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Loss of ion transporters and increased unfolded protein response in Fuchs' dystrophy.

Jalimarada SS, Ogando DG, Bonanno JA - Mol. Vis. (2014)

Bottom Line: Thirteen of the altered genes showed significant changes (p<0.05).The PCR array results were validated by quantitative RT-PCR.FECD samples had evident UPR with significant changes in the expression of the protein processing pathway genes.

View Article: PubMed Central - PubMed

Affiliation: School of Optometry, Indiana University, Bloomington, IN.

ABSTRACT

Purpose: Fuchs' endothelial corneal dystrophy (FECD), which affects approximately 5% of the population over 40 in the U.S.A., is a major cause of corneal transplantation. FECD is associated with mutations of a variety of unrelated genes: SLC4A11, COL8A2, TCF8, and LOXHD1. The current pathological description of the dystrophy includes deficiency of corneal endothelium (CE) pump function and induction of the unfolded protein response (UPR). This study aims to determine the contribution of the two mechanisms by assessing the expression levels of (1) seven endothelial ion transporters known to regulate stromal hydration and (2) UPR related genes in a set of six CE samples obtained from FECD patients compared to that of normal controls.

Methods: CE samples collected during FECD keratoplasty or from an eye bank (normal control) were transferred into an RNA stabilizing agent and refrigerated. Total RNA from each CE specimen was individually extracted. The expression levels of ion transporters and UPR genes were tested using quantitative real-time (RT) PCR and a UPR specific PCR array, respectively.

Results: In normal CE, the comparative expression levels of ion transporters in decreasing order were SLC4A11, Na(+)/K(+) ATPase, pNBCe1, and NHE1, followed by the isoforms of monocarboxylate transporters (MCTs). In FECD samples, Na(+)/K(+) ATPase and MCTs 1 and 4 were significantly downregulated compared to normal controls (p<0.05). The PCR array tested 84 UPR related genes. Data analysis showed upregulation of 39 genes and downregulation of three genes, i.e., approximately 51% of the tested genes had their expression altered in FECD samples with a difference greater than ± twofold regulation. Thirteen of the altered genes showed significant changes (p<0.05). The PCR array results were validated by quantitative RT-PCR.

Conclusions: FECD samples had evident UPR with significant changes in the expression of the protein processing pathway genes. The significant downregulation of ion transporters indicates simultaneous compromised CE pump function in Fuchs' dystrophy.

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Related in: MedlinePlus

Summary of the mechanisms possibly contributing to the clinical symptoms associated with FECD. Evidence of mutations and oxidative stress support the induction of UPR in FECD, which could cause decreased CE cell density and pump function followed by corneal edema. The clinical indications could also occur as a result of compromised pump function caused by the decreased RNA level expression of ion transporters demonstrated in this study, whose mechanism is not known.
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f6: Summary of the mechanisms possibly contributing to the clinical symptoms associated with FECD. Evidence of mutations and oxidative stress support the induction of UPR in FECD, which could cause decreased CE cell density and pump function followed by corneal edema. The clinical indications could also occur as a result of compromised pump function caused by the decreased RNA level expression of ion transporters demonstrated in this study, whose mechanism is not known.

Mentions: Multiple mechanisms have been suggested to describe the pathology of FECD, including apoptotic cell death due to oxidative stress or UPR and loss of CE pump function. This study aimed to analyze two of the proposed mechanisms: loss of corneal endothelial pump function and the induction of UPR by determining the expression levels of ion transporters and UPR related genes in CE from FECD patients. A comparative expression level analysis of the ion transporters of CE in normal CE showed higher expression of SLC4A11 among the seven ion transporters tested, underlining the importance of this transporter. FECD samples showed significant downregulation of Na+/K+ ATPase, which is critical for fluid secretion across CE, along with monocarboxylate transporters MCT1 and 4, which are postulated to have a role in corneal endothelial fluid secretion [1,2]. Simultaneous UPR PCR array analysis, performed for the first time using FECD CE total RNA, denoted significant UPR with more than twofold regulation of about 52% of the genes tested. Although the small amount of tissue available precluded protein level analysis, these results are an indication of compromised ion pump function, along with UPR in FECD. Thus, perhaps both UPR and the loss of ion transport function concurrently play a role in the pathology of FECD. Figure 6 shows a schematic summarizing the mechanisms that could contribute to the occurrence of the clinical symptoms observed in FECD. ER stress in FECD could occur due to identified mutations in associated genes and loci, along with oxidative stress [28]. Accumulation of unfolded protein as a result of ER stress can induce UPR [28,31,36], including the inhibition of protein translation, which could reduce the expression of membrane proteins like Na+/K+ ATPase, leading to inhibition of fluid secretion and corneal edema. In addition to UPR, pump function is also affected in FECD by the downregulation of ion transporters. Concomitantly, uncontrolled UPR induces a loss of endothelial cells due to apoptosis [36].


Loss of ion transporters and increased unfolded protein response in Fuchs' dystrophy.

Jalimarada SS, Ogando DG, Bonanno JA - Mol. Vis. (2014)

Summary of the mechanisms possibly contributing to the clinical symptoms associated with FECD. Evidence of mutations and oxidative stress support the induction of UPR in FECD, which could cause decreased CE cell density and pump function followed by corneal edema. The clinical indications could also occur as a result of compromised pump function caused by the decreased RNA level expression of ion transporters demonstrated in this study, whose mechanism is not known.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4265779&req=5

f6: Summary of the mechanisms possibly contributing to the clinical symptoms associated with FECD. Evidence of mutations and oxidative stress support the induction of UPR in FECD, which could cause decreased CE cell density and pump function followed by corneal edema. The clinical indications could also occur as a result of compromised pump function caused by the decreased RNA level expression of ion transporters demonstrated in this study, whose mechanism is not known.
Mentions: Multiple mechanisms have been suggested to describe the pathology of FECD, including apoptotic cell death due to oxidative stress or UPR and loss of CE pump function. This study aimed to analyze two of the proposed mechanisms: loss of corneal endothelial pump function and the induction of UPR by determining the expression levels of ion transporters and UPR related genes in CE from FECD patients. A comparative expression level analysis of the ion transporters of CE in normal CE showed higher expression of SLC4A11 among the seven ion transporters tested, underlining the importance of this transporter. FECD samples showed significant downregulation of Na+/K+ ATPase, which is critical for fluid secretion across CE, along with monocarboxylate transporters MCT1 and 4, which are postulated to have a role in corneal endothelial fluid secretion [1,2]. Simultaneous UPR PCR array analysis, performed for the first time using FECD CE total RNA, denoted significant UPR with more than twofold regulation of about 52% of the genes tested. Although the small amount of tissue available precluded protein level analysis, these results are an indication of compromised ion pump function, along with UPR in FECD. Thus, perhaps both UPR and the loss of ion transport function concurrently play a role in the pathology of FECD. Figure 6 shows a schematic summarizing the mechanisms that could contribute to the occurrence of the clinical symptoms observed in FECD. ER stress in FECD could occur due to identified mutations in associated genes and loci, along with oxidative stress [28]. Accumulation of unfolded protein as a result of ER stress can induce UPR [28,31,36], including the inhibition of protein translation, which could reduce the expression of membrane proteins like Na+/K+ ATPase, leading to inhibition of fluid secretion and corneal edema. In addition to UPR, pump function is also affected in FECD by the downregulation of ion transporters. Concomitantly, uncontrolled UPR induces a loss of endothelial cells due to apoptosis [36].

Bottom Line: Thirteen of the altered genes showed significant changes (p<0.05).The PCR array results were validated by quantitative RT-PCR.FECD samples had evident UPR with significant changes in the expression of the protein processing pathway genes.

View Article: PubMed Central - PubMed

Affiliation: School of Optometry, Indiana University, Bloomington, IN.

ABSTRACT

Purpose: Fuchs' endothelial corneal dystrophy (FECD), which affects approximately 5% of the population over 40 in the U.S.A., is a major cause of corneal transplantation. FECD is associated with mutations of a variety of unrelated genes: SLC4A11, COL8A2, TCF8, and LOXHD1. The current pathological description of the dystrophy includes deficiency of corneal endothelium (CE) pump function and induction of the unfolded protein response (UPR). This study aims to determine the contribution of the two mechanisms by assessing the expression levels of (1) seven endothelial ion transporters known to regulate stromal hydration and (2) UPR related genes in a set of six CE samples obtained from FECD patients compared to that of normal controls.

Methods: CE samples collected during FECD keratoplasty or from an eye bank (normal control) were transferred into an RNA stabilizing agent and refrigerated. Total RNA from each CE specimen was individually extracted. The expression levels of ion transporters and UPR genes were tested using quantitative real-time (RT) PCR and a UPR specific PCR array, respectively.

Results: In normal CE, the comparative expression levels of ion transporters in decreasing order were SLC4A11, Na(+)/K(+) ATPase, pNBCe1, and NHE1, followed by the isoforms of monocarboxylate transporters (MCTs). In FECD samples, Na(+)/K(+) ATPase and MCTs 1 and 4 were significantly downregulated compared to normal controls (p<0.05). The PCR array tested 84 UPR related genes. Data analysis showed upregulation of 39 genes and downregulation of three genes, i.e., approximately 51% of the tested genes had their expression altered in FECD samples with a difference greater than ± twofold regulation. Thirteen of the altered genes showed significant changes (p<0.05). The PCR array results were validated by quantitative RT-PCR.

Conclusions: FECD samples had evident UPR with significant changes in the expression of the protein processing pathway genes. The significant downregulation of ion transporters indicates simultaneous compromised CE pump function in Fuchs' dystrophy.

Show MeSH
Related in: MedlinePlus