ABSTRACT
We previously generated a cytochrome P450 4F2 (CYP4F2) transgenic mouse model and demonstrated that overexpressed CYP4F2 and overproduced 20-HETE in the kidneys contribute to the increase of blood pressure in the CYP4F2 transgenic mice with normal salt intake. We currently expect to elucidate a potential mechanism of salt-related hypertension whereby diverse levels of 20-HETE interact with dietary salt on Na(+)-K(+)-2Cl(-) cotransporter, isoform 2 (NKCC2) in the kidneys of the transgenic and wild-type mice with high salt intake. High salt intake reduced about 85 % abundance of renal NKCC2 protein in the transgenic mice and about 24 % in the wild-type mice by Western blot. Furthermore, we first found that NKCC2 was ubiquitinated and interacted with Nedd4-2 by immunoprecipitation in the transgenic mice with high salt intake. In addition, inhibition of 20-HETE synthesis or proteasome activity reversed the reduction of NKCC2 expression induced by 20-HETE and high salt intake. These results suggest that 20-HETE and high salt intake synergistically decrease the expression of NKCC2 protein via Nedd4-2-mediated ubiquitin-proteasome pathway, and thereby modulate natriuresis and blood pressure. We propose that diverse levels of 20-HETE have diverse effects on blood pressure in different salt concentrations.
Subject(s)
Blood Pressure/drug effects , Blood Pressure/physiology , Hydroxyeicosatetraenoic Acids/pharmacology , Proteasome Endopeptidase Complex/metabolism , Sodium Chloride, Dietary , Sodium-Potassium-Chloride Symporters/genetics , Ubiquitin/metabolism , Animals , Cytochrome P-450 Enzyme System/genetics , Cytochrome P-450 Enzyme System/metabolism , Endosomal Sorting Complexes Required for Transport/metabolism , Gene Expression Regulation , Male , Mice , Mice, Transgenic , Nedd4 Ubiquitin Protein Ligases , RNA, Messenger/genetics , RNA, Messenger/metabolism , Signal Transduction , Sodium Chloride/metabolism , Sodium-Potassium-Chloride Symporters/metabolism , Solute Carrier Family 12, Member 1 , Ubiquitin-Protein Ligases/metabolism , Vasoconstrictor Agents/pharmacologyABSTRACT
Cytochrome P450 4F2 (CYP4F2) activity is thought to be a factor in the pathogenesis of hypertension through its bioactive metabolite 20-hydroxyeicosatetraenoic acid (20-HETE). We previously found that a gain-in-function CYP4F2 variant in a Chinese cohort was associated with elevated urinary 20-HETE and hypertension. To further explore this association we generated a transgenic mouse model expressing CYP4F2 driven by a modified mouse kidney androgen-regulated protein promoter. This heterologous promoter regulated the expression of luciferase and his-tagged CYP4F2 in transfected HEK 293 cells. In the kidney of transgenic mice, CYP4F2 was localized to renal proximal tubule epithelia and was expressed at a higher level than in control mice, leading to increased urinary 20-HETE excretion. Assessment of CYP4F2 activity by an arachidonic acid hydroxylation assay showed that 20-HETE production was significantly higher in kidney microsomes of transgenic mice compared to control mice, as was their systolic blood pressure. There was a positive correlation of blood pressure with urinary 20-HETE levels. Our results show that increased expression of CYP4F2 in mice enhanced 20-HETE production and elevated blood pressure.
Subject(s)
Cytochrome P-450 Enzyme System/genetics , Cytochrome P-450 Enzyme System/metabolism , Hydroxyeicosatetraenoic Acids/biosynthesis , Hypertension/etiology , Animals , Base Sequence , Blood Pressure/genetics , Blood Pressure/physiology , Cell Line , Cytochrome P450 Family 4 , DNA, Complementary/genetics , Disease Models, Animal , Gene Expression , Humans , Hydroxyeicosatetraenoic Acids/urine , Hypertension/genetics , Hypertension/physiopathology , Kidney Tubules, Proximal/metabolism , Mice , Mice, Transgenic , Promoter Regions, Genetic , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , TransfectionABSTRACT
OBJECTIVE: To investigate the antiestrogenic effect of environment teratogen on the gene expression of insulin-like growth factors (IGFs) family in osteoblast cells during rat skeleton development. METHODS: The fetal rat models with congenital skeleton malformation were constructed by treating 20 female Wistar rats with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on pregnant day 10. The MC-3T3-E1 cells were cultured with estrogen, TCDD, or a combination of the two chemicals for 24 hours. The IGF-II and IGFBP-6 mRNA levels in rat calvaria bone tissue and MC-3T3-E1 cells were detected by reverse transcription-polymerase chain reaction. Flow cytometer was used to determine the cell proliferation. RESULTS: TCDD at the concentration of 5-15 microg/kg induced developmental skeleton defect of fetal rat, and the effect was dose-dependent. The expression of IGF-II mRNA gene was enhanced by estrogen in rat calvaria bone tissue and MC-3T3-E1 cells, whereas IGFBP-6 mRNA was decreased. Estrogen increased the cell proliferation in MC-3T3-E1 cells. TCDD, however, inhibited the effect of estrogen on regulation of IGF-II gene and IGFBP-6 gene as well as MC-3T3-E1 cell proliferation. CONCLUSION: These findings provide the evidence that TCDD can induce congenital fetal skeleton malformation under the condition of high estrogen level in pregnant Wistar rats. TCDD has antiestrogenic effect and hence exerts negative influence on the osteoblast cells through target IGF-II and IGFBP-6 of IGFs family.
