Cytokine-mediated stimulation of laminin expression and cell-growth arrest in a human submandibular gland duct-cell line (HSG)

• Published in: Archives of oral biology Vol. 44; no. 7; pp. 603 - 615
• Main Authors: Daniels, P.J, McArthur, C.P, Heruth, D.P, Rothberg, P.G, Pasztor, L, Wang, Y
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.07.1999
• Subjects: Blotting, Western; Cell Count - drug effects; Cell Division - drug effects; Cell Line; Cell Size - drug effects; Collagen - biosynthesis; Dentistry; Fibronectin; Fibronectins - biosynthesis; GAPDH; Glyceraldehyde-3-Phosphate Dehydrogenases - biosynthesis; HSG; Humans; Immunohistochemistry; Interferon; Interferon-gamma - pharmacology; Intracellular Fluid - metabolism; Karyotype; Karyotyping; Laminin; Laminin - biosynthesis; Precipitin Tests; RNA, Messenger - biosynthesis; Salivary; Submandibular Gland - cytology; Submandibular Gland - drug effects; Submandibular Gland - metabolism; Time Factors; Tumor Necrosis Factor-alpha - pharmacology; Tumour necrosis factor; Type IV collagen; NBT, p-nitro blue tetrazolium chloride; TNF, tumour necrosis factor; BCIP, 5-bromo-4-chloro-3-indoyl phosphate p-toluidine salt; UTP, uridine triphosphate; TBS, Tris-buffered saline; Type IV collagen; ELISA, enzyme-linked immunosorbent assay; Fibronectin; Tumour necrosis factor; Laminin; PBS, phosphate-buffered saline; Salivary; PMSF, phenylmethylsulphonylfluoride; TBE, Tris- borate–EDTA; ICAM, intercellular adhesion molecule; HSG; DMEM, Dulbecco modified Eagle medium; IFN, interferon; SDS–PAGE, sodium dodecyl sulphate–polyacrylamide gel electrophoresis; IL, interleukin; PCR, polymerase chain reaction; Interferon; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; TTBS, Tween–Tris-buffered saline; GAPDH; Karyotype; FCS, fetal calf serum
• ISSN: 0003-9969; 1879-1506
• DOI: 10.1016/S0003-9969(99)00024-2

Increased expression of laminin and various cytokines, including interferon-γ (IFN-γ) and tumour necrosis factor-α (TNF-α) has been demonstrated in minor salivary glands from patients with Sjögren’s syndrome. Previous reports state that exposure of a human salivary-gland cell line (HSG) to IFN-γ results in cellular changes similar to those in vivo Sjögren’s syndrome. To begin studies of the cause of increased laminin expression in salivary glands in Sjögren’s syndrome and laminin’s role in the pathological process, the effects of IFN-γ on laminin expression and growth of HSG cells were examined here. Subconfluent cultures of HSG cells were treated or not with IFN-γ (1000 units/ml) for 1, 3 or 6 days. Immunoprecipitation showed that the expression of cell-associated laminin was significantly greater in IFN-γ-treated cells at 3 or 6 days than in untreated cells, while no significant differences in laminin counts precipitated from the media were evident among any of the IFN-γ-treated or untreated samples. Western blot analysis strongly suggested that this immunoprecipitated product is a dimer of the β- and γ-chains of laminin. Intracellular laminin was demonstrated immunocytochemically in a distinct, perinuclear pattern in both cytokine-treated and untreated cells. However, only faint staining for type IV collagen, and no staining for fibronectin were evident in untreated and cytokine-treated cells. An RNase protection assay showed only slight upregulation of the laminin β-chain mRNA at 3 days, but no significant difference at 6 days of treatment. Taken together, these data suggest enhanced accumulation of a dimer of laminin β- and γ-chains in the cytoplasm of cytokine-treated HSG cells. However, mRNA for glyceraldehyde 3-phosphate dehydrogenase was significantly reduced at 6 days of treatment, suggestive of cytokine-mediated metabolic abnormalities. IFN-γ treatment also resulted in significant reductions in cell numbers over time, in agreement with previous reports. Treatment of HSG cells for 3 days with IFN-γ (1000 U/ml) and TNF-α (20 U/ml) resulted in no significant changes in cell proliferation or laminin protein and/or mRNA species compared to cells treated with IFN-γ alone. Karyotype analysis of HSG cells revealed human chromosomes with triploid chromosome numbers and rearrangements, characteristic of transformed cells. These data demonstrate that IFN-γ increases the amount of intracellular laminin β–γ dimers while decreasing cell growth. Further studies are required to define an interaction between laminin expression and the growth and viability of HSG cells.