Transforming Growth Factor Beta Stimulates Mitogenically Mouse NIH3T3 Fibroblasts and Those Cells Transformed by the EJ-H-ras Oncogene

Abstract

TGF-β1 stimulates thymidine incorporation and the growth rate of mouse NIH3T3 fibroblasts and of those cells transformed by the EJ-H-ras oncogene (TR15 cells), in the presence and the absence of serum. Thymidine incorporation, in serum-deprived cells, is stimulated to a higher degree by 0.1-1 ng/ml of TGF-β in NIH3T3 than in TR15 cells, which have a 10-fold higher basal level of incorporation. In both cell types TGF-β1 is as active, or more active than other mitogens (TGF-α, PDGF-AB, bFGF) at the same concentration. The growth rate of NIH3T3 cells, in low serum or serum-free (S^?) medium, is stimulated by only 10 picograms/ml of TGF-β1, and that of TR15 cells, in S^? medium, by only 1 picogram/m1. In contrast, TGF-β1 inhibits mitogenically unestablished mouse embryo fibroblasts and these fibroblasts immortalized spontaneously and able to grow in S^? medium. It also inhibits the anchorage-independent growth of TR15 cells. NIH3T3 and TR15 cells respond, similarly, to TGF-β activated by acification of their culture medium. The kinetics of thymidine incorporation and of activation of the c-myc proto-oncogene, observed already after 1 hr, in treated NIH3T3 and TR15 cells, suggests a direct mitogenic stimulation. The level of activated c-myc RNA is 2-fold higher at 2 hr, and subsequently decreases relatively less in the TR15 cells.     

Identification and Analysis of Transforming Growth Factor Beta Receptors on Primary Osteoblast-Enriched Cultures Derived from Adult Human Bone

Primary human osteoblast-enriched (PHO) cultures derived from adult trabecular bone were analyzed to determine the presence or absence of transforming growth factor beta (TGF-β) receptors. Saturation binding studies were performed with ¹²⁵I-TGF-β in the absence or presence of 200-fold excess cold TGF-β. Cross-linking experiments utilizing ¹²⁵-I-TGF-β were performed to identify specific cell surface binding proteins for TGF-β. The saturation binding studies demonstrated saturable binding for TGF-β on PHO cells. TGF-β was cross-linked to cell surface binding proteins of 50 to 110 KDa and a high molecular weight component. Thus, these receptors appear to be similar in affinity, number per cell, and molecular weight to those previously identified with other cell types. The potential biological effects of TGF-β on the growth of PHO cultures were evaluated by both ³H-thymidine incorporation and cell number determination. Growth of PHO cells in the presence of TGF-β resulted in an approximately two-fold stimulation in cell number as compared to control cells while the ³H-thymidine experiments demonstrated a two to four-fold increase in thymidine uptake in the presence of TGF-β. Radiographic emulsion studies revealed that the alkaline phosphatase positive and negative cell populations were responsive to the TGF-β mitogenic stimulation. The cumulative findings of saturable binding, specific cell surface binding proteins, and biological effects suggest that functional TGF-β cell surface receptors are present on primary osteoblast-enriched cultures derived from adult human trabecular bone.     

Endothelial Cells Synthesize Basic Fibroblast Growth Factor and Transforming Growth Factor Beta

Abstract

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 NaCI, (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/30⁶ cells in HUVEC, 2.0 ng/10⁶ cells in BCEC, and 13 ng/10⁶ 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 CCI-64 cells. TGF-beta was secreted in an inactive form and required acid activation for detection. HUVEC synthesized 2.0 ng TGF-beta/10⁶ 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.     

Effects of Transforming Growth Factors on Bone Cells

Bone formation results from the anabolic and catabolic functions of osteoblasts and osteoclasts within bone. The activities of these cell populations are controlled by complex interacting effects generated by local (bonederived) and systemic (hormone) growth regulators. One of the more abundant growth regulators produced by bone cells and associated with bone matrix is transforming growth factor β (TGF-β). Recent studies indicate that TGF-β controls the abundance and the biochemical function of osteoblasts and osteoclasts. Also, both TGF-β production by bone cells, and its effects on bone cell activity, can be influenced by other local growth factors and osteotropic hormones.     

The Potential Role of Platelet-Derived Growth Factor as an Autocrine or Paracrine Factor for Human Bone Cells

Platelet-derived growth factor, PDGF, is a potent mitogen for cells of mesenchymal origin such as fibroblasts, smooth muscle cells and glial cells. PDGF is thought to have the potential to act as both a paracrine and an autocrine factor. Studies described here extend these observations to human bone-derived cells. Exogenous PDGF induces biologic activity in two human osteogenic sarcoma cell lines and in one of these, the two PDGF genes, PDGF-1 and PDGF-2/c-sis are expressed. In addition, PDGF stimulates proliferation of normal osteoblastic cells derived from adult human cancellous bone. The expression of the PDGF-1 gene but not the PDGF-2/c-sis gene is demonstrated in normal human adult bone-derived cells by Northern blot analysis and synthesis of PDGF is shown by immunoprecipitation with PDGF antisera. These studies indicate that PDGF has the potential to act as a paracrine or autocrine regulator of bone cells.     

兔脂肪干细胞与软骨细胞微球共培养成软骨分化最适诱导比例的实验研究

通过间接共培养ADSC与软骨细胞微球,得出使ADSC发生软骨向分化的最佳比例。体外分离培养新西兰大白兔ADSC和软骨细胞,制成微球分置于Tranwell上下室,实验分为阳性对照组,阴性对照组,生长因子对照组及不同比例(0.5∶1; 1∶1; 1∶2; 1∶3; 1∶5;1∶7)的ADSC和软骨细胞共培养组,培养28天后分别行组织形态学观察,GAG、COL\|Ⅱ和COL\|Ⅹ的定性定量检测。阿利新蓝\|核固红染色及Ⅱ型胶原免疫组化染色显示比例为0.5∶1和1∶1的阳性较弱,而Ⅹ型胶原在生长因子对照组的表达最为强烈。DMMB法显示总GAG含量在共培养组较阴性对照组均有所增加(P<0.05),且增加量与软骨细胞的比例呈正相关,但是在ADSC:AC比例达到1∶5之后有所下降。OHP检测总胶原蛋白的变化同上,且1∶5组中沉积的胶原量比阴性对照组高出7.3倍。能够使ADSCs最大程度的表达软骨特异性细胞外基质,同时使COL\|Ⅹ等软骨肥大标志无明显上调的ADSC与软骨细胞的比例为1∶5,共培养诱导较生长因子能够抑制ADSC软骨向分化后的肥大问题。     

表皮生长因子受体与他莫西芬耐药机制

雌激素受体及其信号通路在乳腺癌的发生发展中发挥着关键作用.到目前为止,抑制或阻断雌激素信号通路的内分泌治疗尤其是他莫西芬,仍是对雌激素受体阳性乳腺癌患者最有效的治疗手段之一.然而,他莫西芬的耐药问题直接影响了乳腺癌患者的治疗及预后.最近多项研究表明雌激素受体与表皮生长因子受体家族尤其是HER2介导的信号传导通路在多个点上相互交叉,彼此影响,与他莫西芬的耐药密切相关     

Age-Related Loss of Innate Immune Antimicrobial Function of Dermal Fat Is Mediated by Transforming Growth Factor Beta

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氯沙坦对自发性高血压大鼠左心室肥厚和转化生长因子β_1的影响

目的 :探讨转化生长因子 β1 (TGFβ1 )在自发性高血压大鼠(SHR)左心室肥厚中的作用及氯沙坦对左心室肥厚和TGFβ1 水平的影响。方法 :2 0只SHR随机分成氯沙坦治疗组和未治疗组 ,每组10只 ,用免疫组织化学法检测SHR心肌TGFβ1 的表达及氯沙坦治疗后的改变 ,同时观察心肌肥厚的变化 ,并与正常WKY组对照。结果 :正常组心肌细胞胞浆内TGFβ1 表达呈弱阳性 ,SHR对照组呈强阳性 ,氯沙坦治疗组呈阳性反应。用图像分析仪计算其平均吸光度分别为 17.46± 2 .3 8、15 6.81± 2 1.75、67.19± 7.2 4,并且SHR左心室质量指数 (LVWI)高于WKY组 ,氯沙坦治疗组LVWI低于对照组。结论 :TGFβ1 在自发性高血压大鼠心肌中表达增强 ,而氯沙坦治疗可抑制其表达 ,从而可能延缓SHR左心室肥厚的病理学进展。     

骨生长因子在骨修复中的研究进展

骨代谢和损伤后的修复过程高度复杂,涉及到多种生长因子间的相互作用。骨生长因子是指可以增加骨细胞生长活性,调节骨生长、分化以及重塑等多种生物学过程的因子,在骨修复过程中具有重要作用。目前,研究较多的参与骨形成的生长因子包括骨形态发生蛋白(bone morphogenetic protein,BMPs)、转化生长因子β(transforming growth factorβ,TGF-β)、成纤维细胞生长因子(fibroblast growth factors,FGF)、血小板衍生因子(platelet-derived growth factor,PDGF)等。本研究总结国内外相关研究,综述了骨生长因子在骨修复中的作用机制及临床应用,以期为骨生长因子的临床应用提供一定的理论基础。     

各型肝病患者外周血转化生长因子α水平及其临床意义

目的:为探讨转化生长因子α(TCFα)在各型肝病患者中的变化规律.方法:采用非平衡竞争性放射免疫法测定血浆中TCFα水平.结果:172例肝病患者中的TGFα含量(x±SE)分别为:急性肝炎14.19±7.30pg/ml;慢性肝炎11.28±6.84pg/ml);肝硬化代偿期8.14±5.55pg/ml;肝硬化失代偿期16.02±9.81pg/ml;原发性肝癌19.89±9.05pg/ml.各型肝病的TCFα含量与正常(6.86±0.89pg/ml)比较有显著性升高(p<0.05).肝硬化失代偿期与代偿期有显著性差异(p<0.05)结论:TCFα可能与肝病的慢性化程度相关,血清TCFα含量的增加有助于在对肝硬化进展成肝细胞癌的过程的诊断,检测TGFα含量对肝病的临床诊断和预后的估计有一定的作用.     

The Genome of Cowpox Virus Contains a Gene Related to Those Encoding the Epidermal Growth Factor,Transforming Growth Factor Alpha and Vaccinia Growth Factor

Cowpox virus (CPV) is a member of the Orthopoxvirus genus and has the genetic capacity to encode a multitude of genes that interfere with the host inflammatory and immune response or modulate the physiological state of infected and non-infected cells. Among these CPV factors are receptors homologous to interferon and tumor necrosis factor receptors and also a viral cellular serine-proteinase analog. Here we describe the detection of a CPV gene that encodes a protein homologous to epidermal growth factor, transforming growth factor alpha and poxvirus growth factors, such as the vaccinia growth factor (VGF). The VGF and other poxvirus growth factors are produced early in the infection and are secreted into the medium where they bind to the EGF receptors, generating mytotic responses. The cowpox growth factor (CGF) gene was detected in three copies on the virus genome by PCR, and by northern and southern blot hybridization using VGF nucleotide sequences as primers and probes. The CPV gene has a strong nucleotide and predicted amino acid similarity with VGF, and is also produced early in the infection.     

埃他卡林对原代培养人肺动脉平滑肌细胞转化生长因子β1mRNA表达的影响

目的研究新型ATP敏感性钾（KATP）开放剂埃他卡林（IPT）对缺氧诱导原代培养人肺动脉平滑肌细胞转化生长因子β1（TGF—β1）mRNA表达的影响。方法分离3～4级人肺小动脉,按照组织贴块法原代培养人肺动脉平滑肌细胞,应用实时荧光定量逆转录聚合酶链反应（RQ-RT—PCR）技术检测细胞TGF—B,mRNA表达情况。结果缺氧使原代培养人肺动脉平滑肌细胞TGF—β1 mRNA表达增加;相同条件下,在缺氧的同时,分别在培养基中加入IPT 10^-7、10^-6、10^-5 mol·L^-1,IPT呈浓度依赖性地抑制细胞TGF—β1 mRNA表达;加入IPT 10^-5 mol·L^-1前30min,预先加入格列本脲（GLI）10^-7、10^-6、10^-5 mol·L^-1,GLI呈浓度依赖性地拮抗IPT的作用。结论IPT通过激活细胞KATP通道,浓度依赖性地抑制缺氧诱导的原代培养人肺动脉平滑肌细胞TGF-β1 mRNA表达增加。     

Transforming Growth Factor Beta Mediates the Estrogen Induced Inhibition of Umr106 Cell Growth

A mitogenic response to transforming growth factor beta (TGF) occurred in the UMR106 cells cultured in serum-free medium and exposed serially to estradiol and TGF. This mitogenic response was lost when insulin was removed from the medium. TGF inhibited growth and increased the alkaline phosphatase content in the UMR106 cells cultured in medium lacking insulin. Prior exposure of the cells to estradiol enhanced this response. Monoclonal antibodies against TGF blocked the estradiol induced inhibition of growth after a two day incubation in medium devoid of insulin.     

转化生长因子β1及血管内皮细胞因子在颈椎病椎体软骨终板中的表达变化

目的：研究TGF-β1、VEGF在终板软骨细胞中的表达，探讨椎间盘退变的发病机理。方法：应用免疫组化SP染色法检测TGF-β1、VEGF在15例颈椎病及13例正常椎体软骨终板中的表达，并对其阳性表达结果进行比较。结果：TGF-β1在正常终板软骨细胞表达的阳性细胞数为39．0％±1．96％，与颈椎病终板软骨细胞表达的阳性细胞数28．2％±2．18％相比，差异有统计学意义（P〈0．05）。VEGF在正常的终板软骨细胞表达的阳性细胞数为22．2％±2．13％，与颈椎病终板软骨细胞表达阳性细胞数14．3％±1．68％相比，差异有统计学意义（P〈0．05）。结论：TGF-β1和VEGF共同参与了椎间盘退变的形成和发展，在椎间盘退变的发病中可能起重要的作用。调节细胞因子（TGF-β1、VEGF）在终板软骨细胞中的表达，可能为椎间盘退变的治疗提供一种新的途径。     

碱性成纤维细胞生长因子对体外培养的乳兔软骨细胞中p53 mRNA表达的影响

目的 探讨在乳兔关节软骨细胞体外培养过程中,成纤维细胞生长因子（bFGF）对p53mRNA表达水平的影响及意义。方法 原代体外培养乳兔关节软骨细胞并传代,实验组加入10ng／ml的bFGF,光镜下观测各代细胞形态学变化,RT—PCR检测各代细胞中p53基因在mRNA水平上的表达。结果光镜观察体外培养的软骨细胞,对照组第4代培养细胞始出现凋亡细胞,bFGF组第7代出现少量凋亡细胞;RT—PCR检测bFGF组p53的目的基因指数（p53吸光光度值,β—actin吸光光度值）明显低于对照组（P〈0．05）。结论 在体外培养的兔关节软骨细胞中bFGF下调p53基因mRNA水平表达。     

ORIGINAL ARTICLE: Transforming Growth Factor‐Beta1 Gene Polymorphisms in Korean Patients with Pre‐eclampsia

Citation Kim SY, Lim JH, Park SY, Yang JH, Kim MY, Kim MH, Ryu HM. Transforming growth factor‐beta1 (TGF‐β1) gene polymorphisms in Korean patients with pre‐eclampsia. Am J Reprod Immunol 2010; 63: 291–298 Problem The aim of this study was to investigate whether c.869T>C (Leu10Pro) and c.915G>C (Arg25Pro) polymorphisms in exon1 of the transforming growth factor‐beta1 (TGF‐β1) gene are associated with development of pre‐eclampsia (PE) in Korean women. Method of study We analyzed blood samples from 164 patients with PE and 182 healthy pregnant women using the polymerase chain reaction and DNA sequencing. Results The frequencies of the 869CC and combined TC/CC genotypes were higher in patients with PE than in healthy controls. In the PE with intrauterine growth restriction (IUGR), the frequencies of these genotypes were also higher than that in controls. Furthermore, the 869C allele frequency was significantly higher in both PE and IUGR‐complicated PE than in controls. Multivariate analysis showed that the 869TC, CC, and combined TC/CC genotypes were associated with an increased risk of PE compared with the 869TT genotype. In addition, the 869TC, CC, and combined TC/CC genotypes were significantly associated with an increased risk of IUGR‐complicated PE compared with the 869TT genotype. The TGF‐β1 c.915G>C polymorphism was not detected in our population. Conclusion Our findings indicate that the TGF‐β1 c.869T>C polymorphism may be a genetic risk factor for PE and IUGR‐complicated PE.     

成纤维细胞生长因子受体2基因与肿瘤的相关性研究

成纤维细胞生长因子受体2(fibroblast growth factor receptor 2,FGFR2)是人成纤维细胞生长因子受体(FGFRs)家族中的一员,FGFRs在细胞的增殖、分化、血管生成、骨骼发育中起着十分重要的作用.研究表明成纤维细胞生长因子受体2(FGFR2)与肿瘤的发生存在一定相关性.本文就其特点作如下总结.     

Epidermal Growth Factor Receptor Is Related to Poor Survival in Glioblastomas: Single-Institution Experience

Purpose

There are conflicting results surrounding the prognostic significance of epidermal growth factor receptor (EGFR) status in glioblastoma (GBM) patients. Accordingly, we attempted to assess the influence of EGFR expression on the survival of GBM patients receiving postoperative radiotherapy.

Materials and Methods

Thirty three GBM patients who had received surgery and postoperative radiotherapy at our institute, between March 1997 and February 2006, were included. The evaluation of EGFR expression with immunohistochemistry was available for 30 patients. Kaplan-Meier survival analysis and Cox regression were used for statistical analysis.

Results

EGFR was expressed in 23 patients (76.7%), and not expressed in seven (23.3%). Survival in EGFR expressing GBM patients was significantly less than that in non-expressing patients (median survival: 12.5 versus 17.5 months, p=0.013). Patients who received more than 60 Gy showed improved survival over those who received up to 60 Gy (median survival: 17.0 versus 9.0 months, p=0.000). Negative EGFR expression and a higher radiation dose were significantly correlated with improved survival on multivariate analysis. Survival rates showed no differences according to age, sex, and surgical extent.

Conclusion

The expression of EGFR demonstrated a significantly deleterious effect on the survival of GBM patients. Therefore, approaches targeting EGFR should be considered in potential treatment methods for GBM patients, in addition to current management strategies.     

Addition of a C-Terminal Extension Sequence to Transforming Growth Factor-pl Interferes with Biosynthetic Processing and Abolishes Biological Activity

Abstract

Transforming growth factor-β1 (TGF-β1) is synthesized and secreted as a biologically latent complex. It has been proposed that one role of the latent complex is to prevent premature interaction of ligand and receptor intracellularly during biosynthesis (Wakefield et al, J. Cell Biol. (1987) 105, 965-9751. To test this hypothesis, the endoplasmic reticulum retention sequence Lys-Asp-Glu-Leu (KDEL) was added to the C-terminus of the wildtype TGF-β1 coding sequence, and to a construct in which mutagenesis of two cysteine residues in the precursor pro region results in the synthesis and secretion of active, as opposed to latent, TGF-β. Addition of either SEKDEL, or the control sequence SEKDVS to the TGF-β1 protein abolished biological activity. Western blot analysis indicated that the extended gene products are synthesized, but that the extension sequence partially interferes with the normal dimerization of the protein product, and totally inhibits the normal proteolytic processing and glycosylation of the precursor protein. The data suggest that correct folding of the highly conserved C terminus of TGF-PI is critical for subsequent proteolytic cleavage and glycosylation at sites that are quite distant in the primary sequence. Thus molecular strategies for the generation of TGF-β antagonists or superagonists should avoid extensive modification of this region of the molecule. Since synthesis of the endogenous TGF-β1 is unaffected by the presence of the mutated analog, the data further indicate that transfection with the KDEL-extended TGF-β1 sequence cannot be used as a dominant negative mutation to prevent secretion of the endogenous TGF-Pβ protein.