Antitumor effect of vitamin B6 and its mechanisms.

Epidemiological studies have reported an inverse association between vitamin B(6) intake and colon cancer risk. Our recent study has been conducted to examine the effect of dietary vitamin B(6) on colon tumorigenesis in mice. Mice were fed diets containing 1, 7, 14 or 36 mg/kg pyridoxine for 22 weeks, and given a weekly injection of azoxymethane (AOM) for the initial 10 weeks. Compared with the 1 mg/kg pyridoxine diet, 7, 14 and 35 mg/kg pyridoxine diets significantly suppressed the incidence and number of colon tumors, colon cell proliferation and expressions of c-myc and c-fos proteins. Supplemental vitamin B(6) lowered the levels of colonic 8-hydroxyguanosine (8-OHdG), 4-hydroxy-2-nonenal (4-HNE, oxidative stress markers) and inducible nitric oxide (NO) synthase protein. In an ex vivo serum-free matrix culture model using rat aortic ring, supplemental pyridoxine and pyridoxal 5'-phosphate (PLP) had antiangiogenic effect. The results suggest that dietary vitamin B(6) suppresses colon tumorigenesis by reducing cell proliferation, oxidative stress, NO production and angiogenesis.

Expression of Na+/Ca(2+) exchanger (NCX1) gene in the developmental mouse embryo and adult mouse brain.

The Na(+)/Ca(2+) exchanger gene, NCX1, is widely expressed in many tissues, encoding several isoforms through alternative RNA splicing. NCX1 deficient mice are known to be lethal at embryonic day 9-10 (E9-10). However, its expression pattern during embryogenesis is largely unknown. Therefore, to identify and compare the localization and alternatively spliced isoforms of NCX1 mRNA expressed in the developmental stages, we analyzed the mouse embryo. Northern blot analysis demonstrated that NCX1 mRNA was expressed from the earliest stage examined, E7. In situ hybridization analysis revealed that NCX1 mRNA was expressed in the heart alone until E10.5. However, at E14.5 and 16.5, NCX1 mRNA was expressed not only in the heart, but also in neuronal cells. In addition, the expression of NCX1 mRNA in the adult brain was most abundant in the hippocampus. Using reverse transcription-polymerase chain reaction (RT-PCR), we also identified the alternatively spliced isoforms expressed during each developmental stage. The restricted expression of the NCX1 gene suggested that NCX1 may play an important role in the developing mouse embryo.

Human Ca2+/calmodulin-dependent phosphodiesterase PDE1A: novel splice variants, their specific expression, genomic organization, and chromosomal localization.

We report here the identification of novel human PDE1A splice variants, their tissue distribution patterns, genomic structure, and chromosomal localization of the gene. We identified one N-terminus (N3) and one C-terminus (C3) by cDNA library screening and dbEST database search. These N- and C-termini, including the reported N-termini (N1 and N2) and C-termini (C1 and C2), combined to generate nine different PDE1A cDNAs. N1 and N2 are similar to the 5' ends of the bovine PDE1A proteins of 61 kDa and 59 kDa, respectively, and C1 and C2 are the 3' ends of the reported human PDE1A variants. The results of PCR and Southern blot analysis show that nine PDE1A splice variants exhibit distinctive tissue distribution patterns by the difference of the N-terminus. PDE1As with N2 were widely expressed in various tissues, mainly in the kidney, liver, and pancreas. On the other hand, PDE1As with N1 and N3 were particularly expressed at a high level in the brain and testis, respectively. These findings suggest that the distinct expression patterns among PDE1A variants depend on the several promoters situated upstream of exons encoding 5' ends of the variants. The PDE1A gene spans over 120 kb of genomic DNA, and consists of at least 17 exons and 16 introns. The PDE1A gene was located on human chromosome 2q32 by fluorescent in situ hybridization analysis.

Targeted disruption of Na+/Ca2+ exchanger gene leads to cardiomyocyte apoptosis and defects in heartbeat.

Ca(2+), which enters cardiac myocytes through voltage-dependent Ca(2+) channels during excitation, is extruded from myocytes primarily by the Na(+)/Ca(2+) exchanger (NCX1) during relaxation. The increase in intracellular Ca(2+) concentration in myocytes by digitalis treatment and after ischemia/reperfusion is also thought to result from the reverse mode of the Na(+)/Ca(2+) exchange mechanism. However, the precise roles of the NCX1 are still unclear because of the lack of its specific inhibitors. We generated Ncx1-deficient mice by gene targeting to determine the in vivo function of the exchanger. Homozygous Ncx1-deficient mice died between embryonic days 9 and 10. Their hearts did not beat, and cardiac myocytes showed apoptosis. No forward mode or reverse mode of the Na(+)/Ca(2+) exchange activity was detected in null mutant hearts. The Na(+)-dependent Ca(2+) exchange activity as well as protein content of NCX1 were decreased by approximately 50% in the heart, kidney, aorta, and smooth muscle cells of the heterozygous mice, and tension development of the aortic ring in Na(+)-free solution was markedly impaired in heterozygous mice. These findings suggest that NCX1 is required for heartbeats and survival of cardiac myocytes in embryos and plays critical roles in Na(+)-dependent Ca(2+) handling in the heart and aorta.

Isolation and characterization of Na+/Ca2+ exchanger gene and splicing isoforms in mice.

The Na+/Ca2+ exchanger gene NCX1 is ubiquitously expressed in mammalian tissues, and encodes several isoforms through alternative RNA splicing. In this report, we describe the gene structure that gives rise to the multiple isoforms, and the tissue-specific expression of these isoforms in mice. The mouse NCX1 gene contains a cluster of six exons (A, B, C, D, E, and F) which encode a variable region in the large intracellular loop of the protein, as previously reported in rabbits and humans. Using reverse transcription-polymerase chain reaction (RT-PCR), expression of the isoforms was examined in several tissues. We also identified a novel splice variant, which originate from exons A, C, D, and F. These findings provide new insights into the significance of the large repertoire of NCX1 isoforms.

Specific domain of cGMP-dependent protein kinase Ibeta but not Ialpha functions as a transcriptional activator in yeast.

Recently, cyclic GMP-dependent protein kinase (cGK) was shown to translocate to the nucleus and regulate gene transcription. To determine whether cGK I proteins function as transcriptional activators, we produced the constructs of cGK Ialpha or Ibeta fused with the DNA binding domain of the yeast transcriptional activator GAL4. Here, we demonstrate that the amino-terminal region of cGK Ibeta (amino acids 1-107) exhibits transcriptional activation in yeast. However, full-length cGK Ialpha and Ibeta and the amino-terminal region of cGK Ialpha had no transcriptional activation function. Amino acid replacement in the leucine zipper motif of the amino-terminal region of cGK Ibeta substantially reduced transcriptional activation. These results suggest that the Ibeta-specific region in cGK I proteins may interact with other proteins by way of the leucine zipper motif and has a transcriptional activation function.

Insertional mutation of the murine kisimo locus caused a defect in spermatogenesis.

Spermatogenesis is a developmental process that occurs in several phases and is regulated by a large number of gene products. An insertional transgenic mouse mutant (termed kisimo mouse) has been isolated that results in abnormal germ-cell development, showing abnormal elongated spermatids in the lumina of seminiferous tubules. We cloned the disrupted locus of kisimo and identified a novel testis-specific gene, THEG, which is specifically expressed in spermatids and was disrupted in the transgenic mouse. The yeast two-hybrid screening method revealed that THEG protein strongly interacts with chaperonin containing t-complex polypeptide-1epsilon, suggesting that THEG protein functions as a regulatory factor in protein assembly. Our findings indicate that the kisimo locus is essential for the maintenance of spermiogenesis and that a gene expression disorder may be involved in male infertility.

Binding and phosphorylation of a novel male germ cell-specific cGMP-dependent protein kinase-anchoring protein by cGMP-dependent protein kinase Ialpha.

cGMP-dependent protein kinase (cGK) is a major cellular receptor of cGMP and plays important roles in cGMP-dependent signal transduction pathways. To isolate the components of the cGMP/cGK signaling pathway such as substrates and regulatory proteins of cGK, we employed the yeast two-hybrid system using cGK-Ialpha as a bait and isolated a novel male germ cell-specific 42-kDa protein, GKAP42 (42-kDa cGMP-dependent protein kinase anchoring protein). Although the N-terminal region (amino acids 1-66) of cGK-Ialpha is sufficient for the association with GKAP42, GKAP42 could not interact with cGK-Ibeta, cGK-II, or cAMP-dependent protein kinase. GKAP42 mRNA is specifically expressed in testis, where it is restricted to the spermatocytes and early round spermatids. Endogenous cGK-I is co-immunoprecipitated with anti-GKAP42 antibody from mouse testis tissue, suggesting that cGK-I physiologically interacts with GKAP42. Immunocytochemical observations revealed that GKAP42 is localized to the Golgi complex and that cGK-Ialpha is co-localized to the Golgi complex when coexpressed with GKAP42. Although both cGK-Ialpha and -Ibeta, but not cAMP-dependent protein kinase, phosphorylated GKAP42 in vitro, GKAP42 was a good substrate only for cGK-Ialpha in intact cells, suggesting that the association with kinase protein is required for the phosphorylation in vivo. Finally, we demonstrated that the kinase-deficient mutant of cGK-Ialpha stably associates with GKAP42 and that binding of cGMP to cGK-Ialpha facilitates their release from GKAP42. These findings suggest that GKAP42 functions as an anchoring protein for cGK-Ialpha and that cGK-Ialpha may participate in germ cell development through phosphorylation of Golgi-associated proteins such as GKAP42.

Identification of a conserved residue responsible for the autoinhibition of cGMP-dependent protein kinase Ialpha and beta.

We isolated a constitutively active form of cGMP-dependent protein kinase Ialpha (cGK Ialpha) by PCR-driven random mutagenesis. The replacement of Ile-63 by Thr in the autoinhibitory domain results in the enhancement of autophosphorylation and the basal kinase activity in the absence of cGMP. The hydrophobicity at position 63 is essential for the inactive state of cGK Ialpha, and Ile-78 of cGK Ibeta is also required for the autoinhibitory property. Furthermore, cGK Ialpha (Ile-63-Thr) is constitutively active in vivo. These findings suggest that a conserved residue in the autoinhibitory domain was involved in the autoinhibition of both cGK Is.

Alteration of cGMP metabolism during chondrogenic differentiation of chondroprogenitor-like EC cells, ATDC5.

Guanosine 3',5'-cyclic monophosphate (cGMP) has been recently reported to be involved in bone formation. ATDC5 cells were used to investigate cGMP metabolism during chondrogenic differentiation. Natriuretic peptide receptor (NPR)-A and NPR-B coupled with guanylate cyclase (GC) mediate biological functions of NPs, whereas NPR-C uncoupled with GC is thought to be the clearance receptor for NPs. The amounts of NPR-A, NPR-B, and CNP transcripts were increased but the amount of NPR-C transcripts was decreased in association with the chondrogenic differentiation of ATDC5 cells. CNP, a specific ligand for NPR-B lets ATDC5 cells accumulate great amounts of cGMP, revealing NPR-B as a dominant biological receptor through differentiation. cGMP hydrolytic activities of PDE1 and PDE5 existed in ATDC5 cells, and the activity of PDE1, which is stimulated by Ca(2+) and calmodulin (CaM) was major of them. Total cGMP hydrolytic activities as well as the amounts of PDE1 and PDE5 transcripts were enhanced during chondrogenic differentiation. Therefore, cGMP production and hydrolysis, cGMP metabolism was considered to be activated in association with chondrogenic differentiation of ATDC5 cells. These observations may lead to a better understanding of cGMP in the chondrocytes where bone formation occurs.

A novel interaction of cGMP-dependent protein kinase I with troponin T.

cGMP-dependent protein kinase (cGK) is a major intracellular receptor of cGMP and is implicated in several signal transduction pathways. To identify proteins that participate in the cGMP/cGK signaling pathway, we employed the yeast two-hybrid system with cGK Ialpha as bait. cDNAs encoding slow skeletal troponin T (skTnT) were isolated from both mouse embryo and human skeletal muscle cDNA libraries. The skTnT protein interacted with cGK Ibeta but not with cGK II nor cAMP-dependent protein kinase. The yeast two-hybrid and in vitro binding assays revealed that the N-terminal region of cGK Ialpha, containing the leucine zipper motif, is sufficient for the association with skTnT. In vivo analysis, mutations in cGK Ialpha, which disrupted the leucine zipper motif, were shown to completely abolish the binding to skTnT. Furthermore, cGK I also interacted with cardiac TnT (cTnT) but not with cardiac troponin I (cTnI). Together with the observations that cTnI is a good substrate for cGK I and is effectively phosphorylated in the presence of cTnT in vitro, these findings suggest that TnT functions as an anchoring protein for cGK I and that cGK I may participate in the regulation of muscle contraction through phosphorylation of TnI.

Genomic origin and transcriptional regulation of two variants of cGMP-binding cGMP-specific phosphodiesterases.

We have reported alternative splice variants of cGMP-binding cGMP-specific phosphodiesterases (PDE5A), i.e. rat PDE5A2, human PDE5A1, canine PDE5A1 and PDE5A2, which possess distinct N-terminal sequences. In this study, the DNA sequences corresponding to the unique N-terminal portions of PDE5A1 and PDE5A2 were shown to be tandemly located upstream of exons encoding the common region of PDE5A in both human and rat PDE5A genes. The presence of human PDE5A2 and rat PDE5A1 transcripts in lung was confirmed by reverse transcriptase-PCR. These results indicated that two variant forms of PDE5A exist in humans, canines and rats. We examined the tissue distribution of the two variants of human PDE5A in adult and fetal humans. The patterns of expression of the two alternatively spliced transcripts of human PDE5A in human tissues differed. Many putative regulatory elements including cAMP response elements were observed in the 5'-untranslated region and intron of the PDE5A gene. The levels of the PDE5A transcripts, especially the PDE5A2 transcripts, were increased by a cAMP analogue in cultured rat vascular smooth muscle cells, indicating that the PDE5A2 is an inducible variant of PDE5A in rats.

1,25-Dihydroxyvitamin D3 upregulates natriuretic peptide receptor-C expression in mouse osteoblasts.

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3], a key regulator of mineral metabolism, regulates expression of several genes related to bone formation. The present study examined the 1,25(OH)2D3-mediated regulation of natriuretic peptide receptor-C (NPR-C) expression in osteoblasts. 1,25(OH)2D3 treatment significantly increased NPR-C-dependent atrial natriuretic peptide-binding activity and synthesis of the NPR-C protein in mouse osteoblastic cells in a cell-specific manner. Western blot analysis also demonstrated that 1, 25(OH)2D3 upregulated expression of NPR-C protein in slow kinetics. Next, Northern blot analysis revealed a significant increase in the steady-state NPR-C mRNA level by 1,25(OH)2D3. Sequence analysis of the 9 kb of the 5'-flanking region of the mouse NPR-C gene revealed an absence of consensus vitamin D-response elements, and promoter analysis using osteoblastic cells stably transfected with mouse NPR-C promoter-reporter constructs showed a slight increase of promoter activity with 1,25(OH)2D3 treatment. In addition, a nuclear run-on assay exhibited that the transcriptional rate of the NPR-C gene was unchanged by 1,25(OH)2D3, whereas that of the osteopontin gene was increased. Evaluation of NPR-C mRNA half-life demonstrated that 1,25(OH)2D3 significantly increased the NPR-C mRNA stability in osteoblastic cells. 1,25(OH)2D3 attenuated intracellular cGMP production in osteoblastic cells stimulated by C-type natriuretic peptide (CNP) without a significant change of the natriuretic peptide receptor-B mRNA level, suggesting enhancement of the clearance of exogenously added CNP via NPR-C. Furthermore, NPR-C and osteopontin mRNAs in mouse calvariae were significantly increased by administration of 1,25(OH)2D3, and immunohistological analysis demonstrated that NPR-C is actually and strongly expressed in mouse periosteal fibroblasts. These findings suggest that 1,25(OH)2D3 can play a critical role for determination of the natriuretic peptide availability in bones by regulation of NPR-C expression through stabilizing its mRNA.

Novel alternative splice variants of cGMP-binding cGMP-specific phosphodiesterase.

After our recent findings that the amino-terminal portion of rat cGMP-binding, cGMP-specific phosphodiesterase (cGB-PDE) differs from those of bovine and human cGB-PDEs, we found two forms of canine cGB-PDE cDNAs (CFPDE5A1 and CFPDE5A2) in canine lung. Each contained a distinct amino-terminal sequence, CFPDE5A1, possessing an amino-terminal portion with sequence similar to those of bovine and human, and CFPDE5A2, having one similar to that of rat. Other portions coding for the cGMP binding domains and the catalytic domain were conserved. Both CFPDE5A1 and CFPDE5A2 transcripts were detected in the cerebellum, hippocampus, retina, lung, heart, spleen, and thoracic artery. CFPDE5A1 transcripts were particularly abundant in the pylorus, whereas CFPDE5A2 transcripts were quite low in this tissue. CFPDE5A1 and CFPDE5A2 expressed in COS-7 cells had cGMP Km values of 2.68 and 1.97 microM, respectively, and both were inhibited by a low concentration of a cGB-PDE inhibitor, Zaprinast. Both CFPDE5A1 and CFPDE5A2 bound cGMP to their allosteric cGMP binding domains, and this cGMP binding was stimulated by 3-isobutyl-1-methylxanthine. Thus, two types of alternative splice variants of canine cGB-PDE have been identified and shown to have similar biological properties in vitro.

Expression, structure and chromosomal localization of the human cGMP-binding cGMP-specific phosphodiesterase PDE5A gene.

cGMP-binding, cGMP-specific phosphodiesterase which is encoded by the PDE5A gene plays important roles in cardiovascular system, and is a significant target molecule of therapeutic agents. However, little is known about molecular characteristics of the human PDE5A gene. The 4.4-kb cDNA encoding human PDE5A was isolated from lung and placenta cDNA libraries. The deduced amino acid sequence analysis demonstrated that N-terminal amino acid sequence is dissimilar to that of rat PDE5A [Kotera, J., Yanaka, N., Fujishige, K., Imai, Y., Akatsuka, H., Ishizuka, T., Kawashima, K. & Omori, K. (1997) Eur. J. Biochem. 249, 434-442]. Human PDE5A mRNA is produced in high amounts in various tissues such as pancreas, skeletal muscle, placenta, heart, thyroid, adrenal cortex, testis, small intestine and stomach. In addition, the megakaryocyte-like cell line Dami cells and two types of human vascular smooth muscle cells also produce the mRNA. Over 100-kb chromosomal DNA corresponding to the human PDE5A gene was isolated and analyzed. The human PDE5A gene was revealed to contain 21 exons. Comparison of genomic organization with the rod photoreceptor phosphodiesterase beta-subunit gene (PDE6B), which is another kind of cGMP-specific phosphodiesterase, has shown that the PDE5A and PDE6B genes are very similar in their relative exon intron organization. In particular, the evolutionary relatedness of these genes was suggested in the catalytic domain. Furthermore, chromosomal location of the PDE5A gene was defined as being chromosome 4q26 by fluorescent in situ hybridization analysis.

Localization of clearance receptor in rat lung and trachea: association with chondrogenic differentiation.

The lung is rich in atrial natriuretic peptide binding sites, and the majority of them are considered to be the natriuretic peptide clearance receptor (NPR-C). In this study, localization of NPR-C in the rat lung and trachea was investigated by immunohistochemical analysis with the specific antibody. Positive staining was observed in the epithelial cell layers of the trachea and bronchiole and the myocardium surrounding the pulmonary vein. Moreover, expression of NPR-C was seen in mesenchymal cells; it was especially strong in cells in the perichondrium and decreased in chondrocytes in the cartilage. Because mesenchymal cells in the perichondrium differentiate to chondrocytes, NPR-C expression is suggested to be associated with chondrogenic differentiation. The chondrogenic cell line ATDC5 was used to study NPR-C expression during chondrogenic differentiation in vitro. The undifferentiated ATDC5 cells expressed NPR-C at a much higher level than the differentiated ATDC5 cells, in accordance with the observation of the immunohistochemical analysis in the cartilage. These findings suggest that NPR-C expression is differentially regulated in chondrocytes and that the natriuretic peptides may play a role in regulating chondrocyte development in the lung.

TMC-49A, a novel transcriptional up-regulator of low density lipoprotein receptor, produced by Streptomyces sp. AS1345.

Microbial metabolites were screened for a transcriptional up-regulator of low density lipoprotein (LDL) receptor by a reporter assay. TMC-49A was discovered as an up-regulator obtained from the fermentation broth of Streptomyces sp. AS1345. The structure of TMC-49A was elucidated to be butyl N-phenethylcarbamate by spectroscopic analyses. This compound enhanced the synthesis of LDL receptor in human hepatoma HepG2 cells as assessed by a receptor binding assay. Taxonomy of the producing strain is also described.

Isolation and characterization of the 5'-flanking regulatory region of the human natriuretic peptide receptor C gene.

Phenotypic modulation of vascular smooth muscle cells (SMCs) plays a central role in the pathogenesis of atherosclerosis. Natriuretic peptide receptor-C (NPR-C) is highly expressed in vascular SMCs in the experimental arteriosclerotic neointimal area as well as in cultured SMCs, suggesting that increased expression of the NPR-C gene is related to the phenotypic alteration of vascular SMCs. To elucidate the molecular mechanisms and to identify the essential DNA sequences in NPR-C gene expression, a genomic clone containing over 8 kilobases of the 5'-flanking region of the human NPR-C gene has been isolated. Sequence analysis revealed that a number of putative regulatory elements including unusual tandem repeated AP-2-like sequences were observed in the 5'-flanking region. Primer extension and ribonuclease protection analyses revealed that transcription of the human NPR-C gene starts from two major regions. Promoter analysis using deletion constructs in human cells, highly producing NPR-C transcripts, showing that the region (from - 33 to + 13 relative to the transcription start point) had a potential promoter activity suggested that the region from -33 to + 13, containing a pyrimidine-rich stretch composed of four CTTTTT-repeated sequences, is sufficient for the proximal promoter activity. Moreover, three distinct DNA sequences surrounding the transcription start site (P1, from -60 to -33; P2, from + 14 to +40; P3, from +41 to +66) were revealed to be functional as a cis-acting positive enhancer, and a nuclear protein(s) from the human cells was demonstrated to specifically bind to the sequences, respectively. However, promoter analysis has shown that the P2 and P3 sequences could not activate the human NPR-C promoter in a synergistic manner. On the basis of deoxyribonuclease I footprinting analysis showing that a DNA element from +48 to +60 within the P3 sequence is preferentially protected, the P3 sequence appears to contain a potential regulatory element involved in NPR-C gene expression. The present study demonstrated the structure of the 5'-regulatory region of the human NPR-C gene and multiple cis-acting positive sequences closely located around the transcription start points with an important role in regulation of human NPR-C gene expression.

Expression of rat cGMP-binding cGMP-specific phosphodiesterase mRNA in Purkinje cell layers during postnatal neuronal development.

The cDNA encoding rat cGMP-binding, cGMP-specific phosphodiesterase (cGB-PDE) was isolated from a rat lung cDNA library. Although the deduced amino acid sequence showed 93.4% similarity with that of bovine cGB-PDE, the N-terminal portion of rat cGB-PDE was extremely different from that of bovine. Northern blot analysis indicated that cGB-PDE transcripts in rats were expressed not only in aorta and lung, but also in several other tissues including cerebellum. In situ hybridization analysis demonstrated that cerebellar expression of cGB-PDE was confined to Purkinje cell layers in adult rats. To clarify the role of cGB-PDE in the cerebellum, we investigated expression of cGB-PDE mRNA in rats of various ages. cGB-PDE mRNA was not observed in the cerebellum of newborn rats, but levels of a cGB-PDE mRNA were markedly increased between 4 days and 28 days of age and reached a maximum in eight-week-old rats. In this study, we suggest that cGB-PDE plays important roles not only in regulating the relaxation of vascular vessels, but also in establishing neuronal networks in the cerebellum at an early postnatal stage. In addition the NO/cGMP/cGB-PDE pathway appears to be essential for the induction of long-term depression.

Protein kinase C activation down-regulates natriuretic peptide receptor C expression via transcriptional and post-translational pathways.

Natriuretic peptide receptor C (NPR-C) mRNA expression and ANP-binding activity via NPR-C are significantly down-regulated in HeLa cells with phorbol myristate acetate (PMA) treatment. Stabilization of the NPR-C mRNA by PMA indicated that down-regulation of its mRNA was mediated through negative transcriptional regulation. Despite the significant loss of the mRNA, reduction of NPR-C-specific ANP-binding activity after PMA exposure (4 h) was accompanied by a slight decrease in total NPR-C protein (with a 5% loss) and was also produced in the presence of actinomycin D or cycloheximide. The inhibitory effect of a long PMA exposure (18 h) paralleled with a decrease in total NPR-C protein is suggested to be dependent on reduction of de novo NPR-C synthesis. PMA-induced transcriptional and post-translational down-regulation of NPR-C was effectively reversible in the presence of the protein kinase C inhibitor GF109203X. These findings demonstrate that protein kinase C activation down-regulated NPR-C expression through transcriptional and post-translational pathways and that immediate functional receptor loss was mediated via a post-translational mechanism, such as enhanced receptor internalization.