Publicação
Identification of splicing factors with a role in IL-1β secretion
| Resumo: | Inflammation was one of the first immune responses to be reported to exist and has been a focus of intensive research ever since (reviewed in Rocha e Silva, 1978; Medzhitov, 2010). During the course of an inflammatory response several components are involved, such as inflammatory inducers and mediators or different sensors that mediate the detection of a pro-inflammatory stimulus. Amongst the most studied pro-inflammatory mediators is the Interleukin-1β (IL- 1β). Interleukin-1β was one of the first Interleukins to be identified and represents one of the most important mediators of Inflammation and host responses to infection (reviewed in Dinarello, 2004). The large connection between misregulation of IL-1β release and the appearance of inflammatory diseases made this cytokine one of the “hotspots” of intensive research in the past years (reviewed in O'Neill, 2008). Nowadays, several diseases are being treated successfully using different methods that decrease IL-1β circulant levels or block its effects, such as using IL-1β receptor antagonists or by neutralizing IL-1β with monoclonal anti-IL- 1β antibodies (reviewed in Fitzgerald et al., 2005; Kalliolias et al., 2008; Lequerre et al., 2008; Martinon et al., 2009). The IL-1β secretion pathway is a “two-step” fashion mechanism, Production and Processing steps. The cytokine is produced in an inactive pro-form (pro-IL-1mainly upon activation of the nuclear factor-kB (NF-kB) transcription factor and posterior translation into protein. The second and processing step is mediated by active Caspase-1, that will give rise to its mature and secreted form (reviewed in Martinon et al., 2009). Caspase-1 is also synthesized as an inactive form that requires processing by the Inflammasome complex to become active (reviewed in Martinon et al., 2009). Upon Inflammasome formation, Caspase-1 becomes active, leading to subsequent IL-1β processing and posterior release. However, despite the increase knowledge that has been obtained during the past years, a lot still remains to be unveiled concerning the mechanisms involved in the regulation of IL-1β secretion and the effects elicited by this cytokine. Several genes involved in the regulation of IL-1β secretion were shown to be regulated by Alternative Splicing (AS), such as MyD88, IRAK2 and NOD2, among others (reviewed in Leeman et al., 2008). Moreover, the increasing number of cellular processes regulated by AS and the strong correlation between defects in Splicing and disease (reviewed in Leeman et al., 2008; Tazi et al., 2008), strongly suggest that AS may play a role in the initiation and regulation of an inflammatory response, particularly in the secretion of the pro-inflammatory cytokine IL-1β. In an attempt to identify the Splicing Factors and Regulators (SF) with a potential role in the secretion of IL-1β upon an inflammatory stimulus, we performed an RNAi-based screen. Briefly, we used a subset of the TRC Lentiviral Human Library to generate loss-of-function phenotypes for most of SFs. We silenced the expression of 425 genes involved in Splicing with an average 5-fold coverage. After the primary screen and several rounds of phenotypic validation, 19 genes were identified to significantly affect the secretion of IL-1by THP-1 cells after a 24 hours challenge with E. coli, as measured by ELISA. Among the candidates, ASF/SF2 and SRp20, showed a clear negative regulator phenotype, where upon decreased expression of these two candidates, elevated levels of secreted IL-1β secretion were detected, in comparison to control. Thus, we decided to focus our attention in these two SFs. In order to confirm the phenotype observed using the RNAi technology, we overexpressed these two candidates in THP-1 cells. As expected, overexpression decreased the levels of secreted IL-1β, therefore validating the previous results as implicating ASF/SF2 and SRp20 as negative regulators of IL-1β secretion. In addition, using drugs already described to block the Splicing dependant on ASF/SF2 or SRp20, we observed increased levels of IL- 1β upon treatment, therefore, once more confirming the results obtained in the RNAi based screen. Next, we studied the role of ASF/SF2 and SRp20 in the regulation of the two-steps necessary for IL-1β secretion. The levels of IL-1β mRNA expression were increased upon ASF/SF2 or SRp20 knockdown, when compared to control cells. We could then conclude that both SFs are negative regulators of IL-1β production. As mentioned before, the second step necessary for IL-1β secretion is its processing in a Caspase-1 dependant manner. We measured Caspase-1 activation by FACS in cells upon ASF/SF2 or SRp20 knockdown and posterior challenge with E.coli. Knocking-down ASF/SF2 did not show any impact in Caspase-1 activation. In opposition, a remarkable increase in Caspase-1 activation was observed upon SRp20 knockdown, as compared to control cells. Thus, we could conclude that SRp20 acts as a negative regulator of both IL-1β Production and Processing, whereas ASF/SF2 is a negative regulator of IL-1β Production. In addition, increased Caspase-1 mRNA expression was observed in SRp20 knockdown cells as compared to control, consequently suggesting that increased Caspase-1 activation in SRp20 knockdown cells might be due to increased Caspase-1 expression. However further experiments are still required to prove this assumption. In the past years, several reports clearly show the involvement of these two SFs in the regulation of different cellular processes such as transcription, translation and apoptosis (reviewed in Huang et al., 2004; Li et al., 2005a; Long et al., 2009). A recent report implicates ASF/SF2 in Inflammation (Xiong, 2006), as it was shown that ASF/SF2 is downregulated in inflamed muscle or after an inflammatory stimulus, such as TNF. Thus, we decided to check the expression of ASF/SF2 and SRp20 upon E.coli challenge. Decreased expression of ASF/SF2 was observed at the mRNA level, whereas decreased expression of SRp20 was only observed at the protein level after an E.coli challenge. The mechanisms involved in the downregulation of these two SFs upon E.coli challenge are now being investigated. The increased number of genes that are regulated by AS already reported to play a role in Inflammation strongly suggested us to look for their Splicing profiles in ASF/SF2 or SRp20 knockdown cells upon an E.coli challenge. Several PCR experiments were performed to identify differences in the Splicing patterns of the described genes, however with no conclusive results. Consequently, we decided to perform an unbiased and high-throughput analysis of Alternative Splicing Events (ASE) that take place either after an E.coli challenge or after knockdown of ASF/SF2, using the Affymetrix® GeneChip® Human Exon 1.0 ST Arrays platform. Upon data analysis and validation, several genes already reported to undergo AS in different publications were found using our approach, therefore validating our method. We are now focusing on the most relevant candidates to perform follow-up studies. In sum, this work allowed us to identify two novel regulators of IL-1β secretion after an E.coli challenge. Our results clearly show that both ASF/SF2 and SRp20 are negative regulators of IL-1β secretion. While SRp20 is involved in the regulation of the two-steps required of IL-1β secretion, Production and Processing, ASF/SF2 is only involved in the regulation of the first step, Production. In addition to IL-1β, increased Caspase-1 expression was also observed upon SRp20 knockdown, suggesting the involvement of this SF in the regulation of the expression of this inflammatory Caspase. Moreover, the expression of the two SFs was also shown to be altered in the course of an inflammatory response to E.coli challenge, however the mechanism involved has yet to be determined. Several studies are now required to determine the mechanisms by which these two SFs play a role in IL-1β Secretion. |
|---|---|
| Autores principais: | Alves, Pedro Moura, 1981- |
| Assunto: | Processamento alternativo Inflamação Interleucina-1 Interleucina-1beta RNA Teses de doutoramento - 2010 |
| Ano: | 2010 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório da Universidade de Lisboa |
| Resumo: | Inflammation was one of the first immune responses to be reported to exist and has been a focus of intensive research ever since (reviewed in Rocha e Silva, 1978; Medzhitov, 2010). During the course of an inflammatory response several components are involved, such as inflammatory inducers and mediators or different sensors that mediate the detection of a pro-inflammatory stimulus. Amongst the most studied pro-inflammatory mediators is the Interleukin-1β (IL- 1β). Interleukin-1β was one of the first Interleukins to be identified and represents one of the most important mediators of Inflammation and host responses to infection (reviewed in Dinarello, 2004). The large connection between misregulation of IL-1β release and the appearance of inflammatory diseases made this cytokine one of the “hotspots” of intensive research in the past years (reviewed in O'Neill, 2008). Nowadays, several diseases are being treated successfully using different methods that decrease IL-1β circulant levels or block its effects, such as using IL-1β receptor antagonists or by neutralizing IL-1β with monoclonal anti-IL- 1β antibodies (reviewed in Fitzgerald et al., 2005; Kalliolias et al., 2008; Lequerre et al., 2008; Martinon et al., 2009). The IL-1β secretion pathway is a “two-step” fashion mechanism, Production and Processing steps. The cytokine is produced in an inactive pro-form (pro-IL-1mainly upon activation of the nuclear factor-kB (NF-kB) transcription factor and posterior translation into protein. The second and processing step is mediated by active Caspase-1, that will give rise to its mature and secreted form (reviewed in Martinon et al., 2009). Caspase-1 is also synthesized as an inactive form that requires processing by the Inflammasome complex to become active (reviewed in Martinon et al., 2009). Upon Inflammasome formation, Caspase-1 becomes active, leading to subsequent IL-1β processing and posterior release. However, despite the increase knowledge that has been obtained during the past years, a lot still remains to be unveiled concerning the mechanisms involved in the regulation of IL-1β secretion and the effects elicited by this cytokine. Several genes involved in the regulation of IL-1β secretion were shown to be regulated by Alternative Splicing (AS), such as MyD88, IRAK2 and NOD2, among others (reviewed in Leeman et al., 2008). Moreover, the increasing number of cellular processes regulated by AS and the strong correlation between defects in Splicing and disease (reviewed in Leeman et al., 2008; Tazi et al., 2008), strongly suggest that AS may play a role in the initiation and regulation of an inflammatory response, particularly in the secretion of the pro-inflammatory cytokine IL-1β. In an attempt to identify the Splicing Factors and Regulators (SF) with a potential role in the secretion of IL-1β upon an inflammatory stimulus, we performed an RNAi-based screen. Briefly, we used a subset of the TRC Lentiviral Human Library to generate loss-of-function phenotypes for most of SFs. We silenced the expression of 425 genes involved in Splicing with an average 5-fold coverage. After the primary screen and several rounds of phenotypic validation, 19 genes were identified to significantly affect the secretion of IL-1by THP-1 cells after a 24 hours challenge with E. coli, as measured by ELISA. Among the candidates, ASF/SF2 and SRp20, showed a clear negative regulator phenotype, where upon decreased expression of these two candidates, elevated levels of secreted IL-1β secretion were detected, in comparison to control. Thus, we decided to focus our attention in these two SFs. In order to confirm the phenotype observed using the RNAi technology, we overexpressed these two candidates in THP-1 cells. As expected, overexpression decreased the levels of secreted IL-1β, therefore validating the previous results as implicating ASF/SF2 and SRp20 as negative regulators of IL-1β secretion. In addition, using drugs already described to block the Splicing dependant on ASF/SF2 or SRp20, we observed increased levels of IL- 1β upon treatment, therefore, once more confirming the results obtained in the RNAi based screen. Next, we studied the role of ASF/SF2 and SRp20 in the regulation of the two-steps necessary for IL-1β secretion. The levels of IL-1β mRNA expression were increased upon ASF/SF2 or SRp20 knockdown, when compared to control cells. We could then conclude that both SFs are negative regulators of IL-1β production. As mentioned before, the second step necessary for IL-1β secretion is its processing in a Caspase-1 dependant manner. We measured Caspase-1 activation by FACS in cells upon ASF/SF2 or SRp20 knockdown and posterior challenge with E.coli. Knocking-down ASF/SF2 did not show any impact in Caspase-1 activation. In opposition, a remarkable increase in Caspase-1 activation was observed upon SRp20 knockdown, as compared to control cells. Thus, we could conclude that SRp20 acts as a negative regulator of both IL-1β Production and Processing, whereas ASF/SF2 is a negative regulator of IL-1β Production. In addition, increased Caspase-1 mRNA expression was observed in SRp20 knockdown cells as compared to control, consequently suggesting that increased Caspase-1 activation in SRp20 knockdown cells might be due to increased Caspase-1 expression. However further experiments are still required to prove this assumption. In the past years, several reports clearly show the involvement of these two SFs in the regulation of different cellular processes such as transcription, translation and apoptosis (reviewed in Huang et al., 2004; Li et al., 2005a; Long et al., 2009). A recent report implicates ASF/SF2 in Inflammation (Xiong, 2006), as it was shown that ASF/SF2 is downregulated in inflamed muscle or after an inflammatory stimulus, such as TNF. Thus, we decided to check the expression of ASF/SF2 and SRp20 upon E.coli challenge. Decreased expression of ASF/SF2 was observed at the mRNA level, whereas decreased expression of SRp20 was only observed at the protein level after an E.coli challenge. The mechanisms involved in the downregulation of these two SFs upon E.coli challenge are now being investigated. The increased number of genes that are regulated by AS already reported to play a role in Inflammation strongly suggested us to look for their Splicing profiles in ASF/SF2 or SRp20 knockdown cells upon an E.coli challenge. Several PCR experiments were performed to identify differences in the Splicing patterns of the described genes, however with no conclusive results. Consequently, we decided to perform an unbiased and high-throughput analysis of Alternative Splicing Events (ASE) that take place either after an E.coli challenge or after knockdown of ASF/SF2, using the Affymetrix® GeneChip® Human Exon 1.0 ST Arrays platform. Upon data analysis and validation, several genes already reported to undergo AS in different publications were found using our approach, therefore validating our method. We are now focusing on the most relevant candidates to perform follow-up studies. In sum, this work allowed us to identify two novel regulators of IL-1β secretion after an E.coli challenge. Our results clearly show that both ASF/SF2 and SRp20 are negative regulators of IL-1β secretion. While SRp20 is involved in the regulation of the two-steps required of IL-1β secretion, Production and Processing, ASF/SF2 is only involved in the regulation of the first step, Production. In addition to IL-1β, increased Caspase-1 expression was also observed upon SRp20 knockdown, suggesting the involvement of this SF in the regulation of the expression of this inflammatory Caspase. Moreover, the expression of the two SFs was also shown to be altered in the course of an inflammatory response to E.coli challenge, however the mechanism involved has yet to be determined. Several studies are now required to determine the mechanisms by which these two SFs play a role in IL-1β Secretion. |
|---|