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| Author(s): |
Andressa Coope
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | Campinas, SP. |
| Institution: | Universidade Estadual de Campinas (UNICAMP). Faculdade de Ciências Médicas |
| Defense date: | 2011-04-29 |
| Examining board members: |
Licio Augusto Velloso;
Augusto Ducati Luchessi;
Mario José Abdalla Saad;
Heraldo Possolo de Souza;
Silvana Bordin
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| Advisor: | Licio Augusto Velloso |
| Abstract | |
TLR4 activation and the induction of endoplasmic reticulum stress (ERS) are two of the most important mechanisms connecting excessive fat with insulin resistance. Recently, it was shown that activation of TLR4 can, per se, induce ERS, suggesting that TLR4 is a primary event in the induction of the cellular stress that contributes to increased inflammatory gene expression. However, the mechanisms linking these molecular events are unknown. The chaperones GRP78 and GRP94 play an important role during the assembly of newly translated TLR4 molecules. In addition, the chaperone GRP94 escorts the protein to the cell membrane. Under prolonged activation, the demand for newly synthesized TLR4 molecules increases, and thus, the demand for new chaperones. Therefore, we hypothesized that under increased activation of TLR4, the synthesis of the protein would not be matched by the expression of chaperones, thus, triggering ERS. To test this hypothesis, the monocyte cell line THP-1 was incubated with LPS and the expression/activation of proteins involved in ERS was determined by real-time PCR, flow-cytometry, immunoprecipitation and immunoblot. In some experiments, cells were deprived of glucose or treated with siRNA to increase or decrease, respectively, the expression of the chaperones. Time-course experiments revealed that LPS led to a 2.5-fold increase of TLR4 expression starting as early as 8h, peaking after 24h and remaining significantly increased after 48h. The expression of GRP78 underwent a 3-fold increase with a sharp rise at 24h (no increase at 8h), while GRP94 increased by only 1.5-fold with a peak at 2h and an early return to base-line levels. None of the chaperones were increased after 48h. LPS-induced ERS was detected as early as 4h after stimulus as detected by the evaluation of PERK/eIF2?, IRE1 and ATF6 pathways. Strong signals of ERS were still present after 48h. The pre-incubation of THP-1 in glucose-deprived medium produced 2.5 and 11-fold increases of GRP94 and GRP78, respectively. Upon glucosedeprivation, LPS could no longer induce ERS. Inhibition of chaperone expression by siRNA completely abrogated the effect of glucose deprivation to protect cells from LPS-induced ERS. Thus, the hyperexpression of GRP78 and GRP94 protect cells from LPS-induced ERS. Defective TLR4-induced chaperone expression is a mechanism involved in the integration of TLR4 signaling and ERS (AU) | |