<b>Copyright information:</b>Taken from "Identification of a set of KSRP target ... more <b>Copyright information:</b>Taken from "Identification of a set of KSRP target transcripts upregulated by PI3K-AKT signaling"http://www.biomedcentral.com/1471-2199/8/28BMC Molecular Biology 2007;8():28-28.Published online 16 Apr 2007PMCID:PMC1858702. (A) S100 extracts from αT3-1 cells were subjected to gel filtration chromatography on a Superose 6 column. Aliquots of the eluted fractions were analyzed by Western blotting using the indicated antibodies. (B) RNase A-treated S100 extracts from αT3-1 cells were immunoprecipitated with preimmune (lane 2) or anti-KSRP (lane 3) sera and analyzed by immunoblotting with either anti-AUF1 (top) or anti-HnRNPA1 (bottom) antibodies. The arrows mark the position of either AUF1p45 or hnRNPA1, while the asterisk marks a anti-AUF1 cross-reacting band. (C) GST-pulldown of either endogenous AUF1p45 (top) or endogenous hnRNPA1 (bottom) from S100 extracts of αT3-1 cells using either control GST or GST-KSRP. Proteins were analyzed by immunoblotting using the indicated antibodies. The arrows mark the position of either AUF1p45 or hnRNPA1.
Research article KSRP-PMR1-exosome association determines parathyroid hormone mRNA levels and sta... more Research article KSRP-PMR1-exosome association determines parathyroid hormone mRNA levels and stability in transfected cells
Although mammals possess roughly the same number of protein-coding genes as worms, it is evident ... more Although mammals possess roughly the same number of protein-coding genes as worms, it is evident that the non-coding transcriptome content has become far broader and more sophisticated during evolution. Indeed, the vital regulatory importance of both short and long non-coding RNAs (lncRNAs) has been demonstrated during the last two decades. RNA binding proteins (RBPs) represent approximately 7.5% of all proteins and regulate the fate and function of a huge number of transcripts thus contributing to ensure cellular homeostasis. Transcriptomic and proteomic studies revealed that RBP-based complexes often include lncRNAs. This review will describe examples of how lncRNA-RBP networks can virtually control all the post-transcriptional events in the cell.
Long non-coding RNAs (lncRNAs) can affect multiple layers of gene expression to control crucial c... more Long non-coding RNAs (lncRNAs) can affect multiple layers of gene expression to control crucial cellular functions. We have previously demonstrated that the lncRNA EPR, by controlling gene expression at different levels, affects cell proliferation and migration in cultured mammary gland cells and impairs breast tumor formation in an orthotopic transplant model in mice. Here, we used ChIRP-Seq to identify EPR binding sites on chromatin of NMuMG mammary gland cells overexpressing EPR and identified its trans binding sites in the genome. Then, with the purpose of relating EPR/chromatin interactions to the reshaping of the epitranscriptome landscape, we profiled histone activation marks at promoter/enhancer regions by ChIP-Seq. Finally, we integrated data derived from ChIRP-Seq, ChIP-Seq as well as RNA-Seq in a comprehensive analysis and we selected a group of bona fide direct transcriptional targets of EPR. Among them, we identified a subset of EPR targets whose expression is controlle...
Long noncoding RNAs (lncRNAs) are emerging as regulators of fundamental biological processes. Her... more Long noncoding RNAs (lncRNAs) are emerging as regulators of fundamental biological processes. Here we report on the characterization of an intergenic lncRNA expressed in epithelial tissues which we termed EPR (Epithelial cell Program Regulator). EPR is rapidly downregulated by TGF-β and its sustained expression largely reshapes the transcriptome, favors the acquisition of epithelial traits, and reduces cell proliferation in cultured mammary gland cells as well as in an animal model of orthotopic transplantation. EPR generates a small peptide that localizes at epithelial cell junctions but the RNA molecule per se accounts for the vast majority of EPR-induced gene expression changes. Mechanistically, EPR interacts with chromatin and regulates Cdkn1a gene expression by affecting both its transcription and mRNA decay through its association with SMAD3 and the mRNA decay-promoting factor KHSRP, respectively. We propose that EPR enables epithelial cells to control proliferation by modulating waves of gene expression in response to TGF-β.
Resveratrol (RESV) is a natural polyphenolic compound endowed with anti-inflammatory, anti-prolif... more Resveratrol (RESV) is a natural polyphenolic compound endowed with anti-inflammatory, anti-proliferative, as well as pro-apoptotic activities that make it a potential anti-tumor compound. Here we show that RESV counteracts the TGF-β-induced Epithelial to Mesenchymal Transition (EMT) phenotype in mammary gland cells and affects the alternative exon usage of pre-mRNAs that encode crucial factors in adhesion and migration -including CD44, ENAH, and FGFR2- in a panel of immortalized and transformed mammary gland cells. RESV causes a shift from the mesenchymal-specific forms of these factors to the respective epithelial forms and increases the expression of the RNA-binding proteins KHSRP and hnRNPA1. From a mechanistic point of view, we show that the combined silencing of KHSRP and hnRNPA1 prevents the RESV-dependent inclusion of the epithelial-type exons in the Cd44 pre-mRNA. Our findings support an unexpected regulatory mechanism where RESV limits EMT by controlling gene expression at ...
The mechanism (both at the whole body and cellular level) by which metformin improves insulin sen... more The mechanism (both at the whole body and cellular level) by which metformin improves insulin sensitivity has yet to be defined. In the present study, we examined in vivo insulin-mediated whole-body glucose disposal, glycogen synthesis, hepatic glucose production, and insulin secretion, as well as in vitro muscle insulin receptor tyrosine kinase activity in eight control, eight neonatal streptozotocin diabetic rats, and eight diabetic rats before and after treatment with metformin. Ten weeks after birth diabetic rats had higher fasting (132 + 5 Y 101 + 2 mg/dL) and postmeal (231 + 10 v 133 + 3) plasma glucose levels compared with controls (P-C 901). Metformin treatment was followed by a significant decrease in the growth rate and normalized glucose tolerance without enhancing the deficient insulin response. Insulin-mediated glucose uptake in diabetic versus control rats was reduced (P-C .Ol) during the high-dose (15.4 + 0.6 v 18.3 + 1 .O mg/kg. min) insulin clamp study and was increased to values greater (P < .05I than controls following metformin treatment. Muscle glycogen synthetic rate in vivo, measured by incorporation of 3H-3-glucose radioactivity, was diminished by 25% (P < .Ol) in diabetic rats, restored to normal values with metformin, and correlated closely (r = .82, P < .002) with total-body glucose uptake during the insulin clamp in all three groups. Insulin receptor tyrosine kinase activity, measured in partially purified insulin receptors, was reduced in diabetic rats and increased to supernormal levels after metformin. The decrease in muscle tyrosine kinase activity in diabetic versus control animals was entirely accounted for by a reduction in maximal velocity (V,,,) (32 v 45 pmol/mgmin. P < .Ol) and increased to supernormal levels following metformin (91 pmol/mgmin. P < .OOl) without any change in affinity (Km). Muscle tyrosine kinase activity was closely correlated with both the muscle glycogen synthetic rate (r = .82, P < 602) and total-body insulin-mediated glucose disposal (r = 64, P < .Ol) in vivo. The close correlation between in vivo insulin action, muscle glycogen synthesis, and muscle insulin receptor tyrosine kinase activity is consistent with an important role of the enzyme in the insulin resistance of diabetes and its improvement following metformin treatment.
The effect of a polyclonal anti-insulin receptor antibody (pIgG) on the insulin receptor tyrosine... more The effect of a polyclonal anti-insulin receptor antibody (pIgG) on the insulin receptor tyrosine kinase (IRTK) activity toward poly-(Glu-Tyr) was examined using wheat germ agglutinin agarose-purified insulin receptors from rat liver membranes. The main effect of pIgG was a reduction of Vmax (from 60.8 to 31.8 pmol/min/mg), without changes of Km, when IRTK was activated by insulin. In contrast, when IRTK was activated by ATP preincubation, pIgG was unable to affect the reaction, suggesting that IRTK possesses at least two regulatory mechanisms, one of which can be affected by pIgG.
Insulin receptor beta-subunit autophosphorylation, the first event occurring after insulin bindin... more Insulin receptor beta-subunit autophosphorylation, the first event occurring after insulin binding, plays a crucial role in modulation of receptor-associated kinase activity towards exogenous substrates and possibly in the transmission of biological signals of insulin. Receptor autophosphorylation strongly depends on insulin receptor occupancy. Till now the effects of receptor phosphorylation on insulin binding itself have not been clarified. In the present report we demonstrate the absence of any feedback mechanism by which insulin receptor activation by phosphorylation affects binding affinity of insulin receptor itself.
Cell senescence produces changes in the expression of specific genes, suggesting that senescent c... more Cell senescence produces changes in the expression of specific genes, suggesting that senescent cells express an altered pattern of transcription factors. Here we describe how the DNA binding activity of the activator protein 1(AP-1) complex is altered in the fibroblasts of a geroderma osteodysplastica patient in response to extracellular stimuli.
Cultured neoplastic cell lines are widely considered an adequate model for insulin receptor studi... more Cultured neoplastic cell lines are widely considered an adequate model for insulin receptor studies. In this work the insulin receptor of a well-known continuous T-cell line (MOLT 4) was characterized. These cells showed specific insulin receptor with binding properties quite similar to those of other lymphoblastoid cells. Therefore, we used MOLT 4 receptor to evaluate the effect of the sulfonylurea glipizide on insulin binding. After a 24-hour preliminary incubation of cells with glipizide, we found a 44% increase of insulin receptor binding apparently due to an increase of insulin binding sites.
We have studied the ability of mature red cells to regulate the number and affinity of their insu... more We have studied the ability of mature red cells to regulate the number and affinity of their insulin receptors, in vitro. Our data show that mature red cells are not able to change either the number and the affinity of their insulin receptors, after preincubation with high concentrations of insulin alone or insulin and glucose. We conclude that mature red cells possess an insulin receptor system not completely similar to that of major target cells such as hepatocytes and adipocytes, and therefore we suggest some criticism in evaluating these cells in clinical studies, regarding the insulin receptor status.
In vitro the rate of synthesis of the aldiminic linkage between Hb and glucose depends on glucose... more In vitro the rate of synthesis of the aldiminic linkage between Hb and glucose depends on glucose concentration, length of incubation and some other physiological factors. To understand better the regulation of this synthesis and to verify the role of cell age and of basal HbA1 levels on the rate of synthesis of pre-A1, we studied red cells from 7 normal controls and 7 diabetics, with high HbA1 levels. We found that the content of HbA1 (stable glycosylated hemoglobin) is able to negatively affect the rate of synthesis of new pre-A1, according to a curvilinear model. These results suggest that in vitro the glycosylation process is saturable, and that elevated values of HbA1 are able to slow the synthesis of pre-A1 in vitro.
<b>Copyright information:</b>Taken from "Identification of a set of KSRP target ... more <b>Copyright information:</b>Taken from "Identification of a set of KSRP target transcripts upregulated by PI3K-AKT signaling"http://www.biomedcentral.com/1471-2199/8/28BMC Molecular Biology 2007;8():28-28.Published online 16 Apr 2007PMCID:PMC1858702. (A) S100 extracts from αT3-1 cells were subjected to gel filtration chromatography on a Superose 6 column. Aliquots of the eluted fractions were analyzed by Western blotting using the indicated antibodies. (B) RNase A-treated S100 extracts from αT3-1 cells were immunoprecipitated with preimmune (lane 2) or anti-KSRP (lane 3) sera and analyzed by immunoblotting with either anti-AUF1 (top) or anti-HnRNPA1 (bottom) antibodies. The arrows mark the position of either AUF1p45 or hnRNPA1, while the asterisk marks a anti-AUF1 cross-reacting band. (C) GST-pulldown of either endogenous AUF1p45 (top) or endogenous hnRNPA1 (bottom) from S100 extracts of αT3-1 cells using either control GST or GST-KSRP. Proteins were analyzed by immunoblotting using the indicated antibodies. The arrows mark the position of either AUF1p45 or hnRNPA1.
Research article KSRP-PMR1-exosome association determines parathyroid hormone mRNA levels and sta... more Research article KSRP-PMR1-exosome association determines parathyroid hormone mRNA levels and stability in transfected cells
Although mammals possess roughly the same number of protein-coding genes as worms, it is evident ... more Although mammals possess roughly the same number of protein-coding genes as worms, it is evident that the non-coding transcriptome content has become far broader and more sophisticated during evolution. Indeed, the vital regulatory importance of both short and long non-coding RNAs (lncRNAs) has been demonstrated during the last two decades. RNA binding proteins (RBPs) represent approximately 7.5% of all proteins and regulate the fate and function of a huge number of transcripts thus contributing to ensure cellular homeostasis. Transcriptomic and proteomic studies revealed that RBP-based complexes often include lncRNAs. This review will describe examples of how lncRNA-RBP networks can virtually control all the post-transcriptional events in the cell.
Long non-coding RNAs (lncRNAs) can affect multiple layers of gene expression to control crucial c... more Long non-coding RNAs (lncRNAs) can affect multiple layers of gene expression to control crucial cellular functions. We have previously demonstrated that the lncRNA EPR, by controlling gene expression at different levels, affects cell proliferation and migration in cultured mammary gland cells and impairs breast tumor formation in an orthotopic transplant model in mice. Here, we used ChIRP-Seq to identify EPR binding sites on chromatin of NMuMG mammary gland cells overexpressing EPR and identified its trans binding sites in the genome. Then, with the purpose of relating EPR/chromatin interactions to the reshaping of the epitranscriptome landscape, we profiled histone activation marks at promoter/enhancer regions by ChIP-Seq. Finally, we integrated data derived from ChIRP-Seq, ChIP-Seq as well as RNA-Seq in a comprehensive analysis and we selected a group of bona fide direct transcriptional targets of EPR. Among them, we identified a subset of EPR targets whose expression is controlle...
Long noncoding RNAs (lncRNAs) are emerging as regulators of fundamental biological processes. Her... more Long noncoding RNAs (lncRNAs) are emerging as regulators of fundamental biological processes. Here we report on the characterization of an intergenic lncRNA expressed in epithelial tissues which we termed EPR (Epithelial cell Program Regulator). EPR is rapidly downregulated by TGF-β and its sustained expression largely reshapes the transcriptome, favors the acquisition of epithelial traits, and reduces cell proliferation in cultured mammary gland cells as well as in an animal model of orthotopic transplantation. EPR generates a small peptide that localizes at epithelial cell junctions but the RNA molecule per se accounts for the vast majority of EPR-induced gene expression changes. Mechanistically, EPR interacts with chromatin and regulates Cdkn1a gene expression by affecting both its transcription and mRNA decay through its association with SMAD3 and the mRNA decay-promoting factor KHSRP, respectively. We propose that EPR enables epithelial cells to control proliferation by modulating waves of gene expression in response to TGF-β.
Resveratrol (RESV) is a natural polyphenolic compound endowed with anti-inflammatory, anti-prolif... more Resveratrol (RESV) is a natural polyphenolic compound endowed with anti-inflammatory, anti-proliferative, as well as pro-apoptotic activities that make it a potential anti-tumor compound. Here we show that RESV counteracts the TGF-β-induced Epithelial to Mesenchymal Transition (EMT) phenotype in mammary gland cells and affects the alternative exon usage of pre-mRNAs that encode crucial factors in adhesion and migration -including CD44, ENAH, and FGFR2- in a panel of immortalized and transformed mammary gland cells. RESV causes a shift from the mesenchymal-specific forms of these factors to the respective epithelial forms and increases the expression of the RNA-binding proteins KHSRP and hnRNPA1. From a mechanistic point of view, we show that the combined silencing of KHSRP and hnRNPA1 prevents the RESV-dependent inclusion of the epithelial-type exons in the Cd44 pre-mRNA. Our findings support an unexpected regulatory mechanism where RESV limits EMT by controlling gene expression at ...
The mechanism (both at the whole body and cellular level) by which metformin improves insulin sen... more The mechanism (both at the whole body and cellular level) by which metformin improves insulin sensitivity has yet to be defined. In the present study, we examined in vivo insulin-mediated whole-body glucose disposal, glycogen synthesis, hepatic glucose production, and insulin secretion, as well as in vitro muscle insulin receptor tyrosine kinase activity in eight control, eight neonatal streptozotocin diabetic rats, and eight diabetic rats before and after treatment with metformin. Ten weeks after birth diabetic rats had higher fasting (132 + 5 Y 101 + 2 mg/dL) and postmeal (231 + 10 v 133 + 3) plasma glucose levels compared with controls (P-C 901). Metformin treatment was followed by a significant decrease in the growth rate and normalized glucose tolerance without enhancing the deficient insulin response. Insulin-mediated glucose uptake in diabetic versus control rats was reduced (P-C .Ol) during the high-dose (15.4 + 0.6 v 18.3 + 1 .O mg/kg. min) insulin clamp study and was increased to values greater (P < .05I than controls following metformin treatment. Muscle glycogen synthetic rate in vivo, measured by incorporation of 3H-3-glucose radioactivity, was diminished by 25% (P < .Ol) in diabetic rats, restored to normal values with metformin, and correlated closely (r = .82, P < .002) with total-body glucose uptake during the insulin clamp in all three groups. Insulin receptor tyrosine kinase activity, measured in partially purified insulin receptors, was reduced in diabetic rats and increased to supernormal levels after metformin. The decrease in muscle tyrosine kinase activity in diabetic versus control animals was entirely accounted for by a reduction in maximal velocity (V,,,) (32 v 45 pmol/mgmin. P < .Ol) and increased to supernormal levels following metformin (91 pmol/mgmin. P < .OOl) without any change in affinity (Km). Muscle tyrosine kinase activity was closely correlated with both the muscle glycogen synthetic rate (r = .82, P < 602) and total-body insulin-mediated glucose disposal (r = 64, P < .Ol) in vivo. The close correlation between in vivo insulin action, muscle glycogen synthesis, and muscle insulin receptor tyrosine kinase activity is consistent with an important role of the enzyme in the insulin resistance of diabetes and its improvement following metformin treatment.
The effect of a polyclonal anti-insulin receptor antibody (pIgG) on the insulin receptor tyrosine... more The effect of a polyclonal anti-insulin receptor antibody (pIgG) on the insulin receptor tyrosine kinase (IRTK) activity toward poly-(Glu-Tyr) was examined using wheat germ agglutinin agarose-purified insulin receptors from rat liver membranes. The main effect of pIgG was a reduction of Vmax (from 60.8 to 31.8 pmol/min/mg), without changes of Km, when IRTK was activated by insulin. In contrast, when IRTK was activated by ATP preincubation, pIgG was unable to affect the reaction, suggesting that IRTK possesses at least two regulatory mechanisms, one of which can be affected by pIgG.
Insulin receptor beta-subunit autophosphorylation, the first event occurring after insulin bindin... more Insulin receptor beta-subunit autophosphorylation, the first event occurring after insulin binding, plays a crucial role in modulation of receptor-associated kinase activity towards exogenous substrates and possibly in the transmission of biological signals of insulin. Receptor autophosphorylation strongly depends on insulin receptor occupancy. Till now the effects of receptor phosphorylation on insulin binding itself have not been clarified. In the present report we demonstrate the absence of any feedback mechanism by which insulin receptor activation by phosphorylation affects binding affinity of insulin receptor itself.
Cell senescence produces changes in the expression of specific genes, suggesting that senescent c... more Cell senescence produces changes in the expression of specific genes, suggesting that senescent cells express an altered pattern of transcription factors. Here we describe how the DNA binding activity of the activator protein 1(AP-1) complex is altered in the fibroblasts of a geroderma osteodysplastica patient in response to extracellular stimuli.
Cultured neoplastic cell lines are widely considered an adequate model for insulin receptor studi... more Cultured neoplastic cell lines are widely considered an adequate model for insulin receptor studies. In this work the insulin receptor of a well-known continuous T-cell line (MOLT 4) was characterized. These cells showed specific insulin receptor with binding properties quite similar to those of other lymphoblastoid cells. Therefore, we used MOLT 4 receptor to evaluate the effect of the sulfonylurea glipizide on insulin binding. After a 24-hour preliminary incubation of cells with glipizide, we found a 44% increase of insulin receptor binding apparently due to an increase of insulin binding sites.
We have studied the ability of mature red cells to regulate the number and affinity of their insu... more We have studied the ability of mature red cells to regulate the number and affinity of their insulin receptors, in vitro. Our data show that mature red cells are not able to change either the number and the affinity of their insulin receptors, after preincubation with high concentrations of insulin alone or insulin and glucose. We conclude that mature red cells possess an insulin receptor system not completely similar to that of major target cells such as hepatocytes and adipocytes, and therefore we suggest some criticism in evaluating these cells in clinical studies, regarding the insulin receptor status.
In vitro the rate of synthesis of the aldiminic linkage between Hb and glucose depends on glucose... more In vitro the rate of synthesis of the aldiminic linkage between Hb and glucose depends on glucose concentration, length of incubation and some other physiological factors. To understand better the regulation of this synthesis and to verify the role of cell age and of basal HbA1 levels on the rate of synthesis of pre-A1, we studied red cells from 7 normal controls and 7 diabetics, with high HbA1 levels. We found that the content of HbA1 (stable glycosylated hemoglobin) is able to negatively affect the rate of synthesis of new pre-A1, according to a curvilinear model. These results suggest that in vitro the glycosylation process is saturable, and that elevated values of HbA1 are able to slow the synthesis of pre-A1 in vitro.
Uploads
Papers by Roberto Gherzi