Abstract
Epidermal growth factor (EGF) exerts tropic effects on salivary epithelial cells. We examined EGF-mediated signaling pathways in the salivary epithelial cells of patients with Sjögren’s syndrome (SS). We compared the immunohistochemical expression of EGF receptor (EGF-R), phosphatidylinositol 3-kinase (PI3K), Akt and nuclear factor kappa B (NF-κB) in the labial salivary glands of SS patients (n = 6) with those of control subjects (n = 2). EGF-mediated signaling pathways were further studied in vitro (n = 3) using primary salivary epithelial cells; NF-κB p65 nuclear translocation and Akt phosphorylation were examined by immunofluorescence and western blotting, respectively. The phosphorylation of EGF-R and Akt, and the nuclear expression of NF-κB p65, were increased in situ in the salivary epithelial cells of SS patients compared with those of control subjects. Epidermal growth factor induced rapid EGF-R phosphorylation and NF-κB p65 nuclear translocation in primary salivary epithelial cells in vitro. However, EGF also induced late Akt phosphorylation (after 12 h). Chemical inhibition of PI3K-Akt by LY294002/wortmannin did not affect EGF-mediated NF-κB p65 nuclear translocation; and NF-κB inhibition by Bay 11-7082 did not suppress Akt phosphorylation. Our data suggest that EGF stimulates both the PI3K-Akt pathway and NF-κB via distinct mechanisms, promoting tropic effects in SS salivary epithelial cells.
Similar content being viewed by others
Abbreviations
- APS:
-
Aminopropyltriethoxysilane
- DAB:
-
3.3′-diaminobenzidine
- DR4:
-
Death receptor 4
- ECL:
-
Enhanced chemiluminescence
- EGF:
-
Epidermal growth factor
- EGF-R:
-
EGF receptor
- FITC:
-
Fluorescein isothiocyanate
- IKK:
-
IkappaB kinase
- NF-κB:
-
Nuclear factor kappa B
- NGF:
-
Nerve growth factor
- PBS:
-
Phosphate buffered saline
- PDGF:
-
Platelet-derived growth factor
- PFA:
-
Paraformaldehyde
- PI3K:
-
Phosphatidylinositol 3-kinase
- PMSF:
-
Polymethylsulfonylfluoride
- PVDF:
-
Polyvinylidene fluoride
- SDS-PAGE:
-
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
- SS:
-
Sjögren’s syndrome
- TBS:
-
Tris-buffered saline
- TNF:
-
Tumor necrosis factor
- TRITC:
-
Tetramethyl rhodamine isothiocyanate
References
Fox RI, Stern M, Michelson P (2000) Update in Sjögren’s syndrome. Curr Opin Rheumtol 12:391–398
Kong L, Ogawa N, Nakabayashi T, Liu GT, D’Souza E, McGuff HS et al (1997) Fas and Fas ligand expression in the salivary glands of patients with primary Sjögren’s syndrome. Arthritis Rheum 40:87–97
Nakamura H, Koji T, Tominaga M, Kawakami A, Migita K, Kawabe Y et al (1998) Apoptosis in labial salivary glands from Sjögren’s syndrome (SS) patients: comparison with human T lymphotropic virus-I (HTLV-I)-seronegative and -seropositive SS patients. Clin Exp Immunol 114:106–112
Mullighan CG, Heatley S, Lester S, Rischmueller M, Gordon TP, Bardy PG (2004) Fas gene promoter polymorphisms in primary Sjögren’s syndrome. Ann Rheum Dis 63:98–101
Jonsson R, Gordon TP, Konttinen YT (2003) Recent advances in understanding molecular mechanisms in the pathogenesis and antibody profile of Sjögren’s syndrome. Curr Rheumatol Rep 5:311–316
Ohlsson M, Szodoray P, Loro LL, Johannessen AC, Jonsson R (2002) CD40, CD154, Bax and Bcl-2 expression in Sjögren’s syndrome salivary glands: a putative anti-apoptotic role during its effector phases. Scand J Immunol 56:561–571
Polihronis M, Tapinos NI, Theocharis SE, Economou A, Kittas C, Moutsopoulos HM (1998) Modes of epithelial cell death and repair in Sjögren’s syndrome (SS). Clin Exp Immunol 114:485–490
Yamamura Y, Onodera K, Ichinohasama R, Ooya K (2000) A histopathological study of lymphoepithelial island formation in labial salivary glands in patients with primary Sjögren’s syndrome. J Oral Pathol Med 29:110–117
Gorgoulis V, Giatromanolaki A, Iliopoulos A, Kanavaros P, Aninos D, Ioakeimidis D et al (1993) EGF and EGF-R immunoexpression in Sjögren’s syndrome secondary to rheumatoid arthritis. Correlation with EBV expression? Clin Exp Rheumatol 11:623–627
Anest V, Cogswell PC, Baldwin AS Jr (2004) IkappaB kinase alpha and p65/RelA contribute to optimal epidermal growth factor-induced c-fos gene expression independent of IkappaBalpha degradation. J Biol Chem 279:31183–31189
Kitagawa K, Hamada Y, Kato Y, Nakai K, Nishizawa M, Ito S et al (2004) Epidermal growth factor and interleukin-1beta synergistically stimulate the production of nitric oxide in rat intestinal epithelial cells. Am J Physiol Gastrointest Liver Physiol 287:G1188–G1193
Dhar A, Young MR, Colburn NH (2002) The role of AP-1, NF-kappaB and ROS/NOS in skin carcinogenesis: the JB6 model is predictive. Mol Cell Biochem 234–235:185–193
Navolanic PM, Steelman LS, McCubrey JA (2003) EGFR family signaling and its association with breast cancer development and resistance to chemotherapy (Review). Int J Oncol 22:237–252
Franke TF, Hornik CP, Segev L, Shostak GA, Sugimoto C (2003) PI3K/Akt and apoptosis: size matters. Oncogene 22:8983–8998
Viatour P, Merville MP, Bours V, Chariot A (2005) Phosphorylation of NF-kappaB and IkappaB proteins: implications in cancer and inflammation. Trends Biochem Sci 30:43–52
Romashkova JA, Makarov SS (1999) NF-kappaB is a target of AKT in anti-apoptotic PDGF signaling. Nature 401:86–90
Gustin JA, Ozes ON, Akca H, Pincheira R, Mayo LD, Li Q et al (2004) Cell type-specific expression of the IkappaB kinases determines the significance of phosphatidylinositol 3-kinase/Akt signaling to NF-kappa B activation. J Biol Chem 279:1615–1620
Vitali C, Bombardieri S, Jonsson R, Moutsopoulos HM, Alexander EL, Carsons SE et al (2002) Classification criteria for Sjögren’s syndrome: a revised version of the European criteria proposed by the American–European consensus group. Ann Rheum Dis 61:554–558
Ogawa N, Ping L, Zhenjun L, Takada Y, Sugai S (2002) Involvement of the interferon-gamma-induced T cell-attracting chemokines, interferon-gamma-inducible 10 kd protein (CXCL10) and monokine induced by interferon-gamma (CXCL9), in the salivary gland lesions of patients with Sjögren’s syndrome. Arthritis Rheum 46:2730–2741
Ping L, Ogawa N, Sugai S (2005) Novel role of CD40 in fas-dependent apoptosis of cultured salivary epithelial cells from patients with Sjögren’s syndrome. Arthritis Rheum 52:573–581
Nakamura H, Eguchi K, Nakamura T, Mizokami A, Shirabe S, Kawakami A et al (1997) High prevalence of Sjögren’s syndrome in patients with HTLV-I associated myelopathy. Ann Rheum Dis 56:167–172
Nakamura H, Kawakami A, Tominaga M, Migita K, Kawabe Y, Nakamura T et al (1999) Expression of CD40/CD40 ligand and Bcl-2 family proteins in labial salivary glands of patients with Sjögren’s syndrome. Lab Invest 79:261–269
Matsumura R, Kagami M, Tomioka H, Tanabe E, Sugiyama T, Sueishi M et al (1996) Expression of ductal Fas antigen in sialoadenitis of Sjögren’s syndrome. Clin Exp Rheumatol 14:309–311
Ahn JY, Rong R, Liu X, Ye K (2004) PIKE/nuclear PI 3-kinase signaling mediates the antiapoptotic actions of NGF in the nucleus. EMBO J 23:3995–4006
Keates S, Keates AC, Katchar K, Peek RM Jr, Kelly CP (2007) Helicobacter pylori Induces up-regulation of the epidermal growth factor receptor in AGS gastric epithelial cells. J Infect Dis. 196:95–103
Nakamura H, Kawakami A, Yamasaki S, Kawabe Y, Nakamura T, Eguchi K (1999) Expression of mitogen activated protein kinases in labial salivary glands of patients with Sjögren’s syndrome. Ann Rheum Dis. 58:382–385
Ozes ON, Mayo LD, Gustin JA, Pfeffer SR, Pfeffer LM, Donner DB (1999) NF-kappaB activation by tumor necrosis factor requires the Akt serine-threonine kinase. Nature 401:82–85
Dhawan P, Singh AB, Ellis DL, Richmond A (2002) Constitutive activation of Akt/protein kinase B in melanoma leads to up-regulation of nuclear factor-kappaB and tumor progression. Cancer Res 62:7335–7442
Kao SJ, Lei HC, Kuo CT, Chang MS, Chen BC, Chang YC et al (2005) Lipoteichoic acid induces nuclear factor-kappaB activation and nitric oxide synthase expression via phosphatidylinositol 3-kinase, Akt, and p38 MAPK in RAW 264.7 macrophages. Immunology 115:366–374
Meng F, Liu L, Chin PC, D’Mello SR (2002) Akt is a downstream target of NF-kappa B. J Biol Chem 277:29674–29680
Wang HQ, Quan T, He T, Franke TF, Voorhees JJ, Fisher GJ (2003) Epidermal growth factor receptor-dependent, NF-kappaB-independent activation of the phosphatidylinositol 3-kinase/Akt pathway inhibits ultraviolet irradiation-induced caspases-3, -8, and -9 in human keratinocytes. J Biol Chem 278:45737–45745
Ackland ML, Newgreen DF, Fridman M, Waltham MC, Arvanitis A, Minichiello J et al (2003) Epidermal growth factor-induced epithelio-mesenchymal transition in human breast carcinoma cells. Lab Invest 83:435–448
Nakamura H, Kawakami A, Ida H, Koji T, Eguchi K. Epidermal growth factor inhibits Fas-mediated apoptosis in salivary epithelial cells of patients with Sjögren’s syndrome accepted in Clin Exp Rheumatol
Acknowledgments
The authors would like to thank Dr. Noriyoshi Ogawa from the Third Department of Internal Medicine, Hamamatsu Medical University, for advice regarding the primary cell culture of epithelial cells obtained from minor salivary gland biopsy. We would also like to thank Dr. Yoshitaka Hishikawa from the Division of Histology and Cell Biology, Department of Developmental and Reconstructive Medicine, Graduate School of Biomedical Sciences, Nagasaki University, for his kind advice and technical instruction of pixel intensity measurement.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Nakamura, H., Kawakami, A., Ida, H. et al. EGF activates PI3K-Akt and NF-κB via distinct pathways in salivary epithelial cells in Sjögren’s syndrome. Rheumatol Int 28, 127–136 (2007). https://doi.org/10.1007/s00296-007-0411-9
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00296-007-0411-9