Calmodulin-dependent Protein Kinase Kinase 2 Research Articles

EP4-induced mitochondrial localization and cell migration mediated by CALML6 in human oral squamous cell carcinoma

Lymph node metastasis, primarily caused by the migration of oral squamous cell carcinoma (OSCC) cells, stands as a crucial prognostic marker. We have previously demonstrated that EP4, a subtype of the prostaglandin E2 (PGE2) receptor, orchestrates OSCC cell migration via Ca2+ signaling. The exact mechanisms by which EP4 influences cell migration through Ca2+ signaling, however, is unclear. Our study aims to clarify how EP4 controls OSCC cell migration through this pathway. We find that activating EP4 with an agonist (ONO-AE1-473) increased intracellular Ca2+ levels and the migration of human oral cancer cells (HSC-3), but not human gingival fibroblasts (HGnF). Further RNA sequencing linked EP4 to calmodulin-like protein 6 (CALML6), whose role remains undefined in OSCC. Through protein-protein interaction network analysis, a strong connection is identified between CALML6 and calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2), with EP4 activation also boosting mitochondrial function. Overexpressing EP4 in HSC-3 cells increases experimental lung metastasis in mice, whereas inhibiting CaMKK2 with STO-609 markedly lowers these metastases. This positions CaMKK2 as a potential new target for treating OSCC metastasis. Our findings highlight CALML6 as a pivotal regulator in EP4-driven mitochondrial respiration, affecting cell migration and metastasis via the CaMKK2 pathway.

Conditional loss of CaMKK2 in Osterix-positive osteoprogenitors enhances osteoblast function in a sex-divergent manner

Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) is a multi-functional, serine/threonine protein kinase with predominant roles in inflammation, systemic energy metabolism, and bone remodeling. We previously reported that global ablation of CaMKK2 or its systemic pharmacological inhibition led to bone mass accrual in mice by stimulating osteoblasts and inhibiting osteoclasts. However, a direct, cell-intrinsic role for the kinase in the osteoblast lineage has not been established. Here we report that conditional deletion of CaMKK2 from osteoprogenitors, using the Osterix 1 (Osx1) - GFP::Cre (tetracycline-off) mouse line, resulted in increased trabecular bone mass due to an acute stimulation of osteoblast function in male and female mice. The acute simulation of osteoblasts and bone formation following conditional ablation of osteoprogenitor-derived CaMKK2 was sustained only in female mice. Periosteal bone formation at the cortical bone was enhanced only in male conditional knockout mice without altering cortical bone mass or strength. Prolonged deletion of CaMKK2 in early osteoblasts was accompanied by a stimulation of osteoclasts in both sexes, indicating a coupling effect. Notably, alterations in trabecular and cortical bone mass were absent in the doxycycline-removed “Cre-only” Osx1-GFP::Cre mice. Thus, the increase in osteoblast function at the trabecular and cortical bone surfaces following the conditional deletion of CaMKK2 in osteoprogenitors is indicative of a direct but sex-divergent role for the kinase in osteoblasts.

Abstract B033: Targeting CaMKK2 inhibits actin cytoskeletal assembly to suppress cancer metastasis

Abstract Triple-negative breast cancers (TNBCs) tend to become invasive and metastatic at early stages in their development. Despite some treatment successes in early stage localized TNBC, the rate of distant recurrence remains high, and long-term survival outcomes remain poor. In a search for new therapeutic targets for this disease, we observed that elevated expression of the serine/threonine kinase calcium/calmodulin (CaM)-dependent protein kinase kinase 2 (CaMKK2) is highly correlated with tumor invasiveness. In validation studies, genetic disruption of CaMKK2 expression or inhibition of its activity with small molecule inhibitors disrupted spontaneous metastatic outgrowth from primary tumors in murine xenograft models of TNBC. High-grade serous ovarian cancer (HGSOC), a high-risk, poor prognosis ovarian cancer subtype, shares many features with TNBC, and CaMKK2 inhibition effectively blocked metastatic progression in a validated xenograft model of this disease. Mechanistically, CaMKK2 increased the expression of the phosphodiesterase PDE1A which hydrolyzed cyclic guanosine monophosphate (cGMP) to decrease the cGMP-dependent activity of protein kinase G1 (PKG1). Inhibition of PKG1 resulted in decreased phosphorylation of vasodilator stimulated phosphoprotein (VASP), which in its hypophosphorylated state binds to and regulates F-actin assembly to facilitate cell movement. Together, these findings establish a targetable CaMKK2-PDE1A-PKG1-VASP signaling pathway that controls cancer cell motility and metastasis by impacting the actin cytoskeleton. Further, it identifies CaMKK2 as a potential therapeutic target that can be exploited to restrict tumor invasiveness in patients diagnosed with early-stage TNBC or localized HGSOC. Citation Format: Debarati Mukherjee, Rebecca A Previs, Corinne N Haines, Muthana Al Abo, Patrick K Juras, Kyle C Strickland, Binita Chakraborty, Regina Whitaker, Steven R Patierno, Jennifer A Freedman, Ching-Yi Chang, Donald P McDonnell. Targeting CaMKK2 inhibits actin cytoskeletal assembly to suppress cancer metastasis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B033.

Hepatic TRPC3 loss contributes to chronic alcohol consumption-induced hepatic steatosis and liver injury in mice

Abstract Emerging evidence discloses the involvement of calcium channel protein in the pathological process of liver diseases. Transient receptor potential cation channel subfamily C member 3 (TRPC3), a ubiquitously expressed non-selective cation channel protein, controls proliferation, inflammation, and immune response via operating calcium influx in various organs. However, our understanding on the biofunction of hepatic TRPC3 is still limited. The present study aims to clarify the role and potential mechanism(s) of TRPC3 in alcohol-associated liver disease (ALD). We recently found that TRPC3 expression plays an important role in the disease process of ALD. Alcohol exposure led to a significant reduction of hepatic TRPC3 in patients with alcohol-related hepatitis (AH) and ALD models. Antioxidants (N-acetylcysteine and mitoquinone) intervention improved alcohol-induced suppression of TRPC3 via a miR-339-5p-involved mechanism. TRPC3 loss robustly aggravated the alcohol-induced hepatic steatosis and liver injury in mouse liver; this was associated with the suppression of Ca2+/calmodulin-dependent protein kinase kinase 2 (CAMKK2)/AMP-activated protein kinase (AMPK) and dysregulation of genes related to lipid metabolism. TRPC3 loss also enhanced hepatic inflammation and early fibrosis-like change in mice. Replenishing hepatic TRPC3 effectively reversed chronic alcohol-induced detrimental alterations in ALD mice. Briefly, chronic alcohol exposure-induced TRPC3 reduction contributes to the pathological development of ALD via suppression of the CAMKK2/AMPK pathway. Oxidative stress-stimulated miR-339-5p upregulation contributes to alcohol-reduced TRPC3. TRPC3 is the requisite and a potential target to defend alcohol consumption-caused ALD.

Dihydroartemisinin inhibits liver cancer cell migration and invasion by reducing ATP synthase production through CaMKK2/NCLX.

Calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) and mitochondrial sodium/calcium exchanger protein (NCLX) are key regulatory factors in calcium homeostasis. Finding natural drugs that target regulators of calcium homeostasis is critical. Dihydroartemisinin (DHA) is considered to have anticancer effects. The present study aimed to investigate the mechanism of DHA in regulating liver cancer migration and invasion. The present study used HepG2 and HuH-7 cells and overexpressed CaMKK2 and knocked down CaMKK2 and NCLX. The antiproliferative activity of DHA on liver cancer cells was assessed through colony formation and EdU assays. Cell apoptosis was detected through YO-PRO-1/PI staining. The levels of reactive oxygen species (ROS) were measured using a ROS detection kit (DCFH-DA fluorescent probe). Cell migratory and invasive abilities were examined using wound healing and Transwell assays. The ATP production of liver cancer cells was detected using ATP fluorescent probes. Cell microfilaments were monitored for changes using Actin-Tracker Green-488. The effects of DHA on the expression of CaMKK2, NCLX, sodium/potassium-transporting ATPase subunit α-1 (ATP1A1) and ATP synthase subunit d, mitochondrial (ATP5H) were determined by western blotting and reverse transcription-quantitative PCR. The results revealed that DHA significantly inhibited proliferation, reduced ROS levels and promoted apoptosis in liver cancer cells. CaMKK2 overexpression significantly enhanced the invasive and migratory ability of liver cancer cells, whereas DHA inhibited the pro-migratory effects of CaMKK2 overexpression. DHA significantly reduced the mitochondrial ATP production and altered the arrangement of microfilaments in liver cancer cells. In addition, DHA significantly decreased the expression of CaMKK2, NCLX, ATP1A1 and ATP5H. Furthermore, by knockdown experiments of NCLX the results demonstrated that CaMKK2 downregulated the expression of ATP1A1 and ATP5H in liver cancer cells through NCLX. In conclusion, DHA may reduce ATP synthase production via the CaMKK2/NCLX signaling pathway to inhibit the invasive phenotype of liver cancer cells. It is essential to further investigate the effectiveness of DHA in the anticancer mechanism of liver cancer cells.

Neuroprotective effects of vitamin D in an Alzheimer's disease rat model: Improvement of mitochondrial dysfunction via calcium/calmodulin-dependent protein kinase kinase 2 activation of Sirtuin1 phosphorylation.

Mitochondrial dysfunction is an early event in Alzheimer's disease (AD) pathogenesis. To assess the impact of vitamin D3 (Vit.D) on neurogenesis, we investigated its role in mitigating cognitive impairment and mitochondrial dysfunction through calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2)-mediated phosphorylation of Sirtuin1 (SIRT1) in an aluminum-chloride-D-galactose (AlCl3-D-gal)-induced AD rat model. Rats were distributed into four groups: control, AlCl3 + D-gal (10 + 60 mg/kg, ip), Vit.D (500 IU/kg, po), and AlCl3 + D-gal+Vit.D. Novel object recognition (NOR), Morris Water Maze, and passive avoidance (PA) tests were used to measure memory abilities. The hippocampal tissue was used to assess vitamin D3 receptor (VDR) and peroxisome-proliferator-activated-receptor-γ-coactivator-1α (PGC-1α) expression by quantitative real-time polymerase chain reaction (qRT-PCR), CAMKK2, p-SIRT1, phosphorylated-AMP-activated protein kinase (p-AMPK), dynamin-related-protein-1 (Drp1), and mitofusin-1 (Mnf1) proteins by western blot and Ca2+ levels, endothelial nitic oxide synthase (eNOS), superoxide dismutase (SOD), amyloid beta (Aβ), and phospho tau (p-Tau) via enzyme-linked immunosorbent assay(ELISA) in addition to histological and ultrastructural examination of rat's brain tissue. Vit.D-attenuated hippocampal injury reversed the cognitive decline and Aβ aggregation, and elevated p-Tau levels in the AlCl3 + D-gal-induced AD rat model. In AlCl3 + D-gal-exposed rats, Vit.D induced VDR expression, normalized Ca2+ levels, elevated CAMKK2, p-AMPK, p-SIRT1, and PGC-1α expression. Vit.D reduced Drp1, induced Mnf1, increased mitochondrial membrane potential, preserved mitochondrial structure, restored normal mitochondrial function, and retained normal eNOS level and SOD activity in AlCl3 + D-gal rats. In conclusion, our findings proved that Vit.D may ameliorate cognitive deficits in AlCl3 + D-gal-induced AD by restoring normal mitochondrial function and reducing inflammatory and oxidative stress via CAMKK2-AMPK/SIRT1 pathway upregulation.

Inflammatory mechanisms in post-traumatic osteoarthritis: a role for CaMKK2.

Post-traumatic osteoarthritis (PTOA) is a multifactorial disease of the cartilage, synovium, and subchondral bone resulting from direct joint trauma and altered joint mechanics after traumatic injury. There are no current disease-modifying therapies for PTOA, and early surgical interventions focused on stabilizing the joint do not halt disease progression. Chronic pain and functional disability negatively affect the quality of life and take an economic toll on affected patients. While multiple mechanisms are at play in disease progression, joint inflammation is a key contributor. Impact-induced mitochondrial dysfunction and cell death or altered joint mechanics after trauma culminate in inflammatory cytokine release from synoviocytes and chondrocytes, cartilage catabolism, suppression of cartilage anabolism, synovitis, and subchondral bone disease, highlighting the complexity of the disease. Current understanding of the cellular and molecular mechanisms underlying the disease pathology has allowed for the investigation of a variety of therapeutic strategies that target unique apoptotic and/or inflammatory processes in the joint. This review provides a concise overview of the inflammatory and apoptotic mechanisms underlying PTOA pathogenesis and identifies potential therapeutic targets to mitigate disease progression. We highlight Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2), a serine/threonine protein kinase that was recently identified to play a role in murine and human osteoarthritis pathogenesis by coordinating chondrocyte inflammatory responses and apoptosis. Given its additional effects in regulating macrophage inflammatory signaling and bone remodeling, CaMKK2 emerges as a promising disease-modifying therapeutic target against PTOA.

CaMKK2 is not involved in contraction-stimulated AMPK activation and glucose uptake in skeletal muscle

ObjectiveThe 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. MethodsA 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. ResultsSTO-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. ConclusionsWe 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 alleviates myocardial ischemia/reperfusion injury by inhibiting oxidative stress and inflammation via the action on the AMPK-AKT-GSK-3β/Nrf2 signaling cascade.

Calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) can regulate numerous biological processes and is implicated in diverse pathological processes. Yet its role in myocardial ischemia/reperfusion (MI/R) injury remains unknown. This project explored the possible functions and mechanisms of CaMKK2 in MI/R injury. A rat model of MI/R in vivo was established using the left anterior descending coronary artery ligation method. Rat cardiomyocytes were exposed to hypoxia/reoxygenation (H/R) in vitro to establish a cell model. Overexpression of CaMKK2 was achieved by infecting recombinant adeno-associated virus or adenovirus expressing CaMKK2. Real-time quantitative PCR, immunoblotting, TTC staining, TUNEL assay, ELISA, oxidative stress detection assays, flow cytometry, and CCK-8 assay were carried out. A decline in CaMKK2 levels was induced by MI/R in vivo or H/R in vitro. Up-modulation of CaMKK2 in rats ameliorated the cardiac injury evoked by MI/R injury accompanied by suppression of cardiac apoptosis, oxidative stress, and proinflammatory response. Rat cardiomyocytes with CaMKK2 overexpression were also protected from H/R damage by inhibiting apoptosis, oxidative stress, and proinflammatory response. CaMKK2 overexpression led to increased phosphorylation of AMPK, AKT, and GSK-3β, and enhanced activation of Nrf2 under MI/R or H/R conditions. Inhibition of AMPK abolished CaMKK2-mediated Nrf2 activation and relevant cardioprotective effect. Restraint of Nrf2 also diminished CaMKK2-mediated relevant cardioprotective effect. Up-regulation of CaMKK2 provides a therapeutic benefit in the rat model of MI/R injury by boosting the Nrf2 pathway through regulation of AMPK/AKT/GSK-3β, which suggests CaMKK2 as a new molecular target for the treatment of MI/R injury.

Targeting CaMKK2 inhibits actin cytoskeletal assembly to suppress cancer metastasis.

Triple-negative breast cancers (TNBCs) tend to become invasive and metastatic at early stages in their development. Despite some treatment successes in early stage localized TNBC, the rate of distant recurrence remains high, and long-term survival outcomes remain poor. In a search for new therapeutic targets for this disease, we observed that elevated expression of the serine/threonine kinase calcium/calmodulin (CaM)-dependent protein kinase kinase 2 (CaMKK2) is highly correlated with tumor invasiveness. In validation studies, genetic disruption of CaMKK2 expression or inhibition of its activity with small molecule inhibitors disrupted spontaneous metastatic outgrowth from primary tumors in murine xenograft models of TNBC. High-grade serous ovarian cancer (HGSOC), a high-risk, poor prognosis ovarian cancer subtype, shares many features with TNBC, and CaMKK2 inhibition effectively blocked metastatic progression in a validated xenograft model of this disease. Mechanistically, CaMKK2 increased the expression of the phosphodiesterase PDE1A which hydrolyzed cyclic guanosine monophosphate (cGMP) to decrease the cGMP-dependent activity of protein kinase G1 (PKG1). Inhibition of PKG1 resulted in decreased phosphorylation of vasodilator stimulated phosphoprotein (VASP), which in its hypophosphorylated state binds to and regulates F-actin assembly to facilitate cell movement. Together, these findings establish a targetable CaMKK2-PDE1A-PKG1-VASP signaling pathway that controls cancer cell motility and metastasis by impacting the actin cytoskeleton. Further, it identifies CaMKK2 as a potential therapeutic target that can be exploited to restrict tumor invasiveness in patients diagnosed with early-stage TNBC or localized HGSOC.

Abstract 4835: EP4 increases the mitochondrial respiration and promotes cell migration in oral cancer

Abstract Introduction: EP4 is one of the prostaglandin E2 (PGE2) receptors. We previously reported that EP4 promoted cell migration via Ca2+ signaling, however the underlying mechanism has remained unclear. In Ca2+ signaling, Store-operated Ca2+entry (SOCE) is a major mechanism of Ca2+ import from the extracellular to the intracellular space. Orai1, one of the selective Ca2+ channels, plays an important role in SOCE. We focused on the correlation with EP4 and Orai1. Additionally, we found that EP4 promoted the phosphorylation of calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) and AMP-activated protein kinase (AMPK) located downstream of CaMKK2, i.e., cell energy regulators. AMPK activation can control positively or negatively the cellular adenosine triphosphate (ATP) supply. Therefore, we investigated how EP4 regulated the mitochondrial function via Ca2+ and promoted the cell migration in oral cancer cells. Materials and Methods: HSC-3, human tongue squamous cell carcinoma cell line was used. ONO-AE1-437 was used as EP4 agonist. HSC-3 cells were transduced with Orai1 shRNA, and control shRNA using lentivirus. The migration ability was evaluated by scratch assay. Measurement of intracellular Ca2+ concentration was measured using Fura-2. Immunoprecipitation was performed to evaluate the interaction between EP4 and Orai1. XF Cell Mito Stress Test and XF Real-Time ATP Rate Assay were performed to evaluate the mitochondrial aspiration and the ATP production using Extracellular Flux Analyzer. Results: Scratch assay showed EP4 promoted cell migration in oral cancer cells (n=4, p<0.05). In contrast, Orai1 knockdown negated EP4-induced cell migration. EP4 rapidly increased the intracellular Ca2+ concentration, while Orai1 knockdown negated EP4-indued Ca2+ increase (n=4, p<0.05). Immunoprecipitation showed that EP4 was colocalized and formed complexes with Orai1 (n=4, p<0.05). These results indicated that EP4 promoted the cell migration via Ca2+ signaling through Orai1. EP4 increased the maximal respiration 3h after the stimulation (n=6, p<0.05). ATP production rate shifted mitoATP dominant. These data indicated that EP4 increased the mitochondrial function and the respiratory capacity, potentially regulating its energy supply. These results demonstrated that EP4 promoted mitochondria respiration and then increased the energy production from mitochondria in oral cancer cells. Conclusion: We concluded that EP4 regulated the mitochondrial function via Ca2+ through Orai1 and promoted the cell migration in oral cancer cells. Citation Format: Rina Nakakaji, Masanari Umemura, Kohei Osawa, Soichiro Ishikawa, Kenji Mitsudo, Yoshihiro Ishikawa. EP4 increases the mitochondrial respiration and promotes cell migration in oral cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4835.

Calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) inhibitors: a novel approach in small molecule discovery

The calcium/calmodulin dependent protein kinase kinase 2 (CAMKK2) plays a key role in regulation of intracellular calcium levels and signaling pathways. It is involved in activation of downstream signaling pathways that regulate various cellular processes. Dysregulation of CAMKK2 activity has been linked to various diseases including cancer, suggesting that CAMKK2 inhibitors might be beneficial in oncological, metabolic and inflammatory indications. The most pressing issues in small molecule discovery are synthesis feasibility, novel chemical structure and desired biological characteristics. To circumvent this constraint, we employed ‘DrugspaceX’ for rapid lead identification, followed by repositioning seven FDA-approved drugs for CAMKK2 inhibition. Further, first-level transformation (Set1 analogues) was performed in ‘DrugspaceX’, followed by virtual screening. The t-SNE visualization revealed that the transformations surrounding Rucaparib, Treprostinil and Canagliflozin are more promising for developing CAMKK2 inhibitors. Second, using the top-ranked Set1 analogues, Set2 analogues were generated, and virtual screening revealed the top-ranked five analogues. Among the top five Set2 analogues, DE273038_5 had the lowest docking score of −11.034 kcal/mol and SA score of 2.59, retaining the essential interactions with Hotspot residues LYS194 and VAL270 across 250 ns simulation period. When compared to the other four compounds, the ligand effectiveness score was 0.409, and the number of rotatable penalties was only three. Further, DE273038_5 after two rounds of transformations was discovered to be novel and had not been previously described in other databases. These data suggest that the new candidate DE273038_5 is likely to have inhibitory activity at the CAMKK2 active site, implying potential therapeutic use. Communicated by Ramaswamy H. Sarma

Osteocyte-Derived CaMKK2 Regulates Osteoclasts and Bone Mass in a Sex-Dependent Manner through Secreted Calpastatin.

Calcium/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.

CAMKK2 is upregulated in primary human osteoarthritis and its inhibition protects against chondrocyte apoptosis

To investigate the role of calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) in human osteoarthritis. 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. 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. 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.

Geraniin targeting CaMKK2 inhibits lipid accumulation in 3T3-L1 adipocytes by suppressing lipogenesis

Obesity has become a worldwide burden and is associated with severe medical complications. Geraniin is a polyphenolic compound that has a wide range of bioactive properties. There is also evidence to support its pharmacological effects on improving lipid accumulation and obesity. This research investigates the effect of geraniin on lipid accumulation in adipocytes and the underlying mechanism. Mature adipocytes were differentiated from immature 3T3-L1 cells. Oil Red O staining and a triglyceride content determination were conducted to evaluate the intracellular lipid accumulation. Molecular docking studies were performed to determine the interaction between geraniin and the key proteins. Western blotting was used to detect the expression of lipogenic enzymes and transcription factors. Geraniin dose-dependently inhibited lipid accumulation in adipocytes by reducing the expression of fatty acid synthase and increasing the phosphorylation level of acetyl-coenzyme A carboxylase. Moreover, geraniin promoted the phosphorylation of AMP-activated protein kinase (AMPK) and further reduced the expression of lipogenic transcription factors (peroxisome proliferator-activated receptor gamma and CCAAT/enhancer binding protein alpha). The expression of the calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) was increased by the geraniin administration. The molecular docking study demonstrated that geraniin can interact with CaMKK2, which is an upstream kinase of AMPK. A selective CaMKK2 inhibitor reversed the suppressive effect of geraniin on lipogenesis. Geraniin targeted CaMKK2 to inhibit lipid accumulation in 3T3-L1 adipocytes by suppressing lipogenesis, and this supports its potential as a candidate natural anti-obesity drug.

SGC-CAMKK2-1: A Chemical Probe for CAMKK2.

The serine/threonine protein kinase calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) plays critical roles in a range of biological processes. Despite its importance, only a handful of inhibitors of CAMKK2 have been disclosed. Having a selective small molecule tool to interrogate this kinase will help demonstrate that CAMKK2 inhibition can be therapeutically beneficial. Herein, we disclose SGC-CAMKK2-1, a selective chemical probe that targets CAMKK2.

Methyl ferulic acid ameliorates ethanol-induced L02 cell steatosis through microRNA-378b-mediated CaMKK2-AMPK pathway

Alcoholic liver disease(ALD), with its increasing morbidity and mortality, has seriously and extensively affected the health of people worldwide. Methyl ferulic acid(MFA) has been proven to significantly inhibit alcohol-induced lipid production in L02 cells through the AMP-activated protein kinase(AMPK) pathway, but its in-depth mechanism remains unclear. This study aimed to further clarify the mechanism of MFA in improving lipid accumulation in L02 cells through the microRNA-378b(miR-378b)-mediated calcium/calmodulin-dependent protein kinase kinase 2(CaMKK2)-AMPK signaling pathway based on existing researches. L02 cells were induced by 100 mmol·L~(-1) ethanol for 48 h to establish the model of ALD in vitro, and 100, 50, and 25 μmol·L~(-1) concentration of MFA was treated. MiR-378b plasmids(containing the overexpression plasmid-miR-378b mimics, silence plasmid-miR-378b inhibitor, and their respective negative control-miR-378b NCs) were transfected into L02 cells by electroporation to up-regulate or down-regulate the levels of miR-378b in L02 cells. The levels of total cholesterol(TC) and triglyceride(TG) in cells were detected by commercial diagnostic kits and automatic biochemical analyzers. The expression levels of miR-378b in L02 cells were detected by real-time quantitative polymerase chain reaction(qRT-PCR). CaMKK2 mRNA levels were detected by PCR, and protein expressions of related factors involved in lipid synthesis, decomposition, and transport in lipid metabolism were detected by Western blot. The results displayed that ethanol significantly increased TG and TC levels in L02 cells, while MFA decreased TG and TC levels. Ethanol up-regulated the miR-378b level, while MFA effectively inhibited the miR-378b level. The overexpression of miR-378b led to lipid accumulation in ethanol-induced L02 cells, while the silence of miR-378b improved the lipid deposition induced by ethanol. MFA activated the CaMKK2-AMPK signaling pathway by lowering miR-378b, thus improving lipid synthesis, decomposition, and transport, which improved lipid deposition in L02 cells. This study shows that MFA improves lipid deposition in L02 cells by regulating the CaMKK2-AMPK pathway through miR-378b.

Increased CaMKK2 Expression Is an Adaptive Response That Maintains the Fitness of Tumor-Infiltrating Natural Killer Cells.

Calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) is a key regulator of energy homeostasis in several cell types. Expression of this enzyme in tumor cells promotes proliferation and migration, and expression in tumor-associated immune cells facilitates M2 macrophage polarization and the development of myeloid-derived suppressor cells. Thus, there has been interest in developing CaMKK2 inhibitors as potential anticancer therapeutics. One impediment to clinical development of these agents is that the roles of CaMKK2 in other cellular compartments within the tumor immune microenvironment remain to be established. We report herein that CaMKK2 is expressed at low basal levels in natural killer (NK) cells but is upregulated in tumor-infiltrating NK cells where it suppresses apoptosis and promotes proliferation. NK cell-intrinsic deletion of CaMKK2 increased metastatic progression in several murine models, establishing a critical role for this enzyme in NK cell-mediated antitumor immunity. Ablation of the CaMKK2 protein, but not inhibition of its kinase activity, resulted in decreased NK-cell survival. These results indicate an important scaffolding function for CaMKK2 in NK cells and suggest that competitive CaMKK2 inhibitors and ligand-directed degraders (LDD) are likely to have distinct therapeutic utilities. Finally, we determined that intracellular lactic acid is a key driver of CaMKK2 expression, suggesting that upregulated expression of this enzyme is an adaptive mechanism by which tumor-infiltrating NK cells mitigate the deleterious effects of a lactic acid-rich tumor microenvironment. The findings of this study should inform strategies to manipulate the CaMKK2-signaling axis as a therapeutic approach in cancer.

Macrophage Sprouty4 deficiency diminishes sepsis-induced acute lung injury in mice

ObjectiveInflammation and oxidative stress play critical roles in sepsis-induced acute lung injury (ALI). Sprout4 (Spry4) is involved in regulating inflammation and tissue injury; however, its role and mechanism in sepsis-induced ALI remain elusive. MethodsMacrophage-specific Spry4 knockout (Spry4MKO), transgenic (Spry4MTG) mice and matched control littermates were generated and exposed to cecum ligation and puncture (CLP) surgery to establish bacterial sepsis-induced ALI. Bone marrow-derived macrophages (BMDMs) from Spry4MKO or Spry4MTG mice were isolated and subjected to lipopolysaccharide (LPS) stimulation to further validate the role of Spry4 in vitro. To verify the necessity of AMP-activated protein kinase (AMPK), Spry4 and AMPK double knockout mice and compound C were used in vivo and in vitro. BMDMs were treated with STO-609 to inhibit calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2). ResultsWe found that macrophage Spry4 was increased in CLP mice and positively correlated with sepsis-induced ALI. Macrophage Spry4 deficiency prevented, while macrophage Spry4 overexpression exacerbated sepsis-induced inflammation, oxidative stress and ALI in mice and BMDMs. Mechanistic studies revealed that macrophage Spry4 deficiency alleviated sepsis-induced ALI through activating CaMKK2/AMPK pathway. ConclusionOur study identify macrophage Spry4 as a promising predictive and therapeutic target of sepsis-induced ALI.

Immune modulation of calcium/calmodulin dependent protein kinase kinase 2 (CaMKK2) in the ovarian tumor microenvironment (250)

Objectives: Calcium/calmodulin-dependent protein Kinase Kinase 2 (CaMKK2) has been found to be selectively expressed in cancer and immune cells of solid malignancies. Previous work has shown that high expression of CaMKK2 in ovarian cancer is associated with worse survival. Therefore, the goal of this study was to determine the effect of STO-609, a CaMKK2 inhibitor, on the immune response. Methods: Ascites from ovarian cancer patients were cultured with and without STO-609. T cell markers of activation and exhaustion were measured using flow cytometry on a BD LSRFortessa™ Cell Analyzer. Orthotopic and syngeneic murine models of ovarian cancer were utilized to determine the biologic and immunologic effects of CaMKK2 inhibition in vivo. Results: Following a seven-day culture with STO-609, we observed a decrease in co-expression of PD-1 with additional markers of T cell exhaustion (TIGIT, LAG3, and TIM3). In CD4+ T cells, the differences in average frequencies of TIGIT+, LAG3+, and TIM3+ were -4.87 (p=0.23), -3.16 (p=0.33) and -6 (p=0.23), respectively. In CD8+ T cells, the differences in average frequencies of TIGIT+, LAG3+, and TIM3+ were -7.99 (p=0.18), -0.99 (p=0.20), and -4.64 (p=0.28), respectively (Figure 1). Pharmacologic inhibition of CaMMK2 alone and with chemotherapy (cisplatin or paclitaxel) did not significantly decrease tumor growth and metastases in murine models of ovarian cancer (SKOV3ip1 and IG10). In a murine model (ID8), treated with and without STO-609 followed by checkpoint blockade, there was no significant difference in markers of T cell activation, function, or exhaustion. Objectives: Calcium/calmodulin-dependent protein Kinase Kinase 2 (CaMKK2) has been found to be selectively expressed in cancer and immune cells of solid malignancies. Previous work has shown that high expression of CaMKK2 in ovarian cancer is associated with worse survival. Therefore, the goal of this study was to determine the effect of STO-609, a CaMKK2 inhibitor, on the immune response. Methods: Ascites from ovarian cancer patients were cultured with and without STO-609. T cell markers of activation and exhaustion were measured using flow cytometry on a BD LSRFortessa™ Cell Analyzer. Orthotopic and syngeneic murine models of ovarian cancer were utilized to determine the biologic and immunologic effects of CaMKK2 inhibition in vivo. Results: Following a seven-day culture with STO-609, we observed a decrease in co-expression of PD-1 with additional markers of T cell exhaustion (TIGIT, LAG3, and TIM3). In CD4+ T cells, the differences in average frequencies of TIGIT+, LAG3+, and TIM3+ were -4.87 (p=0.23), -3.16 (p=0.33) and -6 (p=0.23), respectively. In CD8+ T cells, the differences in average frequencies of TIGIT+, LAG3+, and TIM3+ were -7.99 (p=0.18), -0.99 (p=0.20), and -4.64 (p=0.28), respectively (Figure 1). Pharmacologic inhibition of CaMMK2 alone and with chemotherapy (cisplatin or paclitaxel) did not significantly decrease tumor growth and metastases in murine models of ovarian cancer (SKOV3ip1 and IG10). In a murine model (ID8), treated with and without STO-609 followed by checkpoint blockade, there was no significant difference in markers of T cell activation, function, or exhaustion.