The Role of Bone in Muscle Wasting

This review describes the role of bone resorption in muscle atrophy as well as in muscle protein anabolism. Both catabolic and anabolic pathways involve components of the proinflammatory cytokine families and release of factors stored in bone during resorption. The juxtaposition of the catabolic and anabolic resorption-dependent pathways raises new questions about control of release of factors from bone, quantity of release in a variety of conditions, and relation of factors released from bone. The catabolic responses involve release of calcium from bone into the circulation resulting in increased inflammatory response in intensity and/or duration. The release of transforming growth factor beta (TGF-β) from bone suppresses phosphorylation of the AKT/mTOR pathway and stimulates ubiquitin-mediated breakdown of muscle protein. In contrast, muscle IL-6 production is stimulated by undercarboxylated osteocalcin, which signals osteoblasts to produce more RANK ligand, stimulating resorptive release of undercarboxylated osteocalcin, which in turn stimulates muscle fiber nutrient uptake and an increase in muscle mass.     

Role of Human Primary Renal Fibroblast in TGF-β1-Mediated Fibrosis-Mimicking Devices

Renal fibrosis is a progressive chronic kidney disease that ultimately leads to end-stage renal failure. Despite several approaches to combat renal fibrosis, an experimental model to evaluate currently available drugs is not ideal. We developed fibrosis-mimicking models using three-dimensional (3D) co-culture devices designed with three separate layers of tubule interstitium, namely, epithelial, fibroblastic, and endothelial layers. We introduced human renal proximal tubular epithelial cells (HK-2), human umbilical-vein endothelial cells, and patient-derived renal fibroblasts, and evaluated the effects of transforming growth factor-β (TGF-β) and TGF-β inhibitor treatment on this renal fibrosis model. The expression of the fibrosis marker alpha smooth muscle actin upon TGF-β1 treatment was augmented in monolayer-cultured HK-2 cells in a 3D disease model. In the vascular compartment of renal fibrosis models, the density of vessels was increased and decreased in the TGF-β-treated group and TGF-β-inhibitor treatment group, respectively. Multiplex ELISA using supernatants in the TGF-β-stimulating 3D models showed that pro-inflammatory cytokine and growth factor levels including interleukin-1 beta, tumor necrosis factor alpha, basic fibroblast growth factor, and TGF-β1, TGF-β2, and TGF-β3 were increased, which mimicked the fibrotic microenvironments of human kidneys. This study may enable the construction of a human renal fibrosis-mimicking device model beyond traditional culture experiments.     

Coordinated Regulation of Mesenchymal Stem Cell Migration by Various Chemotactic Stimuli

Endogenous bone marrow-derived mesenchymal stem cells are mobilized to peripheral blood and injured tissues in response to changes in the expression of various growth factors and cytokines in the injured tissues, including substance P (SP), transforming growth factor-beta (TGF-β), and stromal cell-derived factor-1 (SDF-1). SP, TGF-β, and SDF-1 are all known to induce the migration of bone marrow-derived mesenchymal stem cells (BM-MSCs). However, it is not yet clear how these stimuli influence or interact with each other during BM-MSC mobilization. This study used mouse bone marrow-derived mesenchymal stem cell-like ST2 cells and human BM-MSCs to evaluate whether SP, TGF-β, and SDF-1 mutually regulate their respective effects on the mobilization of BM-MSCs. SP pretreatment of ST2 and BM-MSCs impaired their response to TGF-β while the introduction of SP receptor antagonist restored the mobilization of ST2 and BM-MSCs in response to TGF-β. TGF-β pretreatment did not affect the migration of ST2 and BM-MSCs in response to SP, but downregulated their migration in response to SDF-1. SP pretreatment modulated the activation of TGF-β noncanonical pathways in ST2 cells and BM-MSCs, but not canonical pathways. These results suggest that the migration of mesenchymal stem cells is regulated by complex functional interactions between SP, TGF-β, and SDF-1. Thus, understanding the complex functional interactions of these chemotactic stimuli would contribute to ensuring the development of safe and effective combination treatments for the mobilization of BM-MSCs.     

Correlation between Oxidative Stress and Transforming Growth Factor-Beta in Cancers

The downregulation of reactive oxygen species (ROS) facilitates precancerous tumor development, even though increasing the level of ROS can promote metastasis. The transforming growth factor-beta (TGF-β) signaling pathway plays an anti-tumorigenic role in the initial stages of cancer development but a pro-tumorigenic role in later stages that fosters cancer metastasis. TGF-β can regulate the production of ROS unambiguously or downregulate antioxidant systems. ROS can influence TGF-β signaling by enhancing its expression and activation. Thus, TGF-β signaling and ROS might significantly coordinate cellular processes that cancer cells employ to expedite their malignancy. In cancer cells, interplay between oxidative stress and TGF-β is critical for tumorigenesis and cancer progression. Thus, both TGF-β and ROS can develop a robust relationship in cancer cells to augment their malignancy. This review focuses on the appropriate interpretation of this crosstalk between TGF-β and oxidative stress in cancer, exposing new potential approaches in cancer biology.     

Curcumin Suppresses TGF-β1-Induced Myofibroblast Differentiation and Attenuates Angiogenic Activity of Orbital Fibroblasts

Orbital fibrosis, a hallmark of tissue remodeling in Graves’ ophthalmopathy (GO), is a chronic, progressive orbitopathy with few effective treatments. Orbital fibroblasts are effector cells, and transforming growth factor β1 (TGF-β1) acts as a critical inducer to promote myofibroblast differentiation and subsequent tissue fibrosis. Curcumin is a natural compound with anti-fibrotic activity. This study aims to investigate the effects of curcumin on TGF-β1-induced myofibroblast differentiation and on the pro-angiogenic activities of orbital fibroblasts. Orbital fibroblasts from one healthy donor and three patients with GO were collected for primary cell culture and subjected to myofibroblast differentiation under the administration of 1 or 5 ng/mL TGF-β1 for 24 h. The effects of curcumin on TGF-β1-induced orbital fibroblasts were assessed by measuring the cellular viability and detecting the expression of myofibroblast differentiation markers, including connective tissue growth factor (CTGF) and α-smooth muscle actin (α-SMA). The pro-angiogenic potential of curcumin-treated orbital fibroblasts was evaluated by examining the transwell migration and tube-forming capacities of fibroblast-conditioned EA.hy926 and HMEC-1 endothelial cells. Treatment of orbital fibroblasts with curcumin inhibited the TGF-β1 signaling pathway and attenuated the expression of CTGF and α-SMA induced by TGF-β1. Curcumin, at the concentration of 5 μg/mL, suppressed 5 ng/mL TGF-β1-induced pro-angiogenic activities of orbital fibroblast-conditioned EA hy926 and HMEC-1 endothelial cells. Our findings suggest that curcumin reduces the TGF-β1-induced myofibroblast differentiation and pro-angiogenic activity in orbital fibroblasts. The results support the potential application of curcumin for the treatment of GO.     

Genetics and Epigenetics of Bone Remodeling and Metabolic Bone Diseases

Bone metabolism consists of a balance between bone formation and bone resorption, which is mediated by osteoblast and osteoclast activity, respectively. In order to ensure bone plasticity, the bone remodeling process needs to function properly. Mesenchymal stem cells differentiate into the osteoblast lineage by activating different signaling pathways, including transforming growth factor β (TGF-β)/bone morphogenic protein (BMP) and the Wingless/Int-1 (Wnt)/β-catenin pathways. Recent data indicate that bone remodeling processes are also epigenetically regulated by DNA methylation, histone post-translational modifications, and non-coding RNA expressions, such as micro-RNAs, long non-coding RNAs, and circular RNAs. Mutations and dysfunctions in pathways regulating the osteoblast differentiation might influence the bone remodeling process, ultimately leading to a large variety of metabolic bone diseases. In this review, we aim to summarize and describe the genetics and epigenetics of the bone remodeling process. Moreover, the current findings behind the genetics of metabolic bone diseases are also reported.     

TGF-β Activity of a Demineralized Bone Matrix

Allografts consisting of demineralized bone matrix (DBM) are supposed to retain the growth factors of native bone. However, it is not clear if transforming growth factor β1 (TGF-β1) is maintained in the acid-extracted human bone. To this aim, the aqueous solutions of supernatants and acid lysates of OraGRAFT^® Demineralized Cortical Particulate and OraGRAFT^® Prime were prepared. Exposing fibroblasts to the aqueous solution caused a TGF-β receptor type I kinase-inhibitor SB431542-dependent increase in interleukin 11 (IL11), NADPH oxidase 4 (NOX4), and proteoglycan 4 (PRG4) expression. Interleukin 11 expression and the presence of TGF-β1 in the aqueous solutions were confirmed by immunoassay. Immunofluorescence further confirmed the nuclear translocation of Smad2/3 when fibroblasts were exposed to the aqueous solutions of both allografts. Moreover, allografts released matrix metalloprotease-2 activity and blocking proteases diminished the cellular TGF-β response to the supernatant. These results suggest that TGF-β is preserved upon the processing of OraGRAFT^® and released by proteolytic activity into the aqueous solution.     

MMP9 Differentially Regulates Proteins Involved in Actin Polymerization and Cell Migration during TGF-β-Induced EMT in the Lens

Fibrotic cataracts have been attributed to transforming growth factor-beta (TGF-β)-induced epithelial-to-mesenchymal transition (EMT). Using mouse knockout (KO) models, our laboratory has identified MMP9 as a crucial protein in the TGF-β-induced EMT process. In this study, we further revealed an absence of alpha-smooth muscle actin (αSMA) and filamentous-actin (F-actin) stress fibers in MMP9KO mouse lens epithelial cell explants (LECs). Expression analysis using NanoString revealed no marked differences in αSMA (ACTA2) and beta-actin (β-actin) (ACTB) mRNA between the lenses of TGF-β-overexpressing (TGF-βtg) mice and TGF-βtg mice on a MMP9KO background. We subsequently conducted a protein array that revealed differential regulation of proteins known to be involved in actin polymerization and cell migration in TGF-β-treated MMP9KO mouse LECs when compared to untreated controls. Immunofluorescence analyses using rat LECs and the novel MMP9-specific inhibitor, JNJ0966, revealed similar differential regulation of cortactin, FAK, LIMK1 and MLC2 as observed in the array. Finally, a reduction in the nuclear localization of MRTF-A, a master regulator of cytoskeletal remodeling during EMT, was observed in rat LECs co-treated with JNJ0966 and TGF-β. In conclusion, MMP9 deficiency results in differential regulation of proteins involved in actin polymerization and cell migration, and this in turn prevents TGF-β-induced EMT in the lens.     

Autocrine TGF-β in Cancer: Review of the Literature and Caveats in Experimental Analysis

Autocrine signaling is defined as the production and secretion of an extracellular mediator by a cell followed by the binding of that mediator to receptors on the same cell to initiate signaling. Autocrine stimulation often operates in autocrine loops, a type of interaction, in which a cell produces a mediator, for which it has receptors, that upon activation promotes expression of the same mediator, allowing the cell to repeatedly autostimulate itself (positive feedback) or balance its expression via regulation of a second factor that provides negative feedback. Autocrine signaling loops with positive or negative feedback are an important feature in cancer, where they enable context-dependent cell signaling in the regulation of growth, survival, and cell motility. A growth factor that is intimately involved in tumor development and progression and often produced by the cancer cells in an autocrine manner is transforming growth factor-β (TGF-β). This review surveys the many observations of autocrine TGF-β signaling in tumor biology, including data from cell culture and animal models as well as from patients. We also provide the reader with a critical discussion on the various experimental approaches employed to identify and prove the involvement of autocrine TGF-β in a given cellular response.     

Platelet-Rich Fibrin Increases BMP2 Expression in Oral Fibroblasts via Activation of TGF-β Signaling

Solid platelet-rich fibrin (PRF), consisting of coagulated plasma from fractionated blood, has been proposed to be a suitable carrier for recombinant bone morphogenetic protein 2 (BMP2) to target mesenchymal cells during bone regeneration. However, whether solid PRF can increase the expression of BMPs in mesenchymal cells remains unknown. Proteomics analysis confirmed the presence of TGF-β1 but not BMP2 in PRF lysates. According to the existing knowledge of recombinant TGF-β1, we hypothesized that PRF can increase BMP2 expression in mesenchymal cells. To test this hypothesis, we blocked TGF-β receptor 1 kinase with SB431542 in gingival fibroblasts exposed to PRF lysates. RT-PCR and immunoassays confirmed that solid PRF lysates caused a robust SB431542-dependent increase in BMP2 expression in gingival fibroblasts. Additionally, fractions of liquid PRF, namely platelet-poor plasma (PPP) and the buffy coat (BC) layer, but not heat-denatured PPP (Alb-gel), greatly induced the expression of BMP2 in gingival fibroblasts. Even though PRF has no detectable BMPs, PRF lysates similar to recombinant TGF-β1 had the capacity to provoke canonical BMP signaling, as indicated by the nuclear translocation of Smad1/5 and the increase in its phosphorylation. Taken together, our data suggest that PRF can activate TGF-β receptor 1 kinase and consequently induce the production of BMP2 in cells of the mesenchymal lineage.     

The Dual Role of Smad7 in the Control of Cancer Growth and Metastasis

Smad7 was initially identified as an inhibitor of Transforming growth factor (TGF)-β due mainly to its ability to bind TGF-β receptor type I and prevent TGF-β-associated Smad signaling. More recently, it has been demonstrated that Smad7 can interact with other intracellular proteins and regulate also TGF-β-independent signaling pathways thus making a valid contribution to the neoplastic processes in various organs. In particular, data emerging from experimental studies indicate that Smad7 may differently modulate the course of various tumors depending on the context analyzed. These observations, together with the demonstration that Smad7 expression is deregulated in many cancers, suggest that therapeutic interventions around Smad7 can help interfere with the development/progression of human cancers. In this article we review and discuss the available data supporting the role of Smad7 in the modulation of cancer growth and progression.     

TGF-β Induced CTGF Expression in Human Lung Epithelial Cells through ERK,ADAM17, RSK1, and C/EBPβ Pathways

Background: Lung epithelial cells play critical roles in idiopathic pulmonary fibrosis. Methods: In the present study, we investigated whether transforming growth factor-β (TGF-β)-induced expression of connective tissue growth factor (CTGF) was regulated by the extracellular signal-regulated kinase (ERK)/a disintegrin and metalloproteinase 17 (ADAM17)/ribosomal S6 kinases 1 (RSK1)/CCAAT/enhancer-binding protein β (C/EBPβ) signaling pathway in human lung epithelial cells (A549). Results: Our results revealed that TGF-β-induced CTGF expression was weakened by ADAM17 small interfering RNA (ADAM17 siRNA), TNF-α processing inhibitor-0 (TAPI-0, an ADAM17 inhibitor), U0126 (an ERK inhibitor), RSK1 siRNA, and C/EBPβ siRNA. TGF-β-induced ERK phosphorylation as well as ADAM17 phosphorylation was attenuated by U0126. The TGF-β-induced increase in RSK1 phosphorylation was inhibited by TAPI-0 and U0126. TGF-β-induced C/EBPβ phosphorylation was weakened by U0126, ADAM17 siRNA, and RSK1 siRNA. In addition, TGF-β increased the recruitment of C/EBPβ to the CTGF promoter. Furthermore, TGF-β enhanced fibronectin (FN), an epithelial–mesenchymal transition (EMT) marker, and CTGF mRNA levels and reduced E-cadherin mRNA levels. Moreover, TGF-β-stimulated FN protein expression was reduced by ADAM17 siRNA and CTGF siRNA. Conclusion: The results suggested that TGF-β induces CTGF expression through the ERK/ADAM17/RSK1/C/EBPβ signaling pathway. Moreover, ADAM17 and CTGF participate in TGF-β-induced FN expression in human lung epithelial cells.     

CCN2 Increases TGF-β Receptor Type II Expression in Vascular Smooth Muscle Cells: Essential Role of CCN2 in the TGF-β Pathway Regulation

The cellular communication network factor 2 (CCN2/CTGF) has been traditionally described as a mediator of the fibrotic responses induced by other factors including the transforming growth factor β (TGF-β). However, several studies have defined a direct role of CCN2 acting as a growth factor inducing oxidative and proinflammatory responses. The presence of CCN2 and TGF-β together in the cellular context has been described as a requisite to induce a persistent fibrotic response, but the precise mechanisms implicated in this relation are not described yet. Considering the main role of TGF-β receptors (TβR) in the TGF-β pathway activation, our aim was to investigate the effects of CCN2 in the regulation of TβRI and TβRII levels in vascular smooth muscle cells (VSMCs). While no differences were observed in TβRI levels, an increase in TβRII expression at both gene and protein level were found 48 h after stimulation with the C-terminal fragment of CCN2 (CCN2(IV)). Cell pretreatment with a TβRI inhibitor did not modify TβRII increment induced by CCN2(VI), demonstrating a TGF-β-independent response. Secondly, CCN2(IV) rapidly activated the SMAD pathway in VSMCs, this being crucial in the upregulation of TβRII since the preincubation with an SMAD3 inhibitor prevented it. Similarly, pretreatment with the epidermal growth factor receptor (EGFR) inhibitor erlotinib abolished TβRII upregulation, indicating the participation of this receptor in the observed responses. Our findings suggest a direct role of CCN2 maintaining the TGF-β pathway activation by increasing TβRII expression in an EGFR-SMAD dependent manner activation.     

JNK and p38 Inhibitors Prevent Transforming Growth Factor-β1-Induced Myofibroblast Transdifferentiation in Human Graves’ Orbital Fibroblasts

Transforming growth factor-β1 (TGF-β1)-induced myofibroblast transdifferentiation from orbital fibroblasts is known to dominate tissue remodeling and fibrosis in Graves’ ophthalmopathy (GO). However, the signaling pathways through which TGF-β1 activates Graves’ orbital fibroblasts remain unclear. This study investigated the role of the mitogen-activated protein kinase (MAPK) pathway in TGF-β1-induced myofibroblast transdifferentiation in human Graves’ orbital fibroblasts. The MAPK pathway was assessed by measuring the phosphorylation of p38, c-Jun N-terminal kinase (JNK), and extracellular-signal-regulated kinase (ERK) by Western blots. The expression of connective tissue growth factor (CTGF), α-smooth muscle actin (α-SMA), and fibronectin representing fibrogenesis was estimated. The activities of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) responsible for extracellular matrix (ECM) metabolism were analyzed. Specific pharmacologic kinase inhibitors were used to confirm the involvement of the MAPK pathway. After treatment with TGF-β1, the phosphorylation levels of p38 and JNK, but not ERK, were increased. CTGF, α-SMA, and fibronectin, as well as TIMP-1 and TIMP-3, were upregulated, whereas the activities of MMP-2/-9 were inhibited. The effects of TGF-β1 on the expression of these factors were eliminated by p38 and JNK inhibitors. The results suggested that TGF-β1 could induce myofibroblast transdifferentiation in human Graves’ orbital fibroblasts through the p38 and JNK pathways.     

SB203580—A Potent p38 MAPK Inhibitor Reduces the Profibrotic Bronchial Fibroblasts Transition Associated with Asthma

Subepithelial fibrosis is a component of the remodeling observed in the bronchial wall of patients diagnosed with asthma. In this process, human bronchial fibroblasts (HBFs) drive the fibroblast-to-myofibroblast transition (FMT) in response to transforming growth factor-β₁ (TGF-β₁), which activates the canonical Smad-dependent signaling. However, the pleiotropic properties of TGF-β₁ also promote the activation of non-canonical signaling pathways which can affect the FMT. In this study we investigated the effect of p38 mitogen-activated protein kinase (MAPK) inhibition by SB203580 on the FMT potential of HBFs derived from asthmatic patients using immunocytofluorescence, real-time PCR and Western blotting methods. Our results demonstrate for the first time the strong effect of p38 MAPK inhibition on the TGF-β₁-induced FMT potential throughout the strong attenuation of myofibroblast-related markers: α-smooth muscle actin (α-SMA), collagen I, fibronectin and connexin 43 in HBFs. We suggest the pleiotropic mechanism of SB203580 on FMT impairment in HBF populations by the diminishing of TGF-β/Smad signaling activation and disturbances in the actin cytoskeleton architecture along with the maturation of focal adhesion sites. These observations justify future research on the role of p38 kinase in FMT efficiency and bronchial wall remodeling in asthma.