(Hypertension. 1996;27:514-517.)
© 1996 American Heart Association, Inc.
Articles |
From the Metabolic Research Unit, University of California, San Francisco.
Correspondence to Francisco A.R. Neves, University of California, San Francisco, Metabolic Research Unit, HSW 1141 PO Box 0540, San Francisco, CA 94143-0540.
| Abstract |
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Key Words: renin-angiotensin system enzymes prohormones
| Introduction |
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The enzyme that performs the proteolytic removal of the prosegment in the JG cell secretory granules is not known. Several enzymes have been proposed to be capable of activating prorenin. These include cathepsin B,5 cathepsin D,6 cathepsin G,7 tissue kallikrein,8 PC1,9 10 trypsin,11 and mouse submandibular gland prorenin converting enzyme.12 However, none of these enzymes meet all the criteria necessary for an authentic renal prorenin convertase. There is no evidence that kallikrein, cathepsin G, or trypsin colocalizes with renin in the secretory granules of JG cells; the optimum pHs for kallikrein8 and cathepsin G7 are higher than the pH of secretory granules13 ; trypsin degrades renin11 ; and prorenin converting enzyme, despite processing mouse Ren-2 to prorenin, is unable to activate human prorenin.12
Cathepsin B, however, remains a strong candidate because it cleaves prorenin at the correct site in vitro5 without degrading renin further, and it colocalizes with prorenin in the human5 and rat kidney JG cell14 and human prolactin-producing pituitary cell15 secretory granules. The optimum pH for prorenin processing by cathepsin B is 6, consistent with the pH inside the secretory granule of 5.6.13 Moreover, a thiolprotease purified in the human kidney can accurately cleave the 43-amino-acid prosegment of human recombinant prorenin without degrading renin.16 This enzyme is identical to cathepsin B in its molecular weight and in a partial amino-terminal amino acid sequence.5
Nevertheless, these excellent correlations do not demonstrate that cathepsin B is the renal proreninprocessing enzyme. Of the remaining unanswered questions, it is not known whether in secretory cells containing the regulated secretory pathway, cathepsin B localizes to the appropriate subcellular compartment to allow conversion of prorenin to renin and whether it can function in this capacity in this setting.
Cultured rat pituitary GH4C1 cells serve as a good model to address the latter issue, since they contain a regulated secretory pathway and are known not to process prorenin expressed from a transfected vector.17 In the present studies, we addressed the issue of whether cathepsin B localizes to the appropriate subcellular compartment and processes prorenin by coexpressing prorenin with cathepsin B in GH4C1 cells. The data demonstrate that cathepsin B can activate prorenin into renin in vivo, consistent with a role for this enzyme as a prorenin processing species in the cell.
| Methods |
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Plasmid Constructions
The human preprocathepsin B expression
vector was constructed by
inserting the Pst I/BamHI fragment of plasmid
phCB79-218 (kindly provided by D.F.
Steiner), which encodes human preprocathepsin B downstream of the RSV
long-terminal repeat in plasmid pUC 9. The human prorenin
expression vector pRhR1100 (driven by an RSV promoter),19
human procathepsin D (driven by a CMV promoter)20 (kindly
provided by J.M. Chirgwin), and mouse PC1 (driven by a CMV
promoter)9 (kindly provided by N.G. Seidah) have been
described previously.
Cell Culture and DNA Transfection
Rat GH4C1 cells were
maintained in monolayer culture in DME H21
supplemented with 10% FCS, 2 mmol/L glutamine, 50 U/mL penicillin, and
50 µg/mL streptomycin. Cells were grown at 37°C in 6%
CO2. To increase the number of secretory granules, the
cells were treated for 7 to 9 days before transfection with human
epidermal growth factor (10 nmol/L) (Collaborative Biomedical
Products), estrogen (1 nmol/L), and insulin (300
nmol/L).21 Cells were transfected with 10 µg pRhR1100
and either 40 µg RSV vector (neutral plasmid), 40 µg human
cathepsin B, 40 µg human cathepsin D, or 40 µg mouse PC1. The cells
were electroporated (Bio-Rad Gene Pulser) at 960 µF and 0.3 kV at a
density of approximately 5x106 cells in 0.4 mL PBS
containing 0.1% glucose and 10 µg/mL BioBrene (Applied Biosystems).
Each electroporation was split into two 2.5-cm wells. Twenty-four
to 36 hours after transfection, the cells were washed twice with PBS
and incubated with 1 mL fresh medium either with or without
secretagogues. The secretagogues were 50 mmol/L KCl and 10 µmol/L
forskolin in ethanol.22 23 Control cells without
secretagogue were treated with 50 mmol/L NaCl plus ethanol. After 16
hours, the culture medium was collected for assay of prorenin and
renin.
Radiolabeling and Immunoprecipitation
At 24 to 36 hours after
the transfection, the cells were washed
twice with PBS and grown for 16 hours in originally methionine-free
DME H21 containing 0.15 mCi/mL [35S]methionine (Du
Pont/New England Nuclear) and 5% dialyzed FCS with or
without secretagogue. Cell culture media were collected and
immunoprecipitated with an antibody that recognizes both renin and
prorenin or with an antibody that recognizes only prorenin (a
prosegment-specific antibody) with protein ASepharose (Sigma) as
previously described.19 The precipitates were
analyzed on 12% polyacrylamide gels containing 0.1%
SDS.
Renin and Prorenin Assay
Prorenin and renin levels were
determined by enzymatic assays of
angiotensin I generation (Incstar Corp) as previously
described.19 Briefly, active renin is determined by the
ability of the sample to generate angiotensin I from
nephrectomized sheep serum substrate (kindly provided by R.A.S. Gomes,
Federal University of Uberaba, Minas Gerais, Brazil). Renin levels were
determined before and total renin (renin plus prorenin) was determined
after activation with 50 µg/mL trypsin at 21°C for 1 hour. Prorenin
levels were calculated as the difference between these two values. The
percentage of active renin represents the active renin divided
by total renin times 100.
The percentage of active renin is expressed as mean±SEM; the data were analyzed by the Kruskall-Wallis test, and multiple comparisons were made with Dunn's test. Values of P<.05 were considered statistically significant.
| Results |
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To test the ability of cathepsin B to process prorenin, GH4C1 cells
were cotransfected with preprorenin expression vector and a plasmid
that expresses human preprocathepsin B. As shown in Fig 1
, when
the
cells expressed cathepsin B, the relative amount of renin secreted into
the culture medium in the absence of secretagogues was 8.6% of total
renin, and when the cells were treated with secretagogues, the relative
amount of renin secreted into the medium increased to 12% of total
renin. This result suggests that cathepsin B processed prorenin into
renin in the secretory granules.
Mouse PC1 has been shown to process prorenin to renin in GH4C1
cells.9 10 GH4C1 cells were cotransfected with
vectors
that express prorenin and mouse PC1. As summarized in Fig 1
,
when the
cells expressed PC1, the relative amount of renin secreted into the
cell culture medium by GH4C1 cells in the absence of secretagogues was
12% of total renin; this value increased to 16.5% of total renin with
secretagogue stimulation. By contrast, cotransfection of human
preprorenin and human preprocathepsin D expression vector did not
increase the basal or secretagogue-stimulated release of renin,
which was 6% of total renin in both cases.
Prorenin processing by GH4C1 cells was characterized further by PAGE.
Cells that had been transfected with the prorenin expression
vector or cotransfected with the human prorenin and human procathepsin
B or with the human prorenin and mouse PC1 expression vectors were
labeled with [35S]methionine. Culture media from cells
grown in the presence or absence of secretagogues were
immunoprecipitated with an antibody that recognizes both prorenin and
renin, and the products were analyzed by SDS-PAGE (Fig 2
).
Prorenin, identified as a protein of 47 kD, is
present in all lanes. Renin, identified as a protein of 43 kD, is
observed only in the media of cells that expressed either cathepsin B
or mouse PC1 (lanes 3 and 4 and 7 and 8, respectively). In the presence
of secretagogues, the quantity of the renin protein band is increased,
confirming the renin assay results.
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To confirm that the 43-kD species is renin produced by cleavage of the
prosegment and is not the result of a carboxy-terminal cleavage,
GH4C1 cells were cotransfected with the prorenin and either the
procathepsin B or PC1 expression vectors and labeled with
[35S]methionine. The cells were treated with
secretagogue, and cell culture media were precipitated with either an
antibody that recognizes only the prosegment of prorenin or an antibody
that recognizes both prorenin and renin (see "Methods"). The
immunoprecipitated products were analyzed by SDS-PAGE
(Fig 3
). The antibody that recognizes renin and prorenin
precipitated both the 47- and 43-kD species (lanes 1 and 3). In
contrast, the prosegment-specific antibody precipitated only the
47-kD species (lanes 2 and 4). Taken together, these results suggest
that the 43-kD band is authentic renin produced by the removal of the
amino-terminal prosegment.
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| Discussion |
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Our results also confirm previous results that mouse PC1 can process prorenin into renin in GH4C1 cells.9 10 Although PC1 can process prorenin in GH4C1 cells and may be the enzyme responsible for prorenin processing in mouse pituitary AtT20 cells,24 this enzyme is expressed mainly in neuroendocrine cells25 26 and has not been found in JG cells.10 Thus, PC1 is an unlikely candidate to be the renal prorenin processing enzyme. Recently, another prohormone convertase, mouse PC5, has been proposed to process prorenin into renin in GH4C1 cells, and its RNA has been found in As4.1 JG cells derived from a transgenic mouse kidney tumor.27 However, further studies are necessary to investigate the role of PC5 in the renal processing of prorenin.
The percentages of active renin secreted by GH4C1 cells expressing prorenin and cathepsin B, 12%, or expressing prorenin and PC1, 17%, were not very high. Nevertheless, these differences did represent more than twice the amount of active renin secreted from cells expressing only prorenin, and they were reproducible. The value with PC1 is less than the 25% observed by Benjannet et al.9 This variation may be due to different transfection efficiencies that can result with the calcium phosphate precipitation method. Additionally, we did not measure the fraction of procathepsin B that was correctly targeted to the regulated secretory pathway and converted into active cathepsin B. It is likely that some fraction of the cathepsin B was targeted to lysosomes. Although it has been suggested that procathepsin B is converted into mature cathepsin B by autocatalysis,28 it is possible that inefficient targeting and activation of human procathepsin B in rat pituitary cells could contribute to the low prorenin conversion given by cathepsin B in our experiments.
Unlike the case with cathepsin B and PC1, we were unable to observe increased processing of prorenin with a vector that expressed cathepsin D. The latter colocalizes with renin in secretory granules of JG cells,29 and it has been shown that cathepsin D can activate prorenin in human amniotic fluid.6 However, the optimum pH for this activation (4.8) is less than the range expected of an authentic renal prorenin convertase because the pH inside the secretory granules is 5.6.13 Furthermore, cathepsin D was relatively ineffective in processing human recombinant prorenin at pH 4.6 and did not cleave a synthetic peptide spanning the human prorenin processing site, suggesting that the activation of prorenin by cathepsin D does not occur at the authentic processing site.30 Nevertheless, the data do not exclude the possibility that cathepsin D can function as a prorenin-processing enzyme. We did not prove that cathepsin D was expressed in GH4C1 cells or that it was targeted to the secretory granules with prorenin. Unlike the other members of the aspartic proteinase family, which are mostly secretory proteins, procathepsin D is sorted primarily to the lysosomes,31 and intracellular localization of cathepsin D may differ from one cell to another. Thus, further studies would be needed to define the potential role of cathepsin D in prorenin processing.
The evidence obtained in the present studies that cathepsin B can process prorenin to renin in the cell is complementary to the in vitro evidence cited in the introduction showing that cathepsin B cleaves the prorenin prosegment, does not further degrade renin, has an optimum pH close to the pH of secretory granules, and colocalizes with renin in JG cell secretory granules. This collective evidence is consistent with a role for cathepsin B as a renal proreninprocessing enzyme. Nevertheless, these studies do not demonstrate whether this enzyme is the actual prorenin-processing enzyme in the kidney, and further studies will be needed to address this issue with respect to cathepsin B, PC5, or possibly other enzymes.
| Selected Abbreviations and Acronyms |
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| Acknowledgments |
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| References |
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