(Hypertension. 1996;28:840-846.)
© 1996 American Heart Association, Inc.
Articles |
Prohormone Convertase PC5 Is a Candidate Processing Enzyme for Prorenin in the Human Adrenal Cortex
the Laboratory of Molecular Biochemistry of Hypertension (C.M., I.J., T.L.R.) and the JA DeSeve Laboratory of Biochemical Neuroendocrinology (R.D., N.G.S.), Clinical Research Institute of Montreal (Quebec, Canada).
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Abstract |
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We isolated a cDNA clone encoding the human prohormone convertase PC5 from human adrenal gland mRNA. The deduced protein sequence would encode a 915 amino acid preproPC5 that shares a very high degree of homology with previously cloned rat and mouse homologues. PC5 mRNA was detected in multiple human tissues, including the brain, adrenal and thyroid glands, heart, placenta, lung, and testes. PC5 mRNA was undetectable in the liver and was present at lower levels in skeletal muscle, kidney, pancreas, small intestine, and stomach. Cotransfection of human PC5 and human prorenin expression vectors in cultured GH4C1 cells led to secretion of active renin. The activation of human prorenin by PC5 depended on a pair of basic amino acids at positions 42 and 43 of the prorenin prosegment and occurred only in cells containing dense core secretory granules. Human PC5 was colocalized with renin by immunohistochemistry in the zona glomerulosa of the adrenal gland, suggesting that it could participate in the activation of a local renin-angiotensin system in the human adrenal cortex.
Key Words: renin-angiotensin system • renin • adrenal glands
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Introduction |
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Renin is an aspartyl protease that contributes importantly to cardiovascular physiology and pathophysiology through its key role in the synthesis of the vasoactive octapeptide angiotensin II. Although the kidney is the primary source of circulating active renin, several additional tissues, including the pituitary and adrenal glands, placenta, uterus, ovary, testes, heart, vasculature, and brain, express the renin gene (reviewed in References 1 through 4). The presence of additional components of the RAS in these tissues, including angiotensin-converting enzyme and angiotensin II receptors, has led to the proposal that certain tissues might contain a locally active tissue RAS, although the actual function of the tissue RAS is still largely a matter of conjecture. Renin is first synthesized as an enzymatically inactive precursor, prorenin, which is converted to active renin by the proteolytic removal of a 43–amino acid amino terminal prosegment. The activity of the RAS within any given tissue would therefore depend on the existence of proteolytic enzymes capable of converting prorenin to active renin and on the expression of such PPEs in the same cells that express prorenin. The identity of the enzyme or enzymes responsible for the proteolytic activating human prorenin in vivo is still uncertain. Furthermore, it is possible that multiple PPEs exist in humans, and these may differ among renin-producing tissues. Biochemical and microscopic studies of renin in the kidney suggest that candidate PPEs should be selective for cleavage of human prorenin at Lys42, Arg43 of the prosegment5 and would be active in secretory granules of the juxtaglomerular cells.6 The lysosomal enzyme cathepsin B has been colocalized with human renin/prorenin in the secretory granules of juxtaglomerular cells and human pituitary lactotrophs7 8 and has been shown to cleave human prorenin in vitro with a high affinity and selectivity for the proper cleavage site.9 The prohormone convertase PC1 has also been shown to cleave human prorenin with the correct site and organelle specificity in transfected cells10 and to colocalize with renin in the adrenal medulla and derived tumors11 but not in juxtaglomerular cells.12
In an effort to identify novel PPEs, we recently determined the distribution of processing enzymes in an established renin-expressing tissue culture cell line derived from an oncogene-induced mouse tumor (As4.1 cells13 ). We found one such enzyme, the mouse prohormone convertase PC5, to be capable of partially activating human prorenin (I.J. and T.L.R., unpublished data, 1996). In the current study, we describe the isolation and characterization of a human homologue of this enzyme, hPC5. We demonstrate that hPC5 proteolytically activates human prorenin with the expected site and organelle specificity and that it is coexpressed with prorenin in the zona glomerulosa of the adrenal cortex.
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Methods |
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cDNA Library Construction and Screening
A cDNA library derived from total human adrenal RNA was constructed by Stratagene in the phage vector Uni-Zap XR. Six hundred thousand phage plaques were screened initially with the use of radioactive probes and standard methodologies.14 The initial hybridization probe was a 320-bp DNA fragment derived from RT-PCR of human brain RNA using information derived from an unidentified human cDNA sequence tag in GenBank (accession No. M85522) with a high degree of similarity to the previously cloned mouse PC5.15 Fragment labeling was carried out with [32P]dCTP and a random primer labeling kit (Boehringer-Mannheim Canada) according to the manufacturer's instructions. One positive hybridizing phage (hPC5A) was identified. Its insert was sequenced in its entirety with the dideoxy-chain termination method and found to code for a 1150-bp cDNA with a high degree of sequence similarity to mouse PC5 (data not shown). A 1070-bp fragment (excluding the polyadenylate tail) was excised from hPC5A, labeled, and used to rescreen an additional 600 000 phages from the cDNA library. A second phage clone (hPC5B) was isolated and found to contain a 1807-bp cDNA insert overlapping hPC5A and extending toward the 5' end of the cDNA (Fig 1
).
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RT-PCR
One microgram of polyA+ RNA from total human adrenal (Clontech Laboratories) was subjected to RT-PCR with the use of a published procedure16 and the following oligonucleotides: The forward oligonucleotide was derived from a region corresponding to the signal peptide of mouse PC5.15 An artificial HindIII restriction enzyme cleavage site added to the 5' end of the amplified fragment for the purpose of cloning is underlined: 5'-CCAAGCTTGGCTGCTGTGCGTGCTGGC-3'. The reverse oligonucleotide was derived from the 5' end of the phage hPC5B. An internal BglII restriction enzyme site is underlined: 5'-CTGCCTCAGATCTGTAGTG-3'.
The entire RT-PCR reaction was repeated four times, and four independently derived clones of the amplified fragment were sequenced and the sequences compared. The sequence submitted to GenBank (accession No. U49114) represents the consensus sequence, defined as any nucleotide appearing in three of four clones.
Northern Blot Analysis
Tissue distribution of PC5 mRNA was determined by hybridization of commercially purchased nitrocellulose filters containing aliquots (2 µg) of polyadenylate RNA from various human tissues (Clontech Laboratories). The probe used was a complementary RNA derived from the full-length hPC5 cDNA. Probe labeling and hybridization were carried out as previously described.17
Expression Vector Construction
A cDNA fragment from the Kpn I site (Fig 1
) to just past the stop codon was excised from the phage hPC5B and combined with a Kpn I to HindIII (see above) fragment derived from portions of two independent RT-PCR clones (so as to eliminate errors arising from the Taq polymerase). A region corresponding to the first 12 amino acids of the signal peptide derived from mouse PC5 was attached to the 5' end by overlap-extension PCR.18 Thus, the entire cDNA, encoding amino acids 1 through 16 derived from the mouse PC5 signal peptide and the remainder from hPC5, was subcloned into the expression vector RSV globin,19 which places the cDNA under the control of the Rous sarcoma virus promoter and provides a 3' intron and polyadenylation signal from the rabbit ß-globin gene. The entire subcloned fragment was subsequently verified by DNA sequencing.
Cell Culture and Transfection
GH4C1 cells were plated in six-well culture dishes at a density of 5x105 cells per well. Twenty-four hours later, the medium was changed, and the cells were transfected by the (diethylamino)ethyl-dextran method with the use of a commercial kit (CellPhect Transfection kit, Pharmacia Biotech) according to the manufacturer's instructions. Each well received 0.18 µg of either the hPC5 expression vector or a neutral plasmid vector (pUC18) and 0.18 µg of an expression vector for human prorenin (pRHR1100) or its equivalents in which amino acids 42 or 43 of the prorenin prosegment were mutated to alanine (K/A-2 and R/A-1, respectively20 ). Supernatants were collected 30 hours after transfection and assayed for prorenin and renin content as previously described.20 To verify that conversion of the prorenin occurred in the secretory granules, we stimulated GH4C1 cells transfected with the human prorenin and hPC5 expression vectors to release secretory granules by depolarization using a previously published technique.21 Forty hours after cotransfection, the culture medium in parallel wells of transfected cells was replaced with prewarmed medium supplemented to a final concentration of 50 mmol/L with either NaCl (control) or KCl (secretagogue). The media were collected after 20 minutes and assayed for renin/prorenin. A potassium-dependent increase in the percentage of active renin contained in cell supernatants was taken as an indication of active renin release from the secretory granules of the transfected cells. Results shown in Fig 5 represent the mean of three independent transfection experiments.
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Immunolocalization of hPC5 in Human Tissues
Human tissue was obtained postmortem (kidney and adrenal gland) or postpartum (placental cotyledon), fixed in Bouin's solution, and embedded in paraffin. For immunolocalization, 5-µm sections were mounted on gelatin-coated slides, deparaffinized, and incubated with a 1:50 dilution of a polyclonal rabbit antiserum raised against a peptide corresponding to the N-terminal 16 amino acids of rat PC5 (PC5.MAP antibody) or a 1:200 dilution of a polyclonal rabbit antiserum against recombinant human prorenin. For kidney and placental specimens, immune complexes were revealed by incubation with protein A–colloidal gold (15-nm particles) synthesized from tetrachloroauric acid (BDH) according to the method of Ghitescu and Bendayan.22 Gold particles were enhanced for viewing in the light microscope by incubation with silver (IntenSE M Silver Enhancement kit, Amersham Life Science), and sections were counterstained with hematoxylin and methyl green. Immune complexes on human adrenal sections were detected with a 1:200 dilution of biotin-labeled donkey anti-rabbit IgG and a 1:300 dilution of streptavidin–horseradish peroxidase complex (Amersham Life Science) and were incubated with diaminobenzidine and hydrogen peroxide (Sigma Chemical Co) as chromogen. All positive staining patterns were subsequently verified for specificity by omission of the first antibody.
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Results |
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The primary sequence of hPC5 is shown in Fig 2
. We were unable to clone the extreme 5' end of the cDNA either by the RACE protocol16 or by using oligonucleotides based on the published sequence of mouse PC5,15 23 possibly because of a high G/C content of the cDNA in this region. However, on the basis of the published cDNA sequences for rat and mouse PC5,15 we are confident that we isolated all but the 5'-most portion of the cDNA corresponding to the first 12 amino acids of the signal peptide. By comparison with the published sequence of mouse PC5, we predicted that the cDNA isolated would code for a preproPC5 of 915 amino acids, including a signal peptide and a prosegment of 32 and 84 amino acids, respectively. The deduced sequence of hPC5 was 88% identical to the previously published mouse PC5 cDNA and 96% identical to the mouse PC5 protein.
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Northern analysis of mRNA from a variety of human tissues revealed a major band of approximately 6.6 kb and a minor band of approximately 3.8 kb (Fig 3). PC5 RNA was detected in the brain, heart, placenta, lung, thyroid gland, and testes and at lower levels in the skeletal muscle, kidney, pancreas, small intestine, and stomach. In the adrenal gland, PC5 was particularly enriched in the cortex (Fig 3).
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Because PC5 RNA appears to be expressed in a number of tissues previously reported to contain active renin, we tested the ability of hPC5 to cleave human prorenin in a cell cotransfection assay (Fig 4A). As has been previously reported,10 when cultured rat somatotrophic GH4C1 cells were cotransfected with an expression vector encoding human prorenin and a neutral plasmid vector, only unprocessed prorenin was secreted into the culture supernatant. In contrast, if the human prorenin expression vector was cotransfected with an expression vector encoding hPC5, a portion of the expressed prorenin was secreted as active renin. Coexpression of hPC5 with prorenin mutated at either of the basic residues forming the native cleavage site (lysine 42 or arginine 43) prevented activation. These results suggest that hPC5 activates human prorenin by proteolytic cleavage at the site previously reported for activation of renin in humans.5 Although hPC5 cleaved human prorenin in GH4C1 cells, there was no apparent increase in active renin secretion when cotransfections were carried out in Chinese hamster ovary (CHO) cells (Fig 4B). One obvious difference in the CHO cell line compared with GH4C1 cells is their lack of secretory granules, suggesting that either hPC5, human prorenin, or both require the secretory granule environment for this proteolytic step. This conclusion is supported by the acute increase in active renin detected in the supernatants of cotransfected GH4C1 cells treated for 20 minutes with potassium chloride (Fig 5), a depolarizing agent that causes the release of secretory granules.21
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Using a polyclonal antibody raised against a peptide derived from mouse PC5, we studied the distribution of hPC5 in several human tissues (Fig 6). To date, we have been unable to detect staining for PC5 in the human kidney, although our sections stained positively for renin. In the placental cotyledon, PC5 was located in the syncytiotrophoblast layer of the chorionic villi, and antibody against renin stained primarily the chorionic mesoderm. In the adrenal gland, the antibodies against both renin and PC5 showed a preferential staining of zona glomerulosa cells in the adrenal cortex, with very little staining of the capsule and zona fasciculata. No staining was evident with omission of the first antibody (data not shown). Thus, our immunohistochemical studies would suggest that of the three tissues studied, it is likely that prorenin and PC5 are clearly colocalized only in the zona glomerulosa of the human adrenal cortex.
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Discussion |
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In the present study, we describe the cloning and expression of the human prohormone convertase PC5 and its activity as a human PPE. Cotransfection assays in cultured cells demonstrated that hPC5 activates human prorenin with the expected site specificity and that this cleavage most likely occurs in dense core secretory granules. In addition, immunohistochemistry of human tissues showed colocalization of hPC5 with renin in the zona glomerulosa of the adrenal cortex. Several lines of evidence suggest that the human adrenal gland contains a physiologically important local RAS. First, RNA encoding angiotensinogen and renin has been detected in preparations from the human adrenal zona glomerulosa, fasciculata, and medulla,24 25 confirming that both renin and its substrate are synthesized within the human adrenal gland. Second, angiotensin-converting enzyme inhibition or blockade of angiotensin receptors inhibits aldosterone release from human adrenal tissue explants,26 suggesting that the local RAS plays an active role in the regulation of aldosterone secretion from the adrenal gland. Third, tissue explants of human adrenal cortex and aldosterone-secreting adenomas secrete small quantities of active renin,24 26 27 suggesting that the adrenal cortex expresses a PPE capable of activating human prorenin. Our current results suggest that PC5 could be the PPE responsible for activation of renin in the human adrenal cortex, as both renin and hPC5 were immunodetectable in the zona glomerulosa. Additional circumstantial evidence supports this conclusion. First, centrifugal fractionation of adrenal cortical cells has revealed that renin is contained in the "granular" fraction, which is of intermediate density between vesicles and lysosomes.28 As our current study suggests that PC5 cleaves human prorenin only in cells containing secretory granules, renin would be in the appropriate intracellular compartment to be activated by PC5 in the adrenal cortex. Second, rats transgenic for mouse Ren-2 renin [TGR(mRen-2)27] display fulminant hypertension,29 which correlates best with the expression of the mouse prorenin in the adrenal gland.30 31 32 As previous studies have demonstrated that PC5 is capable of activating mouse Ren-2 prorenin but not rat prorenin (Reference 23 and data not shown), it is possible that the TGR(mRen-2)27 transgenic rat is a model for activation of a tissue RAS by the fortuitous juxtaposition of prorenin with an appropriate PPE in the adrenal cortex. These results also raise the possibility that the tissue distribution of PPEs and their apparent selectivity in activating prorenin from different species could lead to differing functions of the tissue RAS in rodents and humans.
The principal source of circulating active renin in humans is the juxtaglomerular cells of the kidney. Although low levels of hPC5 RNA were detected by Northern blot analysis in a sample of total kidney mRNA (Fig 3), we were unable to localize PC5 immunostaining in kidney sections (Fig 6), raising the possibility that PC5 is expressed at low levels in diffuse cell types in the kidney. Thus, although these results do not formally rule out PC5 as a PPE in the kidney, our inability to detect it in juxtaglomerular cells makes it unlikely that it plays a major role in the production of renal renin. In contrast, relatively abundant amounts of PC5 mRNA and protein were detected in the placenta although evidence suggests that placental cells in culture33 and in vivo34 secrete only prorenin. However, immunostaining revealed that the cells producing PC5 and prorenin in the human placenta are distinct. It is also unlikely that PC5 would activate prorenin once the two proteins are secreted because of the apparent requirement of a granular environment for the cleavage of prorenin by hPC5 in transfected cells. Thus, in contrast to the case in the adrenal gland, it is unlikely that PC5 expressed in the human placenta would activate placental prorenin. In the mouse, two forms of PC5 have been predicted on the basis of cloned cDNAs. The first would be analogous to the hPC5 cDNA described in the present study and to that cloned from rat tissues,15 23 and the second, called PC6B, would be extended at its 3' end because of a differential RNA splicing event.35 Although the hPC5 cDNA we have cloned is only roughly 3 kb in length, the major RNA band seen in human tissues is approximately 6.6 kb long. The identity of the longer band hybridizing to the hPC5 probe is currently unknown. It should be noted that neither of the cDNA clones isolated from a screening of 1.2 million phages from the adrenal library was extended at its 3' end (Fig 1), although the probes used in their isolation cover the region of homology with the mouse PC6B variant.35 In mouse tissues, expression of the PC6B variant is restricted to few tissues,35 whereas the abundance of the 6.6-kb variant detected with the hPC5 probe is directly proportional to the abundance of the 3.8-kb band. Hybridization of RNA blots from rodent tissues with a PC5 probe also has revealed RNA bands of 3.8, 6.5, and 7.5 kb,15 35 and the use of a PC5-specific probe has revealed a band of 6.5 kb. Thus, it is possible that additional PC5 RNA species exist in mammals that are extended at their 5' ends. Alternatively, human tissues may be particularly enriched in a homologue to PC6B that was not picked up in our screenings. Recent data suggest that the alternate C-terminal tail present on PC6B may serve to retain the enzyme in the Golgi network, whereas the "short" form of mouse PC5 is targeted to dense core secretory granules (N.G.S., unpublished observations, 1996). These data and the results of our cotransfection assays (Fig 4) would suggest that the "short" form of hPC5 described here is the form that would be active in renin processing in secretory granules. The PC5 enzymes isolated from humans and mice show a remarkably high degree of conservation at the nucleotide and protein sequence levels. This degree of similarity is higher than that seen for the other mammalian prohormone convertase enzymes that seem to diverge in the C-terminal half of the enzyme.36 37 This high degree of sequence conservation may reflect an essential function of PC5 (and the C-terminus of PC5) in mammals.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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These studies were supported by grants from the Medical Research Council of Canada to T.L.R., N.G.S., and R.D. T.L.R. is the recipient of the Merck-Frosst Chair in Clinical and Molecular Pharmacology. I.J. is funded by a graduate studentship from the National Science and Engineering Research Council of Canada. R.D. is a scholar of the Fonds de la recherche en sante du Quebec (FRSQ). The authors wish to thank Drs C.F. Deschepper and G. Thibault for a critical reading of the manuscript and V. Jodoin for secretarial assistance.
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Footnotes |
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Reprint requests to Timothy L. Reudelhuber, IRCM, 110, avenue des Pins Ouest, Montreal, Quebec, H2W 1R7 Canada.
Received February 23, 1996; first decision April 10, 1996; accepted June 25, 1996.
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