Transient TWEAK overexpression leads to a general salivary epithelial cell proliferation

Abstract

Objectives: Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is a multifunctional cytokine that has pro-apoptotic, pro-angiogenic and pro-inflammatory effects. In liver, TWEAK leads to proliferation of progenitor oval cells, but not of mature hepatocytes. This study evaluated the hypothesis that TWEAK overexpression in salivary glands would lead to the proliferation of a salivary progenitor cell.

Methods: A recombinant, serotype 5 adenoviral vector encoding human TWEAK, AdhTWEAK, was constructed, initially tested in vitro, and then administered to male Balb/c mice via cannulation of Wharton's duct. TWEAK expression in vivo was monitored as protein secreted into saliva and serum by enzyme-linked immunosorbent assays. Salivary cell proliferation was monitored by proliferating cell nuclear antigen staining and apoptosis was monitored using TUNEL staining.

Results: AdhTWEAK administration led to a dose-dependent, transient TWEAK protein expression, detected primarily in saliva. Salivary epithelial cell proliferation was generalized, peaking on approximately days 2 and 3. TWEAK expression had no detectable effect on apoptosis of salivary epithelial cells.

Conclusion: Transient overexpression of TWEAK in murine salivary glands leads to a general proliferation of epithelial cells vs a selective stimulation of a salivary progenitor cell.

Figure 1

Western blot showing secreted hTWEAK from A5 cells in vitro. A5 cells were non-transduced (lane 1), or transduced by an irrelevant vector (AdhLeptin; lane 2) or AdhTWEAK (lane 3) at a multiplicity of infection of 200. Immunoreactive hTWEAK protein is shown in lane 3. The arrows to the left indicate the molecular mass (in kDa) of protein standards. See Materials and methods for additional details

Figure 2

Effect of the AdhTWEAK dose administered on the detection of hTWEAK in murine saliva and serum. Mice were administered AdhTWEAK to their submandibular glands at the indicated doses and hTWEAK was measured by enzyme-linked immunosorbent assays after 72 h in both saliva and serum. Data shown are the mean ± s.e.m. of results from four mice. See Materials and methods for additional details

Figure 3

Time course of hTWEAK detection in saliva and serum after AdhTWEAK administration to murine submandibular glands. AdhTWEAK (10⁹ particles per gland), or saline, was delivered to both submandibular glands and hTWEAK was measured by enzyme-linked immunosorbent assays in both saliva and serum at the indicated times. Data shown are the mean ± s.e.m. of results from 12 mice (saliva) and eight mice (serum). See Materials and methods for additional details

Figure 4

Detection of proliferating cell nuclear antigen (PCNA) staining in submandibular glands of mice following AdhTWEAK administration. AdhTWEAK (10⁹ particles per gland), or saline, was delivered to both submandibular glands (n = 4 mice per group) and PCNA staining performed on gland sections as described in Materials and methods to evaluate cell proliferation. Brown staining represents PCNA-positive nuclei. Sections are counterstained with hematoxylin. (a) Day 0 after saline administration; (b) Day 1 after AdhTWEAK administration; (c) Day 2 after-AdhTWEAK administration; (d) Day 3 after AdhTWEAK administration. Magnification is ×200

Figure 5

Quantification of AdTWEAK-induced salivary epithelial cell proliferation. Three examiners independently counted proliferating cell nuclear antigen (PCNA)-positive nuclei, such as shown in Figure 4, and determined the number of positive ductal, acinar and granular convoluted duct cells, as described in Materials and methods. AdhTWEAK (10⁹ particles per gland), saline or AdhEpo (an irrelevant Ad5 vector encoding human erythropoietin; Voutetakis et al, 2005; 10⁹ particles per gland) was delivered to both submandibular glands. For each animal (n = 4 per group), three different fields per section were evaluated. Data shown are the mean ± s.e.m. of the indicated PCNA positive cell types per field for each treatment group. The level of PCNA staining in AdTWEAK-treated glands on day 2 was significantly different from that seen in AdhEpotreated glands

Figure 6

Detection of TUNEL-positive staining in submandibular glands of mice following AdhTWEAK administration. AdhTWEAK (10⁹ particles per gland), or saline, was delivered to both submandibular glands (n = 4 mice per group) and TUNEL staining performed on gland sections as described in Materials and methods to evaluate apoptosis. Brown staining represents TUNEL-positive (apoptotic) nuclei. Sections are counterstained with methyl green. (a) Day 0 after saline administration; (b) Day 2 after AdhTWEAK administration. Magnification is ×200