TGFß2 mediates oxidative stress-induced epithelial-to-mesenchymal transition of bladder smooth muscle.

Bladder outlet obstruction (BOO) is the primary clinical manifestation of benign prostatic hyperplasia, the most common urinary system disease in elderly men, and leads to associated lower urinary tract symptoms. Although BOO is reportedly associated with increased systemic oxidative stress (OS), the underlying mechanism remains unclear. The elucidation of this mechanism is the primary aim of this study. A Sprague-Dawley rat model of BOO was constructed and used for urodynamic monitoring. The bladder tissue of rats was collected and subjected to real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR), histological examination, and immunohistochemical staining. Through bioinformatics prediction, we found that transforming growth factor ß2 (TGFß2) expression was upregulated in rats with BOO compared with normal bladder tissue. In vitro analyses using primary bladder smooth muscle cells (BSMCs) revealed that hydrogen peroxide (H2O2) induced TGFß2 expression. Moreover, H2O2 induced epithelial-to-mesenchymal transition (EMT) by reducing E-cadherin, an endothelial marker and CK-18, a cytokeratin maker, and increasing mesenchymal markers, including N-cadherin, vimentin, and α-smooth muscle actin (α-SMA) levels. The downregulation of TGFß2 expression in BSMCs using siRNA technology alleviated H2O2-induced changes in EMT marker expression. The findings of the study indicate that TGFß2 plays a crucial role in BOO by participating in OS-induced EMT in BSMCs.

Role of reactive oxygen species in epithelial-mesenchymal transition and apoptosis of human lens epithelial cells.

AIM: To investigate the role of reactive oxygen species (ROS) in epithelial-mesenchymal transition (EMT) and apoptosis of human lens epithelial cells (HLECs). METHODS: Flow cytometry was used to assess ROS production after transforming growth factor ß2 (TGF-ß2) induction. Apoptosis of HLECs after H2O2 and TGF-ß2 interference with or without ROS scavenger N-acetylcysteine (NAC) were assessed by flow cytometry. The corresponding protein expression levels of the EMT marker α-smooth muscle actin (α-SMA), the extracellular matrix (ECM), marker fibronectin (Fn), and apoptosis-associated proteins were detected by using Western blotting in the presence of an ROS scavenger (NAC). Wound-healing and Transwell assays were used to assess the migration capability of HLECs. RESULTS: TGF-ß2 stimulates ROS production within 8h in HLECs. Additionally, TGF-ß2 induced HLECs cell apoptosis, EMT/ECM synthesis protein markers expression, and pro-apoptotic proteins production; nonetheless, NAC treatment prevented these responses. Similarly, TGF-ß2 promoted HLECs cell migration, whereas NAC inhibited cell migration. We further determined that although ROS initiated apoptosis, it only induced the accumulation of the EMT marker α-SMA protein, but not COL-1 or Fn. CONCLUSION: ROS contribute to TGF-ß2-induced EMT/ECM synthesis and cell apoptosis of HLECs; however, ROS alone are not sufficient for EMT/ECM synthesis.

The role of prostate-specific antigen in the osteoblastic bone metastasis of prostate cancer: a literature review.

Prostate cancer is the only human malignancy that generates predominantly osteoblastic bone metastases, and osteoblastic bone metastases account for more than 90% of osseous metastases of prostate cancer. Prostate-specific antigen (PSA) plays an important role in the osteoblastic bone metastasis of prostate cancer, which can promote osteomimicry of prostate cancer cells, suppress osteoclast differentiation, and facilitate osteoblast proliferation and activation at metastatic sites. In the meantime, it can activate osteogenic factors, including insulin-like growth factor, transforming growth factor ß2 and urokinase-type plasminogen activator, and meanwhile suppress osteolytic factors such as parathyroid hormone-related protein. To recapitulate, PSA plays a significant role in the osteoblastic predominance of prostate cancer bone metastasis and bone remodeling by regulating multiple cells and factors involved in osseous metastasis.

LncRNA-MALAT1/miRNA-204-5p/Smad4 Axis Regulates Epithelial-Mesenchymal Transition, Proliferation and Migration of Lens Epithelial Cells.

MATERIALS AND METHODS: LECs were cultured and induced with TGF-ß2 (10 ng/mL). SiRNA against MALAT1 (Si-MALAT1) was transfected into LECs to knockdown the expression of MALAT1. To overexpress or knockdown miR-204-5p, miR-204-5p mimics (miR-204-5p mimics) and anti-miR-204-5p (miR-204-5p inhibitor) were transfected into LECs. We used RNA FISH to identify the location of MALAT1. RNA levels of MALAT1 and miR-204-5p were analyzed by RT-qPCR. Additionally, target protein levels of Smad4, epithelial differentiation and mesenchymal markers were analyzed with Western blot. We employed EdU Labeling to measured cell proliferation and performed Transwell Assay to analyze the cell migration. Dual-luciferase reporter assays in LECs were conducted to verify whether miRNA-204-5p was negatively regulated by MALAT1 and Smad4 was a direct target of miR-204-5p. RESULTS: The expression of MALAT1 was upregulated in PCO specimens. MALAT1 was overexpressed in TGF-ß2 induced LECs, and the knockdown of MALAT1 could attenuate TGF-ß2 induced EMT. Besides, the upregulation of MALAT1 was correlated with the downregulation of miR-204-5p and upregulation of Smad4. Importantly, MALAT1 was revealed to be located in the cytoplasm of LECs. Furthermore, luciferase reporter assays confirmed that MALAT1 could negatively regulate the expression of miR-204-5p and then regulate its direct target Smad4. Finally, the knockdown of MALAT1 could inhibit the EMT, proliferation, and migration of LECs; however, those can be reversed by anti-miR-204-5p. CONCLUSIONS: Our findings reveal that MALAT1 may regulate EMT, proliferation, and migration of LECs as a ceRNA by "sponging" miR-204-5p and targeting Smad4, and serve as a promising therapeutic target in preventing PCO.

Sulforaphane modulates TGFß2-induced conjunctival fibroblasts activation and fibrosis by inhibiting PI3K/Akt signaling.

AIM: To examine the effects of sulforaphane on fibrotic changes of transforming growth factor (TGFß2) induced human conjunctival fibroblast (HConFs). METHODS: HConFs were cultured and divided into control, TGFß2 (1 ng/mL), sulforaphane and TGFß2+sulforaphane groups. Cell viability and apoptosis were detected using the MTT and ApoTox-Glo Triplex assay. Cell migration was detected using scratch and Transwell assay. Real-time quantitative PCR method was used to evaluate mRNA expression of TGFß2, matrix metalloproteinase-2 (MMP2), myosin light chain kinase (MYLK), integrin αV, integrin α5, fibronectin 1 and α-smooth muscle actin (α-SMA). The protein expression of α-SMA, p-PI3K, PI3K, p-Akt, and Akt were detected by Western blot. RESULTS: The proliferation of HConFs was significantly (P<0.05) suppressed by sulforaphane compared to control cells with the increase of the concentration and treatment time. Cell proliferation after 48h incubation was significantly reduced with 100 µmol/L sulforaphane treatment by 17.53% (P<0.05). The Transwell assay showed sulforaphane decreased cell migration by 18.73% compared with TGFß2-induced HConF (P<0.05). TGFß2-induced the increasing expression of fibronectin, type I collagen and α-SMA, and the phosphorylation of PI3K and Akt were all significantly suppressed by sulforaphane pretreatment. CONCLUSION: Sulforaphane inhibits proliferation, migration, and synthesis of the extracellular matrix in HConFs, and inhibiting the PI3K/Akt signaling pathway. Sulforaphane could be a potential therapeutic drug for prevention of scar formation in filtering bleb after trabeculectomy.

Pirfenidone ameliorates the formation of choroidal neovascularization in mice.

The formation and development of choroidal neovascularization (CNV) is accompanied by inflammation and fibrosis. Existing treatments are expensive and can cause irreversible complications. Pirfenidone (PFD) exerts anti­inflammatory and anti­fibrotic effects; however, its applications in the eye remain unclear. Male C57BL/6J mice (aged 6­8 weeks) were used to explore whether PFD can inhibit the formation of laser­induced CNV. The localization of transforming growth factor ß2 (TGFß2) was determined through immunofluorescent staining. After laser photocoagulation, the vehicle and PFD groups were intravitreally injected with 1 µl PBS and 1 µl 0.5% PFD, respectively. At day 7 after intravitreal injection, the expression of TGFß2 and vascular endothelial growth factor (VEGF) was assessed. Fundus fluorescein angiography was performed to investigate the extent of fluorescence leakage, and the CNV areas were analyzed using a choroidal flat mount. The results demonstrated that, on day 7 after photocoagulation, the expression of TGFß2 and VEGF was reduced in the experimental group. In addition, fluorescein angiography showed that the leakage area of CNV was significantly smaller in the PFD injection group than those observed in the control and vehicle groups. Moreover, the areas of CNV in the PFD injection group were smaller compared with those reported in the other two injection groups. Histopathological and TUNEL analyses performed on day 28 revealed that there were no notable abnormalities on the layers of the neural retina of PFD­treated mice. In conclusion, intravitreal injection of PFD inhibited the formation of CNV in mice, likely via the downregulation of VEGF and TGFß2, which did not cause damage to the mouse retina after 28 days of treatment.

Inhibitory role of transforming growth factor ß2 in experimental autoimmune anterior uveitis.

PURPOSE: Experimental autoimmune anterior uveitis (EAAU) is a clinically relevant animal model for human idiopathic anterior uveitis (IAU). The role of the immunomodulator transforming growth factor ß2 (TGF-ß2) in EAAU pathology is unknown. In this study, we investigated the regulatory role of TGF-ß2 in EAAU. METHODS: EAAU was induced in male Lewis rats by footpad injection of melanin-associated antigen (MAA). TGF-ß2 was administered intravenously (iv) in MAA-sensitized rats during the induction of EAAU, or after the clinical onset of uveitis. MAA-sensitized rats injected similarly with an equal volume of PBS served as control. Animals were examined daily between days 7 and 30 post-injection for the clinical signs of uveitis using slit lamp biomicroscopy. Animals were sacrificed at various time points and eyes were harvested for histological analysis to assess the course and severity of inflammation. For histopathological analysis, paraffin sections of harvested eyes were stained with hematoxylin and eosin. Popliteal lymph nodes (LNs) were used for CD4+CD25+FoxP3+ T regulatory (Tregs) population analysis and for CD4+ T cell proliferation assay. RESULTS: Administration of recombinant TGF-ß2 during the early stages of EAAU prevented the induction of uveitis. Compared to PBS, the presence of TGF-ß2 in the cell culture significantly (p < 0.05) inhibited the proliferation of CD4+ T cells in response to MAA. In MAA-sensitized Lewis rats, iv treatment with recombinant TGF-ß2 resulted in significantly (p < 0.05) increased percentage of Tregs compared to animals treated similarly with PBS. Thus, TGF-ß2 inhibited the induction of EAAU by inhibiting CD4+ T cell proliferation and increasing the number of Tregs. Injection of TGF-ß2 in rats with active EAAU resulted in diminished disease activity. Unfortunately, this treatment did not lead to the early resolution of EAAU. CONCLUSIONS: TGF-ß2 plays a critical role in regulation of intraocular inflammation in EAAU. Findings reported in this study improve our understanding of immunopathology of IAU and suggest that recombinant TGF-ß2 may be a promising therapeutic agent for human IAU.

MicroRNA-153 affects nasopharyngeal cancer cell viability by targeting TGF-ß2.

The aim of the present study was to determine the function of microRNA-153 (miR-153) in the viability of nasopharyngeal cancer (NPC) cells and determine the underlying molecular mechanism. The expression of miR-153 in patients with NPC was markedly decreased compared with that in paracarcinoma tissue. miR-153 upregulation observably decreased cell viability, induced apoptosis, increased caspase-3 and -9 activity, and increased the B-cell lymphoma 2 (Bcl-2)-associated X protein/Bcl-2 protein expression ratio in 13-9B cells. miR-153 upregulation also suppressed transforming growth factor-ß2 (TGF-ß2) and Smad2 protein expression in 13-9B cells. TGF-ß2 inhibitor enhanced the effect of miR-153 upregulation on the inhibition of cell viability, induction of apoptosis, increase in caspase-3 and -9 activity, and increase in Bax/Bcl-2 protein expression ratio in 13-9B cells. The results of the present study indicate that miR-153 affects the progression of NPC by targeting the TGF-ß2/Smad2 signaling pathway.

IOP lowering effect of topical trans-resveratrol involves adenosine receptors and TGF-ß2 signaling pathways.

Trans-resveratrol was earlier shown to lower intraocular pressure (IOP) in rats; however, its mechanisms of action remain unclear. It has been shown to modulate adenosine receptor (AR) and TGF-ß2 signaling, both of which play a role in regulating IOP. Hence, we investigated effects of trans-resveratrol on AR and TGF-ß2 signaling. Steroid-induced ocular hypertensive (SIOH) rats were pretreated with A1AR, phospholipase C (PLC) and ERK1/2 inhibitors and were subsequently treated with single drop of trans-resveratrol. Metalloproteinases (MMP)-2 and -9 were measured in aqueous humor (AH). In another set of experiments, effect of trans-resveratrol on AH level of tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA) was determined after single and multiple drop administration in SIOH rats. Effect of trans-resveratrol on ARs expression, PLC and pERK1/2 activation and MMPs, tPA and uPA secretion was determined using human trabecular meshwork cells (HTMC). Further, effect of trans-resveratrol on TGF-ß2 receptors, SMAD signaling molecules and uPA and tPA expression by HTMC was determined in the presence and absence of TGF-ß2. Pretreatment with A1AR, PLC and ERK1/2 inhibitors antagonized the IOP lowering effect of trans-resveratrol and caused significant reduction in the AH level of MMP-2 in SIOH rats. Trans-resveratrol increased A1AR and A2AAR expression, cellular PLC, pERK1/2 levels and MMP-2, tPA and uPA secretion by HTMC. Additionally, it produced TGFßRI downregulation and SMAD 7 upregulation. In conclusion, IOP lowering effect of trans-resveratrol involves upregulation of A1AR expression, PLC and ERK1/2 activation and increased MMP-2 secretion. It downregulates TGFßRI and upregulates SMAD7 hence, inhibits TGF-ß2 signaling.

Transforming growth factor ß2 (TGFß2) signaling plays a key role in glucocorticoid-induced ocular hypertension.

Elevation of intraocular pressure (IOP) is a serious adverse effect of glucocorticoid (GC) therapy. Increased extracellular matrix (ECM) accumulation and endoplasmic reticulum (ER) stress in the trabecular meshwork (TM) is associated with GC-induced IOP elevation. However, the molecular mechanisms by which GCs induce ECM accumulation and ER stress in the TM have not been determined. Here, we show that a potent GC, dexamethasone (Dex), activates transforming growth factor ß (TGFß) signaling, leading to GC-induced ECM accumulation, ER stress, and IOP elevation. Dex increased both the precursor and bioactive forms of TGFß2 in conditioned medium and activated TGFß-induced SMAD signaling in primary human TM cells. Dex also activated TGFß2 in the aqueous humor and TM of a mouse model of Dex-induced ocular hypertension. We further show that Smad3-/- mice are protected from Dex-induced ocular hypertension, ER stress, and ECM accumulation. Moreover, treating WT mice with a selective TGFß receptor kinase I inhibitor, LY364947, significantly decreased Dex-induced ocular hypertension. Of note, knockdown of the ER stress-induced activating transcription factor 4 (ATF4), or C/EBP homologous protein (CHOP), completely prevented Dex-induced TGFß2 activation and ECM accumulation in TM cells. These observations suggested that chronic ER stress promotes Dex-induced ocular hypertension via TGFß signaling. Our results indicate that TGFß2 signaling plays a central role in GC-induced ocular hypertension and provides therapeutic targets for GC-induced ocular hypertension.

Heparin functionalization increases retention of TGF-ß2 and GDF5 on biphasic silk fibroin scaffolds for tendon/ligament-to-bone tissue engineering.

The tendon/ligament-to-bone transition (enthesis) is a highly specialized interphase tissue with structural gradients of extracellular matrix composition, collagen molecule alignment and mineralization. These structural features are essential for enthesis function, but are often not regenerated after injury. Tissue engineering is a promising strategy for enthesis repair. Engineering of complex tissue interphases such as the enthesis is likely to require a combination of biophysical, biological and chemical cues to achieve functional tissue regeneration. In this study, we cultured human primary adipose-derived mesenchymal stem cells (AdMCs) on biphasic silk fibroin scaffolds with integrated anisotropic (tendon/ligament-like) and isotropic (bone/cartilage like) pore alignment. We functionalized those scaffolds with heparin and explored their ability to deliver transforming growth factor ß2 (TGF-ß2) and growth/differentiation factor 5 (GDF5). Heparin functionalization increased the amount of TGF-ß2 and GDF5 remaining attached to the scaffold matrix and resulted in biological effects at low growth factor doses. We analyzed the combined impact of pore alignment and growth factors on AdMSCs. TGF-ß2 and pore anisotropy synergistically increased the expression of tendon/ligament markers and collagen I protein content. In addition, the combined delivery of TGF-ß2 and GDF5 enhanced the expression of cartilage markers and collagen II protein content on substrates with isotropic porosity, whereas enthesis markers were enhanced in areas of mixed anisotropic/isotropic porosity. Altogether, the data obtained in this study improves current understanding on the combined effects of biological and structural cues on stem cell fate and presents a promising strategy for tendon/ligament-to-bone regeneration. STATEMENT OF SIGNIFICANCE: Regeneration of the tendon/ligament-to-bone interphase (enthesis) is of significance in the repair of ruptured tendons/ligaments to bone to improve implant integration and clinical outcome. This study proposes a novel approach for enthesis regeneration based on a biomimetic and integrated tendon/ligament-to-bone construct, stem cells and heparin-based delivery of growth factors. We show that heparin can keep growth factors local and biologically active at low doses, which is critical to avoid supraphysiological doses and associated side effects. In addition, we identify synergistic effects of biological (growth factors) and structural (pore alignment) cues on stem cells. These results improve current understanding on the combined impact of biological and structural cues on the multi-lineage differentiation capacity of stem cells for regenerating complex tissue interphases.

Downregulation of CITED2 contributes to TGFß-mediated senescence of tendon-derived stem cells.

Tendon-derived stem cells (TDSCs) are multipotent adult stem cells with potential applications in tendon and tendon-bone junction repair. However, cellular characteristics change during in vitro passaging. Therefore, elucidation of the molecular and cellular mechanisms of tendon aging will be essential for the development of TDSC-based therapies. The aim of this study is to investigate the effect of CITED2, a nuclear regulator and transforming growth factor ß2 (TGFß2) on TDSC proliferation and senescence by comparing cells derived from Achilles tendon biopsies of young individuals (Y-TDSC) with those of older patients (O-TDSC). Our results showed that CITED2 mRNA and protein expression levels were significantly higher in Y-TDSCs than in O-TDSCs and O-TDSCs displayed decreased proliferation and increased senescence compared with Y-TDSCs. Furthermore, high levels of CITED2 protein expression in Y-TDSCs correlated with the downregulation of SP1 and p21 and the upregulation of MYC, potentially indicating the mechanism by which CITED2 upregulates TDSC proliferation. TGFß2 was found to downregulate the expression of the CITED2 gene and knockdown of CITED2 abolished the effect of TGFß2 on TDSC proliferation and senescence. Thus, the downregulation of CITED2 contributes to TGFß-mediated senescence providing an insight into the molecular and cellular mechanisms that contribute to tendon aging and degeneration. Our findings may aid the development of cell-based therapies for tendon repair.

Genetic variance of transforming growth factor ß2 gene in conotruncal heart defects.

OBJECTIVE: The aim of this study was to evaluate the association between two haplotype-tag single nucleotide polymorphisms (SNPs) (rs6658835 and rs10495098) of TGF-ß2 and conotruncal heart defects (CTDs). METHODS: Two polymorphisms of TGF-ß2 gene were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) from 259 CTDs patients and 310 control subjects. RESULTS: The association between SNP rs6658835 in TGF-ß2 and CTDs has been found. The frequency of G allele in CTDs patients was significantly higher than that in control subjects (52.7% versus 40.3%, p < 0.001, OR =1.649). CONCLUSION: TGF-ß2 gene polymorphisms may serve as a novel genetic marker for the risk of CTDs.

Increased Transforming Growth Factor ß2 in the Neocortex of Alzheimer's Disease and Dementia with Lewy Bodies is Correlated with Disease Severity and Soluble Aß42 Load.

BACKGROUND: Of the three transforming growth factor (TGF)-ß isoforms known, TGFß1 deficits have been widely reported in Alzheimer's disease (AD) and studied as a potential therapeutic target. In contrast, the status of TGFß2, which has been shown to mediate amyloid-ß (Aß)-mediated neuronal death, are unclear both in AD and in Lewy body dementias (LBD) with differential neuritic plaque and neurofibrillary tangle burden. OBJECTIVE: To measure neocortical TGFß2 levels and their correlations with neuropathological and clinical markers of disease severity in a well-characterized cohort of AD as well as two clinical subtypes of LBD, dementia with Lewy bodies (DLB) and Parkinson's disease dementia (PDD), known to manifest relatively high and low Aß plaque burden, respectively. METHODS: Postmortem samples from temporal cortex (BA21) were measured for TGFß2 using a Luminex-based platform, and correlated with scores for neuritic plaques, neurofibrillary tangles, α-synuclein pathology, dementia severity (as measured by annual decline of Mini-Mental State Examination scores) as well as soluble and total fractions of brain Aß42. RESULTS: TGFß2 was significantly increased in AD and DLB, but not in PDD. TGFß2 also correlated with scores for neurofibrillary tangles, Lewy bodies (within the LBD group), dementia severity, and soluble Aß42 concentration, but not with neuritic plaque scores, total Aß42, or monomeric α-synuclein immunoreactivity. CONCLUSIONS: TGFß2 is increased in the temporal cortex of AD and DLB, and its correlations with neuropathological and clinical markers of disease severity as well as with soluble Aß42 load suggest a potential pathogenic role in mediating the neurotoxicity of non-fibrillar Aß. Our study also indicates the potential utility of targeting TGFß2 in pharmacotherapeutic approaches to AD and DLB.

Curcumin Inhibits Transforming Growth Factor ß Induced Differentiation of Mouse Lung Fibroblasts to Myofibroblasts.

Transforming growth factor ß (TGF-ß) induced differentiation of lung fibroblasts to myofibroblasts is a key event in the pathogenesis of pulmonary fibrosis. This study aimed to evaluate the effect of curcumin on TGF-ß induced differentiation of lung fibroblasts to myofibroblasts and explore the underlying mechanism. Mouse lung fibroblasts were cultured and treated with TGF-ß2 and curcumin or rosiglitazone. Cell vitality was examined by MTT assay. The secretion of collagen-1 was assessed by ELISA. α smooth muscle actin (α-SMA) was visualized by immunofluorescence technique. The expression of peroxisome proliferator activated receptor γ (PPAR-γ) and platelet derived growth factor R ß (PDGFR-ß) was detected by PCR and Western blot analysis. We found that curcumin and rosiglitazone inhibited the proliferation and TGF-ß induced differentiation of mouse lung fibroblasts. In addition, curcumin and rosiglitazone inhibited collagen-1 secretion and α-SMA expression in mouse lung fibroblasts. Furthermore, curcumin and rosiglitazone upregulated PPAR-γ and downregulated PDGFR-ß expression in mouse lung fibroblasts. In conclusion, our study reveals novel mechanism by which curcumin inhibits TGF-ß2 driven differentiation of lung fibroblasts to myofibroblasts. Curcumin could potentially be used for effective treatment of pulmonary fibrosis.

TGFB2 and BCL2L11 methylation in male laryngeal cancer patients.

DNA methylation is a major regulatory mechanism of gene expression. The aim of the present study was to test the association of transforming growth factor ß2 (TGFB2) and B cell lymphoma 2-like 11 (BCL2L11) gene methylation with the risk of laryngeal squamous cell carcinoma (LSCC). Using bisulfite pyrosequencing technology, DNA methylation levels of TGFB2 promoter and BCL2L11 gene-body CpG cytosines were measured in 90 LSCC tissues and 90 adjacent normal tissues. Analysis of variance and paired sample t-test were used to determine the association of gene methylation and the risk of LSCC. Our results revealed that there were no differences in TGFB2 and BCL2L11 methylation levels between the LSCC tissues and the paired normal tissues (P>0.05). Further breakdown analyses demonstrated that the association results of the two gene methylation levels and LSCC remained unchanged with the age, smoking history, histological differentiation or clinical stage of the LSCC patients (all adjusted P>0.05). In conclusion, there is no association of TGFB2 promoter and BCL2L11 gene-body methylation with the risk of LSCC in males.

Transforming growth factor ß2 (TGF-ß2)-induced connective tissue growth factor (CTGF) expression requires sphingosine 1-phosphate receptor 5 (S1P5) in human mesangial cells.

Transforming growth factor ß2 (TGF-ß2) is well known to stimulate the expression of pro-fibrotic connective tissue growth factor (CTGF) in several cell types including human mesangial cells. The present study demonstrates that TGF-ß2 enhances sphingosine 1-phosphate receptor 5 (S1P5) mRNA and protein expression in a time and concentration dependent manner. Pharmacological and siRNA approaches reveal that this upregulation is mediated via activation of classical TGF-ß downstream effectors, Smad and mitogen-activated protein kinases. Most notably, inhibition of Gi with pertussis toxin and downregulation of S1P5 by siRNA block TGF-ß2-stimulated upregulation of CTGF, demonstrating that Gi coupled S1P5 is necessary for TGF-ß2-triggered expression of CTGF in human mesangial cells. Overall, these findings indicate that TGF-ß2 dependent upregulation of S1P5 is required for the induction of pro-fibrotic CTGF by TGF-ß. Targeting S1P5 might be an attractive novel approach to treat renal fibrotic diseases.

TGF-ß2 induces transdifferentiation and fibrosis in human lens epithelial cells via regulating gremlin and CTGF.

Transforming growth factor (TGF)-ß2, gremlin and connective tissue growth factor (CTGF) are known to play important roles in the induction of epithelial mesenchymal transition (EMT) and extracellular matrix (ECM) synthesis. However, the complex functional relationship among gremlin, CTGF and TGF-ß2 in the induction of EMT and ECM synthesis in human lens epithelial cells (HLECs) has not been reported. In this study, we found that TGF-ß2, CTGF and gremlin can individually induce the expression of α-smooth muscle actin (α-SMA), fibronectin (Fn), collagen type I (COL-I), Smad2 and Smad3 in HLECs. Blockade of CTGF and gremlin effectively inhibited TGF-ß2-induced expression of α-SMA, Fn, COL-I, Smad2, and Smad3 in HLECs. Furthermore blockade of Smad2 and Smad3 effectively inhibited CTGF and gremlin induced expression of α-SMA, Fn, COL-I in HLECs. In conclusion, TGF-ß2, CTGF and gremlin are all involved in EMT and ECM synthesis via activation of Smad signaling pathway in HLECs. Specifically silencing CTGF and gremlin can effectively block the TGF-ß2-induced EMT, ECM synthesis due to failure in activation of Smad signaling pathway in HLECs.

Effects of synbiotic supplementation on breast milk levels of IgA, TGF-ß1, and TGF-ß2.

BACKGROUND: Effects of probiotics on the immunological composition of breast milk have been investigated in a few previous studies. OBJECTIVES: The aims of this study were to determine the effects of synbiotic (probiotic plus prebiotic) supplementation on immunoglobulin A (IgA), transforming growth factor ß1 (TGF-ß1), and transforming growth factor ß2 (TGF-ß2) levels of breast milk and on diarrhea incidence in infants. METHODS: In this randomized, double-blind, and placebo-controlled trial, we recruited 80 lactating mothers who were exclusively breastfeeding their 3-month-old infants. We randomly divided the mothers into 2 groups to receive a daily synbiotic supplement (n = 40) or a placebo (n = 40) for 30 days. Demographic and clinical data (ie, health status) were obtained through an interview. The IgA levels of breast milk were detected by nephelometry, and the levels of TGF-ß1 and TGF-ß2 were measured using a commercial Platinum ELISA kit. RESULTS: The breast milk IgA increased significantly from 0.41 ± 0.09 to 0.48 ± 0.15 g/L in the supplemented group (P = .018), while in the placebo group, no significant changes were observed. Although the breast milk TGF-ß1 levels did not change significantly, the TGF-ß2 levels of breast milk increased significantly from 270 ± 37.8 to 382 ± 43.7 pg/mL in the supplemented group (P = .043). Also, the incidence of diarrhea in infants decreased significantly in the supplemented group while no significant changes were observed in the placebo group after the experimental period. CONCLUSION: Synbiotic supplementation may have positive effects on the immune composition of breast milk and the reduction of diarrhea incidence in infants.

Effects of transforming growth factor ß2 and connective tissue growth factor on induction of epithelial mesenchymal transition and extracellular matrix synthesis in human lens epithelial cells.

AIM: To investigate the effects of transforming growth factor ß2 (TGF-ß2) and connective tissue growth factor (CTGF) on transdifferentiation of human lens epithelial cells (HLECs) cultured in vitro and synthesis of extracellular matrix (ECM). METHODS: HLECs were treated with TGF-ß2 (0, 0.5, 1.0, 5, 10µg/L) and CTGF (0, 15, 30, 60, 100µg/L) for different times (0, 24, 48, 72h) in vitro and the expression of α-smooth muscle actin (α-SMA), the main component of the extracellular matrix type I collagen (Col-1) and fibronectin (Fn) were measured by using real-time polymerase chain reaction (PCR) and western-blot. RESULTS: TGF-ß2 and CTGF significantly increased expression of α-SMA mRNA and protein (P<0.05, P<0.001), Fn mRNA and protein (P<0.001), Col-1 mRNA and protein (P<0.001). TGF-ß2 could induce HLECs expression of CTGF mRNA and protein in dose-dependent manner (P<0.05, P<0.001). TGF-ß2 and CTGF could induce HLECs to express α-SMA, Fn and Col-1 in time-dependent manner. Each time of TGF-ß2 and CTGF induced HELCs expression of α-SMA, Fn, Col-1 mRNA and protein was significant increase compared with control (P<0.05, P<0.001). CONCLUSION: TGF-ß2 and CTGF could induce HLECs epithelial mesenchymal transition and ECM synthesis.