IU Indianapolis ScholarWorks :: Browsing by Author "Greenfield, Edward M."

Browsing by Author "Greenfield, Edward M."

Now showing 1 - 7 of 7

The Crucial p53-Dependent Oncogenic Role of JAB1 in Osteosarcoma in vivo
(Nature, 2020-06) Samsa, William E.; Mamidi, Murali K.; Bashur, Lindsay A.; Elliott, Robin; Miron, Alexander; Chen, Yuqing; Lee, Brendan; Greenfield, Edward M.; Chan, Ricky; Danielpour, David; Zhou, Guang; Orthopaedic Surgery, School of Medicine
Osteosarcoma (OS) is the most common primary bone cancer and ranks amongst the leading causes of cancer mortality in young adults. Jun activation domain binding protein 1 (JAB1) is overexpressed in many cancers and has recently emerged as a novel target for cancer treatment. However, the role of JAB1 in osteosarcoma was virtually unknown. In this study, we demonstrate that JAB1-knockdown in malignant osteosarcoma cell lines significantly reduced their oncogenic properties, including proliferation, colony formation, and motility. We also performed RNA-sequencing analysis in JAB1-knockdown OS cells and identified 4110 genes that are significantly differentially expressed. This demonstrated for the first time that JAB1 regulates a large and specific transcriptome in cancer. We also found that JAB1 is overexpressed in human OS and correlates with a poor prognosis. Moreover, we generated a novel mouse model that overexpresses Jab1 specifically in osteoblasts upon a TP53 heterozygous sensitizing background. Interestingly, by 13 months of age, a significant proportion of these mice spontaneously developed conventional OS. Finally, we demonstrate that a novel, highly specific small molecule inhibitor of JAB1, CSN5i-3, reduces osteosarcoma cell viability and has specific effects on the ubiquitin-proteasome system in OS. Thus, we show for the first time that the overexpression of JAB1 in vivo can result in accelerated spontaneous tumor formation in a p53-dependent manner. In summary, JAB1 might be a unique target for the treatment of osteosarcoma and other cancers.
Halicin remains active against Staphylococcus aureus in biofilms grown on orthopaedically relevant substrates
(The British Editorial Society of Bone & Joint Surgery, 2024-03-04) Higashihira, Shota; Simpson, Stefanie J.; Morita, Akira; Suryavanshi, Joash R.; Arnold, Christopher J.; Natoli, Roman M.; Greenfield, Edward M.; Orthopaedic Surgery, School of Medicine
Aims: Biofilm infections are among the most challenging complications in orthopaedics, as bacteria within the biofilms are protected from the host immune system and many antibiotics. Halicin exhibits broad-spectrum activity against many planktonic bacteria, and previous studies have demonstrated that halicin is also effective against Staphylococcus aureus biofilms grown on polystyrene or polypropylene substrates. However, the effectiveness of many antibiotics can be substantially altered depending on which orthopaedically relevant substrates the biofilms grow. This study, therefore, evaluated the activity of halicin against less mature and more mature S. aureus biofilms grown on titanium alloy, cobalt-chrome, ultra-high molecular weight polyethylene (UHMWPE), devitalized muscle, or devitalized bone. Methods: S. aureus-Xen36 biofilms were grown on the various substrates for 24 hours or seven days. Biofilms were incubated with various concentrations of halicin or vancomycin and then allowed to recover without antibiotics. Minimal biofilm eradication concentrations (MBECs) were defined by CFU counting and resazurin reduction assays, and were compared with the planktonic minimal inhibitory concentrations (MICs). Results: Halicin continued to exert significantly (p < 0.01) more antibacterial activity against biofilms grown on all tested orthopaedically relevant substrates than vancomycin, an antibiotic known to be affected by biofilm maturity. For example, halicin MBECs against both less mature and more mature biofilms were ten-fold to 40-fold higher than its MIC. In contrast, vancomycin MBECs against the less mature biofilms were 50-fold to 200-fold higher than its MIC, and 100-fold to 400-fold higher against the more mature biofilms. Conclusion: Halicin is a promising antibiotic that should be tested in animal models of orthopaedic infection.
Lysosomal disruption by orthopedic wear particles induces activation of the NLRP3 inflammasome and macrophage cell death by distinct mechanisms
(Wiley, 2021) Fort, Brian P.; Dubyak, George R.; Greenfield, Edward M.; Orthopaedic Surgery, School of Medicine
Wear particles from orthopedic implants cause aseptic loosening, the leading cause of implant revisions. The particles are phagocytosed by macrophages leading to activation of the nod-like receptor protein 3 (NLRP3) inflammasome and release of interleukin-1β (IL-1β) which then contributes to osteoclast differentiation and implant loosening. The mechanism of inflammasome activation by orthopedic particles is undetermined but other particles cause the cytosolic accumulation of the lysosomal cathepsin-family proteases which can activate the NLRP3 inflammasome. Here, we demonstrate that lysosome membrane disruption causes cathepsin release into the cytoplasm that drives both inflammasome activation and cell death but that these processes occur independently. Using wild-type and genetically-manipulated immortalized murine bone marrow derived macrophages and pharmacologic inhibitors, we found that NLRP3 and gasdermin D are required for particle-induced IL-1β release but not for particle-induced cell death. In contrast, phagocytosis and lysosomal cathepsin release are critical for both IL-1β release and cell death. Collectively, our findings identify the pan-cathepsin inhibitor Ca-074Me and the NLRP3 inflammasome inhibitor MCC950 as therapeutic interventions worth exploring in aseptic loosening of orthopedic implants. We also found that particle-induced activation of the NLRP3 inflammasome in pre-primed macrophages and cell death are not dependent on pathogen-associated molecular patterns adherent to the wear particles despite such pathogen-associated molecular patterns being critical for all other previously studied wear particle responses, including priming of the NLRP3 inflammasome.
The master developmental regulator Jab1/Cops5/Csn5 is essential for proper bone growth and survival in mice
(Elsevier, 2021-02) Samsa, William E.; Mamidi, Murali K.; Hausman, Bryan S.; Bashur, Lindsay A.; Greenfield, Edward M.; Zhou, Guang; Orthopaedic Surgery, School of Medicine
Jab1, also known as Csn5/Cops5, is a key subunit of the COP9 Signalosome, a highly conserved macromolecular complex. We previously reported that the conditional knockout of Jab1 in mouse limb buds and chondrocytes results in severely shortened limbs and neonatal lethal chondrodysplasia, respectively. In this study, we further investigated the specific role of Jab1 in osteoblast differentiation and postnatal bone growth by characterizing a novel mouse model, the Osx-cre; Jab1flox/flox conditional knockout (Jab1 cKO) mouse, in which Jab1 is deleted in osteoblast precursor cells. Jab1 cKO mutant mice appeared normal at birth, but developed progressive dwarfism. Inevitably, all mutant mice died prior to weaning age. The histological and micro-computed tomography analysis of mutant long bones revealed severely altered bone microarchitecture, with a significant reduction in trabecular thickness. Moreover, Jab1 cKO mouse tibiae had a drastic decrease in mineralization near the epiphyseal growth plates, and Jab1 cKO mice also developed spontaneous fractures near the tibiofibular junction. Additionally, our cell culture studies demonstrated that Jab1 deletion in osteoblast precursors led to decreased mineralization and a reduced response to TGFβ and BMP signaling. Moreover, an unbiased reporter screen also identified decreased TGFβ activity in Jab1-knockdown osteoblasts. Thus, Jab1 is necessary for proper osteoblast differentiation and postnatal bone growth, likely in part through its positive regulation of the TGFβ and BMP signaling pathways in osteoblast progenitor cells.
Osteocytes directly regulate osteolysis via MYD88 signaling in bacterial bone infection
(Springer Nature, 2022-11-04) Yoshimoto, Tetsuya; Kittaka, Mizuho; Doan, Andrew Anh Phuong; Urata, Rina; Prideaux, Matthew; Rojas, Roxana E.; Harding, Clifford V.; Boom, W. Henry; Bonewald, Lynda F.; Greenfield, Edward M.; Ueki, Yasuyoshi; Biomedical Sciences and Comprehensive Care, School of Dentistry
The impact of bone cell activation on bacterially-induced osteolysis remains elusive. Here, we show that matrix-embedded osteocytes stimulated with bacterial pathogen-associated molecular patterns (PAMPs) directly drive bone resorption through an MYD88-regulated signaling pathway. Mice lacking MYD88, primarily in osteocytes, protect against osteolysis caused by calvarial injections of bacterial PAMPs and resist alveolar bone resorption induced by oral Porphyromonas gingivalis (Pg) infection. In contrast, mice with targeted MYD88 restoration in osteocytes exhibit osteolysis with inflammatory cell infiltration. In vitro, bacterial PAMPs induce significantly higher expression of the cytokine RANKL in osteocytes than osteoblasts. Mechanistically, activation of the osteocyte MYD88 pathway up-regulates RANKL by increasing binding of the transcription factors CREB and STAT3 to Rankl enhancers and by suppressing K48-ubiquitination of CREB/CREB binding protein and STAT3. Systemic administration of an MYD88 inhibitor prevents jawbone loss in Pg-driven periodontitis. These findings reveal that osteocytes directly regulate inflammatory osteolysis in bone infection, suggesting that MYD88 and downstream RANKL regulators in osteocytes are therapeutic targets for osteolysis in periodontitis and osteomyelitis.
Receptor Tyrosine Kinases in Osteosarcoma: 2019 Update
(Springer, 2020) Greenfield, Edward M.; Collier, Christopher D.; Getty, Patrick J.; Orthopaedic Surgery, School of Medicine
The primary conclusions of our 2014 contribution to this series were as follows: Multiple receptor tyrosine kinases (RTKs) likely contribute to aggressive phenotypes in osteosarcoma and, therefore, inhibition of multiple RTKs is likely necessary for successful clinical outcomes. Inhibition of multiple RTKs may also be useful to overcome resistance to inhibitors of individual RTKs as well as resistance to conventional chemotherapies. Different combinations of RTKs are likely important in individual patients. AXL, EPHB2, FGFR2, IGF1R, and RET were identified as promising therapeutic targets by our in vitro phosphoproteomic/siRNA screen of 42 RTKs in the highly metastatic LM7 and 143B human osteosarcoma cell lines. This chapter is intended to provide an update on these topics as well as the large number of osteosarcoma clinical studies of inhibitors of multiple tyrosine kinases (multi-TKIs) that were recently published.
Staphylococcus aureus and Acinetobacter baumannii Inhibit Osseointegration of Orthopedic Implants
(American Society for Microbiology, 2022) Choe, Hyonmin; Tatro, Joscelyn M.; Hausman, Bryan S.; Hujer, Kristine M.; Marshall, Steve H.; Akkus, Ozan; Rather, Phillip N.; Lee, Zhenghong; Bonomo, Robert A.; Greenfield, Edward M.; Orthopaedic Surgery, School of Medicine
Bacterial infections routinely cause inflammation and thereby impair osseointegration of orthopedic implants. Acinetobacter spp., which cause osteomyelitis following trauma, on or off the battlefield, were, however, reported to cause neither osteomyelitis nor osteolysis in rodents. We therefore compared the effects of Acinetobacter strain M2 to those of Staphylococcus aureus in a murine implant infection model. Sterile implants and implants with adherent bacteria were inserted in the femur of mice. Bacterial burden, levels of proinflammatory cytokines, and osseointegration were measured. All infections were localized to the implant site. Infection with either S. aureus or Acinetobacter strain M2 increased the levels of proinflammatory cytokines and the chemokine CCL2 in the surrounding femurs, inhibited bone formation around the implant, and caused loss of the surrounding cortical bone, leading to decreases in both histomorphometric and biomechanical measures of osseointegration. Genetic deletion of TLR2 and TLR4 from the mice partially reduced the effects of Acinetobacter strain M2 on osseointegration but did not alter the effects of S. aureus. This is the first report that Acinetobacter spp. impair osseointegration of orthopedic implants in mice, and the murine model developed for this study will be useful for future efforts to clarify the mechanism of implant failure due to Acinetobacter spp. and to assess novel diagnostic tools or therapeutic agents.

Browsing by Author "Greenfield, Edward M."

Results Per Page

Sort Options