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Identification of the WNK-SPAK/OSR1 signaling pathway in rodent and human lenses
Irene Vorontsova1, Leo Lam1, Eric Delpire2
1Department of Optometry and Vision Science, University of Auckland, New Zealand The New Zealand National Eye Centre, University of Auckland, New Zealand.
Purpose:
To identify whether the kinases that regulate the activity of cation chloride cotransporters (CCC) in other tissues are also expressed in rat and human lenses.
Methods:
The expression of with-no-lysine kinase (WNK 1, 3, 4), oxidative stress response kinase 1 (OSR1), and Ste20-like proline alanine rich kinase (SPAK) were determined at either the transcript or protein levels in the rat and human lenses by reverse-transcriptase PCR and/or Western blotting, respectively. Selected kinases were regionally and subcellularly characterized in rat and human lenses. The transparency, wet weight, and tissue morphology of lenses extracted from SPAK knock-out animals was compared with wild-type lenses.
Results:
WNK 1, 3, 4, SPAK, and OSR1 were identified at the transcript level in rat lenses and WNK1, 4, SPAK, and OSR1 expression confirmed at the protein level in both rat and human lenses. SPAK and OSR1 were found to associate with membranes as peripheral proteins and exhibited distinct subcellular and region-specific expression profiles throughout the lens. No significant difference in the wet weight of SPAK knock-out lenses was detected relative to wild-type lenses. However, SPAK knock-out lenses showed an increased susceptibility to opacification.
Conclusions:
Our results show that the WNK 1, 3, 4, OSR1, and SPAK signaling system known to play a role in regulating the phosphorylation status, and hence activity of the CCCs in other tissues, is also present in the rat and human lenses. The increased susceptibility of SPAK lenses to opacification suggests that disruption of this signaling pathway may compromise the ability of the lens to control its volume, and its ability to maintain its transparency.
Insights
Kinases regulating cation chloride cotransporters (CCCs) are present in the eye lens. Disruption of the Ste20-like proline alanine rich kinase (SPAK) pathway increases lens opacification, impacting transparency.
Area of Science:
- Ophthalmology
- Cell Biology
- Molecular Biology
Background:
- Cation chloride cotransporters (CCCs) are crucial for cell volume regulation.
- Kinase signaling pathways, including With-no-lysine (WNK) kinases, oxidative stress response kinase 1 (OSR1), and Ste20-like proline alanine rich kinase (SPAK), regulate CCC activity in various tissues.
- The role of these kinases in the unique environment of the ocular lens is largely unexplored.
Purpose of the Study:
- To investigate the expression and localization of key regulatory kinases (WNK 1, 3, 4, OSR1, SPAK) in rat and human ocular lenses.
- To determine the functional impact of SPAK on lens transparency and integrity.
Main Methods:
- Reverse-transcriptase PCR and Western blotting were used to detect transcript and protein expression of WNKs, OSR1, and SPAK in rat and human lenses.
- Immunohistochemistry and subcellular fractionation were employed for regional and subcellular localization studies.
- SPAK knockout rat lenses were analyzed for transparency, wet weight, and morphology.
Main Results:
- WNK 1, 3, 4, OSR1, and SPAK transcripts were detected in rat lenses; WNK1, 4, OSR1, and SPAK proteins were confirmed in both rat and human lenses.
- SPAK and OSR1 were identified as peripheral membrane proteins with distinct subcellular and regional expression patterns within the lens.
- SPAK knockout lenses exhibited increased susceptibility to opacification, although wet weight remained unchanged.
Conclusions:
- The WNK-OSR1-SPAK signaling pathway, known to regulate CCCs, is present and functional in the ocular lens.
- Disruption of SPAK signaling compromises lens volume control and transparency, suggesting a role in maintaining ocular health and preventing cataracts.
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