Loss of alpha-tubulin polyglutamylation in ROSA22 mice is associated with abnormal targeting of KIF1A and modulated synaptic function.

Microtubules function as molecular tracks along which motor proteins transport a variety of cargo to discrete destinations within the cell. The carboxyl termini of alpha- and beta-tubulin can undergo different posttranslational modifications, including polyglutamylation, which is particularly abundant within the mammalian nervous system. Thus, this modification could serve as a molecular "traffic sign" for motor proteins in neuronal cells. To investigate whether polyglutamylated alpha-tubulin could perform this function, we analyzed ROSA22 mice that lack functional PGs1, a subunit of alpha-tubulin-selective polyglutamylase. In wild-type mice, polyglutamylated alpha-tubulin is abundant in both axonal and dendritic neurites. ROSA22 mutants display a striking loss of polyglutamylated alpha-tubulin within neurons, including their neurites, which is associated with decreased binding affinity of certain structural microtubule-associated proteins and motor proteins, including kinesins, to microtubules purified from ROSA22-mutant brain. Of the kinesins examined, KIF1A, a subfamily of kinesin-3, was less abundant in neurites from ROSA22 mutants in vitro and in vivo, whereas the distribution of KIF3A (kinesin-2) and KIF5 (kinesin-1) appeared unaltered. The density of synaptic vesicles, a cargo of KIF1A, was decreased in synaptic terminals in the CA1 region of hippocampus in ROSA22 mutants. Consistent with this finding, ROSA22 mutants displayed more rapid depletion of synaptic vesicles than wild-type littermates after high-frequency stimulation. These data provide evidence for a role of polyglutamylation of alpha-tubulin in vivo, as a molecular traffic sign for targeting of KIF1 kinesin required for continuous synaptic transmission

Additional file 3: Figure S1. of EphA receptors and ephrin-A ligands are upregulated by monocytic differentiation/maturation and promote cell adhesion and protrusion formation in HL60 monocytes

Immunofluorescence micrographs showing vinculin and paxillin localization in HL60 cell from the VD-TNF group. HL60 cells from the VD-TNF group were cultured on a coverslip surface on which EphA2-Fc or ephrin-A1-Fc and Fc plus Matrigel were adsorbed. To visualize focal adhesions, cells fixed with 4% paraformaldehyde were incubated with 0.02% Triton X-100 in PBS and then with a mixture of an anti-human vinculin mouse monoclonal antibody (hVIN-1, Sigma-Aldrich) at a dilution of 1:200 and an anti-human paxillin rabbit monoclonal antibody (Y113, Abcam, Cambridge, UK) at a dilution of 1:250 in 1% BSA-PBS for 60 min at 32 °C. After washing with PBS, the cells were incubated with a mixture of Alexa 488-conjugated goat anti-mouse IgG (5 μg/mL; Molecular Probes) and Alexa 568-conjugated donkey anti-rabbit IgG (5 μg/mL; Molecular Probes) in 1% BSA-PBS for 30 min at 32°C. After mounting with PermaFluor (Thermo Fisher Scientific), fluorescence images of vinculin (green) and paxillin immunostaining (red) were photographed. (PPTX 976 kb

Everolimus-induced human keratinocytes toxicity is mediated by STAT3 inhibition

BACKGROUND: Mammalian target of rapamycin (mTOR) inhibitors are associated with dermatological adverse events. The chief aim of this study was to examine the relation between the signal transducer and activator of transcription 3 (STAT3) protein and the dermatological adverse events associated with the mTOR inhibitor everolimus. METHODS: We evaluated the effects of STAT3 activity and related signal transduction activities on everolimus-induced cell growth inhibition in the human keratinocyte HaCaT cell line via a WST-8 assay, and on signal transduction mechanisms involved in everolimus treatments via a western blot analysis. Apoptosis was evaluated using an imaging cytometric assay. RESULTS: The cell growth inhibitory effects of everolimus were enhanced by stattic or STA-21, which are selective inhibitors of STAT3, treatment in HaCaT cells, although such effects were not observed in Caki-1 and HepG2 cells. Phosphorylation at tyrosine 705 of STAT3 was decreased by treatment with everolimus in a dose-dependent manner in HaCaT cells; in contrast, phosphorylation at serine 727 was not decreased by everolimus, but slightly increased. Furthermore, we found that pretreatment of p38 MAPK inhibitor and transfection with constitutively active form of STAT3 in HaCaT cells resisted the cytostatic activity of everolimus. CONCLUSIONS: These findings suggest that STAT3 activity may be a biomarker of everolimus-induced dermatological toxicity

Association of Toxicity of Sorafenib and Sunitinib for Human Keratinocytes with Inhibition of Signal Transduction and Activator of Transcription 3 (STAT3)

Hand-foot skin reaction is a most common multi-kinase inhibitor-related adverse event. This study aimed to examine whether the toxicity of sorafenib and sunitinib for human keratinocytes was associated with inhibiting signal transduction and activator of transcription 3 (STAT3). We studied whether STAT3 activity affects sorafenib- and sunitinib-induced cell growth inhibition in HaCaT cells by WST-8 assay. Stattic enhanced the cell-growth inhibitory and apoptotic effects of sorafenib and sunitinib. HaCaT cells transfected with constitutively-active STAT3 (STAT3C) were resistant to the sorafenib- and sunitinib-induced cell growth inhibition. STAT3 activity decreased after short-term treatment with sorafenib and sunitinib in a dose-dependent manner and recovered after long-term treatment with sorafenib and sunitinib at low doses. Moreover, the expression of survivin and bcl-2 decreased after treatment with sorafenib and sunitinib was concomitant with variations in STAT3 activity. Sorafenib-induced STAT3 inhibition was mediated by regulation via MAPK pathways in HaCaT cells, while sunitinib-induced STAT3 inhibition was not. Thus, STAT3 activation mediating apoptosis suppressors may be a key factor in sorafenib and sunitinib-induced keratinocyte cytotoxicity