Browsing by Author "Bellido, T"

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    Direct cell-to-cell interactions between osteocytes and multiple myeloma (MM) cells up-regulate Sost and down-regulate OPG expression in osteocytes: evidence for osteocytic contributions to MM-induced bone disease
    (Office of the Vice Chancellor for Research, 2014-04-11) Delgado-Calle, J; Bellido, T; Roodman, GD
    Osteocytes are the most abundant bone cells, comprising more than 95% of the cells in bone. They are embedded into the bone matrix, but extensively communicate among themselves and with cells on the bone surface and the bone marrow through the osteocytic lacunar-canalicular network. Osteocytes secrete sclerostin, the product of the Sost gene, an antagonist of Wnt signaling that potently inhibits bone formation. Osteocytes are also a major source of pro- and anti-osteoclastogenic cytokines that regulate osteoclastogenesis and bone resorption, including RANKL and osteoprotegerin (OPG). Recent evidence suggests that the bone remodeling compartment is disrupted in multiple myeloma (MM) allowing close contact of MM cells with bone cells including osteocytes. However, the consequences of these interactions and the contribution of osteocytes to MM bone disease are unclear. Therefore, we determined if interactions between MM cells and osteocytes regulate osteocytic gene expression. We found that co-culture of murine MLO-A5 osteocytic cells with human JJN3 MM cells up-regulated murine Sost mRNA expression 2-3 fold as early as 4h, which remained elevated up to 24h. Consistent with Sost up-regulation induced by MM cells, the expression of OPG, a Wnt target gene, was decreased by 30-50% in MLO-A5 cells, resulting in an increased RANKL/OPG at 4h. Culture of JJN3 cells in the top and MLO-A5 cells in the bottom of Boyden chambers abolished both up-regulation of Sost and down-regulation of OPG mRNA expression in osteocytic cells, demonstrating the requirement of direct contact between MM cells and osteocytic cells. Human Sost and OPG mRNA transcripts were not detected in any of these experiments, demonstrating lack of contribution of MM JJN3 cells. These findings demonstrate that direct interactions between osteocytes and MM cells up-regulate the expression of the bone formation inhibitor Sost in osteocytes, which in turn decreases Wnt signaling, reduces osteocytic OPG expression increasing the RANKL/OPG ratio. We propose that increased Sost/Sclerostin expression contributes to the exacerbated bone resorption and the decreased bone formation that characterizes MM induced bone disease.
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    Prevention of glucocorticoid induced-apoptosis of osteoblasts and osteocytes by protecting against endoplasmic reticulum (ER) stress
    (Office of the Vice Chancellor for Research, 2014-04-11) Sato, A; Plotkin, L; Bellido, T
    Increased oxidative stress, such as with excess of glucocorticoids (GC) or during aging, has been associated with endoplasmic reticulum (ER) stress, due to accumulation of misfolded or unfolded proteins, leading to cellular apoptosis. The double-stranded RNA-activated protein kinase-like ER kinase (PERK) is activated to alleviate ER stress and phosphorylates the eukaryotic translation initiation factor 2 alpha subunit (eIF2α). Phosphorylated eIF2α in turn inhibits global protein translation to provide time to the ER to recover from the unfolded protein load, promoting cell viability. We hypothesized that the pro-apoptotic effect of GC on osteoblasts and osteocytes are at least in part due to induction of ER stress. To test this hypothesis, we used MLO-Y4 osteocytic cells, OB-6 osteoblastic cells, and primary osteoblastic cells derived from neonatal murine calvaria. We found that the synthetic GC dexamethasone (DEX) significantly increased the percentage of apoptotic cells in cultures of MLO-Y4, OB-6, and primary osteoblastic cells. Similarly, the specific ER-stress inducing agents brefeldin A, an inhibitor of ER-golgi apparatus vesicle transport, and tunicamycin, a protein glycosylation inhibitor, significantly increased OB-6 cell apoptosis. We then tested the effect of salubrinal, an agent that protects against ER stress by inhibiting the dephosphorylation of eIF2α, on bone cell apoptosis. Salubrinal blocked apoptosis induced by the ER stressors brefeldin A and tunicamycin in OB-6 cells. Salubrinal was also effective in blocking apoptosis induced by DEX in MLO-Y4, OB-6 and primary osteoblastic cells. Optimal responses were found at 10 μM salubrinal, after either 6 or 24 h. Guanabenz, another inhibitor of eIF2α dephosphorylation, also blocked DEX and tunicamycin-induced apoptosis of primary osteoblastic cells. Furthermore, addition of DEX to mineralizing OB-6 or primary osteoblastic cells markedly decreased mineral deposition and hydroxyapatite formation. In contrast, treatment with guanabenz increased mineralization of OB-6 cell cultures and prevented the inhibitory effect of DEX. We conclude that part of the pro-apoptotic actions of GC on osteoblastic cells are mediated through ER stress and that interventions that prevent dephosphorylation of eIF2α could potentially prevent the deleterious effects of GC on bone.