RESUMO
In this work, we examined the differentiation of oligodendrocytic MO3.13 cells and changes in their gene expression after treatment with phorbol 12-myristate 13-acetate, PMA, or with RNA polymerase I (Pol I) inhibitor, CX-5461. We found that MO3.13 cells changed their morphology when treated with both agents. Interestingly, CX-5461, but not PMA, induced noticeable changes in the integrity of the nucleoli. Then, we analyzed the p53 transcriptional activity in MO3.13 cells and found that it was increased in both cell populations, but particularly in cells treated with PMA. Interestingly, this high p53 transcriptional activity in PMA-treated cells coincided with a lower level of an unmodified (non-phosphorylated) form of this protein. Since morphological changes in MO3.13 cells after PMA and CX-5461 treatment were evident, suggesting that cells were induced to differentiate, we performed RNA-seq analysis of PMA-treated cells, to reveal the direction of alterations in gene expression. The analysis showed that the largest group of upregulated genes consisted of those involved in myogenesis and K-RAS signaling, rather than those associated with oligodendrocyte lineage progression.
Assuntos
Perfilação da Expressão Gênica , Proteína Supressora de Tumor p53 , Humanos , Desenvolvimento Muscular/genética , RNA-Seq , Acetato de Tetradecanoilforbol/farmacologia , Regulação para CimaRESUMO
Cytokine receptors, such as tumor necrosis factor receptor I (TNFRI, also known as TNFRSF1A) and lymphotoxin ß receptor (LTßR), activate inflammatory nuclear factor (NF)-κB signaling upon stimulation. We have previously demonstrated that depletion of ESCRT components leads to endosomal accumulation of TNFRI and LTßR, and their ligand-independent signaling to NF-κB. Here, we studied whether other perturbations of the endolysosomal system could trigger intracellular accumulation and signaling of ligand-free LTßR. While depletion of the CORVET components had no effect, knockdown of Rab7a or HOPS components, or pharmacological inhibition of lysosomal degradation, caused endosomal accumulation of LTßR and increased its interaction with the TRAF2 and TRAF3 signaling adaptors. However, the NF-κB pathway was not activated under these conditions. We found that knockdown of Rab7a or HOPS components led to sequestration of LTßR in intraluminal vesicles of endosomes, thus precluding NF-κB signaling. This was in contrast to the LTßR localization on the outer endosomal membrane that was seen after ESCRT depletion and was permissive for signaling. We propose that the inflammatory response induced by intracellular accumulation of endocytosed cytokine receptors critically depends on the precise receptor topology within endosomal compartments.
Assuntos
Receptor beta de Linfotoxina/metabolismo , NF-kappa B/metabolismo , Endossomos/metabolismo , Técnicas de Silenciamento de Genes , Células HEK293 , Células HeLa , Humanos , Lisossomos/metabolismo , Transporte Proteico , Transdução de Sinais , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/metabolismo , Proteínas de Transporte Vesicular/deficiência , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab de Ligação ao GTP/deficiência , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7RESUMO
BACKGROUND: Lymphotoxin ß receptor (LTßR) is a member of tumor necrosis factor receptor (TNFR) superfamily which regulates the immune response. At the cellular level, upon ligand binding, the receptor activates the pro-inflammatory NF-κB and AP-1 pathways. Yet, the intracellular distribution of LTßR, the routes of its endocytosis and their connection to the signaling activation are not characterized. Here, we investigated the contribution of LTßR internalization to its signaling potential. METHODS: Intracellular localization of LTßR in unstimulated and stimulated cells was analyzed by confocal microscopy. Endocytosis impairment was achieved through siRNA- or CRISPR/Cas9-mediated depletion, or chemical inhibition of proteins regulating endocytic routes. The activation of LTßR-induced signaling was examined. The levels of effector proteins of the canonical and non-canonical branches of the NF-κB pathway, and the phosphorylation of JNK, Akt, ERK1/2, STAT1 and STAT3 involved in diverse signaling cascades, were measured by Western blotting. A transcriptional response to LTßR stimulation was assessed by qRT-PCR analysis. RESULTS: We demonstrated that LTßR was predominantly present on endocytic vesicles and the Golgi apparatus. The ligand-bound pool of the receptor localized to endosomes and was trafficked towards lysosomes for degradation. Depletion of regulators of different endocytic routes (clathrin-mediated, dynamin-dependent or clathrin-independent) resulted in the impairment of LTßR internalization, indicating that this receptor uses multiple entry pathways. Cells deprived of clathrin and dynamins exhibited enhanced activation of canonical NF-κB signaling represented by increased degradation of IκBα inhibitor and elevated expression of LTßR target genes. We also demonstrated that clathrin and dynamin deficiency reduced to some extent LTßR-triggered activation of the non-canonical branch of the NF-κB pathway. CONCLUSIONS: Our work shows that the impairment of clathrin- and dynamin-dependent internalization amplifies a cellular response to LTßR stimulation. We postulate that receptor internalization restricts responsiveness of the cell to subthreshold stimuli. Video Abstract.
Assuntos
Clatrina/metabolismo , Dinaminas/metabolismo , Endocitose , Receptor beta de Linfotoxina/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais , Células A549 , Endossomos/metabolismo , Regulação da Expressão Gênica , Complexo de Golgi/metabolismo , Células HEK293 , Humanos , Ligantes , Lisossomos/metabolismo , ProteóliseRESUMO
BACKGROUND: Lymphotoxin ß receptor (LTßR) plays important roles in the development of the immune system and immune response. At the cellular level, ligand-bound LTßR activates the pro-inflammatory NF-κB pathway but the detailed mechanisms regulating its signaling remain unknown. Understanding them is of high importance since LTßR and its ligands are promising therapeutic targets. Here, we studied the consequences of perturbed cellular cholesterol content on LTßR-induced NF-κB signaling. METHODS: To modulate cholesterol availability and/or level in lung carcinoma A549 and H2228, and endothelial HUVEC cells different treatment regimens with filipin, methyl-ß-cyclodextrin and simvastatin were applied. LTßR localization was studied by confocal microscopy. The activity of LTßR-induced NF-κB pathway was assessed by measuring the levels of NF-κB pathway inhibitor IκBα and phosphorylation of RelA transcription factor by Western blotting. The NF-κB transcriptional response, production of chemokines and adhesion molecules were examined by qRT-PCR, ELISA, and Western blotting, respectively. Adherence of different types of primary immune cells to epithelial A549 cells and endothelial HUVECs was measured fluorometrically. Interactions of LTßR with its protein partners were investigated by immunoprecipitation. RESULTS: We showed that filipin-mediated sequestration of cholesterol or its depletion from the plasma membrane with methyl-ß-cyclodextrin impaired LTßR internalization and potentiated LTßR-dependent activation of the canonical branch of the NF-κB pathway. The latter was manifested by enhanced degradation of IκBα inhibitor, elevated RelA phosphorylation, substantial increase in the expression of NF-κB target genes encoding, among others, cytokines and adhesion molecules known to play important roles in immune response. It was followed by robust secretion of CXCL8 and upregulation of ICAM1, that favored the adhesion of immune cells (NK and T cells, neutrophils) to A549 cells and HUVECs. Mechanistically, we showed that cholesterol depletion stabilized interactions of ligand-stimulated LTßR with modified forms of TRAF2 and NEMO proteins. CONCLUSIONS: Our results showed that the reduction of the plasma membrane content of cholesterol or its sequestration strongly potentiated signaling outcome initiated by LTßR. Thus, drugs modulating cholesterol levels could potentially improve efficacy of LTßR-based therapies. Video abstract.
Assuntos
Colesterol/farmacologia , Receptor beta de Linfotoxina/antagonistas & inibidores , Receptor beta de Linfotoxina/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células A549 , Células Cultivadas , Humanos , Células Jurkat , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismoRESUMO
CacyBP/SIP is a multifunctional protein present in various cells and tissues. However, its expression and role in the epidermis has not been explored so far. In this work, using RT-qPCR, Western blot analysis and three-dimensional (3D) organotypic cultures of HaCaT keratinocytes we show that CacyBP/SIP is present in the epidermis. To investigate the possible role of CacyBP/SIP in keratinocytes we obtained CacyBP/SIP knockdown cells and studied the effect of CacyBP/SIP deficiency on their differentiation and response to viral infection. We found that CacyBP/SIP knockdown results in reduced expression of epidermal differentiation markers in both undifferentiated and differentiated HaCaT cells. Since epidermis is engaged in immune defense, the impact of CacyBP/SIP knockdown on this process was also analyzed. By applying RT-qPCR and Western blot it was found that poly(I:C), a synthetic analog of double-stranded RNA that mimics viral infection, stimulated the expression of genes involved in antiviral response, such as IFIT1, IFIT2 and OASL. Interestingly, following poly(I:C) stimulation, the level of expression of these genes was significantly lower in cells with CacyBP/SIP knockdown than control ones. Since the signaling pathway mediating cellular responses to viral infection involves, among others, the STAT1 transcription factor, we measured its activity using luciferase assay and found that it was lower in CacyBP/SIP knockdown HaCaT cells. Altogether, the presented results indicate that CacyBP/SIP promotes epidermal differentiation and might be involved in response of the skin cells to viral infection.
Assuntos
Queratinócitos , Transdução de Sinais , Proteínas de Ligação ao Cálcio/metabolismo , Diferenciação Celular , Imunidade , Queratinócitos/metabolismo , HumanosRESUMO
The study covered 79 patients after treatment of retinal detachment. Double vision, strabismus and disturbances of eyeballs motility were found. Up to 12 months after intervention, the deterioration of binocular vision was observed in 48.28 to 89.66% of patients, depending on the method used. The majority of disturbances were observed during the first 3 months with tendency to gradual subsidence during consecutive 9 months. A patient, after treatment of retinal detachment, can be qualified to return to work where stereopsis is needed under condition that ophthalmologic examination is done every three months during the first year after operation and than once a year.