The multiple roles of Thy-1 in cell differentiation and regeneration.

Thy-1 is a 25-37 kDa glycophosphatidylinositol (GPI)-anchored cell surface protein that was discovered more than 50 years ago. Recent findings have suggested that Thy-1 is expressed on thymocytes, mesenchymal stem cells (MSCs), cancer stem cells, hematopoietic stem cells, fibroblasts, myofibroblasts, endothelial cells, neuronal smooth muscle cells, and pan T cells. Thy-1 plays vital roles in cell migration, adhesion, differentiation, transdifferentiation, apoptosis, mechanotransduction, and cell division, which in turn are involved in tumor development, pulmonary fibrosis, neurite outgrowth, and T cell activation. Studies have increasingly indicated a significant role of Thy-1 in cell differentiation and regeneration. However, despite recent research, many questions remain regarding the roles of Thy-1 in cell differentiation and regeneration. This review aimed to summarize the roles of Thy-1 in cell differentiation and regeneration. Furthermore, since Thy-1 is an outer leaflet membrane protein anchored by GPI, we attempted to address how Thy-1 regulates intracellular pathways through cis and trans signals. Due to the complexity and mystery surrounding the issue, we also summarized the Thy-1-related pathways in different biological processes, and this might provide novel insights in the field of cell differentiation and regeneration.

Inhibition of mTOR delayed but could not prevent experimental collapsing focal segmental glomerulosclerosis.

Anti-Thy1.1 transgenic mice develop glomerular lesions that mimic collapsing focal segmental glomerulosclerosis (FSGS) in humans with collapse of the glomerular tuft and marked hyperplasia of the parietal epithelial cells (PECs). Immunostaining of phosphor-S6 ribosomal protein (pS6RP) revealed high mTOR activity in PECs of the FSGS lesions of these mice. In this study we questioned whether the mTOR inhibitor rapamycin (sirolimus) could attenuate the development and progression of glomerulosclerotic lesions in the anti-Thy1.1 transgenic mice. We observed reduced mTOR signalling and proliferation in human parietal epithelial cells after rapamycin treatment. Experiments with anti-Thy1.1. mice showed that early treatment with sirolimus reduced the development of glomerular lesions and glomerular cell proliferation at day 4. Levels of albuminuria, podocyte injury and podocyte number were similar in the sirolimus and vehicle treated groups. The initial beneficial effects of sirolimus treatment were not observed at day 7. Late sirolimus treatment did not reduce albuminuria or the progression of glomerulosclerosis. Taken together, rapamycin attenuated PEC proliferation and the formation of early FSGS lesions in experimental FSGS and reduced human PEC proliferation in vitro. However, the initial inhibition of PEC proliferation did not translate into a decline of albuminuria nor in a sustained reduction in sclerotic lesions.

CD90 promotes cell migration, viability and sphere­forming ability of hepatocellular carcinoma cells.

Cluster of differentiation (CD)90 (Thy­1) was proposed as a marker for the liver cancer stem cells that are responsible for tumorigenic activity, however its involvement in the progression of hepatocellular carcinoma (HCC) remains unknown. The aim of the present study was to determine the effect of CD90 on the biological functions of HCC and to investigate the associated circular RNA (circRNA) involved in this process. The analysis of the in vitro data demonstrated that CD90+ cells isolated from SK­Hep­1 cells exhibited increased viability, migration and invasive abilities compared with CD90­ cells. In addition, circRNA expression profiles in CD90+ and CD90­ cells were screened using a microarray assay and hsa_circ_0067531 and hsa_circ_0057096 were identified to be expressed at significantly different levels. It was additionally demonstrated that the expression of hsa_circ_0067531 in HCC tissues was significantly decreased compared with normal adjacent tissues. Overall, the results of the present study suggested that CD90 may be used as a potential biomarker for HCC. Furthermore, it was demonstrated that hsa_circ_0067531 may affect the biological functions of CD90+ HCC cells and may be a promising candidate to aid in the diagnosis and therapy of HCC.

Single-molecule measurements of the effect of force on Thy-1/αvß3-integrin interaction using nonpurified proteins.

Thy-1 and αvß3 integrin mediate bidirectional cell-to-cell communication between neurons and astrocytes. Thy-1/αvß3 interactions stimulate astrocyte migration and the retraction of neuronal prolongations, both processes in which internal forces are generated affecting the bimolecular interactions that maintain cell-cell adhesion. Nonetheless, how the Thy-1/αvß3 interactions respond to mechanical cues is an unresolved issue. In this study, optical tweezers were used as a single-molecule force transducer, and the Dudko-Hummer-Szabo model was applied to calculate the kinetic parameters of Thy-1/αvß3 dissociation. A novel experimental strategy was implemented to analyze the interaction of Thy-1-Fc with nonpurified αvß3-Fc integrin, whereby nonspecific rupture events were corrected by using a new mathematical approach. This methodology permitted accurately estimating specific rupture forces for Thy-1-Fc/αvß3-Fc dissociation and calculating the kinetic and transition state parameters. Force exponentially accelerated Thy-1/αvß3 dissociation, indicating slip bond behavior. Importantly, nonspecific interactions were detected even for purified proteins, highlighting the importance of correcting for such interactions. In conclusion, we describe a new strategy to characterize the response of bimolecular interactions to forces even in the presence of nonspecific binding events. By defining how force regulates Thy-1/αvß3 integrin binding, we provide an initial step towards understanding how the neuron-astrocyte pair senses and responds to mechanical cues.

Multiple roles of CD90 in cancer.

THY1 (CD90) is a 25-37-kDa heavily N-glycosylated, glycophosphatidylinositol (GPI) anchored cell surface protein. It is usually expressed on thymocytes, mesenchymal stem cells, hematopoietic stem cells, natural killer cells, neurons, endothelial cells, renal glomerular mesangial cells, follicular dendritic cells, fibroblasts, and myofibroblasts. It has been found to regulate cell adhesion, migration, apoptosis, axon growth, cell-cell and cell-matrix interactions, T-cell activation, and fibrosis. Several reports have shown that CD90 has an important role in cancer in regulating cancer cell proliferation, metastasis, and angiogenesis. There are also evidences that CD90 is an important prognostic marker in many cancers. Consequently, therapies that target CD90 have great promise in treating many cancers. However, several studies also indicate a contradictory role for CD90, where it acts as a tumor suppressor. In this review, we summarize the expression, function of CD90 in different cancers and its possible use as a biomarker or a therapeutic target in cancer. The challenges and future prospects for the use of CD90 for clinical applications are also discussed in this review.

Rabbit olfactory stem cells. Isolation protocol and characterization.

PURPOSE: To describe a new technique for isolation of a mesenchymal stem cells (MSCs) population from the olfactory mucosa in rabbits. METHODS: Olfactory stem cells (OSCs) were retrieved from under the cribriform plate of the Ethmoid bone. Several assays were accomplished to characterize the cell population and attest its viability in vitro. The cells were submitted to flow cytometry with the antibodies CD34, CD45, CD73, CD79, CD90 and CD105 and also they were induced to differentiate in three lineages. Functional evaluation involved analysis of in vitro growth behavior, colony forming unit like fibroblasts (CFU-f) and cryopreservation response. Further transduction with Green Fluorescent Protein (GFP) was also performed. RESULTS: The OSCs showed mesenchymal features, as positive response to CD34, CD73 and CD90 antibodies and plasticity. Additionally, these cells have high proliferated rate, and they could be cultured through many passages and kept the ability to proliferate and differentiate after cryopreservation. The positive response to the transduction signalizes the possibility of cellular tracking in vivo. This is a desirable feature in case those cells are used for pre-clinical trials. CONCLUSION: The cells harvested were mesenchymal stem cells and the technique described is therefore efficient for rabbit olfactory stem cells isolation.

Rabbit olfactory stem cells. Isolation protocol and characterization

PURPOSE: To describe a new technique for isolation of a mesenchymal stem cells (MSCs) population from the olfactory mucosa in rabbits. METHODS: Olfactory stem cells (OSCs) were retrieved from under the cribriform plate of the Ethmoid bone. Several assays were accomplished to characterize the cell population and attest its viability in vitro. The cells were submitted to flow cytometry with the antibodies CD34, CD45, CD73, CD79, CD90 and CD105 and also they were induced to differentiate in three lineages. Functional evaluation involved analysis of in vitro growth behavior, colony forming unit like fibroblasts (CFU-f) and cryopreservation response. Further transduction with Green Fluorescent Protein (GFP) was also performed. RESULTS: The OSCs showed mesenchymal features, as positive response to CD34, CD73 and CD90 antibodies and plasticity. Additionally, these cells have high proliferated rate, and they could be cultured through many passages and kept the ability to proliferate and differentiate after cryopreservation. The positive response to the transduction signalizes the possibility of cellular tracking in vivo. This is a desirable feature in case those cells are used for pre-clinical trials. CONCLUSION: The cells harvested were mesenchymal stem cells and the technique described is therefore efficient for rabbit olfactory stem cells isolation.

Bleomycin-Treated Chimeric Thy1-Deficient Mice with Thy1-Deficient Myofibroblasts and Thy-Positive Lymphocytes Resolve Inflammation without Affecting the Fibrotic Response.

Lung fibrosis is characterized by abnormal accumulation of fibroblasts in the interstitium of the alveolar space. Two populations of myofibroblasts, distinguished by Thy1 expression, are detected in human and murine lungs. Accumulation of Thy1-negative (Thy1(-)) myofibroblasts was shown in the lungs of humans with idiopathic pulmonary fibrosis (IPF) and of bleomycin-treated mice. We aimed to identify genetic changes in lung myofibroblasts following Thy1 crosslinking and assess the impact of specific lung myofibroblast Thy1-deficiency, in vivo, in bleomycin-injured mouse lungs. Thy1 increased in mouse lung lymphocytes following bleomycin injury but decreased in myofibroblasts when fibrosis was at the highest point (14 days), as assessed by immunohistochemistry. Using gene chip analysis, we detected that myofibroblast Thy1 crosslinking mediates downregulation of genes promoting cell proliferation, survival, and differentiation, and reduces production of extracellular matrix (ECM) components, while concurrently mediating the upregulation of genes known to foster inflammation and immunological functions. Chimeric Thy1-deficient mice with Thy1(+) lymphocytes and Thy1(-) myofibroblasts showed fibrosis similar to wild-type mice and an increased number of CD4/CD25 regulatory T cells, with a concomitant decrease in inflammation. Lung myofibroblasts downregulate Thy1 expression to increase their proliferation but to diminish the in vivo inflammatory milieu. Inflammation is not essential for evolution of fibrosis as was previously stated.

Mechanism of mast cell adhesion to human tenocytes in vitro.

Mast cells and fibroblasts are two key players involved in many fibrotic and degenerative disorders. In the present study we examined the nature of binding interactions between human mast cells and tendon fibroblasts (tenocytes). In the mast cell-fibroblast co-culture model, mast cells were shown to spontaneously bind to tenocytes, in a process that was partially mediated by α5ß1 integrin receptors. The same receptors on mast cells significantly mediated binding of these cells to tissue culture plates in the presence of tenocyte-conditioned media; the tenocyte-derived fibronectin in the media was shown to also play a major role in these binding activities. Upon binding to tenocytes or tissue culture plates, mast cells acquired an elongated phenotype, which was dependent on α5ß1 integrin and tenocyte fibronectin. Additionally, tenocyte-derived fibronectin significantly enhanced mRNA expression of the adhesion molecule, THY1, by mast cells. Our data suggests that α5ß1 integrin mediates binding of mast cells to human tenocyte and to tenocyte-derived ECM proteins, in particular fibronectin.

Thy-1 modulates neurological cell-cell and cell-matrix interactions through multiple molecular interactions.

Thy-1, or CD90, is a glycosylphosphatidylinositol-linked cell surface glycoprotein expressed on multiple cell types, including neurons, thymocytes, fibroblasts, endothelial cells, mesangial cells, and some hematopoietic and stromal stem cells. Thy-1 is developmentally regulated and evolutionarily conserved. Its cellular effects vary between and in some cases within cell types, tissues, and species, indicating that its biological role is context dependent. However, it most often seems to affect cell-cell or cell-matrix interactions and cellular adhesion and migration. In the nervous system, Thy-1 mediates bidirectional cell-cell communication, which modulates cell-matrix adhesion. Neurons express high levels of Thy-1, which interacts with alpha(v)beta3 integrin present in astrocytes and stimulates increased astrocyte adhesion to the underlying surface (trans signaling) and in neurites, the same ligand-receptor association triggers neurite retraction and inhibition of axonal growth (cis signaling). Although Thy-1 lacks a cytoplasmic domain, it affects multiple intracellular signaling cascades through interaction with a number of molecules within lipid raft microdomains. Improved understanding of how this enigmatic adhesion molecule modulates signaling and cell phenotype may yield novel insights into neurodevelopment and nerve recovery after injury.

Thy1-expressing neurons in the basolateral amygdala may mediate fear inhibition.

Research has identified distinct neuronal circuits within the basolateral amygdala (BLA) that differentially mediate fear expression versus inhibition; however, molecular markers of these populations remain unknown. Here we examine whether optogenetic activation of a cellular subpopulation, which may correlate with the physiologically identified extinction neurons in the BLA, would differentially support fear conditioning versus fear inhibition/extinction. We first molecularly characterized Thy1-channelrhodopsin-2 (Thy1-ChR2-EYFP)-expressing neurons as a subpopulation of glutamatergic pyramidal neurons within the BLA. Optogenetic stimulation of these neurons inhibited a subpopulation of medial central amygdala neurons and shunted excitation from the lateral amygdala. Brief activation of these neurons during fear training disrupted later fear memory in male mice. Optogenetic activation during unreinforced stimulus exposure enhanced extinction retention, but had no effect on fear expression, locomotion, or open-field behavior. Together, these data suggest that the Thy1-expressing subpopulation of BLA pyramidal neurons provide an important molecular and pharmacological target for inhibiting fear and enhancing extinction and for furthering our understanding of the molecular mechanisms of fear processing.

Brimonidine protects against loss of Thy-1 promoter activation following optic nerve crush.

BACKGROUND: The loss of RGCs expressing Thy-1 after optic nerve injury has an initial phase of rapid decline followed by a longer phase with slower reduction rate. This study used longitudinal retinal imaging of mice expressing cyan fluorescent protein under control of the Thy-1 promoter (Thy1-CFP mice) to determine how the α2-adrenergic agonist brimonidine influences loss of Thy1 promoter activation. METHODS: Baseline images of the fluorescent retinal neurons in 30 Thy1-CFP mice were obtained using a modified confocal scanning laser ophthalmoscope. Next, brimonidine (100 ug/kg, IP) was administered either one time immediately after optic nerve crush, or immediately after optic nerve crush and then every 2 days for four weeks. A control group received a single saline injection immediately after optic nerve crush. All animals were imaged weekly for four weeks after optic nerve crush. Loss of fluorescent retinal neurons within specific retinal areas was determined by counting. RESULTS: At one week after optic nerve crush, the proportion of fluorescent retinal neurons retaining fluorescence was 44±7% of baseline in control mice, 51±6% after one brimonidine treatment, and 55±6% after brimonidine treatment every other day (P<0.05 for both brimonidine treatment groups compared to the control group). Subsequently, the number of fluorescent retinal neurons in the group that received one treatment differed insignificantly from the control group. In contrast, the number of fluorescent retinal neurons in the group that received repeated brimonidine treatments was greater than the control group by 28% at two weeks after crush and by 32% at three weeks after crush (P<0.05 at both time points). Rate analysis showed that brimonidine slowed the initial rate of fluorescent cell decline in the animals that received multiple treatments (P<0.05). Differences in the rate of loss among the treatment groups were insignificant after the second week. CONCLUSION: Repeated brimonidine treatments protect against loss of fluorescence within fluorescent retinal neurons of Thy1-CFP mice after optic nerve crush. As most of fluorescent retinal neurons in this system are RGCs, these findings indicate that repeated brimonidine treatments may protect RGC health following optic nerve crush.

Canine intra-articular multipotent stromal cells (MSC) from adipose tissue have the highest in vitro expansion rates, multipotentiality, and MSC immunophenotypes.

OBJECTIVE: To identify the optimum intra-articular multipotent stromal cell (MSC) tissue source in the canine stifle. STUDY DESIGN: Experimental. SAMPLE POPULATION: Infrapatellar adipose tissue, synovium lining the joint capsule, and synovium surrounding the cranial cruciate ligament (CrCL) from normal stifles of 6 dogs. METHODS: Nucleated cell density for each tissue was determined, and cell doublings (CD) and doubling times (DT) were quantified for expansion rates. Adipogenic, osteogenic, and chondrogenic differentiation was confirmed with light microscopy. Fibroblastic, adipogenic, and osteogenic colony forming unit frequencies were determined for multipotentiality. Tissue-specific target gene expression was assessed, and percentages of CD29(+) , CD34(+) , CD44(+) , CD45(+) , and CD90(+) cells quantified. RESULTS: Adipose tissue had the highest MSC density (ASC). The CD decreased with increasing passages for all cell types, and ASC values tended to be higher. Multipotentiality decreased with passage, but remained highest in ASC. Tissue-specific target gene expression was higher in induced versus noninduced cells, and ASCs had the highest upregulation across passages. Most cells were CD29(+) , CD44(+) , CD90(+) , and percentages decreased with passage. Within cell types, there were more CD29(+) ASC in early passages and more CD44(+) and CD90(+) ASC in later passages. CONCLUSIONS: ASC had the highest in vitro expansion rates, CFU frequencies, tissue-specific target gene expression, and percentages of MSC immunophenotypes.

Melanoma cells use Thy-1 (CD90) on endothelial cells for metastasis formation.

The cell adhesion molecule Thy-1 (CD90) mediates the adhesion of melanoma cells to activated human endothelial cells (EC) via the interaction with the αvß3-integrin on the tumor cells in vitro. Here, we report a strong expression of Thy-1 on both blood vessel and lymphatic EC in melanoma and melanoma metastases. Vascular endothelial growth factor and tumor necrosis factor-α were identified as inducers of Thy-1 expression on EC in vitro. The physiological role of Thy-1 for lymphogenic and hematogenic metastasis of melanoma cells was substantiated in an experimental metastasis model using B16/F10 melanoma cells. Mice lacking Thy-1 showed markedly diminished experimental lung metastasis after injection of B16/F10 melanoma cells compared to wild-type littermate controls. In addition, on generation of a primary subcutaneous tumor, metastasis to regional lymph nodes was clearly reduced in Thy-1(-/-) mice. However, Thy-1 deletion did not affect subcutaneous primary tumor growth, tumor-induced recruitment of inflammatory cells or T cells, angiogenesis, or T-cell activation. In conclusion, Thy-1 contributes to metastasis of melanoma cells by mechanisms likely involving a Thy-1-mediated adhesion of melanoma cells to EC.

Heligmosomoides polygyrus abrogates antigen-specific gut injury in a murine model of inflammatory bowel disease.

BACKGROUND: Developing countries have a low incidence of inflammatory bowel disease (IBD), perhaps prevented by the high prevalence of helminth infections and other alterations in intestinal flora and fauna. Helminth infections prevent colitis in various murine models of IBD. IBD may be driven by an aberrant immune response to luminal antigen(s). METHODS: We developed a murine model of IBD in which gut injury was induced by a specific antigen to better simulate the IBD disease process and to determine if helminth infections could abolish gut injury induced by an orally administered antigen. The model features pan-enterocolitis triggered by feeding ovalbumin (OVA). RESULTS: The intestinal inflammation is antigen-specific and generates interleukin (IL)-17 and interferon-gamma (IFN-γ), but not IL-4. Full expression of the disease required T cells with defective capacity to make IL-10 and treatment with a noninjurious, low dose of a nonsteroidal antiinflammatory drug. Exposure to Heligmosomoides polygyrus abrogated this antigen-induced gut injury. H. polygyrus colonization induced Foxp3(+) T regulatory cells (Tregs) and mucosal production of IL-10 from non-T cells. Lamina propria mononuclear cells from H. polygyrus-infected mice released less IL-17 and IFN-γ constitutively and when stimulated with OVA or anti-CD3/CD28 monoclonal antibodies. CONCLUSIONS: We developed a murine IBD model featuring antigen-specific enterocolitis and demonstrate for the first time that gut inflammation induced by an antigen could be abrogated by H. polygyrus infection. Protection was associated with suppressed IL-17 and IFN-γ production, induction of Foxp3(+) Tregs, and elevated secretion of non-T-cell-derived IL-10, all of which could be part of the protective processes.

Two lysines in the forkhead domain of foxp3 are key to T regulatory cell function.

BACKGROUND: The forkhead box transcription factor, Foxp3, is master regulator of the development and function of CD4+CD25+ T regulatory (Treg) cells that limit autoimmunity and maintain immune homeostasis. The carboxyl-terminal forkhead (FKH) domain is required for the nuclear localization and DNA binding of Foxp3. We assessed how individual FKH lysines contribute to the functions of Foxp3 in Treg cells. METHODOLOGY/PRINCIPAL FINDINGS: We found that mutation of FKH lysines at position 382 (K17) and at position 393 (K18) impaired Foxp3 DNA binding and inhibited Treg suppressive function in vivo and in vitro. These lysine mutations did not affect the level of expression of Foxp3 but inhibited IL-2 promoter remodeling and had important and differing effects on Treg-associated gene expression. CONCLUSIONS/SIGNIFICANCE: These data point to complex effects of post-translational modifications at individual lysines within the Foxp3 FKH domain that affect Treg function. Modulation of these events using small molecule inhibitors may allow regulation of Foxp3+ Treg function clinically.

Reggie/flotillin and the targeted delivery of cargo.

The two proteins reggie-1/flotillin-2 and reggie-2/flotillin-1 form microdomains at the plasma membrane and at intracellular compartments where src tyrosine kinases associate with them. Specific GPI-anchored proteins, in particular prion protein and Thy-1, co-cluster with reggie microdomains at the plasma membrane and elicit signal transduction in association with reggies which regulates the activation of several GTPases involved in the recruitment of specific membrane proteins from intracellular carriers to target sites of the cell membrane in a cell type-specific manner. For example, prion protein and reggie regulate the recruitment and targeted delivery of the T cell receptor complex to the T cell cap, of E-cadherin to cell-cell contact sites in epithelial cells, and of bulk membrane and growth receptors to the growth cone in developing neurons. Evidence is accumulating that reggies are involved in guiding the cell-type-specific membrane proteins from the intracellular compartments to their target sites at the cell membrane, a function required in all cells which explains why reggies are expressed in many or all cells in invertebrates and vertebrates.

Inhibition of osteoclast function reduces hematopoietic stem cell numbers in vivo.

Osteoblasts play a crucial role in the hematopoietic stem cell (HSC) niche; however, an overall increase in their number does not necessarily promote hematopoiesis. Because the activity of osteoblasts and osteoclasts is coordinately regulated, we hypothesized that active bone-resorbing osteoclasts would participate in HSC niche maintenance. Mice treated with bisphosphonates exhibited a decrease in proportion and absolute number of Lin(-)cKit(+)Sca1(+) Flk2(-) (LKS Flk2(-)) and long-term culture-initiating cells in bone marrow (BM). In competitive transplantation assays, the engraftment of treated BM cells was inferior to that of controls, confirming a decrease in HSC numbers. Accordingly, bisphosphonates abolished the HSC increment produced by parathyroid hormone. In contrast, the number of colony-forming-unit cells in BM was increased. Because a larger fraction of LKS in the BM of treated mice was found in the S/M phase of the cell cycle, osteoclast impairment makes a proportion of HSCs enter the cell cycle and differentiate. To prove that HSC impairment was a consequence of niche manipulation, a group of mice was treated with bisphosphonates and then subjected to BM transplantation from untreated donors. Treated recipient mice experienced a delayed hematopoietic recovery compared with untreated controls. Our findings demonstrate that osteoclast function is fundamental in the HSC niche.

Thy-1 attenuates TNF-alpha-activated gene expression in mouse embryonic fibroblasts via Src family kinase.

Heterogeneous surface expression of Thy-1 in fibroblasts modulates inflammation and may thereby modulate injury and repair. As a paradigm, patients with idiopathic pulmonary fibrosis, a disease with pathologic features of chronic inflammation, demonstrate an absence of Thy-1 immunoreactivity within areas of fibrotic activity (fibroblast foci) in contrast to the predominant Thy-1 expressing fibroblasts in the normal lung. Likewise, Thy-1 deficient mice display more severe lung fibrosis in response to an inflammatory injury than wildtype littermates. We investigated the role of Thy-1 in the response of fibroblasts to the pro-inflammatory cytokine TNF-alpha. Our study demonstrates distinct profiles of TNF-alpha-activated gene expression in Thy-1 positive (Thy-1+) and negative (Thy-1-) subsets of mouse embryonic fibroblasts (MEF). TNF-alpha induced a robust activation of MMP-9, ICAM-1, and the IL-8 promoter driven reporter in Thy-1- MEFs, in contrast to only a modest increase in Thy-1+ counterparts. Consistently, ectopic expression of Thy-1 in Thy-1- MEFs significantly attenuated TNF-alpha-activated gene expression. Mechanistically, TNF-alpha activated Src family kinase (SFK) only in Thy-1- MEFs. Blockade of SFK activation abrogated TNF-alpha-activated gene expression in Thy-1- MEFs, whereas restoration of SFK activation rescued the TNF-alpha response in Thy-1+ MEFs. Our findings suggest that Thy-1 down-regulates TNF-alpha-activated gene expression via interfering with SFK- and NF-kappaB-mediated transactivation. The current study provides a novel mechanistic insight to the distinct roles of fibroblast Thy-1 subsets in inflammation.

Functional characterization of THY1 as a tumor suppressor gene with antiinvasive activity in nasopharyngeal carcinoma.

THY1 was previously identified as a candidate tumor suppressor gene (TSG) associated with lymph node metastases in nasopharyngeal carcinoma (NPC) through functional studies. It was identified by oligonucleotide microarray analysis as an interesting differentially expressed gene. However, direct functional evidence is still lacking for THY1 being a TSG in NPC, as in vivo tumorigenicity assays have not been previously reported in our last study of THY1. In this study, a tetracycline-inducible expression vector, pETE-Bsd, was used to obtain stable transfectants of THY1. The stringent in vivo tumorigenicity assay results show that the activation of THY1 suppresses tumor formation of HONE1 cells in nude mice, and the tumor formation ability was restored in the presence of doxycycline (a tetracycline analog), when the gene is shut off. Functional inactivation of this gene is observed in all the tumors derived from the tumorigenic transfectant. The tumor suppressive effect could be repressed by knockdown of THY1 expression in nontumorigenic microcell hybrids. Further studies indicate that expression of THY1 inhibits HONE1 cell growth in vitro by arresting cells in G(0)/G(1) phase. It greatly reduces the ability for anchorage-independent growth. The invasiveness of HONE1 cells was also inhibited by the expression of THY1. These findings suggest that THY1 is a TSG in NPC, which is involved in invasion and shows an association with tumor metastasis. Taken together, THY1 clearly plays an important functional role in tumor suppression in NPC.