Paola Mirra

In Skeletal Muscle Advanced Glycation End Products (AGEs) Inhibit Insulin Action and Induce the Formation of Multimolecular Complexes Including the Receptor for AGEs

Chronic hyperglycemia promotes insulin resistance at least in part by increasing the formation of advanced glycation end products (AGEs). We have previously shown that in L6 myotubes human glycated albumin (HGA) induces insulin resistance by activating protein kinase Cα (PKCα). Here we show that HGA-induced PKCα activation is mediated by Src. Coprecipitation experiments showed that Src interacts with both the receptor for AGE (RAGE) and PKCα in HGA-treated L6 cells. A direct interaction of PKCα with Src and insulin receptor substrate-1 (IRS-1) has also been detected. In addition, silencing of IRS-1 expression abolished HGA-induced RAGE-PKCα co-precipitation. AGEs were able to induce insulin resistance also in vivo, as insulin tolerance tests revealed a significant impairment of insulin sensitivity in C57/BL6 mice fed a high AGEs diet (HAD). In tibialis muscle of HAD-fed mice, insulin-induced glucose uptake and protein kinase B phosphorylation were reduced. This was paralleled by a 2.5-fold increase in PKCα activity. Similarly to in vitro observations, Src phosphorylation was increased in tibialis muscle of HAD-fed mice, and co-precipitation experiments showed that Src interacts with both RAGE and PKCα. These results indicate that AGEs impairment of insulin action in the muscle might be mediated by the formation of a multimolecular complex including RAGE/IRS-1/Src and PKCα.

Molecular Cloning and Characterization of the Human PED/PEA-15 Gene Promoter Reveal Antagonistic Regulation by Hepatocyte Nuclear Factor 4α and Chicken Ovalbumin Upstream Promoter Transcription Factor II

Overexpression of the ped/pea-15 gene in mice impairs glucose tolerance and leads to diabetes in conjunction with high fat diet treatment. PED/PEA-15 is also overexpressed in type 2 diabetics as well as in euglycemic offspring from these subjects. The cause(s) of this abnormality remains unclear. In the present work we have cloned and localized the promoter region of the human PED/PEA-15 gene within the first 230 bp of the 5®-flanking region. A cis-acting regulatory element located between -320 and -335 bps upstream the PED/PEA-15 gene transcriptional start site (+1) is recognized by both the hepatocyte nuclear factor 4α (HNF-4α) and the chicken ovalbumin upstream promoter transcription factor II (COUP-TFII), two members of the steroid/thyroid superfamily of transcription factors, both of which are involved in the control of lipid and glucose homeostasis. HNF-4α represses PED/PEA-15 expression in HeLa cells, whereas COUP-TFII activates its expression. In hepatocytes, the activation of PED/PEA-15 gene transcription is paralleled by the establishment of a partially dedifferentiated phenotype accompanied by a reduction in mRNA levels encoded by genes normally expressed during liver development. Cotransfection of HeLa cells with a reporter construct containing the PED/PEA-15 response element and various combinations of HNF-4α and COUP-TFII expression vectors indicated that COUP-TFII antagonizes the repression of the PED/PEA-15 gene by HNF-4α. Thus, at least in part, transcription of the PED/PEA-15 gene in vivo is dependent upon the intracellular balance of these positive and negative regulatory factors. Abnormalities in HNF-4α and COUP-TFII balance might have important consequences on glucose tolerance in humans.

Understanding type 2 diabetes: from genetics to epigenetics

The known genetic variability (common DNA polymorphisms) does not account either for the current epidemics of type 2 diabetes or for the family transmission of this disorder. However, clinical, epidemiological and, more recently, experimental evidence indicates that environmental factors have an extraordinary impact on the natural history of type 2 diabetes. Some of these environmental hits are often shared in family groups and proved to be capable to induce epigenetic changes which alter the function of genes affecting major diabetes traits. Thus, epigenetic mechanisms may explain the environmental origin as well as the familial aggregation of type 2 diabetes much easier than common polymorphisms. In the murine model, exposure of parents to environmental hits known to cause epigenetic changes reprograms insulin sensitivity as well as beta-cell function in the progeny, indicating that certain epigenetic changes can be transgenerationally transmitted. Studies from different laboratories revealed that, in humans, lifestyle intervention modulates the epigenome and reverts environmentally induced epigenetic modifications at specific target genes. Finally, specific human epigenotypes have been identified which predict adiposity and type 2 diabetes with much greater power than any polymorphism so far identified. These epigenotypes can be recognized in easily accessible white cells from peripheral blood, indicating that, in the future, epigenetic profiling may enable effective type 2 diabetes prediction. This review discusses recent evidence from the literature supporting the immediate need for further investigation to uncover the power of epigenetics in the prediction, prevention and treatment of type 2 diabetes.

Identification and Functional Characterization of a Novel Mutation in the NKX2-1 Gene: Comparison with the Data in the Literature

BACKGROUND NKX2-1 mutations have been described in several patients with primary congenital hypothyroidism, respiratory distress, and benign hereditary chorea, which are classical manifestations of the brain-thyroid-lung syndrome (BTLS). METHODS The NKX2-1 gene was sequenced in the members of a Brazilian family with clinical features of BTLS, and a novel monoallelic mutation was identified in the affected patients. We introduced the mutation in an expression vector for the functional characterization by transfection experiments using both thyroidal and lung-specific promoters. RESULTS The mutation is a deletion of a cytosine at position 834 (ref. sequence NM_003317) (c.493delC) that causes a frameshift with formation of an abnormal protein from amino acid 165 and a premature stop at position 196. The last amino acid of the nuclear localization signal, the whole homeodomain, and the carboxy-terminus of NKX2-1 are all missing in the mutant protein, which has a premature stop codon at position 196 (p.Arg165Glyfs*32). The p.Arg165Glyfs*32 mutant does not bind DNA, and it is unable to transactivate the thyroglobulin (Tg) and the surfactant protein-C (SP-C) promoters. Interestingly, a dose-dependent dominant negative effect of the p.Arg165Glyfs*32 was demonstrated only on the Tg promoter, but not on the SP-C promoter. This effect was also noticed when the mutation was tested in presence of PAX8 or cofactors that synergize with NKX2-1 (P300 and TAZ). The functional effect was also compared with the data present in the literature and demonstrated that, so far, it is very difficult to establish a specific correlation among NKX2-1 mutations, their functional consequence, and the clinical phenotype of affected patients, thus suggesting that the detailed mechanisms of transcriptional regulation still remain unclear. CONCLUSIONS We describe a novel NKX2-1 mutation and demonstrate that haploinsufficiency may not be the only explanation for BTLS. Our results indicate that NKX2-1 activity is also finely regulated in a tissue-specific manner, and additional studies are required to better understand the complexities of genotype-phenotype correlations in the NKX2-1 deficiency syndrome.

GRP78 Mediates Cell Growth and Invasiveness in Endometrial Cancer

Recent studies have indicated that endoplasmic reticulum stress, the unfolded protein response activation and altered GRP78 expression can play an important role in a variety of tumors development and progression. Very recently we reported for the first time that GRP78 is increased in endometrial tumors. However, whether GRP78 could play a role in the growth and/or invasiveness of endometrial cancer cells is still unknown. Here we report that the silencing of GRP78 expression affects both cell growth and invasiveness of Ishikawa and AN3CA cells, analyzed by the (3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide) and transwell migration assay, respectively. At variance with Ishikawa cells, AN3CA cells showed, besides an endoplasmic reticulum, also a plasma membrane GRP78 localization, evidenced by both immunofluorescence and cell membrane biotinylation experiments. Intriguingly, flow cytometry experiments showed that the treatment with a specific antibody targeting GRP78 C‐terminal domain caused apoptosis in AN3CA but not in Ishikawa cells. Induction of apoptosis in AN3CA cells was not mediated by the p53 pathway activation but was rather associated to reduced AKT phosphorylation. Interestingly, immunofluorescence analysis evidenced that endometrioid adenocarcinoma tissues displayed, similarly to AN3CA cells, also a GRP78 plasma membrane localization. These data suggest that GRP78 and its plasma membrane localization, might play a role in endometrial cancer development and progression and might constitute a novel target for the treatment of endometrial cancer. J. Cell. Physiol. 229: 1417–1426, 2014.

Regulatory T cells, inflammation, and endoplasmic reticulum stress in women with defective endometrial receptivity

OBJECTIVE To investigate immunologic parameters and endoplasmic reticulum (ER) stress associated with unexplained infertility. DESIGN Case-control study. SETTING Academic center. PATIENT(S) Women with no fertility problems (FS) (n = 13), women with recurrent miscarriage (RM) (n = 15) and women with repeated in vitro fertilization failure (RIF) (n = 15). INTERVENTION(S) Endometrial biopsy and collection of peripheral blood during the midsecretory phase of menstrual cycle. MAIN OUTCOME MEASURE(S) Leptin, resistin, soluble tumor necrosis factor receptor (sTNF-R), myeloperoxidase (MPO), soluble intercellular adhesion molecule 1 (sICAM-1), and interleukin 22 (IL-22) concentration in peripheral blood, endometrial CD3(+), CD4(+), CD5(+), CD8(+), and FoxP3(+) T lymphocytes, and endometrial expression of HSPA5, a specific marker of ER stress. RESULT(S) We found an increase of proinflammatory molecules such as resistin, leptin, and IL-22 in both RM and RIF patients; sTNF-R and MPO only in RIF patients when compared with the FS women. We also found in endometria of infertile women a statistically significant increase of CD3(+), CD4(+), CD8(+) in both RM and RIF patients and CD5(+) in RM patients when compared with FS women. This was paralleled by a statistically significant reduction of infiltrating FoxP3(+) regulatory T cells. Finally, endometrial HSPA5 expression levels were statistically significantly up-regulated in both RM and RIF patients. CONCLUSION(S) Women with RM and RIF showed an increase of circulating proinflammatory cytokines, altered endometrial T lymphocytes subsets, and signs of endometrial ER stress.

Circulating miRNAs as intercellular messengers, potential biomarkers and therapeutic targets for Type 2 diabetes

miRNAs have emerged as key epigenetic regulators of metabolism. Their deregulation contributes to metabolic abnormalities, proposing their potential role as therapeutic targets for Type 2 diabetes. The exciting finding that miRNAs exist in the bloodstream suggests that circulating miRNAs may act in a hormone-like fashion. Despite the fact that significant progress has been made in understanding circulating miRNAs, this topic is full of complexities and many questions remain unanswered. The goal of this review is to bring together up-to-date knowledge about circulating miRNAs and their role as intercellular communicators as well as potential biomarkers and therapeutic targets in metabolic diseases, providing examples of possible clinical applications for circulating miRNAs in diabetes and cardiovascular complications.

Hepatocyte nuclear factor (HNF)-4α-driven epigenetic silencing of the human PED gene

Aims/hypothesisOverexpression of PED (also known as PEA15) determines insulin resistance and impaired insulin secretion and may contribute to progression toward type 2 diabetes. Recently, we found that the transcription factor hepatocyte nuclear factor (HNF)-4α binds to PED promoter and represses its transcription. However, the molecular details responsible for regulation of PED gene remain unclear.MethodsHere we used gain and loss of function approaches to investigate the hypothesis that HNF-4α controls chromatin remodelling at the PED promoter in human cell lines.ResultsHNF-4α production and binding induce chromatin remodelling at the −250 to 50 region of PED, indicating that remodelling is limited to two nucleosomes located at the proximal promoter. Chromatin immunoprecipitation assays also revealed concomitant HNF-4α-induced deacetylation of histone H3 at Lys9 and Lys14, and increased dimethylation of histone H3 at Lys9. The latter was followed by reduction of histone H3 Lys4 dimethylation. HNF-4α was also shown to target the histone deacetylase complex associated with silencing mediator of retinoic acid and thyroid hormone receptor, both at the PED promoter, and at GRB14 and USP21 regulatory regions, leading to a reduction of mRNA levels. Moreover, HNF-4α silencing and PED overexpression were accompanied by a significant reduction of hepatic glycogen content.Conclusions/interpretationThese results show that HNF-4α serves as a scaffold protein for histone deacetylase activities, thereby inhibiting liver expression of genes including PED. Dysregulation of these mechanisms may lead to upregulation of the PED gene in type 2 diabetes.

Peroxisome proliferator-activated receptor-γ activation enhances insulin-stimulated glucose disposal by reducing ped/pea-15 gene expression in skeletal muscle cells: evidence for involvement of activator protein-1.

Background: PPARγ modulation of glucoregulatory response in skeletal muscle has been only partially elucidated. Results: PPARγ inhibits the transcription of the diabetes-associated gene ped/pea-15 via AP-1. Conclusion: ped/pea-15 is downstream of a PPARγ-regulated inflammatory network. Significance: These studies further elucidate the gene network responsible for inflammation-induced insulin resistance. The gene network responsible for inflammation-induced insulin resistance remains enigmatic. In this study, we show that, in L6 cells, rosiglitazone- as well as pioglitazone-dependent activation of peroxisome proliferator-activated receptor-γ (PPARγ) represses transcription of the ped/pea-15 gene, whose increased activity impairs glucose tolerance in mice and humans. Rosiglitazone enhanced insulin-induced glucose uptake in L6 cells expressing the endogenous ped/pea-15 gene but not in cells expressing ped/pea-15 under the control of an exogenous promoter. The ability of PPARγ to affect ped/pea-15 expression was also lost in cells and in C57BL/6J transgenic mice expressing ped/pea-15 under the control of an exogenous promoter, suggesting that ped/pea-15 repression may contribute to rosiglitazone action on glucose disposal. Indeed, high fat diet mice showed insulin resistance and increased ped/pea-15 levels, although these effects were reduced by rosiglitazone treatment. Both supershift and ChIP assays revealed the presence of the AP-1 component c-JUN at the PED/PEA-15 promoter upon 12-O-tetradecanoylphorbol-13-acetate stimulation of the cells. In these experiments, rosiglitazone treatment reduced c-JUN presence at the PED/PEA-15 promoter. This effect was not associated with a decrease in c-JUN expression. In addition, c-jun silencing in L6 cells lowered ped/pea-15 expression and caused nonresponsiveness to rosiglitazone, although c-jun overexpression enhanced the binding to the ped/pea-15 promoter and blocked the rosiglitazone effect. These results indicate that PPARγ regulates ped/pea-15 transcription by inhibiting c-JUN binding at the ped/pea-15 promoter. Thus, ped/pea-15 is downstream of a major PPARγ-regulated inflammatory network. Repression of ped/pea-15 transcription might contribute to the PPARγ regulation of muscle sensitivity to insulin.

The SGK1 inhibitor SI113 induces autophagy, apoptosis, and endoplasmic reticulum stress in endometrial cancer cells

Endometrial cancer is often characterized by PI3K/AKT pathway deregulation. Recently it has been suggested that SGK1, a serine/threonine protein kinase that shares structural and functional similarities with the AKT family, might play a role in cancer, since its expression and/or activity has been found to be deregulated in different human tumors. However, the role of SGK1 in endometrial cancer has been poorly investigated. Here, we show that SGK1 expression is increased in tissue specimens from neoplastic endometrium. The SGK1 inhibitor SI113 induced a significant reduction of endometrial cancer cells viability, measured by the (3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide assay. This effect was associated to the increase of autophagy, as revealed by the increase of the markers LC3B‐II and beclin I, detected by both immunofluorescence and western blot analysis. SI113 treatment caused also apoptosis of endometrial cancer cells, evidenced by the cleavage of the apoptotic markers PARP and Caspase‐9. Intriguingly, these effects were associated to the induction of endoplasmic reticulum stress markers GRP78 and CHOP evaluated by both Real‐Time RT‐PCR and Western Blot analysis. Increased expression of SGK1 in endometrial cancer tissues suggest a role for SGK1 in this type of cancer, as reported for other malignancies. Moreover, the efficacy of SI113 in affecting endometrial cancer cells viability, possibly via endoplasmic reticulum stress activation, identifies SGK1 as an attractive molecular target for new tailored therapeutic intervention for the treatment of endometrial cancer.