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NEUROENDOCRINOLOGY |
Department of Molecular Medicine, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371-8512, Japan
Address all correspondence and requests for reprints to: Dr. Tetsuro Izumi, Department of Molecular Medicine, Institute for Molecular and Cellular Regulation, Gunma University, 3-39-15 Showa-machi, Maebashi, Gunma 371-8512, Japan. E-mail: . tizumi{at}showa.gunma-u.ac.jp
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Abstract |
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Introduction |
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Rab27a is known to play a critical role in the intracellular transport of the so-called secretory lysosomes, such as melanosomes in melanocytes, lytic granules in cytotoxic T lymphocytes, and platelet-dense granules (5). The roles of Rab27a in secretory lysosomes are genetically supported because its mutations in mice (6) and humans (7) cause symptoms reflecting the dysfunction of these organelles, such as pigmentary dilution, immunodeficiency, and increased bleeding time. However, endocrine disorders have not been reported in these mice or in human subjects. The Rab27 subfamily consists of Rab27a and Rab27b (8). Although the function of Rab27b is not clear, it may have a role similar to that of Rab27a, as there are highly conserved primary sequences between the two proteins. If this is the case, the apparent lack of endocrine disorders in Rab27a-mutated mice and humans may be due to functional compensation by Rab27b, which may coexist in these tissues.
In the present study we characterized the protein product of Rab27b to verify this hypothesis. We found that Rab27b, like Rab27a, is highly expressed in pituitary tissue. Furthermore, Rab27b associates with secretory granules and granuphilin, an effector protein of Rab27a, in the corticotroph cell line AtT20. Moreover, overexpression of its inactive mutants significantly inhibits ACTH secretion in these cells. These findings support the idea that both Rab27a and Rab27b regulate the exocytosis of dense core granules containing peptide hormones in endocrine cells.
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Materials and Methods |
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Antibodies
Rabbit anti-Rab27b antibodies against GST-fused mouse Rab27b protein were produced as described previously (4). The antibodies were purified from the serum by affinity chromatography on a column of the GST-fused Rab27b protein coupled to Affi-gel 15 (Bio-Rad Laboratories, Inc., Hercules, CA). Rabbit antibodies (
Grp-N) that recognize both granuphilin-a and granuphilin-b have been described previously (4). Anti-Rab27a and anti-Rab3a monoclonal antibodies were purchased from BD Transduction Laboratories, Inc., Lexington, KY. Anti-Xpress monoclonal antibodies were purchased from Invitrogen. Mouse anti-ACTH and goat anti-ß-subunit of TSH antibodies were purchased from Biogenesis (Poole, UK). Rabbit anti-ACTH, guinea pig anti-GH, guinea pig anti-PRL, and mouse anti-ß-subunit of LH antibodies were gifts from Dr. S. Tanaka (Shizuoka University, Shizuoka, Japan). Rabbit anti-ß-subunit of LH antibodies were gifts from Dr. K. Wakabayashi and H. Kobayashi (Gunma University, Maebashi, Japan).
Mice tissue preparation
All animal experiments were performed in accordance with the rules and regulations of animal care and experimentation committee, Gunma University, Showa campus. The C57BL/6J mice were purchased from CLEA Japan, Inc. (Tokyo, Japan). Tissues were immediately excised from 9- to 12-wk-old C57BL/6J mice (18–20 g BW) killed by cervical dislocation and solubilized in lysis buffer containing 50 mM HEPES (pH 7.4), 1% Triton X-100, 150 mM NaCl, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml aprotinin, pepstatin A, and leupeptin. For the pancreatic islet preparation, mice were anesthetized with an ip injection of sodium pentobarbital. Islets were isolated by pancreatic duct injection of 500 U/ml collagenase solution (type XI, Sigma, St. Louis, MO), followed by digestion at 37 C for 40 min with mild shaking as described previously (9).
Immunostaining analyses
The pituitary was excised from C57BL/6J mice that had been anesthetized and perfused for 20 min with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4., and immersed for 1 d at 4 C in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) containing 3% sucrose. After cryoprotection in 10% followed by 20% sucrose PBS and embedding in Tissue-Tek OCT compound (Sakura Finetek U.S.A., Torrance, CA), the specimens were rapidly frozen in liquid nitrogen. Sections (4 µm) were cut and placed on silane-coated slides as described previously (9). AtT20 cells, plated onto eight-well Lab-Tek chamber slides (Nunc, Naperville, IL), were fixed with 4% paraformaldehyde for 30 min on ice and permeabilized with 0.1% Triton X-100. The specimens were incubated overnight at 4 C with the primary antibodies, which were diluted at 1:2000 for anti-TSH, 1:1000 for anti-Rab27b, 1:500 for anti-Xpress, 1:300 for antigranuphilin and anti-ACTH, 1:200 for anti-GH and anti-LH, 1:100 for anti-Rab3a, and 1:50 for anti-Rab27a and anti-PRL antibodies. They were further incubated for 1–2 h at room temperature with corresponding indocarbocyanine-conjugated (1:500 dilution) or fluorescein isothiocyanate-conjugated (1:200 dilution) species-specific anti-IgG secondary antibodies (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). Immunofluorescence was viewed with a BX50 microscope equipped with an epifluorescence attachment (Olympus Corp., Tokyo, Japan). Images were acquired with a SenSys charge-coupled device camera (Photometrics, Tucson, AZ).
In vitro binding assay with GST fusion proteins and immunoprecipitation
GST-fused recombinant proteins were expressed in Escherichia coli XL-1 Blue, and affinity-purified with glutathione-Sepharose 4B (Amersham Pharmacia Biotech). GST-fused proteins immobilized on beads (5 µg each) were incubated at 4 C for 3 h with AtT20 cell extracts (2 mg protein) prepared in the lysis buffer described above. For coimmunoprecipitation experiments, anti-Rab27b or anti-Xpress antibodies (1.5 µg) were incubated with AtT20 cell extracts on ice for 2 h and immobilized on protein G-Sepharose 4FF (Amersham Pharmacia Biotech) at 4 C for 1 h. The beads were washed four times with lysis buffer. The bound proteins were separated by SDS-PAGE and analyzed by immunoblotting with antigranuphilin antibodies (1:1500 dilution). Antibody detection was accomplished using enhanced chemiluminescent Western blotting detection reagents (Amersham Pharmacia Biotech).
Preparation of recombinant adenovirus and measurement of ACTH secretion
Recombinant adenoviruses were prepared as described previously (4). The cDNAs of wild-type and mutant Rab27b with the Xpress epitope tag at the N terminus were subcloned into pAxCAwt (TaKaRa Biomedicals, Kusatsu, Japan). For the ACTH secretion assay, AtT20 cells were seeded at a density of 3 x 105 cells in 12-well dishes (2.5-cm plate). The next day the cells were infected with recombinant adenoviruses. Forty hours later the cells were rinsed twice with serum-free DMEM, then incubated for 1 h in the same medium containing 0.1% BSA in the presence or absence of 10 µM forskolin. The media were collected and centrifuged at 3000 x g for 5 min to remove any cells detached from the dish during incubation. ACTH was measured using an ACTH immunoradiometric assay kit (Yuka Medias, Inashiki-gun, Japan).
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Results |
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Rabbit antibodies were produced against GST-fused mouse Rab27b protein. Because of the highly homologous sequences between Rab27a and Rab27b, anti-Rab27b antibodies as well as the commercially available mouse anti-Rab27a antibodies previously used (4) were first examined for specificity. For this purpose we established AtT20 cells that stably overexpress either Rab27a or Rab27b with the Xpress epitope tag at the respective N termini. Anti-Rab27b antibodies specifically recognized Xpress-tagged Rab27b and a 29-kDa protein, presumably endogenous Rab27b, but not Xpress-tagged Rab27a, in AtT20 cells (Fig. 1
, lanes 5–8). Similarly, anti-Rab27a antibodies specifically detected Xpress-tagged Rab27a and a 28-kDa protein, presumably endogenous Rab27a, but not Xpress-tagged Rab27b (Fig. 1, lanes 1–4). Thus, anti-Rab27a and anti-Rab27b antibodies specifically recognized a corresponding Rab27 isoform. Although AtT20 cells expressed both Rab27a and Rab27b, the pancreatic ß-cell line MIN6 expressed only Rab27a, but not Rab27b (Fig. 1; see also Fig. 2).
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, upper panel). Among the tissues and cells examined, Rab27b was most abundantly expressed in pituitary tissue and AtT20 cells. It was also expressed significantly in brain, spleen, and pancreas tissues and weakly in lung and testis. Compared with the whole pancreas, pancreatic islets faintly express Rab27b, suggesting that it is mainly expressed in the exocrine cells. Consistently, Rab27b was not expressed in MIN6 cells and was only weakly expressed in the pancreatic -cell line TC1.6 (Fig. 2, lower panel). Thus, Rab27a and Rab27b show distinct expression patterns, especially in brain tissue, although both proteins are commonly expressed in pituitary tissues. Because Rab27a regulates melanosome transport in melanocytes (5), we examined the expression of Rab27b in cultured melanocyte cell lines. In contrast to the significant expression of Rab27a in melan-a and B16 melanoma cell lines (4), Rab27b was not expressed in these cells (Fig. 2, lower panel).
The pituitary consists of multiple endocrine cells that express different peptide hormones. To further examine the distribution of Rab27b, we performed an immunohistochemical analysis on the mouse pituitary specimen (Fig. 3). We also examined the distributions of granuphilin, Rab27a, and Rab3a (Fig. 4 and Table 1). Among the five anterior pituitary cell types, both Rab27a and Rab27b were strongly expressed in LH- and PRL-producing cells. Rab27a was not detectable in other cell types, whereas Rab27b was significantly expressed in ACTH- and TSH-producing cells also, but not in GH-producing cells. Rab3a was expressed only in thyrotrophs. Granuphilin was expressed in ACTH-, PRL-, GH-, and TSH-positive cells, but not in LH-positive cells. We cannot, however, exclude the possibility that these proteins are expressed below the detection levels of the antibodies in the negatively stained cells. Nevertheless, we concluded that Rab27a, Rab27b, Rab3a, and granuphilin are not uniformly expressed in pituitary endocrine cells.
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). A similar immunostaining pattern was observed for endogenous Rab27b in AtT20 cells (data not shown). These findings suggest that Rab27b is associated with secretory granules, as in the case of Rab27a in pancreatic ß-cells (4).
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, upper panel). Rab27b immunoprecipitates contained a significant amount of granuphilin, whereas Rab3a/granuphilin complexes were scarcely detectable (Fig. 6A, lower panel). To further investigate whether this interaction occurs under physiological conditions, we next performed coprecipitation assays without overexpression of Rab27b. Rab27b immunoprecipitate from AtT20 cells contained a significant amount of granuphilin (Fig. 6B). The absence of granuphilin in the immunoprecipitates of control serum suggests that the interaction between Rab27b and granuphilin is specific. An in vitro binding assay using GST fusion Rab proteins also showed that granuphilin is specifically bound to either GST-fused Rab27a or Rab27b, but not to GST-fused Rab3a or GST alone (Fig. 6C). All of these findings indicate that Rab27b forms a stable complex with granuphilin-a and -b in the same manner as Rab27a.
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Immunoblotting analysis with anti-Rab27b antibodies indicated that each form of Rab27b is expressed much more abundantly than the endogenous protein (Fig. 7A, upper panel). The immunoprecipitates of anti-Xpress antibodies from cells expressing wild-type Rab27b and Rab27b Q78L contained significant amounts of granuphilin (Fig. 7A, lower panel). In contrast, neither Rab27b T23N nor N133I formed significant complexes with granuphilin, indicating that only active forms of Rab27b interact with granuphilin.
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(Fig. 7B). The addition of forskolin, which stimulates adenylate cyclase and thereby increases the cytosolic concentration of cAMP, caused an approximately 3-fold increase in ACTH release in AtT20 cells. Overexpression of ß-galactosidase, wild-type Rab27b, or Rab27b Q78L did not significantly affect either basal or forskolin-induced ACTH secretion. Although Rab27a T23N and N133I were extremely difficult to express in MIN6 (4) and AtT20 cells (data not shown), corresponding Rab27b mutants were efficiently expressed (Fig. 7A, upper panel). Dominant inhibitory effects of the corresponding mutations in different vesicle transport assay setups have been reported for numerous Rab proteins (11). Although Rab27b T23N caused only weak, but significant, inhibition of forskolin-stimulated ACTH secretion, Rab27b N133I strongly inhibited ACTH secretion in both basal (17% inhibition) and forskolin-stimulated states (35% inhibition; Fig. 7B). Thus, forskolin-inducible ACTH release (forskolin-stimulated minus basal secretion) was decreased by 50% in cells overexpressing Rab27b N133I compared with uninfected AtT20 cells or those expressing ß-galactosidase. This effect is considered specific because the inhibition of ACTH secretion was dependent on the expression levels of Rab27b N133I (Fig. 8). These findings strongly suggest that Rab27b positively regulates ACTH secretion in AtT20 cells.
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Discussion |
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Despite these findings endocrine disorders have not been reported in either ashen mice (6) or patients with Griscelli syndrome (7), where both alleles of the Rab27a gene are mutated, although a detailed analysis of their endocrine functions has yet to be performed. Instead, they exhibit pigmentary dilution of the skin and hair and accumulation of melanosomes in melanocytes (6, 7). These phenotypes might be attributed to the tissue distributions of Rab27a and its homologous protein, Rab27b. We demonstrate that both proteins are highly expressed in pituitary tissues, whereas only Rab27a, and not Rab27b, is expressed in melanocyte cell lines. The dysfunction of Rab27a in ashen mice and Griscelli patients could be overt in melanocytes due to the absence of Rab27b, but could be complemented in pituitary tissue by coexisting Rab27b. It should be noted, however, that Rab27a and Rab27b are not uniformly expressed in pituitary endocrine cells. For example, Rab27a is only expressed in gonadotrophs and lactotrophs. Thus, the mutation of Rab27a in ashen mice and Griscelli patients should not cause the dysfunction of other pituitary endocrine cell types regardless of whether Rab27b is coexpressed. Furthermore, despite the expression of granuphilin, neither Rab27a nor Rab27b is expressed in somatotrophs. Conversely, despite the expression of Rab27a and Rab27b, granuphilin is not significantly expressed in gonadotrophs, suggesting the existence of other Rab27 effector proteins in these cells. The thought of cell-specific Rab27 effectors is supported by recent work discovering another possible Rab27a effector in melanocytes, melanophilin, whose genetic alterations cause pigmentary dilution in leaden mice (12). Although the significance of these complex differential distributions is not known, their differential expression may reflect only quantitative differences, because it was judged solely based on immunohistochemical analysis.
In addition to mutations of Rab27A, those of MYO5A, which encodes myosin Va, cause Griscelli syndrome (13). Patients with mutations of MYO5A, however, have a unique neurological impairment as well as pigmentary dilution of skin and hair (7). Similarly, dilute-lethal mice, whose myosin-Va gene is mutated, show a unique neurological defect in addition to a coat of a lighter color (14). These genetic findings together with the recent biochemical observation that myosin-Va coimmunoprecipitates with Rab27a in extracts from melanocytes (15) suggest a functional link between Rab27a and myosin-Va that probably regulates melanosome transport (5). The presence or absence of neurological phenotypes is well correlated with the expression pattern of the two proteins: expression of myosin-Va in brain tissue has been documented (14, 16), whereas no expression of Rab27a has been detected in brain tissue (4, 8). As Rab27b is significantly expressed in brain tissue, as shown in the present study, it is possible that myosin-Va functions with Rab27b in this tissue.
The efficient expression of Rab27b T23N and N133I allowed us to examine the effects of these mutants, which may act in a dominant negative manner and which could not be assessed for Rab27a because of poor expression of the corresponding mutants (4). Consistent with our expectation, overexpression of Rab27b T23N and N133I did inhibit ACTH secretion in AtT20 cells. However, we could not detect the effect of wild-type Rab27b or its active mutant, Rab27b Q78L, on ACTH secretion in AtT20 cells. This is in contrast to the enhancement of high K+-induced insulin secretion by the corresponding Rab27a proteins in MIN6 cells (4). Although the experimental conditions are different between the two studies, another reason for the discrepancy between them may be the different expression levels of Rab27-interacting proteins between the two cell lines. For example, the expression level of granuphilin in AtT20 cells was much lower than that in MIN6 cells (our unpublished observation). Overexpressed Rab27a could augment hormone secretion in the presence of a high level of granuphilin in MIN6 cells, but could fail to do so with a low level of granuphilin in AtT20 cells. Alternatively, other Rab27 effector proteins may exist and function in AtT20 cells. In any case, these findings indicate that both Rab27a and Rab27b positively regulate peptide hormone secretion.
In summary, the present study demonstrates that Rab27b as well as Rab27a are involved in the exocytosis of dense core granules in endocrine cells, although further studies are required to clarify their differential and/or redundant roles.
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Acknowledgments |
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Footnotes |
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Abbreviations: EST, Expressed sequence tag; GST, glutathione-S-transferase.
Received October 17, 2001.
Accepted for publication January 29, 2002.
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) due to the Xpress tag.
) or presence of 10 µM forskolin (
). ACTH in the medium was measured and corrected by the protein concentration of the cell extract. The results are given as the mean ± SEM of three independent experiments. *, P < 0.05; ***, P < 0.0005 (vs. AtT20 cells infected with the same titer of the virus bearing ß-galactosidase cDNA).
and 
) at different titers (15, 30, and 60 plaque-forming units/cell, multiplicity of infection). The cells were incubated for 1 h in the absence (