Calcium/Calmodulin-Dependent ProteinKinase Kinase 2: Roles in Signaling and Pathophysiology

minireview on functions of CaMKK2 and its potential as a target for therapeutic intervention

Calcium/calmodulin-dependent protein kinase kinase 2 regulates macrophage-mediated inflammatory responses.

Calcium/calmodulin-dependent kinase kinase 2 (CaMKK2) plays a key role in regulating food intake and energy expenditure at least in part by its actions in hypothalamic neurons. Previously, we showed that loss of CaMKK2 protected mice from high-fat diet (HFD)-induced obesity and glucose intolerance. However, although pair feeding HFD to WT mice to match food consumption of CAMKK2-null mice slowed weight gain, it failed to protect from glucose intolerance. Here we show that relative to WT mice, HFD-fed CaMKK2-null mice are protected from inflammation in adipose and remain glucose-tolerant. Moreover, loss of CaMKK2 also protected mice from endotoxin shock and fulminant hepatitis. We explored the expression of CaMKK2 in immune cells and found it to be restricted to those of the monocyte/macrophage lineage. CaMKK2-null macrophages exhibited a remarkable deficiency to spread, phagocytose bacteria, and synthesize cytokines in response to the Toll-like receptor 4 (TLR4) agonist lipopolysaccharide (LPS). Mechanistically, loss of CaMKK2 uncoupled the TLR4 cascade from activation of protein tyrosine kinase 2 (PYK2; also known as PTK2B). Our findings uncover an important function for CaMKK2 in mediating mechanisms that control the amplitude of macrophage inflammatory responses to excess nutrients or pathogen derivatives

Serafino Zappacosta: An Enlightened Mentor and Educator.

With this article, the authors aim to honor the memory of Serafino Zappacosta, who had been their mentor during the early years of their career in science. The authors discuss how the combination of Serafino Zappacosta's extraordinary commitment to teaching and passion for science created a fostering educational environment that led to the creation of the "Ruggero Ceppellini Advanced School of Immunology." The review also illustrates how the research on the MHC and the inspirational scientific context in the Zappacosta's laboratory influenced the authors' early scientific interests, and subsequent professional work as immunologists

CXCL12 prolongs naive CD4 + T lymphocytes survival via activation of PKA, CREB and Bcl2 and BclXl up-regulation

Naive T lymphocytes recirculate through the body, traveling from secondary lymphoid organs through tissues and via lymphatic vessels and peripheral blood into other secondary lymphoid organs and into the bone marrow. In these tissues, lymphocytes are exposed to the chemokine CXCL12 which is abundantly produced in bone marrow and in lymph nodes by stromal cells. CXCL12 is known to drive lymphocytes chemotaxis and, in cells types such as stem cells, an antiapopototic effect has been described. Methods Here we analyzed the effect of CXCL12 exposure on naïve CD4 + T lymphocytes purified from peripheral blood by immunomagnetic negative isolation and cultured in a nutrient poor medium. We also studied, mainly by western blot analysis, the signaling pathways involved in CXCL12 action on naïve CD4 + T lymphocytes. Results We found that CXCL12-exposed cells survived longer than untreated ones and this prolonged lifespan was specific for resting naïve lymphocytes, while in vitro activated lymphoblasts died rapidly despite CXCL12 treatment. We demonstrated that the increased percentage of living cells observed upon CXCL12 administration was not due to induction of proliferation but to a prosurvival effect of this chemokine. Moreover, our data suggest that this prosurvival effect on naïve CD4 + T lymphocytes might likely be mediated by PKA-dependent CREB activation and consequent increased expression of the antiapoptotic factors Bcl2 and BclXl. Conclusions This newly reported activity of CXCL12 might contribute to the maintenance of the naïve T lymphocytes pool in vivo, which is needed to ensure a proper immune response to new antigens

Phylogeny of Toll-Like Receptor Signaling: Adapting the Innate Response

The Toll-like receptors represent a largely evolutionarily conserved pathogen recognition machinery responsible for recognition of bacterial, fungal, protozoan, and viral pathogen associated microbial patterns and initiation of inflammatory response. Structurally the Toll-like receptors are comprised of an extracellular leucine rich repeat domain and a cytoplasmic Toll/Interleukin 1 receptor domain. Recognition takes place in the extracellular domain where as the cytoplasmic domain triggers a complex signal network required to sustain appropriate immune response. Signal transduction is regulated by the recruitment of different intracellular adaptors. The Toll-like receptors can be grouped depending on the usage of the adaptor, MyD88, into MyD88-dependent and MyD88 independent subsets. Herein, we present a unique phylogenetic analysis of domain regions of these receptors and their cognate signaling adaptor molecules. Although previously unclear from the phylogeny of full length receptors, these analyses indicate a separate evolutionary origin for the MyD88-dependent and MyD88-independent signaling pathway and provide evidence of a common ancestor for the vertebrate and invertebrate orthologs of the adaptor molecule MyD88. Together these observations suggest a very ancient origin of the MyD88-dependent pathway Additionally we show that early duplications gave rise to several adaptor molecule families. In some cases there is also strong pattern of parallel duplication between adaptor molecules and their corresponding TLR. Our results further support the hypothesis that phylogeny of specific domains involved in signaling pathway can shed light on key processes that link innate to adaptive immune response

Calcium/calmodulin-dependent kinase kinase 2 regulates hematopoietic stem and progenitor cell regeneration

Hematopoietic stem and progenitor cells (HSPCs) are predominantly quiescent in adults, but proliferate in response to bone marrow (BM) injury. Here, we show that deletion of Ca2+/calmodulin (CaM)-dependent protein kinase kinase 2 (CaMKK2) promotes HSPC regeneration and hematopoietic recovery following radiation injury. Using Camkk2-enhanced green fluorescent protein (EGFP) reporter mice, we found that Camkk2 expression is developmentally regulated in HSPC. Deletion of Camkk2 in HSPC results in a significant downregulation of genes affiliated with the quiescent signature. Accordingly, HSPC from Camkk2 null mice have a high proliferative capability when stimulated in vitro in the presence of BM-derived endothelial cells. In addition, Camkk2 null mice are more resistant to radiation injury and show accelerated hematopoietic recovery, enhanced HSPC regeneration and ultimately a prolonged survival following sublethal or lethal total body irradiation. Mechanistically, we propose that CaMKK2 regulates the HSPC response to hematopoietic damage by coupling radiation signaling to activation of the anti-proliferative AMP-activated protein kinase. Finally, we demonstrated that systemic administration of the small molecule CaMKK2 inhibitor, STO-609, to irradiated mice enhanced HSPC recovery and improved survival. These findings identify CaMKK2 as an important regulator of HSPC regeneration and demonstrate CaMKK2 inhibition is a novel approach to promoting hematopoietic recovery after BM injury

Loss of beta-catenin triggers oxidative stress and impairs hematopoietic regeneration

Accidental or deliberate ionizing radiation exposure can be fatal due to widespread hematopoietic destruction. However, little is known about either the course of injury or the molecular pathways that regulate the subsequent regenerative response. Here we show that the Wnt signaling pathway is critically important for regeneration after radiation-induced injury. Using Wnt reporter mice, we show that radiation triggers activation of Wnt signaling in hematopoietic stem and progenitor cells. β-Catenin-deficient mice, which lack the ability to activate canonical Wnt signaling, exhibited impaired hematopoietic stem cell regeneration and bone marrow recovery after radiation. We found that, as part of the mechanism, hematopoietic stem cells lacking β-catenin fail to suppress the generation of reactive oxygen species and cannot resolve DNA double-strand breaks after radiation. Consistent with the impaired response to radiation, β-catenin-deficient mice are also unable to recover effectively after chemotherapy. Collectively, these data indicate that regenerative responses to distinct hematopoietic injuries share a genetic dependence on β-catenin and raise the possibility that modulation of Wnt signaling may be a path to improving bone marrow recovery after damage

Совершенствование бизнес-процессов на предприятии

Выпускная квалификационная работа содержит 84 страницы, 12 рисунков, 26 таблиц, 20 использованных источников, 5 приложений. Целью работы является разработка практических предложений для оптимизации и совершенствования бизнес-процесса "Оказание платных услуг клиентам" в ООО "КЛДЦ". В результате исследования были предложены мероприятия по совершенствованию бизнес-процесса 슫Оказание платных услуг клиентам슻, а также проведена оценка эффективности реализации данных мероприятий.Graduate qualification work consists of 84 pages, 12 figures, 26 tables, 20 sources used, 5 attachments. The purpose is to create a practice suggestion for the business process «Paid services» LTD «CLDTS» development. As a result of research several measures on the business process «Paid services» LTD «CLDTS» development were elaborated and offered to the company, measures efficiency was evaluated

Diagnosis of prostate cancer with magnetic resonance imaging in men treated with 5-alpha-reductase inhibitors

Purpose The primary aim of this study was to evaluate if exposure to 5-alpha-reductase inhibitors (5-ARIs) modifies the effect of MRI for the diagnosis of clinically significant Prostate Cancer (csPCa) (ISUP Gleason grade >= 2).Methods This study is a multicenter cohort study including patients undergoing prostate biopsy and MRI at 24 institutions between 2013 and 2022. Multivariable analysis predicting csPCa with an interaction term between 5-ARIs and PIRADS score was performed. Sensitivity, specificity, and negative (NPV) and positive (PPV) predictive values of MRI were compared in treated and untreated patients.Results 705 patients (9%) were treated with 5-ARIs [median age 69 years, Interquartile range (IQR): 65, 73; median PSA 6.3 ng/ml, IQR 4.0, 9.0; median prostate volume 53 ml, IQR 40, 72] and 6913 were 5-ARIs naive (age 66 years, IQR 60, 71; PSA 6.5 ng/ml, IQR 4.8, 9.0; prostate volume 50 ml, IQR 37, 65). MRI showed PIRADS 1-2, 3, 4, and 5 lesions in 141 (20%), 158 (22%), 258 (37%), and 148 (21%) patients treated with 5-ARIs, and 878 (13%), 1764 (25%), 2948 (43%), and 1323 (19%) of untreated patients (p < 0.0001). No difference was found in csPCa detection rates, but diagnosis of high-grade PCa (ISUP GG >= 3) was higher in treated patients (23% vs 19%, p = 0.013). We did not find any evidence of interaction between PIRADS score and 5-ARIs exposure in predicting csPCa. Sensitivity, specificity, PPV, and NPV of PIRADS >= 3 were 94%, 29%, 46%, and 88% in treated patients and 96%, 18%, 43%, and 88% in untreated patients, respectively.Conclusions Exposure to 5-ARIs does not affect the association of PIRADS score with csPCa. Higher rates of high-grade PCa were detected in treated patients, but most were clearly visible on MRI as PIRADS 4 and 5 lesions.Trial registration The present study was registered at ClinicalTrials.gov number: NCT05078359

Calmodulin-dependent kinase IV links Toll-like receptor 4 signaling with survival pathway of activated dendritic cells.

Microbial products, including lipopolysaccharide (LPS), an agonist of Toll-like receptor 4 (TLR4), regulate the lifespan of dendritic cells (DCs) by largely undefined mechanisms. Here, we identify a role for calcium-calmodulin–dependent kinase IV (CaMKIV) in this survival program. The pharmacologic inhibition of CaMKs as well as ectopic expression of kinase-inactive CaMKIV decrease the viability of monocyte-derived DCs exposed to bacterial LPS. The defect in TLR4 signaling includes a failure to accumulate the phosphorylated form of the cAMP response element-binding protein (pCREB), Bcl-2, and Bcl-xL. CaMKIV null mice have a decreased number of DCs in lymphoid tissues and fail to accumulate mature DCs in spleen on in vivo exposure to LPS. Although isolated Camk4(−/−) DCs are able to acquire the phenotype typical of mature cells and release normal amounts of cytokines in response to LPS, they fail to accumulate pCREB, Bcl-2, and Bcl-xL and therefore do not survive. The transgenic expression of Bcl-2 in CaMKIV null mice results in full recovery of DC survival in response to LPS. These results reveal a novel link between TLR4 and a calcium-dependent signaling cascade comprising CaMKIV-CREB-Bcl-2 that is essential for DC survival