Renal fibrosis is the hallmark of virtually all progressive kidney diseases and strongly correlates with the deterioration of kidney function. The renin-angiotensin-aldosterone system blockade is central to the current treatment of patients with chronic kidney disease (CKD) for the renoprotective effects aimed to prevent or slow progression to end-stage renal disease (ESRD). However, the incidence of CKD is still increasing, and there is a critical need for new therapeutics. Here, we review novel strategies targeting various components implicated in the fibrogenic pathway to inhibit or retard the loss of kidney function. We focus, in particular, on antifibrotic approaches that target transforming growth factor (TGF)-β1, a key mediator of kidney fibrosis, and exciting new data on the role of autophagy. Bone morphogenetic protein (BMP)-7 and connective tissue growth factor (CTGF) are highlighted as modulators of profibrotic TGF-β activity. BMP-7 has a protective role against TGF-β1 in kidney fibrosis, whereas CTGF enhances TGF-β-mediated fibrosis. We also discuss recent advances in the development of additional strategies for antifibrotic therapy. These include strategies targeting chemokine pathways via CC chemokine receptors 1 and 2 to modulate the inflammatory response, inhibition of phosphodiesterase to restore nitric oxide-cyclic 3',5'-guanosine monophosphate function, inhibition of nicotinamide adenine dinucleotide phosphate oxidase 1 and 4 to suppress reactive oxygen species production, and inhibition of endothelin 1 or tumor necrosis factor α to ameliorate progressive renal fibrosis. Furthermore, a brief overview of some of the biomarkers of kidney fibrosis is currently being explored that may improve the ability to monitor antifibrotic therapies. It is hoped that evidence based on the preclinical and clinical data discussed in this review leads to novel antifibrotic therapies effective in patients with CKD to prevent or delay progression to ESRD.

Kindlin-1 is an integrin tail binding protein that controls integrin activation. Mutations in the FERMT-1 gene, which encodes for Kindlin-1, lead to Kindler syndrome in man, which is characterized by skin blistering, premature skin aging and skin cancer of unknown etiology. Here we show that loss of Kindlin-1 in mouse keratinocytes recapitulates Kindler syndrome and also produces enlarged and hyperactive stem cell compartments, which lead to hyperthickened epidermis, ectopic hair follicle development and increased skin tumor susceptibility. Mechanistically, Kindlin-1 controls keratinocyte adhesion through β1-class integrins and proliferation and differentiation of cutaneous epithelial stem cells by promoting α(v)β(6) integrin-mediated transforming growth factor-β (TGF-β) activation and inhibiting Wnt-β-catenin signaling through integrin-independent regulation of Wnt ligand expression. Our findings assign Kindlin-1 the previously unknown and essential task of controlling cutaneous epithelial stem cell homeostasis by balancing TGF-β-mediated growth-inhibitory signals and Wnt-β-catenin-mediated growth-promoting signals.

Fibrosis is a hallmark of chronic kidney disease, for which there is currently no effective cure. The hormone relaxin is emerging as an effective antifibrotic therapy; however, its mechanism of action is poorly understood. Recent studies have shown that relaxin disrupts the profibrotic actions of transforming growth factor-β1 (TGF-β1) by its cognate receptor, relaxin family peptide receptor 1 (RXFP1), extracellular signal-regulated kinase phosphorylation, and a neuronal nitric oxide synthase-dependent pathway to abrogate Smad2 phosphorylation. Since angiotensin II also inhibits TGF-β1 activity through its AT2 receptor (AT2R), we investigated the extent to which relaxin interacts with the AT2R. The effects of the AT2R antagonist, PD123319, on relaxin activity were examined in primary rat kidney myofibroblasts, and in kidney tissue from relaxin-treated male wild-type and AT2R-knockout mice subjected to unilateral ureteric obstruction. Relaxin's antifibrotic actions were significantly blocked by PD123319 in vitro and in vivo, or when relaxin was administered to AT2R-knockout mice. While heterodimer complexes were formed between RXFP1 and AT2Rs independent of ligand binding, relaxin did not directly bind to AT2Rs but signaled through RXFP1-AT2R heterodimers to induce its antifibrotic actions. These findings highlight a hitherto unrecognized interaction that may be targeted to control fibrosis progression.

Idiopathic pulmonary fibrosis (IPF) is a disease with relentless course and limited therapeutic options. Nintedanib (BIBF-1120) is a multiple tyrosine kinase inhibitor recently approved by the U.S. Food and Drug Administration for the treatment of IPF. The precise antifibrotic mechanism(s) of action of nintedanib, however, is not known. Therefore, we studied the effects of nintedanib on fibroblasts isolated from the lungs of patients with IPF. Protein and gene expression of profibrotic markers were assessed by Western immunoblotting and real-time PCR. Autophagy markers and signaling events were monitored by biochemical assays, Western immunoblotting, microscopy, and immunofluorescence staining. Silencing of autophagy effector proteins was achieved with small interfering RNAs. Nintedanib down-regulated protein and mRNA expression of extracellular matrix (ECM) proteins, fibronectin, and collagen 1a1 while inhibiting transforming growth factor (TGF)-β1-induced myofibroblast differentiation. Nintedanib also induced beclin-1-dependent, ATG7-independent autophagy. Nintedanib's ECM-suppressive actions were not mediated by canonical autophagy. Nintedanib inhibited early events in TGF-β signaling, specifically tyrosine phosphorylation of the type II TGF-β receptor, activation of SMAD3, and p38 mitogen-activated protein kinase. Nintedanib down-regulates ECM production and induces noncanonical autophagy in IPF fibroblasts while inhibiting TGF-β signaling. These mechanisms appear to be uncoupled and function independently to mediate its putative antifibrotic effects.

Mammary tumorigenesis and epithelial-mesenchymal transition (EMT) programs cooperate in converting transforming growth factor-β (TGF-β) from a suppressor to a promoter of breast cancer metastasis. Although previous reports associated β1 and β3 integrins with TGF-β stimulation of EMT and metastasis, the functional interplay and plasticity exhibited by these adhesion molecules in shaping the oncogenic activities of TGF-β remain unknown. We demonstrate that inactivation of β1 integrin impairs TGF-β from stimulating the motility of normal and malignant mammary epithelial cells (MECs) and elicits robust compensatory expression of β3 integrin solely in malignant MECs, but not in their normal counterparts. Compensatory β3 integrin expression also 1) enhances the growth of malignant MECs in rigid and compliant three-dimensional organotypic cultures and 2) restores the induction of the EMT phenotypes by TGF-β. Of importance, compensatory expression of β3 integrin rescues the growth and pulmonary metastasis of β1 integrin-deficient 4T1 tumors in mice, a process that is prevented by genetic depletion or functional inactivation of β3 integrin. Collectively our findings demonstrate that inactivation of β1 integrin elicits metastatic progression via a β3 integrin-specific mechanism, indicating that dual β1 and β3 integrin targeting is necessary to alleviate metastatic disease in breast cancer patients.

Unresolved inflammation underlies the development of fibrosis and organ failure. Here, we investigate the potential of the proresolving eicosanoid lipoxinA₄ (LXA₄) and its synthetic analog benzo-LXA₄ to prophylactically modulate fibrotic and inflammatory responses in a model of early renal fibrosis, unilateral ureteric obstruction (UUO). Male Wistar rats (Animalia, Chordata, Rattus norvegicus) were injected intravenously with vehicle (0.1% ethanol), LXA₄ (45 μg/250-g rat), or benzo-LXA₄ (15 μg/250-g rat) 15 min prior to surgery and sacrificed 3 d postligation. Renal gene and protein expression, collagen deposition, macrophage infiltration, and apoptosis were analyzed using manipulated kidneys from sham operations as control. Lipoxins (LXs) attenuated collagen deposition and renal apoptosis (P<0.05) and shifted the inflammatory milieu toward resolution, inhibiting TNF-α and IFN-γ expression, while stimulating proresolving IL-10. LXs attenuated UUO-induced activation of MAP kinases, Akt, and Smads (P<0.05) in injured kidneys. We explored whether the underlying mechanism reflected LX-induced modulation of fibroblast activation. Using cultured rat renal NRK-49F fibroblasts, we report that LXA₄ (1 nM) inhibits TGF-β1 (10 ng/ml)-induced activation of Smad2 and MAP-kinases (P<0.05), and furthermore, LXA₄ reduced TGF-β1-stimulated PAI-1 luciferase activation (P<0.05) relative to vehicle-stimulated cells. We propose that LXs may represent a potentially useful and novel therapeutic strategy for consideration in the context of renal fibrosis.

Proinflammatory processes have been investigated extensively in the development of type 2 diabetes, but our knowledge on anti-inflammatory proteins is rather limited. This article summarizes studies that investigated associations between circulating levels of anti-inflammatory cytokines and incident type 2 diabetes preferably in prospective epidemiological studies. Adiponectin is the only known anti-inflammatory protein whose circulating levels are decreased before type 2 diabetes. In contrast, concentrations of interleukin-1 receptor antagonist (IL-1RA), transforming growth factor-β1 (TGF-β1) and growth differentiation factor-15 (GDF-15) are increased and indicate the presence of a compensatory, but eventually futile, counter-regulation of proinflammatory stimuli. Importantly, a proof-of-principle study using recombinant IL-1RA to improve metabolic control in patients with type 2 diabetes demonstrated that a more pronounced upregulation of this protein than that found in the natural course of diabetes development may have clinical relevance. Other interesting candidates like omentin (which shows similar associations with metabolic parameters as adiponectin), interleukin-10 (IL-10) and secreted frizzled-related protein-5 (Sfrp5) are currently less well studied with sometimes conflicting results regarding their association with type 2 diabetes. Thus, further research is required to better understand the causal role of proinflammatory cytokines, hypoadiponectinaemia and the upregulation of anti-inflammatory proteins before the onset of type 2 diabetes.

Although the newly developed second-generation anti-androgen drug enzalutamide can repress prostate cancer progression significantly, it only extends the survival of prostate cancer patients by 4-6 months mainly due to the occurrence of enzalutamide resistance. Most of the previous studies on AR antagonist resistance have been focused on AR signaling. Therefore, the non-AR pathways on enzalutamide resistance remain largely unknown. By using C4-2, CWR22Rv1 and LNCaP cell lines, as well as mice bearing CWR22Rv1 xenografts treated with either enzalutamide or metformin alone or in combination, we demonstrated that metformin is capable of reversing enzalutamide resistance and restores sensitivity of CWR22Rv1 xenografts to enzalutamide. We showed that metformin alleviated resistance to enzalutamide by inhibiting EMT. Furthermore, based on the effect of metformin on the activation of STAT3 and expression of TGF-β1, we propose that metformin exerts its effects by targeting the TGF-β1/STAT3 axis. These findings suggest that combination of metformin with enzalutamide could be a more efficacious therapeutic strategy for the treatment of castration-resistant prostate cancer.

Shear stress, especially low shear stress (LowSS), plays an important role in vascular remodeling during atherosclerosis. Endothelial cells (ECs), which are directly exposed to shear stress, convert mechanical stimuli into intracellular signals and interact with the underlying vascular smooth muscle cells (VSMCs). The interactions between ECs and VSMCs modulate the LowSS-induced vascular remodeling. With the use of proteomic analysis, the protein profiles of rat aorta cultured under LowSS (5 dyn/cm(2)) and normal shear stress (15 dyn/cm(2)) were compared. By using Ingenuity Pathway Analysis to identify protein-protein association, a network was disclosed that involves two secretary molecules, PDGF-BB and TGF-β1, and three other linked proteins, lamin A, lysyl oxidase, and ERK 1/2. The roles of this network in cellular communication, migration, and proliferation were further studied in vitro by a cocultured parallel-plate flow chamber system. LowSS up-regulated migration and proliferation of ECs and VSMCs, increased productions of PDGF-BB and TGF-β1, enhanced expressions of lysyl oxidase and phospho-ERK1/2, and decreased Lamin A in ECs and VSMCs. These changes induced by LowSS were confirmed by using PDGF-BB recombinant protein, siRNA, and neutralizing antibody. TGF-β1 had similar influences on ECs as PDGF-BB, but not on VSMCs. Our results suggest that ECs convert the LowSS stimuli into up-regulations of PDGF-BB and TGF-β1, but these two factors play different roles in LowSS-induced vascular remodeling. PDGF-BB is involved in the paracrine control of VSMCs by ECs, whereas TGF-β1 participates in the feedback control from VSMCs to ECs.

Liver fibrosis is an outcome of chronic liver injury of any etiology. It is manifested by extensive deposition of extracellular matrix (ECM) proteins that produce a fibrous scar in the injured liver. Bone marrow (BM) cells may play an important role in pathogenesis and resolution of liver fibrosis. BM cells contribute to the inflammatory response by TGF-β1 secretion and activation of liver resident myofibroblasts. Moreover, BM itself can serve as a source of collagen expressing cells, e.g. BM-derived fibrocytes and mesenchymal progenitors, which in turn, have a potential to in situ differentiate into fibrogenic myofibroblasts and facilitate fibrosis. Finally, BM cells play an active part in resolution of liver fibrosis after cessation of fibrogenic stimuli. While natural killer (NK) cells are implicated in apoptosis of activated hepatic stellate cells/myofibroblasts, cells of myelo-monocitic lineage secrete matrix metalloproteinases to actively degrade the fibrous scar. The focus of this review is on the current understanding of the role of different subsets of BM cells in the onset, development and resolution of liver fibrosis.

In spite of a large number of transforming growth factor β1 (TGF-β1)-regulated genes, the nature of its targets with roles in transformation continues to be poorly understood. Here, we discovered that TGF-β1 stimulates transcription of metastasis-associated protein 1 (MTA1), a dual master coregulator, in epithelial cells, and that MTA1 status is a determinant of TGF-β1-induced epithelial-to-mesenchymal transition (EMT) phenotypes. In addition, we found that MTA1/polymerase II/activator protein-1 (AP-1) co-activator complex interacts with the FosB-gene chromatin and stimulates its transcription, and FosB in turn, utilizes FosB/histone deacetylase 2 complex to repress E-cadherin expression in TGF-β1-stimulated mammary epithelial cells. These findings suggest that TGF-β1 regulates the components of EMT via stimulating the expression of MTA1, which in turn, induces FosB to repress E-cadherin expression and thus, revealed an inherent function of MTA1 as a target and effector of TGF-β1 signaling in epithelial cells.

Despite the known anti-inflammatory effect of amniotic membrane, its action mechanism remains largely unknown. HC-HA complex (HC-HA) purified from human amniotic membrane consists of high molecular weight hyaluronic acid (HA) covalently linked to the heavy chain (HC) 1 of inter-α-trypsin inhibitor. In this study, we show that soluble HC-HA also contained pentraxin 3 and induced the apoptosis of both formyl-Met-Leu-Phe or LPS-activated neutrophils and LPS-activated macrophages while not affecting the resting cells. This enhanced apoptosis was caused by the inhibition of cell adhesion, spreading, and proliferation caused by HC-HA binding of LPS-activated macrophages and preventing adhesion to the plastic surface. Preferentially, soluble HC-HA promoted phagocytosis of apoptotic neutrophils in resting macrophages, whereas immobilized HC-HA promoted phagocytosis in LPS-activated macrophages. Upon concomitant LPS stimulation, immobilized HC-HA but not HA polarized macrophages toward the M2 phenotype by down-regulating IRF5 protein and preventing its nuclear localization and by down-regulating IL-12, TNF-α, and NO synthase 2. Additionally, IL-10, TGF-β1, peroxisome proliferator-activated receptor γ, LIGHT (TNF superfamily 14), and sphingosine kinase-1 were up-regulated, and such M2 polarization was dependent on TLR ligation. Collectively, these data suggest that HC-HA is a unique matrix component different from HA and uses multiple mechanisms to suppress M1 while promoting M2 phenotype. This anti-inflammatory action of HC-HA is highly desirable to promote wound healing in diseases heightened by unsuccessful transition from M1 to M2 phenotypes.

Hypertrophic scars (HTS) and keloids are challenging problems. Their pathogenesis results from an overproduction of fibroblasts and excessive deposition of collagen. Studies suggest a possible anti-scarring effect of basic fibroblast growth factor (bFGF) during wound healing, but the precise mechanisms of bFGF are still unclear. In view of this, we investigated the therapeutic effects of bFGF on HTS animal model as well as human scar fibroblasts (HSF) model. We show that bFGF promoted wound healing and reduced the area of flattened non-pathological scars in rat skin wounds and HTS in the rabbit ear. We provide evidence of a new therapeutic strategy: bFGF administration for the treatment of HTS. The scar elevation index (SEI) and epidermal thickness index (ETI) was also significantly reduced. Histological reveal that bFGF exhibited significant amelioration of the collagen tissue. bFGF regulated extracellular matrix (ECM) synthesis and degradation via interference in the collagen distribution, the α-smooth muscle actin (α-SMA) and transforming growth factor-1 (TGF-β1) expression. In addition, bFGF reduced scarring and promoted wound healing by inhibiting TGFβ1/SMAD-dependent pathway. The levels of fibronectin (FN), tissue inhibitor of metalloproteinase-1 (TIMP-1) collagen I, and collagen III were evidently decreased, and matrix metalloproteinase-1 (MMP-1) and apoptosis cells were markedly increased. These results suggest that bFGF possesses favorable therapeutic effects on hypertrophic scars in vitro and in vivo, which may be an effective cure for human hypertrophic scars.

Re-epithelialization is a complex process that involves migration and proliferation of keratinocytes, in addition to the production of cytokines and growth factors that affect other cells. The induction of transcription factors during these processes is crucial for successful wound healing. The transcription factor forkhead boxO-1 (FOXO1) has recently been found to be an important regulator of wound healing. In particular, FOXO1 has significant effects through regulation of transforming growth factor-beta (TGF-β) expression and protecting keratinocytes from oxidative stress. In the absence of FOXO1, there is increased oxidative damage, reduced TGF-β1 expression, reduced migration and proliferation of keratinocytes and increased keratinocytes apoptosis leading to impaired re-epithelialization of wounds.

OBJECTIVE

Cancer stem cells (CSCs) represent the root of many solid cancers including pancreatic ductal adenocarcinoma, are highly chemoresistant and represent the cellular source for disease relapse. However the mechanisms involved in these processes still need to be fully elucidated. Understanding the mechanisms implicated in chemoresistance and metastasis of pancreatic cancer is critical to improving patient outcomes.

METHODS

Micro-RNA (miRNA) expression analyses were performed to identify functionally defining epigenetic signatures in pancreatic CSC-enriched sphere-derived cells and gemcitabine-resistant pancreatic CSCs.

RESULTS

We found the miR-17-92 cluster to be downregulated in chemoresistant CSCs versus non-CSCs and demonstrate its crucial relevance for CSC biology. In particular, overexpression of miR-17-92 reduced CSC self-renewal capacity, in vivo tumourigenicity and chemoresistance by targeting multiple NODAL/ACTIVIN/TGF-β1 signalling cascade members as well as directly inhibiting the downstream targets p21, p57 and TBX3. Overexpression of miR-17-92 translated into increased CSC proliferation and their eventual exhaustion via downregulation of p21 and p57. Finally, the translational impact of our findings could be confirmed in preclinical models for pancreatic cancer.

CONCLUSIONS

Our findings therefore identify the miR-17-92 cluster as a functionally determining family of miRNAs in CSCs, and highlight the putative potential of developing modulators of this cluster to overcome drug resistance in pancreatic CSCs.

Matrix metalloproteinases (MMPs) are members of the neutral proteinase family. They were previously thought to be anti-fibrotic because of their ability to degrade and remodel of extracellular matrix. However, recent studies have shown that MMPs are implicated in initiation and progression of kidney fibrosis through tubular cell epithelial-mesenchymal transition (EMT) as well as activation of resident fibroblasts, endothelial-mesenchymal transition (EndoMT) and pericyte-myofibroblast transdifferentiation. Interstitial macrophage infiltration has also been shown to correlate with the severity of kidney fibrosis in various chronic kidney diseases. MMPs secreted by macrophages, especially MMP-9, has been shown by us to be profibrotic by induction of tubular cells EMT. EMT is mainly induced by transforming growth factor-β (TGF-β). However, MMP-9 was found by us and others to be up-regulated by TGF-β1 in kidney tubular epithelial cells and secreted by activated macrophages, resulting in EMT and ultimately kidney fibrosis. Therefore, MMP-9 may serve as a potential therapeutic target to prevent kidney fibrosis in chronic kidney disease. This review, by a particular focus on EMT, seeks to provide a comprehensive understanding of MMPs, especially MMP-9, in kidney fibrosis.

Idiopathic pulmonary fibrosis is characterized by myofibroblast accumulation, extracellular matrix (ECM) remodeling, and excessive collagen deposition. ECM-producing myofibroblasts may originate from epithelial cells through epithelial to mesenchymal transition (EMT). TGF-β1 is an inducer of EMT in pulmonary epithelial cells in vitro and in vivo, though the mechanisms are unclear. We hypothesized that TGF-β1 induced EMT through Smad-dependent and -independent processes. To test this hypothesis, we studied the roles and mechanisms of TGF-β1-induced Smad and p38 mitogen-activated protein kinase (MAPK) signaling in EMT-related changes in pulmonary epithelial cells. Exposure of pulmonary epithelial 1HAEo(-) cells to TGF-β1 resulted in morphological and molecular changes of EMT over a 96-h period; loss of cell-cell contact, cell elongation, down-regulation of E-cadherin, up-regulation of fibronectin, and up-regulation of collagen I. Both Smad2/3 and p38 MAPK signaling pathways were activated by TGF-β1. However, neither Smad2/3 nor p38 MAPK were required for the down-regulation of E-cadherin, yet p38 MAPK was associated with fibronectin up-regulation. Both Smad2/3 and p38 MAPK had a role in regulation of TGF-β1-induced collagen expression. Furthermore, these data demonstrate that Smads and p38 MAPK differentially regulate EMT-related changes in pulmonary epithelial cells.

T helper (Th)17 cells and other interleukin (IL)-17-producing cells are supposed to play critical roles in several human immune-mediated diseases, including Crohn's disease (CD) and ulcerative colitis (UC), the main forms of inflammatory bowel diseases (IBD) in man. Th17 cells infiltrate massively the inflamed intestine of IBD patients and in vitro and in vivo studies have shown that Th17-type cytokines may trigger and amplify multiple inflammatory pathways. Nonetheless, some Th17-related cytokines, such as interleukin (IL)-17A and IL-22, may target gut epithelial cells and promote the activation of counter-regulatory mechanisms. This observation together with the demonstration that Th17 cells are not stable and can be converted into either regulatory T cells or Th1 cells if stimulated by immune-suppressive (e.g. TGF-β1) or inflammatory (e.g. IL-12, IL-23) cytokines have contributed to advance our understanding of mechanisms that regulate mucosal homeostasis and inflammation in the gut.

The aim of the present study is to explore possible role of miR-221 in the pathogenesis of HCC. Matched HCC and adjacent non-cancerous samples were assayed for the expression of miR-221 and three G1/S transition inhibitors: p27(Kip1), p21(WAF1/Cip1)and TGF-β1 by in situ hybridization and immunohistochemistry respectively. p27(Kip1) is one of miR-221's proven targets. Real time qRT-PCR was used to investigate miR-221 and p27(Kip1) transcripts in different clinical stages. Western blotting was used to analyze the expression levels of p27(Kip1) protein in different clinical stages. In result, miR-221 and TGF-β1 are frequently up-regulated in HCC, while p27(Kip1) and p21(WAF1/Cip1) proteins are frequently down-regulated. Moreover, miR-221 and p27(Kip1)'s expression correlated with metastasis and miR-221's expression also correlated with tumor size. Both of p21(WAF1/Cip1)and TGF-β1's expression correlated with tumor differentiations. miR-221's upregulation and p27(Kip1)'s downregulation were significantly associated with tumor stages and metastasis. In conclusion, miR-221 is important in tumorigenesis of HCC, possibly by specifically down-regulating p27(Kip1), a cell-cycle inhibitor. These results indicate miR-221 as a new therapeutic target in HCC.

Induction of epithelial-to-mesenchymal transition (EMT) by TGF-β1 requires Ras signaling. We recently identified the transcriptional repressor Blimp-1 (PRDM1) as a downstream effector of the NF-κB, RelB/Bcl-2/Ras-driven pathway that promotes breast cancer cell migration. As the RelB/Blimp-1 pathway similarly required Ras signaling activation, we tested whether Blimp-1 plays a role in TGF-β1-mediated EMT. Here, TGF-β1 treatment of untransformed NMuMG mammary epithelial and MDA-MB-231 breast cancer cells was shown to induce Blimp-1 expression, which promoted an EMT signature and cell migration. TGFB1 and BLIMP1 RNA levels were correlated in patient breast tumors. BLIMP1 gene transcription was activated by TGF-β1 via a c-Raf (RAF1) to AP-1 pathway. Blimp-1 induced expression of the EMT master regulator Snail (SNAI1) via repressing BMP-5, which inhibited Snail expression upon TGF-β1 treatment. Interestingly, a similar cascade was observed during postnatal mouse mammary gland development. RelB expression was detected early in pregnancy followed progressively by Blimp-1 and then Snail; whereas, BMP-5 levels were high in nulliparous and regressing glands. Finally, lower BMP5 RNA levels were detected in patient breast tumors versus normal tissues, and correlated with cancer recurrence. Thus, the Ras effector Blimp-1 plays an essential role in TGF-β1-induced EMT via repression of BMP-5 in breast cancer.

Tenascin-C (TNC), a large hexameric extracellular glycoprotein, is a pleiotropic molecule with multiple domains binding to a variety of receptors mediating a wide range of cellular functions. We earlier reported that TNC induces epithelial-mesenchymal transition (EMT)-like change in breast cancer cells. In the present study, we clarified TNC receptor involvement in this process. Among integrins previously reported as TNC receptors, substantial expression of αv, α2, β1 and β6 subunits was detected by quantitative PCR and immunoblotting in MCF-7 cells. Integrin β6 mRNA was remarkably upregulated by transforming growth factor (TGF)-β1 treatment, and protein expression was prominently increased by additional exposure to TNC. Immunofluorescent labeling demonstrated integrin αvβ6 accumulation in focal adhesions after TNC treatment, especially in combination with TGF-β1. The α2 and β1 subunits were mainly localized at cell-cell contacts, αv being found near cell cluster surfaces. Immunoprecipitation showed increase in αvβ1 heterodimers, but not α2β1, after TNC treatment. Activated β1 subunits detected by an antibody against the Ca(2+)-dependent epitope colocalized with αv in focal adhesion complexes, associated with FAK phosphorylation at tyrosine 925. Neutralizing antibodies against αv and β1 blocked EMT-like change caused by TNC alone. In addition, anti-αv and combined treatment with anti-β1 and anti-αvβ6 inhibited TGF-β1/TNC-induced EMT, whereas either of these alone did not. Integrin subunits αv, β1 and β6, but not α2, bound to TNC immobilized on agarose beads in a divalent cation-dependent manner. Treatments with neutralizing antibodies against β1 and αvβ6 reduced αv subunit bound to the beads. Immunohistochemistry of these receptors in human breast cancer tissues demonstrated frequent expression of β6 subunits in cancer cells forming scattered nests localized in TNC-rich stroma. These findings provide direct evidence that binding of αvβ6 and αvβ1 integrins to TNC as their essential ligand induces EMT-like change in breast cancer cells.

The aim of this study was to assess the effect of extracellular matrix (ECM) deposited by synovium-derived stem cells (SDSCs) on articular chondrocyte expansion and maintenance of differentiation status and redifferentiation capacity. Passage 0 (P0) pig articular chondrocytes were expanded for six passages on plastic flasks (Plastic), SDSC-derived ECM (ECM), or substrate switching from either Plastic to ECM (PtoE) or ECM to Plastic (EtoP). Cell morphology, gene expression profiles, and immunophenotypes at each passage were used to characterize differentiation status of expanded cells. Chondrocytes at P0, P2, and P6 were assessed for redifferentiation capacity in a pellet culture system treated with either TGF-β1- or serum-containing medium for 14 days, using histology, immunohistochemistry, biochemistry, Western blot, and real-time PCR. We found that ECM not only greatly enhanced chondrocyte expansion but also delayed dedifferentiation of expanded chondrocytes. Intriguingly, compared to a dramatic decrease in CD90+/CD105+ cells and CD90+ cells, CD105+ cells dramatically increased when chondrocytes were plated on Plastic; on the contrary, ECM expansion dramatically increased CD90+ cells and delayed the decrease of CD90+/CD105+ cells. Interestingly, expanded chondrocytes on ECM also acquired a strong redifferentiation capacity, particularly in the pellets treated with TGF-β1. In conclusion, the ratio of CD90 to CD105 may serve as a marker indicative of proliferation and redifferentiation capacity of dedifferentiated chondrocytes. ECM deposited by SDSCs provides a tissue-specific three-dimensional microenvironment for ex vivo expansion of articular chondrocytes while retaining redifferentiation capacity, suggesting that ECM may provide a novel approach for autologous chondrocyte-based cartilage repair.

Langerhans cells (LCs) are skin-resident dendritic cells (DC) located in the epidermis that migrate to skin-draining lymph nodes during the steady state and in response to inflammatory stimuli. TGF-β1 is a critical immune regulator that is highly expressed by LCs. The ability to test the functional importance of LC-derived TGF-β1 is complicated by the requirement of TGF-β1 for LC development and by the absence of LCs in mice with an LC-specific ablation of TGF-β1 or its receptor. To overcome these problems, we have engineered transgenic huLangerin-CreER(T2) mice that allow for inducible LC-specific excision. Highly efficient and LC-specific expression was confirmed in mice bred onto a YFP Cre reporter strain. We next generated huLangerin-CreER(T2) × TGF-βRII(fl) and huLangerin-CreER(T2) × TGF-β1(fl) mice. Excision of the TGFβRII or TGFβ1 genes induced mass migration of LCs to the regional lymph node. Expression of costimulatory markers and inflammatory cytokines was unaffected, consistent with homeostatic migration. In addition, levels of p-SMAD2/3 were decreased in LCs from wild-type mice before inflammation-induced migration. We conclude that TGF-β1 acts directly on LCs in an autocrine/paracrine manner to inhibit steady-state and inflammation-induced migration. This is a readily targetable pathway with potential therapeutic implications for skin disease.

CD69 is highly expressed by lymphocytes at mucosal surfaces. We aimed to investigate the role of CD69 in mucosal immune responses. The expression of CD69 by CD4 T cells isolated from the spleen, mesenteric lymph nodes, small intestinal lamina propria, and colonic lamina propria was determined in specific pathogen-free B6 and TCR transgenic animals, as well as in germ-free B6 mice. Transfer colitis was induced by transplanting RAG(-/-) mice with B6 or CD69(-/-)CD45RB(high) CD4 T cells. CD69 expression by CD4 T cells is induced by the intestinal microflora, oral delivery of specific Ag, and type I IFN (IFN-I) signals. CD4 T cells from CD69(-/-) animals produce higher amounts of the proinflammatory cytokines IFN-γ, TNF-α, and IL-21, whereas the production of TGF-β1 is decreased. CD69-deficient CD4 T cells showed reduced potential to differentiate into Foxp3(+) regulatory T cells in vivo and in vitro. The transfer of CD69(-/-)CD45RB(high) CD4 T cells into RAG(-/-) hosts induced an accelerated colitis. Oral tolerance was impaired in CD69(-/-) and IFN-I receptor 1-deficient mice when compared with B6 and OT-II × RAG(-/-) animals. Polyinosinic-polycytidylic acid treatment of RAG(-/-) mice transplanted with B6 but not CD69(-/-) or IFN-I receptor 1-deficient CD45RB(high) CD4 T cells attenuated transfer colitis. CD69 deficiency led to the increased production of proinflammatory cytokines, reduced Foxp3(+) regulatory T cell induction, impaired oral tolerance, and more severe colitis. Hence, the activation Ag CD69 plays an important role in regulating mucosal immune responses.