Age-related expression analysis of mouse liver nuclear protein binding to 3'-untranslated region of Period2 gene.

In mammals, both circadian rhythm and aging play important roles in regulating time-dependent homeostasis. We previously discovered an age-related increase element binding protein, hnRNP A3, which binds to the 3'-untranslated region (UTR) of blood coagulation factor IX (FIX). Here, we describe other members of this protein family, hnRNP C and hnRNP H, which bind to the 3'-UTR of the mouse circadian clock gene Period 2 (mPer2). RNA electrophoretic mobility shift assays using a (32)P-labeled Per2 RNA probe coupled with two-dimensional gel electrophoresis followed by MALDI-TOF/MS peptide mass fingerprint analysis was used to analyze these proteins. Western blotting suggested that the total expression of these proteins in mouse liver cell nuclei does not increase with age. Two-dimensional gel electrophoresis analysis of age-related protein expression showed that many isoforms of these proteins exist in the liver and that each protein exhibits a complex age-related expression pattern. These results suggest that many isoforms of proteins are regulated by different aging systems and that many age regulation systems function in the liver.

Heterogeneous nuclear ribonucleoprotein A3 is the liver nuclear protein binding to age related increase element RNA of the factor IX gene.

BACKGROUND: In the ASE/AIE-mediated genetic mechanism for age-related gene regulation, a recently identified age-related homeostasis mechanism, two genetic elements, ASE (age-related stability element) and AIE (age-related increase element as a stem-loop forming RNA), play critical roles in producing specific age-related expression patterns of genes. PRINCIPAL FINDING: We successfully identified heterogeneous nuclear ribonucleoprotein A3 (hnRNP A3) as a major mouse liver nuclear protein binding to the AIE-derived RNAs of human factor IX (hFIX) as well as mouse factor IX (mFIX) genes. HnRNP A3 bound to the AIE RNA was not phosphorylated at its Ser(359), while hnRNP A3 in the mouse liver nuclear extracts was a mixture of phosphorylated and unphosphorylated Ser(359). HepG2 cells engineered to express recombinant hFIX transduced with adenoviral vectors harboring an effective siRNA against hnRNP A3 resulted in a substantial reduction in hFIX expression only in the cells carrying a hFIX expression vector with AIE, but not in the cells carrying a hFIX expression vector without AIE. The nuclear hnRNP A3 protein level in the mouse liver gradually increased with age, while its mRNA level stayed age-stable. CONCLUSIONS: We identified hnRNP A3 as a major liver nuclear protein binding to FIX-AIE RNA. This protein plays a critical role in age-related gene expression, likely through an as yet unidentified epigenetic mechanism. The present study assigned a novel functional role to hnRNP A3 in age-related regulation of gene expression, opening up a new avenue for studying age-related homeostasis and underlying molecular mechanisms.

An age-related homeostasis mechanism is essential for spontaneous amelioration of hemophilia B Leyden.

Regulation of age-related changes in gene expression underlies many diseases. We previously discovered the first puberty-onset gene switch, the age-related stability element (ASE)/age-related increase element (AIE)-mediated genetic mechanism for age-related gene regulation. Here, we report that this mechanism underlies the mysterious puberty-onset amelioration of abnormal bleeding seen in hemophilia B Leyden. Transgenic mice robustly mimicking the Leyden phenotype were constructed. Analysis of these animals indicated that ASE plays a central role in the puberty-onset amelioration of the disease. Human factor IX expression in these animals was reproducibly nullified by hypophysectomy, but nearly fully restored by administration of growth hormone, being consistent with the observed sex-independent recovery of factor IX expression. Ets1 was identified as the specific liver nuclear protein binding only to the functional ASE, G/CAGGAAG, and not to other Ets consensus elements. This study demonstrates the clinical relevance of the first discovered puberty-onset gene switch, the ASE/AIE-mediated regulatory mechanism.

Age-related effects of regular physical activity on hemostatic factors in men.

BACKGROUND: Age-related changes in blood coagulation and fibrinolytic factors are associated with an increase in risk of thrombotic events. The purpose of this study was to assess the effects of age, regular aerobic exercise and detraining on blood coagulation and fibrinolytic factors in men. METHODS: Initially, 41 sedentary and 42 physically active men (20-64 years) were analyzed for plasma levels of coagulation and fibrinolytic factors. Twelve sedentary men were then subjected to 16-week aerobic exercise training and subsequent 2-week detraining. Their blood samples taken at rest were assayed for activity levels of prothrombin, coagulation factor (F) V, VII, VIII, IX, X, XI and XIII, antithrombin III, protein C and plasminogen, and for antigen levels of fibrinogen, prothrombin fragment 1 + 2 (F1 + 2), FIX, protein C, tissue-type plasminogen activator (tPA), plasminogen activator inhibitor 1 (PAI-1) and tPA/PAI-1 complex. RESULTS: Plasma levels of most coagulation factors, particularly for fibrinogen and FIX antigens as well as FXIII activity significantly increased with aging in sedentary men, while that tendency disappeared in physically active men. By the exercise training, plasma antigen and/or activity levels of most blood coagulation factors except for prothrombin and FIX decreased. These training-effects, however, disappeared after detraining, and in some cases even rebounded to higher levels than those of pre-training. Plasma antigen levels of tPA, PAI-1 and tPA/PAI-1 complex decreased with the training and remained low even after detraining. CONCLUSION: Regular aerobic exercises give complex effects on expression of hemostatic factors, overall favoring the hemostatic balance to less thrombotic, partly cancelling out the age effects.

Technique for performance and evaluation of parapharyngeal hypophysectomy in mice.

Mice at our institution were hypophysectomized to evaluate the effects of growth hormone on the expression of a transfected human factor IX gene. The hypophysectomy was performed in-house by using a parapharyngeal approach modified from previously published surgical techniques. Modifications included: 1) choice of ketamine-xylazine and isoflurane for anesthesia, with butorphanol for postoperative analgesia; 2) use of a V-trough for positioning mice correctly and consistently; 3) selection of increasing sizes of dental burrs to create a foramen in the cranial base through which the pituitary gland was removed; and 4) disuse of a tracheotomy for airway patency. In addition, verification of successful gland removal was assessed by measuring major urinary protein (MUP) in the urine; presence of MUP indicated incomplete hypophysectomy. This assessment enabled antemortem determination of surgical success by using a single urine collection. Each of these modifications contributed to the success of the surgical procedure. We had a safe and reliable anesthetic regimen, consistent positioning of the surgical patient, and smooth and rapid penetration of the cranium. In our experience, the tracheotomy described in previous techniques was unnecessary, as the mice tolerated brief periods of apnea (approximately 5 sec maximum) while the trachea was retracted. Here we seek to provide details that will assist those interested in learning this technique and that will reduce the number of mice needed for practice. Other applications include a method of evaluating the production of growth hormone without euthanizing the animal.

Myocardial fibrosis in mice with overexpression of human blood coagulation factor IX.

Elevated circulatory levels of many blood coagulation factors are known to be a risk factor for deep vein thrombosis in humans. Here we report the first direct demonstration of a close association between elevated circulatory factor IX levels in mice with thrombosis as well as myocardial fibrosis. Transgenic mice overexpressing human factor IX at persistently high levels died at much younger ages than their cohorts expressing lower levels, or nontransgenic control animals. The median survival age of animals was inversely related to the circulatory levels of human factor IX. Prematurely dying animals had focal fibrotic lesions predominantly present in the left ventricular myocardium, and vasculatures in these lesions showed fibrin deposition. Thromboemboli were also present in other organs, including lung and brain. These observations support the hypothesis that persistently high circulatory levels of factor IX are a risk factor not only for thrombosis and/or thromboembolism, but also for myocardial fibrosis mimicking human myocardial infarction.

Limitation in use of heterologous reporter genes for gene promoter analysis. Silencer activity associated with the cloramphenicol acetyltransferase reporter gene.

Various heterologous reporter genes have been widely used for the functional characterization of gene promoters. Many such studies often found weak to very strong silencer activities to be associated with specific parts of the basal promoter or further upstream regions. In this study, we carried out a systematic study on human blood coagulation factor IX (hFIX) and anti-coagulant protein C (hPC) genes, previously shown to have silencer activities associated with their 5'-flanking regions containing promoter sequences. With newly constructed chloramphenicol acetyltransferase (CAT) reporter vectors carrying hFIX or hPC gene promoter sequences, we confirmed the strong silencer activities associated with the regions nt -1895 through nt -416 of the hFIX gene or with the region nt -802 through nt -82 of the hPC gene. However, no such silencer activities associated with the specific regions were found when autologous hFIX cDNA, hFIX minigenes, or hPC minigenes were used as reporters in the expression vector system. Relative levels of CAT, hFIX, and hPC proteins produced in the transient assays correlated well with their mRNA levels. Human FIX minigene constructs containing a simian virus 40 (SV40) 3'-untranslated region (UTR) taken from the CAT reporter gene showed no silencer activity, indicating that SV40 3'-UTR sequence of the CAT reporter gene does not contribute to the silencer activity. Expression vectors constructed with the beta-galactosidase gene under the control of hFIX gene promoter sequences also showed no silencer activity associated with the region nt -1895 through nt -416. These findings indicate that silencer activities associated with specific regions of promoter sequences as analyzed with CAT reporter genes may represent artifacts specific to the CAT reporter genes. Our findings strongly suggest a need for re-examination of promoter characterizations of many eukaryotic genes, which have been studied to date with CAT reporter genes.

Genetic mechanisms of age regulation of protein C and blood coagulation.

Blood coagulation activity in humans increases with age. We previously identified two genetic elements, age-related stability element (ASE; GAGGAAG) and age-related increase element (AIE; unique stretch of dinucleotide repeats), which were responsible for age-related stable and increasing expression patterns, respectively, and together recapitulated normal age regulation of the human factor IX (hFIX) gene. Here we report the age-regulatory mechanisms of human anticoagulant protein C (hPC), which shows an age-stable pattern of circulatory levels. The murine protein C gene showed an age-related stable expression pattern in general agreement with that of the hPC. Through longitudinal analyses of transgenic mice carrying hPC minigenes, the hPC gene was found to have a functional age-related stability element (hPC ASE; CAGGAAG) in the 5'-upstream proximal region but was found to lack any age-related increase element. Three other ASE-like sequences present in the hPC gene, GAGGAAA and (G/C)AGGATG, also bound nuclear proteins but were not active in the age regulation of the hPC gene. Functional hPC ASE and hFIX ASE were apparently generated through convergent evolution, and hFIX ASE can fully substitute for the hPC ASE in conferring age-related stable expression pattern of the hPC gene. In the presence of the hPC ASE, hFIX AIE can convert the age-stable expression pattern of the hPC gene to a hFIX-like age-related increase pattern. These results support the universality of ASE and AIE functions across different genes. Clearance of hPC protein from the circulation was not significantly affected by age. We now have established the basic mechanisms responsible for the age-related increase of blood coagulation activity.