Mutations in the thyroid transcription factor gene NKX2-1 result in decreased expression of SFTPB and SFTPC

Stephanie J Attarian, Sandra L Leibel, Ping Yang, Danielle N Alfano, Brian P Hackett, F Sessions Cole, Aaron Hamvas, Stephanie J Attarian, Sandra L Leibel, Ping Yang, Danielle N Alfano, Brian P Hackett, F Sessions Cole, Aaron Hamvas

Abstract

Background: Mutations in the NK2 homeobox 1 (NKX2-1) gene are associated with lung disease in infants and children. We hypothesize that disruption of normal surfactant gene expression with these mutations contributes to the respiratory phenotypes observed.

Methods: To assess transactivational activity, cotransfection of luciferase reporter vectors containing surfactant protein B or C (SFTPB or SFTPC) promoters with NKX2-1 plasmids was performed and luciferase activity was measured. To assess the binding of mutated proteins to target DNA, electrophoretic mobility shift assays (EMSA) were performed using nuclear protein labeled with oligonucleotide probes representing NKX2-1 consensus binding sequences followed by gel electrophoresis. The effect of overexpression of wild-type (WT) and mutant NKX2-1 on SFTPB and SFTPC was evaluated with quantitative real-time PCR.

Results: Decreased transactivation of the SFTPB promoter by both mutants and decreased transactivation of the SFTPC promoter by the L197P mutation was observed. EMSA demonstrated decreased DNA binding of both mutations to NKX2-1 consensus binding sequences. Transfection of A549 cells with NKX2-1 expression vectors demonstrated decreased stimulation of SFTPB and SFTPC expression by mutant proteins compared with that of WT.

Conclusion: Disruption of transcriptional activation of surfactant protein genes by these DNA-binding domain mutations is a plausible biological mechanism for disruption of surfactant function and subsequent respiratory distress.

Conflict of interest statement

Disclosure: The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
NK2 homeobox 1 (NKX2–1) gene showing three exons and the region encoding the homeodomain (HD) located in exon 3. The two patients described in the manuscript had mutations in the homeodomain at nucleotide 583 (c.583C > T) and nucleotide 590 (c.590T > C), resulting in the R195W and L197P amino-acid substitutions, respectively.
Figure 2
Figure 2
Transactivation of surfactant protein B (SFTPB) (a) and surfactant protein C (SFTPC) (b) promoters by wild-type and mutant NK2 homeobox 1 (NKX2–1). Relative luciferase activity is indicated following correction for transfection efficiency. Error bars indicate standard error of the mean. *P < 0.05 vs. vector alone. **P < 0.05 vs. wild type.
Figure 3
Figure 3
Electrophoretic mobility shift assays were performed with nuclear protein, including wild-type or mutant thyroid transcription factor-1 (TTF-1). Reactions were performed with labeled surfactant protein B (SFTPB) (a) and surfactant protein C (SFTPC) (b) TTF-1 binding sites. In some reactions, unlabeled probe or anti-TTF-1 antibody was included. The white arrow indicates the TTF-1 binding complex; the dark arrow indicates the supershifted binding complex.
Figure 4
Figure 4
Expression of surfactant protein B (SFTPB) (a) and surfactant protein C (SFTPC) (b) by A549 cells in response to wild-type or mutant NK2 homeobox 1 (NKX2–1) expression. Relative transcript abundance is shown compared with that of untransfected cells. Error bars indicate standard error of the mean. *P < 0.05 vs. untransfected cells. **P < 0.05 vs. cells transfected with wild-type vector.

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