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Interleukin-11 mRNA stabilization in phorbol ester-stimulated primate bone marrow stromal cells
L Yang1, C N Steussy, D K Fuhrer
1Department of Biochemistry and Molecular Biology, Walter Oncology Center, Indiana University School of Medicine, Indianapolis, 46202, USA.
Abstract:
12-O-Tetradecanoylphorbol-13-acetate (TPA) stimulation of PU-34 cells, a primate bone marrow stromal cell line, resulted in a prolonged elevation of interleukin-11 (IL-11) mRNA, which can be inhibited by protein synthesis inhibitors. Nuclear run-on assays and actinomycin D experiments demonstrated that the up-regulation of IL-11 gene expression is mainly controlled at the posttranscriptional level through the protein kinase C (PKC) pathway. Inhibition of PKC activity by calphostin C generated an IL-11 mRNA degradation intermediate in TPA-stimulated PU-34 cells. This intermediate retains the 5' untranslated region (5'UTR) and coding region of the IL-11 mRNA but has lost the poly(A) tail and the 3'UTR. The mechanisms underlying IL-11 mRNA stabilization were further investigated by transfections with a variety of chimeric IL-11 constructs and deletion mutants. Two important observations were made from these transient expression experiments: (i) the same 3'UTR of IL-11 mRNA shown to confer instability in one chimeric transcript may not function as a destabilizer in another chimeric RNA, and (ii) the 5'UTR, coding region, and 3'UTR all contribute to IL-11 mRNA decay, and labile IL-11 deletion transcripts are not necessarily stabilized by TPA stimulation. Our study suggests that multiple regions within the IL-11 mRNA are involved in TPA-stimulated IL-11 mRNA stabilization, possibly through a unique RNA folding conformation involving interactions of various RNA sequences within the IL-11 mRNA molecule.
Insights
12-O-Tetradecanoylphorbol-13-acetate (TPA) prolonged interleukin-11 (IL-11) mRNA elevation in primate bone marrow cells. This posttranscriptional regulation involves the protein kinase C (PKC) pathway and multiple IL-11 mRNA regions contributing to stabilization.
Area of Science:
- Molecular Biology
- Cell Biology
- Gene Regulation
Background:
- Interleukin-11 (IL-11) plays a crucial role in various biological processes, including bone metabolism and immune responses.
- Understanding the regulation of IL-11 gene expression is essential for deciphering its physiological functions and potential therapeutic applications.
- Previous studies indicated that IL-11 production can be modulated by external stimuli, but the precise regulatory mechanisms remain incompletely understood.
Purpose of the Study:
- To investigate the posttranscriptional mechanisms regulating interleukin-11 (IL-11) mRNA stability in response to 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulation.
- To elucidate the role of the protein kinase C (PKC) pathway in TPA-induced IL-11 mRNA up-regulation.
- To identify specific RNA elements within the IL-11 mRNA that contribute to its stabilization.
Main Methods:
- PU-34 primate bone marrow stromal cells were stimulated with TPA.
- Nuclear run-on assays and actinomycin D experiments were performed to assess gene expression and mRNA decay rates.
- Protein kinase C (PKC) activity was inhibited using calphostin C.
- Transient transfections with various chimeric IL-11 constructs and deletion mutants were conducted to analyze RNA elements.
Main Results:
- TPA stimulation led to a prolonged elevation of IL-11 mRNA, sensitive to protein synthesis inhibition.
- IL-11 gene expression is primarily controlled posttranscriptionally via the PKC pathway.
- Inhibition of PKC generated an IL-11 mRNA degradation intermediate lacking the poly(A) tail and 3'UTR.
- Both 5'UTR, coding region, and 3'UTR contribute to IL-11 mRNA decay, with TPA stabilization involving multiple regions and potential RNA folding.
Conclusions:
- TPA-induced IL-11 mRNA stabilization in PU-34 cells is a complex process regulated at the posttranscriptional level.
- The protein kinase C (PKC) pathway is a key mediator of this stabilization.
- Multiple regions within the IL-11 mRNA, potentially through intricate RNA folding, are involved in conferring stability upon TPA stimulation.