Browsing by Author "Plotkin, Lillian I."

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    Investigation of the Mechanism of the Gene Regulation of OPG (Osteoprotegerin) by Cx43
    (Office of the Vice Chancellor for Research, 2013-04-05) Hassan, Iraj; Pacheco-Costa, Rafael; Plotkin, Lillian I.
    The main objective is to determine whether the gene regulation of OPG, osteoprotegerin, by Cx43, is at the promoter level. In a recent project, it was found that deletion in Cx43 from osteocytes resulted in a decreased OPG expression. Furthermore, it was found that deletion of Cx43 from osteocytes resulted in enhanced osteoclast differentiation. Osteoprotegerin (OPG), an osteoblast-secreted decoy receptor that modulates osteoclast formation could be directly controlled by Cx43 at the promoter binding sites of p53 and Sp1. Cx43 and OPG in turn are widely up regulated by Wnt, lipid-modified signaling proteins that influence cell proliferation, differentiation, and survival. The activation of Wnt signaling results in the binding of the transcription factor Tcf in gene promoters, which leads to increased gene expression. The investigation was carried out using reporter constructs in which the activation of the promoter resulted in the transcription of the enzyme luciferase. Luciferase activity, in turn, can be measured using a commercially available substrate that emits luminescence when luciferase is present. OPG-Luc and Tcf-Luc were grown in E. coli and purified using a kit from Qiagen. Transfection of OPG 1 and Tcf-Luc reporter constructs on MLO-Y4 osteocyte cells deleted for Cx43 (Cx43shRNA) and Cx37 (Cx37shRNA) was conducted after seeding of the cells a day in advance. For each cell line, regular and Lithium Chloride (to mimic the effects of Wnt) induced medium was used, and cells were cultured for 24h. From the assay, it was deemed that luciferase activity was higher in Wnt induced cells. OPG is a target of Wnt signaling downstream of the transcription factor Tcf. We therefore also measure Tcf-mediated transcription using a Tcf-luciferase construct. Expression of OPG-Luc and Tcf-Luc was higher in cell lines that are not silenced for Cx43 and Cx37. According to ANOVA test, the results did reach statistical significance. However, future trials will be conducted to mimic the results.
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    Molecular Mechanisms Underlying Osteocyte Apoptosis and the Associated Osteoclastogenesis in CX43-Deficiency and Aging
    (2019-06) Davis, Hannah Marie; Plotkin, Lillian I.; Bidwell, Joseph P.; Allen, Matthew R.; Bruzzaniti, Angela
    Old age is associated with increased bone fragility and risk of fracture as a result of skeletal alterations, including low bone density and cortical thinning. Further, apoptotic osteocytes accumulate in old mice and humans. We have previously shown that mice lacking osteocytic connexin (Cx) 43 (Cx43ΔOt) exhibit a phenotype similar to that of the aging skeleton, with elevated osteocyte apoptosis and an associated increase in osteoclastogenesis. These findings suggest that osteocyte apoptosis results in the release of factors that recruit osteoclasts to bone surfaces close to areas that contain apoptotic osteocytes. However, the specific chemotactic signals, the events mediating their release, and the mechanisms of their action remain unknown. Consistent with this notion, we also found that HMGB1 released by Cx43-deficient (Cx43def) MLO-Y4 osteocytic cells enhances osteoclastogenesis in part by increasing osteocytic RANKL, which promotes osteoclastogenesis, and, at the same time, directly stimulating osteoclastogenesis. Further, expression of the pro-survival microRNA (miR), miR21, is low in Cx43def cells and bones from old female mice, and low miR21 levels increase osteocyte apoptosis. However, surprisingly, mice lacking miR21 (miR21ΔOt) have decreased osteoclast number and activity even under conditions of elevated osteocyte apoptosis; suggesting that osteocytic miR21 may mediate osteoclast precursor recruitment/survival induced by apoptotic osteocytes. However, whether HMGB1/miR21 are released by osteocytes, and if the HMGB1 receptors, receptor for advanced glycation end products (RAGE) and/or tolllike receptor (TLR4) are involved in osteoclast recruitment in Cx43ΔOt and old mice is unknown. The overall objectives of this series of studies were to elucidate the mechanisms
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    Neurodegeneration Risk Factor TREM2 R47H Mutation Causes Distinct Sex- and Age- Dependent Musculoskeletal Phenotype
    (2022-05) Essex, Alyson Lola; Plotkin, Lillian I.; Bonetto, Andrea; Allen, Matthew; Landreth, Gary E.
    Triggering Receptor Expressed on Myeloid Cells 2 (TREM2), a receptor expressed in myeloid cells including microglia in brain and osteoclasts in bone has been proposed as a link between brain and bone disease. Previous studies identified an AD-associated mutation (R47H) which is known to confer an increased risk for developing AD. In these studies, we used a heterozygous model of the TREM2 R47H variant (TREM2R47H/+), which does not exhibit cognitive defects, as a translational model of genetic risk factors that contribute to AD, and investigated whether alterations to TREM2 signaling could also contribute to bone and skeletal muscle loss, independently of central nervous system defects. Our study found that female TREM2R47H/+ animals experience bone loss in the femoral mid-diaphysis between 4 and 13 months of age as measured by microCT, which stalls out by 20 months of age. Female TREM2R47H/+ animals also experience significant decreases in the mechanical and material properties of the femur measured by three-point bending at 13 months of age, but not at 4 or 20 months. Interestingly, male TREM2R47H/+ animals do not demonstrate any discernable differences in bone geometry or strength until 20 months of age, where we observed slight changes in the bone volume and material properties of male TREM2R47H/+ bones. Ex vivo osteoclast differentiation assays demonstrate that only male TREM2R47H/+ osteoclasts differentiate more after 7 days with osteoclast differentiation factors compared to WT, but qPCR follow-up showed sexdependent differences in intracellular signaling. However, bone is not the only musculoskeletal tissue affected by the TREM2 R47H variant. Skeletal muscle strength measured by both in vivo plantar flexion and ex vivo contractility of the soleus is increased and body composition is altered in female TREM2R47H/+ mice compared to WT, and this is not likely due to bone-muscle crosstalk. These studies suggests that TREM2 R47H expression in the bone and skeletal muscle are likely impacting each tissue independently. These data demonstrate that AD-associated variants in TREM2 can alter bone and skeletal muscle strength in a sex-dimorphic manner independent of the presence of central neuropathology.