Transforming growth factor-alpha and its role in neoplastic progression.
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Amino Acid Sequence↗
Biomedical subjects
D C Lee
Publications and source records attributed to D C Lee.
Possible role of transforming growth factor alpha in the pathogenesis of Ménétrier's disease: supportive evidence form humans and transgenic mice.
Ménétrier's disease is an uncommon disorder of unknown etiology characterized by enlarged gastric folds with foveolar hyperplasia and cystic dilatation of gastric glands. Biochemical features that are seen frequently include hypoproteinemia, hypochlorhydria, and increased gastric mucus. Because transforming growth factor alpha (TGF alpha) is an epithelial cell mitogen that inhibits gastric acid secretion and increases gastric mucin content, we hypothesized that its altered expression might be involved in the pathogenesis of this disease. Therefore, we characterized TGF alpha immunoreactivity in the gastric mucosa of 4 patients with Ménétrier's disease. In contrast to the normal pattern of TGF alpha immunostaining in which TGF alpha appears most concentrated in parietal cells, there was intense staining in the majority of mucous cells in the gastric mucosa of patients with Ménétrier's disease. In one patient from whom sufficient fresh tissue was obtained to isolate RNA, expression of TGF alpha and the epidermal growth factor receptor was higher in the gastric mucosa relative to a normal control. In addition, metallothionein-TGF alpha transgenic mice, which overexpress TGF alpha in gastric mucosa, show a number of features characteristic of Ménétrier's disease. These include foveolar hyperplasia and glandular cystic dilatation, increased gastric neutral mucin staining, and reduced basal and histamine-stimulated rates of acid production. Taken together, observations derived from the human material and correlation with data from a transgenic mouse model support an important role for TGF alpha in the pathogenesis of Ménétrier's disease.
Animals↗
Anti-tetanus toxoid antibodies in intravenous gamma globulin: an alternative to tetanus immune globulin.
The levels of anti-tetanus toxoid IgG antibodies were measured in 29 lots of intravenous gamma globulin (IVIG). The antibody levels varied from 4 to 90 IU/mL (geometric mean, 18.6; 90% confidence interval, 9.7-35.7). The variation from manufacturer to manufacturer accounted for most of the observed differences among lots; there was relatively little variability among multiple lots from a single manufacturer. IVIG may be an acceptable alternative to horse or human tetanus immune globulin.
Antibodies, Bacterial↗
Induction of transforming growth factor-alpha gene expression in rat decidua is independent of the conceptus.
We have previously shown that decidua is the major site of transforming growth factor-alpha (TGF-alpha) gene expression at the feto-maternal interface during the early stages of pregnancy in the rat. The present study was undertaken to determine whether the presence of TGF-alpha mRNA in the decidua was in direct response to a signal derived from the conceptus. We therefore used the model of pseudopregnancy in rat, inducing decidualization in the absence of a conceptus. TGF-alpha mRNA expression was examined using Northern blot analysis, and TGF-alpha peptide was localized using immunohistochemistry. Pseudopregnancy was produced through standard hormonal priming followed by an artificial stimulus of intrauterine sesame oil or PBS to induce decidualization. Northern blot analysis demonstrated TGF-alpha mRNA expression specifically in the deciduoma beginning on the day of decidual stimulus (Day 4), increasing on Day 6, and continuing through Day 14. In contrast to decidua of normal pregnancy, an additional transcript of 3.0 kb was present in the deciduoma. Immunohistochemistry of tissues from Day 9 of pseudopregnancy revealed specific cytoplasmic immunoreactivity in the majority of stromal type decidual cells of the antimesometrial area, whereas mesometrial decidual cells were not immunoreactive. The results indicate that the induction of decidual TGF-alpha gene expression is not in direct response to a signal derived from the conceptus but is a de novo event associated with the decidual reaction, and that the resulting TGF-alpha peptide is present in the majority of stromal type antimesometrial decidual cells. These findings also suggest that TGF-alpha could be playing a role in proliferation and/or differentiation of the decidua.
Animals↗
Transforming growth factor-alpha is a potential mediator of estrogen action in the mouse uterus.
To better understand the role of peptide growth factors in sex steroid hormone-mediated growth of the female reproductive tract, the effect of estrogen on the expression of transforming growth factor-alpha (TGF alpha) in mouse uterus was investigated. Our results show that estrogen induces the expression of TGF alpha mRNA in the mouse uterus in a dose- and time-dependent manner. The up-regulation of TGF alpha transcripts occurs predominantly in uterine epithelial cells. RIA and Western blot analysis demonstrate that immunoreactive TGF alpha protein is secreted at high levels into mouse uterine luminal fluid after estrogen treatment. The induction of uterine TGF alpha mRNA is specific to estrogen; nonestrogenic steroids did not induce expression. Antibody specific to TGF alpha significantly reduces estrogen-mediated uterine growth, which supports the concept that TGF alpha is a mitogen for the reproductive tract. Analysis of TGF alpha/EGF receptors by binding, affinity labeling, and phosphorylation studies indicates that functional receptors are present in the mouse uterus after estrogen exposure. Thus, our data support a physiological role for TGF alpha and its receptor pathway in the female mouse reproductive tract.
Animals↗
The binding of transcription factor Sp1 to multiple sites is required for maximal expression from the rat transforming growth factor alpha promoter.
Transcription from the rat transforming growth factor alpha (TGF-alpha) promoter initiates at multiple sites within a 200-bp G+C-rich region that lacks TATA and CAAT motifs but contains multiple potential binding sites for the transcription factor Sp1. In the present study, we used deletion analysis to establish the 5' boundary of sequences required for efficient transcription from the predominant -58 start site. We then examined the functional activity of several putative Sp1 binding sites that occur within the transcriptionally important region downstream from -155. In the presence of either crude HeLa cell extract or purified Sp1 protein, two double-stranded oligonucleotides from this region, each of which contains three Sp1 elements, both displayed gel mobility shifts that were specifically inhibited in the presence of excess bona fide Sp1-binding sequence from the hamster dihydrofolate reductase (DHFR) gene. Methylation interference analysis of the major gel-shift complex produced with one of these oligonucleotides revealed a series of critical guanine residues located in, or adjacent to, one of the putative Sp1 recognition elements. The functional importance of Sp1 binding was confirmed by the findings that transcription in vitro from the -58 start site was inhibited in the presence of excess consensus Sp1-binding sequence and that coexpression of Sp1 dramatically induced TGF-alpha promoter-directed chloramphenicol acetyl transferase (CAT) expression in transfected Drosophila Schneider cells that otherwise lack functional Sp1. Additionally, the introduction of mutations into any of several putative Sp1 elements inhibited transcription in vitro from the -58 site, with the most proximal element tested having the largest effect. Sequential mutation of more distal elements produced a synergistic inhibition of transcription, suggesting that cooperative interactions between Sp1 sites are necessary for full expression from the TGF-alpha promoter.
Animals↗
Clonal cosegregation of tumorigenicity with overexpression of c-myc and transforming growth factor alpha genes in chemically transformed rat liver epithelial cells.
Tumorigenicity was correlated with levels of expression of the genes for transforming growth factor alpha (TGF-alpha), epidermal growth factor receptor, c-myc, c-H-ras, and c-K-ras in a series of 16 clonally derived transformed liver epithelial cell lines. The clonal lines, which varied in tumorigenicity from 0 to 97%, were established from a phenotypically heterogeneous population produced by repeated exposure of diploid WB-F344 (WB) cells to N-methyl-N'-nitro-N-nitrosoguanidine. Segregation of gene expression with tumorigenicity among clonal lines was determined by correlating rank orders of gene expression by clones relative to expression by wild-type WB cells. Only the expression of the c-myc gene correlated with tumorigenicity among all transformed clones. TGF-alpha gene expression was not correlated with tumorigenicity among all clones, but it was highly correlated with tumorigenicity among clones that expressed the c-myc gene above the median level for all clones (greater than 5-fold the level of expression by WB cells). Even high levels of expression of the TGF-alpha gene (up to 60-fold the level of expression by WB cells) were not correlated with tumorigenicity among the clones expressing the c-myc gene at levels less than 5-fold the level of expression by WB cells. Clones which simultaneously overexpressed both c-myc and TGF-alpha genes at levels above the median levels for all clones were significantly more tumorigenic than were clones which expressed either or both genes at lower than median levels. These results suggest that overexpressed c-myc and TGF-alpha genes cooperate in their association with tumorigenicity. Most of the highly tumorigenic clones that overexpressed c-myc and TGF-alpha also overexpressed the c-H-ras and/or the c-K-ras genes; clones that overexpressed neither of the c-ras genes nor the genes for c-myc and TGF-alpha were not very tumorigenic, while clones that expressed one or both c-ras genes (but not both c-myc and TGF-alpha) were variably tumorigenic over an intermediate range.
Animals↗
Regional expression of transforming growth factor-alpha mRNA in the rat central nervous system.
The expression of transforming growth factor-alpha (TGF alpha) mRNA in various regions of the adult rat central nervous system (CNS), as well as in selected peripheral tissues was examined using Northern blot analysis. The highest expression was also found in the cerebral spinal cord, with the levels of TGF alpha mRNA being 5- to 6-fold higher in this tissue than in any other examined. Significant expression was also found in the olfactory bulb, anterior olfactory nucleus, corpus striatum, hippocampus, ventral mesencephalon and caudal brainstem. Of the peripheral tissues examined, only adrenal gland expressed TGF alpha mRNA at similar levels. Lower, but detectable, expression was found in prefrontal cortex, cerebral cortex and cerebellum. The latter levels were similar to those observed in lung, liver, kidney and, variable, salivary gland. These data demonstrate a widespread but differential distribution of TGF alpha mRNA throughout the rat CNS, and indicate relatively high levels of expression of this growth factor in central versus peripheral tissues.
Animals↗
Determination of protein secondary structure using factor analysis of infrared spectra.
A method is presented for determining the secondary structural composition of a protein in aqueous solution from its infrared spectrum. A factor analysis approach is used to analyze the infrared spectra of 18 proteins whose crystal structures are known from X-ray studies. Factor analysis followed by multiple linear regression identifies those eigenspectra that correlate with the variation in properties described by the calibration set. The properties of interest in this study are % alpha-helix, % beta-sheet, and % turns. In the analysis of an unknown, the factor loadings required to reproduce its spectrum are substituted in the regression equation for each property to predict its secondary structural composition. The accuracy of the method was determined by removing each standard, in turn, from the calibration set and using a calibration set generated from the remainder to predict its composition. By this method we obtain standard errors of prediction of 3.9% for alpha-helix, 8.3% for beta-sheet, and 6.6% for turns. The method may also be applied to the spectra of proteins in 2H2O. The method has important advantages over those currently in use for the quantitative analysis of the infrared spectra of proteins. Manipulation of the spectrum is kept to a minimum, no curve-fitting is necessary, and the several amide I band components need not be assigned.
Calibration↗
Overexpression of TGF alpha in transgenic mice: induction of epithelial hyperplasia, pancreatic metaplasia, and carcinoma of the breast.
Metallothionein-directed expression of TGF alpha in transgenic mice induced a spectrum of changes in the growth and differentiation of certain adult tissues. First, TGF alpha promoted a uniform epithelial hyperplasia of several organs without otherwise causing major alterations in tissue architecture. Second, in pancreas it promoted proliferation of both acinar cells and fibroblasts and focally altered acinar cell differentiation. The magnitude of this response was proportional to the level of local, tissue-specific TGF alpha expression and was reproduced when expression of TGF alpha was placed under the control of the elastase promoter, implying an autocrine or paracrine mechanism. Third, TGF alpha was oncogenic in vivo. It caused dramatic hyperplasia and dysplasia of the coagulation gland epithelium, which displayed evidence of carcinoma in situ, and in postlactational mammary gland it induced secretory mammary adenocarcinomas. Thus, TGF alpha displays characteristics of both a potent epithelial cell mitogen and an oncogenic protein in vivo.
Animals↗
Generation of transforming growth factor-alpha from the cell surface by an O-glycosylation-independent multistep process.
The precursor for transforming growth factor-alpha (TGF-alpha) is a membrane glycoprotein that can establish contact with epidermal growth factor/TGF-alpha receptors on adjacent cells or can be cleaved to release TGF-alpha that diffuses into the medium. Cleavage of pro-TGF-alpha occurs at Ala/Leu-Ala/Leu-Ala-Val-Val sites located at each end of the mature TGF-alpha sequence. To characterize the cleavage process of pro-TGF-alpha and the role of glycosylation in this process, we have introduced a pro-TGF-alpha expression vector in wild type Chinese hamster ovary (CHO) cells and in the mutant CHO cell clone ldlD that has a reversible defect in protein glycosylation. Analysis of metabolically labeled and cell surface-labeled products immunoprecipitated with antibodies directed against the extracellular TGF-alpha sequence and the cytoplasmic pro-TGF-alpha C-terminal domain shows that cleavage of pro-TGF-alpha in wild type CHO cells occurs in two steps. Both processing steps occur after pro-TGF-alpha reaches the cell surface. In the first step, pro-TGF-alpha rapidly (t1/2 = 30 min) loses the amino-terminal segment that precedes the TGF-alpha sequence. In the second step, pro-TGF-alpha is cleaved at the carboxyl terminus of the TGF-alpha sequence releasing this factor into the medium. This second step is slow (t1/2 = 2 h). The action of pancreatic elastase added to CHO-TGF-alpha cells mimics the first step but not the second one. Synthesis, cell surface exposure, rate of cleavage, and generation of bioactive TGF-alpha in ldlD-TGF-alpha cells are not markedly affected by the lack of N-acetylgalactosamine-dependent protein O-glycosylation or galactose-dependent glycan chain modification. The results indicate that, despite their similarity in amino acid sequence, the two cleavage sites that flank TGF-alpha may be processed with different kinetics which can lead to retention of pro-TGF-alpha on the cell surface.
Amino Acid Sequence↗
Immunologic and physicochemical evidence for conformational changes occurring on conversion of human mast cell tryptase from active tetramer to inactive monomer. Production of monoclonal antibodies recognizing active tryptase.
The catalytic activity of human tryptase, a mast cell neutral endoprotease, is expressed when the enzyme is in its tetrameric form, but is lost under physiologic conditions concomitant with a quaternary structural alteration involving conversion to a monomeric form. The associated changes in the CD spectra noted in the current study indicate accompanying alterations in the secondary structure of the protein. In particular, the progressive disappearance of the negative minimum centered at 228 nm suggests an effect on beta-sheet structure, which may be important for monomer-monomer interaction and/or stabilization of catalytic activity. Dextran sulfate, like heparin, stabilizes the catalytic activity and quaternary structure of tryptase and also maintains the native secondary structure of the enzyme at and beyond a temperature of 40 degrees C. Dextran sulfate-stabilized tryptase therefore was used as an immunogen to which were produced three murine mAb (B2, C11, and G4) recognizing the catalytically active form of the enzyme. Inactive tryptase bound to plastic microtiter wells was not recognized by any of the newly made antibodies, whereas inactive tryptase in solution was recognized by G4, which when biotinylated, could be used as a detector antibody in a sandwich ELISA for tryptase. Each of the newly made mAb recognized the catalytically active form of tryptase. Thus, alterations in epitopes, perhaps reflecting tertiary structural alterations as well as changes in secondary and quaternary conformations, occur with tryptase inactivation. A pragmatic result of these newly generated antibodies is the affinity purification to homogeneity of active tryptase by sequential chromatography with B2 coupled to CH-Sepharose and heparin-agarose. Tryptase purified by this technique had a specific activity with p-tosyl-L-arginine methyl ester of 117 +/- 9 U/mg and had 3.9 +/- 0.3 active sites per molecule of active enzyme (134,000 m.w.) as titrated with p-nitrophenyl-p'-guanidinobenzoate. The spectral and immunologic data in the current study are consistent with concerted conformational alterations in the secondary and tertiary as well as quaternary structures of tryptase associated with loss of catalytic activity. Failure to reverse any of these alterations with dextran sulfate suggests that the pathway of tetramer assembly in vivo is more complicated than simple subunit association.
Antibodies, Monoclonal↗
TGF-alpha: expression and biological activities of the integral membrane precursor.
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Amino Acid Sequence↗
Characterization of the rat transforming growth factor alpha gene and identification of promoter sequences.
We have determined the complete nucleotide sequence of rat transforming growth factor alpha (TGF alpha) mRNA and characterized the six exons that encode this transcript. These six exons span approximately 85 kilobases of genomic DNA, with exons 1 to 3 separated by particularly large introns. What had previously been thought to represent a species-specific difference in the size of the TGF alpha precursor (proTGF alpha) is now shown to be due to microheterogeneity in the splicing of exons 2 and 3. This results from a tandem duplication of the acceptor CAG and gives rise to two alternate forms (159 and 160 amino acids) of the integral membrane precursor. Exon 6, which encodes the 3' untranslated region of TGF alpha mRNA, also encodes, on the opposite strand, a small (approximately 200-nucleotide) transcript whose sequence predicts an open reading frame of 51 amino acids. Expression of this latter transcript does not appear to be coregulated with that of TGF alpha mRNA. Primer extension and S1 nuclease analyses of authentic TGF alpha transcripts revealed two major and multiple minor 5' ends which span more than 200 base pairs of DNA in a G + C-rich region that lacks canonical CCAAT or TATA sequences. The 5' ends of six independently derived cDNAs localized to five different sites in this same region. Restriction fragments that overlap these transcription start sites and extend approximately 300 base pairs in the 5' direction faithfully promote transcription in vitro with HeLa cell nuclear extracts. In addition, they direct the expression of the bacterial chloramphenicol acetyltransferase gene in transient-transfection assays.
Amino Acid Sequence↗
A truncated, secreted form of the epidermal growth factor receptor is encoded by an alternatively spliced transcript in normal rat tissue.
Two independent cDNA clones corresponding to a 2.7-kilobase (kb) epidermal growth factor receptor (EGF-R) mRNA were isolated from a rat liver cDNA library. Sequence analysis revealed 100% homology in the external domain when compared with the full-length rat EGF-R nucleotide sequence and 80 to 90% similarity relative to the human EGF-R. However, the 3'-terminal sequence of these clones did not match EGF-R or any other known sequence(s) and was distinct from the 3' end of the 2.8-kb mRNA, which encodes a truncated EGF-R in A431 cells. The deduced amino acid sequence revealed an open reading frame which is homologous to the external domain of the EGF-R but which terminates prior to the transmembrane region. Southern blot analysis of rat genomic DNA indicated that the 3'-terminal sequence of this transcript is derived from the EGF-R gene. Analysis of a genomic clone containing the 3' end of the 2.7-kb transcript revealed that this sequence is present as a discrete exon in the mid-region of the receptor gene in proximity to the exon encoding the transmembrane domain. Introduction of an expression vector containing the truncated EGF-R cDNA into Chinese hamster ovary (CHO) cells led to the expression of a 95-kilodalton protein which was detected in conditioned media, by using antisera directed against the EGF-R. A similarly sized protein was also detected in the media of WB cells, a continuous, nontransformed line of rat hepatic epithelial cells. Northern (RNA blot) analysis established that the truncated receptor is encoded by a 2.7-kb transcript found in normal rat liver. Furthermore, Northern analysis of rat poly(A)+ RNA showed that the 2.7-kb EGF-R transcript is expressed at differing levels in various fetal and adult tissues. These data indicate that alternative splicing of the EGF-R primary transcript yields a 2.7-kb mRNA which codes for a truncated form of the receptor. This receptor is secreted by rat hepatic epithelial cells in culture, which suggests that it may be secreted by normal rat cells or tissues and perhaps serve an as yet unknown physiological function.
Amino Acid Sequence↗
Sequential changes in epidermal growth factor receptor/ligand function in cultured rat liver epithelial cells transformed chemically in vitro.
Diploid WB rat liver epithelial cells contain abundant, rapidly internalized epidermal growth factor receptors, and respond pleiotropically to ligand binding. Signal transduction pathways downstream from the EGF receptor involve activation of elements that are both dependent on and independent of protein kinase C activation. Neoplastic transformation of wild-type WB rat liver epithelial cells by exposure to N-methyl-N'-nitro-N-nitrosoguanidine is associated with progressive alterations in the responses of affected cells to binding of EGF to EGF receptors, including heightened cell proliferation and the expression of several other phenotypic properties. Tumorigenic rat liver epithelial cells acquire the ability to express transforming growth factor-alpha (TGF-alpha), and to secrete this growth factor in a regulated and then unregulated manner. TGF-alpha expression, together with the presence of abundant EGF receptors, provides affected cells with an autocrine growth cycle. The ability of transformed WB rat liver epithelial cells to produce tumors cosegregates clonally with TGF-alpha expression and with heightened expression of c-myc, c-Ha-ras and c-Ki-ras proto-oncogenes.
Animals↗
Transforming growth factor alpha: expression, regulation and biological action of its integral membrane precursor.
Transforming growth factor alpha (TGF alpha) is a 6 kDa polypeptide mitogen that interacts with the epidermal growth factor receptor and activates its intrinsic tyrosine kinase. The mature 50 amino acid TGF alpha is released from a 159 or 160 amino acid integral membrane glycoprotein precursor, denoted proTGF alpha, via cleavage at both termini by an unknown protease with elastase-like specificity. Rat proTGF alpha is encoded by a 4.5 kb mRNA that is transcribed from a gene containing 6 exons and spanning 85 kb of DNA. Expression of TGF alpha is most prevalent and abundant in transformed cells and tumors, but also detectable at modest levels in a limited number of normal cells and tissues. In many neoplastic cells, proteolytic processing of proTGF alpha is incomplete and/or inefficient, resulting in the preponderance of soluble and/or membrane-bound forms larger than the mature TGF alpha. To characterize the biological activities of the transmembrane TGF alpha precursor in the absence of processing, amino acid substitutions were introduced at the cleavage sites by site-directed mutagenesis of the rat TGF alpha cDNA. Fibroblasts expressing the mutant proTGF alpha constructs did not secrete TGF alpha, but did accumulate proTGF alpha at the cell surface. Coincubation of these cells with A431 cells resulted in binding and autophosphorylation of EGF receptors, and mobilization of intracellular calcium in A431 cells, demonstrating that the transmembrane proTGF alpha can activate EGF receptors on adjacent cells, leading to signal transduction. In addition, rat fibroblasts constitutively expressing the wild-type or mutant proTGF alpha became morphologically transformed in culture, and induced tumors in nude mice. Thus, the interaction between membrane-anchored ligand and receptor triggers mitogenesis that can culminate in neoplastic transformation. To characterize the physiological and pathological effects of TGF alpha in vivo, particularly with respect to epithelial cells, transgenic mice were developed which overexpress the growth factor in multiple or specific tissues. Widespread overexpression of TGF alpha driven by the metallothionein promoter induced epithelial hyperplasia in several organs, including liver and intestine, without disrupting normal tissue architecture. In contrast, the pancreas displayed increased proliferation of both acinar cells and fibroblasts, and focal alteration of acinar cell differentiation. This pancreatic hyperplasia, fibroplasia, and metaplasia were reproduced when TGF alpha expression was placed under control of the elastase promoter, and thus locally restricted to acinar cells, suggesting autocrine and/or paracrine mode of action. Finally, overexpression of TGF alpha promoted neoplastic transformation of certain epithelia. In coagulation gland, there was dramatic hyperplasia and dysplasia with focal evidence of carcinoma in situ.(ABSTRACT TRUNCATED AT 400 WORDS)
Amino Acid Sequence↗
Expression of the TGF alpha integral membrane precursor induces transformation of NRK cells.
TGF alpha is one member of a family of soluble growth factors that are derived from integral-membrane precursors. The mature form of TGF alpha is released from its transmembrane precursor (proTGF alpha) by a protease that, in many tumor cells, is inefficient or limiting. We have previously established that, in the absence of processing, membrane-anchored proTGF alpha is biologically active and can interact with the EGF receptor on adjacent cells, thereby inducing the receptor's intrinsic tyrosine kinase activity. We further showed that this interaction leads to immediate downstream signal transduction as evidenced by Ca2+ mobilization. To extend these observations, and to investigate its transforming potential, we infected normal rat kidney (NRK) cells with retroviral expression vectors that encode mutated forms of proTGF alpha containing amino acid substitutions at the proteolytic cleavage sites. NRK cells harboring these mutant constructs do not secrete mature growth factor, but do express biologically active proTGF alpha on the cell surface as shown by their ability to induce the autophosphorylation of EGF receptor on neighboring A431 cells in co-culture. Expression of the mutant proTGF alpha molecules promoted the anchorage-independent growth of NRK cells in soft agar, and caused them to be tumorigenic when injected into nude mice. These results demonstrate that an interaction between EGF receptor and the integral membrane precursor to TGF alpha can provide a mitogenic stimulus that leads to transformation. They further suggest that the accumulation of proTGF alpha on the surface of some transformed cells has physiological relevance.
Animals↗