Novel AU-rich proximal UTR sequences (APS) enhance CXCL8 synthesis upon the induction of rpS6 phosphorylation

Abstract

The role of ribosomal protein S6 (rpS6) phosphorylation in mRNA translation remains poorly understood. Here, we reveal a potential role in modulating the translation rate of chemokine (C-X-C motif) ligand 8 (CXCL8 or Interleukin 8, IL8). We observed that more CXCL8 protein was being secreted from less CXCL8 mRNA in primary macrophages and macrophage-like HL-60 cells relative to other cell types. This correlated with an increase in CXCL8 polyribosome association, suggesting an increase in the rate of CXCL8 translation in macrophages. The cell type-specific expression levels were replicated by a CXCL8- UTR-reporter (Nanoluc reporter flanked by the 5' and 3' UTR of CXCL8). Mutations of the CXCL8-UTR-reporter revealed that cell type-specific expression required: 1) a 3' UTR of at least three hundred bases; and 2) an AU base content that exceeds fifty percent in the first hundred bases of the 3' UTR immediately after the stop codon, which we dub AU-rich proximal UTR sequences (APS). The 5' UTR of CXCL8 enhanced expression at the protein level and conferred cell type-specific expression when paired with a 3' UTR. A search for other APS-positive mRNAs uncovered TNF alpha induced protein 6 (TNFAIP6), another mRNA that was translationally upregulated in macrophages. The elevated translation of APS-positive mRNAs in macrophages coincided with elevated rpS6 S235/236 phosphorylation. Both were attenuated by the ERK1/2 signaling inhibitors, U0126 and AZD6244. In A549 cells, rpS6 S235/236 phosphorylation was induced by TAK1, Akt or PKA signaling. This enhanced the translation of the CXCL8-UTR-reporters. Thus, we propose that the induction of rpS6 S235/236 phosphorylation enhances the translation of mRNAs that contain APS motifs, such as CXCL8 and TNFAIP6. This may contribute to the role of macrophages as the primary producer of CXCL8, a cytokine that is essential for immune cell recruitment and activation.

Fig 1. CXCL8 translation and expression is elevated in HL-60 macrophages.

(A and B) CXCL8 and IL6 expression levels in HL-60 macrophages (HL-60 Mac), neutrophil-like HL-60 polymorphonuclear leukocytes (HL-60 PMNs), A549 and H1299 lung epithelial carcinoma cells (A549 ECs), and KHYG-1 NK cells. Expression was measured at resting state (not treated, NT) or after overnight activation with 100 ng/mL LPS or 10 ng/mL TNF. Protein levels were determined via ELISA. The mRNA levels were determined via real-time PCR and presented as the ratio of IL6 or CXCL8 divided by the internal control genes, RPL27. (C) Polysome profiles of HL-60 Mac, A549 EC and KHYG-1 NK cells were obtained from a continuous sucrose density gradient (left to right: 10–50% sucrose). Compared to A549 EC and KHYG-1 NK cells, a lower proportion of the global mRNA (detected at A260) of HL-60 Mac was detected (at A260) in the high sucrose density fractions associated with polysomes. This is indicative of a low translation rate which may be due to the tendency of HL-60 cells to clump upon differentiation into macrophage (S1B Fig). From 14 fractions spanning the entire sucrose gradient, the levels of specific mRNAs (CXCL8, RPL27 and ACTB) were quantified via real-time PCR and presented as a percentage of the sum of all fractions. (A to C) Each graph symbol (squares, inverted-triangles or circles) is the result of a replicate experiment. Replicate experiments were performed on different days. ND: Not detected.

Fig 2. The elevated expression of CXCL8 in macrophages is mediated by its 3’ UTR.

(A) CXCL8 expression in cells transfected with the pcDNA control, CXCL8-full or CXCL8-CDS plasmids. CXCL8-full expresses the full CXCL8 transcript while CXCL8-CDS expresses a mutant with the 5’ and 3’ UTRs deleted. CXCL8 protein levels were determined via ELISA. CXCL8 mRNA levels were determined via real-time PCR (with primers that prime within the coding sequences) and presented as the ratio of CXCL8 divided by the internal control gene, RPL27. (B) Cells transfected with the control plasmid were assumed to only express endogenous CXCL8. The CXCL8 expression observed for these control cells were subtracted from CXCL8-full and CXCL8-CDS to obtain the expression level of plasmid-derived CXCL8, as represented by [CXCL8-full] and [CXCL8-CDS]. These values were used to calculate the “CXCL8 fold change” in expression based on the mathematical expression shown. (C, D, E and F) Schematic representations of the coding sequence (CDS) of secreted Nanoluc (Nluc) fused to the 5’ and 3’ UTRs of CXCL8, TNF, IL6 and TIMP1.The expression plasmids for these UTR-Nluc reporters were transfected into parallel cell cultures. The resulting Nluc protein and mRNA expression levels were quantified via luciferase assay and real-time PCR, respectively, after overnight incubation. The ratio of UTR-Nluc over Cntrl-Nluc expression was then determined and presented as the log₂ fold change in Nluc expression. Nluc mRNA levels were standardized to the NeoR gene expressed from the pcDNA3.1(+), which is the expression plasmid for the UTR- and Cntrl-Nluc constructs. Cells were treated with 100 ng/mL LPS or 10 ng/mL TNF, 3 h after transfection in panel E. The amount of UTR- and Cntrl-Nluc plasmid transfected was halved and replaced with empty pcDNA3.1 plasmid in panel E. (G) Mutant variants of the CXCL8-5’+3’ UTR-Nluc reporter and Cntrl-Nluc mRNAs, with deletions in the secretory peptide signals resulting in cytoplasmic expression of Nluc. (A to G) Each graph symbol (squares, triangles, inverted-triangles or circles) is the result of a replicate experiment. Replicate experiments were performed on different days.

Fig 3. A 37-bp conserved sequence within the 5’ UTR of CXCL8 enhances expression.

(A) A 37-bp sequence found within the 5’ UTR of CXCL8 is conserved across the homologs of multiple species. (B) Schematic representations of UTR-Nluc reporters with CXCL8 5’ UTR deletions are shown. The expression plasmids for the UTR-Nluc reporters were transfected into parallel cell cultures. The resulting Nluc protein expression levels were quantified via luciferase assay after overnight incubation. The ratio of UTR-Nluc over Cntrl-Nluc expression was then determined and presented as a log₂ value. (C and D) The truncated 5’ UTR of CXCL8 containing the conserved 37 bp sequence was also fused mCherry red fluorescent protein and firefly lucifease Fluc to form the CXCL8-5’-mCherry and pNFAT-5’-Fluc reporters. Both reporter proteins were expressed into the cytoplasm. Fluc is additionally under the control of a NFAT promoter, which is weaker compared to the CMV promoter upstream of the Nluc and mCherry reporters. Fluorescence micrographs of Cntrl-mCherry and CXCL8-5’-mCherry expression in A549 ECs and the corresponding phase contrast images displayed at the bottom right corner of each fluorescence micrograph are shown in panel C. The ratio of pNFAT-5’-Fluc over pNFAT-Fluc expression, presented as a log₂ value, is shown in panel D. Each graph symbol (squares) is the result of a replicate experiment. Replicate experiments were performed on different days.

Fig 4. The 3’ UTRs of CXCL8 contains AU-rich proximal sequences (APSs).

(A and B) Schematic representations of UTR-Nluc reporters with CXCL8 3’ UTR deletions are shown. The CXCL8-5’+3’ΔARE mutant was generated by deleting known AU-rich elements (AREs) from CXCL8-5’+3’, as shown with red “strikeouts”. The expression plasmids for the UTR-Nluc reporter mRNAs were transfected into parallel cell cultures. The resulting Nluc protein and mRNA expression levels were quantified via luciferase assay and real-time PCR, respectively, after overnight incubation. The ratio of UTR-Nluc over Cntrl-Nluc expression was then determined and presented as log₂ values. Each graph symbol (squares, triangles, or circles) is the result of a replicate experiment. Replicate experiments were performed on different days. (C) Elevated adenine and uracil base content (AU content) in the 3’ UTR of the UTR-Nluc reporters with CXCL8 3’ UTR deletions. (D) The elevated AU content of the CXCL8 3’ UTR is conserved in mammals. (E) Elevated AU content in the first 100 bases (proximal end) of the CXCL8 3’ UTR relative to the ACTB, TNF, IL6 and TIMP1 3’UTRs.

Fig 5. APSs conferred elevated expression in HL-60 macrophages.

(A, B and C) Schematic representations of UTR-Nluc reporters with UTR mutations are shown. The AU content in the first 100 bases of the 3’ UTR of these UTR-Nluc reporter mutants are shown in line charts. Positions 1 to 91 of the 3’ UTR of TNF was inserted in between positions 285 and 286 of the CXCL8-3’ reporter to generate the CXCL8-3’::disTNF-3’(1–91) mutant. The expression plasmids for these UTR-Nluc reporter mRNAs were transfected into parallel cell cultures. The resulting Nluc protein and mRNA expression levels were quantified via luciferase assay and real-time PCR, respectively, after overnight incubation. The ratio of UTR-Nluc over Cntrl-Nluc expression was then determined and presented as log₂ values. Each graph symbol (squares or triangles) is the result of a replicate experiment. Replicate experiments were performed on different days. (D) The 3’ UTR of TNFAIP6, IFNG and IL2 contain putative APSs. (E) From 14 fractions spanning the entire sucrose gradients displayed in Fig 1C, the levels of specific TNFAIP6 mRNA were quantified via real-time PCR and presented as a percentage of the sum of all fractions. Replicate experiments were performed on different days and the data from 2 replicates are shown.

Fig 6. The inhibition of ERK1/2 signaling reduced CXCL8 UTR reporter expression in HL-60 macrophages.

(A) Graphical representations of the plasmid-derived UTR-Nluc reporter mRNAs are shown. These plasmids were transfected into parallel cell cultures. Treatments with DMSO solvent control or ERK1/2 signaling inhibitors (10 μM U0126 or 1 μM AZD6244) were performed three hours after UTR-Nluc reporter transfection. The resulting Nluc protein and mRNA expression levels were quantified via luciferase assay and real-time PCR, respectively, after overnight incubation. The ratio of UTR-Nluc over Cntrl-Nluc expression was then determined and presented as log₂ values. Each graph symbol (squares or circles) is the result of a replicate experiment. Replicate experiments were performed on a different days. (B) Polysome profiles of HL-60 Mac after treatment with 10 μM U0126 or 1 μM AZD6244 for 8 hours. The fractionation was performed on a continuous sucrose density gradient (10–50% sucrose). From 14 fractions spanning the entire sucrose gradient, the levels of specific mRNAs (CXCL8, TNFAIP6 and ACTB) were quantified via real-time PCR and presented as a percentage of the sum of all fractions. The data for the HL-60 Mac controls were previously shown in Fig 1C. Replicate experiments were performed on different days and the data from 2 replicates are shown.

Fig 7. The phosphorylation of rpS6 is elevated in HL-60 macrophages and is sensitive to ERK1/2 signaling inhibition.

(A and B) Western blots displaying the expression levels of rpS6 S235/236+phos, rpS6 S240/244+phos, total rpS6, 4E-BP1 T37/46+phos, total 4E-BP1, eIF4E S209+phos, total eIF4E and β-Tubulin. The expression levels of these proteins were compared between HL-60 Mac, HL-60 PMN, A549 EC, KHYG-1 NK and H1299 EC cells, in panel A. Comparisons were also made between HL-60 cells treated with the solvent control, ERK1/2 signaling inhibitors (10 μM U0126 or 1 μM AZD6244), and mTOR inhibitor (100 nM Torin-1); in panel B. The band signal intensities were quantified and used to calculate the normalized band intensity ratios as described in the mathematical expressions. In panel B, “rpS6+phos fold change” was calculated based on the mathematical expression shown. [Control] and [Inhibitor] are defined as the rpS6+phos/rpS6 ratios of the control and inhibitor (Torin-1, U0126 and AZD6244) treatment cells, respectively, Experimental replicates were repeated over different days and the data from two repeats are shown.

Fig 8. CXCL8 UTR reporter expression is sensitive to the (rpS6 S235/236+phos)/rpS6 ratio.

(A and B) Graphical representations of the CXCL8-5’+3’ Nluc and Cntrl-Nluc mRNAs are shown. These Nluc reporter plasmids were transfected into parallel cell cultures along with plasmids for the expression of TAK1+TAB1, myrAKT, MKK7-JNK2, caPKA or the control empty pcDNA plasmid. In each of these co-transfection experiments, an equimolar ratio of each plasmid species was used. After overnight incubation, the resulting Nluc protein and mRNA expression levels were quantified via luciferase assay and real-time PCR, respectively. The ratio of UTR-Nluc over Cntrl-Nluc expression was then determined and presented as log₂ values. Western blots display the expression levels of ERK1/2 T202/204+phos, total ERK1/2, Akt S473+phos, total Akt, rpS6 S235/236+phos, rpS6 S240/244+phos, total rpS6, 4E-BP1 T37/46+phos, total 4E-BP1, eIF4E S209+phos, total eIF4E and β-Tubulin. The band signal intensities were quantified and used to calculate the normalized band intensity ratios as described in the mathematical expressions. The mRNA levels were determined via real-time PCR. Nluc mRNA levels were standardized to the NeoR gene expressed from the pcDNA3.1(+), which is the expression plasmid for the UTR- and Cntrl-Nluc constructs. Each graph symbol (squares or circles) is the result of a replicate experiment. Replicate experiments were performed on different days. For the western blots, two replicates are shown.

Fig 9. Primary macrophages display an elevated rate CXCL8 translation that is attenuated by ERK1/2 signaling inhibitors.

(A) CXCL8 protein and mRNA expression levels in primary macrophages, neutrophils and NK cells. Expression was measured at resting state (not treated, NT) or after overnight activation with 100 ng/mL LPS or 10 ng/mL TNF. (B) Polysome profiles of primary macrophages and HL-60 Mac were obtained from a continuous sucrose density gradient (10–50% sucrose). From 14 fractions spanning the entire sucrose gradient, the levels of specific mRNAs (CXCL8, TNFAIP6, ACTB and RPL27) in each fraction from the sucrose gradients were quantified via real-time PCR and presented as a percentage of the sum of all fractions. The data for the HL-60 Mac repeats were as previously shown in Figs 1C and 5E. (C) Graphical representations of the CXCL8-5’+3’ UTR-Nluc and Cntrl-Nluc reporter mRNAs are shown. The expression plasmids for these Nluc reporters were transfected into parallel cell cultures. After overnight incubation, the resulting Nluc protein and mRNA expression levels were quantified via luciferase assay and real-time PCR, respectively. The ratio of UTR-Nluc over Cntrl-Nluc expression was then determined and presented as log₂ value. (D and E) Western blots displaying the expression levels of rpS6 S235/236+phos, rpS6 S240/244+phos, total rpS6, 4E-BP1 T37/46+phos, total 4E-BP1, eIF4E S209+phos, total eIF4E and β-Tubulin. The expression levels of these proteins were compared between primary macrophages and NK cells. Comparisons were also made between primary macrophages treated with the solvent control and the ERK1/2 inhibitor, 1 μM AZD6244, The band signal intensities were quantified and used to calculate the normalized band intensity ratios as described in the mathematical expressions. (F) The expression ratio of CXCL8 protein and mRNA expression in primary macrophages and neutrophils upon overnight treatment with ERK1/2 signaling inhibitors (10 μM U0126 or 1 μM AZD6244) relative to solvent control cells. (A to F) Each graph symbol (squares, triangles or circles) is the result of a replicate experiment. Replicate experiments were performed on different days. Cells from different donors were used for each replicate. For the western blots, two replicates are shown. For panels A and F, CXCL8 protein and mRNA levels were determined via ELISA and real-time PCR, respectively. CXCL8 mRNA levels are standardized to RPL27 as the internal control genes and are relative to the control NT samples.

Fig 10. A schematic summary of findings.

Ribosomal protein S6 (rpS6) is directly phosphorylated by the kinases Rsk, S6K1 and PKA [11]. Rsk and S6K1 are activated by ERK1/2 and Akt-mTOR signaling respectively. These signaling pathways induce of rpS6 phosphorylation at S235/236 and enhance the translation of CXCL8 mRNA. The enhancement requires: 1) a 3’ UTR that is at least 300 bases; and 2) an AU base content exceeding fifty percent in the first hundred or so bases of the 3’ UTR immediately after the coding sequence, which we refer to as AU-rich proximal RNA cis-regulatory sequences (APS). This translational control mechanism contributes to the elevated CXCL8 protein expression in macrophages. On its own, the 5’ UTR of CXCL8 is a potent translation enhancer.