Part I: Tristetraprolin (TTP) inhibits poly(A)-tail synthesis in nuclear mRNA that contains AU-rich elements by interacting with poly(A)-binding protein nuclear 1 (PABPN1) Part II: Transcriptional regulation of mitogen-activated protein kinase phosphatase-1 (Mkp-1) expression in LPS-stimulated mouse macrophage RAW264.7 cells
Date Issued
2012
Date
2012
Author(s)
Su, Yu-Lun
Abstract
Part I
Tristetraprolin (TTP) binds mRNA AU-rich elements (ARE) and thereby facilitates the destabilization of mature mRNA. To understand how TTP mechanistically functions, previously our lab biopanned with a phage-display library for proteins that interact with TTP and retrieved, among others, poly(A) binding protein nuclear 1 (PABPN1). PABPN1 assists in the 3’-polyadenylation of mRNA by binding to an immature poly(A) tail and increasing the affinity of poly(A) polymerase, which is directly responsible for polyadenylation. The poly(A) tail is important for mRNA export from the nucleus, mRNA stabilization, and translation efficiency. The TTP/PABPN1 interaction was characterized using co-immunoprecipitation assays. Although TTP and PABPN1 are located in both the cytoplasm and the nucleus, they interacted in vivo only in the nucleus. Expression of a TTP mutant restricted to the nucleus resulted in the downregulation of a TNFα ARE-containing luciferase activity .Interestingly, we found that TTP binds PABPN1 and thereby inhibits polyadenylation of ARE-containing mRNA in vitro. When TNFα mRNA was induced in mouse RAW264.7 cells by lipopolysaccharide treatment, synthesis of a shorter poly(A) tail in nuclear TNFα mRNA and an increased expression of TTP occurred. Consequently, in addition to its known cytosolic mRNA-degrading function, TTP inhibits poly(A) tail synthesis by interacting with PABPN1 in the nucleus to regulate expression of ARE-containing mRNA.
Part II
Innate immune responses rely on the MAPK signaling pathway, especially p38 and Jnk. These kinases are activated by pro-inflammatory agonists, and lead to inflammatory or innate immune responses by regulating the expression of several effector genes. Both p38 and Jnk are negatively regulated through their dephosphorylation by MAPK phosphatase 1 (Mkp-1), a member of dual specificity protein phosphatase (DUSP). Recent reports supported that Mkp-1 is a critical regulator of innate immunity. Previously, we have demonstrated that the mRNA of Mkp-1 was rapidly elevated around 30 to 60 minutes and soon decreased less than half after LPS stimulation for 2 h in RAW264.7 cells. Here we concerned with the molecular mechanism in control of the growth and decline of Mkp-1 mRNA at transcriptional level. Creb1 and Atf3, members of the Atf/Creb family protein, were shown to associate with the Mkp-1 promoter and increase the activity of luciferase reporter that carrying the Mkp-1 promoter. Knockdown of Atf3 in RAW264.7 cells resulted in Mkp-1 mRNA downregulation. Therefore, Creb1 and Atf3 regulate Mkp-1 expression positively. The PI3K signaling pathway, which was activated after LPS stimulation for 2 h, was suggested to play an opposite role to restrict the TLR4 signaling. The Mkp-1 mRNA level was increased in the presence of the PI3K signaling inhibitor, Wortmannin. Using the microarray analysis and real time PCR, the Id3 was identified and confirmed for the candidates that downstream of PI3K signaling to regulate Mkp-1 expression. Further studies are needed to examine the function of Id3 and the molecular mechanism of regulating Mkp-1 expression in LPS-stimulated RAW264.7 cells.
Subjects
Tristetraprolin
AU-rich element
poly(A) binding protein nuclear 1
poly(A) tail
Mkp-1
LPS
Atf3
Creb1
PI3K
transcriptional regulation
Type
thesis
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