Reversible Modulation of Myofibroblast Differentiation in Adipose-Derived Mesenchymal Stem Cells

Unregulated activity of myofibroblasts, highly contractile cells that deposit abundant extracellular matrix (ECM), leads to fibrosis. To study the modulation of myofibroblast activity, we used human adipose-derived mesenchymal stem cells (ADSCs), which have much potential in regenerative medicine. We found that ADSCs treated with TGF-β developed a myofibroblastic phenotype with increases in α-smooth muscle actin (α-SMA), a myofibroblast marker, and ECM proteins type I collagen and fibronectin. In contrast, treatment with bFGF had the opposite effect. bFGF-differentiated ADSCs showed marked down-regulation of α-SMA expression, collagen I, and fibronectin, and loss of focal adhesions and stress fibers. Functionally, bFGF-differentiated ADSCs were significantly more migratory, which correlated with up-regulation of tenascin-C, an anti-adhesive ECM protein, and vimentin, a pro-migratory cytoskeletal protein. On the other hand, TGF-β-differentiated ADSCs were significantly more contractile than bFGF-differentiated cells. Interestingly, cells completely reversed their morphologies, marker expression, signaling pathways, and contractility versus migratory profiles when switched from culture with one growth factor to the other, demonstrating that the myofibroblast differentiation process is not terminal. Cell differentiation was associated with activation of Smad2 downstream of TGF-β and of ERK/MAP kinase downstream of bFGF. Reversibility of the TGF-β-induced myofibroblastic phenotype depends, in part, on bFGF-induced ERK/MAP kinase signaling. These findings show that ADSC differentiation into myofibroblasts and re-differentiation into fibroblast-like cells can be manipulated with growth factors, which may have implications in the development of novel therapeutic strategies to reduce the risk of fibrosis.     

Extracellular matrix as a contextual determinant of transforming growth factor-β signaling in epithelial-mesenchymal transition and in cancer

Extracellular matrix (ECM) provides both structural support and contextual information to cells within tissues and organs. The combination of biochemical and biomechanical signals from the ECM modulates responses to extracellular signals toward differentiation, proliferation, or apoptosis; alterations in the ECM are necessary for development and remodeling processes, but aberrations in the composition and organization of ECM are associated with disease pathology and can predispose to development of cancer. The primary cell surface sensors of the ECM are the integrins, which provide the physical connection between the ECM and the cytoskeleton and also convey biochemical information about the composition of the ECM. Transforming growth factor-β (TGF-β) is an extracellular signaling molecule that is a powerful controller of a variety of cellular functions, and that has been found to induce very different outcomes according to cell type and cellular context. It is becoming clear that ECM-mediated signaling through integrins is reciprocally influenced by TGF-β: integrin expression, activation, and responses are affected by cellular exposure to TGF-β, and TGF-β activation and cellular responses are in turn controlled by signaling from the ECM through integrins. Epithelial-mesenchymal transition (EMT), a physiological process that is activated by TGF-β in normal development and in cancer, is also affected by the composition and structure of the ECM. Here, we will outline how signaling from the ECM controls the contextual response to TGF-β, and how this response is selectively modulated during disease, with an emphasis on recent findings, current challenges, and future opportunities.     

A Novel Role of Matrix Metalloproteinase-8 in Macrophage Differentiation and Polarization

Matrix metalloproteinase-8 (MMP8) has been shown to influence various cellular functions. As monocytes and macrophages (Mφ) express MMP8, we investigated if MMP8 played a role in macrophage differentiation and polarization. MMP8 expression was significantly increased during monocyte differentiation into Mφ. Monocyte-derived Mφ from MMP8-deficient mice expressed higher levels of M1-Mφ markers but lower levels of M2-Mφ markers than monocyte-derived Mφ from wild-type mice. Although Mφ from either MMP8-deficient or wild-type mice were inducible by interferon-γ into M1-Mφ, only wild-type Mφ but not MMP8-deficient Mφ could be induced into M2-Mφ by interleukin-4. However, MMP8-deficient Mφ exposed to conditioned culture media of wild-type Mφ developed a M2-Mφ phenotype. Compared with conditioned culture media of wild-type Mφ, conditioned culture media of MMP8-deficient Mφ contained a lower concentration of active transforming growth factor-β (TGF-β), an M2-Mφ inducer. Moreover, evidence also showed that the degradation of the TGF-β sequester, fibromodulin, was modulated by MMP8. The data indicate a previously unknown role of MMP8 in M2-Mφ polarization by cleaving fibromodulin and therefore increasing the bioavailability of the M2-Mφ inducer TGF-β.     

Oxygen Tension Modulates Differentiation and Primary Macrophage Functions in the Human Monocytic THP-1 Cell Line

The human THP-1 cell line is widely used as an in vitro model system for studying macrophage differentiation and function. Conventional culture conditions for these cells consist of ambient oxygen pressure (∼20% v/v) and medium supplemented with the thiol 2-mercaptoethanol (2-ME) and serum. In consideration of the redox activities of O₂ and 2-ME, and the extensive experimental evidence supporting a role for reactive oxygen species (ROS) in the differentiation and function of macrophages, we addressed the question of whether culturing THP-1 cells under a more physiologically relevant oxygen tension (5% O₂) in the absence of 2-ME and serum would alter THP-1 cell physiology. Comparisons of cultures maintained in 18% O₂ versus 5% O₂ indicated that reducing oxygen tension had no effect on the proliferation of undifferentiated THP-1 cells. However, decreasing the oxygen tension to 5% O₂ significantly increased the rate of phorbol ester-induced differentiation of THP-1 cells into macrophage-like cells as well as the metabolic activity of both undifferentiated and PMA-differentiated THP-1 cells. Removal of both 2-ME and serum from the medium decreased the proliferation of undifferentiated THP-1 cells but increased metabolic activity and the rate of differentiation under either oxygen tension. In differentiated THP-1 cells, lowering the oxygen tension to 5% O₂ decreased phagocytic activity, the constitutive release of β-hexosaminidase and LPS-induced NF-κB activation but enhanced LPS-stimulated release of cytokines. Collectively, these data demonstrate that oxygen tension influences THP-1 cell differentiation and primary macrophage functions, and suggest that culturing these cells under tightly regulated oxygen tension in the absence of exogenous reducing agent and serum is likely to provide a physiologically relevant baseline from which to study the role of the local redox environment in regulating THP-1 cell physiology.     

Differential sensitivity of subclasses of human colon carcinoma cell lines to the growth inhibitory effects of transforming growth factor-β1

In this study we have employed a model system comprising three groups of colon carcinoma cell lines to examine the growth-inhibitory effects of two molecular forms of transforming growth factor-β (TGF-β), TGF-β1 and TGF-β2. Aggressive, poorly differentiated colon carcinoma cells of group I did not respond to growth inhibitory effects of TGF-β1 or TGF-β2, while less aggressive, well-differentiated cells of group III displayed marked sensitivity to both TGF-β1 and TGF-β2 in monolayer culture as well as in soft agarose. One moderately well-differentiated cell line from group II which has intermediate growth characteristics failed to respond to TGF-β1 or TGF-β2, but the growth of two other cell lines in this group was inhibited. TGF-β1 and TGF-β2 were equally potent, 50% growth inhibition for responsive cell lines being observed at a concentration of 1 ng/ml (40 pM). Antiproliferative effects of TGF-β1 and TGF-β2 in responsive cell lines of groups II and III were associated with morphological alterations and enhanced, concentration-dependent secretion of carcinoembryonic antigen. Radiolabeled TGF-β1 bound to all three groups of colon carcinoma cells with high affinity (K_d between 42 and 64 pM). These data indicate for the first time a strong correlation between the degree of differentiation of colon carcinoma cell lines and sensitivity to the antiproliferative and differentiation-promoting effects of TGF-β1 and TGF-β2.     

Inhibition of Plasminogen Activator Inhibitor-1 Attenuates Transforming Growth Factor-β-Dependent Epithelial Mesenchymal Transition and Differentiation of Fibroblasts to Myofibroblasts

Transforming growth factor-β (TGF-β) is central during the pathogenesis of pulmonary fibrosis, in which the plasminogen activator inhibitor-1 (PAI-1) also has an established role. TGF-β is also known to be the strongest inducer of PAI-1. To investigate the link between PAI-1 and TGF-β in fibrotic processes, we evaluated the effect of SK-216, a PAI-1-specific inhibitor, in TGF-β-dependent epithelial-mesenchymal transition (EMT) and fibroblast to myofibroblast differentiation. In human alveolar epithelial A549 cells, treatment with TGF-β induced EMT, whereas co-treatment with SK-216 attenuated the occurrence of EMT. The inhibition of TGF-β-induced EMT by SK-216 was also confirmed in the experiment using murine epithelial LA-4 cells. Blocking EMT by SK-216 inhibited TGF-β-induced endogenous production of PAI-1 and TGF-β in A549 cells as well. These effects of SK-216 were not likely mediated by suppressing either Smad or ERK pathways. Using human lung fibroblast MRC-5 cells, we demonstrated that SK-216 inhibited TGF-β-dependent differentiation of fibroblasts to myofibroblasts. We also observed this inhibition by SK-216 in human primary lung fibroblasts. Following these in vitro results, we tested oral administration of SK-216 into mice injected intratracheally with bleomycin. We found that SK-216 reduced the degree of bleomycin-induced pulmonary fibrosis in mice. Although the precise mechanisms underlying the link between TGF-β and PAI-1 regarding fibrotic process were not determined, PAI-1 seems to act as a potent downstream effector on the pro-fibrotic property of TGF-β. In addition, inhibition of PAI-1 activity by a PAI-1 inhibitor exerts an antifibrotic effect even in vivo. These data suggest that targeting PAI-1 as a downstream effector of TGF-β could be a promising therapeutic strategy for pulmonary fibrosis.     

Prestress in the extracellular matrix sensitizes latent TGF-β1 for activation

Integrin-mediated force application induces a conformational change in latent TGF-β1 that leads to the release of the active form of the growth factor from the extracellular matrix (ECM). Mechanical activation of TGF-β1 is currently understood as an acute process that depends on the contractile force of cells. However, we show that ECM remodeling, preceding the activation step, mechanically primes latent TGF-β1 akin to loading a mechanical spring. Cell-based assays and unique strain devices were used to produce a cell-derived ECM of controlled organization and prestrain. Mechanically conditioned ECM served as a substrate to measure the efficacy of TGF-β1 activation after cell contraction or direct force application using magnetic microbeads. The release of active TGF-β1 was always higher from prestrained ECM as compared with unorganized and/or relaxed ECM. The finding that ECM prestrain regulates the bioavailability of TGF-β1 is important to understand the context of diseases that involve excessive ECM remodeling, such as fibrosis or cancer.     

Myofibroblast contraction activates latent TGF-beta1 from the extracellular matrix

The conjunctive presence of mechanical stress and active transforming growth factor β1 (TGF-β1) is essential to convert fibroblasts into contractile myofibroblasts, which cause tissue contractures in fibrotic diseases. Using cultured myofibroblasts and conditions that permit tension modulation on the extracellular matrix (ECM), we establish that myofibroblast contraction functions as a mechanism to directly activate TGF-β1 from self-generated stores in the ECM. Contraction of myofibroblasts and myofibroblast cytoskeletons prepared with Triton X-100 releases active TGF-β1 from the ECM. This process is inhibited either by antagonizing integrins or reducing ECM compliance and is independent from protease activity. Stretching myofibroblast-derived ECM in the presence of mechanically apposing stress fibers immediately activates latent TGF-β1. In myofibroblast-populated wounds, activation of the downstream targets of TGF-β1 signaling Smad2/3 is higher in stressed compared to relaxed tissues despite similar levels of total TGF-β1 and its receptor. We propose activation of TGF-β1 via integrin-mediated myofibroblast contraction as a potential checkpoint in the progression of fibrosis, restricting autocrine generation of myofibroblasts to a stiffened ECM.     

MT1-MMP releases latent TGF-beta1 from endothelial cell extracellular matrix via proteolytic processing of LTBP-1

Targeting of transforming growth factor beta (TGF-β) to the extracellular matrix (ECM) by latent TGF-β binding proteins (LTBPs) regulates the availability of TGF-β for interactions with endothelial cells during their quiescence and activation. However, the mechanisms which release TGF-β complexes from the ECM need elucidation. We find here that morphological activation of endothelial cells by phorbol 12-myristate 13-acetate (PMA) resulted in membrane-type 1 matrix metalloproteinase (MT1-MMP) -mediated solubilization of latent TGF-β complexes from the ECM by proteolytic processing of LTBP-1. These processes required the activities of PKC and ERK1/2 signaling pathways and were coupled with markedly increased MT1-MMP expression. The functional role of MT1-MMP in LTBP-1 release was demonstrated by gene silencing using lentiviral short-hairpin RNA as well as by the inhibition with tissue inhibitors of metalloproteinases, TIMP-2 and TIMP-3. Negligible effects of TIMP-1 and uPA/plasmin system inhibitors indicated that secreted MMPs or uPA/plasmin system did not contribute to the release of LTBP-1. Current results identify MT1-MMP-mediated proteolytic processing of ECM-bound LTBP-1 as a mechanism to release latent TGF-β from the subendothelial matrix.     

Akt Activation is Required for TGF-β1-Induced Osteoblast Differentiation of MC3T3-E1 Pre-Osteoblasts

Background

We have previously reported that repeated treatment of human periodontal ligament cells and murine pre-osteoblast MC3T3-E1 cells with transforming growth factor-beta 1 (TGF-β1) inhibited their osteoblastic differentiation because of decreased insulin-like growth factor-1 (IGF-1) secretion. We also found that IGF-1/PI3K signaling plays an important role in osteoblast differentiation induced by TGF-β1 treatment; however, the downstream signaling controlling this remains unknown. The aim of this current study is to investigate whether Akt activation is required for osteoblast differentiation.

Methodology/Principal Findings

MC3T3-E1 cells were cultured in osteoblast differentiation medium (OBM) with or without 0.1 ng/mL TGF-β1. OBM containing TGF-β1 was changed every 12 h to provide repeated TGF-β1 administration. MC3T3-E1 cells were infected with retroviral vectors expressing constitutively active (CA) or dominant-negative (DN)-Akt. Alkaline phosphatase (ALP) activity and osteoblastic marker mRNA levels were substantially decreased by repeated TGF-β1 treatment compared with a single TGF-β1 treatment. However, expression of CA-Akt restored ALP activity following TGF-β1 treatment. Surprisingly, ALP activity increased following multiple TGF-β1 treatments as the number of administrations of TGF-β1 increased. Activation of Akt significantly enhanced expression of osteocalcin, but TGF-β1 treatment inhibited this. Mineralization of MC3T3-E1 cells was markedly enhanced by CA-Akt expression under all medium conditions. Exogenous IGF-1 restored the down-regulation of osteoblast-related gene expression by repeated TGF-β1 administration. However, in cells expressing DN-Akt, these levels remained inhibited regardless of IGF-1 treatment. These findings indicate that Akt activation is required for the early phase of osteoblast differentiation of MC3T3-E1 cells induced by TGF-β1. However, Akt activation is insufficient to reverse the inhibitory effects of TGF-β1 in the late stages of osteoblast differentiation.

Conclusions

TGF-β1 could be an inducer or an inhibitor of osteoblastic differentiation of MC3T3-E1 cells depending on the state of Akt phosphorylation. Our results indicate that Akt is the molecular switch for TGF-β1-induced osteoblastic differentiation of MC3T3-E1 cells.     

Osteopontin Is Induced by TGF-β2 and Regulates Metabolic Cell Activity in Cultured Human Optic Nerve Head Astrocytes

The aqueous humor (AH) component transforming growth factor (TGF)-β2 is strongly correlated to primary open-angle glaucoma (POAG), and was shown to up-regulate glaucoma-associated extracellular matrix (ECM) components, members of the ECM degradation system and heat shock proteins (HSP) in primary ocular cells. Here we present osteopontin (OPN) as a new TGF-β2 responsive factor in cultured human optic nerve head (ONH) astrocytes. Activation was initially demonstrated by Oligo GEArray microarray and confirmed by semiquantitative (sq) RT-PCR, realtime RT-PCR and western blot. Expressions of most prevalent OPN receptors CD44 and integrin receptor subunits αV, α4, α 5, α6, α9, β1, β3 and β5 by ONH astrocytes were shown by sqRT-PCR and immunofluorescence labeling. TGF-β2 treatment did not affect their expression levels. OPN did not regulate gene expression of described TGF-β2 targets shown by sqRT-PCR. In MTS-assays, OPN had a time- and dose-dependent stimulating effect on the metabolic activity of ONH astrocytes, whereas TGF-β2 significantly reduced metabolism. OPN signaling via CD44 mediated a repressive outcome on metabolic activity, whereas signaling via integrin receptors resulted in a pro-metabolic effect. In summary, our findings characterize OPN as a TGF-β2 responsive factor that is not involved in TGF-β2 mediated ECM and HSP modulation, but affects the metabolic activity of astrocytes. A potential involvement in a protective response to TGF-β2 triggered damage is indicated, but requires further investigation.     

LOXL4 Is Induced by Transforming Growth Factor β1 through Smad and JunB/Fra2 and Contributes to Vascular Matrix Remodeling

Transforming growth factor β1 (TGF-β1) is a pleiotropic factor involved in the regulation of extracellular matrix (ECM) synthesis and remodeling. In search for novel genes mediating the action of TGF-β1 on vascular ECM, we identified the member of the lysyl oxidase family of matrix-remodeling enzymes, lysyl oxidase-like 4 (LOXL4), as a direct target of TGF-β1 in aortic endothelial cells, and we dissected the molecular mechanism of its induction. Deletion mapping and mutagenesis analysis of the LOXL4 promoter demonstrated the absolute requirement of a distal enhancer containing an activator protein 1 (AP-1) site and a Smad binding element for TGF-β1 to induce LOXL4 expression. Functional cooperation between Smad proteins and the AP-1 complex composed of JunB/Fra2 accounted for the action of TGF-β1, which involved the extracellular signal-regulated kinase (ERK)-dependent phosphorylation of Fra2. We furthermore provide evidence that LOXL4 was extracellularly secreted and significantly contributed to ECM deposition and assembly. These results suggest that TGF-β1-dependent expression of LOXL4 plays a role in vascular ECM homeostasis, contributing to vascular processes associated with ECM remodeling and fibrosis.     

Three-Dimensional Scaffold-Free Fusion Culture: the Way to Enhanced Chondrogenesis of in vitro Propagated Human Articular Chondrocytes

Cartilage regeneration based on isolated and culture-expanded chondrocytes has been studied in various in vitro models, but the quality varies with respect to the morphology and the physiology of the synthesized tissues. The aim of our study was to promote in vitro chondrogenesis of human articular chondrocytes using a novel three-dimensional (3-D) cultivation system in combination with the chondrogenic differentiation factors transforming growth factor beta 2 (TGF-β2) and L-ascorbic acid. Articular chondrocytes isolated from six elderly patients were expanded in monolayer culture. A single-cell suspension of the dedifferentiated chondrocytes was then added to agar-coated dishes without using any scaffold material, in the presence, or absence of TGF-β2 and/or L-ascorbic acid. Three-dimensional cartilage-like constructs, called single spheroids, and microtissues consisting of several spheroids fused together, named as fusions, were formed. Generated tissues were mainly characterized using histological and immunohistochemical techniques. The morphology of the in vitro tissues shared some similarities to native hyaline cartilage in regard to differentiated S100-positive chondrocytes within a cartilaginous matrix, with strong collagen type II expression and increased synthesis of proteoglycans. Finally, our innovative scaffold-free fusion culture technique supported enhanced chondrogenesis of human articular chondrocytes in vitro. These 3-D hyaline cartilage-like microtissues will be useful for in vitro studies of cartilage differentiation and regeneration, enabling optimization of functional tissue engineering and possibly contributing to the development of new approaches to treat traumatic cartilage defects or osteoarthritis.Key words: in vitro cartilage, 3-D cell culture, fusion culture technique, tissue engineering, cell differentiation, extracellular matrix, immunohistochemistry     

Casein kinase 2 promotes the TGF-β-induced activation of α-tubulin acetyltransferase 1 in fibroblasts cultured on a soft matrix

Cell signals for growth factors depend on the mechanical properties of the extracellular matrix (ECM) surrounding the cells. Microtubule acetylation is involved in the transforming growth factor (TGF)-β-induced myofibroblast differentiation in the soft ECM. However, the mechanism of activation of α-tubulin acetyltransferase 1 (α-TAT1), a major α-tubulin acetyltransferase, in the soft ECM is not well defined. Here, we found that casein kinase 2 (CK2) is required for the TGF-β-induced activation of α-TAT1 that promotes microtubule acetylation in the soft matrix. Genetic mutation and pharmacological inhibition of CK2 catalytic activity specifically reduced microtubule acetylation in the cells cultured on a soft matrix rather than those cultured on a stiff matrix. Immunoprecipitation analysis showed that CK2α, a catalytic subunit of CK2, directly bound to the C-terminal domain of α-TAT1, and this interaction was more prominent in the cells cultured on the soft matrix. Moreover, the substitution of alanine with serine, the 236th amino acid located at the C-terminus, which contains the CK2-binding site of α-TAT1, sig-nificantly abrogated the TGF-β-induced microtubule acetylation in the soft matrix, indicating that the successful binding of CK2 and the C-terminus of α-TAT1 led to the phosphorylation of serine at the 236th position of amino acids in α-TAT1 and regulation of its catalytic activity. Taken together, our findings provide novel insights into the molecular mechanisms underlying the TGF-β-induced activation of α-TAT1 in a soft matrix.     

TGF-β3-Induced Chondroitin Sulphate Proteoglycan Mediates Palatal Shelf Adhesion

In mammals, the adhesion and fusion of the palatal shelves are essential mechanisms in the development of the secondary palate. Failure of any of these processes leads to the formation of cleft palate. The mechanisms underlying palatal shelf adhesion are poorly understood, although the presence of filopodia on the apical surfaces of the superficial medial edge epithelial (MEE) cells seems to play an important role in the adhesion of the opposing MEE. We demonstrate here the appearance of chondroitin sulphate proteoglycan (CSPG) on the apical surface of MEE cells only immediately prior to contact between the palatal shelves. This apical CSPG has a functional role in palatal shelf adhesion, as either the alteration of CSPG synthesis by β-d-Xyloside or its specific digestion by chondroitinase AC strikingly alters the in vitro adhesion of palatal shelves. We also demonstrate the absence of this apical CSPG in the clefted palates of transforming growth factor beta 3 (TGF-β₃) null mutant mice, and its induction, together with palatal shelf adhesion, when TGF-β₃ is added to TGF-β₃ null mutant palatal shelves in culture. When chick palatal shelves (that do not adherein vivo nor express TGF-β₃, nor CSPG in the MEE) are cultured in vitro, they do not express CSPG and partially adhere, but when TGF-β₃ is added to the media, they express CSPG and their adhesion increases strikingly. We therefore conclude that the expression of CSPG on the apical surface of MEE cells is a key factor in palatal shelf adhesion and that this expression is regulated by TGF-β₃.