MicroSPECT Imaging-Guided Treatment of Idiopathic Pulmonary Fibrosis in Mice with a Vimentin-Targeting 99mTc-Labeled N-Acetylglucosamine-Polyethyleneimine.

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic disease with poor prognosis. Evidence has shown that vimentin is a key regulator of lung fibrogenesis. 99mTc-labeled N-acetylglucosamine-polyethyleneimine (NAG-PEI), a vimentin-targeting radiotracer, was used for the early diagnosis of IPF, and NAG-PEI was also used as a therapeutic small interfering RNA (siRNA) delivery vector for the treatment of IPF in this study. Single-photon emission-computed tomography (SPECT) imaging of bleomycin (BM)- and silica-induced IPF mice with 99mTc-labeled NAG-PEI was performed to visualize pulmonary fibrosis and monitor the treatment efficiency of siRNA-loaded NAG-PEI, lipopolysaccharide (LPS, a tolerogenic adjuvant), or zymosan (ZYM, an immunostimulant). The lung uptakes of 99mTc-NAG-PEI in the BM- and silica-induced IPF mice were clearly and directly correlated with IPF progression. The lung uptake of 99mTc-NAG-PEI in the NAG-PEI/TGF-ß1-siRNA treatment group or LPS treatment group was evidently lower than that in the control group, while the lung uptake of 99mTc-NAG-PEI was significantly higher in the ZYM treatment group compared to that in the control group. These results demonstrate that NAG-PEI is a potent MicroSPECT imaging-guided theranostic platform for IPF diagnosis and therapy.

Wnt5a Induces Collagen Production by Human Periodontal Ligament Cells Through TGFß1-Mediated Upregulation of Periostin Expression.

Wnt5a, a member of the noncanonical Wnt proteins, is known to play important roles in the development of various organs and in postnatal cell functions. However, little is known about the effects of Wnt5a on human periodontal ligament (PDL) cells. In this study, we examined the localization and potential function of Wnt5a in PDL tissue. Immunohistochemical analysis revealed that Wnt5a was expressed predominantly in rat PDL tissue. Semi-quantitative reverse-transcription polymerase chain reaction and Western blotting analysis demonstrated that human PDL cells (HPDLCs) expressed Wnt5a and its receptors (Ror2, Fzd2, Fzd4, and Fzd5). Removal of occlusal pressure by extraction of opposing teeth decreased Wnt5a expression in rat PDL tissue, and the expression of Wnt5a and its receptors in HPDLCs was upregulated by exposure to mechanical stress. Stimulation with Wnt5a significantly enhanced the proliferation and migration of HPDLCs. Furthermore, Wnt5a suppressed osteoblastic differentiation of HPDLCs cultivated in osteogenic induction medium, while it significantly enhanced the expression of PDL-related genes, such as periostin, type-I collagen, and fibrillin-1 genes, and the production of collagen in HPDLCs cultivated in normal medium. Both knockdown of periostin gene expression by siRNA and inhibition of TGFß1 function by neutralizing antibody suppressed the Wnt5a-induced PDL-related gene expression and collagen production in HPDLCs. Interestingly, in HPDLCs cultured with Wnt5a, TGFß1 neutralizing antibody significantly suppressed periostin expression, while periostin siRNA had no effect on TGFß1 expression. These results suggest that Wnt5a expressed in PDL tissue plays specific roles in inducing collagen production by PDL cells through TGFß1-mediated upregulation of periostin expression.

Effective modulation of transforming growth factor-ß1 expression through engineered microRNA-based plasmid-loaded nanospheres.

BACKGROUND AIMS: Sustained gene delivery could be particularly necessary in modulating gene expression in healing intrasynovial tendons and may be a promising approach for preventing adhesions. METHODS: Polylactic-co-glycolic acid nanospheres were prepared and were then incorporated with plasmids expressing enhanced green fluorescence protein and miRNA for inhibiting the transforming growth factor-ß1 gene expression. RESULTS: We demonstrated that cultured tenocytes could be effectively transfected by means of nanosphere/plasmid. The expression of transforming growth factor-ß1 is significantly downregulated in healing chicken flexor tendon treated with nanosphere/plasmid. Histology analysis did not demonstrate any significant inflammation or necrosis in tendons injected with nanosphere/plasmid after surgery. CONCLUSIONS: Nanosphere/plasmid may be a promising non-viral vector for gene therapy of the injured flexor tendon.

TGF-ß1 and FGF2 stimulate the epithelial-mesenchymal transition of HERS cells through a MEK-dependent mechanism.

Hertwig's epithelial root sheath (HERS) cells participate in cementum formation through epithelial-mesenchymal transition (EMT). Previous studies have shown that transforming growth factor beta 1 (TGF-ß1) and fibroblast growth factor 2 (FGF2) are involved in inducing EMT. However, their involvement in HERS cell transition remains elusive. In this study, we confirmed that HERS cells underwent EMT during the formation of acellular cementum. We found that both TGF-ß1 and FGF2 stimulated the EMT of HERS cells. The TGF-ß1 regulated the differentiation of HERS cells into periodontal ligament fibroblast-like cells, and FGF2 directed the differentiation of HERS cells into cementoblast-like cells. Treatment with TGF-ß1 or FGF2 inhibitor could effectively suppress HERS cells differential transition. Combined stimulation with both TGF-ß1 and FGF-2 did not synergistically accelerate the EMT of HERS. Moreover, TGF-ß1/FGF2-mediated EMT of HERS cells was reversed by the MEK1/2 inhibitor U0126. These results suggest that TGF-ß1 and FGF2 induce the EMT of HERS through a MAPK/ERK-dependent signaling pathway. They also exert their different tendency of cellular differentiation during tooth root formation. This study further expands our knowledge of tooth root morphogenesis and provides more evidence for the use of alternative cell sources in clinical treatment of periodontal diseases.

Molecular events associated with ciclosporin A-induced gingival overgrowth are attenuated by Smad7 overexpression in fibroblasts.

BACKGROUND AND OBJECTIVE: Ciclosporin A (CsA)-induced gingival overgrowth is attributed to an exaggerated accumulation of extracellular matrix, which is mainly due to an increased expression of transforming growth factor-ß1 (TGF-ß1). Herein, the in vitro investigation of effects of overexpression of Smad7, a TGF-ß1 signaling inhibitor, in the events associated with CsA-induced extracellular matrix accumulation was performed. MATERIAL AND METHODS: The effects of Smad7 were assessed by stable overexpression of Smad7 in fibroblasts from normal gingiva. Smad7-overexpressing cells and control cells were incubated with CsA, and synthesis of type I collagen, production and activity of MMP-2 and cellular proliferation were evaluated by ELISA, zymography, growth curve, bromodeoxyuridine incorporation assay and cell cycle analysis. The effects of CsA on cell viability and apoptosis of fibroblasts from normal gingiva were also evaluated. Western blot and immunofluorescence for phospho-Smad2 were performed to measure the activation of TGF-ß1 signaling. RESULTS: Although the treatment with CsA stimulated TGF-ß1 production in both control and Smad7-overexpressing fibroblasts, its signaling was markedly inhibited in Smad7-overexpressing cells, as revealed by low levels of phospho-Smad2. In Smad7-overexpressing cells, the effects of CsA on proliferation, synthesis of type I collagen and the production and activity of MMP-2 were significantly blocked. Smad7 overexpression blocked CsA-induced fibroblast proliferation via p27 regulation. Neither CsA nor Smad7 overexpression induced cell death. CONCLUSION: The data presented here confirm that TGF-ß1 expression is related to the molecular events associated with CsA-induced gingival overgrowth and suggest that Smad7 overexpression is effective in blocking these events, including proliferation, type I collagen synthesis and MMP-2 activity.

Development of a novel gene silencer pyrrole-imidazole polyamide targeting human connective tissue growth factor.

Pyrrole-imidazole (PI) polyamide can bind to specific sequences in the minor groove of double-helical DNA and inhibit transcription of the genes. We designed and synthesized a PI polyamide to target the human connective tissue growth factor (hCTGF) promoter region adjacent to the Smads binding site. Among coupling activators that yield PI polyamides, 1-[bis(dimethylamino)methylene]-5-chloro-1H-benzotriazolium 3-oxide hexafluorophosphate (HCTU) was most effective in total yields of PI polyamides. A gel shift assay showed that a PI polyamide designed specifically for hCTGF (PI polyamide to hCTGF) bound the appropriate double-stranded oligonucleotide. A fluorescein isothiocyanate (FITC)-conjugated PI polyamide to CTGF permeated cell membranes and accumulated in the nuclei of cultured human mesangial cells (HMCs) and remained there for 48 h. The PI polyamide to hCTGF significantly decreased phorbol 12-myristate acetate (PMA)- or transforming growth factor-ß1 (TGF-ß1)-stimulated luciferase activity of the hCTGF promoter in cultured HMCs. The PI polyamide to hCTGF significantly decreased PMA- or TGF-ß1-stimulated expression of hCTGF mRNA in a dose-dependent manner. The PI polyamide to hCTGF significantly decreased PMA- or TGF-ß1-stimulated levels of hCTGF protein in HMCs. These results indicate that the developed synthetic PI polyamide to hCTGF could be a novel gene silencer for fibrotic diseases.

Platelet-derived biomaterials-mediated improvement of bone injury through migratory ability of embryonic fibroblasts: in vitro and in vivo evidence.

Bony injuries lead to compromised skeletal functional ability which further increase in aging population due to decreased bone mineral density. Therefore, we aimed to investigate the therapeutic potential of platelet-derived biomaterials (PDB) against bone injury. Specifically, we assessed the impact of PDB on osteo-inductive characteristics and migration of mouse embryonic fibroblasts (MEFs). Osteogenic lineage, matrix mineralization and cell migration were determined by gene markers (RUNX2, OPN and OCN), alizarin Red S staining, and migration markers (FAK, pFAK and Src) and EMT markers, respectively. The therapeutic impact of TGF-ß1, a key component of PDB, was confirmed by employing inhibitor of TGF-ß receptor I (Ti). Molecular imaging-based in vivo cellular migration in mice was determined by establishing bone injury at right femurs. Results showed that PDB markedly increased expression of osteogenic markers, matrix mineralization, migration and EMT markers, revealing higher osteogenic and migratory potential of PDB-treated MEFs. In vivo cell migration was manifested by expression of migratory factors, SDF-1 and CXCR4. Compared to control, PDB-treated mice exhibited higher bone density and volume. Ti treatment inhibited both migration and osteogenic potential of MEFs, affirming impact of TGF-ß1. Collectively, our study clearly indicated PDB-rescued bone injury through enhancing migratory potential of MEFs and osteogenesis.

Effect of nicotine on gene expression of angiogenic and osteogenic factors in a rabbit model of bone regeneration.

PURPOSE: This study aims to evaluate the influence of nicotine on the gene expression of osteogenic and angiogenic factors in bone regeneration by use of a nicotine-compromised rabbit model of mandibular lengthening. MATERIALS AND METHODS: Thirty adult New Zealand white rabbits were randomly assigned to the nicotine group or the control group. The total nicotine or placebo exposure time for all animals was 7 weeks. Unilateral mandibular distraction osteogenesis was performed. Five animals in each group were sacrificed at day 5, day 11, and day 18, respectively, after commencement of active distraction. The distraction regenerate samples were harvested, and the messenger ribonucleic acid expression of bone transforming growth factor beta(1), platelet-derived growth factor A, and basic fibroblast growth factor was assayed by real-time polymerase chain reaction analysis. RESULTS: The messenger ribonucleic acid expression of transforming growth factor beta(1), platelet-derived growth factor A, and basic fibroblast growth factor was significantly inhibited by nicotine exposure at a variety of time points. CONCLUSIONS: The presence of nicotine inhibited the gene expression of angiogenic and osteogenic factors resulting in compromised bone regeneration.

Insulin impedes osteogenesis of BMSCs by inhibiting autophagy and promoting premature senescence via the TGF-ß1 pathway.

The dysfunction of bone marrow stromal cells (BMSCs) may be a core factor in Type 2 diabetes mellitus (T2DM) associated osteoporosis. However, the underlying mechanism is not well understood. Here, we delineated the critical role of insulin impeding osteogenesis of BMSCs in T2DM. Compared with BMSCs from healthy people (H-BMSCs), BMSCs from T2DM patient (DM-BMSCs) showed decreased osteogenic differentiation and autophagy level, and increased senescent phenotype. H-BMSCs incubated in hyperglycemic and hyperinsulinemic conditions similarly showed these phenotypes of DM-BMSCs. Notably, enhanced TGF-ß1 expression was detected not only in DM-BMSCs and high-glucose and insulin-treated H-BMSCs, but also in bone callus of streptozocin-induced diabetic rats. Moreover, inhibiting TGF-ß1 signaling not only enhanced osteogenic differentiation and autophagy level of DM-BMSCs, but also delayed senescence of DM-BMSCs, as well as promoted mandible defect healing of diabetic rats. Finally, we further verified that it was TGF-ß receptor II (TßRII), not TßRI, markedly increased in both DM-BMSCs and insulin-treated H-BMSCs. Our data revealed that insulin impeded osteogenesis of BMSCs by inhibiting autophagy and promoting premature senescence, which it should be responsible for T2DM-induced bone loss, at least in part. These findings suggest that inhibiting TGF-ß1 pathway may be a potential therapeutic target for T2DM associated bone disorders.

Gingival fibroblast inhibits MMP-7: evaluation in an ex vivo aorta model.

Matrix metalloproteinases (MMP) play a deleterious role in numerous vascular diseases. In contrast, gingival matrix remodelling is adequately regulated by the gingival fibroblast (GF). Here, we aimed to evaluate the GF activity on MMP-7 expression and secretion in coculture with aorta rings. We evaluated MMP-7 transcription and secretion in rabbit aorta rings cultured or not with gingival fibroblasts in collagen gels. GF induced an increase of TIMP-1 transcription and secretion, followed, similarly to other MMPs, by the formation of TIMP-1/MMP-7 complexes. There was also a decrease of MMP-7 mRNA by RT-PCR in aorta rings cocultured with gingival fibroblasts. Interestingly, in contrast with other MMPs (which were not influenced at a transcription level), GF stimulated the release of TGF-beta1, which in turn inhibited the transcription and synthesis of MMP-7, as shown by neutralizing MMP-7 inhibition due to gingival fibroblast by overexpressing decorin (a TGF beta 1 inhibitor) or by silencing TGF beta 1 using siRNA. We showed that healing properties of the GF could be transposed to another organ, i.e., ex vivo aneurism model, implicating a down-regulation of MMP-7.

The effect of locally administered anti-growth factor antibodies on neointimal hyperplasia formation in expanded polytetrafluoroethylene grafts.

The selective blockage of platelet-derived growth factor BB (PDGF-BB), basic fibroblast growth factor (bFGF), and transforming growth factor beta1 (TGF-beta1) by specific antibodies coated into expanded polytetrafluoroethylene (ePTFE) grafts may diminish neointimal hyperplasia. Sixty pigs were divided into two groups (n = 30 each) and then further divided into five subgroups. Group 1 had a bilateral iliac artery ePTFE interposition graft precoated with Matrigel. Three subgroups (A, B, and C) received a specific monoclonal antibody against PDGF-BB, bFGF, or TGF-beta1. One (D) received all antibodies, and one served as control (nonimmune immunoglobulin G [IgG] isotypes) (E). Group 2 had a bilateral iliac artery endothelial cell (EC)-seeded ePTFE interposition graft precoated with Matrigel. Three subgroups (A, B, and C) received a specific antibody against PDGF-BB, bFGF, or TGF-beta1. One (D) received all antibodies, and one served as control (nonimmune IgG isotypes) (E). Light microscopy and immunohistochemical stain showed that neointimal hyperplasia formation was significantly reduced in subgroups D compared to the others (p < 0.05). In subgroups D, the different precoating influenced neointimal hyperplasia formation. It was more pronounced in the prosthesis precoated with EC and Matrigel (p < 0.05). In organ culture, the amount of PDGF-BB, bFGF, and TGF-beta1 release was reduced in subgroup D animals compared to the others (p < 0.05). In subgroups D, the release of PDGF-BB, bFGF, and TGF-beta1 depended on ePTFE seeding. A higher amount of these growth factors was released in the prostheses precoated with EC and Matrigel (p < 0.05), and the bromodeoxyuridine labeling index confirmed higher incorporation in this subgroup (p < 0.001). The combined use of locally administered anti-PDGF-BB, bFGF, and TGF-beta1 monoclonal antibodies reduces neointimal hyperplasia formation.

Mechanical force promotes the proliferation and extracellular matrix synthesis of human gingival fibroblasts cultured on 3D PLGA scaffolds via TGF­ß expression.

Human gingival fibroblasts (HGFs) are responsible for connective tissue repair and scarring, and are exposed to mechanical forces under physiological and pathological conditions. The exact mechanisms underlying gingival tissue reconstruction under mechanical forces remain unclear. The present study aimfed to investigate the effects of mechanical forces on the proliferation and extracellular matrix synthesis in HGFs by establishing a 3­dimensional (3D) HGF culture model using poly(lactide­co­glycolide) (PLGA) scaffolds. HGFs were cultured in 3D PLGA scaffolds and a mechanical force of 0, 5, 15, 25 or 35 g/cm2 was applied to HGFs for 24 h. A mechanical force of 25 g/cm2 induced the highest proliferation rate, and thus was selected for subsequent experiments. Cell viability was determined using the MTT assay at 0, 24, 48 and 72 h. The expression levels of type I collagen (COL­1) and matrix metallopeptidase (MMP)­1 were examined by reverse transcription­quantitative polymerase chain reaction and ELISA, and transforming growth factor (TGF)­ß expression was evaluated by ELISA. The application of mechanical force on HGFs cultured on the 3D PLGA scaffolds resulted in a significant increase in cell proliferation and COL­1 expression, as well as a decrease in MMP­1 expression. A TGF­ß1 inhibitor was also applied, which attenuated the effects of mechanical force on HGF proliferation, and COL­1 and MMP­1 expression, thus suggesting that TGF­ß signaling pathways may mediate the mechanical force­induced alterations observed in HGFs. In conclusion, these findings helped to clarify the mechanisms underlying mechanical force­induced HGF proliferation and ECM synthesis, which may promote the development of targeted therapeutics to treat various diseases, including gingival atrophy caused by orthodontic treatment.

Interaction between macrophages, TGF-beta1, and the COX-2 pathway during the inflammatory phase of skeletal muscle healing after injury.

Inflammation, an important phase of skeletal muscle healing, largely involves macrophages, TGF-beta1, and the COX-2 pathway. To improve our understanding of how these molecules interact during all phases of muscle healing, we examined their roles in muscle cells in vitro and in vivo. Initially, we found that depletion of macrophages in muscle tissue led to reduced muscle regeneration. Macrophages may influence healing by inducing the production of TGF-beta1 and PGE2 in different muscle cell types. We then found that the addition of TGF-beta1 induced PGE2 production in muscle cells, an effect probably mediated by COX-2 enzyme. It was also found that TGF-beta1 enhanced macrophage infiltration in wild-type mice after muscle injury. However, this effect was not observed in COX-2(-/-) mice, suggesting that the effect of TGF-beta1 on macrophage infiltration is mediated by the COX-2 pathway. Furthermore, we found that PGE2 can inhibit the expression of TGF-beta1. PGE2 and TGF-beta1 may be involved in a negative feedback loop balancing the level of fibrosis formation during skeletal muscle healing. In conclusion, our results suggest a complex regulatory mechanism of skeletal muscle healing. Macrophages, TGF-beta1, and the COX-2 pathway products may regulate one another's levels and have profound influence on the whole muscle healing process.

Periostin is essential for the integrity and function of the periodontal ligament during occlusal loading in mice.

BACKGROUND: The ability of the periodontal ligament (PDL) to absorb and distribute forces is necessary for periodontal homeostasis. This adaptive response may be determined, in part, by a key molecule, periostin, which maintains the integrity of the PDL during occlusal function and inflammation. Periostin is primarily expressed in the PDL and is highly homologous to betaig-H3 (transforming growth factor-beta [TGF-beta] inducible gene). Cementum, alveolar bone, and the PDL of periostin-null mice dramatically deteriorate following tooth eruption. The purpose of this study was to determine the role of periostin in maintaining the functional integrity of the periodontium. METHODS: The periodontia from periostin-null mice were characterized followed by unloading the incisors. The effect of substrate stretching on periostin expression was evaluated using a murine PDL cell line. Real-time reverse transcription-polymerase chain reaction was used to quantify mRNA levels of periostin and TGF-beta. TGF-beta1 neutralizing antibodies were used to determine whether the effects of substrate stretching on periostin expression are mediated through TGF-beta. RESULTS: Severe periodontal defects were observed in the periostin-null mice after tooth eruption. The removal of masticatory forces in periostin-null mice rescue the periodontal defects. Periostin expression was increased in strained PDL cells by 9.2-fold at 48 hours and was preceded by a transient increase in TGF-beta mRNA in vitro. Elevation of periostin in response to mechanical stress was blocked by the addition of 2.5 ng/ml neutralizing antibody to TGF-beta1, suggesting that mechanical strain activates TGF-beta to have potential autocrine effects and to increase periostin expression. CONCLUSION: Mechanical loading maintains sufficient periostin expression to ensure the integrity of the periodontium in response to occlusal load.

Antioxidants and NOX1/NOX4 inhibition blocks TGFß1-induced CCN2 and α-SMA expression in dermal and gingival fibroblasts.

TGFbeta induces fibrogenic responses in fibroblasts. Reactive oxygen species (ROS)/nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) may contribute to fibrogenic responses. Here, we examine if the antioxidant N-acetylcysteine (NAC), the NOX inhibitor diphenyleneiodonium (DPI) and the selective NOX1/NOX4 inhibitor GKT-137831 impairs the ability of TGFbeta to induce profibrotic gene expression in human gingival (HGF) and dermal (HDF) fibroblasts. We also assess if GKT-137831 can block the persistent fibrotic phenotype of lesional scleroderma (SSc) fibroblasts. We use real-time polymerase chain reaction and Western blot analysis to evaluate whether NAC and DPI impair the ability of TGFbeta1 to induce expression of fibrogenic genes in fibroblasts. The effects of GKT-137831 on TGFbeta-induced protein expression and the persistent fibrotic phenotype of lesional scleroderma (SSc) fibroblasts were tested using Western blot and collagen gel contraction analyses. In HDF and HGF, TGFbeta1 induces CCN2, CCN1, endothelin-1 and alpha-smooth muscle actin (SMA) in a fashion sensitive to NAC. Induction of COL1A1 mRNA was unaffected. Similar results were seen with DPI. NAC and DPI impaired the ability of TGFbeta1 to induce protein expression of CCN2 and alpha-SMA in HDF and HGF. GKT-137831 impaired TGFbeta-induced CCN2 and alpha-SMA protein expression in HGF and HDF. In lesional SSc dermal fibroblasts, GKT-137831 reduced alpha-SMA and CCN2 protein overexpression and collagen gel contraction. These results are consistent with the hypothesis that antioxidants or NOX1/4 inhibition may be useful in blocking profibrotic effects of TGFbeta on dermal and gingival fibroblasts and warrant consideration for further development as potential antifibrotic agents.

Surface immobilization and bioactivity of TGF-ß1 inhibitor peptides for bone implant applications.

TGF-ß1 is the most related cytokine with the production of fibrotic tissue. It plays an important role on the production of collagen by fibroblasts and other types of cells. The inhibition of this cytokine reduces fibrosis in various types of tissue. Biofunctionalization of dental and orthopedic implants with biomolecules enables modification of the physical, chemical and biochemical properties of their surfaces to improve its biological and clinical performance. Our objective was to develop a reliable method to immobilize oligopeptides on Ti surfaces to obtain a surface with TGF-ß1 inhibitory activity that will potentially minimize fibrotic encapsulation of implants during the process of osseointegration. We covalently immobilized TGF-ß1 inhibitor P17-peptides on Ti surfaces and assessed by characterizing each step of the process that we successfully biofunctionalized the implant surfaces. High amounts of peptides were anchored and homogeneously distributed on the surfaces with mechanical and thermochemical stability after in vitro simulated challenges. Notably, the immobilized peptides retained their TGF-ß1 inhibitory activity in vitro. Thus, these biofunctional coatings are potential candidates for inducing a fast and reliable osseointegration in vivo.

NADPH Oxidase 4 Mediates TGFß1-induced CCN2 in Gingival Fibroblasts.

Transforming growth factor ß (TGFß) plays a central role in the pathogenesis of gingival overgrowth (GO). Connective tissue growth factor (CTGF; or CCN2) is induced by TGFß in human gingival fibroblasts (HGFs) and is overexpressed in GO tissues. CCN2 creates an environment favorable for fibrogenesis and is required for the maximal profibrotic effects of TGFß. We previously showed that Src, JNK, and Smad3 mediate TGFß1-induced CCN2 protein expression in HGFs. Moreover, Src is an upstream signaling transducer of JNK and Smad3. Recent studies suggested that NADPH oxidase (NOX)-dependent redox mechanisms are involved in mediating the profibrotic effects of TGFß. In this study, we demonstrated that TGFß1 upregulated NOX4 protein expression and increased reactive oxygen species (ROS) production in HGFs. Genetic or pharmacologic targeting of NOX4 abrogated TGFß1-induced ROS production; Src, JNK, and Smad3 activation; and CCN2 and type I collagen protein expression in HGFs. Our results indicated that NOX4-derived ROS play pivotal roles in activating Src kinase activity leading to the activation of canonical (Smad3) and noncanonical (JNK) cascades that cooperate to attain maximum CCN2 expression. Furthermore, we demonstrated that curcumin significantly inhibited the TGFß1-induced NOX4 protein expression in HGFs. Curcumin potentially qualifies as an agent to control GO by suppressing TGFß1-induced NOX4 expression in HGFs.

Modulation of the EMT/MET process by pyrrole-imidazole polyamide targeting human transforming growth factor-ß1.

Transforming growth factor-ß1 (TGF-ß1) is a potent induction factor for epithelial-mesenchymal transition (EMT). Mesenchymal-epithelial transition (MET), as the inverse process of EMT, has recently been reported to promote the induction of induced pluripotent stem cells (iPSCs). We have developed pyrrole-imidazole (PI) polyamide, a novel gene regulator that targets human TGF-ß1, and investigated its effects on the EMT/MET process. PI polyamide targeted to TGF-ß1 significantly inhibited the mRNA expression of TGF-ß1 and SNAI1 as an EMT marker and increased mRNA and protein expression of E-cadherin in human epithelial cells. To enhance the induction of iPSCs by the MET process, PI polyamide targeted to TGF-ß1 was applied to human fibroblasts transfected with exogenous reprogramming factors by Sendai virus vector and grown in human iPSCs. The PI polyamide significantly increased the number of alkaline phosphatase-positive colonies. The expression of undifferentiated markers was also observed in these colonies. These results suggest that PI polyamide targeted to human TGF-ß is a novel compound that can control the EMT/MET process of human epithelial cells and enhance the induction of human fibroblasts to iPSCs.

Role of transforming growth factor-beta1 in cyclosporine-induced epithelial-to-mesenchymal transition in gingival epithelium.

BACKGROUND: It has been proposed that cyclosporin A (CsA) may induce epithelial-to-mesenchymal transition (EMT) in gingiva. The aims of the present study are to confirm the notion that EMT occurs in human gingival epithelial (hGE) cells after CsA treatment and to investigate the role of transforming growth factor beta1 (TGF-ß1) on this CsA-induced EMT. METHODS: The effects of CsA, with and without TGF-ß1 inhibitor, on the morphologic changes of primary culture of hGE cells were examined in vitro. The changes of protein and messenger RNA (mRNA) expressions of two EMT markers (E-cadherin and alpha-smooth muscle actin) in the hGE cells after CsA treatment with and without TGF-ß1 inhibitor were evaluated with immunocytochemistry and real-time polymerase chain reaction. RESULTS: The epithelial cells became spindle-like, elongated, and disassociated from neighboring cells and lost their original cobblestone monolayer pattern when CsA was added. However, the epithelial cells stayed in their original cobblestone morphology with treatment of TGF-ß1 inhibitor on top of the CsA treatment. When CsA was given, the protein and mRNA expressions of E-cadherin and α-SMA were significantly altered, and these alterations were significantly reversed with pretreatment of TGF-ß1 inhibitor. CONCLUSIONS: CsA could induce Type 2 EMT in gingiva by changing the morphology of epithelial cells and altering the EMT markers/effectors. The CsA-induced gingival EMT is dependent or at least partially dependent on TGF-ß1.

Inhibition of transforming growth factor ß1/Smad3 signaling decreases hypoxia-inducible factor-1α protein stability by inducing prolyl hydroxylase 2 expression in human periodontal ligament cells.

BACKGROUND: Hypoxia-inducible factor-1α (HIF-1α), the α subunit of the heterodimeric transcription factor HIF-1, maintains oxygen homeostasis by regulating gene expression. Under normoxic conditions, HIF-1α expression is maintained at low steady-state levels by the critical oxygen sensor prolyl hydroxylase 2 (PHD2). Transforming growth factor ß1 (TGF-ß1) activates Smad3 signaling and contributes to HIF-1α stabilization under normoxic conditions. In chronic periodontitis, HIF-1α is expressed highly in gingival fibroblasts and upregulates inflammatory factor transcription, which promotes periodontal inflammation. Here, the authors investigated the effect of TGF-ß1/Smad3 signaling and its blockade by the specific inhibitor of Smad3 (SIS3) on HIF-1α expression and stability in human periodontal ligament cells. METHODS: The authors investigated the effect of TGF-ß1 on HIF-1α protein stability using cycloheximide. Furthermore, they analyzed HIF-1α expression, PHD2 expression, and Smad3 phosphorylation following TGF-ß1 stimulation in the presence or absence of SIS3. RESULTS: The half-life of HIF-1α was prolonged in TGF-ß1-treated cells. TGF-ß1 treatment induced HIF-1α gene expression and enhanced HIF-1α protein stability while decreasing PHD2 expression and activating Smad3 phosphorylation. Notably, HIF-1α protein expression was not detectable prior to TGF-ß1 stimulation. Furthermore, SIS3 treatment abrogated Smad3 phosphorylation, impaired TGF-ß1-induced HIF-1α gene expression and protein stability, and stimulated TGF-ß1-mediated PHD2 inhibition. CONCLUSION: These results demonstrate that HIF-1α transcription and protein synthesis are controlled by TGF-ß1/Smad3 signaling, whereas HIF-1α protein stability is controlled by PHD2, which is regulated by TGF-ß1/Smad3 signaling.