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Tissue-specific expression and regulation by 1,25(OH)2D3 of chick protein kinase inhibitor (PKI) mRNA
1Lineberger Comprehensive Cancer Center, School of Medicine, University of North Carolina, Chapel Hill, USA.
Abstract:
The heat-stable protein kinase inhibitor (PKI) protein is a specific and potent competitive inhibitor of the catalytic subunit of cAMP-dependent protein kinase (PKA). Previously, it has been shown that vitamin D status affects chick kidney PKI activity: a 5- to 10-fold increase in PKI activity was observed in kidneys of chronically vitamin D-deficient chicks and treatment with 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) in cultured kidney cells resulted in a 95% decrease in PKI activity. The authors have recently cloned the cDNA for chick kidney PKI and have used the coding sequence to study the regulation of PKI mRNA. Northern analysis showed the expression of two PKI messages, which are 2.7 and 3.3 kb in size. These mRNAs are expressed in brain, muscle, testis, and kidney, but not in pancreas, liver, or intestine. PKI mRNA steady-state levels are downregulated by 47% in kidneys from vitamin D-replete chicks as compared to vitamin D-deficient chicks. PKI mRNA levels in brain, muscle, and testis are not affected by vitamin D status. Treatment of primary chick kidney cultures treated with 10(-7) M 1,25(OH)2D3 for 24h resulted in a 20-30% decrease in PKI mRNA. 1,25(OH)2D3 treatment does not affect the stability of PKI mRNA as determined by treatment of cell cultures with actinomycin D. This study shows that 1,25(OH)2D3 directly and tissue-specifically downregulates PKI mRNA in the chick kidney.
Insights
Vitamin D directly influences protein kinase inhibitor (PKI) mRNA levels in chick kidneys. This regulation is tissue-specific, impacting kidney function but not other tissues like the brain.
Area of Science:
- Molecular Endocrinology
- Vitamin D Metabolism
- Gene Regulation
Background:
- Protein kinase inhibitor (PKI) regulates cAMP-dependent protein kinase (PKA).
- Vitamin D status significantly alters chick kidney PKI activity.
- 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) reduces PKI activity in cultured kidney cells.
Purpose of the Study:
- To investigate the regulation of PKI mRNA by vitamin D in chick kidney.
- To determine the tissue-specific effects of vitamin D on PKI mRNA expression.
Main Methods:
- Cloning of chick kidney PKI cDNA.
- Northern blot analysis to detect PKI mRNA expression in various tissues.
- Quantitative analysis of PKI mRNA levels in vitamin D-deficient versus replete chicks.
- Primary chick kidney cell culture treated with 1,25[OH]2D3 and actinomycin D.
Main Results:
- Two PKI mRNA transcripts (2.7 and 3.3 kb) were identified, expressed in brain, muscle, testis, and kidney.
- PKI mRNA levels were 47% lower in kidneys of vitamin D-replete chicks compared to deficient chicks.
- 1,25[OH]2D3 treatment decreased PKI mRNA by 20-30% in cultured kidney cells.
- Vitamin D status did not affect PKI mRNA levels in brain, muscle, or testis.
- 1,25[OH]2D3 did not alter PKI mRNA stability.
Conclusions:
- 1,25-dihydroxyvitamin D3 directly and specifically downregulates PKI mRNA in chick kidney.
- Vitamin D-mediated regulation of PKI mRNA is tissue-specific.
- This finding provides insight into the molecular mechanisms of vitamin D action in the kidney.