uPAR Induces Expression of Transforming Growth Factor β and Interleukin-4 in Cancer Cells to Promote Tumor-Permissive Conditioning of Macrophages

Cancer cells condition macrophages and other inflammatory cells in the tumor microenvironment so that these cells are more permissive for cancer growth and metastasis. Conditioning of inflammatory cells reflects, at least in part, soluble mediators (such as transforming growth factor β and IL-4) that are released by cancer cells and alter the phenotype of cells of the innate immune system. Signaling pathways in cancer cells that potentiate this activity are incompletely understood. The urokinase receptor (uPAR) is a cell-signaling receptor known to promote cancer cell survival, proliferation, metastasis, and cancer stem cell–like properties. The present findings show that uPAR expression in diverse cancer cells, including breast cancer, pancreatic cancer, and glioblastoma cells, promotes the ability of these cells to condition co-cultured bone marrow–derived macrophages so that the macrophages express significantly increased levels of arginase 1, a biomarker of the alternatively activated M2 macrophage phenotype. Expression of transforming growth factor β was substantially increased in uPAR-expressing cancer cells via a mechanism that requires uPA-initiated cell signaling. uPAR also controlled expression of IL-4 in cancer cells via a mechanism that involves activation of ERK1/2. The ability of uPAR to induce expression of factors that condition macrophages in the tumor microenvironment may constitute an important mechanism by which uPAR promotes cancer progression.It is well established that certain chronic infections and inflammation predispose to the development of malignancy.^1–3 Once cancer develops, inflammatory cells that infiltrate the tumor may promote disease progression.^4–6 This process is mediated by bidirectional paracrine pathways involving cancer and inflammatory cells. Growth factors and cytokines released by cancer cells are immunosuppressive, and also condition inflammatory cells so that these cells release mediators that support cancer cell growth, survival, metastasis, and angiogenesis.^7–10 Inflammatory cell conditioning is prevalent in breast cancer. These tumors include large numbers of macrophages, dendritic cells, mast cells, and T cells, and the extent to which the tumor is infiltrated by these inflammatory cells correlates with the incidence of metastasis.^11–13 A high density of tumor-associated macrophages (TAMs) is also correlated with higher breast cancer tumor grade and decreased relapse-free and overall survival.^14–17Although macrophages express a wide spectrum of phenotypic properties, these cells are frequently categorized as classically activated (M1) or alternatively activated (M2).^18–21 In response to pathogens, tissue damage, and Th1 cytokines such as IFN-γ and TNF-α, M1-polarized macrophages release cytotoxic compounds and proteins, including nitric oxide, reactive oxygen species, and proinflammatory cytokines (including IL-12, IL-23, and TNF-α). M2-polarized macrophage have been classified into a number of subcategories; in many contexts, these cells demonstrate enhanced activity in the resolution of inflammation, tissue remodeling, and healing.^18–21 Arginase 1 (Arg1), which is expressed selectively by M2-polarized macrophages, diverts substrate from the enzyme systems that produce cytotoxic levels of nitric oxide.^22,23 In general, it is thought that TAMs, which have been conditioned by cancer cells to express tumor-permissive gene products, demonstrate characteristics in common with M2-polarized macrophages, although a recent report highlights phenotypic differences.^18,19,24 Cell-signaling systems in tumor cells that promote the ability of these cells to regulate macrophage phenotype remain incompletely understood.In many forms of cancer, expression of the urokinase receptor [urokinase plasminogen activator receptor (uPAR)] correlates with poor prognosis and shortened survival.^25–28 Originally, the activity of uPAR in cancer was attributed to its ability to bind the serine protease, urokinase-type plasminogen activator (uPA), and activate a cascade of extracellular proteases involved in matrix remodeling and cell migration through tissue boundaries. The current understanding, however, is that uPAR also is a cell-signaling receptor that activates diverse signaling pathways.²⁹ Although uPAR may signal autonomously when expressed at high levels, uPA binding to uPAR robustly activates cell signaling even when the cell-surface abundance of uPAR is low.^29–32 uPAR-initiated cell signaling promotes cancer cell survival, release from states of dormancy, migration, epithelial–mesenchymal transition, cancer stem cell–like properties, and metastasis independently of protease activation.^33–38Here, we show that in multiple forms of cancer, including breast cancer, pancreatic cancer, and glioblastoma (GBM), uPAR expression promotes the ability of the cancer cells to M2-polarize co-cultured macrophages. The mediators that are released selectively by uPAR-expressing cancer cells to regulate macrophage phenotype may vary across different cancer cells; however, we provide evidence that both TGF-β and IL-4 are involved. The ability of cancer-cell uPAR to promote conditioning of inflammatory cells in the tumor microenvironment is a novel mechanism by which uPAR may promote cancer progression.     

Prognostic Significance of Stromal Platelet-Derived Growth Factor β-Receptor Expression in Human Breast Cancer

This study systematically analyzes platelet-derived growth factor (PDGF) receptor expression in six types of common tumors as well as examines associations between PDGF β-receptor status and clinicopathological characteristics in breast cancer. PDGF receptor expression was determined by immunohistochemistry on tumor tissue microarrays. Breast tumor data were combined with prognostic factors and related to outcome endpoints. PDGF α- and β-receptors were independently expressed, at variable frequencies, in the tumor stroma of all tested tumor types. There was a significant association between PDGF β-receptor expression on fibroblasts and perivascular cells in individual colon and prostate tumors. In breast cancer, high stromal PDGF β-receptor expression was significantly associated with high histopathological grade, estrogen receptor negativity, and high HER2 expression. High stromal PDGF β-receptor expression was correlated with significantly shorter recurrence-free and breast cancer-specific survival. The prognostic significance of stromal PDGF β-receptor expression was particularly prominent in tumors from premenopausal women. Stromal PDGF α- and β-receptor expression is a common, but variable and independent, property of solid tumors. In breast cancer, stromal PDGF β-receptor expression significantly correlates with less favorable clinicopathological parameters and shorter survival. These findings highlight the prognostic significance of stromal markers and should be considered in ongoing clinical development of PDGF receptor inhibitors.Platelet-derived growth factor (PDGF) α- and β-tyrosine kinase receptors exert important control functions in mesenchymal cells, such as pericytes, fibroblasts and vascular smooth muscle cells during development.¹ PDGF receptor activation has also been shown to be involved in multiple dimensions of cancer growth.² The clinical relevance of these findings is enhanced by the recent approval of tyrosine kinase inhibitors with PDGF receptor inhibitory activity, eg, imatinib, sunitinib, and sorafenib.PDGF receptor-dependent growth stimulation is well documented in malignant cells of some solid tumors, such as glioblastomas,^3,4,5,6,7 dermatofibrosarcoma protuberans^8,9 and a subset of gastrointestinal stromal tumors.^10,11 Also, in hematological malignancies such as chronic myelomonocytic leukemia and idiopathic eosinophilic syndrome, PDGF α- or β-receptor signaling has been shown to be activated through translocations or deletions of the PDGF receptor genes.^12,13,14 However, in most common solid tumors PDGF receptor signaling appears to be most important for the pericytes of the tumor vessels, and for the fibroblasts of the tumor stroma.Concerning the role of PDGF β-receptor signaling in pericytes, a series of experimental studies have demonstrated that stimulation of PDGF receptors on pericytes increases pericyte coverage of vessels in a manner that is associated with increased vessel function and, in some cases, also increased tumor growth.^15,16,17 Furthermore, vascular endothelial growth factor receptor-targeted antiangiogenic approaches in experimental tumor models appear to be most efficient on immature pericyte-poor vessels.¹⁸ Finally, combinations of vascular endothelial growth factor receptor- and PDGF-receptor inhibitors have been demonstrated to exert synergistic antiangiogenic effects.^19,20Studies in experimental tumor models have demonstrated that paracrine activation of PDGF receptors on fibroblasts acts as a potent signal for tumor stroma recruitment.^21,22 Other studies with PDGF antagonists have also demonstrated direct antitumoral effects of stromal PDGF receptor inhibition,^23,24 as well as beneficial effects on tumor drug uptake.^25,26,27,28The biological effects of PDGF receptors in tumor fibroblasts and pericytes, together with the advent of drugs with PDGF receptor-inhibitory activity thus motivates a systemic characterization of the expression pattern of PDGF α- and β-receptors in human tumors. In this study we have characterized the fibroblast and pericyte expression of PDGF α- and β-receptors in lymphomas and in colon, ovarian, prostate, lung and breast cancers. Furthermore the relationship between stromal PDGF β-receptor status and prognostic parameters and survival was analyzed in breast cancer.     

Dynamic Regulation of Platelet-Derived Growth Factor Receptor α Expression in Alveolar Fibroblasts during Realveolarization

Although the importance of platelet-derived growth factor receptor (PDGFR)-α signaling during normal alveogenesis is known, it is unclear whether this signaling pathway can regulate realveolarization in the adult lung. During alveolar development, PDGFR-α-expressing cells induce α smooth muscle actin (α-SMA) and differentiate to interstitial myofibroblasts. Fibroblast growth factor (FGF) signaling regulates myofibroblast differentiation during alveolarization, whereas peroxisome proliferator-activated receptor (PPAR)-γ activation antagonizes myofibroblast differentiation in lung fibrosis. Using left lung pneumonectomy, the roles of FGF and PPAR-γ signaling in differentiation of myofibroblasts from PDGFR-α-positive precursors during compensatory lung growth were assessed. FGF receptor (FGFR) signaling was inhibited by conditionally activating a soluble dominant-negative FGFR2 transgene. PPAR-γ signaling was activated by administration of rosiglitazone. Changes in α-SMA and PDGFR-α protein expression were assessed in PDGFR-α-green fluorescent protein (GFP) reporter mice using immunohistochemistry, flow cytometry, and real-time PCR. Immunohistochemistry and flow cytometry demonstrated that the cell ratio and expression levels of PDGFR-α-GFP changed dynamically during alveolar regeneration and that α-SMA expression was induced in a subset of PDGFR-α-GFP cells. Expression of a dominant-negative FGFR2 and administration of rosiglitazone inhibited induction of α-SMA in PDGFR-α-positive fibroblasts and formation of new septae. Changes in gene expression of epithelial and mesenchymal signaling molecules were assessed after left lobe pneumonectomy, and results demonstrated that inhibition of FGFR2 signaling and increase in PPAR-γ signaling altered the expression of Shh, FGF, Wnt, and Bmp4, genes that are also important for epithelial-mesenchymal crosstalk during early lung development. Our data demonstrate for the first time that a comparable epithelial-mesenchymal crosstalk regulates fibroblast phenotypes during alveolar septation.     

Can the Nerve Growth Factor promote the reinnervation of the transplanted heart?

The activity of the heart is widely regulated by the autonomous nervous system. This important mechanism of control may be impaired in chronic diseases such as heart failure or lost in those patients who undergo heart transplantation, owing to the surgical interruption of cardiac nerves in the transplanted heart. It has been demonstrated that spontaneous reinnervation can occur in transplanted hearts and is associated with an improvement in cardiac function. However, this process may require many years and the restoration of a proper cardiac innervation and functioning during exercise is never complete. In this perspective, the Nerve Growth Factor (NGF) and other neurotrophic hormones might ameliorate cardiac innervation in the transplanted heart and should be tried in animal models. Endothelial cells engineered with a viral vector to overexpress the NGF might be engrafted in the heart and integrate into cardiac small vessels, thus providing a source of neurotrophic factors which might promote and direct regrowth and axonal sprouting of cardiac nerves.     

Involvement of Transforming Growth Factor β and its Type 1 Receptor in the Development of Breast Cancer

Introduction of recent achievements of molecular biology into clinical practice contributes significantly to both prevention and early detection of breast cancer in women and development of new approaches, including genotyping, to screening for breast cancer. Screening for genetic polymorphisms in genes TGFβ1 and TGFβR1 reveals carriers of sporadic breast cancer. This provides an opportunity to reassess the role and place of preventive measures, also including surgical method, in the fight against breast cancer.     

Effects of Transforming Growth Factor-β1 on Proliferation of Smooth Muscle Cells in Human Aortic Intima and Human Promonocytic Leukemia THP-1 Cells

We studied the effects of transforming growth factor on proliferation of cultured smooth muscle cells from human aortic intima and proliferation and differentiation of human leukemia THP-1 promonocytes. Transforming growth factor inhibited proliferation of these cells, but stimulated differentiation of THP-1 cells. Therefore, transforming growth factor probably modulates proliferation and differentiation of smooth muscle cells and monocytes/macrophages involved in the pathogenesis of atherosclerotic damages.     

High Expression of Insulin-like Growth Factor H (IGF-Ⅱ) Using Bac-to-Bac Expression System

Objective In order to obtain mature insulin-like growth factor- Ⅱ ( IGF- Ⅱ ), we used Bac-to-Bac baculovirus expression system. Methods Firstly the IGF- Ⅱ cDNA was cloned into a donor plasmid pFastBac1 and the recombinant pFastBac1 was then introduced into competent cells DH 10Bac. Recombinant bacmids were constructed by transposing a mini-Tn7 element from a donor plasmid pFastBac1 to the mini-attTn7 attachment site on the bacmid where the Tn7 transposition functions were provided in trans by a helper plasmid, and then used to transfect Sf9 insect cells to get recombinant baculovirus. The recombinant baculovirus was used to infect insect cells. Results Agarose gel analysis showed that recombinant donor plasmid pFastBac1 was constructed successfully; Agarose gel analysis of PCR products confirmed recombinant bacmid ; SDS-PAGE and Western Blotting showed that a 7KD protein band appeared. Conclusion The mature IGF- Ⅱ with immunogenecity has been expressed and produced by using Bac-to-Bac expression system.     

Early Bacterial Colonization Induces Toll-Like Receptor-Dependent Transforming Growth Factor β Signaling in the Epithelium

Colonization of the upper respiratory tract is an initial step that may lead to disease for many pathogens. To prevent compromise of the epithelial barrier, the host must monitor and tightly control bacterial levels on the mucosa. Here we show that innate immune functions of respiratory epithelial cells control colonization by Streptococcus pneumoniae and Haemophilus influenzae in a Toll-like receptor (TLR)-dependent manner. Activation of inflammatory pathways, including mitogen-activated protein kinase signaling, in respiratory epithelial cells was accompanied by the induction of the transforming growth factor β signaling cascade during early colonization. Thus, colonization resulted in upregulation of factors involved in a proinflammatory response (e.g., interleukin-6) as well as factors known to modulate the epithelial barrier (e.g., Snail-1). These in vivo data provided a link between inflammation control and maintenance of the mucosal barrier function during infection and emphasized the importance of TLR-dependent inflammatory responses of the respiratory epithelium.The epithelial surface of the upper respiratory tract is constantly exposed to commensals and potentially pathogenic microbes. For pathogens, colonization of the upper respiratory tract is often the first step in a multistep process leading to disease. Thus, even though the mucosa of the upper respiratory tract is permissive for colonization by a variety of different bacterial microbes, the density and composition of colonization need to be controlled by the host (33). In recent years, it has become evident that the epithelium of the upper respiratory tract not only provides a physical barrier that is highly effective at blocking penetration by most microbes but also has the ability to recognize microbes and to initiate an inflammatory response. However, to prevent excessive colonization and invasion by potential pathogens, the epithelial barrier needs to be a dynamic structure, allowing the egress of immune cells and antimicrobial factors. Insights into the signaling events of the respiratory epithelium in response to bacterial infection, therefore, may provide a better understanding of the initial events of host-pathogen interaction.Numerous in vitro studies have been performed to gain insight into the immune functions of respiratory epithelial cells (9, 10, 26, 30, 37). Respiratory epithelial cells in culture are capable of recognizing microbes through pattern recognition receptors such as Toll-like receptors (TLRs). Haemophilus influenzae, for instance, has been reported to induce immune responses through TLR2 and TLR4, which bind its lipoproteins and lipopolysaccharide (LPS), respectively (6, 40, 43). Streptococcus pneumoniae activates respiratory epithelial cells through the activity of TLR2 via its lipid-modified membrane components and TLR4 via the toxin pneumolysin (22, 37, 38). Recognition of bacterial products by TLRs leads to signaling events that result in activation of NF-κB through the activity of, for instance, mitogen-activated protein kinase (MAPK) signaling cascades, including p38 MAPK (15). Furthermore, in vitro studies suggest that activated respiratory epithelial cells are directly involved in initiating an immune response by releasing cytokines, chemokines, and factors such as antimicrobial peptides that directly mitigate the infectious process (1).Host factors that modulate the barrier function of the respiratory epithelium are less well defined. Transforming growth factor β (TGF-β) signaling through the TGF-β receptor is involved in the remodeling and repair of epithelial surfaces but also mediates epithelial-to-mesenchymal transition, thereby contributing to the progression of complex diseases such as pulmonary fibrosis and cancer (28, 29, 44). Moreover, H. influenzae and S. pneumoniae have been shown to stimulate TGF-β signaling in respiratory cells in vitro (2, 29). We have demonstrated that stimulation of TGF-β signaling in polarized respiratory epithelial cells in culture results in the opening of tight junctions and loss of barrier integrity. These events could be required for the trafficking of immune cells and inflammatory mediators such as neutrophils and complement to the airway lumen, where microbes reside. Opening of the barrier, however, may also result in invasion by bacteria that are not adequately controlled by these host responses (2).Despite the multitude of studies dealing with the immune functions of respiratory epithelial cells in vitro, little is known about whether microbial pathogens activate these pathways during colonization in vivo under physiological conditions. As relatively high numbers of bacteria or concentrations of TLR ligands or other pathogen-associated molecular pattern recognition receptors are needed to activate respiratory tissue in vitro, it is arguable whether colonization of the upper airway by bacterial pathogens is sufficient to evoke an inflammatory response in the respiratory epithelium in vivo. In particular, it is not clear whether the TGF-β signaling pathway is engaged upon bacterial colonization of the respiratory epithelium in vivo.The purpose of this study was to characterize the role of respiratory epithelial cells during bacterial colonization in vivo. We demonstrate innate immune functions of respiratory epithelial cells in vivo by taking advantage of physiologically relevant murine models of colonization of the upper respiratory tract by H. influenzae and S. pneumoniae, the leading gram-negative and gram-positive bacterial pathogens of the human respiratory tract, respectively. We show that epithelial cells not only produce factors that promote an inflammatory response but also activate pathways that modulate barrier and immune functions of the mucosal surface. Furthermore, TLRs are central to both these functions, and deficiency in their signaling leads to decreased control of initial colonization by bacterial pathogens and increased susceptibility to invasive disease.     

Organotypic Culture Model of Pancreatic Cancer Demonstrates that Stromal Cells Modulate E-Cadherin, β-Catenin,and Ezrin Expression in Tumor Cells

Pancreatic cancer is characterized by an intense stromal reaction. Reproducible three-dimensional in vitro systems for exploring interactions of the stroma with pancreatic cancer cells have not previously been available, prompting us to develop such a model. Cancer cells were grown on collagen/Matrigel and embedded with or without stromal cells (hTERT-immortalized human PS-1 stellate cells or MRC-5 fibroblasts) for 7 days. Proliferation and apoptosis, as well as important cell–cell adhesion and cytoskeleton-regulating proteins, were studied. PS-1 cells were confirmed as stellate based on the expression of key cytoskeletal proteins and lipid vesicles. Capan-1, and to a lesser extent PaCa-3, cells differentiated into luminal structures, exhibiting a central apoptotic core with a proliferating peripheral rim and an apico-basal polarity. Presence of either stromal cell type translocated Ezrin from apical (when stromal cells were absent) to basal aspects of cancer cells, where it was associated with invasive activity. Interestingly, the presence of ‘normal’ (not tumor-derived) stromal cells induced total tumor cell number reduction (P < 0.005) associated with a significant decrease in E-cadherin expression (P < 0.005). Conversely, β-catenin expression was up-regulated (P < 0.01) in the presence of stromal cells with predominant cytoplasmic expression. Moreover, patient samples confirmed that these data recapitulated the clinical situation. In conclusion, pancreatic organotypic culture offers a reproducible, bio-mimetic, three-dimensional in vitro model that allows examination of the interactions between stromal elements and pancreatic cancer cells.Pancreatic cancer, with a continuing dismal prognosis despite considerable progress in understanding underlying genetic and molecular events, is characterized by an intense desmoplastic stroma.^1,2,3 It is now appreciated that transformed cells interact with stromal cells, extracellular matrix proteins, and neighboring normal epithelial cells to generate feedback mechanisms essential for tumor progression.^4,5 However, few models exist to enable investigators to dissect out these interactions of cancer cells with their surrounding stroma. Recently, an excellent animal model of pancreatic cancer has been created using transgenic mice with conditional pancreatic expression of mutated K-Ras; producing tumors that mimic human pancreatic intraepithelial neoplasia and full-blown cancers.⁶ However, the long latency period involved makes this model costly and non-amenable to rapid experimental manipulation. For many of the problems needed to be investigated in pancreatic cancer it is possible that organotypic models, where cancer cells are cultured on a “synthetic” stroma comprised of an extracellular matrix gel embedded with stromal cells, can provide a solution.⁷ To our knowledge, such a three-dimensional (3D) in vitro system has not yet been developed for pancreatic cancer. Therefore we aimed to establish such a model in which we could study the effect of stromal cells (pancreatic stellate cells [PSCs] and fibroblasts) on pancreatic cancer cell behavior. We have isolated a PSC line from normal human pancreas and, additionally, have used non-tumorigenic MRC-5 fibroblasts, derived from human fetal lung, which previously were validated as representative stromal cells in the absence of a pancreatic stromal cell line.⁸ The effects of co-culture conditions on proliferation and apoptosis, as well as the expression and subcellular distribution of key proteins regulating cell–cell interactions, such as E-cadherin,⁹ β-catenin,¹⁰ and members of the Ezrin-Radixin-Moesin (ERM) family,¹¹ have been studied in pancreatic cancer cells as a means of investigating the utility of this model.We show here that reproducible quantitative data can be derived from such assays, illuminating the role and mechanisms of epithelial–stromal interactions in modulating pancreatic cancer progression.     

Enhanced bFGF Gene Expression in Response to Transforming Growth Factor-β Stimulation of AKR-2B Cells

Abstract

Treatment of quiescent cultures of mouse embryo-derived AKR-2B cells with transforming growth factor β resulted in an induction of basic fibroblast growth factor (bFGF) mRNA and bFGF protein in the stimulated cells. In contrast to bFGF, acidic fibroblast growth factor (aFGF) was not induced by TGFβ. The mitogenic effect of transforming growth factor β on AKR-2B cells may be mediated by the induction of bFCF in these cells.     

Expression and function of transforming growth factor β in melioidosis

Melioidosis, caused by the Gram-negative bacterium Burkholderia pseudomallei, is an important cause of community-acquired sepsis in Southeast Asia and northern Australia. An important controller of the immune system is the pleiotropic cytokine transforming growth factor β (TGF-β), of which Smad2 and Smad3 are the major signal transducers. In this study, we aimed to characterize TGF-β expression and function in experimental melioidosis. TGF-β expression was determined in 33 patients with culture-proven infection with B. pseudomallei and 30 healthy controls. We found that plasma TGF-β concentrations were strongly elevated during melioidosis. In line with this finding, TGF-β expression in C57BL/6 mice intranasally inoculated with B. pseudomallei was enhanced as well. To assess the role of TGF-β, we inhibited TGF-β using a selective murine TGF-β antibody. Treatment of mice with anti-TGF-β antibody resulted in decreased lung Smad2 phosphorylation. TGF-β blockade appeared to be protective: mice treated with anti-TGF-β antibody and subsequently infected with B. pseudomallei showed diminished bacterial loads. Moreover, less distant organ injury was observed in anti-TGF-β treated mice as shown by reduced blood urea nitrogen (BUN) and aspartate transaminase (AST) values. However, anti-TGF-β treatment did not have an effect on survival. In conclusion, TGF-β is upregulated during B. pseudomallei infection and plays a limited but proinflammatory role during experimental melioidosis.     

Rapamycin Inhibits Transforming Growth Factor β1-Induced Fibrogenesis in Primary Human Lung Fibroblasts

Purpose

The present study was designed to determine whether rapamycin could inhibit transforming growth factor β1 (TGF-β1)-induced fibrogenesis in primary lung fibroblasts, and whether the effect of inhibition would occur through the mammalian target of rapamycin (mTOR) and its downstream p70S6K pathway.

Materials and Methods

Primary normal human lung fibroblasts were obtained from histological normal lung tissue of 3 patients with primary spontaneous pneumothorax. Growth arrested, synchronized fibroblasts were treated with TGF-β1 (10 ng/mL) and different concentrations of rapamycin (0.01, 0.1, 1, 10 ng/mL) for 24 h. We assessed m-TOR, p-mTOR, S6K1, p-S6K1 by Western blot analysis, detected type III collagen and fibronectin secreting by ELISA assay, and determined type III collagen and fibronectin mRNA levels by real-time PCR assay.

Results

Rapamycin significantly reduced TGF-β1-induced type III collagen and fibronectin levels, as well as type III collagen and fibronectin mRNA levels. Furthermore, we also found that TGF-β1-induced mTOR and p70S6K phosphorylation were significantly down-regulated by rapamycin. The mTOR/p70S6K pathway was activated through the TGF-β1-mediated fibrogenic response in primary human lung fibroblasts.

Conclusion

These results indicate that rapamycin effectively suppresses TGF-β1-induced type III collagen and fibronectin levels in primary human lung fibroblasts partly through the mTOR/p70S6K pathway. Rapamycin has a potential value in the treatment of pulmonary fibrosis.     

Biological Feature of Collagen-Chitosan Membrane with Basic Fibroblast Growth Factor for the Culture of Human Fibroblast

INTRODUCTION　　Collagen present in the extracellular matrix is the most promising natural poly-mer for tissue engineering.It has many expected biological features,includinggrowth promotion,biological stability,low antigenicity and cytotoxic properties.Collagen is frequently used material for cell culture carriers in various fields.Al-though collagen possess excellent biocompatibility,the chemical treatment makesthe reconstituted collagen very low tensile strength and easily biodegraded b…     

Transforming Growth Factor-β–Independent Role of Connective Tissue Growth Factor in the Development of Liver Fibrosis

We previously identified transforming growth factor (TGF)-β signaling as a fibronectin-independent mechanism of type I collagen fibrillogenesis following adult liver injury. To address the contribution of TGF-β signaling during the development of liver fibrosis, we generated adult mice lacking TGF-β type II receptor (TGF-βIIR) from the liver. TGF-βIIR knockout livers indeed showed a dominant effect in reducing fibrosis, but fibrosis still remained approximately 45% compared with control and fibronectin knockout livers. Unexpectedly, this was accompanied by significant up-regulation of connective tissue growth factor mRNA levels. Organized type I collagen networks in TGF-βIIR knockout livers colocalized well with fibronectin. We provide evidence that elimination of TGF-βIIR is not sufficient to completely prevent liver fibrosis. Our results indicate a TGF-β–independent mechanism of type I collagen production and suggest connective tissue growth factor as its potent mediator. We advocate combined elimination of TGF-β signaling and connective tissue growth factor as a potential therapeutic target by which to attenuate liver fibrosis.Liver fibrosis is defined as an abnormal response to persistent liver injury, characterized by the excessive accumulation of collagenous extracellular matrices (ECMs).^{1, 2} Liver fibrosis affects tens of millions of people worldwide and is of great clinical significance because normal liver architecture is disrupted and liver function is ultimately impaired. Because there is no effective treatment of liver fibrosis, many patients develop progressive liver cirrhosis, eventually requiring a liver transplant.^{3, 4}There is a long-standing concept that cells in culture cannot form a collagen fibril network without the ECM glycoprotein fibronectin.⁵ We recently established an adult mouse model lacking liver fibronectin and demonstrated that fibronectin-null livers, in fact, formed collagen fibril networks similarly to wild-type mice in response to carbon tetrachloride–induced chronic liver injury. The networks were found to be nucleated by type V collagen, induced by elevated local transforming growth factor (TGF)-β bioavailability.⁶ Therefore, we identified two mechanisms of collagen fibrillogenesis in response to liver injury: both fibronectin and TGF-β–signaling mediated.Early in the fibrogenic process, inflammatory cytokines are important in initiating repair following injury. TGF-β acts as a fibrogenic master cytokine and plays a pivotal role in the progression of a variety of chronic fibrotic diseases by promoting myofibroblastic differentiation, stimulating synthesis of ECMs, and down-regulating ECM degradation.⁷ In TGF-β–mediated signaling, ligand TGF-βs bind to TGF-β type I and type II receptors that form heterotetrameric complexes. On ligand binding, downstream Smad signaling pathways are initiated. Activated (phosphorylated) Smads translocate to the nucleus where they are involved in the regulation of gene expression.^{8, 9} Currently, several monoclonal antibodies and small molecules targeting TGF-β are in the process of clinical application for chronic fibrotic diseases, including liver fibrosis.¹⁰ However, these studies were initiated without knowledge of how TGF-β signaling exerts its action in the development of liver fibrosis.Elevated TGF-β activity in chronic fibrotic diseases is often accompanied by elevated expression of a matricellular protein, connective tissue growth factor (CTGF/CCN2).¹¹ The manifestation of CTGF/CCN2 functions in vivo involves cooperative interactions with costimulatory factors in the microenvironment, such as TGF-β and fibronectin. One hypothesis arising from the in vivo models is that fibrosing liver injuries are exacerbated by the action of TGF-β–mediated CTGF/CCN2.¹² Here, we addressed the extent to which fibrosis is dependent on the TGF-β/CTGF/CCN2 axis in chronic liver injury using an adult mouse model lacking liver TGF-β type II receptors (TGF-βIIR).     

Expression of the CD7 Ligand K-12 in Human Thymic Epithelial Cells: Regulation by IFN-γ

CD7 is an immunoglobulin superfamily molecule expressed on T, NK, and pre-B lymphocytes. Previous studies have demonstrated a role for CD7 in T- and NK-cell activation and cytokine production. Recently, an epithelial cell secreted protein, K12, was identified as a CD7 ligand. Although CD7 is expressed intrathymically, it is not known if K12 is produced in human thymus. To determine roles that K12 might play in the human thymus, we analyzed expression of K12 in human thymocytes, thymic epithelial cells (TE), and thymic fibroblasts. We found that recombinant human K12 bound strongly to soluble hCD7, with a K_eq of 37.6×10^–9M, and this interaction was inhibited by a novel antihuman K12 monoclonal antibody (K12-A1). K12 mRNA was detected by RT–PCR and northern analysis in human TE and thymic fibroblasts, but not in human thymocytes. Expression of K12 in TE cells was upregulated by IFN- . Taken together, these data demonstrated that K12 is produced by human TE cells and thymic fibroblasts, and is regulated in thymus by IFN- . These data suggest a role for thymic microenvironment-produced K12 in regulation of thymocyte signaling and cytokine release, particularly in the setting of thymus pathology where IFN- is upregulated such as myasthenia gravis.     

Interleukin-10- and Transforming Growth Factor β-Independent Regulation of CD8+ T Cells Expressing Type 1 and Type 2 Cytokines in Human Lymphatic Filariasis

Lymphatic filariasis is known to be associated with diminished CD4⁺ Th1 and elevated CD4⁺ Th2 responses to parasite-specific antigens. The roles of cytokine-expressing CD8⁺ T cells in immune responses to filarial infections are not well defined. To study the roles of CD8⁺ T cells expressing type 1, type 2, and type 17 cytokines in filarial infections, we examined the frequencies of these cells in clinically asymptomatic, patently infected (INF) individuals, directly ex vivo and in response to parasite or nonparasite antigens; these frequencies were compared with the results for individuals with filarial lymphedema (i.e., clinical pathology [CP]) and those without active infection or pathology (i.e., endemic normal [EN]). INF individuals exhibited significant decreases in the frequencies of CD8⁺ T cells expressing tumor necrosis factor alpha (TNF-α), gamma interferon (IFN-γ), and interleukin-22 (IL-22) at baseline and/or in response to filarial antigens, compared with CP and EN individuals. In contrast, the same individuals exhibited significant increases in the frequencies of CD8⁺ T cells expressing IL-4, IL-5, IL-9, IL-13, and IL-21, compared with CP and/or EN individuals. Curative treatment resulted in significantly increased frequencies of CD8⁺ T cells expressing IL-2 and significantly decreased frequencies of CD8⁺ T cells expressing type 2 cytokines. Finally, the regulation of these responses appears to be independent of IL-10 and transforming growth factor β (TGF-β), since blockade of IL-10 or TGF-β signaling did not significantly alter the frequencies of type 1 or type 2 cytokine-expressing CD8⁺ T cells. Our findings suggest that alterations in the frequencies of cytokine-expressing CD8⁺ T cells are characteristic features of lymphatic filarial infections.