cDNA Cloning and Gene Expression of Human Type Iα cGMP-Dependent Protein Kinase
Abstract The type I cGMP-dependent protein kinase (cGK) is one of the major pathways for the cGMP cascade and has been demonstrated to inhibit platelet aggregation, relax smooth muscle cells, and control cardiocyte contractility. There are two subtypes of the type I cGK, cGKIα and cGKIβ. The former is more sensitive to cGMP than the latter. In humans, cGKIβ cDNA was isolated, but the full structure and tissue-specific gene expression of cGKIα have not been determined. The significance of cGK in human cardiovascular diseases has not been investigated at the molecular level. In the present study, we isolated the full-length human cGKIα cDNA (−36 to +2177; the translation start site: +1) encoding the 671–amino acid protein. Nucleotides +267 to +2177 of the isolated cDNA were identical to the corresponding nucleotides of human cGKIβ cDNA. Southern blot analysis suggested that human cGKIα and cGKIβ are generated by alternative splicing of a single gene assigned to chromosome 10. By Northern blot analysis, we detected abundant human cGKIα mRNA (7.0 kb) in the aorta, heart, kidneys, and adrenals. In contrast, human cGKIβ mRNA (7.0 kb) was detected abundantly only in the uterus. In cultured vascular smooth muscle cells, the type I cGK mRNA concentration was reduced to 10% of the basal level by 4×10−10 mol/L platelet-derived growth factor. Angiotensin II (10−8 mol/L), transforming growth factor-β (4×10−11 mol/L), and tumor necrosis factor-α (6×10−6 mol/L) also exhibited an inhibitory effect on type I cGK gene expression. These findings suggest a pathophysiological implication of the type I cGK in cardiovascular diseases, including hypertension and atherosclerosis.
- protein kinases
- cloning, molecular
- muscle, smooth, vascular
- platelet-derived growth factor
- angiotensin II
- growth substances
Cyclic GMP–dependent protein kinase (cGK) is a serine-threonine protein kinase selectively activated by cGMP.1 2 cGK regulates cytoplasmic Ca2+ concentration by several pathways: inhibition of inositol 1,4,5-trisphosphate production and receptor activity, activation of Ca2+-ATPase, or modulation of the activity of the Ca2+-dependent K+ channel.3 4 5 Through these mechanisms, cGK is considered to relax VSMCs, reduce cardiocyte contractility, and inhibit platelet aggregation. There are two types of vertebrate cGK: the soluble type I, a homodimer, and the membrane-bound type II, a monomer.1 2 Type I cGK has two subtypes: Iα and Iβ.1 2 The apparent binding affinity of cGKIα for cGMP (10−4 mol/L) is about one tenth that of cGKIβ (1.3×10−3 mol/L).2
The NP system consists of three endogenous ligands—atrial NP, brain NP, and C-type NP—and two types of receptors, the biologically active NP receptor, the particulate guanylate cyclase itself, and the clearance receptor.6 We and other investigators have elucidated that atrial NP and brain NP are mainly secreted from the atrium and ventricle, respectively, to act as cardiac hormones,7 8 whereas C-type NP is synthesized in and secreted from vascular endothelial cells to act as an endothelium-derived relaxing peptide.9 10 11 NPs elicit these biological actions by activating the biologically active NP receptor and elevating intracellular cGMP concentration.1 In contrast, the endothelium-derived relaxing factor, which has been identified as nitric oxide, activates the soluble guanylate cyclase.1 Thus, NP and nitric oxide share the common signaling pathway mediated by cGMP to elicit similar biological activities.
Recently, evidence has accumulated suggesting that the NP or nitric oxide–cGMP-cGK cascade is involved in the regulation of cardiac contractility, vascular tone, and cardiovascular remodeling.12 13 Several pathways of the cGMP signaling cascade have been postulated: a cGMP-gated channel, cGMP-inhibited cAMP-phosphodiesterase, cGMP-stimulated cAMP-phosphodiesterase, and cGK.1 2 Recently in mammalian hearts and VSMCs, cGK was revealed to be the major pathway for the cGMP signaling cascade.14 Thus, the derangement of the cGMP cascade can lead to a number of cardiovascular disorders, including ischemic heart diseases, hypertensive cardiovascular diseases, and atherosclerosis. However, there is no information concerning cGK expression in cardiovascular lesions.
In cows, cGKIα and Iβ cDNAs have been isolated,15 and in humans, cGKIβ cDNA has been isolated.16 However, in humans, the full structure and tissue-specific gene expression of cGKIα have not been determined. Therefore, in the present study, to elucidate the pathophysiological significance of the cGMP cascade in cardiovascular disorders, we isolated the full-length human cGKIα cDNA, determined the chromosomal assignment of the human type I cGK gene, analyzed the gene expression in human tissues, and examined the regulation of the gene expression using cultured VSMCs.
Preparation of RNA and DNA
Total RNA was extracted from human tissues and cultured VSMCs by the guanidinium thiocyanate/cesium chloride method.17 Human tissues were obtained at operation or autopsy. Informed consent was obtained from each patient or family, and the study was approved by the ethics committee on human research of Kyoto University (No. 61-98). Poly(A)+ RNA was extracted from total RNA by oligotex-dT30 Super (Japan Roche). Human genomic DNA was isolated from human white blood cells by the standard method.17
Isolation of Human cGKIα cDNA
Using a DNA synthesizer (model 381A, Applied Biosystems Inc), we synthesized primers P3 through P7 based on the bovine cGKIα cDNA sequence15 as shown in Fig 1A⇓. Nucleotide sequences of these primers are as follows (built-in restriction endonuclease sites are underlined): P3 (sense), 5′-GAG- GTCGACAAGCGGCTGTCAGAGAAG-3′; P4 (antisense), 5′-TTGGTCGACTCTCTGTCGATCACAAGGCA-3′; P5 (sense), 5′-GAGGAATTCGCTATCCTTTACAACTGT-3′; P6 (antisense), 5′-TCTGAATTCCCACAAAAAGTCCATGTTTT-3′; and P7 (antisense), 5′-TGTGAGCTCAGCAATAAGTCCTAATGC-3′. By oligo(dT)-primed reverse transcription and PCR17 with these primers, cDNA fragments B (nucleotides +87 to +936; the translation start site is designated as nucleotide +1), C (+526 to +1539), and D (+526 to +2177) were amplified from the human aortic total RNA (Fig 1A⇓). PCR was carried out under standard conditions (95°C for 0.5 minute, 60°C for 0.5 minute, 72°C for 2 minutes, 35 cycles) with Taq DNA polymerase (Perkin-Elmer Cetus).
To obtain the 5′-terminal region of human cGKIα cDNA, we performed inverse PCR.18 We synthesized antisense primer IP1 (5′-CGATACTAGTCGAGGGCACTGGGAGCACC-3′, +163 to +135) based on the obtained human cGKIα cDNA sequence. The double-stranded cDNA was generated from the human aortic RNA by reverse transcription with primer P4 (Fig 1A⇑) and the Gubler and Hoffman method,17 circularized by intramolecular ligation, and subjected to PCR with primers IP1 and P5 (Fig 1A⇑). Based on the newly obtained sequence, we synthesized primers P1 (sense, 5′-TCAGTCGACAAATGAGCGAGCTAGAGG-3′, −11 to +16) and P2 (antisense, 5′-CTTGTCGACTTGGTGAACTTCCGGAATGC-3′, +266 to +238), and with these primers, we amplified fragment A (−2 to +257, Fig 1A⇑).
PCR products were subcloned into pBluescript vector (Stratagene) and sequenced by the dideoxy chain-termination method.17 To exclude the possibility of misincorporation by Taq DNA polymerase, we sequenced three to seven clones for every fragment.
Southern Blot Analysis
DNA (10 μg) from the human and hamster genomes and human-rodent somatic cell hybrids (Coriell Institute) was digested by EcoRI, Pst I, or HindIII restriction endonucleases and subjected to Southern blot analysis.17 Fragment A of the isolated human cGKIα cDNA (Fig 1A⇑) and the PCR-amplified human cGKIβ cDNA fragment (+1 to +311) specific for cGKIβ16 were labeled with [α-32P]dCTP by the random priming method17 and used as probes for the chromosomal assignment of the gene for cGKIα and Iβ, respectively. The Pst I–HindIII fragment (+925 to +1317) of the isolated human cGKIα cDNA, identical to the corresponding fragment of human cGKIβ cDNA, was used for determination of the copy number of the type I cGK gene in the human genome.
Northern Blot Analysis
Northern blot analysis was performed following the standard method.17 For detection of cGKIα and Iβ mRNAs in human tissues and VSMCs, fragment A (Fig 1A⇑) and the human cGKIβ cDNA fragment (+1 to +311)16 were used as probes, respectively. For detection of the type I cGK mRNA in rat cultured VSMCs, the Pst I–HindIII fragment (+925 to +1317) of the human cGKIα cDNA was used as a probe. For detection of β-actin mRNA, as an internal control, a human β-actin genomic probe (Wako Pure Chemical Industries) was used.
Human aortic VSMCs were purchased from Kurabo and were grown in RPMI-1640 medium (Nissui Pharmaceutical) supplemented with 10% fetal calf serum (Hazleton Biologics) at 37°C in a humidified atmosphere containing 5% CO2. Cells at the sixth passage grown to confluence in 100-mm culture dishes were used for the experiment. The medium was changed to RPMI-1640 with or without 10−3 mol/L 8-bromo-cGMP (Sigma Chemical Co) in the presence of 10% fetal calf serum. After a 12-hour incubation, cells were harvested for RNA preparation. The experiment was performed in triplicate.
Rat VSMCs were isolated from thoracic aortas of male Wistar rats as previously described9 and grown in Dulbecco’s modified Eagle’s medium (GIBCO/BRL) supplemented with 10% fetal calf serum. Cells at the seventh passage grown to subconfluence in 100-mm culture dishes were used for the experiment. The medium was changed to Dulbecco’s modified Eagle’s medium/Ham’s F-12 (GIBCO/BRL) without fetal calf serum containing 8×10−7 mol/L bovine insulin, 6×10−11 mol/L transferrin, and 3×10−8 mol/L sodium selenite. After a 48-hour incubation, 4×10−10 mol/L PDGF-BB (BACHEM Feinchemikalien AG), 10−8 mol/L Ang II (Peptide Institute, Inc), 4×10−11 mol/L transforming growth factor-β (R&D Systems, Inc), 6×10−6 mol/L tumor necrosis factor-α (Dainihon Pharmaceutical Co), or vehicle was added. Before (0 hour) and 6, 24, and 48 hours after the addition of these reagents, cells were harvested for RNA preparation.
Cloning of Human cGKIα cDNA
Using the PCR technique, we obtained cDNA fragments of cGKIα from human aortic total RNA (Fig 1⇑). Fig 1B⇑ shows the nucleotide and deduced amino acid sequences of human cGKIα cDNA. The cDNA obtained in the present study consisted of 2213 bp (−36 to +2177) and contained an open reading frame of 2013 bp. In the coding region, the homology between bovine15 and human cGKIα cDNAs was 94%. Human cGKIα consisted of 671 amino acids with a molecular mass of 76 418 D. There were two amino acid substitutions between bovine15 and human cGKIα: Lys265 to Thr265 and Asn275 to Ser275 (bovine to human sequences). Nucleotides +267 to +2177 of human cGKIα cDNA were identical to nucleotides +312 to +2222 of human cGKIβ cDNA16 (Fig 1B⇑). There was 56% homology between nucleotides −36 to +266 of human cGKIα cDNA and nucleotides −58 to +311 of human cGKIβ cDNA.16 Amino acids 90 to 671 of human cGKIα were identical to amino acids 105 to 685 of human cGKIβ.16 The homology between amino acid sequences of specific regions of human cGKIα (amino acids 1 to 89) and cGKIβ (amino acids 1 to 104)17 was 36%. In the Iα-specific region, the “leucine-isoleucine zipper” and autophosphorylation sites1 2 were conserved between bovine and human cGKIα (Fig 1A⇑).
Chromosomal Assignment and Copy Number of Human Type I cGK Gene
By Southern blot analysis using EcoRI-digested DNAs from the human-rodent somatic cell hybrids with the cGKIα probe (fragment A) or the human cGKIβ probe, the human-specific hybridization signal (10 kb for Iα or 7 kb for Iβ) was detected only in the DNA from the hybrid containing human chromosome 10 (GM10926B, Fig 2⇓). In Southern blot analysis using as a probe the Pst I–HindIII fragment (+925 to +1317) of the isolated human cGKIα cDNA, which is identical to the corresponding fragment of human cGKIβ cDNA, the EcoRI-, Pst I–, and HindIII-digested human genomic DNAs showed 4-, 2-, and 5-kb single hybridization signals, respectively (Fig 3⇓).
Gene Expressions of cGKIα and Iβ in Human Tissues
By Northern blot analysis, cGKIα mRNA was detected as a single 7.0-kb band at high concentrations in the aorta, cardiac atrium and ventricle, kidneys, and adrenals; at moderate concentrations in the cerebellum, lungs, spleen, duodenum, colon, and placenta; and at low concentrations in the ileum and uterus (Fig 4⇓). cGKIα mRNA was not detected in the cerebrum, thymus, and liver (Fig 4⇓). By contrast, cGKIβ mRNA was detected as a single 7.0-kb band at a high concentration in the uterus and at low concentrations in the aorta, kidneys, adrenals, spleen, duodenum, ileum, and colon (Fig 4⇓). In other tissues, cGKIβ mRNA was not detected (Fig 4⇓).
cGK Gene Expression in Cultured VSMCs
In cultured human aortic VSMCs at the sixth passage, cGKIα mRNA was abundantly detected (Fig 5⇓). The cGKIα mRNA concentration in human VSMCs was almost equal to that in human aorta and was not altered by the addition of 10−3 mol/L 8-bromo-cGMP (Fig 5⇓).
In cultured rat aortic VSMCs at the seventh passage, the type I cGK mRNA was detected abundantly, as clearly shown in Fig 6⇓. There were no significant changes in type I cGK mRNA concentrations in vehicle-treated cells from 0 to 48 hours of incubation after the addition (Fig 6⇓). By the addition of 4×10−10 mol/L PDGF-BB, the type I cGK mRNA concentration was drastically decreased to about 10% of the basal level 6 hours after the addition of PDGF-BB and returned to the basal level after a 24-hour incubation (Fig 6⇓). The addition of 10−8 mol/L Ang II, 4×10−11 mol/L transforming growth factor-β, or 6×10−6 mol/L tumor necrosis factor-α also caused significant reductions in the type I cGK mRNA concentration to about 40% of the basal level 6 hours after the addition (Fig 6⇓). Twenty-four hours after the addition of these reagents, the type I cGK mRNA concentration returned to the basal level (Fig 6⇓).
The present study demonstrated that the amino acid sequence from amino acid 90 to the COOH-terminus of human cGKIα is identical to the amino acid sequence from 105 to the COOH-terminus of human cGKIβ. In the NH2-terminal specific region, which contains the dimerization domain and autophosphorylation sites, the homology between amino acid sequences of human cGKIα and Iβ is 36%. The difference in NH2-terminal regions should be responsible for the difference in affinities of cGKIα and Iβ for cGMP.2 There were only two amino acid substitutions between bovine cGKIα reported previously15 and the protein encoded in the cDNA obtained in the present study, and the substituted amino acids were not contained within consensus sequences in the catalytic domain (Fig 1B⇑). This observation suggests that the cDNA obtained in the present study should be human cGKIα cDNA. To verify that the isolated cDNA really encodes cGK, we are now carrying out an overexpression experiment in culture cells using our cDNA clone. The strong conservation in amino acid sequences of bovine and human cGKIα indicates the significance of this enzyme in the cGMP signaling cascade.
Southern blot analysis using DNAs from human-rodent somatic cell hybrids revealed that in humans, the gene for cGKIα and Iβ is assigned to chromosome 10 (Fig 2⇑). Southern blot analysis of the human genome suggested that the human type I cGK gene has only one copy (Fig 3⇑). We showed in the present study that nucleotides +267 to +2177 of human cGKIα cDNA are identical to nucleotides +312 to +2222 of human cGKIβ cDNA. The gene for human cGKIβ was partially isolated, and it has been demonstrated that the specific and identical regions of human cGKIβ are encoded in separate exons.19 These findings suggest that human cGKIα and Iβ are generated by alternative splicing of a single gene assigned to chromosome 10.
The present study revealed that in humans, cGKIα is highly expressed in the aorta, heart, kidneys, and adrenals and moderately expressed in the cerebellum and lungs (Fig 4⇑). These findings are compatible with previous reports that type I cGK activity is detected in VSMCs, somatic smooth muscle cells, cerebellar Purkinje cells, and platelets.2 The present study demonstrated that in human tissues, including cardiovascular tissues, type I cGK is expressed mainly as cGKIα (Fig 4⇑). Only in the uterus was cGKIβ mRNA detected abundantly, and its concentration was higher than the cGKIα mRNA concentration (Fig 4⇑). Further investigation on tissue specificity in the alternative splicing pattern of the type I cGK gene in humans is required.
In the present study, we observed that in cultured human VSMCs, cGKIα gene expression was not reduced by 10−3 mol/L 8-bromo-cGMP (Fig 5⇑). This indicates that in VSMCs, the downregulation of cGKIα gene expression by the elevation of the intracellular cGMP concentration does not substantially occur.
The cGMP signaling cascade inhibits VSMC growth. We and other investigators elucidated that the elevation of intracellular cGMP concentration induced by NPs and nitric oxide inhibits the mitogenesis and proliferation of VSMCs.12 13 The VSMCs transfected with the cDNA encoding the catalytic domain of bovine cGKIα, which is the constitutive active enzyme, have been reported to display morphological changes: synthetic phenotype to contractile phenotype.20 In the present study, we demonstrated that by the addition of PDGF-BB, Ang II, transforming growth factor-β, or tumor necrosis factor-α, the type I cGK gene expression in rat VSMCs was transiently decreased 6 hours after the addition of these reagents and returned to basal levels after 24 hours of incubation. Among these reagents, PDGF-BB potently decreased the type I cGK mRNA concentration to about 10% of the basal level. Therefore, the suppression of the gene expression of the type I cGK, which decelerates VSMC growth, can be one of the common features and/or prerequisites of the stimulation of VSMC growth by these factors. We are currently examining the exact changes of cGK activity along with the type I cGK mRNA concentration. Although further study is required to clarify the molecular mechanisms of the reduction of the cGK gene expression and its significance for actual VSMC growth, the present findings suggest the pathophysiological significance of cGK in vascular remodeling.
In conclusion, we cloned the full-length human cGKIα cDNA, demonstrated cGKIα gene expression in human tissues, and examined the pathophysiological and clinical implication of cGK in human cardiovascular diseases at the molecular level.
Selected Abbreviations and Acronyms
|Ang II||=||angiotensin II|
|cGK||=||cGMP-dependent protein kinase|
|PCR||=||polymerase chain reaction|
|PDGF||=||platelet-derived growth factor|
|VSMC||=||vascular smooth muscle cell|
This work was supported in part by research grants from the Japanese Ministry of Education, Science, and Culture; the Japanese Ministry of Health and Welfare; the Uehara Memorial Foundation; the Salt Science Research Foundation (92041); the Smoking Research Foundation; the Yamanouchi Foundation for Research on Metabolic Disorders; and the Japanese Society for Cardiovascular Research. We thank Dr Yoshihiro Miyamoto for his technical advice. We also thank Mihoko Shida, Hisayo Kitoh, Ayumi Takakoshi, Chizu Kawahara, and Yuko Mori for their secretarial assistance.
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