Cynthia E. Weber

Epithelial-Mesenchymal Transition, TGF-β, and Osteopontin in Wound Healing and Tissue Remodeling After Injury

Epithelial-mesenchymal transition (EMT) is a process essential to wound healing and tissue remodeling after a thermal burn or other injury. EMT is characterized by phenotypic changes in epithelial cells that render them apolar, with decreased cell-cell adhesions, increased motility, and changes in cytoskeletal architecture similar to mesenchymal stem cells. With regard to healing a thermal burn wound, many facets of wound healing necessitate cells to undergo these phenotypic changes; two will be described in the following review. The first is the differentiation of epithelial cells into myofibroblasts that rebuild the extracellular matrix and facilitate wound contraction. The second is reepithelialization by keratinocytes. The primary cytokine signal identified in the literature that triggers EMT is transforming growth factor (TGF)-&bgr;. In addition to its vital role in the induction of EMT, TGF-&bgr; has many other roles in the wound healing process. The following review will provide evidence that EMT is a central event in wound healing. It will also show the importance of a regulated amount of TGF-&bgr; for proper wound healing. Finally, osteopontin will be briefly discussed with its relation to wound healing and its connections to EMT and TGF-&bgr;.

Osteopontin Mediates an MZF1-TGF-β1-Dependent Transformation of Mesenchymal Stem Cells into Cancer Associated Fibroblasts in Breast Cancer

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

Medium and long-term outcomes after pneumatic dilation or laparoscopic Heller myotomy for achalasia: a meta-analysis.

Recent randomized studies comparing outcomes after pneumatic dilation (PD) and laparoscopic Heller myotomy (LHM) for the treatment of achalasia are conflicting and limited to short-term follow-up. Our meta-analysis compared the long-term durability of these approaches, with the hypothesis that LHM offers superior long-term remission compared with PD. We identified 36 studies published between 2001 and 2011 with at least 5 years of follow-up. Those studies describing PD included 3211 patients (mean age, 49.8 y). For PD, the mean 5-year remission rate was 61.9% and the mean 10-year remission rate was 47.9%. Overall, 1526 patients (mean age, 46.3 y) were treated with LHM; 83% received a fundoplication. In contrast, the mean 5- and 10-year remission rates after LHM were 76.1% and 79.6%, respectively. Finally, the perforation rate for LHM was twice that of PD (4.8% vs. 2.4%; P<0.05). We conclude that despite a higher frequency of perforation, LHM affords greater long-term durability.

Gastroesophageal reflux disease in lung transplant patients with cystic fibrosis

BACKGROUND Gastroesophageal reflux disease (GERD) in lung transplant patients is being increasingly investigated because of its reported association with chronic rejection. However, information concerning the characteristics of GERD in cystic fibrosis (CF) patients is scarce. METHODS We compared esophageal pH monitoring, manometry, gastric emptying studies, and barium swallow of 10 lung transplant patients with CF with those of 78 lung transplant patients with other end-stage pulmonary diseases. RESULTS In lung transplant patients with CF, the prevalence of GERD was 90% (vs 54% controls, P = .04), of whom 70% had proximal reflux (vs 29% controls, P = .02). CONCLUSIONS Lung transplant patients with CF have a significantly higher prevalence and proximal extent of GERD than do other lung transplant recipients. These data suggest that CF patients in particular should be routinely screened for GERD after transplantation to identify those who may benefit from antireflux surgery, especially given the risks of GERD-related aspiration and chronic allograft injury.

An MAPK-dependent pathway induces epithelial-mesenchymal transition via Twist activation in human breast cancer cell lines

BACKGROUND Twist is an epithelial-mesenchymal transition (EMT) transcription factor that instigates cell invasion. Our research has shown that osteopontin (OPN) regulates the EMT factor Twist. The underlying signaling pathway is unknown. We hypothesized that OPN activates Twist to induce EMT in human breast cancer. METHODS Potential kinases for Twist were identified using NetPhosK. Inhibitors of MEK1/2, JNK, p38 MAPK, and PI3K were applied to human breast cancer cells MDA-MB231 (OPN high). After 24 h, Twist was immunoprecipitated and incubated with phosphoserine. Expression of the Twist target protein, Bmi-1, was determined following 24-h osteopontin aptamer (APT) treatment; mutant aptamer (MuAPT) was used as the control. Scratch-wound assay was imaged 12, 24, and 48 h after APT and MuAPT treatment. RESULTS MEK1/2 inhibition caused ≈ twofold decrease in Twist serine phosphorylation (P < .05). APT blockade of OPN in MB231 decreased Bmi1 protein twofold (P < .05). Aptamer-treated cells were significantly decreased in cell migration and wound closure in the scratch wound-assay (P < .001). CONCLUSION We demonstrate that OPN extracellular binding to MB231 activates an autocrine MAPK intracellular signaling pathway resulting in Twist activation and promoting Bmi1 expression to further EMT initiation and cellular migration. Our results elucidate a previously undescribed role for OPN as a prime regulator of EMT in human breast cancer cells.

Osteopontin up-regulates critical epithelial-mesenchymal transition transcription factors to induce an aggressive breast cancer phenotype

BACKGROUND Tumor cells undergoing epithelial-mesenchymal transition (EMT) develop cellular properties leading to stroma invasion and intravasation. We have previously shown in a xenograft breast cancer model that blocking osteopontin (OPN), a secreted phosphoprotein, decreases EMT. This study examines OPNs role in EMT initiation through its regulation of EMT transcription factors (TFs) Snail, Slug, and Twist. OPNs role in Twist activation is examined through immunoprecipitation and Western blot. STUDY DESIGN MDA-MB-231 breast cancer cells secreting high levels of OPN were treated with OPN aptamer (APT) or mutant APT. Osteopontin APT binds to and inhibits extracellular OPN. Low-OPN-secreting breast cancer cells, MCF-7, were treated with OPN, OPN+APT, or OPN+mutant APT. Twist was isolated in MDA-MB-231 with immunoprecipitation. Phospho-serine antibody detected activated Twist in Western blot. Activation of Twist was confirmed by chromatin immunoprecipitation. RESULTS Analysis through quantitative polymerase chain reaction demonstrated APT inhibition of OPN in MDA-MB-231 cells caused a decrease in EMT-TF expression (MDA-MB-231 vs MDA-MB-231+APT: *Twist ΔΔCT: 1.0 vs 0.07; *Snail ΔΔCT: 1.0 vs 0.11; *Slug ΔΔCT: 1.0 vs 0.11; *p < 0.001). Mutant APT did not change EMT-TF expression (NS). Treatment of MCF-7 cells with OPN caused an increase in EMT-TF expression (MCF-7 vs MCF-7+OPN: Twist ΔΔCT: 1.0 vs 9.1; *Snail ΔΔCT: 1.0 vs 11.2; *Slug ΔΔCT: 1.0 vs 10.9; *p < 0.001). The EMT-TF expression in MCF-7 treated with OPN+APT did not differ significantly from MCF-7 alone. Phosphorylated Twist protein was reduced 2-fold with APT in MDA-MB-231 compared with MDA-MB-231 and MDA-MB-231+mutant APT. Twist phorphorylation induced binding to the promoter regions of Twist-regulated gene, B lymphoma Mo-MLV insertion region 1 homolog, a critical protein for EMT progression. CONCLUSIONS This study shows that OPN is critical in EMT initiation through activation of Twist via serine phosphorylation. These unique observations indicate that OPN APT can serve a clinical role as a novel therapeutic agent by diminishing breast cancer oncogenesis.

Alcohol Inhibits Osteopontin Dependent Transforming Growth Factor-β1 Expression in Human Mesenchymal Stem Cells

Background: Alcohol (EtOH) exposure has detrimental effects on fracture healing. Results: EtOH inhibits TGF-β1 protein expression via interference with the transcription factor myeloid zinc finger 1. Conclusion: EtOH-induced deficient fracture healing may be related to reduced TGF-β1. Significance: Further understanding of the mechanisms responsible for the effects of EtOH are crucial for the development of interventions aimed at averting morbidities related to EtOH consumption. Alcohol (EtOH) intoxication is a risk factor for increased morbidity and mortality with traumatic injuries, in part through inhibition of bone fracture healing. Animal models have shown that EtOH decreases fracture callus volume, diameter, and biomechanical strength. Transforming growth factor β1 (TGF-β1) and osteopontin (OPN) play important roles in bone remodeling and fracture healing. Mesenchymal stem cells (MSC) reside in bone and are recruited to fracture sites for the healing process. Resident MSC are critical for fracture healing and function as a source of TGF-β1 induced by local OPN, which acts through the transcription factor myeloid zinc finger 1 (MZF1). The molecular mechanisms responsible for the effect of EtOH on fracture healing are still incompletely understood, and this study investigated the role of EtOH in affecting OPN-dependent TGF-β1 expression in MSC. We have demonstrated that EtOH inhibits OPN-induced TGF-β1 protein expression, decreases MZF1-dependent TGF-β1 transcription and MZF1 transcription, and blocks OPN-induced MZF1 phosphorylation. We also found that PKA signaling enhances OPN-induced TGF-β1 expression. Last, we showed that EtOH exposure reduces the TGF-β1 protein levels in mouse fracture callus. We conclude that EtOH acts in a novel mechanism by interfering directly with the OPN-MZF1-TGF-β1 signaling pathway in MSC.

Hiatal hernias: a review of the pathophysiologic theories and implication for research

BackgroundThe pathophysiology of hiatal hernias is incompletely understood. This study systematically reviewed the literature of hiatal hernias to provide an evidence-based explanation of the pathogenetic theories and to identify any risk factors at the molecular and cellular levels.MethodsA systematic search of the Medline and Pubmed databases on the pathophysiology of hiatal hernias was performed to identify English-language citations from the database inception to December 2010.ResultsAlthough few studies have examined the relationship of molecular and cellular changes of the diaphragm to the pathogenesis of hiatal hernias, there appear to be three dominant pathogenic theories: (1) increased intraabdominal pressure forces the gastroesophageal junction (GEJ) into the thorax; (2) esophageal shortening due to fibrosis or excessive vagal nerve stimulation displaces the GEJ into the thorax; and (3) GEJ migrates into the chest secondary to a widening of the diaphragmatic hiatus in response to congenital or acquired molecular and cellular changes, such as the abnormalities of collagen type 3 alpha 1.ConclusionsThe pathogenesis of hiatal hernias at the molecular and cellular levels is poorly described. To date, no single theory has proved to be the definitive explanation for hiatal hernia formation, and its pathogenesis appears to be multifactorial.

Green tea component epigallocatechin-3-gallate decreases expression of osteopontin via a decrease in mRNA half-life in cell lines of metastatic hepatocellular carcinoma

INTRODUCTION Osteopontin (OPN) mediates metastasis and invasion of hepatocellular carcinoma (HCC). Epigallocatechin-3-gallate (EGCG), found in green tea, suppresses HCC tumor growth in vitro. We sought to investigate the role of EGCG in modulating OPN in cell lines of metastatic HCC. METHODS Experimental HCC cell lines included HepG2 and MHCC-97H HCC cells, which express high levels of OPN, and the Hep3B cells, which express lesser levels of OPN. Cells were treated with EGCG (0.02-20 μg/mL) before measurement of OPN with enzyme-linked immunosorbent assay and reverse transcriptase-polymerase chain reaction. Scratch assay measured cell migration. Binding of the OPN promoter to RNA pol II was evaluated by the use of Chromatin-IP assay after EGCG treatment. Transcriptional regulation of OPN was investigated with luciferase reporter plasmids containing various deletion fragments of the human OPN promoter. Measurement of the half-life of OPN mRNA was conducted using actinomycin D. RESULTS Treatment of MHCC-97H and HepG2 cells with 2 μg/mL and 20 μg/mL EGCG caused a ∼6-fold and ∼90-fold decrease in secreted protein levels of OPN (All P < .001). OPN mRNA was decreased with EGCG concentrations of 0.2-20 μg/ml (All P < .001). The 3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (ie, MTT) assay revealed that differences in OPN expression were not due to viability of the HCC cell lines. Promoter assay and chromatin immunoprecipitation analysis revealed no effect of EGCG on the transcriptional regulation of OPN. Posttranscriptionally, EGCG decreased the half-life of OPN mRNA from 16.8 hours (95% confidence interval 9.0-125.1) to 2.5 hours (95% confidence interval 2.1-3.2) (P < .001). Migration was decreased in EGCG treated cells at 24 hours (8.0 ± 2.4% vs 21.2 ± 10.8%, P < .01) and at 48 hours (13.2 ± 3.6% vs 53.5 ± 19.8%, P < .001). CONCLUSION We provide evidence that EGCG decreases OPN mRNA and secreted OPN protein levels by decreasing the half-life of OPN mRNA in MHCC-97H cells. The translatability of EGCG for patients with HCC is promising, because EGCG is an inexpensive, easily accessible chemical with an extensive history of safety.

Current applications of evolving methodologies in gastroesophageal reflux disease testing

Until recently catheter-based 24-h pH monitoring has been the primary methodology for the objective diagnosis of gastroesophageal reflux disease. Yet, this system has some drawbacks, such as patient discomfort, marginal sensitivity, and the inability to detect nonacid reflux. Hampered by these limitations, several new techniques have been recently introduced in clinical practice. In particular, wireless capsule pH monitoring and multichannel intraluminal impedance-pH testing have been forwarded as more sophisticated means of enhancing patient comfort during testing as well as our ability to diagnose gastroesophageal reflux disease, especially in those patients who complain of symptoms of gastroesophageal reflux disease despite adequate acid suppression therapy. The goal of this review is to compare the clinical applicability, advantages and drawbacks of catheter-based 24-h pH testing, wireless capsule pH monitoring, and multichannel intraluminal impedance-pH.