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Published online before print October 2, 2008
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From the Department of Respiratory Medicine,* Kanazawa Medical University, Ishikawa, Japan; Department of Human Pathology, Yamagata University Graduate School of Medicine, Yamagata, Japan; and Program in Cell Biology, National Jewish Medical and Research Center, Denver, Colorado
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Abstract |
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The Rab38 cDNA was originally cloned from a rat lung cDNA library (GenBank Accession No. M94043), and later from a human melanoma cDNA library.5-6 Although many Rab proteins are ubiquitously expressed in a variety of tissues and cells, the expression of some Rab proteins (including Rab38) are selectively regulated and dependent on cell type (epithelial, neuronal, endocrine, or exocrine) and specific cell state (differentiation or polarization). Rab38 mRNA and protein are expressed predominantly in the lung, skin, stomach, liver, and kidney in rats.7 Rab38 is specifically expressed in alveolar type II and Clara cells in rat lung tissue.5 These lung-specific cells synthesize and secrete lung surfactant that lowers alveolar surface tension and thereby maintains alveolar lumen in an open and relatively dry state.
Chocolate (cht) is an autosomal recessive mutation that arose spontaneously in C57BL/6J mice.8 The mutant mouse with its phenotype of oculocutaneous albinism was recently identified as having a point mutation in the Rab38 gene.9 Genomic DNA analysis revealed a G146T transversion that resulted in a replacement of glycine with valine in the conserved GTP/GDP-interacting domain near the N-terminus. Rab38 appears to participate in trafficking of melanogenic enzymes in skin melanocytes10 and retinal pigment epithelial cells.11 This mutation predicts a loss of GTP/GDP-binding activity and a resultant GTP/GDP molecular switch dysfunction. However, molecular characterization of the mutant gene product (Rab38cht) has not been performed. Moreover, because of the high level of lung expression, it seemed probable that the chocolate mutation would also cause lung disease in addition to oculocutaneous albinism.7 Thus, the purpose of this study was to characterize possible effects of the Rab38-G146T on phenotype of the mouse lung and to provide molecular basis of the dysfunction of the mutant gene product (Rab38cht protein).
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Common chemicals were purchased from Sigma (St. Louis, MO) or Wako Chemicals (Osaka, Japan). Rabbit anti-human SP-A, anti-sheep SP-B, anti-mouse SP-D, and mouse anti-glyceraldehyde-3-phosphate dehydrogenase antibody were from Chemicon (Temecula, CA). Horseradish peroxidase-conjugated goat anti-rabbit IgG antibody and anti-mouse IgG antibody were from Bio-Rad (Hercules, CA). A chemiluminescent detection kit, a stripping buffer, and a micro-BCA protein assay kit were from Pierce (Rockford, IL). Autoradiography films and autofluorography films for Western blot analysis were from Kodak (Rochester, NY). The thin-layer chromatography apparatus was from Advantec (Tokyo, Japan), and Silica gel G plates were from Analtec (Uniplate, Newark, DE). Insect cell lines (Sf9 cells), Bac-N-Blue transfection kit, pBlueBacHis2-A plasmid, and Ni++-charged affinity chromatography resin were from Invitrogen (Carlsbad, CA).
Mice
All animal protocols were reviewed and approved by the Institutional Animal Care and Use Committee in Kanazawa Medical University. Two couples of a male chocolate heterozygous (cht/+) mouse and a female chocolate homozygous (cht/cht) mouse were purchased from the Jackson Laboratory (Bar Harbor, ME). Specific-pathogen-free animals were generated by rederivation using an embryo transfer method in Japan Charles River Inc. (Yokohama, Japan). The genotypes of chocolate mice were determined by DNA sequence of PCR products of exon 1 of genomic DNA. Rab38 primer pairs were designed to amplify exon 1: Ex1F (5'-TAGGAAGGAGGATTAAACCCG-3') and Ex1R (5'-GAACTCCTCATGGCTCACTCC-3'), yielding a 428-bp product. The chocolate mutation was confirmed by using primers designed to amplify a 213-bp fragment containing the G146T sequence: cht Ex1F (5'-GGCCTCCAGGATGCAGACACC-3') and cht Ex1R (5'-CCAGCAATGTCCCAGAGCTGC-3').9
Lung Histology
Male mice at 12 and 24 weeks old were sacrificed by severing the abdominal aorta under intraperitoneal anesthesia with sodium pentobarbital (
100 mg/kg body weight) plus sodium heparin (3300 units/kg body weight). The lungs and heart were removed en bloc. The lungs were carefully inflated with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) at a constant hydrostatic pressure of + 25 cmH2O at the height of the carina in the upright position. After the lung volume was measured by water displacement, the right and left lungs were separated, embedded in paraffin, and cut into 5 µm-thick sections in the sagittal plane so as to contain the largest lung area. Care was taken to cut the fixed lungs at the same thickness without any distortion. The lungs were stained using H&E.
Morphometry
An imaginary rectangle whose four sides touched the outline of a lung slice was defined on the microscope stage and was divided into more than 40 fields of the same size. Each field was photographed at 295 x 223 µm2 and contained 35 equidistant test points; two test lines connecting the opposite angle, both 370 µm in length, were drawn on the field. Every test point was classified into one of six categories based on its histological component (alveolar air space, air space in alveolar ducts and sacs, alveolar wall, bronchial and bronchiolar wall, blood vessel wall, and other). Large airways and vessels were excluded from the analysis. The numbers of test points were summed for each of the tissue components, and the volume proportion of the defined tissue component was expressed as a proportion of the total number of points according to the point-counting method.12
Based on the point-counting method modified by Kawakami and associates,13
mean thickness of the alveolar wall (
w), mean linear intercept (Lm), mean chord length of alveoli (la), and mean chord length of ducts and sacs (ld) were calculated. Using another point-counting system, an index of parenchymal destructive change that represents the percentage of destroyed space as a fraction of the total alveolar and duct space, referred to as the destructive index (DI), was calculated.14,15
Lung physiology
Male mice at 24 weeks old were used for measurement of lung pressure-volume relationships. Mice were anesthetized by intraperitoneal injection of sodium pentobarbital (100 mg/kg body weight). The trachea was exposed and cannulated with an 18G catheter attached to a three-way stopcock. The mice were mechanically ventilated with 100% O2 (0.5 ml tidal volume and 60 cpm) (Harvard model 683, South Natick, MA) for 10 minutes, followed by 5 minutes closure of the trachea to completely degas the lungs.16
The mice were exsanguinated and the anterior chest and diaphragm were dissected away. The lungs were attached to a syringe pump (Minato MCIP-III, Tokyo, Japan) and a semiconductor pressure transducer (Kulite XCW-190-5D, Leonia, NJ) through the three-way stopcock, and inflated in stepwise fashion with small increments of air (0.1 ml every 20 seconds) to a maximal pressure of 25 cmH2O. The lungs were deflated in stepwise fashion (2.5 cmH2O decrements every 5 seconds) down to –10 cmH2O. This inflation-deflation cycle was repeated two times, and the third cycle was used for analysis, except for airway opening pressure that was measured from the first cycle inflation. Care was taken not to cause air leak from the lungs. When air leak was found, the lungs were discarded. Pressure-volume curves were plotted and used to assess lung compliance, which was defined as a slope of the linear region of the deflation limb where pressure ranged from 0 to 5 cmH2O. Specific lung compliance was calculated as lung compliance divided by the lung volume at 25 cmH2O. The hysteresis ratio was determined as described by Nardell and Brody.16
Electron Microscopy
Mice at 18 weeks old and 17 days fetal mice were used. The excised lungs were cut into small pieces and fixed for 2 hours with fresh fixative containing 2.5% glutaraldehyde/0.1% picric acid/2% osmium tetroxide/4% sucrose/0.1 M cacodylate buffer (pH 7.4). Blocks were post fixed with 1% aqueous uranyl acetate solution for 1 hour followed by dehydration in a graded series of ethanol and subsequent propylene oxide, and were finally embedded in Epon. Thin sections were counterstained with 2% uranyl acetate, and then 2.66% lead nitrate/3.52% sodium citrate (pH 12). The sections were examined using a Hitachi H-7100 transmission electron microscope.
Lamellar Body Isolation
Male mice at 18 weeks old were used. One ml of saline was infused into the trachea and gently recovered; this procedure was repeated four times and yielded approximately 4.5 ml lavage fluid. The cell-free bronchoalveolar lavage (BAL) fluids were concentrated 10-fold with a centrifugal filter (MWCO5000). The remaining lungs were cut into small pieces and homogenized in 10 mmol/L Hepes (pH 7.4). Small aliquots were saved as lung homogenate. The remaining homogenates were adjusted to 0.9 M sucrose/10 mmol/L Hepes (pH 7.4)/2 mmol/L MgCl2 and layered on a 1.5 M sucrose cushion. Consecutive sucrose steps from 0.8 to 0.2 M were overlaid above the homogenate, and the discontinuous sucrose density gradients were centrifuged at 100,000 x g for 3 hours.17,18 The 0.4 to 0.6 M layers were recovered as the lamellar body (LB) fraction. Protein concentrations were determined by deoxycholate-trichloroacetic acid precipitation and a bicinchoninic acid microprotein assay kit.
Fixed amounts of protein were subjected to 4% to 12% Bis-Tris SDS-polyacrylamide gel electrophoresis (PAGE) under reducing conditions and transferred to a nitrocellulose membrane. The membrane was immunoblotted with a rabbit anti-surfactant protein polyclonal antibody or a mouse anti-glyceraldehyde-3-phosphate dehydrogenase antibody, followed by a horseradish peroxidase-conjugated goat anti-rabbit (or goat anti-mouse) IgG antibody. The chemiluminescent detection assay was performed and exposed on an autofluorography film.
Phospholipid Assay
Male mice at 24 weeks old were used to prepare BAL fluids, lung homogenates, and lamellar body fractions. Total lipids were extracted according to the method of Bligh and Dyer.19 In all samples, 20% of each sample was used for phospholipid phosphorus content determination with the method described by Bartlett.20 The remaining 80% of each sample was used for two-dimensional thin-layer chromatography.21 After development, the silica gel spot corresponding to phosphatidylcholine was scraped off and recovered quantitatively, and the phosphorus content quantified.
Triton X-114 Phase Separation of Mouse Lung Homogenates
Mouse lungs were cut into small pieces and homogenized in 10 mmol/L Hepes (pH 7.4) with a Potter-Elvehjem type homogenizer, followed by centrifugation at 300 x g for 10 minutes. The supernatant was divided into two equal aliquots. One aliquot (0.5 ml) was centrifuged at 100,000 x g for 30 minutes. The volumes of the cytosolic and the pellet fractions were adjusted to be 0.5 ml. The second aliquot (0.5 ml) was processed for Triton X-114 phase separation.22
In brief, it was adjusted to 1% Triton X-114, rotated at 4°C for 2 hours, and centrifuged at 100,000 x g for 30 minutes. The supernatant was layered onto 6% sucrose/PBS/0.06% Triton X-114 in a 1.5 ml-microtube, and warmed up to 30°C for 10 minutes, followed by centrifugation at 300 x g for 10 minutes at room temperature. The upper layer was recovered as the aqueous phase. The oily droplet in the bottom was recovered as the detergent phase. The volumes of the two samples were adjusted to be equal (0.5 ml). The equal volumes of aliquots (12.5 µl) were used for Western blotting.
Protein Production and Purification
The Rab38-cDNA originally cloned from a rat lung cDNA library was inserted into a pBlueBacHis-2A vector at BamHI and HindIII restriction enzyme sites. A forward primer that contains a BamHI restriction site and an initiation codon (5'-CCGGGATCCATGCAGACA-3') and a reverse mutagenic PCR primer that carries the G146T-mutation site and adjacent HaeII restriction site (5'-GTAGCGCTTGATGATGCTGGTCTTGCCTACAACTA-3') were used to amplify the sequence that contains the G146T mutation site. The PCR product was digested with BamHI and HaeII, and inserted into the pBlueBacHis-2A-Rab38 previously digested with BamHI and HaeII. Cationic liposomes were used to co-transfect the recombinant plasmid and a modified baculovirus DNA (Bac-N-Blue DNA) into Sf9 cells. Cells that were 80% to 90% confluent in 150-mm plastic dishes were infected with the recombinant virus at a multiplicity of infection of 10, and cultured for 3 to 4 days. Ten to twenty plates of the infected Sf9 cells lysed in the lysis buffer (1% TritonX-114, 50 mmol/L Hepes, pH 7.4, 150 mmol/L NaCl, and 1.5 mmol/L EGTA) containing protease inhibitors (1 mmol/L PMSF, 10 µg/ml aprotinin, and 10 µg/ml leupeptin) were centrifuged at 100,000 x g for 30 minutes. The supernatant was loaded on a 2-ml bed volume of Ni++-charged affinity column under native conditions. Imidazole step gradients were used to elute the column.
[32P]GTP-Binding Assay
Between 0.5 and 1.5 µg of purified recombinant Rab38 was subjected to SDS-PAGE under reducing conditions. Subsequently, the proteins were electrophoretically transferred to a nitrocellulose membrane. [
-32P]-GTP binding to the proteins immobilized on the membrane was determined.23
The membrane was incubated with 60 nmol/L [-32P]-GTP [specific activity 3000Ci (111TBq)/mmol] (FP-208, Institute of Isotopes Co., Ltd., Budapest, Hungary) in 10 ml of Tris-HCl buffer for 1.5 hours at 25°C with gentle shaking on a platform shaker. The membrane was autoradiographed on an autoradiography film with an intensifying screen at –80°C for overnight. After development of the film, the membrane was washed with PBS and was probed by Western blotting with rabbit anti-rat Rab38 antibody (1:10,000)5,7
for quantifying the amount of protein.
Immunoprecipitation of [3H]-mevalonate-Radiolabeled Rab38
Sf9 cells were seeded at 3 x 106 cells/60-mm dish and infected with the recombinant baculovirus at a multiplicity of infection of 10 and cultured for 24 hours. Next, the cells were radiolabeled with 10 µCi/ml of [3H]mevalonic acid (ART-334, ARC St. Louis, MO) in Grace’s insect media with 10% fetal bovine serum (previously dialyzed against PBS) for 24 hours.24
Ten µg/ml of mevinolin (Calbio 438186) was added 3 hours before adding radioisotope. The cells were lysed with 0.5 ml of 1% Triton X-100/PBS/5 mmol/L MgCl2/antiproteases, followed by centrifugation at 100,000 x g for 30 minutes. A rabbit anti-rat Rab38 antibody5,7
was added at 4 µg/ml and the antigen-antibody complex was immunoprecipitated with 25-µl bed volume of protein A sepharose (CL-4B) beads (Sigma P-3391). The beads were eluted twice with 20 µl of 2x sample buffer containing 0.4 M dithiothreitol. This step typically yielded a total 60 µl eluate sample. A 40 µl aliquot of the sample was used for autoradiography, and the remaining 20 µl was used for Western blot with a mouse anti-Xpress antibody (Invitrogen) for evaluation of the amount of immunoprecipitated Rab38.
Statistics
Data were expressed as mean ± SD. The data were evaluated by Student’s t-test using Statview software (Abacus Concepts Inc, Berkley, CA), and P < 0.05 was considered statistically significant.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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In the C57BL/6J background, mice that are homozygous for chocolate (Rab38cht/cht) have a rich dark brown coat instead of normal black coat (Figure 1A)
. Their skin pigmentation is also lighter, as can be seen in the ear and tail. Although we could not readily distinguish any eye color differences in these mutants, H&E staining of the eyeballs revealed a thinner pigment cell layer and choroid (Figure 1B)
. These mutant mice also showed a visible decrease in the melanin levels in their hair follicles. Hence, these animals exhibit an oculocutaneous albinism phenotype. We found no differences in the bleeding time between wild-type and chocolate mice at 12 weeks (data not shown), consistent with an earlier report.9
The chocolate mice did not show any breeding or growth abnormalities during the 24 weeks observation period of our present study. No visible differences were evident between heterozygotes (Rab38cht/+) and wild-type mice, confirming recessive nature of this phenotype. DNA sequencing of these mutants revealed a point mutation, G146T, in exon 1 of the Rab38 gene, which results in glycine to valine conversion (G19V) in the GTP/GDP-interacting domain of the encoded protein (Figure 1C)
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Chocolate mice showed enlarged distal airspace in comparison with wild-type mice of the same age (Figure 2A)
. Based on the histological evaluation, no significant sign of inflammation was detected in the lung tissue sections. Total protein amounts and total cell numbers in the recovered BAL fluids were not significantly different between wild-type and chocolate mice (data not shown). In both groups, more than 96% of BAL cells were alveolar macrophages without any apparent difference in the appearance between the wild-type and the chocolate.
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, Figure 2B
). In the mutant mice of both age groups, the volume proportion of the alveolar wall was significantly smaller, and the mean linear intercept was significantly larger compared with wild-type mice. At 24 weeks, the volume proportion of alveolar duct and sacs was significantly larger in the chocolate mice. The destructive index significantly increased in the mutant animals of both age groups and was observed to increase with age.
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. Electron microscopy demonstrated increased size and number of lamellar bodies, which completely filled cytoplasm of these cells from chocolate mice (Figure 3B)
. However, despite the increase in size and number of lamellar bodies in the mutant mice, they did not show any structural abnormalities. In contrast to the adult mice, 17-day fetal mice did not show apparent morphological difference in either alveolar type II cells or lamellar bodies when compared between wild-type and chocolate mice (Figure 3, C and D)
.
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Measurement of pressure-volume relationships was performed on the in vivo lungs in the open chests (Table 2)
. Lung volume at 25 cmH2O airway pressure was significantly higher in chocolate mice than in wild-type mice. Static lung compliance was also significantly higher in chocolate mice. However, specific lung compliance, ie, static lung compliance divided by the lung volume at 25 cmH2O airway pressure, was not significantly different. Neither airway opening pressure nor hysteresis ratio was significantly different between wild-type and mutant mice.
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Lamellar bodies, lung homogenates, and BAL fluids from both wild-type and chocolate mice were analyzed by Western blotting for surfactant protein A (SP-A), surfactant protein B (SP-B), surfactant protein D (SP-D), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by sequential immunoblotting and stripping of the same membrane. A representative result is shown in Figure 4A
. SP-A was abundant in the BAL fluid, but no significant differences were evident between wild-type and chocolate mice. In contrast, SP-B was increased in the lung homogenate but decreased in the BAL fluid in chocolate mice. There was no difference in the BAL fluid SP-D. Although SP-A and SP-B appeared to be increased in the lamellar body fraction in chocolate mice, this was not statistically significant (Figure 4B)
. Phosphatidylcholine was increased in the lamellar body fraction and the lung homogenates in chocolate mice, but there was no difference in the BAL fluid. Total phospholipid levels in the samples of the lamellar body and the lung homogenate were also increased in chocolate mice but those in BAL fluids were equivalent (data not shown).
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Native wild-type Rab38 protein was partitioned into both the aqueous and the detergent phases following Triton X-114 phase partitioning of mouse lung homogenates (Figure 5
left). This indicates the protein can have both hydrophilic and hydrophobic properties. Rab38 was found to exist in both membrane-bound and cytosolic form after a series of homogenization and centrifugation treatments of lung tissue (Figure 5
right). However, Rab38cht mutant protein was exclusively partitioned into the aqueous phase and was only present in the cytosolic form. This result is consistent with the previous report of Rab38 in melanocytes derived from chocolate mice.10
Thus, Rab38cht is exclusively hydrophilic and unable to bind to membrane components in vivo.
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-32P]-GTP (Figure 6A)
. Next, Sf9 cells were infected with either of these two recombinant baculoviruses and cultured for 24 hours. These cells were radiolabeled with [3H]-mevalonate in the presence of mevinolin and incubated for a further 24 hours. The cells were lysed and the resulting postnuclear supernatants were immunoprecipitated with a polyclonal rabbit anti-rat Rab38 antibody and protein A-sepharose beads. Although equal amounts of immunoreactive Rab38wt and Rab38cht proteins were obtained, Rab38chtprotein did not undergo posttranslational modification with [3H]-mevalonate (Figure 6B)
. Thus, the mutant Rab38cht protein failed to undergo post translational prenylation.
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Discussion |
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The recombinant Rab38cht protein was unexpectedly found to retain an efficient level of GTP-binding activity, even though the amino acid mutation itself (G19V) was in the conserved GTP/GDP-interacting domain near N-terminus of the native protein.9
Although not an invariant residue within Rab family, glycine 19 is conserved in >50% of Rab proteins. The corresponding amino acid in Rab5 is alanine 30. The fact that a Rab5 A30V mutant shows increased GTP-binding activity25
is consistent with our unexpected finding for Rab38cht (Figure 6A)
. Although Rab38cht protein retains its GTP-binding activity, it is not bound to any membrane components (Figure 5)
. The inability of unprenylated mutant Rab38 to bind to the required membrane components appears to cause its loss of biological activity, as this phenomenon was demonstrated previously for other small GTPase mutants.26
The precise mechanism underlying the failure of the mutant protein to undergo prenylation is currently unknown. It is possible that the mutant cannot interact normally with either the Rab escort protein or Rab geranylgeranyl transferase, and is thereby unavailable for prenyl modification.
We hypothesized that abrogation of Rab38 function would affect the lungs as well as the skin because the mRNA and protein expression levels are highest in this organ.7
Chocolate mice developed aberrant alveolar structures: increased mean linear intercept, destructive index, and volume proportion of alveolar ducts and sacs; and decreased volume proportion of alveolar walls (Figure 2
, Table 1
). However, these morphological changes were mild and not comparable with lung emphysema in cigarette smoke-exposed mice27
or SP-D knockout mice.28
Physiological analysis showed that the chocolate lungs have slightly increased lung compliance, but the difference was abolished after dividing by lung volume, ie, specific lung compliance. This result suggests that chocolate mice have hyperinflationary lungs rather than emphysematous lungs with prominent alveolar destruction. No apparent sign of inflammation was observed in BAL fluids or in the lung tissues in chocolate mice (data not shown). Thus, it is likely that alveolar structural changes in chocolate mice were caused by developmental abnormality rather than inflammatory tissue derangement. However, precise mechanism for development of these alveolar structural abnormalities remains undefined.
Lung surfactant is a complex of several lipids (mainly phosphatidylcholine) and four surfactant apoproteins, SP-A, -B, -C, and -D.29 With the exception of SP-D, these surfactant components are stored in lamellar bodies in alveolar type II cells. Accumulating evidences suggest different intracellular transport pathways for each surfactant component. Newly synthesized phosphatidylcholine is transported to and stored in lamellar bodies via a Golgi-independent pathway.17 Newly synthesized SP-A is transported to the Golgi apparatus and undergoes glycosylation and is then secreted. A portion of the secreted SP-A is subsequently transported into lamellar bodies.18 Newly synthesized SP-B is also transported to the Golgi apparatus, and then to the lamellar bodies to be stored.30 SP-D is synthesized and transported to the Golgi apparatus, and then secreted, but is not routed to the lamellar bodies.31 Both SP-A- and SP-D-deficient mice contain lamellar body organelles in their alveolar type II cells.32,33 Hence, phosphatidylcholine and SP-B appear to be closely associated with biogenesis of lamellar bodies, although the molecular details are not yet fully understood. Given the fact that chocolate alveolar type II cells contain numerous enlarged lamellar bodies, and that phosphatidylcholine and SP-B levels are increased in the cells but not in the BAL fluid, it is likely that the secretory machinery of lamellar bodies that store phosphatidylcholine and SP-B is defective in mutant cells as a consequence of dysfunctional Rab38 protein.
Oculocutaneous albinism and lung disease are closely related to Hermansky-Pudlak syndrome (HPS). HPS is clinically characterized by oculocutaneous albinism, bleeding diathesis, and life-limiting pulmonary fibrosis, although the clinical presentation is heterogeneous.34 The most critical problem for patients with HPS is lung fibrosis, which may lead to death between the fourth and fifth decade of life.34-36 HPS is an autosomal recessive disease resulting from heterogeneous genetic abnormalities.35 Eight genetically distinct forms of HPS have so far been identified in humans (HPS1–8). Most of the mutant genetic products involved in these variant forms of HPS participate in vesicle trafficking that regulates biogenesis of lysosome-related organelles.37 There is accumulating evidence that Rab38 is involved in biogenesis of melanosomes in skin melanocytes and retinal pigment epithelial cells.9-11 Given their high expression of Rab38, there could be a possible link between melanocytes and alveolar type II cells, in which their characteristic cell organelles may be involved. Both cells contain lysosome-related organelles that consist of melanosomes in melanocytes, and lamellar bodies in alveolar type II cells. Lamellar bodies are lung surfactant storage granules and lysosome-related organelles.38
The observed lung changes in chocolate mice are similar to those reported in the mouse homologues of human HPS, pale ear (ep), pearl (pe), and the double mutant ep/ep, pe/pe.39 However, each strain develops emphysematous lung disease instead of pulmonary fibrosis. The discrepancy between mouse and human in the homologous genetic abnormality are not clear. However, recent studies show that HPS mice are more susceptible to bleomycin-induced pulmonary fibrosis than wild-type mice.40 Different environmental conditions between mice and humans may be associated with the phenotype discrepancy. The double mutant ep/ep, pe/pe mice show integrated lung changes with striking abnormalities in alveolar type II cells and share some lung pathological features with human HPS. As seen in the chocolate lungs, alveolar type II cells and lamellar bodies are remarkably enlarged, and lamellar bodies are engorged with surfactant. In the case of the double mutant mice, airspaces of the mutant lungs contain age-related elevations in the numbers of inflammatory cells and foamy macrophages. These features were also shown in the lung pathology of HPS patients.41
In rats, two (Hermansky-Pudlak Syndrome) animal models have been described: Fawn-Hooded rats and Tester-Moriyama rats. Fawn-Hooded rats develop several diseases including oculocutaneous albinism, bleeding diathesis, pulmonary hypertension, systemic hypertension, renal failure, depression, and alcoholism.42 The oculocutaneous albinism and bleeding diathesis that manifests in Fawn-Hooded rats has been attributed to the pleiotropic effects of a single locus, Ruby (R). Recently, the Ruby locus was identified as the Rab38 gene, establishing that rat Ruby and mouse chocolate genes are homologues.43 However, chocolate mice lack platelet storage granule deficiency and subsequent bleeding diathesis. One possible explanation for this is that as the Rab38cht mutant is a full length protein, it can still facilitate platelet storage granules to be formed and secreted. In contrast, the Rab38 Met1Ile substitution found in both Fawn-Hooded and Tester-Moriyama rats is a protein-null defect, as it completely abolishes translation from the mutant allele. It is possible that a complete deficit of Rab38 causes a platelet pool storage deficiency and a prolonged bleeding time.
In summary, Rab38cht-mutant mice develop an abnormal lung phenotype as well as oculocutaneous albinism due to altered homeostasis of systemic lysosome-related organelles. We suggested that genetic abnormality of human Rab38 may also cause the phenotype of HPS in humans.
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Acknowledgements |
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Footnotes |
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Supported by Grant-in-Aid for Scientific Research (C) (2) 15590833 Japan Society for Promotion of Science and Grant for Promoted Research from Kanazawa Medical University (S2005-9).
Accepted for publication July 25, 2008.
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References |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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-32P]GTP to cytosolic and membrane-bound proteins of human platelets correlates with the activation of phospholipase C. Proc Natl Acad Sci USA 1987, 84:2261-2265This article has been cited by other articles:
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  J. Schroder, R. Lullmann-Rauch, N. Himmerkus, I. Pleines, B. Nieswandt, Z. Orinska, F. Koch-Nolte, B. Schroder, M. Bleich, and P. Saftig Deficiency of the Tetraspanin CD63 Associated with Kidney Pathology but Normal Lysosomal Function Mol. Cell. Biol., February 15, 2009; 29(4): 1083 - 1094. [Abstract] [Full Text] [PDF]
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