Blocking transforming growth factor-beta receptor signaling down-regulates transforming growth factor-beta1 autoproduction in keloid fibroblasts.

OBJECTIVE: To study transforming growth factor-beta1 (TGF-beta1) autoproduction in keloid fibroblasts and the regulation effect of blocking TGF-beta intracellular signaling on rhTGF-beta1 autoproduction. METHODS: Keloid fibroblasts cultured in vitro were treated with either rhTGF-beta1 (5 ng/ml) or recombinant adenovirus containing a truncated type II TGF-beta receptor gene (50 pfu/cell). Their effects of regulating gene expression of TGF-beta1 and its receptor I and II were observed with Northern blot. RESULTS: rhTGF-beta1 up-regulated the gene expression of TGF-beta1 and receptor I, but not receptor II. Over-expression of the truncated receptor II down-regulated the gene expression of TGF-beta1 and its receptor I, but not receptor II. CONCLUSIONS: TGF-beta1 autoproduction was observed in keloid fibroblasts. Over-expression of the truncated TGFbeta receptor II decreased TGF-beta1 autoproduction via blocking TGF-beta receptor signaling.     

Effect of transforming growth factor beta and basic fibroblast growth factor on steroid-impaired healing intestinal wounds.

A longitudinal intestinal wound model in the pig was used to assess the effect of parenteral steroids (betamethasone 12 mg 50 kg-1 intramuscularly twice daily) on breaking load. Steroid treatment significantly decreased the breaking load of wounds in the ileum and colon in comparison with wounds from saline-treated animals. In a further group of animals receiving steroids, paired longitudinal wounds were constructed. One wound of a pair was treated with a local application of transforming growth factor beta (TGF-beta) (5 micrograms per wound) or basic fibroblast growth factor (5 micrograms per wound) in a collagen suspension. The other wound was treated with a collagen suspension alone. Ileal wounds treated with TGF-beta were significantly stronger than collagen-treated controls at 7 days. The steroid-induced impairment of breaking load in intestinal wounds is partially reversed by a local application of TGF-beta in a collagen suspension at the time of surgery.     

Gene expression in normal and doxorubicin-impaired wounds: importance of transforming growth factor-beta

The function of transforming growth factor-beta (TGF-beta) in vivo remains unknown despite the fact that it has been identified in numerous biologic processes involving the regulation of cell growth including tissue repair. Doxorubicin is a potent antitumor drug that has been shown to have detrimental effects on wound healing. With specific complementary DNA probes for TFG-beta and type 1 collagen, RNA from wounds of rats treated with saline solution and doxorubicin was analyzed for the expression of each gene at different times after wounding. In a second study, either 2 micrograms exogenous TGF-beta or vehicle was added to wounds of rats treated with doxorubicin, and wound RNA was analyzed in a similar manner. In wounds from rats treated with saline solution, messenger RNA (mRNA) for TGF-beta peaks on day 7 after wounding and is also elevated on days 3 and 10; mRNA for collagen is elevated on days 7 and 10. Doxorubicin decreases mRNA for TGF-beta and collagen on each day. Topical application of TGF-beta to wounds of rats treated with doxorubicin increases collagen mRNA levels to normal or supranormal levels. This study suggests that the impaired healing induced by doxorubicin may be a result of decreased gene expression for TGF-beta and that topical replacement of this growth factor may correct the defect.     

Interaction of basic fibroblast growth factor with bovine growth plate chondrocytes.

The basic fibroblast growth factor (bFGF) family of peptides influences a wide range of cellular actions. To better understand the possible role of bFGF in the growth plate, we have characterized the interaction of this growth factor with isolated bovine growth plate chondrocytes. Basic FGF interacts with two classes of binding sites on these cells. One is consistent with high-affinity bFGF receptors and the other with low-affinity heparin-like binding sites on the chondrocyte surface. Radiolabeled bFGF binding studies revealed approximately 4 x 10(6) binding sites per cell, with a Kd of approximately 42 nM. Graded concentrations of heparin or NaCl competed with [125I]-labeled bFGF in a dose-dependent fashion, reducing [125I]-labeled bFGF binding by 75 and 97%, respectively. The data suggest the presence of a high-capacity, low-affinity class of binding sites with the properties of a heparin-like moiety. Affinity cross-linking of [125I]-labeled bFGF to chondrocytes labeled two principal species with apparent molecular masses of 135 and 160 kDa. Labeled bFGF was specifically displaced from both species by subnanomolar concentrations of unlabeled bFGF. These high-affinity, low-capacity binding sites are characteristic of classical bFGF receptors. Binding of [125I]-labeled bFGF to these sites was also influenced by heparin, consistent with coregulation of binding to the two classes of binding sites. The data suggest that bFGF participates in the regulation of skeletal growth at the growth plate and that this regulation may involve bFGF interaction with at least two distinct classes of binding sites.     

Interaction of epidermal growth factor and transforming growth factor beta in human prostatic epithelial cells in culture.

The present study was conducted to study the interaction between epidermal growth factor (EGF) and transforming growth factor-beta (TGF-beta) in benign human prostatic epithelial cells in culture. Primary cultures of human prostatic epithelial cells were grown in complete WAJC, which consisted of WAJC-404 medium and, in addition to other defined additives, EGF and bovine pituitary extract (BPE). Incomplete WAJC contained the same composition except EGF and BPE were deleted. TGF-beta was added into media at concentrations of 0, 0.1, and 1.0 ng/ml. When cells were grown in complete WAJC, they proliferated rapidly. Cell proliferation was greatly suppressed when incomplete WAJC was used. Addition of TGF-beta to these cultures caused a significant reduction in the final cell number when either complete WAJC or incomplete WAJC was used. In additional experiments, cells were prelabeled with 3H-thymidine for 72 hr prior to treatment with TGF-beta. The percentage of radioactivity released into the medium at the end of a 6-day culture was used as an indication of the extent of cell death. Trypan blue exclusion test was also used to assess the extent of cell death. Addition of TGF-beta into complete WAJC did not significantly affect the extent of cell death beyond what was considered as the result of normal cellular turnover. Addition of TGF-beta into incomplete WAJC, however, caused a significant increase in the percent of cell death in the culture. These results demonstrated an interaction between EGF and TGF-beta in proliferation and cell death in human prostatic epithelia in culture. In the presence of EGF alone in the culture medium, prostatic epithelial cells were stimulated to proliferate. The rate of proliferation was greatly diminished when EGF was deleted from the medium or when TGF-beta was added in the presence of EGF. Finally, cell death was induced when TGF-beta was added into the medium in the absence of EGF.     

Resistance to transforming growth factor-beta occurs in the presence of normal Smad activation

BACKGROUND: Resistance to the growth inhibitory actions of transforming growth factor-beta (TGF-beta) is common in human cancers. This resistance can be a result of decreased expression of TGF-beta receptors. Downregulation of c-Myc by TGF-beta is critical for TGF-beta-mediated growth inhibition. In this study we hypothesized that decreased TGF-beta receptor expression leads to reduced Smad signaling and overexpression of c-Myc in intestinal epithelial (RIE) and transformed intestinal epithelial cells (RIE-Tr) cells. METHODS: RIE (TGF-beta-sensitive) and RIE-Tr (TGF-beta-resistant) cells were treated with and without fetal bovine serum and TGF-beta. Western blot analysis was performed to detect levels of c-Myc, Smad2, Smad4, and phosphorylated Smad2 in RIE and RIE-Tr cells. Smad complex formation was analyzed by immunoprecipitation-coupled Western blotting. RESULTS: c-Myc is overexpressed in RIE-Tr cells. TGF-beta-mediated downregulation of c-Myc is abrogated in RIE-Tr cells. Smad expression and activation is normal in RIE-Tr cells. We found that Smad2, Smad4, and Smad6 expression remained constant in RIE and RIE-Tr cells with or without serum or TGF-beta treatment. In addition, TGF-beta induced similar Smad2 phosphorylation and Smad complex formation in both RIE and RIE-Tr cells. CONCLUSIONS: Our data demonstrate that Smad signaling is preserved in the face of decreased TGF-beta receptor levels. We also demonstrate that Smad signaling is not sufficient for TGF-beta-mediated c-Myc repression.     

Reciprocal balance of hepatocyte growth factor and transforming growth factor-beta 1 in renal fibrosis in mice

BACKGROUND: Transforming growth factor-beta 1 (TGF-beta 1) plays a central role in the pathogenesis of renal fibrosis. In contrast, hepatocyte growth factor (HGF) may be an important molecule for tissue repair. As TGF-beta 1 is a suppressor molecule for HGF expression, we asked whether a decrease in HGF expression would be accompanied by an increase in TGF-beta 1 and whether the progression of renal fibrosis would be modulated. METHODS: We used the ICR strain-derived glomerulonephritis (ICGN) mice as a model of chronic renal disease and examined changes in local HGF expression during the natural course of renal fibrosis. To determine the significance of intrinsic HGF noted during progression of renal fibrosis, we administered an anti-HGF antibody to mice at the early stage of renal fibrosis. RESULTS: At an early stage of renal fibrosis, the mice showed strong peritubular HGF expression, coinciding with tubular proliferation. In the late stages, the renal HGF level was markedly decreased, coinciding with a reduction in proliferative tubular areas. Renal TGF-beta 1 levels were increased in accordance with expansion of fibrotic areas. Notably, the anti-HGF antibody treatment of early-stage mice decreased the HGF level and reduced tubular areas, whereas collagen-deposited areas were expanded in parallel with increased TGF-beta 1 levels. Consequently, in HGF-neutralized mice, there was a rapid progression of renal dysfunction. CONCLUSIONS: Not only an increase in TGF-beta 1 level, but also a decrease in local HGF expression may be responsible for the manifestation of renal fibrosis, particularly tubular destruction.     

Anti-macrophage migration inhibitory factor reduces transforming growth factor-beta 1 expression in experimental IgA nephropathy.

BACKGROUND: In human glomerulonephritis, including immunoglobulin-A nephropathy (IgAN), glomerular expression of macrophage migration inhibitory factor (MIF) is found to correlate with progressive renal injury. We have shown previously that polymeric IgA is capable of inducing MIF production in cultured human mesangial cells, suggesting a role in inducing inflammatory injury in IgAN. Herein, we examined whether IgA deposition and the subsequent renal injury can be ameliorated with anti-MIF treatment in an experimental murine model of IgAN. METHODS: Glomerular IgA deposition was induced in 4-week-old BALB/c mice by intravenous injection of immune complexes consisting of dinitrophenyl-conjugated bovine serum albumin (DNP-BSA) and IgA MOPC-315 myeloma anti-DNP antibodies. To determine the therapeutic effect of anti-MIF, mice were given anti-MIF (5 mg/kg) or isotypic control antibody intravenously 2 h before the immune complexes administration. The mice were sacrificed 48 h after injection of DNP-IgA. Proteinuria and haematuria were determined and the kidneys were removed for histopathology, immunostaining and immunoblotting. The effect of exogenous MIF on production of TGF-beta 1 by cultured mesangial cells was also examined. RESULTS: IgA deposits were detected in glomeruli of all mice receiving the immune complexes while no glomerular deposit was detected in the control mice. Microscopic haematuria and mesangial hypercellularity were present in mice of the three experimental groups and were absent in the control group. Proteinuria was absent in all groups. Anti-MIF treatment also resulted in decreased renal expression of TGF-beta 1. Moreover, the reduction in TGF-beta 1 expression was confined mainly to glomerular mesangium. An in vitro culture experiment demonstrated that MIF increased TGF-beta 1 production in a time- and dose-dependent fashion. MIF-induced TGF-beta 1 synthesis was abolished by incubating cells with neutralizing antibody against MIF. CONCLUSIONS: Our finding shows that anti-MIF treatment can ameliorate kidney injury and reduce glomerular TGF-beta 1 expression in an experimental model of IgAN.     

Role of transforming growth factor-beta in growth and injury response of the pancreatic duct epithelium in vitro

Chronic pancreatitis is characterized by increased levels of expression of transforming growth factor-beta (TGF-β), particularly in and around the ducts. To examine the consequences of elevated exposure to TGF-β on the pancreatic duct epithelium, we cultured segments of the main bovine pancreatic duct in the presence of increasing doses of TGF-β. We also studied the effect of TGF-β on epithelial injury, produced in this model by exposure to a bile acid. The extent of proliferation, migration, and epithelial damage was measured morphometrically on sections stained with hematoxylin and eosin. Proliferation and apoptosis were qualitatively determined by means of immunohistochemical analysis. In this model of duct cell culture, TGF-β stimulated cell migration in areas of the expiants where the native basement membrane of the duct epithelium was absent. In segments where the native basement membrane remained intact, proliferation was inhibited and apoptosis induced. When the expiants were exposed to bile acid, extensive epithelial injury was observed. TGF-β exposure at high doses (1 nmol/L protected epithelial integrity, but cellular morphology was altered and the process of apoptosis appeared to be increased. Our results suggest that increased periductal levels ot TGF-β in the setting of pancreatic injury may be intended to promote repair of acute epithelial damage but may have detrimental long-term effects. Supported by a grant from the Dean’s Research Initiative Fund from the University of Maryland School of Medicine. Presented at the Thirty-Ninth Annual Meeting of The Society for Surgery of the Alimentary Tract, New Orleans, La. May 17–20, 1998.     

Synergistic effect of transforming growth factor beta and fibroblast growth factor on DNA synthesis in chick growth plate chondrocytes

Transforming growth factor beta and fibroblast growth factor are mitogens for chick growth plate chondrocytes. TGF-beta stimulated a 3.5-fold increase, and FGF a 13.5-fold increase in the rate of thymidine incorporation after a 24 h exposure. TGF-beta and FGF were synergistic in chondrocytes, causing a 73-fold stimulation in thymidine incorporation compared with control. This synergistic response was not dependent upon the simultaneous presence of both mitogens. Sequential exposure of chondrocytes to TGF-beta and FGF in either order reproduced in large part the synergistic interaction observed when both growth factors were present simultaneously. The time required for induction of the subsequent synergistic response was brief and, in the case of TGF-beta, corresponded to the time required for [125I]TGF-beta receptor binding. EGF and PDGF were not mitogenic for chondrocytes, and neither of these factors enhanced the response of the cells to either TGF-beta or FGF. Finally, TGF-beta and FGF did not, either alone or in combination, elevate intracellular cAMP levels. These results emphasize the importance of examining growth factor effects in the context of other growth regulators. Furthermore, this specific and dramatic synergistic stimulation of thymidine incorporation may provide a useful tool in elucidating the mitogenic mechanism of the individual growth factors.     

碱性成纤维细胞生长因子拮抗转化生长因子—β1对人α1（I）胶原基因的启动激活

目的　探讨碱性成纤维细胞生长因子 (b FGF)对人α1 ( )胶原基因启动子活性的影响 ,以及与转化生长因子 - β1 (TGF- β1 )之间的相互作用 ,为防治增生性瘢痕提供依据。　方法　正常皮肤及瘢痕成纤维细胞原代、传代培养。采用 Fu GENE转染试剂 ,分别瞬间转染含人α1 ( )胶原基因 5'端序列 - 2 .5 kb与报告基因氯霉素乙酰基转移酶(CAT)的重组体 ph COL2 .5至正常皮肤及瘢痕成纤维细胞。 ELISA法测定 b FGF及 TGF- β1 作用 2 4小时后 ,转染ph COL2 .5的两种成纤维细胞的报告基因 CAT表达量。　结果　b FGF能抑制转染 ph COL2 .5重组体的正常皮肤及瘢痕成纤维细胞 CAT表达量 ,且能拮抗 TGF-β1 对转染 ph COL2 .5重组体的两种成纤维细胞 CAT表达的上调作用。与对照组相比有统计学意义 (P<0 .0 5)。　结论　正常皮肤及瘢痕成纤维细胞中 ,b FGF均能抑制人 α1 ( )胶原基因的启动转录 ,且能拮抗 TGF-β1 对人α1 ( )胶原基因启动活性的上调作用 ,b FGF抗纤维机制有望为增生性瘢痕的防治提供新思路     

Immunohistochemical detection of acidic fibroblast growth factor in bladder transitional cell carcinoma

Summary Acidic fibroblast growth factor (aFGF) is a regulatory peptide which, on account of its structural homologies with the products of oncogenes, is involved in cell proliferation, differentiation, and motility. We previously reported the presence of aFGF in the urine of patients with transitional cell carcinoma (TCC). aFGF can also induce the motility of a rat-derived bladder carcinoma cell line (NBT_II). This immunohistochemical study used polyclonal rabbit antibodies against acidic and basic FGF and peroxidase detection. Native NBT_II nude mice xenografts and aFGF transfected NBT_II (NFS14) nude mice xenografts were used as tissue controls for antibody specificity. The samples included 4 normal urothelia and 12 TCC. In addition, cytospins of 4 different tumoral cell lines of human bladder and normal bladder cells were stained. The results showed strong immunostaining in all tumoral urothelium samples using anti-aFGF and a very low amount of staining or none at all in healthy tissues. A primary analysis suggested that the strongest reaction was obtained in high-grade tumors (3 + vs + for lower-grade tumors). Using bFGF antibody, strong immunohistochemical staining was detected on basal membranes and stromal vessels and none in urothelium. These data confirm aFGF expression in the epithelial cell compartment of bladder cancer and the likely involvement of this regulatory peptide in the biology of TCC.Work supported by Commission de Recherche Clinique de Association Claude Bernard and Université Paris XIIThis paper was selected for publication in Urological Research from the program of the 1991 meeting of the European Society of Urological Oncology and Endocrinology (ESUOE)     

bFGF 、TGFβ-1在膀胱出口梗阻患者逼尿肌细胞中的表达

目的 观察膀胱出口梗阻（BOO）后膀胱平滑肌细胞碱性成纤维细胞生长因子（bFGF）、转化生长因子（TGFβ-1）表达的变化,探讨生长因子在BOO后膀胱平滑肌细胞继发改变中的作用。方法 应用逆转录－聚合酶链反应（RT－PCR）和免疫组织化学SP法在mRNA和蛋白水平检测bFGF、TGFβ-1在30例BOO及4例非BOO膀胱平滑肌组织中的表达。结果 BOO组与非BOO组膀胱平滑肌组织均有bFGF mRNA表达,BO组表达水平高于非BOO组,P＜0．01;TGFβ-1 mRNA在两组未见表达。结论 BOO 后磅胱平滑肌的继发改变与bFGF mRNA的表达有关。     

Pelvi-ureteric junction obstruction in children: the role of urinary transforming growth factor-beta and epidermal growth factor

OBJECTIVES: To investigate the role of transforming growth factor beta(1) (TGF-beta(1)) and epidermal growth factor (EGF) in voided urine for the diagnosis and follow-up of children with pelvi-ureteric junction obstruction (PUJO). PATIENTS, SUBJECTS AND METHODS: The study included 35 children with unilateral PUJO who had a pyeloplasty, and 30 healthy control children. Urine samples were obtained from the bladders of patients before surgery, and as voided samples at 1, 2, 3, 6, 9 and 12 months after surgery. Bladder urine samples were also collected from all 30 children in the control group. TGF-beta(1) and EGF were then measured in all the urine samples. RESULTS: The level of bladder TGF-beta(1) before surgery in the patients was significantly higher than that in the healthy control group. A threshold of 190 pg/mg creatinine gave a sensitivity of 100%, a specificity of 80%, a positive predictive value of 85.4%, negative predictive value of 100% and an overall accuracy of 90.8%. Compared with the value before surgery, urinary TGF-beta(1) was significantly lower at 1 year after pyeloplasty. There was no significant difference between the level of EGF before surgery in the patients and that in the control group, and no significant difference in the level of EGF before and after surgery over the follow-up. CONCLUSION: We do not recommend using EGF levels in voided urine in the routine diagnosis of children with hydronephrosis. The urinary level of TGF-beta(1) is a useful noninvasive tool for the long-term follow-up of children with PUJO treated by pyeloplasty. Further studies with various controls are required to confirm the diagnostic accuracy of TGF-beta(1) in children with PUJO.     

Role of transforming growth factor-beta in bone remodeling

Transforming growth factor-beta (TGF-beta) plays a critical role in bone remodeling. TGF-beta stimulates matrix protein synthesis, has dramatic effects on the bone cells responsible for bone formation and resorption, and is abundant in bone and bone-conditioned media. Multiple sources of TGF-beta have been described. It was initially purified from platelets. Two distinct forms of TGF-beta have been purified from bone. The second form, TGF-beta II, was initially purified from bone but was then identified in platelets and also as the major TGF-beta in the monkey kidney BSC-1 cell line. The two bone-derived factors were called cartilage-inducing Factor A (CIF-A) and cartilage-inducing Factor B (CIF-B), based on their capacity to induce the formation of extracellular matrix proteins, which are characteristic of cartilage. CIF-A is identical to the TGF-beta purified from platelets, which is called TGF-beta I. CIG-B is the same as TGF-beta II, which was sequenced soon after CIF-B was discovered and characterized. There is 70% sequence homology between the two forms. The largest source of TGF-beta in the body is present in bone (200 micrograms/kg tissue), although the most concentrated source is in platelets. TGF-beta has multiple effects on bone cells depending on their phenotype and/or stage of differentiation. Osteoblasts, the cells responsible for formation of new bone and perhaps cellular control of bone remodeling, are directly affected by TGF-beta, which can induce differentiation or proliferation, depending on the osteoblastic cell type examined. TGF-beta inhibits the formation of osteoclast precursors and bone resorption and, in greater concentrations, has inhibitory effects on isolated osteoclasts, the cells responsible for bone resorption. TGF-beta may act as a bone-coupling factor linking bone resorption to bone formation.