Search

We isolated PCR, RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE-PCR)-, and RT-PCR-generated clones from mouse kininogen family transcripts. DNA sequencing indicated that the clones were from two distinct genes. One set (K1) is from the previously reported mouse kininogen gene. The second set (K2) has an open reading frame, is 93% identical to K1 in the overlapping nucleotide sequence, and, unlike T-kininogens in the rat, encodes a bradykinin motif identical to K1. We discovered that K2 exists with two different 5' ends. We used RT-PCR to determine the distribution and relative abundance of K1 and K2 mRNA in mouse tissues. K2 is transcribed and K1 and K2 are generally both expressed in the same tissues; however, they differ in their regulation of the alternative splicing event that yields either low-molecular-weight kininogen (LMWK) or high-molecular-weight kininogen (HMWK). For example, in the liver K1 is expressed as both HMWK and LMWK, whereas K2 is only expressed as LMWK. Conversely, in the kidney K2 is strongly expressed as both HMWK and LMWK, whereas K1 is not expressed as HMWK and expressed only very weakly as LMWK.

Kininogens serve dual functions by forming a scaffold for the assembly of the protein complex initiating the surface-activated blood coagulation cascade and as precursors for the kinin hormones. While rats have three kininogen genes, for mice, cattle, and humans only one gene has been described. Here, we present sequence and expression data of a second mouse kininogen gene. The two genes, kininogen-I and kininogen-II, are located in close proximity on chromosome 16 in a head-to-head arrangement. In liver and kidney, both genes are expressed and for each gene three alternative splice variants are synthesized. Two of them are the expected high and low molecular weight isoforms known from all mammalian kininogens. However, for both genes also a third, hitherto unknown splice variant was detected which lacks part of the high molecular weight mRNA due to splicing from the low molecular weight donor site to alternative splice acceptor sites in exon 10. The physiological functions of the six kininogen isoforms predicted by these findings need to be determined.

Background: The metabolic state effect of liver failure on liver gene regulation was evaluated in a rat model., Methods: Following 70 or 90% hepatectomy and lipopolysaccharide or vehicle treatment at intervals up to 24 h, the liver remnants were analyzed for mRNA levels for acute-phase, liver-specific and growth-related proteins., Results: After 70% hepatectomy mRNA for alpha 1-acid glycoprotein, alpha 2-macroglobulin, thiostatin and fibrinogen, haptoglobin increased three- to sevenfold (P < 0.05), and mRNA for cyclin D and histone 3 increased seven- and 15-fold (P < 0.05), respectively. After lipopolysaccharide injection and 70% hepatectomy were done, mRNA for acute-phase proteins raised significantly (P < 0.05), more to five to 20-fold, while mRNA for growth-related proteins raised significantly (P < 0.05) less to three- to fourfold. After 90% hepatectomy, acute-phase protein mRNA increased five- to ninefold (P < 0.05) more than after 70% hepatectomy, while mRNA for histone 3 and cyclin D did not increase within 24 h, which indicates a delayed growth after 90% hepatectomy. In 90% of hepatectomized rats treated with lipopolysaccharide, acute-phase protein mRNA raised three- to sixfold (P < 0.05) less than after vehicle treatment., Conclusion: In endotoxemia from liver failure, the synthesis of acute-phase proteins is upregulated by gene regulation at the expense of that for regeneration, which may be an appropriate response for immediate survival. In severe liver failure, endotoxin may interfere with the appropriate gene regulation.

To determine the existence of the kallikrein-kinin system in the heart, we have studied in vitro and in vivo whether rat heart expresses kininogens (KGNs). The reverse transcription-polymerase chain reaction (RT-PCR) for KGN mRNAs demonstrated that the cardiac tissue of adult male rats expresses T-KGN mRNA but not high-molecular-weight (H-) KGN mRNA. An intravenous injection of lipopolysaccharide (LPS) resulted in a significant increase in T-KGN mRNA levels of rat heart within 12 h. Polyacrylamide gel electrophoresis of cDNA products generated by RT-PCR from heart mRNA using primers specific for either T- or low-molecular-weigh (L-) KGN revealed that rat heart expressed not only T-KGN gene but also L-KGN gene, and that LPS injection exclusively stimulated the expression of T-KGN but not of L-KGN gene. T-KGN mRNA was also detected in cultured myocytes derived from fetal rat heart, and the expression was markedly enhanced by an addition of LPS to cultures. These results demonstrated that rat cardiomyocytes are the source of T- and L-KGNs but not of H-KGN, and that their expression of T-KGN mRNA is stimulated by LPS, probably via LPS-receptor CD14.

The precursors of kinins, K-kininogens and T-kininogens (KG), are encoded by separate genes that display 90% nucleotide sequence homology. Despite their homology, K-KG and T-KG genes are differentially regulated. The K-KG gene is expressed constitutively and encodes high- and low-molecular-weight KG, the precursors of the vasoactive nonapeptide bradykinin. In contrast, the T-KG gene is inducible, and its protein is a potent thiol-protease inhibitor. Given their potential role in the regulation of blood pressure, renal hemodynamics, and the response to inflammation and tissue injury, K-KG and T-KG gene expression in rats subjected to chronic (1 or 5 wk) unilateral ureteral obstruction (UUO), a maneuver that suppresses renal kallikrein synthesis to 25% of controls, has been examined. Northern and slot blots of total liver and kidney RNA were probed with oligonucleotides complementary to either T-KG or K-KG mRNA under high-stringency conditions. Steady-state levels of hepatic T-KG mRNA were increased in the UUO compared with sham-operated rats--2.7-fold at 1 wk and 4.1-fold at 5 wk (P < 0.05). Western blot analysis revealed that the 68-kd T-KG protein was up-regulated 2.5- to 3-fold in the liver of UUO rats (P < 0.05). In marked contrast, the abundance of high (2.3-kb)- and low (1.6-kb)-molecular-weight splicing transcripts of hepatic pre-K-KG mRNA was not altered at either time after UUO.(ABSTRACT TRUNCATED AT 250 WORDS)

The serum concentration of rat T1 kininogen increases 20-30-fold in response to acute inflammation. This increase, induced in the liver, is regulated primarily at the transcriptional level. In contrast, synthesis of a homologous K kininogen is not induced. In this study, we further analyzed a 321-base pair interleukin (IL)-6 response element in the T1 kininogen promoter and showed that it consists of at least three functionally distinct sequences (A, B, and C boxes). All three sequences were required for full promoter activity. The B box, a strong C/EBP-binding site, was crucial for T1 kininogen's basal expression, whereas A and C boxes resembled the type II IL-6 response elements and were critical for the cytokine response. C/EBP alpha, -beta, and -delta interacted with the B box sequence; however, upon IL-6 stimulation, C/EBP delta binding activity was dramatically induced and became the predominant factor binding to this site. Consistent with these binding studies were the cotransfection experiments, revealing that C/EBP delta was the most potent transactivator under induced conditions and that its transactivation on the T1 kininogen promoter required an intact B box. These findings substantiated the importance of the B box in eliciting the full acute-phase response. A sequence comparison showed the K kininogen promoter contained identical A and B boxes but differed from the T1 kininogen promoter by two nucleotides at the C box. This divergence reduced the IL-6 response by approximately 4-fold, thus contributing to the differential inflammatory response. Our studies demonstrate that evolutionary divergence of a few nucleotides at a critical sequence in the promoter regions can profoundly alter the expression patterns of downstream genes.

Serum concentration of rat T1 kininogen increases 20- to 30-fold in response to acute inflammation, an induced hepatic synthesis regulated primarily at the transcriptional level. We have demonstrated by transient transfection analyses that rat T1 kininogen gene/chloramphenicol acetyltransferase (T1K/CAT) constructs are highly responsive to interleukin-6 and dexamethasone. In these studies we examined the regulation of a highly homologous K kininogen gene promoter and showed that it is minimally induced under identical conditions. The basal expression of the KK/CAT construct was, however, five- to sevenfold higher than that of the analogous T1K/CAT construct. Promoter-swapping experiments to examine the molecular basis of this differentially regulated basal expression showed that at least two K kininogen promoter regions are important for conferring its high basal expression: a distal 19-bp region (C box) constituted a binding site for C/EBP family proteins, and a proximal 66-bp region contained two adjacent binding sites for hepatocyte nuclear factor 3 (HNF-3). While the C box in the K kininogen promoter was able to interact with C/EBP transcription factors, the T1 kininogen promoter C box could not. In addition, HNF-3 binding sites of the K kininogen promoter demonstrated stronger affinities than those of the T1 kininogen promoter. Since C/EBP and HNF-3 are highly enriched in the liver and are known to enhance transcription of liver-specific genes, these differences in their binding activities thus accounted for the K kininogen gene's higher basal expression. Our studies demonstrated that evolutionary divergence of a few critical nucleotides may lead to subtle changes in the binding affinities of a transcription factor to its recognition site, profoundly altering expression of the downstream gene.

Pyrrolizidine monocrotaline (MCT) from plant seed produces pulmonary endothelial cell injury, pulmonary hypertension, and inflammation in rats, providing a useful animal model for studying progressive pulmonary vascular disease. Kininogen is the precursor of proinflammatory kinins and may also exert anti-inflammatory actions by inhibiting cysteine proteinases. Given the potential roles of kininogen in vascular injury and inflammation, we have investigated the regulation of kininogen gene expression in the MCT-induced pulmonary hypertensive rat model. Sprague-Dawley rats, in groups of six, were given a single subcutaneous injection of monocrotatine (60 mg/kg body wt) and sacrificed 10 and 20 days later. Northern blot hybridization using a kininogen cDNA probe showed kininogen gene expression in the liver, lung, and kidney. MCT treatment induced a time-dependent increase in kininogen mRNA levels, whereas it reduced rat alpha 1-antitrypsin and kallikrein-binding protein mRNA levels in the liver. Similarly, kininogen mRNA levels were low in the normal lung and were increased 7.5- and 13.7-fold, respectively, after MCT injection for 10 and 20 days. Immunoreactive kininogen levels in perfused liver and lung extracts of rats receiving MCT injection increased up to 20-fold, as measured by a T-kininogen radioimmunoassay. Western blot analyses showed that a 68-kilodalton immunoreactive kininogen increased in the serum and lung extracts of MCT-treated rats compared to those in the control rats. In control rats, immunostaining for kininogen in the lung was most marked in venous endothelial cells and alveolar macrophages. After MCT treatment, staining for kininogen increased dramatically throughout the lung tissues, often covering the epithelial surfaces of alveoli and bronchi. The present studies have shown that the toxin MCT altered the synthesis and distribution of pulmonary kininogen and suggest that the kininogen/kinin system may be associated with the pulmonary vascular injury, remodeling, and inflammation seen in this animal model.

Kininogens which have multifunctional domains, serve as the precursors of potent vasoactive kinin peptides and also function as cysteine proteinase inhibitors. Given its potential role in blood pressure homeostasis and inflammation, we have examined the regulation of rat kininogen gene expression by sex hormones in vivo. Our studies indicate a differential regulation of kininogen gene expression in rat liver by estrogen and progesterone. Northern and dot blot analysis using a rat low molecular weight kininogen cDNA probe show that kininogen mRNA levels in the liver of female rats are 4-fold higher than those in male rats. Ovariectomy results in a reduction of kininogen transcripts in the liver, while estradiol replacement of the ovariectomized rats increases kininogen mRNA levels. Similarly, Northern blot analysis using a kallikrein cDNA probe shows that estradiol treatment induces an increase of kallikrein gene expression in the kidney of the same animals. In contrast, progesterone treatment of the ovariectomized rats results in an increase in renal kallikrein mRNA levels while it reduces kininogen gene expression as compared to vehicle-treated ovariectomized animals. Immunoreactive kininogen levels in the serum, analyzed by a direct radioimmunoassay and Western blot, are increased by estradiol but slightly decreased by progesterone treatment. Western blot of serum proteins on a two-dimensional polyacrylamide gel reveals that in estradiol-treated ovariectomized rats, the levels of several 68,000 Da kininogens varying in charge are markedly higher than those in ovariectomized rats. The results indicate that estrogen is one of the determinants in regulating low molecular weight kininogen gene expression in vivo. The impact of estrogen-regulated kininogen expression on cardiovascular function awaits further investigation.

Interleukin-6 is a pleiotropic cytokine that has a major role in the coordination of the hepatic acute phase response. In order to more fully understand this role, we have examined the interleukin-6 induction of T kininogen, a cysteine protease inhibitor and a major acute phase reactant in the rat. Using deletional analysis and site-directed mutagenesis of T kininogen-chloramphenicol acetyltransferase fusion constructs transfected into HepG2 hepatoma cells, we have identified two similar interleukin-6 response elements within 250 base pairs of the transcription start site. These two response elements are functionally interdependent. The sequences of these two elements match the consensus sequence for the previously described Type B interleukin-6 response element. Interleukin-6 signal transduction via two Type B elements has not been observed previously in vivo. A DNA fragment encompassing these response elements forms the same protein complex with nuclear extracts from both untreated and interleukin-6-treated cells.

We have constructed a cDNA library from senescent (24-month-old) rat liver mRNA and, by differential screening, have selected clones corresponding to mRNA species with increased abundance in aging rats. Direct sequencing of the inserts indicated that most of the clones (9 of 10) contained sequences coding for T-kininogen, also called major acute-phase protein, cysteine protease inhibitor, or thiostatin. Nuclear elongation experiments showed that the increase in mRNA concentration was controlled at the transcriptional level. RNase mapping and S1 analysis indicated that the age-dependent induction operated preferentially at one of the three transcriptional start sites of the gene(s). The acute-phase reaction (inflammation) is known to also induce these genes at the level of transcription; however, two of the three start sites are induced by inflammation. Transcription from one of these sites was induced by both phenomena, aging and inflammation.

We have examined the regulation of a rat T-kininogen gene by glucocorticoid and estrogen. Expression of the endogenous gene in a rat hepatoma cell line is increased 5-fold and 2-fold in response to dexamethasone and 17 beta-estradiol-3-benzoate, respectively. Various deletion constructs of the 5' region of an isolated T-kininogen gene were fused to a chloramphenicol acetyltransferase (CAT) gene and introduced into the hepatoma cells by electroporation. Analysis of the CAT activity in cell extracts after treatment with glucocorticoid or estrogen revealed that a fragment from -167 to +52 is sufficient to confer full induction. An additional deletion in this region was unresponsive, while a larger fragment (-612 to -100) linked to a heterologous promoter did result in regulated expression. These results suggested that the sequence responsible for the hormonal response was located at -167 to -100 from the transcription start site. This 67 bp region contains a consensus for the core sequence of the glucocorticoid responsive element (GRE) and the estrogen responsive element (ERE). Interestingly these elements are located within 7 bp of each other and both sequences overlap a 16 bp palindrome that may be important in hormone receptor-DNA recognition.

The present investigation using molecular cloning and sequence analysis concerns the examination of the molecular basis for different expression patterns of two types of the rat kininogen genes. We show that the low molecular weight and high molecular weight forms of K kininogens are produced from a single gene through alternative usage of two 3'-coding regions, whereas only the low molecular weight forms of T kininogens are generated as a result of several mutational changes in the high molecular weight-specifying regions of both T-I and T-II kininogen genes. The mutational changes include a nucleotide substitution at the polyadenylation/processing signal site, nucleotide deletions resulting in the frame-shift mutation, and an insertion of the type 2 Alu-equivalent sequence. Because kininogens represent a multifunctional protein comprising the proteinase-inhibitory activity, the kinin moiety, and the clotting activity, these results present evidence indicating the molecular basis for the disappearance of a part of the gene functions. We also show that the K and T kininogen genes as well as the two T kininogen genes are extremely homologous, excluding and including the above mutational changes, respectively. These structural relationships allow us to envisage evolutionary processes for the generation of the rat kininogen gene family, particularly for the disappearance of a part of the gene functions.