ABSTRACT
BACKGROUND: Daily rhythms of mammalian physiology and endocrinology are regulated by circadian pacemakers. The master circadian pacemaker resides in the suprachiasmatic nucleus, which is located in the hypothalamus of the brain, but circadian oscillators also exist in peripheral tissues. Because many studies have demonstrated apparent circadian variations in the frequency of cardiovascular disorders, it is of great interest to investigate a possible relation between circadian gene expression and cardiovascular function. We examined whether a circadian oscillation system exists in the aorta and/or in cultured vascular smooth muscle cells (VSMCs). METHODS AND RESULTS: The mRNA levels of clock genes were assayed by northern blot analysis. The mouse aorta showed a clear circadian oscillation in the expression of mPer2, dbp, and Bmal1. Brief treatment of VSMCs with angiotensin II induced a robust increase in mPer2 gene expression, followed by a marked reduction in mPer2 mRNA levels and subsequent synchronous cycling of mPer2, dbp, and Bmal1 mRNAs. The induction of mPer2 in VSMCs by angiotensin II was completely abolished by treatment with CV11947, a specific angiotensin II type1 receptor antagonist. CONCLUSIONS: The present results demonstrate that the aorta and VSMCs possess a circadian oscillation system which is comparable to that of the suprachiasmatic nucleus and that the circadian gene expression in VSMCs is induced by angiotensin II through the angiotensin II type1 receptor. Our in vitro system will provide a useful tool to further analyze the physiological significance of the peripheral clock in cardiovascular function.
Subject(s)
Angiotensin II/pharmacology , Circadian Rhythm/drug effects , DNA-Binding Proteins , Gene Expression/drug effects , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/metabolism , ARNTL Transcription Factors , Angiotensin Receptor Antagonists , Animals , Aorta, Thoracic/chemistry , Aorta, Thoracic/cytology , Aorta, Thoracic/metabolism , Basic Helix-Loop-Helix Transcription Factors , Benzimidazoles/pharmacology , Biological Clocks/drug effects , Biological Clocks/physiology , Biomarkers/analysis , Biphenyl Compounds , Blotting, Northern , Cell Cycle Proteins , Cells, Cultured , Circadian Rhythm/physiology , Gene Expression/physiology , Imidazoles/pharmacology , Male , Mice , Mice, Inbred BALB C , Muscle, Smooth, Vascular/cytology , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Period Circadian Proteins , Pyridines/pharmacology , RNA, Messenger/analysis , RNA, Messenger/metabolism , Rats , Receptor, Angiotensin, Type 1 , Receptor, Angiotensin, Type 2 , Tetrazoles/pharmacology , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
A subfamily of zinc metalloproteases, represented by Neutral endopeptidase (EC ) and endothelin-converting enzyme, is involved in the metabolism of a variety of biologically active peptides. Recently, we cloned and characterized a novel member of this metalloprotease family termed soluble secreted endopeptidase (SEP), which hydrolyzes many vasoactive peptides. Here we report that alternative splicing of the mouse SEP gene generates two polypeptides, SEP(Delta) and SEP. After synthesis, both isoforms are inserted into the endoplasmic reticulum (ER) as type II membrane proteins. SEP(Delta) then becomes an ER resident, whereas SEP, which differs by only the presence of 23 residues at the beginning of its luminal domain, is proteolytically cleaved by membrane secretase(s) in the ER and transported into the extracellular compartment. An analysis of the chimeric proteins between SEP(Delta) and bovine endothelin-converting enzyme-1b (bECE-1b) demonstrated that the retention of SEP(Delta) in the ER is mediated by the luminal domain. In addition, the dissection of the chimeric bECE-1b/SEP insertion showed that its insertion domain is obviously responsible for its secretion. A series of mutagenesis in this region revealed that the minimal requirement for cleavage was found to be a WDERTVV motif. Our results suggest that the unique subcellular localization and secretion of SEP proteins provide a novel model of protein trafficking within the secretory pathway.
Subject(s)
Alternative Splicing , Endoplasmic Reticulum/metabolism , Metalloendopeptidases/genetics , Amino Acid Sequence , Animals , Aspartic Acid Endopeptidases/metabolism , Base Sequence , CHO Cells , Cattle , Cricetinae , Endothelin-Converting Enzymes , Gene Library , Metalloendopeptidases/metabolism , Mice , Molecular Sequence Data , Mutagenesis, Site-Directed , Plasmids , Protein Processing, Post-TranslationalABSTRACT
One class of zinc metalloproteases, represented by neutral endopeptidase 24.11 and endothelin-converting enzyme, has been shown to be involved in proteolytic activation or inactivation of many regulatory peptides. Here, we report molecular cloning and characterization of a novel member of this type II membrane-bound metalloprotease family, termed soluble secreted endopeptidase (SEP). Alternative splicing results in the generation of another transcript, SEP(Delta), which lacks a 69-base pair nucleotide segment following the transmembrane helix. Both SEP and SEP(Delta) mRNA are detected in all mouse tissues examined. Transfection of an SEP cDNA expression construct resulted in the expression of the membrane-bound form of SEP in the early secretory pathway as well as the soluble secreted form of the enzyme in the culture medium. In contrast, transfection of the SEP(Delta) cDNA only results in the expression of the membrane-bound form. In vitro enzymological analysis of the recombinant soluble form of SEP demonstrated that it hydrolyzes a variety of vasoactive peptides, including endothelin-1, atrial natriuretic peptide, and angiotensin I. This activity of SEP was inhibited by phosphoramidon and the neutral endopeptidase 24.11 specific inhibitor thiorphan, but it was only partially inhibited by the endothelin-converting enzyme specific inhibitor FR901533. These findings suggest that SEP is a novel metalloprotease that possesses a broad substrate specificity and that it may be involved in the metabolism of biologically active peptides intracellulary as well as extracellularly.
