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Frequent recurrence and metastasis caused by cancer stem cells (CSCs) are major challenges in lung cancer treatment. Therefore, identifying and characterizing specific CSC targets are crucial for the success of prospective targeted therapies. In this study, it is found that upregulated TOR Signaling Pathway Regulator-Like (TIPRL) in lung CSCs causes sustained activation of the calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) signaling pathway by binding to CaMKK2, thereby maintaining stemness and survival. CaMKK2-mediated activation of CaM kinase 4 (CaMK4) leads to phosphorylation of cAMP response element-binding protein (CREB) at Ser129 and Ser133, which is necessary for its maximum activation and the downstream constitutive expression of its target genes (Bcl2 and HMG20A). TIPRL depletion sensitizes lung CSCs to afatinib-induced cell death and reduces distal metastasis of lung cancer in vivo. It is determined that CREB activates the transcription of TIPRL in lung CSCs. The positive feedback loop consisting of CREB and TIPRL induces the sustained activation of the CaMKK2-CaMK4-CREB axis as a driving force and upregulates the expression of stemness- and survival-related genes, promoting tumorigenesis in patients with lung cancer. Thus, TIPRL and the CaMKK2 signaling axis may be promising targets for overcoming drug resistance and reducing metastasis in lung cancer.

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Calcium/calmodulin-dependent protein kinase IV (CaMK4) is a versatile serine/threonine kinase involved in various cellular functions. It regulates T-cell differentiation, podocyte function, tumor cell proliferation/apoptosis, ß cell mass, and insulin sensitivity. However, the underlying molecular mechanisms are complex and remain incompletely understood. The aims of this review are to highlight the latest advances in the regulatory mechanisms of CaMK4 underlying T-cell imbalance and parenchymal cell mass in multiple diseases. The structural motifs and activation of CaMK4, as well as the potential role of CaMK4 as a novel therapeutic target are also discussed.

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Calcium/calmodulin-dependent protein kinase IV (CaMK4) serves as a pivotal mediator in the regulation of gene expression, influencing the activity of transcription factors within a variety of immune cells, including T cells. Altered CaMK4 signaling is implicated in autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, and psoriasis, which are characterized by dysregulated immune responses and clinical complexity. These conditions share common disturbances in immune cell functionality, cytokine production, and autoantibody generation, all of which are associated with disrupted calcium-calmodulin signaling. This review underscores the consequences of dysregulated CaMK4 signaling across these diseases, with an emphasis on its impact on Th17 differentiation and T cell metabolism-processes central to maintaining immune homeostasis. A comprehensive understanding of roles of CaMK4 in gene regulation across various autoimmune disorders holds promise for the development of targeted therapies, particularly for diseases driven by Th17 cell dysregulation.

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Previous studies have shown that nuclear binding protein 2 (NUCB2) is expressed in the human placenta and increases with an increase in the syncytialization of trophoblast cells. This study aimed to investigate the role of NUCB2 in the differentiation and fusion of trophectoderm cells. In this study, the expression levels of NUCB2 and E-cadherin in the placentas of rats at different gestation stages were investigated. The results showed that there was an opposite trend between the expression of placental NUCB2 and E-cadherin in rat placentas in different trimesters. When primary human trophoblast (PHT) and BeWo cells were treated with high concentrations of Nesfatin-1, the trophoblast cell syncytialization was significantly inhibited. The effects of NUCB2 knockdown in BeWo cells and Forskolin-induced syncytialization were investigated. These cells showed a significantly decreased cell fusion rate. The mechanism underlying NUCB2-regulated trophoblast cell syncytialization was explored using RNA-Seq and the results indicated that the epidermal growth factor receptor (EGFR)-phospholipase C gamma 1 (PLCG1)-calmodulin-dependent protein kinase IV (CAMK4) pathway might be involved. The results suggested that the placental expression of NUCB2 plays an important role in the fusion of trophoblasts during differentiation via the EGFR-PLCG1-CAMK4 pathway.

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Background: Osteoarthritis (OA) is a common degenerative joint disease with a higher prevalence in females than in males. Sex may be a key factor affecting the progression of OA. This study aimed to investigate critical sex-difference-related genes in patients with OA and confirm their potential roles in OA regulation. Methods: OA datasets GSE12021, GSE55457, and GSE36700 were downloaded from the Gene Expression Omnibus database to screen OA-causing genes that are differentially expressed in the two sexes. Cytoscape was used to construct a protein-protein interaction network and determine hub genes. Synovial tissues of patients (male and female) with OA and female non-OA healthy controls were obtained to confirm the expression of hub genes and screen the key genes among them. Destabilization of the medial meniscus (DMM)-induced OA mice model was established to verify the screened key genes. Hematoxylin and eosin (HE) staining and Safranin O-fast green dye staining were employed to observe synovial inflammation and pathological cartilage status. Results: The abovementioned three datasets were intersected to obtain 99 overlapping differentially expressed genes, of which 77 were upregulated and 22 were downregulated in female patients with OA. The hub genes screened were EGF, AQP4, CDC42, NTRK3, ERBB2, STAT1, and CaMK4. Among them, Ca2+/calmodulin-dependent protein kinase-4 (CaMK4) was identified as a key sex-related gene for OA. It was significantly higher in female OA patients than in the cases of male patients. Moreover, CaMK4 was significantly increased in female patients with OA compared with the female non-OA group. These results suggest that CaMK4 plays an important role in the progression of OA. OA mouse models demonstrated that CaMK4 expression in the mice knee joint synovial tissue elevated after DMM, with aggravated synovial inflammation and significant cartilage damage. Cartilage damage improved after intraperitoneal administration of the CaMK4 inhibitor KN-93. Conclusions: CaMK4 is a key sex-related gene influencing the progression and pathogenesis of OA and may be considered as a new target for OA treatment.

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OBJECTIVE: To investigate the role of calcium/calmodulin-dependent protein kinase IV (CaMK4) in the development of joint injury in a mouse model of arthritis and patients with RA. METHODS: Camk4-deficient, Camk4flox/floxLck-Cre, and mice treated with CaMK4 inhibitor KN-93 or KN-93 encapsulated in nanoparticles tagged with CD4 or CD8 antibodies were subjected to collagen-induced arthritis (CIA). Inflammatory cytokine levels, humoral immune response, synovitis, and T-cell activation were recorded. CAMK4 gene expression was measured in CD4+ T cells from healthy participants and patients with active RA. Micro-CT and histology were used to assess joint pathology. CD4+ and CD14+ cells in patients with RA were subjected to Th17 or osteoclast differentiation, respectively. RESULTS: CaMK4-deficient mice subjected to CIA displayed improved clinical scores and decreased numbers of Th17 cells. KN-93 treatment significantly reduced joint destruction by decreasing the production of inflammatory cytokines. Furthermore, Camk4flox/floxLck-Cre mice and mice treated with KN93-loaded CD4 antibody-tagged nanoparticles developed fewer Th17 cells and less severe arthritis. CaMK4 inhibition mitigated IL-17 production by CD4+ cells in patients with RA. The number of in vitro differentiated osteoclasts from CD14+ cells in patients with RA was significantly decreased with CaMK4 inhibitors. CONCLUSION: Using global and CD4-cell-targeted pharmacologic approaches and conditionally deficient mice, we demonstrate that CaMK4 is important in the development of arthritis. Using ex vivo cell cultures from patients with RA, CaMK4 is important for both Th17 generation and osteoclastogenesis. We propose that CaMK4 inhibition represents a new approach to control the development of arthritis.

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Stroke is a major cause of death and disability throughout the world. A combination of Panax Ginseng and Ginkgo biloba extracts (CGGE) is an effective treatment for nervous system diseases, but the neuroprotective mechanism underlying CGGE remains unclear. Both network analysis and experimental research were employed to explore the potential mechanism of CGGE in treating ischemic stroke (IS). Network analysis identified a total number of 133 potential targets for 34 active ingredients and 239 IS-related targets. What's more, several processes that might involve the regulation of CGGE against IS were identified, including long-term potentiation, cAMP signaling pathway, neurotrophin signaling pathway, and Nod-like receptor signaling pathway. Our studies in animal models suggested that CGGE could reduce inflammatory response by inhibiting the activity of Nod-like receptor, pyrin containing 3 (NLRP3) inflammasome, and maintain the balance of glutamate (Glu)/gamma-aminobutyric acid (GABA) via activating calmodulin-dependent protein kinase type Ⅳ (CAMK4)/cyclic AMP-responsive element-binding protein (CREB) pathway. These findings indicated the neuroprotective effects of CGGE, possibly improving neuroinflammation and excitotoxicity by regulating the NLRP3 inflammasome and CAMK4/CREB pathway.

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Autosomal dominant polycystic kidney disease (ADPKD) is characterized by progressive enlargement of fluid-filled cysts, causing nephron loss and a decline in renal function. Mammalian target of rapamycin (mTOR) is overactive in cyst-lining cells and contributes to abnormal cell proliferation and cyst enlargement; however, the mechanism for mTOR stimulation remains unclear. We discovered that calcium/calmodulin (CaM) dependent kinase IV (CaMK4), a multifunctional kinase, is overexpressed in the kidneys of ADPKD patients and PKD mouse models. In human ADPKD cells, CaMK4 knockdown reduced mTOR abundance and the phosphorylation of ribosomal protein S6 kinase (S6K), a downstream target of mTOR. Pharmacologic inhibition of CaMK4 with KN-93 reduced phosphorylated S6K and S6 levels and inhibited cell proliferation and in vitro cyst formation of ADPKD cells. Moreover, inhibition of calcium/CaM-dependent protein kinase kinase-ß and CaM, two key upstream regulators of CaMK4, also decreased mTOR signaling. The effects of KN-93 were independent of the liver kinase B1-adenosine monophosphate-activated protein kinase (AMPK) pathway, and the combination of KN-93 and metformin, an AMPK activator, had additive inhibitory effects on mTOR signaling and in vitro cyst growth. Our data suggest that increased CaMK4 expression and activity contribute to mTOR signaling and the proliferation of cystic cells of ADPKD kidneys.

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Background: Type II alveolar epithelial cell (AEC II), in addition to its roles in maintaining lung homeostasis, takes an active role in inflammatory response during acute lung injury (ALI). Ca2+/calmodulin-dependent protein kinase IV (CaMK4) activated by Ca2+/calmodulin signaling, has been implicated in immune responses. This study was to investigate the roles of CaMK4 in the development of ALI and the underlying mechanisms. Methods: CaMK4 inhibitor KN-93 was used to investigate the effects of CaMK4 on NLRP3 inflammasome activation. The effects of KN-93 on disease development of lipopolysaccharide (LPS)-induced ALI were also evaluated. The role of CaMK4 on NLRP3 inflammasome activation was explored in human AEC II cell line A549 using KN-93 or CaMK4 siRNA. NLRP3 inflammasome activation was measured by histology immunofluorescence and Western blot. IL-1ß and IL-18 were measured by ELISA. Results: Phosphorylation of CaMK4 and the expression of NLRP3 and Caspase-1 p20 were increased in the lungs of LPS-induced ALI mice, which was suppressed by KN-93 as measured by Western blot. Further, the activation of NLRP3 inflammasome was detected in AEC II from patients with acute respiratory distress syndrome (ARDS) and LPS-induced ALI mice. In vitro, inhibition or silencing CaMK4 in AEC II significantly inhibited NLRP3 inflammasome activation, resulting in reduced IL-1ß production. The inhibition of NLRP3 inflammasome and decreased IL-1ß/IL-18 production by KN-93 led to reduced inflammatory infiltration and ameliorated lung injury in LPS-induced ALI mice. Conclusion: CaMK4 controls the activation of NLRP3 inflammasome in AEC II during LPS-induced ALI. CaMK4 inhibition could be a novel therapeutic approach for the treatment of ALI.

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High fructose has been reported to drive glomerular podocyte oxidative stress and then induce podocyte foot process effacement in vivo, which could be partly regarded as podocyte hypermotility in vitro. Atractylodin possesses anti-oxidative effect. The aim of this study was to explore whether atractylodin prevented against fructose-induced podocyte hypermotility via anti-oxidative property. In fructose-exposed conditionally immortalized human podocytes, we found that atractylodin inhibited podocyte hypermotility, and up-regulated slit diaphragm proteins podocin and nephrin, and cytoskeleton protein CD2-associated protein (CD2AP), α-Actinin-4 and synaptopodin expression, which were consistent with its anti-oxidative activity evidenced by up-regulation of catalase (CAT) and superoxide dismutase (SOD) 1 expression, and reduction of reactive oxygen species (ROS) production. Atractylodin also significantly suppressed expression of transient receptor potential channels 6 (TRPC6) and phosphorylated Ca2+/calmodulin-dependent protein kinase IV (CaMK4) in cultured podocytes with fructose exposure. Additionally, in fructose-exposed podocytes, CaMK4 siRNA up-regulated synaptopodin and reduced podocyte hypermotility, whereas, silencing of TRPC6 by siRNA decreased p-CaMK4 expression, inhibited podocyte hypermotility, showing TRPC6/p-CaMK4 signaling activation in podocyte hypermotility under fructose condition. Just like atractylodin, antioxidant N-acetyl-L-cysteine (NAC) could inhibit TRPC6/p-CaMK4 signaling activation to reduce fructose-induced podocytes hypermotility. These results first demonstrated that the anti-oxidative property of atractylodin may contribute to the suppression of podocyte hypermotility via inhibiting TRPC6/p-CaMK4 signaling and restoring synaptopodin expression abnormality.

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Natural products play significant roles in the development of novel drugs. One of such compounds is vanillin - a natural substance commonly used in food. Anticancer potential of the substance is continually encouraging researchers to conduct further investigations. A rising number of publications describe the role of 4-hydroxy-3-methoxybenzaldehyde (vanillin) in the process of inhibiting tumor growth. Four vanilloid receptors play significant roles in the response of cancer cells to the natural compound. Each of these proteins can be individually affected by vanillin; thus, the substance either leads to inhibition of the cell proliferation or increases the Ca2+ level. The TRPV1, a non-selective cation channel permeable to calcium, acts on cancer development and progression. Thus, vanilloid receptors have the potential to become the target for therapeutical research. Moreover, selective inhibitors of the receptor have proved their efficacy in vitro. CK2α is an antiapoptotic, cancer-sustaining protein and, therefore, the inhibitor of apoptosis. Thus, drugs that exhibit allosteric and ATP-competitive inhibition of the protein might be crucial for cancer therapy. CAMK4 is a protein kinase expression associated with a wide array of cancers. Also, MARK4 is another kinase responsible for the stability of microtubules, overexpressed in many cancer types. Studies concerning this protein revealed that microtubule impairment might be a cancer therapy direction. This review aims to demonstrate the crucial role of described vanilloid receptors in inhibiting the proliferation of cancer cells and to prove the usefulness of using vanillin and its derivatives in the process of drug design.

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Pulmonary microvascular endothelial cells (PMVECs) uniquely express an α1G-subtype of voltage-gated T-type Ca2+ channel. We have previously revealed that the α1G channel functions as a background Ca2+ entry pathway that is critical for the cell proliferation, migration, and angiogenic potential of PMVECs, a novel function attributed to the coupling between α1G-mediated Ca2+ entry and constitutive Akt phosphorylation and activation. Despite this significance, mechanism(s) that link the α1G-mediated Ca2+ entry to Akt phosphorylation remain incompletely understood. In this study, we demonstrate that Ca2+/calmodulin-dependent protein kinase (CaMK) 4 serves as a downstream effector of the α1G-mediated Ca2+ entry to promote the angiogenic potential of PMVECs. Notably, CaMK2 and CaMK4 are both expressed in PMVECs. Pharmacological blockade or genetic knockdown of the α1G channel led to a significant reduction in the phosphorylation level of CaMK4 but not the phosphorylation level of CaMK2. Pharmacological inhibition as well as genetic knockdown of CaMK4 significantly decreased cell proliferation, migration, and network formation capacity in PMVECs. However, CaMK4 inhibition or knockdown did not alter Akt phosphorylation status in PMVECs, indicating that α1G/Ca2+/CaMK4 is independent of the α1G/Ca2+/Akt pathway in sustaining the cells' angiogenic potential. Altogether, these findings suggest a novel α1G-CaMK4 signaling complex that regulates the Ca2+-dominated angiogenic potential in PMVECs.

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BACKGROUND: Atractylodis rhizoma, an aromatic herb for resolving dampness, is used to treat Kidney-related edema in traditional Chinese medicine for thousands years. This herb possesses antioxidant effect. However, it is not yet clear how Atractylodis rhizoma prevents glomerular injury through its anti-oxidation. PURPOSE: Based the analysis of Atractylodis rhizoma water extract (ARE) components and network pharmacology, this study was to explore whether ARE prevented glomerular injury via its anti-oxidation to inhibit oxidative stress-driven transient receptor potential channel 6 (TRPC6) and its downstream molecule calcium/calmodulin-dependent protein kinase IV (CaMK4) signaling. METHODS: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to analyze ARE components. Network pharmacology analysis was preliminarily performed. Male Sprague-Dawley rats were given 10% fructose drinking water (100 mL/d) for 16 weeks. ARE at 720 and 1090 mg/kg was orally administered to rats for the last 8 weeks. Hydrogen peroxide (H2O2) and malondialdehyde (MDA) level, and superoxide dismutase (SOD) activity in rat kidney cortex were detected, respectively. In rat glomeruli, redox-related factors forkhead box O3 (FoxO3), SOD2 and catalase (CAT), podocyte slit diaphragm proteins podocin and nephrin, cytoskeleton proteins CD2-associated protein (CD2AP) and α-Actinin-4, as well as TRPC6, p-CaMK4 and synaptopodin protein levels were analyzed by Western Blotting. SOD2 and CAT mRNA levels were detected by qRT-PCR. RESULTS: 36 components were identified in ARE. Among them, network pharmacology analysis indicated that ARE might inhibit kidney oxidative stress. Accordingly, ARE up-regulated nuclear FoxO3 expression, and then increased SOD2 and CAT at mRNA and protein levels in glomeruli of fructose-fed rats. It reduced H2O2 and MDA levels, and increased SOD activity in renal cortex of fructose-fed rats. Subsequently, ARE down-regulated TRPC6 and p-CaMK4, and up-regulated synaptopodin in glomeruli of fructose-fed rats. Furthermore, ARE increased podocin and nephrin, as well as CD2AP and α-Actinin-4, being consistent with its reduction of urine albumin-to-creatinine ratio and improvement of glomerular structure injury in this animal model. CONCLUSIONS: These results suggest that ARE may prevent glomerular injury in fructose-fed rats possibly by reducing oxidative stress to inhibit TRPC6/p-CaMK4 signaling and up-regulate synaptopodin expression. Therefore, ARE may be a promising drug for treating high fructose-induced glomerular injury in clinic.

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Acute and chronic kidney failure is common in hospitalized patients with COVID-19, yet the mechanism of injury and predisposing factors remain poorly understood. We investigated the role of complement activation by determining the levels of deposited complement components (C1q, C3, FH, C5b-9) and immunoglobulin along with the expression levels of the injury-associated molecules spleen tyrosine kinase (Syk), mucin-1 (MUC1) and calcium/calmodulin-dependent protein kinase IV (CaMK4) in the kidney tissues of people who succumbed to COVID-19. We report increased deposition of C1q, C3, C5b-9, total immunoglobulin, and high expression levels of Syk, MUC1 and CaMK4 in the kidneys of COVID-19 patients. Our study provides strong rationale for the expansion of trials involving the use of inhibitors of these molecules, in particular C1q, C3, Syk, MUC1 and CaMK4 to treat patients with COVID-19.

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Chronic prostatitis and chronic pelvic pain syndrome (CP/CPPS) is a complicated syndrome characterized by genitourinary pain in the absence of bacterial infection. Th17 cell-driven autoimmunity has been proposed as a cause of CP/CPPS. However, the factors that promote Th17-driven autoimmunity in experimental autoimmune prostatitis (EAP) and the molecular mechanisms are still largely unknown. Here, we showed that Th17 cells were excessively activated, and blockade of IL-17A could effectively ameliorate various symptoms in EAP. Furthermore, we revealed that calcium/calmodulin-dependent kinase Ⅳ (CaMK4), especially Thr196 p-CaMK4 was increased in the Th17 cells of the EAP group, which were activated by intracellular cytosolic Ca2+ . Pharmacologic and genetic inhibition of CaMK4 decreased the proportion of Th17 cells, and the protein and mRNA level of IL-17A, IL-22, and RORγt. The phosphorylation of CaMK4 was dependent on the increase in intracellular cytosolic Ca2+ concentration in Th17 cells. A mechanistic study demonstrated that inhibition of CaMK4 reduced IL-17A production by decreasing the phosphorylation of Akt-mTOR, which was well accepted to positively regulate Th17 differentiation. Collectively, our results demonstrated that Ca2+ -CaMK4-Akt/mTOR-IL-17A axis inhibition may serve as a promising therapeutic strategy for CP/CPPS.

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PROBLEM: The balance of the immune microenvironment along the maternal-fetal interface is closely related to pregnancy outcomes, with excessive inflammatory reactions leading to the occurrence of pathological pregnancy outcomes such as abortion. CaMK4 has been reported to play a significant role in autoimmune diseases through the regulation of Th17 cells. However, whether CaMK4 is associated with spontaneous abortion or the immune microenvironment along the maternal-fetal interface remains unclear. METHODS OF STUDY: In this study, we constructed normal pregnancy and LPS-induced abortion models in mice, and a CaMK4 inhibitor called KN-93 was administered to investigate the changes in and mechanisms of the immune response. The expression of CaMK4 was evaluated in the uteroplacental complex and spleen. Furthermore, the infiltration and function of Th17 cells were estimated in peripheral tissues and the uteroplacental complex. RESULTS: The expression of CaMK4 in the uteroplacental complex and spleen was significantly higher in the LPS-treated group than in the normal pregnancy group. KN-93, the CaMK4 inhibitor, reversed fetal resorption and excessive inflammation. In detail, KN-93 led to reduced infiltration of Th17 cells into peripheral tissues and the uteroplacental complex, and the functions of Th17 cells were inhibited. In addition, CaMK4 promoted the AKT/mTOR signaling pathway, which is one of the mechanisms that regulate the immune microenvironment. CONCLUSION: CaMK4 is a critical regulator that promotes the expansion of Th17 cells and enhances their functions through the AKT/mTOR signaling pathway. The inhibition of CaMK4 can reverse the immune imbalance along the maternal-fetal interface and improve pregnancy outcomes.

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Purpose: Beta-boswellic acid (ßBA) may play central roles in neural plasticity. Neural plasticity has significant implications for learning and memory which are governed by strict memoryrelated molecular pathways. To gain insight into the molecular mechanism by which ßBA affects these pathways this study analyzed the expression patterns of Camk2α and Camk4 genes in PC12 cells treated with ßBA. Methods: The cytotoxic effects of different ßBA concentrations on PC12 cells were examined by MTT assay. For gene expression analysis, cells were treated with concentrations of 1 and 10 µM of ßBA for 12, 24, 48, and 72 hours. Total RNA was purified by RNX-Plus solution and reverse transcribed into cDNA using Thermo Scientific Reverse Transcription reagents. The expression patterns of Camk2α and Camk4 genes were quantified by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Results: MTT assay indicated that ßBA reduced PC12 cell viability in a time- and concentrationdependent manner. The 50% inhibitory concentrations for the 48 and 72 hours time points were 35 and 26 µM, respectively; while, the ßBA concentrations up to 100 µM failed to kill 50% of the cells after 24 hours. According to the qRT-PCR data, the Camk2α variant is not expressed in either ßBA-treated or untreated PC12 cells. However, a significant upregulation was observed inCamk4 after 12 hours of treatment with ßBA, which followed by a significant downregulation after 24 hours and a persistent expression equal to the control until 72 hours. Conclusion: these findings indicate that PC12 cells not only does not express Camk2α but also its expression cannot be induced by ßBA. However, ßBA does modulate the expression of Camk4. This result provides further insight into the molecular mechanism by which ßBA affects memory.

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BACKGROUND: Circulatory iron is a hazardous biometal. Therefore, iron is transported in a redox-safe state by a serum glycoprotein - transferrin (TF). Different organs acquire iron from the systemic circulation through a tightly regulated mechanism at the blood-tissue interface which involves receptor-mediated internalization of TF. Thus, abnormal TF trafficking may lead to iron dyshomeostasis associated with several diseases including neurodegeneration. Iron -induced toxicity can cause neuronal damage to iron-sensitive brain regions. Recently, it was discovered that CAMKK2, a calcium (Ca2+)/calmodulin-activated kinase, controls receptor-mediated TF trafficking in mouse tissues, specifically in the brain. The biological function of CAMKK2 is mediated through multiple downstream effectors. Both CAMKK2 and one of its downstream kinase, CAMK4, exhibit overlapping expression in mouse brain. The role of CAMK4 in vesicular transport has been reported and loss of CAMKK2 or CAMK4 leads to cognitive defects in mouse. Therefore, it was hypothesized that CAMKK2-CAMK4 signaling regulates receptor-mediated TF trafficking and iron homeostasis which may be responsible for the neuronal malfunction observed in CAMKK2- or CAMK4-deficient mice. METHODS: CAMK4-/- mouse was used to study tissue-specific turnover of TF, TF-receptor (TFRC) and iron. CRISPR/Cas9-based CAMKK2 and/or CAMK4 deleted human embryonic kidney-derived HEK293 cell clones were used to study the molecular defects in receptor-mediated TF trafficking. Further, a "zero functional G protein" condition in HEK293 cell was exploited to study CAMKK2-CAMK4 signaling-mediated regulation of intracellular Ca2+ homeostasis which was linked to calcium signaling during TF trafficking. RESULTS: Loss of CAMK4 leads to abnormal post-translational modifications (PTMs) and turnover of TF in mouse cerebellum and liver which was associated with iron dyshomeostasis in these tissues. The HEK293 cell-based study revealed that the absence of CAMKK2-CAMK4 signaling altered intracellular Ca2+ homeostasis and lead to abnormal calcium signaling during TF trafficking. Also, CAMKK2-CAMK4 signaling deficiency affected the molecular interaction of TF and TF-receptor-associated protein complexes which indicated a potential failure in the recruitment of interacting proteins due to differential PTMs in TF. CONCLUSION: Overall, this study established a novel mechanistic link between intracellular Ca2+ level, receptor-mediated TF trafficking, and iron homeostasis, all regulated by CAMKK2-CAMK4 signaling. Video Abstract.

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