• Introduction
• Materials and Methods
• Animal sacrifice
• Administration of CsA
• Denervation of the chorda‐lingual nerve
• Microscopic observation
• Total RNA preparation and PCR
• Immunohistochemistry
• Immunoblotting
• Results
• Immunohistochemical Findings
• Expression of CypA
• Discussion
• Acknowledgements

Introduction

 Cyclosporine A (CsA) is a hydrophobic cyclic peptide composed of 11 amino acids (MW=1202.6) which is naturally produced by the fungus  Tolypocladium inflatum [1]. CsA forms a heterotrimeric complex with cyclophilin and calcineurin, the calcium, calmodulin-depen dant serine/ threonine phosphatase [2,3]. It inhibits calcineurin activity in T-cells, a critical step in activation of T‐cells, inhibiting IL-2 tyrosine kinase in T cells [4,5]. Since it is known as a safe and potent immunosuppressive agent, it is widely used not only in dealing with a variety of autoimmune disorders of immunologic origin but also in the manage-ment of post-transplantation patients to prevent unwanted rejection phenomena of transplanted organs [6,7].

 Cyclophilin A (CypA), a CsA binding protein is a ubiquitously distributed intracellular protein [8] for peptidy-lprolyl cis-trans-isomerization [9] and has been regarded as an immunophilin because of its ability to bind cyclos-porine A [10,1]. The molecule is secreted in response to in-flammatory stimuli [12,13], initiating signaling response in target cells [14] and chemoattracting neutrophil [12] and eo-sinophil [13] and T cells chemoattractant [15,16] in vitro and in vivo.

CypA increases endothelial cell proliferation, migra-tion, invasive capacity and tubulogenesis at low concen-trations (10 to 100 ng/ml). To the contrary, it decreases endothelial cell migration and viability at a high con-centration (2  μg/ml). The molecule is also released from cardiac myocytes in response to hypoxia/reoxygenation, protecting them from oxidative stress-uced apoptosis [17, 18]. Thus, CypA has been considered as a novel paracrine and autocrine modulator of endothelial cell functions in immune mediated vascular diseases [19]. The major sali-vary glands undergo complex processes for growth and dif-ferentiation for branching morphogenesis during prenatal and postnatal periods [20]. This morphogenesis is regu-lated by hormones, elements in the extracellular matrix (ECM), and the nervous system. At birth, the submandibular gland in rodents acquires a ramifying ductal system ending in terminal tubules which consist of terminal tubule cells and proacinar cells. Until day 2, the terminal tubules mostly consist of buds of proacinar cells, which give rise to acinar cells during the first week of development. At the end of the first week, these buds become composed predominantly of acinar cells and the terminal tubules become the site of future intercalated ducts [21].

 Although CsA is a potent immunosuppressant, its action is not restricted to the lymph system. This molecule stimu-lates proliferation and endocrine function of thymic epit-helial cells in human and mice [22]. In the exocrine glands including the salivary and lacrimal glands, CsA induces functional and morphological changes in the salivary glan-ds [23], increasing fluid secretion of tear and saliva [24] and also altering salivary composition. However, its mode of ac-tion for exocrine secretion is still largely unknown, although it has been suggested that CsA increases salivation by re-leasing neurotransmitters [24,25] and/or by preventing lym-phocyte-induced apoptosis of acinar cells in Sjὃgren syndrome [26,27].

 Those reported clinical effects of CypA on salivation strongly suggest that CsA may act on acinar or tubular cells via its receptor, CypA. Consequently, it is expected that CypA will be subject to changes in expression for the next downstream events. The present study was performed to describe how CsA acts on ductal cells in the salivary glands via modulating expression of its receptor, CypA.

Materials and Methods

Animal sacrifice

 Sprague-Dawley rats were raised in Association for Ac-creditation and Assessment of Laboratory Animal Care- Approved Facilities and provided with regular food and tap water. Pups at postnatal days 1, 4, 7 and 10 and adults were sacrificed by decapitation.

Administration of CsA

 CsA (Chongkundang Pharm, Seoul, Korea) was disso-lved in olive oil and subcutaneously injected daily into adult and postnatal day 1 rats at a concentration of 10 mg/kg body weight for 10 days.

Denervation of the chorda‐lingual nerve

Adult rats approximately weighing 200 g were anes-thetized by intraperitoneal injection of ketamine (50 mg/ kg). After a vertical skin incision was made along the main duct of the left submandibular gland, the chorda-lingual nerve was carefully exposed at the level of the hypoglossal nerve, where the chorda‐lingual crosses over, and severed under the mylohyoid muscle. The right submandibular glands were sham operated and used as a control.  

Microscopic observation

 Pup rats were sacrificed by decapitation without the perfusion. The adult rats were sacrificed by perfusion fi-xation using 4% paraformaldehyde solution at postope-rative days 5 and 13. The glands were further immer-sion-fixed in the same solution overnight. They were then dehydrated in a graded ethanol series and embedded in paraffin. Sections were cut 5 μm thick and stained with H-E for morphological characteristics.

Total RNA preparation and PCR

 The isolated submandibular glands were immediately frozen in liquid nitrogen. Total RNA was extracted using a Trizol^® Reagent (Invitrogen) according to manufacturer's instructions. The extracted RNA samples were quantified using UV spectrophotometer. CypA primers were custom- designed (GenBank accession no. XM_344509.1). The sequences of the forward and the reverse primers of CypA were 5' AGA CAA AGT TCC AAA GAC AG 3' and 5' GAG AGC AGA GAT TAC AGG G 3' respectively, generating an expected PCR product of 521 bp. The housekeeping gene GAPDH (GenBank accession no. AF_106860) was also amplified using primers of specific sequences of 5' CCA TGG AGA AGG CTG GGG 3' for the forward and 5' CAA AGT TGT CAT GGA TGA CC 3' for the reverse, generating an expected product of 195 bp. DNase I (Gibco BRL, MD, USA) was treated before cDNA synthesis was performed. First strand cDNA synthesis was performed using Superscript II (Gibco BRL, MD, USA). For the first reactions, mixtures of 25 mM Oligo (dT)_12-18 (Gibco BRL, MD, USA) and 1 μg of RNA, were incubated at 70℃ for 10 min. For the second reactions, First strand buffer, DTT, dNTP mix and RNAse inhibitor (Gibco BRL, MD, USA) were gently mixed to the first reactions for incubation at 42℃  for 2 min, followed by adding two hundred units of Superscript II for incubation for 50 min at 42℃. The reaction was inactivated by heating at 70℃ for 15 min and followed by the addition of RNase H and subsequent incubation at 37℃ for 20 min. RT controls were carried out using the same RT reaction mixtures except substituting cDNA for DEPC‐treated H₂O. Thirty PCR cycles were performed in a Perkin‐Elmer GeneAmp PCR system 2400 with the following profile: denaturation for 1 min at 95℃, annealing for 1 min at 60℃ for both CypA and GAPDH primers and 1 min extension step at 72℃. The last cycle was followed by a final extension step of 10 min at 72℃. Preliminary studies were performed to determine the optimum number of cycles for quantization. PCR products were resolved on a 1.2% agarose gel and visu-alized using ethidium bromide. The size was confirmed using 1 kb DNA ladder (Gibco BRL, MD, USA).

Immunohistochemistry

 Immunohistochemical reaction was performed using Vectastain Elite ABC Kit (Vector Laboratories, Burlin-game, CA, USA). Purified mouse monoclonal anti-CypA and anti‐PCNA (Santa Cruz biotechnology, Inc. USA) were used as primary antibodies. For the negative control of the reaction, normal horse serum was used to substitute the primary antibodies. Blocking of endogenous peroxidase and subse-quent non-specific reactions were undertaken by incubating sections in 0.3% H₂O₂ for 20 min and in the blocking serum for 30 min, respectively. Sections were reacted in the pri-mary antibodies at 4℃ overnight, followed by incubation in biotinylated secondary antibody for 2 hrs. They were then reacted in avidine-biotin peroxidase complex for 30 min and developed with AEC for microscopic observation.

Immunoblotting

 Total proteins were prepared from the submandibular glands. The tissues were cut into small pieces and ho-mogenized in a buffer containing 20 mM Tris‐HCl at pH 8.0, 150 mM NaCl, 2 mM  EDTA, 10% glycerol, 0.5% Nonidet P-40, 1 mM dithiothreitol, 1 mM phenylmet-hylsulfonyl fluoride, 5 µg/ml leupeptin, 5 µg/ml aprotinin and 5 µg/ml pepstatin. The homogenates were centrifuged at 12,000 rpm at 4℃. Protein concentrations of the supernatants were determined using the Bradford protein assay reagent (Bio-Rad, CA, USA). Extracts containing total proteins were aliquotted and stored at 80℃. Protein lysates were boiled for 5 min in a denaturing sample buffer and 10 µg of total protein was loaded into each well of 12% SDS‐polya-crylamide gel. They were then electrophoresed at 100 V for 50-60 min at 4℃ and electrotransferred to Hybond ECL membranes (Amersham Pharmacia Biotech, USA), which were then blocked with TBS‐T buffer (10 mM Tris-buffered isotonic saline, pH 7.0, 0.1% merthiolate, and 0.1% Tween-20) containing 5% nonfat dry milk for 1 hr at room tempe-rature with shaking. The membranes were then incubated with the polyclonal antibody to CypA (Calbiochem, CA, USA) diluted 1:500 in TBS-T buffer for 24 hrs at 4℃ with gentle shaking. The membrane was washed twice with the same buffer for 10 min and incubated using appropriate horse-radish peroxidase-conjugated secondary antibody (CST, USA) diluted 1:5000. Immunodetection was performed using the ECL substrate (Amersham Pharmacia Biotech, USA) accor-ding to the manufacturer's instructions. The protein markers were the Prestained SDS -PAGE Standards which is ranged from 6 to 194 kD (Bio- Rad, CA, USA).

Results

Immunohistochemical Findings

 CypA immunoreactivities were assessed in the sub-mandibular glands. At postnatal day 10, CypA immu-noreactivities were observed in most ducts, but not in acini (Fig. 1a). To evaluate effects of CsA treatment on CypA expression, immunoreactivities of CypA were also mea-sured. Stronger intensities were seen in the CsA treated glands than those in the normal control (Fig. 1b).

Fig. 1. (a) At postnatal day 10, CypA immunoreactivities are observed in ducts of the normal submandibular glands, not in acini. (b) Stronger CypA intensities than those in the normal control are demonstrated in the glands treated with CsA for 10 days. (c) At postnatal day 10, PCNA immunoreactive cells are rarely seen in ducts of the normal glands. (d) Many PCNA immunoreactive cells are demonstrated in nuclei of ductal cells in the glands treated with CsA for 10 days. (e) Negative control omitting the primary antibodies shows no immunoreactivities.

 To evaluate effects of CsA treatment on cell proli-feration, immunoreactivities against PCNA were also as-sayed. At postnatal day 10 in the normal control glands, PCNA immunoreactivities were rarely observed in the nuclei in ducts (Fig. 1c). However, immunoreactive nuclei were frequently found in the CsA treated glands for 10 days (Fig. 1d). Negative controls omitting primary anti-body were not stained in all the reactions (Fig. 1e).

Expression of CypA

 CypA mRNA was found in a single band of 521 bp as expected from the primer design. To evaluate the deve-lopmental changes of expression of CypA mRNA in the submandibular glands, RT‐PCR was performed at 5 deve-lopmental points: postnatal days 1, 4, 7 and 10 and adult. Its expression level was maintained from postnatal day 1 (P1) to 7 (P7), and then decreased in a time- dependent manner (Fig. 2).

Fig. 2. Levels of CypA mRNA expression were determined at 5 developmental time points by RT‐PCR.

 To determine the effects of  denervation of the chorda- lingual nerve, secretomotor fibers in the submandibular glands, on CypA expression, its mRNA level was de-termined by RT- PCR at 13 and 16 days after dener-vation. The CypA mRNA expression seemed to be slightly decreased at day 13, but did not show changes at day 16 by the denervation (Fig. 3).

Fig. 3. To evaluate effects of denervation of the chorda‐lingual on the expression of CypA, its mRNA was determined by RT‐PCR. Its mRNA level was not affected by the denervation at postoperation days 13 and 16. C: control, E: experiment.

 To determine the effect of CsA on the expression of CypA mRNA, its level was elucidated by RT‐PCR at 10 days after CsA administration. Its expression level was inc-reased two fold by the CsA administration (Fig. 4). Also, this result was confirmed at the protein level by immuno-blotting (Fig. 5).

Fig. 4. To elucidate effects of CsA on the expression of CypA, its mRNA was determined by RT‐PCR. Expression of CypA mRNA was increased by the treatment for 10 days.

Fig. 5. Increased expression of CypA by 10 day treatment of CsA was confirmed by immunoblotting.

Discussion

 The parenchyma of the mature submandibular gland in rats consists of acini, intercalated ducts, granular convo-luted tubules and striated ducts. The striated ducts are continuous with interlobular ducts that form the common excretory duct (ED), leading to the oral cavity [28,29]. The three major glands share a common developmental pattern in rodents, in that their full branching morphogenesis of the ductal system is accomplished during postnatal period [20]. The submandibular gland at birth in rodents consists of a ramifying ductal system which ends in terminal tubules consisting of terminal tubule cells which become the site of future intercalated ducts and proacinar cells which give rise to acinar cells during the first week [21,30].

During the morphogenesis in the salivary gland, complex processes for growth and differentiation are regulated by hormones, elements in the extracellular matrix (ECM), and the autonomic nervous system. Of note, CypA increases endo-thelial cell proliferation, migration, invasive capacity and tubulogenesis at low concentrations (10 to 100 ng/ml). To the contrary, it decreases migration and viability at high con-centration (2 µg/ ml). The molecule is released from cardiac myocytes in response to hypoxia/reoxygenation, protecting them from oxidative stress‐induced apoptosis [17]. It has been considered a novel paracrine and autocrine modulator of endothelial cell functions in immune mediated vascular diseases [19].  

 Regarding the role of CypA in the salivary gland, re-search thus far is sparse. In the present study, to evaluate its posacsible role during salivation, the chorda- lingual nerve, secretomotor fibers the submandibular gland, was cut to block of salivation. The expression of CypA was not changed at days 13 and 16 after the operation. These results suggested that CypA is not involved in either salivation or changes of composition in saliva.

 A possibility that CypA may be implicated in branching morphogenesis was tested by monitoring the expression levels of CypA during the development of the gland. Its level was maintained up to postnatal day 7, gradually dec-reasing at postnatal day 10 and adult. These expression pat-terns raise a possibility that CypA may be involved in ductal morphogenesis in the salivary gland.

The function of CsA in the salivary gland and its mode of action for exocrine secretion is still largely unknown. Ho-wever, several reports suggested that the molecule can inc-rease fluid secretion of tear  and saliva [24] and also alter composition of saliva. This implies that CsA can induce functional and morphological changes in the salivary glands [23]. In the present study, CsA increased CypA expression by short term treatment of the CsA. This result raised a pos-sibility that CsA can induce ductal morphogenesis, con-sequentially resulting in changes in salivation and com-position of saliva. 

The effects of CsA on branching morphogenesis in the salivary gland was also immunohistochemically confirmed by staining proliferating cell nuclear antigen (PCNA) which is synthesized in early G1 and S phase of the cell cycle for cell cycle progression [31]. In the present study, PCNA posi-tive cells were increased in nuclei of duct cells by the 10 days' treatment of CsA. These results were in agreement with the report that CsA stimulated proliferation of thymic epithelial cells in human and mice [22].  

In summation, the present study showed that CsA treat-ment increased CypA at the translation and transcription levels. Considering the previous reports that CypA is involved in cell proliferation  and tubulogenesis in endot-helial cells, it is assumed that CsA can directly act on duc-tal cells in the salivary glands, modulating expression of its receptor, CypA. The molecular mechanism of CypA release is not known thus far. Important issues for future studies may include the in vivo conditions that stimulate the secretion of CypA. Answers to these questions will help to understand how CsA affects salivation and tearing in the salivary and lacri-mal glands respectively.

Acknowledgements

 This research was supported by the Chonnam National University Research Institute of Clinical Medicine (CRI-10051-1) and the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. 2011-0030760).