Different secretory pathways of renin from mouse cells transfected with the human renin gene

Mammalian cells in culture, transfected with human renin gene, can provide a useful tool for studying renin biosynthesis and secretion. We transfected fibroblast cells (mouse L929 and Chinese hamster ovary cells) and pituitary tumor cells (mouse AtT-20) with the human renin gene and a selectable plasmid (pSV2Neo). Transfected fibroblasts synthesize prorenin only. Prorenin is secreted by fibroblasts constitutively and the secretion is not influenced by 8-bromo-cAMP. On the other hand, transfected AtT-20 cells synthesized both prorenin and mature active renin. Transfected AtT-20 cells release prorenin by constitutive secretion but mature renin is secreted by a regulated mechanism since the secretion of the former is not influenced by 8-bromo-cAMP but the release of the latter is significantly stimulated. Our studies demonstrate that human renin may be secreted by at least two cellular pathways: prorenin by a constitutive pathway and mature renin by a regulated pathway. These transfected cells may provide useful models for studies of human renin synthesis, processing, and secretion.     

A targeting sequence for dense secretory granules resides in the active renin protein moiety of human preprorenin

Human renin plays an important role in blood pressure homeostasis and is secreted in a regulated manner from the juxtaglomerular apparatus of the kidney in response to various physiological stimuli. Many aspects of the regulated release of renin (including accurate processing of prorenin to renin, subcellular targeting of renin to dense secretory granules, and regulated release of active renin) can be reproduced in mouse pituitary AtT-20 cells transfected with a human preprorenin expression vector. Using protein engineering, we have attempted to define the roles of various structures in prorenin that affect its production and trafficking to dense core secretory granules, resulting in its activation and regulated secretion. Replacement of the native signal peptide of human preprorenin with that of a constitutively secreted protein (immunoglobulin M) had no apparent effect on either the constitutive secretion of prorenin or the regulated secretion of active renin in transfected AtT-20 cells. Removal of the pro segment resulted in a marked reduction in total renin secretion, but did not prevent renin from entering the regulated secretory pathway. Single or combined mutations in the two glycosylation sites of human renin did not prevent its regulated secretion; however, the complete elimination of glycosylation resulted in a significant increase in the ratio of renin/prorenin secreted by the transfected cells. Thus, these results suggest that 1) at least one of the sequences that target human renin to dense secretory granules lies within the protein moiety of active renin; 2) the presence of the pro segment is important for efficient prorenin and renin production; and 3) glycosylation can quantitatively affect the proportion of active renin secreted.     

Modulation of active renin secretion by renin-binding protein (RnBP) in mouse pituitary AtT-20 cells transfected with human renin and RnBP cDNAs.

To investigate the role of renin-binding protein (RnBP) in renin metabolism, RnBP expression plasmid, which was constructed to express human RnBP under the control of mouse mammary tumor virus long terminal repeat, was transfected into mouse pituitary AtT-20 cells together with the expression plasmid encoding human renin. The transfectant secreted prorenin and active renin, whereas RnBP was expressed only in the presence of dexamethasone and without secretion into the medium. The secretion of active renin was stimulated by forskolin, and the stimulation was repressed by dexamethasone. The secretion of prorenin, however, was insensitive to forskolin irrespective of the presence or absence of dexamethasone. Moreover, the forskolin-stimulated release of active renin was hardly repressed by dexamethasone in AtT-20 cells transfected with the renin expression plasmid and a selectable plasmid pMAMneo. Coexistence of RnBP and renin mRNAs in human Wilms' tumor G-401 cells was shown by means of polymerase chain reaction of respective cDNAs from the cells. These results suggest that RnBP modulates the release of active renin in renin-producing cells.     

The molecular biology of human renin and its gene

The molecular biology of renin, prorenin, and the renin gene have been studied. A tissue-specific pattern of expression was found in rat and human tissues. In the human placenta, the transfected and endogenous renin promoters are active, and renin mRNA levels and transfected promoter activity are increased by a calcium ionophore plus cAMP. Cultured pituitary AtT-20 cells transfected with a preprorenin expression vector mimick renal renin release by converting prorenin to renin and releasing renin in response to 8Br-cAMP. Studies with mutant renin genes suggest that the body of renin directs renin to the regulated secretory pathway, and renin glycosylation affects its trafficking. Chinese hamster ovary cells were used to produce recombinant prorenin. Infused prorenin was not converted to renin in monkeys. Renin crystals were used to determine its three-dimensional structure. Renin resembles other aspartyl proteases in the active site and core, but it differs in other regions that probably explain renin's unique substrate specificity. Based on structural and mutational analysis, a model for human prorenin was built that suggests lysine -2 of the prosegment interacts with active site aspartate residues, and that the prosegment inactivation of renin is stabilized by binding of an amino terminal beta strand into a groove on renin.     

mGK-6-derived true tissue kallikrein is synthesized, processed, and targeted through a regulated secretory pathway in mouse pituitary AtT-20 cells.

mGK-6-derived true tissue kallikrein was shown to be synthesized in mouse pituitary AtT-20 cells. This cell line, which is capable of processing other prohormones, only partially processed the proform of kallikrein to its active form, secreting it predominantly as the proform. The secretion of the active form was stimulated in response to a secretagogue, 8-bromo-cyclic AMP. These results imply that not only cellular elements capable of directing the processing of the proform to the active form and the intracellular transport of the kallikrein, but also a pathway that regulates the release of the active form may be present in the AtT-20 cells, thus the availability of this cell line for investigation of biosynthetic and secretory processes for tissue kallikrein in vivo being suggested.     

N-linked glycosylation affects the processing of mouse submaxillary gland prorenin in transfected AtT20 cells

Most mouse inbred strains carry two renin genes, Ren-1 and Ren-2, Renin-2, the product of the Ren-2 gene, is highly expressed in the submaxillary gland. It is a renin isoenzyme 96% similar to kidney renin-1, but unglycosylated. In order to investigate if glycosylation of prorenin affects its processing and/or secretion we have introduced two potential N-linked glycosylation sites into preprorenin-2 cDNA using site-directed mutagenesis. Expression plasmids were derived from wild-type and mutant renin-2 cDNA and were transfected into AtT20 cells. Both transfected cells, expressing glycosylated or unglycosylated forms, secreted prorenin and renin by the constitutive and regulated pathways, respectively. Prorenin was correctly processed to active renin but the second maturation site was not cleaved in AtT20 cells. The comparison of glycosylated and unglycosylated renin expression showed a diminished secretion of glycosylated active renin. Prevention of glycosylation with tunicamycin resulted in an improved secretion of active renin. Moreover, the efficiency of the trypsin activation in vitro was reduced for glycosylated prorenin and it was restored when the activation was performed on mutant renin secreted from tunicamycin-treated cells. It is proposed that the bulky carbohydrates attached to prorenin constitute a steric hindrance to proteolysis by maturation enzymes.     

Prorenin in plasma and kidney

Circulating prorenin is an enzymatically inactive form of renin, also present in kidney, which can be activated in vitro. Its biochemical properties and physiological behavior suggest that it may be a biosynthetic precursor of active renin. However, in contrast to typical prohormones, the normal plasma concentrations of prorenin are much higher than the active hormone. The purposes and functions of prorenin are unclear. It may have no further role after its secretion into the circulation. On the other hand, it may be a transport form of renin that can enter or exit cells more easily than the active form. It is also possible that the activity of the renin-angiotensin system may be regulated by the conversion of prorenin to renin in the kidney (which may be under beta-adrenergic control) or at other possible sites. Irreversible activation of prorenin appears to be a proteolytic process. In addition, acidification causes reversible activation, perhaps through a change in molecular conformation. Such reversible activation might occur in vivo by unknown mechanisms. Future studies are needed to define the biochemical processes by which increased physiological demand for renin is translated into the production of more active enzyme.     

Sta!le and transient expression of mouse submaxillary gland renin cDNA in AtT20 cells: proteolytic processing and secretory pathways

Apart from kidney, where renin synthesis takes place in all mammals, the submaxillary gland (SMG) of most mouse strains constitutes an important source of an isoenzyme, renin-2, that is highly homologous to renal renin, but unglycosylated [(1982) Nature 298, 90-92]. This unique phenotype is due to the presence of an extra copy of th renin gene. A puzzling observation is that (pro)renin-2 cannot be detected in the kidney of these animals, although both mRNAs accumulate at similar levels [(1985) Proc. Natl. Acad. Sci. USA 82, 6196-6200]. In order to investigate whether (pro)renin-2 expression is detectable in mouse heterologous cell lines we transfected the renin-2 cDNA into AtT20 (pituitary corticotrope) and BTG9A (hepatoma) cells. Stable clones expressing renin were obtained in both cases. BTG9A cells secreted only prorenin while AtT20 cells secreted prorenin and active renin. In addition, in AtT20 cells the secretion of active renin was stimulated by 8-Br cAMP. Our results show that unglycosylated (pro)renin-2 can be expressed and secreted in two murine cell lines. Moreover, it is correctly processed to active renin and secreted upon stimulation in AtT20 cells.     

Purification and characterization of human truncated prorenin.

Posttranslational processing of enzymatically inactive prorenin to an active form participates in the control of the activity of a key system involved in blood pressure regulation, growth, and other important functions. The issue is complicated because renin can be produced by a number of tissues throughout the body, in addition to the kidney, but the mechanism by which they process prorenin to renin is unknown and difficult to determine because of the small amounts of renin present. In the juxtaglomerular cell of the kidney, a 43 amino acid prosegment is cleaved from the amino terminus of prorenin to generate renin of molecular weight 44,000 [Do, Y. S., Shinagawa, T., Tam, H., Inagami, T., & Hsueh, W. A. (1987) J. Biol. Chem. 262, 1037-1043]. Using human uterine lining or a recombinant human prorenin system, we employed the same approach as that used in kidney, ammonium sulfate precipitation at pH 3.1 followed by pepstatin and H-77 affinity chromatography or gel filtration, to purify to homogeneity a 45,500-MW totally active renin. The specific activity of the active truncated prorenin was 850 Goldblatt units (GU)/mg of protein for chorion-decidua renin and 946 GU/mg of protein for recombinant renin, both similar to that reported for pure human renal renin. Both forms of renin cross-reacted with an antibody generated against 44,00-MW pure human renal renin and with an antibody generated against a peptide identical to the carboxy-terminal one-third of the prosegment.(ABSTRACT TRUNCATED AT 250 WORDS)     

(Pro)renin promotes fibrosis gene expression in HEK cells through a Nox4-dependent mechanism

The (pro)renin receptor (PRR) has recently been demonstrated to bind equally well renin and its precursor, prorenin, leading to a similar intracellular signaling independent of angiotensin II. In this study, we report that human embryonic kidney cells (HEK) exposed to renin or prorenin for 24 h in the presence of a blocking concentration of the angtiotensin-converting enzyme inhibitor perindoprilate increased superoxide anion production as measured by luminescence (lucigenin) and electron spin resonance spectroscopy (hydroxylamine radical transition). Also, both renin and prorenin increased Nox4 expression while Nox2, p47(phox), and p67(phox) remained unchanged. In an investigation of the effects of renin and prorenin on fibrosis genes, it appeared that both proteins stimulated transforming growth factor-β (TGF-β), fibronectin, and plasminogen activator inhibitor type 1 (PAI-1) expression and therefore participated to an overall switch toward a profibrotic state of the kidney cells. When the cells were transfected with a siRNA targeting the PRR, Nox4 expression was efficiently prevented as well as the increase in superoxide production, TGF-β, fibronectin, and PAI-1. Finally, we demonstrated that transfection of the cells with a Nox4-specific small interfering (si) RNA also prevented fibrosis gene expression following treatment with renin or prorenin. The results demonstrate that renin and prorenin, through their specific membrane receptor and independently of angiotensin II, promote fibrosis gene expression via a Nox4-dependent mechanism.     

Observations on the renal processing and sorting of prorenin.

Human prorenin is the biosynthetic precursor of renin. In general, prorenin is enzymatically inactive until it is converted to renin. The kidney is the major source of renin in the circulation, and is also an important source of circulating prorenin. The mechanisms of prorenin sorting and processing to renin in the juxtaglomerular cell may be a determinant of renal renin production. Therefore, our studies have focused on renal enzymes involved in "limited proteolysis" of prorenin to renin and on the morphology of prorenin sorting in the human juxtaglomerular cell.     

Biochemical and immunological differences between plasma inactive renin from normal and nephrectomized rats.

Using immunological techniques, we have demonstrated that about half the trypsin-activatable renin in normal rat plasma is prorenin, while the other is not, and that inactive renin in nephrectomized rat plasma is not prorenin. In the present study, the trypsin-induced angiotensin I generating activity not related to prorenin from normal rat plasma disappeared after HPLC on G3000SW. HPLC analysis of trypsin-treated plasma showed the generation of active renin by trypsin for normal rat plasma, while it did not for nephrectomized rat plasma. These results indicate that trypsin treatment of crude plasma results in the generation of angiotensin I generating activity not due to prorenin, as well as activation of prorenin. HPLC on G3000SW is a useful tool for the determination of plasma prorenin.     

Human placental chorionic renin: production, purification and characterization

Native human renin, produced from the culture of human chorionic trophoblasts, has been purified to homogeneity on a milligram scale using a five-step purification scheme. The chorion cells secrete 50-200 milliGoldblatt Units of trypsin-activatable prorenin per ml into the medium. The pro-enzyme is partially purified by ammonium sulfate fractionation and chromatographies on QAE-Sephadex and cibracon blue-agarose. Following conversion of prorenin to the active enzyme by porcine trypsin, the renin is purified to homogeneity by affinity chromatography and gel filtration. Chorionic prorenin has a molecular weight of 43,000; the active enzyme 40,000. Both proteins exist as a single polypeptide chain as determined by SDS-polyacrylamide gel electrophoresis under reducing conditions. The average specific activity of six different preparations was found to be 1072 Goldblatt Units/mg. The amino acid composition and N-terminal sequence of the active enzyme has been determined and is identical to the human kidney enzyme. Microheterogeneity of chorionic renin was demonstrated by isoelectrofocusing analysis. The physical characterization of chorionic renin is compared with that reported for the human kidney enzyme.     

Sequence requirements for prohormone processing in mouse pituitary AtT-20 cells. Analysis using prorenins as model substrates

Although cleavage of peptides at sites marked by paired basic amino acids is a common feature of prohormone processing, little is known about the properties of endoprotease(s) responsible for cleavage of the precursor. To examine the cleavage specificity of a processing endoprotease, we have altered the Lys-Arg cleavage site of human prorenin to Arg-Arg, Lys-Lys and Arg-Lys by site-directed mutagenesis, and expressed the native and mutated precursors in mouse pituitary AtT-20 cells which are known to process foreign prohormones, including prorenin, at paired basic sites during the regulated secretory process. All native and mutated human prorenins were sorted into the regulated secretory pathway. The mutated precursor with Arg-Arg instead of the Lys-Arg native pair was processed at about half the efficiency of the native one, while the Lys-Lys and Arg-Lys mutants were not processed. Rat prorenin, which naturally has a Lys-Lys pair, was not processed in the cells. In addition, mouse Ren2 prorenin, which has a Ser residue next to the Lys-Arg pair, but not mouse Ren1 prorenin, which has a Pro residue next to the pair, was processed. These results suggest that the Arg residue at the COOH side of the basic pair is essential for cleavage of prorenins by a processing enzyme during the regulated secretory process in AtT-20 cells, although the NH2-side Lys residue also plays a role. The results also demonstrate that the processing enzyme cannot cleave the Arg-Pro peptide bond.     

Human prorenin structure sheds light on a novel mechanism of its autoinhibition and on its non-proteolytic activation by the (pro)renin receptor

Antibodies and prorenin mutants have long been used to structurally characterize prorenin, the inactive proenzyme form of renin. They were designed on the basis of homology models built using other aspartyl protease proenzyme structures since no structure was available for prorenin. Here, we present the first X-ray structure of a prorenin. The current structure of prorenin reveals that, in this zymogene, the active site of renin is blocked by the N-terminal residues of the mature version of the renin molecule, which are, in turn, covered by an Ω-shaped prosegment. This prevents access of substrates to the active site. The departure of the prosegment on activation induces an important global conformational change in the mature renin molecule with respect to prorenin: similar to other related enzymes such as pepsin or gastricsin, the segment that constitutes the N-terminal β-strand in renin is displaced from the renin active site by about 180° straight into the position that corresponds to the N-terminal β-strand of the prorenin prosegment. This way, the renin active site will become completely exposed and capable of carrying out its catalytic functions. A unique inactivation mechanism is also revealed, which does not make use of a lysine against the catalytic aspartates, probably in order to facilitate pH-independent activation [e.g., by the (pro)renin receptor].     

Isolation and characterization of renin-expressing cell lines from transgenic mice containing a renin-promoter viral oncogene fusion construct

We constructed transgenic mice containing a renin-promoter SV40 T antigen fusion transgene with the intention of inducing neoplasia in renin-expressing cells and isolating renin-expressing cell lines in vitro. We examined six kidney tumors from mice representing three different transgenic lines and found they expressed their endogenous renin gene. Initially, five nonclonal kidney tumor-derived cell lines were established which expressed their endogenous renin gene in addition to the transgene. They retained active renin intracellularly and constitutively secreted an inactive form of renin (prorenin). One of these cell lines was cloned to homogeneity. This line maintained high level expression of renin mRNA throughout 3 months of continuous culture. Although the cells contained an equal proportion of active and inactive renin, the species constitutively secreted into the media was predominantly (95%) prorenin. However, active renin secretion was stimulated 2.3- and 4.6-fold by treatment with 8-bromo-cAMP after 4 and 15 h, respectively. In addition, the presence of multiple secretory granules was confirmed by ultrastructural analysis. These cells, which express renin mRNA and can regulate secretion of active renin, should provide an excellent tool for studying renin gene regulation and secretion. Furthermore, these mice should provide a useful source for the establishment of renin-expressing cell lines from a variety of renin-expressing tissues.     

Renin processing studied by immunogold localization of prorenin and renin in granular juxtaglomerular cells in mice treated with enalapril

Summary Immunogold techniques were used to investigate renin processing within granular juxtaglomerular cells following short-term (6 h and 1 day) and long-term (4 weeks) enalapril treatment in female BALB/c mice. In control animals, renin protein labelling was localized to all types of granules (proto-, polymorphous, intermediate and mature) and to transport vesicles, whilst prorenin labelling was found in all these sites except mature granules, confirming that active renin is localized to mature granules only. Following short-term enalapril treatment, the exocytosis of renin protein from mature granules was increased. Long-term enalapril treatment resulted in increased numbers of transport vesicles and all types of granules, consistent with increased synthesis and storage of renin. More large intermediate granules contained discrete regions labelled for prorenin. Renin protein was exocytosed from individual and multiple granules, whilst prorenin was exocytosed from protoand intermediate granules. It is concluded that under normal conditions prorenin is secreted constitutively by bulk flow from transport vesicles. On the other hand, active renin is secreted regulatively from mature granules. In conditions of intense stimulation (angiotensin-converting enzyme inhibition treatment), increased synthesis of prorenin leads to enhanced secretion of prorenin by both constitutive and regulative pathways. Under these conditions, the conversion of prorenin to active renin is increased, with increased secretion of active renin occurring in a regulative manner. Furthermore, the localization of prorenin to one discrete region of large intermediate granules leads us to conclude, that cleavage of the prosegment of renin occurs with the transition of intermediate to mature granules.     

Prorenins activation by an enzyme from rat plasma (PreR-Co)

The aim of the present research was to explore the capacity of PreR-Co to process prorenin purified from kidney and corpora lutea (CL) and to study its action on extrarenal tissues. The PreR-Co was obtained from plasma as a single electrophoretic band by (NH4)2SO4 precipitation, gel filtration, anti-rat albumin immunoaffinity, and ion-exchange chromatography. Prorenin free of renin was obtained after (NH4)2SO4 precipitation, gel filtration, and ion-exchange chromatography by a passage through an affinity gel of H-77 Sepharose. SDS-PAGE of supernatant and of acidic elution from gel, exhibited a single band of 43 kDa and 35 kDa, respectively; both recognized by the specific anti rat renin antibody. The isolated renin was not attacked by PreR-Co; on the contrary prorenin was completely activated. The product of PreR-Co-activated prorenin showed an analogous MW to that of renin and was recognized by the specific antibody. In addition to processing kidney prorenin, PreR-Co was able to cleave inactive renin from ovary, CL, uterus and adrenal gland homogenates. However, the amount of active renin generated from these tissues was lower than those produced by trypsin activation. PreR-Co is a good candidate for the role of the enzyme involved in tissues prorenin activation.     

Extrarenal production and activation of human plasma prorenin: the evidence after venous occlusion

Venous occlusion of the left arm in consenting men was induced for 10 or 20 min to stimulate local fibrinolytic and other proteases, thereby favouring the conversion of prorenin to renin. Using the two techniques cryoactivation and tryptic activation, we found that plasma active renin increased significantly after such occlusion (10 and 20 min) while prorenin rose more convincingly and progressively from 10 to 20 min. The renin increase can be partially attributed to hemoconcentration, but in vivo production and (or) local activation of prorenin to renin cannot be excluded. The prorenin rise can apparently be attributed to local extrarenal production, and not to hemoconcentration or influx, since it was progressive and neither prorenin nor renin levels were raised at all in blood circulating outside the occluded arm. Prekallikrein and plasminogen levels were elevated in occlusion plasmas, but responsibility of these enzyme systems for any enhanced activation of prorenin was not established. The trypsin inhibitory capacity was also elevated, increasing the requirement of trypsin to achieve optimal activation of prorenin, but not changing the prorenin estimate itself. Thus, prorenin appears to be released extrarenally, within the vasculature of an occluded arm, while in vitro evidence suggests that the mechanisms for its activation were stimulated. The importance of such extrarenal production and activation of prorenin for renin production under other physiological or pathophysiological conditions remains to be determined.     

Renin gene expression, biosynthesis, and cellular pathways of secretion

The molecular biology of the human renin gene is reviewed. This 12.5 kb gene contains 10 exons and 9 introns. In its 5' flanking region, major control elements are present. These include promoters and enhancers as well as regulatory elements. The combined action of these elements would result in tissue specific expression and regulation of the gene. In addition to the control at the gene expression level, renin is also regulated at the posttranslational and secretory levels. The translational product of renin mRNA is preprorenin, which is cotranslationally cleaved to prorenin, an inactive precursor of renin. The majority of new synthesized human prorenin is constitutively secreted. However, prorenin is also processed intracellularly to the mature single chain active renin which is stored in secretory granules. Active renin is released by a regulated mechanism which can be stimulated by cAMP and other secretagogues. Studies are under way to examine the responses of renin gene expression, biosynthesis and secretion to various physiological conditions.