Maintenance of the enteric stem cell niche by bacterial lipopolysaccharides? Evidence and perspectives

The enteric nervous system (ENS) has to respond to continuously changing microenvironmental challenges within the gut and is therefore dependent on a neural stem cell niche to keep the ENS functional throughout life. In this study, we hypothesize that this stem cell niche is also affected during inflammation and therefore investigated lipopolysaccharides (LPS) effects on enteric neural stem/progenitor cells (NSPCs). NSPCs were derived from the ENS and cultured under the influence of different LPS concentrations. LPS effects upon proliferation and differentiation of enteric NSPC cultures were assessed using immunochemistry, flow cytometry, western blot, Multiplex ELISA and real‐time PCR. LPS enhances the proliferation of enteric NSPCs in a dose‐dependent manner. It delays and modifies the differentiation of these cells. The expression of the LPS receptor toll‐like receptor 4 on NSPCs could be demonstrated. Moreover, LPS induces the secretion of several cytokines. Flow cytometry data gives evidence for individual subgroups within the NSPC population. ENS‐derived NSPCs respond to LPS in maintaining at least partially their stem cell character. In the case of inflammatory disease or trauma where the liberation and exposure to LPS will be increased, the expansion of NSPCs could be a first step towards regeneration of the ENS. The reduced and altered differentiation, as well as the induction of cytokine signalling, demonstrates that the stem cell niche may take part in the LPS‐transmitted inflammatory processes in a direct and defined way.     

Cell–cell contact and signaling in the muscle stem cell niche

Muscle stem cells (also called satellite cells or SCs) rely on their local niche for regulatory signals during homeostasis and regeneration. While a number of cell types communicate indirectly through secreted factors, here we focus on the significance of direct contact between SCs and their neighbors. During quiescence, SCs reside under a basal lamina and receive quiescence-promoting signals from their adjacent skeletal myofibers. Upon injury, the composition of the niche changes substantially, enabling the formation of new contacts that mediate proliferation, self-renewal, and differentiation. In this review, we summarize the latest work in understanding cell–cell contact within the satellite cell niche and highlight areas of open questions for future studies.     

Transient expression of human interleukin-2 and interferon-γ genes is regulated by interaction between distinct cell subsets

The level of transient expression of human IL-2 and IFN-γ genes, we show, is regulated by dynamic interaction between two functionally distinct cell populations. One is able to express these genes, while the other, bearing one of several specific surface markers, actively inhibits their expression. Defined cell subsets were isolated from PBMC and tonsil cells using immunomagnetic beads coated with monoclonal antibodies directed against surface markers. Depletion of CD8, CD11a (Leu15), or Leu8 subsets led to a pronounced superinduction of IL-2 and IFN-γ gene expression when the remaining cell population was stimulated with mitogen (PHA) or antigen (SEB). Thus, a 10-fold increase in production of IFN-γ was observed after removal of CD11a (Leu15) cells constituting only a small percentage of the total cell population. By contrast, depletion of cells expressing CD19, a B cell marker, did not yield any superinduction. Conversely, CD8, CD11a (Leu15), or Leu8 cell subsets, but not CD19 cells, each inhibited the induction of IL-2 and IFN-γ gene expression almost completely in depleted or total cell populations from which they were derived. Gene expression occurring within one cell subset could be effectively inhibited by cells from a second subset. Introduction of inhibitory cells (Leu8) into a population that actively expressed IL-2 and IFN-γ mRNA resulted in an immediate cessation of gene expression. This suppression involves a soluble mediator, since the culture medium in which such cells were activated exerted a similarly effective inhibition.     

Basal expression of copper transporter 1 in intestinal epithelial cells is regulated by hypoxia-inducible factor 2α

Hypoxia, via stabilization of HIF2α, regulates the expression of the intestinal iron transporters DMT1 and ferroportin. Here we investigated whether the intestinal copper importer Ctr1 was also regulated by hypoxia. Copper uptake and Ctr1 mRNA expression were significantly increased in Caco-2 cells exposed to hypoxia. To determine whether HIF2α was involved in regulation of Ctr1 expression, we employed three models of HIF2α knockdown (chemical suppression of HIF2α translation in Caco-2 cells; HIF2α-siRNA-treated HuTu80 cells; HIF2α-intestinal knockout mice); Ctr1 mRNA expression was decreased in all three models under normoxic conditions. HIF2α translational inhibitor did not alter Ctr1 expression under hypoxic conditions. We conclude that basal expression of Ctr1 is regulated by HIF2α; however, the induction by hypoxia is a HIF2α-independent event.     

Soy peptide-induced stem cell proliferation: involvement of ERK and TGF-β1

This study was conducted to investigate the proliferative effect of vegetable soy peptides on adult stem cells (ASCs) in the absence of serum and their possible mechanisms of action. The proliferation of human adipose tissue-derived mesenchymal stem cells (ADSCs) and cord blood-derived mesenchymal stem cells (CB-MSCs) treated with soy peptides was found to increase significantly upon 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and Click-iT 5-ethynyl-2'-deoxyuridine flow cytometry assay. In addition, soy peptides led to stepwise phosphorylation of the p44/42 MAPK (ERK), mammalian target of rapamycin (mTOR), p70 S6 kinase, S6 ribosomal protein (S6RP) and eukaryotic initiation factor 4E (eIF4E) in ADSCs. Furthermore, quantitative analysis of the cytokines revealed that the production of transforming growth factor-beta1 (TGF-β1), vascular endothelial growth factor and interleukin-6 increased significantly in response to treatment with soy peptides in both ADSCs and CB-MSCs. Similarly, soy peptide-induced phosphorylation of the ERK/mTOR/S6RP/eIF4E pathway was blocked in response to pretreatment with PD98059, a specific ERK inhibitor. Moreover, inhibition of TGF-β1 through PD98059 pretreatment and a consecutive decrease in ADSC proliferation revealed that TGF-β1 induces the phosphorylation of mTOR/S6RP/eIF4E. Collectively, the results of this study indicate that ERK-dependent production of TGF-β1 plays a crucial role in the soy peptide-induced proliferation of ADSCs under serum-free conditions.     

Preproendothelin-1 expression is negatively regulated by IFNγ during hepatic stellate cell activation

Endothelin-1 (ET-1), a powerful vasoconstrictor peptide, is produced by activated hepatic stellate cells (HSC) and promotes cell proliferation, fibrogenesis, and contraction, the latter of which has been thought to be mechanistically linked to portal hypertension in cirrhosis. Interferon-γ (IFNγ), a Th1 cytokine produced by T cells, inhibits stellate cell proliferation, fibrogenesis, and muscle-specific gene expression. Whether IFNγ-induced inhibitory effects are linked to regulation of ET-1 expression in activated stellate cells remains unknown. Here we examined IFNγ's effects on preproET-1 mRNA expression and the signaling pathways underlying this process. We demonstrated that preproET-1 mRNA expression in HSCs was prominently increased during cell culture-induced activation; IFNγ significantly inhibited both preproET-1 mRNA expression and ET-1 peptide production. Similar results were found in an in vivo model of liver injury and intraperitoneal administration of IFNγ. PreproET-1 promoter analysis revealed that IFNγ-induced inhibition of preproET-1 mRNA expression was closely linked to the AP-1 and Smad3 signaling pathways. Furthermore, IFNγ reduced JNK phosphorylation, which tightly was associated with decreased phosphorylation of downstream factors c-Jun and Smad3 and decreased binding activity of c-Jun and Smad3 in the preprpET-1 promoter. Importantly, IFNγ reduced both c-Jun mRNA and protein levels. Given the important role of ET-1 in wound healing, our results suggest a novel negative signaling network by which IFNγ inhibits preproET-1 expression, highlighting one potential molecular mechanism for IFNγ-induced host immunomodulation of liver fibrogenesis.     

Regulation of S100A2 expression by TGF-β-induced MEK/ERK signalling and its role in cell migration/invasion

The present study demonstrates the important structural features of ceramide required for proper regulation, binding and identification by both pro-apoptotic and anti-apoptotic Bcl-2 family proteins. The C-4=C-5 trans-double bond has little influence on the ability of Bax and Bcl-xL to identify and bind to these channels. The stereochemistry of the headgroup and access to the amide group of ceramide is indispensible for Bax binding, indicating that Bax may interact with the polar portion of the ceramide channel facing the bulk phase. In contrast, Bcl-xL binding to ceramide channels is tolerant of stereochemical changes in the headgroup. The present study also revealed that Bcl-xL has an optimal interaction with long-chain ceramides that are elevated early in apoptosis, whereas short-chain ceramides are not well regulated. Inhibitors specific for the hydrophobic groove of Bcl-xL, including 2-methoxyantimycin A3, ABT-737 and ABT-263 provide insights into the region of Bcl-xL involved in binding to ceramide channels. Molecular docking simulations of the lowest-energy binding poses of ceramides and Bcl-xL inhibitors to Bcl-xL were consistent with the results of our functional studies and propose potential binding modes.     

Teratogenicity of retinoic acid and its effects on TGF-β2 expression in the developing cerebral cortex of the rat

Vitamin A metabolites are potent teratogens in a wide variety of species, including man. Transforming growth factor betas (TGF-s) are involved in several mammalian prenatal developmental processes. The aim of this study was to determine the effects of exogenous and excessive all-trans retinoic acid on TGF2 expression in the developing cerebral cortex of the rat. Many of the malformations including exencephaly, exophtalmus, abdominal wall defects, extremity reduction defects observed in this study were dependent on the time of administration of retinoic acid. TGF-2 was diversely expressed, as revealed immunohistochemically, in the cerebral cortex and plexus choroideus. The diversity depended on the gestational day and the was affected by the administration of retinoic acid. In the 15-day-old fetus from mothers who had been fed by gavage a single dose of 60mg/kg body weight of all-trans retinoic acid on the 8th day of gestation, TGF-2 immunoreactivity in the brain was decreased. However, by the 18th day of gestation, TGF-2 expression increased. The expression of TGF-2 in fetuses whose mothers had been given all-trans retinoic acid after the neurulation period (on day 12 of gestation) was generally similar to that in a control group. We conclude that all-trans retinoic acid leads to severe congenital malformations if administered before neurulation whereas if given after neurulation, it is not so teratogenic. Further, retinoic acid has a variable effect on the expression of TGF-2.     

Matrix and TGF-β-related gene expression during human dental pulp stem cell (DPSC) mineralization

We have recently reported the induction of dental pulp stem cells (DPSCs) into dentin-secreting odontoblast-like cells after stimulation by isolated dentin matrix components, thus mimicking the nature of tissue regeneration seen after tooth disease and injury. After confluency, the cells were further cultured for 21 d in the 10% fetal bovine serum (FBS) Dulbecco’s modified Eagle’s medium (DMEM) (control), and in this medium, with the addition of dentin extract (DE) and the mineralization supplement (MS) of ascorbic acid and β-glycerophosphate (treatment). To identify genes associated with this process, specimens were analyzed with a HG-U133A human gene chip and Arrayassist software. A total of 425 genes, among them 21 matrix and eight TGF-β-related genes, were either up- or downregulated in the experimental group in which the cells showed odontoblast-like differentiation and mineralization. Expression of selected genes was further confirmed by real-time polymerase chain reaction (PCR) analysis. Of the extracellular matrix (ECM)-related genes, two types of collagen genes were upregulated and seven others downregulated. Other ECM-related genes, for example fibulin-1, tenascin C, and particularly thrombospondin 1, were upregulated, and fibulin-2 was downregulated. Most noticeably, the matrix metalloproteinase 1 was induced by the treatment. In the TGF-β superfamily, upregulation of the type II receptor, endoglin, and growth/differentiation factor 5 was coordinated with the downregulation of activin A, TGF-β2, and TGF-β1 itself. This study identifies the matrix and TGF-β-related gene profiles during the DPSC cell mineralization in which several genes are reported for the first time to be associated with this process, thus greatly expanding our molecular knowledge of the induced disease repair process.     

Enhanced chondrogenic marker expression of human mesenchymal stem cells by interaction with both TGF-β3 and hyaluronic acid

This study was designed to evaluate the additive effects of transforming growth factor-beta3 (TGF-β3) and hyaluronic acid (HA) on chondrogenic differentiation of human mesenchymal stem cells (hMSCs). The hMSCs were cultured on collagen type I-, HA-, or fibronectin-coated cell culture dishes with or without TGF-β3 added to the culture medium. Four weeks after cell culture, chondrogenic differentiation of hMSCs was determined by evaluating the expression of cartilage-specific markers using real-time polymerase chain reaction, immunocytochemistry, and Western blot analysis. hMSCs cultured on HA-coated dishes with TGF-β3 supplementation revealed a prominent increase in collagen type II, aggrecan, and Sox9. When hMSCs were cultured without TGF-β3 supplementation, only hMSCs cultured on HA-coated dishes showed prominent expression of the cartilage-specific markers. This study shows that chondrogenic differentiation of hMSCs can be enhanced additively by interactions with both a specific cell-adhesion matrix and a soluble growth factor.     

Effects of TGF-β1 on the proliferation and differentiation of human periodontal ligament cells and a human periodontal ligament stem/progenitor cell line

Periodontal ligament (PDL) is a specialized connective tissue that influences the lifespan of the tooth. Transforming growth factor-β1 (TGF-β1) is a multifunctional cytokine, but little is known about the effects of TGF-β1 on PDL cells. Our aim has been to demonstrate the expression of TGF-β1 in rat PDL tissues and to evaluate its effects on the proliferation and gene expression in human PDL cells (HPLCs) and a human PDL stem/progenitor cell line, line 1-11, that we have recently developed. The expression of TGF-β1 in the entire PDL tissue was confirmed immunohistochemically, and both HPLCs and cell line 1-11 expressed mRNA from the TGF-β1, TGF-β type I receptor, and TGF-β type II receptor genes. Although exogenous TGF-β1 stimulated the proliferation of HPLCs, it did not upregulate the expression of alpha-smooth muscle actin (α-SMA), type I collagen (Col I), or fibrillin-1 (FBN1) mRNA or of α-SMA protein in HPLCs, whereas expression for these genes was attenuated by an anti-TGF-β1 neutralizing antibody. In contrast, exogenous TGF-β1 reduced the proliferation of cell line 1-11, although it upregulated the expression of α-SMA, Col I, and FBN1 mRNA and of α-SMA protein in this cell line. In addition, interleukin-1 beta stimulation significantly reduced the expression of TGF-β1 mRNA and protein in HPLCs. Thus, TGF-β1 seems to play an important role in inducing fibroblastic differentiation of PDL stem/progenitor cells and in maintaining the PDL apparatus under physiological conditions.     

TGF-β1 expression is associated with invasion and metastasis of intrahepatic cholangiocarcinoma

Background

Transforming growth factor (TGF)-β is involved in many physiologic processes, it often promotes metastasis, and its high expression is correlated with poor prognosis. In the present study, we analyzed the correlation between transforming growth factor beta 1 (TGF-β1) expression and prognosis in intrahepatic cholangiocarcinoma.

Results

We examined the expression of TGF-β1 in 78 intrahepatic cholangiocarcinomas by immunohistochemistry and correlated the expression with clinicopathological parameters. TGF-β1 was expressed in 37 of 78 (47.4 %) intrahepatic cholangiocarcinomas. The expression of TGF-β1 was significantly correlated with lymph node metastasis, distant metastasis, and tumour recurrence. Patients with TGF-β1-positive tumours had significantly shorter survival time. In a multivariant analysis, the expression of TGF-β1 and the tumour stage were independent prognostic factors.

Conclusions

Our data suggest that expression of TGF-β1 is a novel prognostic marker for intrahepatic cholangiocarcinoma.     

Star-PAP control of BIK expression and apoptosis is regulated by nuclear PIPKIα and PKCδ signaling

BIK protein is an initiator of mitochondrial apoptosis, and BIK expression is induced by proapoptotic signals, including DNA damage. Here, we demonstrate that 3' end processing and expression of BIK mRNA are controlled by the nuclear PI4,5P(2)-regulated poly(A) polymerase Star-PAP downstream of DNA damage. Nuclear PKCδ is a key mediator of apoptosis, and DNA damage stimulates PKCδ association with the Star-PAP complex where PKCδ is required for Star-PAP-dependent BIK expression. PKCδ binds the PI4,5P(2)-generating enzyme PIPKIα, which is essential for PKCδ interaction with the Star-PAP complex, and PKCδ activity is directly stimulated by PI4,5P(2). Features in the BIK 3' UTR uniquely define Star-PAP specificity and may block canonical PAP activity toward BIK mRNA. This reveals a nuclear phosphoinositide signaling nexus where PIPKIα, PI4,5P(2), and PKCδ regulate Star-PAP control of BIK expression and induction of apoptosis. This pathway is distinct from the Star-PAP-mediated oxidative stress pathway indicating signal-specific regulation of mRNA 3' end processing.     

Regulated expression of the integrin α9β1 in the epithelium of the developing human gut and in intestinal cell lines: Relation with cell proliferation

The integrin α9β1 is one of the recently identified integrins whose expression is restricted to specialized tissues. Its exact function is still unknown. In the present study, we have analyzed the expression of the α9 subunit in human fetal and adult small intestinal and colonic epithelia as well as in intestinal cell lines by indirect immunofluorescence, immunoprecipitation, Western blot, and Northern blot. In intact tissues, the antigen was restricted to the basolateral domain of epithelial cells in intestinal crypts at the fetal stage and was absent in the adult. The α9β1 integrin was also detected in the intestinal cell lines HIEC-6 and Caco-2/15. The presence of α9β1 in HIEC-6 was found to be consistent with their proliferative crypt-like status. In Caco-2/15 cells, the integrin was present at high levels in proliferating cells but was downregulated when cells cease to grow and undertake their differentiation. EGF treatment, which is known to maintain Caco-2/15 cells in a proliferative state, resulted in higher levels of α9 as compared to control cells. Taken together, these observations suggest a relation between integrin α9β1 expression and proliferation in human intestinal cells. J. Cell. Biochem. 71:536–545, 1998. © 1998 Wiley-Liss, Inc.