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.     

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.     

Human renin is correctly processed and targeted to the regulated secretory pathway in mouse pituitary AtT-20 cells

Renin is formed by intracellular processing of prorenin and catalyzes the conversion of angiotensinogen to angiotensin I, the precursor to angiotensin II. Several tissues synthesize prorenin. However, in man, the kidney is the only known source of circulating renin, raising the possibility that the processing enzyme is unique to that tissue. We have transfected a gene that directs prorenin synthesis in pituitary AtT-20 cells, which are capable of processing other prohormones. The results demonstrate that transfected AtT-20 cells can secrete inactive prorenin, accurately process prorenin to active renin, and be stimulated to release active renin in response to a secretagogue. These data imply that cellular elements capable of directing the processing of prorenin to renin and its correct subcellular compartmentalization may be present in nonrenal cell types and that critical elements of the regulated release of renin that occur in the kidney can be reconstituted in cells in culture.     

A study on renin binding protein (RnBP) in the human kidney

We purified, from human kidney, a protein that reacts with rabbit anti-porcine kidney renin binding protein (RnBP) antiserum by trapping with porcine kidney renin. The purified preparation showed a single protein peak on gel filtration by high performance liquid chromatography (HPLC) and two protein bands on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The latter two kinds of protein were identified as the porcine renin and human kidney protein from their electrophoretic mobilities and reactivity toward rabbit anti-porcine kidney renin and RnBP antisera. The molecular weights of the purified preparation and the human kidney protein were estimated to be 56,000 by HPLC and 43,000 by SDS-PAGE, respectively. The specific activity of porcine renin in the purified preparation was 8.6 mg angiotensin I per mg of protein per h at 37 degrees C and pH 6.5. This specific activity was about one-fifth that of free porcine renin. Therefore, it is suggested from the reactivity toward the anti-porcine RnBP antiserum and inhibitory action toward porcine renin that the human kidney protein is RnBP and that the human RnBP is purified as a complex with porcine renin.     

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.     

Effects of nucleotides on the interaction of renin with GlcNAc 2-epimerase (renin binding protein, RnBP)

Renin binding protein (RnBP), a cellular renin inhibitor, was identified as an enzyme, GlcNAc 2-epimerase. Recombinant RnBP inhibited porcine renin activity in a dose dependent manner. However, the inhibition was neutralized by nucleotides, such as ATP, dATP, dGTP, dCTP or dTTP. Moreover, ATP inhibited the formation of hetero-complex of renin with RnBP, called high molecular weight (HMW) renin. On the other hand, N-ethylmaleimide (NEM), a SH-alkylating reagent inhibited the GlcNAc 2-epimerase activity concomitant with the decaying of the dimer to the monomer of the enzyme. The inhibition was modulated in the presence of ATP. These results indicate that nucleotides stabilize the dimeric form RnBP (GlcNAc 2-epimerase) and inhibited the formation of the renin-RnBP hetero complex, HMW renin.     

Purification and characterization of renin binding protein (RnBP) from porcine kidney

Renin binding protein (RnBP) was purified from porcine kidney using pepstatin affinity column chromatography, DEAE-Sepharose column chromatography, gel filtration on Ultrogel-AcA 34, aminohexyl-Sepharose 4B column chromatography, and high performance liquid chromatography (HPLC) on TSK-gel G-3000 SW. The purified preparation was homogeneous as judged by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, polyacrylamide disc gel electrophoresis and isoelectric focusing on polyacrylamide gel. The isoelectric point was at pH 4.85, and the apparent molecular weight of RnBP was estimated to be 42,000 by SDS-polyacrylamide gel electrophoresis. The preparation did not show any renin activity and was stable for 30 min at 37 degrees C between pH 5.0 and 9.0 or on storage for 4 weeks at 4 degrees C or -80 degrees C. The activity of renin was greatly inhibited by RnBP. From the kinetic analysis of the inhibition we roughly estimated the dissociation constant between renin and RnBP to be about 0.2 nM, assuming that the stoichiometry in the complex, i.e., high molecular weight (HMW) renin, is one to one, and that the complex is inactive. The inhibitory activity of RnBP was lost by acidification at pH 3.0 and the activity of renin was restored. The purified RnBP formed a single precipitin line with the antiserum prepared with the purified HMW renin as antigen, which is RnBP-renin complex (Takahashi, S., et al. (1983) J. Biochem. 93, 265-274), and this line fused with one of the two precipitin lines formed between HMW renin and anti-HMW renin antiserum. The other of the two lines was between renin and anti-HMW renin antiserum. The purified preparation was thus identified as RnBP. The HMW renin was reconstituted with the purified RnBP and renin, and the apparent molecular weight of the reconstituted specimen was estimated to be 60,000 by gel filtration on Ultrogel AcA 44.     

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.     

The pro-peptide is not necessary for active renin secretion from transfected mammalian cells

Cultured mouse myeloma cells were transfected with expression vectors encoding the aspartyl proteinase, human renin. The full construct, encoding the renin precursor prorenin, allows transfected cells to secrete the enzymically inactive pro-protein. Activity is detectable only following trypsin treatment which mimics the physiological activation step. Accordingly, it appears that myeloma cells do not contain detectable levels of an appropriate activating proteinase. However, when these cells are transfected with a construct from which the pro-peptide coding sequence has been deleted, they secrete an apparently fully active enzyme which is indistinguishable from mature renin. We conclude that expression of the pro-peptide is not necessary to allow correct folding of the molecule and its passage through the secretory pathway.     

Expression of a deletion mutant of the prosegment of human prorenin in Chinese hamster ovary cells

Expression plasmids encoding native human preporenin and a mutant deleted in its entire prosegment were transfected into Chinese hamster ovary cells. The cells transfected with the expression plasmid of native preporenin secreted exclusively inactive prorenin, while the cells transfected with the mutant secreted the active enzyme. The secreted amount of renin from the latter cells was much lower than that of prorenin from the former ones, although these two enzymes had little difference in specific activity after trypsin activation. These results suggest that the prosegment plays an important role in the secretory process of renin, although the fully active enzyme can be formed in its absence.     

Purification of high molecular weight (HMW) renin from porcine kidney and direct evidence that the HMW renin is a complex of renin with renin binding protein (RnBP)

The high molecular weight (HMW) renin was purified from porcine kidney by a procedure involving extraction with a buffer system containing protease inhibitors, ammonium sulfate fractionation, pepstatin-aminohexyl-Sepharose 4B column chromatography, gel filtration on Ultrogel AcA 44 and aminohexyl-Sepharose 4B column chromatography. The resulting preparation showed a single band on isoelectric focusing, exhibiting an isoelectric point at pH 5.25, and was stable on storage at -80 degrees C for 4 months. The specific activity was 3.97 mg of angiotensin I formed/mg of protein per h at 37 degrees C and at pH 6.5 with porcine angiotensinogen as the substrate. When the HMW renin was exposed to acid, renin activity increased by about 5-fold and the free form of fully active renin was recovered from the acidified HMW renin, leaving an insoluble aggregate of protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the HMW renin showed two protein bands, of which one was identified as renin from the electrophoretic mobility and the other was the protein, assigned as renin binding protein (RnBP), that was insolubilized by acidification. The purified HMW renin is a complex of renin with RnBP, and the molecular weights of RnBP and renin in the HMW renin were estimated to be 39,000 and 32,000, respectively, by gel permeation liquid chromatography in 6 M guanidine-HCl. A modified rapid method for purification of renin is also presented.     

Identification of renin and renin messenger RNA sequence in rat ovary and uterus

An increase in plasma prorenin during pregnancy suggests that prorenin might be synthesized in the ovary and the secretion of renin or prorenin may be stimulated by an ovarian steroid-mediated process. Recently, renin and angiotensinogen have been identified in human ovarian follicular fluid. However, there is considerable controversy over whether renin is synthesized in the ovary or derived from circulation. In the present study, we confirmed the presence of renin and renin mRNA in rat ovary and uterus by Northern blot analysis with rat renin cRNA as a hybridization probe. Our data show that ovarian or uterine renin is synthesized in the same cells. This suggests that the function of renin might be closely linked to the reproductive process.     

Tubular morphogenesis and mesenchymal interactions affect renin expression and secretion in SIMS mouse submandibular cells

We have previously immortalized a mouse submandibular gland (SMG) ductal epithelial cell line, SIMS, from pubertal male mice transgenic for the SV40 large T antigen under the control of the adenovirus 5 E1A promoter. Here we demonstrate the role of the extracellular environment in directing not only the morphogenetic behavior of the cells, but also their functional differentiation in terms of renin expression and secretion. First, we measured renin activity of polarized SIMS cells. Low levels of renin are secreted from both the apical and the basolateral domains; the mechanism appears to be direct as no renin was found to be transcytosed across the cell. Second, we studied homotypic and heterotypic mesenchymal cell interactions with SIMS cells. We found that epithelial-mesenchymal coculture in collagen I gels results in branching tubular morphogenesis of SIMS cells and that significant amounts of renin are secreted, probably into the lumen, as the precursor form, prorenin. Third, we investigated the effects of the basement membrane on SIMS cell morphology and function and found that this structure alone is sufficient to allow expression and secretion of both prorenin and active renin. Finally, we established that SIMS cells can express androgen-regulated genes in a transient transfection assay. In addition, in Matrigel cultures androgen receptor expression appears to be induced, suggesting that the SIMS cell line will be useful for further studies on the molecular basis of the observed high-level expression of SMG-specific genes in male mice.     

Secretion of enzymatically active human renin from mammalian cells using an avian retroviral vector

Recombinant plasmid-based retroviral expression vectors were constructed using a modified spleen necrosis virus (SNV) containing the Herpes simplex virus thymidine kinase gene promoter controlling the expression of the Tn5 neomycin phosphotransferase II gene (NPTII gene). The human renin (HRn) gene (hrn) was inserted into the 5' end of the SNV sequences such that in concatemeric plasmid DNA its expression was controlled by the strong promoter in the SNV long terminal repeat (LTR). Dog cells transfected with the concatemeric plasmid DNA secreted a small amount of a HRn-like 43-kDa protein. After cotransfection of chicken cells with concatemeric plasmid DNA and proviral DNA of reticuloendotheliosis virus strain A, infectious stocks of viruses were recovered. Cells infected with the virus carrying the viral LTR-hrn gene oriented for expression secreted the 43-kDa HRn-like protein at about 100-fold higher levels than the cells transfected with the plasmid DNAs. Biological activity of secreted HRn was determined by measuring levels of angiotensin I generated by incubating culture media with either a porcine or human angiotensinogen substrate. Infected dog cells produce about 40 ng of enzymatically active HRn per 10(6) cells per 24 h. These data indicate that retroviral expression vectors provide a good system for obtaining the secretion of high levels of enzymatically active heterologous proteins from mammalian cells.     

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.     

Renin is sorted to the regulated secretory pathway in transfected PC12 cells by a mechanism which does not require expression of the pro-peptide

The rat pheochromocytoma cell line PC12 targets secretory proteins into two distinct pathways. When DNA encoding human prorenin was transfected into PC12 cells, the protein was sorted into the regulated secretory pathway and released with similar kinetics to noradrenaline upon carbachol stimulation. To determine whether information for targeting prorenin lies within the pro-peptide we have transfected PC12 cells with a construct lacking the pro-peptide coding sequence. The transformed line secretes an apparently fully active enzyme and responds to carbachol stimulation with a rapid release of renin activity. We conclude that the pro-peptide of renin is not essential for targeting the protein to the regulated pathway in PC12 cells.     

Normal blood pressure and plasma renin activity in mice lacking the renin-binding protein, a cellular renin inhibitor

In renal extracts, some renin is present as "high molecular weight renin," a heterodimeric complex of renin with the 46-kDa renin-binding protein (RnBP), also known as N-acyl-D-glucosamine 2-epimerase. Because RnBP specifically inhibits renin activity, the protein was proposed to play an important role in the regulation of the renin-angiotensin system (RAS). Using gene targeting, we have generated mice lacking RnBP and tested this hypothesis in vivo. In particular, we analyzed biosynthesis, secretion, and activity of renin and other components of the RAS in mice lacking RnBP. Despite extensive investigations, we were unable to detect any major effects of RnBP deficiency on the plasma and renal RAS or on blood pressure regulation. Contrary to previous hypotheses, we conclude that RnBP does not play a significant role in the regulation of renin activity in plasma or kidney. However, RnBP knockout mice excrete an abnormal pattern of carbohydrates in the urine, indicating a role of the protein in renal carbohydrate metabolism.     

Mouse submaxillary renin has a protease activity and converts human plasma inactive prorenin to an active form

The activation of inactive prorenin by active renin was investigated. Inactive prorenin extensively purified from human plasma was activated by active renin which had been purified from mouse submaxillary glands by multiple chromatographic steps. The apparent lack of protease activity in renin was puzzling in view of the close similarity of its active site structure with that of acid proteases. After a series of affinity chromatographic steps designed to eliminate minute contaminants, renin was found to contain a very low but finite level of a neutral protease activity which was equivalent to 1/40,000 of that of cathepsin D tested by hemoglobinolytic activity. The protease activity was considered as intrinsic to renin since it co-purified with renin persistently at a constant ratio to the renin activity, was precipitated by a monoclonal antibody specific for renin, showed a neutral pH optimum of the enzyme activity in the same pH range as that of renin, and was inhibited by pepstatin. The neutral protease activity is likely to mediate the activation of inactive prorenin.     

Biosynthesis of a renin binding protein

The biosynthesis of a porcine renin binding protein (RnBP), which specifically binds to renin and forms an inactive high molecular weight renin, was investigated. mRNAs from various porcine tissues were used to investigate in vitro protein synthesis. The kidney mRNA directed the synthesis of a high level of RnBP, whereas the liver, adrenal and pituitary gland mRNAs gave as low but significant level of it. The in vitro synthesized RnBP as well as the immunologically detected RnBP synthesized in vivo had the same molecular weight, 42,000, as that of the purified protein. Moreover, both the human and rat kidney mRNAs directed the synthesis of this protein identified with an anti-porcine RnBP antibody. These results strongly indicate that RnBP, present in various mammalian species, is synthesized in renin-producing tissues as the mature size and undergoes binding with renin without proteolytic processing.