Avian leukosis virus (ALV)-induced osteopetrosis is a proliferative disorder of the bone affecting the growth and differentiation of osteoblasts. Osteopetrosis is a polyclonal disease in which cells of the bone contain, on average, multiple viral DNA copies. Osteopetrotic bone is also characterized by the accumulation of unintegrated viral DNA, suggesting an atypical life cycle of the virus in the infected osteoblasts. To better understand virus-host interactions in the induction of osteopetrosis by ALVs, infected chick osteoblast cultures and osteopetrotic bone were examined for aspects of the virus life cycle and effects of infection on osteoblast function. Levels of infection and virus expression were compared in cultured osteoblasts and osteopetrotic bone. Osteopetrotic bone contained higher levels of viral DNA and correspondingly higher levels of viral proteins than infected osteoblast cultures, suggesting a higher viral load in the diseased bone. A significant level of mature Gag protein was present in the bone, suggesting the accumulation of mature virus particles in the diseased bone. It is possible that the accumulation of virus could facilitate the high levels of infection observed in the diseased bone. The mechanism by which unintegrated viral DNA persisted in osteopetrotic bone was investigated by examining the susceptibility of infected osteoblasts to superinfection. The results indicated that, in culture, infected osteoblasts were able to establish interference to superinfection. This suggests that the persistence of unintegrated viral DNA in osteopetrotic bone may not result from the continuing infection of productively infected osteoblasts. The effect of virus infection on osteoblast function was examined in the diseased bone and in osteoblast cultures. In infected chickens, osteoblast activity, as evidenced by the expression of osteoblast phenotypic markers, was increased only in chickens developing severe osteopetrosis. In culture, virus infection had no apparent effect on either the proliferation or differentiation of osteoblasts. This indicates that infection was itself not sufficient to perturb osteoblast function. Furthermore, it suggested that additional components of the bone may be required for ALV infection to induce the abnormal activity of osteoblasts observed in osteopetrosis.

Indiana University-Purdue University Indianapolis (IUPUI) / Pyk2: Potential Regulator of Post-Menopausal Bone Loss H.W. LARGURA1,2*, P. ELENISTE2, S. HUANG2, S. LIU1, M. ALLEN3, A. BRUZZANITI2. 1Indiana University School of Dentistry Department Orthodontics and Oral Facial Development, 2Indiana University School of Dentistry Department of Oral Biology, 3Indiana University School of Medicine Department of Anatomy and Cell Biology, Indianapolis, Indiana, USA Osteoporosis is a pathologic condition of bone, commonly found in post-menopausal women, which occurs from an imbalance between bone formation and resorption. Following menopause, the bone resorbing activity of osteoclasts exceeds bone formation by osteoblasts, resulting in decreased trabecular and cortical bone and a subsequent decrease in bone mass. Reduced bone mass increases the risk of pathologic fracture of bones. Due to adverse effects associated with current treatment protocols for bone loss, alternative treatment modalities with reduced adverse effects are needed. Estrogen plays a role in maintaining balance in the bone remodeling cycle by controlling remodeling activation, osteoblast and osteoclast numbers, and their respective effectiveness in formation and resorption. With declining estrogen levels, this elegantly balanced interaction is altered and bone resorption exceeds bone formation, resulting in bone loss and increased bone fragility. Pyk2 is a protein tyrosine kinase that plays an important role in regulating bone resorption by osteoclasts, as well as osteoblast proliferation and differentiation. Deletion of the Pyk2 gene in mice leads to an increase in bone mass, in part due to dysfunctional osteoclast and osteoblast activity. In this study, we examined the role of Pyk2 in the effects of estrogen on bone mass. We used wild type (WT) and Pyk2 knock-out (KO) mice that had been ovariectomized (OVX) and treated with or without estrogen (E2)-releasing pellets. Control mice included sham OVX surgery receiving placebo pellet. We found that deletion of Pyk2 conferred increased bone mass in sham, OVX and OVX+E2 mice. In addition, Pyk2 KO mice supplemented with 17estradiol exhibited a marked increase in bone volume/trabecular volume, trabecular number, and trabecular thickness, but not cortical bone parameters compared to WT mice. Results of this study provide evidence for the role of Pyk2 in the effects of estrogen on bone mass. Understanding the role of Pyk2 in bone could lead to the development of new pharmaceutical targets for the treatment of bone loss associated with osteoporosis.

L’osteopetrosi autosomale recessiva (ARO) è un gruppo di malattie dovute a un difettoso funzionamento degli osteoclasti che preclude un rimodellamento osseo corretto. Nel 50% dei casi umani il difetto è dovuto ad una delezione nel gene Tcirg1. Il modello murino mutante oc/oc porta lo stesso difetto genetico e fenotipico umano. Nel lavoro di tesi si è dimostrato che gli epatociti fetali di 12.5 giorni di gestazione trapiantati in utero in feti mutati di 13.5 giorni di gestazione sono in grado di curare il fenotipo malato. Si è inoltre derivata una sottolinea di cellule staminali embrionali murine transgeniche per il costrutto plasmidico GOF18eGFP. Si vuole utilizzare la GFP sotto il controllo del promotore del gene Oct-4 come marcatore del livello di staminalità cellulare per microiniettare le ESC in blastocisti murine mutate oc/oc. / Autosomal recessive osteopetrosis (ARO) is a group of genetic disorders due to defects that preclude normal function of osteoclasts. In half the cases, human ARO is due to mutations in the Tcirg1 gene. The oc/oc mutant mouse closely recapitulates human Tcirg1-dependent ARO. In ths work we demonstrate that in utero injection of allogenic fetal liver cells on 12.5 days into oc/oc fetuses at 13.5 day post coitum completely rescue the osteopetrotic phenotype. Moreover, an embryonic stem cells line transgenic for GOF18eGFP was produced. The goal is to use the GFP under the transcriptional control of the Oct-4 promoter as a marker of pluripotency of the ESC that are to microinject into oc/oc blastocysts.

L’ostéopétrose est une pathologie osseuse caractérisée par des os denses et fragiles principalement due à l’incapacité des ostéoclastes, cellules d’origine hématopoïétique, à résorber le tissu osseux. La forme la plus sévère de cette maladie génétique est l’ostéopétrose autosomale récessive infantile due à une mutation du gène Ostm1 (Protéine transmembranaire de type 1 associée à l'ostéopétrose). Le gène Ostm1 est exprimé principalement dans la lignée des cellules hématopoïétiques, mais aussi dans le système nerveux central et les mélanocytes. Cette mutation développe plusieurs symptômes comme l’apparition d’une couleur de pelage gris chez la souris, une anémie sévère, une sensibilité aux infections et des troubles neuronaux chez l’homme et la souris. Afin de mieux comprendre cette maladie, nous avons généré des souris transgéniques sur un fond génétique grey-lethal (gl) dans lesquelles l’expression d’Ostm1 est ciblée à un tissu spécifique. Nous avons caractérisé le gène Ostm1 responsable de la mutation ostéopétrotique spontanée gl chez la souris. La complémentation fonctionnelle des défauts hématopoïétiques a été obtenue dans les souris transgéniques PU.1-Ostm1-gl/gl mais ces souris meurent prématurément avec une neurodegénérescence sévère. Cette perte cellulaire affecte le système nerveux central dans son ensemble incluant la rétine. Ce mémoire porte sur le but d’établir le profil d’expression du gène Ostm1 dans la rétine puisque la perte du gène entraine une dégénérescence rétinienne. Pour définir le rôle d’Ostm1 dans la rétine, nous avons caractérisé son expression dans ce tissu (organe). Des analyses PCR, démontrent une expression d’Ostm1 dans l’œil total et enrichie dans la neurorétine et dans l’épithélium pigmentée (RPE). Après avoir caractérisé avec des marqueurs protéiques spécifiques les sous populations cellulaires de la rétine, in situ hybridation détecte l’expression préférentielle d’Ostm1 dans l’épithélium pigmentée (RPE) et la couche nucléaire interne (INL). Basé sur ce profil d’expression, nous avons induit dans un premier temps la perte de fonction d’Ostm1 spécifiquement dans le RPE. Dans un premier temps nous avons vérifié que l’expression de la recombinasse Cre seule n’est pas toxique. Nous avons ensuite induit la perte d’expression d’Ostm1 dans ces cellules et démontré que la perte d’Ostm1 dans le RPE se traduit par une perte graduelle des photorécepteurs avec l’âge. Ces résultats préliminaires suggèrent que l’expression post-natale d’Ostm1 dans le RPE est essentielle au maintien de l’homéostasie des photorécepteurs dans la rétine. / Osteopetrosis is a disease characterized by high bone density and fragility principally caused by impaired activity of osteoclasts, which are cells that reside in bone and dissolve bone tissue. The most severe form of osteopetrosis is infantile autosomal recessive osteopetrosis (ARO) which is caused by mutations in genes Ostm1. As Ostm1(osteopetrosis-associated transmembrane protein 1) is expressed in multiple hematopoietic stem cell lineages, melanocytes and the nervous system, mutations in Ostm1 can cause coat color change in mice as well as bone fragility, anemia, infections and neuronal disorders in humans and mice. To further the understanding of these conditions linked with Ostm1 loss, multiple tissue specific Ostm1 transgenic mice over an Ostm1 knockout (gl/gl) background were constructed. To better understand this disease, we characterized the Ostm1 gene responsible for the spontaneous osteopetrotic mutation grey- lethal (gl) in mice. Functional complementation of hematopoietic defects was obtained in PU.1-Ostm1-gl/gl transgenic mice, but these mice die prematurely with severe neurodegeneration. This indicates that Ostm1 has a crucial role in neuronal and retinal health. As a result, we wished to establish an expression profile of Ostm1 in all the layers of the retina to further decipher the role of Ostm1 in the retina. Polymerase chain reaction (PCR) of reverse-transcribed mRNA of separated sections of the eye demonstrate that Ostm1 is expressed in the whole eye, neuroretina and retinal pigmented epithelium (RPE). Further specific expression analyses were performed by in- situ hybridization which showed that Ostm1 is expressed specifically in the inner nuclear layer of the neuroretina as well as in the RPE. Based on this tissue expression pattern, we have constructed, for the first time, an RPE specific knockdown of Ostm1 expression and verified that the expression of Cre recombinase in this tissue is not toxic. The reduction of Ostm1 in the RPE of the eye resulted in gradual loss of photoreceptors of the retina. These preliminary results suggest that the post-natal expression of Ostm1 in the RPE is essential for maintaining the homeostasis of the photoreceptors of the retina.