Persistence of TGF-β1 induction of increased fibroblast contractility

Fibroblast contraction of collagen gels is regarded as a model of wound contraction. Transforming growth factor (TGF)-beta added to such gels can augment contraction consistent with its suggested role as a mediator of fibrotic repair. Since fibroblasts isolated from fibrotic tissues have been suggested to express a "fibrotic phenotype," we hypothesized that TGF-beta exposure may lead to a persistent increase in fibroblasts' contractility. To evaluate this question, confluent human fetal lung fibroblasts were treated with serum-free Dulbecco modified Eagle medium (DMEM), with or without 100 pM [corrected] TGF-beta1, TGF-beta2, or TGF-beta3 for 48 h. Fibroblasts were then trypsinized and cast into gels composed of native type I collagen isolated from rat tail tendons. After 20 min for gelation, the gels were released and maintained in serum-free DMEM. TGF-beta-pretreated fibroblasts caused significantly more rapid gel contraction (52.5+/-0.6, 50.9+/-0.2, and 50.3+/-0.5% by TGF-beta1, -beta2, and -beta3 pretreated fibroblasts, respectively) than control fibroblasts (74.0+/-0.3%, P < 0.01). This effect is concentration dependent (50-200 nM), and all three isoforms had equal activity. The effect of TGF-beta1, however, persisted for only a short period of time following the removal of TGF-beta, and was lost with sequential passage. These observations suggest that the persistent increase in collagen-gel contractility, mediated by fibroblasts from fibrotic tissues, would not appear to be solely due to previous exposure of these cells to TGF-beta.     

TGFβ2-mediated production of hyaluronan is important for the induction of epicardial cell differentiation and invasion

In the developing heart, the epicardium is a major source of progenitor cells that contribute to the formation of the coronary vessel system. These epicardial progenitors give rise to the different cellular components of the coronary vasculature by undergoing a number of morphological and physiological changes collectively known as epithelial to mesenchymal transformation (EMT). However, the specific signaling mechanisms that regulate epicardial EMT are yet to be delineated. In this study we investigated the role of TGFβ2 and hyaluronan (HA) during epicardial EMT and how signals from these two molecules are integrated during this important process. Here we show that TGFβ2 induces MEKK3 activation, which in turn promotes ERK1/2 and ERK5 phosphorylation. TGFβ2 also increases Has2 expression and subsequent HA production. Nevertheless, inhibition of MEKK3 kinase activity, silencing of ERK5 or pharmacological disruption of ERK1/2 activation significantly abrogates this response. Thus, TGFβ2 promotes Has2 expression and HA production through a MEKK3/ERK1/2/5-dependent cascade. Furthermore, TGFβ2 is able to induce epicardial cell invasion and differentiation but not proliferation. However, inhibition of MEKK3-dependent pathways, degradation of HA by hyaluronidases or blockade of CD44, significantly impairs the biological response to TGFβ2. Taken together, these findings demonstrate that TGFβ2 activation of MEKK3/ERK1/2/5 signaling modulates Has2 expression and HA production leading to the induction of EMT events. This is an important and novel mechanism showing how TGFβ2 and HA signals are integrated to regulate changes in epicardial cell behavior.     

In vitro differentiation of ciliated cells in ALI-cultured human airway epithelium – The framework for functional studies on airway differentiation in ciliopathies

Primary cultures of the human airway epithelium (AE) cells are an indispensable tool in studies of pathophysiology of genetic and environmental pulmonary diseases, including cystic fibrosis (CF), primary ciliary dyskinesia (PCD) and chronic obstructive pulmonary disease (COPD). Air-liquid interface (ALI) culture is the best method to follow the differentiation of ciliated cells, whose dysfunction forms the basis of PCD. Here, we used custom-designed Taqman Low Density Array (TLDA), qRT-PCR-based assay, to analyze expression of 14 AE genes in cells from healthy donors, cultured in ALI settings using Pneumacult medium, with the focus on genes involved in cilia differentiation and in PCD pathogenesis. The results of TLDA assay were compared with the bulk RNAseq analysis, and placed in the cellular context using immunofluorescent staining (IF) of ALI cultured cells.Expression analysis revealed culture time-related upregulation of the majority of cilia-related genes, followed by the appearance of respective protein signals visualized by IF. Strong correlation of TLDA with RNAseq results indicated that TLDA assay is a reliable and scalable approach to analyze expression of selected genes specific for different AE cell types. Characterization of temporal and inter-donor changes in the expression of these genes, performed in healthy donors and in well-defined ALI/Pnemacult culture conditions, provides a useful reference relevant for a broad spectrum of functional studies where the in vitro AE differentiation is in focus.     

In vitro articular cartilage growth with sequential application of IGF-1 and TGF-β1 enhances volumetric growth and maintains compressive properties

In vitro cultures with insulin-like growth factor-1 (IGF-1) and transforming growth factor-β1 (TGF-β1) have previously been shown to differentially modulate the growth of immature bovine articular cartilage. IGF-1 stimulates expansive growth yet decreases compressive moduli and increases compressive Poisson's ratios, whereas TGF-β1 maintains tissue size, increases compressive moduli, and decreases compressive Poisson's ratios. The current study's hypothesis was that sequential application of IGF-1 and TGF-β1 during in vitro culture produces geometric and compressive mechanical properties that lie between extreme values produced when using either growth factor alone. Immature bovine articular cartilage specimens were harvested and either untreated (D0, i.e., day zero) or cultured in vitro for either 6 days with IGF-1 (D6 IGF), 12 days with IGF-1 (D12 IGF), or 6 days with IGF-1 followed by 6 days with TGF-β1 (D12 SEQ, i.e., sequential). Following treatment, all specimens were tested for geometric, biochemical, and compressive mechanical properties. Relative to D0, D12 SEQ treatment enhanced volumetric growth, but to a lower value than that for D12 IGF. Furthermore, D12 SEQ treatment maintained compressive moduli and Poisson's ratios at values higher and lower, respectively, than those for D12 IGF. Considering the previously described effects of 12 days of treatment with TGF-β1 alone, D12 SEQ induced both growth and mechanical property changes between those produced with either IGF-1 or TGF-β1 alone. The results suggest that it may be possible to vary the durations of select growth factors, including IGF-1 and TGF-β1, to more precisely modulate the geometric, biochemical, and mechanical properties of immature cartilage graft tissue in clinical repair strategies.     

Ebsulfur as a potent scaffold for inhibition and labelling of New Delhi metallo-β-lactamase-1 in vitro and in vivo

The superbug infection caused by New Delhi metallo-β-lactamase (NDM-1) has grown into an emerging threat, labelling and inhibition of NDM-1 has proven challenging due to its shuttling between pathogenic bacteria. Here, we report a potent covalent scaffold, ebsulfur, for targeting the protein in vitro and in vivo. Enzymatic kinetic study indicated that eighteen ebsulfurs gained except 1a–b and 1f inhibited NDM-1, exhibiting an IC₅₀ value ranging of 0.16–9 μM, and 1g was found to be the best, dose- and time-dependent inhibitor with an IC₅₀ of 0.16 μM. Also, these ebsulfurs effectively restored the antibacterial activity of cefazolin against E. coli expressing NDM-1, and the best effect was observed to be from 1g, 1i and 1n, resulting in an 256-fold reduction in MIC of the antibiotic at a dose of 16 μg/mL. The equilibrium dialysis study implied that the ebsulfur disrupted the coordination of one Zn(II) ion at active site of NDM-1. Labelling of NDM-1 using a constructed fluorescent ebsulfur Ebs-R suggested that the inhibitor covalently bound to the target through SDS-PAGE analysis in vitro. Also, labelling NDM-1 in living E. coli cells with Ebs-R by confocal microscopic imaging showed the real-time distribution change process of intracellular recombinant protein NDM-1. Moreover, the cytotoxicity of these ebsulfurs against L929 mouse fibroblastic cells was tested, and their capability to restore antibacterial activity of antibiotic against clinical strains E. coli EC08 producing NDM-1 was determined. The ebsulfur scaffold proposed here is valuable for development of the covalent irreversible inhibitors of NDM-1, and also for labelling the target in vitro and in vivo.     

Oxidative stress contributes to the induction and persistence of TGF-β1 induced pulmonary fibrosis

Oxidative stress with reactive oxygen species (ROS) can contribute to the pathogenesis of idiopathic pulmonary fibrosis. Antioxidant enzymes, such as extracellular superoxide dismutase (ECSOD), may modulate the injury and repair components of the fibrogenic response. Here we determined whether ECSOD could attenuate experimental TGF-β1-induced persistent lung fibrosis. In this study, primary human lung fibroblasts, MRC-5 fibroblasts and A549 epithelial cells were exposed to recombinant active TGF-β1. An adenovirus vector that expresses human ECSOD (AdECSOD) was constructed and rats were endotracheally intubated with an adenoviral vector encoding active TGF-β1 (AdTGF-β1), AdECSOD or a control vector (AdDL70) alone or in combinations AdTGF-β1/AdDL70 or AdTGF-β1/AdECSOD. TGF-β1 alone induced fibrotic responses and significantly down-regulated endogenous ECSOD gene expression both in vitro and in vivo and caused oxidative stress in rat lung, associated with increased levels of activated TGF-β1 in lung fluid and tissue. ECSOD protein was markedly reduced in the interstitium and fibrotic foci in TGF-β1 induced experimental lung fibrosis. The fibrotic response caused by AdTGF-β1 was markedly attenuated by concomitant gene transfer using AdECSOD, detected by lung function measurements, histologic and morphometric analysis, hydroxyproline content and fibrosis-related gene expression. In addition, the oxidative stress and increased presence of activated TGF-β1 in rat lung induced by AdTGF-β1 was significantly reduced by ECSOD gene transfer. These findings suggest a substantial role for oxidative stress in the pathogenesis of TGF-β1 driven persistent pulmonary fibrosis and enhanced presence of ECSOD can inhibit latent TGF-β1 activation by ROS and diminish subsequent fibrotic responses.     

TGF-β1 reverses PDGF-stimulated migration of human aortic smooth muscle cells in vitro

Summary Platelet-derived growth factor (PDGF) and transforming growth factor beta-1(TGF-β1) were tested separately or together for the ability to stimulate migration of human aortic vascular smooth muscle cells (VSMC). PDGF (10 ng/ml) stimulated migration of VSMC over a 48-h period. TGF-β1 (10 ng/ml) had no effect on migration during the same period. VSMC exposed simultaneously to both TGF-β1 and PDGF exhibited diminished migration (50%) when compared to cells treated only with PDGF. Cells that migrated in the presence of PDGF possessed short actin cables that extended from cellular processes at the leading edge of migrating cells; focal adhesions containing the α_vβ₃/β₅ integrins localized to the same region. Cells grown in the presence of TGF-β1 exhibited long, intensely stained actin filaments that spanned the entire length of the cell and were similar to untreated control VSMC. Focal adhesions containing α_vβ₃/β₅ distributed evenly on the basal surface in both TGF-β1-treated cells and control cultures. Cellular responses to PDGF were mitigated when TGF-β1 was present in the culture medium. VSMC grown in the presence of both PDGF and TGF-β1 exhibited elongated actin filaments that were similar to nonmotile TGF-β1-treated cultures. Concomitant exposure of VSMC to PDGF and TGF-β1 resulted in focal adhesions that distributed evenly on the lower cell surface. This study suggests that TGF-β1 can partially reverse the stimulatory effect of PDGF on VSMC migration in vitro by modifying the actin cytoskeleton and the distribution of the α _vβ₃/β₅ integrins.     

Receptor-mediated nonproteolytic activation of prorenin and induction of TGF-β1 and PAI-1 expression in renal mesangial cells

While elevated plasma prorenin levels are commonly found in diabetic patients and correlate with diabetic nephropathy, the pathological role of prorenin, if any, remains unclear. Prorenin binding to the (pro)renin receptor [(p)RR] unmasks prorenin catalytic activity. We asked whether elevated prorenin could be activated at the site of renal mesangial cells (MCs) through receptor binding without being proteolytically converted to renin. Recombinant inactive rat prorenin and a mutant prorenin that is noncleavable, i.e., cannot be activated proteolytically, are produced in 293 cells. After MCs were incubated with 10(-7) M native or mutant prorenin for 6 h, cultured supernatant acquired the ability to generate angiotensin I (ANG I) from angiotensinogen, indicating both prorenins were activated. Small interfering RNA (siRNA) against the (p)RR blocked their activation. Furthermore, either native or mutant rat prorenin at 10(-7) M alone similarly and significantly induced transforming growth factor-β(1), plasminogen activator inhibitor-1 (PAI-1), and fibronectin mRNA expression, and these effects were blocked by (p)RR siRNA, but not by the ANG II receptor antagonist, saralasin. When angiotensinogen was also added to cultured MCs with inactive native or mutant prorenin, PAI-1 and fibronectin were further increased significantly compared with prorenin or mutant prorenin alone. This effect was blocked partially by treatment with (p)RR siRNA or saralasin. We conclude that prorenin binds the (p)RR on renal MCs and is activated nonproteolytically. This activation leads to increased expression of PAI-1 and transforming growth factor-β(1) via ANG II-independent and ANG II-dependent mechanisms. These data provide a mechanism by which elevated prorenin levels in diabetes may play a role in the development of diabetic nephropathy.     

Ets-1 is essential for connective tissue growth factor (CTGF/CCN2) induction by TGF-β1 in osteoblasts

Background

Ets-1 controls osteoblast differentiation and bone development; however, its downstream mechanism of action in osteoblasts remains largely undetermined. CCN2 acts as an anabolic growth factor to regulate osteoblast differentiation and function. CCN2 is induced by TGF-β1 and acts as a mediator of TGF-β1 induced matrix production in osteoblasts; however, the molecular mechanisms that control CCN2 induction are poorly understood. In this study, we investigated the role of Ets-1 for CCN2 induction by TGF-β1 in primary osteoblasts.

Results

We demonstrated that Ets-1 is expressed and induced by TGF-β1 treatment in osteoblasts, and that Ets-1 over-expression induces CCN2 protein expression and promoter activity at a level similar to TGF-β1 treatment alone. Additionally, we found that simultaneous Ets-1 over-expression and TGF-β1 treatment synergize to enhance CCN2 induction, and that CCN2 induction by TGF-β1 treatment was impaired using Ets-1 siRNA, demonstrating the requirement of Ets-1 for CCN2 induction by TGF-β1. Site-directed mutagenesis of eight putative Ets-1 motifs (EBE) in the CCN2 promoter demonstrated that specific EBE sites are required for CCN2 induction, and that mutation of EBE sites in closer proximity to TRE or SBE (two sites previously shown to regulate CCN2 induction by TGF-β1) had a greater effect on CCN2 induction, suggesting potential synergetic interaction among these sites for CCN2 induction. In addition, mutation of EBE sites prevented protein complex binding, and this protein complex formation was also inhibited by addition of Ets-1 antibody or Smad 3 antibody, demonstrating that protein binding to EBE motifs as a result of TGF-β1 treatment require synergy between Ets-1 and Smad 3.

Conclusions

This study demonstrates that Ets-1 is an essential downstream signaling component for CCN2 induction by TGF-β1 in osteoblasts, and that specific EBE sites in the CCN2 promoter are required for CCN2 promoter transactivation in osteoblasts.     

Periodontal ligament-associated protein-1 knockout mice regulate the differentiation of osteoclasts and osteoblasts through TGF-β1/Smad signaling pathway

It has been known that periodontal ligament-associated protein-1 (PLAP-1/Asporin) not only inhibits cartilage formation in osteoarthritis, but it also influences the healing of skull defect. However, the effect and mechanism of PLAP-1/Asporin on the mutual regulation of osteoclasts and osteoblasts in periodontitis are not clear. In this study, we utilized a PLAP-1/Asporin gene knockout (KO) mouse model to research this unknown issue. We cultured mouse bone marrow mesenchymal stem cells with Porphyromonas gingivalis lipopolysaccharide (P.g. LPS) for osteogenic induction in vitro. The molecular mechanism of PLAP-1/Asporin in the regulation of osteoblasts was detected by immunoprecipitation, immunofluorescence, and inhibitors of signaling pathways. The results showed that the KO of PLAP-1/Asporin promoted osteogenic differentiation through transforming growth factor beta 1 (TGF-β1)/Smad3 in inflammatory environments. We further found the KO of PLAP-1/Asporin inhibited osteoclast differentiation and promoted osteogenic differentiation through the TGF-β1/Smad signaling pathway in an inflammatory coculture system. The experimental periodontitis model was established by silk ligation and the alveolar bone formation in PLAP-1/Asporin KO mice was promoted through TGF-β1/Smad3 signaling pathway. The subcutaneous osteogenesis model in nude mice also confirmed that the KO of PLAP-1/Asporin promoted bone formation by the histochemical staining. In conclusion, PLAP-1/Asporin regulated the differentiation of osteoclasts and osteoblasts through TGF-β1/Smad signaling pathway. The results of this study lay a theoretical foundation for the further study of the pathological mechanism underlying alveolar bone resorption, and the prevention and treatment of periodontitis.     

Latency Associated Peptide Has In Vitro and In Vivo Immune Effects Independent of TGF-β1

Latency Associated Peptide (LAP) binds TGF-β1, forming a latent complex. Currently, LAP is presumed to function only as a sequestering agent for active TGF-β1. Previous work shows that LAP can induce epithelial cell migration, but effects on leukocytes have not been reported. Because of the multiplicity of immunologic processes in which TGF-β1 plays a role, we hypothesized that LAP could function independently to modulate immune responses. In separate experiments we found that LAP promoted chemotaxis of human monocytes and blocked inflammation in vivo in a murine model of the delayed-type hypersensitivity response (DTHR). These effects did not involve TGF-β1 activity. Further studies revealed that disruption of specific LAP-thrombospondin-1 (TSP-1) interactions prevented LAP-induced responses. The effect of LAP on DTH inhibition depended on IL-10. These data support a novel role for LAP in regulating monocyte trafficking and immune modulation.     

Proteomic identification of ADAM12 as a regulator for TGF-β1-induced differentiation of human mesenchymal stem cells to smooth muscle cells

Background

Transforming growth factor-β1 (TGF-β1) induces the differentiation of human adipose tissue-derived mesenchymal stem cells (hASCs) into smooth muscle cells. Lipid rafts are cholesterol-rich microdomains in cell membranes that reportedly play a key role in receptor-mediated signal transduction and cellular responses. In order to clarify whether lipid rafts are involved in TGF-β1-induced differentiation of hASCs into smooth muscle cells, we analyzed the lipid raft proteome of hASCs.

Methods and Results

Pretreatment of hASCs with the lipid raft disruptor methyl-β-cyclodextrin abrogated TGF-β1-induced expression of α-smooth muscle actin, a smooth muscle cell marker, suggesting a pivotal role of lipid rafts in TGF-β1-induced differentiation of hASCs to smooth muscle cells. Sucrose density gradient centrifugation along with a shotgun proteomic strategy using liquid chromatography-tandem mass spectrometry identified 1002 individual proteins as the lipid raft proteome, and 242 of these were induced by TGF-β1 treatment. ADAM12, a disintegrin and metalloproteases family member, was identified as the most highly up-regulated protein in response to TGF-β1 treatment. TGF-β1 treatment of hASCs stimulated the production of both ADAM12 protein and mRNA. Silencing of endogenous ADAM12 expression using lentiviral small hairpin RNA or small interfering RNA abrogated the TGF-β1-induced differentiation of hASCs into smooth muscle cells.

Conclusions

These results suggest a pivotal role for lipid raft-associated ADAM12 in the TGF-β1-induced differentiation of hASCs into smooth muscle cells.     

MicroRNA-98 inhibits TGF-β1-induced differentiation and collagen production of cardiac fibroblasts by targeting TGFBR1

To investigate the effects of miR-98 on TGF-β1-induced cardiac fibrosis in human cardiac fibroblasts (HCFs), and to establish the mechanism underlying these effects, HCFs were transfected with miR-98 inhibitor or mimic, and then treated with or without TGF-β1. The level of miR-98 was determined by qRT-PCR in TGF-β1-induced HCFs. Cell differentiation and collagen accumulation of HCFs were detected by qRT-PCR and Western blot assays, respectively. The mRNA and protein expressions of TGFBR1 were determined by qRT-PCR and Western blotting. In this study, the outcomes showed that TGF-β1 could dramatically decrease the level of miR-98 in a time- and concentration-dependent manner. Upregulation of miR-98 dramatically improved TGF-β1-induced increases in cell differentiation and collagen accumulation of HCFs. Moreover, bioinformatics analysis predicted that the TGFBR1 was a potential target gene of miR-98. Luciferase reporter assay demonstrated that miR-98 could directly target TGFBR1. Inhibition of TGFBR1 had the similar effect as miR-98 overexpression. Downregulation of TGFBR1 in HCFs transfected with miR-98 inhibitor partially reversed the protective effect of miR-98 overexpression on TGF-β1-induced cardiac fibrosis in HCFs. Upregulation of miR-98 ameliorates TGF-β1-induced differentiation and collagen accumulation of HCFs by downregulation of TGFBR1. These results provide further evidence for protective effect of miR-98 overexpression on TGF-β1-induced cardiac fibrosis.