Browsing by Author "Schipani, Ernestina"

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    ATF4 and HIF-1α in bone: an intriguing relationship
    (Wiley, 2013-09) Schipani, Ernestina; Mangiavini, Laura; Merceron, Christophe; Department of Medicine, IU School of Medicine
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    Control of bone mass and remodeling by PTH receptor signaling in osteocytes
    (2008-08-13) O'Brien, Charles A.; Plotkin, Lilian I.; Galli, Carlo; Goellner, Joseph J.; Gortazar, Arancha R.; Allen, Matthew R.; Robling, Alexander G.; Bouxsein, Mary; Schipani, Ernestina; Turner, Charles H.; Jilka, Robert L.; Weinstein, Robert S.; Manolagas, Stavros C.; Bellido, Teresita
    Osteocytes, former osteoblasts buried within bone, are thought to orchestrate skeletal adaptation to mechanical stimuli. However, it remains unknown whether hormones control skeletal homeostasis through actions on osteocytes. Parathyroid hormone (PTH) stimulates bone remodeling and may cause bone loss or bone gain depending on the balance between bone resorption and formation. Herein, we demonstrate that transgenic mice expressing a constitutively active PTH receptor exclusively in osteocytes exhibit increased bone mass and bone remodeling, as well as reduced expression of the osteocyte-derived Wnt antagonist sclerostin, increased Wnt signaling, increased osteoclast and osteoblast number, and decreased osteoblast apoptosis. Deletion of the Wnt co-receptor LDL related receptor 5 (LRP5) attenuates the high bone mass phenotype but not the increase in bone remodeling induced by the transgene. These findings demonstrate that PTH receptor signaling in osteocytes increases bone mass and the rate of bone remodeling through LRP5-dependent and -independent mechanisms, respectively.
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    Erythropoietin stimulates murine and human fibroblast growth factor-23, revealing novel roles for bone and bone marrow
    (Ferrata Storti Foundation, 2017-11) Clinkenbeard, Erica L.; Hanudel, Mark R.; Stayrook, Keith R.; Appaiah, Hitesh Nidumanda; Farrow, Emily G.; Cass, Taryn A.; Summers, Lelia J.; Ip, Colin S.; Hum, Julia M.; Thomas, Joseph C.; Ivan, Mircea; Richine, Briana M.; Chan, Rebecca J.; Clemens, Thomas L.; Schipani, Ernestina; Sabbagh, Yves; Xu, Linlin; Srour, Edward F.; Alvarez, Marta B.; Kacena, Melissa A.; Salusky, Isidro B.; Ganz, Tomas; Nemeth, Elizabeta; White, Kenneth E.; Medical and Molecular Genetics, School of Medicine
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    Loss of VHL in mesenchymal progenitors of the limb bud alters multiple steps of endochondral bone development
    (Elsevier, 2014-09-01) Mangiavini, Laura; Merceron, Christophe; Araldi, Elisa; Khatri, Richa; Gerard-O'Riley, Rita; Wilson, Tremika LeShan; Rankin, Erinn B.; Giaccia, Amato J.; Schipani, Ernestina; Department of Medicine, Division of General Internal Medicine, IU School of Medicine
    Adaptation to low oxygen tension (hypoxia) is a critical event during development. The transcription factors Hypoxia Inducible Factor-1α (HIF-1α) and HIF-2α are essential mediators of the homeostatic responses that allow hypoxic cells to survive and differentiate. Von Hippel Lindau protein (VHL) is the E3 ubiquitin ligase that targets HIFs to the proteasome for degradation in normoxia. We have previously demonstrated that the transcription factor HIF-1α is essential for survival and differentiation of growth plate chondrocytes, whereas HIF-2α is not necessary for fetal growth plate development. We have also shown that VHL is important for endochondral bone development, since loss of VHL in chondrocytes causes severe dwarfism. In this study, in order to expand our understanding of the role of VHL in chondrogenesis, we conditionally deleted VHL in mesenchymal progenitors of the limb bud, i.e. in cells not yet committed to the chondrocyte lineage. Deficiency of VHL in limb bud mesenchyme does not alter the timely differentiation of mesenchymal cells into chondrocytes. However, it causes structural collapse of the cartilaginous growth plate as a result of impaired proliferation, delayed terminal differentiation, and ectopic death of chondrocytes. This phenotype is associated to delayed replacement of cartilage by bone. Notably, loss of HIF-2α fully rescues the late formation of the bone marrow cavity in VHL mutant mice, though it does not affect any other detectable abnormality of the VHL mutant growth plates. Our findings demonstrate that VHL regulates bone morphogenesis as its loss considerably alters size, shape and overall development of the skeletal elements.
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    Oxygen-sensing PHDs regulate bone homeostasis through the modulation of osteoprotegerin
    (CSH Press, 2015-04-15) Wu, Colleen; Rankin, Erinn B.; Castellini, Laura; Fernandez-Alcudia, Javier; LaGory, Edward L.; Andersen, Rebecca; Rhodes, Steven D.; Wilson, Tremika L.S.; Mohammad, Khalid S.; Castillo, Alesha B.; Guise, Theresa; Schipani, Ernestina; Giaccia, Amato J.; Department of Medicine, IU School of Medicine
    The bone microenvironment is composed of niches that house cells across variable oxygen tensions. However, the contribution of oxygen gradients in regulating bone and blood homeostasis remains unknown. Here, we generated mice with either single or combined genetic inactivation of the critical oxygen-sensing prolyl hydroxylase (PHD) enzymes (PHD1–3) in osteoprogenitors. Hypoxia-inducible factor (HIF) activation associated with Phd2 and Phd3 inactivation drove bone accumulation by modulating osteoblastic/osteoclastic cross-talk through the direct regulation of osteoprotegerin (OPG). In contrast, combined inactivation of Phd1, Phd2, and Phd3 resulted in extreme HIF signaling, leading to polycythemia and excessive bone accumulation by overstimulating angiogenic–osteogenic coupling. Wealso demonstrate that genetic ablation of Phd2 and Phd3 was sufficient to protect ovariectomized mice against bone loss without disrupting hematopoietic homeostasis. Importantly,we identify OPG as a HIF target gene capable of directing osteoblast-mediated osteoclastogenesis to regulate bone homeostasis. Here, we show that coordinated activation of specific PHD isoforms fine-tunes the osteoblastic response to hypoxia, thereby directing two important aspects of bone physiology: cross-talk between osteoblasts and osteoclasts and angiogenic–osteogenic coupling.
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    Regulation of HIF1 under Hypoxia by APE1/Ref-1 Impacts CA9 Expression: Dual-Targeting in Patient-Derived 3D Pancreatic Cancer Models
    (Office of the Vice Chancellor for Research, 2016-04-08) Logsdon, Derek P.; Grimard, Michelle; Shahda, Safi; Zyromski, Nicholas; Schipani, Ernestina; Carta, Fabrizio; Supuran, Claudiu T.; Korc, Murray; Ivan, Mircea; Kelley, Mark R.; Fishel, Melissa L.
    Abstract Half of all patients diagnosed with pancreatic ductal adenocarcinoma (PDAC) die within a year despite extensive surgery and/or a highly aggressive chemotherapy regimen. Several mechanisms are proposed to play a role in PDAC therapeutic resistance, including reactive stroma and hypoxia. Hypoxia signaling creates a more aggressive phenotype with increased metastatic potential and impaired therapeutic efficacy. Carbonic anhydrase IX (CA9) functions as part of the cellular hypoxia response to regulate intracellular pH, promoting cell survival. Apurinic/Apyrimidinic Endonuclease-1-Reduction/oxidation Effector Factor 1 (APE1/Ref-1) is a multi-functional protein with two major activities: an endonuclease function in DNA base excision repair and a redox signaling function that reduces oxidized transcription factors, enabling them to bind to their DNA target sequences. APE1/Ref-1 regulates several transcription factors involved in survival mechanisms, tumor growth, and hypoxia signaling. We explored the mechanisms underlying PDAC cell responses to hypoxia and modulation of APE1/Ref-1 redox signaling control of hypoxia inducible factor 1 alpha (HIF1), a critical factor in hypoxiainduced CA9 transcription. We hypothesized that obstructing the HIF-CA9 axis at two points via APE1/Ref-1 inhibition and CA9 inhibition will result in enhanced PDAC cell killing under hypoxic conditions. Methods: We performed qRT-PCR and Western Blots to confirm changes in CA9 expression in PDAC cells following APE1/Ref-1 inhibition and hypoxia exposure. Proliferation assays were used to assess cell killing following inhibition of APE1/Ref-1 and CA9 under hypoxia. Ex vivo 3-Dimensional co-culture models including both tumor and CAFs were used to examine whether we could enhance the efficacy of APE1/Ref-1 and/or CA9 inhibition with a dual-targeting approach to kill tumor spheroids. Results: HIF1-mediated induction of CA9 is significantly diminished in PDAC cells following APE1/Ref-1 redox inhibition. Additionally, dual-targeting of APE1/Ref-1 and CA9 reduces PDAC tumor cell growth under hypoxic conditions and in 3D tumor co-cultures.
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    Regulation of HIF1α under Hypoxia by APE1/Ref-1 Impacts CA9 Expression: Dual Targeting in Patient-Derived 3D Pancreatic Cancer Models
    (AACR, 2016-11-01) Logsdon, Derek P.; Grimard, Michelle; Luo, Meihua; Shahda, Safi; Jiang, Yanlin; Tong, Yan; Yu, Zhangsheng; Zyromski, Nicholas; Schipani, Ernestina; Carta, Fabrizio; Supuran, Claudiu T.; Korc, Murray; Ivan, Mircea; Kelley, Mark R.; Fishel, Melissa L.; Department of Pediatrics, School of Medicine
    Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related mortality in the United States. Aggressive treatment regimens have not changed the disease course, and the median survival has just recently reached a year. Several mechanisms are proposed to play a role in PDAC therapeutic resistance, including hypoxia, which creates a more aggressive phenotype with increased metastatic potential and impaired therapeutic efficacy. AP Endonuclease-1/Redox Effector Factor 1 (APE1/Ref-1) is a multifunctional protein possessing a DNA repair function in base excision repair and the ability to reduce oxidized transcription factors, enabling them to bind to their DNA target sequences. APE1/Ref-1 regulates several transcription factors involved in survival mechanisms, tumor growth, and hypoxia signaling. Here, we explore the mechanisms underlying PDAC cell responses to hypoxia and modulation of APE1/Ref-1 redox signaling activity, which regulates the transcriptional activation of hypoxia-inducible factor 1 alpha (HIF1α). Carbonic anhydrase IX (CA9) is regulated by HIF1α and functions as a part of the cellular response to hypoxia to regulate intracellular pH, thereby promoting cell survival. We hypothesized that modulating APE1/Ref-1 function will block activation of downstream transcription factors, STAT3 and HIF1α, interfering with the hypoxia-induced gene expression. We demonstrate APE1/Ref-1 inhibition in patient-derived and established PDAC cells results in decreased HIF1α–mediated induction of CA9. Furthermore, an ex vivo three-dimensional tumor coculture model demonstrates dramatic enhancement of APE1/Ref-1–induced cell killing upon dual targeting of APE1/Ref-1 and CA9. Both APE1/Ref-1 and CA9 are under clinical development; therefore, these studies have the potential to direct novel PDAC therapeutic treatment.
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    Severe Extracellular Matrix Abnormalities and Chondrodysplasia in Mice Lacking Collagen Prolyl 4-Hydroxylase Isoenzyme II in Combination with a Reduced Amount of Isoenzyme I
    (American Society for Biochemistry and Molecular Biology, 2015-07-03) Aro, Ellinoora; Salo, Antti M.; Khatri, Richa; Finnilä, Mikko; Miinalainen, Ilkka; Sormunen, Raija; Pakkanen, Outi; Holster, Tiina; Soininen, Raija; Prein, Carina; Clausen-Schaumann, Hauke; Aszódi, Attila; Tuukkanen, Juha; Kivirikko, Kari I.; Schipani, Ernestina; Myllyharju, Johanna; Department of Anatomy & Cell Biology, IU School of Medicine
    Collagen prolyl 4-hydroxylases (C-P4H-I, C-P4H-II, and C-P4H-III) catalyze formation of 4-hydroxyproline residues required to form triple-helical collagen molecules. Vertebrate C-P4Hs are α2β2 tetramers differing in their catalytic α subunits. C-P4H-I is the major isoenzyme in most cells, and inactivation of its catalytic subunit (P4ha1(-/-)) leads to embryonic lethality in mouse, whereas P4ha1(+/-) mice have no abnormalities. To study the role of C-P4H-II, which predominates in chondrocytes, we generated P4ha2(-/-) mice. Surprisingly, they had no apparent phenotypic abnormalities. To assess possible functional complementarity, we established P4ha1(+/-);P4ha2(-/-) mice. They were smaller than their littermates, had moderate chondrodysplasia, and developed kyphosis. A transient inner cell death phenotype was detected in their developing growth plates. The columnar arrangement of proliferative chondrocytes was impaired, the amount of 4-hydroxyproline and the Tm of collagen II were reduced, and the extracellular matrix was softer in the growth plates of newborn P4ha1(+/-);P4ha2(-/-) mice. No signs of uncompensated ER stress were detected in the mutant growth plate chondrocytes. Some of these defects were also found in P4ha2(-/-) mice, although in a much milder form. Our data show that C-P4H-I can to a large extent compensate for the lack of C-P4H-II in proper endochondral bone development, but their combined partial and complete inactivation, respectively, leads to biomechanically impaired extracellular matrix, moderate chondrodysplasia, and kyphosis. Our mouse data suggest that inactivating mutations in human P4HA2 are not likely to lead to skeletal disorders, and a simultaneous decrease in P4HA1 function would most probably be required to generate such a disease phenotype.