The p38/MAPK pathway regulates microtubule polymerization through phosphorylation of MAP4 and Op18 in hypoxic cells

Abstract

In both cardiomyocytes and HeLa cells, hypoxia (1% O(2)) quickly leads to microtubule disruption, but little is known about how microtubule dynamics change during the early stages of hypoxia. We demonstrate that microtubule associated protein 4 (MAP4) phosphorylation increases while oncoprotein 18/stathmin (Op18) phosphorylation decreases after hypoxia, but their protein levels do not change. p38/MAPK activity increases quickly after hypoxia concomitant with MAP4 phosphorylation, and the activated p38/MAPK signaling leads to MAP4 phosphorylation and to Op18 dephosphorylation, both of which induce microtubule disruption. We confirmed the interaction between phospho-p38 and MAP4 using immunoprecipitation and found that SB203580, a p38/MAPK inhibitor, increases and MKK6(Glu) overexpression decreases hypoxic cell viability. Our results demonstrate that hypoxia induces microtubule depolymerization and decreased cell viability via the activation of the p38/MAPK signaling pathway and changes the phosphorylation levels of its downstream effectors, MAP4 and Op18.

Fig. 1

Hypoxia-induced microtubule depolymerization. Immunofluorescent confocal micrographs and immunoblot analysis of microtubule dynamics in neonatal rat cardiomyocytes (CMs) and HeLa cells. a Micrographs of CMs and HeLa cells, showing control cells and cells after 15, 30, and 60 min of hypoxia. All the cells were stained for α-tubulin (green) and the nuclear stain DAPI (blue). Bar 25 μm. The boxed areas are shown at higher magnification in the inserts to illustrate details of MTs. Bar 6 μm. The immunoblots in (b) were prepared from CMs and HeLa cells. Free and polymerized tubulin fractions were prepared and probed with anti-α-tubulin antibody; GAPDH in the cytosol fractions was chosen as the internal control for free tubulin and VDAC in mitochondrial fractions was chosen as the control for polymerized tubulin. Graph represents the relative integrated signal from densitometer readings for three separate experiments. The data represent the (n = 3). *P < 0.05, **P < 0.01 versus controls, one-way ANOVA followed by Tukey comparison tests

Fig. 2

Effect of hypoxia on MAP4 and Op18 phosphorylation. Western blot analysis of phospho-MAP4 (p-MAP4), MAP4, phospho-Op18 (p-Op18) and Op18 in neonatal rat CMs and HeLa cells under normoxic and hypoxic conditions. Representative western blots are shown for the two groups. Graph represents the (n = 3) of the relative integrated signals. *P < 0.05, **P < 0.01 versus controls

Fig. 3

Hypoxia activated p38/MAPK signaling pathway. Representative blots and data summary of phospho-p38 (p-p38) and p38 in CMs and HeLa cells under normoxic and hypoxic conditions. Representative western blots are shown for the two groups. Graph represents the (n = 3) of the relative integrated signals *P < 0.05, **P < 0.01 versus controls

Fig. 4

Effect of the p38/MAPK inhibitor, SB203580, on the morphology and quantification of MT dynamics in HeLa cells and neonatal rat CMs. a Immunofluorescent confocal micrographs of CMs and HeLa cells under normoxic and hypoxic [1% O₂ for 30 min (H 30)] conditions with and without the addition of SB203580 (SB). Cells were stained for α-tubulin (green) and with DAPI for the nuclei (blue). Bar, 25 μm. The boxed areas are shown at higher magnification in the inserts to illustrate details of MTs. Bar 6 μm. b Immunoblots of CMs and HeLa cells. Polymerized tubulin fractions were prepared and probed with an anti-α-tubulin antibody, and VDAC was used as a standard control. Graph represents the (n = 3) of the relative integrated signals. *P < 0.05, **P < 0.01 versus controls

Fig. 5

Effect of MKK(Glu) overexpression on the morphology and quantification of MT dynamics in HeLa cells and neonatal rat CMs. a In the immunofluorescent confocal micrographs all the cells were divided into control, GFP transduction and MKK6(Glu) transduction groups and were stained for α-tubulin (red) and the nuclear stain DAPI (blue). Bar, 25 μm. The boxed areas are shown at higher magnification in the upper inserts to illustrate details of MTs. Bar 6 μm. The lower inserts show the cells containing GFP after transduction (note the lack of staining in the control group). Bar 25 μm. b Immunoblots of cardiomyocytes and HeLa cells. Polymerized tubulin fractions of the cells were prepared and probed with anti-α-tubulin antibody, and VDAC was used as a control. Graph represents the (n = 3) of the relative integrated signals. *P < 0.05, **P < 0.01 versus controls

Fig. 6

Involvement of SB203580 in hypoxia-induced MAP4 phosphorylation and Op18 dephosphorylation. Western blot analysis of phospho-MAP4 (p-MAP4), MAP4, phospho-Op18 (p-Op18) and Op18 of CMs and HeLa cells in the presence or absence of the p38/MAPK inhibitor SB203580 (5 μM, SB) under normoxic and hypoxic (1% O₂ for 30 min, H 30) conditions. Cell lysates were immunoblotted with antibodies that recognize p-MAP4 (Ser768), MAP4, p-Op18 (Ser16), Op18 and GAPDH. A representative western blot is shown. Graph represents the (n = 3) of the relative integrated signals. *P < 0.05, versus controls; ^# P < 0.05, versus H 30 group

Fig. 7

Involvement of MKK6(Glu) in hypoxia-induced MAP4 phosphorylation and Op18 dephosphorylation. CMs and HeLa cells were divided into control, GFP transduction and MKK6(Glu) transduction groups. Cell lysates were immunoblotted with antibodies that recognize phospho-MAP4 (p-MAP4; Ser768), MAP4, phospho-Op18 (p-Op18; Ser16), Op18 and GAPDH. A representative western blot is shown. Graph represents the (n = 3) of the relative integrated signals. *P < 0.05, versus GFP group

Fig. 8

The interaction between phospho-p38 (p-p38) and MAP4 detected by immunoprecipitation under normoxic (N) and hypoxic [1% O₂ for 30 min (H)] conditions. The representative blots were immunoprecipitated by anti-phospho-p38 and anti-MAP4 antibodies in CMs and HeLa cells and then immunoblotted with anti-MAP4 or anti-phospho-p38 antibody. IP Immunoprecipitation, IB immunoblotting, IC isotype control

Fig. 9

Cell viability. CMs and HeLa cells were pretreated with the p38 inhibitor SB203580 (5 μM) or MKK6(Glu) overexpression and then subjected to 30 min of hypoxia. Graph represents the (n = 6) of the relative integrated signals. *P < 0.01, versus normoxic control group; ^# P < 0.01, versus hypoxic control group; one-way ANOVA followed by Tukey’s post-hoc tests

Fig. 10

A schematic representation of how hypoxia signaling promotes MT disruption. Hypoxia-activated p38/MAPK phosphorylates the downstream effector MAP4, which promotes MT disruption. It also suggests a link between p38/MAPK activation and Op18 dephosphorylation, which also promotes MT disruption. The mechanism by which p38/MAPK causes Op18 dephosphorylation and MT disruption remains unknown