Browsing by Author "Li, Yong"
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Item Dynamic MTORC1-TFEB feedback signaling regulates hepatic autophagy, steatosis and liver injury in long-term nutrient oversupply(Taylor & Francis, 2018) Zhang, Hao; Yan, Shengmin; Khambu, Bilon; Ma, Fengguang; Li, Yong; Chen, Xiaoyun; Puertollano, Rosa; Li, Yu; Chalasani, Naga; Yin, Xiao-Ming; Martina, Jose A.; Pathology & Laboratory Medicine, IU School of MedicineNormal metabolism requires a controlled balance between anabolism and catabolism. It is not completely known how this balance can be retained when the level of nutrient supply changes in the long term. We found that in murine liver anabolism, as represented by the phosphorylation of RPS6KB (ribosomal protein S6 kinase), was soon elevated while catabolism, as represented by TFEB (transcription factor EB)-directed gene transcription and lysosomal activities, was downregulated after the administration of a high-fat diet (HFD). Surprisingly, neither the alteration in RPS6KB phosphorylation nor that in TFEB functions was static over the long course of HFD feeding. Instead, the 2 signals exhibited dynamic alterations in opposite directions, which could be explained by the dependence of MTORC1 (MTOR complex 1) activation on TFEB-supported lysosome function and the feedback suppression of TFEB by MTORC1. Disruption of the dynamics by enforced expression of TFEB in HFD-fed mice at the peaks of MTORC1 activation restored lysosome function. Consistently, interference of MTORC1 activation with rapamycin or with a constitutively activated RRAGA mutant at the peak or nadir of MTORC1 oscillation enhanced or reduced the lysosome function, respectively. These treatments also improved or exacerbated hepatic steatosis and liver injury, respectively. Finally, there was a significant inverse correlation between TFEB activation and steatosis severity in the livers of patients with non-alcohol fatty liver diseases, supporting the clinical relevance of TFEB-regulated events. Thus, maintaining catabolic function through feedback mechanisms during enhanced anabolism, which is caused by nutrient oversupply, is important for reducing liver pathology.Item EBV-positive DLBCL frequently harbors somatic mutations associated with clonal hematopoiesis of indeterminate potential(American Society of Hematology, 2023) Li, Yong; Xu-Monette, Zijun Y.; Abramson, Jeremy; Sohani, Aliyah R.; Bhagat, Govind; Tzankov, Alexandar; Visco, Carlo; Zhang, Shanxiang; Dybkaer, Karen; Pan, Zenggang; Xu, Min; Tam, Wayne; Zu, Youli; Hsi, Eric D.; Hagemeister, Fredrick B.; Go, Heounjeong; van Krieken, J. Han; Winter, Jane N.; Ponzoni, Maurilio; Ferreri, Andrés J. M.; Møller, Michael B.; Piris, Miguel A.; Wang, Yingjun; Zhang, Mingzhi; Young, Ken H.; Pathology and Laboratory Medicine, School of MedicineItem The Generation of Closed Femoral Fractures in Mice: A Model to Study Bone Healing(JoVE, 2018-08-16) Williams, Justin N.; Li, Yong; Kambrath, Anuradha Valiya; Sankar, Uma; Anatomy and Cell Biology, School of MedicineBone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies that promote efficient bone healing are non-existent and in high demand. Effective and reproducible animal models of fractures healing are needed to understand the complex biological processes associated with bone regeneration. Many animal models of fracture healing have been generated over the years; however, murine fracture models have recently emerged as powerful tools to study bone healing. A variety of open and closed models have been developed, but the closed femoral fracture model stands out as a simple method for generating rapid and reproducible results in a physiologically relevant manner. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice and facilitate a post-fracture stabilization of the femur by inserting an intramedullary steel rod. Although devices such as a nail or a screw offer greater axial and rotational stability, the use of an intramedullary rod provides a sufficient stabilization for consistent healing outcomes without producing new defects in the bone tissue or damaging nearby soft tissue. Radiographic imaging is used to monitor the progression of callus formation, bony union, and subsequent remodeling of the bony callus. Bone healing outcomes are typically associated with the strength of the healed bone and measured with torsional testing. Still, understanding the early cellular and molecular events associated with fracture repair is critical in the study of bone tissue regeneration. The closed femoral fracture model in mice with intramedullary fixation serves as an attractive platform to study bone fracture healing and evaluate therapeutic strategies to accelerate healing.Item Hepatic Autophagy Deficiency Compromises FXR Functionality and Causes Cholestatic Injury(AASLD, 2019) Khambu, Bilon; Li, Tiangang; Yan, Shengmin; Yu, Changshun; Chen, Xiaoyun; Goheen, Michael; Li, Yong; Lin, Jingmei; Cummings, Oscar W.; Lee, Youngmin A.; Friedman, Scott; Dong, Zheng; Feng, Gen-Sheng; Wu, Shangwei; Yin, Xiao-Ming; Pathology and Laboratory Medicine, School of MedicineAutophagy is important for hepatic homeostasis, nutrient regeneration and organelle quality control. We investigated the mechanisms by which liver injury occurred in the absence of autophagy function. We found that mice deficient in autophagy due to the lack of Atg7 or Atg5, key autophagy‐related genes, manifested intracellular cholestasis with increased levels of serum bile acids, a higher ratio of TMCA/TCA in the bile, increased hepatic bile acid load, abnormal bile canaliculi and altered expression of hepatic transporters. In determining the underlying mechanism, we found that autophagy sustained and promoted the basal and upregulated expression of Fxr in the fed and starved conditions, respectively. Consequently, expression of Fxr and its downstream genes, particularly Bsep, and the binding of FXR to the promoter regions of these genes, were suppressed in autophagy‐deficient livers. In addition, co‐deletion of Nrf2 in autophagy deficiency status reversed the FXR suppression. Furthermore, the cholestatic injury of autophagy‐deficient livers was reversed by enhancement of FXR activity or expression, or by Nrf2 deletion.Item HMGB1 promotes ductular reaction and tumorigenesis in autophagy-deficient livers(American Society for Clinical Investigation, 2018-06-01) Khambu, Bilon; Huda, Nazmul; Chen, Xiaoyun; Antoine, Daniel J.; Li, Yong; Dai, Guoli; Köhler, Ulrike A.; Zong, Wei-Xing; Waguri, Satoshi; Werner, Sabine; Oury, Tim D.; Dong, Zheng; Yin, Xiao-Ming; Pathology and Laboratory Medicine, School of MedicineAutophagy is important for liver homeostasis, and the deficiency leads to injury, inflammation, ductular reaction (DR), fibrosis, and tumorigenesis. It is not clear how these events are mechanistically linked to autophagy deficiency. Here, we reveal the role of high-mobility group box 1 (HMGB1) in two of these processes. First, HMGB1 was required for DR, which represents the expansion of hepatic progenitor cells (HPCs) implicated in liver repair and regeneration. DR caused by hepatotoxic diets (3,5-diethoxycarbonyl-1,4-dihydrocollidine [DDC] or choline-deficient, ethionine-supplemented [CDE]) also depended on HMGB1, indicating that HMGB1 may be generally required for DR in various injury scenarios. Second, HMGB1 promoted tumor progression in autophagy-deficient livers. Receptor for advanced glycation end product (RAGE), a receptor for HMGB1, was required in the same two processes and could mediate the proliferative effects of HMBG1 in isolated HPCs. HMGB1 was released from autophagy-deficient hepatocytes independently of cellular injury but depended on NRF2 and the inflammasome, which was activated by NRF2. Pharmacological or genetic activation of NRF2 alone, without disabling autophagy or causing injury, was sufficient to cause inflammasome-dependent HMGB1 release. In conclusion, HMGB1 release is a critical mechanism in hepatic pathogenesis under autophagy-deficient conditions and leads to HPC expansion as well as tumor progression.Item Inhibition of CaMKK2 Enhances Fracture Healing by Stimulating Indian Hedgehog Signaling and Accelerating Endochondral Ossification(Wiley, 2018) Williams, Justin N.; Kambrath, Anuradha Valiya; Patel, Roshni B.; Kang, Kyung Shin; Mével, Elsa; Li, Yong; Cheng, Ying-Hua; Pucylowski, Austin J.; Hassert, Mariah A.; Voor, Michael J.; Kacena, Melissa A.; Thompson, William R.; Warden, Stuart J.; Burr, David B.; Allen, Matthew R.; Robling, Alexander G.; Sankar, Uma; Anatomy and Cell Biology, School of MedicineApproximately 10% of all bone fractures do not heal, resulting in patient morbidity and healthcare costs. However, no pharmacological treatments are currently available to promote efficient bone healing. Inhibition of Ca2+/calmodulin (CaM)-dependent protein kinase kinase 2 (CaMKK2) reverses age-associated loss of trabecular and cortical bone volume and strength in mice. In the current study, we investigated the role of CaMKK2 in bone fracture healing and show that its pharmacological inhibition using STO-609 accelerates early cellular and molecular events associated with endochondral ossification, resulting in a more rapid and efficient healing of the fracture. Within 7 days postfracture, treatment with STO-609 resulted in enhanced Indian hedgehog signaling, paired-related homeobox (PRX1)-positive mesenchymal stem cell (MSC) recruitment, and chondrocyte differentiation and hypertrophy, along with elevated expression of osterix, vascular endothelial growth factor, and type 1 collagen at the fracture callus. Early deposition of primary bone by osteoblasts resulted in STO-609–treated mice possessing significantly higher callus bone volume by 14 days following fracture. Subsequent rapid maturation of the bone matrix bestowed fractured bones in STO-609–treated animals with significantly higher torsional strength and stiffness by 28 days postinjury, indicating accelerated healing of the fracture. Previous studies indicate that fixed and closed femoral fractures in the mice take 35 days to fully heal without treatment. Therefore, our data suggest that STO-609 potentiates a 20% acceleration of the bone healing process. Moreover, inhibiting CaMKK2 also imparted higher mechanical strength and stiffness at the contralateral cortical bone within 4 weeks of treatment. Taken together, the data presented here underscore the therapeutic potential of targeting CaMKK2 to promote efficacious and rapid healing of bone fractures and as a mechanism to strengthen normal bones.Item Osteocyte-Derived CaMKK2 Regulates Osteoclasts and Bone Mass in a Sex-Dependent Manner through Secreted Calpastatin(MDPI, 2023-03-01) Williams, Justin N.; Irwin, Mavis; Li, Yong; Kambrath, Anuradha Valiya; Mattingly, Brett T.; Patel, Sheel; Kittaka, Mizuho; Collins, Rebecca N.; Clough, Nicholas A.; Doud, Emma H.; Mosley, Amber L.; Bellido, Teresita; Bruzzaniti, Angela; Plotkin, Lilian I.; Trinidad, Jonathan C.; Thompson, William R.; Bonewald, Lynda F.; Sankar, Uma; Anatomy, Cell Biology and Physiology, School of MedicineCalcium/calmodulin (CaM)-dependent protein kinase kinase 2 (CaMKK2) regulates bone remodeling through its effects on osteoblasts and osteoclasts. However, its role in osteocytes, the most abundant bone cell type and the master regulator of bone remodeling, remains unknown. Here we report that the conditional deletion of CaMKK2 from osteocytes using Dentine matrix protein 1 (Dmp1)-8kb-Cre mice led to enhanced bone mass only in female mice owing to a suppression of osteoclasts. Conditioned media isolated from female CaMKK2-deficient osteocytes inhibited osteoclast formation and function in in vitro assays, indicating a role for osteocyte-secreted factors. Proteomics analysis revealed significantly higher levels of extracellular calpastatin, a specific inhibitor of calcium-dependent cysteine proteases calpains, in female CaMKK2 null osteocyte conditioned media, compared to media from female control osteocytes. Further, exogenously added non-cell permeable recombinant calpastatin domain I elicited a marked, dose-dependent inhibition of female wild-type osteoclasts and depletion of calpastatin from female CaMKK2-deficient osteocyte conditioned media reversed the inhibition of matrix resorption by osteoclasts. Our findings reveal a novel role for extracellular calpastatin in regulating female osteoclast function and unravel a novel CaMKK2-mediated paracrine mechanism of osteoclast regulation by female osteocytes.Item PET imaging with [68Ga]-labeled TGFβ-targeting peptide in a mouse PANC-1 tumor model(Frontiers Media, 2023-09-15) Li, Yong; Zhao, Hong; Hu, Shan; Zhang, Xichen; Chen, Haojian; Zheng, Qihuang; Radiology and Imaging Sciences, School of MedicinePurpose: Transforming growth factor β (TGFβ) is upregulated in many types of tumors and plays important roles in tumor microenvironment construction, immune escape, invasion, and metastasis. The therapeutic effect of antibodies and nuclide-conjugated drugs targeting TGFβ has not been ideal. Targeting TGFβ with small-molecule or peptide carriers labeled with diagnostic/therapeutic nuclides is a new development direction. This study aimed to explore and confirm the imaging diagnostic efficiency of TGFβ-targeting peptide P144 coupled with [68Ga] in a PANC-1 tumor model. Procedures: TGFβ-targeting inhibitory peptide P144 with stable activity was prepared through peptide synthesis and screening, and P144 was coupled with biological chelator DOTA and labeled with radionuclide [68Ga] to achieve a stable TGFβ-targeting tracer [68Ga]Ga-P144. This tracer was first used for positron emission tomography (PET) molecular imaging study of pancreatic cancer in a mouse PANC-1 tumor model. Results: [68Ga]Ga-P144 had a high targeted uptake and relatively long uptake retention time in tumors and lower uptakes in non-target organs and backgrounds. Target pre-blocking experiment with the cold drug P144-DOTA demonstrated that the radioactive uptake with [68Ga]Ga-P144 PET in vivo, especially in tumor tissue, had a high TGFβ-targeting specificity. [68Ga]Ga-P144 PET had ideal imaging efficiency in PANC-1 tumor-bearing mice, with high specificity in vivo and good tumor-targeting effect. Conclusion: [68Ga]Ga-P144 has relatively high specificity and tumor-targeted uptake and may be developed as a promising diagnostic tool for TGFβ-positive malignancies.Item Preliminary Efficacy of a Stroke Caregiver Intervention Program for Reducing Depressive Symptoms(Office of the Vice Chancellor for Research, 2013-04-05) Bakas, Tamilyn; Austin, Joan K.; Buelow, Janice M.; Habermann, Barbara; Li, Yong; McLennon, Susan M.; Weaver, Michael T.; Williams, Linda S.Background and Purpose: Stroke caregivers who lack skills because of unmet needs are at increased risk for depressive symptoms; which can impede rehabilitation of the survivor and increase the survivor’s risk for costly, long-term institutionalization. The Telephone Assessment and Skill-Building Kit (TASK) is an 8-week follow-up program based on individualized assessment of caregiver needs. The purpose of this study was to explore preliminary efficacy of the TASK program for improving stroke caregiver depressive symptoms. Methods: A subgroup of 15 caregivers who screened positive for mild to severe depressive symptoms at baseline (PHQ-9 > 5) were randomized to the TASK program (n=8) or an attention control group (n=7). Data were analyzed using Univariate ANCOVA, controlling for baseline scores and number of minutes spent with the nurse for each timepoint (4, 8, and 12 weeks after baseline). Partial ƞ2 was used to estimate effect sizes (< .08 small, .09-.24 medium, >.25 large). Results: Although not statistically significant because of the small sample size, medium to large improvements based on effect sizes were found in depressive symptoms for the TASK group relative to the control group at 4 weeks [F(1,11) = 4.15, p=.07, ƞ2=.27], 8 weeks [F(1,11) = 1.66, p=.22, ƞ2=.13], and 12 weeks after baseline [F(1,11) = 1.47, p=.25, ƞ2 =.12]. Adjusted PHQ-9 means for the TASK group at 4, 8, and 12 weeks ranged from 4.9 to 5.9; adjusted PHQ-9 means for the control group at 4, 8, and 12 weeks ranged from 9.0 to 10.8. Conclusions: Caregivers in the TASK group reduced their depressive symptoms to the mild range, while caregivers in the control group maintained their scores primarily in the moderately depressed range. Further testing of the TASK program in a larger randomized controlled clinical trial is warranted and is currently underway.Item Systemic Inhibition or Global Deletion of CaMKK2 Protects Against Post-Traumatic Osteoarthritis(Elsevier, 2022) Mével, Elsa; Shutter, Jennifer A.; Ding, Xinchun; Mattingly, Brett T.; Williams, Justin N.; Li, Yong; Huls, Anthony; Kambrath, Anuradha Valiya; Trippel, Stephen B.; Wagner, Diane; Allen, Matthew R.; O’Keefe, Regis; Thompson, William R.; Burr, David B.; Sankar, Uma; Anatomy, Cell Biology and Physiology, School of MedicineObjective: To investigate the role of Ca2+/calmodulin-dependent protein kinase 2 (CaMKK2) in post-traumatic osteoarthritis (PTOA). Methods: Destabilization of the medial meniscus (DMM) or sham surgeries were performed on 10-week-old male wild-type (WT) and Camkk2-/- mice. Half of the DMM-WT mice and all other cohorts (n = 6/group) received tri-weekly intraperitoneal (i.p.) injections of saline whereas the remaining DMM-WT mice (n = 6/group) received i.p. injections of the CaMKK2 inhibitor STO-609 (0.033 mg/kg body weight) thrice a week. Study was terminated at 8- or 12-weeks post-surgery, and knee joints processed for microcomputed tomography imaging followed by histology and immunohistochemistry. Primary articular chondrocytes were isolated from knee joints of 4-6-day-old WT and Camkk2-/- mice, and treated with 10 ng/ml interleukin-1β (IL)-1β for 24 or 48 h to investigate gene and protein expression. Results: CaMKK2 levels and activity became elevated in articular chondrocytes following IL-1β treatment or DMM surgery. Inhibition or absence of CaMKK2 protected against DMM-associated destruction of the cartilage, subchondral bone alterations and synovial inflammation. When challenged with IL-1β, chondrocytes lacking CaMKK2 displayed attenuated inflammation, cartilage catabolism, and resistance to suppression of matrix synthesis. IL-1β-treated CaMKK2-null chondrocytes displayed decreased IL-6 production, activation of signal transducer and activator of transcription 3 (Stat3) and matrix metalloproteinase 13 (MMP13), indicating a potential mechanism for the regulation of inflammatory responses in chondrocytes by CaMKK2. Conclusions: Our findings reveal a novel function for CaMKK2 in chondrocytes and highlight the potential for its inhibition as an innovative therapeutic strategy in the prevention of PTOA.