Correction: Linking bacterial enterotoxins and alpha defensin 5 expansion in the Crohn's colitis: A new insight into the etiopathogenetic and differentiation triggers driving colonic inflammatory bowel disease.

[This corrects the article DOI: 10.1371/journal.pone.0246393.].

Lysosome-dependent FOXA1 ubiquitination contributes to luminal lineage of advanced prostate cancer.

Changes in FOXA1 (forkhead box protein A1) protein levels are well associated with prostate cancer (PCa) progression. Unfortunately, direct targeting of FOXA1 in progressive PCa remains challenging due to variations in FOXA1 protein levels, increased FOXA1 mutations at different stages of PCa, and elusive post-translational FOXA1 regulating mechanisms. Here, we show that SKP2 (S-phase kinase-associated protein 2) catalyzes K6- and K29-linked polyubiquitination of FOXA1 for lysosomal-dependent degradation. Our data indicate increased SKP2:FOXA1 protein ratios in stage IV human PCa compared to stages I-III, together with a strong inverse correlation (r = -0.9659) between SKP2 and FOXA1 levels, suggesting that SKP2-FOXA1 protein interactions play a significant role in PCa progression. Prostate tumors of Pten/Trp53 mice displayed increased Skp2-Foxa1-Pcna signaling and colocalization, whereas disruption of the Skp2-Foxa1 interplay in Pten/Trp53/Skp2 triple-null mice demonstrated decreased Pcna levels and increased expression of Foxa1 and luminal positive cells. Treatment of xenograft mice with the SKP2 inhibitor SZL P1-41 decreased tumor proliferation, SKP2:FOXA1 ratios, and colocalization. Thus, our results highlight the significance of the SKP2-FOXA1 interplay on the luminal lineage in PCa and the potential of therapeutically targeting FOXA1 through SKP2 to improve PCa control.

Medroxyprogesterone Acetate (MPA) Enhances HIV-1 Accumulation and Release in Primary Cervical Epithelial Cells by Inhibiting Lysosomal Activity.

Medroxyprogesterone acetate (MPA) is one of the most widely used contraceptives in the world. Epidemiologic studies have uncovered a possible link between the use of MPA and an increased risk of HIV-1 transmission. However, the understanding of the mechanism is still limited. Our previous publication demonstrated that the lysosomal activity in human vaginal epithelial cells attenuated the trafficking of viral particles during HIV-1 transcytosis. In this study, we show that treating human primary cervical epithelial cells with MPA led to a reduction in lysosomal activity. This reduction caused an increase in the intracellular HIV-1 accumulation and, consequently, an increase in viral release. Our study uncovers a novel mechanism by which MPA enhances HIV-1 release in primary cervical epithelial cells, thus providing vital information for HIV intervention and prevention.

Linking bacterial enterotoxins and alpha defensin 5 expansion in the Crohn's colitis: A new insight into the etiopathogenetic and differentiation triggers driving colonic inflammatory bowel disease.

Evidence link bacterial enterotoxins to apparent crypt-cell like cells (CCLCs), and Alpha Defensin 5 (DEFA5) expansion in the colonic mucosa of Crohn's colitis disease (CC) patients. These areas of ectopic ileal metaplasia, positive for Paneth cell (PC) markers are consistent with diagnosis of CC. Retrospectively, we: 1. Identified 21 patients with indeterminate colitis (IC) between 2000-2007 and were reevaluation their final clinical diagnosis in 2014 after a followed-up for mean 8.7±3.7 (range, 4-14) years. Their initial biopsies were analyzed by DEFA5 bioassay. 2. Differentiated ulcer-associated cell lineage (UACL) analysis by immunohistochemistry (IHC) of the CC patients, stained for Mucin 6 (MUC6) and DEFA5. 3. Treated human immortalized colonic epithelial cells (NCM460) and colonoids with pure DEFA5 on the secretion of signatures after 24hr. The control colonoids were not treated. 4. Treated colonoids with/without enterotoxins for 14 days and the spent medium were collected and determined by quantitative expression of DEFA5, CCLCs and other biologic signatures. The experiments were repeated twice. Three statistical methods were used: (i) Univariate analysis; (ii) LASSO; and (iii) Elastic net. DEFA5 bioassay discriminated CC and ulcerative colitis (UC) in a cohort of IC patients with accuracy. A fit logistic model with group CC and UC as the outcome and the DEFA5 as independent variable differentiator with a positive predictive value of 96 percent. IHC staining of CC for MUC6 and DEFA5 stained in different locations indicating that DEFA5 is not co-expressed in UACL and is therefore NOT the genesis of CC, rather a secretagogue for specific signature(s) that underlie the distinct crypt pathobiology of CC. Notably, we observed expansion of signatures after DEFA5 treatment on NCM460 and colonoids cells expressed at different times, intervals, and intensity. These factors are key stem cell niche regulators leading to DEFA5 secreting CCLCs differentiation 'the colonic ectopy ileal metaplasia formation' conspicuously of pathogenic importance in CC.

KDM5B Is Essential for the Hyperactivation of PI3K/AKT Signaling in Prostate Tumorigenesis.

KDM5B (lysine[K]-specific demethylase 5B) is frequently upregulated in various human cancers including prostate cancer. KDM5B controls H3K4me3/2 levels and regulates gene transcription and cell differentiation, yet the contributions of KDM5B to prostate cancer tumorigenesis remain unknown. In this study, we investigated the functional role of KDM5B in epigenetic dysregulation and prostate cancer progression in cultured cells and in mouse models of prostate epithelium-specific mutant Pten/Kdm5b. Kdm5b deficiency resulted in a significant delay in the onset of prostate cancer in Pten-null mice, whereas Kdm5b loss alone caused no morphologic abnormalities in mouse prostates. At 6 months of age, the prostate weight of Pten/Kdm5b mice was reduced by up to 70% compared with that of Pten mice. Pathologic analysis revealed Pten/Kdm5b mice displayed mild morphologic changes with hyperplasia in prostates, whereas age-matched Pten littermates developed high-grade prostatic intraepithelial neoplasia and prostate cancer. Mechanistically, KDM5B governed PI3K/AKT signaling in prostate cancer in vitro and in vivo. KDM5B directly bound the PIK3CA promoter, and KDM5B knockout resulted in a significant reduction of P110α and PIP3 levels and subsequent decrease in proliferation of human prostate cancer cells. Conversely, KDM5B overexpression resulted in increased PI3K/AKT signaling. Loss of Kdm5b abrogated the hyperactivation of AKT signaling by decreasing P110α/P85 levels in Pten/Kdm5b mice. Taken together, our findings reveal that KDM5B acts as a key regulator of PI3K/AKT signaling; they also support the concept that targeting KDM5B is a novel and effective therapeutic strategy against prostate cancer. SIGNIFICANCE: This study demonstrates that levels of histone modification enzyme KDM5B determine hyperactivation of PI3K/AKT signaling in prostate cancer and that targeting KDM5B could be a novel strategy against prostate cancer.

A rapidly growing human papillomavirus-positive oral tongue squamous cell carcinoma in a 21-year old female: A case report.

Oral tongue squamous cell carcinoma (OTSCC) has a median age at diagnosis of 62 years. The incidence of OTSCC in young adults has been increasing, and the reason is unclear. The present study describes a case, and molecular analysis, of OTSCC in a 21-year-old female. Clinical and pathological information were collected from medical records. Formalin-fixed paraffin-embedded biopsy tissue from the patient was reassessed using standard hematoxylin & eosin staining, and immunohistochemistry was used to assess the expression of cellular p16, MutL homolog (MLH)1, MLH2, MutS homolog 6 (MSH6) and PMS1 homolog 2 (PMS2). The human papilloma virus (HPV) genome was detected by PCR analysis of the extracted DNA. The young age of the patient with OTSCC was unusual. The original pathology report indicated koilocytotic atypia, a cellular abnormality associated with HPV. Although HPV-positive oral cancer tends to occur in 'younger' individuals, 21 years is unusual. The confirmation of biologically active HPV in the tumor was obtained via the observation of strong positive staining for cellular p16. The patient described a maternal family cluster of rare cancer types, thus the possibility that this rapidly growing cancer resulted from HPV infection combined with an underlying genetic mutation causing decreased DNA-mismatch repair was explored. However, MSH1, MSH2, MSH6 and PSM2, proteins that are associated with Lynch Syndrome, were expressed at normal levels. A rapidly growing OTSCC of a 21-year-old female was determined to be HPV-positive. The patient underwent combination chemotherapy and radiation and has experienced long-term survival without recurrence. The reason this tumor grew so quickly in such a young individual remains unknown. These types of cases warrant additional genomic and proteomic studies to improve understanding of this phenomenon.

Correction: Human alpha defensin 5 is a candidate biomarker to delineate inflammatory bowel disease.

[This corrects the article DOI: 10.1371/journal.pone.0179710.].

Human alpha defensin 5 is a candidate biomarker to delineate inflammatory bowel disease.

Inability to distinguish Crohn's colitis from ulcerative colitis leads to the diagnosis of indeterminate colitis. This greatly effects medical and surgical care of the patient because treatments for the two diseases vary. Approximately 30 percent of inflammatory bowel disease patients cannot be accurately diagnosed, increasing their risk of inappropriate treatment. We sought to determine whether transcriptomic patterns could be used to develop diagnostic biomarker(s) to delineate inflammatory bowel disease more accurately. Four patients groups were assessed via whole-transcriptome microarray, qPCR, Western blot, and immunohistochemistry for differential expression of Human α-Defensin-5. In addition, immunohistochemistry for Paneth cells and Lysozyme, a Paneth cell marker, was also performed. Aberrant expression of Human α-Defensin-5 levels using transcript, Western blot, and immunohistochemistry staining levels was significantly upregulated in Crohn's colitis, p< 0.0001. Among patients with indeterminate colitis, Human α-Defensin-5 is a reliable differentiator with a positive predictive value of 96 percent. We also observed abundant ectopic crypt Paneth cells in all colectomy tissue samples of Crohn's colitis patients. In a retrospective study, we show that Human α-Defensin-5 could be used in indeterminate colitis patients to determine if they have either ulcerative colitis (low levels of Human α-Defensin-5) or Crohn's colitis (high levels of Human α-Defensin-5). Twenty of 67 patients (30 percent) who underwent restorative proctocolectomy for definitive ulcerative colitis were clinically changed to de novo Crohn's disease. These patients were profiled by Human α-Defensin-5 immunohistochemistry. All patients tested strongly positive. In addition, we observed by both hematoxylin and eosin and Lysozyme staining, a large number of ectopic Paneth cells in the colonic crypt of Crohn's colitis patient samples. Our experiments are the first to show that Human α-Defensin-5 is a potential candidate biomarker to molecularly differentiate Crohn's colitis from ulcerative colitis, to our knowledge. These data give us both a potential diagnostic marker in Human α-Defensin-5 and insight to develop future mechanistic studies to better understand crypt biology in Crohn's colitis.

Fetuin-A (alpha 2HS glycoprotein) modulates growth, motility, invasion, and senescence in high-grade astrocytomas.

Glioblastomas (high-grade astrocytomas) are highly aggressive brain tumors with poor prognosis and limited treatment options. In the present studies, we have defined the role of fetuin-A, a liver-derived multifunctional serum protein, in the growth of an established glioblastoma cell line, LN229. We hereby demonstrate that these cells synthesize ectopic fetuin-A which supports their growth in culture in the absence of serum. We have demonstrated that a panel of tissue microarray (TMA) of glioblastomas also express ectopic fetuin-A. Knocking down fetuin-A using shRNA approach in LN229, significantly reduced their in vitro growth as well as growth and invasion in vivo. The fetuin-A knockdown subclones of LN229 (A and D) also had reduced motility and invasive capacity. Treatment of LN229 cells with asialofetuin (ASF), attenuated their uptake of labeled fetuin-A, and induced senescence in them. Interestingly, the D subclone that had ~90% reduction in ectopic fetuin-A, underwent senescence in serum-free medium which was blunted in the presence of purified fetuin-A. Uptake of labeled exosomes was attenuated in fetuin-A knockdown subclones A and D. Taken together, the studies demonstrate the impact of fetuin-A as significant node of growth, motility, and invasion signaling in glioblastomas that can be targeted for therapy.

Accurate preterm labor diagnosis using a CD55-TLR4 combination biomarker model.

We previously demonstrated immune activation in the maternal peripheral circulation associated with preterm labor (PTL). There was an elevation in WBC mRNA of anti-inflammatory complement decay-accelerating factor (CD55) and the innate-immune response activating toll-like receptor 4 (TLR4). These findings suggested that collectively, these two molecules might serve as useful biomolecules to aid in the diagnosis of PTL. In this study, we used a combined marker approach to determine whether a dual marker model utilizing both CD55 and TLR4 mRNA levels to classify PTL would increase diagnostic accuracy compared to either molecule alone. Two methods were evaluated; a linear discriminant (LD) method and a distribution free (DF) method, in order to find the optimal linear combination of TLR4 and CD55 data to diagnose PTL accurately. Our results indicated that a combined CD55-TLR4 dual marker model could provide statistically significant improvements compared to CD55 or TLR4 single marker models for PTL classification performance.

1,25-Dihydroxyvitamin D3 promotes a sustained LPS-induced NF-κB-dependent expression of CD55 in human monocytic THP-1 cells.

The vitamin D3 system imposes immunosuppressive effects on monocytic cells, in part, by inhibiting NF-κB-dependent expression of proinflammatory mediators. CD55, a cell surface complement regulatory protein that promotes protective and anti-inflammatory properties, is reportedly an NF-κB target gene transiently induced in monocytic cells by the bacterial endotoxin LPS. CD55 is elevated on white cells in women experiencing preterm labor (a pathophysiology commonly associated with bacterial infection) and failure to maintain CD55 was associated with subsequent preterm delivery. We examined the influence of vitamin D3 signaling on LPS-induced expression of CD55 in human monocytic THP-1 cells using quantitative PCR, immunoblot, immunohistochemistry, and NF-κB activation pathway inhibitors. Non-NF-κB targets CD14 and CD11b, which modulate bacterial surveillance and eradication, respectively, were also examined. LPS produced a rapid transient 1.6-fold increase in CD55 mRNA. 1,25-D3 alone did not affect CD55 mRNA expression within the first 48 h. However, in 1,25-D3 pretreated cells, LPS produced a >4-fold immediate and sustained increase in CD55 mRNA and protein expression, which was blocked by NF-κB inhibitors. Our results unexpectedly suggest that vitamin D3 signaling may promote an anti-inflammatory response through an NF-κB-dependent increase in CD55 expression. As expected, LPS or 1,25-D3 alone led to sustained increases in CD14 and CD11b expression. In 1,25-D3 pretreated cells, LPS differentially regulated protein expression - CD14 (21-fold increase) and CD11b (a transient 2-fold decrease) - principally at the posttranscriptional level. The coordinated temporal expression of CD55, CD14 and CD11b would contribute to an anti-inflammatory response by providing protection against complement-mediated cell lysis during pathogen recognition and eradication. Overall, the vitamin D3 system may play a role coordinating an anti-inflammatory response pattern of the host complement immune system. This may be particularly important when considering the high rates of preterm births in blacks, a population that exhibits reduced circulating vitamin D3 levels.

Effects of progesterone treatment on expression of genes involved in uterine quiescence.

An important action of progesterone during pregnancy is to maintain the uterus in a quiescent state and thereby prevent preterm labor. The causes of preterm labor are not well understood, so progesterone action on the myometrium can provide clues about the processes that keep the uterus from contracting prematurely. Accordingly, we have carried out Affymetrix GeneChip analysis of progesterone effects on gene expression in immortalized human myometrial cells cultured from a patient near the end of pregnancy. Progesterone appears to inhibit uterine excitability by a number of mechanisms, including increased expression of calcium and voltage-operated K(+) channels, which dampens the electrical activity of the myometrial cell, downregulation of agents, and receptors involved in myometrial contraction, reduction in cell signal components that lead to increased intracellular Ca(2+) concentrations in response to contractile stimuli, and downregulation of proteins involved in the cross-linking of actin and myosin filaments to produce uterine contractions.

Spontaneous preterm labor is associated with an increase in the proinflammatory signal transducer TLR4 receptor on maternal blood monocytes.

BACKGROUND: Localized inflammation and increased expression of TLR4 receptors within the uterus has been implicated in the pathogenesis of preterm labor. It remains unclear whether intrauterine inflammatory responses activate the maternal peripheral circulatory system. Therefore we determined whether increased TLR4 expression is present in the peripheral maternal white blood cells of women with spontaneous preterm labor. METHODS: This is a cross-sectional study of 41 preterm labor cases and 41 non-preterm controls. For each case and control sample, RNA was purified from white blood cells and TLR4 mRNA pool size was evaluated by quantitative PCR. Protein expression levels were determined by flow cytometry. Statistical evaluation using multiple linear regressions was used to determine any significant differences between the cases and controls. The purpose was to determine association prevalence of TLR4 levels and preterm labor. RESULTS: Adjusted mean TLR4 mRNA levels of 0.788 ± 0.037 (standard error) for preterm labor and 0.348 ± 0.038 for the corresponding pregnant control women were statistically significantly different (P = 0.002). Using the lower 95% confidence interval of the mean expression level in PTL subjects (0.7) as a cutoff value for elevated TLR4 mRNA levels, 25/41 (60.9%) of PTL patients expressed elevated TLR4 mRNA as compared to 0/41 (0%) in control subjects. The TLR4 receptor levels in the granulocyte fraction of white blood cells from preterm labor and pregnant controls were similar. However, TLR4+/CD14+monocytes were 2.3 times more frequent (70% vs. 30%) and TLR4 also had a 2.6-fold higher density (750 vs. 280 molecules per cell) in preterm labor women compared with pregnant controls. There was no difference in the levels of TLR4 in patients at term. CONCLUSIONS: Patients with preterm labor exhibited elevated levels of CD14+ maternal blood monocytes each bearing enhanced expression of TLR4, indicating that the peripheral circulatory system is activated in patients with preterm labor. Elevated leukocyte TLR4 levels may be a useful biomarker associated with preterm labor.

Preterm labor: CD55 in maternal blood leukocytes.

PROBLEM: Intrauterine inflammation is a frequent and significant factor associated with the pathogenesis of preterm labor/birth (PTL/PTB). However, it remains unclear whether the intrauterine inflammatory responses activate the maternal peripheral circulation. We explored the association between PTL/PTB and the 'activation' of the peripheral circulatory system by determining whether CD55 mRNA expression within peripheral WBCs differed between PTL and control patients not in labor. METHOD OF STUDY: RNA was purified from white blood cells collected from pregnant women with preterm labor (n = 45), and from pregnant (n = 30) control women. CD55 gene expression was evaluated by quantitative PCR. RESULTS: The mean CD55 mRNA level within the PTL group (0.77 +/- 0.03) was 1.48-fold higher than that observed (0.52 +/- 0.02) within the control group (P < 0.0001); 71% of PTL patients and only 6.7% of control subjects expressed elevated CD55 mRNA. The receiver operating characteristics (with 95% CI) of CD55 as a marker for PTL were as follows: Sensitivity, 69% (53-82%); Specificity, 93% (78-99%); Positive Predictive Value, 94% (80-99%); and Negative Predictive Value, 67% (51-80%). In the patient population that delivered prematurely (before 37 weeks), 81% expressed elevated CD55 mRNA levels with a mean of 0.78 +/- 0.03 and 95% CI of 0.71-0.84. The receiver operating characteristics were as follows: Sensitivity, 73% (54-88%); Specificity, 86% (71-95%); Positive Predictive Value, 81.5% (62-94%); and Negative Predictive Value, 80% (64-91%). CONCLUSION: Here we report for the first time that CD55 mRNA expression was elevated in the peripheral WBCs of subjects with preterm labor compared with control gestationally-matched pregnant woman and that elevated leukocyte CD55 may be a useful predictor of subsequent PTB.

Progesterone-induced sphingosine kinase-1 expression in the rat uterus during pregnancy and signaling consequences.

Sphingosine 1-phosphate (Sph-1-P), a product of sphingomyelin metabolism, can act via a family of cognate G protein-coupled receptors or as an intracellular second messenger for agonists acting through their membrane receptors. In view of the general growth promoting and developmental effects of Sph-1-P on target cells, we hypothesized that it plays a role in adaptation of the uterus to pregnancy. We analyzed its potential role and that of the related lysophospholipid lysophosphatidic acid in the pregnant rat uterus by examining changes in mRNA levels of cognate receptors and enzymes involved in their turnover. Of these, only sphingosine kinase-1 (SphK1) was markedly changed ( approximately 30-fold increase), being localized in the glandular epithelium, vasculature, and the myometrium. Uterine SphK1 mRNA and protein levels paralleled those of serum progesterone, and treatment with progesterone or an antagonist elevated or reduced SphK1 mRNA expression, respectively. Progesterone also increased SphK1 mRNA steady-state levels in a rat myometrial/leiomyoma cell line (ELT3). Overexpressing human SphK1 in these cells resulted in increased levels of the cell cycle regulator cyclin D1 and increased myosin light-chain phosphorylation. Ectopic expression of SphK1 also resulted in increased proliferation rates, possibly in conjunction with increased cyclin D1 expression. These studies suggest that the uterine expression of SphK1 mediates processes involved in growth and differentiation of uterine tissues during pregnancy.

Interleukin-1-induced NF-kappaB recruitment to the oxytocin receptor gene inhibits RNA polymerase II-promoter interactions in cultured human myometrial cells.

The myometrial oxytocin receptor (OTR) is highly regulated during pregnancy, reaching maximal concentrations near term. These levels are then abruptly reduced in advanced labour and the post-partum period. Our goal was to examine the molecular basis for this reduction, using chromatin immunoprecipitation (ChIP). Interleukin-1alpha (IL1A) treatment of cultured human myometrial cells has previously been shown to reduce steady-state levels of OTR mRNA. We show further that IL1A reduced RNA polymerase II cross-linking to the otr promoter, as reflective of transcriptional inhibition. IL1A also increased the recruitment of nuclear factor kappaB (NF-kappaB) to a site 955 bp upstream from the transcriptional start site. Inhibition of NF-kappaB activation negated the effects of IL1A on polymerase II dissociation, indicating a causal relationship, at least in part, between recruitment of NF-kappaB and detachment of polymerase from the otherwise constitutively active otr promoter. IL1A treatment also resulted in increased histone H4 acetylation in the otr promoter region. Whereas NF-kappaB recruitment and histone acetylation are generally associated with activation of gene expression, our findings show that both processes can be involved in dissociation of RNA polymerase II from an active promoter. The results of these studies suggest that the elevation of IL1 in the myometrium occurring at the end of pregnancy initiates the process of down-regulation of OTRs in advanced labour, resulting in the desensitization of the myometrium to elevated levels of OT in the blood during lactation.

Immortalization and characterization of human myometrial cells from term-pregnant patients using a telomerase expression vector.

An examination of cellular processes involved in myometrial function has been greatly assisted by the use of human myometrial cells in primary culture. However, these cells can be used only for several passages before they senesce, and responses to various agents change with time in culture. The use of transformed cells is limited, as they can be polynucleated and can lose or gain chromosomes. We have developed three telomerase-immortalized cell lines from term-pregnant human myometrium to eliminate variability between passage numbers and allow genetic manipulations of myometrial cells to fully characterize signal pathways. These cells have a normal karyotype and were verified to be uterine smooth muscle by immunocytochemical staining for smooth muscle cell-specific alpha-actin and high affinity oxytocin antagonist binding sites. The three cell lines and the cells in primary culture from which they were derived were examined by cDNA microarray analysis. Of >10 000 expressed genes, there were consistent changes in the expression of approximately 1% in the three immortalized cell lines. We were unable to detect any significant differences between primary and immortalized cells in signal pathways such as epidermal growth factor-stimulated epidermal growth factor receptor phosphorylation, insulin-stimulated Akt phosphorylation, oxytocin and lysophosphatidic acid-stimulated extracellular signal-regulated kinase 1 and 2 phosphorylation, myosin light chain phosphorylation, and interleukin-1 induction of IkappaBalpha degradation. The immortalized cells should be useful for a range of studies, including high throughput analyses of the effects of environmental agents on the human myometrium.