scholarly journals Predicting a Potential Link to Antidepressant Effect: Neuroprotection of Zhi-zi-chi Decoction on Glutamate-induced Cytotoxicity in PC12 Cells

2021 ◽  
Vol 11 ◽  
Author(s):  
Yin Zhang ◽  
Yusha Luo ◽  
Dongqi Zhang ◽  
Bo Pang ◽  
Jun Wen ◽  
...  
Keyword(s):  
Cell Viability ◽  
Pc12 Cells ◽  
Synergistic Effects ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Antidepressant Effect ◽  
Mild Stress ◽  
Antidepressant Effects

Zhi-zi-chi Decoction (ZZCD), composed of Fructus Gardeniae (Zhizi in Chinese, ZZ in brief) and Semen sojae praeparatum (Dandouchi in Chinese, DDC in brief), has been used as a drug therapy for depression for thousands of years in China. However, the antidepressant mechanism of ZZCD still remains unknown. This study was aimed at exploring antidepressant effects of ZZCD from the aspect of neuroprotection based on herb compatibility. Glutamate-treated PC12 cells and chronic unpredictable mild stress (CUMS)-induced rats were established as models of depression in vitro and in vivo respectively. Cell viability, lactate dehydrogenase (LDH), apoptosis rate, reactive oxygen species (ROS), glutathione reductase (GR) and superoxide dismutase (SOD), and the expressions of Bax, Bcl-2 and cyclic adenosine monophosphate-response element binding protein (CREB) were measured to compare neuroprotection among single herbs and the formula in vitro. Behavior tests were conducted to validate antidepressant effects of ZZCD in vivo. Results showed that the compatibility of ZZ and DDC increased cell viability and activities of GR and SOD, and decreased the levels of LDH, apoptosis cells and ROS. Besides, the expressions of Bcl-2 and CREB were up-regulated while that of Bax was down-regulated by ZZCD. Furthermore, the compatibility of ZZ and DDC reversed abnormal behaviors in CUMS-induced rats and displayed higher efficacy than any of the single herbs. This study revealed that the antidepressant effects of ZZCD were closely associated with neuroprotection and elucidated synergistic effects of the compatibility of ZZ and DDC based on it.

scholarly journals Resveratrol Enhances Inhibition Effects of Cisplatin on Cell Migration and Invasion and Tumor Growth in Breast Cancer MDA-MB-231 Cell Models In Vivo and In Vitro

Molecules ◽  
2021 ◽  
Vol 26 (8) ◽  
pp. 2204
Author(s):  
Meng-Die Yang ◽  
Yang Sun ◽  
Wen-Jun Zhou ◽  
Xiao-Zheng Xie ◽  
Qian-Mei Zhou ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Migration ◽  
Tumor Growth ◽  
Cell Viability ◽  
Synergistic Effects ◽  
Migration And Invasion ◽  
Cell Migration And Invasion ◽  
Tgf Β1

Triple-negative breast cancer (TNBC) is a refractory type of breast cancer that does not yet have clinically effective drugs. The aim of this study is to investigate the synergistic effects and mechanisms of resveratrol combined with cisplatin on human breast cancer MDA-MB-231 (MDA231) cell viability, migration, and invasion in vivo and in vitro. In vitro, MTS assays showed that resveratrol combined with cisplatin inhibits cell viability as a concentration-dependent manner, and produced synergistic effects (CI < 1). Transwell assay showed that the combined treatment inhibits TGF-β1-induced cell migration and invasion. Immunofluorescence assays confirmed that resveratrol upregulated E-cadherin expression and downregulated vimentin expression. Western blot assay demonstrated that resveratrol combined with cisplatin significantly reduced the expression of fibronectin, vimentin, P-AKT, P-PI3K, P-JNK, P-ERK, Sma2, and Smad3 induced by TGF-β1 (p < 0.05), and increased the expression of E-cadherin (p < 0.05), respectively. In vivo, resveratrol enhanced tumor growth inhibition and reduced body weight loss and kidney function impairment by cisplatin in MDA231 xenografts, and significantly reduced the expressions of P-AKT, P-PI3K, Smad2, Smad3, P-JNK, P-ERK, and NF-κB in tumor tissues (p < 0.05). These results indicated that resveratrol combined with cisplatin inhibits the viability of breast cancer MDA231 cells synergistically, and inhibits MDA231 cells invasion and migration through Epithelial-mesenchymal transition (EMT) approach, and resveratrol enhanced anti-tumor effect and reduced side of cisplatin in MDA231 xenografts. The mechanism may be involved in the regulations of PI3K/AKT, JNK, ERK and NF-κB expressions.

239 USE OF CYCLIC ADENOSINE MONOPHOSPHATE MODULATORS IN IN VITRO PRODUCTION OF BOVINE EMBRYOS

2015 ◽  
Vol 27 (1) ◽  
pp. 209
Author(s):  
T. Fanti ◽  
N. M. Ortega ◽  
R. Garaguso ◽  
M. J. Franco ◽  
C. Herrera ◽  
...  
Keyword(s):  
Phosphodiesterase Inhibitor ◽  
Basal Medium ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Control Group ◽  
Hatching Rate ◽  
Bovine Embryos ◽  
Oocyte Competence

In vitro embryo production systems (IVP) try to emulate and enhance molecular events that occur in in vivo reproductive systems in order to increase, not only the number of embryos generated, but also their quality. Despite advances, IVP processes are still inefficient compared with in vivo systems. Several studies have attributed this deficiency to a lack of oocyte competence due to spontaneous premature resumption of meiotic maturation in the oocyte following the removal from its follicular environment. Therefore, our objective was to increase oocyte competence avoiding premature resumption of meiosis by using cyclic adenosine monophosphate modulators. Cumulus-oocyte complexes (COC) were obtained from ovaries of slaughterhouses, washed, and randomly allocated in 2 culture systems. Oocytes in the control group (IVM) were cultured for a period of 24 h in basal medium TCM-199 with EGF (1 µg mL–1) supplemented with rhFSH (25 mIU mL–1). Oocytes in the biphasic in vitro maturation (b-IVM) group were cultured for 2 h in a basal medium supplemented with a phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX, 500 µM), and an activator of adenylate cyclase (forskolin, 100 µM). Subsequently, COC were washed and cultured in basal medium supplemented with cilostamide (20 µM) and rhFSH (25 mIU mL–1) for 24 h. Maturation rates were analysed and IVF was performed with a dose of 1 × 106 sperm cells mL–1 in IVF-SOF medium. The presumptive zygotes were cultured in continuous-single-culture medium (Irvine) supplemented with 8 mg mL–1 of BSA until they reached the blastocyst stage. No significant differences in maturation, cleavage, and cryotolerance were observed between b-IVM and IVM groups (P > 0.05; Table 1). This study showed that b-IVM produced a significant increase in IVP compared with the control (IVM) at Days 7 and 8 (P < 0.01). Blastocyst hatching rate was significant (P < 0.05) for both treatment and day of analysis. The b-IVM group yielded an increase of 10 and 7.5% at Days 7 and 8, respectively, of IVP. The biphasic maturation showed an improvement in quality regarding the control group, in the timing analysis of production, and hatching percentages, and these results show that the use of cyclic adenosine monophosphate modulators in the oocyte maturation process enhances oocyte competence, which is reflected in increased productivity and embryo quality. We propose this treatment as an alternative to the standard protocols currently used in IVP of bovine embryos. Table 1.Effect of treatment on maturation, cleavage, and cryotolerance

159 EFFECT OF 3-ISOBUTYL-1-METHYLXANTHINE (IBMX) ON THE GERMINAL VESICLE STAGE OF PORCINE OOCYTES DURING OOCYTE COLLECTION IN VITRO

2014 ◽  
Vol 26 (1) ◽  
pp. 193
Author(s):  
R. Appeltant ◽  
J. Beek ◽  
D. Maes ◽  
A. Van Soom
Keyword(s):  
Germinal Vesicle ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Cumulus Cells ◽  
Guanosine Monophosphate ◽  
Developmental Competence ◽  
Meiotic Arrest ◽  
Oocyte Collection

When using modern maturation conditions for in vitro maturation, pig oocytes yield ~20% blastocysts only. One problem is that cumulus cells, which are normally connected with the immature oocyte by cellular projections penetrating through the zona pellucida and with the oolemma via gap junctions, are prematurely losing these connections after the cumulus–oocyte complex is removed from the follicle. The oocyte possesses a type 3 phosphodiesterase, which degrades 3′,5′-cyclic adenosine monophosphate (cAMP), and this activity is inhibited by supply of 3′,5′-cyclic guanosine monophosphate (cGMP) to the oocyte via the cumulus cells. Consequently, cAMP levels, which are typically high during early stages of oocyte maturation in vivo, decrease, leading to spontaneous nuclear maturation and oocytes of low developmental competence. Therefore, the maintenance of these cumulus-oocyte connections is important to keep cAMP high and the oocyte under meiotic arrest. One way to prevent this drop in cAMP is using N6, 2′-o-dibutyryladenosine 3′,5′-cyclic monophosphate sodium (dbcAMP) that causes an arrest at germinal vesicle (GV) stage II (Funahashi et al. 1997 Biol. Reprod. 57, 49–53). Another option is collecting the oocytes in a medium containing the phoshodiesterase inhibitor, IBMX. The present study investigated the influence of IBMX on the progression of the GV of the oocyte after collection, just before the start of the maturation procedure. The GV stage was defined according to Sun et al. (2004 Mol. Reprod. Dev. 69, 228–234). In parallel with the findings on dbcAMP, we hypothesised an arrest at GV II by the presence of IBMX during collection. One group of oocytes were collected in HEPES-buffered TALP without IBMX (n = 375) and another group in the same medium containing 0.5 mM IBMX (n = 586). An average incubation time of 140 min was applied in both groups, and 3 replicates were performed. The proportions of oocytes before or at GV II and beyond GV II were compared in both groups using logistic regression analysis. The proportion of oocytes was included as dependent variable and group (IBMX addition or not) as independent variable. Replicate was also included in the model. The proportion of oocytes before or at GV II was not statistically significant between the group without and the group with IBMX (59.2 v. 58.7% respectively; P > 0.05). In conclusion, the use of IBMX during oocyte collection did not influence the state of the germinal vesicle of the oocyte during collection, indicating that IBMX did not cause a meiotic arrest in the oocytes during collecting in vitro.

scholarly journals Glycine Protects against Hypoxic-Ischemic Brain Injury by Regulating Mitochondria-Mediated Autophagy via the AMPK Pathway

2019 ◽  
Vol 2019 ◽  
pp. 1-29 ◽  
Author(s):  
Chen-chen Cai ◽  
Jiang-hu Zhu ◽  
Li-xia Ye ◽  
Yuan-yuan Dai ◽  
Ming-chu Fang ◽  
...  
Keyword(s):  
Pc12 Cells ◽  
Adenosine Monophosphate ◽  
Ischemic Brain Injury ◽  
Hypoxic Stress ◽  
Ischemic Brain ◽  
Ampk Pathway ◽  
Amp Activated Protein Kinase ◽  
Ischemic Encephalopathy

Hypoxic-ischemic encephalopathy (HIE) is detrimental to newborns and is associated with high mortality and poor prognosis. Thus, the primary aim of the present study was to determine whether glycine could (1) attenuate HIE injury in rats and hypoxic stress in PC12 cells and (2) downregulate mitochondria-mediated autophagy dependent on the adenosine monophosphate- (AMP-) activated protein kinase (AMPK) pathway. Experiments conducted using an in vivo HIE animal model and in vitro hypoxic stress to PC12 cells revealed that intense autophagy associated with mitochondrial function occurred during in vivo HIE injury and in vitro hypoxic stress. However, glycine treatment effectively attenuated mitochondria-mediated autophagy. Additionally, after identifying alterations in proteins within the AMPK pathway in rats and PC12 cells following glycine treatment, cyclosporin A (CsA) and 5-aminoimidazole-4-carboxamide-1-b-4-ribofuranoside (AICAR) were administered in these models and indicated that glycine protected against HIE and CoCl2 injury by downregulating mitochondria-mediated autophagy that was dependent on the AMPK pathway. Overall, glycine attenuated hypoxic-ischemic injury in neurons via reductions in mitochondria-mediated autophagy through the AMPK pathway both in vitro and in vivo.

scholarly journals Cilostazol Improves Hyperglycemia-Induced Impairment of Angiogenesis by Stimulating Adiponectin/Adiponectin Receptor1/Sirtuin1

2021 ◽  
Author(s):  
Shih-Ya Tseng ◽  
Hsien-Yuan Chang ◽  
Yi-Heng Li ◽  
Ting-Hsing Chao
Keyword(s):  
Endothelial Cells ◽  
Signaling Pathway ◽  
Vascular Endothelial Cells ◽  
Umbilical Vein ◽  
Antiplatelet Agent ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Sirtuin 1

Abstract Background: Cilostazol is an antiplatelet agent with vasodilating effects that functions by increasing the intracellular concentration of cyclic adenosine monophosphate. However, the effect of cilostazol on adiponectin is still unclear. Purpose: We investigated the effects of cilostazol on adiponectin/adiponectin receptors and the Sirtuin 1 (SIRT1)/AMP-activated protein kinase (AMPK) signaling pathway to prevent high glucose (HG)-induced impairment of angiogenesis in vitro and in vivo. Methods and Results: Human umbilical vein endothelial cells (HUVECs) and human aortic smooth muscle cells (HASMCs) were cocultured in HG conditions. Adiponectin concentrations in the supernatant were significantly increased when HASMCs were treated with cilostazol but not significantly changed when only HUVECs were treated with cilostazol. Cilostazol treatment restored the expression of the adipoR1 and SIRT1 proteins and upregulated the phosphorylation of AMPKa1 in the HUVECs treated with HG but not adipoR2. Cilostazol prevented apoptosis and stimulated proliferation, chemotactic motility and capillary-like tube formation in HG-treated HUVECs through the adipoR1/AMPK/SIRT1 signaling pathway. In cilostazol-treated mice, recovery of the blood flow ratio after hindlimb ischemia and circulating CD34+CD45dim cells were significantly attenuated by adipoR1 knockdown but not adipoR2 knockdown. The expression of SIRT1, phosphorylation of AMPKa1/acetyl-CoA carboxylase and Akt/endothelial nitric oxide synthase in ischemic muscles were significantly attenuated by gene knockdown of adipoR1. Conclusions: Cilostazol prevents HG-induced endothelial dysfunction in vascular endothelial cells and enhances angiogenesis in hyperglycemic mice by upregulating the expression of adiponectin/adipoR1 and its SIRT1/AMPK downstream signaling pathway.

Effects of Supraphysiologic Levels of Estradiol on Endometrial Decidualization, sFlt1, and HOXA10 Expression

2019 ◽  
Vol 26 (12) ◽  
pp. 1626-1632 ◽  
Author(s):  
Hanh N. Cottrell ◽  
Venkataraman Deepak ◽  
Jessica B. Spencer ◽  
Neil Sidell ◽  
Augustine Rajakumar
Keyword(s):  
Growth Factor ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Controlled Ovarian Hyperstimulation ◽  
Vascular Endothelial ◽  
Endometrial Stromal Cells ◽  
Vitro Fertilization ◽  
Endothelial Growth Factor

Objective: Supraphysiologic estradiol (E2) levels associated with controlled ovarian hyperstimulation in high in vitro fertilization (IVF) responders may alter implantation and placentation and increase the risk of preeclampsia. Our hypothesis is that elevated E2 levels in vitro significantly alter endometrial decidualization, sFlt1, and HOXA10 expression. Methods: Human endometrial stromal cells were treated with a decidualization cocktail of medroxyprogesterone, cyclic adenosine monophosphate, and 3 concentrations of E2 10 nM (standard), 100 nM (intermediate), or 1000 nM E2 (high). Effects on sFlt1, prolactin (PRL), insulin-like growth factor binding protein 1 (IGFBP-1), vascular endothelial growth factor (VEGF), and HOXA10 were studied. Results: Prolactin, IGFBP-1, and VEGF significantly increased at all 3 E2 concentrations. While IGFBP-1 and VEGF did not change with increasing E2, PRL was less with high E2 (6.0 ng/mL ± 1.4 standard error of the mean) compared to standard (21.4 ± 3.2) and intermediate (19.8 ± 3.8). sFlt1 decrease was similar at all E2 concentrations. HOXA10 was lower at standard (10%) and intermediate (30%) as expected, but did not change with high E2. Conclusions: Supraphysiologic E2 levels associated with high IVF responders that exceed in vivo levels may impair in vitro endometrial decidualization. Although PRL did increase with high E2, the levels were, however, attenuated and 3.4-fold lower than standard and intermediate E2. sFlt1 was decreased under all 3 conditions with no differences between concentrations. Reduced HOXA10 was not observed with high E2. These findings suggest that elevated E2 levels in vitro may alter endometrial decidualization and subsequently affect implantation and placentation.

A new adenylyl cyclase, putative disease-resistance RPP13-like protein 3, participates in abscisic acid-mediated resistance to heat stress in maize

2020 ◽  
Author(s):  
Hao Yang ◽  
Yulong Zhao ◽  
Ning Chen ◽  
Yanpei Liu ◽  
Shaoyu Yang ◽  
...  
Keyword(s):  
Heat Stress ◽  
Disease Resistance ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Wild Type ◽  
In Vivo Experiments ◽  
In The Wild ◽  
Shock Proteins

Abstract In plants, 3´,5´-cyclic adenosine monophosphate (cAMP) is an important second messenger with varied functions; however, only a few adenylyl cyclases (ACs) that synthesize cAMP have been identified. Moreover, the biological roles of ACs/cAMP in response to stress remain largely unclear. In this study, we used quantitative proteomics techniques to identify a maize heat-induced putative disease-resistance RPP13-like protein 3 (ZmRPP13-LK3), which has three conserved catalytic AC centres. The AC activity of ZmRPP13-LK3 was confirmed by in vitro enzyme activity analysis, in vivo RNAi experiments, and functional complementation in the E. coli cyaA mutant. ZmRPP13-LK3 is located in the mitochondria. The results of in vitro and in vivo experiments indicated that ZmRPP13-LK3 interacts with ZmABC2, a possible cAMP exporter. Under heat stress, the concentrations of ZmRPP13-LK3 and cAMP in the ABA-deficient mutant vp5 were significantly less than those in the wild-type, and treatment with ABA and an ABA inhibitor affected ZmRPP13-LK3 expression in the wild-type. Application of 8-Br-cAMP, a cAMP analogue, increased heat-induced expression of heat-shock proteins in wild-type plants and alleviated heat-activated oxidative stress. Taken together, our results indicate that ZmRPP13-LK3, a new AC, can catalyse ATP for the production of cAMP and may be involved in ABA-regulated heat resistance.

scholarly journals Ketamine Increases Proliferation of Human iPSC-Derived Neuronal Progenitor Cells via Insulin-Like Growth Factor 2 and Independent of the NMDA Receptor

Cells ◽  
2019 ◽  
Vol 8 (10) ◽  
pp. 1139 ◽  
Author(s):  
Alessandra Grossert ◽  
Narges Zare Mehrjardi ◽  
Sarah J. Bailey ◽  
Mark A. Lindsay ◽  
Jürgen Hescheler ◽  
...  
Keyword(s):  
Nmda Receptor ◽  
Growth Factor ◽  
Progenitor Cells ◽  
Neural Progenitor Cells ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Nmda Receptor Antagonist ◽  
Insulin Like Growth Factor

The N-methyl-D-aspartate (NMDA) receptor antagonist ketamine offers promising perspectives for the treatment of major depressive disorder. Although ketamine demonstrates rapid and long-lasting effects, even in treatment-resistant patients, to date, the underlying mode of action remains elusive. Thus, the aim of our study was to investigate the molecular mechanism of ketamine at clinically relevant concentrations by establishing an in vitro model based on human induced pluripotent stem cells (iPSCs)-derived neural progenitor cells (NPCs). Notably, ketamine increased the proliferation of NPCs independent of the NMDA receptor, while transcriptome analysis revealed significant upregulation of insulin-like growth factor 2 (IGF2) and p11, a member of the S100 EF-hand protein family, which are both implicated in the pathophysiology of depression, 24 h after ketamine treatment. Ketamine (1 µM) was able to increase cyclic adenosine monophosphate (cAMP) signaling in NPCs within 15 min and cell proliferation, while ketamine-induced IGF2 expression was reduced after PKA inhibition with cAMPS-Rp. Furthermore, 24 h post-administration of ketamine (15 mg/kg) in vivo confirmed phosphorylation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) in the subgranular zone (SGZ) of the hippocampus in C57BL/6 mice. In conclusion, ketamine promotes the proliferation of NPCs presumably by involving cAMP-IGF2 signaling.

258 IMPACT OF MILRINONE AND FORSKOLIN ON THE EFFICIENCY AND QUALITY OF BOVINE OOCYTE IN VITRO MATURATION

2013 ◽  
Vol 25 (1) ◽  
pp. 277
Author(s):  
E. Stachowiak ◽  
K. Papis ◽  
J. Karasiewicz ◽  
J. A. Modlinski
Keyword(s):  
In Vitro Maturation ◽  
Combined Treatment ◽  
Phosphodiesterase Inhibitor ◽  
Microscopic Analysis ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Experimental Conditions ◽  
Oocyte Meiosis

The efficiency of in vitro maturation (IVM) of bovine oocytes remains inferior compared with maturation in vivo. Recently, some modifications of in vitro maturation (IVM) procedures have been proposed, such as simulated physiological maturation (Gilchrist 2011 Reprod. Fertil. Dev. 23, 23–31). In our experiment, a comparison of the traditional IVM efficiency with maturation after oocyte meiosis inhibition using roscovitine or with a modified two-step maturation using forskolin (cyclic adenosine monophosphate stimulator) and milrinone (type-3 phosphodiesterase inhibitor) was performed. Control oocytes obtained from slaughterhouse-derived ovaries were subjected to the traditional 24-h maturation in TCM-199 medium supplemented with sodium pyruvate, l-glutamine, gentamicin, 10% FCS, and hormones (pregnant mare’s serum gonadotropin and hCG, PG 600, Intervet, Kenilworth, NJ, USA). The roscovitine (50 µM, 24 h) inhibitory treatment was accomplished in the same medium (without hormones) and subsequently, traditional 24-h IVM was performed. The same TCM-199 medium (with hormones) supplemented with forskolin (100 µM) and milrinone (50 µM) was used for the first step (17 h) of the two-step maturation, whereas the second step (7 h) was performed in the same TCM-199 medium devoid of forskolin and milrinone. Fertilization with frozen sperm processed using TALP media was performed in TALP supplemented with heparin, penicillamine, hypotaurine, epinephrine, and BSA. In vitro culture of presumptive zygotes was performed in CR1aa medium. Portions of oocytes from all treatments after maturation and after fertilization procedures were stained and subjected to microscopic analysis. There were no differences in terms of maturation and fertilization rates between treatments. However, roscovitine-mediated inhibition of maturation performed in our experimental conditions was efficient and reversible, but harmful for subsequent embryo development. On the other hand, two-step maturation was equally as efficient as (but not better than) traditional IVM in all aspects examined in the present study (Table 1). In conclusion, the forskolin and milrinone combined treatment during the IVM procedure gives hope for fully efficient IVM. However, to achieve this goal, more research is necessary. Table 1.Development of embryos after different oocyte maturation procedures1

Identification and characterization of CKLiK, a novel granulocyte Ca++/calmodulin-dependent kinase

Blood ◽  
2000 ◽  
Vol 96 (9) ◽  
pp. 3215-3223 ◽  
Author(s):  
Sandra Verploegen ◽  
Jan-Willem J. Lammers ◽  
Leo Koenderman ◽  
Paul J. Coffer
Keyword(s):  
Messenger Rna ◽  
Kinase Activity ◽  
Myeloid Cell ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Reading Frame ◽  
Effector Functions ◽  
Calcium Calmodulin Dependent

Human granulocytes are characterized by a variety of specific effector functions involved in host defense. Several widely expressed protein kinases have been implicated in the regulation of these effector functions. A polymerase chain reaction–based strategy was used to identify novel granulocyte-specific kinases. A novel protein kinase complementary DNA with an open reading frame of 357 amino acids was identified with homology to calcium-calmodulin–dependent kinase I (CaMKI). This has been termed CaMKI-like kinase (CKLiK). Analysis of CKLiK messenger RNA (mRNA) expression in hematopoietic cells demonstrated an almost exclusive expression in human polymorphonuclear leukocytes (PMN). Up-regulation of CKLiK mRNA occurs during neutrophilic differentiation of CD34+ stem cells. CKLiK kinase activity was dependent on Ca++ and calmodulin as analyzed by in vitro phosphorylation of cyclic adenosine monophosphate responsive element modulator (CREM). Furthermore, CKLiK- transfected cells treated with ionomycin demonstrated an induction of CRE- binding protein (CREB) transcriptional activity compared to control cells. Additionally, CaMK-kinaseα enhanced CKLiK activity. In vivo activation of CKLiK was shown by addition of interleukin (IL)-8 to a myeloid cell line stably expressing CKLiK. Furthermore inducible activation of CKLiK was sufficient to induce extracellular signal-related kinase (ERK) mitogen-activated protein (MAP) kinase activity. These data identify a novel Ca++/calmodulin-dependent PMN- specific kinase that may play a role in Ca++-mediated regulation of human granulocyte functions.

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