43 results on '"Yik, Jasper H. N."'
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2. Biomimetic Bone-Like Composite Hydrogel Scaffolds Composed of Collagen Fibrils and Natural Hydroxyapatite for Promoting Bone Repair
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Yang, Wentao, primary, Ni, Weiyu, additional, Yu, Congcong, additional, Gu, Tianyuan, additional, Ye, Lin, additional, Sun, Rongtai, additional, Ying, Xiaozhang, additional, Yik, Jasper H. N., additional, Haudenschild, Dominik R., additional, Yao, Shasha, additional, and Hu, Ziang, additional
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- 2024
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3. Current trends in the prevention of adhesions after zone 2 flexor tendon repair.
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Vinitpairot, Chaiyos, Yik, Jasper H. N., Haudenschild, Dominik R., Szabo, Robert M., and Bayne, Christopher O.
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Treating flexor tendon injuries within the digital flexor sheath (commonly referred to as palmar hand zone 2) presents both technical and logistical challenges. Success hinges on striking a delicate balance between safeguarding the surgical repair for tendon healing and initiating early rehabilitation to mitigate the formation of tendon adhesions. Adhesions between tendon slips and between tendons and the flexor sheath impede tendon movement, leading to postoperative stiffness and functional impairment. While current approaches to flexor tendon repair prioritize maximizing tendon strength for early mobilization and adhesion prevention, factors such as pain, swelling, and patient compliance may impede postoperative rehabilitation efforts. Moreover, premature mobilization could risk repair failure, necessitating additional surgical interventions. Pharmacological agents offer a potential avenue for minimizing inflammation and reducing adhesion formation while still promoting normal tendon healing. Although some systemic and local agents have shown promising results in animal studies, their clinical efficacy remains uncertain. Limitations in these studies include the relevance of chosen animal models to human populations and the adequacy of tools and measurement techniques in accurately assessing the impact of adhesions. This article provides an overview of the clinical challenges associated with flexor tendon injuries, discusses current on‐ and off‐label agents aimed at minimizing adhesion formation, and examines investigational models designed to study adhesion reduction after intra‐synovial flexor tendon repair. Understanding the clinical problem and experimental models may serve as a catalyst for future research aimed at addressing intra‐synovial tendon adhesions following zone 2 flexor tendon repair. [ABSTRACT FROM AUTHOR]
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- 2024
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4. CircGNB1 drives osteoarthritis pathogenesis by inducing oxidative stress in chondrocytes
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Liang, Yi, primary, Shen, Lifeng, additional, Ni, Weiyu, additional, Ding, Yuhong, additional, Yang, Wentao, additional, Gu, Tianyuan, additional, Zhang, Chenfeng, additional, Yik, Jasper H. N., additional, Haudenschild, Dominik R., additional, Fan, Shunwu, additional, Shen, Shuying, additional, and Hu, Ziang, additional
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- 2023
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5. Closed Joint ACL Disruption Murine Model of PTA
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Christiansen, Blaine A., Yik, Jasper H. N., Haudenschild, Dominik R., Olson, MD, Steven A., editor, and Guilak, PhD, Farshid, editor
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- 2015
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6. Acute bone loss following SARS‐CoV‐2 infection in mice
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Haudenschild, Anne K., primary, Christiansen, Blaine A., additional, Orr, Sophie, additional, Ball, Erin E., additional, Weiss, Christopher M., additional, Liu, Hongwei, additional, Fyhrie, David P., additional, Yik, Jasper H. N., additional, Coffey, Lark L., additional, and Haudenschild, Dominik R., additional
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- 2023
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7. Production of Inhalable Ultra-Small Particles for Delivery of Anti-Inflammation Medicine via a Table-Top Microdevice
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Owen, Matthew J., primary, Celik, Umit, additional, Chaudhary, Subash K., additional, Yik, Jasper H. N., additional, Patton, John S., additional, Kuo, Mei-chang, additional, Haudenschild, Dominik R., additional, and Liu, Gang-yu, additional
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- 2022
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8. The Influence of Endplate Morphology on Cage Subsidence in Patients With Stand-Alone Oblique Lateral Lumbar Interbody Fusion (OLIF).
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Hu, Ziang, He, Dengwei, Gao, Jiawei, Zeng, Zhongyou, Jiang, Chao, Ni, Weiyu, Yik, Jasper H. N., Zhao, Xing, and Fan, Shunwu
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RADIOGRAPHY ,PERIOPERATIVE care ,HEALTH outcome assessment ,BIOMECHANICS ,LORDOSIS ,VISUAL analog scale - Abstract
Study Design: A retrospective study of prospectively collected radiographic and clinical data. Objective: This study aims to investigate the relationship between endplate morphology parameters and the incidence of cage subsidence in patients with mini-open single-level oblique lateral lumbar interbody fusion (OLIF). Methods: We included 119 inpatients who underwent OLIF from February 2015 to December 2017. A total of 119 patients with single treatment level of OLIF were included. Plain anteroposterior and lateral radiograph were taken preoperatively, postoperatively, and during follow-up. The correlation between disc height, endplate concave angle/depth, cage position and cage subsidence were investigated. Functional rating index (Visual Analogue Scale for pain, and Roland Morris Disability Questionnaire) were employed to assess clinical outcomes. Results: Cage subsidence was more commonly seen at the superior endplates (42/119, 35.29%) than at the inferior endplates (6/119, 5.04%) (p < 0.01). More importantly, cage subsidence was significantly less in patients with superior endplates that were without concave angle (3/20, 15%) than with concave angle (37/99, 37.37%) (p < 0.05). Cage subsidence correlated negatively with preoperative anterior disc height (r = −0.21, p < 0.05), but positively with disc distraction rate (r = 0.27, p < 0.01). Lastly, the distance of cage to the anterior edges of the vertebral body showed a positive correlation (r = 0.26, p < 0.01). Conclusions: This study for the first time demonstrated that endplate morphology correlates with cage subsidence after OLIF. Since relatively flat endplates with smaller concave angle significantly diminish the incidence of subsidence, the morphology of cage surface should be taken into consideration when designing the next generation of cage. In addition, precise measurement of the disc height to avoid over-distraction, and more anteriorly placement of the cage is suggested to reduce subsidence. [ABSTRACT FROM AUTHOR]
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- 2023
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9. Reply
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Haudenschild, Dominik R. and Yik, Jasper H. N.
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- 2014
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10. Cyclin-Dependent Kinase 9 Inhibition Protects Cartilage From the Catabolic Effects of Proinflammatory Cytokines
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Yik, Jasper H. N., Hu, Ziʼang, Kumari, Ratna, Christiansen, Blaine A., and Haudenschild, Dominik R.
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- 2014
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11. The Influence of Endplate Morphology on Cage Subsidence in Patients With Stand-Alone Oblique Lateral Lumbar Interbody Fusion (OLIF)
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Hu, Ziang, primary, He, Dengwei, additional, Gao, Jiawei, additional, Zeng, Zhongyou, additional, Jiang, Chao, additional, Ni, Weiyu, additional, Yik, Jasper H. N., additional, Zhao, Xing, additional, and Fan, Shunwu, additional
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- 2021
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12. Cyclin-Dependent Kinase 9 (CDK9) Inhibitor Atuveciclib Suppresses Intervertebral Disk Degeneration via the Inhibition of the NF-κB Signaling Pathway
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Ni, Weiyu, primary, Zhang, Feizhou, additional, Zheng, Lin, additional, Wang, Lili, additional, Liang, Yi, additional, Ding, Yuhong, additional, Yik, Jasper H. N., additional, Haudenschild, Dominik R., additional, Fan, Shunwu, additional, and Hu, Ziang, additional
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- 2020
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13. Tat competes with HEXIM1 to increase the active pool of P-TEFb for HIV-1 transcription
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Barboric, Matjaz, Yik, Jasper H. N., Czudnochowski, Nadine, Yang, Zhiyuan, Chen, Ruichuan, Contreras, Xavier, Geyer, Matthias, Peterlin, B. Matija, and Zhou, Qiang
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- 2007
14. Acute Changes in NADPH Oxidase 4 in Early Post‐Traumatic Osteoarthritis
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Wegner, Adam M., primary, Campos, Nestor R., additional, Robbins, Michael A., additional, Haddad, Andrew F., additional, Cunningham, Hailey C., additional, Yik, Jasper H. N., additional, Christiansen, Blaine A., additional, and Haudenschild, Dominik R., additional
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- 2019
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15. Label-Free and Direct Visualization of Multivalent Binding of Bone Morphogenetic Protein-2 with Cartilage Oligomeric Matrix Protein
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Tran, Victoria, primary, Karsai, Arpad, additional, Fong, Michael C., additional, Cai, Weiliang, additional, Yik, Jasper H. N., additional, Klineberg, Eric, additional, Haudenschild, Dominik R., additional, and Liu, Gang-yu, additional
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- 2018
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16. Flavopiridol Protects Bone Tissue by Attenuating RANKL Induced Osteoclast Formation
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Hu, Zi’ang, primary, Chen, Yilei, additional, Song, Lijiang, additional, Yik, Jasper H. N., additional, Haudenschild, Dominik R., additional, and Fan, Shunwu, additional
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- 2018
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17. Inhibition of early response genes prevents changes in global joint metabolomic profiles in mouse post-traumatic osteoarthritis.
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Haudenschild, D.R., Carlson, A.K., Zignego, D.L., Yik, J.H.N., Hilmer, J.K., June, R.K., Haudenschild, Dominik R, Carlson, Alyssa K, Zignego, Donald L, Yik, Jasper H N, Hilmer, Jonathan K, and June, Ronald K
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Objective: Although joint injury itself damages joint tissues, a substantial amount of secondary damage is mediated by the cellular responses to the injury. Cellular responses include the production and activation of proteases (MMPs, ADAMTSs, Cathepsins), and the production of inflammatory cytokines. The trajectory of cellular responses is driven by the transcriptional activation of early response genes, which requires Cdk9-dependent RNA Polymerase II phosphorylation. Our objective was to determine whether inhibition of cdk9-dependent early response gene activation affects changes in the joint metabolome.Design: To model post-traumatic osteoarthritis, we subjected mice to non-invasive Anterior Cruciate Ligament (ACL)-rupture joint injury. Following injury, mice were treated with flavopiridol - a potent and selective inhibitor of Cdk9 kinase activity - to inhibit Cdk9-dependent transcriptional activation, or vehicle control. Global joint metabolomics were analyzed 1 h after injury.Results: We found that injury induced metabolomic changes, including increases in Vitamin D3 metabolism, anandamide, and others. Inhibition of primary response gene activation immediately after injury largely prevented the global changes in the metabolomics profiles. Cluster analysis of joint metabolomes identified groups of injury-induced and drug-responsive metabolites.Conclusions: Metabolomic profiling provides an instantaneous snapshot of biochemical activity representing cellular responses. We identified two sets of metabolites that change acutely after joint injury: those that require transcription of primary response genes, and those that do not. These data demonstrate the potential for inhibition of early response genes to alter the trajectory of cell-mediated degenerative changes following joint injury, which may offer novel targets for cell-mediated secondary joint damage. [ABSTRACT FROM AUTHOR]- Published
- 2019
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18. Label-Free and Direct Visualization of Multivalent Binding of Bone Morphogenetic Protein‑2 with Cartilage Oligomeric Matrix Protein.
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Tran, Victoria, Karsai, Arpad, Fong, Michael C., Cai, Weiliang, Yik, Jasper H. N., Klineberg, Eric, Haudenschild, Dominik R., and Liu, Gang-yu
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- 2019
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19. Brd4 and HEXIM1: Multiple Roles in P-TEFb Regulation and Cancer
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Chen, Ruichuan, Yik, Jasper H. N., Lew, Qiao Jing, and Chao, Sheng-Hao
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Article Subject - Abstract
Bromodomain-containing protein 4 (Brd4) and hexamethylene bisacetamide (HMBA) inducible protein 1 (HEXIM1) are two opposing regulators of the positive transcription elongation factor b (P-TEFb), which is the master modulator of RNA polymerase II during transcriptional elongation. While Brd4 recruits P-TEFb to promoter-proximal chromatins to activate transcription, HEXIM1 sequesters P-TEFb into an inactive complex containing the 7SK small nuclear RNA. Besides regulating P-TEFb’s transcriptional activity, recent evidence demonstrates that both Brd4 and HEXIM1 also play novel roles in cell cycle progression and tumorigenesis. Here we will discuss the current knowledge on Brd4 and HEXIM1 and their implication as novel therapeutic options against cancer.
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- 2014
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20. The Yin and Yang of P-TEFb Regulation: Implications for Human Immunodeficiency Virus Gene Expression and Global Control of Cell Growth and Differentiation
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Zhou, Qiang, primary and Yik, Jasper H. N., additional
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- 2006
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21. A Human Immunodeficiency Virus Type 1 Tat-Like Arginine-Rich RNA-Binding Domain Is Essential for HEXIM1 To Inhibit RNA Polymerase II Transcription through 7SK snRNA-Mediated Inactivation of P-TEFb
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Yik, Jasper H. N., primary, Chen, Ruichuan, additional, Pezda, Andrea C., additional, Samford, Craig S., additional, and Zhou, Qiang, additional
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- 2004
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22. Compensatory Contributions of HEXIM1 and HEXIM2 in Maintaining the Balance of Active and Inactive Positive Transcription Elongation Factor b Complexes for Control of Transcription.
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Yik, Jasper H. N., Ruichuan Chen, Pezda, Andrea C., and Qiang Zhou
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PROTEINS , *CELLULAR control mechanisms , *GENETIC transcription , *PROTEIN kinases , *RNA polymerases , *RNA - Abstract
Human positive transcriptional elongation factor b (P-TEFb), consisting of a cyclin-dependent kinase 9-cyclin T heterodimer, stimulates general and disease-specific transcriptional elongation by phosphorylating RNA polymerase II. The HEXIM1 protein, aided by the 7SK snRNA, sequesters P-TEFb into an inactive 7SK·HEXIM1·P-TEFb small nuclear ribonucleic acid particle for inhibition of transcription and, consequently, cell proliferation. Here we show that, like HEXIM1, a highly homologous protein named HEXIM2 also possesses the ability to inactivate P-TEFb to suppress transcription through a 7SK-mediated interaction with PTEFb. Furthermore, HEXIM1 and HEXIM2 can form stable homo- and hetero-oligomers (most likely dimers), which may nucleate the formation of the 7SK small nuclear ribonucleic acid particle. Despite their similar functions, HEXIM1 and HEXIM2 exhibit distinct expression patterns in various human tissues and established cell lines. In HEXIM1-knocked down cells, HEXIM2 can functionally and quantitatively compensate for the loss of HEXIM1 to maintain a constant level of the 7SK/ HEXIM-bound P-TEFb. Our results demonstrate that there is a tightly regulated cellular process to maintain the balance between active and inactive P-TEFb complexes, which controls global transcription as well as cell growth and differentiation. [ABSTRACT FROM AUTHOR]
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- 2005
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23. Intra-articular injection of an extended-release flavopiridol formulation represents a potential alternative to other intra-articular medications for treating equine joint disease.
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Katzman, Scott A., Cissell, Derek, Leale, Dustin, Perez-Nogues, Marcos, Hall, Mark D., Bloom, Grace, Hamamoto-Hardman, Briana, Chun-Yi Wu, Haudenschild, Anne K., Gang-yu Liu, Yik, Jasper H. N., and Haudenschild, Dominik R.
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SYNOVIAL fluid , *INTRA-articular injections , *JOINT diseases , *ANTI-inflammatory agents , *DETECTION limit - Abstract
OBJECTIVE To establish the pharmacokinetics of the cyclin-dependent kinase-9 inhibitor flavopiridol in equine middle carpal joints, using an extended-release poly lactic-co-glycolic acid (PLGA) microparticle formulation. ANIMALS 4 healthy horses without evidence of forelimb lameness. METHODS A 6-week longitudinal pharmacokinetic study was conducted in 2 phases (6 weeks each) in 4 healthy horses. The PLGA microparticles containing 122 μg flavopiridol in 3 mL saline were administered by intra-articular injection into 1 middle carpal joint, with empty PLGA microparticles injected into the contralateral joint as a control. Synovial fluid and plasma were collected at time points out to 6 weeks, and drug concentrations in synovial fluid and plasma were determined using validated protocols. Synovial fluid total protein and total nucleated cell count and differential, CBC, serum biochemistry, and lameness exams were performed at each of the time points. RESULTS Synovial fluid flavopiridol averaged 19 nM at week 1, gradually reduced to 1.4 nM by 4 weeks, and was generally below the detection limit at 5 and 6 weeks. There was no detectable flavopiridol in the plasma samples, and no adverse effects were observed at any time point. CLINICAL RELEVANCE Intra-articular injection of PLGA microparticle-encapsulated flavopiridol was well tolerated in horses, with detectable levels of flavopiridol in the synovial fluid out to 4 weeks with negligible systemic exposure. Flavopiridol is a cyclin-dependent kinase-9 inhibitor with potent anti-inflammatory and analgesic activity. The extended-release microparticle formulation promotes intra-articular retention of the drug and it may be an alternative to other intraarticular medications for treatment of equine joint disease. [ABSTRACT FROM AUTHOR]
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- 2024
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24. Nonpalmitoylated human asialoglycoprotein receptors recycle constitutively but are defective in coated pit-mediated endocytosis, dissociation, and delivery of ligand to lysosomes.
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Yik, Jasper H N, Saxena, Amit, Weigel, Janet A, and Weigel, Paul H
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The hepatic asialoglycoprotein receptor (ASGP-R) internalizes desialylated glycoproteins via the clathrin-coated pit pathway and mediates their delivery to lysosomes for degradation. The human ASGP-R contains two subunits, H1 and H2. Cytoplasmic residues Cys(36) in H1, as well as Cys(54) and Cys(58) in H2 are palmitoylated (Zeng, F.-Y., and Weigel, P. H. (1996) J. Biol. Chem. 271, 32454). In order to study the function(s) of ASGP-R palmitoylation, we mutated these Cys residues to Ser and generated stably transfected SK-Hep-1 cell lines expressing either wild-type or nonpalmitoylated ASGP-Rs. Compared with wild-type ASGP-Rs, palmitoylation-defective ASGP-Rs showed normal ligand binding, intracellular distribution and trafficking patterns, and pH-induced dissociation profiles in vitro. However, continuous ASOR uptake, and the uptake of prebound cell surface ASOR were slower in cells expressing palmitoylation-defective ASGP-Rs than in cells expressing wild-type ASGP-Rs. Unlike native ASGP-Rs in hepatocytes or hepatoma cells, which mediate endocytosis via the clathrin-coated pit pathway and are almost completely inhibited by hypertonic medium, only approximately 40% of the ASOR uptake in SK-Hep-1 cells expressing wild-type ASGP-Rs was inhibited by hyperosmolarity. This result suggests the existence of an alternate nonclathrin-mediated internalization pathway, such as transcytosis, for the entry of ASGP-R.ASOR complexes into these cells. In contrast, ASOR uptake mediated by cells expressing palmitoylation-defective ASGP-Rs showed only a marginal difference under hypertonic conditions, indicating that most of the nonpalmitoylated ASGP-Rs were not internalized and processed normally through the clathrin-coated pit pathway. Furthermore, cells expressing wild-type ASGP-Rs were able to degrade the internalized ASOR, whereas ASOR dissociation was impaired and degradation was barely detectable in cells expressing nonpalmitoylated ASGP-Rs. We conclude that palmitoylation of the ASGP-R is required for its efficient endocytosis of ligand by the clathrin-dependent endocytic pathway and, in particular, for the proper dissociation and delivery of ligand to lysosomes.
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- 2002
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25. H2, the minor subunit of the human asialoglycoprotein receptor, trafficks intracellularly and forms homo-oligomers, but does not bind asialo-orosomucoid.
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Saxena, Amit, Yik, Jasper H N, and Weigel, Paul H
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The functional human hepatic asialoglycoprotein receptor (ASGP-R) is a hetero-oligomer composed of two subunits, designated H1 and H2, which are highly homologous. Despite their extensive homology, the major H1 subunit is stably expressed by itself, whereas in the absence of H1 most of the H2 subunits are degraded in the ER. In this study, we were able to investigate the capability of the minor ASGP-R subunit, H2, to function independently of H1, because it was apparently stabilized by fusing its NH(2) terminus with an epitope tag. We could thus create stable cell lines in hepatoma-derived SK-Hep-1 cells that expressed the H2 subunit alone. H2 was expressed on the cell surface and was internalized, predominantly through the clathrin-coated pit pathway. Since the internal pool of H2 was also able to traffick to the cell surface, we conclude that H2 recycles between the surface and intracellular compartments, similar to the constitutive recycling of hetero-oligomeric ASGP-R complexes. However, the rate of H2 recycling and internalization was approximately 25-33% that of H1. Similar to H1, the H2 polypeptides were also able to self-associate to form homo-oligomers, including trimers and tetramers. However, unlike H1, which can bind the ligand asialo-orosomucoid (ASOR) when overexpressed in COS-7 cells, H2 failed to bind or endocytose ASOR. In summary, the H2 subunit of the human ASGP-R contains functional, although weak, signal(s) for endocytosis and recycling and has the ability to oligomerize. H2 homo-oligomers, however, do not create binding sites for desialylated glycoproteins, such as ASOR, that contain tri- and tetra-antennary N-linked oligosaccharides. Nonetheless, these results raise the intriguing possibility that naturally occurring H2 homo-oligomers may exist in human hepatocytes and have an as yet undiscovered function.
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- 2002
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26. The Yin and Yang of P-TEFb Regulation: Implications for Human Immunodeficiency Virus Gene Expression and Global Control of Cell Growth and Differentiation.
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Qiang Zhou and Yik, Jasper H. N.
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PHOSPHORYLATION , *RNA polymerases , *HIV , *GENOMES , *HYPERTROPHY , *BREAST cancer , *HIV infections - Abstract
The positive transcription elongation factor b (P-TEFb) stimulates transcriptional elongation by phosphorylating the carboxy-terminal domain of RNA polymerase II and antagonizing the effects of negative elongation factors. Not only is P-TEFb essential for transcription of the vast majority of cellular genes, but it is also a critical host cellular cofactor for the expression of the human immunodeficiency virus (HIV) type 1 genome. Given its important role in globally affecting transcription, P-TEFb's activity is dynamically controlled by both positive and negative regulators in order to achieve a functional equilibrium in sync with the overall transcriptional demand as well as the proliferative state of cells. Notably, this equilibrium can be shifted toward either the active or inactive state in response to diverse physiological stimuli that can ultimately affect the cellular decision between growth and differentiation. In this review, we examine the mechanisms by which the recently identified positive (the bromodomain protein Brd4) and negative (the noncoding 7SK small nuclear RNA and the HEXIM1 protein) regulators of P-TEFb affect the P-TEFb-dependent transcriptional elongation. We also discuss the consequences of perturbations of the dynamic associations of these regulators with P-TEFb in relation to the pathogenesis and progression of several major human diseases, such as cardiac hypertrophy, breast cancer, and HIV infection. [ABSTRACT FROM AUTHOR]
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- 2006
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27. Enhanced Activity of Transforming Growth Factor β1 (TGF-β1) Bound to Cartilage Oligomeric Matrix Protein.
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Haudenschild, Dominik R., Hong, Eunmee, Yik, Jasper H. N., Chromy, Brett, Mörgelin, Matthias, Snow, Kaylene D., Acharya, Chitrangada, Takada, Yoshikazu, and Cesare, Paul E. Di
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EXTRACELLULAR matrix proteins , *CARTILAGE , *COLLAGEN , *PROTEOGLYCANS , *BIOCHEMICAL research - Abstract
Cartilage oligomeric matrix protein (COMP) is an important non-collagenous cartilage protein that is essential for the structural integrity of the cartilage extracellular matrix. The repeated modular structure of COMP allows it to “bridge” and assemble multiple cartilage extracellular matrix components such as collagens, matrilins, and proteoglycans. With its modular structure, COMP also has the potential to act as a scaffold for growth factors, thereby affecting how and when the growth factors are presented to cell-surface receptors. However, it is not known whether COMP binds growth factors. We studied the binding interaction between COMP and TGF-β1 in vitro and determined the effect of COMP on TGF-β1-induced signal transduction in reporter cell lines and primary cells. Our results demonstrate that mature COMP protein binds to multiple TGF-β1 molecules and that the peak binding occurs at slightly acidic pH. These interactions were confirmed by dual polarization interferometry and visualized by rotary shadow electron microscopy. There is cation-independent binding of TGF-β1 to the C-terminal domain of COMP. In the presence of manganese, an additional TGF-β-binding site is present in the TSP3 repeats of COMP. Finally, we show that COMP-bound TGF-β1 causes increased TGF-β1-dependent transcription. We conclude that TGF-β1 binds to COMP and that TGF-β1 bound to COMP has enhanced bioactivity. [ABSTRACT FROM AUTHOR]
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- 2011
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28. A two-stage digestion of whole murine knee joints for single-cell RNA sequencing.
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Leale DM, Li L, Settles ML, Mitchell K, Froenicke L, Yik JHN, and Haudenschild DR
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Objective: Single-cell RNA sequencing (scRNA-seq) is a powerful technology that can be applied to the cells populating the whole knee in the study of joint pathology. The knee contains cells embedded in hard structural tissues, cells in softer tissues and membranes, and immune cells. This creates a technical challenge in preparing a viable and representative cell suspension suitable for use in scRNA-seq in minimal time, where under-digestion may exclude cells in hard tissues, over-digestion may damage soft tissue cells, and prolonged digestion may induce phenotypic drift. We developed a rapid two-stage digestion protocol to overcome these difficulties., Design: A two-stage digest consisting of first collagenase IV, an intermediate cell recovery, then collagenase II on the remaining hard tissue. Cells were sequenced on the 10x Genomics platform., Results: We observed consistent cell numbers and viable single cell suspensions suitable for scRNA-seq analysis. Comparison of contralateral knees and separate mice showed reproducible cell yields and gene expression patterns by similar cell-types. A diverse collection of structural and immune cells were captured with a majority from immune origins. Two digestions were necessary to capture all cell-types., Conclusions: The knee contains a diverse mixture of stromal and immune cells that may be crucial for the study of osteoarthritis. The two-stage digestion presented here reproducibly generated highly viable and representative single-cell suspension for sequencing from the whole knee. This protocol facilitates transcriptomic studies of the joint as a complete organ., (© 2022 The Authors.)
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- 2022
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29. Intra-articular injection of flavopiridol-loaded microparticles for treatment of post-traumatic osteoarthritis.
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Sangsuwan R, Yik JHN, Owen M, Liu GY, Haudenschild DR, and Lewis JS
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- Animals, Biocompatible Materials, Cyclin-Dependent Kinase 9, Flavonoids, Inflammation pathology, Injections, Intra-Articular, Piperidines, Rats, Cartilage, Articular pathology, Osteoarthritis drug therapy, Osteoarthritis pathology
- Abstract
Rapid joint clearance of small molecule drugs is the major limitation of current clinical approaches to osteoarthritis and its subtypes, including post-traumatic osteoarthritis (PTOA). Particulate systems such as nano/microtechnology could provide a potential avenue for improved joint retention of small molecule drugs. One drug of interest for PTOA treatment is flavopiridol, which inhibits cyclin-dependent kinase 9 (CDK9). Herein, polylactide-co-glycolide microparticles encapsulating flavopiridol were formulated, characterized, and evaluated as a strategy to mitigate PTOA-associated inflammation through the inhibition of CDK9. Characterization of the microparticles, including the drug loading, hydrodynamic diameter, stability, and release profile was performed. The mean hydrodynamic diameter of flavopiridol particles was ∼15 µm, indicating good syringeability and low potential for phagocytosis. The microparticles showed no cytotoxicity in-vitro, and drug activity was maintained after encapsulation, even after prolonged exposure to high temperatures (60 °C). Flavopiridol-loaded microparticles or blank (unloaded) microparticles were administered by intraarticular injection in a rat knee injury model of PTOA. We observed significant joint retention of flavopiridol microparticles compared to the soluble flavopiridol, confirming the sustained release behavior of the particles. Matrix metalloprotease (MMP) activity, an indicator of joint inflammation, was significantly reduced by flavopiridol microparticles 3 days post-injury. Histopathological analysis showed that flavopiridol microparticles reduced PTOA severity 28 days post-injury. Taken altogether, this work demonstrates a promising biomaterial platform for sustained small molecule drug delivery to the joint space as a therapeutic measure for post-traumatic osteoarthritis. STATEMENT OF SIGNIFICANCE: Post-traumatic osteoarthritis (PTOA) begins with the deterioration of subchondral bone and cartilage after acute injuries. In spite of the prevalence of PTOA and its associated financial and psychological burdens, therapeutic measures remain elusive. A number of small molecule drugs are now under investigation to replace FDA-approved palliative measures, including cyclin-dependent kinase 9 (CDK9) inhibitors which work by targeting early inflammatory programming after injury. However, the short half-life of these drugs is a major hurdle to their success. Here, we show that biomaterial encapsulation of Flavopiridol (CDK9 inhibitor) in poly (lactic-co-glycolic acid) microparticles is a promising route for direct delivery and improved drug retention time in the knee joint. Moreover, administration of the flavopiridol microparticles reduced the severity of PTOA., Competing Interests: Declaration of Competing Interest D.R.H and J.H.N.Y report a relationship with Tesio Pharmaceuticals, Inc. that includes stock ownership and board membership. D.R.H, J.H.N.Y, and J.S.L report patents (issued and pending) assigned to Regents of the University of California and Licensed by Tesio Pharmaceuticals. There is an institutionally approved plan for managing any potential conflicts arising from these relationships., (Copyright © 2022. Published by Elsevier Ltd.)
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- 2022
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30. CircSLC7A2 protects against osteoarthritis through inhibition of the miR-4498/TIMP3 axis.
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Ni W, Jiang C, Wu Y, Zhang H, Wang L, Yik JHN, Haudenschild DR, Fan S, Shen S, and Hu Z
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- Animals, Apoptosis, Cartilage, Articular metabolism, Cartilage, Articular pathology, Down-Regulation, Gene Expression Regulation, Humans, Mice, MicroRNAs genetics, Osteoarthritis pathology, RNA, Circular analysis, Tissue Inhibitor of Metalloproteinase-3 analysis, Osteoarthritis genetics, RNA, Circular genetics, Tissue Inhibitor of Metalloproteinase-3 genetics
- Abstract
Objectives: Circular RNAs (circRNAs) are noncoding RNAs that compete against other endogenous RNA species, such as microRNAs, and have been implicated in many diseases. In this study, we investigated the role of a new circRNA (circSLC7A2) in osteoarthritis (OA)., Materials and Methods: The relative expression of circSLC7A2 was significantly lower in OA tissues than it was in matched controls, as shown by real-time quantitative polymerase chain reaction (RT-qPCR). Western blotting, RT-qPCR and immunofluorescence experiments were employed to evaluate the roles of circSLC7A2, miR-4498 and TIMP3. The in vivo role and mechanism of circSLC7A2 were also conformed in a mouse model., Results: circSLC7A2 was decreased in OA model and the circularization of circSLC7A2 was regulated by FUS. Loss of circSLC7A2 reduced the sponge of miR-4498 and further inhibited the expression of TIMP3, subsequently leading to an inflammatory response. We further determined that miR-4498 inhibitor reversed circSLC7A2-knockdown-induced OA phenotypes. Intra-articular injection of circSLC7A2 alleviated in vivo OA progression in a mouse model of anterior cruciate ligament transection (ACLT)., Conclusions: The circSLC7A2/miR-4498/TIMP3 axis of chondrocytes catabolism and anabolism plays a critical role in OA development. Our results suggest that circSLC7A2 may serve as a new therapeutic target for osteoarthritis., (© 2021 The Authors. Cell Proliferation Published by John Wiley & Sons Ltd.)
- Published
- 2021
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31. Direct Visualization of the Binding of Transforming Growth Factor Beta 1 with Cartilage Oligomeric Matrix Protein via High-Resolution Atomic Force Microscopy.
- Author
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Tran V, Karsai A, Fong MC, Cai W, Fraley JG, Yik JHN, Klineberg E, Haudenschild DR, and Liu GY
- Subjects
- Binding Sites, Cartilage Oligomeric Matrix Protein, Microscopy, Atomic Force, Signal Transduction, Transforming Growth Factor beta1
- Abstract
This work reports the first direct observations of binding and complex formation between transforming growth factor beta 1 (TGF-β1) and cartilage oligomeric matrix protein (COMP) using high-resolution atomic force microscopy (AFM). Each COMP molecule consists of pentamers whose five identical monomeric units bundle at N-termini. From this central point, the five monomers' flexible arms extend outward with C-terminal domains at the distal ends, forming a bouquet-like structure. In commonly used buffer solutions, TGF-β1 molecules typically form homodimers (majority), double dimers (minority), and aggregates (trace amount). Mixing TGF-β1 and COMP leads to rapid binding and complex formation. The TGF-β1/COMP complexes contain one to three COMP and multiple TGF-β1 molecules. For complexes with one COMP, the structure is more compact and less flexible than that of COMP alone. For complexes with two or more COMP molecules, the conformation varies to a large degree from one complex to another. This is attributed to the presence of double dimers or aggregates of TGF-β1 molecules, whose size and multiple binding sites enable binding to more than one COMP. The number and location of individual TGF-β1 dimers are also clearly visible in all complexes. This molecular-level information provides a new insight into the mechanism of chondrogenesis enhancement by TGF-β1/COMP complexes, i.e., simultaneous and multivalent presentation of growth factors. These presentations help explain the high efficacy in sustained activation of the signaling pathway to augment chondrogenesis.
- Published
- 2020
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32. In-vitro and in-vivo imaging of MMP activity in cartilage and joint injury.
- Author
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Fukui T, Tenborg E, Yik JH, and Haudenschild DR
- Subjects
- Animals, In Vitro Techniques, Male, Mice, Mice, Inbred BALB C, Real-Time Polymerase Chain Reaction, Cartilage enzymology, Knee Injuries enzymology, Matrix Metalloproteinases metabolism
- Abstract
Non-destructive detection of cartilage-degrading activities represents an advance in osteoarthritis (OA) research, with implications in studies of OA pathogenesis, progression, and intervention strategies. Matrix metalloproteinases (MMPs) are principal cartilage degrading enzymes that contribute to OA pathogenesis. MMPSense750 is an in-vivo fluorimetric imaging probe with the potential to continuously and non-invasively trace real-time MMP activities, but its use in OA-related research has not been reported. Our objective is to detect and characterize the early degradation activities shortly after cartilage or joint injury with MMPSense750. We determined the appropriate concentration, assay time, and linear range using various concentrations of recombinant MMPs as standards. We then quantified MMP activity from cartilage explants subjected to either mechanical injury or inflammatory cytokine treatment in-vitro. Finally, we performed in-vivo MMP imaging of a mouse model of post-traumatic OA. Our in-vitro results showed that the optimal assay time was highly dependent on the MMP enzyme. In cartilage explant culture media, mechanical impact or cytokine treatment increased MMP activity. Injured knees of mice showed significantly higher fluorescent signal than uninjured knees. We conclude that MMPSense750 detects human MMP activities and can be used for in-vitro study with cartilage, as well as in-vivo studies of knee injury, and can offering real-time insight into the degradative processes that occurring within the joint before structural changes become evident radiographically., (Copyright © 2015 Elsevier Inc. All rights reserved.)
- Published
- 2015
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33. Reply: To PMID 24470357.
- Author
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Haudenschild DR and Yik JH
- Subjects
- Humans, Cartilage, Articular drug effects, Chondrocytes drug effects, Cyclin-Dependent Kinase 9 antagonists & inhibitors, Cytokines pharmacology, Flavonoids pharmacology, Inflammation prevention & control, Piperidines pharmacology, Protein Kinase Inhibitors pharmacology
- Published
- 2014
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34. Cartilage oligomeric matrix protein and its binding partners in the cartilage extracellular matrix: interaction, regulation and role in chondrogenesis.
- Author
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Acharya C, Yik JH, Kishore A, Van Dinh V, Di Cesare PE, and Haudenschild DR
- Subjects
- Humans, Models, Biological, Musculoskeletal Abnormalities genetics, Protein Binding, Protein Interaction Mapping, Protein Structure, Tertiary, Cartilage Oligomeric Matrix Protein metabolism, Chondrogenesis physiology, Extracellular Matrix Proteins metabolism, Musculoskeletal Abnormalities metabolism
- Abstract
Thrombospondins (TSPs) are widely known as a family of five calcium-binding matricellular proteins. While these proteins belong to the same family, they are encoded by different genes, regulate different cellular functions and are localized to specific regions of the body. TSP-5 or Cartilage Oligomeric Matrix Protein (COMP) is the only TSP that has been associated with skeletal disorders in humans, including pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED). The pentameric structure of COMP, the evidence that it interacts with multiple cellular proteins, and the recent reports of COMP acting as a 'lattice' to present growth factors to cells, inspired this review of COMP and its interacting partners. In our review, we have compiled the interactions of COMP with other proteins in the cartilage extracellular matrix and summarized their importance in maintaining the structural integrity of cartilage as well as in regulating cellular functions., (Copyright © 2014 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.)
- Published
- 2014
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35. The Oncogene LRF Stimulates Proliferation of Mesenchymal Stem Cells and Inhibits Their Chondrogenic Differentiation.
- Author
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Yik JH, Li H, Acharya C, Kumari R, Fierro F, Haudenschild DR, Nolta J, and Di Cesare PE
- Abstract
Objective: The oncogene leukemia/lymphoma-related factor (LRF) enhances chondrosarcoma proliferation and malignancy. This study aimed to investigate the roles of LRF in chondrogenic differentiation of primary human bone marrow-derived mesenchymal stem cells (BMSCs)., Design: LRF was overexpressed in BMSC by lentiviral transduction. Chondrogenic differentiation of BMSC was induced by high-density pellet culture. Western blotting and real-time polymerase chain reaction were used to investigate changes in protein and mRNA levels, respectively, during chondrogenesis. Safranin-O staining, immunohistochemistry, and glycoaminoglycan contents were used to assess cartilage matrix deposition. BMSC proliferation was determined by mitochondrial dehydrogenase activity and cell counting. Cell cycle profiling was performed by flow cytometry., Results: LRF overexpression effectively inhibited protein and mRNA expression of chondrocyte markers and cartilage matrix deposition during chondrogenesis of BMSC. Endogenous LRF expression was constitutively high in undifferentiated BMSC but remained low in primary articular chondrocytes. Endogenous LRF protein was downregulated in a time-dependent manner during chondrogenesis. BMSCs overexpressing LRF had higher proliferation rates and cell population in the S phase. LRF suppressed p53 expression during chondrogenesis and this might prevent differentiating chondrocytes from entering a quiescent state., Conclusion: Our data showed that LRF is important for stimulating stem cell proliferation and cell cycle progression. It is known that LRF is highly expressed in the mouse limb buds prior to overt chondrogenesis; thus, LRF might function to prevent premature chondrogenic differentiation of stem cells.
- Published
- 2013
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36. Cartilage oligomeric matrix protein enhances osteogenesis by directly binding and activating bone morphogenetic protein-2.
- Author
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Ishida K, Acharya C, Christiansen BA, Yik JH, DiCesare PE, and Haudenschild DR
- Subjects
- Animals, Bone Morphogenetic Protein Receptors metabolism, Cations pharmacology, Cell Line, Choristoma metabolism, Choristoma pathology, Disease Models, Animal, Humans, Hydrogen-Ion Concentration drug effects, Luciferases metabolism, Manganese pharmacology, Mesenchymal Stem Cells cytology, Mesenchymal Stem Cells drug effects, Mesenchymal Stem Cells metabolism, Mice, Protein Binding drug effects, Rats, Signal Transduction drug effects, Smad Proteins metabolism, Transcription, Genetic drug effects, Transforming Growth Factor beta metabolism, Bone Morphogenetic Protein 2 metabolism, Matrilin Proteins metabolism, Osteogenesis drug effects
- Abstract
Bone morphogenetic proteins (BMPs) are effective for bone regeneration, and are used clinically. However, supraphysiological doses are required, which limits their use. Cartilage oligomeric matrix protein is an extracellular matrix protein, which we have previously shown can bind to growth factors of the TGFs family, suggesting that COMP may also bind to BMP-2. Rather than being a passive component of the matrix, COMP may serve as an "instructive matrix" component capable of increasing local growth factor concentration, slowing the diffusion of growth factors, and promoting their biological activity. The purpose of this investigation was to determine whether COMP binds to BMP-2, and whether it promotes the biological activity of BMP-2 with respect to osteogenesis. We found that COMP binds BMP-2, and characterized the biochemical nature of the binding interaction. COMP binding enhanced BMP-2-induced intracellular signaling through Smad proteins, increased the levels of BMP receptors, and up-regulated the luciferase activity from a BMP-2-responsive reporter construct. COMP binding enhanced BMP-2-dependent osteogenesis in vitro, in the C2C12 cell line and in primary human bone mesenchymal stem cells, as measured by alkaline phosphatase activity, matrix mineralization, and gene expression. Finally, we found that COMP enhanced BMP-2-dependent ectopic bone formation in a rat model assessed histologically, by alkaline phosphatase activity, gene expression, and micro-CT. In summary, this study demonstrates that COMP enhances the osteogenic activity of BMP-2, both in-vitro and in-vivo., (Copyright © 2013 Elsevier Inc. All rights reserved.)
- Published
- 2013
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37. The oncogene LRF is a survival factor in chondrosarcoma and contributes to tumor malignancy and drug resistance.
- Author
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Kumari R, Li H, Haudenschild DR, Fierro F, Carlson CS, Overn P, Gupta L, Gupta K, Nolta J, Yik JH, and Di Cesare PE
- Subjects
- Antibiotics, Antineoplastic pharmacology, Blotting, Western, Bone Neoplasms drug therapy, Bone Neoplasms metabolism, Cell Adhesion drug effects, Cell Cycle drug effects, Cell Movement drug effects, Cell Proliferation drug effects, Chondrosarcoma drug therapy, Chondrosarcoma metabolism, Cyclin-Dependent Kinase Inhibitor p21 metabolism, DNA-Binding Proteins antagonists & inhibitors, DNA-Binding Proteins genetics, Humans, Immunoenzyme Techniques, Neoplasm Grading, Prognosis, RNA, Small Interfering genetics, Tissue Array Analysis, Transcription Factors antagonists & inhibitors, Transcription Factors genetics, Tumor Cells, Cultured, Tumor Suppressor Protein p53 metabolism, Apoptosis drug effects, Bone Neoplasms pathology, Chondrosarcoma pathology, DNA-Binding Proteins metabolism, Doxorubicin pharmacology, Drug Resistance, Neoplasm, Oncogenes, Transcription Factors metabolism
- Abstract
Chondrosarcoma is a form of malignant skeletal tumor of cartilaginous origin. The non-malignant form of the disease is termed chondroma. Correctly distinguishing between the two forms is essential for making therapeutic decisions. However, due to their similar histological appearances and the lack of a reliable diagnostic marker, it is often difficult to distinguish benign tumors from low-grade chondrosarcoma. Therefore, it is necessary to search for a potential marker that has diagnostic and prognostic values in chondrosarcoma. In this study, we demonstrated by immunohistochemistry that elevated leukemia/lymphoma-related factor (LRF) expression was associated with increased malignancy in human chondrosarcoma tissue microarrays. Moreover, siRNA depletion of LRF drastically reduced proliferation of chondrosarcoma cell lines and effectively induced senescence in these cells. This could be attributed to the observation that LRF-depleted cells were arrested at the G(1) phase, and had increased p53 and p21 expression. Moreover, LRF depletion not only drastically reduces the cellular migration and invasion potentials of chondrosarcoma cells but also sensitized these cells to the apoptosis-inducing chemotherapeutic agent doxorubicin. We conclude that LRF is a survival factor in chondrosarcomas and its expression correlates with tumor malignancy and chemoresistance. Our data implicate the potential role of LRF as both a diagnostic marker and therapeutic target for chondrosarcomas.
- Published
- 2012
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38. Recruitment of P-TEFb for stimulation of transcriptional elongation by the bromodomain protein Brd4.
- Author
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Yang Z, Yik JH, Chen R, He N, Jang MK, Ozato K, and Zhou Q
- Subjects
- Base Sequence, Cyclin-Dependent Kinase 9 genetics, Cyclin-Dependent Kinase 9 metabolism, Gene Products, tat genetics, HIV-1 genetics, HeLa Cells, Humans, Molecular Sequence Data, Nuclear Proteins, Oncogene Proteins, Fusion genetics, Transcription Factors, Transcription, Genetic, tat Gene Products, Human Immunodeficiency Virus, Oncogene Proteins, Fusion metabolism, Positive Transcriptional Elongation Factor B metabolism, RNA Polymerase II metabolism
- Abstract
The cyclinT1/Cdk9 heterodimer that constitutes core P-TEFb is generally presumed to be the transcriptionally active form for stimulating RNA polymerase II elongation. About half of cellular P-TEFb also exists in an inactive complex with the 7SK snRNA and the HEXIM1 protein. Here, we show that the remaining half associates with the bromodomain protein Brd4. In stress-induced cells, the 7SK/HEXIM1-bound P-TEFb is quantitatively converted into the Brd4-associated form. The association with Brd4 is necessary to form the transcriptionally active P-TEFb, recruits P-TEFb to a promoter, and enables P-TEFb to contact the Mediator complex, a potential target for the Brd4-mediated recruitment. Although generally required for transcription, the P-TEFb-recruitment function of Brd4 can be substituted by that of HIV-1 Tat, which recruits P-TEFb directly for activated HIV-1 transcription. Brd4, HEXIM1, and 7SK are all implicated in regulating cell growth, which may result from their dynamic control of the general transcription factor P-TEFb.
- Published
- 2005
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- View/download PDF
39. Inhibition of P-TEFb (CDK9/Cyclin T) kinase and RNA polymerase II transcription by the coordinated actions of HEXIM1 and 7SK snRNA.
- Author
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Yik JH, Chen R, Nishimura R, Jennings JL, Link AJ, and Zhou Q
- Subjects
- Acetamides pharmacology, Binding Sites genetics, Gene Expression Regulation, Enzymologic drug effects, Gene Expression Regulation, Enzymologic genetics, HeLa Cells, Humans, Positive Transcriptional Elongation Factor B antagonists & inhibitors, Positive Transcriptional Elongation Factor B genetics, RNA Polymerase II genetics, RNA-Binding Proteins genetics, RNA-Binding Proteins pharmacology, Stress, Physiological chemically induced, Stress, Physiological enzymology, Stress, Physiological genetics, Transcription Factors, Transcription, Genetic drug effects, Positive Transcriptional Elongation Factor B metabolism, RNA Polymerase II metabolism, RNA, Small Nuclear genetics, RNA-Binding Proteins metabolism, Transcription, Genetic genetics
- Abstract
The positive transcriptional elongation factor b (P-TEFb), consisting of CDK9 and cyclin T, stimulates transcription by phosphorylating RNA polymerase II. It becomes inactivated when associated with the abundant 7SK snRNA. Here, we show that the 7SK binding alone was not sufficient to inhibit P-TEFb. P-TEFb was inhibited by the HEXIM1 protein in a process that specifically required 7SK for mediating the HEXIM1:P-TEFb interaction. This allowed HEXIM1 to inhibit transcription both in vivo and in vitro. P-TEFb dissociated from HEXIM1 and 7SK in cells undergoing stress response, increasing the level of active P-TEFb for stress-induced transcription. P-TEFb was the predominant HEXIM1-associated protein factor, and thus likely to be the principal target of inhibition coordinated by HEXIM1 and 7SK. Since HEXIM1 expression is induced in cells treated with hexamethylene bisacetamide, a potent inducer of cell differentiation, targeting the general transcription factor P-TEFb by HEXIM1/7SK may contribute to the global control of cell growth and differentiation.
- Published
- 2003
- Full Text
- View/download PDF
40. The position of cysteine relative to the transmembrane domain is critical for palmitoylation of H1, the major subunit of the human asialoglycoprotein receptor.
- Author
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Yik JH and Weigel PH
- Subjects
- Alanine chemistry, Amino Acid Motifs, Amino Acid Sequence, Animals, Asialoglycoprotein Receptor chemistry, COS Cells, Cytoplasm metabolism, DNA, Complementary metabolism, Humans, Molecular Sequence Data, Mutation, Palmitic Acid metabolism, Plasmids metabolism, Protein Binding, Protein Structure, Tertiary, Sequence Homology, Amino Acid, Transfection, Asialoglycoprotein Receptor metabolism, Cell Membrane metabolism, Cysteine chemistry, Palmitic Acids chemistry
- Abstract
The mammalian hepatic asialoglycoprotein receptor (ASGP-R) is an endocytic recycling receptor that mediates the internalization of desialylated glycoproteins and their delivery to lysosomes where they are degraded. The human ASGP-R is a hetero-oligomeric complex composed of two subunits designated H1 and H2. Both subunits are palmitoylated at the cytoplasmic Cys residues near their transmembrane domains (TMD). The cytoplasmic Cys(36) in H1 is located at a position that is five amino acids from the transmembrane junction. Because the sequences of subunits in all mammalian ASGP-R species are highly conserved especially at the region near the palmitoylated Cys, we sought to identify a recognition signal for the palmitoylation of H1. Various types of H1 mutants were created by site-directed or deletion mutagenesis including alteration of the amino acids surrounding Cys(36), replacing portions of the TMD with that of a different protein and partial deletion of the cytoplasmic domain as well as transposing the palmitoylated Cys to positions further away from the TMD. Mutant H1 cDNAs were transiently expressed in COS-7 cells, and the H1 proteins were analyzed after metabolic labeling with [(3)H]palmitate. The results indicate that neither the native amino acid sequence surrounding Cys(36) nor the majority of the cytoplasmic domain sequence is critical for palmitoylation. Palmitoylation was also not dependent on the native TMD of H1. In contrast, the attachment of palmitate was abolished if the Cys residue was transposed to a position that was 30 amino acids away from the transmembrane border. We conclude that the spacing of a Cys residue relative to the TMD in the primary protein sequence of H1 is the major determinant for successful palmitoylation.
- Published
- 2002
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41. Palmitoylation-defective asialoglycoprotein receptors are normal in their cellular distribution and ability to bind ligand, but are defective in ligand uptake and degradation.
- Author
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Yik JH, Saxena A, Weigel JA, and Weigel PH
- Subjects
- Asialoglycoprotein Receptor genetics, Binding Sites, Biological Transport, Cell Line, Humans, Iodine Radioisotopes, Kinetics, Ligands, Microscopy, Confocal, Mutagenesis, Site-Directed, Osmolar Concentration, Radioisotope Dilution Technique, Recombinant Proteins chemistry, Recombinant Proteins metabolism, Transfection, Tumor Cells, Cultured, Asialoglycoprotein Receptor metabolism, Asialoglycoproteins metabolism, Palmitic Acid metabolism
- Abstract
The hepatic asialoglycoprotein receptor (ASGP-R) is an endocytic recycling receptor that mediates the endocytosis of desialylated glycoproteins. The human ASGP-R is composed of two homologous subunits, H1 and H2, and the cytoplasmic Cys residues in both subunits are palmitoylated. To study the effects of palmitoylation on ASGP-R activity and function, we generated four types of stably transfected cell lines in SK-Hep-1 hepatoma cells, expressing wild-type, or partially or completely palmitoylation-defective ASGP-Rs containing Cys-to-Ser mutations in either one or both subunits. Scatchard analysis showed that all four stable cell lines expressed a similar number of binding sites for asialo-orosomucoid, with comparable dissociation constants of approximately 1-3nM. Immunofluorescence confocal microscopy indicated a normal distribution of the palmitoylation-defective H1 and H2 subunits compared to the wild-type. However, cell lines expressing palmitoylation-defective ASGP-Rs had markedly reduced rates of ligand uptake and degradation compared to cells expressing wild-type ASGP-Rs. We conclude that failure to palmitoylate Cys residues in either or both subunits of human ASGP-Rs results in very inefficient uptake and degradation of ligands.
- Published
- 2002
- Full Text
- View/download PDF
42. Glycans as endocytosis signals: the cases of the asialoglycoprotein and hyaluronan/chondroitin sulfate receptors.
- Author
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Weigel PH and Yik JH
- Subjects
- Animals, Asialoglycoprotein Receptor, Cloning, Molecular, Coated Pits, Cell-Membrane metabolism, Glycoconjugates metabolism, Humans, Hyaluronan Receptors chemistry, Hyaluronan Receptors genetics, Ligands, Receptors, Cell Surface chemistry, Receptors, Cell Surface genetics, Signal Transduction, Endocytosis, Hyaluronan Receptors physiology, Lectins physiology, Liver metabolism, Polysaccharides physiology, Receptors, Cell Surface physiology
- Abstract
Animal cells internalize specific extracellular macromolecules (ligands) by using specialized cell surface receptors that operate through a complex and highly regulated process known as receptor-mediated endocytosis, which involves the binding, internalization, and transfer of ligands through a series of distinct intracellular compartments. For the uptake of a variety of carbohydrate-containing macromolecules, such as glycoproteins, animal cells use specialized membrane-bound lectins as endocytic receptors that recognize different sugar residues or carbohydrate structures present on various ligands. The hepatic asialoglycoprotein receptor, which recognizes glycoconjugates containing terminal galactose or N-acetylgalactosamine residues, was the first membrane lectin discovered and has been a classical system for studying receptor-mediated endocytosis. Studies of how the asialoglycoprotein receptor functions have led to the discovery of two functionally distinct, parallel pathways of clathrin-mediated endocytosis (called the State 1 and State 2 pathways), which may also be utilized by all the other endocytic recycling receptor systems. Another endocytic membrane lectin, the hyaluronan/chondroitin sulfate receptor, which has recently been purified and cloned, is responsible for the turnover in mammals of these glycosaminoglycans, which are important components of extracellular matrices. We discuss the characteristics and physiological importance of these two proteins as examples of how lectins can function as endocytic receptors.
- Published
- 2002
- Full Text
- View/download PDF
43. The minor subunit splice variants, H2b and H2c, of the human asialoglycoprotein receptor are present with the major subunit H1 in different hetero-oligomeric receptor complexes.
- Author
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Yik JH, Saxena A, and Weigel PH
- Subjects
- Amino Acid Sequence, Animals, Asialoglycoprotein Receptor, Base Sequence, COS Cells, Carcinoma, Hepatocellular metabolism, Cytoplasm metabolism, DNA, Complementary metabolism, Endocytosis, Glycosylation, Humans, Hydrogen-Ion Concentration, Kinetics, Models, Genetic, Molecular Sequence Data, Oligonucleotides metabolism, Plasmids metabolism, Protein Binding, Protein Isoforms, Protein Structure, Tertiary, RNA metabolism, Rats, Rats, Sprague-Dawley, Reverse Transcriptase Polymerase Chain Reaction, Sequence Homology, Nucleic Acid, Time Factors, Transfection, Tumor Cells, Cultured, Alternative Splicing, Receptors, Cell Surface chemistry
- Abstract
The hepatic asialoglycoprotein receptor (ASGP-R) is an endocytic receptor that mediates the internalization of desialylated glycoproteins and their delivery to lysosomes. The human ASGP-R is a hetero-oligomeric complex composed of H1 and H2 subunits. There are three naturally occurring H2 splice variants, designated H2a, H2b, and H2c, although the expression of the H2c protein had not been reported. Following deglycosylation of purified ASGP-R, we detected the H2b and H2c proteins in HepG2 and HuH-7 hepatoma cells, using an antibody directed against a COOH-terminal peptide common to all H2 isoforms (anti-H2-COOH) and another antibody against a 19-amino acid cytoplasmic insert found only in H2b (anti-H2-Cyto19). H1 and both H2b and H2c were co-purified by affinity chromatography, using asialo-orosomucoid (ASOR)-, anti-H1-, or anti-H2-COOH-Sepharose, whereas only H1 and H2b were immunoprecipitated with anti-H2-Cyto19. These results indicate that H2b and H2c are not present in the same ASGP-R complexes with H1. Similar to the H2b isoform, H2c was also palmitoylated, indicating that the 19-residue cytoplasmic insert does not regulate palmitoylation. Stably transfected SK-Hep-1 cell lines expressing ASGP-R complexes containing H1 and either H2b or H2c had similar binding affinities for ASOR and endocytosed and degraded ASOR at similar rates. The pH dissociation profiles of ASOR.ASGP-R complexes were also identical for complexes containing either H2b or H2c. We conclude that the H2b and H2c isoforms are both functional but are not present with H1 in the same hetero-oligomeric ASGP-R complexes. This structural difference between two functional subpopulations of ASGP-Rs may provide a molecular basis for the existence of two different pathways, designated State 1 and State 2, by which several types of recycling receptors mediate endocytosis.
- Published
- 2002
- Full Text
- View/download PDF
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