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K C Flanders

Publications and source records attributed to K C Flanders.

At least 91 records · Page 5Linked to original sources
PubMed1991

Expression of transforming growth factor-beta s 1-4 in chicken embryo chondrocytes and myocytes.

cDNA probes and antibodies for TGF-beta s 1, 2, 3, and 4 were used to study the expression of these different TGF-beta isoforms in cultured chicken embryo chondrocytes and cardiac myocytes, as well as in developing cartilage and heart tissues. TGF-beta s 2, 3, and 4 mRNAs, but not TGF-beta 1 mRNA, were detected in cultured chondrocytes and myocytes. Expression of TGF-beta s 2 and 4 mRNAs increased with age, while expression of TGF-beta 3 mRNA was independent of age in chondrocytes cultured from 12- to 17-day-old embryos. In contrast, expression of TGF-beta s 2, 3, and 4 mRNAs was constitutive in myocytes cultured from 7- to 9-day-old embryonic hearts; expression of TGF-beta s 3 and 4 mRNAs increased, while expression of TGF-beta 2 mRNA remained unchanged in myocytes from 10-day-old embryos. Immunoprecipitation studies demonstrated expression of TGF-beta in both the conditioned media and the cell lysates of metabolically labeled chondrocyte and myocyte cell cultures. Immunohistochemical staining of cultured chondrocytes and myocytes and of cartilage and heart tissues of developing chicken embryos with antibodies specific for each TGF-beta isoform showed immunoreactive TGF-beta s 1, 2, 3, and 4. Our results demonstrate coordinate expression of these four TGF-beta isoforms in chicken embryo chondrocytes and myocytes, both in vitro and in vivo, with expression of TGF-beta s 2, 3, and 4 mRNA and protein more prominent than that of TGF-beta 1.

Amino Acid Sequence↗
PubMed1991

Transforming growth factor beta isoforms in the adult rat central and peripheral nervous system.

The distribution of transforming growth factor-beta isoforms 1, 2 and 3 and transforming growth factor-beta 2 and 3 mRNAs in adult rat central and peripheral nervous system was examined using Northern blotting and isoform specific antibodies for immunocytochemistry. Transforming growth factor-beta 2 and 3 mRNA were present in all brain areas including cerebral cortex, hippocampus, striatum, cerebellum and brainstem. In sciatic nerve, transforming growth factor-beta 3 mRNA was highly expressed, but transforming growth factor-beta 2 mRNA was not detectable. Transforming growth factor-beta 1-like immunoreactivity was confined to meninges and choroid plexus in the brain and connective tissue in peripheral ganglia and nerves. Transforming growth factor-beta 2 and 3 immunoreactivity entirely overlapped and, in general, were found in large multipolar neurons. Highest densities of immunoreactive neuronal perikarya were present in spinal cord and brainstem motor nuclei, hypothalamus, amygdaloid complex, hippocampus and cerebral cortical layers II, III and V. Most thalamic nuclei, superior colliculi, periaqueductal gray and striatum were almost devoid of transforming growth factor-beta 2- and 3-immunoreactive neurons. Fibrous astrocytes in white matter areas were intensely immunostained. Most dorsal root ganglionic neurons, their satellite cells and Schwann cells in peripheral nerves were also labeled. Transforming growth factor-beta 2- and 3-immunoreactive neurons were localized in brain regions that have been shown to contain neurons synthesizing and/or storing basic fibroblast growth factor suggesting possible opposing or synergistic effects of these peptide growth factors. However, the precise functions of local synthesis and storage of the transforming growth factor-beta isoforms in the nervous system are as yet unknown.

Animals↗
PubMed1991

Localization of transforming growth factor-beta 1 in mitochondria of murine heart and liver.

Using both electron microscopic immunohistochemistry and cell fractionation techniques, we show that transforming growth factor-beta 1 (TGF-beta 1) is found in mitochondria of rat and mouse cardiac myocytes and rat hepatocytes. Four different polyclonal antibodies, raised against various epitopes encompassing the mature portion of the TGF-beta 1 molecule as well as the pro-region of its precursor, were used for the electron microscopy studies. The localization of TGF-beta 1 in mitochondria was confirmed by detection of the native peptide in mitochondria isolated from rat heart and liver; the majority of native TGF-beta 1 found in liver homogenates was recovered in highly pure mitochondrial fractions. The functional role of TGF-beta in the mitochondrion is unknown at present.

Animals↗
PubMed1991

Increased production and immunohistochemical localization of transforming growth factor-beta in idiopathic pulmonary fibrosis.

Transforming growth factor-beta (TGF-beta) can regulate cell growth and differentiation as well as production of extracellular matrix proteins. Elevated production of TGF-beta has been associated with human and rodent chronic inflammatory and fibrotic diseases. Using immunohistochemical staining, we have examined lung sections of patients with advanced idiopathic pulmonary fibrosis (IPF), a disease characterized by chronic inflammation and fibrosis and demonstrated a marked and consistent increase in TGF-beta production in epithelial cells and macrophages when compared to patients with nonspecific inflammation and those with no inflammation or fibrosis. In patients with advanced IPF, intracellular staining with anti-LC (1-30) TGF-beta antibody was seen prominently in bronchiolar epithelial cells. In addition, epithelial cells of honeycomb cysts and hyperplastic type II pneumocytes stained intensely. Anti-CC (1-30) TGF-beta antibody, which reacts with extracellular TGF-beta, was localized in the lamina propria of bronchioles and in subepithelial regions of honeycomb cysts in areas of dense fibroconnective tissue deposition. The close association of subepithelial TGF-beta to the intracellular form in advanced IPF suggests that TGF-beta was produced and secreted primarily by epithelial cells. Because of the well-known effects of TGF-beta on extracellular matrix formation and on epithelial cell differentiation, the increased production of TGF-beta in advanced IPF may be pathogenic to the pulmonary fibrotic and regenerative responses seen in this disease.

Biopsy↗
PubMed1991

Complex regulation of TGF beta expression by retinoic acid in the vitamin A-deficient rat.

We report the results of a histochemical study, using polyclonal antipeptide antibodies to the different TGF beta isoforms, which demonstrates that retinoic acid regulates the expression of TGF beta 2 in the vitamin A-deficient rat. Basal expression of TGF beta 2 diminished under conditions of vitamin A deficiency. Treatment with retinoic acid caused a rapid and transient induction of TGF beta 2 and TGF beta 3 in the epidermis, tracheobronchial and alveolar epithelium, and intestinal mucosa. Induction of TGF beta 1 expression was also observed in the epidermis. In contrast to these epithelia, expression of the three TGF beta isoforms increased in vaginal epithelium during vitamin A deficiency, and decreased following systemic administration of retinoic acid. Our results show for the first time the widespread regulation of TGF beta expression by retinoic acid in vivo, and suggest a possible mechanism by which retinoics regulate the functions of both normal and pre-neoplastic epithelia.

Animals↗
PubMed1991

Localization and actions of transforming growth factor-beta s in the embryonic nervous system.

We present evidence for unique localization and specific biological activities for transforming growth factor-beta s (TGF-beta s) 2 and 3, as compared to TGF-beta 1, in the nervous system of the 12-18 day mouse embryo. Each TGF-beta isoform was localized immunohistochemically by specific antibodies raised to peptides corresponding to unique sequences in the respective TGF-beta proteins. Staining for TGF-beta 1 was principally in the meninges, while TGF-beta s 2 and 3 co-localized in neuronal perikarya and axons, as well as in radial glial cells. In the central nervous system, staining was most prominent in zones where neuronal differentiation occurs and less intense in zones of active proliferation, while in the peripheral nervous system, many nerve fibers as well as their cell bodies were strongly immunoreactive for TGF-beta s 2 and 3. Functionally, we have also found that in the presence of an extract of chick eye tissue, TGF-beta s 2 and 3 inhibit survival of cultured embryonic chick ciliary ganglionic neurons in a dose-dependent fashion; TGF-beta 1 shows no inhibitory effects. Our data suggest that TGF-beta s 2 and 3 may play a role in regulation of neuronal migration and differentiation, as well as in glial cell proliferation and differentiation.

Animals↗
PubMed1991

Regulated expression and growth inhibitory effects of transforming growth factor-beta isoforms in mouse mammary gland development.

Transforming Growth Factor-beta 1 (TGF-beta 1) was previously shown to inhibit reversibly the growth of mouse mammary ducts when administered in vivo by miniature slow-release plastic implants. We now report a comparative analysis of three TGF-beta isoforms with respect to gene expression and localization of protein products within the mouse mammary gland. Our studies revealed overlapping expression patterns of TGF-beta 1, TGF-beta 2 and TGF-beta 3 within the epithelium of the actively-growing mammary end buds during branching morphogenesis, as well as within the epithelium of growth-quiescent ducts. However, TGF-beta 3 was the only isoform detected in myoepithelial progenitor cells (cap cells) of the growing end buds and myoepithelial cells of the mature ducts. During pregnancy, TGF-beta 2 and TGF-beta 3 transcripts increased to high levels, in contrast to TGF-beta 1 transcripts which were moderately abundant; TGF-beta 2 was significantly transcribed only during pregnancy. Molecular hybridization in situ revealed overlapping patterns of expression for the three TGF-beta isoforms during alveolar morphogenesis, but showed that, in contrast to the patterns of TGF-beta 1 and TGF-beta 2 expression, TGF-beta 3 is expressed more heavily in ducts than in alveoli during pregnancy. Developing alveolar tissue and its associated ducts displayed striking TGF-beta 3 immunoreactivity which was greatly reduced during lactation. All three isoforms showed dramatically reduced expression in lactating tissue. The biological effects of active, exogenous TGF-beta 2 and TGF-beta 3 were tested with slow-release plastic implants. These isoforms, like TGF-beta 1, inhibited mammary ductal elongation in situ by causing the disappearance of the proliferating stem cell layer (cap cells) and rapid involution of ductal end buds. None of the isoforms were active in inhibiting alveolar morphogenesis. We conclude that under the limited conditions of these tests, the three mammalian isoforms are functionally equivalent. However, striking differences in patterns of gene expression and in the distribution of immunoreactive peptides suggest that TGF-beta isoforms may have distinct roles in mammary growth regulation, morphogenesis and functional differentiation.

Animals↗
PubMed1990

Murine pregnancy decidua produces a unique immunosuppressive molecule related to transforming growth factor beta-2.

Non-T small lymphocytic suppressor cells in murine allopregnancy release a potent immunosuppressive factor in vitro that is neutralized by rabbit anti-transforming growth factor (TGF)-beta. Previous studies have suggested that the decidual suppressor factor (DSF) is smaller than TGF-beta 1, and in this paper, we show that DSF on HPLC-sieving columns also elutes later than TGF-beta 2. Nevertheless, DSF has the ability to promote anchorage-independent growth of NRK fibroblasts similar to TGF-beta s. Using turkey antibodies specific for TGF-beta 1 or beta 2, we show that DSF is related to TGF-beta 2 rather than TGF-beta 1, and this relationship was confirmed by using a panel of murine mAb to TGF-subtypes. PAGE and Western blotting showed that the TGF-beta 2-reactive molecules in HPLC-purified DSF was slightly smaller than TGF-beta 2 and approximately 20 to 23 kDa. The DSF molecule is therefore closely related to TGF-beta 2 but as released from decidua, differs in size. The TGF-beta 2-related decidual suppressor factor was also obtained from the decidua of synpregnant C.B.-17 severe combined immune deficiency (SCID) and pregnant SCID-BG (C57BL/6 background) mice, confirming the lack of T or B cell dependence of DSF production and the generality of production of a TGF-beta-related suppressor factor by decidua associated with successful pregnancy in mice.

Animals↗
PubMed1990

Transforming growth factor-beta: multifunctional regulator of differentiation and development.

Transforming growth factors-beta (TGF-beta) are 25 kilodalton (kDa) homodimeric peptides with multifunctional actions controlling the growth, differentiation and function of a broad range of target cells of both epithelial and mesenchymal derivation. They are expressed early in embryogenesis and their tissue-specific and developmentally dependent expression is strongly suggestive of an essential role in particular morphogenetic and histogenetic events. Five distinct TGF-beta s have been characterized so far, with 65-80% homology to each other. By using both molecular biological and immunohistochemical techniques, we are currently attempting to define specific sites of expression of the different TGF-beta s and to determine whether TGF-beta s 1-5 might have unique functions in development and in the mature organism. Comparative study of the promoter regions for the different TGF-beta s and for any particular TGF-beta in different species is also underway. Mechanistically, TGF-beta s act to control gene expression of their target cells, many of their actions converging on a complex, multifaceted scheme of control of matrix proteins and their interactions with cells; these effects on matrix are thought to mediate many of the effects of TGF-beta on development.

Amino Acid Sequence↗
PubMed1990

Cell type-specific expression of transforming growth factor-beta 1 in the mouse uterus during the periimplantation period.

Immunohistochemistry and in situ and Northern blot hybridization were employed to determine temporal and spatial expression of transforming growth factor-beta 1 (TGF beta 1) in the mouse uterus during the periimplantation period. The polyclonal antisera anti-LC-(1-30) and anti-CC-(1-30), raised against two different preparations of a peptide corresponding to the amino-terminal 30 amino acids of TGF beta 1, were used for histochemical analyses because of their distinct staining patterns. Anti-LC shows intracellular staining, while staining by anti-CC is primarily extracellular. The colocalization of intracellular staining by anti-LC with in situ hybridization of TGF beta 1 mRNA in the luminal and glandular epithelia on days 1-4 of pregnancy (day 1 = vaginal plug) indicates that the epithelial cells are the primary sites of TGF beta 1 synthesis during the preimplantation period. On the other hand, staining of the extracellular matrix of the stroma by anti-CC during this period suggests an active accumulation of TGF-beta 1 that is synthesized in and secreted from the epithelia. While intracellular staining and accumulation of TGF-beta 1 mRNA in the epithelia were clearly evident on days 1-4, the extracellular staining showed temporal fluctuations. The clear extracellular staining of the stroma that was observed on day 1 was absent on day 2; moderate staining was again visualized in the stroma on day 3 and was markedly increased on day 4.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Colocalization of TGF-beta 1 and collagen I and III, fibronectin and glycosaminoglycans during lung branching morphogenesis.

The possible in vivo role of TGF-beta 1 in regulating various proteins of the extracellular matrix, including fibronectin, collagen I and III, and glycosaminoglycans, was examined by immunohistochemical methods during critical stages of lung morphogenesis in the 11- to 18-day-old mouse embryo. Sections of Bouin-fixed, paraffin-embedded whole embryos were exposed to polyclonal antibodies specific to synthetic peptides present in the precursor part of TGF-beta 1 (pro-TGF-beta 1), in the processed TGF-beta 1 (antibody CC), collagen I and III, fibronectin, followed by the PAP or ABC technique to visualize the location of the antibody. GAG were stained with Alcian Blue 8GX. Our results indicate colocalization of TGF-beta 1 expression and that of matrix proteins in the developing lung when branching morphogenesis (cleft formation) and tissue stabilization occur. The presence of TGF-beta 1 at the epithelial-mesenchymal interfaces of stalks and clefts at a time when matrix proteins can first be visualized in these areas, suggests a direct participation of the growth factor in the development of the basic architecture of the lung.

Animals↗
PubMed1990

Antibodies to transforming growth factor-beta 2 peptides: specific detection of TGF-beta 2 in immunoassays.

Polyclonal antibodies have been raised to synthetic peptides corresponding to several regions of transforming growth factor-beta 2 (TGF-beta 2). All antisera were tested for their ability to react with either the native or reduced forms of both TGF-beta 1 and TGF-beta 2 in enzyme-linked immunosorbent assays, Western blots, and immunoprecipitation assays. On Western blots, antisera raised to a peptide corresponding to residues 50-75 of TGF-beta 2 specifically detected 5 ng TGF-beta 2, while antisera raised to regions 1-30 and 79-108 cross-reacted with TGF-beta 1. Anti-P 50-75(2) also localized TGF-beta 2 in murine placenta in immunohistochemical studies. In immunoprecipitation assays with either iodinated TGF-beta s or with media conditioned by cells labeled with [35S]cysteine, both anti-P 50-75(2) and anti-P 79-108(2) specifically immunoprecipitated TGF-beta 2 under reducing conditions only, while anti-P 79-108(2) also reacted with TGF-beta 1. None of the TGF-beta 2 peptide antibodies was able to block receptor binding of either TGF-beta 1 or 2. Analysis of the cross-reactivity patterns of these peptide antibodies suggests conformational differences between TGF-beta 1 and TGF-beta 2.

Amino Acid Sequence↗
PubMed1990

Transforming growth factor-beta expression in fibropapillomas induced by bovine papillomavirus type 1, in normal bovine skin, and in BPV-1-transformed cells.

There is substantial evidence to suggest that transforming growth factor-beta (TGF-beta) plays an important role in wound healing and tissue repair as well as in carcinogenesis. It has also been observed that naturally occurring bovine papillomavirus type 1 (BPV-1)-induced bovine fibropapillomas occur predominantly at traumatized sites of the body, suggesting that humoral factors released in wounds might be important for papillomavirus infection. We have therefore investigated the possible role of TGF-beta 1 in BPV-1 infections. Two antipeptide antibodies which recognize different epitopes in the N-terminus of TGF-beta 1 were used to localize TGF-beta 1 in bovine fibropapillomas and normal bovine skin using immunohistochemical methods. Staining by anti-LC(1-30) is intracellular in suprabasal keratinocytes of the epidermis as well as the hair follicles and sebaceous glands and correlates with known sites of TGF-beta 1 mRNA synthesis. Anti-CC(1-30) staining is extracellular in the immediately underlying dermis. Neither the pattern nor intensity of TGF-beta 1 staining was affected by BPV-1 infection. C127 cells and BPV-1-transformed C127 cells were compared for TGF-beta 1 mRNA expression and secretion of TGF-beta 1 peptide. Although the levels of messenger RNA and secreted TGF-beta 1 peptide were similar in both cell types, five- to six-fold greater amounts of TGF-beta-like activity per cell was detected in media conditioned by the uninfected cells. TGF-beta 1 treatment had no effect on the growth rate of either cell type or on BPV-1 gene expression in the transformed cells.

Animals↗
PubMed1990

Histochemical localization of transforming growth factor-beta 1 in developing rat molars using antibodies to different epitopes.

In this study transforming growth factor-beta 1 (TGF-beta 1) has been immunolocalized in developing rat molars using two well characterized polyclonal antibodies, Anti-CC and Anti-LC, that recognize extracellular and intracellular TGF-beta 1, respectively. With immunohistochemical methods and the ABC-peroxidase system of detection, the growth factor was immunolocalized within the ectodermally derived enamel organ and the neural crest-derived dental papilla at the early and advanced bell stages of development. With Anti-CC, widespread and abundant extracellular TGF-beta 1 was found associated with the stellate reticulum and within central and apical regions of dental papilla mesenchyme. In contrast, Anti-LC localized TGF-beta 1 intensely within the cells of the outer dental epithelium. Moderate immunostaining for TGF-beta 1 with Anti-LC was also evident within the apical cytoplasm of inner dental epithelial cells and odontoblasts. These findings support the hypothesis that TGF-beta 1 may play a paracrine role in tooth development by regulating the epithelial-mesenchymal interactions that influence growth and cytodifferentiation events.

Animals↗
PubMed1990

Transforming growth factor-beta 1 specifically localizes in elastin during synovial inflammation: an immunoelectron microscopic study.

We report here that extracellular TGF-beta 1 is associated exclusively with microfibrils of elastin which are present in the extracellular matrix of the inflamed articular joint of the rat. Inflammation was initiated by bacterial cell walls localized in the synovium following intraperitoneal injection of the bacterial components. This synovitis is associated with both destruction of connective tissue components and matrix deposition. The growth factor was localized by using a polyclonal antibody raised to a synthetic peptide corresponding to amino terminal 30 amino acids of TGF-beta 1 in conjunction with a gold-labeled secondary antibody. The results suggest a close association of TGF-beta 1 with proteoglycans which are known to be a major component of the microfibrils in elastin. Proteoglycan-mediated binding and concentration of TGF-beta 1 in specific areas of the extracellular matrix may constitute a mechanism whereby the growth factor could be targeted to specific sites of action.

Animals↗
PubMed1989

Transforming growth factor-beta production by synovial tissues from rheumatoid patients and streptococcal cell wall arthritic rats. Studies on secretion by synovial fibroblast-like cells and immunohistologic localization.

The growth of synovial fibroblast-like cells from patients with rheumatoid arthritis and rats with streptococcal cell wall (SCW)-induced arthritis in vitro under anchorage-independent conditions is inhibited by transforming growth factor-beta (TGF-beta). Because this growth factor is present in rheumatoid synovial fluids, we studied whether this cytokine might be secreted by cells in rheumatoid synovial tissue. We show that synovial tissues from patients with rheumatoid arthritis and osteoarthritis, and rats with SCW-induced arthritis, contain TGF-beta-1 mRNA. TGF-beta, predominantly type 1, was spontaneously secreted in vitro by synovial tissue explants and synovial fibroblast-like cells. In addition, TGF-beta could be detected immunohistochemically in cells throughout rheumatoid and SCW-induced arthritic rat synovial tissues. Finally, exogenous TGF-beta induced collagen and inhibited collagenase mRNA levels by cultured synoviocytes. These data support an autocrine role for TGF-beta in the regulation of synoviocytes in rheumatoid arthritis and, in light of its demonstrated effects on the immune system, suggest that TGF-beta might also have important paracrine effects on infiltrating inflammatory cells.

Animals↗
PubMed1989

Immunodetection and quantitation of the two forms of transforming growth factor-beta (TGF-beta 1 and TGF-beta 2) secreted by cells in culture.

Transforming growth factor beta (TGF-beta), a potent modulator of cell growth, differentiation, and the expression of extracellular matrix components in a variety of cell types, exists as two distinct homodimers (TGF-beta 1 and TGF-beta 2), sharing 71% sequence homology. Radioreceptor and previously described radioimmunological assays using rabbit antibodies have not been able to distinguish between these two forms. We have developed antisera in turkeys against native TGF-beta 1 and TGF-beta 2, each of which specifically blocks both the receptor binding and biological activity of each of these peptides. With these immunological reagents we describe sensitive and specific immunological assays for TGF-beta 1 and TGF-beta 2 in complex biological fluids. Using these assays we show that both TGF-beta 1 and TGF-beta 2 are secreted by a variety of cultured cells, but that some cells secrete predominantly either TGF-beta 1 or TGF-beta 2 while others secrete both peptides in nearly equal amounts. Our results demonstrate that the expression of each of the two forms of TGF-beta is independently regulated.

Animals↗