Regulation of monocyte induced cell migration by the RNA binding protein, FXR1

Abstract

FXR1 belongs to a family of RNA-binding proteins that play critical roles in post-transcriptional regulation of gene expression in immunity, development and cancer. FXR1 is associated with regulation of specific mRNAs in myocytes and macrophages. In quiescent cells (> 24 h of extended serum-starvation, ∼30-48 h or more), a spliced isoform of FXR1, FXR1a, promotes translation of the cytokine TNFα, independent of the effects of RNA levels. Here we examined the role of FXR1 in THP1 human monocytic leukemic cells that were grown in serum, as well as in early (24 h) serum-starvation conditions that demonstrates differences in gene expression mechanisms and is distinct from quiescent (> 24 h extended serum-starvation) cells. Global RNA profiling, conducted to investigate the role of FXR1 on mRNA levels, revealed that FXR1 affects levels of specific mRNAs in serum-grown and in early 24 h serum-starvation conditions. FXR1 decreases levels of several mRNAs, including as previously identified, CDKN1A (p21CIP1 or p21) mRNA in serum-grown cells. Interestingly, we find that FXR1 positively regulates mRNA levels of specific cytokines and chemokines in serum-grown and in early 24 h serum-starvation conditions. These include IL1β and CCL2 that control cell migration. Accordingly, depletion and overexpression of FXR1 decreased and increased levels of CCL2 mRNA. Consistent with the reduced levels of IL1β, CCL2 and other chemokines upon FXR1 depletion, our data reveal that depletion of FXR1 decreases the ability of these cells to induce cell migration of neighboring monocytic cells. These data reveal a new role of FXR1 in controlling induction of monocyte migration.

Keywords: FXR1; cell migration; chemokines; gene expression; mRNA; monocyte.

Figure 1.

FXR1 knockdown in serum grown and serum-starved THP1 monocytic leukemic cells. (A) Western blot analysis of FXR1 in THP1 stable cell lines expressing control shRNA (shCtrl) or shRNA against FXR1 (shFXR1) after 3 d of doxycycline induction in serum containing media (S+) or serum-free media for 24 h (S- 24 h). (B) Quantitation of multiple Western blots using image J. The data represented are the mean and SD values of 6 experiments. p-values were calculated by one tailed paired t test. (C) RNA level after knockdown of FXR1 evaluated by qRT-PCR. The data represent the mean and SD of 5 experiments p-values were calculated by one-tailed paired t-test.

Figure 2.

Global gene expression profiling of FXR1 regulated mRNAs by microarray analysis in control and FXR1 knockdown THP1 monocytes. (A) (Left) Heatmap analysis of microarray data (Table S1) from THP1 control (shCtrl) and FXR1 knockdown (shFXR1) cells grown in serum containing media (S+) or serum-starved for 24 h (S-24 h). Heatmap representation and clustering (Euclidean) was done using GENE-E software. (Right) The logarithmic value of individual RNAs in FXR1 knockdown cells (shFXR1) was plotted against those values in control cells (shCtrl), grown in serum containing media (S+) or serum-starved for 24 h (S-24 h). R square and curve equations were calculated using linear regression model with prism6. (B-C) Fold change values from qRT-PCR of a subset of targets identified from the microarray from serum grown (S+) and serum-starvation (S- 24h) conditions (Tables S2A-B). The data plotted are the mean and SD of 4 biological replicates. Statistical significance was determined with t test comparison using the Holm-Sidak method.

Figure 3.

Immune response genes, including cytokines and chemokines, are regulated by FXR1. (A) Gene Ontology (GO) analysis for differentially expressed genes was performed using the DAVID tool, as previously conducted. Immune response associated genes in serum-starved cells (S-24 h) are observed to be significantly affected by FXR1 depletion (Fig. S2, Tables S2B, S3). Analysis of RNA expression for a subset of mRNAs related to immune response in serum-starved FXR1 depleted cells compared to serum-starved shCtrl cells: the log base 2 of the fold change values for qRT-PCR (black dot) and microarray (red dot) are plotted. (B) qRT-PCR analysis of select cytokine and chemokine mRNAs in serum-starved cells showing the fold decrease upon FXR1 knockdown. (C) qRT-PCR analysis of CCL2 mRNA normalized to tRNA-lys RNA levels in control (shCtrl) and FXR1 knockdown (shFXR1) cells grown in serum (S+) and 24 h serum-starvation (S-) conditions. The average of 3 technical replicates is shown with SEM as error bars. p-values were calculated by 2-tailed paired t-test. (D) Western blot analysis of FXR1 in THP1 stable cell lines without (WT) or with FXR1a constitutive overexpression (FXR1a ox), grown in serum (S+) and 24 h serum-starvation (S- 24 h) conditions. (E) qRT-PCR analysis of CCL2 mRNA normalized to tRNA-lys RNA levels in control (WT) and FXR1 overexpression (FXR1a ox) cells grown in serum (S+) and 24 h serum-starvation (S-) conditions. The average of 3 technical replicates is shown with SEM as error bars. p-values were calculated by 2-tailed paired t-test.

Figure 4.

Il1β levels are decreased upon FXR1 knockdown. (A-B) Western blot analysis of IL1β and TNFα upon FXR1 depletion. Cell lysate and medium from 24 h serum-starved cells were concentrated using Strataclean resin and the eluates were analyzed by Western blotting. Western blot analysis of (A) IL1β and (B) TNFα, with actin as loading control. The average of 3 replicates is shown with SEM as error bars. p-values were calculated by 2-tailed unpaired t-test.

Figure 5.

FXR1 knockdown leads to decreased induction of cell migration by THP1 monocytes Cell migration assays were performed in transwells to test the migration of a THP1 monocytic cell line that stably expresses GFP for visualization. Cell migration assay was performed using THP1 cell lines expressing (A) control shRNA (shCtrl) or (B) shRNA against FXR1 (shFXR1), which were grown in serum-starvation conditions (S-24 h), and then placed in the bottom chambers and supplemented with 10% serum. The top chamber was filled with a THP1 monocyte cell line that stably expresses GFP. (C) Graph representing cell migration count using imageJ of GFP positive cells that migrated through the membrane filter in (A-B). p-values were calculated by one-tailed unpaired t-test.