TGF-beta: regulation of extracellular matrix.
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Animals↗
Biomedical subjects
M B Sporn
Publications and source records attributed to M B Sporn.
Transforming growth factor-beta: recent progress and new challenges.
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Protein Conformation↗
Role of transforming growth factor-beta in maintenance of function of cultured neonatal cardiac myocytes. Autocrine action and reversal of damaging effects of interleukin-1.
The three isoforms of transforming growth factor-beta (TGF-beta) have previously been implicated in embryonic development of the heart as well as in repair of myocardial damage after ischemia/reperfusion injury. TGF-beta 1 has also been localized intracellularly to both mitochondria and contractile filaments of cardiac myocytes, although its role in these structures has not been defined. We now report that exogenous TGF-beta stabilizes the beating rate of neonatal rat cardiac myocytes cultured on fibroblast matrix, and sustains their spontaneous rhythmic beating in serum-free medium. Moreover, using blocking antibodies to TGF-beta, we show that endogenous TGF-beta secreted by these myocytes acts in an autocrine fashion to maintain their beating rate. In contrast, IL-1 beta, an inflammatory mediator secreted by immune cells during myocardial injury, inhibits the beating of cardiac myocytes, and TGF-beta can overcome this inhibition. The antagonistic effects of TGF-beta and IL-1 were not observed when the myocytes were cultured on gelatin, as compared to native fibroblast matrix. The data indicate that TGF-beta is an important regulator of contractile function of the heart and have significant implications for understanding cardiac physiology in health and disease.
Animals↗
Role of nitric oxide in antagonistic effects of transforming growth factor-beta and interleukin-1 beta on the beating rate of cultured cardiac myocytes.
We have recently shown that transforming growth factor-beta (TGF beta) acts in an autocrine manner to maintain the beating rate of neonatal rat cardiac myocytes cultured in serum-free medium on cardiac fibroblast matrix. Interleukin-1 beta (IL-1 beta) suppresses the myocyte-beating rate, and TGF beta antagonizes this effect. We now show that TGF beta and IL-1 beta also have antagonistic effects on the secretion of nitric oxide (NO) by these myocytes, and that NO secretion, the activity of NO synthase (NOS), and expression of the inducible form of NOS correlate inversely with the effects of these two agents on the beating rate. Western blot analysis shows that treatment of myocytes with TGF beta antagonizes the induction of NOS after treatment with IL-1 beta. Release of NO, induced by IL-1 beta, is dependent upon the availability of the substrate, L-arginine, and is suppressed by a competitive inhibitor, NG-monomethyl-L-arginine. L-Arginine (> 0.25 mM) also suppresses, and NG-monomethyl-L-arginine (> 0.5 mM) enhances the myocyte-beating rate. Treatment with IL-1 beta, but not TGF beta, increases cellular cGMP, presumably by activation of guanylate cyclase by NO. Methylene blue, an inhibitor of guanylate cyclase, reverses the suppression of beating caused by IL-1 beta. Bacterial lipopolysaccharide, present in the serum-free medium, is a coinducer of NO secretion. The suppressive effects of NO on the beating rate can be overcome by altering either the set of cytokines employed to induce NO or the matrix on which the myocytes are cultured, demonstrating that additional parameters are also involved in regulation of the beating rate.
Amino Acid Oxidoreductases↗
Identification and characterization of the chicken transforming growth factor-beta 3 promoter.
The promoter regions of the three mammalian transforming growth factor-beta genes (TGF-beta s 1, 2, and 3) have been recently cloned and characterized. The sequences show little similarity, suggesting different mechanisms of transcriptional control of these genes. To study differences in transcriptional regulation of mammalian and avian TGF-beta, we have cloned and sequenced the 5'-flanking region of chicken TGF-beta 3. Characterization of this region showed a TATA box and cAMP-responsive element (CRE) and AP-2 binding site consensus sequences starting at 12 and 28 base pairs, respectively, upstream from the TATA box. Moreover, four additional AP-2-like sites, 10 binding sites for the transcription factor Sp1, as well as two AP-1-like sites were also identified. Except for 32 base pairs of identity centered around the TATA box and CRE site and four other relatively small regions of identity, the chicken TGF-beta 3 promoter was found to be structurally very different from the human TGF-beta 3 promoter. Promoter fragments were cloned into a chloramphenicol acetyltransferase reporter plasmid to study functional activity. Basal transcriptional activity of the promoter was regulated in quail fibrosarcoma QM7 cells and in human adenocarcinoma A375 cells by multiple upstream elements including the TATA, CRE, and AP-2 sites. As in the human TGF-beta 3 promoter, the CRE site showed activation by forskolin, an effect which could be shown by expression of TGF-beta 3 mRNA in cultured chicken and quail cells as well. Our results indicate a complex pattern of transcriptional regulation of the chicken TGF-beta 3 gene and suggest that differences in the regulation of expression of the genes for mammalian and avian TGF-beta 3 may result in part from the unique structure of their 5'-flanking regions.
Amino Acid Sequence↗
Regulation of expression of transforming growth factor-beta 2 by transforming growth factor-beta isoforms is dependent upon cell type.
The effect of three different isoforms of transforming growth factor-beta (TGF-beta) on the expression of TGF-beta 2 mRNA was studied in several continuous tumor cell lines. As previously reported for the mouse fibroblast cell line AKR-2B, the expression of TGF-beta 2 mRNA transcripts of 5.4, 4.7, 3.7 and 3.0 kb was decreased after a 24 hr treatment with 5 ng/ml of TGF-beta 1, TGF-beta 2 or TGF-beta 3. In A549, HBL-100 and BSC-1 epithelial cell lines, five distinct TGF-beta 2 mRNA transcripts of 5.8, 5.1, 4.0, 3.8 and 2.8 kb were detected by Northern blot analysis. Treatment of these cells with TGF-beta 1, TGF-beta 2 or TGF-beta 3 for 24 hr resulted in a 2-3 fold increase in the 5.8, 4.0 and 3.8 kb transcripts, with little detectable change in abundance of the 5.1 and 2.8 kb transcripts. The effect of the TGF-beta proteins was dose (5 ng/ml) and time (3-6 hr) dependent. A similar 2-3 fold increase in the level of secreted TGF-beta 2 was observed following treatment of A549 cells with TGF-beta 1. Basal level and induced expression of TGF-beta 2 mRNA in response to TGF-beta isoforms was decreased in the presence of actinomycin D. In all cell lines studied, the expression of the 2.5 kb TGF-beta 1 mRNA was relatively unchanged or markedly increased in response to treatment with TGF-beta.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Distribution of transforming growth factor beta in a two-week-old human embryo.
Using immunohistochemical methods we have investigated the presence of transforming growth factors beta 1, beta 2 and beta 1 precursor in a two-week-old bilaminar human embryo. TGF-beta 1 precursor was seen in both the epiblast and the hypoblast. In contrast to the widespread localization of TGF-beta 1 precursor in the embryo proper, antibodies to the mature TGF-beta 1 peptide localized preferentially to the hypoblast with only weak staining in the epiblast. Staining with antibodies to TGF-beta 2 was generally weak in both the epiblast and hypoblast layers of the embryo proper. These results show that TGF-beta 1 and beta 2 peptides are detectable as early as the second week of human development.
Embryo, Mammalian↗
Mechanistic interrelationships between two superfamilies: the steroid/retinoid receptors and transforming growth factor-beta.
The fields of endocrinology and peptide growth factors can no longer be studied independently, nor can hypotheses about the modes of action of hormones and growth factors be constrained by outmoded classic definitions. Although much is still to be learned about the mechanisms of induction of TGF-beta activity by steroids/retinoids, about the involvement of specific nuclear receptors and about the factors regulating the differential effects of steroids/retinoids on the various isoforms of TGF-beta, it is clear that the identification of the interrelationships between these two superfamilies of multi-functional effectors has heralded a new era of discovery of the underlying regulatory events controlling cellular proliferation and differentiation.
Animals↗
Carcinogenesis and cancer: different perspectives on the same disease.
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Cell Differentiation↗
Prevention of primary prostate cancer in Lobund-Wistar rats by N-(4-hydroxyphenyl)retinamide.
We report for the first time that a synthetic retinoid, N-(4-hydroxyphenyl)retinamide, can prevent the development of both primary and metastatic tumors in an animal model of metastasizing primary prostate cancer. Prostatic adenocarcinomas were induced in high incidence in Lobund-Wistar rats by initiation with methylnitrosourea i.v. and promotion with testosterone. Feeding of N-(4-hydroxyphenyl)retinamide to these rats during the latency period markedly diminished the final incidence of both primary and metastatic prostate carcinomas.
Adenocarcinoma↗
Regulation of transforming growth factor beta 1 gene expression by the product of the retinoblastoma-susceptibility gene.
Transforming growth factor beta (TGF-beta) isoforms inhibit the growth of many cell types and block progression of the cell cycle by inhibiting events in late G1 phase. The retinoblastoma gene product, RB, also has properties of a cell-cycle regulatory factor. It remains underphosphorylated in the presence of TGF-beta and has been shown to repress the activity of the c-fos promoter, resulting in inhibition of transit through the cell cycle. These observations led us to examine effects of human RB on the expression of the human TGF-beta 1 gene. Using chimeric TGF-beta 1 promoter-chloramphenicol acetyltransferase gene constructs, we show that RB induces TGF-beta 1 gene expression in CCL-64 mink lung epithelial cells and A-549 human lung adenocarcinoma cells but represses its expression in NIH 3T3 and AKR-2B mouse cells. Several sequences homologous to the c-fos RB control element were identified in the TGF-beta 1 promoter. These results demonstrate that human RB can regulate TGF-beta 1 gene expression negatively or positively depending on the cell type.
Animals↗
Introduction: what is TGF-beta?
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Humans↗
Differential regulation of the expression of transforming growth factor-beta s 1 and 2 by retinoic acid, epidermal growth factor, and dexamethasone in NRK-49F and A549 cells.
Although most biological activities of transforming growth factor-beta s 1 and 2 (TGF-beta 1 and TGF-beta 2) examined in vitro are similar or identical, recent studies suggest that each of these factors may be independently regulated in vivo. In this study we have used highly sensitive and specific sandwich enzyme-linked immunosorbent assays for TGF-beta 1 and TGF-beta 2 to examine the effects of a variety of treatments on expression of these two TGF-beta isoforms. We show that epidermal growth factor (EGF) induces secretion of TGF-beta 1 and not TGF-beta 2, whereas retinoic acid (RA) induces secretion of TGF-beta 2 and not TGF-beta 1 in NRK-49F normal rat kidney fibroblasts and A549 human lung carcinoma cells. Moreover, treatment with EGF diminishes the levels of TGF-beta 2, while RA decreases the levels of TGF-beta 1 in both cell lines. Dexamethasone (Dex), on the other hand, inhibits the secretion of both TGF-beta 1 and TGF-beta 2 in A549 cells, while selectively inhibiting TGF-beta 1 secretion in NRK-49F cells. The interactive effects of EGF, RA, and Dex on the production of TGF-beta 1 and TGF-beta 2, which were studied on NRK-49F cells, demonstrate that EGF blocks the induction of TGF-beta 2 mRNA and peptide by RA, while Dex inhibits the induction of TGF-beta 1 mRNA and peptide by EGF. These results demonstrate that RA, EGF and Dex are each unique, differential, and interactive regulators of the expression of TGF-beta s 1 and 2.
Animals↗
Altered regulation of TGF-beta 1 and TGF-alpha in primary keratinocytes and papillomas expressing v-Ha-ras.
The influence of an oncogenic v-Ha-ras gene on the expression of TGF-beta and TGF-alpha by mouse keratinocytes and derived tumors has been investigated. Normal mouse keratinocytes cultured as basal cells in 0.05 mM Ca2+ secreted low levels of TGF-beta 2 peptide, and this increased markedly following culture in 1.4 mM Ca2+, retinoic acid, or phorbol esters. In contrast, introduction of a v-Ha-ras gene into normal keratinocytes increased basal expression and secretion of TGF-beta 1 (rather than TGF-beta 2) in response to all three agents. The selective secretion of TGF-beta 1 in v-Ha-ras keratinocytes in response to 1.4 mM Ca2+ occurred even though the four TGF-beta 2 transcripts were induced and the TGF-beta 1 transcript decreased, suggesting that the activated v-Ha-ras gene product regulates expression of the TGF-beta isoforms at the posttranscriptional level. Immunohistochemical analysis of papillomas formed following skin grafting of v-Ha-ras keratinocytes onto nude mice indicated that TGF-beta 1 was abundant in the basal and spinous layers, while there was no expression of TGF-beta 1 in normal skin. In contrast, both normal and neoplastic tissues expressed TGF-beta 2 and TGF-beta 3 in the granular layers. Furthermore, TGF-alpha mRNA expression was also elevated fivefold in cultured v-Ha-ras keratinocytes, and TGF-alpha protein was overexpressed in the grafted papillomas, but there was no detectable expression in normal skin. Elevated expression of both TGF-beta 1 and TGF-alpha in the basal and spinous layers of benign tumors may be important for the high proliferation rate in these tumors as well as for increased proliferation in the suprabasal layer.
Animals↗
Workshop on growth factors.
The 'Workshop on Growth Factors' which took place at the Lugano Lymphoma Conference on June 8, 1990, included a presentation by Michael Sporn on the concept that loss of inhibitory control mechanisms may be important in the development and growth of human cancer. Examples illustrating this were taken from current experimental biology research into transforming growth factor beta (TGF-beta) interactions. Brian Durie presented recent data on the biology of interleukin-6 (IL-6) and its putative role in plasma cell diseases. These studies have culminated in the first clinical study of the role of an antibody to a growth factor as therapy for a human cancer (anti-IL-6 antibody as therapy for patients with myeloma). Derek Crowther presented data concerning the current clinical role of the haematopoietic growth factors in patients undergoing chemotherapy for cancer. Recent clinical research has established the role of granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in improving the safety of high-dose or accelerated chemotherapy, and their use is associated with enhanced neutrophil recovery following ablative therapy and bone marrow rescue. This session was followed by the presentation of three papers concerning the use of G-CSF and GM-CSF in association with chemotherapy for patients with malignant lymphoma.
Animals↗
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↗
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↗
A second messenger RNA species of transforming growth factor beta 1 in infarcted rat heart.
Transforming growth factor-beta 1 (TGF-beta 1) is encoded predominantly by a 2.4-kb mRNA in most tissues. However, an additional transcript of 1.9 kb can be detected in rat heart after experimental myocardial infarction caused by ligation of the left coronary artery. This transcript level is significantly higher in infarcted heart tissue than in normal heart tissue, suggesting an important role for this mRNA species in response to injury. Structural characterization of the 1.9-kb mRNA showed that it included the entire coding sequence present in the 2.4-kb TGF-beta 1 mRNA, but also contained an additional nonhomologous 3'-untranslated region (UTR). The junction between the shared and unique 3' sequence in the 1.9-kb mRNA occurred only two nucleotides before the proposed polyadenylation site of the rat TGF-beta 1 2.4-kb mRNA. The unique 3'-UTR and the deduced shortened 5'-UTR in the novel 1.9-kb TGF-beta 1 mRNA suggest different transcriptional and translational regulatory mechanisms under conditions of tissue injury.
Amino Acid Sequence↗