We examined the localization of transforming growth factor (TGF)-beta in first-trimester and term human decidua and chorionic villi and explored the role of this factor on the proliferation and differentiation of cultured trophoblast cells. Two antibodies, 1D11.16.8, a mouse monoclonal neutralizing antibody capable of recognizing both TGF-beta 1 and TGF-beta 2 and CL-B1/29, a rabbit polyclonal antibody capable of recognizing TGF-beta 2, were used to immunolocalize TGF-beta in fixed, paraffin-embedded, or fixed, frozen sections of placenta and decidua, providing similar results. Intense labeling was observed in the extracellular matrix (ECM) of the first-trimester decidua and cytoplasm of term decidual cells. Syncytiotrophoblast cell cytoplasm as well as the ECM in the core of the chorionic villi of both first-trimester and term placentas exhibited a moderate degree of labeling. Strong cytoplasmic labeling was observed in the cytotrophoblastic shell of the term placenta. To examine the role of TGF-beta on trophoblast proliferation and differentiation, early passage cultures of first-trimester and primary cultures of term trophoblast cells were established and characterized on the basis of numerous immunocytochemical and functional markers. These cells expressed cytokeratin, placental alkaline phosphatase, urokinase-type plasminogen activator, and pregnancy-specific beta glycoprotein, but not factor VIII or 63D3; they also produced hCG and collagenase type IV. Exposure of first-trimester trophoblast cultures to TGF-beta 1 significantly inhibited proliferation in a dose-dependent manner. An antiproliferative effect was also noted in the presence of TGF-beta 2. These effects were abrogated in the presence of the neutralizing anti-TGF-beta antibody (1D11.16.8) in a concentration-dependent manner. In a 3-day culture, exogenous TGF-beta 1 stimulated formation of multinucleated cells by the first trimester as well as term trophoblast cells. Addition of neutralizing anti-TGF-beta antibody to first-trimester trophoblast cells stimulated proliferation beyond control levels in a 24-h culture and reduced formation of multinucleated cells in a 3-day culture, indicating the presence of endogenous TGF-beta activity. These results indicate that TGF-beta produced at the human fetal-maternal interface plays a major regulatory role in the proliferation and differentiation of the trophoblast.

OBJECTIVE

Angiotensin II (AngII) is involved in extracellular matrix (ECM) accumulation contributing to heart failure. Periostin, a 90 kDa ECM protein, is a key regulator of cardiac fibrosis, and its expression is significantly higher in failing hearts. We determined the modulatory effect of AngII on periostin level and explored the possible signal transduction mechanism.

RESULTS

AngII (400 ng/kg/min) or normal saline was infused subcutaneously for 28 days into rats; AngII antagonism was with losartan (10 mg/kg/day orally). AngII infusion induced cardiac fibrosis and increased periostin expression, which was attenuated by losartan. In cultured adult rat cardiac fibroblasts, AngII promoted the mRNA and protein expression of periostin. AngII provoked activation of cAMP response element-binding protein (CREB), and CREB small interfering RNA (siRNA) suppressed AngII-induced periostin expression. Inhibition of p38 mitogen-activated protein kinase (p38 MAPK) with SB202190 attenuated AngII-induced CREB activation and periostin expression. Transfection with Ras guanyl-releasing protein 1 siRNA or RasN17 dominant-negative plasmid prevented AngII-induced p38 MAPK phosphorylation and periostin expression. Transforming growth factor (TGF)-β1 antibody decreased the stimulatory effect of AngII on periostin expression. The extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor PD98059 attenuated AngII-induced TGF-β1 expression, Smad2/3 nuclear accumulation, and periostin expression.

CONCLUSIONS

The activation of the Ras/p38 MAPK/CREB pathway is required for AngII-induced periostin expression. ERK1/2 also participates in AngII-induced periostin expression by regulating TGF-β1/Smad signalling.

BACKGROUND

Ovarian cancer (OvCa) is one of the most common malignant diseases of the female reproductive system in the world. The majority of OvCa is diagnosed with metastasis in the abdominal cavity. Epithelial-to-mesenchymal transition (EMT) plays a key role in tumor cell metastasis. However, it is still unclear whether long non-coding RNA (lncRNA) is implicated in EMT and influences cell invasion and metastasis in OvCa.

RESULTS

In this study, using bioinformatcis analysis, we constructed a lncRNA-mediated competing endogenous RNA (ceRNA) network for mesenchymal OvCa and identified lncRNA AP000695.4, which we named pro-transition associated RNA (PTAR). PTAR was significantly up-regulated in the mesenchymal subtype samples compared with the epithelial subtype samples from the TCGA OvCa data sets. In addition, our study showed that PTAR expression was positively correlated with the expression level of ZEB1 in the mesenchymal OvCa samples. Meanwhile, we found that silencing miR-101 promoted cell migration, whereas the overexpression of miR-101 suppressed EMT and cell migration in OvCa cell lines through the regulation of ZEB1. Further analysis showed that enhanced expression of PTAR promoted EMT and metastasis through the regulation of miR-101, whereas silencing PTAR led to the attenuation of TGF-β1-induced tumorigenicity in ovarian cancer cells. Mechanistically, we found that PTAR acted as a ceRNA of miR-101, as forced expression of PTAR reduced the expression and activity of miR-101. More importantly, the knockdown of PTAR reduced tumorigenicity and metastasis in vivo.

CONCLUSIONS

Taken together, the results from our study highlight a role for the PTAR-miR-101-ZEB1 axis in OvCa, which offers novel strategies for the prevention of metastasis in OvCa.

In vivo studies have demonstrated that the state of dispersion of carbon nanotubes (CNTs) plays an important role in generating adverse pulmonary effects. However, little has been done to develop reproducible and quantifiable dispersion techniques to conduct mechanistic studies in vitro. This study was to evaluate the dispersion of multiwalled carbon nanotubes (MWCNTs) in tissue culture media, with particular emphasis on understanding the forces that govern agglomeration and how to modify these forces. Quantitative techniques such as hydrophobicity index, suspension stability index, attachment efficiency, and dynamic light scattering were used to assess the effects of agglomeration and dispersion of as-prepared (AP), purified (PD), or carboxylated (COOH) MWCNTs on bronchial epithelial and fibroblast cell lines. We found that hydrophobicity is the major factor determining AP- and PD-MWCNT agglomeration in tissue culture media but that the ionic strength is the main factor determining COOH-MWCNT suspendability. Bovine serum albumin (BSA) was an effective dispersant for MWCNTs, providing steric and electrosteric hindrances that are capable of overcoming hydrophobic attachment and the electrostatic screening of double layer formation in ionic media. Thus, BSA was capable of stabilizing all tube versions. Dipalmitoylphosphatidylcholine (DPPC) provided additional stability for AP-MWCNTs in epithelial growth medium (BEGM). While the dispersion state did not affect cytotoxicity, improved dispersion of AP- and PD-MWCNTs increased TGF-β1 production in epithelial cells and fibroblast proliferation. In summary, we demonstrate how quantitative techniques can be used to assess the agglomeration state of MWCNTs when conducting mechanistic studies on the effects of dispersion on tissue culture cells.

Primary hepatocellular carcinoma (HCC) is one of the most common forms of malignant cancer with the fourth highest mortality rate worldwide. Major risk factors for the development of HCC include chronic infections with the hepatitis B or C virus, alcohol consumption, exposure to dietary aflatoxin B1, hereditary liver disease or liver cirrhosis of any etiology. Recent studies have discovered changes in the insulin-like growth factor (IGF) axis that affect the molecular pathogenesis of HCC, including the autocrine production of IGFs, IGF binding proteins (IGFBPs), IGFBP proteases, and IGF receptor expression. Characteristic alterations detected in HCC and hepatoma cell lines comprise the overexpression of IGF-II and the IGF-I receptor emerging as critical events in malignant transformation and growth of tumors. Simultaneous reduction of IGFBP expression and the increase in proteolytic cleavage of IGFBPs result in an excess of bioactive IGFs. Finally, defective functions of the IGF-II/mannose 6-phosphate receptor involved in degradation of IGF II, the activation of the growth inhibitor TGF-beta1, and the lysosomal targeting of cathepsin proteases capable to degrade extracellular matrix proteins may contribute to the development of HCC.

T cell activation levels, viral load and CD4(+) T cell counts at early stages of HIV-1 infection are predictive of the rate of progression towards AIDS. We evaluated whether the inflammatory profile during primary HIV-1 infection is predictive of the virological and immunological set-points and of disease progression. We quantified 28 plasma proteins during acute and post-acute HIV-1 infection in individuals with known disease progression profiles. Forty-six untreated patients, enrolled during primary HIV-1 infection, were categorized into rapid progressors, progressors and slow progressors according to their spontaneous progression profile over 42 months of follow-up. Already during primary infection, rapid progressors showed a higher number of increased plasma proteins than progressors or slow progressors. The plasma levels of TGF-β1 and IL-18 in primary HIV-1 infection were both positively associated with T cell activation level at set-point (6 months after acute infection) and together able to predict 74% of the T cell activation variation at set-point. Plasma IP-10 was positively and negatively associated with, respectively, T cell activation and CD4(+) T cell counts at set-point and capable to predict 30% of the CD4(+) T cell count variation at set-point. Moreover, plasma IP-10 levels during primary infection were predictive of rapid progression. In primary infection, IP-10 was an even better predictor of rapid disease progression than viremia or CD4(+) T cell levels at this time point. The superior predictive capacity of IP-10 was confirmed in an independent group of 88 HIV-1 infected individuals. Altogether, this study shows that the inflammatory profile in primary HIV-1 infection is associated with T cell activation levels and CD4(+) T cell counts at set-point. Plasma IP-10 levels were of strong predictive value for rapid disease progression. The data suggest IP-10 being an earlier marker of disease progression than CD4(+) T cell counts or viremia levels.

Based on the tetracycline-regulated gene expression system, a double-transgenic mouse model for liver fibrosis was established in which the expression of transforming growth factor beta1 (TGF-beta1) can be regulated deliberately by addition or removal of doxycycline hydrochloride to the drinking water. TGF-beta1 plasma levels in induced double-transgenic mice reached values ranging from 250 to 1,200 ng/mL, being 10 to 30 times above the normal plasma levels. By applying a cyclic induction-deinduction protocol, deleterious effects of the high plasma TGF-beta1 levels were overcome. By using this protocol, liver fibrosis occurred within a few cycles and progressed further to an intermediary fibrosis when cyclic induction was continued. On histochemical staining, a marked perisinusoidal deposition of extracellular matrix was detected accompanied by the activation of hepatic stellate cells as shown by alpha-smooth muscle actin (alpha-SMA) expression. Apoptosis of hepatocytes was prominent in TGF-beta1 high producers, leading to a decreasing number of TGF-beta1-expressing cells with time. No compensatory proliferation of hepatocytes could be detected. In advanced stages, fibrogenesis could be stopped by switching off TGF-beta1 production and reversal of fibrosis could be shown by (immuno)histochemistry within 6 to 21 days. Determination of messenger RNA (mRNA) levels of procollagen I and III, laminin (B1), matrix metalloproteinase (MMP)-2, -9, and -13, and tissue inhibitor of matrix metalloproteinase (TIMP)-1 and -2 by real-time reverse-transcription polymerase chain reaction (RT-PCR) provided insight into some mechanistic details of the fibrogenic process and its reversal. In conclusion, this model will enable the analysis of fibrogenesis at progressive stages and help in elucidating the cellular changes during development and regression of liver fibrosis caused by elevated TGF-beta1 expression.

Interactions between tumor cells and cancer-associated fibroblasts (CAFs) in the tumor microenvironment significantly influence cancer growth and metastasis. Transforming growth factor-β (TGF-β) is known to be a critical mediator of the CAF phenotype, and osteopontin (OPN) expression in tumors is associated with more aggressive phenotypes and poor patient outcomes. The potential link between these two pathways has not been previously addressed. Utilizing in vitro studies using human mesenchymal stem cells (MSCs) and MDA-MB231 (OPN+) and MCF7 (OPN-) human breast cancer cell lines, we demonstrate that OPN induces integrin-dependent MSC expression of TGF-β1 to mediate adoption of the CAF phenotype. This OPN-TGF-β1 pathway requires the transcription factor, myeloid zinc finger 1 (MZF1). In vivo studies with xenotransplant models in NOD-scid mice showed that OPN expression increases cancer growth and metastasis by mediating MSC-to-CAF transformation in a process that is MZF1 and TGF-β1 dependent. We conclude that tumor-derived OPN engenders MSC-to-CAF transformation in the microenvironment to promote tumor growth and metastasis via the OPN-MZF1-TGF-β1 pathway.

Proliferating hepatic stellate cells (HSCs) respond to liver damage by secreting collagens that form fibrous scar tissue, which can lead to cirrhosis if in appropriately regulated. Advancement of microRNA (miRNA) hepatic therapies has been hampered by difficulties in delivering miRNA to damaged tissue. However, exosomes secreted by adipose-derived mesenchymal stem cells (ADSCs) can be exploited to deliver miRNAs to HSCs. ADSCs were engineered to overexpress miRNA-181-5p (miR-181-5p-ADSCs) to selectively home exosomes to mouse hepatic stellate (HST-T6) cells or a CCl4-induced liver fibrosis murine model and compared with non-targeting control Caenorhabditis elegans miR-67 (cel-miR-67)-ADSCs. In vitro analysis confirmed that the transfer of miR-181-5p from miR-181-5p-ADSCs occurred via secreted exosomal uptake. Exosomes were visualized in HST-T6 cells using cyc3-labelled pre-miRNA-transfected ADSCs with/without the exosomal inhibitor, GW4869. The effects of miRNA-181-5p overexpression on the fibrosis associated STAT3/Bcl-2/Beclin 1 pathway and components of the extracellular matrix were assessed. Exosomes from miR181-5p-ADSCs down-regulated Stat3 and Bcl-2 and activated autophagy in the HST-T6 cells. Furthermore, the up-regulated expression of fibrotic genes in HST-T6 cells induced by TGF-β1 was repressed following the addition of isolated miR181-5p-ADSC exosomes compared with miR-67-ADSCexosomes. Exosome therapy attenuated liver injury and significantly down-regulated collagen I, vimentin, α-SMA and fibronectin in liver, compared with controls. Taken together, the effective anti-fibrotic function of engineered ADSCs is able to selectively transfer miR-181-5p to damaged liver cells and will pave the way for the use of exosome-ADSCs for therapeutic delivery of miRNA targeting liver disease.

Tissue homeostasis requires an effective, limited wound-healing response to injury. In chronic disease, failure to regenerate parenchymal tissue leads to the replacement of lost cellular mass with a fibrotic matrix. The mechanisms that dictate the balance of cell regeneration and fibrogenesis are not well understood. Here we report that fibrogenic hepatic stellate cells (HSCs) in the liver are negative regulators of hepatocyte regeneration. This negative regulatory function requires stimulation of the 5-hydroxytryptamine 2B receptor (5-HT(2B)) on HSCs by serotonin, which activates expression of transforming growth factor β1 (TGF-β1), a powerful suppressor of hepatocyte proliferation, through signaling by mitogen-activated protein kinase 1 (ERK) and the transcription factor JunD. Selective antagonism of 5-HT(2B) enhanced hepatocyte growth in models of acute and chronic liver injury. We also observed similar effects in mice lacking 5-HT(2B) or JunD or upon selective depletion of HSCs in wild-type mice. Antagonism of 5-HT(2B) attenuated fibrogenesis and improved liver function in disease models in which fibrosis was pre-established and progressive. Pharmacological targeting of 5-HT(2B) is clinically safe in humans and may be therapeutic in chronic liver disease.

Fibroblasts residing in connective tissues throughout the body are responsible for extracellular matrix (ECM) homeostasis and repair. In response to tissue damage, they activate to become myofibroblasts, which have organized contractile cytoskeletons and produce a myriad of proteins for ECM remodeling. However, persistence of myofibroblasts can lead to fibrosis with excessive collagen deposition and tissue stiffening. Thus, understanding which signals regulate de-activation of myofibroblasts during normal tissue repair is critical. Substrate modulus has recently been shown to regulate fibrogenic properties, proliferation and apoptosis of fibroblasts isolated from different organs. However, few studies track the cellular responses of fibroblasts to dynamic changes in the microenvironmental modulus. Here, we utilized a light-responsive hydrogel system to probe the fate of valvular myofibroblasts when the Young's modulus of the substrate was reduced from ~32 kPa, mimicking pre-calcified diseased tissue, to ~7 kPa, mimicking healthy cardiac valve fibrosa. After softening the substrata, valvular myofibroblasts de-activated with decreases in α-smooth muscle actin (α-SMA) stress fibers and proliferation, indicating a dormant fibroblast state. Gene signatures of myofibroblasts (including α-SMA and connective tissue growth factor (CTGF)) were significantly down-regulated to fibroblast levels within 6 hours of in situ substrate elasticity reduction while a general fibroblast gene vimentin was not changed. Additionally, the de-activated fibroblasts were in a reversible state and could be re-activated to enter cell cycle by growth stimulation and to express fibrogenic genes, such as CTGF, collagen 1A1 and fibronectin 1, in response to TGF-β1. Our data suggest that lowering substrate modulus can serve as a cue to down-regulate the valvular myofibroblast phenotype resulting in a predominantly quiescent fibroblast population. These results provide insight in designing hydrogel substrates with physiologically relevant stiffness to dynamically redirect cell fate in vitro.

Cardiac dysfunction in CKD is characterized by aberrant cardiac remodeling with hypertrophy and fibrosis. CKD is a state of severe systemic Klotho deficiency, and restoration of Klotho attenuates vascular calcification associated with CKD. We examined the role of Klotho in cardiac remodeling in models of Klotho deficiency-genetic Klotho hypomorphism, high dietary phosphate intake, aging, and CKD. Klotho-deficient mice exhibited cardiac dysfunction and hypertrophy before 12 weeks of age followed by fibrosis. In wild-type mice, the induction of CKD led to severe cardiovascular changes not observed in control mice. Notably, non-CKD mice fed a high-phosphate diet had lower Klotho levels and greatly accelerated cardiac remodeling associated with normal aging compared with those on a normal diet. Chronic elevation of circulating Klotho because of global overexpression alleviated the cardiac remodeling induced by either high-phosphate diet or CKD. Regardless of the cause of Klotho deficiency, the extent of cardiac hypertrophy and fibrosis correlated tightly with plasma phosphate concentration and inversely with plasma Klotho concentration, even when adjusted for all other covariables. High-fibroblast growth factor-23 concentration positively correlated with cardiac remodeling in a Klotho-deficient state but not a Klotho-replete state. In vitro, Klotho inhibited TGF-β1-, angiotensin II-, or high phosphate-induced fibrosis and abolished TGF-β1- or angiotensin II-induced hypertrophy of cardiomyocytes. In conclusion, Klotho deficiency is a novel intermediate mediator of pathologic cardiac remodeling, and fibroblast growth factor-23 may contribute to cardiac remodeling in concert with Klotho deficiency in CKD, phosphotoxicity, and aging.

Elevated p53 expression is associated with several kidney diseases including diabetic nephropathy (DN). However, the mechanisms are unclear. We report that expression levels of transforming growth factor-β1 (TGF-β), p53, and microRNA-192 (miR-192) are increased in the renal cortex of diabetic mice, and this is associated with enhanced glomerular expansion and fibrosis relative to nondiabetic mice. Targeting miR-192 with locked nucleic acid-modified inhibitors in vivo decreases expression of p53 in the renal cortex of control and streptozotocin-injected diabetic mice. Furthermore, mice with genetic deletion of miR-192 in vivo display attenuated renal cortical TGF-β and p53 expression when made diabetic, and have reduced renal fibrosis, hypertrophy, proteinuria, and albuminuria relative to diabetic wild-type mice. In vitro promoter regulation studies show that TGF-β induces reciprocal activation of miR-192 and p53, via the miR-192 target Zeb2, leading to augmentation of downstream events related to DN. Inverse correlation between miR-192 and Zeb2 was observed in glomeruli of human subjects with early DN, consistent with the mechanism seen in mice. Our results demonstrate for the first time a TGF-β-induced feedback amplification circuit between p53 and miR-192 related to the pathogenesis of DN, and that miR-192-knockout mice are protected from key features of DN.

How TGF-β1-mediated signaling pathways are finely tuned to orchestrate the generation of carcinoma-associated fibroblasts (CAFs) is poorly understood. Here, we demonstrate that miR-21 and the signaling of its target Smad 7 determine TGF-β1-induced CAF formation. In primary cultured fibroblasts, mature miR-21 increases after TGF-β1 treatment, whereas the Smad 7 protein level decreases. MiR-21 binds to the 3' UTR of Smad7 mRNA and inhibits its translation, rather than causing its degradation. Most importantly, Smad 7 is bound to Smad 2 and 3, which are thought to competitively bind to TGFBR1, and prevents their activation upon TGF-β1 stimulation. The depletion of miR-21 or the overexpression of Smad 7 blocks TGF-β1-induced CAF formation, whereas the overexpression of miR-21 or the depletion of Smad 7 promotes CAF formation, even without TGF-β1 stimulation. Collectively, these findings clearly demonstrate that miR-21 and Smad7 are critical regulators of TGF-β1 signaling during the induction of CAF formation.

The mechanism by which TGF-β regulates renal inflammation and fibrosis is largely unclear; however, it is well accepted that its biological effects are mediated through Smad2 and Smad3 phosphorylation. Following activation, these Smads form heteromeric complex with Smad4 and translocate into the nucleus to bind and regulate the expression of target genes. Here we studied the roles of Smad4 to regulate TGF-β signaling in a mouse model of unilateral ureteral obstruction using conditional Smad4 knockout mice and in isolated Smad4 mutant macrophages and fibroblasts. Disruption of Smad4 significantly enhanced renal inflammation as evidenced by a greater CD45(+) leukocyte and F4/80(+) macrophage infiltration and upregulation of IL-1β, TNF-α, MCP-1, and ICAM-1 in the obstructed kidney and in IL-1β-stimulated macrophages. In contrast, deletion of Smad4 inhibited renal fibrosis and TGF-β1-induced collagen I expression by fibroblasts. Further studies showed that the loss of Smad4 repressed Smad7 transcription, leading to a loss of functional protein. This, in turn, inhibited IκBα expression but enhanced NF-κB activation, thereby promoting renal inflammation. Interestingly, deletion of Smad4 influenced Smad3-mediated promoter activities and the binding of Smad3 to the COL1A2 promoter, but not Smad3 phosphorylation and nuclear translocation, thereby inhibiting the fibrotic response. Thus, Smad4 may be a key regulator for the diverse roles of TGF-β1 in inflammation and fibrogenesis by interacting with Smad7 and Smad3 to influence their transcriptional activities in renal inflammation and fibrosis.

OBJECTIVE

In calcific aortic valve disease, myofibroblasts and activation of the transforming growth factor-β1 (TGF-β1) and Wnt/β-catenin pathways are observed in the fibrosa, the stiffer layer of the leaflet, but their association is unknown. We elucidated the roles of β-catenin and extracellular matrix stiffness in TGF-β1-induced myofibroblast differentiation of valve interstitial cells (VICs).

RESULTS

TGF-β1 induced rapid β-catenin nuclear translocation in primary porcine aortic VICs in vitro through TGF-β receptor I kinase. Degrading β-catenin pharmacologically or silencing it with small interfering RNA inhibited TGF-β1-induced myofibroblast differentiation without altering Smad2/3 activity. Conversely, increasing β-catenin availability with Wnt3A alone did not induce differentiation. However, combining TGF-β1 and Wnt3A caused greater myofibroblast differentiation than TGF-β1 treatment alone. Notably, in VICs grown on collagen-coated PA gels with physiological stiffnesses, TGF-β1-induced β-catenin nuclear translocation and myofibroblast differentiation occurred only on matrices with fibrosa-like stiffness, but not ventricularis-like stiffness. In diseased aortic valves from pigs fed an atherogenic diet, myofibroblasts colocalized with increased protein expression of Wnt3A, β-catenin, TGF-β1, and phosphorylated Smad2/3 in the fibrosa.

CONCLUSIONS

Myofibroblast differentiation of VICs involves matrix stiffness-dependent crosstalk between TGF-β1 and Wnt signaling pathways and may explain in part why the stiffer fibrosa is more susceptible to disease.

The homing properties of adipose tissue-derived mesenchymal stem cells (AdMSCs) have stimulated intravenous applications for their use in stem cell therapy. However, the soluble factors and corresponding cellular receptors responsible for inducing chemotaxis of AdMSCs have not yet been reported. In the present study, the migration capacity of human AdMSCs (hAdMSCs) toward various cytokines or growth factors (GFs) and the expression of their receptors were determined. In a conventional migration assay, PDGF-AB, TGF-β1, and TNF-α showed the most effective chemoattractant activity. When AdMSCs were preincubated with various chemokines or GF, and then allowed to migrate toward medium containing 10% FBS, those preincubated with TNF-α showed the highest migratory activity. Next, hAdMSCs were either preincubated or not with TNF-α, and allowed to migrate in response to various GFs or chemokines. Prestimulation with TNF-α increased the migration activity of hAdMSCs compared to unstimulated hAdMSCs. When analyzed by FACS and RT-PCR methods, hAdMSCs were found to express C-C chemokine receptor type 1 (CCR1), CCR7, C-X-C chemokine receptor type 4 (CXCR4), CXCR5, CXCR6, EGF receptor, fibroblast growth factor receptor 1, TGF-β receptor 2, TNF receptor superfamily member 1A, PDGF receptor A and PDGF receptor B at both the protein and the mRNA levels. These results indicate that the migration capacity of hAdMSCs is controlled by various GFs and chemokines. Prior in vitro modulation of the homing capacity of hAdMSCs could stimulate their movement into injured sites in vivo when administered intravenously, thereby improving their therapeutic potential.

Probiotic organisms are claimed to offer several functional properties including stimulation of immune system. This review is presented to provide detailed informations about how probiotics stimulate our immune system. Lactobacillus rhamnosus GG, Lactobacillus casei Shirota, Bifidobacterium animalis Bb-12, Lactobacillus johnsonii La1, Bifidobacterium lactis DR10, and Saccharomyces cerevisiae boulardii are the most investigated probiotic cultures for their immunomodulation properties. Probiotics can enhance nonspecific cellular immune response characterized by activation of macrophages, natural killer (NK) cells, antigen-specific cytotoxic T-lymphocytes, and the release of various cytokines in strain-specific and dose-dependent manner. Mixture and type (gram-positive and gram-negative) of probiotic organisms may induce different cytokine responses. Supplementation of probiotic organisms in infancy could help prevent immune-mediated diseases in childhood, whereas their intervention in pregnancy could affect fetal immune parameters, such as cord blood interferon (IFN)-γ levels, transforming growth factor (TGF)-β1 levels, and breast milk immunoglobulin (Ig)A. Probiotics that can be delivered via fermented milk or yogurt could improve the gut mucosal immune system by increasing the number of IgA(+) cells and cytokine-producing cells in the effector site of the intestine.

OBJECTIVE

Krüppel-like factor 8 (KLF8), a downstream transcription factor of transforming growth factor-β1 (TGF-β1), has a role in tumorigenesis, tumor progress and epithelial-to-mesenchymal transition (EMT) induction. Recent studies mainly focused on its role in breast cancer and hepatocellular carcinoma; however, little is studied in gastric cancer. Here, we aim to explore whether KLF8 is involved in TGF-β1-induced EMT in gastric cancer cells.

METHODS

Western blot and real-time PCR assays were used to detect the expression of KLF8, E-cadherin and vimentin in gastric cancer cell line SGC7901 treated with or without TGF-β1. The lentivirus-mediated RNA interference technique was used to knock down the expression of KLF8 in gastric cancer cell line SGC7901. In vitro, the ability of cell migration and invasion were measured by transwell and wound healing assays; the cell motility was detected by high content screening assay.

RESULTS

TGF-β1 could induce EMT via down-regulating E-cadherin and up-regulating vimentin expression in gastric cancer cells. Further study found that TGF-β1 could induce KLF8 expression at the protein and mRNA levels in gastric cancer cells (P < 0.05). Western blot and real-time PCR assays found that small interference RNA (siRNA)-mediated KLF8 silence blocked TGF-β1-induced EMT-like transformation and subsequently reversed the loss of E-cadherin and gain of vimentin. In vitro, inhibition of KLF8 decreased TGF-β1-prompted cell migration, invasion and motility.

CONCLUSIONS

KLF8, a transcription factor, is involved in TGF-β1-induced EMT in gastric cancer cells and may be a novel therapeutic target for the treatment of gastric cancer.

Tribbles homolog 3 (TRB3, also known as TRIB3, NIPK and SKIP3), a human homolog of Drosophila Tribbles, has been found to interact with a variety of signaling molecules to regulate diverse cellular functions. Here, we report that TRB3 is a novel SMAD3-interacting protein. Expression of exogenous TRB3 enhanced the transcriptional activity of SMAD3, whereas knocking down endogenous TRB3 reduced the transcriptional activity of SMAD3. The kinase-like domain (KD) of TRB3 was responsible for the interaction with SMAD3 and the regulation of SMAD3-mediated transcriptional activity. In addition, TGF-β1 stimulation or overexpression of SMAD3 enhanced the TRB3 promoter activity and expression, suggesting that there is a positive feedback loop between TRB3 and TGF-β-SMAD3 signaling. Mechanistically, TRB3 was found to trigger the degradation of SMAD ubiquitin regulatory factor 2 (Smurf2), which resulted in a decrease in the degradation of SMAD2 and phosphorylated SMAD3. Moreover, TRB3-SMAD3 interaction promoted the nuclear localization of SMAD3 because of the interaction of TRB3 with the MH2 domain of SMAD3. These effects of TRB3 were responsible for potentiating the SMAD3-mediated activity. Furthermore, knockdown of endogenous TRB3 expression inhibited the migration and invasion of tumor cells in vitro, which were associated with an increase in the expression of E-cadherin and a decrease in the expression of Twist-1 and Snail, two master regulators of epithelial-to-mesenchymal transition, suggesting a crucial role for TRB3 in maintaining the mesenchymal status of tumor cells. These results demonstrate that TRB3 acts as a novel SMAD3-interacting protein to participate in the positive regulation of TGF-β-SMAD-mediated cellular biological functions.

OBJECTIVE

When used as an alternative substrate following bare sclera removal of pterygium and other ocular surface diseases, amniotic membrane transplantation can reduce scarring on the reconstructed conjunctival surface. This study was carried out to determine if the amniotic membrane (AM) suppresses the expression of the TGFb signaling system in cultured normal conjunctival (HCF) and pterygial body fibroblasts (PBF).

METHODS

HCF and PBF were cultured on AM and plastic wells in serum-containing and serum-free DMEM with or without TGF-beta1. Total RNA was extracted and subjected to Northern hybridization with probes of TGF-beta1, b2 and b3; TGF-beta receptors (TGF- beta R) type I, II and III; a-smooth muscle actin (alpha-SM), b1-integrin, CD44, fibroblast growth factor receptor 1 (FGF-R1/ flg) and platelet-derived growth factor receptor b (PDGFR-beta); and GAPDH as a loading control. MTT assay was used for cell proliferation.

RESULTS

Amniotic membrane markedly suppressed the transcript expression of TGF-beta2, b3 and all three types of TGF-beta receptors by both fibroblasts as compared to their cultures on plastic surface. In addition, expression of CD44 transcript was also markedly suppressed while that of b1 integrin, a-SM actin, and FGFR1/flg was mildly suppressed. In contrast, expression of TGF-beta1 and PDGFR-beta remained largely unchanged. The cell proliferation of HCF and PBF grown on AM was also significantly suppressed.

CONCLUSIONS

Amniotic membrane matrix uniquely suppresses TGF- beta signaling in both types of fibroblasts. It may also suppress signaling via CD44, b1 integrin and FGFR1/flg. As a result, the phenotype may become less mitogenic, contractile and fibrogenic. These data support in part why amniotic membrane transplantation has an anti-scarring effect for conjunctival surface reconstruction.

Long noncoding RNAs (lncRNAs) are emerging as important regulatory molecules at the transcriptional and post-transcriptional levels and may play essential roles in the differentiation of human bone marrow mesenchymal stem cell (hMSC). However, their roles and functions remain unclear. Here, we showed that lncRNA H19 was significantly upregulated after the induction of osteoblast differentiation. Overexpression of H19 promoted osteogenic differentiation of hMSCs in vitro and enhanced heterotopic bone formation in vivo, whereas knockdown of H19 inhibited these effects. Subsequently, we found that miR-675, encoded by exon1 of H19, promoted osteoblast differentiation of hMSCs and was partially responsible for the pro-osteogenic effect of H19. Investigating the underlying mechanism, we demonstrated that H19/miR-675 inhibited mRNA and protein expression of transforming growth factor-β1 (TGF-β1). The downregulation of TGF-β1 subsequently inhibited phosphorylation of Smad3. Meanwhile, H19/miR-675 downregulated the mRNA and protein levels of histone deacetylase (HDAC) 4/5, and thus increased osteoblast marker gene expression. Taken together, our results demonstrated that the novel pathway H19/miR-675/TGF-β1/Smad3/HDAC regulates osteogenic differentiation of hMSCs and may serve as a potential target for enhancing bone formation in vivo.

Following myocardial infarction, tissue repair is mediated by the recruitment of monocytes and their subsequent differentiation into macrophages. Recent findings have revealed the dynamic changes in the presence of polarized macrophages with pro-inflammatory (M1) and anti-inflammatory (M2) properties during the early (acute) and late (chronic) stages of cardiac ischemia. Mesenchymal stem cells (MSCs) delivered into the injured myocardium as reparative cells are subjected to the effects of polarized macrophages and the inflammatory milieu. The present study investigated how cytokines and polarized macrophages associated with pro-inflammatory (M1) and anti-inflammatory (M2) responses affect the survival of MSCs. Human MSCs were studied using an in vitro platform with individual and combined M1 and M2 cytokines: IL-1β, IL-6, TNF-α, and IFN-γ (for M1), and IL-10, TGF-β1, TGF-β3, and VEGF (for M2). In addition, polarization molecules (M1: LPS and IFN-γ; M2: IL-4 and IL-13) and common chemokines (SDF-1 and MCP-1) found during inflammation were also studied. Indirect and direct co-cultures were conducted using M1 and M2 polarized human THP-1 monocytes. M2 macrophages and their associated cytokines supported the growth of hMSCs, while M1 macrophages and their associated cytokines inhibited the growth of hMSCs in vitro under certain conditions. These data imply that an anti-inflammatory (M2) environment is more accommodating to the therapeutic hMSCs than a pro-inflammatory (M1) environment at specific concentrations.

Cellular transplantation strategies for repairing the injured spinal cord have shown consistent benefit in preclinical models, and human clinical trials have begun. Interactions between transplanted cells and host tissue remain poorly understood. Trophic factor secretion is postulated a primary or supplementary mechanism of action for many transplanted cells, however, there is little direct evidence to support trophin production by transplanted cells in situ. In the present study, trophic factor expression was characterized in uninjured, injured-untreated, injured-treated with transplanted cells, and corresponding control tissue from the adult rat spinal cord. Candidate trophic factors were identified in a literature search, and primers were designed for these genes. We examined in vivo trophin expression in 3 paradigms involving transplantation of either brain or spinal cord-derived neural precursor cells (NPCs) or bone marrow stromal cells (BMSCs). Injury without further treatment led to a significant elevation of nerve growth factor (NGF), leukemia inhibitory factor (LIF), insulin-like growth factor-1 (IGF-1), and transforming growth factor-β1 (TGF-β1), and lower expression of vascular endothelial growth factor isoform A (VEGF-A) and platelet-derived growth factor-A (PDGF-A). Transplantation of NPCs led to modest changes in trophin expression, and the co-administration of intrathecal trophins resulted in significant elevation of the neurotrophins, glial-derived neurotrophic factor (GDNF), LIF, and basic fibroblast growth factor (bFGF). BMSCs transplantation upregulated NGF, LIF, and IGF-1. NPCs isolated after transplantation into the injured spinal cord expressed the neurotrophins, ciliary neurotrophic factor (CNTF), epidermal growth factor (EGF), and bFGF at higher levels than host cord. These data show that trophin expression in the spinal cord is influenced by injury and cell transplantation, particularly when combined with intrathecal trophin infusion. Trophins may contribute to the benefits associated with cell-based repair strategies for spinal cord injury.