Inhibition Of Epithelial Cell Proliferation Research Articles

Metformin enhances epithelial cell growth inhibition via the protein kinase–insulin-like growth factor binding protein-1 pathway

Background Abnormal stromal-epithelial cell communication is a pathogenic mechanism in endometriosis, and metformin can modulate it. Insulin-like growth factor binding protein-1 (IGFBP1) plays a role in endometriosis, but the exact mechanism is unknown. IGFBP1 is reportedly a downstream target of metformin in some diseases. We aimed to investigate the role of IGFBP1 in endometriosis development, whether it is associated with abnormal communication, and whether metformin affects IGFBP1 expression. Methods Patients who underwent surgical treatment for endometriosis or other diseases were enrolled. Ten patients with ovarian-type endometriosis and eight patients each who underwent surgical treatment for other lesions with or without endometriosis were selected, and their tissues taken for cell proliferation, western blotting, polymerase chain reaction, and knockdown experiments. Results Ectopic and eutopic stromal cells (EcSCs and EuSCs) lost their ability to inhibit epithelial cell proliferation, and IGFBP1 expression was downregulated in both groups of stromal cells compared to that in normal stromal cells (NSCs; 1.09 vs. 0.25, p = .0002 1.09 vs. 0.57, p = .0029). In an EcSC IGFBP1 overexpression model, the ability of EcSCs to inhibit epithelial cell proliferation was enhanced (EdU positivity decreased from 38% to 25%, p = .0001). Furthermore, adenosine 5’-monophosphate-activated protein kinase (AMPK) phosphorylation was downregulated in EcSCs and EuSCs compared to that in NSCs (0.99 vs. 0.42, p = .0006/0.99 vs. 0.57, p = 0.0032). Treatment of EcSCs with metformin increased AMPK phosphorylation (0.47 vs. 1.04, p = .0107) while upregulating IGFBP1 expression (0.69 vs. 1.01, p = .0164), whereas pre-treatment with an AMPK phosphorylation inhibitor abrogated metformin-induced IGFBP1 upregulation. Conclusions IGFBP1 mediates aberrant stromal-epithelial communication in endometriosis. Metformin can upregulate IGFBP1 expression in EcSCs by activating AMPK, and upregulated IGFBP1 enhances the inhibition of epithelial cell proliferation. IGFBP1 is expected to be a therapeutic target for endometriosis.

Upregulation of IGSF3 expression by iron chelation leads to the inhibition of epithelial cell proliferation

Background/Significance We recently demonstrated that the Immunoglobulin Superfamily-3 (IGSF3), a transmembrane tetraspanin interacting protein, is required for scratch wound repair, cell migration, and barrier function, suggesting a potential key role in lung injury repair. However, the role of IGSF3 in epithelial cell proliferation remain unknown. Furthermore, it is unclear how IGSF3 expression is affected in acute respiratory distress syndrome (ARDS), characterized by hypoxia, loss of lung epithelial barrier, and insufficient cell repair. We hypothesized that hypoxia alters IGSF3 expression and that IGSF3 is mechanistically involved in the effects of hypoxia on human bronchial epithelial cell proliferation. Methods To mimic hypoxia, we exposed human bronchial epithelial cells (Beas2B) submerged in cell culture media to iron chelator deferoxamine (DFO, 100uM, 16-24hr). Proliferation was measured using CCK8 assay. We stably knocked down IGSF3 in Beasw2B using CRISPR-CAS9. Tetraspanin enriched microdomains (TEMs) were isolated using density gradient ultracentrifugation. Single cell RNA-seq (scRNA-seq) was performed on deidentified human ARDS (n=1) and non-diseased (n=3) lungs. Results DFO caused a significant decrease in epithelial cell proliferation (by~ 25%, p<0.001) concomitant to a markedly increased IGSF3 gene and protein expression (by 2- and 2.5-fold, respectively, p<0.01) that was prevented by treatment with the HIF-1a inhibitor, KCF72 (p<0.01). IGSF3-deficient Beas2B exhibited increased cell proliferation at baseline (by~20-50% vs control cells, 24hr; p<0.01) and following treatment with DFO (by ~ 200% vs DFO-treated control cells; 24hr, p<0.01). Compared to non-diseased lungs, scRNA-seq of ARDS lungs revealed marked enrichment of IGSF3 in basal epithelial cell. Interestingly, a similar pattern was observed in Tspan5, an IGSF3 binding tetraspanin induced by iron chelation and in CD147, a hypoxia-inducible Immuhnoglobulin Superfamily protein (Basigin, EMMPRIN). We confirmed that IGSF3 co-localized with Tspan5 within TEMs in Beas2B and, interestingly found that both proteins co-localized with CD147. Conclusions IGSF3 may be induced by hypoxia in lung epithelial cells such as basal cells where it may inhibit cell proliferation and therefore repair. Interestingly, loss of IGSF3 has also a detrimental effect on processes involved in repair such as cell migration and barrier function. This suggests that IGSF3 homeostasis is important in epithelial cell function, possibly through interactions with membrane complexes such as tetraspanins and other transmembrane proteins such as the matrix-regulating CD147.

MTORC1 Prevents Epithelial Damage During Inflammation and Inhibits Colitis-Associated Colorectal Cancer Development

Epithelial cells lining the intestinal mucosa constitute a selective-semipermeable barrier acting as first line of defense in the organism. The number of those cells remains constant during physiological conditions, but disruption of epithelial cell homeostasis has been observed in several pathologies. During colitis, epithelial cell proliferation decreases and cell death augments. The mechanism responsible for these changes remains unknown. Here, we show that the pro-inflammatory cytokine IFNγ contributes to the inhibition of epithelial cell proliferation in intestinal epithelial cells (IECs) by inducing the activation of mTORC1. Activation of mTORC1 in response to IFNγ was detected in IECs present along the crypt axis and in colonic macrophages. mTORC1 inhibition enhances cell proliferation, increases DNA damage in IEC. In macrophages, mTORC1 inhibition strongly reduces the expression of pro-inflammatory markers. As a consequence, mTORC1 inhibition exacerbated disease activity, increased mucosal damage, enhanced ulceration, augmented cell infiltration, decreased survival and stimulated tumor formation in a model of colorectal cancer CRC associated to colitis. Thus, our findings suggest that mTORC1 signaling downstream of IFNγ prevents epithelial DNA damage and cancer development during colitis.

Actomyosin contractility provokes contact inhibition in E-cadherin-ligated keratinocytes

Confluence-dependent inhibition of epithelial cell proliferation, termed contact inhibition, is crucial for epithelial homeostasis and organ size control. Here we report that among epithelial cells, keratinocytes, which compose the stratified epithelium in the skin, possess a unique, actomyosin-dependent mechanism for contact inhibition. We have observed that under actomyosin-inhibited conditions, cell-cell contact itself through E-cadherin promotes proliferation of keratinocytes. Actomyosin activity in confluent keratinocytes, however, inhibits nuclear localization of β-catenin and YAP, and causes attenuation of β-catenin- and YAP-driven cell proliferation. Confluent keratinocytes develop E-cadherin-mediated punctate adhesion complexes, to which radial actin cables are connected. Eliminating the actin-to-E-cadherin linkage by depleting α-catenin increases proliferation of confluent keratinocytes. By contrast, enforced activation of RhoA-regulated actomyosin or external application of pulling force to ligated E-cadherin attenuates their proliferation, suggesting that tensile stress at E-cadherin-mediated adhesion complexes inhibits proliferation of confluent keratinocytes. Our results highlight actomyosin contractility as a crucial factor that provokes confluence-dependent inhibition of keratinocyte proliferation.

TIPE2, a negative regulator of TLR signaling, regulates p27 through IRF4-induced signaling.

Targeted inhibition of specific toll-like receptor (TLR) pathways may provide an effective strategy to prevent the development of selected gastric malignancies. Tumor necrosis factor (TNF)-α-induced protein 8-like-2 (TIPE2) was identified as a novel negative regulator of TLR signaling. Our previous study identified TIPE2 as an inhibitor of gastric cancer cell growth; it promotes p27 expression, which leads to restored control of the cell cycle and cell division. However, the molecular mechanism by which TIPE2 regulates p27 remains unclear. In the present study, we examined the expression patterns of TIPE2 in serial clinical gastritis tissues as well as gastric cancer, and found a negative correlation between TIPE2 expression and progression of gastritis to gastric cancer. This negative correlation verified the role of TIPE2 in preventing the occurrence and development of gastric cancer, suggesting that TIPE2 may be a potential biomarker for gastric cancer progression. To determine the mechanism employed by TIPE2 in gastric cell carcinogenesis, a TIPE2-expressing plasmid was introduced into gastric cell lines, and microarray and western blot analysis revealed that TIPE2 selectively upregulates the expression of interferon regulatory factor 4 (IRF4). Variations in IRF4 expression were additionally verified in knockout mice. Next, the effect of IRF4 on p27 expression was tested by an IRF4 siRNA interference assay. Finally, we explored the signaling pathways used by TIPE2 to regulate IRF4. An experiment using pathway inhibitors and a nuclear factor κ-light-chain enhancer of activated B cells (NF-κB) luciferase reporter assay showed that NF-κB plays a crucial role in regulating IRF4 expression. Our data provide evidence that TIPE2, a potential biomarker for gastric cancer progression, stimulates an IRF4-associated signaling cascade that promotes p27 expression and controls cell growth. To the best of our knowledge, this is the first study to demonstrate that IRF4 acts as an inhibitor of epithelial cell proliferation and mediates the expression of TIPE2, a negative regulator of TLR signaling, to control cell growth.

Inhibition of Aerobic Glycolysis Attenuates Disease Progression in Polycystic Kidney Disease.

Dysregulated signaling cascades alter energy metabolism and promote cell proliferation and cyst expansion in polycystic kidney disease (PKD). Here we tested whether metabolic reprogramming towards aerobic glycolysis (“Warburg effect”) plays a pathogenic role in male heterozygous Han:SPRD rats (Cy/+), a chronic progressive model of PKD. Using microarray analysis and qPCR, we found an upregulation of genes involved in glycolysis (Hk1, Hk2, Ldha) and a downregulation of genes involved in gluconeogenesis (G6pc, Lbp1) in cystic kidneys of Cy/+ rats compared with wild-type (+/+) rats. We then tested the effect of inhibiting glycolysis with 2-deoxyglucose (2DG) on renal functional loss and cyst progression in 5-week-old male Cy/+ rats. Treatment with 2DG (500 mg/kg/day) for 5 weeks resulted in significantly lower kidney weights (-27%) and 2-kidney/total-body-weight ratios (-20%) and decreased renal cyst index (-48%) compared with vehicle treatment. Cy/+ rats treated with 2DG also showed higher clearances of creatinine (1.98±0.67 vs 1.41±0.37 ml/min), BUN (0.69±0.26 vs 0.40±0.10 ml/min) and uric acid (0.38±0.20 vs 0.21±0.10 ml/min), and reduced albuminuria. Immunoblotting analysis of kidney tissues harvested from 2DG-treated Cy/+ rats showed increased phosphorylation of AMPK-α, a negative regulator of mTOR, and restoration of ERK signaling. Assessment of Ki-67 staining indicated that 2DG limits cyst progression through inhibition of epithelial cell proliferation. Taken together, our results show that targeting the glycolytic pathway may represent a promising therapeutic strategy to control cyst growth in PKD.

Neonatal Estrogen Receptor β Is Important in the Permanent Inhibition of Epithelial Cell Proliferation in the Mouse Uterus.

Estrogen receptor α (ERα) plays a pivotal role in the mouse uterine and vaginal epithelial cell proliferation stimulated by estrogen, whereas ERβ inhibits cell proliferation. ERβ mRNA is expressed in neonatal uteri and vaginae; however, its functions in neonatal tissues have not been ascertained. In this study, we investigated the ontogenic mRNA expression and localization of ERβ, and its roles in cell proliferation in neonatal uteri and vaginae of ERβ knockout (βERKO) mice. ERβ mRNA and protein were abundant in the uterine and vaginal epithelia of 2-day-old mice and decreased with age. In uterine and vaginal epithelia of 2-day-old βERKO mice, cell proliferation was greater than that in wild-type animals and in uterine epithelia of 90- and 365-day-old βERKO mice. In addition, p27 protein, known as a cyclin-dependent kinase inhibitor, was decreased in the uteri of 90- and 365-day-old βERKO mice. Inhibition of neonatal ERs by ICI 182780 (an ER antagonist) treatment stimulated cell proliferation and decreased p27 protein in the uterine luminal epithelium of 90-day-old mice but not in the vaginal epithelium. These results suggest that neonatal ERβ is important in the persistent inhibition of epithelial cell proliferation with accumulation of p27 protein in the mouse uterus. Thus, suppression of ERβ function in the uterine epithelium during the neonatal period may be responsible for a risk for proliferative disease in adults.

Steviol retards renal cyst growth through reduction of CFTR expression and inhibition of epithelial cell proliferation in a mouse model of polycystic kidney disease (690.2)

Cyst growth in ADPKD is associated with cAMP‐activated proliferation of cyst‐lining epithelial cells and transepithelial fluid secretion into the cyst lumen via CFTR chloride channel. It will eventually lead to renal insufficiency and renal failure for which no effective treatment is currently available. We previously reported that steviol retards MDCK cyst growth by inhibiting CFTR channel activity and promoting proteasomal‐mediated CFTR degradation. However, the MDCK cells do not carry the mutated PKD genes and do not recapitulate the complexity of PKD pathogenesis. Therefore, it is imperative to examine the effect of steviol in animal model that closely resembles PKD. In this study, we determined the effect of steviol on renal cyst growth in an orthologous mouse model of human ADPKD (Pkd1flox/flox :Pkhd1‐Cre). The results showed that daily treatment with both 200 mg/kg BW of steviol and 1,000 mg/kg BW of stevioside for 14 days markedly decreased kidney weight and cystic index in these mice. Surprisingly, only steviol markedly reduced blood urea nitrogen and creatinine values. It also reduced cell proliferation but had no effect on cell apoptosis. In addition, steviol suppressed CFTR and mTOR/S6K expressions but stimulated AMP‐activated protein kinase (AMPK) in renal cyst‐lining epithelial cells. These findings indicate that steviol slows cyst progression in PKD mouse model, in part, through the activation of AMPK which subsequently inhibits both CFTR expression and renal epithelial cell proliferation via mTOR/S6K pathway. Most importantly, steviol could markedly improve kidney function in a mouse model of PKD. Steviol thus has potential application for further development as therapeutic compound for the treatment of polycystic kidney disease.Grant Funding Source: Supported by Thailand Research Fund and Faculty of Science, Mahidol University, Bangkok, Thailand

Steviol retards renal cyst growth through reduction of CFTR expression and inhibition of epithelial cell proliferation in a mouse model of polycystic kidney disease

Cyst enlargement in autosomal dominant polycystic kidney disease (ADPKD) is associated with cAMP-activated proliferation of cyst-lining epithelial cells and transepithelial fluid secretion into the cyst lumen via cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel leading to renal failure for which no effective treatment is currently available. We previously reported that steviol retards Madin–Darby canine kidney (MDCK) cyst enlargement by inhibiting CFTR channel activity and promoting proteasomal-mediated CFTR degradation. It is imperative to examine the effect of steviol in animal models of ADPKD. Therefore, we examined the effect of steviol on renal cyst growth in an orthologous mouse model of human ADPKD (Pkd1flox/flox:Pkhd1-Cre). The results showed that daily treatment with both 200mg/kg BW of steviol and 1000mg/kg BW of stevioside for 14 days markedly decreased kidney weight and cystic index in these mice. However, only steviol markedly reduced blood urea nitrogen and creatinine values. Steviol also reduced cell proliferation but had no effect on cell apoptosis. In addition, steviol suppressed CFTR and mTOR/S6K expression in renal cyst-lining epithelial cells. Interestingly, steviol was found to stimulate AMP-activated protein kinase (AMPK). Our findings indicate that steviol slows cyst progression in ADPKD mouse model, in part, through the activation of AMPK which subsequently inhibits CFTR chloride channel expression and inhibits renal epithelial cell proliferation via mTOR/S6K pathway. Most importantly, steviol could markedly improve kidney function in a mouse model of ADPKD. Steviol thus has potential application for further development as a therapeutic compound for the treatment of polycystic kidney disease.

New Roles of Serotonin and Tachykinins in Intestinal Mucositis?

The paper by Matsumoto et al. [1] published in this issue of Digestive Diseases and Sciences reports an investigation of expression of 5-hydroxytrypamine (5HT) and tachykinins and their cognate receptors in an experimental model of mucositis of the mouse jejunum induced by the chemotherapeutic agent 5-fluorouracil (5-FU). Mucositis is a serious adverse effect of common chemotherapeutic treatments. Damage to mucosa, prominent in the oral cavity (oral mucositis), also occurs in the esophagus, stomach, and small and large intestines (gastrointestinal mucositis). Gastrointestinal symptoms include pain, nausea and vomiting, ulceration, diarrhea, and rectal bleeding. Patients with mucositis often have to temporarily withdraw from chemotherapy, which can contribute to reduced cancer survival [2]. Current guidelines for treatment of gastrointestinal mucositis include use of the oral histamine H2-receptor antagonist ranitidine or a proton pump inhibitor to reduce gastric injury. Loperamide is recommended for control of diarrhea; if loperamide fails, subcutaneous octreotide is also recommended [3]. Mucosal damage was originally believed to result primarily from inhibition of epithelial cell proliferation, which reduces protection and repair of the mucosa. More recent evidence shows that chemotherapeutic agents stimulate nuclear factor-jB production; subsequent production of proinflammatory cytokines significantly induces mucositis [4]. Therapy limiting intestinal mucosal damage is not in common use, primarily because suitable molecular targets have not been identified. The publication by Matsumoto et al. builds on their previous findings that 5HT3 receptor antagonists reduce damage to mucosa, mucosal cytokine production, and enterocyte apoptosis caused by 5-FU in mice [5]. Their latest paper reports that 5HT3 and neurokinin (NK) NK1 receptor expression were upregulated, mostly in lamina propria macrophage-like cells (CD11?) of the jejunum, of mice injected intraperitoneally with 5-FU for 5 days, investigated on the 5th day. The study also reports increased 5HT and tachykinin (substance P) content of small intestine homogenate, although the number of cells expressing these compounds was unchanged. The number of mucosal 5HT3 receptor immunoreactive nerve fibers also increased. An important unanswered question is whether the results are clinically relevant, given that numerous studies have reported that 5HT3 receptors help modulate the immune– inflammatory axis. Rodents have only two genes encoding 5HT3 subunits whereas the human genome contains five 5-HT3 receptor-encoding genes, although the 5HT3C, 5HT3D, and 5HT3E subunits only express functional channels when co-expressed with 5HT3A [6]. It is currently unclear whether any of the 5HT3 receptor subtypes are expressed on human macrophages. 5HT2B and 5HT7 were the only serotonin receptor-encoding genes expressed above background levels in human macrophages produced by treatment of peripheral monocytes with GM-CSF or M-CSF (granulocyte macrophage and macrophage colonystimulating factors) [7]. Kapellar et al. [6] detected immunoreactivity to 5HT3A, 5HT3C, 5HT3D, and 5HT3E in lamina propria immune cells in the human colon, although their identity was not verified. There is, however, good evidence that 5HT3 receptors are expressed on human B. Callaghan J. B. Furness (&) Department of Anatomy and Neuroscience, University of Melbourne, Parkville, VIC, Australia e-mail: j.furness@unimelb.edu.au

In Situ Hybridization probing by Transforming Growth Factor β1 marker during prenatal period for rat's primary visual cortex

The objective of this study is to recount the prenatal developmental changes of the primary visual cortex with the space and time appearance of the activity ofthe studied marker through In Situ Hybridization technique. TGF-β1 has a multitude of functions, including both promoting the formation and inhibiting thebreakdown of connective tissue with inhibition of epithelial cell proliferation. Eighteen embryos were used; three embryos for each of the following gestational days (G15, G16, G17, G18, G19 and G20). The reaction was gradual and followed an inside-out gradient from deep to superficial laminae of the rat's primary visual cortex.Cell migration was altered by TGF-β1 in a concentration-dependent manner: at low concentrations, cell migration was promoted whereas at high concentrations, migration was impeded. Therefore, it is an important regulator of the cell behavior throughout prenatal age.&#x0D;

Nucleolar Sik-Similar Protein (Sik-SP) Is Required for the Maintenance of Uterine Estrogen Signaling Mechanism Via ERα

Sik-similar protein (Sik-SP), a small nucleolar ribonucleoprotein, has been shown to be primarily involved in ribosome biogenesis. However, its role in the hormone-directed nuclear receptor signaling is largely unknown. Here, we provide novel evidence that Sik-SP is required for appropriate regulation of estrogen receptor (ER)α-mediated estradiol-17β (E2)-dependent uterine physiologic responses in mice. Studies by Western blotting using the newly developed antibodies for Sik-SP showed that this protein is up-regulated in both the ovariectomized wild-type and ERα null uteri by E2. Immunohistochemical analyses in uterine sections showed that this protein is induced in the epithelial and stromal cells. Coimmunoprecipitation studies revealed that E2 directs molecular interaction between Sik-SP and ERα. Furthermore, gel-mobility shift and chromatin immunoprecipitation analyses provided evidence that Sik-SP is recruited with ERα to estrogen-responsive uterine gene promoters. Overexpression of Sik-SP in vitro demonstrated a role for Sik-SP in cellular growth and viability. In a primary uterine epithelial-stromal coculture system, E2 exhibited early induction of Sik-SP in both the epithelial and stromal cells. Interestingly, suppression of Sik-SP in this coculture model, for the stromal but not epithelial cells, caused perturbation of E2-dependent proliferation in the epithelial cell layer. Similarly, in vivo uterine suppression of Sik-SP also caused inhibition of epithelial cell proliferation and aberrant prolongation of water imbibition in the late phase by E2. Finally, studies showed that Sik-SP is physiologically important during the onset of implantation by E2. In conclusion, Sik-SP, an early E2-responsive nucleolar protein, is necessary to induce E2-dependent ERα-mediated appropriate physiologic responses in the uterus.

Mouse Primary Uterine Cell Coculture System Revisited: Ovarian Hormones Mimic the Aspects of in Vivo Uterine Cell Proliferation

Previously, the uterine epithelial-stromal coculture system had limited success mimicking in vivo ovarian hormone-dependent cell-specific proliferation. Here, we established a mouse primary uterine coculture system, in which cells collected in pseudopregnancy specifically on d 4 are conducive to supporting hormone-induced cell-specific proliferation. When two cell types are placed in coculture without direct contact via cell culture inserts (nonadjacent), as opposed to with contact (adjacent), epithelial cells exhibit significant proliferation by estradiol-17β (E2), whereas progesterone in combination with E2 caused inhibition of epithelial cell proliferation and a major shift in proliferation from epithelial to stromal cells. Epithelial cell integrity, with respect to E-cadherin expression, persisted in nonadjacent, but not adjacent, conditions. In subsequent studies of nonadjacent cocultures, localization of estrogen receptor (ER)α and progesterone receptor (PR), but not ERβ, appeared to be abundant, presumably indicating that specific ER or PR coregulator expression might be responsible for this difference. Consistently, an agonist of ERα, but not ERβ, was supportive of proliferation, and antagonists of ER or PR totally eliminated cell-specific proliferation by hormones. RT-PCR analyses also revealed that hormone-responsive genes primarily exhibit appropriate regulation. Finally, suppression of immunoglobulin heavy chain binding protein, a critical regulator of ERα signaling, in epithelial and/or stromal cells caused dramatic inhibition of E2-dependent epithelial cell proliferation, suggesting that a molecular perturbation approach is applicable to mimic in vivo uterine control. In conclusion, our established coculture system may serve as a useful alternative model to explore in vivo aspects of cell proliferation via communication between the epithelial and stromal compartments under the direction of ovarian hormones.

Interferon-γ modulates intestinal epithelial cell function in-vitro through a TGFβ-dependent mechanism

Introduction Interferon γ (IFNγ) has been originally identified by its anti-viral activity and has been demonstrated to act as potent modulator of the immune system with a range of target cells limited largely to immune cell populations. Although IFNγ has been shown to directly affect the barrier function of intestinal epithelial cells, only limited information is available about other functional effects of IFNγ on intestinal epithelial cells. Methods The effects on intestinal epithelial cell migration were studied using a previously described in-vitro model of epithelial restitution in confluent IEC-6 cell monolayers. Intestinal epithelial cell proliferation rates were assessed in various human and rat intestinal and colon epithelial cell lines using colorimetric MTT assays. Apoptosis of IEC-6 cells exposed to IFNγ was assessed by flow cytometry. In addition, transforming growth factor β mRNA expression after IFNγ treatment of IEC-6 cells was assessed by Northern blot analysis. Results IFNγ significantly stimulated intestinal epithelial cell migration in an in-vitro wounding model. Furthermore, IFNγ caused a significant dose-dependent inhibition of epithelial cell proliferation in non-transformed small intestinal IEC-6 cells and human colon cancer-derived HT-29 cells and no significant rates of apoptosis were detected in the exposed epithelial cells. The effect of IFNγ on epithelial cell migration and proliferation could be completely blocked by neutralizing antibodies against TGFβ indicating that these effects are mediated through a TGFβ dependent pathway. In addition, increased expression of TGFβ1 mRNA by IEC-6 cells after treatment with IFNγ supports the hypothesis that IFNγ modulates intestinal epithelial cell function through a TGFβ-dependent pathway. Conclusion These studies suggest that IFNγ produced by constituents of the mucosal immune system modulates epithelial cell functions with relevance for intestinal wound healing and may play a role in preserving the integrity of the intestinal epithelium following various forms of injuries.

Antiproliferative factor decreases Akt phosphorylation and alters gene expression via CKAP4 in T24 bladder carcinoma cells

BackgroundUrinary bladder cancer is a common malignancy worldwide, and outcomes for patients with advanced bladder cancer remain poor. Antiproliferative factor (APF) is a potent glycopeptide inhibitor of epithelial cell proliferation that was discovered in the urine of patients with interstitial cystitis, a disorder with bladder epithelial thinning and ulceration. APF mediates its antiproliferative activity in primary normal bladder epithelial cells via cytoskeletal associated protein 4 (CKAP4). Because synthetic asialo-APF (as-APF) has also been shown to inhibit T24 bladder cancer cell proliferation at nanomolar concentrations in vitro, and because the peptide segment of APF is 100% homologous to part of frizzled 8, we determined whether CKAP4 mediates as-APF inhibition of proliferation and/or downstream Wnt/frizzled signaling events in T24 cells.MethodsT24 cells were transfected with double-stranded siRNAs against CKAP4 and treated with synthetic as-APF or inactive control peptide; cells that did not undergo electroporation and cells transfected with non-target (scrambled) double-stranded siRNA served as negative controls. Cell proliferation was determined by 3H-thymidine incorporation. Expression of Akt, glycogen synthase kinase 3β (GSK3β), β-catenin, p53, and matrix metalloproteinase 2 (MMP2) mRNA was determined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Akt, GSK-3β, MMP2, β-catenin, and p53 protein expression, plus Akt, GSK-3β, and β-catenin phosphorylation, were determined by Western blot.ResultsT24 cell proliferation, MMP2 expression, Akt ser473 and thr308 phosphorylation, GSK3β tyr216 phosphorylation, and β-catenin ser45/thr41 phosphorylation were all decreased by APF, whereas p53 expression, and β-catenin ser33,37/thr41 phosphorylation, were increased by APF treatment in non-electroporated and non-target siRNA-transfected cells. Neither mRNA nor total protein expression of Akt, GSK3β, or β-catenin changed in response to APF in these cells. In addition, the changes in cell proliferation, MMP2/p53 mRNA and protein expression, and Akt/GSK3β/β-catenin phosphorylation in response to APF treatment were all specifically abrogated following CKAP4 siRNA knockdown.ConclusionsSynthetic as-APF inhibits cell proliferation in T24 bladder carcinoma cells via the CKAP4 receptor. The mechanism for this inhibition involves regulating phosphorylation of specific cell signaling molecules (Akt, GSK3β, and β-catenin) plus mRNA and protein expression of p53 and MMP2.

Mouse Primary Uterine Cell Coculture System Revisited: Ovarian Hormones Mimic the Aspects of In Vivo Uterine Cell Proliferation.

Previously, the uterine epithelial-stromal coculture system had limited success mimicking in vivo ovarian hormone-dependent cell-specific proliferation. Here, we established a mouse primary uterine coculture system, in which cells collected in pseudopregnancy specifically on d 4 are conducive to supporting hormone-induced cell-specific proliferation. When two cell types are placed in coculture without direct contact via cell culture inserts (nonadjacent), as opposed to with contact (adjacent), epithelial cells exhibit significant proliferation by estradiol-17β (E2), whereas progesterone in combination with E2 caused inhibition of epithelial cell proliferation and a major shift in proliferation from epithelial to stromal cells. Epithelial cell integrity, with respect to E-cadherin expression, persisted in nonadjacent, but not adjacent, conditions. In subsequent studies of nonadjacent cocultures, localization of estrogen receptor (ER)α and progesterone receptor (PR), but not ERβ, appeared to be abundant, presumably indicating that specific ER or PR coregulator expression might be responsible for this difference. Consistently, an agonist of ERα, but not ERβ, was supportive of proliferation, and antagonists of ER or PR totally eliminated cell-specific proliferation by hormones. RT-PCR analyses also revealed that hormone-responsive genes primarily exhibit appropriate regulation. Finally, suppression of immunoglobulin heavy chain binding protein, a critical regulator of ERα signaling, in epithelial and/or stromal cells caused dramatic inhibition of E2-dependent epithelial cell proliferation, suggesting that a molecular perturbation approach is applicable to mimic in vivo uterine control. In conclusion, our established coculture system may serve as a useful alternative model to explore in vivo aspects of cell proliferation via communication between the epithelial and stromal compartments under the direction of ovarian hormones.

TGF-Beta Auto-Induction and Connective Tissue Growth Factor Expression in Human Renal Tubule Epithelial Cells Requires N-Ras

Background/Aims: Transforming growth factor (TGF) β is strongly implicated in the progression of renal fibrosis. TGFβ1 is reported to cause epithelial-mesenchymal transition, inhibition of epithelial cell proliferation, increased apoptosis, auto-induction of TGFβ production and induction of secondary mediators of tissue fibrosis such as connective tissue growth factor (CTGF, CCN2). The aims of this study were to investigate the role of the Ras/MAP kinase pathway in TGFβ1 inhibition of proliferation, TGFβ auto-induction and TGFβ1-induced CTGF expression in HKC human renal tubule epithelial cells. Methods and Results: TGFβ1 (0–25 ng/ml) inhibited proliferation of HKC cells and at 25 ng/ml also induced apoptosis. After 5–10 min of incubation, TGFβ1 increased cellular levels of phospho-ERK1/2 and phospho-AKT with a bell-shaped dose-response curve with a maximally effective concentration of 2.5 ng/ml. TGFβ3 caused an increase in extracellular TGFβ1, which was significantly reduced in the presence of PD 98059. TGFβ1 increased cellular and secreted CTGF protein in HKC cells in a MEK-dependent manner. To identify the Ras isoform involved, specific antisense oligonucleotides targeted to Ha-Ras, Ki-Ras and N-Ras were employed. Only inhibition of N-Ras resulted in a significant reduction of auto-induced TGFβ1 secretion and TGFβ1-induced cellular and secreted CTGF. Conclusion: These results establish that the Ras/MAP kinase pathway, specifically through N-Ras, mediates TGFβ1 auto-induction and TGFβ1-induced CTGF expression in human renal tubule epithelial cells.

Loss of expression of TGF-β1, TβRI, and TβRII correlates with differentiation in human oral squamous cell carcinomas

Despite advances in biological and molecular characteristics, the prognosis of oral squamous cell carcinomas is still very unfavourable and is based on the classical clinicopathological parameters. However, tumors with similar clinicopathological characteristics may differ dramatically in their clinical outcome. Thus, the identification of novel prognostic factors is necessary to improve prognostic and therapeutic approaches. Transforming growth factor-beta1 (TGF-beta1) is a potent growth inhibitor of epithelial cell proliferation, thus, inactivation of TGF-beta1 signalling may play a role in cancer. The expression levels of TGF-beta1 and its type I and type II receptors (TbetaRI and TbetaRII) were assessed by immunohistochemical and Western blot analyses in 22 oral squamous cell carcinoma lesions, in their normal adjacent mucosa and in the squamous carcinoma cell lines FaDu and CAL27. Immunohistochemistry on 22 oral carcinomas and case-matched normal oral mucosae demonstrated that TGF-beta1, TbetaRI, and TbetaRII were intensively and homogeneously expressed in all normal epithelia. In contrast, TGF-beta1 and its receptors were significantly reduced in poorly (G3) differentiated tumors as compared to moderately (G2) and well differentiated (G1) lesions (p=2.8 x 10(-3), p=1.3 x 10(-3), p=2.8 x 10(-3) and p=1.3 x 10(-3), respectively). The progressive reduction of the expression levels was confirmed by Western blotting. The oral squamous carcinoma cell lines Cal27 and FaDu demonstrated a reduced and a lack of TbetaRI expression, respectively. A significant decrease of TbetaRII expression, as compared to Cal27 cells, was shown in FaDu cells. Thus, the decreased expression of TbetaRII combined with the absence of TbetaRI could account for the resistance of FaDu cells to the growth-inhibiting effect of TGF-beta1. TGF-beta1 and TGF-beta1 receptor expression significantly decreased as tumors became less differentiated and thus more aggressive, suggesting a functional role of these molecules in oral tumor progression.

Mechanisms in TGF-beta action.

The various isoforms of TGF-beta are multifunctional. We are exploring pathways of cellular regulation by TGF-beta that lead to suppression of cell proliferation, modulation of cell adhesion and control of cell differentiation. These cellular responses appear to be activated by binding of TGF-beta to a similar set of receptor glycoproteins in all cell types. TGF-beta receptor types I and II are specifically lost in cell mutants that are resistant to TGF-beta. The concomitant loss of these two receptors in certain mutants suggests that they are components of the TGF-beta signal-transducing receptor complex. Inhibition of epithelial cell proliferation by TGF-beta is linked to retention of the retinoblastoma growth suppressor gene product in an underphosphorylated state that is presumed to have growth suppressive activity. Inhibition of myogenic differentiation by TGF-beta involves a block in the expression of the master myogenic differentiation genes, such as myogenin, but appears also to involve up-regulation of extracellular matrix production. Expression of components of the cell adhesion apparatus--cell adhesion receptors and extracellular matrix proteins--is controlled by TGF-beta in an array of cell types. This response could have a great impact on the ability of cells to migrate, home to specific tissue locations and differentiate during development, invasion and metastasis.

Transforming Growth Factor-β Can Suppress Tumorigenesis through Effects on the Putative Cancer Stem or Early Progenitor Cell and Committed Progeny in a Breast Cancer Xenograft Model

The transforming growth factor-beta (TGF-beta) pathway has tumor-suppressor activity in many epithelial tissues. Because TGF-beta is a potent inhibitor of epithelial cell proliferation, it has been widely assumed that this property underlies the tumor-suppressor effect. Here, we have used a xenograft model of breast cancer to show that endogenous TGF-beta has the potential to suppress tumorigenesis through a novel mechanism, involving effects at two distinct levels in the hierarchy of cellular progeny that make up the epithelial component of the tumor. First, TGF-beta reduces the size of the putative cancer stem or early progenitor cell population, and second it promotes differentiation of a more committed, but highly proliferative, progenitor cell population to an intrinsically less proliferative state. We further show that reduced expression of the type II TGF-beta receptor correlates with loss of luminal differentiation in a clinical breast cancer cohort, suggesting that this mechanism may be clinically relevant. At a molecular level, the induction of differentiation by TGF-beta involves down-regulation of Id1, and forced overexpression of Id1 can promote tumorigenesis despite persistence of the antiproliferative effect of TGF-beta. These data suggest new roles for the TGF-beta pathway in regulating tumor cell dynamics that are independent of direct effects on proliferation.