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Mucosal expression of genes encoding possible upstream regulators of Na+ transport during pneumococcal otitis media
Ha-Sheng Li1, William J Doyle, J Douglas Swarts
1Department of Pediatric Otolaryngology, Children's Hospital of Pittsburgh Pennsylvania 15213, USA. shengli@pitt.edu
Objective:
Recently, we reported that gene transcripts encoding 3 Na+ transport proteins (pump, channel and exchanger) in the middle ear mucosa (MEM) were simultaneously suppressed at 12 and 48 h after Streptococcus pneumoniae (SP) challenge of rat middle ears.
Material And Methods:
From cDNA microarray screening of those specimens, several gene clusters, including Nos2 and the transcription factors Fos, Fosl1, Jun and Nfkb1, were identified as possible upstream regulators of Na+ transport protein expression. The altered expression of those genes in MEM was validated and quantified using real-time polymerase chain reaction and MEM protein expression for Atp1a1, Nos2 and Nfkb1 was studied using Western blot and/or immunohistochemistry assays.
Results:
At both time-points. Atp1a1 mRNA and protein were decreased and Nos2 mRNA and protein were increased in MEM. While Nfkb1 protein was decreased at those times. the corresponding mRNA was increased at 12 h but decreased at 48 h. Gene expression for Fos was suppressed at both times, while that for Fosl1 and Jun was augmented at 12 h and suppressed at 48 h. Immunohistochemical study of specimens challenged with SP showed a swollen MEM with infiltration of inflammatory cells that stained positive for Nos2.
Conclusion:
Given the known activities of Nos2, these results can be interpreted as evidencing a transcriptional suppression of Na+ transport protein synthesis secondary to upregulated Nos2 expression during SP infection of the rat MEM. This proposed signaling pathway does not require the continuous upregulation of Nfkb1 or the other assayed transcription factors as early as 12 h after middle ear infection.
Insights
Streptococcus pneumoniae infection suppresses middle ear Na+ transport proteins. Upregulated Nos2 expression appears to drive this suppression, independent of other transcription factors.
Area of Science:
- Otolaryngology
- Molecular Biology
- Infectious Disease
Background:
- Middle ear mucosa (MEM) Na+ transport proteins are crucial for middle ear homeostasis.
- Previous studies reported simultaneous suppression of these proteins after Streptococcus pneumoniae (SP) challenge.
Purpose of the Study:
- To identify upstream regulators of Na+ transport protein expression in response to SP infection.
- To elucidate the signaling pathway involved in SP-induced suppression of Na+ transport proteins in rat MEM.
Main Methods:
- cDNA microarray screening to identify potential upstream regulators.
- Real-time PCR and Western blot/immunohistochemistry to validate gene and protein expression changes.
- Analysis of Nos2, Atp1a1, Nfkb1, Fos, Fosl1, and Jun expression.
Main Results:
- SP challenge led to decreased Atp1a1 mRNA and protein, and increased Nos2 mRNA and protein in MEM.
- Nfkb1 showed complex mRNA and protein level changes; Fos expression was suppressed.
- Inflammatory cell infiltration and Nos2 positivity were observed in infected MEM.
Conclusions:
- Upregulated Nos2 expression likely causes transcriptional suppression of Na+ transport proteins during SP infection.
- This pathway may not require sustained upregulation of Nfkb1 or other tested transcription factors early in infection.