Journal of Undergraduate Research
Volume 2, Issue 11 - August 2001

Effects of Anti-Muscarinic Receptor Antibodies on Salivary Acinar Cell Proliferation and Protein Kinase C Expression Involved in Intracellular Signal Transduction Pathway of Sjögren's Syndrome

Joanna Sadowska

ABSTRACT

Sjögren's syndrome is an autoimmune disease in which the immune system attacks the exocrine tissue (salivary and lacrimal glands), resulting in loss of secretory capacity and clinical manifestations of oral and ocular dryness. Exocrine hypofunction associated with the disease has been proposed to be caused by autoantibodies against the muscarinic3 receptor (M3R) on the acinar cell surface. In vitro studies were conducted in order to determine how particular autoantibodies might affect autoimmune pathogenesis by altering signal transduction and inducting apoptosis in the exocrine tissue. The study was conducted using rat submandibular C6 acinar cells treated with purified monoclonal antibodies to the M3R and the IgM isotype control antibodies. The cells were grown for three days in the presence of the antibodies and then incubated with radioactive 3H-thymidine. The membranes were isolated from another set of cells treated with antibodies and/or carbachol for subsequent Western blot detection of Protein Kinase C (PKC). The results showed a distinctive decline in cell proliferation rate of the cells treated with the anti-M3R antibodies, but not IgM or saliva controls, with increasing antibody concentration, suggesting the antagonistic role of anti-M3R antibodies. PKC detection revealed a significant upregulation of PKC isoform y, indicating that it has a potentially important role in a signal transduction pathway leading to apoptosis and Sjögren's syndrome pathogenesis.

INTRODUCTION

Sjögren's syndrome is a chronic inflammatory autoimmune disease exhibiting focal lymphocytic infiltration in the submandibular and lacrimal glands. The results of the defective function of immune cells are expressed as a loss of secretory function, with clinical manifestations of oral and ocular dryness. Exocrine hypofunction of the disorder is characterized by mononuclear cells forming focal infiltrates in the exocrine glands leading to eventual glandular destruction and production of a variety of autoantibodies recognizing many antigens. Recent studies suggest that the functional impairment of the secretory capacity of the salivary and lacrimal glands may be linked to the action of autoantibodies against the muscarinic3 receptor (M3R) on the acinar cell surface of the salivary and lacrimal glands [1]. M3R, the acetylcholine receptor responsible for the fluid secretion, represents a potentially insightful autoantigen due to its role in the stimulation of lacrimal and salivary exocrine glands. The secretory suppression that might be caused by the autoantibodies towards the M3R is a direct consequence of intracellular signaling cascade, which activates programmed cell death and induces apoptosis in fluid-secreting cells of the exocrine glands.

Antagonistic function of the pathogenic autoantibodies has been suggested based on the in vitro study showing inhibited bladder smooth muscle contraction in the presence of anti-M3R autoantibodies compared to the control [6]. In addition, impairment of the translocation of aquaporin-5 as a consequence of the antagonistic effects of the autoantibodies could further explain the diminished excretory function of the affected glands [4]. As another possibility, repeated stimulation of the receptor by the autoantibodies may lead to a downregulation of a cell surface receptor density and subsequent activation of downstream molecules involved in fluid secretion from acinar cells of the exocrine glands.

Protein Kinase C (PKC) is one of the proteins involved in signal transduction pathway stimulated by cholinergic neurotransmitters acting on M3R. PKC has been shown to play an important role in the regulation of cell proliferation and differentiation as well as apoptosis [3]. Different isoforms exhibit variations in substrate specificity and in subcellular localization, indicating the possibility of diverse roles of PKC isoforms in the downstream signaling pathway. Previous studies on submandibular acinar cells demonstrated the presence of PKC a, b, d, and E [2]. Depending on the signals that are present at the time of activation, overexpression of constitutively utilized PKC isoforms can enhance growth rate or can drive apoptosis. Since the expression of PKC isoforms tends to be altered in patients with Sjögren's syndrome [5], it is reasonable to suspect that PKC is a contributor to the pathogenesis of this disorder.

To elucidate the possible pro-apoptotic effects of the anti-M3R antibodies on submandibular rat acinar cells, the growth rates of the cells were measured in vitro using 3H-thymidine radioactivity assay. In order to determine how anti-M3R antibodies generated in mice with autoimmune exocrinopathy might alter downstream signaling pathways, the expression of PKC isoforms was examined using four different antibodies specific to four types of PKC isoforms (a, b, g, and e). It is hypothesized that anti-M3R antibodies will inhibit submandibular rat acinar cell proliferation in vitro due to possible downstream apoptotic events. It is also predicted that autoantibodies towards M3R will have antagonistic effects on the receptor sites as opposed to the M3R agonist carbachol in the acinar submandibular cells.

METHODS

Antibody purification

Hybridoma cell lines producing monoclonal IgM and anti-M3R antibodies were cultured in Iscove's Modified Dulbecco's Medium with 4mM 1-glutamine adjusted to contain 1.5 g/L sodium bicarbonate and 10% fetal bovine serum. The antibodies were collected and purified using Sephadex G-200 size-exclusion chromatography and 10mM solution of Tris (pH 7.4) as a buffer. The collected fractions were then quantified with a Bio-Rad protein assay. SDS-PAGE was used on the eluted fractions in order to determine which fractions contained the specific IgM antibodies. The fractions containing the antibody of interest were combined, dialyzed overnight, and resuspended the following day in 1mL of 10mM Tris (pH 7.4). The final concentration was determined after repeated dialysis for future use in the experiment.

Culture of rat submandibular rat acinar cells

Transformed C6 rat submandibular acinar cells were a generous gift from Dr. David O. Quissell at the University of Colorado. The cells were cultured using Dulbecco's Modified Eagle Medium with 5% fetal bovine serum and nutrients adequate for optimal growth. Previously purified antibodies were added in specified concentrations (1x10-6 M, 5x10-6 M, and 1x10-5 M) to the submandibular acinar cells that reached 60-70% confluence and incubated for 3 days. 1 µL of 3H-thymidine was then added to each well and incubated for 4 hours at 37º C. After thymidine incorporation, the cells were washed with 2mL of 1 x PBS, followed by 2-3 ml of c-PEG for 15-20 min in an incubator, and then frozen at -80ºC. The cells were thawed and the 3H-thymidine was quantified using scintillation counter in order to determine cell proliferation rate based on the incorporation of radioactively labeled thymidine into the DNA synthesizing cells that are undergoing replication. Separate submandibular rat acinar cells were treated with purified antibodies in specified concentrations (5x10-8 M, 1x10-7 M, and 2x10-7 M) in the absence of radioactive 3H-thymidine and kept for subsequent PKC analysis.

PKC detection and analy

The cells that were only stimulated with purified antibodies in the absence of 3H-thymidine were lysed by centrifugation at 15,000 rpm for 20 min at 4ºC and the membrane fractions were isolated. Anti-PKC isoform antibodies against a, b, g, and e at 1:1000 dilutions as a primary antibody and goat anti-mouse IgG at 1:10,000 as a secondary antibody were used for the detection of PKCs by Western blot analysis.

PKC Western blotting with alkaline phosphatase conjugates

Protein concentrations were obtained using Bradford assay with a kit from BioRad (Emoryville, CA).15-30 µg of protein per lane was separated on 12% SDS poly-acrylamide gel. 3-5µg of rat brain lysate was used as a positive control. The gels were transferred to nitrocellulose membranes, placed in the blocking buffer (5% non-fat dry milk, 10mM Tris pH 7.5, 100mM NaCl, 0.1% Tween 20) for 2 hours at room temperature, then incubated with 1:1000 dilutions of anti-mouse PKC antibodies as a primary antibody and 1:10,000 dilutions of goat anti-mouse antibodies as a secondary antibody at room temperature for 2 hours. The protein expression was visualized by the addition of NBT/BCIP substrate.

RESULTS

3H-thymidine findings show that submandibular rat acinar cells growth rate increased at first with the lowest anti-M3R antibody concentration (1x10-6) as compared to the baseline. However, as the concentration of the autoantibody was increased to 5x10-6 and 1x10-5, the corresponding growth rate decreased significantly. Varying concentrations of the isotype control IgM reveal insignificant fluctuations of the growth curve (Figure 1).

Figure 1. Radioactive 3H-thymidine incorporation rates for submandibular rat acinar cells incubated with isotype IgM control antibodies and anti-M3R antibodies of increasing concentrations.

Figure 1. Radioactive 3H-thymidine incorporation rates for submandibular rat acinar cells incubated with isotype IgM control antibodies and anti-M3R antibodies of increasing concentrations.

Increased concentration of PKC g was detected in the cell membranes incubated with anti-M3R antibodies in comparison with the cells incubated with the isotype IgM control (Figure 2). Similar amount of PKC a associated with the plasma membrane was detected in the cells based on the densitometer analysis of the bands indicating that PKC a may not be a significant contributor to the downstream signal transduction pathway (Figure 3). No membrane-associated protein was detected for PKC b in the transformed cell lines incubated with the antibodies. There were no significant differences found for the expression of PKC e stimulated with isotype IgM control and anti-M3R antibodies (Figure 4).

Figure 2. In vitro membrane-associated Protein Kinase C ≥ (PKC ≥) following stimulation of the M3R and signal transduction pathway with carbachol.

Figure 2. In vitro membrane-associated Protein Kinase C γ (PKC γ) following stimulation of the M3R and signal transduction pathway with carbachol. Submandibular rat acinar cells were incubated with the increasing concentrations of the isotope control IgM and the anti-M3 receptor autoantibody (∝ M3R). Incubation with antibodies were harvested and lysed by freeze-thawing. Cell membranes were collected for subsequent Western blotting PKC analysis.

Figure 3. In vitro membrane-associated Protein Kinase C ë (PKC ë) following stimulation of the M3R with carbachol.

Figure 3. In vitro membrane-associated Protein Kinase C α (PKC α) following stimulation of the M3R with carbachol. Refer to Figure 2. for detailed explanation.

Figure 4. In vitro membrane-associated Protein Kinase C ó (PKC) ó following stimulation of the M3R with carbachol.

Figure 4. In vitro membrane-associated Protein Kinase C Η (PKC Η) following stimulation of the M3R with carbachol. Refer to Figure 2. for detailed explanation.

DISCUSSION

The muscarinic receptors can trigger activation of the G protein, which activates phospholipase C. Phospholipase C cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) to inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DAG). The IP3 diffuses through the cytosol and releases stored Ca2+ ions from the endoplasmic reticulum. Released calcium and DAG in the membrane activate protein kinase C (PKC). The activation of PKC in different cell types results in phosphorylation of various substrate proteins that are involved in an array of cellular events. In addition, PKC phosphorylates various transcription factors, such as NF-kB, regulating the transcription of certain genes and thus controlling cell proliferation or apoptosis.

In the current study, the increasing concentrations of the anti-M3R antibody led to the reduced cell proliferation rate in vitro. This finding confirms our hypothesis, which suggests that the anti-M3R autoantibodies act as antagonists for the receptor sites and might trigger pro-apoptotic events in downstream regulatory pathway. The increase in the cell proliferation rate at the low anti-M3R antibody concentration may be due to the partial blockage of the receptors on the acinar cell surface following the stimulation with carbachol.

Upregulation of PKC g was detected in the cells stimulated with anti-M3R antibodies in comparison with the cells incubated with the isotype IgM control in vitro. This suggests that PKC g might play a role in the signal transduction pathway, but additional analyses are recommended to explain the exact role of PKC g in the array of events, since it has been previously noted that induction of apoptosis represents the integration of the activation of both pro- and anti-apoptotic isoforms. Thus, PKC g may be targeted by caspases involved in signal transduction leading to fluid secretion, which could potentially induce blockage of crucial intracellular metabolic events exacting its toll as exocrine hypofunction. Autoantigens are generated following apoptosis via the exposure to cryptic antigens as a consequence of cleavage by activated caspases.

Our current study suggests that anti-M3R antibody acts as an antagonist for the M3R, competing with the agonist carbachol for the receptor binding sites. This antagonistic effect of the antibody can result in the alterations of downstream signaling pathway involved in PKC activation. Therefore, reduced plasma membrane binding may be due to alteration in the membrane dynamics/receptor-ligand interactions when autoantibodies to the M3R are present. Alterations in the expression of PKC isoforms could lead to the final outcome, which is represented as cell proliferation or apoptosis. Studies investigating the outcome of changes in signaling pathway mediated by PKCs need to be further conducted by using cell proliferation assays.

In conclusion, anti-M3R antibodies have a significant influence on the downstream signal transduction pathway by blocking the ligand-binding sites, leading to alterations in membrane translocation of different PKC isoforms, which may have potential consequences of promoting either cell proliferation or cell death.

REFERENCES

  1. Backman, S., Sterin-Borda, L., Camusso, J.J., Arana, R., Hubscher, O., & Borda, E. (1996). Circulating antibodies against rat parotid gland M3 muscarinic receptors in primary Sjögren's syndrome. Clinical Experimental Immunology 104 (3): 454-9.

  2. Fleming, N., & Mellow, L. (1995). Distribution and translocation of isoforms of protein kinase C in rat submandibular acinar cells. Life Sci 57: 2003-2010.

  3. Leszczynski, D. (1996). The role of protein kinase C in regulation of apoptosis: a brief overview of the controversy. Cancer Journal 9: 308-313.

  4. Nguyen, K.H., Brayer, J., Cha, S., Diggs, S., Yasunari, U. et al. (2000). Anti-muscarinic acetylcholine receptor antibodies interfere with salivary gland function in NOD mice. Arthritis Rheum 43(10): 2297-2306.

  5. Tornwall, J., Konttinen, Y., Tuominen, R., & Tornwall, M. (1997). Protein kinase C expression in salivary gland acinar epithelial cells in Sjögren's syndrome. Lancet. 349: 1814-1815.

  6. Waterman, S.A., Gordon, T.P., & Rischmueller, M. (2000). Inhibitory effects of muscarinic receptor autoantibodies on parasympathetic neurotransmission in Sjögren's syndrome. Arthritis Rheum 43(7): 1647-54.

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