Modulation of human β-defensin-2 expression by 17β-estradiol and progesterone in vaginal epithelial cells

We investigated the expression of HBD-1 and -2 in vaginal epithelial cells treated with lipopolysaccharide (LPS) and the effects on HBD-2 expressions by 17β-estradiol and progesterone. Primary vaginal epithelial cells were isolated from a segment of normal anterior vaginal wall obtained during vaginoplasty and were cultured in keratinocyte growth medium and were allowed to undergo their 3rd passage. Expression of HBD-1 and -2 by different stimuli using LPS 0.5 μg/ml, 17β-estradiol 2 nM and progesterone 1 μM was measured by RT-PCR, ELISA and real-time RT-PCR, respectively. HBD-1 was produced constitutively in vaginal epithelial cells and the production of HBD-1 was not influenced by LPS, 17β-estradiol and progesterone, but the production of HBD-2 was increased inducibly by LPS. 17β-Estradiol and progesterone did not change the production of HBD-2 in normal state, but 17β-estradiol increased the production of HBD-2 and progesterone suppressed the production of HBD-2 under the circumstances with infection. The HBD-2 plays an important role at innate host defense on genitourinary tract. The lacks of estrogen during menopause or uses of a progesterone-based oral contraceptive in sexually active women may influence production of HBD-2 in vaginal epithelium and may increase susceptibility to bacterial vaginitis or recurrent UTI.     

Modulation of actin mRNAs in cultured vascular cells by matrix components and TGF-β1

Summary Alpha-smooth muscle actin is currently considered a marker of smooth muscle cell differentiation. However, during various physiologic and pathologic conditions, it can be expressed, sometimes only transiently, in a variety of other cell types, such as cardiac and skeletal muscle cells, as well as in nonmuscle cells. In this report, the expression of actin mRNAs in cultured rat capillary endothelial cells (RFCs) and aortic smooth muscle cells (SMCs) has been studied by Northern hybridization in two-dimensional cultures seeded on individual extracellular matrix proteins and in three-dimensional type I collagen gels. In two-dimensional cultures, in addition to cytoplasmic actin mRNAs which are normally found in endothelial cell populations, RFCs expressed α-smooth muscle (SM) actin mRNA at low levels. α-SM actin mRNA expression is dramatically enhanced by TGF-β₁. In addition, double immunofluorescence staining with anti-vWF and anti-α-SM-1 (a monoclonal antibody to α-SM actin) shows that RFCs co-express the two proteins. In three dimensional cultures, RFCs still expressed vWF, but lost staining for α-SM actin, whereas α-SM actin mRNA became barely detectable. In contrast to two-dimensional cultures, the addition of TGF-β₁ to the culture media did not enhance α-SM actin mRNA in three-dimensional cultures, whereas it induced rapid capillary tube formation. Actin mRNA expression was modulated in SMCs by extracellular matrix components and TGF-β₁ with a pattern very different from that of RFCs. Namely, the comparison of RFCs with other cell types such as bovine aortic endothelial cells shows that co-expression of endothelial and smooth muscle cell markers is very unique to RFCs and occurs only in particular culture conditions. This could be related to the capacity of these microvascular endothelial cells to modulate their phenotype in physiologic and pathologic conditions, particularly during angiogenesis, and could reflect different embryologic origins for endothelial cell populations. Supported by a Post-Doctoral Fellowship from the Swiss National Science Foundation (OK) and grant HL-RO1-28373 (JAM) from the Department of Human Services, Public Health Service, Washington, D.C.     

Accelerated epithelial cell senescence in IPF and the inhibitory role of SIRT6 in TGF-β-induced senescence of human bronchial epithelial cells

Reepithelialization of remodeled air spaces with bronchial epithelial cells is a prominent pathological finding in idiopathic pulmonary fibrosis (IPF) and is implicated in IPF pathogenesis. Recent studies suggest that epithelial senescence is a risk factor for development of IPF, indicating such reepithelialization may be influenced by the acceleration of cellular senescence. Among the sirtuin (SIRT) family, SIRT6, a class III histone deacetylase, has been demonstrated to antagonize senescence. We evaluated the senescence of bronchiolization in association with SIRT6 expression in IPF lung. Senescence-associated β-galactosidase staining and immunohistochemical detection of p21 were performed to evaluate cellular senescence. As a model for transforming growth factor (TGF)-β-induced senescence of abnormal reepithelialization, we used primary human bronchial epithelial cells (HBEC). The changes of SIRT6, p21, and interleukin (IL)-1β expression levels in HBEC, as well as type I collagen expression levels in fibroblasts, were evaluated. In IPF lung samples, an increase in markers of senescence and SIRT6 expression was found in the bronchial epithelial cells lining cystically remodeled air spaces. We found that TGF-β induced senescence in primary HBEC by increasing p21 expression, and, whereas TGF-β also induced SIRT6, it was not sufficient to inhibit cellular senescence. However, overexpression of SIRT6 efficiently inhibited TGF-β-induced senescence via proteasomal degradation of p21. TGF-β-induced senescent HBEC secreted increased amounts of IL-1β, which was sufficient to induce myofibroblast differentiation in fibroblasts. These findings suggest that accelerated epithelial senescence plays a role in IPF pathogenesis through perpetuating abnormal epithelial-mesenchymal interactions, which can be antagonized by SIRT6.     

TGF-β2 induces transdifferentiation and fibrosis in human lens epithelial cells via regulating gremlin and CTGF

Transforming growth factor (TGF)-β2, 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-β2 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-β2, 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-β2-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-β2, 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-β2-induced EMT, ECM synthesis due to failure in activation of Smad signaling pathway in HLECs.     

Activation of extracellular signal-regulated kinase by TGF-β1 via TβRII and Smad7 dependent mechanisms in human bronchial epithelial BEP2D cells

Transforming growth factor-β1 (TGF-β1) can activate mitogen-activated protein kinases (MAPKs) in many types of cells. The mechanism of this activation is not well elucidated. Here, we explore the role of TGF-β/Smads signaling compounds in TGF-β1-mediated activation of extracellular signal-regulated kinase (ERK) MAPK in human papillomavirus (HPV)-18 immortalized human bronchial epithelial cell line BEP2D and the role of TGF-β1-induced phosphorylation of ERK in proliferation and apoptosis of BEP2D. The cell models of siRNA-mediated silencing of TGF-β receptor type II (TβRII), Smad2, Smad3, Smad4, and Smad7 were employed in this study. Our results demonstrate that TGF-β1 activates ERK in a time-dependent manner with a maximum effect at 60 min; overexpression of Smad7 increased this TGF-β1-mediated phosphorylation of the ERK; and siRNA-mediated silencing of TβRII, Smad3, Smad4, and Smad7 abrogated this effect. Moreover, we observed that overexpression of Smad7 restored TGF-β1-mediated ERK phosphorylation in Smad4 knockdown cells but not in TβRII knockdown cells. In BEP2D cells, TGF-β1 treatment effectively inhibited cells’ proliferation and induced their apoptosis. Pretreatment with U0126, an inhibitor of ERK1/2, significantly enhanced the TGF-β1-mediated antiproliferative and apoptosis induction effects in BEP2D cells. These data revealed that TβRII and Smad7 play the critical roles in TGF-β1-mediated activation of ERK; Smad3 and Smad4 can play an indirect role through up-regulating Smad7 expression; and TGF-β1-induced phosphorylation of ERK may participate in BEP2D cell proliferation and apoptosis regulation.     

TGF-β1 signaling plays a dominant role in the crosstalk between TGF-β1 and the aryl hydrocarbon receptor ligand in prostate epithelial cells

Crosstalk between the aryl hydrocarbon receptor (AhR) and transforming growth factor-β1 (TGF-β1) signaling has been observed in various experimental models. However, both molecular mechanism underlying this crosstalk and tissue-specific context of this interaction are still only partially understood. In a model of human non-tumorigenic prostate epithelial cells BPH-1, derived from the benign prostatic hyperplasia, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) persistently activates the AhR signaling pathway and induces expression of xenobiotic metabolizing enzymes, such as CYP1A1 or CYP1B1. Here we demonstrate that TGF-β1 suppresses the AhR-mediated gene expression through multiple mechanisms, involving inhibition of AhR expression and down-regulation of nuclear AhR, via a SMAD4-dependent pathway. In contrast, TCDD-induced AhR signaling does not affect either TGF-β1-regulated gene expression or epithelial-to-mesenchymal transition. These observations suggest that, in the context of prostate epithelium, TGF-β1 signaling plays a dominant role in the crosstalk with AhR signaling pathway. Given the importance of TGF-β1 signaling in regulation of prostate epithelial tissue homeostasis, as well as the recently revealed role of AhR in prostate development and tumorigenesis, the above findings contribute to our understanding of the mechanisms underlying the crosstalk between the two signaling pathways in the prostate-specific context.     

Modulation of the TGF-β1-induced epithelial to mesenchymal transition (EMT) mediated by P1 and P2 purine receptors in MDCK cells

Epithelial to mesenchymal transition (EMT) occurs during embryogenesis or under pathological conditions such as hypoxia, injury, chronic inflammation, or tissue fibrosis. In renal tubular epithelial cells (MDCK), TGF-β1 induces EMT by reducing or increasing epithelial or mesenchymal marker expression, respectively. In this study, we confirmed that the cAMP analogues, 8-CPT-cAMP or N6-Ph-cAMP, inhibited the TGF-β1-driven overexpression of the mesenchymal markers ZEB-1, Slug, Fibronectin, and α-SMA. Furthermore, we showed that A1, A2A, P2Y1, P2Y11, and P2X7 purine receptor agonists modulated the TGF-β1-induced EMT through the involvement of PKA and/or MAPK/ERK signaling. The stimulation of A2A receptor reduced the overexpression of the EMT-related markers, mainly through the cAMP-dependent PKA pathway, as confirmed by cell pre-treatment with Myr-PKI. Both A1 and P2Y1 receptor stimulation exacerbated the TGF-β1-driven effects, which were reduced by cell pre-treatment with the MAPK inhibitor PD98059, according to the increased ERK1/2 phosphorylation upon receptor activation. The effects induced by P2Y11 receptor activation were oppositely modulated by PKA or MAPK inhibition, in line with the dual nature of the Gs- and Gq-coupled receptor. Differently, P2X7 receptor induced, per se, similar and not additive effects compared to TGF-β1, after prolonged cell exposure to BzATP. These results suggest a putative role of purine receptors as target for anti-fibrotic agents.     

Proteome changes of human bronchial epithelial cells in response to pro-inflammatory mediator leukotriene E4 and pro-remodelling factor TGF-β1

Inflammatory environment chronically activates bronchial epithelial cells to stimulate airway cells including epithelial cells themselves by secreting pro-inflammatory and regulatory factors. Proteomic approach is most relevant to screen the epithelial pathways following the inflammatory stimuli. We compared protein expression of the human bronchial epithelial cells exposed to leukotriene E₄ (LTE₄) and transforming growth factor-β₁ (TGF-β₁) with that of non-stimulated cells. The proteins were separated by 2-DE and the differentially expressed proteins were identified by MALDI-TOF MS and TOF/TOF tandem MS/MS. This approach allowed identification of 31 proteins, of which 26 corresponded to different proteins. β-tubulin, significantly down-regulated by LTE₄, was confirmed as a ciliated cell marker β-tubulin IV, whose decrease by LTE₄ was further corroborated by flow cytometry and RT-qPCR. This refers to a contribution of cysteinyl leukotrienes to epithelial remodelling and initiation of epithelial-mesenchymal transition in conducting airways. Of the affected proteins by TGF-β₁, clinically most relevant ones were up-regulated antioxidant enzyme superoxide dismutase 1, pro-fibrotic enzyme protein disulfide-isomerase and heat shock 70 kDa protein 9B. The changed protein profiles from this study add novel aspects to improve our understanding of the airway pathobiology, provide hints for further directed airway research and may contribute to selecting targets for future therapeutics.     

Modulation of LXR-α and the effector genes by Ascorbic acid and Statins in psoriatic keratinocytes

Following the degradative pathway, vesicles loaded with extracellular material, eventually, dock and fuse with lysosomes, acquiring specific membrane markers of these organelles and acid hydrolases responsible for digest their content. The lysosomal-associated membrane protein 2 (LAMP-2), the best characterized lysosomal membrane protein, is found in late stages of endosome maturation and may be used as a marker of lysosome-associated membranes. Lysosomal storage disorders (LSDs) are described by the absence or deficiency in hydrolase activity leading to substrate accumulation within lysosomal components and to the onset of several diseases. It is known that lymphocytes infected by Epstein–Barr virus (EBV) are able to form cytoplasmic vacuoles, which work as a storage compartment for lysosomal acidic hydrolases. At the present study, we validate the EBV as a transforming agent of B lymphocytes in stability studies of long-term stored samples, since the methods used to keep samples in liquid nitrogen and thaw them have all proven to be efficient in samples frozen for up to 2 years. To confirm and investigate some of the most prevalent LSDs in the South of Brazil—Pompe, Fabry and Gaucher diseases—we first measured the enzymatic activity of α-glicosidase, α-galactosidase, and β-glicosidase in those cytoplasmic-formed vacuoles and then looked to LAMP-2 immunoreactivity by employing confocal microscopy techniques.     

SKIP is required for TGF-β1-induced epithelial mesenchymal transition and migration in transformed keratinocytes

Transforming growth factor-β1 (TGF-β1) potently induces the epithelial-mesenchymal transition (EMT) during tumoral progression. Although Sky-interacting protein (SKIP) regulates TGF-β1-induced Smad activation, its role in the induction of cell malignance remains uncertain. We found that TGF-β1 increases SKIP expression in PDV cells. In cells stably transfected with SKIP antisense, AS-S, Smad3 activation decreased, along with an inhibition of TGF-β1-induced EMT, and the cells were sensitized to the TGF-β1-dependent inhibition of proliferation. Also, AS-S cells showed a weaker migration and invasion response. Moreover, TGF-β1-induced urokinase-type plasminogen activator expression was inhibited, concomitantly with a TGF-β1-independent increment of the plasminogen-activator inhibitor-1 expression. Thus, these results suggest that SKIP is required for EMT and invasiveness induced by TGF-β1 in transformed cells.     

Lefty A attenuates the TGF-β1-induced epithelial to mesenchymal transition of human renal proximal epithelial tubular cells

The epithelial to mesenchymal transition (EMT) is a crucial event for renal fibrosis that can be elicited by TGF-β1/Smads signaling and its downstream mediator connective tissue growth factor (CTGF). As a distinct member of the TGF-β superfamily, Lefty A has been shown to be significantly downregulated in the kidneys of patients with severe ureteral obstruction, suggesting its role in renal fibrosis induced by obstructive nephropathy. In order to determine whether Lefty A prevents TGF-β1-induced EMT, human proximal tubule epithelial cells (HK-2) were stably transfected with Lefty A or control vectors and stimulated with 10 ng/ml TGF-β1 for 48 h. The results show that stimulation with TGF-β1 led to EMT including cell morphology changes, Smad2/3 signaling pathway activation, increased α-SMA, collagen type I, and CTGF expression, and decreased E-cadherin expression in mock-transfected HK-2 cells. Overexpression of Lefty A efficiently blocked p-Smad2/3 activation and attenuated all these EMT changes induced by TGF-β1. This finding suggests that Lefty A may serve as a potential new therapeutic target to inhibit or even reverse EMT during the process of renal fibrosis.     

TGF-β mediates proinflammatory seminal fluid signaling in human cervical epithelial cells

The cervix is central to the female genital tract immune response to pathogens and foreign male Ags introduced at coitus. Seminal fluid profoundly influences cervical immune function, inducing proinflammatory cytokine synthesis and leukocyte recruitment. In this study, human Ect1 cervical epithelial cells and primary cervical cells were used to investigate agents in human seminal plasma that induce a proinflammatory response. TGF-β1, TGF-β2, and TGF-β3 are abundant in seminal plasma, and Affymetrix microarray revealed that TGF-β3 elicits changes in Ect1 cell expression of several proinflammatory cytokine and chemokine genes, replicating principal aspects of the Ect1 response to seminal plasma. The differentially expressed genes included several induced in the physiological response of the cervix to seminal fluid in vivo. Notably, all three TGF-β isoforms showed comparable ability to induce Ect1 cell expression of mRNA and protein for GM-CSF and IL-6, and TGF-β induced a similar IL-6 and GM-CSF response in primary cervical epithelial cells. TGF-β neutralizing Abs, receptor antagonists, and signaling inhibitors ablated seminal plasma induction of GM-CSF and IL-6, but did not alter IL-8, CCL2 (MCP-1), CCL20 (MIP-3α), or IL-1α production. Several other cytokines present in seminal plasma did not elicit Ect1 cell responses. These data identify all three TGF-β isoforms as key agents in seminal plasma that signal induction of proinflammatory cytokine synthesis in cervical cells. Our findings suggest that TGF-β in the male partner's seminal fluid may influence cervical immune function after coitus in women, and potentially be a determinant of fertility, as well as defense from infection.     

TGF-β1 suppresses IL-6-induced STAT3 activation through regulation of Jak2 expression in prostate epithelial cells

Chronic inflammation plays an important role in the initiation and progression of various human diseases including benign prostatic hyperplasia or prostate cancer. Here we show that the proinflammatory cytokine interleukin-6 (IL-6) has prosurvival effects and chronically activates the Jak2/STAT3 signalling pathway in a model of benign prostatic hyperplasia (BPH-1). We demonstrate that the antiinflammatory cytokine transforming growth factor-β1 (TGF-β1), which also permanently activates its canonical signalling pathway through SMAD proteins in BPH-1 cells, modifies the effects of IL-6 on cell proliferation. Importantly, TGF-β1 inhibits IL-6 signal transduction by decreasing the phosphorylation levels of STAT3. This effect is associated with decreased expression of Jak2 at both mRNA and protein levels. Moreover, we showed that TGF-β1 inhibits IL-6-induced expression of the cancer-associated gene MUC1. These observations demonstrated a novel interaction between TGF-β1 and IL-6 signalling and suggested another mechanism of how defects in TGF-β signalling, frequently associated with prostate pathologies, can contribute to the disruption of tissue homeostasis.     

Rac1 modulates TGF-β1-mediated epithelial cell plasticity and MMP9 production in transformed keratinocytes

Transforming growth factor-β1 (TGF-β1) activates Rac1 GTPase in mouse transformed keratinocytes. Expression of a constitutively active Q61LRac1 mutant induced an epithelial to mesenchymal transition (EMT) linked to stimulation of cell migration and invasion. On the contrary, expression of a dominant-negative N17TRac1 abolished TGF-β1-induced cell scattering, migration and invasion. Moreover, Q61LRac1 enhanced metalloproteinase-9 (MMP9) production to levels comparable to those induced by TGF-β1, while N17TRac1 was inhibitory. TGF-β1-mediated EMT involves the expression of the E-cadherin repressor Snail1, regulated by the Rac1 and mitogen-activated protein kinase (MAPK) pathways. Furthermore, MMP9 production was MAPK-dependent, as the MEK inhibitor PD98059 decreased TGF-β1-induced MMP9 expression and secretion in Q61LRac1 expressing cells. We propose that regulation of TGF-β1-mediated plasticity of transformed keratinocytes requires the cooperation between the Rac1 and MAPK signalling pathways.     

TGF-β1 induced epithelial to mesenchymal transition (EMT) in human bronchial epithelial cells is enhanced by IL-1β but not abrogated by corticosteroids

Background

Chronic persistent asthma is characterized by ongoing airway inflammation and airway remodeling. The processes leading to airway remodeling are poorly understood, and there is increasing evidence that even aggressive anti-inflammatory therapy does not completely prevent this process. We sought to investigate whether TGFβ₁ stimulates bronchial epithelial cells to undergo transition to a mesenchymal phenotype, and whether this transition can be abrogated by corticosteroid treatment or enhanced by the pro-inflammatory cytokine IL-1β.

Methods

BEAS-2B and primary normal human bronchial epithelial cells were stimulated with TGFβ₁ and expression of epithelial and mesenchymal markers assessed by quantitative real-time PCR, immunoblotting, immunofluorescence microscopy and zymography. In some cases the epithelial cells were also incubated with corticosteroids or IL-1β. Results were analyzed using non-parametric statistical tests.

Results

Treatment of BEAS-2B or primary human bronchial epithelial cells with TGFβ₁ significantly reduced the expression level of the epithelial adherence junction protein E-cadherin. TGFβ₁ then markedly induced mesenchymal marker proteins such as collagen I, tenascin C, fibronectin and α-smooth muscle actin mRNA in a dose dependant manner. The process of mesenchymal transition was accompanied by a morphological change towards a more spindle shaped fibroblast cell type with a more motile and invasive phenotype. Corticosteroid pre-treatment did not significantly alter the TGFβ₁ induced transition but IL-1β enhanced the transition.

Conclusion

Our results indicate, that TGFβ₁ can induce mesenchymal transition in the bronchial epithelial cell line and primary cells. Since asthma has been strongly associated with increased expression of TGFβ₁ in the airway, epithelial to mesenchymal transition may contribute to the contractile and fibrotic remodeling process that accompanies chronic asthma.     

γ-glutamyltransferase in human diploid fibroblasts and other mammalian cells

Summary γ-Glutamyltransferase was determined in WI-38 human diploid fibroblasts and compared to enzyme levels determined in several other mammalian cell lines including: fibroblast-like cells from human skin, tibia and foreskin; epithelial-like cells from human, bovine and monkey kidney; and transformed cells (Chinese hamster ovary, HeLa S₃ and SV-40 transformed WI-38). Transformed cells had the lowest activity found followed in increasing order by fibroblasts, human and bovine epithelial cells and monkey kidney epithelial cells. The enzyme isolated from the plasma membrane of WI-38 cells, like the enzyme from kidney and brain, was found to be irreversibly inhibited by iodoacetamide, reversibly by serine-borate, and had a strong specificity for certain amino acids. The possibility exists that γ-glutamyltransferase could be involved in transport of amino acids into cells in culture; and glutamine, used in media, is an excellent substrate for the enzyme. Preliminary reports of some of this work were presented at meetings of The American Society of Biological Chemists in Minneapolis (Abstracts Fed. Proc. 33: 957, 1974) and at Atlantic City (Abstracts Fed. Proc. 34: 2243, 1975). This work was supported by Grant NIH 1 P01 HD 07173. The WI-38 starter cultures and cell pack used in these studies were obtained through Contract M01 HD 42828 to Stanford University from the National Institute of Aging.