Salivary Gland Protein Expression after Bion-M1 and Space Shuttle STS-135 Missions

Understanding the effects of weightlessness during travel in space is important in order to institute countermeasures if the findings are detrimental. Catecholamine hormone-controlled cellular events are altered during spaceflight (Mednieks et al., 1998; Mednieks et al., 2000; Mednieks et al., 2014), indicating an environmental effect or a response to microgravity. In salivary glands, β-adrenergically regulated responses modify exocytosis and protein secretion (Bdolah and Schramm, 1965; Butcher and Putney, 1980; Mednieks and Hand, 1982; Horio et al., 1984). Protein expression, mediated via cyclic AMP pathways, is particularly affected and may serve as an index of physiological or disease-related changes (Gold et al., 2013) [e.g., in diabetes (Szczepanski et al., 1998; Mednieks et al., 2009)], as well as responses to mechanical (Burke et al., 2002) or environmental stimuli (Beavo and Brunton, 2002).

Figure 1 is a diagram of the components of exocytotic stimulus pathways with the β-adrenergic receptor (βAR) activating adenylate cyclase (AC), formation of cyclic AMP [with phosphodiesterase (PDE) to lower intracellular levels], activation of cyclic AMP-dependent protein kinase (PKA) by subunit dissociation, and the eventual nuclear reactivity of the type II regulatory subunit (RII) via the action of DNA binding A-kinase anchor proteins (AKAPS) (Dodge et al., 2001; Herberg et al., 2000). The metabolic interplay of these components provides an internal stability to environmental changes at the cellular level of organization. The individual molecular components of this pathway are well-studied (Daniel et al., 1998; Gold et al., 2013) and provide a series of biomarkers for biochemically measuring stress responses. Serum levels of these components are difficult to analyze and dependent on a variety of internal variations, and the collection of blood and urine for bio-fluid analysis is not convenient. However, the RII is secreted in saliva (Mednieks and Hand, 1984) and is an easily measurable biochemical indicator of environmental stress.

Figure 1.

Numerous other salivary secretory proteins with known function may be indices of metabolic pathways and associated physiologic or pathologic functions (Mandel, 1993; Ruhl, 2012). Among those considered in this study are alpha-amylase (α-amylase) (Valdez and Fox, 1991), the proline-rich proteins (PRPs) (Mehansho et al., 1985; Carlson et al., 1991), demilune cell and parotid protein (DCPP) (Bekhor et al., 1994), and parotid secretory protein (PSP) (Owerbach and Hjörth, 1980; Ball et al., 2003). Microarray analysis of mouse salivary gene expression was carried out comparing flight with control data (Mednieks et al., 2014). By considering this initial array of secretory protein responses, a number of criteria can be established to assess the effects of the length of microgravity exposure and eventual transience or permanence of these effects upon return to Earth.

Human and rodent salivary glands are morphologically and functionally similar. In both species, secretory proteins produced by salivary glands are stored in granules and released into saliva where they can be measured. The mouse model is, therefore, appropriate for studies of stimulated secretion. This system avoids the difficulties associated with obtaining tissue from human subjects and saliva from test animals. Our objectives, therefore, are: 1) To study the effects of weightlessness at the tissue, cell, and molecular level; 2) to identify specific molecular mechanisms affected by space travel; 3) to propose the design of a simple, economic device to measure changes in salivary proteins that can be used to monitor the physiological status of astronauts in space, or patients in Earth-based clinics.

Thirty C57Bl/6J adult female mice, nine weeks old at launch, were housed in Animal Enclosure Modules (AEMs) and flown on the space shuttle Atlantis STS-135 mission, launched from Kennedy Space Center on July 8, 2011, with landing on July 21, 2011. Fifteen mice housed in AEMs served as ground controls. Fifteen additional control mice were housed under standard vivarium conditions (for experiment details see Gridley et al., 2013). The flight mice and AEM ground control mice were fed identical diets [NASA rodent food bars (Sun et al., 2014)] and had access to water ad libitum. The vivarium control mice were fed pelleted rodent chow. Tissues from seven of the flight mice were obtained within 2-5 hours after landing. Tissues from the vivarium control mice were obtained one day prior to landing, and tissues from the AEM control mice were obtained 48 hours after landing. All animal procedures were approved by the Institutional Animal Care and Use Committee at the National Aeronautics and Space Administration (NASA), and conducted in compliance with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health.

Mice from the Bion-M1 mission were C57BL/6N specific pathogen-free males, 19-20 weeks old at launch on April 19, 2013. Samples were obtained from six flight mice between 13-16.5 hours after landing on May 19, 2013, and from eight vivarium control mice immediately afterwards. Samples were also obtained from seven asynchronous control mice, housed two months after the mission in flight habitats for 30 days under environmental conditions simulating the flight conditions, and seven asynchronous vivarium control mice. The flight mice and the asynchronous habitat control mice were fed a paste diet based on standard rodent chow, with water and casein added as a gelling agent. Vivarium control mice were fed pelleted rodent chow. Details of the experimental conditions can be found in Andreev-Andrievsky et al. (2014). All animal procedures were approved by the Institutional Animal Care and Use Committee of Moscow State University Institute of Mitoengineering and the Biomedical Ethics Commission of the Institute for Biomedical Problems, and conducted in compliance with the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes.

After euthanasia, the salivary glands were excised and placed in cold phosphate buffered saline (PBS). A portion of each gland was rapidly frozen in a protease inhibitor mixture for protein assays and in RNAlater or Allprotect (Qiagen, Hilden, Germany) for RNA analyses. The remaining tissues were minced and fixed in 2.5% glutaraldehyde-2% paraformaldehyde for electron microscopy, or 4% paraformaldehyde for immunocytochemistry. The samples were shipped by overnight express to the University of Connecticut Health Center.

The specific secretory proteins studied were α-amylase, PSP, DCPP, PRPs, the regulatory subunit of type II protein kinase A (PKA RII) (regulatory protein), salivary androgen binding protein alpha (SABPα), epidermal growth factor (EGF), and nerve growth factor (NGF). Antibodies used were: anti-RII (Szczepanski et al., 1998; Mednieks et al., 2008), anti-α-amylase and anti-EGF (Sigma-Aldrich, St. Louis, MO), anti-PSP (Ball et al., 1988; L. Mirels, unpublished), anti-PRPs (D.M. Carlson, unpublished), anti-DCPP and anti-SABPα (L. Mirels, unpublished), and anti-NGF (Santa Cruz Biotechnology, Dallas, TX).

Parotid glands were postfixed in 1% osmium tetroxide, treated with 1% aqueous uranyl acetate, dehydrated in ethanol, and embedded in epoxy resin following standard procedures (Hand, 1995). Thin sections were stained with uranyl acetate and lead citrate, and examined in a Hitachi H7650 TEM.

Tissues were embedded in LR Gold resin (London Resin Co., Ltd., Electron Microscopy Sciences, Hatfield, PA) at -20°C. Thin sections were collected on Formvar-coated nickel grids, treated with 1% BSA/5% NGS in PBS to block nonspecific binding, and incubated with primary antibody overnight at 4°C. Bound antibodies were visualized with gold-labeled goat anti-rabbit IgG (Aurion, Electron Microscopy Sciences) or gold-labeled protein-A (BB International, Ted Pella, Redding, CA). The sections were stained with uranyl acetate and lead citrate and examined in the TEM. Digital images were taken at 10,000X using an AMT XR41C camera (Advanced Microscopy Techniques, Woburn, MA).

Quantification of immunogold labeling was done on the TEM images. Using Photoshop (CS2 v. 9.0.2 or CS4 Extended v. 11.0.2), a grid pattern with a separation between grid lines corresponding to 0.5 μm was superimposed on the image. Grid intersections lying over the organelle of interest were counted and divided by 4 to estimate area in μm². Gold particles labeling the organelle were counted and divided by the area to give a labeling density (gold particles/μm²). Significance was assessed using ANOVA and a two-tailed T-test.

Tissue samples were homogenized in PBS containing 12.5 mM benzamidine and mM phenylmethyl sulfonyl fluoride. The homogenate was centrifuged at 10,000xG for 10 min at 4°C and the supernatant was collected. A light microscope smear was made of the pellet to ascertain effective homogenization of cells and organelles. Concentration of total proteins in the soluble fraction was determined by densitometric analysis of Ponceau S dye intensity on slot blots, compared to those of standards of known protein concentration. The sample proteins were adjusted to identical protein concentrations per lane and subjected to SDS–polyacrylamide gel electrophoresis (PAGE) using pre-cast 10% polyacrylamide gels. The proteins were electrotransferred to nitrocellulose membranes, stained with Ponceau S, scanned, and digitized to visualize electrophoretic banding patterns. The membranes were then washed to remove the dye, blocked with a 3% milk solution in PBS - 0.01% Tween20, and incubated with specific primary antibodies. Horseradish peroxidase-conjugated secondary antibodies were applied, and the Western blot developed with 4-chloro-1-naphthol-H₂O₂ solution, or Vector NovaRED substrate (Vector Laboratories, Burlingame, CA). Protein concentrations and protein banding pattern profiles were determined by scanning and measuring the digitized images using ImageJ (v. 1.43u or 1.48; Public domain) software and compared to the densities of standard proteins of known concentration. A standard curve was prepared for each experiment. Average values obtained by scanning of duplicate lanes were used for comparisons among the groups. Due to the small sample size, additional Western blotting was carried out using a slot blotting method (HYBRI-SLOT™ MANIFOLD, Life Technologies Inc., Gaithersburg, MD). Duplicate samples were on the two slot blot lanes of the same membrane; antibody incubations, color development, and digitized sample analysis were carried out as described above for the electrophoretically separated protein samples.

Individual tissue samples — eight control and seven flight from STS-135, and seven control and five flight from Bion-M1 — were submitted for RNA isolation and microarray analyses by Phalanx Biotech Group (San Diego/Belmont, CA) using Mouse OneArray 2 Gene Expression Profiling. All samples had high (>6) Agilent 6000 Nano Assay RNA integrity (RIN) numbers. Information about the mouse microarray can be found at http://www.phalanxbiotech.com/products/MOA.php (accessed 5/25/15), and their gene expression service at http://www.phalanxbiotech.com/services/service_genome.php (accessed 5/25/15).

The flight mice from both STS-135 and Bion-M1 appeared healthy upon landing. The flight mice and the AEM (habitat) control mice from STS-135 mission lost an average of 2.3 and 1.3 grams of body weight, respectively (Table 1). The flight mice and habitat control mice from the Bion-M1 mission both gained weight, 2.6 and 2.4 grams, respectively. There were no significant differences between the body weights of the flight mice and their respective habitat or vivarium controls, thus indicating a similar nutritional status.

The morphology of a parotid acinar cell of a STS-135 flight mouse is shown in Figure 2, Panel A. The cells have a basally located nucleus, abundant rough endoplasmic reticulum, a prominent Golgi complex, and numerous secretory granules stored in the apical cytoplasm. The secretory granules typically have a variable density, with a peripheral dense region and a lighter central region. Sometimes a dense area is present in the middle of the light region. The apical surface of the cells faces the central lumen of the acinus, and finger-like extensions of the lumen — intercellular canaliculi — are located along the lateral sides of the cells. Junctional complexes join the cells at the apical ends of the lateral intercellular spaces and seal the intercellular canaliculi from the intercellular space. The basic structure of the acinar cells of STS-135 and Bion-M1 flight mice were similar, and not different from that of habitat or vivarium control mice.

Figure 2.

There were, however, some specific changes observed in the glands of flight animals. Figure 2, Panel B, shows an intercalated duct of a STS-135 flight mouse. Intercalated ducts are the first component of the duct system connecting the acini with the striated ducts, which are the major ductal component and are involved in electrolyte reabsorption and secretion. The cells of both intercalated and striated ducts — especially in STS-135 samples, but also in Bion-M1 samples — had large apical vacuoles containing acinar secretory proteins endocytosed from the ductal lumen. The inset in Panel B shows immunogold labeling of a vacuole in a striated duct cell for PSP, an acinar secretory protein. The acinar cells of mice from both flights also showed an increased number of autophagic vacuoles and an increase in the number of apoptotic cells was observed. Panel C shows autophagic vacuoles in a Bion-M1 flight mouse containing secretory proteins and other organelles undergoing degradation. Gold particles indicating the presence of PSP, an acinar secretory protein, label the content of one autophagic vacuole and other secretory granules in the cell. Panel D illustrates two apoptotic acinar cells engulfed by a macrophage in a Bion-M1 flight mouse. The structure of the surrounding acinar cells appears normal.

Quantitative immunogold labeling was done to assess secretory protein expression in acinar cells of STS-135 and Bion-M1 flight and habitat control mice. Figure 3, Panels A and B, shows PRP labeling of secretory granules in STS-135 flight and AEM ground control samples, respectively. As shown in the micrographs, the labeling density (gold particles/μm²) of the flight granules was greater than that of the control granules, indicating greater expression of PRP in the flight mice. Panels C and D show amylase labeling in Bion-M1 flight and habitat ground control samples, respectively, demonstrating increased amylase expression in flight mice compared to habitat controls. A summary of the immunogold labeling data for flight mice, and their respective habitat controls from both STS-135 and Bion-M1, is shown in Table 2 and Figure 4. The longer flight duration of Bion-M1 resulted in some specific differences in secretory protein expression. Amylase expression was decreased in STS-135 flight samples, but increased in Bion-M1 flight samples. Expression of PSP was slightly, but significantly, increased in both flights. Expression of PRP was increased in STS-135 flight samples, but slightly decreased in Bion-M1 flight samples. On the other hand, expression of DCPP was slightly decreased in both STS-135 and Bion-M1 flight samples, and PKA RII expression was essentially unchanged from habitat controls. Table 2 also shows results of immunogold labeling of submandibular glands of Bion-M1 flight and habitat control mice. Expression of the acinar cell proteins SABPα and PRP, and the granular duct cell proteins EGF and NGF, was significantly increased in flight mice compared to the controls.

Figure 3.

Figure 4.

The Bion-M1 Western blotting results showed no difference between RII in Bion-M1 flight and vivarium control mice, seen in Figure 5, Panel A. Electrophoretic banding patterns of the flight and control animals, shown in Figure 5, Panel B, were virtually identical. Similar electrophoretic banding patterns and densitometry profiles were seen in STS-135. However, as shown in the top profile of Figure 6, Panel A, the Western blotting RII band of the STS-135 flight samples was significantly smaller than the corresponding band in either vivarium or habitat controls, middle and bottom profiles, respectively. These results indicate that on the shorter, STS-135 flight the expression of RII was decreased, while in the longer Bion-M1 flight an apparent stabilization or adjustment to the microgravity environment had occurred and the RII levels were not different from those of either of the controls. Both Bion-M1 and STS-135 flight and both control samples show a significant, faster-moving fragment (R_fr) that is recognized by the anti-RII antibody. Protease inhibitors were used and samples frozen rapidly after dissection, nevertheless the faster moving component seen in the Western blot (WB) may be due to proteolysis, as may the relatively high backgrounds. Table 3 shows the ratios of the flight R_fr to total proteins are not significantly different for either the STS-135 or Bion-M1 samples compared to controls. The decrease, therefore, in RII for the STS-135 flight may be due to flight conditions rather than to increased degradation. However, the ratio of RII/protein in flight samples compared with both vivarium and habitat control samples in STS-135 is significantly lower than that of Bion-M1. These results are consistent with those from immunocytochemistry experiments and microarray analyses. Figure 6, Panel B, shows that the expression of α-amylase is significantly reduced in the flight parotid, and also in the habitat control when compared to vivarium controls.

Figure 5.

Figure 6.

Changes in the expression of selected genes associated with cyclic AMP signaling pathways in the flight animals compared with habitat control mice are shown in Figure 7. The flight duration (STS-135, 13 days; Bion-M1, 30 days) had a significant effect on the expression of most of these genes. The expression of the type II PKA regulatory subunit (Prkar2a) was decreased in STS-135 flight mice, but was near control levels in Bion-M1 flight mice. Phosphodiesterase 4a (Pde4a) also was decreased, more so in Bion-M1 flight mice than STS-135 flight mice. The two flights showed opposite effects on the expression of A-kinase anchor protein 13 (Akap13) and adrenergic receptor beta 2 (Adrb2); the former increased in STS-135 mice and decreased in Bion-M1 mice, whereas the latter decreased in STS-135 mice and increased in Bion-M1 mice. Adenylate cyclase 3 (Adcy3) expression was increased in STS-135 flight mice, but was similar to that of controls in Bion-M1 flight mice. Expression of amylase 1 (Amy1), which is regulated by catecholamine and insulin signaling, was slightly increased in mice from both flights.

Figure 7.

Table 4 shows changes in the expression of secretory protein genes in flight animals compared with habitat controls. The increased flight duration of Bion-M1 had opposite effects on the expression of two secretory protein genes, increasing lysozyme (Lyz1) and decreasing PRP (Prpmp5). In contrast, the expression of the genes for parotid secretory protein (Bpifa2), lactoperoxidase (Lpo), and prolactin inducible protein (Pip) showed only small changes from controls and did not differ between the two flights. These results show individual secretory protein expression responds to travel in space, and the microgravity environment has specific gene regulatory effects on secretory and signaling pathway proteins. Additionally, there is a flight duration dependent component where initial responses may show opposite changes or readjust to basal levels.