Analysis of gene expression and correlation with clinical parameters has the potential to become an important factor in therapeutic decision making. The ability to analyze gene expression in archived tissues, for which clinical followup is already available, will greatly facilitate research in this area. A major obstacle to this approach, however, has been the uncertainty about whether gene expression analyses from routinely archived tissues accurately reflect expression before fixation. In the present study we have optimized the RNA isolation and reverse transcription steps for quantitative reverse transcription-polymerase chain reaction (RT-PCR) on archival material. Using tissue taken directly from the operating room, mRNAs with half-lives from 10 minutes to >8 hours were isolated and reverse transcribed. Subsequent real-time quantitative PCR methodology (TaqMan) on these cDNAs gives a measurement of gene expression in the fixed tissues comparable to that in the fresh tissue. In addition, we simulated routine pathology handling and demonstrate that this method of mRNA quantitation is insensitive to pre-fixation times (time from excision to fixation) of up to 12 hours. Therefore, it should be feasible to analyze gene expression in archived tissues where tissue collection procedures are largely unknown.

Recent progress in chemotherapy has significantly increased its efficacy, yet the development of chemoresistance remains a major drawback. In this study, we show that GFRA1/GFRa1 (GDNF family receptor a 1), contributes to cisplatin-induced chemoresistance by regulating autophagy in osteosarcoma. We demonstrate that cisplatin treatment induced GFRA1 expression in human osteosarcoma cells. Induction of GFRA1 expression reduced cisplatin-induced apoptotic cell death and it significantly increased osteosarcoma cell survival via autophagy. GFRA1 regulates AMPK-dependent autophagy by promoting SRC phosphorylation independent of proto-oncogene RET kinase. Cisplatin-resistant osteosarcoma cells showed NFKB1/NFkB-mediated GFRA1 expression. GFRA1 expression promoted tumor formation and growth in mouse xenograft models and inhibition of autophagy in a GFRA1-expressing xenograft mouse model during cisplatin treatment effectively reduced tumor growth and increased survival. In cisplatin-treated patients, treatment period and metastatic status were associated with GFRA1-mediated autophagy. These findings suggest that GFRA1-mediated autophagy is a promising novel target for overcoming cisplatin resistance in osteosarcoma.

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Estrogen receptor-related receptor ␥ (ERR␥/ERR3/NR3B3) is a member of the orphan nuclear receptor with important functions in development and homeostasis. Recently it has been reported that ERR␣ is involved in osteoblast differentiation and bone formation. In the present study we examined the role of ERR␥ in osteoblast differentiation. Here, we showed that ERR␥ is expressed in osteoblast progenitors and primary osteoblasts, and its expression is increased temporarily by BMP2. Overexpression of ERR␥ reduced BMP2-induced alkaline phosphatase activity and osteocalcin production as well as calcified nodule formation, whereas inhibition of ERR␥ expression significantly enhanced BMP2-induced osteogenic differentiation and mineralization, suggesting that endogenous ERR␥ plays an important role in osteoblast differentiation. In addition, ERR␥ significantly repressed Runx2 transactivity on osteocalcin and bone sialoprotein promoters. We also observed that ERR␥ physically interacts with Runx2 in vitro and in vivo and competes with p300 to repress Runx2 transactivity. Notably, intramuscular injection of ERR␥ strongly inhibited BMP2-induced ectopic bone formation in a dose-dependent manner. Taken together, these results suggest that ERR␥ is a novel negative regulator of osteoblast differentiation and bone formation via its regulation of Runx2 transactivity.Bone formation is a series of well orchestrated lineage-specific differentiation events (1). Osteoblasts, which play key roles in bone formation, are derived from pluripotent mesenchymal stem cells that have the capacity to differentiate into myocytes, adipocytes, and chondrocytes (2). Osteoblasts possess the necessary components to form bone matrix, which allows subsequent mineralization. Several hormones, growth factors, cytokines, and nuclear receptor proteins regulate these sequential events to trigger a complex network of signaling pathways.Bone morphogenetic proteins (BMPs) 5 are members of the transforming growth factor ␤ family and were originally identified by their capacity to induce ectopic bone formation (3, 4). Among the BMP family members, the action of BMP2 has been studied extensively in embryonic skeletal development, postnatal bone remodeling, and bone repair (5, 6). BMP2 promotes the commitment of pluripotent mesenchymal cells to the osteoblast lineage by regulating the signals that stimulate the specific transcriptional programs required for bone formation (5, 7).A master regulator of osteoblasts, Runx2, is indispensable for a skeletal development and maturation. Targeted disruption of Runx2 results in a complete lack of functional osteoblasts (8, 9). Runx2 directly regulates osteoblast-specific genes such as osteocalcin (OC), bone sialoprotein (BSP), osteopontin, and type I collagen through binding to specific DNA enhancer elements of its target gene promoters (4). In addition, Runx2 interacts with a variety of transcription factors (10) and recruits both co-activators (11-13) and co-repressors (14, 15) to form a complex on its target promoter. Theref...

Background: COUP-TFII, an orphan nuclear receptor, regulates the differentiation process in various cell types during development. Results: COUP-TFII inhibits Runx2-dependent osteocalcin transcription through physical interaction with Runx2 and matrix mineralization. Conclusion: COUP-TFII is a negative regulator of osteoblast differentiation. Significance: COUP-TFII has therapeutic potential for controlling bone-related disease.

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