Involvement of non-coding RNAs and transcription factors in the induction of Transglutaminase isoforms by ATRA.

The multifunctional protein Transglutaminase type 2, is associated with cancer epithelial mesenchymal transition, invasiveness, stemness and drugs resistance. Several variant isoforms and non-coding RNAs are present in cancer and this report explored the expression of these transcripts of the TGM2 gene in cancer cell lines after induction with all-trans retinoic acid. The expression of truncated variants along with two long non-coding RNAs, was demonstrated. One of these is coded from the first intron and the Last Exon Variant is constituted by a sequence corresponding to the last three exons and the 3'UTR. Analysis of ChIP-seq data, from ENCODE project, highlighted factors interacting with intronic sequences, which could interfere with the progression of RNApol II at checkpoints, during the elongation process. Some relevant transcription factors, bound in an ATRA-dependent way, were found by RNA immunoprecipitation, notably GATA3 mainly enriched to Last Exon Variant non-coding RNA. The involvement of NMD in the regulation of the ratio among these transcripts was observed, as the prevalent recovering of Last Exon Variant to phUPF1-complexes, with decrease of the binding towards other selective targets. This study contributes to identify molecular mechanisms regulating the ratio among the variants and improves the knowledge about regulatory roles of the non-coding RNAs of the TGM2 gene.

Spotlight on the transglutaminase 2 gene: a focus on genomic and transcriptional aspects.

The type 2 isoenzyme is the most widely expressed transglutaminase in mammals displaying several intra- and extracellular activities depending on its location (protein modification, modulation of gene expression, membrane signalling and stabilization of cellular interactions with the extracellular matrix) in relation to cell death, survival and differentiation. In contrast with the appreciable knowledge about the regulation of the enzymatic activities, much less is known concerning its inducible expression, which is altered in inflammatory and neoplastic diseases. In this context, we first summarize the gene's basic features including single-nucleotide polymorphism characterization, epigenetic DNA methylation and identification of regulatory regions and of transcription factor-binding sites at the gene promoter, which could concur to direct gene expression. Further aspects related to alternative splicing events and to ncRNAs (microRNAs and lncRNAs) are involved in the modulation of its expression. Notably, this important gene displays transcriptional variants relevant for the protein's function with the occurrence of at least seven transcripts which support the synthesis of five isoforms with modified catalytic activities. The different expression of the TG2 (type 2 transglutaminase) variants might be useful for dictating the multiple biological features of the protein and their alterations in pathology, as well as from a therapeutic perspective.

A long non-coding RNA inside the type 2 transglutaminase gene tightly correlates with the expression of its transcriptional variants.

The long non-coding RNAs (lncRNAs) are matter of intense investigation as potential regulators of gene expression. In the case of the transglutaminase 2 gene (TGM2) the databases of genome sequence indicate location of a lncRNA (LOC107987281) within the first intron. This lncRNA is 1000 bp long, arises from 2 exons and starts few nucleotides 3' of the first splicing site of translated TGM2. We have analysed correlations between expression of LOC107987281 lncRNA and TGM2 mRNA by real-time PCR in K562 cell line untreated or treated with the anticancer drugs TPA (12-O-tetradecanoylphorbol-13-acetate), Docetaxel and Doxorubicin. In the treated cells the lncRNA increase follows the trend of TGM2 transcript. To validate this finding we used HumanExon1_0ST Affymetrix; chip data were background-adjusted, quantile-normalized and summarized using robust multi-array average analysis implemented in the R package. The probesets recognize sequences inside each exon, near intronic splicing sites and others located in the untranslated regions of TGM2 gene. The analysis of total RNA samples in GEO datasets from K562, HL-60, THP-1 and U937 cell lines, untreated or treated with TPA in replicated experiments confirmed our earlier results. These demonstrate correlation between LOC107987281 and TGM2 mRNA in the cell lines (K562, HL60 and THP-1) where increased levels of TGM2 mRNA are produced. Additional array study on 358 samples of several normal and paired tumor tissues leads to the same conclusions, indicating a correlation between full-length TGM2 mRNA and LOC107987281 lncRNA in relation to the development of several tumors.

Oxidative damage and the pathogenesis of menopause related disturbances and diseases.

The postmenopausal phase of life is frequently associated in women with subjective symptoms (e.g. vasomotor) and real diseases (atherosclerosis with coronary ischemia, osteoporosis, Alzheimer-type neurodegeneration, urogenital dystrophy), which together determine the post-menopausal syndrome. Observations that oxidative damage by reactive oxygen/nitrogen species in experimental models can contribute to the pathogenesis of these disturbances stimulated research on the relationships between menopause, its endocrine deficiency, oxidative balance and the "wellness" in postmenopausal life. The connection among these events is probably due to the loss of protective actions exerted by estrogens during the fertile life. Most recent studies have revealed that estrogens exert an antioxidant action not by direct chemical neutralization of reactants as it was expected until recently but by modulating the expression of antioxidant enzymes that control levels of biological reducing agents. Also nutritional antioxidants apparently act by a similar mechanism. From this perspective it is conceivable that a cumulative control of body oxidant challenges and biological defenses could help in monitoring between "normal" and "pathological" menopause. However, as clinical studies failed to confirm this scenario in vivo, we have decided to review the existing literature to understand the causes of this discrepancy and whether this was due to methodologic reasons or to real failure of the basic hypothesis.

PON-1 and ferroxidase activities in older patients with mild cognitive impairment, late onset Alzheimer's disease or vascular dementia.

BACKGROUND: A large body of evidence suggests that not only cerebral but also systemic oxidative stress (OxS) might be involved in the pathogenesis of late onset Alzheimer's disease (LOAD) and vascular dementia (VAD), as well as of the prodromal phase of dementia, the so-called mild cognitive impairment (MCI). In the present study, we evaluated whether paraoxonase 1 (PON-1) and ferroxidase (FeOx) activities, because of their well acknowledged effectiveness as systemic antioxidants, might be associated with dementia and/or MCI. METHODS: Serum arylesterase and paraoxonase of PON-1, along with FeOx I (ceruloplasmin-related) and II activities were assessed in 223 MCI, 162 LOAD, 65 VAD patients, and in 143 older normal cognitive controls. RESULTS: Among the enzymatic activities examined, only arylesterase significantly changed across the groups (ANOVA: p<0.001), with similar lower levels in MCI, LOAD, and VAD compared to controls. By multivariate logistic regression analysis we showed that, in respect to controls, low levels (under the median value) of serum arylesterase were independently associated with an increase in the likelihood of being affected by LOAD [odds ratio (OR) 2.8, 95% confidence interval (CI) 1.5-5.0], VAD (OR 2.7, 95% CI 1.2-6.2), or MCI (OR 2.3, 95% CI 1.3-3.8). CONCLUSIONS: Overall, our results suggest that depression of PON-1, and in particular, of arylesterase activity, in serum might be an early feature of dementia-related diseases. Further longitudinal exploration of the role of this enzyme in the onset and progression of these disorders are required.

Serum levels of hydroperoxides and multimorbidity among older patients with mild cognitive impairment or late-onset Alzheimer's disease.

BACKGROUND: Oxidative stress (OxS) might be involved in the pathogenesis of late-onset Alzheimer disease (LOAD); noteworthy, the presence of multimorbidity is regarded as a common denominator of OxS and dementia. AIM: To evaluate the contribution of multimorbidity to OxS in LOAD and mild cognitive impairment (MCI). METHODS: Serum hydroperoxides and multimorbidity (CIRS-CI scale) were evaluated in 46 Controls, 104 MCI and 75 LOAD. RESULTS: A trend toward an increase of hydroperoxides from Controls to MCI to LOAD was observed (LOAD vs Controls p = 0.01). This OxS marker was positively and significantly correlated with CIRS-CI in Controls (p = 0.002) and patients with MCI (p = 0.005) but not in those with LOAD (p = 0.104). CONCLUSIONS: Multimorbidity is associated with systemic OxS but only in elderly people with either no or mild cognitive impairment. Although OxS is elevated in LOAD patients, its association with multimorbidity seems to be negligible, confirming the existence of strong disease-specific pro-oxidant mechanisms.

Waist circumference and dual-energy X-ray absorptiometry measures of overall and central obesity are similarly associated with systemic oxidative stress in women.

Growing evidence suggests that overall and abdominal obesity might lead to oxidative stress (OxS), which, in turn, plays a key role in the pathogenesis of a wide spectrum of diseases. In this study, for the first time, we compared the correlations of indirect, i.e. anthropometric, and direct, by dual energy X-ray absorptiometry (DXA), measures of body fat with circulatory OxS markers in women. To address this issue, we assessed central and total body fat mass (FM) by DXA, and serum levels of advanced oxidation protein products (AOPP), thiols and hydroperoxides in 275 healthy women (age 21-65 years; body mass index [BMI] 21.1-32.0 kg/m(2); waist circumference [WC] 60.1-109.9 cm). Among the markers considered in the study, only hydroperoxides levels, i.e. by-products of lipid peroxidation, were significantly (p < 0.05 for all) and positively correlated to body fat accumulation after controlling for confounding factors. In particular, this marker was found to be similarly associated with DXA-derived total FM, total FM % and trunk FM as well as with WC. Of note, hydroperoxides appeared to be correlated with abdominal but not with general obesity, as classified according to standard WC and BMI cut-offs, respectively. In conclusion, taken together our data demonstrated that, at least in women, the measurement of body FM by DXA has no advantage over the simpler and cheaper WC with regard to their associations with systemic OxS markers. Moreover, WC emerged as a superior potential predictor of OxS compared to the other most commonly used anthropometric measures (including BMI and waist to hip ratio).

Transcriptomics Studies Reveal Functions of Transglutaminase 2 in Breast Cancer Cells Using Membrane Permeable and Impermeable Inhibitors.

Transglutaminase 2 (TG2) performs many functions both under physiological and pathological conditions. In cancer, its expression is associated with aggressiveness, propensity to epithelial-mesenchymal transition, and metastasis. Since TG2 performs key functions both outside and inside the cell, using inhibitors with different membrane permeability we analyzed the changes in the transcriptome induced in two triple-negative cell lines (MDA-MB-436 and MDA-MB-231) with aggressive features. By characterizing pathways and gene networks, we were able to define the effects of TG2 inhibitors (AA9, membrane-permeable, and NCEG2, impermeable) in relation to the roles of the enzyme in the intra- and extracellular space within the context of breast cancer. The deregulated genes revealed p53 and integrin signaling to be the common pathways with some genes showing opposite changes in expression. In MDA-MB-436, AA9 induced apoptosis, modulated cadherin, Wnt, gastrin and cholecystokinin receptors (CCKR) mediated signaling, with RHOB and GNG2 playing significant roles, and affected the Warburg effect by decreasing glycolytic enzymes. In MDA-MB-231 cells, AA9 strongly impacted HIF-mediated hypoxia, including AKT and mTOR pathway. These effects suggest an anti-tumor activity by blocking intracellular TG2 functions. Conversely, the use of NCEG2 stimulated the expression of ATP synthase and proteins involved in DNA replication, indicating a potential promotion of cell proliferation through inhibition of extracellular TG2. To effectively utilize these molecules as an anti-tumor strategy, an appropriate delivery system should be evaluated to target specific functions and avoid adverse effects. Additionally, considering combinations with other pathway modulators is crucial.

The side chain of glutamine 13 is the acyl-donor amino acid modified by type 2 transglutaminase in subunit T of the native rabbit skeletal muscle troponin complex.

Subunit T of the native muscle troponin complex is a recognised substrate of transglutaminase both in vitro and in situ with formation of isopeptide bonds. Using a proteomic approach, we have now determined the precise site of in vitro labelling of the protein. A preparation of troponin purified from ether powder from mixed rabbit skeletal muscles was employed as transglutaminase substrate. The only isoform TnT2F present in our preparation was recognised as acyl-substrate by human type 2 transglutaminase which specifically modified glutamine 13 in the N-terminal region. During the reaction, the troponin protein complex was polymerized. Results are discussed in relation to the structure of the troponin T subunit, in the light of the role of troponins in skeletal and cardiac muscle diseases, and to the rules governing glutamine side chain selection by tissue transglutaminase.

Bone mass density selectively correlates with serum markers of oxidative damage in post-menopausal women.

BACKGROUND: Post-menopausal osteoporosis (PO) affecting a large fraction of elderly women, is triggered by the decline in 17ß-estradiol (E2) level. Experimental studies in animal models and cell cultures have suggested that the fall in E2 might contribute to developing oxidative stress (OS) which in turn is believed to play an important role in PO pathogenesis. The scarcity of human studies focusing on this issue prompted us to investigate the effects of the reproductive and post-reproductive phase of women's life on OS and bone health. METHODS: Serum parameters of oxidative challenge (lipid hydroperoxides and protein advanced oxidation products) and antioxidant defence (total serum antioxidants levels) along with bone mineral density (BMD) at femoral neck and lumbar spine were assessed in a sample of 191 women (98 pre- and 93 post-menopausal, of whom 30 osteoporotic). RESULTS: Pearson's correlation analysis unveiled that spinal BMD was negatively correlated with lipid hydroperoxides in overall postmenopausal subsample (r=-0.251, p=0.012), while no significant link between these two variables was detected in women in reproductive age (r=-0.022, p=0.833). Noteworthy, stepwise multiple regression analysis showed that the association found in post-menopausal women retained significance after adjusting for potential confounding factors (p=0.001). CONCLUSIONS: Our data showed that markers of oxidative challenge are associated with bone loss in women in post-menopausal status. We suggest that menopause-related estrogen withdrawal might contribute to make bone more vulnerable to oxidative injury thereby increasing the risk of PO development.

Accumulation of central fat correlates with an adverse oxidative balance in non-obese postmenopausal women.

The aim of the present study was to investigate whether accumulation of central fat is correlated with systemic oxidative stress (OxS) in non-obese apparently healthy postmenopausal women. Serum parameters of OxS (hydroperoxides and non-enzymatic antioxidants) along with body fat distribution, as assessed by dual-energy-X-ray absorptiometry (DXA), were evaluated in 134 non-obese postmenopausal women. Multiple regression analysis showed that central (trunk) fat significantly correlated with both markers of OxS independently of confounding factors (i.e. BMI, smoking, age, hypertension, legs and arms fat mass). In specific, the standardized regression coefficient was positive for hydroperoxides (ß = 0.324, p < 0.05) and negative for antioxidants (ß = -0.495, p < 0.01) level. In conclusion, the current data showed that the increase in central fat is an independent predictor of OxS condition among non-obese women in postmenopausal status. The possible pro-oxidant effects of the excess in central adiposity might be more harmful among post- than among pre-menopausal women, due to the postulated ability of E2 to contrast oxidative challenge and the related diseases.

Characterization by Gene Expression Analysis of Two Groups of Dopaminergic Cells Isolated from the Mouse Olfactory Bulb.

The olfactory bulb (OB) is one of two regions of the mammalian brain which undergo continuous neuronal replacement during adulthood. A significant fraction of the cells added in adulthood to the bulbar circuitry is constituted by dopaminergic (DA) neurons. We took advantage of a peculiar property of dopaminergic neurons in transgenic mice expressing eGFP under the tyrosine hydroxylase (TH) promoter: while DA neurons located in the glomerular layer (GL) display full electrophysiological maturation, eGFP+ cells in the mitral layer (ML) show characteristics of immature cells. In addition, they also display a lower fluorescence intensity, possibly reflecting different degrees of maturation. To investigate whether this difference in maturation might be confirmed at the gene expression level, we used a fluorescence-activated cell sorting technique on enzymatically dissociated cells of the OB. The cells were divided into two groups based on their level of fluorescence, possibly corresponding to immature ML cells and fully mature DA neurons from the GL. Semiquantitative real-time PCR was performed to detect the level of expression of genes linked to the degree of maturation of DA neurons. We showed that indeed the cells expressing low eGFP fluorescence are immature neurons. Our method can be further used to explore the differences between these two groups of DA neurons.

Metabolic characterisation of transglutaminase 2 inhibitor effects in breast cancer cell lines.

Transglutaminase 2 (TG2), which mediates post-translational modifications of multiple intracellular enzymes, is involved in the pathogenesis and progression of cancer. We used 1 H-NMR metabolomics to study the effects of AA9, a novel TG2 inhibitor, on two breast cancer cell lines with distinct phenotypes, MCF-7 and MDA-MB-231. AA9 can promote apoptosis in both cell lines, but it is particularly effective in MD-MB-231, inhibiting transamidation reactions and decreasing cell migration and invasiveness. This metabolomics study provides evidence of a major effect of AA9 on MDA-MB-231 cells, impacting glutamate and aspartate metabolism, rather than on MCF-7 cells, characterised by choline and O-phosphocholine decrease. Interestingly, AA9 treatment induces myo-inositol alteration in both cell lines, indicating action on phosphatidylinositol metabolism, likely modulated by the G protein activity of TG2 on phospholipase C. Considering the metabolic deregulations that characterise various breast cancer subtypes, the existence of a metabolic pathway affected by AA9 further points to TG2 as a promising hot spot. The metabolomics approach provides a powerful tool to monitor the effectiveness of inhibitors and better understand the role of TG2 in cancer.

Effects of the regulatory ligands calcium and GTP on the thermal stability of tissue transglutaminase.

Tissue transglutaminase undergoes thermal inactivation with first-order kinetics at moderate temperatures, in a process which is affected in opposite way by the regulatory ligands calcium and GTP, which stabilize different conformations. We have explored the processes of inactivation and of unfolding of transglutaminase and the effects of ligands thereon, combining approaches of differential scanning calorimetry (DSC) and of thermal analysis coupled to fluorescence spectroscopy and small angle scattering. At low temperature (38-45°C), calcium promotes and GTP protects from inactivation, which occurs without detectable disruption of the protein structure but only local perturbations at the active site. Only at higher temperatures (52-56°C), the protein structure undergoes major rearrangements with alterations in the interactions between the N- and C-terminal domain pairs. Experiments by DSC and fluorescence spectroscopy clearly indicate reinforced and weakened interactions of the domains in the presence of GTP and of calcium, and different patterns of unfolding. Small angle scattering experiments confirm different pathways of unfolding, with attainment of limiting values of gyration radius of 52, 60 and 90 Å in the absence of ligands and in the presence of GTP and calcium. Data by X-rays scattering indicate that ligands influence retention of a relatively compact structure in the protein even after denaturation at 70°C. These results suggest that the complex regulation of the enzyme by ligands involves both short- and long-range effects which might be relevant for understanding the turnover of the protein in vivo.

Machine Learning Algorithms Highlight tRNA Information Content and Chargaff's Second Parity Rule Score as Important Features in Discriminating Probiotics from Non-Probiotics.

Probiotic bacteria are microorganisms with beneficial effects on human health and are currently used in numerous food supplements. However, no selection process is able to effectively distinguish probiotics from non-probiotic organisms on the basis of their genomic characteristics. In the current study, four Machine Learning algorithms were employed to accurately identify probiotic bacteria based on their DNA characteristics. Although the prediction accuracies of all algorithms were excellent, the Neural Network returned the highest scores in all the evaluation metrics, managing to discriminate probiotics from non-probiotics with an accuracy greater than 90%. Interestingly, our analysis also highlighted the information content of the tRNA sequences as the most important feature in distinguishing the two groups of organisms probably because tRNAs have regulatory functions and might have allowed probiotics to evolve faster in the human gut environment. Through the methodology presented here, it was also possible to identify seven promising new probiotics that have a higher information content in their tRNA sequences compared to non-probiotics. In conclusion, we prove for the first time that Machine Learning methods can discriminate human probiotic from non-probiotic organisms underlining information within tRNA sequences as the most important genomic feature in distinguishing them.

Dysregulation of Transglutaminase type 2 through GATA3 defines aggressiveness and Doxorubicin sensitivity in breast cancer.

The role of transglutaminase type 2 in cell physiology is related to protein transamidation and signal transduction (affecting extracellular, intracellular and nuclear processes) aided by the expression of truncated isoforms and of two lncRNAs with regulatory functions. In breast cancer TG2 is associated with disease progression supporting motility, epithelial-mesenchymal transition, invasion and drug resistance. The aim of his work is to clarify these issues by emphasizing the interconnections among TGM2 variants and transcription factors associated with an aggressive phenotype, in which the truncated TGH isoform correlates with malignancy. TGM2 transcripts are upregulated by several drugs in MCF-7, but only Doxorubicin is effective in MDA-MB-231 cells. These differences reflect the expression of GATA3, as demonstrated by silencing, suggesting a link between this transcription factor and gene dysregulation. Of note, NC9, an irreversible inhibitor of enzymatic TG2 activities, emerges to control NF-ĸB and apoptosis in breast cancer cell lines, showing potential for combination therapies with Doxorubicin.

A Multidisciplinary Approach Establishes a Link between Transglutaminase 2 and the Kv10.1 Voltage-Dependent K+ Channel in Breast Cancer.

Since the multifunctionality of transglutaminase 2 (TG2) includes extra- and intracellular functions, we investigated the effects of intracellular administration of TG2 inhibitors in three breast cancer cell lines, MDA-MB-231, MDA-MB-436 and MDA-MB-468, which are representative of different triple-negative phenotypes, using a patch-clamp technique. The first cell line has a highly voltage-dependent a membrane current, which is low in the second and almost absent in the third one. While applying a voltage protocol to responsive single cells, injection of TG2 inhibitors triggered a significant decrease of the current in MDA-MB-231 that we attributed to voltage-dependent K+ channels using the specific inhibitors 4-aminopyridine and astemizole. Since the Kv10.1 channel plays a dominant role as a marker of cell migration and survival in breast cancer, we investigated its relationship with TG2 by immunoprecipitation. Our data reveal their physical interaction affects membrane currents in MDA-MB-231 but not in the less sensitive MDA-MB-436 cells. We further correlated the efficacy of TG2 inhibition with metabolic changes in the supernatants of treated cells, resulting in increased concentration of methyl- and dimethylamines, representing possible response markers. In conclusion, our findings highlight the interference of TG2 inhibitors with the Kv10.1 channel as a potential therapeutic tool depending on the specific features of cancer cells.

The Motility and Mesenchymal Features of Breast Cancer Cells Correlate with the Levels and Intracellular Localization of Transglutaminase Type 2.

We have investigated motility in breast cancer cell lines in association with the expression of Transglutaminase type 2 (TG2) as well as upon the administration of Doxorubicin (Dox), an active cytotoxic agent that is employed in chemotherapy. The exposure of MCF-7 cells to the drug increased TG2 levels, triggering epithelial-mesenchymal transition (EMT), thereby supporting cell motility. The effects of Dox on the movement of MCF-7 cells were counteracted by treatment with NC9, a TG2 inhibitor, which induced morphological changes and also reduced the migration of MDA-MB-231 cells exhibiting high levels of TG2. The physical association of TG2 with the cytoskeletal component vimentin appeared pivotal both in drug-treated MCF-7 and in MDA-MB-231 cells and seemed to be independent of the catalytic activity of TG2. NC9 altered the subcellular distribution of TG2 and, consequently, the co-localization of TG2 with vimentin. Furthermore, NC9 induced a nuclear accumulation of TG2 as a prelude to TG2-dependent gene expression modifications. Since enzyme activity can affect both motility and nuclear functions, targeting of this protein could represent a method to improve therapeutic interventions in breast tumors, particularly those to control progression and to limit drug resistance.

Inhibition of the lncRNA Coded within Transglutaminase 2 Gene Impacts Several Relevant Networks in MCF-7 Breast Cancer Cells.

Long non-coding RNAs are nucleotide molecules that regulate transcription in numerous cellular processes and are related to the occurrence of many diseases, including cancer. In this regard, we recently discovered a polyadenylated long non-coding RNA (named TG2-lncRNA) encoded within the first intron of the Transglutaminase type 2 gene (TGM2), which is related to tumour proliferation in human cancer cell lines. To better characterize this new biological player, we investigated the effects of its suppression in MCF-7 breast cancer cells, using siRNA treatment and RNA-sequencing. In this way, we found modifications in several networks associated to biological functions relevant for tumorigenesis (apoptosis, chronic inflammation, angiogenesis, immunomodulation, cell mobility, and epithelial-mesenchymal transition) that were originally attributed only to Transglutaminase type 2 protein but that could be regulated also by TG2-lncRNA. Moreover, our experiments strongly suggest the ability of TG2-lncRNA to directly interact with important transcription factors, such as RXRα and TP53, paving the way for several regulatory loops that can potentially influence the phenotypic behaviour of MCF-7 cells. These considerations imply the need to further investigate the relative relevance of the TG2 protein itself and/or other gene products as key regulators in the organization of breast cancer program.