IU Indianapolis ScholarWorks :: Browsing by Author "Sankar, Uma"

Browsing by Author "Sankar, Uma"

Now showing 1 - 10 of 29

A Reproducible Cartilage Impact Model to Generate Post-Traumatic Osteoarthritis in the Rabbit
(MyJove Corporation, 2023-11-21) Dilley, Julian; Noori-Dokht, Hessam; Seetharam, Abhijit; Bello, Margaret; Nanavaty, Aaron; Natoli, Roman M.; McKinley, Todd; Bault, Zachary; Wagner, Diane; Sankar, Uma; Anatomy, Cell Biology and Physiology, School of Medicine
Post-traumatic osteoarthritis (PTOA) is responsible for 12% of all osteoarthritis cases in the United States. PTOA can be initiated by a single traumatic event, such as a high-impact load acting on articular cartilage, or by joint instability, as occurs with anterior cruciate ligament rupture. There are no effective therapeutics to prevent PTOA currently. Developing a reliable animal model of PTOA is necessary to better understand the mechanisms by which cartilage damage proceeds and to investigate novel treatment strategies to alleviate or prevent the progression of PTOA. This protocol describes an open, drop tower-based rabbit femoral condyle impact model to induce cartilage damage. This model delivered peak loads of 579.1 ± 71.1 N, and peak stresses of 81.9 ± 10.1 MPa with a time-to-peak load of 2.4 ± 0.5 ms. Articular cartilage from impacted medial femoral condyles (MFCs) had higher rates of apoptotic cells (p = 0.0058) and possessed higher Osteoarthritis Research Society International (OARSI) scores of 3.38 ± 1.43 compared to the non-impacted contralateral MFCs (0.56 ± 0.42), and other cartilage surfaces of the impacted knee (p < 0.0001). No differences in OARSI scores were detected among the non-impacted articular surfaces (p > 0.05).
Androgen Receptor-CaMKK2 Axis in Prostate Cancer and Bone Microenvironment
(Frontiers Media, 2018-06-18) Dadwal, Ushashi C.; Chang, Eric S.; Sankar, Uma; Anatomy and Cell Biology, School of Medicine
The skeletal system is of paramount importance in advanced stage prostate cancer (PCa) as it is the preferred site of metastasis. Complex mechanisms are employed sequentially by PCa cells to home to and colonize the bone. Bone-resident PCa cells then recruit osteoblasts (OBs), osteoclasts (OCs), and macrophages within the niche into entities that promote cancer cell growth and survival. Since PCa is heavily reliant on androgens for growth and survival, androgen-deprivation therapy (ADT) is the standard of care for advanced disease. Although it significantly improves survival rates, ADT detrimentally affects bone health and significantly increases the risk of fractures. Moreover, whereas the majority patients with advanced PCa respond favorably to androgen deprivation, most experience a relapse of the disease to a hormone-refractory form within 1-2 years of ADT. The tumor adapts to surviving under low testosterone conditions by selecting for mutations in the androgen receptor (AR) that constitutively activate it. Thus, AR signaling remains active in PCa cells and aids in its survival under low levels of circulating androgens and additionally allows the cancer cells to manipulate the bone microenvironment to fuel its growth. Hence, AR and its downstream effectors are attractive targets for therapeutic interventions against PCa. Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2), was recently identified as a key downstream target of AR in coordinating PCa cell growth, survival, and migration. Additionally, this multifunctional serine/threonine protein kinase is a critical mediator of bone remodeling and macrophage function, thus emerging as an attractive therapeutic target downstream of AR in controlling metastatic PCa and preventing ADT-induced bone loss. Here, we discuss the role played by AR-CaMKK2 signaling axis in PCa survival, metabolism, cell growth, and migration as well as the cell-intrinsic roles of CaMKK2 in OBs, OCs, and macrophages within the bone microenvironment.
CaMKK2 is not involved in contraction-stimulated AMPK activation and glucose uptake in skeletal muscle
(Elsevier, 2023) Negoita, Florentina; Addinsall, Alex B.; Hellberg, Kristina; Bringas, Conchita Fraguas; Hafen, Paul S.; Sermersheim, Tyler J.; Agerholm, Marianne; Lewis, Christopher T. A.; Ahwazi, Danial; Ling, Naomi X. Y.; Larsen, Jeppe K.; Deshmukh, Atul S.; Hossain, Mohammad A.; Oakhill, Jonathan S.; Ochala, Julien; Brault, Jeffrey J.; Sankar, Uma; Drewry, David H.; Scott, John W.; Witczak, Carol A.; Sakamoto, Kei; Anatomy, Cell Biology and Physiology, School of Medicine
Objective: The AMP-activated protein kinase (AMPK) gets activated in response to energetic stress such as contractions and plays a vital role in regulating various metabolic processes such as insulin-independent glucose uptake in skeletal muscle. The main upstream kinase that activates AMPK through phosphorylation of α-AMPK Thr172 in skeletal muscle is LKB1, however some studies have suggested that Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) acts as an alternative kinase to activate AMPK. We aimed to establish whether CaMKK2 is involved in activation of AMPK and promotion of glucose uptake following contractions in skeletal muscle. Methods: A recently developed CaMKK2 inhibitor (SGC-CAMKK2-1) alongside a structurally related but inactive compound (SGC-CAMKK2-1N), as well as CaMKK2 knock-out (KO) mice were used. In vitro kinase inhibition selectivity and efficacy assays, as well as cellular inhibition efficacy analyses of CaMKK inhibitors (STO-609 and SGC-CAMKK2-1) were performed. Phosphorylation and activity of AMPK following contractions (ex vivo) in mouse skeletal muscles treated with/without CaMKK inhibitors or isolated from wild-type (WT)/CaMKK2 KO mice were assessed. Camkk2 mRNA in mouse tissues was measured by qPCR. CaMKK2 protein expression was assessed by immunoblotting with or without prior enrichment of calmodulin-binding proteins from skeletal muscle extracts, as well as by mass spectrometry-based proteomics of mouse skeletal muscle and C2C12 myotubes. Results: STO-609 and SGC-CAMKK2-1 were equally potent and effective in inhibiting CaMKK2 in cell-free and cell-based assays, but SGC-CAMKK2-1 was much more selective. Contraction-stimulated phosphorylation and activation of AMPK were not affected with CaMKK inhibitors or in CaMKK2 null muscles. Contraction-stimulated glucose uptake was comparable between WT and CaMKK2 KO muscle. Both CaMKK inhibitors (STO-609 and SGC-CAMKK2-1) and the inactive compound (SGC-CAMKK2-1N) significantly inhibited contraction-stimulated glucose uptake. SGC-CAMKK2-1 also inhibited glucose uptake induced by a pharmacological AMPK activator or insulin. Relatively low levels of Camkk2 mRNA were detected in mouse skeletal muscle, but neither CaMKK2 protein nor its derived peptides were detectable in mouse skeletal muscle tissue. Conclusions: We demonstrate that pharmacological inhibition or genetic loss of CaMKK2 does not affect contraction-stimulated AMPK phosphorylation and activation, as well as glucose uptake in skeletal muscle. Previously observed inhibitory effect of STO-609 on AMPK activity and glucose uptake is likely due to off-target effects. CaMKK2 protein is either absent from adult murine skeletal muscle or below the detection limit of currently available methods.
CAMKK2 is Upregulated in Primary Human Osteoarthritis and its Inhibition Protects Against Chondrocyte Apoptosis
(Elsevier, 2023) Dilley, Julian E.; Seetharam, Abhijit; Ding, Xinchun; Bello, Margaret A.; Shutter, Jennifer; Burr, David B.; Natoli, Roman M.; McKinley, Todd O.; Sankar, Uma; Anatomy, Cell Biology and Physiology, School of Medicine
Objective: To investigate the role of calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) in human osteoarthritis. Materials and methods: Paired osteochondral plugs and articular chondrocytes were isolated from the relatively healthier (intact) and damaged portions of human femoral heads collected from patients undergoing total hip arthroplasty for primary osteoarthritis (OA). Cartilage from femoral plugs were either flash frozen for gene expression analysis or histology and immunohistochemistry. Chondrocyte apoptosis in the presence or absence of CAMKK2 inhibition was measured using flow cytometry. CAMKK2 overexpression and knockdown in articular chondrocytes were achieved via Lentivirus- and siRNA-mediated approaches respectively, and their effect on pro-apoptotic and cartilage catabolic mechanisms was assessed by immunoblotting. Results: CAMKK2 mRNA and protein levels were elevated in articular chondrocytes from human OA cartilage compared to paired healthier intact samples. This increase was associated with elevated catabolic marker matrix metalloproteinase 13 (MMP-13), and diminished anabolic markers aggrecan (ACAN) and type II collagen (COL2A1) levels. OA chondrocytes displayed enhanced apoptosis, which was suppressed following pharmacological inhibition of CAMKK2. Levels of MMP13, pSTAT3, and the pro-apoptotic marker BAX became elevated when CAMKK2, but not its kinase-defective mutant was overexpressed, whereas knockdown of the kinase decreased the levels of these proteins. Conclusions: CAMKK2 is upregulated in human OA cartilage and is associated with elevated levels of pro-apoptotic and catabolic proteins. Inhibition or knockdown of CAMKK2 led to decreased chondrocyte apoptosis and catabolic protein levels, whereas its overexpression elevated them. CAMKK2 may be a therapeutic target to prevent or mitigate human OA.
CaMKK2 Knockout Bone Marrow Cells Collected/Processed in Low Oxygen (Physioxia) Suggests CaMKK2 as a Hematopoietic Stem to Progenitor Differentiation Fate Determinant
(Springer, 2022) Broxmeyer, Hal E.; Ropa, James; Capitano, Maegan L.; Cooper, Scott; Racioppi, Luigi; Sankar, Uma; Microbiology and Immunology, School of Medicine
Little is known about a regulatory role of CaMKK2 for hematopoietic stem (HSC) and progenitor (HPC) cell function. To assess this, we used Camkk2−/− and wild type (WT) control mouse bone marrow (BM) cells. BM cells were collected/processed and compared under hypoxia (3% oxygen; physioxia) vs. ambient air (~21% oxygen). Subjecting cells collected to ambient air, even for a few minutes, causes a stress that we termed Extra Physiological Shock/Stress (EPHOSS) that causes differentiation of HSCs and HPCs. We consider physioxia collection/processing a more relevant way to assess HSC/HPC numbers and function, as the cells remain in an oxygen tension closer physiologic conditions. Camkk2−/− cells collected/processed at 3% oxygen had positive and negative effects respectively on HSCs (by engraftment using competitive transplantation with congenic donor and competitor cells and lethally irradiated congenic recipient mice), and HPCs (by colony forming assays of CFU-GM, BFU-E, and CFU-GEMM) compared to WT cells processed in ambient air. Thus, with cells collected/processed under physioxia, and therefore never exposed and naïve to ambient air conditions, CaMKK2 not only appears to act as an HSC to HPC differentiation fate determinant, but as we found for other intracellular mediators, the Camkk−/− mouse BM cells were relatively resistant to effects of EPHOSS. This information is of potential use for modulation of WT BM HSCs and HPCs for future clinical advantage.
CaMKK2 Signaling in Metabolism and Skeletal Disease: a New Axis with Therapeutic Potential
(Springer Nature, 2019-08) Williams, Justin N.; Sankar, Uma; Anatomy and Cell Biology, School of Medicine
PURPOSE OF REVIEW: Age and metabolic disorders result in the accumulation of advanced glycation endproducts (AGEs), oxidative stress, and inflammation, which cumulatively cause a decline in skeletal health. Bone becomes increasingly vulnerable to fractures and its regenerative capacity diminishes under such conditions. With a rapidly aging population in the USA and the global increase in diabetes, efficacious, multi-dimensional therapies that can treat or prevent skeletal diseases associated with metabolic dysfunction and inflammatory disorders are acutely needed. RECENT FINDINGS: Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) is a key regulator of nutrient intake, glucose metabolism, insulin production, and adipogenesis. Recent studies suggest a pivotal role for CaMKK2 in bone metabolism, fracture healing, and inflammation. Aside from rekindling previous concepts of CaMKK2 as a potent regulator of whole-body energy homeostasis, this review emphasizes CaMKK2 as a potential therapeutic target to treat skeletal diseases that underlie metabolic conditions and inflammation.
CaMKK2 Signaling in Metabolism and Skeletal Disease: A New Axis with Therapeutic Potential
(2022-07) Williams, Justin N.; Sankar, Uma; Evans-Molina, Carmella; Bonewald, Lynda; Burr, David; Allen, Matthew
Type 2 diabetes mellitus (T2DM) is a growing problem globally and is associated with increased fracture risk and delayed bone healing. Novel approaches are needed in the treatment of T2DM and the resulting diabetic osteopathy. Recent studies highlight the role of bone as an endocrine organ producing factors that communicate with distant tissues to modulate systemic glucose metabolism. Ca2+/calmodulin (CaM)-dependent protein kinase kinase 2 (CaMKK2) is a potent regulator of whole-body energy metabolism, inflammation, bone remodeling and fracture healing. Genetic ablation of CaMKK2 protects from diet-induced obesity, insulin resistance and inflammation, while enhancing pancreatic β cell survival and insulin secretion. Deletion or inhibition of CaMKK2 promotes bone accrual by stimulating osteoblast-mediated bone formation and suppressing osteoclast-mediated bone resorption; however, its specific role in osteocytes, the master regulator of bone remodeling remains unknown. Here we demonstrate that conditional deletion of CaMKK2 from osteocytes enhances bone mass in 3-month-old female, but not male mice, due to suppression of osteoclasts. Conditioned media experiments and proteomics analysis revealed that female osteocytes lacking CaMKK2 suppressed osteoclast formation and function through enhanced secretion of calpastatin, a potent inhibitor of calpains, which are calciumdependent cysteine proteases that support osteoclasts. Further, to determine if CaMKK2- deficient osteocytes regulate whole-body glucose homeostasis, we placed these mice on a high-fat diet (HFD) for a period of 16 weeks. Although the diet did not significantly impact bone mass or strength, we found that conditional deletion of CaMKK2 in osteocytes enhanced bone microarchitecture in 6-month-old male and female mice. We also observed that conditional deletion of CaMKK2 from osteocytes protected male and female mice from HFD-induced obesity and insulin insensitivity. Taken together, these findings highlight CaMKK2 as a potent regulator of osteocyte-mediated modulation of bone remodeling and whole-body energy metabolism.
Characterization of a Novel Hunk Inhibitor in HER2+ Breast Cancer
(2024-07) Dilday, Tinslee Y.; Yeh, Elizabeth; Fehrenbacher, Jill; Brustovetsy, Nickolay; Safa, Ahmad; Sankar, Uma
Human Epidermal Growth Factor Receptor 2 (HER2)-targeted agents have proven to be effective, however, the development of resistance to these agents has become an obstacle in treating HER2+ breast cancer. Prior evidence implicates Hormonally Upregulated Neu-associated Kinase (HUNK) as an anti-cancer target for primary and resistant HER2+ breast cancers. An inhibitor Staurosporine (STU) has been identified as a HUNK inhibitor in HER2+ breast cancer. While STU was determined as a promising tool for inhibiting HUNK, it is a broad-spectrum kinase inhibitor and has not moved forward clinically. Therefore, identifying a more selective inhibitor of HUNK could be critical for targeting HUNK in HER2+ breast and understanding mechanisms by which HUNK promotes resistance to HER2-inhibitors. Specifically, HUNK has been implicated in promoting autophagy as a resistance mechanism in HER2+ breast cancer. Previously, we have identified that HUNK binds and phosphorylates an autophagy inhibitory protein, Rubicon, at Serine (S) 92 in 293T cells. This phosphorylation event causes Rubicon to switch to being an autophagy promoter. However, the role that Rubicon S92 plays in HER2+ breast cancer has yet to be examined. In this study, a novel inhibitor of HUNK is characterized alongside Rubicon S92 phosphorylation. This study establishes that HUNK-mediated phosphorylation of Rubicon at S92 promotes tumorigenesis in HER2/neu+ breast cancer. HUNK inhibition prevents S92 Rubicon phosphorylation in HER2/neu+ breast cancer models and inhibits both autophagy and tumorigenesis. This study characterizes a downstream phosphorylation event as a measure of HUNK activity and identifies a novel HUNK inhibitor that has meaningful efficacy toward HER2+ breast cancer.
A complete map of the Calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) signaling pathway
(Springer, 2021-06) Najar, Mohd Altaf; Rex, D.A.B.; Modi, Prashant Kumar; Agarwal, Nupur; Dagamajalu, Shobha; Karthikkeyan, Gayathree; Vijayakumar, Manavalan; Chatterjee, Aditi; Sankar, Uma; Prasad, T.S. Keshava; Anatomy and Cell Biology, School of Medicine
Calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) is a serine/threonine-protein kinase belonging to the Ca2+/calmodulin-dependent protein kinase subfamily. CAMKK2 has an autocatalytic site, which gets exposed when Ca2+/calmodulin (CAM) binds to it. This results in autophosphorylation and complete activation of CAMKK2. The three major known downstream targets of CAMKK2 are 5'-adenosine monophosphate (AMP)-activated protein kinase (AMPKα), calcium/calmodulin-dependent protein kinase 1 (CAMK1) and calcium/calmodulin-dependent protein kinase 4 (CAMK4). Activation of these targets by CAMKK2 is important for the maintenance of different cellular and physiological processes within the cell. CAMKK2 is found to be important in neuronal development, bone remodeling, adipogenesis, and systemic glucose homeostasis, osteoclastgensis and postnatal myogensis. CAMKK2 is reported to be involved in pathologies like Duchenne muscular dystrophy, inflammation, osteoporosis and bone remodeling and is also reported to be overexpressed in prostate cancer, hepatic cancer, ovarian and gastric cancer. CAMKK2 is involved in increased cell proliferation and migration through CAMKK2/AMPK pathway in prostate cancer and activation of AKT in ovarian cancer. Although CAMKK2 is a molecule of great importance, a public resource of the CAMKK2 signaling pathway is currently lacking. Therefore, we carried out detailed data mining and documentation of the signaling events associated with CAMKK2 from published literature and developed an integrated reaction map of CAMKK2 signaling. This resulted in the cataloging of 285 reactions belonging to the CAMKK2 signaling pathway, which includes 33 protein-protein interactions, 74 post-translational modifications, 7 protein translocation events, and 22 activation/inhibition events. Besides, 124 gene regulation events and 25 activator/inhibitors involved in CAMKK2 activation were also cataloged. The CAMKK2 signaling pathway map data is made freely accessible through WikiPathway database ( https://www.wikipathways.org/index.php/Pathway:WP4874 ). We expect that data on a signaling map of CAMKK2 will provide the scientific community with an improved platform to facilitate further molecular as well as biomedical investigations on CAMKK2 and its utility in the development of biomarkers and therapeutic targets.
Decreased SIRT1 Activity Is Involved in the Acute Injury Response of Chondrocytes to Ex Vivo Injurious Mechanical Overload
(MDPI, 2023-03-30) Karnik, Sonali; Noori-Dokht, Hessam; Williams, Taylor; Joukar, Amin; Trippel, Stephen B.; Sankar, Uma; Wagner, Diane R; Mechanical and Energy Engineering, School of Engineering and Technology
A better understanding of molecular events following cartilage injury is required to develop treatments that prevent or delay the onset of trauma-induced osteoarthritis. In this study, alterations to SIRT1 activity in bovine articular cartilage explants were evaluated in the 24 h following a mechanical overload, and the effect of pharmacological SIRT1 activator SRT1720 on acute chondrocyte injury was assessed. SIRT1 enzymatic activity decreased as early as 5 min following the mechanical overload, and remained suppressed for at least 24 h. The chondrocyte injury response, including apoptosis, oxidative stress, secretion of inflammatory mediators, and alterations in cartilage matrix expression, was prevented with pharmacological activation of SIRT1 in a dose-dependent manner. Overall, the results implicate SIRT1 deactivation as a key molecular event in chondrocyte injury following a mechanical impact overload. As decreased SIRT1 signaling is associated with advanced age, these findings suggest that downregulated SIRT1 activity may be common to both age-related and injury-induced osteoarthritis.

Browsing by Author "Sankar, Uma"

Results Per Page

Sort Options