Effect of vitamin D and plasma calcium upon proteoglycan size in chick growth cartilage.

Proteoglycan subunit isolated from the growth cartilage of rachitic chicks is of much smaller size than the corresponding preparation from vitamin D-treated chicks. The size change can be related to differences in plasma calcium concentration and not to the vitamin D status of the chicks.     

Induction of fibronectin gene expression by transforming growth factor beta-1 is attenuated in bronchial epithelial cells by ADP-ribosyltransferase inhibitors.

Transforming growth factor-beta (TGF-beta) exerts several effects on cultured airway epithelial cells including inhibition of proliferation and stimulation of fibronectin gene expression. ADP-ribosylation is one potential regulatory mechanism of gene expression by TGF-beta. We tested this possibility by exposing cultured bovine bronchial epithelial cells to the chemical inhibitor of ADP-ribosyl transferase enzymes, 3-aminobenzamide (3-AB) and, for comparison, 3-aminobenzoic acid (3-ABA), which is structurally similar to 3-AB but which does not inhibit ADP-ribosyl transferases. Exponential cell growth rate (1.2 doublings/day) or cellular morphology observed by phase contrast microscopy were not affected by 3 mM 3-AB or 3-ABA. Neither compound antagonized inhibition of cell division or induction of squamous morphology by TGF-beta 1. In contrast, the sixfold stimulation of fibronectin production by exposure of cells to 30 pM TGF-beta 1 for 48 h was reduced by 50% in the presence of 3 mM 3-AB, whereas 3 mM 3-ABA had no effect. The antagonistic effect was augmented by administration of 3-AB 24 h prior to induction by TGF-beta 1. Northern blot hybridization analyses demonstrated that 3-AB, but not 3-ABA, attenuated the induction of fibronectin mRNA by TGF-beta 1 by up to 50%. These observations may implicate a role of cellular ADP-ribosylation in the regulation of some gene expression by TGF-beta.     

Basic fibroblast growth factor and transforming growth factor beta-1: Synergistic mediators of angiogenesis in vitro

We investigated the relative roles of basic fibroblast growth factor (bFGF) and transforming growth factor beta-1 (TGF-b) on bovine aortic endothelial cell mitogenesis and morphogenesis using two-dimensional Petri dish cultures and a threedimensional hydrated collagen gel. bFGF alone stimulated endothelial cell proliferation with an EC₅₀ of 0.5 ng/ml. At bFGF levels greater than 2.5 ng/ml, morphologic alterations in confluent monolayers predominated; cells changed from a cobblestone morphology to an elongated cell pattern and showed enhanced migration into a denuded area of a Petri dish. In the three-dimensional model, exposure of endothelial cell monolayers to high bFGF levels stimulated minor cell migration directly under the monolayer but no invasion into the gel matrix. In combination with bFGF, heparin potentiated morphogenic changes, but not mitogenesis. bFGF, modification of the antiproliferative effect of TGF-b in confluent cultures was evidenced by induction of endothelial cell sprouting in response to 0.5 ng/ml TGF-b and 10–20 ng/ml bFGF in two-dimensional cultures. On collagen gels, endothelial cells migrated into the deep layers of the gel in a dose-dependent manner: invasion was maximal at 0.3–0.7 ng/ml TGF-b with decreased invasion at higher concentrations. The optimal collagen concentration that supported cell invasion was 0.075% collagen with the number of invading cells decreasing with increasing collagen gel density. By scanning electron microscopy, invading endothelial cells assumed a fibroblast-like appearance with slender cell extensions. We concluded that bFGF and TGF-b had independent effects on endothelial cell morphology and mitogenesis in culture. In combination at specific doses, these agents stimulated sprouting in the two-dimensional model and cell invasion in a collagen gel model. Morphogenic changes may be the primary event in determining angiogenesis. © 1993 Wiley-Liss, Inc.     

Induction of apoptosis in porcine thyroid follicles by transforming growth factor beta1 and epidermal growth factor.

For thyroid cells in culture DNA fragmentation and morphological changes related to apoptosis were first described in dog thyroid cells after deprivation of serum, epidermal growth factor or thyrotropin. With intact porcine thyroid follicles in three-dimensional culture, the effect of deprivation of growth factors and of incubation with transforming growth factor beta1 (TGF-beta1), epidermal growth factor (EGF), thyrotropin (TSH) or insulin-like growth factor I (IGF-I) on the incidence of apoptosis was studied. Thyroid follicles were embedded in growth factor-depleted Matrigel and cultured in serum-free medium with or without growth factors for 7 days followed by incubation for 4, 24 and 72 h with TGF-beta1 (2 or 5 ng/mL). The percentage of apoptotic cells was determined by direct counting in electron-microscopy. Approximately 1% of apoptotic bodies could be detected in unstimulated follicles. This was unchanged in the presence of TSH (1 mU/mL) or IGF (10 ng/mL) but significantly increased up to 3.99 +/- 1.24% with 2 ng/mL of EGF. After incubation with TGF-beta apoptosis increased dose-dependently to 4.05 +/- 0.67% with 2 ng/mL TGF-beta1 and 5.16 +/- 1.75% with 5 ng/mL TGF-beta1. The incidence of necrotic cells remained constant at about 1 to 2%. Preincubation of follicles with 2 ng/mL of EGF followed by incubation with 5 ng/mL TGF-beta1 increased the rate of apoptic bodies up to 13.19 +/- 1.9%. We conclude that growth factor depletion in thyroid follicles in three-dimensional culture does not lead to apoptosis. TGF-beta1, however, induces apoptosis even in quiescent thyroid follicular cells and is significantly more pronounced in growing thyroid cells. EGF, which is a dedifferentiating growth factor for thyroid cells, also induces apoptosis. As EGF enhances TGF-beta1 mRNA and protein in thyroid follicular cells, the induction of apoptosis by EGF might also be due to TGF-beta1.     

小鼠早期胚胎发育期间TGF—β免疫组织化学定位

The distribution of transforming growth factor beta-1 (TGF-beta-1) in the early developing mouse embryos between day 1 and day 12 of gestation was examined by immunohistochemical techniques. Polyclonal rabbit antiserum raised against a synthetic oligopeptide identical to the N-terminal residues 1-29 of TGF-beta-1 from human platelets was used. The following results were obtained: 1. Embryonic cells of early cleavage stages (2, 4 and 8 cells) and late morulae showed positive immunofluorescent reaction without any difference in staining intensity (Plate I, Figs. 1-4). 2. Marked staining of blastocysts in toto or sections with anti-TGF-beta-1 antibodies by either immunofluorescence or immunoperoxidase reaction was also observed. Inner cell mass (ICM) cells and trophoectoderm cells were both reacted, but more intense staining was found in primary endoderm cells differentiated from ICM cells adjacent to blastocoele (Plate II, Fig. 5). 3. Scattered granules stained strongly with immunoperoxidase reaction were present in embryonic ectoderm and visceral endoderm surrounding the forming mesoderm which was only slightly stained (Plate II, Fig. 6). 4. Intense immunoperoxidase staining was also present in mesoderm of visceral yolk sac of day 8 and day 10 embryos (Plate II, Fig. 7). 5. During the formation of somites, neural tube and limb bud, remarkable staining was found in mesenchyme, individual cells of somites, mucous layer of gut tubes, heart and limb buds (Plate III, Figs. 8-10). No significant staining was seen in neural cells per se except the inner surface of neural tube. The results of present studies indicate that abundant TGF-beta-1 is present in preimplantation mouse embryos including cleavage, morulae and blastocyst stages. In postimplantation embryos, TGF-beta-1 appears to play an important role in the differentiation of endoderm and mesoderm, particularly in the development of extraembryonic tissues, and in later morphogenetic and histogenetic events involving mainly mesoderm or mesenchyme cells.     

Basic fibroblast growth factor and transforming growth factor beta-1 expression in the developing dura mater correlates with calvarial bone formation

Numerous studies have found dura mater-calvarial mesenchyme interactions during calvarial bone induction; however, the exact molecular mechanisms governing these inductive events remain unknown. Recent studies have implicated basic fibroblast growth factor (FGF-2) and transforming growth factor-beta1 (TGF-beta1) in regulating bone formation. The purpose of this study was, therefore, to investigate the expression of FGF-2 and TGF-beta1 during calvarial bone formation in rats. Eight rats were killed on embryonic days 14, 18, and 20 and neonatal day 1 (n = 32). Four animals at each time point were analyzed by in situ hybridization, and the remainder were analyzed by immunohistochemistry. The results indicated that the dura mater underlying the developing calvarial bone strongly expressed FGF-2 and TGF-beta1 mRNA at all time points examined. In contrast, minimal growth factor expression was noted in the overlying calvarial mesenchyme until embryonic day 18, but it increased significantly with increasing age. Importantly, FGF-2 and TGF-beta1 mRNA expression in the dura mater underlying the developing calvarium preceded and was significantly greater than expression in the calvarium mesenchyme (p < 0.05). Interestingly, minimal expression of FGF-2 and TGF-beta1 mRNA was noted for all time points in the dura mater underlying the posterior frontal suture and within the posterior frontal suture connective tissue (p < 0.01 when compared with the dura mater underlying the developing calvarium). Immunohistochemical findings closely paralleled mRNA expression, with intense staining for FGF-2 and TGF-beta1 in the dura mater underlying the developing calvarial mesenchyme. Increasing FGF-2 and TGF-beta1 staining was noted within calvarial osteoblasts with increasing age, particularly in cells located near the endocranial surface (i.e., in contact with the developing dura mater). These findings, together with the known biologic functions of FGF-2 and TGF-beta1, implicate these growth factors in the regulation of calvarial bone growth by the developing dura mater. The possible mechanisms of this interaction are discussed.     

Effect of transforming growth factor beta-1 on ovine satellite cell proliferation and fusion

We have evaluated the effect of transforming growth factor beta-1 (TGF beta-1) on proliferation and fusion of cultured ovine satellite cells isolated from 5-month-old wether lambs. The isolation and culture protocols were validated by clonal analysis of the original cell preparation and assessment of proliferation and fusion of control cultures. Approximately 85% of the original cells isolated were myogenic as assessed by clonal analysis. The ovine cells doubled approximately every 18 hours during their exponential growth period and achieved a maximum percent fusion of 39.5% after 144 hours in culture. TGF beta-1 inhibited fusion of these cells in a dose-dependent manner with half-maximal inhibition occurring at .08 ng/ml. Maximal inhibition (95% suppression) occurred between .1 and .5 ng/ml. TGF Beta-1 (.05-3.0 ng/ml) did not inhibit proliferation of cultured ovine satellite cells in serum-containing medium or in serum-free defined medium. In contrast, TGF beta-1 did significantly suppress serum-stimulated proliferation of either porcine or bovine satellite cells that were isolated by using a procedure identical to that used to isolate the ovine satellite cells. Thus, proliferation of ovine satellite cells appears to respond differently to TGF beta-1 than does proliferation of either porcine or bovine satellite cells.     

Insulin-like growth factor 1 can decrease degradation and promote synthesis of proteoglycan in cartilage exposed to cytokines.

A model system of explanted cartilage has been used in vitro to determine whether insulin-like growth factor 1 (IGF 1), which promotes matrix formation is effective in the presence of cytokines such as interleukin 1 (IL1) and tumour necrosis factor (TNF), which induce net matrix depletion. IGF 1 induced a dose-dependent 2.5-fold stimulation of proteoglycan synthesis, with a half-maximal dose of 25 ng/ml. A similar relative increase occurred in response to IGF 1 (10-100 ng/ml) in cartilage cultured also with IL1 or TNF (5-500 pM). There was no detectable qualitative change in the average molecular size or charge of the aggregating proteoglycan synthesized by explants exposed to IGF 1 alone or with IL1 or TNF. The increased production of prostaglandin E2, which is initiated when IL1 or TNF bind to the chondrocytes, was the same in the presence or absence of IGF 1. The time taken for 50% of pre-labelled proteoglycan to be released from the explants (t1/2) increased in the presence of IGF 1 (100 ng/ml) from 21 to 32 days in control cultures and from 8 to 26 days in cartilage cultured with IL1 (50 pM). It is concluded that IGF 1 enhances the synthesis of aggregating proteoglycan in cartilage exposed to cytokines and can directly decrease both the basal and the cytokine-stimulated degradation of proteoglycan in cartilage.     

Aging, osteoarthritis and transforming growth factor-beta signaling in cartilage

Osteoarthritis is a common malady of the musculoskeletal system affecting the articular cartilage. The increased frequency of osteoarthritis with aging indicates the complex etiology of this disease, which includes pathophysiology and joint stability including biomechanics. The balance between anabolic morphogens and growth factors and catabolic cytokines is at the crux of the problem of osteoarthritis. One such signal is transforming growth factor-beta (TGF-beta). The impaired TGF-beta signaling has been identified as a culprit in old mice in a recent article in this journal. This commentary places this discovery in the context of anabolic and catabolic signals and articular cartilage homeostasis in the joint.     

Human transforming growth factor beta.alpha 2-macroglobulin complex is a latent form of transforming growth factor beta

125I-Labeled human platelet-derived transforming growth factor beta (125I-TGF-beta) and human alpha 2-macroglobulin (alpha 2M) formed a complex as demonstrated by 5% native polyacrylamide gel electrophoresis. The 125I-TGF-beta.alpha 2M complex migrated at a position identical to that of the fast migrating form of alpha 2M. Most of the 125I-TGF-beta.alpha 2M complex could be dissociated by acid or urea treatment. When 125I-TGF-beta was incubated with serum, the high molecular weight form of 125I-TGF-beta could be immunoprecipitated by anti-human alpha 2M anti-sera as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. alpha 2M purified from platelet-rich plasma also showed the latent transforming growth factor activity and immunoreactivity of TGF-beta. These results suggest that TGF-beta.alpha 2M complex is a latent form of TGF-beta.     

The effect of interleukin-10 and transforming growth factor beta-1 on HLA-DR expression in colonic epithelial cells.

The aim of this study was to assess whether interleukin-10 (IL-10) and/or transforming growth factor beta-1 (TGFbeta1) downregulate HLA-DR expression using the HT29 cell line as a model of colonic epithelial cells. HLA-DR expression was induced in HT29 cells with gamma-interferon. The effects of IL-10 alone, TGFbeta1 alone, and IL-10 and TGFbeta1 in combination were studied. HLA-DR expression was assessed using flow cytometric analysis. Gamma-interferon induced HLA-DR expression in a dose-dependent fashion. In the absence of gamma-interferon, neither IL-10 nor TGFbeta1 induced HLA-DR expression. In isolation, neither IL-10 nor TGFbeta1 downregulated HLA-DR expression. When IL-10 and TGFbeta1 were added in combination, small (6-30%) statistically significant reductions in HLA-DR expression were seen. The biological significance is unclear.     

Monoclonal antibodies recognizing transforming growth factor-beta. Bioactivity neutralization and transforming growth factor beta 2 affinity purification

Four mAb able to recognize transforming growth factor-beta 2 (TGF-beta)2 were obtained. One of these mAb, 1D11.16, was able to neutralize the biological activity of both TGF-beta 1 and beta 2 in vitro. This was demonstrated in an Il-1, PHA-dependent thymocyte mitogenic assay that is inhibitable by TGF-beta in a dose-dependent manner. All four mAb recognized the dimeric form of TGF-beta 2 in Western blots. The mAb were also found to immunoprecipitate [125I]-TGF-beta 2. mAb 3C7.14 coupled to Sepharose could efficiently immunoaffinity purify TGF-beta 2 from a complex mixture of proteins. Affinity constants were determined for the four mAb and they ranged from 3.4 x 10(8) to 1.6 x 10(7) L/mol.     

Modulation of a fibrotic process induced by transforming growth factor beta-1 in dermal equivalents

Why initially normal wound healing sometimes shifts toward an impaired cicatrization is poorly understood. Collagen gels with incorporated fibroblasts constitute valuable in vitro models to study mechanisms of connective tissue reorganization. Such 1-week-old, partially contracted normal dermal equivalents were treated with concentrations of TGF-₁ ranging from 1 to 10 ng/ml. The cytokine was applied in a single dose or four times at regular intervals, over a 2-week period. Dose-dependent activation of fibroblasts was observed after treatment. The cytokine induced a myofibroblastic transformation of dermal cells, significantly enhanced the process of dermal contraction, and stimulated synthesis of such proteins as cellular fibronectin, tenascin and smooth-muscle actin. Our approach is more informative than models using pathological or pretreated dermal cells, since it demonstrates newly induced modulation of fibrotic transformation in an initially normal dermal equivalent. This in vitro assay will enable us to study mechanisms involved in the shift between normal and impaired fibrotic transformation during wound healing.     

Stimulation of cartilage-matrix proteoglycan synthesis by morphologically transformed chondrocytes grown in the presence of fibroblast growth factor and transforming growth factor-beta

The effects of transforming growth factor-beta (TGF-beta) on the synthesis of cartilage-matrix proteoglycan by cultured rabbit chondrocytes were examined. Rabbit chondrocytes were seeded at low density and exposed to a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium supplemented with 0.5% fetal bovine serum, 1% bovine serum albumin, 50 micrograms/ml ascorbic acid, and 2 x 10(-7) M hydrocortisone (Medium A). Various combinations of TGF-beta, insulin-like growth factor-I (IGF-I), and fibroblast growth factor (FGF) were also added to Medium A, and the chondrocytes were grown to confluency. Chondrocytes grown with TGF-beta or FGF alone became flat or fibroblastic, those grown with FGF and TGF-beta became very elongated and formed distinct foci, and those grown with FGF and IGF-I showed the spherical configuration characteristic of overtly differentiated chondrocytes. Nevertheless, the incorporation of 3H with glucosamine into the large, chondroitin sulfate proteoglycan synthesized by cultures with FGF and TGF-beta was similar to that in cells grown with FGF and IGF-I and five times that in cells cultured with FGF alone. The increases in incorporation of 3H reflected real increases in proteoglycan synthesis, because chemical analyses showed an increase in the accumulation of macromolecules containing uronic acid in cultures with FGF and TGF-beta or with FGF and IGF-I. However, FGF in combination with either TGF-beta or IGF-I had little effect on the incorporation of 3H into small proteoglycans or hyaluronic acid. These results indicate that chondrocytes morphologically transformed with TGF-beta and FGF fully express the differentiated proteoglycan phenotype rather than the transformed glycosaminoglycan phenotype.     

Neuroprotection of microglial conditioned medium on 6-hydroxydopamine-induced neuronal death: role of transforming growth factor beta-2

Microglia, the immune cells of the CNS, play essential roles in both physiological and pathological brain states. Here we have used an in vitro model to demonstrate neuroprotection of a 48 h-microglial conditioned medium (MCM) towards cerebellar granule neurons (CGNs) challenged with the neurotoxin 6-hydroxydopamine, which induces a Parkinson-like neurodegeneration, and to identify the protective factor(s). MCM nearly completely protects CGNs from 6-hydroxydopamine neurotoxicity and at least some of the protective factor(s) are peptidic in nature. While the fraction of the medium containing molecules < 30 kDa completely protects CGNs, fractions containing molecules < 10 kDa or > 10 kDa are not neuroprotective. We further demonstrate that microglia release high amounts of transforming growth factor-β2 (TGF-β2) and that its exogenous addition to the fraction of the medium not containing it (< 10 kDa) fully restores the neuroprotective action. Moreover, MCM neuroprotection is significantly counteracted by an inhibitor of TGF-β2 transduction pathway. Our results identify TGF-β2 as an essential neuroprotective factor released by microglia in its culture medium that requires to be fully effective the concomitant presence of other factor(s) of low molecular weight.     

Induction of c-fos and c-myc proto-oncogene expression by epidermal growth factor and transforming growth factor alpha is calcium-independent

Intracellular calcium has been proposed to be an important mediator of signal transduction by various growth factors. We have studied the role of intracellular calcium in the mitogenic stimulation of C3H 10T1/2 mouse fibroblasts by epidermal growth factor and transforming growth factor alpha. We have found that both these peptides can cause a marked, transient increase in intracellular calcium levels. This rise occurs only in the presence of extracellular calcium. However, this calcium transient is not involved in the accumulation of c-fos and c-myc mRNAs which are elicited by these growth factors, since mRNA induction is observed to an equivalent degree in the absence or presence of extracellular calcium. These results demonstrate that although these growth factors cause an increase in intracellular calcium, the calcium second messenger system is not responsible for the induction of c-fos and c-myc mRNAs in C3H 10T1/2 fibroblasts.