The autophagy protein Ambra1 regulates gene expression by supporting novel transcriptional complexes.

Ambra1 is considered an autophagy and trafficking protein with roles in neurogenesis and cancer cell invasion. Here, we report that Ambra1 also localizes to the nucleus of cancer cells, where it has a novel nuclear scaffolding function that controls gene expression. Using biochemical fractionation and proteomics, we found that Ambra1 binds to multiple classes of proteins in the nucleus, including nuclear pore proteins, adaptor proteins such as FAK and Akap8, chromatin-modifying proteins, and transcriptional regulators like Brg1 and Atf2. We identified biologically important genes, such as Angpt1, Tgfb2, Tgfb3, Itga8, and Itgb7, whose transcription is regulated by Ambra1-scaffolded complexes, likely by altering histone modifications and Atf2 activity. Therefore, in addition to its recognized roles in autophagy and trafficking, Ambra1 scaffolds protein complexes at chromatin, regulating transcriptional signaling in the nucleus. This novel function for Ambra1, and the specific genes impacted, may help to explain the wider role of Ambra1 in cancer cell biology.

Hypoxic tumor microenvironment activates GLI2 via HIF-1α and TGF-ß2 to promote chemoresistance in colorectal cancer.

Colorectal cancer patients often relapse after chemotherapy, owing to the survival of stem or progenitor cells referred to as cancer stem cells (CSCs). Although tumor stromal factors are known to contribute to chemoresistance, it remains not fully understood how CSCs in the hypoxic tumor microenvironment escape the chemotherapy. Here, we report that hypoxia-inducible factor (HIF-1α) and cancer-associated fibroblasts (CAFs)-secreted TGF-ß2 converge to activate the expression of hedgehog transcription factor GLI2 in CSCs, resulting in increased stemness/dedifferentiation and intrinsic resistance to chemotherapy. Genetic or small-molecule inhibitor-based ablation of HIF-1α/TGF-ß2-mediated GLI2 signaling effectively reversed the chemoresistance caused by the tumor microenvironment. Importantly, high expression levels of HIF-1α/TGF-ß2/GLI2 correlated robustly with the patient relapse following chemotherapy, highlighting a potential biomarker and therapeutic target for chemoresistance in colorectal cancer. Our study thus uncovers a molecular mechanism by which hypoxic colorectal tumor microenvironment promotes cancer cell stemness and resistance to chemotherapy and suggests a potentially targeted treatment approach to mitigating chemoresistance.

Transcriptional Analysis of Endothelial Cell Alternation Induced by Atrial Natriuretic Polypeptide in Human Umbilical Vein Endothelial Cells.

The aim of this study was to explore how atrial natriuretic polypeptide (ANP) affects the properties and function of endothelial cells. Gene expression data GSE56976 generated at 0, 1, and 6 hours after ANP incubation in human umbilical vein endothelial cells (HUVEC) was used. Microarray data were preprocessed for differentially expressed genes (DEGs) in each time-dependent group. Next, gene ontology (GO), pathway analysis, and transcriptional regulation were performed. Co-expression clustering analysis of DEGs and functional enrichment analysis of co-expression modules were processed. RT-PCR analysis was performed to validate gene expression. DEGs were obtained and their counts were increased from 0 hours to 6 hours. No overlapping DEGs were obtained among the 3 groups. The DEGs of ANP_6hours, including TGFB2 (transforming growth factor, beta 2), LTF (lactotransferrin/lactoferrin), and ETV7 (Ets variant 7) were mainly related with cell apoptosis and immune responses. The DEGs in the network of ANP_0hour were mainly associated with epithelial ion transport processes. In addition, 3 co-expressed modules were detected. CSF2 (colony stimulating factor 2) and PF4 (platelet factor 4) of the blue module were related with cytolysis, while FXYD1 (FXYD domain containing ion transport regulator 1) and TGFB2 of the yellow module were mainly enriched in ion transport and the ovulation cycle. The expression of TGFB2 obtained by microarray analysis was consistent with that of RT-PCR. Ion transport could be affected promptly after ANP treatment, and subsequently, the cytolysis of vein endothelial cells may be promoted and endothelial permeability would be enhanced, followed by activated immune responses.

Foxf2 is required for secondary palate development and Tgfß signaling in palatal shelf mesenchyme.

The secondary palate separates the oral from the nasal cavity and its closure during embryonic development is sensitive to genetic perturbations. Mice with deleted Foxf2, encoding a forkhead transcription factor, are born with cleft palate, and an abnormal tongue morphology has been proposed as the underlying cause. Here, we show that Foxf2(-/-) maxillary explants cultured in vitro, in the absence of tongue and mandible, failed to close the secondary palate. Proliferation and collagen content were decreased in Foxf2(-/-) palatal shelf mesenchyme. Phosphorylation of Smad2/3 was reduced in mutant palatal shelf, diagnostic of attenuated canonical Tgfß signaling, whereas phosphorylation of p38 was increased. The amount of Tgfß2 protein was diminished, whereas the Tgfb2 mRNA level was unaltered. Expression of several genes encoding extracellular proteins important for Tgfß signaling were reduced in Foxf2(-)(/)(-) palatal shelves: a fibronectin splice-isoform essential for formation of extracellular Tgfß latency complexes; Tgfbr3 - or betaglycan - which acts as a co-receptor and an extracellular reservoir of Tgfß; and integrins αV and ß1, which are both Tgfß targets and required for activation of latent Tgfß. Decreased proliferation and reduced extracellular matrix content are consistent with diminished Tgfß signaling. We therefore propose that gene expression changes in palatal shelf mesenchyme that lead to reduced Tgfß signaling contribute to cleft palate in Foxf2(-)(/)(-) mice.

Effect of tamoxifen on fibrosis, collagen content and transforming growth factor-ß1, -ß2 and -ß3 expression in common bile duct anastomosis of pigs.

End-to-end anastomosis in the treatment for bile duct injury during laparoscopic cholecystectomy has been associated with stricture formation. The aim of this study was to experimentally investigate the effect of oral tamoxifen (tmx) treatment on fibrosis, collagen content and transforming growth factor-ß1, -ß2 and -ß3 expression in common bile duct anastomosis of pigs. Twenty-six pigs were divided into three groups [sham (n = 8), control (n = 9) and tmx (n = 9)]. The common bile ducts were transected and anastomosed in the control and tmx groups. Tmx (40 mg/day) was administered orally to the tmx group, and the animals were euthanized after 60 days. Fibrosis was analysed by Masson's trichrome staining. Picrosirius red was used to quantify the total collagen content and collagen type I/III ratio. mRNA expression of transforming growth factor (TGF)-ß1, -ß2 and -ß3 was quantified using real-time polymerase chain reaction (qRT-PCR). The control and study groups exhibited higher fibrosis than the sham group, and the study group showed lower fibrosis than the control group (P = 0.011). The control and tmx groups had higher total collagen content than the sham group (P = 0.003). The collagen type I/III ratio was higher in the control group than in the sham and tmx groups (P = 0.015). There were no significant differences in the mRNA expression of TGF-ß1, -ß2 and -ß3 among the groups (P > 0.05). Tmx decreased fibrosis and prevented the change in collagen type I/III ratio caused by the procedure.

Endothelial-to-mesenchymal transition in lipopolysaccharide-induced acute lung injury drives a progenitor cell-like phenotype.

Pulmonary vascular endothelial function may be impaired by oxidative stress in endotoxemia-derived acute lung injury. Growing evidence suggests that endothelial-to-mesenchymal transition (EndMT) could play a pivotal role in various respiratory diseases; however, it remains unclear whether EndMT participates in the injury/repair process of septic acute lung injury. Here, we analyzed lipopolysaccharide (LPS)-treated mice whose total number of pulmonary vascular endothelial cells (PVECs) transiently decreased after production of reactive oxygen species (ROS), while the population of EndMT-PVECs significantly increased. NAD(P)H oxidase inhibition suppressed EndMT of PVECs. Most EndMT-PVECs derived from tissue-resident cells, not from bone marrow, as assessed by mice with chimeric bone marrow. Bromodeoxyuridine-incorporation assays revealed higher proliferation of capillary EndMT-PVECs. In addition, EndMT-PVECs strongly expressed c-kit and CD133. LPS loading to human lung microvascular endothelial cells (HMVEC-Ls) induced reversible EndMT, as evidenced by phenotypic recovery observed after removal of LPS. LPS-induced EndMT-HMVEC-Ls had increased vasculogenic ability, aldehyde dehydrogenase activity, and expression of drug resistance genes, which are also fundamental properties of progenitor cells. Taken together, our results demonstrate that LPS induces EndMT of tissue-resident PVECs during the early phase of acute lung injury, partly mediated by ROS, contributing to increased proliferation of PVECs.

Silibinin inhibits triple negative breast cancer cell motility by suppressing TGF-ß2 expression.

Transforming growth factor-beta (TGF-ß) is a multifunctional cytokine that regulates many biological events including cell motility and angiogenesis. Here, we investigated the role of elevated TGF-ß2 level in triple negative breast cancer (TNBC) cells and the inhibitory effect of silibinin on TGF-ß2 action in TNBC cells. Breast cancer patients with high TGF-ß2 expression have a poor prognosis. The levels of TGF-ß2 expression increased significantly in TNBC cells compared with those in non-TNBC cells. In addition, cell motility-related genes such as fibronectin (FN) and matrix metalloproteinase-2 (MMP-2) expression also increased in TNBC cells. Basal FN, MMP-2, and MMP-9 expression levels decreased in response to LY2109761, a dual TGF-ß receptor I/II inhibitor, in TNBC cells. TNBC cell migration also decreased in response to LY2109761. Furthermore, we observed that TGF-ß2 augmented the FN, MMP-2, and MMP-9 expression levels in a time- and dose-dependent manner. In contrast, TGF-ß2-induced FN, MMP-2, and MMP-9 expression levels decreased significantly in response to LY2109761. Interestingly, we found that silibinin decreased TGF-ß2 mRNA expression level but not that of TGF-ß1 in TNBC cells. Cell migration as well as basal FN and MMP-2 expression levels decreased in response to silibinin. Furthermore, silibinin significantly decreased TGF-ß2-induced FN, MMP-2, and MMP-9 expression levels and suppressed the lung metastasis of TNBC cells. Taken together, these results suggest that silibinin suppresses metastatic potential of TNBC cells by inhibiting TGF-ß2 expression in TNBC cells. Thus, silibinin may be a promising therapeutic drug to treat TNBC.

Rho GTPase signaling promotes constitutive expression and release of TGF-ß2 by human trabecular meshwork cells.

Elevated intraocular pressure (IOP) is causally implicated in the pathophysiology of primary open-angle glaucoma (POAG). The molecular mechanisms responsible for elevated IOP remain elusive, but may involve aberrant expression and signaling of transforming growth factor (TGF)-ß2 within the trabecular meshwork (TM). Consistent with previously published studies, we show here that exogenous addition of TGF-ß2 to cultured porcine anterior segments significantly attenuates outflow facility in a time-dependent manner. By comparison, perfusing segments with a TGFßRI/ALK-5 antagonist (SB-431542) unexpectedly elicited a significant and sustained increase in outflow facility, implicating a role for TM-localized constitutive expression and release of TGF-ß2. Consistent with this thesis, cultured primary or transformed (GTM3) quiescent human TM cells were found to constitutively express and secrete measurable amounts of biologically-active TGF-ß2. Disrupting monomeric GTPase post-translational prenylation and activation with lovastatin or GGTI-298 markedly reduced constitutive TGF-ß2 expression and release. Specifically, inhibiting the Rho subfamily of GTPases with C3 exoenzyme similarly reduced constitutive expression and secretion of TGF-ß2. These findings suggest that Rho GTPase signaling, in part, regulates constitutive expression and release of biologically-active TGF-ß2 from human TM cells. Localized constitutive expression and release of TGF-ß2 by TM cells may promote or exacerbate elevation of IOP in POAG.

Endothelial lineage differentiation from induced pluripotent stem cells is regulated by microRNA-21 and transforming growth factor ß2 (TGF-ß2) pathways.

Finding a suitable cell source for endothelial cells (ECs) for cardiovascular regeneration is a challenging issue for regenerative medicine. In this paper, we describe a novel mechanism regulating induced pluripotent stem cells (iPSC) differentiation into ECs, with a particular focus on miRNAs and their targets. We first established a protocol using collagen IV and VEGF to drive the functional differentiation of iPSCs into ECs and compared the miRNA signature of differentiated and undifferentiated cells. Among the miRNAs overrepresented in differentiated cells, we focused on microRNA-21 (miR-21) and studied its role in iPSC differentiation. Overexpression of miR-21 in predifferentiated iPSCs induced EC marker up-regulation and in vitro and in vivo capillary formation; accordingly, inhibition of miR-21 produced the opposite effects. Importantly, miR-21 overexpression increased TGF-ß2 mRNA and secreted protein level, consistent with the strong up-regulation of TGF-ß2 during iPSC differentiation. Indeed, treatment of iPSCs with TGFß-2 induced EC marker expression and in vitro tube formation. Inhibition of SMAD3, a downstream effector of TGFß-2, strongly decreased VE-cadherin expression. Furthermore, TGFß-2 neutralization and knockdown inhibited miR-21-induced EC marker expression. Finally, we confirmed the PTEN/Akt pathway as a direct target of miR-21, and we showed that PTEN knockdown is required for miR-21-mediated endothelial differentiation. In conclusion, we elucidated a novel signaling pathway that promotes the differentiation of iPSC into functional ECs suitable for regenerative medicine applications.

Hairless Up-Regulates Tgf-ß2 Expression via Down-Regulation of miR-31 in the Skin of "Hairpoor" (HrHp) Mice.

Hairless (HR) has been shown to regulate hair follicle (HF) morphogenesis and hair cycling. The Hr mutant hair loss mouse referred to as "hairpoor" (Hr(Hp)) displays overexpression of the HR protein through translational derepression. In this study, we found that 64 miRNAs were differentially expressed between the skin of Hr(Hp)/Hr(Hp) and wild type mice at P7 using miRNA-microarray analysis and miR-31 displayed the most reduced expression in Hr(Hp)/Hr(Hp) skin. In vivo observation and investigation using an in vitro reporter expression system revealed that miR-31 and pri-miR-31 were consistently down-regulated in the HR over-expressed condition. In addition, we found that the transforming growth factor ß2 (Tgf-ß2), a known catagen inducer, is the putative target of miR-31. Furthermore, Tgf-ß2 level was also increased in HR over-expressed keratinocyte and Hr(Hp)/Hr(Hp) mice. These study results suggest that HR controls Tgf-ß2 expression via regulation of miR-31, thus causing abnormal hair cycle in Hr(Hp)/Hr(Hp) mice.

Association of TGF-ß2 levels in breast milk with severity of breast biopsy diagnosis.

PURPOSE: TGF-ß plays a dual role in breast carcinogenesis, acting at early stages as tumor-suppressors and later as tumor-promoters. TGF-ß isoforms are expressed in breast tissues and secreted in milk, suggesting that analysis of levels in milk might be informative for breast cancer risk. Accordingly, we assessed TGF-ß2 levels in milk from women who had undergone a breast biopsy and related the concentrations to diagnosis. METHODS: Milk donated by women who had undergone or were scheduled for a breast biopsy was shipped on ice for processing and testing. Breast cancer risk factors were obtained through a self-administered questionnaire, and biopsy diagnoses were extracted from pathology reports. TGF-ß2 levels in milk, assessed as absolute levels and in relation to total protein, were analyzed in bilateral samples donated by 182 women. Linear regression was used to estimate relationships of log-transformed TGF-ß2 levels and TGF-ß2/ total protein ratios to biopsy category. RESULTS: Milk TGF-ß2 levels from biopsied and non-biopsied breasts within women were highly correlated (r (2) = 0.77). Higher mean TGF-ß2 milk levels (based on average of bilateral samples) were marginally associated with more severe breast pathological diagnosis, after adjusting for duration of nursing current child (adjusted p trend = 0.07). CONCLUSIONS: Our exploratory analysis suggests a borderline significant association between higher mean TGF-ß2 levels in breast milk and more severe pathologic diagnoses. Further analysis of TGF-ß signaling in milk may increase understanding of postpartum remodeling and advance efforts to analyze milk as a means of assessing risk of breast pathology.

Effects of ranibizumab on TGF-ß1 and TGF-ß2 production by human Tenon's fibroblasts: An in vitro study.

PURPOSE: Inhibiting exaggerated wound healing responses, which are primarily mediated by human Tenon's fibroblast (HTF) migration and proliferation, has become the major determining factor for a successful trabeculectomy. Antivascular endothelial growth factor (anti-VEGF) has showed promising results as a potential antifibrotic candidate for use concurrently in trabeculectomy. Preliminary cohort studies have revealed improved bleb morphology following trabeculectomy augmented with ranibizumab. However, the effects on HTFs remain unclear. This study was conducted to understand the effects of ranibizumab on transforming growth factor (TGF)-ß1 and transforming growth factor (TGF)-ß2 expression by HTFs. METHODS: The effect of ranibizumab on HTF proliferation and cell viability was determined using 3-(4,5-dimethylthiazone-2-yl)-2,5-diphenyl tetrazolium (MTT) assay. Ranibizumab at concentrations ranging from 0.01 to 0.5 mg/ml were administered for 24, 48, and 72 h in serum and serum-free conditions. Supernatants and cell lysates from samples were assessed for TGF-ß1 and TGF-ß2 mRNA and protein levels using quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA). RESULTS: At 48 h, 0.5 mg/ml of ranibizumab significantly induced cell death under serum-free culture conditions (p<0.05). Ranibizumab caused a significant reduction in TGF-ß1 mRNA, but not for TGF-ß2. However, the total protein production of TGF-ß1 and TGF-ß2 was unaffected by this anti-VEGF treatment. CONCLUSIONS: Exposure of HTFs to an intravitreal dose of ranibizumab significantly suppresses cell viability in vitro; however, the application seemed unable to affect the ultimate production of TGF-ß. Therefore, we highlighted ranibizumab as a potential antiscarring agent that acts via a different mechanism when used synergistically with another antifibrotic agent. Understanding the mechanism of actions of ranibizumab offers an additional view of a possible new rational therapeutic strategy.

Tumor suppressive microRNA-200a inhibits renal cell carcinoma development by directly targeting TGFB2.

A large body of evidence indicates that microRNAs play a critical role in tumor initiation and progression by negatively regulating oncogenes or tumor suppressor genes. Here, we report that the expression of miR-200a was notably downregulated in 45 renal cell carcinoma (RCC) samples. Restoration of miR-200a suppressed cell proliferation, migration, and invasion in two RCC cell lines. Furthermore, we used an epithelial-to-mesenchymal transition PCR array to explore the putative target genes of miR-200a. By performing quantitative real-time PCR, ELISA, and luciferase reporter assays, transforming growth factor beta2 (TGFB2) was validated as a direct target gene of miR-200a. Moreover, siRNA-mediated knockdown of TGFB2 partially phenocopied the effect of miR-200a overexpression. These results suggest that miR-200a suppresses RCC development via directly targeting TGFB2, indicating that miR-200a may present a novel target for diagnostic and therapeutic strategies in RCC.

Participation of miR-200a in TGF-ß1-mediated hepatic stellate cell activation.

Hepatic stellate cell (HSC) activation is a pivotal event in the initiation and progression of hepatic fibrosis since it mediates transforming growth factor beta 1 (TGF-ß1)-driven extracellular matrix (ECM) deposition. MicroRNAs (miRNAs), small non-coding RNAs modulating messenger RNA (mRNA) and protein expression, have emerged as key factors to regulate cell proliferation, differentiation, and apoptosis. Although the function of miR-200a has been discussed in many cancers and fibrotic diseases, its role in hepatic fibrosis is still poorly understood. The aim of this study is to investigate whether miR-200a could attenuate hepatic fibrosis partly through Wnt/ß-catenin and TGF-ß-dependant mechanisms. Our study found that the expression of endogenous miR-200a was decreased in vitro in TGF-ß1-induced HSC activation as well as in vivo in CCl4-induced rat liver fibrosis. Overexpression of miR-200a significantly inhibited α-SMA activity and further affected the proliferation of TGF-ß1-dependent activation of HSC. In addition, we identified ß-catenin and TGF-ß2 as two functional downstream targets for miR-200a. Interestingly, miR-200a specifically suppressed ß-catenin in the protein level, whereas miR-200a-mediated suppression of TGF-ß2 was shown on both mRNA and protein levels. Our results revealed the critical regulatory role of miR-200a in HSC activation and implied miR-200a as a potential candidate for therapy by deregulation of Wnt/ß-catenin and TGFß signaling pathways, at least in part, via decreasing the expression of ß-catenin and TGF-ß2.

Mycophenolate antagonizes IFN-γ-induced catagen-like changes via ß-catenin activation in human dermal papilla cells and hair follicles.

Recently, various immunosuppressant drugs have been shown to induce hair growth in normal hair as well as in alopecia areata and androgenic alopecia; however, the responsible mechanism has not yet been fully elucidated. In this study, we investigate the influence of mycophenolate (MPA), an immunosuppressant, on the proliferation of human dermal papilla cells (hDPCs) and on the growth of human hair follicles following catagen induction with interferon (IFN)-γ. IFN-γ was found to reduce ß-catenin, an activator of hair follicle growth, and activate glycogen synthase kinase (GSK)-3ß, and enhance expression of the Wnt inhibitor DKK-1 and catagen inducer transforming growth factor (TGF)-ß2. IFN-γ inhibited expression of ALP and other dermal papillar cells (DPCs) markers such as Axin2, IGF-1, and FGF 7 and 10. MPA increased ß-catenin in IFN-γ-treated hDPCs leading to its nuclear accumulation via inhibition of GSK3ß and reduction of DKK-1. Furthermore, MPA significantly increased expression of ALP and other DPC marker genes but inhibited expression of TGF-ß2. Therefore, we demonstrate for the first time that IFN-γ induces catagen-like changes in hDPCs and in hair follicles via inhibition of Wnt/ß-catenin signaling, and that MPA stabilizes ß-catenin by inhibiting GSK3ß leading to increased ß-catenin target gene and DP signature gene expression, which may, in part, counteract IFN-γ-induced catagen in hDPCs.

Biliary epithelial senescence and plasticity in acute cellular rejection.

Biliary epithelial cells (BEC) are important targets in some liver diseases, including acute allograft rejection. Although some injured BEC die, many can survive in function compromised states of senescence or phenotypic de-differentiation. This study was performed to examine changes in the phenotype of BEC during acute liver allograft rejection and the mechanism driving these changes. Liver allograft sections showed a positive correlation (p < 0.0013) between increasing T cell mediated acute rejection and the number of BEC expressing the senescence marker p21(WAF1/Cip) or the mesenchymal marker S100A4. This was modeled in vitro by examination of primary or immortalized BEC after acute oxidative stress. During the first 48 h, the expression of p21(WAF1/Cip) was increased transiently before returning to baseline. After this time BEC showed increased expression of mesenchymal proteins with a decrease in epithelial markers. Analysis of TGF-ß expression at mRNA and protein levels also showed a rapid increase in TGF-ß2 (p < 0.006) following oxidative stress. The epithelial de-differentiation observed in vitro was abrogated by pharmacological blockade of the ALK-5 component of the TGF-ß receptor. These data suggest that stress induced production of TGF-ß2 by BEC can modify liver allograft function by enhancing the de-differentiation of local epithelial cells.

HoxA10 regulates transcription of the gene encoding transforming growth factor beta2 (TGFbeta2) in myeloid cells.

HoxA10 is a homeodomain transcription factor that is maximally expressed in myeloid progenitor cells. HoxA10 is overexpressed in a poor prognosis subset of human acute myeloid leukemia (AML) and in vivo overexpression of HoxA10 in murine bone marrow induces myeloid leukemia. HoxA10 contributes to myeloid progenitor expansion and differentiation block, but few target genes have been identified that explain the influence of HoxA10 on these processes. The current study identifies the gene encoding transforming growth factor ß2 (TGFß2) as a HoxA10 target gene. We found that HoxA10 activated TGFß2 transcription by interacting with tandem cis elements in the promoter. We also determined that HoxA10 overexpression in myeloid progenitor cells increased Tgfß2 production by the cells. Tgfß2 stimulates proliferation of hematopoietic stem and progenitor cells. Therefore, these studies identified autocrine stimulation of myeloid progenitors by Tgfß2 as one mechanism by which HoxA10 expands this population. Because HoxA proteins had not been previously known to influence expression of pro-proliferative cytokines, this has implications for understanding molecular mechanisms involved in progenitor expansion and the pathobiology of AML.

Transforming growth factor-ß enhances invasion and metastasis in Ras-transfected human malignant epidermal keratinocytes.

Transforming growth factor-ß (TGF-ß) is known to act as a tumour suppressor early in carcinogenesis, but then switches to a pro-metastatic factor in some late stage cancers. However, the actions of TGF-ß are context dependent, and it is currently unclear how TGF-ß influences the progression of human squamous cell carcinoma (SCC). This study examined the effect of overexpression of TGF-ß1 or TGF-ß2 in Ras-transfected human malignant epidermal keratinocytes that represent the early stages of human SCC. In vitro, the proliferation of cells overexpressing TGF-ß1 or TGF-ß2 was inhibited by exogenous TGF-ß1; cells overexpressing TGF-ß1 also grew more slowly than controls, but the growth rate of TGF-ß2 overexpressing cells was unaltered. However, cells that overexpressed either TGF-ß1 or TGF-ß2 were markedly more invasive than controls in an organotypic model of SCC. The proliferation of the invading TGF-ß1 overexpressing cells in the organotypic assays was higher than controls. Similarly, tumours formed by the TGF-ß1 overexpressing cells following transplantation to athymic mice were larger than tumours formed by control cells and proliferated at a higher rate. Our results demonstrate that elevated expression of either TGF-ß1 or TGF-ß2 in cells that represent the early stages in the development of human SCC results in a more aggressive phenotype.

Cutting edge: human latency-associated peptide+ T cells: a novel regulatory T cell subset.

Regulatory T cells (Tregs) play an important role in the maintenance of peripheral tolerance. Several molecules including TGF-beta have been linked to the function and differentiation of Tregs. In this study, we describe a unique population of T cells expressing a membrane bound form of TGF-beta, the latency-associated peptide (LAP), and having regulatory properties in human peripheral blood. These CD4(+)LAP(+) T cells lack Foxp3 but express TGF-betaR type II and the activation marker CD69. CD4(+)LAP(+) T cells are hypoproliferative compared with CD4(+)LAP(-) T cells, secrete IL-8, IL-9, IL-10, IFN-gamma, and TGF-beta upon activation, and exhibit TGF-beta- and IL-10-dependent suppressive activity in vitro. The in vitro activation of CD4(+)LAP(-) T cells results in the generation of LAP(+) Tregs, which is further amplified by IL-8. In conclusion, we have characterized a novel population of human LAP(+) Tregs that is different from classic CD4(+)Foxp3(+)CD25(high) natural Tregs.

TGF-ß1 mediated activation of Rho kinase induces TGF-ß2 and endothelin-1 expression in human hepatic stellate cells.

BACKGROUND & AIMS: TGF-ß1 a key pro-fibrotic factor activates signaling via the canonical ALK/SMAD as well as the Rho GTPase pathways. Rho kinase is a major downstream effector of Rho GTPase signaling. To understand the contribution of Rho kinase activation towards the synthesis of fibrotic mediators by hepatic stellate cells (HSC), we first profiled activated HSC and fibrotic liver tissues to identify common transcripts that were most significantly up-regulated across all samples. We then applied a pharmacologic as well as a genomics approach in a TGF-ß1 activated human HSC line (LX-2) to study the involvement of Rho kinase signaling in the expression of a subset of these up-regulated fibrotic genes. METHODS: Total RNA was profiled using microarray chips. Data analysis was performed using Ingenuity Pathway Analysis software. LX-2 cells were activated with 10 ng/ml of TGF-ß1 for 24 h. Activation of downstream pathways was assessed by Western blotting with phospho-specific target biomarker antibodies. Targeted knockdown of Rho kinase isoforms 1 and 2 was achieved with RNAi. Secreted levels of endothelin-1, TGF-ß2, and thrombospondin-1 were measured by ELISA. RESULTS: TGF-ß1 activated Rho kinase and Smad pathways in LX-2 cells. The syntheses of endothelin-1 and TGF-ß2 were significantly inhibited in TGF-ß1 treated LX-2 cells, by isoform non-selective Rho kinase inhibitors. siRNA knockdown of each isoform suggested that endothelin-1 synthesis was largely mediated by the Rho kinase-1 isoform, while both isoforms contributed to the synthesis of TGF-ß2. CONCLUSIONS: The TGF-ß1 mediated secretion of endothelin-1 and TGF-ß2 is mediated by Rho kinase activation in human HSC.