Increased expression of basic fibroblast growth factor in hyperoxic-injured mouse lung

Basic fibroblast growth factor (bFGF) is a mitogenic polypeptide for a wide variety of cell types and has been immunolocalized in the rodent and human lung. We investigated the mRNA and protein expression of bFGF in hyperoxic-injured adult mouse lungs using northern blot analysis and immunohistochemistry. Mece (6–8weeks) were continuously exposed to 80% osygen up to 4 days. Levels of bFGF mRNA were increased from room air control on days 3 and 4 of hyperoxia. mRNA levels of acidic fibroblast growth factor (aFGF), fibronectin, and transin/stromelysin were also examined in this injury model. Similar to bFGF, the fibronectin and transin/stromelysin mRNA levels were increased after 3 days of hyperoxia. In contrast, the aFGF mRNA levels were gradually reduced on each day of hyperoxia. A rabbit polyclonal anti-bFGF antibody was used to determine the distribution and levels of expression in the hyperoxic-injured lungs. The room air control and day 1 hyperoxic-exposed lungs exhibited staining for bFGF in the basement membranes of the blood vessels, airways, and alveoli. Patchy but intense alveolar staining was prominent on day 4 of hyperoxia. The bFGF immunoreactivity of blood vessels and airways unaffected by the hyperoxia exposure. These results suggest that bFGF may play a role in the alveolar response to hyperoxic-induced injury by virtye of the altered mRNA levels and protein distribution in this injury model.     

Effects of basic fibroblast growth factor on endothelial cells under conditions of simulated microgravity

Fibroblast growth factors interact with appropriate endothelial cell (EC) surface receptors and initiate intracellular signal cascades, which participate in modulating blood vessel growth. EC, upon exposure to basic fibroblast growth factors (bFGFs) undergo profound functional alterations, which depend on their actual sensitivity and involve gene expression and de novo protein synthesis. We investigated the effects of bFGF on signaling pathways of EA.hy926 cells in different environments. EC were cultured under normal gravity (1 g) and simulated microgravity (micro g) using a three-dimensional (3D) clinostat. Microgravity induced early and late apoptosis, extracellular matrix proteins, endothelin-1 (ET-1) and TGF-beta(1) expression. Microgravity reduced eNOS mRNA within 24 h. Moreover, a six- to eightfold higher amount of IL-6 and IL-8 was secreted within 24 h micro g. In addition, microgravity induced a duplication of NF-kappaB p50, while p65 was quadrupled. At 1 g, bFGF application (4 h) reduced ET-1, TGF-beta(1) and eNOS gene expression. After 24 h, bFGF enhanced fibronectin, VEGF, Flk-1, Flt-1, the release of IL-6, IL-8, and TGF-beta(1). Furthermore, bFGF promoted apoptosis, reduced NFkB p50, but enhanced NFkB p65. After 4 h micro g, bFGF decreased TGF-beta(1), eNOS, and ET-1 gene expression. After 24 h micro g, bFGF elevated fibronectin, Flk-1 and Flt-1 protein, and reduced IL-6 and IL-8 compared with vehicle treated micro g cultures. In micro g, bFGF enhanced NF-KappaB p50 by 50%, Bax by 25% and attenuated p65, activation of caspase-3 and annexin V-positive cells. bFGF differently changes intracellular signals in ECs depending whether it is applied under microgravity or normal gravity conditions. In microgravity, bFGF contributes to protect the EC from apoptosis.     

Characterization of an endooligopeptidase A-like protein in PC12 cells: Activity modulation by cAMP but not by basic fibroblast growth factor

Endooligopeptidase A is a putative neuropeptide-metabolizing enzyme. It converts small enkephalin-containing peptides into the corresponding enkephalins and inactivates biopeptides such as bradykinin and neurotensin in vitro. We investigated the presence of endooligopeptidase A in PC12 cells. This cell line was derived from a rat pheochromocytoma tumor and resembles fetal chromaffin cell. Depending on the supplements added to the cell culture, this cell line can be differentiated into mature chromaffin cell or sympathetic neuron-like cell. Endooligopeptidase A activity was measured in soluble cellular extracts using a specific fluorogenic substrate QF-ERP7. The PC12 endooligopeptidase A-like activity shared similar but not identical biochemical properties with rabbit brain endooligopeptidase A. Similarly to rabbit brain endooligopeptidase A, the PC12 endooligopeptidase A-like activity was enhanced by DTT, totally inhibited by DTNB and 1-10 Phenanthroline, partially inhibited by cFP-AAF-pAb, and not affected by PMSF. Furthermore, the PC12 endooligopeptidase A-like activity displayed identical elution profile as rabbit brain endooligopeptidase A in gel filtration and anion-exchange chromatography. In addition, an antiserum raised against rabbit brain endooligopeptidase A cross-reacted with a 71 kDa component from PC12 cell extracts in Western blotting and was also able to partially neutralize the PC12 endooligopeptidase A-like activity. Treatment of PC12 cells with basic fibroblast growth factor (bFGF), a neurotrophic factor for this cell line, did not modify the specific activity of this enzyme. However, cAMP analogs decreased the specific activity of the enzyme. These results indicate the presence of an endooligopeptidase A-like activity in PC12 cells which is modulated by cAMP but not by bFGF.     

C2C12成肌细胞诱导肌性分化过程中miR-101a的表达

以C2C12成肌细胞为模型,在分化培养基中诱导C2C12建立体外肌性细胞分化模型.以poly (A)3′-端加尾和实时定量PCR方法研究miR-101a在C2C12细胞分化过程中的表达情况.结果发现,在细胞转入分化培养基进行肌性分化的1-5 d中,miR-101a的表达量逐渐增加,提示miR-101a可能在肌肉发生中发挥调控作用.     

碱性成纤维细胞生长因子对脑微血管内皮细胞血管新生基因谱和环加氧酶-2表达的影响

本文研究了碱性成纤维细胞生长因子（basic fibroblast growth factor, bFGF）对小鼠脑微血管内皮细胞（microvascular endothelial cell, MVEC）株bEnd．3中血管新生相关基因表达谱的改变,并重点从mRNA、蛋白质和细胞水平检测bFGF对血管新生旁观分子环加氧酶-2（cyclooxygenase-2,COX-2）表达的影响。用特异性小鼠血管新生基因芯片高通量检测bEnd．3细胞基因谱表达的改变,分析促血管新生基因及抑制血管新生的基因表达谱的变化;用RT—PCR、Western blot、免疫细胞化学等方法分别从mRNA、蛋白质和细胞水平检测COX-2表达变化及细胞内的定位。结果发现用10ng／ml的bFGF刺激bEnd．3细胞2h后多种促血管新生基因表达明显上调,如Adamtsl、MMP-9、Ang-1、PDGFB、G—CSF、FGFl6、IGF-1等分别上调3、8、120、5．2、4．5、1．7、2．7倍。与此同时,多种抑制血管新生的基因表达相应下调,如TSP-3、TIMP-2、TGFβ1等表达分别下调3．4、1．5和3．5倍。RT-PCR和Western blot的结果证实,bFGF可以上调COX-2mRNA的表达和蛋白质的合成。免疫组化的结果表明,COX-2主要分布在胞浆。以上结果提示：bFGF具有上调促血管新生基因表达,下调抑制血管新生基因表达的作用,两者协同作用,促进血管新生。同时bFGF还可以明显促进血管新生旁观分子COX-2mRNA的表达和蛋白质的合成。本文讨论了bFGF引起MVEC内COX-2表达上调的意义。     

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

Summary To determine if fibroblasts could be a source of fibroblast growth factor (FGF) in tissue, cells were initiated in culture from newborn human foreskin. Cells were studied in Passages 2 to 8. 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. Supported in part by grant DK 31063 from the National Institutes of Health, Bethesda, MD.     

吸入糖皮质激素对大鼠肺纤维化的干预作用及bFGF表达的影响

目的:研究吸入糖皮质激素对大鼠肺纤维化模型的干预作用及可能的机制。方法:雌性Wistar大鼠40只,体重180～250g,按照随机数字表法将大鼠随机分为4组(n=10):①对照组(C组);②模型组(M组);③布地奈德组(B组);④地塞米松组(D组)。M组、B组、D组给大鼠气管内吸入博莱霉素(5mg/kgbw,8mg)复制肺纤维化模型,C组气管内吸入同等剂量的生理盐水作对照,B组于次日给予雾化吸入等效剂量布地奈德,D组于次日腹腔内注射地塞米松。上述各组均于注药后第1、4周各宰杀5只。通过苏木素-伊红染色观察肺泡炎、Masson胶原染色观察肺纤维化、用免疫组化及酶联免疫吸附测定(ELISA)法检测bFGF蛋白在大鼠肺组织,血清及肺泡灌洗液(BALF)的表达。结果:1、4周时M组表现为肺泡炎及肺间质炎症,B组、D组肺泡炎及肺纤维化程度较M组减轻。1、4周时血清、肺组织、BALF中M组的bFGF表达高于C组(P0.01),B、D组低于M组(P0.01)。结论:吸入糖皮质激素可减轻博莱霉素诱导的肺纤维化,其抗纤维化作用的机制与抑制bFGF表达有关。     

Expression of fibroblast growth factor receptor 3 by fibroblast growth factor 2 in cultured chick embryo chondrocytes

Although fibroblast growth factor 2 (FGF2) and fibroblast growth factor receptor 3 (FGFR3) both inhibit longitudinal bone growth, little is known about the relationship between FGF2 and FGFR3. Accordingly, the current study examined the expression of FGFR3 mRNA after the administration of FGF2 using cultured chondrocytes from day 17 chick embryos to evaluate the relationship between FGF2 and FGFR3. The chondrocytes were isolated from the caudal one-third portion (LS) of sterna, peripheral regions (USP) and central core regions (USC) of the cephalic portion of the sterna, and lower portion of the proximal tibial growth plate (Ti) of day 17 chick embryo. The expression of FGFR1, FGFR3, and type II and X collagen mRNA in the chondrocytes from the LS, USP, USC, and Ti was determined. FGFR1 was not expressed in the LS and USP chondrocytes, yet strongly expressed in the USC and Ti chondrocytes. With a treatment of FGF2, the expression of FGFR1 slightly increased in the USC chondrocytes and was not related with the concentration of FGF2 in the Ti chondrocytes. FGFR3 was expressed in all the chondrocyte types, yet strongly increased in the LS, USC, USP, and Ti in that order according to the concentration of FGF2. For the LS and USP chondrocytes, the expression of FGFR3 with FGF2 increased in a 4-day culture, yet decreased in a 6-day culture, whereas for the USC chondrocytes, the expression of FGFR3 mRNA with FGF2 increased in a 2-day culture, yet decreased in a 4-day culture, suggesting that the hypertrophic chondrocytes were more numerous and sensitive compared to the proliferative chondrocytes. For all the chondrocyte types, FGF2 appeared to be up-regulated to FGFR3, as the expression of FGFR3 mRNA increased with a higher concentration of FGF2 until a peak level. In conclusion, FGF2 was found to up-regulate to FGFR3 until the peak level of FGFR3 mRNA expression, while in hypertrophic chondrocytes, FGFR3 appeared to cause the differentiaton of chondrocytes, resulting in the inhibition of longitudinal bone growth after the peak level of FGFR3 mRNA expression.     

Basic fibroblast growth factor is cardioprotective in ischemia-reperfusion injury

To examine whether basic fibroblast growth factor (bFGF) administered to the heart by perfusion can improve cardiac resistance to injury we employed an isolated rat heart model of ischemia-reperfusion injury and determined the extent of functional recovery in bFGF-treated and control hearts. Global ischemia was simulated by interruption of flow for 60 min. Recovery of developed force of contraction (DF), recorded after reestablishment of flow for 30 min, reached 63.8±1.5% and 96.5±3.5% of preischemic levels in control and bFGF-treated hearts (10 g/heart), respectively, indicating that bFGF induced significantly improved recovery of mechanical function. Recoveries of the rates of contraction or relaxation were also significantly improved in bFGF-treated hearts. Extent of myocardial injury, assessed by determination of phosphocreatine kinase in the effluent, was reduced as a result of bFGF treatment. As a first step towards understanding the mechanism and direct cellular target(s) of bFGF-induced cardioprotection, we investigated its fate after perfusion. Perfusion of 10 g bFGF/heart resulted in a 4-fold increase in bFGF associated with the heart compared to control levels, as estimated by biochemical fractionation and immunoblotting. Immunofluorescent staining of the bFGF-perfused hearts revealed intense anti-bFGF staining in association with blood vessels as well as the periphery of cardiomyocytes, suggesting that the latter may be a target for direct bFGF action. In conclusion, our findings of bFGF-induced increases in cardiac resistance to, and improved functional recovery from, ischemia-reperfusion injury indicate that bFGF may have clinical applications in the treatment of ischemic heart disease.     

Ceramide 1-phosphate stimulates proliferation of C2C12 myoblasts

Recent studies have established specific cellular functions for different bioactive sphingolipids in skeletal muscle cells. Ceramide 1-phosphate (C1P) is an important bioactive sphingolipid that has been involved in cell growth and survival. However its possible role in the regulation of muscle cell homeostasis has not been so far investigated. In this study, we show that C1P stimulates myoblast proliferation, as determined by measuring the incorporation of tritiated thymidine into DNA, and progression of the myoblasts through the cell cycle. C1P induced phosphorylation of glycogen synthase kinase-3β and the product of retinoblastoma gene, and enhanced cyclin D1 protein levels. The mitogenic action of C1P also involved activation of phosphatidylinositol 3-kinase/Akt, ERK1/2 and the mammalian target of rapamycin. These effects of C1P were independent of interaction with a putative G(i)-coupled C1P receptor as pertussis toxin, which maintains G(i) protein in the inactive form, did not affect C1P-stimulated myoblast proliferation. By contrast, C1P was unable to inhibit serum starvation- or staurosporine-induced apoptosis in the myoblasts, and did not affect myogenic differentiation. Collectively, these results add up to the current knowledge on cell types targeted by C1P, which so far has been mainly confined to fibroblasts and macrophages, and extend on the mechanisms by which C1P exerts its mitogenic effects. Moreover, the biological activities of C1P described in this report establish that this phosphosphingolipid may be a relevant cue in the regulation of skeletal muscle regeneration, and that C1P-metabolizing enzymes might be important targets for developing cellular therapies for treatment of skeletal muscle degenerative diseases, or tissue injury.     

农杆菌介导的碱性成纤维细胞生长因子（bFGF）转化金针菇的研究

构建了金针菇表达载体p139035S-bFGF,并将重组质粒转入到根癌农杆菌EHA105中。以白金针菇Flammulina velutipes菌丝球为受体材料,用根癌农杆菌介导转入碱性成纤维细胞生长因子（bFGF）。金针菇菌株对潮霉素（Hyg）抗性测验结果表明,在PDA固体培养基上的潮霉素筛选浓度为9μg/mL,在液体培养基中为6μg/mL。探索头孢霉素（Cefotaxime）对菌丝的毒性实验、农杆菌的菌液浓度、侵染时间、乙酰丁香酮（AS）的添加及其浓度的控制、共培养的时间等因素对转化效率的影响。结果表明,Cef对金针菇菌丝几乎无抑制作用,最佳抑菌浓度为400μg/mL;农杆菌的菌液浓度OD600为0.5,侵染时间在30min左右,在AS为200μmol/L的共培养基上共培养72h,转化率最高。PCR与Southern杂交结果证明bFGF基因已整合到金针菇的基因组中。在无选择压力的PDA固体培养基上继代培养5次后仍检测到bFGF基因的存在,表明bFGF基因在转基因金针菇中稳定遗传。     

Scintigraphic detection of xenografted tumors producing human basic fibroblast growth factor

A murine monoclonal antibody 3H3 recognizes the basic fibroblast growth factor (FGF) and inhibits the growth of human glioblastoma cells both in vitro and in vivo. We studied the potential of a scintigraphic technique using the 3H3 antibody to detect tumors that produce basic FGF.¹²⁵I- and¹¹¹In-labeled 3H3 bound to U87MG human glioblastoma cells in vitro. U87MG cells were inoculated subcutaneously into nude mice. After development of the tumor, radiolabeled 3H3 was injected into the subcutaneous space surrounding the tumor. A high level of radioactivity from 3H3 was retained at the tumor, whereas an irrelevant antibody cleared rapidly from the injected site. Radiolabeled 3H3 was not retained in tumors that did not produce basic FGF. Scintigraphic detection of tumors expressing basic FGF would be valuable for the therapeutic application of the antibody.     

Constant expression of mouse calpastatin isoforms during differentiation in myoblast cell line, C2C12

C2C12 is a myoblast cell line which is used to studydifferentiation into multinucleated cells in vitro. Addition of calpain inhibitors, calpeptin orE-64d, to the culture medium prevented the myoblasticfusion of C2C12 cells. Immunoblot studies usingaffinity-purified antibody, revealed that the expressedlevels of mouse calpastatin remained unaltered duringC2C12 cell fusion. The detected calpastatin migratedas a protein of 130 kDa on SDS-polyacrylamide gelelectrophoresis. The estimated molecular mass wassomewhat greater than that in mouse liver anderythrocytes, and much greater than that reported inrat myoblasts. The 130 kDa isoform may contain anadditional N-terminal region designated XL domainfound in bovine calpastatin.     

Oncogenic K-Ras and basic fibroblast growth factor prevent Fas-mediated apoptosis in fibroblasts through activation of mitogen-activated protein kinase

By an expression cloning method using Fas-transgenic Balb3T3 cells, we tried to obtain inhibitory genes against Fas-mediated apoptosis and identified proto-oncogene c-K-ras. Transient expression of K-Ras mutants revealed that oncogenic mutant K-Ras (RasV12) strongly inhibited, whereas dominant-inhibitory mutant K-Ras (RasN17) enhanced, Fas-mediated apoptosis by inhibiting Fas-triggered activation of caspases without affecting an expression level of Fas. Among the target molecules of Ras, including Raf (mitogen-activated protein kinase kinase kinase [MAPKKK]), phosphatidylinositol 3 (PI-3) kinase, and Ral guanine nucleotide exchange factor (RalGDS), only the constitutively active form of Raf (Raf-CAAX) could inhibit Fas-mediated apoptosis. In addition, the constitutively active form of MAPKK (SDSE-MAPKK) suppressed Fas-mediated apoptosis, and MKP-1, a phosphatase specific for classical MAPK, canceled the protective activity of oncogenic K-Ras (K-RasV12), Raf-CAAX, and SDSE-MAPKK. Furthermore, physiological activation of Ras by basic fibroblast growth factor (bFGF) protected Fas-transgenic Balb3T3 cells from Fas-mediated apoptosis. bFGF protection was also dependent on the activation of the MAPK pathway through Ras. All the results indicate that the activation of MAPK through Ras inhibits Fas-mediated apoptosis in Balb3T3 cells, which may play a role in oncogenesis.     

Progranulin compensates for blocked IGF-1 signaling to promote myotube hypertrophy in C2C12 myoblasts via the PI3K/Akt/mTOR pathway

It is well known that growth hormone (GH)-induced IGF-1 signaling plays a dominant role in postnatal muscle growth. Our previous studies have identified a growth factor, progranulin (PGRN), that is co-induced with IGF-1 upon GH administration. This result prompted us to explore the function of PGRN and its association with IGF-1. In the present study, we demonstrated that, similar to IGF-1, PGRN can promote C2C12 myotube hypertrophy via the PI(3)K/Akt/mTOR pathway. Moreover, PGRN can rescue the muscle atrophy phenotypes in C2C12 myotube when IGF-1 signaling is blocked. This result shows that PGRN can substitute for IGF-1 signaling in the regulation of muscle growth. Our findings provide new insights into IGF-1-modulated complicated networks that regulate muscle growth.     

Stretch activation of GTP-binding proteins in C2C12 myoblasts

Mechanical stimulation has been proposed as a fundamental determinant of muscle physiology. The mechanotransduction of strain and strain rate in C2C12 myoblasts were investigated utilizing a radiolabeled GTP analogue to detect stretch-induced GTP-binding protein activation. Cyclic uniaxial strains of 10% and 20% at a strain rate of 20% s(-1) rapidly (within 1 min) activated a 25-kDa GTPase (183 +/- 17% and 186 +/- 19%, respectively), while 2% strain failed to elicit a response (109 +/- 11%) relative to controls. One, five, and sixty cycles of 10% strain elicited 187 +/- 20%, 183 +/- 17%, and 276 +/- 38% increases in activation. A single 10% stretch at 20% s(-1), but not 0.3% s(-1), resulted in activation. Insulin activated the same 25-kDa band in a dose-dependent manner. Western blot analysis revealed a panel of GTP-binding proteins in C2C12 myoblasts, and tentatively identified the 25-kDa GTPase as rab5. In separate experiments, a 40-kDa protein tentatively identified as Galpha(i) was activated (240 +/- 16%) by 10% strain at 1 Hz for 15 min. These results demonstrate the rapid activation of GTP-binding proteins by mechanical strain in myoblasts in both a strain magnitude- and strain rate-dependent manner.