Emerging roles of Keap1/Nrf2 signaling in the thyroid gland and perspectives for bench-to-bedside translation.

The signaling pathway centered on the transcription factor nuclear erythroid factor 2-like 2 (Nrf2) has emerged during the last 15 years as a target for the prevention and treatment of diseases broadly related with oxidative stress such as cancer, neurodegenerative and metabolic diseases. The roles of Nrf2 are expanding beyond general cytoprotection, and they encompass its crosstalk with other pathways as well as tissue-specific functions. The thyroid gland relies on reactive oxygen species for its main physiological function, the synthesis and secretion of thyroid hormones. A few years ago, Nrf2 was characterized as a central regulator of the antioxidant response in the thyroid, as well as of the transcription and processing of thyroglobulin, the major thyroidal protein that serves as the substrate for thyroid hormone synthesis. Herein, we summarize the current knowledge about the roles of Nrf2 in thyroid physiology, pathophysiology and disease. We focus specifically on the most recent publications in the field, and we discuss the implications for the preclinical and clinical use of Nrf2 modulators.

The Keap1/Nrf2 Signaling Pathway in the Thyroid-2020 Update.

The thyroid gland has a special relationship with oxidative stress. On the one hand, like all other tissues, it must defend itself against reactive oxygen species (ROS). On the other hand, unlike most other tissues, it must also produce reactive oxygen species in order to synthesize its hormones that contribute to the homeostasis of other tissues. The thyroid must therefore also rely on antioxidant defense systems to maintain its own homeostasis in the face of continuous self-exposure to ROS. One of the main endogenous antioxidant systems is the pathway centered on the transcription factor Nuclear factor erythroid 2-related factor 2 (Nrf2) and its cytoplasmic inhibitor Kelch-like ECH-associated protein 1 (Keap1). Over the last few years, multiple links have emerged between the Keap1/Nrf2 pathway and thyroid physiology, as well as various thyroid pathologies, including autoimmunity, goiter, hypothyroidism, hyperthyroidism, and cancer. In the present mini-review, we summarize recent studies shedding new light into the roles of Keap1/Nrf2 signaling in the thyroid.

The Transcriptomic Response of the Murine Thyroid Gland to Iodide Overload and the Role of the Nrf2 Antioxidant System.

BACKGROUND: Thyroid follicular cells have physiologically high levels of reactive oxygen species because oxidation of iodide is essential for the iodination of thyroglobulin (Tg) during thyroid hormone synthesis. Thyroid follicles (the functional units of the thyroid) also utilize incompletely understood autoregulatory mechanisms to defend against exposure to excess iodide. To date, no transcriptomic studies have investigated these phenomena in vivo. Nuclear erythroid factor 2 like 2 (Nrf2 or Nfe2l2) is a transcription factor that regulates the expression of numerous antioxidant and other cytoprotective genes. We showed previously that the Nrf2 pathway regulates the antioxidant defense of follicular cells, as well as Tg transcription and Tg iodination. We, thus, hypothesized that Nrf2 might be involved in the transcriptional response to iodide overload. METHODS: C57BL6/J wild-type (WT) or Nrf2 knockout (KO) male mice were administered regular water or water supplemented with 0.05% sodium iodide for seven days. RNA from their thyroids was prepared for next-generation RNA sequencing (RNA-Seq). Gene expression changes were assessed and pathway analyses were performed on the sets of differentially expressed genes. RESULTS: Analysis of differentially expressed messenger RNAs (mRNAs) indicated that iodide overload upregulates inflammatory-, immune-, fibrosis- and oxidative stress-related pathways, including the Nrf2 pathway. Nrf2 KO mice showed a more pronounced inflammatory-autoimmune transcriptional response to iodide than WT mice. Compared to previously published datasets, the response patterns observed in WT mice had strong similarities with the patterns typical of Graves' disease and papillary thyroid carcinoma (PTC). Long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) also responded to iodide overload, with the latter targeting mRNAs that participate mainly in inflammation pathways. CONCLUSIONS: Iodide overload induces the Nrf2 cytoprotective response and upregulates inflammatory, immune, and fibrosis pathways similar to autoimmune hyperthyroidism (Graves' disease) and PTC.

Rare and common genetic variations in the Keap1/Nrf2 antioxidant response pathway impact thyroglobulin gene expression and circulating levels, respectively.

Nuclear factor, erythroid 2-like 2 (Nrf2) is a transcription factor that has been gaining attention in the field of pharmacology and especially in the chemoprevention of diseases such as cancer, metabolic and neurodegenerative diseases, etc. This is because natural compounds such as sulforaphane, which is found in broccoli sprout extracts, can activate Nrf2. The repertoire of the roles of Nrf2 is ever increasing; besides its traditional antioxidant and cytoprotective effects, Nrf2 can have other functions as a transcription factor. We have recently shown that Nrf2 directly regulates the expression of thyroglobulin (Tg), which is the most abundant thyroidal protein and the precursor of thyroid hormones. Two functional binding sites for Nrf2 (antioxidant response elements, AREs) were identified in the regulatory region of the TG gene. Interestingly, we then observed that one of these AREs harbors a rare single-nucleotide polymorphism (SNP). Also recently, we performed the first genome-wide association study (GWAS) for common SNPs that impact the circulating levels of Tg. Based on these investigations, we were triggered (i) to investigate whether common SNPs in the Nrf2 pathway correlate with circulating Tg levels; and (ii) to examine whether the rare SNP in one of the TG regulatory AREs may affect gene expression. To address the first question, we analyzed GWAS data from a general population and its two subpopulations, one with thyroid disease and/or abnormal thyroid function tests and the other without, in which circulating Tg levels had been measured. Statistically significant associations with Tg levels were observed in the genes encoding Nrf2 and Keap1, including, notably, a known functional SNP in the promoter of the gene encoding Nrf2. Regarding the rare SNP (rs778940395) in the proximal ARE of the TG enhancer, luciferase reporter gene expression studies in PCCL3 rat thyroid follicular cells showed that this SNP abrogated the basal and sulforaphane- or TSH-induced luciferase activity, behaving as a complete loss-of-function mutation. Thus, both rare and common genetic variation in the Keap1/Nrf2 pathway can impact TG expression and Tg circulating levels, respectively.

Keap1/Nrf2 Signaling: A New Player in Thyroid Pathophysiology and Thyroid Cancer.

The Keap1/Nrf2 pathway is a key mediator of general redox and tissue-specific homeostasis. It also exerts a dual role in cancer, by preventing cell transformation of normal cells but promoting aggressiveness, and drug resistance of malignant ones. Although Nrf2 is well-studied in other tissues, its roles in the thyroid gland are only recently emerging. This review focuses on the involvement of Keap1/Nrf2 signaling in thyroid physiology, and pathophysiology in general, and particularly in thyroid cancer. Studies in mice and cultured follicular cells have shown that, under physiological conditions, Nrf2 coordinates antioxidant defenses, directly increases thyroglobulin production and inhibits its iodination. Increased Nrf2 pathway activation has been reported in two independent families with multinodular goiters due to germline loss-of-function mutations in KEAP1. Nrf2 pathway activation has also been documented in papillary thyroid carcinoma (PTC), due to somatic mutations, or epigenetic modifications in KEAP1, or other pathway components. In PTC, such Nrf2-activating KEAP1 mutations have been associated with tumor aggressiveness. Furthermore, polymorphisms in the prototypical Nrf2 target genes NQO1 and NQO2 have been associated with extra-thyroidal extension and metastasis. More recently, mutations in the Nrf2 pathway have also been found in Hürthle-cell (oncocytic) thyroid carcinoma. Finally, in in vitro, and in vivo models of poorly-differentiated, and undifferentiated (anaplastic) thyroid carcinoma, Nrf2 activation has been associated with resistance to experimental molecularly-targeted therapy. Thus, Keap1/Nrf2 signaling is involved in both benign and malignant thyroid conditions, where it might serve as a prognostic marker or therapeutic target.

Impact of Antioxidant Natural Compounds on the Thyroid Gland and Implication of the Keap1/Nrf2 Signaling Pathway.

BACKGROUND: Natural compounds with potential antioxidant properties have been used in the form of food supplements or extracts with the intent to prevent or treat various diseases. Many of these compounds can activate the cytoprotective Nrf2 pathway. Besides, some of them are known to impact the thyroid gland, often with potential side-effects, but in other instances, with potential utility in the treatment of thyroid disorders. OBJECTIVE: In view of recent data regarding the multiple roles of Nrf2 in the thyroid, this review summarizes the current bibliography on natural compounds that can have an effect on thyroid gland physiology and pathophysiology, and it discusses the potential implication of the Nrf2 system in the respective mechanisms. METHODS & RESULTS: Literature searches for articles from 1950 to 2018 were performed in PubMed and Google Scholar using relevant keywords about phytochemicals, Nrf2 and thyroid. Natural substances were categorized into phenolic compounds, sulfur-containing compounds, quinones, terpenoids, or under the general category of plant extracts. For individual compounds in each category, respective data were summarized, as derived from in vitro (cell lines), preclinical (animal models) and clinical studies. The main emerging themes were as follows: phenolic compounds often showed potential to affect the production of thyroid hormones; sulfur-containing compounds impacted the pathogenesis of goiter and the proliferation of thyroid cancer cells; while quinones and terpenoids modified Nrf2 signaling in thyroid cell lines. CONCLUSION: Natural compounds that modify the activity of the Nrf2 pathway should be evaluated carefully, not only for their potential to be used as therapeutic agents for thyroid disorders, but also for their thyroidal safety when used for the prevention and treatment of non-thyroidal diseases.

Broccoli sprout beverage is safe for thyroid hormonal and autoimmune status: Results of a 12-week randomized trial.

Sulforaphane is a redox-active natural product present in cruciferous vegetables like broccoli. Broccoli sprout-derived products are promising agents for the prevention of oxidative stress-related diseases, but some have long been suspected of thyroidal toxicity. Recent findings also raise the possibility that long-term exposure to sulforaphane, or to other natural substances or drugs that modulate the activity of the transcription factor Nrf2 (NFE2-related factor 2) may lead to thyroid dysfunction or thyroid autoimmune disease, questioning the safety of trials with sulforaphane-containing products. Previous studies addressing possible effects of sulforaphane-related compounds from natural product extracts on the thyroid were quite short and/or inconsistent. To investigate whether long-term exposure to a beverage enriched with sulforaphane and its precursor glucoraphanin may affect thyroid function, we analyzed biochemical measures of thyroid function and thyroid autoimmunity in 45 female participants in a randomized clinical trial at baseline and after 84 days of beverage administration. Serum levels of thyroid-stimulating hormone, free thyroxine and thyroglobulin were not affected by the treatment, and neither was the thyroid autoimmunity status of participants. These results provide evidence in favor of the safety of chemoprevention strategies that target the activation of Nrf2 to protect against environmental exposures and other oxidative stress-related pathologies.

CRISPR/Cas9 genome-wide screening identifies KEAP1 as a sorafenib, lenvatinib, and regorafenib sensitivity gene in hepatocellular carcinoma.

Sorafenib is the first-line drug used for patients with advanced hepatocellular carcinoma (HCC). However, acquired sorafenib resistance in cancer patients limits its efficacy. Here, we performed the first genome-wide CRISPR/Cas9-based screening on sorafenib-treated HCC cells to identify essential genes for non-mutational mechanisms related to acquired sorafenib resistance and/or sensitivity in HCC cells. KEAP1 was identified as the top candidate gene by Model-based Analysis of Genome-wide CRISPR/Cas9 Knockout (MAGeCK). KEAP1 disrupted HCC cells were less sensitive than wild-type cells in short- and long-term sorafenib treatments. Compared to wild-type cells, KEAP1-disrupted cells showed lower basal and sorafenib-induced reactive oxygen species (ROS) levels and were more resistant to oxidative stress-induced cell death. The absence of KEAP1 led to increased activity of Nrf2, a key transcription factor controlling antioxidant responses, as further evidenced by increased expression of Nrf2-controlled genes including NQO1, GPX2 and TXNRD1, which were positively associated with chemoresistance. In addition, KEAP1 disruption counteracted the reduction of cell viability and the elevation of ROS caused by lenvatinib, a drug that recently showed clinical efficacy as a first-line treatment for unresectable HCC. Finally, Keap1 disruption also increased the resistance of cells to regorafenib, a recently approved drug to treat HCC as a second line therapy. Taken together, our data indicate that deregulation of the KEAP1/Nrf2 pathway following KEAP1 inactivation contributes to sorafenib, lenvatinib, and regorafenib resistance in human HCC cells through up-regulation of Nrf2 downstream genes and decreased ROS levels.

Raman spectroscopy for the preoperative diagnosis of thyroid cancer and its subtypes: An in vitro proof-of-concept study.

OBJECTIVE: In 2016, there were an estimated 56 870 new cases of thyroid cancer (TC) in the USA. Fine needle aspiration cytology (FNAC) is the most safe, accurate and cost-effective method for the initial investigation of thyroid nodules. FNAC is limited by the inability to diagnose malignancy in follicular-patterned lesions accurately and, as a result, 20%-30% of cases under investigation for TC are classified as cytologically indeterminate, illustrating a problem with current FNAC procedure. Raman spectroscopy has shown promising results for the detection of many cancers; however, to date there has been no report on the performance of Raman spectroscopy on thyroid cytological samples. The aim of this study was to examine whether Raman spectroscopy could be used to correctly classify cell lines representing benign thyroid cells and various subtypes of TC. METHODS: A benign thyroid cell line and seven TC cell lines were prepared as ThinPrep® cytology slides and analysed with Raman spectroscopy. Principal components analysis and linear discriminant analysis were implemented to develop effective diagnostic algorithms for classification of Raman spectra of different TC subtypes. RESULTS: The spectral differences separating benign and TC cell lines were assigned to differences in the composition of nucleic acids, lipids, carbohydrates and protein in the benign and cancer cells. Good sensitivities (74%-85%), specificities (65%-93%) and diagnostic accuracies (71%-88%) were achieved for the identification of TC. CONCLUSION: These findings suggest that Raman spectroscopy has potential for preoperative TC diagnosis on FNAC samples.

Nrf2 is commonly activated in papillary thyroid carcinoma, and it controls antioxidant transcriptional responses and viability of cancer cells.

CONTEXT: The antioxidant transcription factor NFE2-related factor 2 (Nrf2), encoded by NFE2L2, has been implicated as mediator of thyroid cancer cell line resistance to proteasome inhibitors. However, the activity status of the Nrf2 pathway in human thyroid cancer remains unknown. OBJECTIVE: The aims of this study were assessment of the activity status of the Nrf2 pathway in papillary thyroid carcinoma (PTC) and investigation of its role(s) in antioxidant transcriptional responses and viability of cancer cells. DESIGN AND SETTING: We conducted retrospective immunohistochemical analyses of PTC specimens, adjacent normal tissue, and benign lesions; assays of viability and gene expression in the PTC cell lines K1 and TPC-1 after genetic/pharmacological manipulation of Nrf2; and DNA sequencing at an academic medical center. PATIENTS: The study included 42 PTC and 42 benign lesions (24 adenomas and 18 nodular hyperplasias). MAIN OUTCOME MEASURES: We assessed the abundance of Nrf2, Nqo1, Keap1, and 4HNE; cell line viability and mRNA expression of Nrf2, Nqo1, and Trdx1; and the sequence of NFE2L2, KEAP1, and BRAF. RESULTS: Nrf2 and its target Nqo1 were undetectable in normal tissue; their levels were significantly higher in PTC than in benign lesions (P < .0001 and P = .024, respectively). The Nrf2 inhibitor Keap1 was variably abundant in PTC, and its levels did not correlate with Nrf2 (P = .37), arguing against decreased levels as the mechanism for Nrf2 activation. The oxidized lipid 4HNE was more abundant in PTC than normal tissue (P < .001), indicating oxidative stress. Nrf2 mediated transcriptional antioxidant responses in both the PTC cell lines K1 and TPC-1 and in the nontransformed cell line TAD2, but it conferred a viability advantage specifically in the PTC cell lines. CONCLUSIONS: The high activity of Nrf2 in PTC warrants further exploration of this pathway's potential diagnostic, prognostic, and/or therapeutic utility in PTC.

Rare types of rotaviruses isolated from children with acute gastroenteritis in Patras, Greece.

BACKGROUND: Acute diarrhea remains a major public health issue worldwide, with gastroenteritis agents associated with a high morbidity, especially in infants and young children. A 1-year study (2009-2010) of rotavirus (RV)-caused pediatric diarrhea was performed on hospitalized children admitted with symptoms of acute diarrhea to the University Hospital of Patras, Greece. METHODS: Twenty-nine fecal samples were investigated for RVs, adenoviruses (AdVs), and enteroviruses (EVs) in an attempt to characterize these enteric viruses, which have been implicated in hospitalized diarrhea. RESULTS: A 44.8% (13/29) incidence of viral infection was reported for the viral targets. Monoinfections accounted for 31% (9/29), while bi-infections accounted for 13.4% (4/29). Sequencing of positive samples allowed identification of RVs G4P[8] (2/29), G9P[8] (1/29), and interestingly of the rare type G12P[8] (2/29). AdV types 1, 2, and 6 were also identified in 4/29, 1/29, and 1/29 of the samples, respectively. Regarding the EVs, EV71 (2/29), coxsackievirus A4 (1/29), echovirus 11 (1/29), and EV96 (1/29) were typed. CONCLUSION: The results of the present study, and specifically the detection of rare RV G12 and EV71 strains, address the need for continuous epidemiological surveys to provide epidemiological pictures of pediatric viral infections circulating in the community.

Differential expression of microRNAs in adipose tissue after long-term high-fat diet-induced obesity in mice.

Obesity is a major health concern worldwide which is associated with increased risk of chronic diseases such as metabolic syndrome, cardiovascular disease and cancer. The elucidation of the molecular mechanisms involved in adipogenesis and obesogenesis is of essential importance as it could lead to the identification of novel biomarkers and therapeutic targets for the development of anti-obesity drugs. MicroRNAs (miRNAs) have been shown to play regulatory roles in several biological processes. They have become a growing research field and consist of promising pharmaceutical targets in various fields such as cancer, metabolism, etc. The present study investigated the possible implication of miRNAs in adipose tissue during the development of obesity using as a model the C57BLJ6 mice fed a high-fat diet.C57BLJ6 wild type male mice were fed either a standard (SD) or a high-fat diet (HFD) for 5 months. Total RNA was prepared from white adipose tissue and was used for microRNA profiling and qPCR.Twenty-two of the most differentially expressed miRNAs, as identified by the microRNA profiling were validated using qPCR. The results of the present study confirmed previous results. The up-regulation of mmu-miR-222 and the down-regulation of mmu-miR-200b, mmu-miR-200c, mmu-miR-204, mmu-miR-30a*, mmu-miR-193, mmu-miR-378 and mmu-miR-30e* after HFD feeding has also been previously reported. On the other hand, we show for the first time the up-regulation of mmu-miR-342-3p, mmu-miR-142-3p, mmu-miR-142-5p, mmu-miR-21, mmu-miR-146a, mmu-miR-146b, mmu-miR-379 and the down-regulation of mmu-miR-122, mmu-miR-133b, mmu-miR-1, mmu-miR-30a*, mmu-miR-192 and mmu-miR-203 during the development of obesity. However, future studies are warranted in order to understand the exact role that miRNAs play in adipogenesis and obesity.

Nrf2 activation diminishes during adipocyte differentiation of ST2 cells.

Adipocyte differentiation (adipogenesis) is a highly controlled process known to be affected, among other factors, by the redox status of the cell. Nrf2 (NFE2-related factor 2) is a transcription factor that orchestrates the expression of a battery of antioxidant and detoxification genes under both basal and stress conditions. The present study investigated the activation of Nrf2 during adipocyte differentiation using as a model the mouse bone marrow-derived ST2 cell line. Treatment of ST2 cells with a differentiation cocktail containing IBMX, indomethacin, hydrocortisone and insulin induced differentiation into adipocytes over 5 days. During adipogenesis, the intracellular glutathione redox potential, which is an indicator of oxidative stress levels, became steadily more oxidized, as shown by real-time measurement in differentiating ST2 cells stably transfected with a redox-sensitive Grx1-roGFP2 fusion protein. The nuclear abundance of Nrf2 was assessed by Western immunoblotting and its DNA binding activity by EMSA (electrophoretic mobility shift assay) performed on nuclear protein extracts prepared every 24 h. The nuclear abundance of Nrf2 continuously decreased during adipogenesis in ST2 cells. Its DNA binding activity reached a nadir during the first two days of differentiation, after which it increased slightly without approaching its initial level. The pattern of Nrf2 DNA binding corresponded to its transcriptional activity as assessed in ST2 cells stably transfected with a reporter construct bearing a Nrf2 bind site upstream of the luciferase gene. In conclusion, the activation of Nrf2 decreased significantly during adipogenesis. The observed changes might lead to increased oxidative stress levels that could facilitate the differentiation process. These findings could shed new light on the pathogenesis of obesity, in which the adipose tissue and oxidative stress play prominent roles.

Development of an optimized method for the detection of airborne viruses with real-time PCR analysis.

BACKGROUND: Airborne viruses remain one of the major public health issues worldwide. Detection and quantification of airborne viruses is essential in order to provide information regarding public health risk assessment. FINDINGS: In this study, an optimized new, simple, low cost method for sampling of airborne viruses using Low Melting Agarose (LMA) plates and a conventional microbial air sampling device has been developed. The use of LMA plates permits the direct nucleic acids extraction of the captured viruses without the need of any preliminary elution step. Molecular detection and quantification of airborne viruses is performed using real-time quantitative (RT-)PCR (Q(RT-)PCR) technique. The method has been tested using Adenoviruses (AdVs) and Noroviruses (NoVs) GII, as representative DNA and RNA viruses, respectively. Moreover, the method has been tested successfully in outdoor experiments, by detecting and quantifying human adenoviruses (HAdVs) in the airborne environment of a wastewater treatment plant. CONCLUSIONS: The great advantage of LMA is that nucleic acids extraction is performed directly on the LMA plates, while the eluted nucleic acids are totally free of inhibitory substances. Coupled with QPCR the whole procedure can be completed in less than three (3) hours.

Molecular detection of multiple viral targets in untreated urban sewage from Greece.

BACKGROUND: Urban sewage virological analysis may produce important information about the strains that cause clinical and subclinical infections in the population, thus supporting epidemiological studies. METHODS: In the present study, a twenty one-month survey (November 2007 to July 2009) was conducted in order to evaluate the presence of human adenoviruses (hAdV), hepatitis A viruses (HAV), hepatitis E viruses (HEV), Noroviruses (NoV), and human Polyomaviruses (hPyV) in untreated sewage samples collected from the inlet of Patras' municipal biological wastewater treatment plant, located in southwestern Greece. Nucleic acid amplification techniques were applied for viral nucleic acid detection. Positive samples were confirmed by sequencing and comparative phylogenetic analysis was performed on the isolated viral strains. RESULTS: In total, viruses were detected in 87.5% (42/48) of sewage samples. AdVs, PyVs, HAV, and NoVs were detected in 45.8% (22/48), 68.8% (33/48), 8.3% (4/48), and 6.3% (3/48) of the samples collected from the plant's inlet, while HEV was not detected at all. Adenovirus types 8 (Ad8), 40 (Ad40) and 41 (Ad41) were recognized, while JC and BK polyomaviruses were recorded. Noroviruses were identified as GII.4. HAV was typed as genotype IA. CONCLUSIONS: Our study demonstrates the advantages of environmental surveillance as a tool to elucidate the molecular epidemiology of community circulating viruses. We underline the need of environmental surveillance programs in countries such as Greece with inadequate and problematic epidemiological surveillance system and no environmental surveillance system currently in action.

Simvastatin lowers reactive oxygen species level by Nrf2 activation via PI3K/Akt pathway.

The beneficial effects of HMG-CoA (3-hydroxy-3-methyl-glutaryl-CoA) reductase inhibitors (statins) have been attributed not only to their cholesterol lowering effect but also to their pleiotropic actions and especially to their anti-oxidant activity. Nrf2 (NF-E2-related factor 2) is a transcription factor that orchestrates the transcriptional response of cells to oxidative stressors and electrophilic xenobiotics. In this study, primary mouse embryonic fibroblasts from wild type or Nrf2 knock out C57BL6J mice and ST-2 cells were used to investigate the implication of Nrf2 in the mediation of the anti-oxidant effects of statins and the possible involvement of PI3K/Akt pathway in this process. We show for the first time that simvastatin lowers reactive oxygen species (ROS) by activating Nrf2 through the PI3K/Akt pathway.

Disease-related anemia in chronic lymphocytic leukemia is not due to intrinsic defects of erythroid precursors: a possible pathogenetic role for tumor necrosis factor-alpha.

BACKGROUND/AIMS: Disease-related anemia in chronic lymphocytic leukemia (CLL) occurs when the obvious causes are excluded while its pathogenesis is still obscure. We investigated its underlying mechanisms in 56 untreated patients with CLL. METHODS: Bone marrow (BM) lymphocytic infiltration was estimated in trephine biopsies. Serum erythropoietin (EPO) and tumor necrosis factor-alpha (TNF-alpha) levels were measured by ELISA. The potential of BM CD34+ to differentiate into erythroid cells was evaluated by methylcellulose-based assays and in liquid cultures supplemented with EPO, SCF, IL-3 +/- TNF-alpha. The response of erythroid precursors to EPO +/- TNF-alpha was assessed by detecting activated key proteins of EPO-EPO receptor signalling pathway using Western Blot and EMSA. RESULTS: Bone marrow lymphocytic infiltration was not exclusively responsible for disease-related anemia and CD34+ cells were intrinsically capable of generating erythroid precursors. Also, no deficiency of serum erythropoietin (EPO) or defective intracellular response of erythroid precursors to EPO +/- TNF-alpha stimulation was observed. Serum TNF-alpha levels were found increased in anemic CLL patients and TNF-alpha appeared to directly inhibit the erythroid development in early stages of erythropoiesis. CONCLUSION: We concluded that CLL-related anemia was not due to intrinsic defects of erythroid precursors, but might result from the direct suppressive effect of TNF-alpha on the erythroid production.

Runx2: of bone and stretch.

Runx2 is a key transcriptional modulator of osteoblast differentiation that plays a fundamental role in osteoblast maturation and homeostasis. Runx2-null mice despite normal skeletal patterning have no osteoblasts and consequently bone tissue. Mutations of the runx2 gene in humans cause cleidocranial dysplasia. As a member of the Runx family of transcription factors, Runx2 operates by binding to the osteoblast-specific cis-acting element 2 (OSE2), which is found in the regulatory region of all main osteoblast-related genes controlling their expression. Its expression and/or activity are dictated by a number of different external cues while multiple signalling pathways that affect osteoblast function merge to and are integrated by Runx2. Among the various stimuli that modulate Runx2 activity, mechanical loading (strain/stretching) has been revealed to be one of the most critical signals that connect Runx2 with osteoblast function and bone remodelling through mechanotransduction.

Combined effect of mutations of the GH1 gene and its proximal promoter region in a child with growth hormone neurosecretory dysfunction (GHND).

Mutational analysis of the growth hormone 1 (GH1) gene and its promoter in a patient with GH neurosecretory dysfunction (GHND) revealed a heterozygous new deletion of one base 7-bp downstream from the 3'-splice site of exon 4 (IVS4'del+7) of the GH1 gene and two new heterozygous mutations at sites -135 and -138 of the GH1 promoter. In addition, two polymorphisms at sites -301 and -308 of the GH1 promoter were observed. All other family members had either the -301/-308 polymorphisms or the IVS4'del+7 mutation, but none had both. The IVS4'del+7 mutation located close to the splice donor site possibly interferes with the success of the splicing process, or the mutant transcripts are highly unstable because of nonsense-mediated mRNA decay. The -135/-138 mutations, albeit in close proximity to a putative Pit-1 recognition site, do not seem to affect binding of this transcription factor. The combination of the two polymorphisms, -301/-308, results in significantly reduced DNA-binding activity as monitored by electrophoretic mobility-shift assay. Transcription factor recognition site analysis of the GH1 promoter (MatInspector) revealed that HES1, one of the effectors of the Notch signalling system, is the only transcription factor whose binding is expected to be disrupted by each haplotype or by their combination. We provide evidence that the combination of -301/-308 polymorphisms with the IVS4'del+7 mutation in a GHND patient probably accounts for the reduced amount of growth hormone spontaneously secreted from his pituitary gland and for the severe growth delay.

Growth hormone attenuates the transcriptional activity of Runx2 by facilitating its physical association with Stat3beta.

UNLABELLED: We document that GH controls osteoblast function by modulating the biological activity of the osteospecific transcription factor Runx2. Evidence is provided for a physical interaction between Runx2 and Stat3beta, which is enhanced by GH and downregulates the transcriptional properties of this key osteogenic regulator. INTRODUCTION: Growth hormone (GH) signals to bone either through insulin-like growth factor-1 or directly by influencing the function of osteoblasts, the bone-forming cells. This study aimed at exploring the molecular events that underlie the direct biological action of GH on osteoblastic cells, and specifically, the effects that it might exert on the function of the bone-specific transcriptional regulator Runx2. MATERIALS AND METHODS: The GH-responsive human osteoblastic cell line Saos-2 was used as our experimental system. Western blot analyses were used to monitor the presence of several parameters known to be affected by GH in these cells (i.e., downregulation of GH receptor, induction of STATs, and extracellular signal-regulated kinase [ERK] mitogen-activated protein kinase [MAPK] pathways). Electrophoretic mobility shift assays were used to assess Runx2 and Stat3 binding activity on an osteoblast-specific element (OSE2) after GH treatment. A combination of yeast two-hybrid and co-immunoprecipitation assays were performed to test for the existence of a physical Runx2.Stat3beta association. Finally, co-transfection experiments were used to investigate the interplay of the two transcription factors on the activity of a p6OSE2-Luc promoter after GH stimulation. RESULTS: We show that GH signaling through Stat3/ERK MAPK potentiates the DNA binding activity of Runx2 but, at the same time, restrains its transcriptional potential. Moreover, a novel physical interaction of Runx2 with transcription factor Stat3beta, which is enhanced by GH stimulation, was documented both in vitro and in vivo. Importantly, this interaction impairs the transcriptional activity of Runx2 without affecting its DNA binding capacity. CONCLUSION: Our data provide the first evidence that GH modulates the transcriptional function of Runx2 in osteoblastic cells by promoting its inhibitory interaction with Stat3beta. Shedding light on such mechanisms will contribute to a better understanding of GH effects on skeletal homeostasis that may impact on decisions at the clinical level, especially in diseases affecting bone quantity and quality (e.g., osteoporosis).