Orthovanadate both mimics and antagonizes the transforming growth factor β action on normal rat kidney cells

Normal rat kidney [NRK] cells grown in the presence of epidermal growth factor (EGF) or platelet-derived growth factor (PDGF) have a normal phenotype and undergo density-dependent growth inhibition, whereas in the presence of multiple growth factors, density arrest is lost and the cells become phenotypically transformed. We studied the influence of the protein tyrosine phosphatease (PTPase) inhibitor sodium orthovanadate on the mitogenic stimulation of NRK cells by growth factors and on transformation-linked properties as loss of density-dependent growth inhibition and anchorage-independent growth. The fraction of cells in serum-deprived monolayer cultures that is induced to proliferate upon mitogenic stimulation by EGF or PDGF is only slightly enhanced upon addition of low concentrations (25–50 μM) of vanadate. Addition of vanadate per se induces proliferation of only a very limited amount of cells, but results in a shift of the dose-response curves for other growth factors to lower concentrations. Vanadate added in combination with EGF or PDGF is able to mimic the effect of transforming growth factor β (TGFβ) in inducing phenotypic transformation. In monolayer cultures density-dependent growth inhibition is lost and anchorage-independent proliferation is observed on dishes coated with poly(2-hydroxy-ethyl methacrylate) (polyHEMA). The extent of these changes is similar to that induced by TGFβ. However, the morphology of the obtained colonies in polyHEMA-coated dishes is quite different. Cells transformed by TGFβ in the presence of EGF form rather amorphous colonies, whereas in the presence of orthovanadate colonies are formed that tend to fall apart in loose cells. The effect of vanadate on cell transformation is dependent on the growth factor conditions in a bimodal way. When a suboptimal dose of growth factor(s) is used, 25 μM vanadate is very effective in preventing density-induced growth inhibition and stimulating anchorage-independent proliferation. However, the same concentration of vandate is inhibitory when cells are maximally stimulated and antagonizes the transforming effect of TGFβ added in combination with other growth factors. It is hypothesized that vanadate acts on a set of different protein tyrosine phosphatases. Some of these are positive and others negative regulators of growth. © 1993 Wiley-Liss, Inc.     

Transforming growth factor-beta and retinoic acid modulate phenotypic transformation of normal rat kidney cells induced by epidermal growth factor and platelet-derived growth factor

In this study we have investigated the ability of epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and transforming growth factor-beta (TGF beta) together with retinoic acid (RA) at saturating concentrations to induce phenotypic transformation of normal rat kidney (NRK) cells in a growth factor-defined medium. This medium contains serum in which all growth factor activity has been chemically inactivated, thereby eliminating the effects of growth factors from serum in the assay. It is shown that neither TGF eta nor a ligand binding to the EGF receptor is essential for phenotypic transformation of NRK cells, since anchorage-independent growth is also induced by EGF in combination with RA and by PDGF in combination with RA and TGF beta. Our data indicate strong similarities between TGF beta and RA in their ability to act as modulators for phenotypic transformation. In addition, both agents enhance the number of EGF receptors in NRK cells, without affecting the number of PDGF receptors. On the other hand, TGF beta has mitogenic effects on a number of non-transformed cell lines, such as Swiss 3T3 fibroblasts, particularly when assayed in the absence of insulin, whereas RA is mitogenic for these cells only in the presence of insulin. These data demonstrate that phenotypic transformation of NRK cells requires specific combinations of polypeptide growth factors and modulating agents, but that this process can be induced under many more conditions than previously described. Moreover, our data point toward both parallels and differences in the activities of TGF beta and RA.     

Effect of serum and growth factors on heat sensitivity in Swiss mouse 3T3 cells

Quiescent Swiss mouse 3T3 cells react to a heat treatment at 46°C for 20 min by changing their flat, well-extended morphology to a round appearance with retracted cytoplasmic processes during the subsequent 2 h at 37°C. The percentage of morphologically changed cells was used to quantify changes in heat sensitivity, or resistance, in response to mitogenic stimulation. Stimulating quiescent cells with serum or with the specific growth factors epidermal growth factor (EGF) and prostaglandin F_2α (PGF_2α) markedly increased the heat resistance to a 46°C treatment, but only when the heat treatment, but only when the heat treatment was applied within 2–3 h after the addition. When insulin (which is not mitogenic, but synergistic with EGF and PGF_2α in these cells) was added alone or in combination with either EGF or PGF_2α, it had no effect on the development of heat resistance. Neither did cycloheximide nor tunicamycin inhibit heat resistance induced by EGF, and cycloheximide even enhanced it after 2–4 h. However, adding colcemid before or at the beginning of the heat treatment abolished the increased heat resistance. The results indicate that the resistance to a single heat treatment at 46°C may be related to changes in the metabolic state after mitogenic stimulation, even though these changes need not be reflected in the rate of entry into S phase. Furthermore, the cytoskeletal organization appears to be a crucial component in heat resistance of Swiss 3T3 cells.     

Response to transforming growth factor α (TGFα) and epidermal growth factor (EGF) in hepatocytes: Lower EGF receptor affinity of TGFα is associated with more sustained activation of p42/p44 mitogen-activated protein kinase and greater efficacy in stimulation of DNA synthesis

The epidermal growth factor (EGF) receptor mediates the effects of both EGF and transforming growth factor α (TGFα). Recent data suggested that EGF acts as a partial agonist/antagonist in hepatocytes, TGFα exerting a larger maximal stimulation of DNA synthesis than EGF. To further study the mechanisms involved in mediating the different effects of EGF and TGFα, we have examined receptor binding of the two growth factors and their action on the p42/p44 mitogen-activated protein (MAP) kinase activity in hepatocytes. Single-ligand concentration curves and competition experiments showed that the binding affinity to a common population of surface binding sites was about 20-fold lower for TGFα than for EGF. MAP kinase activity responded to EGF and TGFα with different kinetics. While the two agents produced almost identical acute (5 min) stimulation (peak about fivefold), TGFα produced a more sustained MAP kinase activity than EGF. The difference between EGF and TGFα was still detectable 24 h after growth factor addition. The results show that in hepatocytes a lower receptor affinity of TGFα, as compared to EGF, is associated with a more sustained activation of the MAP kinase and a greater efficacy in the stimulation of DNA synthesis. This suggests that differential interaction of these two agents with the EGF receptor results in differences in the downstream events elicited at a given level of receptor occupancy. The data also are compatible with a role of a prolonged MAP kinase activity in the mitogenic effects of EGF and TGFα. J. Cell. Physiol. 175:10–18, 1998. © 1998 Wiley-Liss, Inc.     

Epidermal growth factor receptor-induced activator protein 1 activity controls density-dependent growth inhibition in normal rat kidney fibroblasts

Density-dependent growth inhibition secures tissue homeostasis. Dysfunction of the mechanisms, which regulate this type of growth control is a major cause of neoplasia. In confluent normal rat kidney (NRK) fibroblasts, epidermal growth factor (EGF) receptor levels decline, ultimately rendering these cells irresponsive to EGF. Using an activator protein (AP)-1 sensitive reporter construct, we show that AP-1 activity is strongly decreased in density-arrested NRK cells, but is restored after relaxation of density-dependent growth inhibition by removing neighboring cells. EGF could not induce AP-1 activity or S-phase entry in density-arrested cells, but could do so after pretreatment with retinoic acid, which enhances EGF receptor expression. Our results support a model in which the EGF receptor regulates density-dependent growth control in NRK fibroblasts, which is reflected by EGF-induced mitogenic signaling and consequent AP-1 activity.     

The effect of serum,EGF, PGF2α and insulin on S6 phosphorylation and the initiation of protein and DNA synthesis

To test the connection between S6 phosphorylation and the activation of protein and DNA synthesis, we compared the effects of serum, epidermal growth factor (EGF), prostaglandin F_2α (PGF_2α) and insulin (which is not mitogenic in these cells). Increasing concentrations of serum or EGF produced roughly parallel effects on all three processes, though the maximum response elicited by EGF (10^?9 M) was only a portion of that caused by saturating levels of serum (7.5% to 10%). PGF_2α (8.5 × 10^?7 M) alone acted similarly to EGF (10^?9 M) and with EGF produced a synergistic effect on all three processes. Insulin (10^?9 M) alone stimulated both S6 phosphorylation and protein synthesis to approximately the same level as EGF or PGF_2α, but had no effect on initiation of DNA synthesis. Thus neither stimulation of S6 phosphorylation nor activation of protein synthesis is sufficient for initiation of DNA synthesis. The requirement for S6 phosphorylation could not be dissociated from the activation of protein synthesis. Ribosomes containing the most highly phosphorylated forms of S6 appear to have a selective advantage in entering polysomes.     

Epidermal growth factor (EGF)-nonresponsive variants of normal rat kidney cell line: response to EGF and transforming growth factor-beta

Anchorage-independent growth in soft agar of normal rat kidney (NRK) fibroblasts depends on both transforming growth factor-beta (TGF-beta) and epidermal growth factor (EGF) (or TGF-alpha). We have isolated two EGF-nonresponsive cell lines, N-3 and N-9, from chemically mutagenized NRK cells, after selection of mitogen-specific nonproliferative variants in the presence of EGF and colchicine. Saturation binding kinetics with 125I-EGF showed one-half or fewer EGF receptors in N-3 and N-9 than in their parental NRK. Cellular uptake of 2-deoxy-D-glucose was enhanced in all NRK, N-3, and N-9 cell lines by TGF-beta treatment, whereas treatment with EGF significantly enhanced the cellular uptake of the glucose analog in NRK cells, but not in N-3 and N-9 cells. DNA synthesis of NRK during the quiescent state, but not that of N-3 and N-9, was stimulated by EGF. Anchorage-independent growth of N-9 could not be observed even in the presence of both EGF and TGF-beta, whereas that of N-3 was significantly enhanced by TGF-beta alone. EGF stimulated phosphorylation of a membrane protein with molecular size 170 kDa of NRK, but not of N-3, when immunoprecipitates reacting with anti-phosphotyrosine antibody were analyzed. Exposure of NRK cells to EGF increased cellular levels of TGF-beta mRNA, but there appeared little expression of TGF-beta mRNA in N-3 and N-9 cells. Exposure of N-3 cells to EGF or TGF-beta enhanced the secretion of EGF into culture medium, but exposure of NRK or N-9 cells did not. Altered response to EGF of N-3 or N-9 might be related to their aberrant growth behaviors.     

Stimulation of hepatocyte DNA synthesis by prostaglandin E2 and prostaglandin F2α additivity with the effect of norepinephrine,and synergism with epidermal growth factor

Previous data obtained in vivo and in vitro suggest that both prostaglandins (PGs) and catecholamines may have a role in promoting hepatocyte proliferation, and PGE₂ and PGFF_2α have also been implicated as mediators of the mitogenic actions of epidermal growth factor (EGF) (and transforming growth factor alpha [TGFα]). We have studied the effects of PGs and norepinephrine on DNA synthesis in serum-free primary cultures of rat hepatocytes, and compared the PG effects with those of norepinephrine. PGE₂, PGF_2α, PGD₂, and the synthetic analog dimethyl-PGE₂ markedly enhanced the DNA synthesis. A more quantitative analysis of the effects of PGE₂ and PGF_2α on the DNA synthesis, in the presence and absence of EGF, indicated that these PGs interacted in an essentially multiplicative manner with the effect of EGF. The effects of PGE₂ and PGF_2α showed almost complete additivity with the stimulation of DNA synthesis produced by maximally effective concentrations of norepinephrine. The data suggest (a) that PGE₂ and PGF_2α facilitate and synergize with, rather than mediate, the actions of EGF in hepatocytes, and (b) that this effect of the PGs occurs by mechanisms that are at least partly distinct from those of norepinephrine. © 1994 wiley-Liss, Inc.     

Receptor-mediated effects on ligand availability influence relative mitogenic potencies of epidermal growth factor and transforming growth factor α

Epidermal growth factor (EGF) and transforming growth factor α (TGFα) elicit quantitatively different cell proliferation responses even though they act via a common receptor, the epidermal growth factor receptor (EGFR). We hypothesized that differential cellular trafficking of available ligand is responsible for the different mitogenic responses elicited by EGF and TGFα. Mitogenesis and ligand depletion were determined simultaneously in NR6 mouse fibroblasts expressing either wild-type (WT) or internalization-deficient cytoplasmic domain-truncated (c'973) EGFR. Thus we could determine the effects of both ligand-induced and low level constitutive ligand/receptor processing. For a given initial amount of growth factor, TGFα is a weaker stimulus than EGF in cells expressing either form of the EGFR. This difference in the mitogenic potencies correlates with increased depletion of TGFα observed during the growth assays. When this difference in ligand depletion is accounted for, or minimized, EGF and TGFα elicit quantitatively similar growth responses. Therefore, the relative mitogenic potencies of EGF and TGFα depend on ligand availability, as determined by the cellular trafficking of these ligands in conjunction with environmental circumstances. Interestingly, our data demonstrate that TGFα can be a less potent mitogenic stimulus than EGF under conditions where ligand availability is limited. Further, in our assays, differences in ligand processing are sufficient to explain the different mitogenic potencies of these growth factors in either of the receptor trafficking scenarios. Our results suggest a model of regulation of hormone responsiveness which favors dissociative ligands (such as TGFα) in receptor-limited situations and non-dissociative ligands (such as EGF) in the face of high receptor levels. © 1996 Wiley-Liss, Inc.     

Phenotypic transformation of normal rat kidney cells by transforming growth factor beta is not paralleled by enhanced production of a platelet-derived growth factor.

Phenotypic transformation of normal rat kidney (NRK) cells requires the concerted action of multiple polypeptide growth factors. Serum-deprived NRK cells cultured in the presence of epidermal growth factor (EGF) become density-inhibited at confluence, but they can be restimulated by a number of defined polypeptide growth factors, resulting in phenotypic cellular transformation. Kinetic data show that restimulation by transforming growth factor beta (TGF-beta) and retinoic acid is delayed when compared to induction by platelet-derived growth factor (PDGF), indicating that both TGF beta and retinoic acid may exert their growth-stimulating action by an indirect mechanism. Northern blot analysis shows that NRK cells express the genes for various polypeptide growth factors, including TGF beta 1, PDGF A-chain and basic fibroblast growth factor, but that the levels of expression are not affected by TGF beta or retinoic acid treatment. NRK cells also secrete low amounts of a PDGF-like growth factor into their extracellular medium, but the levels of secretion are insufficient to induce mitogenic stimulation and are unaffected by agents inducing phenotypic transformation. In combination with studies on the effects of anti-PDGF antibodies, it is concluded that phenotypic transformation of NRK cells by TGF beta and retinoic acid is not the result of enhanced production of a PDGF-like growth factor.     

Phenotypic transformation of normal rat kidney fibroblasts by endothelin-1. Different mode of action from lysophosphatidic acid, bradykinin, and prostaglandin f2alpha

In the present study, we compared the effects of endothelin (ET)-1 on cell proliferation and second messenger induction in normal rat kidney (NRK) fibroblasts, with those of other activators of G-protein-coupled receptors such as prostaglandin (PG)-F2alpha, bradykinin (BK), and lysophosphatidic acid (LPA). LPA is mitogenic by itself, while the other factors require the presence of EGF. In density-arrested NRK cells, ET-1 and LPA induce phenotypic transformation rapidly, with similar kinetics as retinoic acid (RA) and transforming growth factor (TGF)-beta, while BK and PGF2alpha only do so with delayed kinetics. ET-1 and PGF2alpha are strong inducers of anchorage-independent growth, with a similar level of induction as TGFbeta, in contrast to LPA and BK. When investigating the second messenger generation, we found that ET-1 is the strongest activator of arachidonic acid release and phosphatidylinositol diphosphate hydrolysis. Only in the case of ET-1 the cell depolarization is not reversible upon removal of the factor. Similarly, only the ET-1-induced transient enhancement of intracellular calcium concentration is paralleled by both homologous and heterologous desensitization. In conclusion, these data show that ET-1 is a potent inducer of second messengers and phenotypic transformation in NRK cells, with characteristics that clearly differ from those of other activators of G-protein-coupled receptors, most likely as a result of prolonged receptor activation.     

The role of EGF receptor transmodulation in embryonal carcinoma-derived growth factor-induced mitogenesis.

Exposure of quiescent 10T1/2 fibroblast cells to embryonal carcinoma-derived growth factor (ECDGF) results in a rapid temperature and ECDGF concentration-dependent inhibition of [125I]EGF binding to the epidermal growth factor (EGF) receptor (transmodulation). ECDGF predominantly inhibits the association of [125I]EGF with a high affinity subclass of EGF receptors, and induces increased phosphorylation of the EGF receptor on serine and threonine residues. No mitogenic effect of EGF can be detected in the presence of ECDGF concentrations which induce maximal EGF receptor transmodulation. ECDGF-induced EGF receptor transmodulation is sensitive to phorbol ester-induced desensitization whereas ECDGF-induced DNA synthesis is unaffected by prolonged pre-treatment with biologically active phorbol ester. These findings suggest that EGF receptor transmodulation is not essential for ECDGF mitogenicity but may inhibit EGF-induced DNA synthesis.     

Reinitiation of DNA synthesis in quiescent mouse keratinocytes; Regulation by polypeptide hormones,cholera toxin,dexamethasone, and retinoic acid

Summary Cloned mouse keratinocytes (MK-1 cells) display density-dependent growth arrest when reaching confluency in a serum-free medium with a calcium concentration <0.1 mM, supplemented only with insulin and transferrin. In this quiescent state, greater than 95% of the cell population is in the G_0/1 phase of the cell cycle. Treatment of quiescent MK-1 cells with 1 to 10 ng/ml epidermal growth factor (EGF) resulted in a sharp burst of DNA synthetic activity. Both insulin and cholera toxin potentiated the mitogenic effect of EGF, but neither agent was necessary or sufficient to induce thymidine incorporation into DNA. Dexamethasone abolished the effect of insulin, but not the mitogenic effect of EGF alone. In contrast, retinoic acid (RA) did not possess any mitogenic effect for quiescent MK-1 cells, nor did it modulate the actions of EGF or dexamethasone. A number of commercially available crude extracts of bovine brain and pituitary were also capable of initiating DNA synthesis in resting MK-1 cells. Finally, transforming growth factor type beta (TGFβ) proved to be a potent inhibitor of the mitogen-induced DNA synthesis in MK-1 cells (IC₅₀∶10pM). This defined culture system is eminently suited to study the regulation of DNA synthesis of epidermal cells. In addition, it can be used as a sensitive bioassay for the detection of epidermal mitogens, as well as inhibitors of DNA synthesis such as TGFβ. Supported by PHS Award CA-41556 from the National Cancer Institute, Bethesda, MD.     

Cadmium produces a delayed mitogenic response and modulates the EGF response in quiescent NRK cells

Recent studies have shown that cadmium, at subtoxic levels, may induce a response characteristic of that elicited by a type of growth factor that supports the anchorage independent growth of cells that are not fully transformed. That is, Cd⁺⁺ was found to replace transforming growth factor beta in supporting soft agar growth of NRK-49F cells. To tes the extent to which Cd⁺⁺ further mimics transforming growth factor beta in its effects and to establish response patterns that suggest possible molecular mechnisms of action, we have determined the effects of Cd⁺⁺ and/or epidermal growth factor (EGF) on DNA synthesis in quiescent NRK-49F cells. We found that subtoxic doses of Cd⁺⁺ modulate EGF-induced DNA synthesis in a dose-dependent fashion. Although Cd⁺⁺ effects on early (16–24 hr) EGF-induced DNA synthesis are primarily inhibitory, later effects involve stimulation as well. Subtoxic doses of Cd⁺⁺ did not stimulate DNA synthesis in quiescent cells within 24 hr of addition. At later times (40 or 64 hr), however, an increase in DNA synthesis of up to threefold was induced by 0.25 M Cd⁺⁺. This pattern of mitogenic response, involving inhibition of early growth-factor induced DNA synthesis and stimulation of late DNA synthesis, is consistent with that reported to be effected in some instances by transforming growth factor beta. Because a defined pattern of gene expression also is associated with the mitogenic responses to transforming growth factor beta, future studies at the molecular level can definitively test the degree to which Cd⁺⁺ and transforming growth factor beta effects are common.Abbreviations CFE colony forming efficiency - EGF epidermal growth factor - MT metallothionein - PGDF paltelet derived growth factor - TGF transforming growth factor     

Phenotypic transformation of normal rat kidney cells in a growth-factor-defined medium: induction by a neuroblastoma-derived transforming growth factor independently of the EGF receptor

Polypeptide growth factor activity in serum can be destroyed by treatment with dithiothreitol. When such growth-factor-inactivated serum is used as a supplement of culture media instead of regular serum, normal rat kidney (NRK) cells become quiescent unless defined polypeptide growth factors like insulin and epidermal growth factor (EGF) are added. On this basis a growth-factor-defined medium has been developed for NRK cells, which permits cell proliferation as rapidly as in media supplemented with serum, even at low cell densities. Moreover, cells can be serially passaged in this medium. NRK cells can be induced to grow in semisolid media when incubated with transforming growth factors. The growth-factor-defined medium permits soft agar growth experiments of NRK cells, without interference from polypeptide growth factors in serum. Using this assay system we have shown that EGF alone is unable to induce any degree of anchorage-independent growth in NRK cells. However, a recently identified transforming growth factor from mouse neuroblastoma cells which does not compete with EGF for receptor binding is able to induce progressively growing colonies of NRK cells in soft agar, even without additional EGF.     

Type beta transforming growth factor from feline sarcoma virus-transformed rat cells. Isolation and biological properties

We have isolated a strongly mitogenic, type beta transforming growth factor (beta TGF) released by Snyder-Theilen feline sarcoma virus-transformed rat embryo (FeSV-Fre) cells that induces phenotypic transformation of normal NRK cells when they are concomitantly stimulated by analogues of epidermal growth factor (EGF). Molecule filtration chromatography separates beta TGF from an EGF-like TGF (eTGF) which is also present in acid extracts from medium conditioned by FeSV-Fre cells (J. Massagué, (1983) J. Biol. Chem. 258, 13606-13613). Final purification of beta TGF is achieved by reverse phase high pressure liquid chromatography (HPLC) on octadecyl support, molecular filtration HPLC, and nonreducing dodecyl sulfate-polyacrylamide gel electrophoresis steps, yielding a 300,000-fold purified polypeptide with a final recovery of 21%. The purified rat beta TGF consists of two Mr = 11,000-12,000 polypeptide chains disulfide-linked as a Mr = 23,000 dimer. Induction of anchorage-independent proliferation of NRK cells by rat beta TGF depends on the simultaneous presence of eTGF or EGF. In the presence of a saturating (300 pM) concentration of either rat eTGF or mouse EGF, half-maximal anchorage-independent proliferation of NRK cells is obtained with 4-6 pM rat beta TGF. In the presence of a saturating (20 pM) concentration of rat beta TGF, half-maximal anchorage-independent proliferation of NRK cells is obtained with either rat eTGF or mouse EGF at a 50-70 pM concentration. Rat beta TGF is also able to induce DNA synthesis and cell proliferation on growth-arrested NRK, human lung, and Swiss mouse 3T3 fibroblast monolayers, this effect being half-maximal at 2-3 pM beta TGF for NRK cells. These results identify eTGF and beta TGF as the two synergistically acting factors responsible for the transforming action of culture fluids from FeSV-Fre cells.     

Cytokine regulation of adult human osteoblast-like cell prostaglandin biosynthesis

Prostaglandin (PG) biosynthesis by cytokine stimulated normal adult human osteoblast-like (hOB) cells was evaluated by thin layer chromatography, high performance liquid chromatography, and specific immunoassays. PGE₂ was the predominant PG formed under all incubation conditions tested. Control samples produced measurable amounts of PGE₂, and the measured level of this metabolite increased by 22-fold (from 7 to 152 ng/ml) following a 20 h treatment with the combination of TGFβ and tumor necrosis factor-α(TNF). The production of 6-keto-PGF_1α (the stable metabolite of prostacyclin) and of PGF_2α were each increased by about five-fold (from about 0.5 to 2.5 ng/ml) in samples treated with the cytokines. Thus, TGFβ and TNF exerted a regulation of hOB cell PG biosynthesis that was principally directed towards an increased PGE₂ biosynthesis, with lesser effects on the production of other PG metabolites. COX-2 mRNA levels were increased within 2 h of cytokine stimulation, reached a maximum at 6–12 h, and levels had appreciably diminished by 24 h after treatment. Both TGFβ and TNF could independently increase COX-2 mRNA levels and PG biosynthesis. However, the increased production of PGE₂ resulting from TNF stimulation was blocked by the addition of an interleukin-1β (IL-1β) neutralizing antibody, suggesting that TNF regulation of hOB cell PG synthesis was secondary to its capacity to increase hOB cell IL-1β production. TGFβ regulation of PG production was not affected by the addition of the neutralizing antibody. These studies support the proposition that PGs can be important autocrine/paracrine mediators of bone biology, whose production by hOB cells is responsively regulated by osteotropic cytokines. J. Cell. Biochem. 64:618–631. © 1997 Wiley-Liss, Inc.     

EGF receptor activities in mammalian development

The receptor for epidermal growth factor (EGF) and its analog transforming growth factor alpha (TGF alpha) is ubiquitous, implying quite general roles for EGF/TGF alpha in cell viability and tissue maintenance in adult tissues. There is also evidence that the EGF receptor is active in promoting wound healing and tissue regeneration in adult organs, such as skin, liver, and intestinal epithelium. It is likely that EGF receptors have more specific roles during the gestation period. For example, we have detected EGF receptors on the 3.5-day blastocyst (trophectoderm) surface and since TGF alpha-like mRNA sequences and peptides have been detected at this time (Rappolee et al., Science 241:1823, 1988), there is a strong implication for autocrine stimulation in pre- and peri-implantation stage embryos. Paracrine stimulation between the embryo and maternal tissues is also likely since both receptors and TGF alpha are present in decidual cells. Therefore EGF receptors may take part in growth regulation of the early embryo and in the process of implantation. Other examples where EGF receptors may play specific roles during embryonic development are discussed.     

Mitogenic response to epidermal growth factor (EGF) modulated by platelet-derived growth factor in cultured fibroblasts

The mitotic effects of epidermal growth factor (EGF) were investigated in two cultured fibroblast lines, BALB/c-3T3 and C3H 10T1/2 cells. EGF (30 ng/ml) added to quiescent 3T3 cells in medium containing either platelet-poor plasma or 10(-5) M insulin caused only minimal increases in the percentage of cells stimulated to initiate DNA synthesis. In contrast, EGF acted synergistically with either insulin or plasma to stimulate DNA synthesis in quiescent cultures of 10T1/2 cells, although the maximum effects of EGF were measured at concentrations several-fold greater than those found in either serum or plasma. In either 3T3 or 10T1/2 cells a transient preexposure to platelet-derived growth factor (PDGF) caused over a 10-fold increase in the sensitivity to the mitogenic effects of EGF. It is therefore possible that a primary action of PDGF is to increase the sensitivity of fibroblasts to EGF, independent of whether EGF alone is found to be mitogenic.     

Evidence for epidermal growth factor (EGF)-induced intermolecular autophosphorylation of the EGF receptors in living cells.

In response to epidermal growth factor (EGF) stimulation, the intrinsic protein tyrosine kinase of EGF receptor is activated, leading to tyrosine phosphorylation of several cellular substrate proteins, including the EGF receptor molecule itself. To test the mechanism of EGF receptor autophosphorylation in living cells, we established transfected cell lines coexpressing a kinase-negative point mutant of EGF receptor (K721A) with an active EGF receptor mutant lacking 63 amino acids from its carboxy terminus. The addition of EGF to these cells caused tyrosine phosphorylation of the kinase-negative mutant by the active receptor molecule, demonstrating EGF receptor cross-phosphorylation in living cells. After internalization the kinase-negative mutant and CD63 have separate trafficking pathways. This limits their association and the extent of cross-phosphorylation of K721A by CD63. The coexpression of the kinase-negative mutant together with active EGF receptors in the same cells suppressed the mitogenic response toward EGF as compared with that in cells that express active receptors alone. The presence of the kinase-negative mutant functions as a negative dominant mutation suppressing the response of active EGF receptors, probably by interfering with EGF-induced signal transduction. It appears, therefore, that crucial events of signal transduction occur before K721A and active EGF receptors are separated by their different endocytic itineraries.