117 genes, 861 being up-regulated and 1256 down-regulated, using a threshold of twofold. Eight of nine candidate genes, validated by real-time quantitative polymerase chain reaction (qPCR), were correlated well with the array data. The GenMAPP and MappFinder software packages were further used for pathway analysis of these significantly altered genes. In support of multiple biological functions for FGF10, several gene pathways were found to be involved in processes of cell cycle, DNA repair, apoptosis, development, and wound healing. These data also provide a basis to further investigation of FGF10 molecular mechanisms.     

Cloning and expression of cDNA encoding human basic fibroblast growth factor

A cDNA encoding human basic fibroblast growth factor (bFGF) was isolated from a human foreskin fibroblast cDNA library. The cDNA, 4 kilobases in size, had a coding sequence, 5' and 3' untranslated regions, and a poly(A) chain. Isolation of additional cDNA clones that had a short 3' untranslated region suggested the presence of multiple mRNA forms. By Northern blot analysis, at least five bFGF mRNA species were detected in cultured fibroblast cells. Transfection of the cDNA to COS cells resulted in the detection of mitogenic activity in the culture medium of the transfected cells, suggesting that a part of the synthesized protein might be secreted from cells despite its unusual short signal sequence.     

Effects of 3-D clino-rotation on gene expression in human fibroblast cells

Continuous variation in the direction of the gravity vector leads to various cellular responses including modulation of gene expression. Complementary DNA (cDNA) array analyses are available to observe the variation of gene expression under different conditions. In this study, expression levels of 588 representative genes were compared using the Atlas human cDNA expression array in human fibroblast cells with and without 3-dimensional (3-D) clinostat. Five upregulated and 8 downregulated genes were detected. Among these genes, upregulation of XRCC1, and downregulation of ERB-B2 and p21(Cip1/Waf1) were confirmed by RT-PCR. These results suggested that the gene expression levels of XRCC1, ERB-B2 and p21(Cip1/Waf1) were modulated by vector-averaged microgravity induced by 3-D clinostat in human fibroblast cells. Our findings may be a basis for the biological study of 3-D culture systems.     

The effect of recombinant human basic fibroblast growth factor rhfgf-2 on human osteoblast in growth and phenotype expression

This paper describes the studies of human recombinant basic fibroblast growth factor (rhFGF-2) for its effects on human osteoblast growth and phenotype expression. During a 24-h period of treatment, rhFGF-2 highly stimulated DNA synthesis in a dose-related fashion with a maximum stimulation of 150% for 1 ng/ml. On the other hand, rhFGF-2 decreases alkaline phosphatase activity, synthesis of type I collagen, and cumulative amount of osteocalcin. Moreover, rhFGF-2 provoked a threefold increase in the amount of intracellular cAMP. Scatchard plots show the presence of two classes of [¹²⁵I] rhFGF-2 receptors. This data suggests that rhFGF-2 which stimulate cell replication may act indirectly as an anabolic agent and stimulate some of the phenotypic expression markers.     

Chemical synthesis of a human fibroblast interferon gene and its expression in Escherichia coli

Using the solid phase phosphotriester method, a gene coding for human fibroblast interferon consisting of 166 amino acid residues was chemically synthesized. The gene obtained by ligation of 61 synthesized oligodeoxyribonucleotide fragments was inserted into the downstream of tryptophan promoter, and expressed in E. coli. Lysate of this E. coli showed the antiviral activity which was specifically neutralized by anti-fibroblast interferon antibody. No particular advantage was observed in the expression efficiency by the synthetic gene over that by the native gene.     

High-level expression of human acidic fibroblast growth factor and basic fibroblast growth factor in silkworm (Bombyx mori L.) using recombinant baculovirus

A hybrid of Autographa californica nuclear polyhedrosis virus and Bombyx mori nuclear polyhedrosis virus, which is infectious to both Spodoptera frugiperda and Bombyx mori, was prepared in our previous study. Two recombinant hybrid baculoviruses, carrying cDNAs of human acidic and basic fibroblast growth factors, respectively, were successfully constructed in this study, for the large-scale production of human aFGF and bFGF using silkworm as host. These recombinant viruses were used to inoculate silkworm larvae. After the infection, the recombinant proteins were not found in the hemolymph. Such nonsecretion from cells has also been observed in the established insect cell lines, Sf21 and Tn-5. Tissue distribution analysis indicated that the expressed products were mainly located in fat body and the production of the recombinant aFGF and bFGF was maximal at around 80 h postinfection. Therefore, silkworm larvae infected with recombinant viruses were dissected and fat bodies were collected for the purification of recombinant aFGF and bFGF. The expression levels in both cases were estimated to be as high as approximately 600-700 microg per larva. Furthermore, the recombinant proteins were characterized and their biological activities were evaluated by in vitro bioassay using cell culture.     

The period length of fibroblast circadian gene expression varies widely among human individuals

Mammalian circadian behavior is governed by a central clock in the suprachiasmatic nucleus of the brain hypothalamus, and its intrinsic period length is believed to affect the phase of daily activities. Measurement of this period length, normally accomplished by prolonged subject observation, is difficult and costly in humans. Because a circadian clock similar to that of the suprachiasmatic nucleus is present in most cell types, we were able to engineer a lentiviral circadian reporter that permits characterization of circadian rhythms in single skin biopsies. Using it, we have determined the period lengths of 19 human individuals. The average value from all subjects, 24.5 h, closely matches average values for human circadian physiology obtained in studies in which circadian period was assessed in the absence of the confounding effects of light input and sleep–wake cycle feedback. Nevertheless, the distribution of period lengths measured from biopsies from different individuals was wider than those reported for circadian physiology. A similar trend was observed when comparing wheel-running behavior with fibroblast period length in mouse strains containing circadian gene disruptions. In mice, inter-individual differences in fibroblast period length correlated with the period of running-wheel activity; in humans, fibroblasts from different individuals showed widely variant circadian periods. Given its robustness, the presented procedure should permit quantitative trait mapping of human period length.     

Transcriptional regulation of basic fibroblast growth factor gene expression in capillary endothelial cells.

The growth of capillary endothelial cells (BCE) is an important regulatory step in the formation of capillary blood vessels. In vivo, the proliferation of these cells is stringently controlled. In vitro they can be stimulated by polypeptide growth factors, such as acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF). Since bFGF is synthesized and stored by vascular endothelial cells, this mitogen may play an important role in an autocrine growth regulation during angiogenesis. Here, evidence is presented for induction of the mRNA of bFGF by bFGF itself. A similar increase of bFGF mRNA was observed in response to thrombin and after treatment with phorbol ester. These results suggest that an autocrine loop may exist that may serve to modulate the mitogenic response in BCE under various physiological conditions, (e.g., wound healing and new capillary formation).     

Effects of collagen density on cardiac fibroblast behavior and gene expression

Interactions between cells and the extracellular matrix (ECM) play essential roles in modulating cell behavior during development and disease. The myocardial ECM is composed predominantly of interstitial collagen type I and type III. The composition, organization, and accumulation of these collagens are altered concurrent with cardiovascular development and disease. Changes in these parameters are thought to play significant roles in myocardial function. While a number of studies have examined how changes in the ECM affect myocardial function as a whole, much less is known regarding the response at the cellular level to changes in the collagenous ECM. Experiments were carried out to determine the effects of alterations in collagen density and ECM stiffness on the behavior of isolated heart fibroblasts. In vitro bioassays were performed to measure the effects of changes in collagen concentration (0.75-1.25 mg/ml) on adhesion, migration, spreading, and gene expression by heart fibroblasts. Increased density of collagen in 3-dimensional gels resulted in more efficient adhesion, spreading, and migration by heart fibroblasts. These experiments indicated that the density of the collagen matrix has a significant impact on fibroblast function. These studies begin to elucidate the effects of ECM density at the cellular level in the myocardium.     

Effects of interleukin-33 on cardiac fibroblast gene expression and activity

Interleukin-33 (IL-33) is a recently described member of the interleukin-1 (IL-1) family. It is produced by diverse cell types in response to a variety of stresses including hemorrhage and increased mechanical load. Though only relatively recently discovered, IL-33 has been shown to participate in several pathological processes including promoting type 2 T helper cell-associated autoimmune diseases. In contrast, IL-33 has been also found to have protective effects in cardiovascular diseases. Recent studies have illustrated that IL-33 attenuates cardiac fibrosis induced by increased cardiovascular load in mice (transaortic constriction). Since cardiac fibrosis is largely dependent on increased production of extracellular matrix by cardiac fibroblasts, we hypothesized that IL-33 directly inhibits pro-fibrotic activities of these cells. Experiments have been carried out with isolated rat cardiac fibroblasts to evaluate the effects of IL-33 on the modulation of cardiac fibroblast gene expression and function to test this hypothesis. The expression of the IL-33 receptor, interleukin-1 receptor-like 1 (ST2), was detected at the mRNA and protein levels in isolated adult rat cardiac fibroblasts. Subsequently, the effects of IL-33 treatment (0-100 ng/ml) on the expression of extracellular matrix proteins and pro-inflammatory cytokines/chemokines were examined as well as the effects on rat cardiac fibroblast activities including proliferation, collagen gel contraction and migration. While IL-33 did not directly inhibit collagen I and collagen III production, it yielded a dose-dependent increase in the expression of interleukin-6 and monocyte chemotactic protein-1. Treatment of rat cardiac fibroblasts with IL-33 also impaired the migratory activity of these cells. Further experiments illustrated that IL-33 rapidly activated multiple signaling pathways including extracellular signal-regulated kinases, p38 mitogen-activated protein kinase, c-Jun N-terminal kinases and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) in a dose-dependent manner. Experiments were carried out with pharmacological inhibitors to determine the role of specific signaling pathways in the response of fibroblasts to IL-33. These experiments illustrated that the activation of p38 mitogen-activated protein kinase and extracellular signal-regulated kinases are critical to the increased production of interleukin-6 and monocyte chemotactic protein-1 in response to IL-33. These studies suggest that IL-33 has an important role in the modulation of fibroblast function and gene expression. Surprisingly, IL-33 had no effect on the expression of genes encoding extracellular matrix components or on proliferation, markers typical of fibrosis. The major effects of IL-33 detected in these studies included inhibition of cell migration and activation of cytokine/chemokine expression. The previously reported inhibition of cardiac fibrosis may include more complicated mechanisms that involve other cardiac cell types. Future studies aimed at determining the effects of IL-33 on other cardiac cell types are warranted.     

Effects of interleukin-18 on cardiac fibroblast function and gene expression

Fibroblasts are the primary cell type responsible for synthesis and remodeling of the extracellular matrix in the heart. A number of factors including growth factors, hormones and mechanical forces have been identified that modulate the production of extracellular matrix by cardiac fibroblasts. Inflammatory mediators including pro-inflammatory cytokines and chemokines also impact fibrosis of the heart. Recent studies have illustrated that interleukin-18 promotes a pro-fibrotic response in cardiac fibroblasts; however the effects of this cytokine on other aspects of fibroblast function have not been examined. While fibroblasts have long been known for their role in production and remodeling of the extracellular matrix, other functions of these cells are only now beginning to be appreciated. We hypothesize that exposure to interleukin-18 will stimulate other aspects of fibroblast behavior important in myocardial remodeling including proliferation, migration and collagen reorganization. Fibroblasts were isolated from adult male rat hearts and bioassays performed to determine the effects of interleukin-18 on fibroblast function. Treatment of fibroblasts with interleukin-18 (1-100ng/ml) resulted in increased production of extracellular matrix components and remodeling or contraction of three-dimensional collagen scaffolds by these cells. Furthermore, exposure to interleukin-18 stimulated fibroblast migration and proliferation. Treatment of heart fibroblasts with interleukin-18 resulted in the rapid activation of the c-Jun N-terminal kinase (JNK) and phosphoinositide 3-kinase (PI3-kinase) pathways. Studies with pharmacological inhibitors illustrated that activation of these pathways is critical to interleukin-18 mediated alterations in fibroblast function. These studies illustrate that interleukin-18 plays a role in modulation of cardiac fibroblast function and may be an important component of the inflammation-fibrosis cascade during pathological myocardial remodeling.     

Pro-inflammatory cytokines downregulate platelet derived growth factor-α receptor gene expression in human osteoblastic cells

Platelet derived growth factor (PDGF) is thought to play a significant role in bone repair and regeneration. We previously demonstrated that PDGF-AA binding can be modulated by interleukin-1 (IL-1). We now report that TNF-α significantly reduces PDGF-AA binding by decreasing the number of PDGF-α receptor subunits on the surface of normal human osteoblastic cells. This inhibition is likely due to a decrease in synthesis of PDGF-α receptors since TNF-α causes a relatively rapid decrease in PDGF-α receptor mRNA levels as determined by Northern blot analysis. The physiologic importance of this inhibition is demonstrated by a TNF-α induced decrease in PDGF-AA stimulated tyrosine kinase activity. When saturating concentrations of TNF-α were used, the addition of IL-1 further inhibited PDGF-AA binding and further decreased surface expression of PDGF-α receptors. In contrast, other mediators such as IL-6, PTH, 1,25(OH)₂ vit D₃, hydrocortisone, PGE₂, bFGF, and IGF-1 had no effect. These results suggest that binding to the PDGF-α receptor is decreased by the strong pro-inflammatory cytokines such as IL-1β and TNF-α rather than as a general response to mediators important in bone resorption or bone formation. TNF-α and IL-1 are often co-expressed during destructive inflammatory processes. Thus, TNF-α and IL-1 may work in concert to limit the response of osteoblastic cells to PDGF-AA during periods of osseous inflammation. © 1996 Wiley-Liss, Inc.     

Effect of Alpinia galanga extract on cartilage degradation and on gene expression in human chondrocyte and synovial fibroblast metabolism

We investigated the effects of A. galanga extract on metabolism and gene expression involved in the interleukin-1β (IL-1β) response of human chondrocyte and synovial fibroblast. A. galanga extract inhibited IL-1β enhanced matrix breakdown of the cartilage explants in a dose-dependent manner. It suppressed uronic acid loss from the tissue and decreased the release of sulfated GAG and hyaluronan into the medium. MMP-2 and MMP-9 activity in the culture medium of chondrosarcomas and synovial fibroblasts were significantly reduced in the presence of A. galanga extract, which also suppressed the production of MMP-1,-3 and-13. The A. galanga extract also significantly increased type II collagen, SOX9 and aggrecan gene expression, suggesting an ability to enhance anabolic activity. At a high dose of A. galanga extract there was a down-regulation of aggrecan gene expression. Comparison with Diacerein® showed its general anti-inflammatory potential to be similar. The A. galanga extract was shown to inhibit IL-1β-stimulated cartilage matrix degradation in both systems. Additionally, the extract showed the potential to up-regulate certain chondrocyte anabolic genes. It may, therefore, offer some cartilage protective and anti-inflammatory properties as a therapeutic agent in arthritis.     

Acidic fibroblast growth factor modulates gene expression in the rat thyroid in vivo.

We have recently demonstrated that the iv administration of acidic fibroblast growth factor (a-FGF) to rats for 6 days results in a marked increase in thyroid weight with colloid accumulation and flat, quiescent follicular cells. Whereas a-FGF administration consistently increases thyroid weight, there are only minor alterations in serum TSH and thyroid hormones, and no change in intrathyroidal metabolism of 125I metabolism. In the present work, we studied the effects of 1 or 6 daily injections of a-FGF (60 micrograms/kg BW) or vehicle on the mRNA levels for histone, c-fos, actin, type I 5' deiodinase (5'D-I), thyroid peroxidase, and thyroglobulin and cathepsin D in the thyroid, liver and bone. Rats were sacrificed 0.5, 2, 4, 8 and 24 h after the 1st or the 6th a-FGF injection and thyroid, liver, and calvarium were removed. The relative amounts of mRNAs were determined by slot blot analysis. There was a 43% increase in thyroid weight in rats treated with a-FGF for 6 days compared to vehicle-treated rats. We observed an increase in c-fos mRNA content in the thyroid gland 0.5 to 4 h after 1 or 6 injections of a-FGF. In contrast, treatment with a-FGF for 1 or 6 days did not affect histone mRNA content, a marker of proliferative activity or actin mRNA levels. Treatment with a-FGF caused a marked decrease in thyroid 5' D-I mRNA content in the thyroid. The decrease was present 2 h after the first injection and reached a nadir 8 h later.(ABSTRACT TRUNCATED AT 250 WORDS)     

Altered cell growth and induction of an interferon-responsive gene in fibroblast lines derived from patients with familial Alzheimer's disease

Cell growth and response to interferon (IFN) have been used to assess alteration in properties of cultured skin fibroblasts from patients with familial Alzheimer's Disease (AD). A comparison of the proliferative characteristics between "low" (7-11) and "high" (18-21) passage diploid fibroblasts from control and AD individuals shows that AD cells become severely restricted in their proliferative potential earlier than matched controls as a result of "in vitro aging". In addition, while control cells grow adequately in a serum-free, chemically-defined media (Nutridoma) (90-95% compared to FBS-supplemented media), AD cells grow poorly in Nutridoma (45-53% compared to FBS-supplemented media). An interferon (IFN)-inducible enzyme, 2'5'-oligoadenylate (2-5A) synthetase, is significantly reduced in IFN-treated AD cells compared to age-matched control cells in two cell passage-dependent manner. These data suggest that an intrinsic property of AD cells is the progressive reduced ability to interact with normal biologic signals provided by proteins such as interferons which in turn may contribute to the pathogenesis of AD.