Keratinocyte Growth Factor Expression in the Mesenchymal Cells of Human Amnion*
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Abstract Amnion epithelial and mesenchymal cells were separated by differential protease treatment, and the separated cells were maintained in monolayer culture. Keratinocyte growth factor (KGF) messenger RNA (mRNA) was readily detected by Northern analysis of amnion mesenchymal cell total RNA (10 μg) but not in amnion epithelial cells. Treatment of the amnion mesenchymal cells in serum-free medium with tetradecanoyl phorbol acetate (1 nm) caused an increase in the level of KGF mRNA. Forskolin treatment also caused an increase in KGF mRNA but not to the levels attained with tetradecanoyl phorbol acetate treatment. Dexamethasone (1 nm) treatment of these cells effected a reduction in the level of KGF mRNA. Prolonged maintenance of mesenchymal cells in serum-free medium also was associated with an increase in the level of KGF mRNA. Treatment with a variety of other agents, viz., interleukin (IL)-1, IL-6 plus or minus IL-6 soluble receptor, IL-11, oncostatin M , epidermal growth factor (EGF), and transforming growth factor-β did not modify the level of KGF mRNA. Treatment of amnion epithelial cells with KGF caused an increase in the rate of [3H]thymidine incorporation, but the rate of cell replication induced by KGF was less than that induced by treatment with EGF. Transforming growth factor-β treatment inhibited basal and EGF- and KGF-stimulated amnion epithelial cell replication. The findings of this study are indicative that KGF is expressed in human amnion mesenchymal cells, and that KGF may act on the epithelial cells of this tissue.Keywords:
Keratinocyte growth factor
Amnion
Amnion
Cyclic adenosine monophosphate
Aquaporin 3
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Newborn rat astroglia cells possess epidermal growth factor (EGF) and insulin-like growth factor (IGF) receptors, which suggests that these growth factors regulate their growth and development. To determine the relative roles and interactions between the two growth factors on astroglial growth, primary cultures of astroglial cells from newborn rats (1 day postnatal) were treated with pure peptides, singly or in combination in various concentrations, and the growth response was determined by DNA synthesis ([3H]thymidine incorporation). EGF, IGF-I, and IGF-II, as single peptides, stimulated DNA synthesis, with half-maximal stimulatory concentrations of 0.25 ng/ml for EGF, 2.0 ng/ml for IGF-I, and 25 ng/ml for IGF-II, respectively. These findings indicate that astroglial cells are responsive to these growth factors in physiological concentrations, with the relative sensitivity of EGF greater than IGF greater than IGF-II. When EGF and IGF-I were added in combination, the growth stimulatory effect was greater than the additive effects of each growth factor added alone, indicating that the two growth factors act in synergism with each other. In particular, addition of increasing concentrations of EGF from 0.25-10 ng/ml to a constant concentration of 50 ng/ml IGF-I resulted in significant potentiation of [3H]thymidine incorporation of astroglial cells. To determine if the synergistic effect was due to a local synthesis of IGF-I by astroglia, a specific monoclonal antibody against IGF-I (Sm 1.2) was added to the peptides. Sm 1.2 decreased not only IGF-I-stimulated DNA synthesis, but also EGF-stimulated DNA synthesis, suggesting that the effects of EGF were contributed to in part by the local synthesis of IGF-I by astroglial cells. Analysis of conditioned medium from cells treated with EGF revealed a significant increase (approximately 2-fold) in IGF-I (from 4.5 to 8.8 ng/ml), but not IGF-II. To determine if the EGF effect on IGF synthesis was at the level of IGF-I mRNA transcription, stable IGF-I mRNA levels were determined in the astroglial cells before and after stimulation with EGF, using Northern analysis and quantification by densitometry. Astroglia expressed four IGF-I mRNA transcripts as in the adult and fetal liver, but only one (3.6 kilobases) IGF-II mRNA.
Thymidine
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Platelet-derived growth factor
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Keratinocyte growth factor
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Serially transplantable rat mammary tumor (RMT) cells are not dependent on exogenous epidermal growth factor (EGF) and insulin-like growth factor-I for continuous growth in serum-free medium. Previously, we found that conditioned medium obtained from these cells contained EGF-like mitogenic activity and stimulated tyrosine phosphorylation of a 185-kDa protein in EGF-dependent mammary epithelial cells. This protein is distinct from the EGF receptor and resembles a 185-kDa tyrosine-phosphorylated protein present in RMT cells themselves. The results of the studies reported here indicate that the tyrosine-phosphorylated p185 detected in growth factor-independent RMT cells and in human mammary epithelial cells exposed to RMT-conditioned medium was activated erbB-2 protein. Partial purification of the activating factor present in RMT-conditioned medium yielded a heparin-binding growth factor with biochemical properties similar to those of neu differentiation factor/heregulin (NDF/HRG). RNA-polymerase chain reaction analysis demonstrated that RMT cells expressed mRNA for NDF/HRG, and western-blot analysis confirmed the presence of the 45-kDa secreted form of NDF/HRG in conditioned medium from the growth factor-independent RMT cells. The biological activity of partially purified rat NDF/HRG was examined and found to be the same as that of the pure growth factor. In addition, we found that RMT-conditioned medium, fractionated on an anion-exchange column and by reverse-phase high-pressure liquid chromatography, contained a potent EGF-like growth factor that was distinct from NDF/HRG. This factor competes with 125I-EGF for binding to EGF receptors and has an apparent molecular mass of 6600 Da. This factor copurifies by high-pressure liquid chromotography with pure transforming growth factor-α (TGF-α), and the cells are positive for TGF-α mRNA. Thus, growth factor-independent RMT cells also synthesize and secrete TGF-α. These results indicate that growth factor-independent RMT cells secrete two growth factors with overlapping biological activities and suggest that autocrine loops mediated by these factors are important in the growth factor-independent proliferation of the RMT cells. © 1996 Wiley-Liss, Inc.
A431 cells
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The effects of various growth factors on the production of immunoreactive insulin-like growth factor I (iIGF-I) in short term (3-day) cultures of porcine granulosa cells was investigated. Epidermal growth factor (EGF) was shown to be a potent dose-dependent stimulator of iIGF-I production, achieving a 3.6-fold stimulation at a dose of 10 ng/ml. Transforming growth factor-alpha (10 ng EGF equivalents/ml) was also stimulatory. Platelet-derived growth factor (10 ng/ml) had no effect of its own, but enhanced EGF-stimulated iIGF-I production. The acidic and basic fibroblast growth factors (100 ng/ml) had no effect alone or in combination with EGF. Transforming growth factor-beta (10 ng/ml) had no effect of its own, but inhibited EGF-stimulated iIGF-I production. The interactive effects of EGF and FSH (200 ng/ml) on iIGF-I production were investigated in short term and longer term (7-day) cultures. In short term cultures under conditions optimized for EGF-dependent iIGF-I production, FSH had no effect of its own and inhibited EGF action. Conversely, in longer term cultures optimized for FSH-dependent iIGF-I production, EGF had no effect of its own and inhibited FSH action. Thus IGF production by cultured porcine granulosa cells is regulated in a complex manner and is highly dependent on the culture conditions. Our results suggest that IGF production in the ovary may also be regulated in a complex manner which is dependent on the developmental state of the follicle.
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Keratinocyte growth factor
Gene transfer
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Human ectocervical epithelial cells are a primary target for infection by oncogenic papillomaviruses, which are strongly implicated as causative agents in the genesis of cervical cancer. Growth factors have been implicated as agents that stimulate proliferation and enhance the possibility of malignant transformation. In the present study we utilize several human papillomavirus (HPV) type 16-immortalized ectocervical epithelial cell lines to investigate the effects of epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) on cell proliferation and the production of IGF binding proteins (IGFBPs). ECE16-1 cells, an HPV16-immortalized/nontumorigenic cell line, maintained in defined medium, produce and release high levels of IGFBP-3 (38/42 kDa) as well as smaller amounts of a 24-kDa IGFBP. Supplementation of defined medium with EGF causes a dose-dependent increase in cell growth and a concomitant decrease in the levels of IGFBP-3 released into the culture medium. EGF suppression of IGFBP-3 is maintained even when EGF-stimulated cell growth is suppressed 67% due to the simultaneous presence of 3 ng/ml of TGF beta 1, indicating that EGF suppression of IGFBP-3 levels is independent of EGF effects on cell growth. EGF suppression of IGFBP-3 production is correlated with a reduction in IGFBP-3 mRNA level. In the presence of EGF, the growth response of the cells to ng amounts of IGF-I is significantly enhanced. Moreover, the simultaneous presence of both EGF and IGF-I reduces the level of IGFBP-3 more efficiently than EGF alone. We also observe that the IGFBP-3 level is decreased and the 24-kDa IGFBP level is increased in HPV16-positive tumorigenic versus nontumorigenic cell lines. This is the first report of EGF acting as a positive regulator of IGF-I action via the IGFBPs. On the basis of these findings, we propose that EGF stimulates ECE16-1 cell growth via a dual-action mechanism by (a) stimulating growth directly via the EGF mitogenic pathway and (b) stimulating growth indirectly by reducing the levels of inhibitory IGFBPs and thereby potentiating the effects of IGF-I. In addition, the observation that more highly transformed cell types produce lower levels of IGFBP-3 and higher levels of 24-kDa IGFBP suggests that tumor cells in more advanced cervical cancers may have an altered response to IGF-I.
Immortalised cell line
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Cholera toxin
Growth inhibition
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Introduction: Growth factors have been shown to affect the complex cascade of wound healing; however, interaction between different growth factors during dermal and epidermal regeneration are still not entirely defined. In the present study we determined the interaction between keratinocyte growth factor (KGF), an epidermal growth factor, administered as liposomal cDNA, with other dermal and epidermal growth factors and collagen types I, III, and IV synthesis in an acute wound. Method: Adult male Sprague-Dawley rats received 30% total body surface area scalds under general anesthesia. They were then divided into two groups to receive weekly subcutaneous injections of liposomes plus the Lac Z gene (0.2 μg, vehicle), or liposomes plus the KGF cDNA (2.2 μg) and Lac Z gene (0.2 μg), for 4 weeks. Immunological assays, histological, and immunohistochemical techniques were used to determine growth factor concentrations and different types of collagen (I, III, and IV), rate of reepithelialization and dermal morphology, after KGF cDNA gene transfer. Results: KGF cDNA transfer significantly increased IGF-I (Insulin-like Growth Factor-I), IGFBP-3 (Insulin-like Growth Factor Binding Protein-3), and FGF (Fibroblast Growth Factor) and decreased TGF-β(Transforming Growth Factor–beta) levels (p < 0.05). KGF had no effect on PDGF (Platelet-Derived Growth Factor). KGF cDNA significantly increased collagen type IV (p < 0.02) at the wound edge as well as the wound bed, while it had no effect on collagen type I and III. KGF cDNA significantly increased reepithelialization compared to the control group. Conclusion: Exogenous KGF cDNA increases IGF-I, IGF-BP3, FGF expression and decreases TGF-β concentration in an acute wound. It accelerates re-epithelialization, improves dermal morphology and increases basement membrane formation, without a concomitant increase in inflammation or scarring. Acknowledgments: Clayton Foundation for Research
Keratinocyte growth factor
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