Inhibition of parathyroid hormone-responsive adenylate cyclase in clonal osteoblast-like cells by transforming growth factor alpha and epidermal growth factor

The effects of transforming growth factor alpha (TGF-alpha) and epidermal growth factor (EGF) on parathyroid hormone (PTH)-responsive adenylate cyclase were examined in clonal rat osteosarcoma cells (UMR-106) with the osteoblast phenotype. Recombinant TFG-alpha and EGF incubated with UMR-106 cells for 48 h each produced concentration-dependent inhibition of PTH-responsive adenylate cyclase, with maximal inhibition of 38-44% at 1-3 ng/ml of either growth factor. TGF-alpha and EGF also inhibited beta-adrenergic agonist (isoproterenol)-stimulated adenylate cyclase by 32%, but neither growth factor affected enzyme response to prostaglandin or basal (unstimulated) activity. Nonreceptor-mediated activation of adenylate cyclase by forskolin and cholera toxin was inhibited 18-20% by TGF-alpha and EGF. Pertussis toxin augmented PTH-stimulated adenylate cyclase, suggesting modulation of PTH response by a functional inhibitory guanine nucleotide-binding regulatory component of the enzyme. However, pertussis toxin had no effect on TGF-alpha inhibition of PTH response. Growth factor inhibition of PTH response was time-dependent, with maximal inhibition by 4-12 h of TGF-alpha exposure, and was reduced by prior treatment of UMR-106 cells with cycloheximide. TGF-alpha was not mitogenic for UMR-106 cells. The results indicate that TGF-alpha and EGF selectively impair PTH- and beta-adrenergic agonist-responsive adenylate cyclase of osteoblast-like cells. Growth factor inhibition of adenylate cyclase may be exerted at the receptor for stimulatory agonist and at nonreceptor components excluding pertussis toxin-sensitive guanine nucleotide-binding regulatory proteins. The inhibitory action of growth factors may also require protein synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of autophosphorylation of epidermal growth factor receptor by a small peptide not employing an ATP-competitive mechanism

Previously we found that short peptides surrounding major autophosphorylation sites of EGFR (VPEY(1068)INQ, DY(1148)QQD, and ENAEY(1173)LR) suppress phosphorylation of purified EGFR to 30-50% at 4000 microM. In an attempt to improve potencies of the peptides, we modified the sequences by substituting various amino acids for tyrosine or by substituting Gln and Asn for Glu and Asp, respectively. Among the modified peptides, Asp/Asn- and Glu/Gln-substitution in DYQQD (NYQQN) and ENAEYLR (QNAQYLR), respectively, improved inhibitory potencies. The inhibitory potency of NYQQN was not affected by the concentration of ATP, while that of QNAQYLR was affected. Docking simulations showed different mechanisms of inhibition for the peptides: inhibition by binding to the ATP-binding site (QNAQYLR) and inhibition by binding to a region surrounded by alphaC, the activation loop, and the catalytic loop and interfering with the catalytic reaction (NYQQN). The inhibitory potency of NYQQN for insulin receptor drastically decreased, whereas QNAQYLR inhibited autophosphorylation of insulin receptor as well as EGFR. In conclusion, NYQQN is not an ATP-competitive inhibitor and the binding site of this peptide appears to be novel as a tyrosine kinase inhibitor. NYQQN could be a promising seed for the development of anti-cancer drugs having specificity for EGFR.     

Isolation and characterization of a novel bladder cancer cell line: Inhibition by epidermal growth factor

Summary A novel continuous cell line, designated BC3c, was established from a surgical biopsy of an invasive solid transitional cell carcinoma of the bladder derived from an 82-yr-old Caucasian female. BC3c cells were near-triploid bearing multiple structural and numerical chromosome anomalies. The epithelial origin of the cancer cells was indicated by the expression of cytokeratins 8 and 19 as well as by the absence of mesenchymal markers. Polymerase chain reaction-restriction-fragment length polymorphisms and single-strand conformation polymorphism mutation detection assays did not reveal any mutations in H-ras codon 12 and K-ras codons 12 and 13. In addition, no mutation in specific hot-spot codons of the p53 gene and no accumulation of the p53 protein were observed. BC3c cells grew rapidly in vitro, even in the absence of exogenous growth factors, because they were found to stimulate their growth in an autocrine manner. BC3c cells were found to express the epidermal growth factor-receptor (EGF-r) abundantly, but in contrast to other established bladder cancer cell lines, human recombinant epidermal growth factor inhibited the cells’ proliferation in vitro. These features render the newly established bladder cancer cell line BC3c a useful tool for further experimentation.     

The solution structures of epidermal growth factor and transforming growth factor alpha

The structures of human epidermal growth factor (EGF) and human transforming growth factor alpha (TGFα) have been determined in solution using nuclear magnetic resonance techniques. The features of each structure are described and similarities and differences between them are discussed. The structures are combined with information from sequence homologies to produce a model of the receptor-recognition sites of EGF and TGFα, which can be tested in a site-directed mutagenesis programme. The model assists in explaining previous observations of sequence-activity relationships. The TGFα and EGF structures also serve as models for homologous modules in other extracellular proteins.     

Modulation of transforming growth factor alpha-dependent expression of epidermal growth factor receptor gene by transforming growth factor beta, triiodothyronine, and retinoic acid

We have investigated the actions of transforming growth factor (TGF) type alpha on epidermal growth factor (EGF) receptor mRNA expression in MDA-468 human mammary carcinoma cells in serum-free media. We found that exposure of MDA-468 cells to TGF alpha results in elevated levels of EGF receptor mRNA. This increase in mRNA accumulation showed time and dose dependence. Addition of TGF beta 1 enhanced the accumulation of EGF receptor mRNA induced by TGF alpha in a time- and dose-dependent manner. We also found that triiodothyronine at physiological concentrations exerts synergistic control on the action of TGF alpha alone, or in association with TGF beta 1, on EGF receptor mRNA expression. Similarly, retinoic acid treatment also enhanced in a time- and dose-dependent manner the TGF alpha-dependent response of EGF receptor mRNA and acted synergistically with TGF beta 1. The results described here suggest that optimum regulation of EGF receptor gene expression by TGF alpha is a complex process involving synergistic interactions with heterologous growth factors and hormones.     

Inhibition of stimulus-dependent epidermal growth factor receptor and transforming growth factor-alpha mRNA accumulation by the protein kinase C inhibitor staurosporine

The ability of staurosporine, a potent inhibitor of protein kinase C, to block certain cellular events initiated by 12-O-tetradecanoylphorbol-13-acetate (TPA) and epidermal growth factor (EGF) was examined. Treatment of MDA468 breast cancer cells with TPA decreases EGF binding to the cell surface and this effect is blocked by pretreatment with staurosporine with an IC50 of 30 nM. Either 10(-9) M EGF or 100 ng/ml TPA stimulated the accumulation of both EGF receptor and TGF-alpha mRNA and staurosporine (50 nM) completely abolished these mRNA accumulations. Staurosporine did not block EGF-stimulated tyrosine phosphorylation of its receptor as measured by immunoblotting with anti-phosphotyrosine antibodies. The ability of staurosporine to block the mRNA responses of either EGF or TPA suggests that these two agents have common signaling pathways and it implies a role for protein kinase C in the control of EGF receptor and TGF-alpha expression.     

1,25-Dihydroxyvitamin D3 increases epidermal growth factor receptors and transforming growth factor beta-like activity in a bone-derived cell line

To investigate possible mechanisms through which 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) affects cell proliferation and differentiation, we have studied the effects of 1,25-(OH)2D3 on the binding and mitogenic activity of epidermal growth factor (EGF) in RCJ 1.20 cells, an established, non-tumorigenic cell line derived from 21-day-old fetal rat calvaria. 1,25-(OH)2D3 caused a dose- and time-dependent 2- to 3-fold increase in the number of receptors for EGF. The 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 metabolites of vitamin D3 were ineffective in eliciting changes in EGF binding. Saturation and Scatchard analyses indicated that an increase in available unoccupied high affinity EGF binding sites was responsible for the 1,25-(OH)2D3-induced EGF binding. In addition, 1,25-(OH)2D3 enhanced EGF-dependent growth of RCJ 1.20 cells in soft agar. The potentiation of EGF effects on RCJ 1.20 cell growth by 1,25-(OH)2D3 may be related to the 1,25-(OH)2D3 regulation of EGF binding. However, the induction of anchorage-independent growth by 1,25-(OH)2D3 appears to be due to the stimulation of transforming growth factor beta-like activity. These results provide a possible explanation for the mechanism whereby the effects of 1,25-(OH)2D3 on cell proliferation and bone metabolism may be mediated.     

Mouse Balb/c3T3 cell mutant with low epidermal growth factor receptor activity: induction of stable anchorage-independent growth by transforming growth factor beta

A mutant clone (MO-5) was originally isolated as a clone resistant to Na+/K+ ionophoric antibiotic monensin from mouse Balb/c3T3 cells. MO-5 was found to show low receptor-endocytosis activity for epidermal growth factor (EGF): binding activity for EGF in MO-5 was less than one tenth of that in Balb/c3T3. Anchorage-independent growth of MO-5 was compared to that of Balb/c3T3 when assayed by colony formation capacity in soft agar. Coadministration of EGF and TGF-beta efficiently enhanced anchorage-independent growth of normal rat kidney (NRK) cells, but neither factor alone was competent to promote the anchorage-independent growth. The frequency of colonies appearing in soft agar of MO-5 or Balb/c3T3 was significantly enhanced by TGF-beta while EGF did not further enhance that of MO-5 or Balb/c3T3. Colonies of Balb/c3T3 formed in soft agar in the presence of TGF-beta showed low colony formation capacity in soft agar in the absence of TGF-beta. Colonies of MO-5 formed by TGF-beta in soft agar, however, showed high colony formation capacity in soft agar in the absence of TGF-beta. Pretreatment of MO-5 with TGF-beta induced secretion of TGF-beta-like activity from the cells, while the treatment of Balb/c3T3 did not induce the secretion of a significant amount of TGF-beta-like activity. The loss of EGF-receptor activity in the stable expression and maintenance of the "transformed" phenotype in MO-5 is discussed.     

Regulation of cell proliferation by epidermal growth factor

Epidermal Growth Factor (EGF) is a 6045 dalton polypeptide which stimulates the proliferation of various cell types in vitro and in vivo. EGF binds to diffusely distributed membrane receptors which rapidly cluster primarily on coated pits areas on the plasma membrane. Subsequently, the EGF-receptor complexes are endocytosed and degraded by lysosomal enzymes. The lateral diffusion coefficient (D) of EGF-receptor complexes on cultured cells increases gradually from D = 2.8 X 10(-10) cm2/sec at 5 degrees C to 8.5 X 10(-10) cm2/sec at 37 degrees C. In the same range of temperature the rotational correlation times change from 25 to 50 microseconds to approximately 350 microseconds. Hence, at 4 degrees C, the occupied EGF receptors translate and rotate rapidly in the plane of the membrane. At 37 degrees C, EGF receptors form microclusters composed of 10 to 50 molecules. Moreover, it is concluded that both at 4 degrees C and 37 degrees C lateral diffusion of the occupied receptors is not the rate determining step for either receptor clustering or internalization. EGF receptor is a 150,000 to 170,000 dalton glycoprotein. The receptor is in close proximity to an EGF-sensitive, cAMP-independent, tyrosine-specific protein kinase which also phosphorylates the receptor molecules itself. The EGF sensitive kinase is similar to the kinase activity which is associated with certain RNA tumor viruses. The fact that the non-mitogenic cyanogen-bromide cleaved EGF is as potent as native EGF in stimulating phosphorylation suggests that EGF-induced, protein phosphorylation is a necessary but insufficient signal for the induction of DNA synthesis by EGF. EGF receptor serves also as the binding site for Transforming Growth Factors (TGF) which compete with EGF and induce anchorage-independent growth of normal cells in soft agar. Tumor promoters such as phorbol ester effect the binding of EGF to its membrane receptors and its ability to stimulate DNA synthesis. EGF itself has also some tumor promoting activity. Hence, the membrane receptor for EGF seems to participate in the regulation of normal and neoplastic growth. Monoclonal antibodies against EGF receptor (IgM) induce various early and delayed effects of EGF, while their monovalent Fab' fragments are devoid of biological activity. These observations support the notions that EGF receptor rather than EGF itself is the active moiety and that the role of the hormone is to perturb the receptor in the appropriate way, probably by inducing the microaggregation of EGF receptors.     

Inhibition of iodine-125-labeled human follitropin binding to testicular receptor by epidermal growth factor and synthetic peptides

Two tetrapeptide sequence homologies between mouse epidermal growth factor precursor (mEGFP) and human follitropin (FSH) were revealed by a computer program that identifies identical residues among polypeptide sequences. The two tetrapeptides, Lys-Thr-Cys-Thr (KTCT) and Thr-Arg-Asp-Leu (TRDL), are present in the hormone-specific beta subunit of FSH from all species studied. These tetrapeptides are not present in the alpha subunit, which is common to all pituitary glycoprotein hormones. Both tetrapeptides are also found in mEGFP, and one tetrapeptide, TRDL, is located within the 53-residue form of mEGF purified from mouse submaxillary glands. Computer-generated hydropathy profiles predicted that both tetrapeptides are located in hydrophilic portions of the FSH beta subunit and that TRDL is in a hydrophilic portion of commercially available mEGF. Therefore, the tetrapeptides might be accessible to receptor binding sites for FSH. We report that mEGF inhibits binding of 125I-labeled human FSH to receptors in testis by 50% (I50) at a concentration of 1.8 X 10(-5) M. No binding inhibition was observed by GnRH or arginine-vasopressin at 10(-4) M, neither of which contain the tetrapeptide sequences. FSH beta subunit, which contains both tetrapeptides, also inhibited binding (I50 = 9 X 10(-8) M) of 125I-labeled human FSH to testis receptor. Thus, it appears that FSH beta subunit and mEGF are capable of inhibiting binding of FSH to testicular FSH receptors, presumably through interactions that include the homologous tetrapeptides. This presumption was supported by the observation that the synthetic tetrapeptides (KTCT or TRDL) were also active in inhibiting binding of 125I-labeled human FSH to testis receptor.     

Inhibition of Nb2 T-lymphoma cell growth by transforming growth factor-beta.

1. Mitochondria isolated from the gut-dwelling nematodes Nippostrongylus brasiliensis and Ascaridia galli (muscle and gut + reproductive tissue) were examined for cytochromes, and it was observed that N. brasiliensis and A. galli muscle tissue mitochondria contained a-, b- and c-type cytochromes, but their stoichiometries were quite different (1:2:1.9 and 1:11.4:13.6 respectively); A. galli gut + reproductive-tissue mitochondria, however, only contained b and c cytochromes, in a ratio of 1:0.8. 2. CO difference spectra showed the presence of CO-reacting b-type cytochrome(s) in all three types of mitochondria; the fast-reacting species comprised 30, 44 and 39% of the total in N. brasiliensis, A. galli muscle and A. galli gut + reproductive-tissue mitochondria respectively. 3. Cytochrome aa3 was observed in N. brasiliensis mitochondria and in those from A. galli muscle, but was below the level of detectability (less than 0.005 nmol/mg of protein) for A. galli gut + reproductive-tissue mitochondria. 4. Photochemical action spectra for the reversal of CO inhibition of the endogenous respiration of whole worms (at 24 microM- and 40 microM-O2 respectively for N. brasiliensis and A. galli) gave maxima at 598 and 542-543 nm, corresponding to the alpha- and beta-absorption maxima of cytochrome aa3, and at 567 nm (b-type cytochrome) for both worms. These results suggest that cytochrome aa3 is the major functional oxidase in N. brasiliensis, whereas the CO-reacting b-type cytochrome dominates in A. galli.     

Epidermal growth factor and transforming growth factor alpha bind differently to the epidermal growth factor receptor

Epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) compete with each other for binding to the EGF receptor. These two growth factors have similar actions, but there are distinguishable differences in their biological activities. It has never been clear how this one receptor can mediate different responses. A monoclonal antibody to the EGF receptor (13A9) has been identified which has only small effects on the binding of EGF to the EGF receptor, but which has very large effects on the binding of TGF alpha to the EGF receptor; 5 micrograms/mL antibody has been shown to totally block 0.87 microM TGF alpha from binding to purified EGF receptor and to lower both the high- and low-affinity binding constants of TGF alpha binding to EGF receptor on A431 cells by about 10-fold. The 13A9 antibody causes a 2.5-fold stimulation of the tyrosine kinase activity of partially purified EGF receptor, compared to a 4.0-fold stimulation of the tyrosine kinase activity by EGF under the same conditions. The data suggest either that the antibody stabilizes a conformation of the EGF receptor which is not favorable for TGF alpha binding or that it blocks a part of the surface of the receptor which is necessary for TGF alpha binding but not EGF binding.     

Deciphering vascular endothelial cell growth factor/vascular permeability factor signaling to vascular permeability. Inhibition by atrial natriuretic peptide

Vascular endothelial cell growth factor (VEGF) was originally described as a potent vascular permeability factor (VPF) that importantly contributes to vascular pathobiology. The signaling pathways that underlie VEGF/VPF-induced permeability are not well defined. Furthermore, endogenous vascular peptides that regulate this important VPF function are currently unknown. We report here that VPF significantly enhances permeability in aortic endothelial cells via a linked signaling pathway, sequentially involving Src, ERK, JNK, and phosphatidylinositol 3-kinase/AKT. This leads to the serine/threonine phosphorylation and redistribution of actin and the tight junction (TJ) proteins, zona occludens-1 and occludin, and the loss of the endothelial cell barrier architecture. Atrial natriuretic peptide (ANP) inhibited VPF signaling, TJ protein phosphorylation and localization, and VPF-induced permeability. This involved both guanylate cyclase and natriuretic peptide clearance receptors. In vivo, transgenic mice that overexpress ANP showed significantly less VPF-induced kinase activation and vascular permeability compared with non-transgenic littermates. Thus, ANP acts as an anti-permeability factor by inhibiting the signaling functions of VPF that we define here and by preserving the endothelial cell TJ functional morphology.     

Solution structure and dynamics of epidermal growth factor and transforming growth factor alpha.

Circular dichroism (CD) and Fourier transform infrared spectroscopic studies have shown that the secondary structure of transforming growth factor alpha (TGF-alpha) is very similar to that of epidermal growth factor (EGF). The infrared spectra revealed a minor difference between the two proteins, in particular in the beta-sheet structure. A large difference was observed with CD between the two proteins in the apparent conformation each adopts when the disulfide bonds are reduced. Reduced TGF-alpha showed a distinct alpha-helical conformation only at a high trifluoroethanol concentration, whereas reduced EGF assumed an alpha-helical conformation in the absence of trifluoroethanol. This indicates that these two proteins adopt different secondary structures in the absence of disulfide bonds, although they assume similar folding structures in their presence. These data suggest that the disulfide bonds to a large degree dictate the conformation of these two proteins. Additionally, differences in the dynamic behavior between EGF and TGF-alpha were also observed. Infrared experiments showed that the hydrogen-deuterium exchange rate is much higher for TGF-alpha than for EGF, indicating that TGF-alpha is a more flexible molecule. The rate of reduction of the disulfide bonds by dithiothreitol was also faster for TGF-alpha. Therefore, it can be concluded that although EGF and TGF-alpha have a similar overall conformation, TGF-alpha is a more flexible molecule than EGF.     

Multidomain binding of transforming growth factor alpha to the epidermal growth factor receptor.

Solubilized epidermal growth factor receptor (EGF-R) has been used in an extension of the Geysen epitope mapping protocol in order to provide additional insight into the amino acid residues in human transforming growth factor alpha (hTGF alpha) which are critical to recognition and binding. Overlapping heptapeptides which encompassed the 50 amino acid primary sequence of hTGF alpha were synthesized on a polyethylene solid phase, and the amount of detergent-solubilized EGF-R bound to each peptide was measured using ELISA. EGF-R appeared to bind reproducibly to four heptapeptides cognate to sequences in both the N- and C-domains of hTGF alpha (residues 22-28, 28-34, 36-42, and 44-50). Visualization of these four regions on three-dimensional solution phase structures of hTGF alpha, derived from 1H NMR measurements [Kline, T.-P., Brown, F.K., Brown, S.C., Jeffs, P.W., Kopple, K.D., & Mueller, L. (1990) Biochemistry 29, 7805-7813], indicated that the peptide segments are located on a single face of the protein and suggested the presence of a potential receptor binding cavity. If peptide segments within both the N- and C-domains of hTGF alpha are involved in binding to EGF-R, then this has direct consequences for possible molecular mechanisms by which receptor activation might take place. For example, the observed conformational flexibility in the six NMR-derived hTGF alpha structures due to variations in the main-chain torsion angles of Val-33, in combination with the involvement of residues from both domains in the proposed binding cavity, may imply that receptor activation results from interdomain reorientation in the protein ligand.(ABSTRACT TRUNCATED AT 250 WORDS)