1 LSU Eye Center, Lions Eye Research Laboratories, Laboratory for the Molecular Biology of the Ocular Surface; Louisiana State University Health Sciences Center, New Orleans, Louisiana, 70112
2 LSU GeneChip Bioinformatics Core, Department of Genetics, Louisiana State University Health Sciences Center, New Orleans, Louisiana, 70112
3 Singapore Eye Research Institute, Singapore 168751
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ABSTRACT |
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muscarinic; dry eye
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INTRODUCTION |
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We have previously shown that sensory innervation plays a role in this functional unit in the rabbit (41, 57, 71) and rat (50). In the rabbit, sensory input was eliminated by ablating a portion of the trigeminal ganglion containing the cell bodies whose axons receive sensory input from the surface of the eye. This resulted in structural changes in the organization of the LG and increased secretory responsiveness to in vitro stimulation of the LG acinar cells with carbachol, a cholinergic agonist (41, 57). Interrupting the neural outflow of the functional unit by permanently severing the preganglionic parasympathetic outflow from the CNS produced more severe structural alterations of the LG, as well as significantly decreased tear flow within 24 h and pathological changes to the ocular surface by 7 days (71).
In the rat, the overall effect of the functional unit was tested by sectioning the preganglionic nerve, which resulted in reduced tear flow and clinical symptoms including disruption of the integrity of the surface of the eye (50). LG structure was also affected.
In our model of ocular surface homeostasis, constant muscarinic stimulation of LG secretion was suggested by regulation of the physiological activity of the parasympathetic ganglion from the CNS. It was also found that, without constant stimulation, the LG had no intrinsic ability to secrete (71). Interruption of neural outflow for tear secretion from the LG also affected components of translation and could lead to alteration in protein synthesis and processing (49). Thus we hypothesized that muscarinic stimulation controls a network of genes responsible for maintaining the secretory function of the LG epithelial cell.
Tissues are known to show adaptive changes in response to innervational status. Our model of the relationship between the innervation of the ocular surface and the target/secretory tissues suggests that this type of relationship may also hold true for the expression of genes. Modulation of gene expression in skeletal systems is probably the best-studied tissue model, and neural influences of both normal and aged muscle have been described (16, 24, 30). More recent studies on the autonomic innervation of the salivary and other glands have shown a significant but diverse range of effects on function and morphology (20, 21, 60, 69). Rossi et al. (55) reported reduced tear production with dry eye-like symptoms in neurturin-related neurotrophic factor-deficient mice lacking parasympathetic innervation to the harderian glands and LG. Additionally, in mice lacking the M3 muscarinic receptor (M3R), the salivary secretory response to cholinergic stimulation was reduced (40). The functional importance of muscarinic parasympathetic input to lacrimal secretion is well established; however, the role of muscarinic parasympathetic innervation in the regulation of genes associated with secretory function in exocrine tissues is unclear.
With DNA microarray technology, it is possible to examine the conditional regulation of many genes simultaneously, thus gaining a better understanding of gene function, interactions, and regulation. Because there is no functional cross-innervation between the two LGs of an individual animal and both exist under the same physiological conditions, gene expression in the contralateral LG provides an appropriate baseline level for determining muscarinic parasympathetic control of gene expression in the LG (28). DNA microarrays have been used in the LG to profile gene expression patterns associated with gender and Sjögren syndrome (4, 54).
In this study, we carried out gene expression profiling in the rat LG after removal of the preganglionic parasympathetic control of secretion. The identification of patterns of gene expression provides insights into genes that are conditionally linked to parasympathetic secretory control.
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MATERIALS AND METHODS |
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Animals were killed with an overdose of pentobarbital sodium, and the LG was removed through a skin incision from the Ctla and Px sides. A 2 mm x 2 mm fragment of LG tissue was fixed for light microscopy; the remaining tissue was frozen immediately in liquid nitrogen. For structural analysis, the tissues from the Ctla and Px sides were placed in a mixed aldehyde solution containing 2% glutaraldehyde and 1% paraformaldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) containing 0.2% picric acid for 4 h, and washed overnight in buffer. Plastic sections (1-µm thick) were prepared and stained with toluidine blue and photographed using a Nikon E600 microscope (41).
RNA extraction and microarray analysis.
Total RNA was extracted from the experimental (n = 5) and control (n = 5) LGs [contralateral control (n = 3), normal unoperated control (n = 1), sham-operated control (n = 1)] using the RNeasy Mini Kit (Qiagen, Valencia, CA). RNA integrity was determined by spectrophotometry and by formaldehyde gel electrophoresis. Procedures for cDNA synthesis, labeling, and hybridization were carried out as described at http://www.affymetrix.com/support/technical/manual/expression_manual.affx (Affymetrix, Santa Clara, CA). All experiments were performed using Affymetrix RG U34A GeneChips as described at http://www.affymetrix.com/products/arrays/specific/rgu34.affx. Briefly, 8 µg of total RNA was used for first-strand synthesis using HPLC-purified T7-(dT)24 primer. Synthesis of biotinylated-labeled cRNA was carried out using the ENZO RNA transcript labeling kit (Affymetrix) and processed for hybridization. For overnight hybridization, 15 µg of fragmented cDNA was used in an Affymetrix GeneChips Hybridization Oven 640, washed, stained with streptavidin-phycoerythrin using a microfluidics workstation (Affymetrix), and scanned with a confocal laser scanner (Agilent Technologies, Palo Alto, CA). Further description of the methodology according to MIAME ("minimum information about a microarray experiment") guidelines (http://www.mged.org/Workgroups/MIAME/miame.html) is provided in the Supplementary Material (available at the Physiological Genomics web site).1
Microarray quantification and statistical analysis.
Signal and background intensities were quantitated by pixel intensity, and expression signals were analyzed using Affymetrix Microarray Suite 5.0 (MAS 5.0). All array images and quality control measurements were within acceptable limits. Details of quality control measurements are provided in the Supplementary Material. Absolute expression transcript levels were normalized for each array by globally scaling all probe sets to a target signal intensity of 2,500. Three statistical algorithms [detection, change call, signal log ratio (SLR)] were then used to identify differential gene expression in control and experimental samples. The detection metric (present, marginal, or absent) was assigned to each transcript using default parameters in MAS 5.0. For comparison expression analysis, the control samples were used as a baseline, and batch analyses were performed in MAS 5.0, in which pair-wise comparisons between individual experimental and control arrays were made to generate an SLR value for each transcript.
Data and statistical analysis and data visualization were performed with LIMS 3.0 and Data Mining Tools (DMT) 3.0 (Affymetrix). Transcripts that were found absent in three of five experiments in both the control and experimental groups were eliminated from further analysis. Significant gene expression was analyzed using the Mann-Whitney test to compare the signal intensity between the Px LG and the Ctla LG. Two criteria were used to group significant changes in gene expression. First, we defined a positive change call as one in which more than 50% of the transcripts had a call of "increased" (I) or "marginally increased" (MI) for upregulated genes, and "decreased" (D) or "marginally decreased" (MD) for downregulated genes. The median value of the SLR from each comparison file was calculated using DMT 3.0. Second, genes with statistically significant changes were compared based on a percentage of present detection calls (>50%) in the five control or experimental LGs. Genes with median SLR values of >1 or less than 1 were grouped as upregulated and downregulated genes, respectively. Finally, genes were grouped based on their biological functions using Affymetrix NetAffx, NCBI UniGene, and LocusLink. Complete microarray expression data are available at NCBI Gene Expression Omnibus (GEO) database (GEO submissions GSM12935 through GSM12953, GSE844, NCBI tracking system 15029722) at http://www.ncbi.nlm.nih.gov/geo.
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RESULTS AND DISCUSSION |
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The dramatic decline in tear secretion clearly points to the absence of the muscarinic stimulation necessary to elicit tear flow. Thus, although the innervation is intact from the pterygopalatine ganglion to the LG, the results indicate that, as hypothesized, the neural signals required for the release of neurotransmitter are not available. As shown below, the results of DNA expression profiling suggest that the absence of muscarinic stimulation leads to substantial changes in levels of expression among networks of acinar cell genes.
Microarray data analysis.
Based on the detection call comparison between the control and experimental LG, the number of genes expressed in the Px LG (2,792 present genes) was greater than the number in the Ctla LG (2,272 present genes). The greater numbers of expressed genes in the Px LG suggest an elevated gene transcription program in response to alterations in the organization and structure of the LG. The upregulated gene expression pattern may be a compensatory or stress response by acinar cells, which make up 80% of the LG cellular mass. The loss of the major parasympathetic innervation may have an impact on LG tissue homeostasis and trigger transcriptional responses from ductal and interstitial cells.
On the basis of present to absent detection calls for comparison between the Ctla and Px LG, 18 genes were found to be significantly downregulated, only one of which was not detected in any of the experimental LG samples. However, only five genes had a median SLR value less than 1 (Table 1). Conversely, 78 genes were found to be significantly upregulated and 41 of these 78 genes were not detected in any of the five Ctla LG samples, a substantially greater number of genes than was found in the downregulated group. Twenty of the upregulated genes had a median SLR value >1 (Table 2). Given the known biological functions associated with these upregulated genes, it appeared that, without parasympathetic input to the LG, expression of proinflammatory and proapoptotic transcripts increases. Experimental comparison of statistically significant genes based on change call criteria found 45 downregulated genes and 97 upregulated genes in the Px LG (Tables 3 and 4).
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Cellular mechanisms controlling mRNA translation operate most efficiently at the initiation step of protein synthesis. The gene for PHAS-I, a heat- and acid-stable protein also known as eIF4E-binding protein 1, was significantly downregulated (Table 3). PHAS-I is a regulator of the initiation step of protein translation (51). However, regulation of secretory proteins is thought to occur at the elongation step of protein synthesis and involve the Ca2+/calmodulin-dependent elongation factor-2 kinase, which was not found to be significantly changed in the Px LG (31). These results suggest that either the initiation step of translation was generally downregulated and/or that selective translation of mRNA is occurring in the Px LG. Accordingly, decreased expression of the L-type amino acid transporter 1 (LAT1) and 4F2hc surface antigen suggests that protein synthesis is decreased in the Px LG (Table 3). As such, it is possible that downstream pathways for secretory protein maturation involving protein folding, processing, and modification in the endoplasmic reticulum (ER) and Golgi may be affected by the lack of muscarinic cholinergic stimulation.
As shown in Table 3, 17 genes associated with protein translocation, folding, and glycosylation in the ER and Golgi were found to have significant changes, and 9 of these 17 genes were downregulated more than twofold. The majority of these downregulated genes are ER-resident proteins, which suggests that protein folding and processing are severely affected by removal of the secretory input.
Furin is a serine endopeptidase localized mainly in the trans-Golgi network. It is associated with proteolytic processing and maturation of precursor proteins into their bioactive form and may also play a role in protein processing of improperly folded membrane proteins in the ER (5, 48). Some of the substrates for furin include transforming growth factor-ß1 (TGF-ß1), matrix metalloproteinase 1 (MMP1), ß-nerve growth factor, and insulin-like growth factor (48, 72). Another ER-Golgi protein processing activity that may be affected is the UDP-galactose transporter. This Golgi-associated protein is involved in the posttranslational modification of proteoglycan, a critical and rate-limiting step necessary for proper localization and insertion into the plasma membrane and extracellular matrix (62, 70).
A number of other genes involved in protein translocation, folding and processing, and transport were found to be significantly downregulated, but less than twofold, including ribophorin II, translocon-associated protein (TRAP), p34, sec61, dolichyl-di-phosphooligosaccharide-protein glycotransferase, and rab26 (Table 3). Overall, based on the biological processes associated with these genes, synthesis of secretory proteins does not appear to be affected, probably because translation of mRNA for secretory proteins is regulated at the elongation step (31).
By contrast, protein quality control and translocation and transport through the lumen of the ER and Golgi do appear to be adversely affected by the lack of muscarinic stimulation, which reduces the capacity for regulated secretion of protein in the LG acinar cells. However, expression of ribosomal protein was upregulated in the Px LG, suggesting an increase in transcription of ribosomal DNA and ribosomal protein genes (Table 4). This situation is counterintuitive, as protein synthesis is directly related to the status of ribosomal genesis. It is more likely that the expression pattern of upregulated ribosomal protein genes found in the Px LG is a consequence of posttranslational stabilization of the messages that may be correlated with a reduction in protein synthesis.
The development of denervation-induced supersensitivity in target tissues has been reported to involve changes in receptor number in the target tissues, including the rat salivary gland (67). In the rabbit LG, supersensitivity was seen following preganglionic parasympathetic and sensory denervation. Stimulation of LG fragments in vitro with carbachol (a cholinergic agonist) resulted in a significant increase in the amount of protein released (41, 49). In the present study, however, the expression of M3R found predominantly in acinar cells was not significantly different 7 days after the GSPN was severed. Similarly, no significant change in the vasoactive intestinal peptide receptor (VIP-R) was found, even though VIP-R colocalizes with acetylcholine in the parasympathetic nerve terminal. Real-time RT-PCR also confirmed that the change in M3R expression was not statistically significant. (Details of M3R expression are available in the Supplementary Materials.) The absence of an increase in the expression of M3R in the parotid gland after unilateral postganglionic parasympathetic denervation has been reported, suggesting that other changes in the parotid gland may contribute to the supersensitivity response (2, 67). The reported experimental procedure was somewhat different from that of the present study in that the nerves to the LG remained intact. Since the expression of these receptors was not changed in the present study, it is possible that components of the intracellular signaling pathways associated with either muscarinic or VIP receptor activation may become more sensitized or coupled more efficiently to downstream targets. On the other hand, as in the case of the Ins(1,4,5)P3/Ca2+ and protein kinase C signal cascade, one branch may be preferentially activated after the loss of muscarinic stimulation.
Calcium released from intracellular stores stimulates secretion and serves as an important signaling and sensor molecule in the cytosol (8). Depletion of Ca2+ from the ER leads to accumulation of misfolded proteins, activates expression of ER chaperones, and inhibits protein translation (9). Removal of parasympathetic control, which would have the effect of decreasing muscarinic parasympathetic stimulation, may in fact serve as a trigger to mediate a cellular stress response. Thus one would expect that without stimulation-induced Ca2+ release, secretion would be reduced and expression of cytoplasmic and ER chaperones would not be altered. We found that mRNAs for protein disulfide isomerase (PDI), calreticulin, immunoglobulin heavy chain binding protein (BiP) [also known as the glucose-regulated protein-78 (GRP78)], and FK506 binding protein were significantly decreased, most greater than twofold. Cyclophilin B, a peptidyl prolyl isomerase that is also found in the ER, was also significantly decreased (Table 3). PDI catalyzes disulfide formation critical for folding and assembly of secretory proteins (19, 26, 43). In the absence of muscarinic activation, mobilization and depletion of Ca2+ stores in the ER do not take place, and the molecular chaperones and disulfide isomerases necessary for proper maturation of secretory and membrane proteins are not needed.
In the LG, as well as in the salivary gland and pancreas, Ca2+ is released from the ER store by activation of the Ins(1,4,5)P3 receptors and then taken up by SERCA, a Ca2+-ATPase of the ER, after the secretory signal ceases (33). The Ca2+ storage capacity in the ER is modulated and enhanced by Ca2+-binding proteins to maintain cellular Ca2+ homeostasis. Several Ca2+-binding proteins were found to be significantly downregulated, including calreticulin, oncomodulin (also known as parvalbumin-ß), and calmodulin (Table 3). These Ca2+-binding proteins also serve as modulators of Ca2+-mobilizing stimuli and as regulators of the cytoskeletal contractile apparatus (8, 44). Additionally, calreticulin has been shown to modulate transcriptional activity of nuclear hormone receptors such as the androgen receptor and retinoic acid receptor, which are known to have a profound effect in maintaining LG function (15, 46). Calmodulin, an effector of Ca2+-mediated secretion of the Ins(1,4,5)P3/Ca2+ pathway, is also a member of this superfamily; its expression was also significantly decreased. The downregulation of these Ca2+-binding proteins and effectors affects the cells ability to respond to agonist-mediated inositol-dependent Ca2+ from the ER, with limited consequences on Ca2+ storage capacity in the ER (15, 36). Additionally, expression of a Ca2+-independent phospholipase A2 was significantly downregulated in the Px LG (Table 1), which may affect membrane lipid hydrolysis and transmembrane ion flux (38). The expression of the Ca2+-binding proteins may be under parasympathetic control, and the decreased expression of these proteins resulting from the lack of parasympathetic input may lead to the inability or reduced capacity of the Px LG to respond to hormonal stimulation.
Annexin represents a class of Ca2+-binding proteins also known as the lipocortin protein family (8). In this study, expression of annexin I and annexin II were both upregulated (Table 4). Annexins are cytosolic and associated with the membrane or the cytoskeleton, binding phospholipid in a calcium-dependent manner. The annexin family of proteins plays a role in mediation of the steroid anti-inflammatory response, membrane aggregation and fusion, endocytosis and exocytosis, and apoptosis (3, 7, 52). Since these proteins bind cellular phospholipids, it is plausible that there may be an increase in membrane phospholipid metabolism, thereby affecting exocytotic and endocytotic processes (7). Similarly, increased expression of annexin I has been implicated in the membrane clustering mediation of the phagocytosis of apoptotic cells (3). In cells where annexin I was overexpressed, calcium release was abrogated on stimulation of purinergic or bradykinin receptors. However, basal calcium and calcium stores in the ER and mitochondria were not affected (27). The increase in annexin I expression and activity may affect Ca2+ release from Ins(P)3-sensitive stores, an impairment that may be attributed to the lack of phospholipase C activity from loss of muscarinic cholinergic stimulation in the Px LG.
Initially, the LG acinar cell secretion is an NaCl-rich, plasma-like fluid. This fluid is modified by LG ductal cells, primarily by NaCl absorption, to produce a final KCl-rich fluid (47). Accordingly, stimulation via Ca2+ agonists enhances fluid exchange and transport by upregulating acinar NKCC1 cotransporter and Na+/K+ exchange activity (22). This cotransporter has been found in both acinar cells and duct cells and plays an important role in volume regulation. Treatment with a specific blocker of NKCC1 has been shown to reduce LG fluid secretion in response to carbachol (72). In the salivary glands of mice deficient in NKCC1, a severe deficit in saliva secretion in vivo was reported (23). This may be the result of a deficiency in Cl uptake as acinar Cl influx was reduced in vitro. However, in the present study, NKCC1 expression was upregulated in the absence of parasympathetic activation. This contradicts the expectation that downregulation of NKCC1 would occur in the face of diminished tear secretion in the Px LG as measured by the Schirmer tear test (71). It is possible that this is a compensatory mechanism responding to osmotic challenge and that other compensatory mechanisms may also exist and be activated in response to a decreased muscarinic signaling system.
The hormonal milieu of the LG also plays an important role in modulating LG secretion and function. Melanocortin-5 receptor (MC5R) is abundantly expressed in exocrine tissues, specifically in the acinar cells of the LG (10, 34). In this study, expression of MC5R was significantly downregulated in the Px LG. Melanocortin-stimulating hormone (MSH) and adrenocorticotropic thyroid hormone (ACTH) increase cAMP level in the LG and peroxidase secretion (13, 32). However, in mice deficient in MC5R, stimulation with physiological levels of MSH and ACTH did not stimulate protein secretion (10).
Another pituitary-derived peptide hormone with an important modulating role in the LG is prolactin. Prolactin has been shown to increase expression of muscarinic receptors, maintain acinar cell morphology, and affect secretion (46). Prolactin and prolactin receptors are also synthesized in the LG, and prolactin is a secretory product of acinar cells (74). Expression of prolactin receptor and MC5R was significantly decreased, with a greater than twofold reduction, in the Px LG (Tables 1 and 3). The decreased expression of the prolactin receptor may compromise prolactin-mediated modulation of LG prolactin levels and muscarinic receptors. Previous studies suggest that this may alter the immunoregulatory environment of the LG (46).
Pathological changes in the ocular surface and LG with and without associated autoimmune components correlate with increased lymphocytic infiltration, cytokine synthesis, and apoptosis (25). In the present study, expression of many genes coding proinflammatory cytokines, complements, and proteolytic enzymes, as well as apoptotic-related gene expression, was found to be upregulated in the Px LG (Tables 1 and 4). Interestingly, a considerable number of genes coding for the major histocompatibility complex (MHC) class II were also upregulated (Table 4). Generally, normal LG acinar cells do not express MHC-related genes in vivo; however, carbachol stimulation can induce expression of MHC class II genes in the LG in NZB/W mice at the age of onset of autoimmune disease and in acinar cells grown in culture (46). It is plausible that removal or loss of extracellular matrix attachment, perturbation of normal cytosolic and ER Ca2+ homeostasis, or a dramatic change in transcriptional activity may induce MHC gene expression, thus transforming normal cells into antigen-presenting cells. Moreover, protein fragments derived from lysosomal cathepsin proteolysis and engulfed cellular contents that have entered the endosomal sorting may be bound by MHC class II molecules and presented at the cell surface as antigens (68). Expression of proinflammatory and apoptotic caspases 1, 2, and 6 was significantly upregulated (Table 2). Caspase 6 is the major caspase in apoptotic cells and has been found to cleave poly(ADP-ribose) polymerase and nuclear lamin A (12). DNA microarray analysis of MRL/lpr mice, one type of model of Sjögren syndrome, also showed increased expression of inflammation- and apoptosis-associated genes. However, the LGs of the mice used in that study had severe lymphocytic infiltration and represented a late stage of the disease (4). The proinflammatory and proapoptotic genes found in our study suggest an early event in the inflammatory process, in that light microscopy results did not reveal any lymphocytic infiltration.
Extracellular matrix proteins and regulatory mediators of synthesis, maintenance, and degradation were generally found to be upregulated in the LG after loss of muscarinic stimulation. Upregulated extracellular matrix proteins and secreted proteins included fibronectin, decorin, secreted acidic cysteine-rich glycoprotein (SPARC), amyloidogenic glycoprotein, and pulmonary surfactant protein (SP-D) (Table 4). SP-D, also called collectin-7, was abundantly present in the luminal contents of secretory cells, including the LG. SP-D binds carbohydrates and lipids in a calcium-dependent manner and may have a role in the innate host defense against pathogens at sites of entry into various organs (63). Cystatin C, another lipid-binding protein found to be upregulated, may be related to the lipocalin family of lipid-binding proteins and may have anti-cysteine protease properties (1, 17, 53, 56). In our study, the common salivary protein 1 (CSP1) was the most highly expressed gene found in the Px LG (SLR = 3.51). CSP1 is secreted by the salivary gland and has been localized to the intercalated duct cells of the parotid, the submandibular glands, and demilune cells of the sublingual gland (29, 61). As CSP1 had not been previously detected in the LG (29), the identification and differential expression of CSP1 found in this study may be the result of a difference in the sensitivity of the molecular technical procedure. Ebnerin, another gene that was upregulated in this experiment, is homologous to the deleted gene in malignant brain tumor 1 (DMBT1), CRT-ductin, and Hensin (6, 11, 35, 66). It has been found in association with SP-D and is also found in tear fluid (39, 58). Ebnerins function in the LG is also unclear, but it may, like SP-D, affect the innate immune response and, possibly, mediation of cell proliferation (6, 11, 58).
In the present study, an interesting though not surprising finding was the increased expression of genes associated with cell structure and tissue remodeling. Expression of structural proteins, including smooth muscle SM22, myosin regulatory light chain isoform C, tropomyosin, actin binding protein coronin 1A, and fibronectin, suggests increased myoepithelial and parenchymal cell activity (47). Coronin regulates nonmuscle cell cytoskeleton remodeling, as well as cell adhesion and migration (14, 18). The conditions for normal tissue remodeling are favorable when the expression and activity of proteases, protease inhibitors, and extracellular matrix-associated proteins are coordinately regulated. Three genes were significantly upregulated more than twofold in the Px LG: SPARC, tissue inhibitor of metalloproteinase type 2 (TIMP-2), and plasma protease C1 inhibitor precursor (similar to -1 antitrypsin). This suggests a shift in the balance of protease and protease inhibitors to a more inhibitory condition that may lead to abnormal tissue maintenance. Finally, intracellularly, expression of lysosomal membrane glycoprotein 1, lysosomal acid lipase, and proteasome subunit RC1 was upregulated, suggesting an increase in lysosomal activity and protein proteolysis.
Overall, DNA microarray analysis showed gene expression patterns shifted toward greater gene activation, possibly as part of the survival genetic response. In the absence of activation of the M3 muscarinic receptor, genes coding for components of the M3 muscarinic receptor signal transduction cascade were repressed, as were genes associated with protein synthesis and posttranslational processing. This illustrates the importance of the main neural input in protein synthesis and directly links quality control to protein processing (protein folding and glycosylation) in a secretory tissue. Precursor proteins that transit through the secretory pathway often require proteolytic cleavage to release their bioactive entities. A loss of or reduction in the capacity for this process may negatively affect the regulated secretory pathway and the proper expression of specific proteins and secretory granule genesis. Perturbation of Ca2+ homeostasis decreases the capacity of acinar cells to respond to stimuli, affects quality control of protein processing and maturation, and represents a form of stress that may trigger apoptosis. Additionally, by modulating the expression of receptors for hormones, parasympathetic innervation affects the responsiveness of acinar cells to circulating hormones such as ACTH and prolactin. The MC5R may have a direct, trophic role in maintaining lacrimal function and secretion as the MC5R-deficient rat develops alacrimia; this dysfunction was hypothesized to be the result of impairment of MC5R-coupled signal transduction pathways (34). On the other hand, parasympathetic input may be required for the maintenance of acinar epithelial differentiation and overall tissue organization, in that the current study demonstrated an increase in interstitial content including extracellular matrix production, inflammatory mediators, induction of immunity-associated genes, and proteolytic activation. Loss of parasympathetic activation of acinar cells may trigger glandular atrophy, followed by induction of apoptosis and targeting of acinar cells for destruction by infiltrating lymphocytes.
In conclusion, parasympathetic innervation and, by extension, muscarinic cholinergic stimulation are critical elements in maintaining LG structure and cellular organization. The consequences of loss of innervation may be an early step in the pathogenesis of dry eye in the clinical setting.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Address for reprint requests and other correspondence: R. W. Beuerman, LSU Eye Center, 2020 Gravier St., Suite B, New Orleans, LA 70112 (E-mail: rbeuer{at}lsuhsc.edu).
1 The Supplementary Material for this article is available online at http://physiolgenomics.physiology.org/cgi/content/full/00011.2004/DC1.
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REFERENCES |
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