(Hypertension. 1996;27:491-494.)
© 1996 American Heart Association, Inc.
Articles |
Functional Analysis of Human Tissue Kallikrein in Transgenic Mouse Models
From the Department of Biochemistry and Molecular Biology, Medical University of South Carolina (Charleston).
Correspondence to Julie Chao, PhD, Department of Biochemistry and Molecular Biology, Medical University of South Carolina, 171 Ashley Ave, Charleston, SC 29425.
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Abstract |
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 Top Abstract IntroductionStructure of the HTK...Expression of the HTK...Expression of the HTK...Tissue-Specific Expression of...Chronic Hypotension in...Somatic Gene Delivery of...Conclusions References |
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Abstract Clinical studies show that an inverse correlation exists between blood pressure and urinary kallikrein levels. It has been postulated that the tissue kallikrein-kinin system contributes to the maintenance of normal blood pressure. To test this hypothesis, we have established transgenic mice that overexpress human tissue kallikrein under the promoter control of the mouse metallothionein gene and a liver-targeted albumin gene. These animals secrete human tissue kallikrein in plasma at levels 10- to 40-fold higher than that found in normal human serum, and they are chronically hypotensive. This hypotensive effect can be reversed by the injection of aprotinin, a potent tissue kallikrein inhibitor, or Hoe 140, a specific bradykinin receptor antagonist. Transgenic mice overexpressing human tissue kallikrein show a sustained reduction in blood pressure throughout their life spans, indicating the lack of sufficient compensatory mechanisms to reverse the hypotensive effect of kallikrein. Somatic gene delivery of rat kallikrein-binding protein by muscle injection increases the blood pressure of the hypotensive transgenic mice to levels comparable with those in normotensive control mice. These results indicate that a direct link exists between kallikrein gene expression and alterations in blood pressure. In addition, we have developed normotensive transgenic mice that harbor the human tissue kallikrein gene containing 801 bp of its native promoter. The tissue distribution pattern of human kallikrein in these transgenic mice is similar to that in human tissues, with the highest level in the pancreas and much lower levels in the kidney and salivary gland. These transgenic mice provide new animal models for investigating the tissue-specific regulation of tissue kallikrein and its role in altering blood pressure.
Key Words: kallikrein • kallikrein-binding protein • mice, transgenic • somatic gene delivery • aprotinin
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Introduction |
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TopAbstractIntroductionStructure of the HTK...Expression of the HTK...Expression of the HTK...Tissue-Specific Expression of...Chronic Hypotension in...Somatic Gene Delivery of...Conclusions References |
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The tissue kallikrein-kinin system has been postulated to play a role in the homeostasis of normal blood pressure, and defects in this system could contribute to the pathogenesis of clinical hypertension. Tissue kallikrein (EC 3.4.21.35) is a serine proteinase that is capable of cleaving low-molecular-weight kininogen to produce the vasoactive kinin peptide.1 2 The binding of kinin to its specific receptor produces a broad spectrum of biological effects: smooth muscle contraction and relaxation, increased vascular permeability, vasodilatation, electrolyte and glucose transport, and pain induction.3 4 Previous studies have shown that the tissue kallikrein-kinin system is involved in many physiological and pathological processes, such as blood pressure homeostasis, renal sodium excretion, allergy, and inflammatory disorders.1 4 Extensive epidemiological studies have documented that an inverse correlation exists between blood pressure and urinary kallikrein levels.1 5 6 The notion that tissue kallikrein may contribute to the regulation of blood pressure was supported by a large Utah family pedigree study, which shows that a dominant allele expressed as high urinary kallikrein excretion may be associated with a decreased risk of essential hypertension.7 Reduced urinary or renal kallikrein levels also have been described in a number of genetically hypertensive rats.8 9 The rat tissue kallikrein gene has been linked with hypertension by restriction fragment length polymorphisms and cosegregation studies in a hypertensive rat model.10 11 Collectively, these studies suggest that a deficiency in renal kallikrein activity may contribute to the pathogenesis of hypertension and that high tissue kallikrein levels could have a protective effect against high blood pressure.
Reduced kallikrein activity could be attributed to a deficiency in protein synthesis, accelerated degradation, or increased activity of kallikrein inhibitors. Tissue kallikrein levels are regulated at both transcriptional and posttranslational levels. At the posttranslational level, the activity and bioavailability of tissue kallikrein may be controlled by endogenous kallikrein inhibitors or kallikrein-binding proteins. To understand the regulatory mechanisms of tissue kallikrein, we have purified and cloned a new kallikrein-binding protein from rat, mouse, and human tissues.12 13 14 15 The kallikrein-binding protein rapidly binds to tissue kallikrein and inhibits its activity in vitro.13 16 The transgenic animal model expressing either tissue kallikrein or kallikrein-binding protein will be valuable for examining the role of the kallikrein-kinin system in blood pressure regulation.
The creation of transgenic mice by germ line transfer of exogenous genes has been very useful in cardiovascular and hypertension research. For example, Mullins et al17 reported that the mouse Ren-2 transgene in rats caused fulminant hypertension. Another study showed that elevated blood pressure was observed in transgenic mice coexpressing both rat and human renin and angiotensinogen genes but not in animals expressing either one of the two genes.18 19 Steinhelper et al20 reported that hepatic expression of atrial natriuretic peptide in transgenic mice caused hypotension. To analyze the function of tissue kallikrein and the regulation of kallikrein gene expression, we have developed transgenic mouse lines expressing the HTK gene under the control of several promoters.21 22 We have shown that transgenic mice overexpressing HTK are chronically hypotensive, and we have provided a new animal model for investigating the role of the tissue kallikrein-kinin system in blood pressure regulation.
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Structure of the HTK Transgene |
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TopAbstractIntroductionStructure of the HTK...Expression of the HTK...Expression of the HTK...Tissue-Specific Expression of...Chronic Hypotension in...Somatic Gene Delivery of...Conclusions References |
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Fig 1
shows HTK transgene constructs that have been
used to generate transgenic animal models. All constructs contain the
promoterless HTK gene (5.6 kb), including the entire coding sequence
and 300 bp of the 3'-flanking sequence. The filled and open blocks
represent the five exons and four introns, respectively. The
three constructs that were produced are (1) the HTK gene under the
control of an MRE promoter of the mouse
metallothionein gene (MRE-pHK; the shaded arrow
represents the MRE promoter),21 (2) the HTK gene
under the control of the mouse albumin promoter and enhancer
(ALB-pHK), and (3) the HTK gene under the control of 801 bp of its own
promoter (pHK).22
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Expression of the HTK Transgene Under the Control of Metallothionein Promoter |
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TopAbstractIntroductionStructure of the HTK...Expression of the HTK...Expression of the HTK...Tissue-Specific Expression of...Chronic Hypotension in...Somatic Gene Delivery of...Conclusions References |
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We have established two transgenic mouse lines that overexpress the HTK gene under the control of the metallothionein gene MRE promoter (MRE-pHK).21 Expression of HTK in these transgenic mice was identified by a specific radioimmunoassay and an ELISA for HTK. Immunoreactive human kallikrein reached high levels in the serum and urine (Table
). It was also detected in the liver,
pancreas, salivary gland, kidney, and spleen of these animals but not
in the control littermates. HTK levels in the serum of these transgenic
mice were 10- to 40-fold higher than those in normal human serum. The
expression of HTK transcript in the transgenic mice was further
confirmed by Northern blot and RT-PCR–Southern blot analyses.
Transgenic mice overexpressing HTK showed a sustained reduction in
blood pressure. Both lines of transgenic mice (line 483 and line 519)
had significantly lower blood pressures (86.4±5.1 mm Hg
[mean±SEM], n=8, P<.05, and 78±4.7
mm Hg, n=8,
P<.01, respectively) than control mice (100.9±1.9 mm Hg,
n=8). Administration of aprotinin, a tissue kallikrein
inhibitor, or Hoe 140, a bradykinin receptor blocker,
restored blood pressure to normal levels in the transgenic mice without
affecting control littermates. These studies suggest that a continuous
supply of HTK can have a prolonged effect on blood pressure reduction.
These results are consistent with previous clinical studies
showing that repeated oral administration of porcine pancreatic
kallikrein in hypertensive patients reduces blood
pressure.23 24 25 However, when enzyme
therapy is terminated,
blood pressure of these kallikrein-treated patients quickly
reverses to the hypertensive state.
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Expression of the HTK Transgene Under the Control of Albumin Promoter |
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TopAbstractIntroductionStructure of the HTK...Expression of the HTK...Expression of the HTK...Tissue-Specific Expression of...Chronic Hypotension in...Somatic Gene Delivery of...Conclusions References |
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In another approach, we targeted HTK expression to the liver to create a unique animal model that maintains a high level of HTK in the circulation. Three independent transgenic mouse lines were obtained. Liver-targeted expression of HTK in these animals was accomplished by placing the HTK gene under the control of the mouse albumin promoter (ALB-pHK) (Fig 1
). Expression of human kallikrein in
these
animals was identified by an ELISA for HTK and by Northern blot
analyses and RT-PCR followed by Southern blot analysis.
The results showed that the HTK transgene directed by the
albumin promoter was expressed primarily in the liver. Also,
these animals maintained high levels of HTK in the circulation and were
chronically hypotensive. The average systolic blood pressure of
these animals was about 17 mm Hg less than that of the control
littermates. Administration of aprotinin or Hoe 140 to these transgenic
mice restored blood pressure to normal levels, but it did not affect
control littermates. Neither HTK nor its mRNA was detected in the
control mice. |
Tissue-Specific Expression of the HTK Gene Under the Control of Its Own Promoter |
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TopAbstractIntroductionStructure of the HTK...Expression of the HTK...Expression of the HTK...Tissue-Specific Expression of...Chronic Hypotension in...Somatic Gene Delivery of...Conclusions References |
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HTK gene expression is tissue specific and is regulated by hormones and trans-acting factors in a complex manner. It is virtually impossible to study its complex regulation using tissue culture systems. To analyze the mechanisms of kallikrein gene regulation and function, we developed three lines of transgenic mice that carry the HTK gene containing 801 bp of its native promoter (Fig 1
).22 The tissue distribution pattern of HTK in
these
transgenic mice is similar to that in human tissues (Table).
Likewise,
Northern blot analysis shows that the major site of HTK
synthesis is in human pancreas, with a lower expression level in the
kidney.26 Immunoreactive HTK cannot be detected in the
serum of transgenic animals harboring HTK under the control of its own
promoter. In contrast, the hypotensive transgenic mice that overexpress
HTK under the control of the MRE or albumin promoter secrete
high levels of HTK into the circulation. The transgenic mouse line
harboring the HTK gene under the control of its native promoter is
normotensive. Moreover, the results show that 801 bp in the promoter
region is sufficient to direct tissue-specific expression of the
HTK gene. |
Chronic Hypotension in Transgenic Mice Overexpressing HTK |
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TopAbstractIntroductionStructure of the HTK...Expression of the HTK...Expression of the HTK...Tissue-Specific Expression of...Chronic Hypotension in...Somatic Gene Delivery of...Conclusions References |
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Blood pressure regulation is affected by a number of factors. It is therefore important to determine whether the elevated kallikrein levels in the transgenic animals are subject to a feedback control mechanism(s) that could eventually abolish the hypotensive phenotype. The reduction of blood pressure of the transgenic mice overexpressing HTK was observed in young adult mice when compared with control littermates.21 The blood pressure of the transgenic and control mice was monitored every month for more than 21 months (Fig 2
). The blood pressure of the transgenic
mice at 21 months of age (85.4±2.2 mm Hg, n=10) was still
significantly lower than that in the control mice (98.2±2.8 mm Hg,
n=10, P<.01). The hypotensive effect in these transgenic
mice was maintained throughout the lifetime of these animals. These
results indicate that during the life span of these animals there is no
intrinsic compensatory mechanism(s) to reverse kallikrein-induced
hypotension in vivo or that the mechanism, if it exists, is too weak to
exert much of an effect. It is not known whether hypotension produced
in these transgenic animals is caused by the action of kinin in the
vasculature or its action on the kidney. Additional experiments with
kidney-targeted tissue kallikrein gene constructs may be needed to
provide a clear answer to this question.
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Somatic Gene Delivery of Rat KBP Reverses Hypotension in Transgenic Mice Expressing HTK |
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TopAbstractIntroductionStructure of the HTK...Expression of the HTK...Expression of the HTK...Tissue-Specific Expression of...Chronic Hypotension in...Somatic Gene Delivery of...Conclusions References |
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Tissue kallikrein binds to KBP (kallistatin) in vitro,12 13 16 and its activity and function may be regulated by kallistatin in vivo.27 This notion was tested by intramuscular injection of the rat KBP gene into hypotensive transgenic mice overexpressing HTK under the control of the metallothionein gene (MRE-pHK).28 Expression of the rat KBP gene was detected in the serum and skeletal muscle of the transgenic mice at 10, 20, 30, and 40 days after injection by a rat KBP-specific ELISA, Western blot analysis, and RT-PCR–Southern blot analysis. Rat KBP protein or its mRNA was not detected in the control mice receiving vector DNA. The levels of rat KBP mRNA and immunoreactive protein in these mice were detectable at 10 days after injection and increased significantly at 20 and 30 days. During this period, somatic delivery of the rat KBP gene significantly increased systolic blood pressure in these hypotensive transgenic mice to a level comparable with that of the control mice. Delivery of the rat KBP gene or the vector DNA alone had no significant effect on the blood pressure of normotensive control mice. Since the difference between the transgenic mice and their control littermates is overproduction of HTK in the former, these results indicate that the reversal of hypotension by KBP in the transgenic mice is mediated by interaction of expressed KBP and HTK. Furthermore, purified HTK and rat KBP forms a covalently linked complex in vitro.28 Therefore, the in vivo studies using transgenic mice overexpressing foreign kallikrein suggest that the increase in blood pressure by somatic gene delivery of rat KBP may be mediated by inhibition of activity of tissue kallikrein.
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Conclusions |
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TopAbstractIntroductionStructure of the HTK...Expression of the HTK...Expression of the HTK...Tissue-Specific Expression of...Chronic Hypotension in...Somatic Gene Delivery of...Conclusions References |
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Transgenic technology has been instrumental in providing new insight into the mechanisms of development, pattern formation, regulation, and the physiological functions of genes.29 Transgenic mice created by germ line transfer of exogenous genes offer valuable animal models for studying cardiovascular and hypertensive diseases. Hypertension is a multigenetic and multifactorial disease. There is a large body of clinical studies suggesting a correlation between reduced renal kallikrein levels and essential hypertension. However, molecular evidence establishing a direct link between a kallikrein gene and alteration of blood pressure is lacking because of the difficulty in maintaining high levels of kinin or kallikrein in experimental animals. The finding that the transgenic mice overexpressing HTK transgenes (MRE-pHK or ALB-pHK) are chronically hypotensive supports the notion that the tissue kallikrein-kinin system contributes to the maintenance of normal blood pressure. These transgenic mice are useful animal models for analyzing the regulation and function of tissue kallikrein in vivo.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by National Institutes of Health grants HL-29397 and HL-44083.
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References |
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TopAbstractIntroductionStructure of the HTK...Expression of the HTK...Expression of the HTK...Tissue-Specific Expression of...Chronic Hypotension in...Somatic Gene Delivery of...Conclusions References |
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