A cascade of Ca2+/calmodulin-dependent protein kinases regulates the differentiation and functional activation of murine neutrophils

Abstract

Objective-The function of neutrophils as primary mediators of innate immunity depends on the activity of granule proteins and critical components of the NADPH oxidase complex. Expression of their cognate genes is regulated during neutrophil differentiation by a complex network of intracellular signaling pathways. In this study we have investigated the role of two members of the calcium/calmodulin-dependent protein kinase (CaMK) signaling cascade, CaMKI-like kinase (CKLiK) and CaMKKα, in regulating neutrophil differentiation and functional activation.Materials and Methods-Mouse myeloid cell lines were used to examine the expression of a CaMK cascade in developing neutrophils and to examine the effects of constitutive activation versus inhibition of CaMKs on neutrophil maturation.Results-Expression of CaMKKα was shown to increase during neutrophil differentiation in multiple cell lines, whereas expression of CKLiK increased as multipotent progenitors committed to promyelocytes but then decreased as cells differentiated into mature neutrophils. Expression of constitutively active CKLiKs did not affect morphologic maturation, but caused dramatic decreases in both respiratory burst responses and chemotaxis. This loss of neutrophil function was accompanied by reduced secondary granule and gp91 phox gene expression. The CaMK inhibitor KN93 attenuated cytokine-stimulated proliferative responses in promyelocytic cell lines, and inhibited the respiratory burst. Similar data were observed with the CaMKKα inhibitor, STO-609.Conclusions-Overactivation of a cascade of CaMKs inhibits neutrophil maturation, suggesting that these kinases play an antagonistic role during neutrophil differentiation, but at least one CaMK is required for myeloid cell expansion and functional activation.

Lamin B Receptor Regulates the Growth and Maturation of Myeloid Progenitors via its Sterol Reductase Domain: Implications for Cholesterol Biosynthesis in Regulating Myelopoiesis

“…We began by transducing a full-length version of Lbr into EML- ic / ic cells to demonstrate that expression of wild-type Lbr proteins can rescue the observed phenotypes. For expression of full-length Lbr, a mouse cDNA that encodes a FLAG-tagged version of Lbr was cloned into the pMSCV-puro retroviral expression vector, previously shown to drive abundant protein expression in the EML cell line (53). Both EML-+/ ic and - ic / ic cells were transduced with the expression vector or an empty vector.…”

Activation of the Aryl Hydrocarbon Receptor by the Calcium/Calmodulin-Dependent Protein Kinase Kinase Inhibitor 7-Oxo-7H-benzimidazo[2,1-a]benz[de]isoquinoline-3-carboxylic Acid (STO-609)

“…It is interesting to note that the half-maximal effective concentration (EC 50 ) of STO-609-inducing CYP1A1 activity in MCF-7 cells, i.e., 3.4 M, is close to STO-609 concentrations used for inhibiting CaMKK activity in cell cultures, which ranged from 2.5 to 25 M (Tokumitsu et al, 2002;Schmitt et al, 2004;Gaines et al, 2008). In addition, STO-609 has been described as an efficient inhibitor of purified CaMKK␣ and ␤, with IC 50 values of 0.32 and 0.106 M, respectively.…”

Cooperative Activity of GABP with PU.1 or C/EBPε Regulates Lamin B Receptor Gene Expression, Implicating Their Roles in Granulocyte Nuclear Maturation

“…an estrogen regulated-Hoxb8 chimeric protein). The resulting cells are maintained in an undifferentiated state when cultured in media supplemented with SCF and β-estradiol (which constitutively activates the ER-Hoxb8 chimeric protein), but withdrawal of β-estradiol in the presence of SCF causes differentiation into fully mature neutrophils (19, 44). We analyzed this model for Lbr transcription and discovered that the undifferentiated cells expressed high levels of Lbr , despite low levels of Cebpe and undetectable Spi1 (Fig.…”

“…The total DNA concentration was equalized for each reaction by supplementing with pCMV empty vector DNA. Each transfection mixture also contained 0.2 μg of pCMV-β-galactosidase as an internal control to measure the transfection efficiency via β-galactosidase assays as described previously (19). After 48–72 h of transfection, cells were washed with ice cold PBS and then lysed by adding 200 μl of reporter lysis buffer (Promega) according to the manufacturer’s recommendations.…”

“…Importantly, use of the dominant-negative RARα403 expression system allows for the generation of EML-like myeloid cell lines from bone marrow of mice with specific genetic defects, such as deficiency of Lbr or C/EBPε (15, 18). Moreover, the neutrophils derived from EML-like cells generated with this technique express both primary and secondary granule proteins, and exhibit upregulation of transcription factors well-known to regulate neutrophil differentiation (19, 20). …”