Involvement of calmodulin and calmodulin kinase II in tumor necrosis factor alpha-induced survival of bone marrow derived macrophages

Abstract

We previously showed that survival signaling in TNFα-treated, human THP1-derived macrophages (TDMs) has an obligatory requirement for constitutive Ca(2+) influx through a mechanism involving calmodulin/calmodulin kinase II (CAM/CAMKII). We also demonstrated that such requirement also applies to the protective actions of TNFα in murine bone marrow-derived macrophages (BMDMs) and that TRPC3 channels mediate constitutive Ca(2+) influx. Using a pharmacological approach we here examined if in BMDMs, similarly to TDMs, TNFα-induced survival signaling also involves CAM/CAMKII. In BMDMs, TNFα induced rapid activation of the survival pathways NFκB, AKT and p38MAPK. All these routes were activated in a PI3K-dependent fashion. Activation of AKT and NFκB, but not that of p38MAPK, was abrogated by the CAM inhibitor W7, while KN-62, a CAMKII inhibitor, prevented activation of AKT and p38MAPK but not that of NFκB. Inhibition of CAM or CAMKII completely prevented the protective actions of TNFα. Our observations indicate that in BMDMs CAM and CAMKII have differential contributions to the components of TNFα-dependent survival signaling and underscore a complex interplay among canonical survival routes. These findings set a signaling framework to understand how constitutive Ca(2+) influx couples to macrophage survival in BMDMs.

Figure 1

A) Bone marrow-derived macrophages were incubated for 24 hours in complete growth medium (CM), serum-free RPMI medium (RPMI) or RPMI containing TNFα (TNF, 10 ng/ml) in the presence or absence of selective inhibitors of PI3K (LY294002 or LY, 10 μM), CAM (W7, 10 μM), CAMKII (KN62, 25 μM), p38MAPK (SB203580 or SB, 10 μM) or IkBα (hypoestoxide, “Hypo”, 50 μM). Alternatively, cells were incubated (24 h) in RPMI containing those inhibitors but in the absence of TNFα. Following treatments macrophages were processed for evaluation of apoptosis by TUNEL assay (see details in Materials and Methods). *P<0.05, **P<0.0001, respect to RPMI; ***P<0.0003 respect to RPMI+TNF. “ns”: not statistically significant difference. Averages are from four independent experiments. B) Bone marrow-derived macrophages were incubated for 24 hours in serum-free RPMI medium (RPMI) or RPMI containing inhibitors of survival pathways at the concentrations indicated in panel “A”. Following treatments cells were processed for immunodetection of cleaved PARP (89 kDa) in whole cell lysates. Membranes were reprobed for GAPDH to control for protein loading. Shown is a blot representative of three independent experiments and its corresponding densitometric analysis.

Figure 1

A) Bone marrow-derived macrophages were incubated for 24 hours in complete growth medium (CM), serum-free RPMI medium (RPMI) or RPMI containing TNFα (TNF, 10 ng/ml) in the presence or absence of selective inhibitors of PI3K (LY294002 or LY, 10 μM), CAM (W7, 10 μM), CAMKII (KN62, 25 μM), p38MAPK (SB203580 or SB, 10 μM) or IkBα (hypoestoxide, “Hypo”, 50 μM). Alternatively, cells were incubated (24 h) in RPMI containing those inhibitors but in the absence of TNFα. Following treatments macrophages were processed for evaluation of apoptosis by TUNEL assay (see details in Materials and Methods). *P<0.05, **P<0.0001, respect to RPMI; ***P<0.0003 respect to RPMI+TNF. “ns”: not statistically significant difference. Averages are from four independent experiments. B) Bone marrow-derived macrophages were incubated for 24 hours in serum-free RPMI medium (RPMI) or RPMI containing inhibitors of survival pathways at the concentrations indicated in panel “A”. Following treatments cells were processed for immunodetection of cleaved PARP (89 kDa) in whole cell lysates. Membranes were reprobed for GAPDH to control for protein loading. Shown is a blot representative of three independent experiments and its corresponding densitometric analysis.

Figure 2

Bone marrow-derived macrophages were treated for the indicated times with TNFα (TNF, 10 ng/ml) in the absence (A, B, E) or presence (C, D, F) of LY294002 (LY, 10 μM) and then processed for immunodetection of phospho-IκBα (Ser32/36, 40 kDa, A, D), phospho-AKT (Ser473; 60 kDa, B, C), or phospho-p38MAPK (pP38, Thr180/Tyr182; 43 kDa, E, F) in whole cell lysates. Membranes were reprobed for GAPDH, total AK or total p38MAPK (P38) to control for protein loading. Blots are representative from three independent experiments.

Figure 2

Bone marrow-derived macrophages were treated for the indicated times with TNFα (TNF, 10 ng/ml) in the absence (A, B, E) or presence (C, D, F) of LY294002 (LY, 10 μM) and then processed for immunodetection of phospho-IκBα (Ser32/36, 40 kDa, A, D), phospho-AKT (Ser473; 60 kDa, B, C), or phospho-p38MAPK (pP38, Thr180/Tyr182; 43 kDa, E, F) in whole cell lysates. Membranes were reprobed for GAPDH, total AK or total p38MAPK (P38) to control for protein loading. Blots are representative from three independent experiments.

Figure 3

Bone marrow-derived macrophages were pre-incubated for 15 min with the calmodulin inhibitor W7 (25 μM), treated with TNFα (TNF, 10 ng/ml) for the indicated times and then processed for immunodetection of phospho-AKT (Ser473; 60 kDa, A), phospho-IκBα (Ser32/36, 40 kDa, B) or phospho-p38MAPK (pP38, Thr180/Tyr182Ser473; 43 kDa, C) in whole cell lysates. Membranes were reprobed for total AKT, GAPDH or total p38MAPK to control for protein loading. Blots are representative from three independent experiments.

Figure 4

Bone marrow-derived macrophages were pre-incubated for 15 min with the calmodulin-dependent kinase II inhibitor KN62 (10 μM), treated with TNFα (TNF, 10 ng/ml) for the indicated times and then processed for immunodetection of phospho-AKT (Ser473; 60 kDa, A), phospho-IκBα (Ser32/36, 40 kDa, B) or phospho-p38MAPK (pP38, Thr180/Tyr182Ser473; 43 kDa, C) in whole cell lysates. Membranes were reprobed for total AKT, GAPDH or total p38MAPK to control for protein loading. Blots are representative from three independent experiments.