Interstitial flow emanates from tumors into the microenvironment where it promotes tumor cell invasion. Fibroblasts are key constituents of the tumor stroma that modulate the mechanical environment by matrix remodeling and contraction. Here, we explore how interstitial fluid flow affects fibroblast-tumor cell interactions. Using a 3-dimensional invasion assay and MDA-MB-435S cells cocultured with dermal fibroblasts in a collagen matrix, we showed a synergistic enhancement of tumor cell invasion by fibroblasts in the presence of interstitial flow. Interstitial flow also drove transforming growth factor (TGF)-β1 and collagenase-dependent fibroblast migration, consistent with previously described mechanisms in which flow promotes invasion through autologous chemotaxis and increased motility. Concurrently, migrating fibroblasts enhanced tumor cell invasion by matrix priming via Rho-mediated contraction. We propose a model in which interstitial flow promotes fibroblast migration through increased TGF-β1 activation and collagen degradation, positioning fibroblasts to locally reorganize collagen fibers via Rho-dependent contractility, in turn enhancing tumor cell invasion via mechanotactic cues. This represents a novel mechanism in which interstitial flow causes fibroblast-mediated stromal remodeling that facilitates tumor invasion.

Peroxisome proliferator-activated receptor-γ (PPARγ) agonists ameliorate renal fibrotic lesions in diabetic nephropathy. However, the effects of the agonists on the epithelial-mesenchymal transition (EMT) linked to membrane transport dysfunction are unknown. The present study aimed to verify the effects of the PPARγ agonist troglitazone on high glucose (HG)-induced EMT in primary cultured renal proximal tubular epithelial cells (PTCs). HG (25 mM) as well as hydrogen peroxide (H(2)O(2)) and transforming growth factor-β1 (TGF-β1) decreased expression of epithelial cell marker E-cadherin and increased the expression of the mesenchymal markers vimentin and α-smooth muscle actin (α-SMA). HG, H(2)O(2), and TGF-β1 decreased Na(+)/H(+) exchangers (NHEs) or Na(+)-glucose cotransporters (SGLTs) and glucose uptake, showing membrane transport dysfunction. HG stimulated the production of cellular reactive oxygen species (ROS), and antioxidants blocked the HG-induced increase in phosphatidylinositol 3-kinase (PI3K)/Akt activation. Antioxidants and inhibitors of PI3K/Akt reversed HG-induced EMT protein expression. Inhibition of PI3K/Akt also blocked HG-induced glycogen synthase kinase-3β (GSK-3β) phosphorylation. HG and lithium chloride (GSK-3β inhibitor) blocked Snail1 and β-catenin activation. Moreover, transfection with Snail1 or β-catenin small interfering RNA (siRNA) reversed HG-induced EMT protein expression. Importantly, HG decreased PPARγ activation and troglitazone reversed HG-induced expression of PI3K/Akt, GSK-3β, Snail1, and β-catenin as well as EMT proteins. Finally, inhibitors of PI3K/Akt, Snail1/β-catenin siRNA, and troglitazone blocking the HG-induced EMT restored glucose uptake in PTCs. In conclusion, HG induces EMT through ROS, PI3K/Akt, GSK-3β, Snail, and β-catenin. Subsequently, HG-induced EMT may result in SGLT dysfunction that is restored by the PPARγ agonist troglitazone in primary cultured PTCs.

Osteoarthritis (OA) is a highly prevalent disease affecting more than 20% of American adults. Predispositions include joint injury, heredity, obesity, and aging. Biomechanical alterations are commonly involved. However, the molecular mechanisms of this disease are complex, and there is currently no effective disease-modifying treatment. The initiation and progression of OA subtypes is a complex process that at the molecular level probably involves many cell types, signaling pathways, and changes in extracellular matrix. Ex vivo studies with tissue derived from OA patients and in vivo studies with mutant mice have suggested that pathways involving receptor ligands such as TGF-β1, WNT3a, and Indian hedgehog; signaling molecules such as Smads, β-catenin, and HIF-2a; and peptidases such as MMP13 and ADAMTS4/5 are probably involved to some degree. This review focuses on molecular mechanisms of OA development related to recent findings.

Engineering complex tissues is important but difficult to achieve in tissue regeneration. Osteochondral tissue engineering for the repair of osteochondral defect, involving simultaneous regeneration of bone and cartilage, has attracted considerable attention and also serves as an optimal model system for developing effective strategies aimed at regenerating complex tissues. In the present study, we formulated a bilayered gene-activated osteochondral scaffold consisting of plasmid TGF-β1-activated chitosan-gelatin scaffold for chondrogenic layer and plasmid BMP-2-activated hydroxyapatite/chitosan-gelatin scaffold for osteogenic layer. Mesenchymal stem cells seeded in each layer of the bilayered gene- activated osteochondral scaffold showed significant cell proliferation, high expression of TGF-β1 protein and BMP-2 protein respectively. The results showed that spatially controlled and localized gene delivery system in the bilayered integrated scaffolds could induce the mesenchymal stem cells in different layers to differentiate into chondrocytes and osteoblasts in vitro, respectively, and simultaneously support the articular cartilage and subchondral bone regeneration in the rabbit knee ostochondral defect model. This study gives the evidence that multi-tissue regeneration through the combination of biomimetic and multi-phasic scaffold design, spatially controlled and localized gene delivery system and multi-lineage differentiation of a single stem cell population represents a promising strategy for facilitating the development of complex tissue or organ systems.

BACKGROUND

IL-9-secreting (T(H)9) T cells are thought to represent a distinct T-cell subset. However, evidence for their functionality in disease is uncertain.

OBJECTIVE

To define a functional phenotype for T(H)9-driven pathology in vivo.

METHODS

We used fluorescence-activated cell sorting to identify circulating T(H)9 cells in atopic and nonatopic subjects. In mice we utilized a model of allergic airways disease induced by house dust mite to determine T(H)9 cell function in vivo and the role of activin A in T(H)9 generation.

RESULTS

Allergic patients have elevated T(H)9 cell numbers in comparison to nonatopic donors, which correlates with elevated IgE levels. In a murine model, allergen challenge with house dust mite leads to rapid T(H)9 differentiation and proliferation, with much faster kinetics than for T(H)2 cell differentiation, resulting in the specific recruitment and activation of mast cells. The TGF-β superfamily member activin A replicates the function of TGF-β1 in driving the in vitro generation of T(H)9 cells. Importantly, the in vivo inhibition of T(H)9 differentiation induced by allergen was achieved only when activin A and TGF-β were blocked in conjunction but not alone, resulting in reduced airway hyperreactivity and collagen deposition. Conversely, adoptive transfer of T(H)9 cells results in enhanced pathology.

CONCLUSIONS

Our data identify a distinct functional role for T(H)9 cells and outline a novel pathway for their generation in vitro and in vivo. Functionally, T(H)9 cells promote allergic responses resulting in enhanced pathology mediated by the specific recruitment and activation of mast cells in the lungs.

BACKGROUND

Recent studies indicate that circular RNA (circRNA) plays a pivotal role in cancer progression. Here, we sought to investigate its role in breast cancer.

METHODS

CircANKS1B (a circRNA originated from exons 5 to 8 of the ANKS1B gene, hsa_circ_0007294) was identified by RNA-sequencing and validated by qRT-PCR and Sanger sequencing. Clinical breast cancer samples were used to evaluate the expression of circANKS1B and its associations with clinicopathological features and prognosis. Gain- and loss-of-function experiments in cell lines and mouse xenograft models were performed to support clinical findings and elucidate the function and underlying mechanisms of circANKS1B in breast cancer.

RESULTS

CircANKS1B was significantly up-regulated in triple-negative breast cancer (TNBC) compared with non-TNBC tissues and cell lines. Increased circANKS1B expression was closely associated with lymph node metastasis and advanced clinical stage and served as an independent risk factor for overall survival of breast cancer patients. Functional studies revealed that circANKS1B promoted breast cancer invasion and metastasis both in vitro and in vivo by inducing epithelial-to-mesenchymal transition (EMT), while had no effect on breast cancer growth. Mechanistically, circANKS1B abundantly sponged miR-148a-3p and miR-152-3p to increase the expression of transcription factor USF1, which could transcriptionally up-regulate TGF-β1 expression, resulting in activating TGF-β1/Smad signaling to promote EMT. Moreover, we found that circANKS1B biogenesis in breast cancer was promoted by splicing factor ESRP1, whose expression was also regulated by USF1.

CONCLUSIONS

Our data uncover an essential role of the novel circular RNA circANKS1B in the metastasis of breast cancer, which demonstrate that therapeutic targeting of circANKS1B may better prevent breast cancer metastasis.

On the basis of our previous studies on RhoA signaling in smooth muscle cells (SMC), we hypothesized that RhoA-mediated nuclear translocalization of the myocardin-related transcription factors (MRTFs) was important for regulating SMC phenotype. MRTF-A protein and MRTF-B message were detected in aortic SMC and in many adult mouse organs that contain a large SMC component. Both MRTFs upregulated SMC-specific promoter activity as well as endogenous SM22alpha expression in multipotential 10T1/2 cells, although to a lesser extent than myocardin. We used enhanced green fluorescent protein (EGFP) fusion proteins to demonstrate that the myocardin factors have dramatically different localization patterns and that the stimulation of SMC-specific transcription by certain RhoA-dependent agonists was likely mediated by increased nuclear translocation of the MRTFs. Importantly, a dominant-negative form of MRTF-A (DeltaB1/B2) that traps endogenous MRTFs in the cytoplasm inhibited the SM alpha-actin, SM22alpha, and SM myosin heavy chain promoters in SMC and attenuated the effects of sphingosine 1-phosphate and transforming growth factor (TGF)-beta on SMC-specific transcription. Our data confirmed the importance of the NH(2)-terminal RPEL domains for regulating MRTF localization, but our analysis of MRTF-A/myocardin chimeras and myocardin RPEL2 mutations indicated that the myocardin B1/B2 region can override this signal. Gel shift assays demonstrated that myocardin factor activity correlated well with ternary complex formation at the SM alpha-actin CArGs and that MRTF-serum response factor interactions were partially dependent on CArG sequence. Taken together, our results indicate that the MRTFs regulate SMC-specific gene expression in at least some SMC subtypes and that regulation of MRTF nuclear localization may be important for the effects of selected agonists on SMC phenotype.

Transforming growth factor (TGF)-β has been shown to play a central role in the development of tubulointerstitial fibrosis, which can be corrected via treatment with paclitaxel. The biology of microRNA (miR) can be modulated by paclitaxel. We hypothesized that paclitaxel may attenuate renal fibrosis in a rat model of remnant kidney disease by inhibiting TGF-β induced-miRs. Rats in groups of 12 were subjected to 5/6 nephrectomy and received low-dose intraperitoneal injection of paclitaxel. Renal functions were assessed at 8 weeks. The TGF-β signalling cascade and ECM proteins were evaluated by real-time polymerase chain reaction (TRT-PCR) and immunofluorescence microscopy. Animals with remnant kidneys developed hypertension, which was not relieved with paclitaxel treatment. However, paclitaxel treatment resulted in dampening the proteinuric response, reduction in serum BUN, creatinine levels and urine protein : creatinine ratio and normalization of creatinine clearance. These effects were accompanied by the inhibition of Smad2/3 activation, attenuation of renal fibrosis and normalization of integrin-linked kinase (ILK), COL(I)A1, COL(IV)A2 and α-SMA expression. Also, paclitaxel down-regulated the expression of miR-192, miR-217 and miR -377, while miR-15 was up-regulated in the remnant kidney. In vitro, in tubular epithelial cells (NRK-52E), paclitaxel also inhibited TGF-β1-induced Smad2/3 activation and normalized ILK, COL(I)A1, COL(IV)A2 and α-SMA expression. Furthermore, ChIP analyses indicated that Taxol suppressed Smad3-mediated miR-192 transcriptional activity. Over-expression of miR-192 in NRK-52E mimicked the changes seen in the remnant kidney, while inclusion of miR-192 inhibitor in the culture medium blocked TGF-β1-induced COL(I)A1 and COL(IV)A2 expression, while ILK and α-SMA were unaffected. These data suggest that low-dose paclitaxel ameliorates renal fibrosis via modulating miR-192 pathobiology and TGF-β/Smad signalling.

The stem cell-related transcription factor Oct4 regulates tumor proliferation and apoptosis, but its role in tumor migration and invasion is still undefined. Here, we compared Oct4 expression in MCF-7 and MDA-MB-231 cells, two breast cancer cell lines with similar epithelial origins, but distinct invasive and metastatic characteristics. We found MCF-7 cells to express very high levels of Oct4, while no obvious expression was detected in MDA-MB-231 cells. We then downregulated Oct4 expression using small interfering RNA (siRNA) to explore its effects on migration and invasion. Transwell assays showed that silencing Oct4 in MCF-7 cells improved their migration and invasion capabilities. Reverse-transcriptase PCR and western blots showed that E-cadherin expression decreased, and α-smooth muscle actin expression increased with Oct4 downregulation, which suggests that epithelial-to-mesenchymal transition (EMT) occurred. A potent EMT stimulus, TGF-β1, significantly inhibited Oct4 expression in both dose- and time course-dependent manners. Silencing Oct4 also upregulated expression of two major components of store-operated Ca(2+) entry channels (SOCs), STIM1 and Orai1, and enhanced SOC-directed Ca(2+) influx. Silencing STIM1 blocked the Ca(2+) influx and rescued the EMT initiated by Oct4 downregulation. In conclusion, silencing Oct4 promotes invasion and metastasis in breast cancer cells by inducing EMT. This effect may be related to SOCs-directed enhancement of Ca(2+) influx.

We performed a phase I trial of FANG vaccine, an autologous tumor-based product incorporating a plasmid encoding granulocyte-macrophage colony-stimulating factor (GMCSF) and a novel bifunctional short hairpin RNAi (bi-shRNAi) targeting furin convertase, thereby downregulating endogenous immunosuppressive transforming growth factors (TGF) β1 and β2. Patients with advanced cancer received up to 12 monthly intradermal injections of FANG vaccine (1 × 10(7) or 2.5 × 10(7) cells/ml injection). GMCSF, TGFβ1, TGFβ2, and furin proteins were quantified by enzyme-linked immunosorbent assay (ELISA). Safety and response were monitored. Vaccine manufacturing was successful in 42 of 46 patients of whom 27 received ≥1 vaccine. There were no treatment-related serious adverse events. Most common grade 1, 2 adverse events included local induration (n = 14) and local erythema (n = 11) at injection site. Post-transfection mean product expression GMCSF increased from 7.3 to 1,108 pg/10(6) cells/ml. Mean TGFβ1 and β2 effective target knockdown was 93.5 and 92.5% from baseline, respectively. Positive enzyme-linked immunospot (ELISPOT) response at month 4 was demonstrated in 9 of 18 patients serially assessed and correlated with survival duration from time of treatment (P = 0.025). Neither dose-adverse event nor dose-response relationship was noted. In conclusion, FANG vaccine was safe and elicited an immune response correlating with prolonged survival. Phase II assessment is justified.

Despite the fact that androgen deprivation therapy (ADT) can effectively reduce prostate cancer (PCa) size, its effect on PCa metastasis remains unclear. We examined the existing data on PCa patients treated with ADT plus anti-androgens to analyze ADT effects on primary tumor size, prostate-specific antigen (PSA) values, and metastatic incidence. We found that the current ADT with anti-androgens might lead to primary tumor reduction, with PSA decreased yet metastases increased in some PCa patients. Using in vitro and in vivo metastasis models with four human PCa cell lines, we evaluated the effects of the currently used anti-androgens, Casodex/bicalutamide and MDV3100/enzalutamide, and the newly developed anti-AR compounds, ASC-J9® and cryptotanshinone, on PCa cell growth and invasion. In vitro results showed that 10 μm Casodex or MDV3100 treatments suppressed PCa cell growth and reduced PSA level yet significantly enhanced PCa cell invasion. In vivo mice studies using an orthotopic xenograft mouse model also confirmed these results. In contrast, ASC-J9® led to suppressed PCa cell growth and cell invasion in in vitro and in vivo models. Mechanism dissection indicated these Casodex/MDV3100 treatments enhanced the TGF-β1/Smad3/MMP9 pathway, but ASC-J9® and cryptotanshinone showed promising anti-invasion effects via down-regulation of MMP9 expression. These findings suggest the potential risks of using anti-androgens and provide a potential new therapy using ASC-J9® to battle PCa metastasis at the castration-resistant stage.

Cardiac fibrosis is characterized by aberrant proliferation of cardiac fibroblasts and exaggerated deposition of extracellular matrix (ECM) in the myocardial interstitial, and ultimately impairs cardiac function. It is still controversial whether microRNA-21 (miR-21) participates in the process of cardiac fibrosis. Our previous study confirmed that transforming growth factor beta receptor III (TGFβRIII) is a negative regulator of TGF-β pathway. Here, we aimed to decipher the relationship between miR-21 and TGFβRIII in the pathogenic process of myocardial fibrosis. We found that TGF-β1 and miR-21 were up-regulated, whereas TGFβRIII was down-regulated in the border zone of mouse hearts in response to myocardial infarction. After transfection of miR-21 into cardiac fibroblasts, TGFβRIII expression was markedly reduced and collagen content was increased. And, luciferase results confirmed that TGFβRIII was a target of miR-21. It suggests that up-regulation of miR-21 could increase the collagen content and at least in part through inhibiting TGFβRIII. Conversely, we also confirmed that overexpression of TGFβRIII could inhibit the expression of miR-21 and reduce collagen production in fibroblasts. Further studies showed that overexpression of TGFβRIII could also deactivate TGF-β1 pathway by decreasing the expression of TGF-β1 and phosphorylated-Smad3 (p-Smad3). TGF-β1 has been proven as a positive regulator of miR-21. Taken together, we found a novel reciprocal loop between miR-21 and TGFβRIII in cardiac fibrosis caused by myocardial infarction in mice, and targeting this pathway could be a new strategy for the prevention and treatment of myocardial remodeling.

In this study, we show that neuroblastoma (NB) cell conditioning affects the chemokine receptor repertoire of human resting NK cells. In particular, NB cells upregulated the expression of CXCR4 and CXCR3 in all NK cells and downregulated CX3CR1 in the CD56(dim) subset. On the contrary, the expression of CXCR1 and CCR7 remained unaltered. The phenomenon was dependent on the release by NB cells of TGF-β1, and rTGF-β1 induced a chemokine receptor repertoire identical to that of NB-conditioned NK cells. The immune modulatory role of TGF-β1 appears to be dose dependent because low amounts of the cytokine were sufficient to modulate CXCR4 and CX3CR1 expression, intermediate amounts modified that of CXCR3, and high amounts were necessary to downregulate the expression of the NKp30 activating receptor. Notably, a similar receptor modulation was observed in rTGF-β2-conditioned NK cells. Finally, the analysis of NK cells from patients with stage 4 NB suggests that NB conditioning could exert in vivo an immune modulatory effect resembling that emerged from in vitro experiments. Altogether our data propose a novel tumor escape-mechanism based on the modulation of chemokine receptors that play pivotal roles in NK cells bone marrow homing, egress, or recruitment into peripheral tissues.

CONCLUSIONS

Myofibroblasts are responsible for wound closure that occurs in healed acute wounds. However, their actions can result in disfiguring scar contractures, compromised organ function, and a tumor promoting stroma. Understanding the mechanisms regulating their contractile machinery, gene expression, and lifespan is essential to develop new therapies to control their function.

UNASSIGNED

Mechanical stress and transforming growth factor beta-1 (TGF-β1) regulate myofibroblast differentiation from mesenchymal progenitors. As these precursor cells differentiate, they assemble a contractile apparatus to generate the force used to contract wounds. The mechanisms by which mechanical stress promote expression of contractile genes through the TGF-β1 and serum response factor pathways and offer therapeutic targets to limit myofibroblast function are being elucidated.

RESULTS

Emerging evidence suggests that the integration of mechanical cues with intracellular signaling pathways is critical to myofibroblast function via its effects on gene expression, cellular contraction, and paracrine signaling with neighboring cells. In addition, while apoptosis is clearly one pathway that can limit myofibroblast lifespan, recent data suggest that pathogenic myofibroblasts can become senescent and adopt a more beneficial phenotype, or may revert to a quiescent state, thereby limiting their function.

CONCLUSIONS

Given the important role that myofibroblasts play in pathologies as disparate as cutaneous scarring, organ fibrosis, and tumor progression, knowledge gained in the areas of intracellular signaling networks, mechanical signal transduction, extracellular matrix biology, and cell fate will support efforts to develop new therapies with a wide impact.

Muscular dystrophies are inherited myogenic disorders accompanied by progressive skeletal muscle weakness and degeneration. We previously showed that resveratrol (3,5,4'-trihydroxy-trans-stilbene), an antioxidant and activator of the NAD(+)-dependent protein deacetylase SIRT1, delays the progression of heart failure and prolongs the lifespan of δ-sarcoglycan-deficient hamsters. Because a defect of dystroglycan complex causes muscular dystrophies, and δ-sarcoglycan is a component of this complex, we hypothesized that resveratrol might be a new therapeutic tool for muscular dystrophies. Here, we examined resveratrol's effect in mdx mice, an animal model of Duchenne muscular dystrophy. mdx mice that received resveratrol in the diet for 32 weeks (4 g/kg diet) showed significantly less muscle mass loss and nonmuscle interstitial tissue in the biceps femoris compared with mdx mice fed a control diet. In the muscles of these mice, resveratrol significantly decreased oxidative damage shown by the immunostaining of nitrotyrosine and 8-hydroxy-2'-deoxyguanosine and suppressed the up-regulation of NADPH oxidase subunits Nox4, Duox1, and p47(phox). Resveratrol also reduced the number of α-smooth muscle actin (α-SMA)(+) myofibroblast cells and endomysial fibrosis in the biceps femoris, although the infiltration of CD45(+) inflammatory cells and increase in transforming growth factor-β1 (TGF-β1) were still observed. In C2C12 myoblast cells, resveratrol pretreatment suppressed the TGF-β1-induced increase in reactive oxygen species, fibronectin production, and expression of α-SMA, and SIRT1 knockdown blocked these inhibitory effects. SIRT1 small interfering RNA also increased the expression of Nox4, p47(phox), and α-SMA in C2C12 cells. Taken together, these findings indicate that SIRT1 activation may be a useful strategy for treating muscular dystrophies.

BACKGROUND

The goals of this study were to characterize urine-derived stem cells obtained from the upper urinary tract (uUSC), induce these cells to differentiate into urothelial and smooth muscle cells, and determine whether they could serve as a potential stem cell source for bladder tissue engineering.

METHODS

Urine samples were collected from five patients with normal upper urinary tracts during renal pyeloplasty. Cells were isolated from this urine and extensively expanded in vitro.

RESULTS

The mean population doubling of uUSC was 46.5±7.7. The uUSC expressed surface markers associated with mesenchymal stem cells and pericytes. These cells could differentiate into smooth muscle-like cells that expressed smooth muscle-specific gene transcripts and proteins, including α-smooth muscle actin, desmin, and myosin, when exposed to TGF-β1 and PDGF-BB. In a collagen lattice assay, these myogenic-differentiated uUSC displayed contractile function that was similar to that seen in native smooth muscle cells. Urothelial-differentiated uUSC expressed urothelial-specific genes and proteins such as uroplakin-Ia and -III, cytokeratin (CK)-7, and CK-13.

CONCLUSIONS

uUSC possess expansion and differentiation (urothelial and myogenic) capabilities, and can potentially be used as an alternative cell source in bladder tissue engineering for patients needing cystoplasty.

BACKGROUND

Epidermal growth factor receptor- tyrosine kinase inhibitors (EGFR-TKIs) benefit Non-small cell lung cancer (NSCLC) patients, and an EGFR-TKIi erlotinib, is approved for patients with recurrent NSCLC. However, resistance to erlotinib is a major clinical problem. Earlier we have demonstrated the role of Hedgehog (Hh) signaling in Epithelial-to-Mesenchymal transition (EMT) of NSCLC cells, leading to increased proliferation and invasion. Here, we investigated the role of Hh signaling in erlotinib resistance of TGF-β1-induced NSCLC cells that are reminiscent of EMT cells.

METHODS

Hh signaling was inhibited by specific siRNA and by GDC-0449, a small molecule antagonist of G protein coupled receptor smoothened in the Hh pathway. Not all NSCLC patients are likely to benefit from EGFR-TKIs and, therefore, cisplatin was used to further demonstrate a role of inhibition of Hh signaling in sensitization of resistant EMT cells. Specific pre- and anti-miRNA preparations were used to study the mechanistic involvement of miRNAs in drug resistance mechanism.

RESULTS

siRNA-mediated inhibition as well as pharmacological inhibition of Hh signaling abrogated resistance of NSCLC cells to erlotinib and cisplatin. It also resulted in re-sensitization of TGF-β1-induced A549 (A549M) cells as well the mesenchymal phenotypic H1299 cells to erlotinib and cisplatin treatment with concomitant up-regulation of cancer stem cell (CSC) markers (Sox2, Nanog and EpCAM) and down-regulation of miR-200 and let-7 family miRNAs. Ectopic up-regulation of miRNAs, especially miR-200b and let-7c, significantly diminished the erlotinib resistance of A549M cells. Inhibition of Hh signaling by GDC-0449 in EMT cells resulted in the attenuation of CSC markers and up-regulation of miR-200b and let-7c, leading to sensitization of EMT cells to drug treatment, thus, confirming a connection between Hh signaling, miRNAs and drug resistance.

CONCLUSIONS

We demonstrate that Hh pathway, through EMT-induction, leads to reduced sensitivity to EGFR-TKIs in NSCLCs. Therefore, targeting Hh pathway may lead to the reversal of EMT phenotype and improve the therapeutic efficacy of EGFR-TKIs in NSCLC patients.

Neuropathic pain remains a pressing clinical problem. Here, we demonstrate that a local, intrathecal (i.t.) injection of bone marrow stromal cells (BMSCs) following lumbar puncture alleviates early- and late-phase neuropathic pain symptoms, such as allodynia and hyperalgesia, for several weeks in murine chronic constriction injury (CCI) and spared nerve injury models. Moreover, i.t. BMSCs reduced CCI-induced spontaneous pain and axonal injury of dorsal root ganglion (DRG) neurons and inhibited CCI-evoked neuroinflammation in DRGs and spinal cord tissues. BMSCs secreted TGF-β1 into the cerebrospinal fluid, and neutralization of TGF-β1, but not IL-10, reversed the analgesic effect of BMSCs. Conversely, i.t. administration of TGF-β1 potently inhibited neuropathic pain. TGF-β1 acted as a powerful neuromodulator and rapidly (within minutes) suppressed CCI-evoked spinal synaptic plasticity and DRG neuronal hyperexcitability via TGF-β receptor 1-mediated noncanonical signaling. Finally, nerve injury upregulated CXCL12 in lumbar L4-L6 DRGs, and this upregulation caused migration of i.t.-injected BMSCs to DRGs through the CXCL12 receptor CXCR4, which was expressed on BMSCs. BMSCs that migrated from the injection site survived at the border of DRGs for more than 2 months. Our findings support a paracrine mechanism by which i.t. BMSCs target CXCL12-producing DRGs to elicit neuroprotection and sustained neuropathic pain relief via TGF-β1 secretion.

The expression of smooth muscle actin-α (SMA-α) by fibroblasts defines phenotypic transition to myofibroblasts and is a primary contributor to contractile force generation by these differentiated cells. Although the regulation of SMA-α expression has been the focus of many studies, there is presently only limited information concerning miRNA regulation of lung myofibroblast differentiation and the involvement of these miRNAs in pulmonary fibrosis. To determine the role of miR-145 in regulating lung myofibroblast differentiation and pulmonary fibrosis. Wild-type and miR-145(-/-) mice were studied. Lung fibrosis models and cell culture systems were employed. miR-145 mimics or inhibitors were transfected into pulmonary fibroblasts. Fibrogenic and contractile activities of lung fibroblasts were determined. We found that miR-145 expression is upregulated in TGF-β1-treated lung fibroblasts. miR-145 expression is also increased in the lungs of patients with idiopathic pulmonary fibrosis as compared to in normal human lungs. Overexpression of miR-145 in lung fibroblasts increased SMA-α expression, enhanced contractility, and promoted formation of focal and fibrillar adhesions. In contrast, miR-145 deficiency diminished TGF-β1 induced SMA-α expression. miR-145 did not affect the activity of TGF-β1, but promoted the activation of latent TGF-β1. miR-145 targets KLF4, a known negative regulator of SMA-α expression. Finally, we found that miR-145(-/-) mice are protected from bleomycin-induced pulmonary fibrosis. miR-145 plays an important role in the differentiation of lung myofibroblasts. miR-145 deficiency is protective against bleomycin-induced lung fibrosis, suggesting that miR-145 may be a potential target in the development of novel therapies to treat pathological fibrotic disorders.

Autophagy can lead to cell death in response to stress, but it can also act as a protective mechanism for cell survival. We show that TGF-β1 induces autophagy and protects glomerular mesangial cells from undergoing apoptosis during serum deprivation. Serum withdrawal rapidly induced autophagy within 1 h in mouse mesangial cells (MMC) as determined by increased microtubule-associated protein 1 light chain 3 (LC3) levels and punctate distribution of the autophagic vesicle-associated-form LC3-II. We demonstrate that after 1 h there was a time-dependent decrease in LC3 levels that was accompanied by induction of apoptosis, evidenced by increases in cleaved caspase 3. However, treatment with TGF-β1 resulted in induction of the autophagy protein LC3 while suppressing caspase 3 activation. TGF-β1 failed to rescue MMC from serum deprivation-induced apoptosis upon knockdown of LC3 by siRNA and in MMC from LC3 null (LC3(-/-)) mice. We show that TGF-β1 induced autophagy through TAK1 and Akt activation, and inhibition of PI3K-Akt pathway by LY294002 or dominant-negative Akt suppressed LC3 levels and enhanced caspase 3 activation. TGF-β1 also up-regulated cyclin D1 and E protein levels while down-regulating p27, thus stimulating cell cycle progression. Bafilomycin A1, but not MG132, blocked TGF-β1 down-regulation of p27, suggesting that p27 levels were regulated through autophagy. Taken together, our data indicate that TGF-β1 rescues MMC from serum deprivation-induced apoptosis via induction of autophagy through activation of the Akt pathway. The autophagic process may constitute an adaptive mechanism to glomerular injury by inhibiting apoptosis and promoting mesangial cell survival.

Tolerance induction in T cells takes place in most tumors and is thought to account for tumor evasion from immune eradication. Production of the cytokine TGF-β is implicated in immunosuppression, but the cellular mechanism by which TGF-β induces T cell dysfunction remains unclear. With a transgenic model of prostate cancer, we showed that tumor development was not suppressed by the adaptive immune system, which was associated with heightened TGF-β signaling in T cells from the tumor-draining lymph nodes. Blockade of TGF-β signaling in T cells enhanced tumor antigen-specific T cell responses and inhibited tumor development. Surprisingly, T cell- but not Treg cell-specific ablation of TGF-β1 was sufficient to augment T cell cytotoxic activity and blocked tumor growth and metastases. These findings reveal that T cell production of TGF-β1 is an essential requirement for tumors to evade immunosurveillance independent of TGF-β produced by tumors.

BACKGROUND

Preterm and term newborns are at high risk of brain damage as well as subsequent cerebral palsy and learning disabilities. Indeed, hypoxia-ischemia (HI), pathogen exposures, and associated intracerebral increase of pro-inflammatory cytokines have all been linked to perinatal brain damage. However, the developmental effects of potential variations of pro- and anti-inflammatory cytokine ratios remain unknown.

METHODS

Using rat models of perinatal brain damage induced by exposures to lipopolysaccharide (LPS) and/or HI at distinct levels of maturity, we compared cytokine expression at stages of cerebral development equivalent to either preterm (postnatal day 1, P1) or term (P12) newborns.

RESULTS

At P1, expression of anti-inflammatory cytokine within the brain was either not modulated (IL-6, IL-10) or down-regulated (IL-1ra, TGF-β1) by HI, LPS or LPS+HI. In contrast, there was at P12 an up-regulation of all anti-inflammatory cytokines studied in HI or LPS+HI condition, but not after LPS exposure. Interestingly, IL-1β was the main pro-inflammatory cytokine up-regulated moderately at P1, and strongly at P12, with a weak co-expression of TNF-α observed mainly at P12. These age-dependant inflammatory reactions were also accompanied, under HI and LPS+HI conditions, at P12 only, by combined: (i) expression of chemokines CINC-1 and MCP-1, (ii) blood-brain barrier (BBB) leakage, and (iii) intracerebral recruitment of systemic immune cells such as neutrophils. In contrast, sole LPS induced IL-1β responses mainly within white matter at P1 and mainly within gray matter at P12, that were only associated with early MCP-1 (but no CINC-1) induction at both ages, without any recruitment of neutrophils and CD68+ cells.

CONCLUSIONS

HI and LPS+HI induce pro-inflammatory oriented immune responses in both preterm and term like brains, with a maximal inflammatory response triggered by the combination of LPS+HI. The profile of these neuroinflammatory responses presented striking variations according to age: no or down-regulated anti-inflammatory responses associated with mainly IL-1β release in preterm-like brains (P1), in sharp contrast to term-like brains (P12) presenting stronger anti-and pro-inflammatory responses, including both IL-1β and TNF-α releases, and BBB leakage. These developmental-dependant variations of neuroinflammatory response could contribute to the differential pattern of brain lesions observed across gestational ages in humans. This also highlights the necessity to take into consideration the maturation stage, of both brain and immune systems, in order to develop new anti-inflammatory neuroprotective strategies.

OBJECTIVE

The dysfunction of miRNAs has been demonstrated to participate in the development of various tumors. However, whether miRNAs are involved in metastasis and progression of gallbladder carcinoma (GBC) remains unknown.

METHODS

A new designed gain-of-function miRNA screening technology was applied to filter out pro-metastatic miRNAs in GBC. Their expression in GBC tissues was validated by real-time PCR. The biological functions of miRNAs were intensively studied by transwell, immunoblot, immunohistochemical, and in situ hybridization assays. Tumorigenicity and liver metastasis were further examined in nude mice.

RESULTS

Of 880 miRNAs, 17 were filtered out as the prominent metastatic inducers of GBCs. Among them, the upregulation of pro-metastatic miR-20a was closely associated with local invasion, distant metastasis, and poor prognosis of 67 followed-up GBC patients, clinically. Patients with higher miR-20a expression exhibited worse overall survival (OS and median OS time was 5 and 20 months, respectively) than the lower expression group. A dramatically increased TGF-β1 level was found in GBC patients, which was responsible for the elevation of miR-20a. The ectopic expression of miR-20a could induce epithelial-mesenchymal transition and enhance metastasis of GBC cells in vitro and in vivo, by directly targeting the 3' UTR of Smad7, and subsequently promoting nuclear translocation of β-catenin. Conversely, the blockage of miR-20a by specific antagomir effectively restored the expression of Smad7 and attenuated TGF-β-induced cell metastasis.

CONCLUSIONS

TGF-β1-mediated activation of the miR-20a/Smad7/β-catenin axis plays a pivotal role in the pathogenesis and worse prognosis of GBCs and may serve as a potential therapeutic target in the future.

While transforming growth factor-β (TGF-β1)-induced SMAD2/3 signaling is a critical event in the progression of chronic kidney disease, the role of non-SMAD mechanisms in the orchestration of fibrotic gene changes remains largely unexplored. TGF-β1/SMAD3 pathway activation in renal fibrosis (induced by ureteral ligation) correlated with epidermal growth factor receptor(Y845) (EGFR(Y845)) and p53(Ser15) phosphorylation and induction of disease causative target genes plasminogen activator inhibitor-1 (PAI-1) and connective tissue growth factor (CTGF) prompting an investigation of the mechanistic involvement of EGFR and tumor suppressor p53 in profibrotic signaling. TGF-β1, PAI-1, CTGF, p53 and EGFR were co-expressed in the obstructed kidney localizing predominantly to the tubular and interstitial compartments. Indeed, TGF-β1 activated EGFR and p53 as well as SMAD2/3. Genetic deficiency of either EGFR or p53 or functional blockade with AG1478 or Pifithrin-α, respectively, effectively inhibited PAI-1and CTGF induction and morphological transformation of renal fibroblasts as did SMAD3 knockdown or pretreatment with the SMAD3 inhibitor SIS3. Reactive oxygen species (ROS)-dependent mechanisms initiated by TGF-β1 were critical for EGFR(Y845) and p53(Ser15) phosphorylation and target gene expression. The p22(Phox) subunit of NADPH oxidase was also elevated in the fibrotic kidney with an expression pattern similar to p53 and EGFR. EGF stimulation alone initiated, albeit delayed, c-terminal SMAD3 phosphorylation (that required the TGF-β1 receptor) and rapid ERK2 activation both of which are necessary for PAI-1 and CTGF induction in renal fibroblasts. These data highlight the extensive cross-talk among SMAD2/3, EGFR and p53 pathways essential for expression of TGF-β1-induced fibrotic target genes.