ALK-5 mediates endogenous and TGF-beta1-induced expression of connective tissue growth factor in embryonic lung

Transforming growth factor-beta1 (TGF-beta1) has been implicated as a major negative regulator of lung branching morphogenesis. Since connective tissue growth factor (CTGF) is a downstream mediator of TGF-beta1 effects on mesenchymal cells, we hypothesized that TGF-beta1 induces CTGF expression in mouse embryonic lung explants and that CTGF mediates TGF-beta1 inhibition of branching morphogenesis. We show that addition of TGF-beta1 to the serum-free medium of embryonic day (E)12.5 lung explant cultures inhibited branching morphogenesis and induced CTGF mRNA expression in time- and dose-dependent manners. In contrast to basal endogenous CTGF protein, which was exclusively localized in the distal airway epithelium, TGF-beta1-induced CTGF protein was localized in both the epithelium and the mesenchyme. Addition of exogenous CTGF to culture medium directly inhibited branching morphogenesis. To identify the signal transduction pathway through which TGF-beta1 induces CTGF, we used SB431542, a specific inhibitor for TGF-beta type I receptor (TbetaRI)/ALK-5 to block TGF-beta1-induced Smad2/3 phosphorylation. Consequently, SB431542 stimulated normal branching morphogenesis and blocked TGF-beta1 inhibition of branching. Furthermore, SB-431542 blocked both endogenous and TGF-beta1-induced expression of CTGF mRNA and protein. These results demonstrate for the first time that TGF-beta1 induces CTGF expression in mouse embryonic lung explants, that CTGF inhibits branching morphogenesis, and that both endogenous and TGF-beta1-induced CTGF expression are mediated by the TbetaRI/ALK-5-dependent Smad2 signaling pathway.     

Density-dependent inhibition of cell growth by transforming growth factor-beta 1 in normal human fibroblasts

We report here that transforming growth factor-beta 1 (TGF-beta 1) inhibits platelet-derived growth factor (PDGF)-induced DNA synthesis in normal human fibroblasts in a cell density-dependent manner; no inhibition was seen in sparse cultures, approximately 50% inhibition in confluent cell cultures, and an almost total inhibition in dense cultures. The PDGF-inducible genes c-myc and c-fos were induced also by TGF-beta 1. Simultaneous addition of TGF-beta 1 and PDGF resulted in sustained, rather than transient, expression of c-fos mRNA; c-fos mRNA was detected as long as 24 hr after addition of PDGF and TFG-beta 1. TGF-beta 1 also induced mRNA for the A chain, but not the B chain, of PDGF. Conversely, PDGF induced TGF-beta 1 mRNA in sparse but not in dense cultures. These data indicate the existence of a complex interdependent regulation of PDGF and TGF-beta mRNA expression which is influenced by the cell density.     

川芎嗪和siRNA沉默转化生长因子β1干预大鼠肝星状细胞 结缔组织生长因子的表达

背景：作者前期研究发现川芎嗪可以通过抑制肝星状细胞的增殖、阻抑p38MARK信号通路、下调结缔组织生长因子的表达等多途径发挥抗肝纤维化的作用。一般认为转化生长因子β1是促进纤维化发生最重要的细胞因子,结缔组织生长因子是转化生长因子β1的下游效应介质,siRNA靶向抑制转化生长因子β1可能成为治疗肝纤维化的新方法。 目的：观察川芎嗪及化学合成的siRNA转化生长因子β1对大鼠肝星状细胞结缔组织生长因子表达的影响。 方法：体外培养大鼠肝星状细胞,从3条siRNA转化生长因子β1中筛选一条有效的基因沉默片段,然后利用脂质体转染试剂共同转染培养24 h的细胞作为转染组,并设计空白对照组、转染试剂组、川芎嗪组及联合治疗组。 结果与结论：与空白对照组相比,转染组、川芎嗪组、联合治疗组Ⅰ、Ⅲ型胶原及结缔组织生长因子mRNA表达均下调(P < 0.05)。在转染组、川芎嗪组及联合治疗组,结缔组织生长因子蛋白表达均下调(P < 0.05);培养上清液中Ⅰ型胶原和Ⅲ型胶原含量均降低(P < 0.05)。结果表明,siRNA 沉默转化生长因子β1基因能下调结缔组织生长因子的表达,减少Ⅰ、Ⅲ型胶原的表达。川芎嗪也能下调结缔组织生长因子的表达,但对Ⅰ、Ⅲ型胶原表达的抑制作用更加显著,提示川芎嗪抗肝纤维化可能是多种作用靶点。     

Hypoxia induces expression of connective tissue growth factor in scleroderma skin fibroblasts

Connective tissue growth factor (CTGF) plays a role in the fibrotic process of systemic sclerosis (SSc). Because hypoxia is associated with fibrosis in several profibrogenic conditions, we investigated whether CTGF expression in SSc fibroblasts is regulated by hypoxia. Dermal fibroblasts from patients with SSc and healthy controls were cultured in the presence of hypoxia or cobalt chloride (CoCl(2)), a chemical inducer of hypoxia-inducible factor (HIF)-1alpha. Expression of CTGF was evaluated by Northern and Western blot analyses. Dermal fibroblasts exposed to hypoxia (1% O(2)) or CoCl(2) (1-100 microM) enhanced expression of CTGF mRNA. Skin fibroblasts transfected with HIF-1alpha showed the increased levels of CTGF protein and mRNA, as well as nuclear staining of HIF-1alpha, which was enhanced further by treatment of CoCl(2). Simultaneous treatment of CoCl(2) and transforming growth factor (TGF)-beta additively increased CTGF mRNA in dermal fibroblasts. Interferon-gamma inhibited the TGF-beta-induced CTGF mRNA expression dose-dependently in dermal fibroblasts, but they failed to hamper the CoCl(2)-induced CTGF mRNA expression. In addition, CoCl(2) treatment increased nuclear factor (NF)-kappaB binding activity for CTGF mRNA, while decreasing IkappaBalpha expression in dermal fibroblasts. Our data suggest that hypoxia, caused possibly by microvascular alterations, up-regulates CTGF expression through the activation of HIF-1alpha in dermal fibroblasts of SSc patients, and thereby contributes to the progression of skin fibrosis.     

转化生长因子-β1诱导大鼠毛乳头细胞转分化为成纤维细胞的研究

目的 研究大鼠触须毛乳头细胞在转化生长因子-β1（TGF-β1）诱导作用下转分化为成纤维细胞的可能性,从新的角度探讨增生性瘢痕的形成机制.方法 消化收集第4代毛乳头细胞,处理组以 TGF-β1（10 ng/mL）处理细胞4 d;对照组加入正常培养基（不含胎牛血清的DMEM/F12培养液）,每隔24 h观察两组细胞生长形态及生长方式;分别用实时一步法RT-PCR和流式细胞仪检测细胞α平滑肌肌动蛋白（α-SMA）、波形蛋白（Vimentin）的mRNA表达和蛋白表达,Western blotting检测成纤维细胞特异蛋白（FSP1）的表达.结果流式细胞仪检测TGF-β1诱导48 h、72 h后α-SMA表达明显低于对照组（P〈0.01）;TGF-β1诱导48 h、72 h、96 h后Vimentin表达明显高于对照组（P〈0.01）.实时一步法RT-PCR检测Vimentin的mRNA表达逐渐增强,α-SMA的mRNA表达在48 h后明显下降（P〈0.01）,但96 h后表达又有所增加;Western blotting法检测FSP1表达在诱导48 h后逐渐增加（P〈0.01）.结论 毛乳头细胞经TGF-β1诱导后,失去其典型的细胞形态及生长方式,而表现出成纤维细胞的细胞形态及生长方式;其相对特异性标记物α-SMA的表达下降,而成纤维细胞的相对特异性标记物Vimentin和FSP1表达增加.故TGF-β1可诱导大鼠毛乳头细胞转分化为成纤维细胞.     

Immunolocalization of transforming growth factor-beta1, connective tissue growth factor,phosphorylated-SMAD2/3, and phosphorylated-ERK1/2 during mouse incisor development

Background/aims: Connective tissue growth factor (CTGF) is a downstream mediator of transforming growth factor-beta 1 (TGF-β₁) and TGF-β₁-induced CTGF expression is regulated through SMAD and mitogen-activated protein kinase (MAPK) signaling pathways. However, little is known about the localization of CTGF and TGF-β₁ signaling cascades during incisor development. Therefore, we aimed to investigate the distribution pattern of TGF-β₁, CTGF, phosphorylated-SMAD2/3 (p-SMAD2/3), and phosphorylated-ERK1/2 (p-ERK1/2) in the developing mouse incisors.

Materials and methods: ICR mice heads of embryonic (E) day 16.5, postnatal (PN) day 0.5 and PN3.5 were processed for immunohistochemistry.

Results: From E16.5 to PN3.5, moderate to strong staining for TGF-β₁ and CTGF was localized in stellate reticulum (SR), transit amplifying (TA) cells, outer enamel epithelium (OEE), preameloblasts (PA), preodontoblasts (PO), and dental papilla (DP). p-SMAD2/3 was weakly positive in SR and OEE at E16.5 and PN0.5 but was strongly positive in SR and OEE at PN3.5. Particularly, in the stem cell niche, p-SMAD2/3 was only localized in SR cells adjacent to OEE. There was no staining for p-SMAD2/3 in TA cells, PA and PO, although weak to moderate staining for p-SMAD2/3 was seen in DP. From E16.5 to PN3.5, p-ERK1/2 was negative in TA cells, OEE, PA and PO, whereas weak to moderate staining for p-ERK1/2 was observed in SR. DP was moderately stained for p-ERK1/2.

Conclusions: TGF-β₁ and CTGF show a similar expression, while p-SMAD2/3 and p-ERK1/2 exhibit differential distribution pattern, which indicates that CTGF and TGF-β₁ signaling cascades might play a regulatory role in incisor development.     

The accumulation of type I collagen mRNAs in human embryonic lung fibroblasts stimulated by transforming growth factor-beta

We examined the expression of type I collagen mRNAs (alpha 1(I) and alpha 2 (I)) by embryonic lung fibroblasts in cultures treated with transforming growth factor-beta (TGF-beta). TGF-beta caused a concentration dependent increase in the expression of alpha 1(I) mRNA for type I collagen. TGF-beta at low concentration (0.1 ng/ml) slightly increased the level of alpha 1(I) mRNA. Higher concentrations of TGF-beta (1.0 and 5.0 ng/ml) further increased the amount of alpha 1(I) mRNA. The increase in alpha 1(I) mRNA was associated with a marked increase in production of intact type I collagen molecules. TGF-beta did not increase expression of alpha 2(I) mRNA. The alpha 2(I) mRNA levels in human lung fibroblast cultures were not affected by varying the duration of exposure to TGF-beta nor the concentration of TGF-beta. In contrast, TGF-beta increased the amount of both alpha 1(I) and alpha 2(I) mRNA in NIH3T3 cells. These data suggest that the amount of alpha 2(I) mRNA is not rate limiting with respect to type I collagen production during TGF-beta stimulation in human lung fibroblast cultures.     

周期性张应力下人牙周膜成纤维细胞结缔组织生长因子的表达

背景：前期研究发现,周期性张应力在一定时间内可以诱导人牙周膜成纤维细胞增殖。 目的：观察周期性张应力对人牙周膜成纤维细胞表达结缔组织生长因子的影响;明确JNK、p38MAPK、PI3K信号通路在周期性张应力诱导人牙周膜成纤维细胞表达结缔组织生长因子过程中的作用。 方法：采用多通道细胞牵张应力加载系统对体外培养的人牙周膜成纤维细胞分别给予周期性张应力刺激1,6,12,24 h,并以未加力组为对照组。对加力12 h的细胞分别添加JNK、p38MAPK、PI3K信号通路特异性抑制剂预处理,并与未加抑制剂的细胞作对比。应用ELISA法检测培养细胞分泌到上清液中的结缔组织生长因子蛋白;应用荧光定量PCR技术检测细胞结缔组织生长因子mRNA的表达。 结果与结论：加载周期性张应力组与对照组相比较,1 h人牙周膜成纤维细胞表达结缔组织生长因子开始增强、6 h表达明显增强,12 h达最高峰值、24 h表达开始下降。加入JNK信号通路特异性抑制剂后,人牙周膜成纤维细胞表达结缔组织生长因子出现下降;而加入p38MAPK信号通路和PI3K信号通路的特异性抑制剂后结缔组织生长因子表达未发生明显改变。提示在一定时间范围内,周期性张应力引起结缔组织生长因子mRNA与蛋白水平的表达与时间呈依赖性升高;其后随着时间的延长,结缔组织生长因子的表达则开始下降。周期性张应力通过JNK通路的介导调控人牙周膜成纤维细胞结缔组织生长因子的表达。中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

结缔组织生长因子在转化生长因子β诱导的肾小管上皮细胞转分化中的作用

目的：探讨结缔组织生长因子（CTGF）在转化生长因子-β1（TGF-β1）诱导的肾小管上皮细胞表型转化中的可能作用。方法： 将NRK52E肾小管上皮细胞分组处理，光镜、扫描电镜、透射电镜观察细胞形态的改变，细胞免疫组化检测ɑ-平滑肌肌动蛋白（α-SMA）和细胞角蛋白-18的表达，RT-PCR和Western blot检测Ⅰ型胶原的表达。 结果：TGF-β1 10 μg/L作用3 d，NRK52E小管上皮细胞失去正常的椭圆形，变得肥大，胞体拉长，扫描电镜下，见成纤维细胞状，失去上皮细胞特有的顶端-基底极性和表面的微绒毛结构，透射电镜下胞浆中见到微丝和致密体结构，骨架标志上肾小管上皮细胞较具特征性的细胞角蛋白-18表达减少,肌成纤维细胞标志性的α-SMA表达增多，Ⅰ型胶原分泌增多；加入TGF-β1中和抗体和CTGF反义寡核苷酸可以大部分阻断TGF-β的作用，而正义的CTGF寡核苷酸不能阻断TGF-β的作用。 结论： NRK52E细胞中，CTGF作为TGF-β的下游效应因子，介导了TGF-β诱导的肾小管上皮细胞转分化。     

Inhibition of human neutrophil degranulation by transforming growth factor-beta1

Neutrophils enter tissues including the uterus and are found in the endometrium in increased numbers prior to menses. In this environment, they are exposed to transforming growth factor (TGF)-beta1 produced by endometrial stromal and epithelial cells. We observed that incubation of neutrophils in vitro with TGF-beta1 at 1 pg/ml significantly reduced their secretion of lactoferrin in response to lipopolysaccharide (LPS). This effect was achieved with as little as 15 min of pretreatment with TGF-beta1. Inhibition of lactoferrin release by TGF-beta1 was observed irrespective of whether neutrophils were stimulated by ligands for Toll-like receptor (TLR)-2, TLR-4 or FPR, the G protein-coupled receptor for formylated peptides. Inhibition by TGF-beta1 was negated by SB-431542, a small molecule inhibitor that specifically blocks the kinase activity of the type I TGF-beta receptor (ALK5) In contrast to lactoferrin release, another important neutrophil function, interleukin (IL)-8 driven chemotaxis, was not affected by TGF-beta1 at 1 pg/ml or 100 pg/ml. We conclude that in tissues of the female reproductive tract, TGF-beta1 inhibition of neutrophil degranulation may prevent these cells from initiating an inflammatory response or releasing degradative enzymes that could potentially damage the oocyte or fetus.     

Thy-1 expression regulates the ability of rat lung fibroblasts to activate transforming growth factor-beta in response to fibrogenic stimuli

Distinct subpopulations of fibroblasts contribute to lung fibrosis, although the mechanisms underlying fibrogenesis in these subpopulations are not clear. Differential expression of the glycophosphatidylinositol-linked protein Thy-1 affects proliferation and myofibroblast differentiation. Lung fibroblast populations selected on the basis of Thy-1 expression by cell sorting were examined for responses to fibrogenic stimuli. Thy-1 (-) and Thy-1 (+) fibroblast populations were treated with platelet-derived growth factor-BB, interleukin-1beta, interleukin-4, or bleomycin and assessed for activation of transforming growth factor (TGF)-beta, Smad3 phosphorylation, and alpha-smooth muscle actin and fibronectin expression. Thy-1 (-) fibroblasts responded to these stimuli with increased TGF-beta activity, Smad3 phosphorylation, and expression of alpha-smooth muscle actin and fibronectin, whereas Thy-1 (+) fibroblasts resisted stimulation. The unresponsiveness of Thy-1 (+) cells is not because of defective TGF-beta signaling because both subsets respond to exogenous active TGF-beta. Rather, Thy-1 (-) fibroblasts activate latent TGF-beta in response to fibrogenic stimuli, whereas Thy-1 (+) cells fail to do so. Defective activation is common to multiple mechanisms of TGF-beta activation, including thrombospondin 1, matrix metalloproteinase, or plasmin. Thy-1 (-) lung fibroblasts transfected with Thy-1 also become resistant to fibrogenic stimulation, indicating that Thy-1 is a critical biological response modifier that protects against fibrotic progression by controlling TGF-beta activation. These studies provide a molecular basis for understanding the differential roles of fibroblast subpopulations in fibrotic lung disease through control of latent TGF-beta activation.     

In situ expression of connective tissue growth factor in human crescentic glomerulonephritis

Connective tissue growth factor (CTGF) has recently been recognized as an important profibrotic factor and is up-regulated in various renal diseases with fibrosis. The present study describes the sequential localization of CTGF mRNA and its association with transforming growth factor (TGF)-1 in human crescentic glomerulonephritis (CRGN). Furthermore, we examined the phenotype of CTGF-expressing cells using serial section analysis. Kidney biopsy specimens from 18 CRGN patients were examined using in situ hybridization and immunohistochemistry. CTGF mRNA was expressed in the podocytes and parietal epithelial cells (PECs) in unaffected glomeruli. In addition, it was strongly expressed in the cellular and fibrocellular crescents, particularly in pseudotubule structures. Serial sections revealed that the majority of CTGF mRNA-positive cells in the crescents co-expressed the epithelial marker cytokeratin, but not a marker for macrophages. Moreover, TGF-1, its receptor TGF- receptor-I, and extracellular matrix molecules (collagen type I and fibronectin) were co-localized with CTGF mRNA-positive crescents. Our results suggest that CTGF is involved in extracellular matrix production in PECs and that it is one of the mediators promoting the scarring process in glomerular crescents.     

人转化生长因子β3、结缔组织生长因子和基质金属蛋白酶抑制剂1基因重组慢病毒多表达质粒的构建及活性检测*

背景：前期试验显示单个病毒载体介导的多基因共表达系统能够显著提高椎间盘退变转基因疗效。 目的：构建人转化生长因子β3、结缔组织生长因子和基质金属蛋白酶抑制剂1基因重组慢病毒多表达质粒。 方法：应用全基因合成技术,以“自剪切多肽2A”串联目的基因,并克隆到慢病毒表达质粒构建重组慢病毒质粒Lenti-TGF-β3-P2A-CTGF-T2A-TIMP1。转染293细胞后,应用RT-PCR和Western-Blot技术分别检测转染后不同时间点目的基因mRNA和蛋白质表达水平。 结果与结论：RT-PCR和Western-blot技术检测结果显示重组质粒成功表达了3种目的基因,并于转染后48 h左右达到峰值,“2A”结构下游基因蛋白质表达量约为上游基因的80%。说明成功构建了携带人转化生长因子β3、结缔组织生长因子和基质金属蛋白酶抑制剂1基因的慢病毒多表达质粒。     

ERK 1/2介导结缔组织生长因子刺激系膜细胞产生MCP-1

目的检测结缔组织生长因子（CTGF）是否刺激大鼠肾小球系膜细胞分泌单核细胞趋化蛋白-1（MCP-1）,并探讨其作用机制。方法应用CTGF刺激静息的培养的系膜细胞,在刺激后不同时间点应用RT-PCR方法测定MCP-1的mRNA表达,应用酶联免疫吸附试验（ELISA）测定上清液中MCP-1,应用趋化试验测定上清液对单核细胞（THP-1）的趋化作用。应用Western blot测定CTGF对细胞外信号调节激酶（ERK）1／2磷酸化的作用。应用ERK1／2抑制剂PD98059预处理,观察CTGF对上清液中MCP-1分泌的影响。结果应用CTGF刺激后,系膜细胞的MCP-1的mRNA表达上升,上清液中分泌量增加。MCP-1抗体可部分阻止上清液对单核细胞的趋化作用。CTGF诱导ERK1／2磷酸化,而PD98059可抑制这一作用,并部分抑制CTGF诱导的上清液中MCP-1的分泌。结论CTGF可引起系膜细胞分泌MCP-1,其作用机制部分依赖于ERK1／2的磷酸化。     

Distribution of transforming growth factor-beta 1 in human astrocytomas.

We used immunohistochemical techniques to study the distribution of transforming growth factor-beta 1 (TGF-beta 1) and infiltrating lymphocytes and macrophages in human astrocytomas. Thirteen of 15 grade 4 astrocytomas (glioblastomas) showed staining with anti-TGF-beta 1 antibody, predominantly in proliferating endothelial complexes and surrounding small and medium-sized blood vessels. Brain tissue microscopically free of tumor cells (n = 8) and more differentiated astrocytomas of varying grade (1 to 3; n = 6) devoid of endothelial proliferation did not stain with anti-TGF-beta 1. Normal brain contained only rare lymphoreticular cells. The majority of astrocytomas studied, however, contained T lymphocytes and macrophages with smaller numbers of B lymphocytes. The lymphoreticular infiltrates were concentrated primarily in close proximity to blood vessels. Within an individual tumor perivascular regions staining for TGF-beta 1 never contained more than occasional T lymphocytes. Perivascular regions not staining for TGF-beta 1 frequently contained low to high numbers of T lymphocytes. The inverse relationship in the distribution of TGF-beta 1 and lymphocyte infiltrates is compatible with a functional relationship between this cytokine and an immune effector cell response to glioblastomas.