MicroRNA-24 regulates the processing of latent TGFβ1 during cyclic mechanical stress in human trabecular meshwork cells through direct targeting of FURIN

Cyclic mechanical stress (CMS) leads to alterations of cellular functions in the trabecular meshwork (TM), including the up-regulation of transforming growth factor beta 1 (TGFβ1), that can potentially contribute to the pathogenesis of glaucoma. Although microRNAs (miRNAs) are known to play important roles in many biological functions, little is known about their potential involvement in the cellular responses elicited by mechanical stress. Here we analyzed changes in miRNA expression induced by CMS, and examined the possible role of miR-24 in the response of human TM cells to CMS. CMS induced the expression of miR-24 that led to the down regulation of the subtilisin-like proprotein convertase FURIN, which is known to play a major role in the processing of TGFβ1. FURIN was confirmed as a novel target of miR-24 by 3' UTR luciferase assay and western blot. Overexpression of miR-24 resulted in a significant decrease in activated TGFβ1. This effect was mimicked by down regulation of FURIN by siRNA. Conversely, inhibition of miR-24 expression with a specific antagomir led to a small but significant increase in TGFβ1. Furthermore, the increase in active TGFβ1 induced by CMS in HTM cells was prevented by miR-24. Altogether, our results suggest that miRNAs might contribute to the regulation of responses to CMS in TM cells. Specifically, miR-24 might play an important role in modulating the induction of TGFβ1 mediated by CMS through direct targeting of FURIN.     

Isofraxidin attenuates IL-1β-induced inflammatory response in human nucleus pulposus cells

Inflammation has been demonstrated to be the key factor for intervertebral disc degeneration (IVD), which remains a major public health problem. Isofraxidin is a coumarin compound that possesses strong anti-inflammatory activity. However, the role of isofraxidin in IVD remains unclear. The aim of this study was to evaluate the effects of isofraxidin on inflammatory response in human nucleus pulposus cells (NPCs) exposed to interleukin-1β (IL-1β). The results proved that isofraxidin attenuated the IL-1β-induced significant increases in inflammatory mediators and cytokines including nitric oxide (NO), inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2), tumor necrosis factor alpha (TNF-α), and IL-6. Besides, isofraxidin also inhibited the induction effect of IL-1β on matrix metalloproteinases (MMP)-3 and MMP-13. Moreover, the NF-κB activation caused by IL-1β was significantly inhibited by isofraxidin treatment. These findings suggested that isofraxidin alleviates IL-1β-induced inflammation in NPCs. Our work provided an idea that isofraxidin might act as a novel preventive role in IVD.     

A novel TRPC6-dependent mechanism of TGF-β-induced migration and invasion of human hepatocellular carcinoma cells

正Mechanisms on cancer cell migration and invasion have been major topics of cancer research and anti-cancer therapy development. Among the multiple cell signaling pathways involved in cell migration, those elicited by transforming growth factorβ(TGF-β) have attracted tremendous attention. The TGF-βpolypeptide cytokines include four isoforms:TGF-β1, TGF-β2, TGF-β3, and TGF-β4, which are secreted mainly from cells of white blood cell lineage, such as macrophages, T cells and platelets. However, TGF-βcan also be synthesized and released from fibroblasts and cancer     

TGFβ-induced GRK2 expression attenuates AngII-regulated vascular smooth muscle cell proliferation and migration

Through diametric actions, the transforming growth factor β (TGFβ) and Angiotensin II (AngII) play important roles in regulating various biological responses such as cell proliferation and migration. Signaling initiated by TGFβ and AngII occurs through two structurally and functionally distinct receptor super families, the serine/threonine kinase and G protein-coupled receptors (GPCRs). Previously, we identified the G protein-coupled receptor kinase-2 (GRK2), a key regulatory factor in the desensitization of GPCRs, as a direct downstream target of the TGFβ signaling cascade. GRK2 acts through a negative feed-back loop mechanism to terminate TGFβ-induced smad signaling. To investigate the impact of TGFβ-induced GRK2 expression on GPCR signaling, we examined its effect on AngII signaling in vascular smooth muscle cells (VSMCs). In this study, we show that activation of the TGFβ signaling cascade in VSMCs results in increased GRK2 expression levels, which consequently inhibits AngII-induced ERK phosphorylation and antagonizes AngII-induced VSMC proliferation and migration. Moreover, the inhibitory effect of TGFβ on AngII signaling occurs at the Mek-Erk interface and is abrogated when an anti-sense oligonucleotide directed against GRK2 is used. Thus, we conclude that TGFβ signaling antagonizes AngII-induced VSMC proliferation and migration through the inhibition of ERK phosphorylation and that GRK2 is a key factor mediating the cross-talk between these two receptor super families.     

Lefty A attenuates the TGF-β1-induced epithelial to mesenchymal transition of human renal proximal epithelial tubular cells

The epithelial to mesenchymal transition (EMT) is a crucial event for renal fibrosis that can be elicited by TGF-β1/Smads signaling and its downstream mediator connective tissue growth factor (CTGF). As a distinct member of the TGF-β superfamily, Lefty A has been shown to be significantly downregulated in the kidneys of patients with severe ureteral obstruction, suggesting its role in renal fibrosis induced by obstructive nephropathy. In order to determine whether Lefty A prevents TGF-β1-induced EMT, human proximal tubule epithelial cells (HK-2) were stably transfected with Lefty A or control vectors and stimulated with 10 ng/ml TGF-β1 for 48 h. The results show that stimulation with TGF-β1 led to EMT including cell morphology changes, Smad2/3 signaling pathway activation, increased α-SMA, collagen type I, and CTGF expression, and decreased E-cadherin expression in mock-transfected HK-2 cells. Overexpression of Lefty A efficiently blocked p-Smad2/3 activation and attenuated all these EMT changes induced by TGF-β1. This finding suggests that Lefty A may serve as a potential new therapeutic target to inhibit or even reverse EMT during the process of renal fibrosis.     

Antifibrotic effect of methylated quercetin derivatives on TGFβ-induced hepatic stellate cells

Quercetin (QCT) and isorhamnetin (ISO), natural flavonoids, were both shown to possess antifibrotic activity in in vivo and in vitro models of hepatic fibrosis. Although ISO is a direct metabolite of QCT differing by a methyl group, it has been reported to be absorbed more adequately and eliminated slower than QCT after oral administration. Our aim of the study was to investigate biological effect of mono-methylated QCT derivatives against fibrosis using rat hepatic stellate cells (HSC-T6). All test derivatives were synthesized from QCT. HSC-T6 cells were induced by TGFβ and treated with derivatives followed by cell proliferation assay, immunofluorescence staining of αSMA, and gene expression analysis of fibrosis markers. All compounds showed a dose- and time-dependent antiproliferation effect. ISO, 3-O-methylquercetin (3MQ), and rhamnetin (RHA) reduced αSMA mRNA; 3MQ prevented the augmentation of collagen I mRNA; and compounds, except azaleatin and 3MQ, reduced Timp1 mRNA expression in TGFβ-induced HSCs. In conclusion, each compound had singular effect against different features of fibrosis depending on the position of methyl group although the further mechanism of action of compounds during fibrosis development remains to be investigated. These findings suggest that antifibrotic effect of quercetin can be enhanced by adding methyl group on functionally important position.     

Salidroside attenuates interleukin-1β-induced inflammation in human osteoarthritis chondrocytes

Salidroside, a bioactive constituent isolated from Rhodiola rosea, has been reported to have anti-inflammatory effects. However, the effects of salidroside on interleukin (IL)-1β-stimulated osteoarthritis (OA) chondrocytes remain to be elucidated. Thus, this study aimed to evaluate the anti-inflammatory effects of salidroside on IL-1β-stimulated human OA chondrocytes and explore its underlying mechanisms. Our results showed that salidroside significantly inhibited the production of nitric oxide and prostaglandin E-2, as well as suppressed the expression of inducible nitric oxide synthase and cyclooxygenase-2 in IL-1β-stimulated chondrocytes ( P < .05). In addition, salidroside also suppressed IL-1β-induced matrix metalloproteinases production in human OA chondrocytes ( P < .05). Furthermore, pretreatment with salidroside prevented IL-1β-induced NF-κB activation in OA chondrocytes ( P < .05). In conclusion, the current study demonstrated that salidroside inhibited the IL-1β-induced inflammatory response in OA chondrocytes via inhibition of NF-κB activation.     

Simvastatin inhibits TGFβ1-induced fibronectin in human airway fibroblasts

Background

Bronchial fibroblasts contribute to airway remodelling, including airway wall fibrosis. Transforming growth factor (TGF)-β1 plays a major role in this process. We previously revealed the importance of the mevalonate cascade in the fibrotic response of human airway smooth muscle cells. We now investigate mevalonate cascade-associated signaling in TGFβ1-induced fibronectin expression by bronchial fibroblasts from non-asthmatic and asthmatic subjects.

Methods

We used simvastatin (1-15 μM) to inhibit 3-hydroxy-3-methlyglutaryl-coenzyme A (HMG-CoA) reductase which converts HMG-CoA to mevalonate. Selective inhibitors of geranylgeranyl transferase-1 (GGT1; GGTI-286, 10 μM) and farnesyl transferase (FT; FTI-277, 10 μM) were used to determine whether GGT1 and FT contribute to TGFβ1-induced fibronectin expression. In addition, we studied the effects of co-incubation with simvastatin and mevalonate (1 mM), geranylgeranylpyrophosphate (30 μM) or farnesylpyrophosphate (30 μM).

Results

Immunoblotting revealed concentration-dependent simvastatin inhibition of TGFβ1 (2.5 ng/ml, 48 h)-induced fibronectin. This was prevented by exogenous mevalonate, or isoprenoids (geranylgeranylpyrophosphate or farnesylpyrophosphate). The effects of simvastatin were mimicked by GGTI-286, but not FTI-277, suggesting fundamental involvement of GGT1 in TGFβ1-induced signaling. Asthmatic fibroblasts exhibited greater TGFβ1-induced fibronectin expression compared to non-asthmatic cells; this enhanced response was effectively reduced by simvastatin.

Conclusions

We conclude that TGFβ1-induced fibronectin expression in airway fibroblasts relies on activity of GGT1 and availability of isoprenoids. Our results suggest that targeting regulators of isoprenoid-dependent signaling holds promise for treating airway wall fibrosis.     

Asymmetric dimethylarginine exacerbates Aβ-induced toxicity and oxidative stress in human cell and Caenorhabditis elegans models of Alzheimer disease

Growing evidence suggests a strong association between cardiovascular risk factors and incidence of Alzheimer disease (AD). Asymmetric dimethylarginine (ADMA), the endogenous nitric oxide synthase inhibitor, has been identified as an independent cardiovascular risk factor and is also increased in plasma of patients with AD. However, whether ADMA is involved in the pathogenesis of AD is unknown. In this study, we found that ADMA content was increased in a transgenic Caenorhabditis elegans β-amyloid (Aβ) overexpression model, strain CL2006, and in human SH-SY5Y cells overexpressing the Swedish mutant form of human Aβ precursor protein (APPsw). Moreover, ADMA treatment exacerbated Aβ-induced paralysis and oxidative stress in CL2006 worms and further elevated oxidative stress and Aβ secretion in APPsw cells. Knockdown of type 1 protein arginine N-methyltransferase to reduce ADMA production failed to show a protective effect against Aβ toxicity, but resulted in more paralysis in CL2006 worms as well as increased oxidative stress and Aβ secretion in APPsw cells. However, overexpression of dimethylarginine dimethylaminohydrolase 1 (DDAH1) to promote ADMA degradation significantly attenuated oxidative stress and Aβ secretion in APPsw cells. Collectively, our data support the hypothesis that elevated ADMA contributes to the pathogenesis of AD. Our findings suggest that strategies to increase DDAH1 activity in neuronal cells may be a novel approach to attenuating AD development.     

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.     

Analysis and characterization of the functional TGFβ receptors required for BMP6-induced osteogenic differentiation of mesenchymal progenitor cells

Although BMP6 is highly capable of inducing osteogenic differentiation of mesenchymal progenitor cells (MPCs), the molecular mechanism involved remains to be fully elucidated. Using dominant negative (dn) mutant form of type I and type II TGFβ receptors, we demonstrated that three dn-type I receptors (dnALK2, dnALK3, dnALK6), and three dn-type II receptors (dnBMPRII, dnActRII, dnActRIIB), effectively diminished BMP6-induced osteogenic differentiation of MPCs. These findings suggested that ALK2, ALK3, ALK6, BMPRII, ActRII and ActRIIB are essential for BMP6-induced osteogenic differentiation of MPCs. However, MPCs in this study do not express ActRIIB. Moreover, RNA interference of ALK2, ALK3, ALK6, BMPRII and ActRII inhibited BMP6-induced osteogenic differentiation in MPCs. Our results strongly suggested that BMP6-induced osteogenic differentiation of MPCs is mediated by its functional TGFβ receptors including ALK2, ALK3, ALK6, BMPRII, and ActRII. [BMB Reports 2013; 46(2): 107-112]     

Shear stress modulation of IL-1β-induced E-selectin expression in human endothelial cells

Background

Endothelial cells (ECs) are continuously exposed to hemodynamic forces imparted by blood flow. While it is known that endothelial behavior can be influenced by cytokine activation or fluid shear, the combined effects of these two independent agonists have yet to be fully elucidated.

Methodology

We investigated EC response to long-term inflammatory cues under physiologically relevant shear conditions via E-selectin expression where monolayers of human umbilical vein ECs were simultaneously exposed to laminar fluid shear and interleukin-1ß (shear-cytokine activation) in a parallel plate flow chamber.

Results and Conclusion

Naïve ECs exposed to shear-cytokine activation display significantly higher E-selectin expression for up to 24 hr relative to ECs activated in static (static-cytokine). Peak E-selectin expression occurred after 8–12 hr of continuous shear-cytokine activation contrary to the commonly observed 4–6 hr peak expression in ECs exposed to static-cytokine activation. Cells with some history of high shear conditioning exhibited either high or muted E-selectin expression depending on the durations of the shear pre-conditioning and the ensuing shear-cytokine activation. Overall, the presented data suggest that a high laminar shear enhances acute EC response to interleukin-1ß in naïve or shear-conditioned ECs as may be found in the pathological setting of ischemia/reperfusion injury while conferring rapid E-selectin downregulation to protect against chronic inflammation.     

Selenium supplementation attenuates procollagen-1 and interleukin-8 production in fat-loaded human C3A hepatoblastoma cells treated with TGFβ1

Background

Non-alcoholic fatty liver disease (NAFLD) is associated with obesity, insulin resistance and hepatic steatosis. Non-alcoholic steatohepatitis (NASH) is a serious consequence of NAFLD where chronic tissue damage and inflammation result in fibrosis which may progress to cirrhosis. Transforming growth factor β1 (TGFβ1), proinflammatory cytokines and oxidative stress are thought to play crucial roles in the pathogenesis of these conditions. The contributions of individual liver cell types to fibrogenesis remain controversial and the influence of selenium status has not been investigated.

Methods

In this study we have used a cell culture model of fat-loading using oleate-treated human hepatoblastoma (C3A) cells to investigate how fat-loading and selenium status might influence the production of collagen in response to TGFβ1. The secretion of inflammatory cytokines was also investigated, together with the epithelial character of the treated cells.

Results

We found that in response to treatment with TGFβ1, C3A cells produced mRNA encoding the pro-αI chain of procollagen I, secreted procollagen I peptide, up-regulated production of the proinflammatory cytokine interleukin-8 (IL-8) and the mesenchymal marker vimentin, and down-regulated albumin production. Most of these responses were considerably enhanced when cells were fat-loaded with oleate and were attenuated by selenium addition at a dose that optimised the expression of thioredoxin reductase and glutathione peroxidase.

Conclusions

Our data establish that both fat-loading and suboptimal selenium status enhance collagen and IL-8 production by C3A hepatocytes in response to TGFβ1, possibly as part of an epithelial to mesenchymal transition.

General significance

These findings suggest that the hepatocyte may be an important contributor to the pathogenesis of fibrosis associated with NAFLD.     

TGFβ2-induced tenogenesis impacts cadherin and connexin cell-cell junction proteins in mesenchymal stem cells

Tenogenic differentiation of stem cells is needed for tendon tissue engineering approaches. A current challenge is the limited information on the cellular-level changes during tenogenic induction. Tendon cells in embryonic and adult tendons possess an array of cell-cell junction proteins that include cadherins and connexins, but how these proteins are impacted by tenogenic differentiation is unknown. Our objective was to explore how tenogenic induction of mesenchymal stem cells (MSCs) using the transforming growth factor (TGF)β2 impacted protein markers of tendon differentiation and protein levels of N-cadherin, cadherin-11 and connexin-43. MSCs were treated with TGFβ2 for 21 days. At 3 days, TGFβ2-treated MSCs developed a fibroblastic morphology and significantly decreased levels of N-cadherin protein, which were maintained through 21 days. Similar decreases in protein levels were found for cadherin-11. Connexin-43 protein levels significantly increased at 3 days, but then decreased below control levels, though not significantly. Protein levels of scleraxis and tenomodulin were significantly increased at day 14 and 21, respectively. Taken together, our results indicate that TGFβ2 is an inducer of tendon marker proteins (scleraxis and tenomodulin) in MSCs and that tenogenesis alters the protein levels of N-cadherin, cadherin-11 and connexin-43. These findings suggest a role for connexin-43 early in tenogenesis, and show that early-onset and sustained decreases in N-cadherin and cadherin-11 may be novel markers of tenogenesis in MSCs.     

Role of aldose reductase in TGF-β1-induced fibronectin synthesis in human mesangial cells

Accumulation of glomerular extracellular matrix (ECM) may result in glomerulosclerosis. Several lines of evidence indicate a key role for transforming growth factor-β1 (TGF-β1) in glomerular ECM synthesis and degradation, such as fibronectin (FN). Aldose reductase (AR) was proven to be one of the TGF-β1 responsive genes in cultured rat mesangial cells using the SSH–PCR method and there were positive correlation between the AR and TGF-β1 in our previous studies. So we assumed that AR could regulate FN synthesis. In this study, we explored the role of AR in FN production and possible mechanism involved. The expression of AR, FN and c-Jun proteins were analyzed by Western blot and the activity of activator protein-1 (AP-1) was assessed by electrophoretic mobility shift assay (EMSA). Our results showed that AR could mediate the TGF-β1-induced FN production, which may associate with AP-1 activation.