Separation, purification, and sequence identification of TGF-β1 and TGF-β2 from bovine milk

Cox and Bürk (Eur. J. Biochem., 1991) reported the partial characterization of Milk Growth Factor (MGF) which stimulated the migration of fibroblasts. We have fractionated the partially purified sample by RP-HPLC and obtained the separation of two peaks of activity. The two active components were isolated as pure MGF-a and MGF-b by RP-HPLC and preparative SDS-PAGE. The purified MGF-a, consisting of a single band by gel electrophoresis and a single peak on an HPLC reversed-phase C-4 column, has the same specific activity as TGF-2 in the fibroblast migration assay. MGF-a was digested by endoprotease Asp-N and the cleaved peptides were analyzed by Edman degradation and plasma desorption mass spectrometry (PDMS). The whole sequence of MGF-a determined by automated sequenator and PDMS of S-pyridylethylated protein and selected fragments was found to be identical to that of TGF-2. MGF-b protein mixture separated by SDS-PAGE was electrophoretically transferred onto a Biometra Glassybond membrane, and the blotted MGF-b protein was directly sequenced on an automated sequenator. The identified 29 amino acids sequence of MGF-b was identical to the amino-terminal sequence of TGF-1. Our study demonstrates that MGF is composed of both TGF-1 and TGF-2. TGF-2 (85%) is the predominant form.     

Short communication: Concentration of TGF-β1, IL-10 and IL-6 in boar seminal plasma and TGF-β1 level in different fractions of ejaculates

Levels of the cytokines transforming growth factor (TGF)-β1, interleukin (IL)-10 and IL-6 in the boar seminal plasma (SP) as well as TGF-β1 level in different fractions of ejaculate were studied. These cytokines was chosen because of their expected effect on tissue immune response, i.e. suppressive (TGF-β1 and IL-10) and pro-inflammatory (IL-6). Three whole ejaculates from five boars A-E, (n=15) were sampled weekly to evaluate the levels of seminal plasma TGF-β1, IL-10 and IL-6 as well as their fluctuations over time. The effect of different storage temperatures, -20°C or -80°C, on the level of seminal plasma TGF β1 was also tested (three boars, two fractions in one ejaculate). In addition, in 4 different fractions of ejaculates: the pre-sperm-rich (Pre-SRF), first 10 ml of sperm-rich (10SRF), the rest of the sperm-rich fraction (Rest-SRF) and the rest of the ejaculate (RE) fraction, were collected from three boars (A-C) on four different occasions for TGF-β1 evaluation. In the whole ejaculates (n=15), a wide range in the concentration of the cytokines TGF-β1 (20.4 - 766.5 pg/mL) and IL-10, (73.7 - 837.3 pg/mL), was found. For IL-6, the concentration was low (range 11.5 - 30.9 pg/ml) and only detected in four out of 15 collections (from two boars). The mean levels of TGF-β1 and IL-10 between individual boars varied but were not statistical different. The level of TGF-β1 in Pre-SRF, Rest-SRF and RE fractions was significantly lower in boar A than the other boars. A significantly higher concentration of TGF-β1 was found in the 10SRF than in the other fractions. Different storage temperatures (-20°C or -80°C) did not affect the seminal plasma TGF-β1 level after one year of storage. To conclude: Boar seminal plasma contained TGF- β1 and IL-10 but with high individual variation. IL-6 was low or undetectable. The TGF- β1 level was highest in the first 10 mL of the sperm-rich fraction of the ejaculate. Further studies are needed on the role of different levels of cytokine in boar semen on porcine female reproductive tissue, especially for TGF- β1.     

IL-1β, IL-6, and RANTES as Biomarkers of Chikungunya Severity

Background

Little is known about the immunopathogenesis of Chikungunya virus. Circulating levels of immune mediators and growth factors were analyzed from patients infected during the first Singaporean Chikungunya fever outbreak in early 2008 to establish biomarkers associated with infection and/or disease severity.

Methods and Findings

Adult patients with laboratory-confirmed Chikungunya fever infection, who were referred to the Communicable Disease Centre/Tan Tock Seng Hospital during the period from January to February 2008, were included in this retrospective study. Plasma fractions were analyzed using a multiplex-microbead immunoassay. Among the patients, the most common clinical features were fever (100%), arthralgia (90%), rash (50%) and conjunctivitis (40%). Profiles of 30 cytokines, chemokines, and growth factors were able to discriminate the clinical forms of Chikungunya from healthy controls, with patients classified as non-severe and severe disease. Levels of 8 plasma cytokines and 4 growth factors were significantly elevated. Statistical analysis showed that an increase in IL-1β, IL-6 and a decrease in RANTES were associated with disease severity.

Conclusions

This is the first comprehensive report on the production of cytokines, chemokines, and growth factors during acute Chikungunya virus infection. Using these biomarkers, we were able to distinguish between mild disease and more severe forms of Chikungunya fever, thus enabling the identification of patients with poor prognosis and monitoring of the disease.     

The Effects of IL-1β on Astrocytes are Conveyed by Extracellular Vesicles and Influenced by Age

Neurochemical Research - The aging brain is associated with significant pathophysiological changes reflected in changes in astrocyte function. In this study, we hypothesized that the response of...     

Effects of IL-13 on TGF-β and MMP-1 in periodontitis

Periodontitis is an inflammatory disease of the supporting tissues of the teeth. Interleukin (IL)-13 is a multifunctional T-helper type2 (Th2) cytokine that can diminish inflammatory responses. I investigated using ELISA the effects of IL-13 on transforming growth factor-beta (TGF-β) and matrix metalloproteinase-1 (MMP-1). MMP-1 was detected using immunohistochemistry. Gingival fibroblasts were stimulated with IL-13 or together with tumor necrosis factor-α (TNF-α). I found that macrophage-like cells, fibroblast-like cells, vascular endothelial cells and gingival epithelial cells were stained more intensely for MMP-1 and were observed more frequently in the periodontitis affected group than in the control group. The cultured gingival fibroblasts with IL-13 produced more TGF-β than unstimulated cells. After stimulation with additional TNF-α, MMP-1 production was diminished. IL-13 may play a role in regulating collagen homeostasis in gingival fibroblasts. IL-13 induces both up-regulation of TGF-β, a cytokine known to stimulate production of collagen, and down-regulation of collagen-destroying MMP-1 production. This effect may be strong during periodontitis when Th2 cells assist T cells.     

The Interaction of Endothelin-1 and TGF-β1 Mediates Vascular Cell Remodeling

Background

Pulmonary arterial hypertension is characterized by increased thickness of pulmonary vessel walls due to both increased proliferation of pulmonary arterial smooth muscle cell (PASMC) and deposition of extracellular matrix. In patients suffering from pulmonary arterial hypertension, endothelin-1 (ET-1) synthesis is up-regulated and may increase PASMC activity and vessel wall remodeling through transforming growth factor beta-1 (TGF-β1) and connective tissue growth factor.

Objective

To assess the signaling pathway leading to ET-1 induced proliferation and extracellular matrix deposition by human PASMC.

Methods

PASMC were serum starved for 24 hours before stimulation with either ET-1 and/or TGF-β1. ET-1 was inhibited by Bosentan, ERK1/2 mitogen activated protein kinase (MAPK) was inhibited by U0126 and p38 MAPK was inhibited by SB203580.

Results

ET-1 increased PASMC proliferation when combined with serum. This effect involved the mitogen activated protein kinases (MAPK) ERK1/2 MAPK and was abrogated by Bosentan which caused a G1- arrest through activation of p27^(Kip). Regarding the contribution of extracellular matrix deposition in vessel wall remodeling, TGF-β1 increased the deposition of collagen type-I and fibronectin, which was further increased when ET-1 was added mainly through ERK1/2 MAPK. In contrast, collagen type-IV was not affected by ET-1. Bosentan dose-dependently reduced the stimulatory effect of ET-1 on collagen type-I and fibronectin, but had no effect on TGF-β1.

Conclusion and Clinical Relevance

ET-1 alone does not induce PASMC proliferation and extracellular matrix deposition. However, ET-1 significantly up-regulates serum induced proliferation and TGF-β1 induced extracellular matrix deposition, specifically of collagen type-I and fibronectin. The synergistic effects of ET-1 on serum and TGF-β1 involve ERK1/2 MAPK and may thus present a novel mode of action in the pathogenesis of pulmonary arterial hypertension.     

TGF-β1/Smad Signaling Pathway Regulates Epithelial-to-Mesenchymal Transition in Esophageal Squamous Cell Carcinoma: In Vitro and Clinical Analyses of Cell Lines and Nomadic Kazakh Patients from Northwest Xinjiang,China

Invasion and metastasis are the major causes of death in patients with esophageal squamous cell carcinoma (ESCC). Epithelial-mesenchymal transition (EMT) is a critical step in tumor progression and transforming growth factor-β1 (TGF-β1) signaling has been shown to play an important role in EMT. In this study, we investigated how TGF-β1 signaling pathways contributed to EMT in three ESCC cell lines as well as 100 patients of nomadic ethnic Kazakhs residing in northwest Xinjiang Province of China. In vitro analyses included Western blotting to detect the expression of TGF-β1/Smad and EMT-associated proteins in Eca109, EC9706 and KYSE150 cell lines following stimulation with recombinant TGF-β1 and SB431542, a potent inhibitor of ALK5 that also inhibits TGF-β type II receptor. TGF-β-activated Smad2/3 signaling in EMT was significantly upregulated as indicated by mesenchymal markers of N-cadherin and Vimentin, and in the meantime, epithelial marker, E-cadherin, was markedly downregulated. In contrast, SB431542 addition downregulated the expression of N-cadherin and Vimentin, but upregulated the expression of E-cadherin. Moreover, the TGF-β1-induced EMT promoted invasion capability of Eca109 cells. Tumor cells undergoing EMT acquire fibroblastoid-like phenotype. Expressed levels of TGF-β1/Smad signaling molecules and EMT-associated proteins were examined using immunohistochemical analyses in 100 ESCC tissues of Kazakh patients and 58 matched noncancerous adjacent tissues. The results showed that ESCC tissues exhibited upregulated expression of TGF-β1/Smad. We also analyzed the relationship between the above proteins and the patients'' clinicopathological characteristics. The TGF-β1/Smad signaling pathway in human Eca109 ESCC cells may carry similar features as in Kazakh ESCC patients, suggesting that TGF-β1/Smad signaling pathway may be involved in the regulation of EMT in ethnic Kazakh patients with ESCC from Xinjiang, China.     

Expression of TGF-β1, SNAI1 and MMP-9 is associated with lymph node metastasis in papillary thyroid carcinoma

TGF-β1, SNAI1 and MMP-9 are implicated in tumor invasion and metastasis. The purpose of this study was to examine TGF-β1, SNAI1 and MMP-9 expression in papillary thyroid carcinoma (PTC), and to assess association of TGF-β1, SNAI1 and MMP-9 expression with several clinicopathological indicators of PTC. TGF-β1, SNAI1 and MMP-9 protein expression in 83 PTCs and their matched normal thyroid specimens were analyzed using immunohistochemistry. The mRNA expression levels of TGF-β1, SNAI1 and MMP-9 in 12 fresh PTC specimens with lymph node metastasis (LNM), 12 fresh PTC specimens without LNM and their matched normal thyroid specimens were assessed by real-time RT-PCR. The results showed that the mRNA and protein expression levels of TGF-β1, SNAI1 and MMP-9 were significantly higher in PTCs than in their matched normal thyroid tissues. There were not significant differences in TGF-β1, SNAI1 and MMP-9 protein expression relative to age, gender, tumor size and TNM stage, except for MMP-9 whose protein expression correlated with tumor size. However, high mRNA and protein expression levels of TGF-β1, SNAI1 and MMP-9 were significantly correlated with LNM. Furthermore, TGF-β1, SNAI1 and MMP-9 protein expression were significantly correlated with one another. Concomitant expression of any two or all of the three molecules had stronger correlation with LNM than did each alone. Collectively, the present results indicate that immunohistochemical and real-time RT-PCR evaluation of TGF-β1, SNAI1 and MMP-9 expression in PTC may be useful to predict the risk of LNM in PTC patients.     

β Cell-Specific Deletion of the IL-1 Receptor Antagonist Impairs β Cell Proliferation and Insulin Secretion

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Dominance of the α1B-Adrenergic Receptor and its Subcellular Localization in Human and TRAMP Prostate Cancer Cell Lines

The function and distribution of α₁-adrenergic receptor (AR) subtypes in prostate cancer cells is well characterized. Previous studies have used RNA localization or low-avidity antibodies in tissue or cell lines to determine the α₁-AR subtype and suggested that the α_{1 A}-AR is dominant. Two androgen-insensitive, human metastatic cancer cell lines DU145 and PC3 were used as well as the mouse TRAMP C1-C3 primary and clonal cell lines. The density of α₁-ARs was determined by saturation binding and the distribution of the different α₁-AR subtypes was examined by competition-binding experiments. In contrast to previous studies, the major α₁-AR subtype in DU145, PC3 and all of the TRAMP cell lines is the α_1B-AR. DU145 cells contained 100% of the α_1B-AR subtype, whereas PC3 cells were composed of 21% α_{1 A}-AR and 79% α_1B-AR. TRAMP cell lines contained between 66% and 79% of the α_1B-AR with minor fractions of the other two subtypes. Faster doubling time in the TRAMP cell lines correlated with decreasing α _1B-AR and increasing α_{1 A}- and α_1D-AR densities. Transfection with EGFP-tagged α_1B-ARs revealed that localization was mainly intracellular, but the majority of the receptors translocated to the cell surface after extended preincubation (18 hr) with either agonist or antagonist. Localization was confirmed by ligand-binding studies and inositol phosphate assays where prolonged preincubation with either agonist and/or antagonist increased the density and function of α ₁-ARs, suggesting that the native receptors were mostly intracellular and nonfunctional. Our studies indicate that α_1B-ARs are the major α₁-AR subtype expressed in DU145, PC3, and all TRAMP cell lines, but most of the receptor is localized in intracellular compartments in a nonfunctional state, which can be rescued upon prolonged incubation with any ligand.     

Cell adhesion to anosmin via α5β1, α4β1, and α9β1 integrins

Anosmin is an extracellular matrix protein, and genetic defects in anosmin result in human Kallmann syndrome. It functions in neural crest formation, cell adhesion, and neuronal migration. Anosmin consists of multiple domains, and it has been reported to bind heparan sulfate, FGF receptor, and UPA. In this study, we establish cell adhesion/spreading assays for anosmin and use them for antibody inhibition analyses to search for an integrin adhesion receptor. We find that α5β1, α4β1, and α9β1 integrins are needed for effective adhesive receptor function in cell adhesion and cell spreading on anosmin; adhesion is inhibited by both RGD and α4β1 CS1-based peptides. This identification of anosmin-integrin adhesion receptors should facilitate studies of anosmin function in cell and developmental biology.     

2型糖尿病干眼症患者泪液IL-6、MMP-9、TGF-β1表达及其临床意义

摘要 目的：探讨2型糖尿病（T2DM）合并干眼症（DE）患者泪液白细胞介素-6（IL-6）、基质金属蛋白酶-9（MMP-9）、转化生长因子-β1（TGF-β1）表达及其临床意义。方法：选择2021年5月至2023年10月本院收治的T2DM患者97例,根据是否合并DE分为T2DM组和T2DM合并DE组,另选择同期体检的健康对象45例为对照组。采用酶联免疫吸附法（ELISA）检测三组泪液IL-6、MMP-9、TGF-β1表达水平,比较不同病情严重程度DE患者泪液IL-6、MMP-9、TGF-β1表达水平,采用单因素及多因素Logistics回归分析法分析T2DM并发DE的影响因素,采用受试者工作特征曲线（ROC）分析泪液IL-6、MMP-9、TGF-β1对T2DM并发DE的预测价值。结果：T2DM合并DE组泪液IL-6、MMP-9、TGF-β1表达水平高于T2DM组,T2DM组高于对照组（P＜0.05）。重度组泪液IL-6、MMP-9、TGF-β1表达水平及眼表疾病指数量表（OSDI）评分高于中度组,中度组高于轻度组（P＜0.05）;重度组泪膜破裂时间（BUT）、泪液分泌试验（SIT）低于中度组,中度组低于轻度组（P＜0.05）。单因素分析结果显示,年龄、空腹血糖（FBG）、空腹胰岛素（FINS）、超敏C反应蛋白（hs-CRP）、BUT、SIT、OSDI积分均是T2DM并发DE的影响因素（P＜0.05）,多因素分析结果显示,OSDI积分及泪液IL-6、MMP-9、TGF-β1升高是T2DM并发DE的独立危险因素（P＜0.05）,BUT、SIT升高是保护因素（P＜0.05）。泪液IL-6、MMP-9、TGF-β1及联合检测预测T2DM并发DE的曲线下面积（AUC）分别为0.815、0.806、0.725和0.905,联合检测的预测价值高于单独检测。结论：T2DM合并DE患者泪液IL-6、MMP-9、TGF-β1表达升高,可引起DE病情加重,三项指标联合检测对T2DM并发DE的风险具有较高的预测价值。     

Comparison of metabolic effects of EGF,TGF-α, and TGF-β1 in primary culture of fetal bovine myoblasts and rat L6 myoblasts

• 1.1. Comparative studies of EGF, TGF-α, and TGF-βl action on the synthesis of DNA and cellular proteins in rat L6 myogenic cells and fetal bovine myoblasts demonstrated considerable differences between particular growth factors, dependent on dose and target cells.
• 2.2. Among examined growth factors only EGF exerted mitostimulatory action, more pronounced at lower concentrations. EGF, progressively with dose, stimulated protein synthesis much more effectively in fetal bovine myoblasts than in L6 cells.
• 3.3. The dynamics of stimulation of protein synthesis by TGF-α was greater than by EGF in both examined types of cell cultures.
• 4.4. The maximal response of fetal bovine myoblasts to TGF-α in a concentration of 100 ng/ml reached 370%, whereas EGF in a 10 times higher concentration stimulated protein synthesis only to 123% of control.
• 5.5. In contrast to EGF, TGF-α significantly inhibits DNA synthesis. Inhibition of the mitogenic response with simultaneous stimulation of protein synthesis by TGF-α may indicate changes toward cell differentiation.
• 6.6. TGF-β 1 in smallest concentration inhibits both DNA and protein synthesis. The suppressive action of TGF-β 1 was more distinct in fetal bovine myoblasts than in the L6 cell line.
• 7.7. Increasing concentrations of TGF-β l diminished its inhibitory effect, even leading to stimulation of protein synthesis at higher doses in L6 myoblasts.

     

Immunohistochemistry of IL-1β, IL-6 and TNF-α in spleens of mice treated with gold nanoparticles

Gold nanoparticles (AuNPs) possess considerable biocompatibility and therefore gaining more attention for their biomedical applications. Previous studies have shown the transient increase in pro-inflammatory cytokines expression in different organs of rats and mice exposed to AuNPs. Structural changes in the spleen of mice treated with AuNPs have also been reported. This investigation was aimed to study the immunostaining of IL-1β, IL-6 and TNF-α in mice treated with different sizes of AuNPs. The animals were divided into 7 groups of 4 animals in each group. One group received saline and served as control. Two sets of three groups were treated with 5 nm, 20 nm and 50 nm diameter AuNPs. One set was sacrificed on day 1 and the other on day 7 following the AuNPs injections. Spleens were dissected out and promptly fixed in formalin for 3 days and then processed for IL-1β, IL-6 and TNF-α immunostaining using target-specific antibodies. The immunoreactivities of IL-1β and IL-6 were increased with the increase of AuNP size. The immunostaining of IL-1β in spleen of 20 nm AuNP treated mice was subsequently decreased on day 7 whereas it persisted in 50 nm AuNP group. The increase in the immunoreactivity of IL-6 on day 1 was decreased on day 7 in the spleens of mice treated with 20 nm or 50 nm AuNPs. The immunostaining of TNF-α was found to be negative in all the treatment groups. In conclusion, the size of AuNPs plays an important role in the expression of proinflammatory cytokines in mouse spleen; small size (5 nm) AuNPs caused minimal effect, whereas larger (50 nm) AuNPs produced intense immunostaining.     

GSK-3 mediates the release of IL-1β, TNF-α and IL-10 from cortical glia

Neuroinflammation has been shown to contribute to neurodegenerative and psychiatric disorders such as Alzheimer's disease and major depression due to the inappropriate release of pro-inflammatory cytokines from activated microglia. The precise molecular events that mediate cytokine release from glia remain unknown but we suggest that the serine/threonine kinase glycogen synthase kinase-3 (GSK-3) may be involved. The aim of this study therefore was to investigate the effect of lipopolysaccharide (LPS) on expression and activity of the GSK-3β isoform in glia, and to assess if GSK-3 mediates the LPS-induced change in inflammatory cytokine levels in culture medium from rat glial-enriched cortical cultures. GSK-3β was expressed in microglia and astrocytes, and stimulation of these cultures with LPS induced an increase in GSK-3β expression and activity, and in pro-inflammatory cytokine levels in culture media. We show that GSK-3 inhibition using a small molecule inhibitor SB216763 or the mood stabiliser lithium chloride reduced the LPS-induced elevated levels of pro-inflammatory cytokines present in culture media from rat glial-enriched cortical cultures. These results demonstrate a role for GSK-3 as a modulator of inflammatory cytokine levels in the brain, and contribute to a mechanistic insight into neurological disorders in which neuroinflammation is a characteristic feature.     

Pancreatic Cancer Cell Glycosylation Regulates Cell Adhesion and Invasion through the Modulation of α2β1 Integrin and E-Cadherin Function

In our previous studies we have described that ST3Gal III transfected pancreatic adenocarcinoma Capan-1 and MDAPanc-28 cells show increased membrane expression levels of sialyl-Lewis x (SLe^x) along with a concomitant decrease in α2,6-sialic acid compared to control cells. Here we have addressed the role of this glycosylation pattern in the functional properties of two glycoproteins involved in the processes of cancer cell invasion and migration, α2β1 integrin, the main receptor for type 1 collagen, and E-cadherin, responsible for cell-cell contacts and whose deregulation determines cell invasive capabilities. Our results demonstrate that ST3Gal III transfectants showed reduced cell-cell aggregation and increased invasive capacities. ST3Gal III transfected Capan-1 cells exhibited higher SLe^x and lower α2,6-sialic acid content on the glycans of their α2β1 integrin molecules. As a consequence, higher phosphorylation of focal adhesion kinase tyrosine 397, which is recognized as one of the first steps of integrin-derived signaling pathways, was observed in these cells upon adhesion to type 1 collagen. This molecular mechanism underlies the increased migration through collagen of these cells. In addition, the pancreatic adenocarcinoma cell lines as well as human pancreatic tumor tissues showed colocalization of SLe^x and E-cadherin, which was higher in the ST3Gal III transfectants. In conclusion, changes in the sialylation pattern of α2β1 integrin and E-cadherin appear to influence the functional role of these two glycoproteins supporting the role of these glycans as an underlying mechanism regulating pancreatic cancer cell adhesion and invasion.