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Effect of Schiff base formation on the function of the calcitriol receptor
S R Patel1, R J Koenig, C H Hsu
1Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, USA.
Abstract:
The genomic action of calcitriol is mediated through the interaction of the calcitriol receptor (VDR) with vitamin D response elements (VDREs) of the target genes. We have shown that the interaction of VDRs with VDREs is inhibited by uremic toxins. We hypothesize that uremic toxins form Schiff bases with the lysine residues of the VDR DNA binding domain and inhibit the VDR interaction with the VDRE. In this study, pyridoxal 5'-phosphate was used as a probe to test Schiff base formation as the inhibitory mechanism, since it forms Schiff bases with steroid receptors. Pyridoxal 5'-phosphate inhibited the VDR binding to the VDREs and chemically modified the DNA binding domain of the VDR in vitro. The inhibition was reversed when pyridoxal 5'-phosphate was preincubated with lysine. Further, this chemical agent also blocked the production of chloramphenicol acetyltransferase (CAT) enzyme induced by calcitriol in cells transfected with a constructed VDRE attached to a CAT reporter gene. This finding is consistent with the hypothesis that pyridoxal 5'-phosphate could interact with the VDR and impair its DNA binding within cells. Since induction of 24-hydroxylase synthesis is a receptor mediated process, we studied the effect of pyridoxal 5'-phosphate on the synthesis of renal 24-hydroxylase in rats. When pyridoxal 5'-phosphate was infused to rats, renal 24-hydroxylase activity was suppressed, consequently, degradation of calcitriol was also reduced in these animals. Thus, chemicals capable of Schiff base formation potentially could alter the physiological function of VDR and calcitriol.
Insights
Uremic toxins may inhibit vitamin D receptor (VDR) function by forming Schiff bases. Pyridoxal 5'-phosphate confirmed this mechanism, blocking VDR-DNA binding and calcitriol-induced gene expression, impacting VDR
Area of Science:
- Molecular Endocrinology
- Biochemistry
- Renal Physiology
Background:
- Calcitriol (active vitamin D) genomic effects rely on the vitamin D receptor (VDR) binding to vitamin D response elements (VDREs).
- Uremic toxins are known to inhibit VDR-VDRE interactions, but the precise mechanism remains unclear.
Purpose of the Study:
- To investigate if Schiff base formation between uremic toxins and VDR lysine residues mediates the inhibition of VDR-VDRE binding.
- To assess the functional consequences of this potential inhibition on calcitriol's genomic actions.
Main Methods:
- Utilized pyridoxal 5 -phosphate, a Schiff base-forming agent, as a probe to test VDR modification.
- Assessed VDR-VDRE binding inhibition and chemical modification of the VDR DNA-binding domain in vitro.
- Evaluated calcitriol-induced reporter gene (CAT) expression in transfected cells.
- Measured renal 24-hydroxylase activity in rats infused with pyridoxal 5 -phosphate.
Main Results:
- Pyridoxal 5 -phosphate inhibited VDR binding to VDREs and modified the VDR DNA-binding domain.
- This inhibition was reversible upon preincubation with lysine.
- Pyridoxal 5 -phosphate blocked calcitriol-induced CAT gene expression in cells.
- In vivo, pyridoxal 5 -phosphate infusion suppressed renal 24-hydroxylase activity and reduced calcitriol degradation in rats.
Conclusions:
- Schiff base formation is a plausible mechanism by which uremic toxins inhibit VDR function.
- Chemical agents forming Schiff bases can impair VDR DNA binding and alter calcitriol's physiological actions.
- These findings highlight a potential molecular basis for VDR dysfunction in uremia.