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A2217 - Reduced Expression of BCL9 and Disruption of the Beta-Catenin-BCL9 Complex May Affect the Regenerative Capacity of Alveolar Epithelial Cells
Author Block: K. Rydell-Tormanen1, G. Westergren Thorsson2; 1Lung Biology, Dept. Experimental Medical Science, Lund University, Lund, Sweden, 2Lung Biology, Dept. Experimental Medical Science, Lund University, Lund S 22184, Sweden.
Introduction
β-catenin-induced transcription is essential in regeneration, and IPF is characterized by defect regeneration in the lung parenchyma. β-catenin interacts with different co-factors to induce transcription of specific genes, and one of these co-factors is BCL9. We have previously shown that BCL9 is decreased in IPF compared to healthy lungs. β-catenin has a multitude of target genes, but expression is related to its transcriptional co-factors, such as BCL9.
We therefore hypothesized that reduced availability of BCL9 in alveolar epithelial cells will decrease the transcription of genes induced by the β-catenin-BCL9 complex. This may skew the transcriptional profile into a more fibrotic phenotype.
Methods
A549 cells were used to investigate decreased BCL9 signaling: by silencing and disruption of the β-catenin-BCL9 complex.
BCL9 was silenced with two different siRNA (Silencer Select, Thermo Fischer Scientific), using a lipofectamine protocol, and the β-catenin-BCL9 complex was disrupted by LATS2, whose expression was increased by Nocodazole (1 ng/ml).
The effect of BCL9-deficiency was investigated on a palette of known β-catenin/β-catenin-BCL9 complex target genes (AXIN1, VIM, CDH1/E-cadherin, CDH2/N-cadherin, NKD1/Naked1, MMP7) by RT-PCR. Each in vitro experiment was repeated 3-4 separate times.
Results
Down regulation by two different siRNA decreased total BCL9 mRNA by 76%. The decreased BCL9 resulted in increased expression of NKD1 (900%), whereas MMP7, CDH2, AXIN1 and VIM were increased 33-58%. In contrast, the expression of CDH1 decreased ≈20%.
Induction of LATS2 increases the disruption of the β-catenin-BCL9 complex, and thus decreases transcription. In similarity to the silencing of BCL9, we found the expression of NKD1 and CDH2 to increase 117% and 70% respectively, whereas CDH1 again decreased (by 30%).
Conclusions
Data obtained by direct or indirectly decreased BCL9 are in accordance with each other, as well as our previous investigations on diagnostic biopsies, where we found significant increases in protein expression of Naked1.
Decreased CDH1 combined with increased CDH2, as well as increased expression of vimentin, are considered a hallmarks of EMT, whereas NKD1 is considered a marker of de-differentiation. Our results thus suggest that decreased expression of BCL9 may have severe effects on the general regenerative capacity of epithelial cells.
Furthermore, as defect regeneration is believed to be crucial in IPF, BCL9 may be directly involved in the pathogenesis. Hence, identification of the reason behind decreased BCL9 expression may provide an important piece in the understanding of IPF.