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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Constitutive and basal secretion from the endocrine cell line, AtT-20

A variant of the ACTH-secreting pituitary cell line, AtT-20, has been isolated that does not make ACTH, sulfated proteins characteristic of the regulated secretory pathway, or dense-core secretory granules but retains constitutive secretion. Unlike wild type AtT-20 cells, the variant cannot store or release on stimulation, free glycosaminoglycan (GAG) chains. In addition, the variant cells cannot store trypsinogen or proinsulin, proteins that are targeted to dense core secretory granules in wild type cells. The regulated pathway could not be restored by transfecting with DNA encoding trypsinogen, a soluble regulated secretory protein targeted to secretory granules. A comparison of secretion from variant and wild type cells allows a distinction to be made between constitutive secretion and basal secretion, the spontaneous release of regulated proteins that occurs in the absence of stimulation.     

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.     

The fate of prorenin during granulopoiesis in epithelioid cells

Summary Comparative immunocytochemical experiments with antisera directed against renin and three synthetical peptides (Pro 1, Pro 2 A and Pro 3) covering almost the entire span of human renin prosegment were performed on human kidney tissue. With anti-Pro 1, i.e. the antiserum which recognizes the NH₂ terminus of human prorenin, no clear immunolabeling of juxtaglomerular epithelioid cell secretory granules could be obtained. It is therefore concluded that the corresponding portion of human prorenin may be cleaved off in the Golgi complex.After application of anti-Pro 3, the antiserum which recognizes the COOH terminus of the prosegment, only the juvenile secretory granules of epithelioid cells were consistently labeled, whereas, in contrast, some of the intermediate and most of the mature secretory granules were anti-Pro 3-negative. As the immunoreactivity of mature renin increased remarkably from protogranules to mature secretory granules, it is suggested that the cleavage of the COOH terminus of the prosegment, i.e. the activation of renin, takes place in juvenile and intermediate granules during condensation of the enzyme.The immunoreactivity of Pro 2A, corresponding to the middle portion of the prosegment, disappeared in a some-what earlier stage of granulopoiesis than that of Pro 3. It is therefore concluded that the corresponding segmental cleavage, the result of which is a truncated version of intact prorenin, occurs in the protogranules of epithelioid cells.The data presented are consistent with the assumption that the secretion of active renin takes place by the exocytosis of mature secretory granules, while the secretion of inactive renin, which is a truncated version of intact prorenin, is mediated by the exocytosis of juvenile and intermediate granules.These studies were supported by the German Research Foundation within the Forschergruppe Niere/Heidelberg     

Targeting of secretory vesicles to cytoplasmic domains in AtT-20 and PC- 12 cells

Organelles are not uniformly distributed throughout the cytoplasm but have preferred locations that vary between tissues and during development. To investigate organelle targeting to cytoplasmic domains we have taken advantage of the mouse pituitary cell line, AtT-20, which, when induced to extend long processes, accumulates dense core secretory granules at the tips of the processes. During mitosis, these secretory granules accumulate along the plane of division. Protein synthesis is not mandatory for such redistribution of secretory granules. To explore the specificity of the redistribution we have used transfected AtT-20 cells that express the immunoglobulin kappa light chain. While the endogenous hormone ACTH is found in secretory granules, the kappa chain is a marker for organelles involved in constitutive secretion. By immunofluorescence, kappa also accumulates at the tips of growing processes, and along the midline of dividing cells, suggesting that the redistribution of vesicles is not specific for dense-core secretory granules. Since there is evidence for selective organelle transport along processes in neuronal cells, the rat pheochromocytoma cell PC-12 was transfected with DNA encoding markers for regulated and constitutive secretory vesicles. Again regulated and constitutive vesicles co-distribute, even in cells grown in the presence of nerve growth factor. We suggest that at least in the cells studied here, cytoskeletal elements normally carry exocytotic organelles to the surface; when the cytoskeletal elements coalesce in an extending process, exocytotic organelles of both the constitutive and regulated pathway are transported nonselectively to the tips of the cytoskeletal elements where they accumulate.     

Expression of individual forms of peptidylglycine alpha-amidating monooxygenase in AtT-20 cells: endoproteolytic processing and routing to secretory granules

Peptidylglycine alpha-amidating monooxygenase (PAM: EC 1.14.17.3) is a bifunctional protein which catalyzes the COOH-terminal amidation of bioactive peptides; the NH2-terminal monooxygenase and mid-region lyase act in sequence to perform the peptide alpha-amidation reaction. Alternative splicing of the single PAM gene gives rise to mRNAs generating PAM proteins with and without a putative transmembrane domain, with and without a linker region between the two enzymes, and forms containing only the monooxygenase domain. The expression, endoproteolytic processing, storage, and secretion of this secretory granule-associated protein were examined after stable transfection of AtT-20 mouse pituitary cells with naturally occurring and truncated PAM proteins. The transfected proteins were examined using enzyme assays, subcellular fractionation, Western blotting, and immunocytochemistry. Western blots of crude membrane and soluble fractions of transfected cells demonstrated that all PAM proteins were endoproteolytically processed. When the linker region was present between the monooxygenase and lyase domains, monofunctional soluble enzymes were generated from bifunctional PAM proteins; without the linker region, bifunctional enzymes were generated. Soluble forms of PAM expressed in AtT-20 cells and soluble proteins generated through selective endoproteolysis of membrane-associated PAM were secreted in an active form into the medium; secretion of the transfected proteins and endogenous hormone were stimulated in parallel by secretagogues. PAM proteins were localized by immunocytochemistry in the perinuclear region near the Golgi apparatus and in secretory granules, with the greatest intensity of staining in the perinuclear region in cell lines expressing integral membrane forms of PAM. Monofunctional and bifunctional PAM proteins that were soluble or membrane-associated were all packaged into regulated secretory granules in AtT-20 cells.     

The purification and characterization of recombinant human renin expressed in the human kidney cell line 293

The cDNA encoding human preprorenin has been introduced into the adenovirus-transformed human kidney cell line 293. The recombinant 293 cells expressed and secreted prorenin; trypsin was used to activate the secreted prorenin to renin in vitro. The recombinant protein was purified to homogeneity by a single affinity chromatographic step. Using synthetic tetradecapeptide, the Km was 57.1 +/- 9.3 microM and the kcat was (7.48 +/- 1.57) x 10(3)/hr. Activation with trypsin resulted in a secondary cleavage between Arg53 and Leu54 generating a two chain form held together via a disulfide between Cys51 and Cys58. This secondary cleavage did not affect enzyme activity as determined by the ability of renin to degrade a synthetic tetradecapeptide substrate. Our paper demonstrates the potential for producing large quantities of renin from human kidney cells and also suggests that the use of trypsin, which has been widely used to convert prorenin to renin in vitro, causes a secondary cleavage in the renin peptide chain.     

Development and fate of the secretory granules of juxtaglomerular epithelioid cells

Summary The development and fate of the secretory granules in murine, rat and human juxtaglomerular epithelioid cells were examined using ultrastructural and immunocytochemical methods. The formation of mature renin granules occurs by fusion of rhomboid protogranules followed by coalescence of their paracrystalline contents, and by the fusion of roundish juvenile granules having an amorphous internum. Protogranules with paracrystalline contents are prominent in animals with stimulated renin synthesis, indicating an overcharge in processing and/or packaging of the secretory product, renin, under these conditions. Various similarities between lysosomes/multivesicular bodies (MVBs) and juvenile renin granules have been observed. With the exception of small MVBs, no renin-negative organelles that could be regarded as lysosomes were found in epithelioid cells of mice and rats. Therefore, we suggest that renin granules are modified lysosomes. Immunocytochemical findings indicate that juvenile secretory granules of epithelioid cells represent the converting and activating compartment for prorenin. Endocytosed foreign tracers such as HRP or cationized ferritin are preferentially internalized by juvenile renin granules, which hence appear to be outstanding by their fusogeneity. Consequently, juvenile granules are probably responsible for the secretion of prorenin, and mature granules for that of active renin.These studies were supported by the German Research Foundation within the Forschergruppe Niere/Heidelberg     

Cell type-specific storage of dopamine beta-monooxygenase

Expression of dopamine beta-monooxygenase (DBM), the enzyme that converts dopamine into norepinephrine, is limited to adrenal chromaffin cells and a small population of neurons. We studied DBM trafficking to regulated granules by stably expressing rat DBM in AtT-20 corticotrope tumor cells, which contain regulated granules, and in Chinese hamster ovary (CHO) cells, which lack regulated granules. The behavior of exogenous DBM in both cell lines was compared with endogenous DBM in adrenal chromaffin cells. CHO cells secreted active DBM, indicating that production of active enzyme does not require features unique to neuroendocrine cells. Pulse-chase experiments indicated that early steps in DBM maturation followed a similar time course in AtT-20, CHO, and adrenal chromaffin cells. Use of a conformation-sensitive DBM antiserum indicated that acquisition of a folded structure occurred with a similar time course in all three cell types. Cell type-specific differences in DBM trafficking became apparent only when storage in granules was examined. As expected, DBM was stored in secretory granules in chromaffin cells; CHO cells failed to store DBM. Despite the fact that AtT-20 cells have regulated granules, exogenous DBM was not stored in these granules. Thus storage of DBM in secretory granules requires cell type specific factors.     

In vitro mutagenesis of trypsinogen: role of the amino terminus in intracellular protein targeting to secretory granules

The mouse anterior pituitary tumor cell line, AtT-20, targets secretory proteins into two distinct intracellular pathways. When the DNA that encodes trypsinogen is introduced into AtT-20 cells, the protein is sorted into the regulated secretory pathway as efficiently as the endogenous peptide hormone ACTH. In this study we have used double-label immunoelectron microscopy to demonstrate that trypsinogen colocalizes in the same secretory granules as ACTH. In vitro mutagenesis was used to test whether the information for targeting trypsinogen to the secretory granules resides at the amino (NH2) terminus of the protein. Mutations were made in the DNA that encodes trypsinogen, and the mutant proteins were expressed in AtT-20 cells to determine whether intracellular targeting could be altered. Replacing the trypsinogen signal peptide with that of the kappa-immunoglobulin light chain, a constitutively secreted protein, does not alter targeting to the regulated secretory pathway. In addition, deletion of the NH2-terminal "pro" sequence of trypsinogen has virtually no effect on protein targeting. However, this deletion does affect the signal peptidase cleavage site, and as a result the enzymatic activity of the truncated trypsin protein is abolished. We conclude that neither the signal peptide nor the 12 NH2-terminal amino acids of trypsinogen are essential for sorting to the regulated secretory pathway of AtT-20 cells.     

Proprotein conversion is determined by a multiplicity of factors including convertase processing, substrate specificity, and intracellular environment. Cell type-specific processing of human prorenin by the convertase PC1.

Proprotein and prohormone processing at pairs of basic residues is generally thought to be both tissue- and precursor-specific and to be developmentally regulated. Furin, PC1 (also called PC3), and PC2 represent three recently discovered subtilisin-like proteinases which cleave a number of precursors at the same pairs of basic residues normally processed in vivo. Using human prorenin as a model, we show that PC1 can process it to active renin in cells containing secretory granules, such as the somatomammotroph cell line GH4, but not in cells which lack granules, such as the Chinese hamster ovary or African green monkey kidney epithelial (BSC-40) cell lines. In contrast, in both cell types, human prorenin is not activated by either PC2 or furin. Using the vaccinia virus expression system, biosynthetic labeling experiments demonstrated that PC1 and PC2 are themselves cleaved intracellularly at pairs of basic residues and that these two proenzymes are processed to different extents independent of whether the cell line contains dense core secretory granules. Furthermore, we also show that the cells mostly secrete the cleaved forms of PC1 and PC2, and that intracellularly the pro- form of PC2 predominates. Our data demonstrate that propeptide removal from these enzymes, possibly leading to their activation, is not the only criterion which governs precursor processing.