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Ca2+/calmodulin-dependent kinase kinase alpha is expressed by monocytic cells and regulates the activation profile.

Guest CB, Deszo EL, Hartman ME, York JM, Kelley KW, Freund GG - PLoS ONE (2008)

Bottom Line: Inhibition of CaMKKalpha, enhanced PMA-dependent CD86 expression and reduced CD11b expression.In addition, inhibition was associated with decreased translocation of CaMKKalpha to the nucleus.Taken together, these findings indicate an important new role for CaMKKalpha in the differentiation of monocytic cells.

View Article: PubMed Central - PubMed

Affiliation: Division of Nutritional Sciences, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America.

ABSTRACT
Macrophages are capable of assuming numerous phenotypes in order to adapt to endogenous and exogenous challenges but many of the factors that regulate this process are still unknown. We report that Ca(2+)/calmodulin-dependent kinase kinase alpha (CaMKKalpha) is expressed in human monocytic cells and demonstrate that its inhibition blocks type-II monocytic cell activation and promotes classical activation. Affinity chromatography with paramagnetic beads isolated an approximately 50 kDa protein from nuclear lysates of U937 human monocytic cells activated with phorbol-12-myristate-13-acetate (PMA). This protein was identified as CaMKKalpha by mass spectrometry and Western analysis. The function of CaMKKalpha in monocyte activation was examined using the CaMKKalpha inhibitors (STO-609 and forskolin) and siRNA knockdown. Inhibition of CaMKKalpha, enhanced PMA-dependent CD86 expression and reduced CD11b expression. In addition, inhibition was associated with decreased translocation of CaMKKalpha to the nucleus. Finally, to further examine monocyte activation profiles, TNFalpha and IL-10 secretion were studied. CaMKKalpha inhibition attenuated PMA-dependent IL-10 production and enhanced TNFalpha production indicating a shift from type-II to classical monocyte activation. Taken together, these findings indicate an important new role for CaMKKalpha in the differentiation of monocytic cells.

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The CaMKKα inhibitor STO-609 enhances PMA-dependent up-regulation of CD86 but blocks CD11b expression and CaMKKα nuclear localization.(A and B) U937 cells were pretreated with or without 5 µg/ml STO-609 for 6 h prior to activation with 100 nM PMA for 48 h, as indicated. Surface expression of CD11b and CD86 was quantified by flow cytometry using FITC-conjugated CD11b and PE-conjugated CD86 antibodies. Results significantly different from PMA at α = 0.05 are indicated by (*). Results represent an average of three independent experiments±SEM. (C) Cells were treated as in panel A and CaMKKα was quantified by Western analysis of nuclear lysates. Results are representative of three independent experiments. (D) U937 cells were treated as in panel A and ERK1/2 phosphorylation (ERK-p) and mass (ERK) were measured by Western analysis in whole cell lysates. Results are representative of three independent experiments.
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pone-0001606-g003: The CaMKKα inhibitor STO-609 enhances PMA-dependent up-regulation of CD86 but blocks CD11b expression and CaMKKα nuclear localization.(A and B) U937 cells were pretreated with or without 5 µg/ml STO-609 for 6 h prior to activation with 100 nM PMA for 48 h, as indicated. Surface expression of CD11b and CD86 was quantified by flow cytometry using FITC-conjugated CD11b and PE-conjugated CD86 antibodies. Results significantly different from PMA at α = 0.05 are indicated by (*). Results represent an average of three independent experiments±SEM. (C) Cells were treated as in panel A and CaMKKα was quantified by Western analysis of nuclear lysates. Results are representative of three independent experiments. (D) U937 cells were treated as in panel A and ERK1/2 phosphorylation (ERK-p) and mass (ERK) were measured by Western analysis in whole cell lysates. Results are representative of three independent experiments.

Mentions: To determine the role of CaMKKα in PMA-dependent monocytic cell activation, STO-609 inhibition studies were performed. Figure 3A shows that, when U937 cells were pre-treated with STO-609 for 6 h prior to PMA addition for 48 h, cell surface CD86 expression was increased nearly 77% over PMA treatment alone (P<0.05). In contrast, STO-609 reduced PMA-dependent CD11b cell surface expression by 50% (P<0.05) (Figure 3B). Next, the consequence of STO-609 on PMA-dependent CaMKKα nuclear localization was examined. Western analysis (Figure 3C) demonstrated that STO-609 reduced CaMKKα nuclear localization by 50% (P<0.05). Similar to Figure 1C, PMA and/or STO-609 treatment had no impact on whole cell recovered CaMKKα (data not shown). Importantly, we have shown that ERK1/2 activation regulates CD86 and CD11b expression [17]. Interestingly, inhibition of CaMKKα with STO-609 had no impact on PMA-dependent activation of ERK1/2 (Figure 3D). These findings indicate that STO-609 inhibited PMA-induced up-regulation of CD11b and augmented PMA-stimulated CD86 expression. STO-609 also appears to inhibit PMA-dependent CaMKKα nuclear localization.


Ca2+/calmodulin-dependent kinase kinase alpha is expressed by monocytic cells and regulates the activation profile.

Guest CB, Deszo EL, Hartman ME, York JM, Kelley KW, Freund GG - PLoS ONE (2008)

The CaMKKα inhibitor STO-609 enhances PMA-dependent up-regulation of CD86 but blocks CD11b expression and CaMKKα nuclear localization.(A and B) U937 cells were pretreated with or without 5 µg/ml STO-609 for 6 h prior to activation with 100 nM PMA for 48 h, as indicated. Surface expression of CD11b and CD86 was quantified by flow cytometry using FITC-conjugated CD11b and PE-conjugated CD86 antibodies. Results significantly different from PMA at α = 0.05 are indicated by (*). Results represent an average of three independent experiments±SEM. (C) Cells were treated as in panel A and CaMKKα was quantified by Western analysis of nuclear lysates. Results are representative of three independent experiments. (D) U937 cells were treated as in panel A and ERK1/2 phosphorylation (ERK-p) and mass (ERK) were measured by Western analysis in whole cell lysates. Results are representative of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2229650&req=5

pone-0001606-g003: The CaMKKα inhibitor STO-609 enhances PMA-dependent up-regulation of CD86 but blocks CD11b expression and CaMKKα nuclear localization.(A and B) U937 cells were pretreated with or without 5 µg/ml STO-609 for 6 h prior to activation with 100 nM PMA for 48 h, as indicated. Surface expression of CD11b and CD86 was quantified by flow cytometry using FITC-conjugated CD11b and PE-conjugated CD86 antibodies. Results significantly different from PMA at α = 0.05 are indicated by (*). Results represent an average of three independent experiments±SEM. (C) Cells were treated as in panel A and CaMKKα was quantified by Western analysis of nuclear lysates. Results are representative of three independent experiments. (D) U937 cells were treated as in panel A and ERK1/2 phosphorylation (ERK-p) and mass (ERK) were measured by Western analysis in whole cell lysates. Results are representative of three independent experiments.
Mentions: To determine the role of CaMKKα in PMA-dependent monocytic cell activation, STO-609 inhibition studies were performed. Figure 3A shows that, when U937 cells were pre-treated with STO-609 for 6 h prior to PMA addition for 48 h, cell surface CD86 expression was increased nearly 77% over PMA treatment alone (P<0.05). In contrast, STO-609 reduced PMA-dependent CD11b cell surface expression by 50% (P<0.05) (Figure 3B). Next, the consequence of STO-609 on PMA-dependent CaMKKα nuclear localization was examined. Western analysis (Figure 3C) demonstrated that STO-609 reduced CaMKKα nuclear localization by 50% (P<0.05). Similar to Figure 1C, PMA and/or STO-609 treatment had no impact on whole cell recovered CaMKKα (data not shown). Importantly, we have shown that ERK1/2 activation regulates CD86 and CD11b expression [17]. Interestingly, inhibition of CaMKKα with STO-609 had no impact on PMA-dependent activation of ERK1/2 (Figure 3D). These findings indicate that STO-609 inhibited PMA-induced up-regulation of CD11b and augmented PMA-stimulated CD86 expression. STO-609 also appears to inhibit PMA-dependent CaMKKα nuclear localization.

Bottom Line: Inhibition of CaMKKalpha, enhanced PMA-dependent CD86 expression and reduced CD11b expression.In addition, inhibition was associated with decreased translocation of CaMKKalpha to the nucleus.Taken together, these findings indicate an important new role for CaMKKalpha in the differentiation of monocytic cells.

View Article: PubMed Central - PubMed

Affiliation: Division of Nutritional Sciences, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America.

ABSTRACT
Macrophages are capable of assuming numerous phenotypes in order to adapt to endogenous and exogenous challenges but many of the factors that regulate this process are still unknown. We report that Ca(2+)/calmodulin-dependent kinase kinase alpha (CaMKKalpha) is expressed in human monocytic cells and demonstrate that its inhibition blocks type-II monocytic cell activation and promotes classical activation. Affinity chromatography with paramagnetic beads isolated an approximately 50 kDa protein from nuclear lysates of U937 human monocytic cells activated with phorbol-12-myristate-13-acetate (PMA). This protein was identified as CaMKKalpha by mass spectrometry and Western analysis. The function of CaMKKalpha in monocyte activation was examined using the CaMKKalpha inhibitors (STO-609 and forskolin) and siRNA knockdown. Inhibition of CaMKKalpha, enhanced PMA-dependent CD86 expression and reduced CD11b expression. In addition, inhibition was associated with decreased translocation of CaMKKalpha to the nucleus. Finally, to further examine monocyte activation profiles, TNFalpha and IL-10 secretion were studied. CaMKKalpha inhibition attenuated PMA-dependent IL-10 production and enhanced TNFalpha production indicating a shift from type-II to classical monocyte activation. Taken together, these findings indicate an important new role for CaMKKalpha in the differentiation of monocytic cells.

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