a rat model of supraspinatus overuse has suggested mechanisms governing tendon degeneration; howe... more a rat model of supraspinatus overuse has suggested mechanisms governing tendon degeneration; however, delineating which changes are pathologic or simply physiologic adaptations to increased loading remains a question. The objective of this study was to develop and characterize a rat exercise model that induces systemic and local shoulder adaptations without mechanical injury to the supraspinatus tendon. exercise rats completed a treadmill training protocol for 12 weeks. Body, fat pad, and heart weights were determined. Supraspinatus tendon collagen content, cross-sectional area, and mechanical properties were measured. Supraspinatus muscle cross-sectional area, weight, and the expression of mitochondrial oxidative phosphorylation (OXPHOS) proteins were measured. Humeri were analyzed with μCT and mechanically tested. exercise decreased fat pad mass. Supraspinatus muscle hypertrophied and had increased OXPHOS proteins. Humerus trabecular bone had increased anisotropic orientation, and...
Cyclin D1 is an important molecular driver of human breast cancer but better understanding of its... more Cyclin D1 is an important molecular driver of human breast cancer but better understanding of its oncogenic mechanisms is needed, especially to enhance efforts in targeted therapeutics. Currently, pharmaceutical initiatives to inhibit cyclin D1 are focused on the catalytic component since the transforming capacity is thought to reside in the cyclin D1/CDK activity. We initiated the following study to directly test the oncogenic potential of catalytically inactive cyclin D1 in an in vivo mouse model that is relevant to breast cancer. Herein, transduction of cyclin D1-/- mouse embryonic fibroblasts (MEFs) with the kinase dead KE mutant of cyclin D1 led to aneuploidy, abnormalities in mitotic spindle formation, autosome amplification, and chromosomal instability (CIN) by gene expression profiling. Acute transgenic expression of either cyclin D1WT or cyclin D1KE in the mammary gland was sufficient to induce a high CIN score within 7 days. Sustained expression of cyclin D1KE induced mamm...
ABSTRACT Cyclin D1 is an important molecular driver of human breast cancer but better understandi... more ABSTRACT Cyclin D1 is an important molecular driver of human breast cancer but better understanding of its oncogenic mechanisms is needed, especially to enhance efforts in targeted therapeutics. Currently, pharmaceutical initiatives to inhibit cyclin D1 are focused on the catalytic component since the transforming capacity is thought to reside in the cyclin D1/CDK activity. We initiated the following study to directly test the oncogenic potential of catalytically inactive cyclin D1 in an in vivo mouse model that is relevant to breast cancer. Herein, transduction of cyclin D1-/- mouse embryonic fibroblasts (MEFs) with the kinase dead KE mutant of cyclin D1 led to aneuploidy, abnormalities in mitotic spindle formation, autosome amplification, and chromosomal instability (CIN) by gene expression profiling. Acute transgenic expression of either cyclin D1WT or cyclin D1KE in the mammary gland was sufficient to induce a high CIN score within 7 days. Sustained expression of cyclin D1KE induced mammary adenocarcinoma with similar kinetics to that of the wild-type cyclin D1. ChIP-Seq studies demonstrated recruitment of cyclin D1WT and cyclin D1KE to the genes governing CIN. We conclude that the CDK-activating function of cyclin D1 is not necessary to induce either chromosomal instability or mammary tumorigenesis.
a rat model of supraspinatus overuse has suggested mechanisms governing tendon degeneration; howe... more a rat model of supraspinatus overuse has suggested mechanisms governing tendon degeneration; however, delineating which changes are pathologic or simply physiologic adaptations to increased loading remains a question. The objective of this study was to develop and characterize a rat exercise model that induces systemic and local shoulder adaptations without mechanical injury to the supraspinatus tendon. exercise rats completed a treadmill training protocol for 12 weeks. Body, fat pad, and heart weights were determined. Supraspinatus tendon collagen content, cross-sectional area, and mechanical properties were measured. Supraspinatus muscle cross-sectional area, weight, and the expression of mitochondrial oxidative phosphorylation (OXPHOS) proteins were measured. Humeri were analyzed with μCT and mechanically tested. exercise decreased fat pad mass. Supraspinatus muscle hypertrophied and had increased OXPHOS proteins. Humerus trabecular bone had increased anisotropic orientation, and...
The pathogenesis of Myotonic Dystrophy type 1 (DM1) is linked to unstable CTG repeats in the DMPK... more The pathogenesis of Myotonic Dystrophy type 1 (DM1) is linked to unstable CTG repeats in the DMPK gene which induce the mis-splicing to fetal/neonatal isoforms of many transcripts, including those involved in cellular Ca2+ homeostasis. Here we monitored the splicing of three genes encoding for Ca2+ transporters and channels (RyR1, SERCA1 and CACN1S) during maturation of primary DM1 muscle cells in parallel with the functionality of the Excitation-Contraction (EC) coupling machinery. At 15 days of differentiation, fetal isoforms of SERCA1 and CACN1S mRNA were significantly higher in DM1 myotubes compared to controls. Parallel functional studies showed that the cytosolic Ca2+ response to depolarization in DM1 myotubes did not increase during the progression of differentiation, in contrast to control myotubes. While we observed no differences in the size of intracellular Ca2+ stores, DM1 myotubes showed significantly reduced RyR1 protein levels, uncoupling between the segregated ER/SR Ca2+ store and the voltage-induced Ca2+ release machinery, parallel with induction of endoplasmic reticulum (ER) stress markers. In conclusion, our data suggest that perturbed Ca2+ homeostasis, via activation of ER stress, contributes to muscle degeneration in DM1 muscle cells likely representing a premature senescence phenotype.
Biased segregation of mitochondrial DNA variants has been widely documented, but little was known... more Biased segregation of mitochondrial DNA variants has been widely documented, but little was known about its molecular basis. We set out to test the hypothesis that altering the balance between mitochondrial fusion and fission could influence the segregation of mutant and wild-type mtDNA variants, because it would modify the number of organelles per cell. Therefore human cells heteroplasmic for the pathological A3243G mitochondrial DNA mutation were transfected with constructs designed to silence Drp1 or hFis1, whose gene products are required for mitochondrial fission. Drp1 and hFis1 gene silencing were both associated with increased levels of mutant mitochondrial DNA. Thus, the extent of the mitochondrial reticular network appears to be an important factor in determining mutant load. The fact that the level of mutant and wild-type mitochondrial DNA can be manipulated by altering the expression of nuclear encoded factors involved in mitochondrial fission suggests new interventions for mitochondrial DNA disorders.
Cyclin D1 overexpression is found in more than 50% of human breast cancers and causes mammary can... more Cyclin D1 overexpression is found in more than 50% of human breast cancers and causes mammary cancer in transgenic mice. Dysregulation of cyclin D1 gene expression or function contributes to the loss of normal cell cycle control during tumorigenesis. Recent studies have demonstrated that cyclin D1 conducts additional specific functions to regulate gene expression in the context of local chromatin, promote cellular migration and inhibit mitochondrial metabolism. It is anticipated that these additional functions contribute to the pathology associated with dysregulated cyclin D1 abundance. This article discusses evidence that examines the significance of cyclin D1 in breast cancer with emphasis on its role in breast cancer stem cell expansion.
a rat model of supraspinatus overuse has suggested mechanisms governing tendon degeneration; howe... more a rat model of supraspinatus overuse has suggested mechanisms governing tendon degeneration; however, delineating which changes are pathologic or simply physiologic adaptations to increased loading remains a question. The objective of this study was to develop and characterize a rat exercise model that induces systemic and local shoulder adaptations without mechanical injury to the supraspinatus tendon. exercise rats completed a treadmill training protocol for 12 weeks. Body, fat pad, and heart weights were determined. Supraspinatus tendon collagen content, cross-sectional area, and mechanical properties were measured. Supraspinatus muscle cross-sectional area, weight, and the expression of mitochondrial oxidative phosphorylation (OXPHOS) proteins were measured. Humeri were analyzed with μCT and mechanically tested. exercise decreased fat pad mass. Supraspinatus muscle hypertrophied and had increased OXPHOS proteins. Humerus trabecular bone had increased anisotropic orientation, and...
Cyclin D1 is an important molecular driver of human breast cancer but better understanding of its... more Cyclin D1 is an important molecular driver of human breast cancer but better understanding of its oncogenic mechanisms is needed, especially to enhance efforts in targeted therapeutics. Currently, pharmaceutical initiatives to inhibit cyclin D1 are focused on the catalytic component since the transforming capacity is thought to reside in the cyclin D1/CDK activity. We initiated the following study to directly test the oncogenic potential of catalytically inactive cyclin D1 in an in vivo mouse model that is relevant to breast cancer. Herein, transduction of cyclin D1-/- mouse embryonic fibroblasts (MEFs) with the kinase dead KE mutant of cyclin D1 led to aneuploidy, abnormalities in mitotic spindle formation, autosome amplification, and chromosomal instability (CIN) by gene expression profiling. Acute transgenic expression of either cyclin D1WT or cyclin D1KE in the mammary gland was sufficient to induce a high CIN score within 7 days. Sustained expression of cyclin D1KE induced mamm...
ABSTRACT Cyclin D1 is an important molecular driver of human breast cancer but better understandi... more ABSTRACT Cyclin D1 is an important molecular driver of human breast cancer but better understanding of its oncogenic mechanisms is needed, especially to enhance efforts in targeted therapeutics. Currently, pharmaceutical initiatives to inhibit cyclin D1 are focused on the catalytic component since the transforming capacity is thought to reside in the cyclin D1/CDK activity. We initiated the following study to directly test the oncogenic potential of catalytically inactive cyclin D1 in an in vivo mouse model that is relevant to breast cancer. Herein, transduction of cyclin D1-/- mouse embryonic fibroblasts (MEFs) with the kinase dead KE mutant of cyclin D1 led to aneuploidy, abnormalities in mitotic spindle formation, autosome amplification, and chromosomal instability (CIN) by gene expression profiling. Acute transgenic expression of either cyclin D1WT or cyclin D1KE in the mammary gland was sufficient to induce a high CIN score within 7 days. Sustained expression of cyclin D1KE induced mammary adenocarcinoma with similar kinetics to that of the wild-type cyclin D1. ChIP-Seq studies demonstrated recruitment of cyclin D1WT and cyclin D1KE to the genes governing CIN. We conclude that the CDK-activating function of cyclin D1 is not necessary to induce either chromosomal instability or mammary tumorigenesis.
a rat model of supraspinatus overuse has suggested mechanisms governing tendon degeneration; howe... more a rat model of supraspinatus overuse has suggested mechanisms governing tendon degeneration; however, delineating which changes are pathologic or simply physiologic adaptations to increased loading remains a question. The objective of this study was to develop and characterize a rat exercise model that induces systemic and local shoulder adaptations without mechanical injury to the supraspinatus tendon. exercise rats completed a treadmill training protocol for 12 weeks. Body, fat pad, and heart weights were determined. Supraspinatus tendon collagen content, cross-sectional area, and mechanical properties were measured. Supraspinatus muscle cross-sectional area, weight, and the expression of mitochondrial oxidative phosphorylation (OXPHOS) proteins were measured. Humeri were analyzed with μCT and mechanically tested. exercise decreased fat pad mass. Supraspinatus muscle hypertrophied and had increased OXPHOS proteins. Humerus trabecular bone had increased anisotropic orientation, and...
The pathogenesis of Myotonic Dystrophy type 1 (DM1) is linked to unstable CTG repeats in the DMPK... more The pathogenesis of Myotonic Dystrophy type 1 (DM1) is linked to unstable CTG repeats in the DMPK gene which induce the mis-splicing to fetal/neonatal isoforms of many transcripts, including those involved in cellular Ca2+ homeostasis. Here we monitored the splicing of three genes encoding for Ca2+ transporters and channels (RyR1, SERCA1 and CACN1S) during maturation of primary DM1 muscle cells in parallel with the functionality of the Excitation-Contraction (EC) coupling machinery. At 15 days of differentiation, fetal isoforms of SERCA1 and CACN1S mRNA were significantly higher in DM1 myotubes compared to controls. Parallel functional studies showed that the cytosolic Ca2+ response to depolarization in DM1 myotubes did not increase during the progression of differentiation, in contrast to control myotubes. While we observed no differences in the size of intracellular Ca2+ stores, DM1 myotubes showed significantly reduced RyR1 protein levels, uncoupling between the segregated ER/SR Ca2+ store and the voltage-induced Ca2+ release machinery, parallel with induction of endoplasmic reticulum (ER) stress markers. In conclusion, our data suggest that perturbed Ca2+ homeostasis, via activation of ER stress, contributes to muscle degeneration in DM1 muscle cells likely representing a premature senescence phenotype.
Biased segregation of mitochondrial DNA variants has been widely documented, but little was known... more Biased segregation of mitochondrial DNA variants has been widely documented, but little was known about its molecular basis. We set out to test the hypothesis that altering the balance between mitochondrial fusion and fission could influence the segregation of mutant and wild-type mtDNA variants, because it would modify the number of organelles per cell. Therefore human cells heteroplasmic for the pathological A3243G mitochondrial DNA mutation were transfected with constructs designed to silence Drp1 or hFis1, whose gene products are required for mitochondrial fission. Drp1 and hFis1 gene silencing were both associated with increased levels of mutant mitochondrial DNA. Thus, the extent of the mitochondrial reticular network appears to be an important factor in determining mutant load. The fact that the level of mutant and wild-type mitochondrial DNA can be manipulated by altering the expression of nuclear encoded factors involved in mitochondrial fission suggests new interventions for mitochondrial DNA disorders.
Cyclin D1 overexpression is found in more than 50% of human breast cancers and causes mammary can... more Cyclin D1 overexpression is found in more than 50% of human breast cancers and causes mammary cancer in transgenic mice. Dysregulation of cyclin D1 gene expression or function contributes to the loss of normal cell cycle control during tumorigenesis. Recent studies have demonstrated that cyclin D1 conducts additional specific functions to regulate gene expression in the context of local chromatin, promote cellular migration and inhibit mitochondrial metabolism. It is anticipated that these additional functions contribute to the pathology associated with dysregulated cyclin D1 abundance. This article discusses evidence that examines the significance of cyclin D1 in breast cancer with emphasis on its role in breast cancer stem cell expansion.
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Papers by Emanuele Loro