Combined effects of somatomedin-like growth factors with fibroblast growth factor or epidermal growth factor in DNA synthesis in rabbit chondrocytes

Summary The somatomedin-like growth factors cartilage-derived factor (CDF) and multiplication-stimulating activity (MSA) stimulate DNA synthesis and proliferation of rabbit costal chondrocytes under serum-free conditions. Previously, we suggeted that CDF and MSA act on chondrocytes in an early G₁ phase to stimulate DNA synthesis. CDF and MSA have synergistic effects with epidermal growth factor (EGF) or fibroblast growth factor (FGF) in stimulating DNA synthesis of the cells. The mode of combined action of CDF or MSA with EGF or FGF in chondrocytes was studied by sequential treatments with these agents. EGF or FGF had synergistic effects with CDF or MSA in stimulating DNA synthesis, even when added 10 h after the latter. Synergism was also observed in cells pretreated with CDF or MSA; That is, the cultures were treated for 5 h with CDF or MSA and then washed, and treated with FGF or EGF. However, when CDF or MSA was added more than 5 h after EGF or FGF, no synergism of effects was observed. These findings suggest that the cultured chondrocytes become activated to interact with FGF or EGF for commitment to DNA synthesis when they are exposed to somatomedin-like growth factors at an early stage in the G₁ phase. Thus chondrocytes are under a different mechanism of growth control from fibroblastic cells.Abbreviations CDF cartilage-derived factor - MSA multiplication-stimulating activity - EGF epidermal growth factor - FGF fibroblast growth factor     

Epidermal growth factor induced apoptosis

Apoptosis, or programmed cell death, plays an important role in the pathogenesis of a number of human diseases, including cancers, autoimmune diseases, and neurodegenerative disorders. Recent evidence suggests that EGF induced signal transduction pathways which govern cell proliferation and cell cycle progression also mediate antiproliferative effects leading to increased apoptosis in cells that express high levels of epidermal growth factor receptors. Treatments designed to increase apoptosis have potential to change the natural progression of cancer and eventually lead to its successful control.     

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.     

Density-induced down regulation of epidermal growth factor receptors

Summary Previous studies have shown that cell density can regulate the binding of several growth factors. To determine whether cell density exerts a uniform effect on the expression of epidermal growth factor (EGF) receptors, seven cell lines were examined in detail. For each cell line, EGF binding was found to decrease as cell density increases. Scatchard analysis of the binding data reveals that decreases in EGF binding are due to reductions in the number of cell surface EGF receptors. The only apparent exception is the effect of cell density on the binding of EGF to A-431 cells. For these cells, increases in cell density lead to two effects: decreases in the number of high affinity EGF receptors and increases in the total number of EGF receptors. In addition to the effects of cell density on EGF receptors, it was determined that increases in cell density can coordinately down-regulate receptors for as many as four different growth factors. Overall, the findings described in this report for EGF and those previously described for transforming growth factor type-β (TGF-β) and fibroblast growth factor (FGF) demonstrate the existence of a common mechanism for down-regulating growth factor receptors. This work was supported by grants from the Nebraska Department of Health (89-51), the National Cancer Institute (Laboratory Research Center Support Grant, CA36727), and the American Cancer Society (Core Grant ACS SIG-16). EDITOR'S STATEMENT The paper by Rizzino et al. demonstrates that receptor number decreases as a function of cell density. This may represent a mechanism by which cell proliferation is reduced as cell density increases.     

Immunoreactive fibroblast growth factor (FGF) in a transplantable chondrosarcoma: inhibition of tumor growth by antibodies to FGF

Using a radioimmunoassay for bovine pituitary fibroblast growth factor (FGF), we have established the presence of the immunoreactive mitogen in extracts of a transplantable mouse chondrosarcoma. Both neutral and acidic extracts of the tumor contain an immunoreactive FGF (ir-FGF) that cross-reacts in a parallel and dose-dependent fashion in the radioimmunoassay. The ir-FGF is retained on heparin-Sepharose affinity columns and can be detected in the same molecular weight forms as rat pituitary FGF. Mice (C57/Bl) inoculated with the tumor (10 mg, im) show a decreased rate of tumor growth when passively immunized with the antiserum to FGF. The results establish the presence of FGF in this tumor and implicate its role in the etiology of its development.     

Hepatocyte growth factor induces epithelial cell motility through transactivation of the epidermal growth factor receptor

Hepatocyte growth factor (HGF) is a potent inducer of motility in epithelial cells. Since we have previously found that activation of the epidermal growth factor receptor (EGFR) is an absolute prerequisite for induction of motility of corneal epithelial cells after wounding, we investigated whether induction of motility in response to HGF is also dependent on activation of the EGFR. We now report that HGF induces transactivation of the EGFR in an immortalized line of corneal epithelial cells, in human skin keratinocytes, and in Madin-Darby canine kidney cells. EGFR activation is unconditionally required for induction of motility in corneal epithelial cells, and for induction of a fully motile phenotype in Madin-Darby canine kidney cells. Activation of the EGFR occurs through amphiregulin and heparin-binding epidermal growth factor-like growth factor. Early after HGF stimulation, blocking EGFR activation does not inhibit extracellular-signal regulated kinase 1/2 (ERK1/2) activation by HGF, but the converse is seen after approximately 1 h, indicating the existence of EGFR-dependent and -independent routes of ERK1/2 activation. In summary, HGF induces transactivation of the EGFR in epithelial cells, and this is a prerequisite for induction of full motility.     

Nanoparticle mediated controlled delivery of dual growth factors

Peripheral nerve functional recovery after nerve injury generally requires multiple growth factors by synergistic effect.However,the optical combination of multiple synergistic growth factors for axonal regeneration has been scarcely considered up to now.Meanwhile,the use of growth factors in promoting nerve regeneration was limited by its short biological half-life in vivo,its vulnerability to structure disruption or hydrolyzation,leading to loss of bioactivity.Herein,a novel polymeric nanoparticle delivery system composed of heparin andε-poly-L-lysine(PL)was prepared for control release of nerve growth factor(NGF)and basic fibroblast growth factor(bFGF).The nanoparticles were synthesized by polyelectrolyte complexation in aqueous solution at room temperature,followed by cross-linking with biological genipin.The obtained nanoparticles had a spherical shape,with a mean diameter of about 246 nm,and high growth factors encapsulation efficiency as well as good stability.NGF and bFGF were encapsulated in the nanoparticles and showed a continuous and slow release behavior in vitro.The bioactivities of the released growth factors were evaluated,and exhibited the synergistic effect.The controlled release of the dual synergistic growth factors would improve the treatment of peripheral nerve injury to mimic the natural cellular microenvironments.     

血小板源生长因子受体与肿瘤

血小板源生长因子(platelet-derived growth factor,PDGF)经由其受体(platelet-derived growth fac tor receptor,PDGFR)表现细胞效应。PDGF和PDGFR涉及多种肿瘤的发病机制并在血管生成中起重要作用。PDGF在肿瘤中的自分泌刺激、PDGFR的过表达或过度活化或者刺激肿瘤内血管生成都会促进肿瘤生长;PDGFR的阻断可以降低实体瘤中组织间质液压而增强药物传送。这些机制可能提示在肿瘤治疗中PDGFR抑制剂单用、与化疗药物或者和其他靶点药物联合用药的可能性和可行性。随着PDGFR拮抗剂,如imatinib的上市,PDGFR作为抗肿瘤药物的靶点备受瞩目。     

Effect of recombinant growth hormone on expression of growth hormone receptor,insulin-like growth factor mRNA and serum level of leptin in growing pigs

Sixteen Large White × Landrace castrated male pigs were allotted into treatment and control group. The treatment group was injected intramuscularly with recombinant porcine growth hormone (rpGH, 4 mg d⁻¹) and the control group with vehicle for 28 days. Animals were slaughtered 4 h after final injection for liver, longissimus dorsi (LD) muscle and blood sampling. Serum concentration of insulin-like growth factor 1 (IGF-I) and leptin were determined by RIA. The total RNA was extracted from tissues to measure the abundance of growth hormone receptor (GHR), IGF-I mRNA by RT-PCR with 18S rRNA internal standard. Results showed that rpGH enhanced the average daily weight gain by 26.1% (P < 0.05), the serum IGF-I concentration by 70.94% (P < 0.01), decreased serum leptin by 34.8% (P < 0.01). The relative abundance of GHR and IGF-I mRNA in liver were increased by 24.45% (P < 0.05) and 45.30% (P < 0.01), respectively, but no difference of GHR (P > 0.05) and IGF-I mRNA (P > 0.05) in LD between GH treated and control group was found. These results suggest that rpGH can up-regulate hepatic GHR and IGF-I gene expression and improve animal growth. However the effect of rpGH on GHR and IGF-I gene expression are tissue-specific.     

Effect of recombinant growth hormone on expression of growth hormone receptor, insulin-like growth factor mRNA and serum level of leptin in growing pigs

Growth hormone (GH) plays an important role in regulation of animal growth, metabolism and lactation[1]. Numerous studies have shown that exogenous somatotropin (ST) can increase average daily weight gain, improve feed efficiency, stimulate protein deposition and muscle growth and decrease lipid accretion rate[1]. The original somatomedin hypothesis suggested that the effect of GH on postnatal growth was mediated by insulin-like growth hormone factor 1 (IGF-I) which was thought to be deriv…     

Osteopontin contributes to hepatocyte growth factor-induced tumor growth and metastasis formation

The cytokine hepatocyte growth factor (HGF)/scatter factor-1 and its cognate receptor, Met, are involved in the etiology and progression of many types of cancer. Despite recent advances in understanding the signal transduction pathways activated by HGF, the mechanism by which HGF exerts its tumorigenic effect is not well understood. To identify proteins that may be involved in mediating HGF-induced cell motility, invasiveness, and tumorigenesis, we used two separate differential display screening methods to identify changes in gene expression that are initiated by HGF in an epithelial cell culture system. Among several known and unknown genes whose expression was modified, osteopontin (OPN), a protein previously associated with tumorigenesis, was found to be upregulated within 6 h following HGF stimulation. OPN expression was dependent on activation of the PI-3 kinase pathway. Autocrine secretion of HGF resulted in sustained expression of OPN. Downregulation of opn expression by stable antisense transfection attenuated OPN expression and repressed HGF-induced invasiveness in vitro and decreased HGF-mediated tumor growth and metastasis formation in vivo. Constitutive expression of OPN in itself exerted partial invasiveness in vitro, but its expression itself was not sufficient to initiate tumor growth or metastasis formation in vivo. Thus, together with other molecules, OPN activity contributes to HGF-induced tumor growth and invasiveness.     

Nerve growth factor-induced reduction in epidermal growth factor responsiveness and epidermal growth factor receptors in PC12 cells: an aspect of cell differentiation.

The rat pheochromocytoma clone PC12 responds to nerve growth factor through the expression of a number of differentiated neuronal properties. One of the most rapid changes is a large, transient increase in the activity of ornithine decarboxylase. These cells also show an increase in ornithine decarboxylase activity in response to the mitogen, epidermal growth factor, but do not respond morphologically as they do to nerve growth factor. Specific, high-affinity epidermal growth factor receptors are present on the cells. When the cells are differentiated with nerve growth factor, the response to epidermal growth factor is markedly diminished and there is a marked reduction in the binding of epidermal growth factor to the cells.     

Characterization of a variety of neutralizing anti-heparin-binding epidermal growth factor-like growth factor monoclonal antibodies by different immunization methods

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a member of the epidermal growth factor family. The accumulated evidence on the tumor-progressing roles of HB-EGF has suggested that HB-EGF-targeted cancer therapy is expected to be promising. However, the generation of neutralizing anti-HB-EGF monoclonal antibodies (mAbs) has proved difficult. To overcome this difficulty, we performed a hybridoma approach using mice from different genetic backgrounds, as well as different types of HB-EGF immunogens. To increase the number of hybridoma clones to screen, we used an electrofusion system to generate hybridomas and a fluorometric microvolume assay technology to screen anti-HB-EGF mAbs. We succeeded in obtaining neutralizing anti-HB-EGF mAbs, primarily from BALB/c and CD1 mice, and these were classified into 7 epitope bins based on their competitive binding to the soluble form of HB-EGF (sHB-EGF). The mAbs showed several epitope bin-dependent characteristics, including neutralizing and binding activity to human sHB-EGF, cross-reactivity to mouse/rat sHB-EGF and binding activity to the precursor form of HB-EGF. The neutralizing activity was also validated in colony formation assays. Interestingly, we found that the populations of mAb bins and the production rates of the neutralizing mAbs were strikingly different by mouse strain and by immunogen type. We succeeded in generating a variety of neutralizing anti-HB-EGF mAbs, including potent sHB-EGF neutralizers that may have potential as therapeutic agents for treating HB-EGF-dependent cancers. Our results also suggest that immunization approaches using different mouse strains and immunogen types affect the biological activity of individual neutralizing antibodies.     

Characterization of a variety of neutralizing anti-heparin-binding epidermal growth factor-like growth factor monoclonal antibodies by different immunization methods

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a member of the epidermal growth factor family. The accumulated evidence on the tumor-progressing roles of HB-EGF has suggested that HB-EGF-targeted cancer therapy is expected to be promising. However, the generation of neutralizing anti-HB-EGF monoclonal antibodies (mAbs) has proved difficult. To overcome this difficulty, we performed a hybridoma approach using mice from different genetic backgrounds, as well as different types of HB-EGF immunogens. To increase the number of hybridoma clones to screen, we used an electrofusion system to generate hybridomas and a fluorometric microvolume assay technology to screen anti-HB-EGF mAbs. We succeeded in obtaining neutralizing anti-HB-EGF mAbs, primarily from BALB/c and CD1 mice, and these were classified into 7 epitope bins based on their competitive binding to the soluble form of HB-EGF (sHB-EGF). The mAbs showed several epitope bin-dependent characteristics, including neutralizing and binding activity to human sHB-EGF, cross-reactivity to mouse/rat sHB-EGF and binding activity to the precursor form of HB-EGF. The neutralizing activity was also validated in colony formation assays. Interestingly, we found that the populations of mAb bins and the production rates of the neutralizing mAbs were strikingly different by mouse strain and by immunogen type. We succeeded in generating a variety of neutralizing anti-HB-EGF mAbs, including potent sHB-EGF neutralizers that may have potential as therapeutic agents for treating HB-EGF-dependent cancers. Our results also suggest that immunization approaches using different mouse strains and immunogen types affect the biological activity of individual neutralizing antibodies.