Embryonic expression of the transforming growth factor beta ligand and receptor genes in chicken

Results: Here we report the embryonic mRNA expression patterns in chicken embryos of the canonical TGFβ ligands (TGFB1, TGFB2, and TGFB3) and receptors (TGFBR1, TGFBR2, TGFBR3), plus the Activin A receptor, type 1 (ACVR1) and co receptor Endoglin (ENG) that also transduce TGFβ signaling. 相似文献   

Attenuated TGF‐β1 responsiveness of dendritic cells and their precursors in atopic dermatitis

The responsiveness of DCs and their precursors to transforming growth factor beta1 (TGF‐β1) affects the nature of differentiating DC subsets, which are essential for the severity of atopic dermatitis (AD). To evaluate TGF‐β signaling in monocytes and monocyte‐derived DCs of AD patients compared with that of controls, in vitro generated Langerhans cell (LC) like DCs, expression of TGF‐β receptors, phospho‐Smad2/3 and Smad7 were evaluated. Furthermore, TNF‐α expression and synergistic effects of TNF‐α upon TGF‐β signaling and DC generation were evaluated. We found LC‐like DC differentiation of monocytes from AD patients in response to TGF‐β1 was remarkably reduced and TGF‐β1 receptor expression was significantly lower compared with that of healthy controls. Attenuated TGF‐β1 responsiveness mirrored by lower phospho‐Smad2/3 expression after TGF‐β1 stimulation and higher expression of inhibitory Smad7 was observed in monocytes from AD patients. During DC generation, mRNA expression of Smad7 was relatively higher in LC‐like DCs of AD patients. Lower TNF‐α expression of monocytes from AD patients might further contribute to attenuated TGF‐β signaling in the disease since TNF‐α had synergistic effects on TGF‐β1 signaling and LC generation through mediating the degradation of Smad7. Our results demonstrate alleviated TGF‐β1 signaling together with the amount of soluble co‐factors might direct the nature of differentiating DCs.     

Increased expression of pro-inflammatory cytokines and metalloproteinase-1 by TGF-beta1 in synovial fibroblasts from rheumatoid arthritis and normal individuals

Transforming growth factor (TGF)-beta1 is expressed abundantly in the rheumatoid synovium. In this study, the inflammatory effect of TGF-beta1 in rheumatoid arthritis (RA) was investigated using cultured fibroblast-like synoviocytes (FLS) from RA and osteoarthritis (OA) patients, as well as non-arthritic individuals. mRNA expressions of IL-1beta, tumour necrosis factor (TNF)-alpha, IL-8, macrophage inflammatory protein (MIP)-1alpha and metalloproteinase (MMP)-1 were increased in RA and OA FLS by TGF-beta1 treatment, but not in non-arthritic FLS. Enhanced protein expression of IL-1beta, IL-8 and MMP-1 was also observed in RA FLS. Moreover, TGF-beta1 showed a synergistic effect in increasing protein expression of IL-1beta and matrix metalloproteinase (MMP)-1 with TNFalpha and IL-1beta, respectively. Biological activity of IL-1 determined by mouse thymocyte proliferation assay was also enhanced by 50% in response to TGF-beta1 in the culture supernatant of RA FLS. DNA binding activities of nuclear factor (NF)-kappaB and activator protein (AP)-1 were shown to increase by TGF-beta1 as well. These results suggest that TGF-beta1 contributes for the progression of inflammation and joint destruction in RA, and this effect is specific for the arthritic synovial fibroblasts.     

The relation between anti‐TGBFR1 immunohistochemical reaction and low Ki67, small tumor size and high estrogen receptor expression in invasive breast cancer

Most breast cancers are derived from the luminal epithelium, which composes the inside of the breast ductal structure. Ductal carcinoma in situ (DCIS) leads to invasive ductal carcinoma, but noncancerous intraductal proliferative lesions are also a risk factor for ductal carcinoma. The transforming growth factor beta (TGFB) signaling pathway behaves as a tumor suppressor in the early stage of cancer, and conversely as a tumor growth factor in invasive stages in several cancers. In this study, we performed immunohistochemistry with an antibody that detects the cytoplasmic region of TGFB receptor 1 (TGFBR1) and elucidated TGFBR1 protein expression in luminal epithelial cells of noncancerous breast ducts and in several cases of DCIS and invasive carcinoma. TGFBR1 expression was higher in noncancerous breast tissue than in cancerous tissue, and a difference in expression was also seen among histological subtypes. Comparing the expression level of TGFBR1 in cancer cells and clinico‐pathological parameters, cases expressing low TGFBR1 tended to show low estrogen receptor expression, large tumor size (≥10 mm), and a high Ki67 labeling index. These data suggested that TGFBR1 protein expression may be related to the suppression of breast cancer cell growth.     

ROCK1 Expression is Regulated by TGFβ3 and ALK2 During Valvuloseptal Endocardial Cushion Formation

During early heart development at the looped heart stage, endothelial cells in the outflow tract and atrioventricular (AV) regions transform into mesenchyme to generate endocardial cushion tissue. This endocardial epithelial–mesenchymal transition (EMT) is regulated by several regulatory pathways, including the transforming growth factor‐beta (TGFβ), bone morphogenetic protein (BMP), and Rho‐ROCK pathways. Here, we investigated the spatiotemporal expression pattern of ROCK1 mRNA during EMT in chick and examined whether TGFβ or BMP could induce the expression of ROCK1. At the onset of EMT, ROCK1 expression was up‐regulated in endothelial/mesenchymal cells. A three‐dimensional collagen gel assay was used to examine the mechanisms regulating the expression of ROCK1. In AV endocardium co‐cultured with associated myocardium, ROCK1 expression was inhibited by either anti‐TGFβ3 antibody, anti‐ALK2 antibody or noggin, but not SB431542 (ALK5 inhibitor). In cultured preactivated AV endocardium, TGFβ3 protein induced the expression of ROCK1, but BMP did not. AV endothelial cells that were cultured in medium supplemented with TGFβ3 plus anti‐ALK2 antibody failed to express ROCK1. These results suggest that the expression of ROCK1 is up‐regulated at the onset of EMT and that signaling mediated by TGFβ3/ALK2 together with BMP is involved in the expression of ROCK1. Anat Rec, 291:845‐857, 2008. © 2008 Wiley‐Liss, Inc.     

Expression,Structure, and Function of Enamel Proteinases

Proteinases serve two important functions during dental enamel formation: They (a) process and (b) degrade enamel proteins. Different enzymes carry out these functions. Enamelysin (MMP-20) is the foremost enamel matrix-processing enzyme. Its expression initiates prior to the onset of dentin mineralization and continues throughout the secretory stage of amelogenesis. In vitro, enamelysin catalyzes all of the amelogenin cleavages that are known to occur during the secretory stage in vivo, and it is probably the enzyme responsible for the processing of all enamel proteins. There is evidence suggesting that enamelysin activity is critical for proper enamel formation. Uncleaved and processed enamel proteins often segregate into different compartments within the developing enamel layer, suggesting that they may have different functions. Intact ameloblastin and its C-terminal cleavage products localize in the superficial rod and interrod enamel, while its N-terminal cleavage products congregate in the sheath space. Intact enamelin is only present at the mineralization front within a micrometer of the enamel surface, while its cleavage products concentrate in the rod and interrod enamel. Processed enamel proteins accumulate during the secretory stage, but disappear early in the maturation stage. Enamel matrix serine proteinase 1 (EMSP1), now officially designated kallikrein 4 (KLK4), is believed to be the predominant degradative enzyme that clears enamel proteins from the matrix during maturation. KLK4 expression initiates during the transition stage and continues throughout maturation. KLK4 concentrates at the enamel surface when the enamel matrix disappears, and aggressively degrades amelogenin in vitro. During tooth development, proteinases are secreted by ameloblasts into the extracellular space, where they cleave enamel proteins by catalyzing the hydrolysis of peptide bonds. Enamel proteinases are present in low abundance and are not likely to participate directly in the mineralization process. Two major enamel proteinases have been identified: enamelysin (MMP20) and kallikrein 4 (KLK4). These proteinases are expressed at different times and have different functions. Their roles are to modify and/or to eliminate enamel matrix proteins, which affects the way enamel proteins interact with each other and with the developing enamel crystallites. A brief review of dental enamel formation is presented, followed by a more detailed analysis of enamelysin and KLK4 expression, structure, and function.     

Differential expression of TGFBR3 (betaglycan) in mouse ovary and testis during gonadogenesis

TGFBR3 is an accessory receptor that binds to and modulates the activities of both transforming growth factor-beta (TGFβ) and inhibin, two members of the TGFβ superfamily of growth factors that regulate many aspects of reproductive biology. Tgfbr3 is known to be expressed in adult testis and ovary, but little is known about this receptor during gonadogenesis. Herein, we describe Tgfbr3 expression in the male and female fetal and neonatal murine gonad. Real-time PCR analysis revealed that Tgfbr3 mRNA was expressed at higher levels in the developing testis compared to ovary. TGFBR3 was expressed within the fetal testis interstitium, predominantly by Leydig cells, but expression shifted inside the seminiferous cords at birth. In contrast, TGFBR3 was detected in both the somatic and germ cell lineages in the fetal and neonatal ovary. This differential expression pattern suggests divergent roles for this TGFBR3 in developing testis and ovary.     

Characterization of transforming growth factor-beta 1 gene expression in porcine immune cells.

We have investigated the regulation of transforming growth factor beta 1 gene expression in a variety of porcine immune cell populations, including peripheral blood mononuclear cells (PBMC), peripheral blood monocytes, alveolar macrophages and lymphoid cells from various swine lymphoid tissues. Using porcine transforming growth factor beta 1 cDNA probes in Northern blot assays, messages of 2.5 and 3.5 kb TGF beta 1 mRNA were detected in the cells investigated. A variety of mitogenic and immunomodulatory substances were examined for their ability to induce TGF beta 1 mRNA expression. These include phorbol 12-myristate 13-acetate (PMA), phytohemagglutinin (PHA), concanavalin A (Con A), lipopolysaccharide (LPS), dexamethasone (Dex), tumor necrosis factor (TNF) and interleukin (IL)-1 alpha. While low level constitutive expression of TGF beta 1 mRNA was detected from all cells investigated, PMA treatment of PBMC and alveolar macrophages resulted in a more than 10-fold increase in the steady-state level of TGF beta 1 mRNA within 2 hr of PMA addition. Also, the effect of opiate drugs, methadone (Md) and morphine (Mor), on TGF beta 1 gene expression was determined. Cells treated with opiates expressed the same levels of TGF beta 1 mRNA as untreated cells. Since TGF beta 1 biological activity can be induced by opiates, the regulation of TGF beta 1 gene expression likely involves mechanisms that do not cause changes in mRNA levels.     

Kallikrein-related peptidase 14 acts on proteinase-activated receptor 2 to induce signaling pathway in colon cancer cells

Serine proteinases participate in tumor growth and invasion by cleaving and activating proteinase-activated receptors (PARs). Recent studies have implicated PAR-1 and PAR-4 (activated by thrombin) and PAR-2 (activated by trypsin but not by thrombin) in human colon cancer growth. The endogenous activators of PARs in colon tumors, however, are still unknown. We hypothesize that the kallikrein-related peptidase (KLK) family member KLK14, a known tumor biomarker, is produced by colonic tumors and signals to human colon cancer cells by activating PARs. We found that i) KLK14 mRNA was present in 16 human colon cancer cell lines, ii) KLK14 protein was expressed and secreted in colon cancer cell lines, and iii) KLK14 (0.1 μmol/L) induced increases in intracellular calcium in HT29, a human colon cancer-derived cell line. KLK14-induced calcium flux was associated with internalization of KLK14-mediated activation of PAR-2. Furthermore, KLK14 induced significant extracellular signal-regulated kinases 1 and 2 (ERK1/2) phosphorylation and HT29 cell proliferation, presumably by activating PAR-2. A PAR-2 cleavage and activation-blocking antibody dramatically reduced KLK14-induced ERK1/2 signaling. Finally, ectopic expression of KLK14 in human colon adenocarcinomas and its absence in normal epithelia was demonstrated by IHC analysis. These results demonstrate, for the first time, the aberrant expression of KLK14 in colon cancer and its involvement in PAR-2 receptor signaling. Thus, KLK14 and its receptor, PAR-2, may represent therapeutic targets for colon tumorigenesis.     

Transforming growth factor beta 1 messenger RNA in Reed-Sternberg cells in nodular sclerosing Hodgkin's disease.

AIMS--To determine the cellular origin of the most potent cytokine present in Hodgkin's disease, transforming growth factor (TGF) beta, the polycellular population of Hodgkin's tissue was studied using in situ hybridisation. METHODS--A biotin labelled oligo-complementary DNA (cDNA) was constructed according to the previously determined sequence for TGF beta 1 cDNA. Forty three frozen and paraffin wax embedded tissue samples replaced by Hodgkin's disease or non-Hodgkin's lymphoma, three Reed-Sternberg cell lines, one Ki1 positive lymphoma cell line, and an epithelial cell line were studied for expression of TGF beta 1 messenger RNA (mRNA) as well as secretion of the TGF beta 1 protein and expression of the CD30 epitope. RESULTS--The results obtained with the 24 frozen tissue samples confirmed that the TGF beta antigen is found predominantly in the nodular sclerosing Hodgkin's disease (NSHD) subtype. Nineteen paraffin wax embedded tissue samples were used to measure the simultaneous expression of CD30 and TGF beta 1 mRNA. The latter was found in eight of eight NSHD samples, two of six mixed cellularity samples, and two of five non-Hodgkin's lymphoma samples. No evidence of fibroblast expression of TGF beta 1 mRNA was noted. CONCLUSIONS--Activated lymphocytes in NSHD express TGF beta 1 mRNA, but binucleate Reed-Sternberg cells and mononuclear Hodgkin's cells are the primary sources of activated TGF beta in Hodgkin's disease.     

山柰酚-3-O-芸香糖苷对血管平滑肌细胞增殖、迁移及TGFBR1信号通路活化的影响

目的:探索山柰酚-3-O-芸香糖苷(KR)对血管平滑肌细胞(VSMC)增殖、迁移及转化生长因子β受体1(TGFBR1)信号通路活化的影响。方法:将KR(10、20和40μmol/L)孵育大鼠VSMC细胞系A7R5 24 h,或将40μmol/L KR孵育A7 R5细胞24、48和72 h后,MTT法检测细胞活力,Ed U染色检测细胞增殖情况,Transwell小室实验检测细胞迁移能力的变化,Western blot检测细胞迁移相关蛋白基质金属蛋白酶2(MMP2)及基质金属蛋白酶9(MMP9)的表达;分子对接技术探索KR与TGFBR1之间的相互关系,Western blot检测TGFBR1及其下游的Smad2和Smad3的激活情况。结果 :KR剂量和时间依赖性地降低细胞活力,剂量依赖性地减少Ed U染色阳性细胞数量,降低迁移细胞数量,减少迁移相关蛋白MMP2和MMP9的表达(P0.05)。KR与TGFBR1发生分子对接的结合力为-9.804 kcal/mol,与TGBFR1的SER-280、ARG-215、ASP-290和LYS-335氨基酸残基形成氢键连接。KR剂量依赖性地降低TGFBR1及其下游Smad2和Smad3的激活(P0.05)。结论:KR能抑制VSMC的增殖和迁移,其机制可能与抑制TGFBR1信号通路相关。     

JNK1 and JNK2 regulate α‐SMA in hepatic stellate cells during CCl4‐induced fibrosis in the rat liver

Following liver injuries, hepatic stellate cells (HSCs) express α‐SMA. Mitogen activated protein kinase (MAPK) signaling pathways mediate α‐SMA expression in distinct cell types. However, the regulation of α‐SMA expression by MAPKs in HSCs has been rarely studied. We aimed to study the role of MAPKs in the activation of HSCs during liver fibrosis. Liver fibrosis of rats was induced by carbon tetrachloride. HSC‐T6 cells, murine embryonic fibroblasts, JNK1^?/? and JNK2^?/? cells were used for in vitro studies. Immunohistochemistry and immunoblot analysis were used. We have found that the expression of JNK and α‐SMA co‐localized in HSCs during liver fibrosis, but ERK and p38 expressed in macrophages. The expression of α‐SMA was up‐regulated by JNK1 and JNK2 in non‐stress condition. Under TGF‐β stimulation, however, the level α‐SMA expression was increased by only JNK1, but not significantly changed by JNK2. We suggest that JNKs are responsible for α‐SMA regulation, and especially JNK1 has a major role in up‐regulation of α‐SMA expression in HSCs under stress condition induced by TGF‐β during liver fibrosis.     

Expression,structure, and function of enamel proteinases

Proteinases serve two important functions during dental enamel formation: They (a) process and (b) degrade enamel proteins. Different enzymes carry out these functions. Enamelysin (MMP-20) is the foremost enamel matrix-processing enzyme. Its expression initiates prior to the onset of dentin mineralization and continues throughout the secretory stage of amelogenesis. In vitro, enamelysin catalyzes all of the amelogenin cleavages that are known to occur during the secretory stage in vivo, and it is probably the enzyme responsible for the processing of all enamel proteins. There is evidence suggesting that enamelysin activity is critical for proper enamel formation. Uncleaved and processed enamel proteins often segregate into different compartments within the developing enamel layer, suggesting that they may have different functions. Intact ameloblastin and its C-terminal cleavage products localize in the superficial rod and interrod enamel, while its N-terminal cleavage products congregate in the sheath space. Intact enamelin is only present at the mineralization front within a micrometer of the enamel surface, while its cleavage products concentrate in the rod and interrod enamel. Processed enamel proteins accumulate during the secretory stage, but disappear early in the maturation stage. Enamel matrix serine proteinase 1 (EMSP1), now officially designated kallikrein 4 (KLK4), is believed to be the predominant degradative enzyme that clears enamel proteins from the matrix during maturation. KLK4 expression initiates during the transition stage and continues throughout maturation. KLK4 concentrates at the enamel surface when the enamel matrix disappears, and aggressively degrades amelogenin in vitro. During tooth development, proteinases are secreted by ameloblasts into the extracellular space, where they cleave enamel proteins by catalyzing the hydrolysis of peptide bonds. Enamel proteinases are present in low abundance and are not likely to participate directly in the mineralization process. Two major enamel proteinases have been identified: enamelysin (MMP20) and kallikrein 4 (KLK4). These proteinases are expressed at different times and have different functions. Their roles are to modify and/or to eliminate enamel matrix proteins, which affects the way enamel proteins interact with each other and with the developing enamel crystallites. A brief review of dental enamel formation is presented, followed by a more detailed analysis of enamelysin and KLK4 expression, structure, and function.     

Notch1 modulates mesenchymal stem cells mediated regulatory T‐cell induction

Notch1 signaling is involved in regulatory T (Treg)‐cell differentiation. We previously demonstrated that, when cocultured with CD3⁺ cells, mesenchymal stem cells (MSCs) induced a T‐cell population with a regulatory phenotype. Here, we investigated the molecular mechanism underlying MSC induction of human Treg cells. We show that the Notch1 pathway is activated in CD4⁺ T cells cocultured with MSCs. Inhibition of Notch1 signaling through GSI‐I or the Notch1 neutralizing antibody reduced expression of HES1 (the Notch1 downstream target) and the percentage of MSC‐induced CD4⁺CD25^highFOXP3⁺ cells in vitro. Moreover, we demonstrate that FOXP3 is a downstream target of Notch signaling in human cells. No crosstalk between Notch1 and TGF‐β signaling pathways was observed in our experimental system. Together, these findings indicate that activation of the Notch1 pathway is a novel mechanism in the human Treg‐cell induction mediated by MSCs.     

Blocking Dishevelled signaling in the noncanonical Wnt pathway in sea urchins disrupts endoderm formation and spiculogenesis,but not secondary mesoderm formation

Dishevelled (Dsh) is a phosphoprotein key to beta‐catenin dependent (canonical) and beta‐catenin independent (noncanonical) Wnt signaling. Whereas canonical Wnt signaling has been intensively studied in sea urchin development, little is known about other Wnt pathways. To examine roles of these beta‐catenin independent pathways in embryogenesis, we used Dsh‐DEP, a deletion construct blocking planar cell polarity (PCP) and Wnt/Ca²⁺ signaling. Embryos overexpressing Dsh‐DEP failed to gastrulate or undergo skeletogenesis, but produced pigment cells. Although early mesodermal gene expression was largely unperturbed, embryos exhibited reduced expression of genes regulating endoderm specification and differentiation. Overexpressing activated beta‐catenin failed to rescue Dsh‐DEP embryos, indicating that Dsh‐DEP blocks endoderm formation downstream of initial canonical Wnt signaling. Because Dsh‐DEP‐like constructs block PCP signaling in other metazoans, and disrupting RhoA or Fz 5/8 in echinoids blocks subsets of the Dsh‐DEP phenotypes, our data suggest that noncanonical Wnt signaling is crucial for sea urchin endoderm formation and skeletogenesis. Developmental Dynamics 238:1649–1665, 2009. © 2009 Wiley‐Liss, Inc.     

I型膜型基质金属蛋白酶在癌细胞系中的表达及其与明胶酶A活化关系的研究

目的探讨Ｉ型膜型基质金属蛋白酶（ＭＴ１ＭＭＰ）在人肿瘤细胞系中的表达情况及其与明胶酶Ａ（ＭＭＰ２）的分泌和活化的关系。方法运用逆转录巢式ＰＣＲ、ＲＮＡ印迹分析和蛋白质印迹分析的方法,检测ＭＴ１ＭＭＰｍＲＮＡ和ＭＭＰ２蛋白在４个黑色素瘤、２个肺癌和２个前列腺癌细胞系中的表达水平。结果ＭＴ１ＭＭＰｍＲＮＡ在所有癌细胞系中均有表达,但表达水平很不一致：在４个黑色素瘤细胞系中的表达水平明显高于肺癌和前列腺癌细胞系;仅在黑色素瘤细胞系的无血清条件培养液中,检测到了活化型ＭＭＰ２,而人肺癌细胞系ＰＧ虽然分泌酶原型ＭＭＰ２,但不产生活化型ＭＭＰ２。结论ＭＴ１ＭＭＰｍＲＮＡ的表达具有一定的组织特异性,并与ＭＭＰ２的活化密切相关。