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ItemAvenanthramides Prevent Osteoblast and Osteocyte Apoptosis and Induce Osteoclast Apoptosis in Vitro in an Nrf2-Independent Manner(MDPI, 2016-07-11) Pellegrini, Gretel G.; Morales, Cynthya C.; Wallace, Taylor C.; Plotkin, Lilian I.; Bellido, Teresita; Department of Anatomy & Cell Biology, IU School of MedicineOats contain unique bioactive compounds known as avenanthramides (AVAs) with antioxidant properties. AVAs might enhance the endogenous antioxidant cellular response by activation of the transcription factor Nrf2. Accumulation of reactive oxygen species plays a critical role in many chronic and degenerative diseases, including osteoporosis. In this disease, there is an imbalance between bone formation by osteoblasts and bone resorption by osteoclasts, which is accompanied by increased osteoblast/osteocyte apoptosis and decreased osteoclast apoptosis. We investigated the ability of the synthethic AVAs 2c, 2f and 2p, to 1-regulate gene expression in bone cells, 2-affect the viability of osteoblasts, osteocytes and osteoclasts, and the generation of osteoclasts from their precursors, and 3-examine the potential involvement of the transcription factor Nrf2 in these actions. All doses of AVA 2c and 1 and 5 µM dose of 2p up-regulated collagen 1A expression. Lower doses of AVAs up-regulated OPG (osteoprotegerin) in OB-6 osteoblastic cells, whereas 100 μM dose of 2f and all concentrations of 2c down-regulated RANKL gene expression in MLO-Y4 osteocytic cells. AVAs did not affect apoptosis of OB-6 osteoblastic cells or MLO-Y4 osteocytic cells; however, they prevented apoptosis induced by the DNA topoisomerase inhibitor etoposide, the glucocorticoid dexamethasone, and hydrogen peroxide. AVAs prevented apoptosis of both wild type (WT) and Nrf2 Knockout (KO) osteoblasts, demonstrating that AVAs-induced survival does not require Nrf2 expression. Further, KO osteoclast precursors produced more mature osteoclasts than WT; and KO cultures exhibited less apoptotic osteoclasts than WT cultures. Although AVAs did not affect WT osteoclasts, AVA 2p reversed the low apoptosis of KO osteoclasts. These in vitro results demonstrate that AVAs regulate, in part, the function of osteoblasts and osteocytes and prevent osteoblast/osteocyte apoptosis and increase osteoclast apoptosis; further, these regulatory actions are independent of Nrf2. ItemContribution of rankl regulation to bone resorption induced by PTH receptor activation in osteocytes(2012-10-19) Ben-awadh, Abdullah Nasser; Bellido, Teresita M.; Plotkin, Lilian I.; Allen, Matthew R.PTH increases osteoclasts by upregulating RANKL in cells of the osteoblastic lineage, but the precise differentiation stage of the PTH target cell remains undefined. Recent findings demonstrate that PTH regulates gene expression in osteocytes and that these cells are an important source of RANKL. We therefore investigated whether direct regulation of the RANKL gene by PTH in osteocytes is required to stimulate osteoclastic bone resorption. To address this question, we examined bone resorption and RANKL expression in transgenic mice in which PTH receptor signaling is activated only in osteocytes (DMP1-caPTHR1) crossed with mice lacking the distal control region regulated by PTH in the RANKL gene (DCR -/-). Longitudinal analysis of circulating C-terminal telopeptide (CTX) in male mice showed elevated resorption in growing mice that progressively decreased to plateau at 3-5 month of age. Resorption was significantly higher (~100%) in DMP1-caPTHR1 mice and non-significantly lower (15-30%) in DCR -/-mice, versus wild type littermates (WT) across all ages. CTX in compound DMP1-caPTHR1; DCR -/-mice was similar to DMP1-caPTHR1 mice at 1 and 2 months of age, but by 3 months of age, was significantly lower compared to DMP1-caPTHR1 mice (50% higher than WT), and by 5 months, it was undistinguishable from WT mice. Micro-CT analysis revealed lower tissue material density in the distal femur of DMP1-caPTHR1 mice, indicative of high remodeling, and this effect was partially corrected in compound vi mice. The increased resorption exhibited by DMP1-caPTHR1 mice was accompanied by elevated RANKL mRNA in bone at 1 and 5 months of age. RANKL expression levels displayed similar patterns to CTX levels in DMP1-caPTHR1; DCR -/-compound mice at 1 and 5 month of age. The same pattern of expression was observed for M-CSF. We conclude that resorption induced by PTH receptor signaling requires direct regulation of the RANKL gene in osteocytes, but this dependence is age specific. Whereas DCR-independent mechanisms involving gp130 cytokines or vitamin D 3 might operate in the growing skeleton, DCR-dependent, cAMP/PKA/CREB-activated mechanisms mediate resorption induced by PTH receptor signaling in the adult skeleton. ItemExpression and Function of the PRL Family of Protein Tyrosine Phosphatase(2012-12) Dumaual, Carmen Michelle; Stauffacher, Cynthia; Randall, Stephen Karl, 1953-; Malkova, Anna; Sandusky, George Earl, 1945-The PRL family of enzymes constitutes a unique class of protein tyrosine phosphatase, consisting of three highly homologous members (PRL-1, PRL-2, and PRL-3). Family member PRL-3 is highly expressed in a number of tumor types and has recently gained much interest as a potential prognostic indicator of increased disease aggressiveness and poor clinical outcome for multiple human cancers. PRL-1 and PRL-2 are also known to promote a malignant phenotype in vitro, however, prior to the present study, little was known about their expression in human normal or tumor tissues. In addition, the biological function of all three PRL enzymes remains elusive and the underlying mechanisms by which they exert their effects are poorly understood. The current project was undertaken to expand our knowledge surrounding the normal cellular function of the PRL enzymes, the signaling pathways in which they operate, and the roles they play in the progression of human disease. We first characterized the tissue distribution and cell-type specific localization of PRL-1 and PRL-2 transcripts in a variety of normal and diseased human tissues using in situ hybridization. In normal, adult human tissues we found that PRL-1 and PRL-2 messages were almost ubiquitously expressed. Only highly specialized cell types, such as fibrocartilage cells, the taste buds of the tongue, and select neural cells displayed little to no expression of either transcript. In almost every other tissue and cell type examined, PRL-2 was expressed strongly while PRL-1 expression levels were variable. Each transcript was widely expressed in both proliferating and quiescent cells indicating that different tissues or cell types may display a unique physiological response to these genes. In support of this idea, we found alterations of PRL-1 and PRL-2 transcript levels in tumor samples to be highly tissue-type specific. PRL-1 expression was significantly increased in 100% of hepatocellular and gastric carcinomas, but significantly decreased in 100% of ovarian, 80% of breast, and 75% of lung tumors as compared to matched normal tissues from the same subjects. Likewise, PRL-2 expression was significantly higher in 100% of hepatocellular carcinomas, yet significantly lower in 54% of kidney carcinomas compared to matched normal specimens. PRL-1 expression was found to be associated with tumor grade in the prostate, ovary, and uterus, with patient gender in the bladder, and with patient age in the brain and skeletal muscle. These results suggest an important, but pleiotropic role for PRL-1 and PRL-2 in both normal tissue function and in the neoplastic process. These molecules may have a tumor promoting effect in some tissue types, but inhibit tumor formation or growth in others. To further elucidate the signaling pathways in which the PRLs operate, we focused on PRL-1 and used microarray and microRNA gene expression profiling to examine the global molecular changes that occur in response to stable PRL-1 overexpression in HEK293 cells. This analysis led to identification of several molecules not previously associated with PRL signaling, but whose expression was significantly altered by exogenous PRL-1 expression. In particular, Filamin A, RhoGDIalpha, and SPARC are attractive targets for novel mediators of PRL-1 function. We also found that PRL-1 has the capacity to indirectly influence the expression of target genes through regulation of microRNA levels and we provide evidence supporting previous observations suggesting that PRL-1 promotes cell proliferation, survival, migration, invasion, and metastasis by influencing multi-functional molecules, such as the Rho GTPases, that have essential roles in regulation of the cell cycle, cytoskeletal reorganization, and transcription factor function. The combined results of these studies have expanded our current understanding of the expression and function of the PRL family of enzymes as well as of the role these important signaling molecules play in the progression of human disease. ItemSignaling Pathways Involved in Mechanical Stimulation and ECM Geometry in Bone Cells(2010-07-27T20:24:41Z) Jiang, Chang; Yokota, Hiroki, 1955-; Liu, Yunlong; Ji, JulieThe proliferation and differentiation of osteoblasts are influenced by mechanical and geometrical growth environments. A specific aim of my thesis was the elucidation of signaling pathways involved in mechanical stimulation and geometric alterations of the extracellular matrix (ECM). A pair of questions addressed herein was (a) Does mechanical stimulation modulate translational regulation through the phosphorylation of eukaryotic initiation factor 2 (eIF2)? (b) Do geometric alterations affect the phosphorylation patterns of mitogen-activated protein kinase (MAPK) signaling? My hypothesis was mechanical stress enhances the proliferation and survival of osteoblasts through the reduction in phosphorylation of eIF2, while 3-dimensional (3D) ECM stimulates differentiation of osteoblasts through the elevation of phosphorylation of p38 MAPK. First, mechanical stimulation reduced the phosphorylation of eIF2. Furthermore, flow pre-treatment reduced thapsigargin-induced cell mortality through suppression of phosphorylation of protein kinase RNA-like ER kinase (Perk). However, H2O2-driven cell mortality, which is not mediated by Perk, was not suppressed by mechanical stimulation. Second, in the ECM geometry study, the expression of the active (phosphorylated) form of p130Cas, focal adhesion kinase (FAK) and extracellular signal-regulated protein kinase (ERK) was reduced in cells grown in the 3D matrix. Conversely, phosphorylation of p38 MAPK was elevated in the 3D matrix and its up-regulation was linked to an increase in mRNA levels of dentin matrix protein 1 and bone sialoprotein. In summary, our observations suggest the pro-survival role of mechanical stimulation and the modulation of osteoblastic fates by ECM geometry. ItemTesting the reliability and selectivity of different bone-cell-specific Cre- expressing mouse models for studying bone cell metabolism(2015-05) Kambrath, Anuradha Valiya; Robling, Alexander G.; Goebl, Mark G.; Harrington, Maureen A.The Cre/loxP system is a tool for targeted recombination of DNA. For applying Cre recombinase-mediated genome modifications, there is a requirement for reliable, high-fidelity, and specific transgenic expression of the Cre recombinase. This study focuses on the reliability of different bone cell specific Cre models in the Cre/loxP system. In this study, DMP1-Cre transgenic mouse which has a transgene driven by DMP1 promotor that allows Cre-expression only in late stage osteoblasts and osteocytes was used. Ctsk-Cre mouse with a driven by Ctsk promoter was used so that only osteoclasts would undergo Cre-mediated recombination. E2A-Cre mouse where the Cre recombinase is driven by a global promoter E2A was also included in this study as a control line to test the Cre reporter line Ai9. Dmp1-Cre, Ctsk-Cre and E2A-Cre mice were crossed to the fluorescent Cre-reporter line—Ai9, which harbors a floxed stop codon, followed by the fluorophoremTomato, inserted into the Rosa26 locus. This construct is expected to give red fluorescence when it recombines with Cre-expressing mouse cells and no fluorescence in non-recombinant mouse cells. Double positive (Ai9+/Cre+) offspring selected by PCR were perfused, and 5mu-m thick section of bone and soft tissues were examined for red fluorescent expression. Cre positive cells were quantitated using ‘ImageJ’ software program. The DMP1-vi Cre mouse results showed significant expression in the targeted osteocytes and osteoblasts. In addition, skeletal muscle tissue also showed significant Cre- expression. Ctsk-Cre mice showed significant expression in targeted osteoclasts. But brain tissue was positive in Cre-expression. Bone-Cre mouse models are expected to express Cre recombinase only in their respective bone cells and they have been used for gene deletion studies in bone cells. However, this study has revealed that the bone cell specific Cre mouse models DMP1-Cre and Ctsk-Cre have unexpected expression in muscle and brain respectively. In order to use these models for targeted gene deletion in bone cells, further testing and studies have to be conducted.