Abstract
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.
Highlights
The first element was located at nt Ϫ832 through Ϫ826 (CAGGAAG; designated as human anticoagulant protein C (hPC) agerelated stability element (ASE)), which is consistent with the PEA-3 consensus motif and different from the human factor IX (hFIX) ASE (GAGGAAG) by one nucleotide
The second series of hPC minigenes were constructed with hFIX-derived ASE or age-related increase element (AIE) (Fig. 2B)
The presence of ASE did not have any significant effect on transient hPC expression in HepG2 cells, while the presence of AIE in hPC minigenes lowered their transient expression activities by ϳ30% (Fig. 2B) in agreement with our previous observations on hFIX minigenes containing AIE [12]
Summary
Materials—Restriction enzymes and DNA modification enzymes were purchased from Invitrogen and New England Biolabs. The 3Ј-end portion of the resultant intermediate construct, from the internal Sse8387I site at nt ϩ10497 in the 3Ј-UTR to the EcoRI site outside of the poly(A) attachment site (nt ϩ10783), was released by Sse8387I/ EcoRI double digestion This portion was replaced by a PCRamplified fragment with Sse8387I/EcoRI sticky ends (612 bp in length, spanning nt ϩ10497 through ϩ11108 with a 325-bp extension into the 3Ј-flanking region sequence of the hPC gene), generating minigene Ϫ1462hPCm1. Human FIX minigenes, ASEmin/Ϫ416FIXm1 and AIE/Ϫ416FIXm1/ASE, were constructed by inserting a single copy of the 17-bp minimum ASE sequence (ASEmin: nt Ϫ795 through Ϫ779 of the hFIX gene) or a copy of the above AIE into minigenes Ϫ416FIXm1 and Ϫ416FIXm1/ASE [12] at the 5Ј-end SphI site, respectively. Circulatory hPC levels were determined by ELISA using serum samples, which were prepared from blood samples (ϳ50-l aliquots) collected via snipped tails at 10 min and at 2, 6, 12, 18, 24, 30, 36, and 48 h after protein injection
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