Dataset Information

AKT-independent activation of p38 MAP kinase promotes vascular calcification.

ABSTRACT: Vascular calcification is prevalent in patients with atherosclerosis, and oxidative stress promotes pathogenesis of atherosclerosis. We have previously reported that activation of AKT by oxidative stress induces vascular calcification. Using sodium dichloroacetate (DCA), a previously reported small molecule inhibitor of AKT, the present studies uncovered an AKT-independent mechanism in regulating vascular calcification. We found that DCA dose-dependently induced calcification of vascular smooth muscle cells (VSMC) in vitro and aortic rings ex vivo. Furthermore, DCA markedly enhanced vascular calcification in atherosclerotic ApoE knockout mice in vivo. DCA-induced VSMC calcification was associated with increased Runx2, but not via activation of AKT, a key upstream signal that upregulates Runx2 during VSMC calcification. In contrast, DCA inhibited AKT activation and induced activation of p38 MAPK in calcified atherosclerotic lesions in vivo and calcified VSMC in vitro. Using a pharmacological inhibitor and shRNA for p38 MAPK, we demonstrated that inhibition of p38 MAPK blocked DCA-induced Runx2 upregulation and VSMC calcification. Furthermore, Runx2 deletion attenuated DCA-induced VSMC calcification. Immunoprecipitation analysis revealed association of p38 MAPK with Runx2, which was enhanced by DCA treatment. Knockdown p38 MAPK inhibited DCA-induced Runx2 transactivity, supporting the function of p38 MAPK in regulating Runx2 transactivity. Our studies have uncovered a new function of DCA in regulating vascular calcification, via AKT-independent activation of p38 MAPK. Furthermore, we have identified novel interaction between p38 MAPK and Runx2 enhances Runx2 transactivity, thus promoting VSMC calcification. These results revealed a novel signaling mechanism underlying DCA-induced vascular calcification, and offer opportunities to identify new therapeutic targets.

SUBMITTER: Yang Y

PROVIDER: S-EPMC5952999 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:TFEB (transcription factor EB) and TFE3 (transcription factor E3) are "master regulators" of autophagy and lysosomal biogenesis. The stress response p38 mitogen-activated protein (MAP) kinases affect multiple intracellular responses including inflammation, cell growth, differentiation, cell death, senescence, tumorigenesis, and autophagy. Small molecule p38 MAP kinase inhibitors such as SB202190 are widely used in dissection of related signal transduction mechanisms including redox biology and autophagy. Here, we initially aimed to investigate the links between p38 MAP kinase and TFEB/TFE3-mediated autophagy and lysosomal biogenesis. Unexpectedly, we found that only SB202190, rather than several other p38 inhibitors, promotes TFEB and TFE3 to translocate from the cytosol into the nucleus and subsequently enhances autophagy and lysosomal biogenesis. In addition, siRNA-mediated Tfeb and Tfe3 knockdown effectively attenuated SB202190-induced gene expression and lysosomal biogenesis. Mechanistical studies showed that TFEB and TFE3 activation in response to SB202190 is dependent on PPP3/calcineurin rather than on the inhibition of p38 or MTOR signaling, the main pathway for regulating TFEB and TFE3 activation. Importantly, SB202190 increased intracellular calcium levels, and calcium chelator BAPTAP-AM blocked SB202190-induced TFEB and TFE3 activation as well as autophagy and lysosomal biogenesis. Moreover, endoplasmic reticulum (ER) calcium is required for TFEB and TFE3 activation in response to SB202190. In summary, we identified a previously uncharacterized role of SB202190 in activating TFEB- and TFE3-dependent autophagy and lysosomal biogenesis via ER calcium release and subsequent calcium-dependent PPP3/calcineurin activation, leading to dephosphorylation of TFEB and TFE3. Given the importance of p38 MAP kinase invarious conditions including oxidative stress, the findings collectively indicate that SB202190 should not be used as a specific inhibitor for elucidating the p38 MAP kinase biological functions due to its potential effect on activating autophagy-lysosomal axis.

Project description:Vascular calcification is a serious cardiovascular complication that contributes to the increased morbidity and mortality of patients with diabetes mellitus. Hyperglycemia, a hallmark of diabetes mellitus, is associated with increased vascular calcification and increased modification of proteins by O-linked N-acetylglucosamine (O-GlcNAcylation).We sought to determine the role of protein O-GlcNAcylation in regulating vascular calcification and the underlying mechanisms.Low-dose streptozotocin-induced diabetic mice exhibited increased aortic O-GlcNAcylation and vascular calcification, which was also associated with impaired aortic compliance in mice. Elevation of O-GlcNAcylation by administration of Thiamet-G, a potent inhibitor for O-GlcNAcase that removes O-GlcNAcylation, further accelerated vascular calcification and worsened aortic compliance of diabetic mice in vivo. Increased O-GlcNAcylation, either by Thiamet-G or O-GlcNAcase knockdown, promoted calcification of primary mouse vascular smooth muscle cells. Increased O-GlcNAcylation in diabetic arteries or in the O-GlcNAcase knockdown vascular smooth muscle cell upregulated expression of the osteogenic transcription factor Runx2 and enhanced activation of AKT. O-GlcNAcylation of AKT at two new sites, T430 and T479, promoted AKT phosphorylation, which in turn enhanced vascular smooth muscle cell calcification. Site-directed mutation of AKT at T430 and T479 decreased O-GlcNAcylation, inhibited phosphorylation of AKT at S473 and binding of mammalian target of rapamycin complex 2 to AKT, and subsequently blocked Runx2 transactivity and vascular smooth muscle cell calcification.O-GlcNAcylation of AKT at 2 new sites enhanced AKT phosphorylation and activation, thus promoting vascular calcification. Our studies have identified a novel causative effect of O-GlcNAcylation in regulating vascular calcification in diabetes mellitus and uncovered a key molecular mechanism underlying O-GlcNAcylation-mediated activation of AKT.

Project description:BackgroundConventional p38α inhibitors have limited efficacy in rheumatoid arthritis, possibly because p38 blockade suppresses the counter-regulatory mechanisms that limit inflammation. In contrast, targeting the upstream MAP kinase kinases, MKK3 and MKK6, partially maintains p38-mediated anti-inflammatory responses in bone marrow-derived macrophages (BMDM). In this study, we explored the mechanisms that preserve anti-inflammatory gene expression by evaluating differential regulation of IL-10 and p38-dependent anti-inflammatory genes in MKK3-/-, MKK6-/-, and p38 inhibitor-treated wildtype cells.MethodsBMDM from wild type (WT), MKK3-/-, and MKK6-/- mice were pre-treated with p38 inhibitor SB203580 (SB), JNK inhibitor SP600125 (SP), and/or ERK inhibitor PD98059 (PD) and stimulated with LPS. Supernatant protein levels were measured by multiplex bead immunoassay. mRNA expression was determined by qPCR and protein expression by Western blot analysis. De novo IL-10 mRNA synthesis was quantified in cells treated with ethynyl-uridine and LPS followed by reverse transcription and qPCR. mRNA half-life was measured in LPS-treated cells that were then incubated with actinomycin D ± SB203580.ResultsPre-treatment of WT BMDM with p38 inhibitor significantly reduced IL-10 production in the three groups, while ERK and JNK inhibitors had minimal effects. IL-10 production was significantly decreased in MKK3-/- BMDM compared with either WT or MKK6-/- cells. IL-10 mRNA expression was modestly reduced in MKK3-/- BMDM but was preserved in MKK6-/- cells compared with WT. De novo IL-10 mRNA synthesis was inhibited in MKK3-/- and p38 inhibitor pre-treated cells, but not MKK6-/- cells compared with WT. IL-10 mRNA half-life was markedly reduced in p38 inhibitor-treated WT cells while MKK-deficiency had minimal effect. DUSP1 mRNA levels were preserved in MKK-deficient cells but not in p38 inhibitor-treated WT cells. Tristetraprolin mRNA and protein levels were reduced in p38 inhibitor-treated WT cells compared with MKK6-/- cells.ConclusionUnlike p38-inhibition, the absence of MKK6 mostly preserves IL-10 and TTP protein expression in BMDM. MKK6-deficiency also spares DUSP1 and IL-1RA, which are key negative regulators of the inflammatory response. Together, these data suggest that MKK6 is a potential therapeutic target in RA.

Dataset Information

AKT-independent activation of p38 MAP kinase promotes vascular calcification.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets