Porous silicon and silica carriers for delivery of peptide therapeutics.

Peptides have gained tremendous popularity as biological therapeutic agents in recent years due to their favourable specificity, diversity of targets, well-established screening methods, ease of production, and lower cost. However, their poor physiological and storage stability, pharmacokinetics, and fast clearance have limited their clinical translation. Novel nanocarrier-based strategies have shown promise in overcoming these issues. In this direction, porous silicon (pSi) and mesoporous silica nanoparticles (MSNs) have been widely explored as potential carriers for the delivery of peptide therapeutics. These materials possess several advantages, including large surface areas, tunable pore sizes, and adjustable pore architectures, which make them attractive carriers for peptide delivery systems. In this review, we cover pSi and MSNs as drug carriers focusing on their use in peptide delivery. The review provides a brief overview of their fabrication, surface modification, and interesting properties that make them ideal peptide drug carriers. The review provides a systematic account of various studies that have utilised these unique porous carriers for peptide delivery describing significant in vitro and in vivo results. We have also provided a critical comparison of the two carriers in terms of their physicochemical properties and short-term and long-term biocompatibility. Lastly, we have concluded the review with our opinion of this field and identified key areas for future research for clinical translation of pSi and MSN-based peptide therapeutic formulations.

Molecular interaction of quercetin and its derivatives against nucleolin in breast cancer: in-silico and in-vitro study.

Nucleolin, a multifaceted RNA binding domain protein is overexpressed in various cancers leading to dysfunction of several cellular signaling pathways. Quercetin, a distinctive bioactive molecule, along with its derivatives have shown exclusive physio-chemical properties which makes them appealing choices for drug development, yet their role in targeted cancer therapy is limited. Here, the RBD domain structure of Nucleolin was modeled and stabilized by MD simulations for a period of 1000 ns. Molecular docking was performed to determine the binding capability of ligands with the target. To determine the stability of the ligand inside the binding pocket of the protein, MD simulation was performed for a period of 250 ns each for Quercetin-4'-o'-Glucoside, Quercetin_9 and Quercetin complexes. Further, in-vitro studies including cytotoxicity and RT-PCR assays were performed to validate quercetin against Nucleolin. Molecular docking and MD Simulation studies suggested a potential mechanism of interaction of Quercetin-4'-o'-Glucoside, Querectin_9 and Quercetin with Nucleolin with the binding free energy of -63.653, -58.86 and -46.9 kcal/mol, respectively. Moreover, Lys 348 and Glu379 were identified as important amino acids in ligand interaction located at the RRM2 motif of Nucleolin. In-vitro studies showed significant downregulation in Nucleolin expression by 15.18 and 2.51-fold at 48h and 72h respectively in MCF-7 cells with Quercetin (IC50 = 160 µM). Our findings suggested the potential role of specific RRM motifs in interaction with natural compounds targeting Nucleolin. This could be an effective strategy in the identification of potential molecules in targeting Nucleolin which can be further explored for developing targeted therapies for breast cancer.Communicated by Ramaswamy H. Sarma.

Tumor tissue-specific bacterial biomarker panel for colorectal cancer: Bacteroides massiliensis, Alistipes species, Alistipes onderdonkii, Bifidobacterium pseudocatenulatum, Corynebacterium appendicis.

Human microbiome studies have shown diversity to exist among different ethnic populations. However, studies pertaining to the microbial composition of CRC among the Indian population have not been well explored. We aimed to decipher the microbial signature in tumor tissues from North Indian CRC patients. Next-generation sequencing of tumor and adjacent tissue-derived bacterial 16S rRNA V3-V4 hypervariable regions was performed to investigate the abundance of specific microbes. The expression profile analysis deciphered a decreased diversity among the tumor-associated microbial communities. At the phyla level, Proteobacteria was differentially expressed in CRC tissues than adjacent normal. Further, DeSeq2 normalization identified 4 out of 79 distinct species (p < 0.005) only in CRC, Bacteroides massiliensis, Alistipes onderdonkii, Bifidobacterium pseudocatenulatum, and Corynebacterium appendicis. Thus, the findings suggest that microbial signatures can be used as putative biomarkers in diagnosis, prognosis and treatment management of CRC.

Current and future strategies for diagnostic and management of obstructive sleep apnea.

INTRODUCTION: Obstructive sleep apnea (OSA) is a common sleep disorder with multiple comorbidities including hypertension, diabetes, and cardiovascular disorders. Detected based on an overnight sleep study is called polysomnography (PSG); OSA still remains undiagnosed in majority of the population mainly attributed to lack of awareness. To overcome the limitations posed by PSG such as patient discomfort and overnight hospitalization, newer technologies are being explored. In addition, challenges associated with current management of OSA using continuous positive airway pressure (CPAP), etc. presents several pitfalls. AREAS COVERED: Conventional and modern detection/management techniques including PSG, CPAP, smart wearable/pillows, bio-motion sensors, etc., have both pros and cons. To fulfill the limitations in OSA diagnostics, there is an imperative need for new technology for screening of symptomatic and more importantly asymptomatic OSA patients to reduce the risk of several associated life-threatening comorbidities. In this line, molecular marker-based diagnostics have shown great promises. EXPERT OPINION: A detailed overview is presented on the OSA management and diagnostic approaches and recent advances in the molecular screening methods. The potentials of biomarker-based detection and its limitations are also portrayed and a comparison between the standard, current modern approaches, and promising futuristic technologies for OSA diagnostics and management is set forth.ABBREVIATIONS AHI: Apnea hypopnea index; AI: artificial intelligence; CAM: Cell adhesion molecules; CPAP: Continuous Positive Airway Pressure; COVID-19: Coronavirus Disease 2019; CVD: Cardiovascular disease; ELISA: Enzyme linked immunosorbent assay; HSAT: Home sleep apnea testing; IR-UWB: Impulse radio-ultra wideband; MMA: maxillomandibular advancement; PSG: Polysomnography; OSA: Obstructive sleep apnea; SOD: Superoxide dismutase; QD: Quantum dot.

The Synergistic Role of Tip α, Nucleolin and Ras in Helicobacter pylori Infection Regulates the Cell Fate Towards Inflammation or Apoptosis.

Infection with Helicobacter pylori (H. pylori) leads to a fork in the road situation where it is critical and complex to judge the fate of the cell. We propose for the first time an in silico representation of a protein level network model that can unfold the mystery behind the cell fate decision between inflammation or cell proliferation or cell death. Upon infection TNF inducible protein α (Tip α) is internalised after binding with the cell surface receptor Nucleolin which is overexpressed on the cell surface thereby activating the Ras pathway. Tip α, Nucleolin and Ras decides the cell fate for apoptosis or abnormal cell proliferation along with ulcers in the gastric tract, hence we term it as the "death triad", which otherwise triggers the inflammatory pathway through downstream signalling of NF-κß. A series of proteins involved in the signalling cascade are portrayed through compartmentalization of the bacteria and the gut wall. The depicted network works synchronously toward an overarching goal of deciding between apoptosis or inflammation or proliferation. The model has been validated by simulating it with existing transcriptomic data along with clinical findings from patients infected with H. pylori across different regions in India. The results clearly indicate that for a short period of time there is increased binding of Tip α to Nucleolin and the receptor starts to saturate. This increases the tenacity of binding and the cell triggers an inflammatory cascade reaction which involves proinflammatory cytokines such as TNF α thereby progressing to inflammation by activating NF-κß downstream. On the other hand, Ras involved in interaction with nucleolin can be present both in its activated or inactivated state. Binding of Tip α as a monomer leads to desensitization of Nucleolin leading to cell survival and proliferation.

Prevalence of tumor necrosis factor alpha inducing protein (tipα) gene of Helicobacter pylori and its association with upper gastrointestinal diseases in India.

Helicobacter pylori (H. pylori) is known to cause several gastroduodenal diseases including chronic Gastritis, Peptic Ulcer disease and Gastric Cancer. Virulent genes of H. pylori like cagA, vacA are known to be responsible for the disease pathogenesis. However, these virulence genes are not always found to be associated with disease outcome in all populations around the world. Tumor necrosis factor alpha inducing protein tipα is a newly discovered virulence gene of H. pylori and is an inducer of certain cytokines and chemokines that are responsible for causing stomach cancer. Therefore, we conducted a study, which aims to find the prevalence of tipα gene in the Indian patients with gastroduodenal symptoms, and its association with H. pylori related gastroduodenal diseases. 267 clinical H. pylori isolates are included in our study for finding the prevalence of tipα gene and its association with cagA and vacA gene using PCR assay. The current study shows that the prevalence rate of tipα gene is 59.9%. Our study has found a significant association (p < 0.05) of tipα gene with Non Ulcer Dyspepsia (NUD) and an association of cagA and vacAs1m1 with Gastritis and Duodenal Ulcer. Our study demonstrates for the first time the presence of tipα as virulence factor of H. pylori strain in Indian population isolated from patients suffering from gastroduodenal diseases. Further, tipα is significantly associated with NUD but not with other gastroduodenal diseases in India.

Separation of distinct exosome subpopulations: isolation and characterization approaches and their associated challenges.

Exosomes are nano-sized extracellular vesicles that serve as a communications system between cells and have shown tremendous promise as liquid biopsy biomarkers in diagnostic, prognostic, and even therapeutic use in different human diseases. Due to the natural heterogeneity of exosomes, there is a need to separate exosomes into distinct biophysical and/or biochemical subpopulations to enable full interrogation of exosome biology and function prior to the possibility of clinical translation. Currently, there exists a multitude of different exosome isolation and characterization approaches which can, in limited capacity, separate exosomes based on biophysical and/or biochemical characteristics. While notable reviews in recent years have reviewed these approaches for bulk exosome sorting, we herein present a comprehensive overview of various conventional technologies and modern microfluidic and nanotechnological advancements towards isolation and characterization of exosome subpopulations. The benefits and limitations of these different technologies to improve their use for distinct exosome subpopulations in clinical practices are also discussed. Furthermore, an overview of the most commonly encountered technical and biological challenges for effective separation of exosome subpopulations is presented.

Effect of Yuzu (Citrus junos) Seed Limonoids and Spermine on Intestinal Microbiota and Hypothalamic Tissue in the Sandhoff Disease Mouse Model.

The effect of limonoids and spermine (Spm) extracted from yuzu (Citrus junos) seeds on the gut and the brain in a mouse model with Sandhoff disease (SD) was investigated. Wild-type and SD mice were fed a normal diet, or a diet supplemented with limonoid, Spm, or limonoid + Spm for 14-18 weeks, and then 16S rRNA gene amplicon sequencing with extracted DNA from their feces was executed. For SD control mice, intestinal microbiota was mostly composed of Lactobacillus and linked to dysbiosis. For SD and wild-type mice fed with limonoids + Spm or limonoids alone, intestinal microbiota was rich in mucin-degrading bacteria, including Bacteroidetes, Verrucomicrobia, and Firmicutes, and displayed a higher production of short-chain fatty acids and immunoglobulin A. Additionally, SD mice fed with limonoids + Spm or limonoids alone had less inflammation in hypothalamic tissues and displayed a greater number of neurons. Administration of limonoids and/or Spm improved the proportions of beneficial intestinal microbiota to host health and reduced neuronal degeneration in SD mice. Yuzu seed limonoids and Spermine may help to maintain the homeostasis of intestinal microbiota and hypothalamic tissue in the SD mouse model.

In-silico investigations of selective miRNA-gene targets and their validation studies in obstructive sleep apnea (OSA) patient cohorts.

BACKGROUND: Obstructive sleep apnoea (OSA) is a prevalent form of sleep disordered breathing which results in sleep fragmentation and deprivation. Obesity and cardiovascular disorders are the major risk factors associated with OSA. Molecular analysis of the factors associated with OSA could demarcate the clinical analysis pattern in a population. OBJECTIVE: This study pertains to in-silico analyses of miRNA and their gene targets with validation for their potential role in OSA as putative biomarker candidates. METHODS: miRDB, TargetScan and miRanda databases were used to identify targets of miR-27 and let-7 that have documented role in OSA and co-related obesity and cardiovascular disorders. Quantitative PCR was used to analyze expression pattern of miR-27 and let-7 in obese and non-obese OSA patient cohorts with respective controls. In-silico analysis was done using PatchDoc to obtain atomic contact energy (ACE) scores that indicated the docked gene targets to the predicted miRNA structures. The docked structures were analysed using Maestro Suite 11 for the hydrogen and aromatic interactions. RESULTS: Downregulation of miR-27 and let-7 in OSA compared to controls was observed. In-silico data analysis was performed for gene targets (TGFBR1, TGFBR2, SMAD2, SMAD4, CRY2 and CNR1) of the selected miRNAs (miR-27 and let-7). Among all, CNR1 and CRY2 were found to be better targets for miR-27 and let-7 respectively as per ACE scores, ROC scores and expression fold change in OSA. CONCLUSION: Our study gives insights to the expression profiling of miR-27 and let-7 and explore a set of potential target genes (CNR1 and CRY2) of these two miRNAs for a promising clinical relevance in OSA.

Permanent superhydrophilic surface modification in microporous polydimethylsiloxane sponge for multi-functional applications.

Polydimethylsiloxane (PDMS) is one of the most preferred material in microfluidic device/biomedical applications because of its unique properties. However, improvement in surface wettability of PDMS is highly desired for microfluidic and biomedical applications as its surface is inherently hydrophobic in nature that restricts flow of aqueous fluid or adherence of biomolecules onto its surface. In spite of several surface modification techniques, prompt recurrence of hydrophobic properties is quite typical in PDMS. Here, we demonstrate a facile and a permanent conversion of a hydrophobic PDMS sponge onto a superhydrophilic state. PDMS sponge was prepared using an eco-friendly sugar leaching method and modified by an ultra-thin coating of polyacrylic acid (PAA). The resultant PDMS-PAA hybrid sponge was found to have highly stable and sustained superhydrophilic property for more than 18 months with water absorption efficiency as high as 89%. Valuable applications like, portable pressure pump in a microfluidic device and as a bioactive matrix for microbial cell immobilization for biodegradation of distillery industry effluent treatment has been demonstrated using these surface modified PDMS sponges.

Canvassing the aetiology, prognosis and molecular signatures of obstructive sleep apnoea.

CONTEXT: Obstructive sleep apnoea (OSA) is a sleep respiration disorder with multiple pathophysiological risks. The study of OSA is important from a public health perspective due to increased risk of cardiovascular morbidity and metabolic disorders. OBJECTIVE: The review content contributes to amalgamate the clinico-molecular analysis of microRNAs, single-nucleotide polymorphisms (SNPs), transcriptome profiles and epigenetics in the prognosis of OSA. The conceptual focus here is to demarcate the involvement of regulatory players like miRNAs that have most probable contributions towards other changes in patients leading to clinical outcomes. METHODS: Literature survey was done by retrieving data from databases such as Google Scholar, PubMed and Science Direct. RESULTS: Abundant reports have suggested the involvement and role of biomarkers such as CRP, TNF-α, IL-6, IL-8 and CAMs but there are interspersed speculations about the involvement of epigenetics in OSA. CONCLUSIONS: miRNA and transcriptome profiling, DNA methylation and SNPs are some of the less researched aspects that aim to bridge the gap in the molecular mechanisms underlying the pathophysiology of OSA. The quest for biomarkers in OSA is now distinctly achieving new heights. In the context of diagnosis, the above mentioned epigenetic regulators are likely to emerge as viable contenders.

Regulation of nucleolin expression by miR-194, miR-206, and HuR.

Nucleolin is a proliferation-associated protein that is overexpressed in multiple types of cancer. The mechanisms leading to overexpression of nucleolin in specific cancers are not fully understood. This study found that nucleolin is notably elevated in breast cancer cell lines MCF-7 and MDA-231 compared to nonmalignant breast epithelial MCF-10A cells. In silico analyses revealed the presence of putative binding sites for microRNAs miR-194 and miR-206 in the 3'-untranslated region (3'-UTR) of Ncl mRNA. Transfection of the three cell lines with pre-miR-194 or pre-miR-206 specifically decreased the Ncl mRNA and protein expression. Treatments of the cells with antagomiR-194 or antagomiR-206 upregulated nucleolin expression ~2- to 3-fold. Co-transfection of cells with a reporter vector containing the Ncl 3'-UTR downstream from the Renilla luciferase gene and pre-miR-194 or pre-miR-206 led to a ~3-fold decrease in Renilla/firefly luciferase activity. Cytoplasmic levels of the RNA-binding protein HuR were higher in MCF-7 and MDA-231 cells than those in MCF-10A cells. RNA immunoprecipitation assays demonstrated that HuR binds to Ncl mRNA in all the three cell types. ShRNA-mediated knock-down of HuR induced a decrease in nucleolin expression, while exogenous expression of HuR led to upregulation of nucleolin expression. Analysis of the polysome-monosome distribution of Ncl mRNA in HuR knock-down cells demonstrated that HuR enhances the translation efficiency of Ncl mRNA. These findings demonstrate that nucleolin expression is down-regulated by miR-194 and miR-206 and upregulated by HuR.

Regulation of expression of microRNAs by DNA methylation in lung cancer.

Differential expression of miRNAs has been linked with lung carcinogenesis. Recent studies have indicated that DNA hypermethylation can lead to silencing of tumor suppressor miRNA-encoding genes. Restoration of tumor suppressor miRNAs using inhibitors of DNA methyltransferases has been shown to suppress cell proliferation, angiogenesis, invasion and metastasis implying that modulation of methylation of specific miRNAs can be used as novel therapeutic targets in lung cancer. In this review, we highlight tremendous progress which has been made in the identification of methylation-mediated silencing of miRNAs and their contribution in lung carcinogenesis along with the clinical utility of methylated miRNAs.

A Pit-1 Binding Site Adjacent to E-box133 in the Rat PRL Promoter is Necessary for Pulsatile Gene Expression Activity.

Recent evidence reveals that prolactin gene expression (PRL-GE) in mammotropes occurs in pulses, but the molecular process(es) underlying this phenomenon remains unclear. Earlier, we have identified an E-box (E-box133) in the rat PRL promoter that binds several circadian elements and is critical for this dynamic process. Preliminary analysis revealed a Pit-1 binding site (P2) located immediately adjacent to this E-box133 raising the possibility that some type of functional relationship may exist between these two promoter regions. In this study, using serum shocked GH3 cell culture system to synchronize PRL-GE activity, we determined that Pit-1 gene expression occurred in pulses with time phases similar to that for PRL. Interestingly, EMSA analysis not only confirmed Pit-1 binding to the P2 site, but also revealed an interaction with factor(s) binding to the adjacent E-box133 promoter element. Additionally, down-regulation of Pit-1 by siRNA reduced PRL levels during pulse periods. Thus, using multiple evidences, our results demonstrate clearly that the Pit-1 P2 site is necessary for PRL-GE elaboration. Furthermore, the proximity of this critical Pit-1 binding site (P2) and the E-box133 element coupled with the evidences of a site-to-site protein interactions suggest that the process of PRL-GE pulse activity might involve more dynamic and intricate cross-talks between promoter elements that may span some, or all, of the proximal region of the PRL promoter in driving its pulsatile expression.

Episodes of prolactin gene expression in GH3 cells are dependent on selective promoter binding of multiple circadian elements.

Prolactin (PRL) gene expression in mammotropes occurs in pulses, but the mechanism(s) underlying this dynamic process remains obscure. Recent findings from our laboratory of an E-box in the rat PRL promoter (E-box133) that can interact with the circadian factors, circadian locomoter output cycles kaput (CLOCK) and brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein (BMAL)-1, and was necessary for pulse activity raised the intriguing possibility that the circadian system may be central to this oscillatory process. In this study, we used serum-shocked GH(3) cells, established previously to synchronize PRL pulses between cells in culture, to reveal that pulses of PRL mRNA are linked temporally to the expression of bmal1, cry1, per1, and per3 mRNA in these cells. Moreover, we found that each of these circadian factors binds to the rat PRL promoter by chromatin immunoprecipitation analysis. Using EMSA analysis, we observed that two sites present in the proximal promoter region, E-box133 and E-box10, bind circadian factors differentially (E-box133 interacted with BMAL1, cryptochrome-1, period (PER)-1, and PER3 but not PER2 and E-box10 bound BMAL1, cryptochrome-1, PER2, PER3 but not PER1). More importantly, down-regulation of any factor binding E-box133 significantly reduced PRL mRNA levels during pulse periods. Our results demonstrate clearly that certain circadian elements binding to the E-box133 site are required for episodes of PRL mRNA expression in serum-shocked GH(3) cultures. Moreover, our findings of binding-related differences between functionally distinct E-boxes demonstrate not only that E-boxes can bind different components but suggest that the number and type of circadian elements that bind to an E-box is central in dictating its function.

Administration of connexin43 siRNA abolishes secretory pulse synchronization in GnRH clonal cell populations.

GnRH is released from hypothalamic neurons in coordinated pulses, but the cellular basis for this process is poorly understood. Previously, we found that secretory pulses from GT1-7 cells became synchronized with time in culture. Using this culture model, we investigated whether the gap junction proteins connexin43 (Cx43) and/or connexin26 (Cx26) are involved in this synchronization. Our results reveal that cytoplasmic densities immunoreactive for Cx43, and mRNA or protein for Cx43 increase with time in culture. Also, microinjection of day-3 cultures with siRNA for Cx43 abolished synchronized activity at day 7. Interestingly, cytoplasmic plaques, mRNA, or protein for Cx26 remained stable with culture time and Cx26 siRNA administration did not alter secretory activity. Our findings demonstrate that Cx43, but not Cx26 is necessary for synchronized secretory activity in these GT1-7 cultures and raise the possibility that Cx43-related gap junctions may be important in GnRH neuronal coordination in the hypothalamus.

Angiotensin II up-regulates PAX2 oncogene expression and activity in prostate cancer via the angiotensin II type I receptor.

BACKGROUND: Paired homeobox 2 gene (PAX2) is a transcriptional regulator, aberrantly expressed in prostate cancer cells and its down-regulation promotes cell death in these cells. The molecular mechanisms of tumor progression by PAX2 over-expression are still unclear. However, it has been reported that angiotensin-II (A-II) induces cell growth in prostate cancer via A-II type 1 receptor (AT1R) and is mediated by the phosphorylation of mitogen activated protein kinase (MAPK) as well as signal transducer and activator of transcription 3 (STAT3). METHODS: Here we have demonstrated that A-II up-regulates PAX2 expression in prostate epithelial cells and prostate cancer cell lines resulting in increased cell growth. Furthermore, AT1R receptor antagonist losartan was shown to inhibit A-II induced PAX2 expression in prostate cancer. Moreover, analysis using pharmacological inhibitors against MEK1/2, ERK1/2, JAK-II, and phospho-STAT3 demonstrated that AT1R-mediated stimulatory effect of A-II on PAX2 expression was regulated in part by the phosphorylation of ERK1/2, JAK II, and STAT3 pathways. In addition, we have showed that down-regulation of PAX2 by an AT1R antagonist as well as JAK-II and STAT3 inhibitors suppress prostate cancer cell growth. RESULTS: Collectively, these findings show for the first time that the renin-angiotensin system (RAS) may promote prostate tumorigenesis via up-regulation of PAX2 expression. CONCLUSIONS: Therefore, PAX2 may be a novel therapeutic target for the treatment of carcinomas such as prostate cancer via the down-regulation of its expression by targeting the AT1R signaling pathways.

PAX2 oncogene negatively regulates the expression of the host defense peptide human beta defensin-1 in prostate cancer.

Human beta defensin-1 (hBD1) is a component of the immune system which links the innate and adaptive immune responses. We have demonstrated that hBD1 induces rapid cytolysis of prostate cancer cells and that it may also possess tumor suppressive abilities. In addition, there is a high frequency of cancer-specific loss of hBD1 expression which further suggests its potential role in tumor progression. However, the factors responsible for the loss of hBD1 expression are not known. PAX2, a transcriptional regulator normally expressed during early development, has been implicated as an oncogene in carcinomas of the kidney, prostate, breast and ovary. It is known that expression of PAX2 in these tumor cells mediates the evasion of cell death through the suppression of cell death pathways involving the p53 tumor suppressor. However, we have demonstrated that knock-down of PAX2 expression results in cell death independent of p53 status, thus suggesting that additional cell death pathways are negatively regulated by PAX2. Here we describe a novel pathway in which PAX2 represses hBD1 expression through binding of the PAX2 homeodomain to the hBD1 promoter. Furthermore, knock-down of PAX2 expression results in the re-expression of hBD1, and subsequently prostate cancer cell death. These findings are the first to demonstrate that the PAX2 oncogene suppresses hBD1 expression in cancer and further implicate PAX2 as a novel therapeutic target for prostate cancer treatment.

Specific GATA-binding elements in the GnRH promoter are required for gene expression pulse activity: role of GATA-4 and GATA-5 in this intermittent process.

Recent evidence reveals that several GATA factors act as versatile transcriptional modulators in neuroendocrine gene expression. The rat GnRH promoter is expressed in an episodic fashion that requires a portion of the promoter termed the neuron-specific enhancer (NSE) for activity. In this study, we examined whether certain GATA regulatory elements in the NSE are necessary for this intermittent activity. When injected into individual living GT1-7 cells, luciferase reporter constructs containing mutations of either GATA-A- or GATA-B-binding sites resulted in a marked reduction in gene expression pulse frequency, while mutations of both sites virtually abolished pulses. In subsequent studies, RT-PCR and western blot analysis revealed for the first time that GATA-5 and GATA-6 were expressed in GT1-7 cells, but electrophoretic mobility shift assays demonstrated further that GATA-5 bound to one of these GATA sites: GATA-A. Chromatin immunoprecipitation analysis revealed that all three factors, GATA-4, GATA-5, and GATA-6, were associated with the GnRH promoter in vivo. Interestingly though, immunoneutralization of GATA-5 or GATA-4 (reported to bind GATA-B) abolished gene expression pulses, but injection of GATA-6 antibody did not, indicating that of these factors just GATA-5 and GATA-4 are critical for intermittent activity. Finally, gel shift competition experiments revealed an interaction between proteins binding at the GATA-A site and those associating with an adjacent OCT1 site, previously shown to be necessary for pulse formation. These findings indicate that episodic GnRH gene expression pulses are mediated by GATA-5 and GATA-4, likely acting through the GATA-binding sites in the GnRH NSE region. Moreover, our observations that factors associated with GATA sites may also interact with OCT1 sites and that both are critical for pulse activity raise the intriguing possibility that GnRH pulse elaboration is a highly complex process that may require the coordinated interaction of several NSE-binding elements of the GnRH promoter.

Oncogenic role of engrailed-2 (en-2) in prostate cancer cell growth and survival.

Prostate cancer is the second leading cause of cancer death among men in the United States of America. However, the molecular mechanisms underlying the disease remain largely unknown. Therefore, the identification of tumor specific molecules that serve as targets for the development of new cancer drugs is considered to be a major goal in cancer research. The mouse Engrailed-2 (En-2) gene, which is a homeobox-containing transcription factor was recently identified as a candidate oncogene in breast cancer. Here, we demonstrate that En-2 is over-expressed in human prostate cancer cells as compared to normal prostate epithelial cells. In addition, our data suggests that EN2 expression may be positively modulated by PAX2 transcription factor. Furthermore, down-regulation of EN2 expression by siRNA resulted in a decrease in PAX2 expression. We also provide evidence that down-regulation of EN2 expression causes a dramatic decrease in prostate cancer cell proliferation. Therefore, from our studies we conclude that En-2 is a candidate oncogene in prostate cancer and its PAX2-regulated expression contributes to prostate cancer cell growth.