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K C Flanders

Publications and source records attributed to K C Flanders.

At least 109 records · Page 6Linked to original sources
PubMed1989

Transforming growth factor-beta 1: histochemical localization with antibodies to different epitopes.

We have localized transforming growth factor-beta (TGF-beta) in many cells and tissues with immunohistochemical methods, using two polyclonal antisera raised to different synthetic preparations of a peptide corresponding to the amino-terminal 30 amino acids of TGF-beta 1. These two antibodies give distinct staining patterns; the staining by anti-CC(1-30) is intracellular. This differential staining pattern is consistently observed in several systems, including cultured tumor cells; mouse embryonic, neonatal, and adult tissues; bovine fibropapillomas; and human colon carcinomas. The extracellular staining by anti-CC(1-30) partially resembles that seen with an antibody to fibronectin, suggesting that extracellular TGF-beta may be bound to matrix proteins. The intracellular staining by anti-LC(1-30) is similar to that seen with two other antibodies raised to peptides corresponding to either amino acids 266-278 of the TGF-beta 1 precursor sequence or to amino acids 50-75 of mature TGF-beta 1, suggesting that anti-LC(1-30) stains sites of TGF-beta synthesis. Results from RIA and ELISAs indicate that anti-LC(1-30) and anti-CC(1-30) recognize different epitopes of this peptide and of TGF-beta 1 itself.

Animals↗
PubMed1989

Expression of transforming growth factor-beta 1 in specific cells and tissues of adult and neonatal mice.

We have used immunohistochemical techniques to detect transforming growth factor-beta 1 (TGF-beta 1) in many tissues of adult and neonatal mice. Each of two antibodies raised to the amino-terminal 30 amino acids of TGF-beta 1 selectively stained this molecule in either intracellular or extracellular locations. Strong intracellular staining was found in adrenal cortex, megakaryocytes and other cells of the bone marrow, cardiac myocytes, chondrocytes, renal distal tubules, ovarian glandular cells, and chorionic cells of the placenta. Marked staining of extracellular matrix was found in cartilage, heart, pancreas, placenta, skin, and uterus. Staining was often particularly intense in specialized cells of a given tissue, suggesting unique roles for TGF-beta within that tissue. Levels of expression of mRNA for TGF-beta 1 and its histochemical staining did not necessarily correlate in a given tissue, as in the spleen. The present data lend further support to the concept that TGF-beta has an important role in controlling interactions between epithelia and surrounding mesenchyme.

Adrenal Glands↗
PubMed1989

In vitro and in vivo association of transforming growth factor-beta 1 with hepatic fibrosis.

Despite extensive efforts, little progress has been made in identifying the factors that induce hepatic fibrosis. Transforming growth factor-beta (TGF-beta) has been shown to enhance collagen production, therefore its role in hepatic fibrosis was investigated. Treatment of cultured hepatic cells with TGF-beta 1 increased type I procollagen mRNA levels 13-fold due to post-transcriptional gene regulation. When two animal models of hepatic fibrosis, murine schistosomiasis and CCl4-treated rats, were examined, they both exhibited increased levels of TGF-beta 1 gene expression at times that somewhat preceded the increase in collagen synthesis. In contrast, in murine schistosomiasis, mRNA levels of tumor necrosis factor and interleukin-1 peaked early in the fibrogenic process. Immunohistochemical analysis showed TGF-beta 1 to be present in normal mouse liver and to be markedly increased in mice infected with schistosomiasis. TGF-beta 1 appeared in the hepatic parenchyma, primarily in hepatocytes. These findings strongly suggest a role for TGF-beta 1 in a pathophysiological state.

Animals↗
PubMed1989

Retinoic acid induces transforming growth factor-beta 2 in cultured keratinocytes and mouse epidermis.

We have studied the functional interaction between retinoic acid and transforming growth factor-beta (TGF-beta), using the mouse epidermis as a model system. Treatment with retinoic acid increases expression of TGF-beta 2 in cultured keratinocytes in vitro, as well as in the epidermis in vivo. This TGF-beta 2 is secreted in a biologically active form that can bind to surface receptors, in contrast to most other conditions in which TGF-beta is secreted in a latent form. Specific antibodies to TGF-beta 2 partially reverse the ability of retinoic acid to inhibit DNA synthesis in cultured keratinocytes. The regulation of TGF-beta 2 expression by retinoic acid may have important physiological and pharmacological roles in the maintenance of epidermal homeostasis.

Animals↗
PubMed1989

Expression of tumor necrosis factor-alpha and transforming growth factor-beta 1 in acute liver injury.

Tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta 1 (TGF-beta 1) have a number of in vitro functions that could be important in vivo in acute liver injury and repair. Therefore, we investigated these two cytokines in acute liver damage. Northern blots of RNA isolated from rats sacrificed at various time intervals after a single oral dose of CCl4 revealed that TNF-alpha mRNA levels were elevated within 6 hr of CCl4 administration and returned to control values by 24-32 hr. In contrast, TGF-beta 1 mRNA levels started to rise significantly at 24 hr, peaked at 48 hr, and approached baseline levels by 72 hr. Identical changes in TNF-alpha and TGF-beta 1 mRNA levels were also seen with D-galactosamine-induced hepatotoxicity. Immunohistochemical analysis using a TGF-beta 1 antibody demonstrated increased hepatic staining in CCl4-treated rats, at times corresponding to the increases in TGF-beta 1 gene expression. Therefore, there is a differential expression of these cytokines in acute CCl4 and galactosamine hepatotoxicity with an early rise in TNF-alpha, suggesting that this cytokine may affect inflammation and cell toxicity, while TGF-beta 1 peaks later, when it may regulate hepatocyte proliferation and extracellular matrix repair.

Acute Disease↗
PubMed1988

Latent transforming growth factor-beta from human platelets. A high molecular weight complex containing precursor sequences.

Human platelets, when induced to degranulate by thrombin, secrete transforming growth factor-beta (TGF-beta) in a biologically latent form. In this form, TGF-beta cannot bind to its cellular receptor, nor can it be immunoprecipitated by polyclonal antisera to TGF-beta, suggesting that the receptor-binding site and other TGF-beta epitopes may be masked. Western blot analysis of the platelet secretate indicates that the latent form of TGF-beta is a 220-235 kDa complex, in which mature TGF-beta (25 kDa) is noncovalently associated with sequences from the remainder of the precursor (74 kDa), and a third unidentified entity (approximately 135 kDa). The third component is immunologically unrelated to other growth factor binding proteins. The complex is glycosylated, and gel filtration analysis suggests it may exist in solution as higher molecular weight aggregates. Further chromatographic analysis indicates that in its latent form, the platelet TGF-beta cannot bind to alpha 2-macroglobulin (alpha 2M), but that if the platelet latent TGF-beta is activated by transient acidification, the released active TGF-beta will bind to alpha 2M. We have previously identified the latent form of TGF-beta found in serum as an alpha 2M.TGF-beta complex (O'Connor-McCourt, M. D., and Wakefield, L. M. (1987) J. Biol. Chem. 262, 14090-14099). We now propose that the latent TGF-beta secreted by platelets may be a cellular delivery complex, whereas the latent form found in serum may represent a clearance complex. Thus alpha 2M may scavenge excess TGF-beta that is released when the platelet latent form is activated, possibly by the clotting process. Finally, we have shown that the latent form of TGF-beta secreted by a variety of cell types in culture is similar, if not identical to that secreted by platelets.

Amino Acid Sequence↗
PubMed1988

Transforming growth factor beta 1 positively regulates its own expression in normal and transformed cells.

Transforming growth factor beta 1 (TGF-beta 1) regulates the growth, differentiation, or function of nearly all cell types. We now report that TGF-beta 1 increases steady-state levels of its own message in six different normal and transformed cells in culture. Accumulation of TGF-beta 1 mRNA can be detected by Northern blot analysis within 3 h of addition of the peptide to cells, and enhanced message levels persist as long as TGF-beta 1 is present in the culture medium. This autoinduction is half-maximal at approximately 10 PM TGF-beta 1, and maximal stimulation corresponds to a 2-3-fold increase in transcript levels. In normal rat kidney cells, the rise in TGF-beta 1 mRNA is actinomycin D-sensitive and is accompanied by a parallel (approximately 3-fold) increase in secretion of TGF-beta 1 protein in the culture medium of treated cells, as detected by immunoprecipitation of biosynthetically labeled 35S-labeled TGF-beta 1 using specific anti-TGF-beta 1 antibodies. Treatment of normal rat kidney cells with either epidermal growth factor or platelet-derived growth factor also results in an increase in TGF-beta 1 mRNA (2-3-fold), although epidermal growth factor and TGF-beta 1 appear to act via distinct mechanisms since their combined effects are greater than additive.

Cell Line↗
PubMed1988

Antibodies to peptide determinants in transforming growth factor beta and their applications.

Polyclonal antibodies have been raised to a series of synthetic peptides which correspond to essentially all regions of the transforming growth factor beta 1 (TGF-beta 1) molecule. All antisera were evaluated for their abilities to react with TGF-beta 1 and TGF-beta 2 in either the native or reduced form in enzyme-linked immunosorbent assays, Western blots, and immunoprecipitation assays. While all antisera demonstrated some ability to recognize TGF-beta 1 in these systems, there was limited cross-reactivity with TGF-beta 2, suggesting that substantial sequence or conformational differences exist between the two growth factors. On Western blots 5-10 ng of purified human platelet TGF-beta 1 could be detected when probed with affinity-purified peptide antisera generated against peptides corresponding to residues 48-77, 50-75, and 78-109 of the 112 amino acid TGF-beta 1 monomer. Antisera raised against peptides 50-75 and 78-109 were most effective in immunoprecipitating reduced and native 125I-TGF-beta 1, respectively. The antisera also were tested for their effectiveness in blocking the binding of 125I-TGF-beta 1 to its receptor. Anti-peptide 78-109 and anti-peptide 50-75 blocked 80% and 40% of the binding, respectively, while antibodies against amino-terminal peptides were without effect. These data suggest that the carboxyl-terminal region of TGF-beta 1 may play a significant role in the binding of the native ligand to its receptor.

Animals↗
PubMed1988

Accumulation, localization, and compartmentation of transforming growth factor beta during endochondral bone development.

Endochondral bone formation was induced in postnatal rats by implantation of demineralized rat bone matrix. Corresponding control tissue was generated by implanting inactive extracted bone matrix, which did not induce bone formation. At various times, implants were removed and sequentially extracted with guanidine hydrochloride, and then EDTA and guanidine hydrochloride. Transforming growth factor beta (TGF beta) in the extracts was quantitated by a radioreceptor assay. TGF beta was present in demineralized bone matrix before implantation, and the concentration had decreased by 1 d after implantation. Thereafter, TGF beta was undetectable by radioreceptor assay until day 9. From day 9-21 the TGF beta was extracted only after EDTA demineralization, indicating tight association with the mineralized matrix. During this time, the content of TGF beta per milligram soluble protein rose steadily and remained high through day 21. This increased concentration correlated with the onset of vascularization and calcification of cartilage. TGF beta was detected only between days 3-9 in the controls; i.e., non-bone-forming implants. Immunolocalization of TGF beta in bone-forming implants revealed staining of inflammatory cells at early times, followed later by staining of chondrocytes in calcifying cartilage and staining of osteoblasts. The most intense staining of TGF beta was found in calcified cartilage and mineralized bone matrix, again indicating preferential compartmentalization of TGF beta in the mineral phase. In contrast to the delayed expression of TGF beta protein, northern blot analysis showed TGF beta mRNA in implants throughout the sequence of bone formation. The time-dependent accumulation of TGF beta when cartilage is being replaced by bone in this in vivo model of bone formation suggests that TGF beta may play a role in the regulation of ossification during endochondral bone development.

Animals↗
PubMed1988

Endothelial cells synthesize basic fibroblast growth factor and transforming growth factor beta.

Endothelial cells, including human umbilical vein endothelial cells (HUVEC), bovine aortic endothelial cells (BAEC), and bovine capillary endothelial cells (BCEC) in culture synthesize basic fibroblast growth factor (bFGF) and transforming growth factor type beta (TGF-beta). Basic FGF was cell-associated and synthesis was demonstrated by (i) the presence of bFGF mRNA species, (ii) binding to heparin-Sepharose and elution at 1.5 M NaCl, (iii) cross-reactivity with anti-bFGF antibodies when analyzed by electrophoretic blotting, and (iv) biological activity. Basic FGF was found in cell lysates at 2.3 ng/10(6) cells in HUVEC, 2.0 ng/10(6) cells in BCEC, and 13 ng/10(6) cells in BAEC. TGF-beta was secreted into media, and synthesis was demonstrated by (i) presence of TGF-beta mRNA species, (ii) cross-reactivity with anti-TGF-beta antibodies when analyzed by immunoprecipitation, (iii) competitive binding with authentic human platelet-derived TGF-beta that was blocked by TGF-beta specific blocking antibodies, and (iv) inhibition of [3H]TdR incorporation in CCl-64 cells. TGF-beta was secreted in an inactive form and required acid activation for detection. HUVEC synthesized 2.0 ng TGF-beta/10(6) cells per 12 hr; BCEC, 3.5 ng; and BAEC, 3.5 ng. HUVEC proliferation was not affected by treatment with exogenous TGF-beta, while BCEC proliferation was decreased by treatment with TGF-beta. Vascular endothelium is thus a source for these two potent multifunctional regulatory molecules, both of which may affect the growth of endothelium and neighboring fibroblasts, smooth muscle cells and white blood cells. The activation or release of these factors by endothelium may be a precipitating event in important cellular processes such as wound healing, organogenesis, and angiogenesis.

Animals↗
PubMed1988

Transforming growth factor beta-1 in acute myocardial infarction in rats.

TGF-beta 1 has been examined in the heart during myocardial infarction caused by ligation of the left coronary artery. Infarcted and uninfarcted myocardium have been compared by immunohistochemical staining of TGF-beta 1 and by Northern blot analysis of mRNA. Normal ventricular myocytes are strongly stained by an antibody to TGF-beta 1. Progressive loss of staining of these myocytes begins within 1 hr after coronary ligation. However, by 24-48 hr after ligation, intense staining of myocytes at the margin of infarcted areas is seen. Northern blots of infarcted myocardium 48 hr after ligation show a 3- to 4-fold increase in the principal 2.4 kb TGF-beta 1 mRNA; there is also a marked increase in a minor 1.9 kb transcript. In the same tissue samples, there is a 2-fold decrease in the mRNA for the glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase. The results indicate a significant role for TGF-beta in the response of the heart to injury.

Animals↗
PubMed1988

Increased expression of growth factor mRNAs accompanies viral transformation of rodent cells.

Transforming growth factors-alpha and beta (TGF-alpha and -beta) and platelet-derived growth factor (PDGF), three distinct peptide hormones, acting together, are able to potentiate the phenotypic transformation of normal rat kidney (NRK) cells. Cells transformed by retroviruses have been shown to secrete increased levels of TGF-alpha, TGF-beta and PDGF. We report here that Harvey sarcoma virus-transformed NIH-3T3 cells and Moloney sarcoma virus-transformed NRK cells show increased expression of the mRNAs for TGF-alpha, TGF-beta, PDGF A-chain and nerve growth factor (NGF) compared to their untransformed counterparts. No amplification or rearrangement in the genomic DNA is seen in the transformed cells. In tumor tissue formed by subcutaneous injection of Ha-3T3 cells, this enhanced level of TGF-beta mRNA returns to the control level of the untransformed cells. The increase in TGF-beta mRNA in the transformed cells is paralleled by an increase in the level of TGF-beta protein as shown by immunoprecipitation and Western blotting procedures using specific TGF-beta antibodies.

Animals↗
PubMed1987

Evidence that transforming growth factor-beta is a hormonally regulated negative growth factor in human breast cancer cells.

The hormone-dependent human breast cancer cell line MCF-7 secretes transforming growth factor-beta (TGF-beta), which can be detected in the culture medium in a biologically active form. These polypeptides compete with human platelet-derived TGF-beta for binding to its receptor, are biologically active in TGF-beta-specific growth assays, and are recognized and inactivated by TGF-beta-specific antibodies. Secretion of active TGF-beta is induced 8 to 27-fold under treatment of MCF-7 cells with growth inhibitory concentrations of antiestrogens. Antiestrogen-induced TGF-beta from MCF-7 cells inhibits the growth of an estrogen receptor-negative human breast cancer cell line in coculture experiments; growth inhibition is reversed with anti-TGF-beta antibodies. We conclude that in MCF-7 cells, TGF-beta is a hormonally regulated growth inhibitor with possible autocrine and paracrine functions in breast cancer cells.

Breast Neoplasms↗
PubMed1987

Semisynthetic D-His1,N epsilon-acetimidoglucagon: structure-function relationships.

The histidine residue at the amino terminus of lysine-12 protected glucagon was replaced by its D-isomer by an established semisynthetic strategy to extend a stepwise series of replacements at this position. The product was examined for its secondary structure and its function. Circular dichroism spectra obtained at concentrations from 0.25 to 1.09 mg/ml at pH 10.2 in 0.2 M phosphate buffer were similar to those obtained with native hormone. Competitive binding assays and adenylate cyclase activation assays with partially purified rat liver plasma membranes show this D-His1 analog of glucagon to be a full agonist, causing the same maximum activation of adenylate cyclase as native hormone; but both binding and activation assays show the binding affinity to be diminished about 10-fold. The data suggest that the adjustment of the bonding of the imidazole group to the receptor to bring about transduction results in constraints on the conformation along the peptide sequence which interfere with the peptide adopting the same binding conformation achieved by the native hormone.

Adenylyl Cyclases↗
PubMed1987

Osteoblasts synthesize and respond to transforming growth factor-type beta (TGF-beta) in vitro.

Transforming growth factor-type beta (TGF-beta) has been identified as a constituent of bone matrix (Seyedin, S. M., A. Y. Thompson, H. Bentz, D. M. Rosen, J. M. McPherson, A. Conti, N. R. Siegel, G. R. Gallupi, and K. A. Piez, 1986, J. Biol. Chem. 261:5693-5695). We used both developing bone and bone-forming cells in vitro to demonstrate the cellular origin of this peptide. TGF-beta mRNA was detected by Northern analysis in both developing bone tissue and fetal bovine bone-forming cells using human cDNA probes. TGF-beta was shown to be synthesized and secreted by metabolically labeled bone cell cultures by immunoprecipitation from the medium. Further, TGF-beta activity was demonstrated in conditioned media from these cultures by competitive radioreceptor and growth promotion assays. Fetal bovine bone cells (FBBC) were found to have relatively few TGF-beta receptors (5,800/cell) with an extremely low Kd of 2.2 pM (high binding affinity). In contrast to its inhibitory effects on the growth of many cell types including osteosarcoma cell lines, TGF-beta stimulated the growth of subconfluent cultures of FBBC; it had little effect on the production of collagen by these cells. We conclude that bone-forming cells are a source for the TGF-beta that is found in bone, and that these cells may be modulated by this factor in an autocrine fashion.

Animals↗
PubMed1987

Role of transforming growth factor-beta in the development of the mouse embryo.

Using immunohistochemical methods, we have investigated the role of transforming growth factor-beta (TGF-beta) in the development of the mouse embryo. For detection of TGF-beta in 11-18-d-old embryos, we have used a polyclonal antibody specific for TGF-beta type 1 and the peroxidase-antiperoxidase technique. Staining of TGF-beta is closely associated with mesenchyme per se or with tissues derived from mesenchyme, such as connective tissue, cartilage, and bone. TGF-beta is conspicuous in tissues derived from neural crest mesenchyme, such as the palate, larynx, facial mesenchyme, nasal sinuses, meninges, and teeth. Staining of all of these tissues is greatest during periods of morphogenesis. In many instances, intense staining is seen in mesenchyme when critical interactions with adjacent epithelium occur, as in the development of hair follicles, teeth, and the submandibular gland. Marked staining is also seen when remodeling of mesenchyme or mesoderm occurs, as during formation of digits from limb buds, formation of the palate, and formation of the heart valves. The presence of TGF-beta is often coupled with pronounced angiogenic activity. The histochemical results are discussed in terms of the known biochemical actions of TGF-beta, especially its ability to control both synthesis and degradation of both structural and adhesion molecules of the extracellular matrix.

Animals↗