Abstract
Purpose :
Mutations in SLC4A11 are associated with Congenital Hereditary Endothelial Dystrophy. Given the proposed role of SLC4A11 in facilitating glutamine metabolism in corneal endothelium (CEn), we investigated the metabolic changes resulting from reduction of SLC4A11 in CEn.
Methods :
Transcriptome profiles and the phosphorylation status of selected proteins essential for regulating cell metabolism, p53 and AMPK, were investigated. Total RNA, isolated from primary cultured human corneal endothelial cells (pHCEnC) after treatment with either siRNA targeting SLC4A11 or scrambled RNA control (SLC4A11 KD pHCEnC and scRNA pHCEnC; n=3 each); passages 6/7 and 39/40 of mouse corneal endothelial cell lines (MCEnC) with either Slc4a11 knockout or wild-type control (early and late passage Slc4a11-/- and Slc4a11+/+ MCEnC; n=4 each), was subject to sequencing and differential gene expression (DGE) analysis (cutoff: adjusted p-value ≤ 0.05). Pathway and biological function enrichment analysis were performed using Ingenuity Pathway analysis (Qiagen). Protein isolated from SLC4A11 KD pHCEnC and scRNA pHCEnC, was analyzed on an automated Western blot machine (Wes; ProteinSimple). The following antibodies (Cell Signaling) were used: Ser15 phosphorylated p53, p53, Thr172 phosphorylated AMPKα, AMPKα, Ser182 phosphorylated AMPKβ and AMPKβ.
Results :
In the transcriptome of SLC4A11 KD pHCEnC, early and late passage Slc4a11-/- MCEnC, we identified 3714 genes that are consensually differentially expressed compared to scRNA pHCEnC or Slc4a11+/+ controls, with 1041 genes changed in the same direction. Pathway analysis of the 3714 consensus DGE showed a generalized inhibition of multiple metabolic pathways, including glycolysis, gluconeogenesis, GDP-mannose biosynthesis, acetyl-CoA biosynthesis and TCA cycle. Analysis of biological function enrichment in the consensus DGE identified inhibition in “transport of molecule” and “mitochondrial dysfunction”. Western blot showed increased levels of phosphorylated p53 and phosphorylated AMPKα in SLC4A11 KD pHCEnC than scRNA pHCEnC, while phosphorylated AMPKβ was not changed.
Conclusions :
Reduction of SLC4A11 expression in pHCEnC and MCEnC leads to inhibition of ion transport function, mitochondria dysfunction and a generalized downregulation of genes involved in multiple metabolic pathways. In pHCEnC, AMPKα and p53 phosphorylation are involved in regulating such metabolic changes.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.