Epithelial–mesenchymal transition of A549 cells is enhanced by co-cultured with THP-1 macrophages under hypoxic conditions

Epithelial–mesenchymal transition (EMT) is associated with pulmonary fibrosis, including idiopathic pulmonary fibrosis (IPF). In this study, we investigated EMT of human pulmonary epithelial-derived cells (A549). A549 cells was either cultured by itself or co-cultured with THP-1 macrophages under normoxic (21% O₂) and hypoxic (2% O₂) conditions. We evaluated the presence of EMT by determining the expression of EMT markers, E-cadherin, vimentin, and fibronectin. To determine the role of TGF-β1 and IL-1β in EMT of the A549 cells, we analyzed the effects of blocking their activity with TGF-β1 inhibitor or IL-1β neutralizing antibody respectively. The A549 cells presented EMT when they were co-cultured with THP-1 macrophages. The EMT of the A549 cells co-cultured with THP-1 macrophages was exacerbated under hypoxia. In addition, the EMT were prevented by the addition of TGF-β1 type I receptor kinase inhibitor. The hypoxic condition increased the mRNA levels of TGF-β1 in A549 cells and THP-1 macrophages and that of IL-1β in THP-1 macrophages when each cells were co-cultured. Anti-IL-1β neutralizing antibody attenuated TGF-β1 secretion in co-culture media under hypoxic conditions. Thus, the IL-1β from THP-1 macrophages up-regulated the TGF-β1 from A549 cells and THP-1 macrophages, and then the TGF-β1 from both cells induced and promoted the EMT of A549 cells when they were co-cultured under hypoxia. Together, these results demonstrate that the interaction between type II pneumocytes and macrophages under hypoxia is necessary for the development of pulmonary fibrosis.     

Interleukin-2 induces extracellular matrix synthesis and TGF-β2 expression in retinal pigment epithelial cells

Macular fibrosis is a vital obstacle of vision acuity improvement of age-related macular degeneration patients. This study was to investigate the effects of interleukin 2 (IL-2) on epithelial-mesenchymal transition (EMT), extracellular matrix (ECM) synthesis and transforming growth factor β2 (TGF-β2) expression in retinal pigment epithelial (RPE) cells. 10 μg/L IL-2 was used to induce fibrosis in RPE cells for various times. Western blot was used to detect the EMT marker α-smooth muscle actin (α-SMA), ECM markers fibronectin (Fn) and type 1 collagen (COL-1), TGF-β2, and the activation of the JAK/STAT3 and NF-κB signaling pathway. Furthermore, JAK/STAT3 and NF-κB signaling pathways were specifically blocked by WP1066 or BAY11-7082, respectively, and the expression of α-SMA, COL-1, Fn and TGF-β2 protein were detected. Wound healing and Transwell assays were used to measure cell migration ability of IL-2 with or without WP1066 or BAY11-7082. After induction of IL-2, the expressions of Fn, COL-1, TGF-β2 protein were significantly increased, and this effect was correlated with IL-2 treatment duration, while α-SMA protein expression did not change significantly. Both WP1066 and BAY11-7082 could effectively downregulate the expression of Fn, COL-1 and TGF-β2 induced by IL-2. What's more, both NF-κB and JAK/STAT3 inhibitors could suppress the activation of the other signaling pathway. Additionally, JAK/STAT3 inhibitor WP1066 and NF-κB inhibitor BAY 11-7082 could obviously decrease RPE cells migration capability induced by IL-2. IL-2 promotes cell migration, ECM synthesis and TGF-β2 expression in RPE cells via JAK/STAT3 and NF-κB signaling pathways, which may play an important role in proliferative vitreoretinopathy.     

ALK1 heterozygosity increases extracellular matrix protein expression,proliferation and migration in fibroblasts

Fibrosis is a pathological situation in which excessive amounts of extracellular matrix (ECM) are deposited in the tissue. Myofibroblasts play a crucial role in the development and progress of fibrosis as they actively synthesize ECM components such as collagen I, fibronectin and connective tissue growth factor (CTGF) and cause organ fibrosis. Transforming growth factor beta 1 (TGF-β1) plays a major role in tissue fibrosis. Activin receptor-like kinase 1 (ALK1) is a type I receptor of TGF-β1 with an important role in angiogenesis whose function in cellular biology and TGF-β signaling is well known in endothelial cells, but its role in fibroblast biology and its contribution to fibrosis is poorly studied. We have recently demonstrated that ALK1 regulates ECM protein expression in a mouse model of obstructive nephropathy. Our aim was to evaluate the role of ALK1 in several processes involved in fibrosis such as ECM protein expression, proliferation and migration in ALK1^+/+ and ALK1^+/− mouse embryonic fibroblasts (MEFs) after TGF-β1 stimulations and inhibitors. ALK1 heterozygous MEFs show increased expression of ECM proteins (collagen I, fibronectin and CTGF/CCN2), cell proliferation and migration due to an alteration of TGF-β/Smad signaling. ALK1 heterozygous disruption shows an increase of Smad2 and Smad3 phosphorylation that explains the increases in CTGF/CCN2, fibronectin and collagen I, proliferation and cell motility observed in these cells. Therefore, we suggest that ALK1 plays an important role in the regulation of ECM protein expression, proliferation and migration.     

Accelerated epithelial cell senescence in IPF and the inhibitory role of SIRT6 in TGF-β-induced senescence of human bronchial epithelial cells

Reepithelialization of remodeled air spaces with bronchial epithelial cells is a prominent pathological finding in idiopathic pulmonary fibrosis (IPF) and is implicated in IPF pathogenesis. Recent studies suggest that epithelial senescence is a risk factor for development of IPF, indicating such reepithelialization may be influenced by the acceleration of cellular senescence. Among the sirtuin (SIRT) family, SIRT6, a class III histone deacetylase, has been demonstrated to antagonize senescence. We evaluated the senescence of bronchiolization in association with SIRT6 expression in IPF lung. Senescence-associated β-galactosidase staining and immunohistochemical detection of p21 were performed to evaluate cellular senescence. As a model for transforming growth factor (TGF)-β-induced senescence of abnormal reepithelialization, we used primary human bronchial epithelial cells (HBEC). The changes of SIRT6, p21, and interleukin (IL)-1β expression levels in HBEC, as well as type I collagen expression levels in fibroblasts, were evaluated. In IPF lung samples, an increase in markers of senescence and SIRT6 expression was found in the bronchial epithelial cells lining cystically remodeled air spaces. We found that TGF-β induced senescence in primary HBEC by increasing p21 expression, and, whereas TGF-β also induced SIRT6, it was not sufficient to inhibit cellular senescence. However, overexpression of SIRT6 efficiently inhibited TGF-β-induced senescence via proteasomal degradation of p21. TGF-β-induced senescent HBEC secreted increased amounts of IL-1β, which was sufficient to induce myofibroblast differentiation in fibroblasts. These findings suggest that accelerated epithelial senescence plays a role in IPF pathogenesis through perpetuating abnormal epithelial-mesenchymal interactions, which can be antagonized by SIRT6.     

TGF-β1-induced collagen promotes chicken ovarian follicle development via an intercellular cooperative pattern

Follicle development is a complex process under strict regulation of diverse hormones and cytokines including transforming growth factor β (TGF-β) superfamily members. TGF-β is pivotal for the regulation of ovarian functions under physiological and pathological conditions. In this study, effect of TGF-β1 on chicken follicle development was examined through investigating the accumulation and action of collagen, an indispensable member of the extracellular matrix (ECM) involved in this process. The granulosa cells (GCs) and theca cells (TCs) were separated from growing follicles of the laying chicken for treatment of TGF-β1 and analysis of expression of ECM components and key proteins in intracellular signaling pathways. Results showed that collagen was mainly distributed in the follicular theca layer and was produced with the formation of the granulosa layer during ovarian development. Collagen accumulation increased with follicle growth and treatment of GCs with TGF-β1 elicited an increased expression of collagen. After production from GCs, collagen was transferred to the neighboring TCs to promote cell proliferation and inhibit apoptosis. Treatment of collagen remarkably increased expression of p-ERK, mitogen-activated protein kinase (MAPK), and p-MAPK, but treatment with hydroxylase inhibitor (to break collagen structure) reversed these alterations. In conclusion, during follicle growth collagen was secreted by GCs under TGF-β1 stimulation and was subsequently collaboratively transferred to neighboring TCs to increase cell proliferation and thus to promote follicle development via an intercellular cooperative pattern during development of chicken growing follicles.     

TGF-β1 induces type I collagen deposition in granulosa cells via the AKT/GSK-3β signaling pathway-mediated MMP1 down-regulation

Type I collagen is the most abundant extracellular matrix (ECM) protein in the mammalian ovary, and comprises two COL1A1 subunits and one COL1A2 subunit. Matrix metalloproteinase 1 (MMP1) is a typical collagenase of type I collagen, that can be detected in ovarian follicles and early corpus luteum. Previous studies demonstrated that MMP1-mediated degradation of type I collagen plays a functional role in regulating corpus luteum formation, and transforming growth factor β1 (TGF-β1) inhibits luteinization and progesterone production in granulosa cells (GCs). Whether TGF-β1 regulates the expression of MMP1, COL1A1, or the deposition of type I collagen during corpus luteum formation remains to be elucidated. This study aimed to investigate the molecular mechanisms through which TGF-β1 regulates MMP1 expression and type I collagen deposition in GCs. Our results show that TGF-β1 upregulates COL1A1 expressions and downregulates MMP1 expression. Inhibition approaches, including pharmacological inhibitors such as p38 inhibitor (SB203580), ERK1/2 inhibitor (U0126), AKT inhibitor (LY294002), and GSK-3β inhibitor (LiCl), as well as knockdown using siRNA specific to these genes, were used. Our results suggest that TGF-β1 decreases MMP1 production via an ALK5-mediated AKT/GSK-3β-dependent signaling pathway, and a decrease in MMP1 levels and an increase in COL1A1 levels synergistically promote type I collagen deposition in GCs. Collectively, these findings provide novel insights into the underlying molecular mechanisms by which TGF-β1 upregulates type I collagen deposition in GCs.     

Effects of rhDecorin on TGF-β1 induced human hepatic stellate cells LX-2 activation

Decorin is a small leucine-rich extracellular matrix proteoglycan composed of a core protein with a single glycosaminoglycan (GAG) chain near the N-terminus and N-glycosylated at three potential sites. Decorin is involved in the regulation of formation and organization of collagen fibrils, modulation of the activity of growth factors such as transforming growth factor β (TGF-β), and exerts other effects on cell proliferation and behavior. Increasing evidences show that decorin plays an important role in fibrogenesis by regulating TGF-β, a key stimulator of fibrosis, and by directly modulating the degradation of extracellular matrix (ECM) from activated hepatic stellate cells (HSCs). In this study, the core protein of human decorin was cloned and expressed in Escherichia coli. The purified recombinant human decorin (rhDecorin) significantly inhibited the proliferation of LX-2 cells, a human HSC cell line, stimulated by TGF-β1. RT-PCR result showed that the expression of metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-1 (TIMP-1) were reduced by rhDecorin in LX-2 cells stimulated by TGF-β1. Furthermore, the protein expression of smooth muscle-α-actin (α-SMA), collagen type III and phosphorylated Smad2 (p-Smad2) was significantly decreased in the presence of rhDecorin. rhDecorin also reduced fibrillogenesis of collagen type I in a dose-dependent manner. Gene expression profiles of LX-2 cells stimulated by TGF-β1 in the presence and the absence of rhDecorin were obtained by using cDNA microarray technique and differentially expressed genes were identified to provide further insight into the molecular action mechanism of decorin on LX-2 cells.     

The antifibrotic effects of TGF-β1 siRNA on hepatic fibrosis in rats

Background/aims: Hepatic fibrosis results from the excessive secretion of matrix proteins by hepatic stellate cells (HSCs), which proliferate during fibrotic liver injury. Transforming growth factor (TGF)-β1 is the dominant stimulus for extracellular matrix (ECM) production by stellate cells. Our study was designed to investigate the antifibrotic effects of using short interference RNA (siRNA) to target TGF-β1 in hepatic fibrosis and its mechanism in rats exposed to a high-fat diet and carbon tetrachloride (CCL4). Methods: A total of 40 healthy, male SD (Sprague–Dawley) rats were randomly divided into five even groups containing of eight rats each: normal group, model group, TGF-β1 siRNA 0.125 mg/kg treatment group, TGF-β1 siRNA 0.25 mg/kg treatment group and TGF-β1 siRNA negative control group (0.25 mg/kg). CCL4 and a high-fat diet were used for 8 weeks to induce hepatic fibrosis. All the rats were then sacrificed to collect liver tissue samples. A portion of the liver samples were soaked in formalin for Hematoxylin–Eosin staining, classifying the degree of liver fibrosis, and detecting the expression of type I and III collagen and TGF-β1; the remaining liver samples were stored in liquid nitrogen to be used for detecting TGF-β1 by Western blotting and for measuring the mRNA expression of type I and III collagen and TGF-β1 by quantitative real-time polymerase chain reaction. Results: Comparing the TGF-β1 siRNA 0.25 mg/kg treatment group to the model group, the TGF-β1 siRNA negative control group and the TGF-β1 siRNA 0.125 mg/kg treatment group showed significantly reduced levels of pathological changes, protein expression and the mRNA expression of TGF-β1, type I collagen and type III collagen (P < 0.01). Conclusions: Using siRNA to target TGF-β1 can inhibit the expression of TGF-β1 and attenuate rat hepatic fibrosis induced by a high-fat diet and CCL4. A possible mechanism is through the down-regulation of TGF-β1 expression, which could inhibit HSC activation, as well as the proliferation and collagen production of collagen reducing, so that collagen deposition in the liver is reduced.     

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.     

Extracellular matrix production and regulation in micropatterned endothelial cells

Production and maintenance of extracellular matrix (ECM) is an essential aspect of endothelial cell (EC) function. ECM surfaces composed of collagen type IV and laminin support an atheroprotective endothelium, while fibronectin may encourage an atheroprone endothelium through inflammation or wound repair signaling. ECs maintain this underlying structure through regulation of protein production and degradation, yet the role of cytoskeletal alignment on this regulation is unknown. To examine the regulation and production of ECM by ECs with an atheroprotective phenotype, ECs were micropatterned onto lanes, which created an elongated EC morphology similar to that seen with unidirectional fluid shear stress application. Collagen IV and fibronectin protein production were measured as were gene expression of collagen IV, fibronectin, laminin, MMP2, MMP9, TIMP1, TIMP2, and TGF-β1. ECs were also treated with TNF to simulate an injury model. Micropattern-induced elongation led to significant increases in collagen IV and fibronectin protein production, and collagen IV, laminin, and TGF-β1 gene expression, but no significant changes in the MMP or TIMP genes. TNF treatment significantly increased collagen IV gene and protein production. These results suggest that the increase in ECM synthesis in micropattern-elongated ECs is likely regulated with TGF-β1, and this increase in ECM could be relevant to the atheroprotection needed for maintenance of a healthy endothelium in vivo.     

Circular RNA circRNA_15698 aggravates the extracellular matrix of diabetic nephropathy mesangial cells via miR-185/TGF-β1

Circular RNAs (circRNAs) are a novel type of noncoding RNAs that modulate the pathogenesis of multiple diseases. Nevertheless, the role of circRNAs in diabetic nephropathy (DN) pathogenesis is still ambiguous. In the current study, our team aims to investigate the expression profiles of circRNAs in DN and identify the function of circRNA on mesangial cells. CircRNAs microarray analysis revealed dysregulated circRNA in db/db DN mice, and circRNA_15698 was validated to be upregulated in both db/db mice and mouse mesangial cells (SV40-MES13) that were exposed to high glucose (25 mM) using real-time polymerase chain reaction. Loss-of-functional experiments showed that circRNA_15698 knockdown significantly inhibited the expression levels of collagen type I (Col. I), collagen type IV (Col. IV), and fibronectin. Moreover, the cellular localization of circRNA_15698 was mainly in the cytoplasm. Bioinformatics tools and luciferase reporter assay confirmed that circRNA_15698 acted as a ‘sponge’ of miR-185, and then positively regulated the transforming growth factor-β1 (TGF-β1) protein expression, suggesting a circRNA_15698/miR-185/TGF-β1 pathway. Further validation experiments validated that circRNA_15698/miR-185/TGF-β1 promoted extracellular matrix (ECM)-related protein synthesis. In summary, our study preliminarily investigates the role of circRNAs in mesangial cells and ECM accumulation, providing a novel insight for DN pathogenesis.     

Halofuginone prevents extracellular matrix deposition in diabetic nephropathy

Transforming growth factor-β (TGF-β) is known to promote the accumulation of extracellular matrix (ECM) and the development of diabetic nephropathy. Halofuginone, an analog of febrifugine, has been shown to block TGF-β₁ signaling and subsequent type I collagen production. Here, the inhibitory effect of halofuginone on diabetic nephropathy was examined. Halofuginone suppressed Smad2 phosphorylation induced by TGF-β₁ in cultured mesangial cells. In addition, the expression of TGF-β type 2 receptor decreased by halofuginone. Halofuginone showed an inhibitory effect on type I collagen and fibronectin expression promoted by TGF-β₁. An in vivo experiment using db/db mice confirmed the ability of halofuginone to suppress mesangial expansion and fibronectin overexpression in the kidneys. Moreover, an analysis of urinary 8-OHdG level and dihydroethidium fluorescence revealed that halofuginone reduced oxidative stress in the glomerulus of db/db mice. These data indicate that halofuginone prevents ECM deposition and decreases oxidative stress, thereby suppressing the progression of diabetic nephropathy.     

Regulation of TGF-β storage and activation in the human idiopathic pulmonary fibrosis lung

Idiopathic pulmonary fibrosis (IPF) is a progressive disease of unknown cause. The pathogenesis of the disease is characterized by fibroblast accumulation and excessive transforming growth factor-β (TGF-β) activation. Although TGF-β activation is a complex process involving various protein interactions, little is known of the specific routes of TGF-β storage and activation in human lung. Here, we have systematically analyzed the expression of specific proteins involved in extracellular matrix targeting and activation of TGF-β. Latent TGF-β-binding protein (LTBP)-1 was found to be significantly upregulated in IPF patient lungs. LTBP-1 expression was especially high in the fibroblastic foci, in which P-Smad2 immunoreactivity, indicative of TGF-β signaling activity, was less prominent. In cultured primary lung fibroblasts and epithelial cells, short-interfering-RNA-mediated downregulation of LTBP-1 resulted in either increased or decreased TGF-β signaling activity, respectively, suggesting that LTBP-1-mediated TGF-β activation is dependent on the cellular context in the lung. Furthermore, LTBP-1 was shown to colocalize with fibronectin, fibrillin-1 and fibrillin-2 proteins in the IPF lung. Fibrillin-2, a developmental gene expressed only in blood vessels in normal adult lung, was found specifically upregulated in IPF fibroblastic foci. The TGF-β-activating integrin β8 subunit was expressed at low levels in both control and IPF lungs. Alterations in extracellular matrix composition, such as high levels of the TGF-β storage protein LTBP-1 and the re-appearance of fibrillin-2, probably modulate TGF-β availability and activation in different pulmonary compartments in the fibrotic lung.     

Catalpol inhibits TGF-β1-induced epithelial-mesenchymal transition in human non–small-cell lung cancer cells through the inactivation of Smad2/3 and NF-κB signaling pathways

Catalpol, one of the main active ingredients isolated from Rehmannia glutinosa, was reported to possess anticancer activity. However, the role of catalpol in transforming growth factor β1 (TGF-β1)-induced epithelial-mesenchymal transition (EMT) in human non–small-cell lung cancer (NSCLC) cells has not been elucidated. The objective of this study was to investigate the effect of catalpol on EMT in human NSCLC cells. Our results showed that catalpol significantly inhibited the TGF-β1-induced cell migration and invasion of A549 cells, as well as repressed matrix metalloproteinase (MMP)2 and MMP9 expression induced by TGF-β1 in A549 cells. In addition, catalpol markedly repressed the EMT process in A549 cells in response to TGF-β1. Furthermore, catalpol prevented the activation of Smad2/3 and nuclear factor κB (NF-κB) signaling pathways induced by TGF-β1 in A549 cells. In conclusion, these findings indicated that catalpol inhibits TGF-β1-induced EMT in human NSCLC cells through the inactivation of Smad2/3 and NF-κB signaling pathways. Thus, catalpol may be a promising agent for the treatment of NSCLC.     

TGF-β-induced epithelial-mesenchymal transition of A549 lung adenocarcinoma cells is enhanced by pro-inflammatory cytokines derived from RAW 264.7 macrophage cells

Cancer cells undergo epithelial-mesenchymal transition (EMT) during invasion and metastasis. Although transforming growth factor-β (TGF-β) and pro-inflammatory cytokines have been implicated in EMT, the underlying molecular mechanisms remain to be elucidated. Here, we studied the effects of proinflammatory cytokines derived from the mouse macrophage cell line RAW 264.7 on TGF-β-induced EMT in A549 lung cancer cells. Co-culture and treatment with conditioned medium of RAW 264.7 cells enhanced a subset of TGF-β-induced EMT phenotypes in A549 cells, including changes in cell morphology and induction of mesenchymal marker expression. These effects were increased by the treatment of RAW 264.7 cells with lipopolysaccharide, which also induced the expression of various proinflammatory cytokines, including TNF-α and IL-1β. The effects of conditioned medium of RAW 264.7 cells were partially inhibited by a TNF-α neutralizing antibody. Dehydroxy methyl epoxyquinomicin, a selective inhibitor of NFκB, partially inhibited the enhancement of fibronectin expression by TGF-β, TNF-α, and IL-1β, but not of N-cadherin expression. Effects of other pharmacological inhibitors also suggested complex regulatory mechanisms of the TGF-β-induced EMT phenotype by TNF-α stimulation. These findings provide direct evidence of the effects of RAW 264.7-derived TNF-α on TGF-β-induced EMT in A549 cells, which is transduced in part by NFκB signalling.     

Ascochlorin suppresses TGF-β1-induced PAI-1 expression through the inhibition of phospho-EGFR in rat kidney fibroblast cells

Fibrosis is induced by the excessive and abnormal deposition of extracellular matrix (ECM) with various growth factors in tissues. Transforming growth factor-β1 (TGF-β1), the growth factor involved in fibrosis, modulates ECM synthesis and accumulation. TGF-β1 enhances the production of stimulators of ECM synthesis such as plasminogen activator inhibitor type 1 (PAI-1). As such, PAI-1 expression directly influences the proteolysis, invasion, and accumulation of ECM. It was shown in this study that ascochlorin, a prenylpenl antiobiotic, prevents the expression of profibrotic factors, such as PAI-1 and collagen type I, and that the TGF-β1-induced PAI-1 promoter activity is inhibited by ascochlorin. Ascochlorin abolishes the phosphorylation of the EGFR-MEK-ERK signaling pathway to regulate the TGF-β1-induced expression of PAI-1 without the inhibition of TβRII phosphorylation. Furthermore, the MEK inhibitor and EGFR siRNA block PAI-1 expression, and the Raf-1, MEK, and ERK signaling pathways for the regulation of PAI-1 expression. Ascochlorin suppresses the matrix metalloproteinases (MMPs) activity to activate the heparin-binding EGF-like growth factor (HB-EGF), to induce the phosphorylation of EGFR, and the MMPs inhibitor suppresses EGFR phosphorylation and the PAI-1 mRNA levels. These results suggest that ascochlorin prevents the expression of PAI-1 via the inhibition of an EGFR-dependent signal transduction pathway activated by MMPs.     

TGF-β1 diminishes collagen production during long-term cyclic stretching of engineered connective tissue: implication of decreased ERK signaling

Cyclic stretching and growth factors like TGF-β have been used to enhance extracellular matrix (ECM) production by cells in engineered tissue to achieve requisite mechanical properties. In this study, the effects of TGF-β1 were evaluated during long-term cyclic stretching of fibrin-based tubular constructs seeded with neonatal human dermal fibroblasts. Samples were evaluated at 2, 5, and 7 weeks for tensile mechanical properties and ECM deposition. At 2 weeks, +TGF-β1 samples had 101% higher collagen concentration but no difference in ultimate tensile strength (UTS) or modulus compared to -TGF-β1 samples. However, at weeks 5 and 7, -TGF-β1 samples had higher UTS/modulus and collagen concentration, but lower elastin concentration compared to +TGF-β1 samples. The collagen was better organized in -TGF-β1 samples based on picrosirius red staining. Western blot analysis at weeks 5 and 7 showed increased phosphorylation of ERK in -TGF-β1 samples, which correlated with higher collagen deposition. The TGF-β1 effects were further evaluated by western blot for αSMA and SMAD2/3 expression, which were 16-fold and 10-fold higher in +TGF-β1 samples, respectively. The role of TGF-β1 activated p38 in inhibiting phosphorylation of ERK was evaluated by treating samples with SB203580, an inhibitor of p38 activation. SB203580-treated cells showed increased phosphorylation of ERK after 1 hour of stretching and increased collagen production after 1 week of stretching, demonstrating an inhibitory role of activated p38 via TGF-β1 signaling during cyclic stretching. One advantage of TGF-β1 treatment was the 4-fold higher elastin deposition in samples at 7 weeks. Further cyclic stretching experiments were thus conducted with constructs cultured for 5 weeks without TGF-β1 to obtain improved tensile properties followed by TGF-β1 supplementation for 2 weeks to obtain increased elastin content, which correlated with a reduction in loss of pre-stress during preconditioning for tensile testing, indicating functional elastin. This study shows that a sequential stimulus approach - cyclic stretching with delayed TGF-β1 supplementation - can be used to engineer tissue with desirable tensile and elastic properties.     

Phenotypic consequences of transforming growth factor β1 gene ablation in murine embryonic fibroblasts: Autocrine control of cell proliferation and extracellular matrix biosynthesis

The profound effects of transforming growth factor β1 (TGF-β1) on the immune system, cardiogenesis, in yolk sac hematopoeisis and in differentiation of endothelium have been demonstrated by detailed analyses of TGF-β1 knockout mice during embryogenesis. We have systematically examined the autocrine and paracrine roles of TGF-β1 in cell proliferation and in its ability to modulate the gene expression of selected components of extracellular matrix (ECM) using embryonic fibroblasts from TGF-β1 null mice (TGF-β1^−/−). The rates of cell proliferation of embryonic fibroblasts from normal mice (TGF-β1^+/+) and TGF-β1 null mice were compared by cell counting, by ³H thymidine incorporation, and by measuring the fraction of cells in the G1, S, and G2/M phases of the cell cycle by fluorescent activated cell sorting (FACS). Concurrently, the expression of pro-α1(I) collagen, fibronectin, and plasminogen activator inhibitor-1 (PAI-1) was also quantified by hybridization of total mRNA from TGF-β1^+/+ and TGF-β1^−/− embryonic fibroblasts. We report that TGF-β1^−/− cells proliferated at about twice the rate of TGF-β1^+/+ cells. Further, TGF-β1 null fibroblasts accumulated and synthesized lower constitutive levels of pro-α1(I) collagen, fibronectin, and PAI-1 mRNA. The quantitative differences in the rates of cell proliferation and ECM gene expression between TGF-β1^+/+ and TGF-β1^−/− cells could be eliminated by treatment of TGF-β1^+/+ cells with a neutralizing antibody of TGF-β1. Thus, our results are consistent with the hypothesis that TGF-β1 acts as a negative autocrine regulator of growth and a positive autocrine regulator of ECM biosynthesis in embryonic fibroblasts. 176:67–75, 1998. Published 1998 Wiley-Liss, Inc.

1 This article was prepared by a group of United States government employees and non-United States government employees, and as such is subject to 17 U.S.C. Sec. 105.