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© 2000 American Heart Association, Inc.
Brief Review |
Genetic Mechanisms of Age Regulation of Blood Coagulation
Factor IX Model
From the Department of Human Genetics, University of Michigan Medical School, Ann Arbor.
Correspondence to Kotoku Kurachi, PhD, Department of Human Genetics, University of Michigan Medical School, M4804 Medical Science II Building, Ann Arbor, MI 48109-0618. E-mail kkurachi{at}umich.edu
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Abstract |
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 Top Abstract IntroductionhFIX Gene and Age...Characteristics of hFIX...Characteristics of hFIX Minigene...ConclusionReferences |
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Abstract—Blood coagulation capacity increases with age in healthy individuals, apparently because of increases in the plasma concentration of most procoagulant factors. This phenomenon may play an important role in the advancing age–associated increase of cardiovascular diseases and thrombosis. Through longitudinal analyses of transgenic mice, we recently identified 2 critical age-regulatory elements, AE5' and AE3', which are together essential for age regulation of the normal human factor IX (hFIX) gene. AE5', present in the long interspersed repetitive element–derived sequence of the 5' upstream region, containing polyomavirus enhancer activator-3 or a closely related element, is responsible for age-stable expression of the gene and functions in a position-independent manner. AE3', present in the middle of the 3' untranslated region, is responsible for age-associated elevation of hFIX mRNA levels in the liver. Presence of both AE5' and AE3' is needed to recapitulate normal age regulation of the hFIX gene. Because factor IX clearance from the circulation is not significantly affected by age, age regulation of hFIX levels is achieved primarily by a combination of stabilization of gene transcription and age-dependent increases in the mRNA levels, which are presumably due to increasing mRNA stabilization. The stage is now set for further systematic studies of the genetic and molecular mechanisms of age regulation of other key coagulation and anticoagulation factors in hopes of understanding the overall age regulation of blood coagulation.
Key Words: aging • homeostasis • factor IX • hemophilia • cardiovascular disease • thrombosis
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Introduction |
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TopAbstractIntroductionhFIX Gene and Age...Characteristics of hFIX...Characteristics of hFIX Minigene...ConclusionReferences |
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Blood coagulation plays a critical role not only in homeostasis but also in many physiological and pathological conditions.1 Blood coagulation potential in humans as well as other mammals reaches a young adult level around the time of weaning, followed by a gradual increase during young adulthood and an almost 2-fold increase by old age.2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 This advancing age–associated increase in coagulation potential takes place in healthy centenarians,18 indicating that the increase is a normal age-associated phenomenon. However, it is conceivable that in the general human population, such increases in blood coagulation potential may substantially contribute to the development and progression of age-associated cardiovascular and thrombotic disorders.16 17 19 20 21 This increase in blood coagulation potential is apparently due to the collective effects of increases in the plasma level of procoagulant factors, combined with only marginal increases or even decreases in the plasma levels of anticoagulation factors (such as antithrombin III and protein C) or of factors involved in fibrinolysis.16 17 19 20 21 The age-associated increase in blood coagulation potential represents a net increase in the coagulation activity and appears to be a strictly controlled phenomenon rather than a consequence of general aging-associated dysregulation of hemostasis. Literally nothing was known about the genetic and molecular mechanisms responsible for this important phenomenon until the recent findings regarding the human (h) factor IX (FIX) gene.22 Why this controlled age-associated increase in the blood coagulation potential exists and how such age regulation of blood coagulation is maintained are two fascinating and intriguing questions.
Toward understanding of this phenomenon, we recently carried out systematic studies of the age regulation of hFIX, a key coagulation factor and the first model analyzed in depth.22 The transgenic mouse approach taken in these studies has proven to be highly effective in gaining critical insight into the mechanisms of age regulation of hFIX.
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hFIX Gene and Age Regulation |
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TopAbstractIntroductionhFIX Gene and Age...Characteristics of hFIX...Characteristics of hFIX Minigene...ConclusionReferences |
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FIX, a plasma protease precursor, is one of the key procoagulant factors. FIX occupies a key position in the blood coagulation cascade, where 2 major initiation pathways, intrinsic and extrinsic, merge.1 23 FIX is synthesized in the liver with high tissue specificity, and its deficiency results in the bleeding disorder hemophilia B. FIX is 1 of 2 blood coagulation factors, along with factor VIII, whose genes are on the X chromosome; thus, hemophilia B is primarily seen in males and only rarely in females. The hFIX gene is
40 kb in size and is composed of 8 exons (Figure 1
). Its contiguous complete
nucleotide sequence was reported in 1985,25
and >1500 abnormal genes have been analyzed to date to their
molecular level.26 This enormous knowledge that has been
accumulated regarding FIX, its genes, and their structure-function
relation, physiology, and pathology makes hFIX one of the most
extensively studied mammalian proteins and, thus, highly suited for
studying the complex mechanisms of age-dependent regulation.
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With advancing age, hFIX increases its circulatory levels to levels approximately twice those found at young ages, as does blood coagulation potential.27 Similarly, mouse FIX (mFIX) activity in the circulation also increases with age,22 28 which is directly correlated with an increase in the liver mFIX mRNA level.28 Mice are used as a well-established mammalian model system for various in vivo testing and have a blood coagulation system similar to that in humans. The transgenic mouse approach, therefore, is appropriate and is used in analyzing the molecular mechanisms of age regulation of the hFIX gene.22
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Characteristics of hFIX Expression In Vitro From Minigenes |
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TopAbstractIntroductionhFIX Gene and Age...Characteristics of hFIX...Characteristics of hFIX Minigene...ConclusionReferences |
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A series of hFIX minigene expression vectors were constructed and first tested with the HepG2 cell (human hepatoma cell line) assay system22 (Figure 2
). These
minigenes contain a common hFIX coding region derived from cDNA with a
shortened, but fully functional, first intron inserted at the natural
position,29 which was linked with variously extended 5'
promoter regions up to nucleotide -2231. They also carry
the 3' untranslated region (UTR) with or without its middle portion
deleted. The middle portion of the 3' UTR contains a 102-bp stretch of
inverted AT, GT, and GC dinucleotide repeats, which can
potentially form stem-loop structures in mRNA25 and are
known to affect the stability of mRNA.30
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Systematic testing of these minigenes with HepG2 cells unraveled unique
characteristics of the hFIX gene. The HepG2 cell assay system with the
transfection reagent FuGene 6 was invaluable for successful transient
expression analysis,31 allowing all hFIX minigenes
tested to produce recombinant hFIX at high levels (50
ng/106 cells per 48 hours).22 One of
the unexpected findings was that no hFIX minigene carrying the 5'
upstream region (nucleotide -802 up through
nucleotide -1900) showed the significant silencer
activity22 that was previously observed when a
chloramphenicol acetyltransferase gene was used as a
reporter.32 Use of such heterologous reporter genes to
analyze unrelated genes, such as the FIX gene, may very likely
give irrelevant observations, significantly skewed from the natural
gene regulation. This may presumably be due to the absence of critical
structural elements in the heterologous reporter gene that are needed
for regulation of the native genes in concert with their own 5'
promoters. Conversely, structural elements in the heterologous reporter
genes may affect the transcriptional regulation of the studied gene.
Irrelevant observations may also be due to the cell lines used.
All minigenes containing the entire 3' UTR reproducibly showed activity that was 25% to 30% lower than counterpart minigenes missing the middle portion of the 3' UTR.22 This moderately suppressive effect appears only in the presence of the middle portion of the 3' UTR. No mechanistic explanations for the suppressive activity are currently available, but this finding is intriguing when the in vivo observations discussed below are taken into account.
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Characteristics of hFIX Minigene Expression In Vivo: Observations Involving Transgenic Mice |
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TopAbstractIntroductionhFIX Gene and Age...Characteristics of hFIX...Characteristics of hFIX Minigene...ConclusionReferences |
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Critical insight into the regulatory mechanisms of the hFIX gene was obtained only from the longitudinal analysis of the entire life span of circulatory hFIX in hundreds of individual transgenic mice carrying hFIX minigenes22 (Figure 3
). The prepuberty levels of systemic
hFIX in these animals largely varied, primarily because of transgene
positional effects and zygosity status, from very low levels (<10
ng/mL serum) to high levels similar to the plasma level of the natural
gene (4 to 5 µg/mL serum). Regardless of the initial prepubertal hFIX
levels in the circulation, comparison of age patterns of hFIX levels in
the circulation among animals with different minigene transgenes
provided the most valuable information and proved the robustness of the
transgenic mouse approach for analyzing the complex age-regulatory
mechanisms of the hFIX gene.
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In the present review, we will focus our discussion on a few
selected animal lines. At 1 month of age (prepubertal stage),
transgenic animals carrying -416 FIXm1 minigenes, which contain the 5'
promoter region up to nucleotide -416 and the 3' UTR with
its middle portion deleted, produced hFIX at a wide range of levels, as
mentioned above (Figure 3A
). Importantly, independent of the
initial prepubertal level, circulatory hFIX levels in these animals
rapidly declined during puberty and during the subsequent 2 to 3 months
to lower, stable levels. This rapid age-dependent decline in the
circulatory hFIX level is observed regardless of the founder line,
initial prepubertal hFIX level, generation, sex, or zygosity status of
the transgenes and is directly related to a similar decline in the
steady-state liver hFIX mRNA. The hFIX transgene copy number level does
not change significantly with age. Transgenic mice with -416FIXm1/1.4,
which, unlike -416FIXm1, contains the entire 3'UTR, also showed a
similar, though much less steep, age-dependent decline in hFIX levels,
and expression levels were stabilized at significantly higher levels
than those observed for -416FIXm1 (Figure 3B). These results
indicated that the presence or absence of the complete 3' UTR
containing the extensive dinucleotide repeat structure
cannot prevent an age-associated decline in hFIX expression. Similar
age-associated regulation was observed for all animals carrying
minigenes with proximal promoter regions up to nucleotide
-770, indicating that the essential element or elements required for
normal age-associated regulation of the hFIX gene are not contained in
these minigenes. The age-dependent decline in the circulatory hFIX in
animals with these minigenes again correlates with the decline in liver
hFIX mRNA, which is presumably due to a decline in promoter
activity.22
Surprisingly, animals carrying the minigene -802FIXm1 (Figure 4A), which has an extra 5' stretch of 32
bp extended beyond nucleotide -770 (Figure 2)
showed grossly different patterns of age-associated regulation of hFIX
production.22 These animals invariably showed
remarkably age-stable circulatory hFIX levels until death, usually for
up to 20 to 24 months of age. Although age-stable plasma hFIX levels
are also consistent with age-stable liver hFIX mRNA levels,
interestingly, the circulatory hFIX protein turnover time does not
change significantly in vivo with increasing age. These observations
were further supported by the age-stable hFIX expression observed in
mice carrying -2231FIXm1. Thus, a critical structural element required
for age-stable expression of the hFIX gene was located in a small
region spanning nucleotides -770 through -802, which was
designated the "age-regulatory element in the 5' end"
(AE5'). This region contains a sequence element, GAGGAAG
(nucleotides -784 to -790), which matches the consensus
motif of polyomavirus enhancer activator-3 (PEA-3), a
member of the Ets family of transcriptional
factors.33 As demonstrated by footprint and bandshift
analyses, a specific nuclear protein binds to this element in
an age-dependent manner.22 Whether the protein is PEA-3 or
a closely related protein, which can bind to the PEA-3 element, has yet
to be determined. It is noteworthy that the presence of AE5' is
required for strict liver-specific expression of the hFIX gene, whereas
in its absence, high, but incomplete, liver-specific expression was
observed.
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AE5', which contains the PEA-3 element identified, is located in the 5' flanking region derived from a long interspersed repetitive element (LINE-1 or L1, retrotransposable element). Interestingly, this region has a twintron-like LINE-1 organization (2 LINE-1–derived sequences overlapped).34 Apparently, this was generated by 2 successive events of retrotransposition with the second LINE-1 retrotransposed into the middle of the originally recruited LINE-1, thus dividing it into 2 parts.34 AE5' is present in the proximal region of the originally inserted LINE-1. Because the modern retrotransposable LINE-135 does not have an AE5' (PEA-3)–like structure at the corresponding region, AE5' of the hFIX gene must have been generated through substantial mutations of the inserted LINE-1 sequence. The mFIX gene, which has an age-regulation pattern similar to that of hFIX,28 also contains a LINE-1–derived sequence in its 5' upstream position similar to that in the hFIX gene and has multiple PEA-3 consensus elements.34 In evolution, therefore, retrotransposition of LINE-1 has played, and will continue to play, critical roles not only in inactivating functional genes but also in generating normal gene function.
The most critical observations were obtained from mice with
-802FIXm1/1.4 as well as -2231FIXm1/1.4, both containing the complete
3' UTR. These mice showed an advancing age–associated increase in
circulatory hFIX levels, very similar to those observed for the hFIX
gene (Figure 4B). This age-associated increase in circulatory
hFIX levels was directly correlated with increased liver hFIX mRNA
levels, resulting in discovery of the second age-regulatory element,
designated AE3', located in the middle of the 3' UTR. Only with both
AE5' and AE3' in the minigenes could the characteristic age regulation
of the normal hFIX gene be recapitulated. The unique concerted actions
of AE5' and AE3' are again independent of founder line, initial
expression levels at 1 month of age, sex, generation, or zygosity of
the animals.22 AE3' has been delineated to an 300-bp
middle region of the 3' UTR, where a stretch of
dinucleotide repeats are present, although complete
delineation will require further animal studies (S.K., K.K.,
unpublished data, 1999). AE3' does not function as a
position-independent enhancer but raises the liver hFIX mRNA level in
direct correlation with the age-dependent increase in circulatory hFIX
levels (Figure 4B). Observations made to date suggest that the
function of AE3' is to increase hFIX mRNA stability with age, although
other possible mechanisms for AE3' action have yet to be explored. This
in vivo observation appears to be contradictory to the moderate
suppression observed in vitro with the HepG2 cell assay system and
further suggests that use of in vitro assay systems alone in studying
gene regulation may give skewed and misleading results.
Finally, it is noteworthy that animals expressing increasingly high
levels of hFIX (>1500 ng/mL serum) in addition to normal level mFIX
over some months die at much earlier ages than control animals or those
producing lower levels of hFIX (see animals labeled "d" in Figure 4). This strongly suggests a possibility that substantially
elevated levels of FIX may be a risk factor for thrombosis and/or
cardiovascular diseases.
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Conclusion |
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TopAbstractIntroductionhFIX Gene and Age...Characteristics of hFIX...Characteristics of hFIX Minigene...ConclusionReferences |
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The basic genetic and molecular mechanisms of age regulation (homeostasis) of hFIX have now been established. In addition to the basic structures, such as the basal promoter, coding region, and introns, the mechanisms involve 2 critical elements (AE5' and AE3') that regulate hFIX gene expression at the transcription step and modulate hFIX mRNA stability (Figure 5
).
Success of the study of age regulation of hFIX is due to the systematic
and longitudinal analyses of hFIX expression in a large number
of individual transgenic animals during their life spans. This
analysis was possible primarily because FIX is a plasma
protein, thus allowing longitudinal analysis of many individual
animals without euthanizing animals at various ages for
analysis. If intracellular gene products ought to be used
as a reporter for analyzing age regulation, frequent euthanasia of
animals would be required, making any quantitative study extremely
difficult, if not impossible. A stage is now set for investigating many
other individual procoagulant and anticoagulant factors. It is of
particular interest to determine whether AE5' and AE3' also function in
other genes or whether each gene requires different mechanisms for
age-dependent regulation. Eventually, we may be able to provide a
rational explanation as to why such an increase exists in a healthy
individual and its correlation with the aging-associated increase in
thrombosis and/or cardiovascular diseases.
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Acknowledgments |
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This review was supported by National Institutes of Health (NIH) grants HL-38644 and HL-53713, the University of Michigan Nathan Shock Center for the Basic Biology of Aging Award (National Institute on Aging grant AG-13283), Pepper Center for Aging Research Award (grant AG-08808), the Multipurpose Arthritis and Musculoskeletal Disease Center (NIH grant 5-P60-AR-20557), and the General Clinical Research Center (grant M01-RR-00042). We thank J. Huo for critical reading of the manuscript.
Received October 7, 1999; accepted December 3, 1999.
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TopAbstractIntroductionhFIX Gene and Age...Characteristics of hFIX...Characteristics of hFIX Minigene...ConclusionReferences |
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