Lipopolysaccharide triggers nuclear import of Lpcat1 to regulate inducible gene expression in lung epithelia

Abstract

Aim: To report that Lpcat1 plays an important role in regulating lipopolysaccharide (LPS) inducible gene transcription.

Methods: Gene expression in Murine Lung Epithelial MLE-12 cells with LPS treatment or Haemophilus influenza and Escherichia coli infection was analyzed by employing quantitative Reverse Transcription Polymerase Chain Reaction techniques. Nucleofection was used to deliver Lenti-viral system to express or knock down Lpcat1 in MLE cells. Subcellular protein fractionation and Western blotting were utilized to study Lpcat1 nuclear relocation.

Results: Lpcat1 translocates into the nucleus from the cytoplasm in murine lung epithelia (MLE) after LPS treatment. Haemophilus influenza and Escherichia coli, two LPS-containing pathogens that cause pneumonia, triggered Lpcat1 nuclear translocation from the cytoplasm. The LPS inducible gene expression profile was determined by quantitative reverse transcription polymerase chain reaction after silencing Lpcat1 or overexpression of the enzyme in MLE cells. We detected that 17 out of a total 38 screened genes were upregulated, 14 genes were suppressed, and 7 genes remained unchanged in LPS treated cells in comparison to controls. Knockdown of Lpcat1 by shRNA dramatically changed the spectrum of the LPS inducible gene transcription, as 18 genes out of 38 genes were upregulated, of which 20 genes were suppressed or unchanged. Notably, in Lpcat1 overexpressed cells, 25 genes out of 38 genes were reduced in the setting of LPS treatment.

Conclusion: These observations suggest that Lpcat1 relocates into the nucleus in response to bacterial infection to differentially regulate gene transcriptional repression.

Keywords: Epigenetic code; Escherichia coli; Gene expression; Haemophilus influenza; Lipopolysaccharide; Lung epithelia; Lysophosphatidylcholine acyltransferase 1; Nuclear import; Quantitative reverse transcription polymerase chain reaction.

Figures

Figure 1

Lpcat1 shifts into the nucleus in lipopolysaccharide treated lung epithelia. MLE cells were treated with 10 μg/mL of LPS for 2 h. The harvested cells were processed for cytosolic and nuclear protein fractionation and the fractions were subjected to immunoblotting with different antibodies as indicated. “C” donates to cytosolic fraction and “N” represents the nuclear fraction. Lamin A/C is used as a nuclear fraction protein marker and β-actin is the cytosolic fraction protein marker. The results represent three independent experiments. LPS: Lipopolysaccharide.

Figure 2

Haemophilus influenzae and Escherichia coli trigger Lpcat1 nuclear translocation. MLE cells were exposed to LPS-containing bacteria at a concentration of 109/mL for 4 h. The infected cells were then washed and cytosolic and nuclear protein fractionations were prepared. Immunoblotting analysis was performed using antibodies as indicated. The results represent three independent experiments. “C” donates to cytosolic fraction and “N” represents the nuclear fraction. Lamin A/C is used as a nuclear fraction protein marker and β-actin is the cytosolic fraction protein marker. LPS: Lipopolysaccharide.

Figure 3

Lipopolysaccharide inducible gene expression profiles after Lpcat1 knockdown. Protein levels were determined with Lpcat1 immunoblotting in wild type, LPS treated wild type, and LPS treated Lpcat1 knockdown MLE cells as showed in the imbedded image. Total cellular RNA was extracted from cells and quantitative reverse transcription-polymerase chain reaction analysis was performed. The relative mRNA levels of the genes in LPS treated samples were normalized by the same gene from untreated MLE cells. The data represents the mean value (mean + SEM) from three independent experiments. LPS: Lipopolysaccharide.

Figure 4

Overexpressed Lpcat1 suppresses lipopolysaccharide inducible gene expression. Lpcat1 protein levels were determined by immunoblotting in wild type, LPS treated wild type, and LPS treated Lpcat1 overexpressed MLE cells as in the imbedded image. Quantitative reverse transcription-polymerase chain reaction analysis was performed by using total cellular RNA extracted from wild type, LPS treated wild type, and LPS treated Lpcat1 overexpressed cells. The relative mRNA levels of the genes in LPS treated samples were normalized by the same gene from untreated MLE cells. The data represents the mean value (mean + SEM) from three independent experiments. LPS: Lipopolysaccharide.