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. Author manuscript; available in PMC: 2010 Mar 1.
Published in final edited form as: Dev Dyn. 2009 Mar;238(3):724–731. doi: 10.1002/dvdy.21875

A Role for Notch Signaling in Salivary Acinar Cell Growth and Differentiation

Howard Dang 1, Alan L Lin 2, Binxian Zhang 3,4, Hong-Mei Zhang 3,5, Michael S Katz 3, Chih-Ko Yeh 2,4
PMCID: PMC2671016  NIHMSID: NIHMS90545  PMID: 19235730

Abstract

The Notch pathway is crucial for stem/progenitor cell maintenance, growth and differentiation in a variety of tissues. The Notch signaling is essential for Drosophila salivary gland development but its role in mammalian salivary gland remains unclear. The human salivary epithelial cell line, HSG, was studied to determine the role of Notch signaling in salivary epithelial cell differentiation. HSG expressed Notch 1 to 4, and the Notch ligands Jagged 1 and 2 and Delta 1. Treatment of HSG cells with inhibitors of γ-secretase, which is required for Notch cleavage and activation, blocked vimentin expression, an indicator of HSG differentiation. HSG differentiation was also associated with Notch downsteam signal Hes-1 expression, and Hes-1 expression was inhibited by γ-secretase inhibitors. siRNA corresponding to Notch 1 to 4 was used to show that silencing of all four Notch receptors was required to inhibit HSG differentiation. Normal human submandibular gland expressed Notch 1 to 4, Jagged 1 and 2, and Delta 1, with nuclear localization indicating Notch signaling in vivo. Hes-1 was also expressed in the human tissue, with staining predominantly in the ductal cells. In salivary tissue from rats undergoing and recovering from ductal obstruction, we found that Notch receptors and ligands were expressed in the nucleus of the regenerating epithelial cells. Taken together, these data suggest that Notch signaling is critical for normal salivary gland cell growth and differentiation.

Keywords: Salivary Gland, Epithelial Cell Differentiation, Notch, Hes1

Introduction

Branching morphogenesis plays a critical role in the development of many organs including the lung, kidney, mammary gland, and salivary gland (Tucker, 2007). The development of salivary glands from simple precursor epithelial buds to functional glands is a highly complex and dynamic process. Numerous studies have shown that salivary gland branching morphogenesis requires processes such as cell-cell and cell-matrix interactions (Hieda and Nakanishi, 1997), cell proliferation (Morita and Nogawa, 1999) and cell migration (Larsen et al., 2006); various growth factors [e.g., epidermal growth factor (EGF) (Kashimata and Gresik, 1997; Morita and Nogawa, 1999) and several members of the fibroblast growth factor (FGF) family (Hoffman et al., 2002)]; and extracellular matrix (ECM) components (e.g., laminin and fibronectin) (Nogawa and Takahashi, 1991; Larsen et al., 2006).

One approach to understanding salivary gland morphogenesis is to identify molecules that are involved with gland differentiation and development. An important family of molecules in salivary gland differentiation is Notch, which was first identified in fruit flies. Drosophila Notch mutants (Hartenstein et al., 1992; Lammel and Saumweber, 2000) and flies lacking the Notch ligand Ser, do not form salivary glands (Fleming et al., 1997a; Hukriede et al., 1997). Notch homologues have since been identified in numerous other organisms including mammals (Fleming et al., 1997b; Yan et al., 2004). While Notch signaling has been clearly linked in directing cell differentiation during embyrogenesis and self-renewing in many organs (Katsube and Sakamoto, 2005; Blanpain et al., 2007; Fiuza and Arias, 2007), the significance of Notch signaling during mammalian salivary gland differentiation remains to be elucidated.

The Notch receptor mammalian family consists of four members: Notch1 through Notch 4. Notch is a single-pass transmembrane receptor that is a heterodimer comprised of two noncovalently bound subunits. Notch proteins are initially synthesized as full-length unprocessed proteins, following transport through the secretory pathway to the trans-Golgi network, Notch is cleaved at a site referred to as the S1 cleavage site to generate two Notch subunits, one extracellular domain and one with the reminder of the extracellular domain and the complete transmembrane and intracellular domains (Fiuza and Arias, 2007). Similar to Notch, Notch ligands are single-pass transmembrane proteins expressing on neighboring cell surfaces. In mammals, five structurally similar Notch ligands have been identified in mammals, including Jagged1/2 and Delta-like (Dll)1/3/4 (Katsube and Sakamoto, 2005; Blanpain et al., 2007). The cell-cell Notch ligand to Notch receptor interaction initiates successive proteolytic cleavages of Notch by extracellular metalloprotease (S2 cleavage) and γ-secretase (S3/S4 cleavages), resulting in formation of the Notch intracellular domain (Notch IC). Notch IC subsequently translocates to the nucleus where it associates with DNA-binding protein CSL transcription factor, of which the mastermind adaptor is an essential complex component (Chiba, 2006). The binding of Notch IC turns the CSL complex from a transcriptional repressor to a transcriptional activator. The hairy enhancer of split (Hes) family are among the best known of downstream target genes of the Notch IC -CLS complex (Blanpain et al., 2007).

Saliva provides the main oral defense mechanism against oral infection and diseases. Compromised salivary function not only causes severe dental diseases but also adversely affects eating, speech, and overall quality of life (Llena-Puy, 2006). When salivary gland is damaged by an inflammatory (i.e., Sjogren’s syndrome) or physical (i.e., radiation therapy) assault, gland function is usually irreversibly lost. Currently, there is no adequate treatment for patients with such irreversible gland damage. Therefore, the rationale for salivary gland re-engineering or regeneration is to provide better treatment for salivary gland loss. One approach to understand salivary gland re-engineering and regeneration is to identify molecules that are involved in gland differentiation and development. This study shows for the first time that the Notch signaling pathway is involved in expression of differentiation marker vimentin and cystatin S in HSG cells and is upregulated in a rat salivary gland injury/recovery model. The presence of Notch signaling elements in human salivary tissue signifies the importance of Notch signaling in growth and differentiation of adult salivary precursor cells and branching morphogenesis.

Results

Previous studies have shown that the HSG cell line can differentiate into acinus-like structures and express differentiation markers (i.e., vimentin, cystatin and amylase) when grown on an extracellular matrix (Hoffman et al., 1996; Lafrenie et al., 1998; Dang et al., 2006). In the present work, Western blot analysis (Figure 1A) showed that vimentin and cystatin S expression was induced as early as 2 hrs in HSG cells grown on Matrigel®, an extracellular matrix derivative. To investigate the role of Notch signaling during HSG cell differentiation, we first examined the expression of Notch receptor and ligand family members during differentiation on Matrigel®. HSG cells expressed all four Notch receptors and the three Notch ligands (i.e., Jagged 1 and 2, and Delta 1) studied. Growth on Matrigel® had had differential effects on Notch receptor and ligand expression (Figure 1B and C). For example, Notch 2–4 expression was increased after 2 hrs on Matrigel® but Notch 1 expression remained unchanged over time. In addition, Jagged 2 expression was elevated while the expression of Jagged 1 and Delta 1 did not change. Activated Notch 1 receptor (Notch IC) was further examined in HSG cells. Notch IC was minimally detectable in HSG cells grown on plastic culture surface but was clearly evident in cells cultured on Matrigel® (Figure 1F).

Figure 1.

Figure 1

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Expression of Notch receptors and ligands in HSG cells. HSG cells were cultured on plastic or Matrigel® for 2 or 12 hrs. Cell lysates were obtained, and vimentin and cystatin S (A), Notch receptors (B), ligands (D) and Notch IC (F) expression was determined by Western blot analysis. β-Actin expression served as a loading control. Receptor and ligand band intensities were quantified and standardized against β-Actin (C and E, respectively). Data points represent the mean ± SD of 3–5 separate experiments.

Notch receptors require cleavage by γ-secretase in order to generate activated Notch IC (De Strooper et al., 1999). The effect of γ-secretase inhibitors, DAPT and DAPTM, on vimentin and cystatin S expression in HSG cells was determined (Figure 2). Both DAPT and DAPM inhibited Matrigel®-induced vimentin and cystatin S expression in a dose-dependent manner. At 1.0 µM, DAPT and DAPM inhibited vimentin and cystatin S expression to baseline levels (Figure 2A). DAPM and DAPT also prevented Notch IC formation (Figure 2B). Notch pathway activation is tightly associated with the transcriptional repressor Hes-1 (Ohtsuka et al., 1999). As with vimentin, Hes-1 was not expressed in HSG cells grown on plastic but was present when cells were grown on Matrigel® (Figure 2C). Consistent with Hes-1 being Notch pathway dependent, the presence of DAPT or DAPM inhibited Hes-1 expression. These results suggest that HSG differentiation is dependent upon γ-secretase activity and Notch activation.

Figure 2.

Figure 2

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γ-secretase inhibitors, DAPT and DAPTM, blocks vimentin, cystatin S,, Notch IC formation, and Hes-1 induction in HSG cells. HSG cells were cultured on plastic or Matrigel® for 12 hrs. Some cultures contained the γ-secretase inhibitor DAPT and DAPM. Cell lysates were obtained, and vimentin and cystatin S (A), Notch IC (B) and Hes-1 (C) expression was determined by Western blot analysis. β-Actin expression served as a loading control. For panel A, band density was quantified and the vimentin/cystatin S: β-Actin ratios were presented.

To further confirm that Notch signaling is associated with HSG cell differentiation, experiments were performed in which HSG cells were transfected with siRNA corresponding to Notch 1 to 4. Figure 3 shows that knockdown of only a single Notch receptor was not sufficient to inhibit vimentin induction. When HSG was transfected with siRNAs corresponding to all four Notch members, vimentin expression was inhibited suggesting that the four Notch signaling receptors have redundant function.

Figure 3.

Figure 3

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Suppression of Notch expression inhibits vimentin induction in HSG cells. HSG cells were transfected with siRNA corresponding to Notch 1 to 4 and cultured on Matrigel® for 12 hrs. Cell lysates were obtained, and Notch receptor and vimentin expression was assayed by Western blot analysis. Control (Ctl) cells transfected with a scrambled siRNA. β-Actin expression served as a loading control.

Rat ductal obstruction induces salivary acinar cell atrophy whereas the removal of that obstruction results in spontaneous acinar cell recovery (Burford-Mason et al., 1993; Scott et al., 1999). It is thought that during the recovery phase that either the precursor cells are activated to repopulate the gland, or redifferentiation occurs from dedifferentiated acinar cells (Tamarin, 1971a; Tamarin, 1971b; Denny et al., 1997; Takahashi et al., 1998; Scott et al., 1999). In the current study the excretory ducts of rat parotid glands were ligated for 7 days resulting in the loss of acinar cells. The Notch signaling was examined after ductal ligation induced acinar cell loss and at 7 and 30 days post ligature removal. In sham-operated rat parotid glands, Notch 1 to 4, Jagged 1 and 2, and Delta 1 were expressed with much more intense staining in the ductal cells than in the acinar cells (Figure 4). Nuclear staining was observed in both cell types suggesting that both receptor and ligand may be involved in differentiation (Bland et al., 2003). Upon the induction of atrophy all the cells in the gland stained intensely for both Notch receptor and ligands. Gradual gland recovery showed that the recovering acinar cells stained more positively for all the Notch receptors and ligands studied than did acinar cells from the sham-operated group. Coinciding with Notch receptor and ligand expression was Hes-1 (Figure 4). Hes-1 was present in the ductal cells of normal tissues, but was present in the regenerating acinar cells of recovering glands post ligature removal with an expression pattern similar to that of Notch receptors and ligands. These results further suggest in vivo Notch signaling during salivary gland growth and differentiation. Figure 5 confirms that both Notch receptor and ligand co-localizes in the atrophied acinar cell.

Figure 4.

Figure 4

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Notch receptor and ligand, and Hes-1 expression in rat parotid glands undergoing ductal obstruction. The parotid glands were removed from sham-operated rats (Sham), rats undergoing parotid ductal obstruction for 7 days, and rats in which 7-day ductal obstruction was followed by a 7- or 30-day recovery period post-ligation release. The glands were subjected to immunohistochemistry for Notch 1 and 4, Jagged 1 and 2, Delta 1, and Hes-1 with polyclonal rabbit antibodies. Staining pattern and intensity for Notch 2 and 3 were similar to that seen with anti-Notch 1 and 4 antibody (data not shown). (original magnification shown in parenthesis)

Figure 5.

Figure 5

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Notch receptors and ligands co-localizes in salivary acinar cells. Parotid tissue sections from rats that were sham-operated or had undergone ductal ligation for 7 days were stained for Notch 1 and Jagged 1. The immunoconjugate for Notch 1 and Jagged 1 was FITC and Cy3, respectively. The merged image show nuclear co-localization in acinar cells (A) in glands that had undergone ductal ligation and weak staining in tissues from sham-operated animals. There was moderate staining in the ducts (D).

Finally, to demonstrate that our findings can be extended to the clinical setting, we studied the expression of Notch and Notch ligands in normal human submandibular gland (Figure 6). Immunohistochemical staining showed that the human gland expressed Notch 1 to 4, Jagged 1 and 2, and Delta 1. In general, Notch receptors and ligands were stained with more intensity in the ductal cells compared to the acinar cells. Some but not all acinar cells stained positively for the Notch receptors and ligands. In those acinar cells that were positive for the Notch receptor and ligand, the staining was localized predominantly in the nucleus, suggesting active Notch signaling in these cells. In human salivary tissue, all ductal cells stained intensely for Hes-1 whereas an occasional acinar cell displayed weakly positive staining (Figure 6). The strong expression of Notch signal components in ductal cells suggests that these cells indeed are the reservoirs of stem cell/progenitors for human salivary gland replenishment and/or regeneration (Ihrler et al., 2002).

Figure 6.

Figure 6

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Expression of Notch receptors and ligands, and Hes-1 in a normal human salivary gland. Submandibular salivary tissue was derived from a 59 yr old male, fixed in buffered formalin and embedded in paraffin. Tissue sections were stained for Notch 1 to 4 (A), Jagged 1 and 2, Delta 1 (B), and Hes-1 (C) with polyclonal rabbit antibodies. Note the intense staining of the ductal cells and nuclear staining in some of the acinar cells. (original magnification: 400x)

Discussion

Notch receptors and their ligands transduce crucial signals underlying cell to cell interactions in various tissues, and have been conserved across species. The present study shows that the salivary HSG cell line, as well as human and rat salivary glands express all four Notch receptors and many of its cognate ligands. Upon HSG differentiation to acinar-like features, the Notch pathway is activated inducing Hes-1 expression. Furthermore, HSG differentiation is inhibited by γ-secretase inhibitors and siRNA mediated silencing of all four Notch receptors indicating that Notch signaling is involved in HSG differentiation. Finally, Notch signaling is present in the human salivary gland and the regenerating/recovering rat acinar cells suggesting that the Notch pathway has in vivo relevance.

The Notch gene was first identified in fruit flies having “notched” wings and is evolutionarily conserved from worms to humans. The role of the Notch signaling system has been extensively studied in Drosophila development (Katsube and Sakamoto, 2005; Fiuza and Arias, 2007). Both Notch receptors and Notch ligands are transmembrane proteins and, accordingly the Notch signaling is initiated upon interaction of adjacent cells. Notch signaling plays an instructional role for embryo development by providing lateral inhibition, asymmetric cell fate assignment, and boundary formation in Drosophila or somitogenesis in vertebrates (Katsube and Sakamoto, 2005; Fiuza and Arias, 2007). Intact Notch signaling is crucial for embryogenesis/organogenesis of multiple organs and tissues, e.g., central nervous and vascular systems, pancreas, liver, and kidney (Chiba, 2006). In Drosophila, Notch mutants or flies with knockdown of Notch ligand Ser do not develop salivary glands (Hartenstein et al., 1992; Kashimata and Gresik, 1996; Hukriede et al., 1997; Lammel and Saumweber, 2000). In post-natal stages, Notch signaling determines proliferation/differentiation/survival of stem cells or progenitors in organs undergoing active physiological self-renewal such as skin, hair follicle bulge, intestine, and mammary glands (Chiba, 2006; Blanpain et al., 2007; Fiuza and Arias, 2007). In the current study, we have shown that all four Notch receptors (Notch 1 – Notch 4) and all Notch ligands tested (i.e., Jagged 1, Jagged 2 and Delta 1) are expressed in a salivary cell line, rat parotid glands, and human salivary glands indicating that multiple Notch signaling systems are present in the mammalian salivary tissue.

Extracellular matrix is known to cause epithelial cell differentiation mediated through the interaction with cell surface integrin receptor. The HSG cell line contains the characteristics of salivary ductal epithelial cells and shows morphological differentiation with expression of biomarkers vinmentin, salivary cystatin and α-amylase when cultured on a complex extracellular matrix (Hoffman et al., 1996; Zheng et al., 1998). We have used vimentin and cystatin as a marker to evaluate the relationship between Notch signaling and in vitro salivary cell differentiation on Matrigel®. Growth of HSG cells on Matrigel® differentially alter the protein expression of Notch receptors and Notch ligands and triggered the activation of Notch signal pathways as evident by an increase in active Notch IC in the cells. This result is consistent with previous studies showing crosstalk between integrin and Notch pathways (Katsube and Sakamoto, 2005; Campos et al., 2006). Currently, the mechanisms by which integrin activates Notch signaling pathway and the relationships among Notch, Notch ligands and integrin are not clearly defined (Katsube and Sakamoto, 2005; Campos et al., 2006).

Activation of Notch receptor signaling involves multiple proteolytic processes in which γ-secretase play as the key mediator (Shih Ie and Wang, 2007). The membrane γ-secretase complex consists of a catalytic subunit (presenilin-1 or presenilin-2) and accessory subunits. Its substrates consist of amyloid precursor protein (APP, a protein associated with Alzheimer’s disease), CD44, ErbB4 and Notch receptors. As in other reports, in the present study HSG cells grown on Matrigel® were differentiated with expression of vimentin within 2 hr. We have further demonstrated that inhibition of γ-secretase by dipeptides DAPT and DAPM abolish vimentin expression and a Notch downstream effector Hes-1 in cells on Matrigel®. Hes-1, a member of the basic-helix-loop-helix (bHLH) family of DNA binding transcription factors, serves as either a gene repressor or activator (Ross et al., 2006; Liu et al., 2007). Although the direct molecular events linking Hes-1 to vimentin and cystatin S expression is unknown, activation of Notch pathway is clearly involved in regulation of vimentin expression in salivary cells as demonstrated in the current study. The role of the Notch receptor was further substantiated in HSG cells in which all four Notch receptors were downregulated using siRNAs. Interestingly, HSG cells contain redundant Notch signal pathways leading to upregulation of vimentin expression since reduction of individual Notch receptor subtypes by siRNA had no effect on vimentin expression.

Rat salivary duct obstruction and release model has been used widely for studying salivary tissue injury, regeneration/repair and development (Dang et al., 2008). The role of Notch signaling in regeneration in a variety of nonsalivary tissues has been well documented including in exocrine cells (Carlson et al., 2007). For example, during pancreatic injury and repair the Notch signaling pathway is activated (Kashimata and Gresik, 1996). Likewise, in our study expression of Notch 1–4, Notch ligands Jagged 1 and 2, and Delta and Hes-1 was enhanced in atrophic rat parotid gland following ductal ligation and gradually returned to normal levels after release of the ductal obstruction. Nuclear staining of Notch receptors and ligands in the regenerating gland suggests that both are involved in acinar cell differentiation and supports our results in HSG cells.

Recent work from our laboratory has shown that vimentin expression is also upregulated in this ligation/release model indicating that Notch signaling may play a role in vimentin expression in vivo (Dang et al., 2008). A possible mechanism by which Notch signaling might lead to salivary regeneration/recovery is suggested in studies of salivary gland tumors. Fitzgerald et al (Fitzgerald et al., 2000) reported that Notch signaling can activate the Ras pathway in salivary solid tumors derived from a transgenic mouse expressing constitutive active Notch 4. In these sold tumors, Ras in turn results in the activation of ERK 1 and 2 leading to oncogenesis. In the mouse, epidermal growth factor (EGF)-mediated salivary gland branching morphogenesis involves ERK 1 and 2 (ERK1/2) phosphorylation (Kashimata et al., 2000). It is interesting to note that not only are ERK1/2 activated by Matrigel® in HSG cells (Jung et al., 2000; Dang et al., 2006), but also the expression and activation of ERK1/2 and EGF receptors are increased in the rat parotid duct obstruction/release model (Dang et al., 2008). Integrated regulation of EGF and Notch signaling pathways has been shown to be critical for salivary gland and other organ development/renewal as well as tumor development (Haberman et al., 2003; Stenman, 2005; Li et al., 2007; Liu et al., 2007). A causal relationship between Notch activation and the EGF receptor pathway in salivary gland development and repair/regeneration warrants further investigations.

Proliferative markers have been demonstrated in all parenchymal compartments of adult salivary gland tissue (i.e., acini, intercalated ducts, myoepithelial cells, oxyphilic cells and basal cells) (Man et al., 2001; Ihrler et al., 2002). The intercalated duct cells are considered as progenitor/stem cell pools to replenish acini (secretory end pieces), secretory ducts and myoepithelial cells in normal adult salivary glands (Eversole, 1971; Man et al., 2001). In the present work, immunohistochemical staining of rat and human salivary tissues demonstrate that Notch receptors and ligands as well as Hes-1 are expressed mainly in the ductal and intercalated duct cells with few nuclear staining present in some acinar cells, suggestive of involvement of Notch signaling in normal salivary gland replenishment. Preservation or expansion of these ductal stem cell populations is a primary therapeutic target for the development of future strategies to manage salivary gland diseases and radiation-induced damages in the future.

Experimental Procedures

Reagents and Antibodies

γ-secretase inhibitors, N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester (DAPT) and N-[N-3,5-difluorophenacetyl]-L-alanyl-S-phenylglycine methyl ester (DAPM), were purchased from CalBiochem (San Diego, CA) and dissolved in DMSO at a concentration of 5 mM. Rabbit antibodies to vimentin, cystatin S, Notch 1 to 4, Jagged 1 and 2, Delta 1, and Notch 1 intracellular domain (Notch IC); and goat antibody to Jagged 1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and used at a 1:1 000 and 1:100 dilutions for Western blot analysis and immunohistochemical staining, respectively. Small interfering RNA (siRNA) against Notch 1–4 was purchased from Santa Cruz Biotechnology.

Cell Culture

The HSG cell line (Shirasuna et al., 1981) was used throughout theses studies. It was kindly provided by Dr. Bruce Baum (NIDCR, Bethesda, MD). Cells were grown in DMEM/F12 (1:1 mixture) medium containing 5% fetal calf serum, non-essential amino acids, pyruvate, and antibiotics at an initial density of 5×105/ml. In some experiments cells were grown on Matrigel® (BD Bioscience, San Jose, CA) prepared according to the manufacturer’s instructions.

siRNA Transfection

HSG cells were plated at a density of 2 × 105/ml in 35 mm plates and cultured overnight in DMEM/F12 (1:1 mixture) medium. The cells were washed three times with DMEM, followed by incubation with 800 µl of DMEM containing siRNA (60nM) and Lipofectamine™ RNAi Max (Invitrogen, Carlsbad, CA) according the manufacturer’s recommendation for 8 hrs at 37°C. Cells were then washed twice with phosphate buffered saline and cultured in DMEM supplemented with 5% fetal bovine serum and penicillin/streptomycin for an additional 72 hrs. Afterwards, transfected cells were re-cultured on plates with or without Matrigel®.

Ductal Obstruction/Release Rat Model

Ductal obstruction of the rat parotid glands was performed as previously described (Dang et al., 2008) with the approval of the Institutional Animal Care and Use Committee of the University of Texas Health Science Center at San Antonio (UTHSCSA). Three month old male Sprague Dawley rats (Harlan, Indianapolis, IN) were used in these studies. An incision was made over the lateral, posterior mandibular area. The parotid salivary duct was dissected from the facial nerve and surrounding structures. A 3-0 silk ligature was passed through the skin, through a sterile 5–7 mm plastic disc around the duct, back through the disc and through the skin approximately 2–3 mm from the original entry site. The suture was pulled to occlude the duct against the plastic disc tied to subcutaneous skin. This procedure permits ligature removal without another surgical procedure. Animals were given Penicillin B&G postoperatively to prevent infection and buprenorphine for pain relief. For sham-operated animals, the skin was immediately sutured after the initial incision was made.

Human Tissue

A normal submandibular salivary gland was obtained from a 56-year old male undergoing head and neck surgery (National Disease Research Interchange, Philadelphia, PA). Consent was obtained from the National Disease Research Interchange and the protocol was approved by the institutional review board of the UTHSCSA.

Immunohistochemistry

Formalin-fixed tissues were embedded in paraffin and sectioned (4 µm). Paraffin sections were deparaffinized with xylene and hydrated with a series of graded ethanol washes. Endogenous peroxidase was inactivated by incubation for 30 min in 0.3% H2O2. The primary antibody was added for 1 hr at room temperature. Tissue sections were treated with VECTASTAIN® ABC-Peroxidase Kit (Vector Laboratories, Burlingame, CA). The DAB Substrate Kit (Vector Laboratories) was used as the enzyme substrate. Sections were counterstained with CAT Hematoxylin (Biocare Medical, Concord, CA).

Immunofluorescent Staining

Deparaffinized and hydrated tissue sections were first incubated with rabbit anti-Notch 1 antibody followed by biotinylated goat antibody to rabbit IgG (DAKO, Carpinteria, CA) and FITC-conjugated streptavidin. Thereafter, sections were incubated with goat antibody to Jagged 1 followed by biotinylated rabbit antibody to goat IgG (DAKO) and Cy3-conjugated streptavidin (Invitrogen). Prior the initial staining and after the addition of conjugated streptavidin, sections were treated with an avidin/biotin blocking reagent (Invitrogen). Double-labeled tissue images were acquired using a Bio-Red confocal laser scan imaging system. The excitation laser beams used were 488 and 554 nm for FITC and Cy3, respectively. Fluorescent images were sequentially recorded from the same field with 515nm band-pass and 570nm long-pass filters.

Western Blot Analysis

Immunoblot analysis was performed as described previously (Dang et al., 2006). HSY cells were washed three times with cold PBS, lysed in a buffer containing 50 mM Tris·HCl (pH 7.4), 150 mM NaCl, 0.1% NP-40, protease and phosphatase inhibitor cocktails (RIPA buffer) at 4°C for 30 min. After centrifugation at 10 000 × g for 15 min at 4°C, supernatant protein samples were added to 15 µl of 4X Laemmli sample buffer with β-mercaptoethanol. The protein samples were separated on 10% SDS-PAGE gels and transferred to polyvinylidene difluoride membranes (Millipore Corp, Bedford, MA). The membranes were incubated with primary antibody [1:1 000] followed by a secondary horseradish peroxidase conjugated goat antibody (1:1 000). Antibody bound bands were visualized by an enhanced chemiluminescence system (SuperSignal West Pico Chemiluminescent Substrate; Pierce Biotechnology, Rockford, IL).

Acknowledgments

This research was supported by National Institute of Dental and Craniofacial Research R21 DE15381 (C.-K. Yeh), National Heart Lung and Blood Institute R01 HL75011 (B. Zhang) and Department of Veterans Affairs Merit Awards (C.-K. Yeh).

Grant Sponsor: National Institute of Dental and Craniofacial Research, National Heart Lung and Blood Institute, NIH; and the Department of Veterans Affairs

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