Effects of TGF-β1 on the proliferation and differentiation of human periodontal ligament cells and a human periodontal ligament stem/progenitor cell line

Periodontal ligament (PDL) is a specialized connective tissue that influences the lifespan of the tooth. Transforming growth factor-β1 (TGF-β1) is a multifunctional cytokine, but little is known about the effects of TGF-β1 on PDL cells. Our aim has been to demonstrate the expression of TGF-β1 in rat PDL tissues and to evaluate its effects on the proliferation and gene expression in human PDL cells (HPLCs) and a human PDL stem/progenitor cell line, line 1-11, that we have recently developed. The expression of TGF-β1 in the entire PDL tissue was confirmed immunohistochemically, and both HPLCs and cell line 1-11 expressed mRNA from the TGF-β1, TGF-β type I receptor, and TGF-β type II receptor genes. Although exogenous TGF-β1 stimulated the proliferation of HPLCs, it did not upregulate the expression of alpha-smooth muscle actin (α-SMA), type I collagen (Col I), or fibrillin-1 (FBN1) mRNA or of α-SMA protein in HPLCs, whereas expression for these genes was attenuated by an anti-TGF-β1 neutralizing antibody. In contrast, exogenous TGF-β1 reduced the proliferation of cell line 1-11, although it upregulated the expression of α-SMA, Col I, and FBN1 mRNA and of α-SMA protein in this cell line. In addition, interleukin-1 beta stimulation significantly reduced the expression of TGF-β1 mRNA and protein in HPLCs. Thus, TGF-β1 seems to play an important role in inducing fibroblastic differentiation of PDL stem/progenitor cells and in maintaining the PDL apparatus under physiological conditions.     

TGF-β-Operated Growth Inhibition and Translineage Commitment into Smooth Muscle Cells of Periodontal Ligament-Derived Endothelial Progenitor Cells through Smad- and p38 MAPK-Dependent Signals

The periodontal ligament (PDL) is a fibrous connective tissue that attaches the tooth to the alveolar bone. We previously demonstrated the ability of PDL fibroblast-like cells to construct an endothelial cell (EC) marker-positive blood vessel-like structure, indicating the potential of fibroblastic lineage cells in PDL tissue as precursors of endothelial progenitor cells (EPCs) to facilitate the construction of a vascular system around damaged PDL tissue. A vascular regeneration around PDL tissue needs proliferation of vascular progenitor cells and the subsequent differentiation of the cells. Transforming growth factor-β (TGF-β) is known as an inducer of endothelial-mesenchymal transition (EndMT), however, it remains to be clarified what kinds of TGF-β signals affect growth and mesenchymal differentiation of PDL-derived EPC-like fibroblastic cells. Here, we demonstrated that TGF-β1 not only suppressed the proliferation of the PDL-derived EPC-like fibroblastic cells, but also induced smooth muscle cell (SMC) markers expression in the cells. On the other hand, TGF-β1 stimulation suppressed EC marker expression. Intriguingly, overexpression of Smad7, an inhibitor for TGF-β-induced Smad-dependent signaling, suppressed the TGF-β1-induced growth inhibition and SMC markers expression, but did not the TGF-β1-induced downregulation of EC marker expression. In contrast, p38 mitogen-activated protein kinase (MAPK) inhibitor SB 203580 suppressed the TGF-β1-induced downregulation of EC marker expression. In addition, the TGF-β1-induced SMC markers expression of the PDL-derived cells was reversed upon stimulation with fibroblast growth factor (FGF), suggesting that the TGF-β1 might not induce terminal SMC differentiation of the EPC-like fibroblastic cells. Thus, TGF-β1 not only negatively controls the growth of PDL-derived EPC-like fibroblastic cells via a Smad-dependent manner but also positively controls the SMC-differentiation of the cells possibly at the early stage of the translineage commitment via Smad- and p38 MAPK-dependent manners.     

Functions of beta2-adrenergic receptor in human periodontal ligament cells

Adrenergic receptors (ARs) are receptors of noradrenalin and adrenalin, of which there are nine different subtypes. In particular, β2 adrenergic receptor (β2-AR) is known to be related to the restoration and maintenance of homeostasis in bone and cardiac tissues; however, the functional role of signaling through β2-AR in periodontal ligament (PDL) tissue has not been fully examined. In this report, we investigated that β2-AR expression in PDL tissues and their features in PDL cells. β2-AR expressed in rat PDL tissues and human PDL cells (HPDLCs) derived from two different patients (HPDLCs-2G and -3S). Rat PDL tissue with occlusal loading showed high β2-AR expression, while its expression was downregulated in that without loading. In HPDLCs, β2-AR expression was increased exposed to stretch loading. The gene expression of PDL-related molecules was investigated in PDL clone cells (2-23 cells) overexpressing β2-AR. Their gene expression and intracellular cyclic adenosine monophosphate (cAMP) levels were also investigated in HPDLCs treated with a specific β2-AR agonist, fenoterol (FEN). Overexpression of β2-AR significantly promoted the gene expression of PDL-related molecules in 2 to 23 cells. FEN led to an upregulation in the expression of PDL-related molecules and increased intracellular cAMP levels in HPDLCs. In both HPDLCs, inhibition of cAMP signaling by using protein kinase A inhibitor suppressed the FEN-induced gene expression of α-smooth muscle actin. Our findings suggest that the occlusal force is important for β2-AR expression in PDL tissue and β2-AR is involved in fibroblastic differentiation and collagen synthesis of PDL cells. The signaling through β2-AR might be important for restoration and homeostasis of PDL tissue.     

New molecular approaches to tissue analysis.

The completion of the Human Genome Project will produce new opportunities for analysis of genes and their products in human tissue. The emergence of new technologies will enable investigators to directly examine human tissues for gene deletion, transposition, and amplification. In addition, we will be able to assess the complete gene expression of a tissue by examining the mRNA species using microarray chips. The emerging technologies of laser capture microdissection and RNA amplification enables these procedures to be carried out on groups of a few hundred cells, which will facilitate the examination of heterogeneous lesions. Finally, the application of tissue arrays and the capability of obtaining protein sequences in samples of only a few femtomoles of protein using desorption mass spectroscopy will revolutionize the analysis of protein expression.     

TGF-β1 和CTGF 在大鼠高动力性肺动脉高压模型中的表达及意义*

目的:研究大鼠高动力性肺动脉高压模型转化生长因子-β1(transforming growth factor-beta 1,TGF-β1)和结缔组织生长因子(connective tissue growing factor,CTGF)的表达变化及意义。方法:45只SD大鼠随机分成左肺全切组(A)、假手术组(B)和对照组(C),每组15只。手术后6周测各组大鼠肺动脉平均压(mPAP),右心室肥厚指数(RVH),光镜下检测肺肌型小动脉占肺小血管百分比(SMA%)。免疫组化观察TGF-β1和CTGF在肺组织中的表达,RT-PCR检测肺组织TGF-β1mRNA和CTGF mRNA的表达水平。结果:①左肺全切后复制了高动力性肺动脉高压模型,引起mPAP、RVH和SMA%明显增高(P<0.05)。②左肺全切组TGF-β1和CTGF蛋白及mRNA表达均较假手术组和对照组显著性增加(P<0.05)。而假手术组和对照组之间各指标没有显著性差异。结论:TGF-β1和CTGF的过度表达是高动力性肺动脉高压发生发展的重要因素,可能共同促进了肺血管重构。     

Eplerenone inhibits atrial fibrosis in mutant TGF-β1 transgenic mice

The purpose of the present study was to study the impacts of eplerenone(EPL), an antagonist of mineralocorticoid receptors(MR), on atrial fibrosis in a mouse model with selective fibrosis in the atrium, and to explore the possible mechanisms. Using mutant TGF-β1 transgenic(Tx) mice, we first demonstrated that EPL inhibited atrial fibrosis specifically and decreased macrophage accumulation in the atria of these mice. Results from immunohistochemistry and western blotting showed that EPL attenuated protein expression of fibrosis-related molecules such as connective tissue growth factor(CTGF) and fibronectin in the atria of Tx mice. In culture, EPL inhibited gene expression of fibrosis-related molecules such as fibronectin, α-SMA, and CTGF in TGF-β1-stimulated atrial fibroblasts. Finally, using a co-culture system, we showed that TGF-β1-stimulated atrial fibroblasts induced migration of macrophages and this was blocked by EPL. EPL also blocked TGF-β1-induced gene expression of intedeukin-6(IL-6) in atrial fibroblasts. Therefore, we conclude that EPL attenuated atrial fibrosis and macrophage infiltration in Tx mice. TGF-β1 and IL-6 were involved in the impacts of EPL on activation of atrial fibroblasts and interactions between fibroblasts and macrophages.     

TGF-β1诱导人牙周膜细胞细胞骨架重排的机制

目的：牙周病是由多种因素引起的,特别是人牙周膜细胞的缺失。转化生长因子-β1（TGF—β1）是一种多功能细胞因子,在治疗牙周病中发挥重要的作用,但很少有人清楚地研究TGF-β1对人牙周膜细胞的影响。因此,本研究的目的是探讨TGF—p1诱导人牙周膜细胞细胞骨架重排的信号通路。方法：人牙周膜细胞取自健康的前磨牙,并向同步化处理的细胞中加入10ng／m1的TGF-β1,并通过相差显微镜观察它们的形态学变化。通过免疫组化和共聚焦显微镜观察F-肌动蛋白重排。用Westernblot分析蛋白表达情况。结果：我们发现TGF-β1诱导人牙周膜细胞细胞骨架重排,激活ROCK蛋白的表达,并增加p-IIMK和p-cofilin的蛋白表达。ROCK抑制剂Y-27632使ROCK,p-IIMK和p-cofilin的蛋白表达下降。结论：TGF-β1可以诱导人牙周膜细胞细胞骨架重排,并且是通过上凋ROCK,P．IlMK和p-cofilin的活性完成的。本研究可以增强对TGF-β1在治疗牙周疾病方面的作用机制的了解。     

Abberant methylation of p16, HIC1, N33 and GSTP1 genes in tumor epitelium and tumor-associated stromal cells of prostate cancer

The methylation status of four genes significant in prostate carcinogenesis p16, HIC1, N33 and GSTP1, were evaluated using quantitative methylationsensitive polymerase chain reaction. Tumor epithelia, tumor-associated stroma, normal epithelia, foci of PIN and benign prostate hyperplasia, and stroma adjacent to tumor tissues were isolated from whole-mount prostatectomy specimens of patients with localized prostate cancer by using laser capture microdissection. We found high levels of gene methylation in the tumor epithelium and tumor-associated stromal cells and some methylation in both hyperplastic epithelium and stromal cells in normal-appearing tissues located adjacent to tumors. Promoter methylation in the non-neoplastic cells of the prostate tumor microenvironment may play an important role in cancer development and progression. We examined the promoter methylation status of pl6, HIC1, N33 and GSTP1 in prostate biopsy fragments and prostate tissues after radical prostatectomy from patients with adenocarcinoma without laser capture microdissection. Methylation frequencies of all genes in tumor samples were considerably lower than frequencies in microdissected tumour samples (HIC1, 71 versus 89%; p16, 22 versus 78%; GSTP1, 32 versus 100%; N33, 20 versus 33%). The laser capture microdissection is required procedure in methylation studies taking into account multifocality and heterogenity of prostate cancer tissue.     

Micropreparation of single secretory glands from the carnivorous plant Nepenthes

A rapid mechanical micropreparation technique has been developed to isolate multicellular glands, here from Nepenthes pitchers, based on a microdissection platform. The method is an alternative to laser capture dissection because fresh plant tissue can be used directly without previous fixation. Subsequent experiments, such as polymerase chain reaction (PCR)-based detection of an individual gene encoding a thaumatin-like protein and RNA extraction for gene expression analysis, have been successfully added to prove the quality of the prepared biological material. The procedure described is adaptable to a broad range of plant species and should find wide application in the preparation of multicellular glands or other tissues.     

HSPA1A is upregulated in periodontal ligament at early stage of tooth movement in rats

Heat shock proteins (HSPs) are molecular chaperones that maintain intracellular protein homeostasis and ensure survival of cells. Continuous orthodontic force on the tooth is considered to be a type of physical stress loaded to the periodontal ligament (PDL). However, little is known about the role of HSPs during tooth movement. This study was performed to examine the expression of HSPs in the PDL during tooth movement using laser microdissection, microarray analysis, real-time RT-PCR and immunohistochemistry. Gene expression of HSPA1A in the pressure zone of the PDL was higher during 6 h of tooth movement than in the control group. Expression of HSPA1A decreased with time. HSPA1A was also detected in the pressure zone of the PDL at the protein level 24 h after the initial tissue change. These results strongly suggest that expression of HSPA1A in the PDL during early stages of tooth movement is a critical factor for tissue reaction.     

NLRP1 promotes TGF-β1-induced myofibroblast differentiation in neonatal rat cardiac fibroblasts

Nuclear localization leucine-rich-repeat protein 1 (NLRP1) is a member of Nod-like receptors (NLRs) family. Recent studies have reported that NLRP1 is involved in various diseases, especially in cardiovascular diseases. However, the effect of NLRP1 on cardiac fibrosis remains unclear. In this study, NLRP1 overexpression and NLRP1 silencing constructs were transfected into neonatal rat cardiac fibroblasts induced by TGF-β1 for 48 h to investigate the effect of NLRP1 in cardiac fibrosis and its molecular mechanisms. Cardiac fibroblasts were transfected with NLRP1 and then cultured in the presence and absence of TGF-β1and Smad3 inhibitor (SIS3). Our data indicated that NLRP1 not only promoted fibroblast activation and myofibroblast differentiation, but also upregulated the mRNA and protein levels of α-SMA in the TGF-β1-treated neonatal rat cardiac fibroblasts. Overexpressing NLRP1 in TGF-β1-induced cardiac fibroblasts upregulated the mRNA and protein levels of Collagen I, Collagen III, and connective tissue growth factor. Moreover, NLRP1 upregulated the protein levels of Smad2, Smad3, and Smad4 in nuclei of fibroblasts, and attenuated levels of phosphorylated Smad2 and Smad3 in the cytoplasm of fibroblasts induced by TGF-β1. In addition, the increase in fibrotic genes and Smad proteins was significantly reduced in the presence of SIS3. Our findings illustrated that NLRP1 promoted myofibroblast differentiation and excessive ECM production in TGF-β1-induced neonatal cardiac fibroblasts through directly targeting TGF-β1/Smad signaling pathways.     

Exposure to transforming growth factor-β1 after basic fibroblast growth factor promotes the fibroblastic differentiation of human periodontal ligament stem/progenitor cell lines

Basic fibroblast growth factor (bFGF) is a cytokine that promotes the regeneration of the periodontium, the specialized tissues supporting the teeth. bFGF, does not, however, induce the synthesis of smooth muscle actin alpha 2 (ACTA2), type I collagen (COL1), or COL3, which are principal molecules in periodontal ligament (PDL) tissue, a component of the periodontium. We have suggested the feasibility of using transforming growth factor-β1 (TGFβ1) to induce fibroblastic differentiation of PDL stem/progenitor cells (PDLSCs). Here, we investigated the effect of the subsequent application of TGFβ1 after bFGF (bFGF/TGFβ1) on the differentiation of PDLSCs into fibroblastic cells. We first confirmed the expression of bFGF and TGFβ1 in rat PDL tissue and primary human PDL cells. Receptors for both bFGF and TGFβ1 were expressed in the human PDLSC lines 1-11 and 1-17. Exposure to bFGF for 2 days promoted vascular endothelial growth factor gene and protein expression in both cell lines and down-regulated the expression of ACTA2, COL1, and COL3 mRNA in both cell lines and the gene fibrillin 1 (FBN1) in cell line 1-11 alone. Furthermore, bFGF stimulated cell proliferation of these cell lines and significantly increased the number of cells in phase G2/M in the cell lines. Exposure to TGFβ1 for 2 days induced gene expression of ACTA2 and COL1 in both cell lines and FBN1 in cell line 1-11 alone. BFGF/TGFβ1 treatment significantly up-regulated ACTA2, COL1, and FBN1 expression as compared with the group treated with bFGF alone or the untreated control. This method might thus be useful for accelerating the generation and regeneration of functional periodontium.     

Influence of Micropatterning on Human Periodontal Ligament Cells’ Behavior

The periodontal ligament (PDL) is highly ordered connective tissue located between the alveolar bone and cementum. An aligned and organized architecture is required for its physiological function. We applied micropatterning technology to arrange PDL cells in 10- or 20-μm-wide extracellular protein patterns. Cell and nuclear morphology, cytoskeleton, proliferation, differentiation, and matrix metalloproteinase system expression were investigated. Micropatterning clearly elongated PDL cells with a low cell-shape index and low spreading area. The nucleus was also elongated as nuclear height increased, but the nuclear volume remained intact. The cytoskeleton was rearranged to form prominent bundles at cells’ peripheral regions. Moreover, proliferation was promoted by 10- and 20-μm micropatterning. Osteogenesis and adipogenesis were each inhibited, but micropatterning increased PDL cells’ stem cell markers. β-catenin was expelled to cytoplasm. YAP/TAZ nuclear localization and activity both decreased, which might indicate their role in micropatterning-regulated differentiation. Collagen Ι expression increased in micropatterned groups. It might be due to the decreased expression of matrix metalloproteinase-1, 2 and the tissue inhibitor of metalloproteinase-1 gene expression elevation in micropatterned groups. The findings of this study provide insight into the effects of a micropatterned surface on PDL cell behavior and may be applicable in periodontal tissue regeneration.     

Angiotensin II-induced pro-fibrotic effects require p38MAPK activity and transforming growth factor beta 1 expression in skeletal muscle cells

Fibrotic disorders are typically characterised by excessive connective tissue and extracellular matrix (ECM) deposition that preclude the normal healing of different tissues. Several skeletal muscle dystrophies are characterised by extensive fibrosis. Among the factors involved in skeletal muscle fibrosis is angiotensin II (Ang-II), a key protein of the renin-angiotensin system (RAS). We previously demonstrated that myoblasts responded to Ang-II by increasing the ECM protein levels mediated by AT-1 receptors, implicating an Ang-II-induced reactive oxygen species (ROS) by a NAD(P)H oxidase-dependent mechanism. In this paper, we show that in myoblasts, Ang-II induced the increase of transforming growth factor beta 1 (TGF-β1) and connective tissue growth factor (CTGF) expression through its AT-1 receptor. This effect is dependent of the NAD(P)H oxidase (NOX)-induced ROS, as indicated by a decrease of the expression of both pro-fibrotic factors when the ROS production was inhibited via the NOX inhibitor apocynin. The increase in pro-fibrotic factors levels was paralleled by enhanced p38MAPK and ERK1/2 phosphorylation in response to Ang-II. However, only the p38MAPK activity was critical for the Ang-II-induced fibrotic effects, as indicated by the decrease in the Ang-II-induced TGF-β1 and CTGF expression and fibronectin levels by SB-203580, an inhibitor of the p38MAPK, but not by U0126, an inhibitor of ERK1/2 phosphorylation. Furthermore, we showed that the Ang-II-dependent p38MAPK activation, but not the ERK1/2 phosphorylation, was necessary for the NOX-derived ROS. In addition, we demonstrated that TGF-β1 expression was required for the Ang-II-induced pro-fibrotic effects evaluated by using SB-431542, an inhibitor of TGF-βRI kinase activity, and by knocking down TGF-β1 levels by shRNA technique. These results strongly suggest that the fibrotic response to Ang-II is mediated by the AT-1 receptor and requires the p38MAPK phosphorylation, NOX-induced ROS, and TGF-β1 expression increase mediated by Ang-II in skeletal muscle cells.