Proteomic Analysis of Human Transplanted Submandibular Gland in Patients with Epiphora after Transplantation

Abstract

Submandibular gland autotransplantation is effective for treating severe dry eye syndrome. However, more than 40% of patients show epiphora within 3-6 months after treatment. The mechanism underlying the hypersecretion in epiphora remains to be elucidated for developing novel interventions. Since salivary gland secretion is dependent on a variety of proteins, we analyzed the changes in protein expression in transplanted glands of epiphora patients with 2-D gel electrophoresis and electrospray ionization quadrupole/time-of-flight mass spectrometry and evaluated their possible roles in epiphora. There were 23 proteins that showed altered expression in the glands of epiphora patients, 15 being up-expressed and 8 being down-expressed. The expression of secretory proteins was decreased in these glands, including alpha-amylase, cystatin S, SA, and SN. In contrast, cytoskeletal proteins were all up-regulated, including actin and vimentin. Immunofluorescence revealed that the intensity ratio of F-actin in apical and lateral cytoplasm to total F-actin in acini was decreased in the glands of epiphora patients. Carbachol stimulation induced a similar redistribution of F-actin in the control glands. Phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) was increased in both carbachol-stimulated and epiphora glands. Preincubation of submandibular glands with ERK1/2 inhibitors PD98059 or U0126 inhibited carbachol-induced F-actin redistribution. These results indicated that differentially expressed proteins participated in the hypersecretion of transplanted submandibular glands and the redistribution of F-actin might be involved in this hypersecretion in an ERK1/2-dependent manner.

Adiponectin Increases Secretion of Rat Submandibular Gland via Adiponectin Receptors-Mediated AMPK Signaling

“…The submandibular gland tissues and cultured cells were homogenized with lysis buffer (containing 50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1% Triton X-100, 0.1% SDS, and 0.1% sodium deoxycholate, pH 7.2) using a polytron homogenizer as previously described [23]. The homogenates were centrifuged at 1000 g for 10 min at 4°C.…”

“…Gland tissues were sectioned 5 μm thick and fixed in cold acetone for 15 min as previously reported [23]. SMG-C6 cells were plated on coverslips and fixed in 4% paraformaldehyde for 10 min.…”

Morphological Features of the Porcine Lacrimal Gland and Its Compatibility for Human Lacrimal Gland Xenografting

“…This procedure is capable of improving comfort, but the ocular surface remains damaged due to the salivary character of the new tear film [6], [7], [8], [9], [10], [11], [12], [13], [14], [15]. Also, more than 40% of patients show epiphora within 3–6 months after transplantation [16]. A graft for DES treatment has to meet several requirements.…”

β-adrenoceptor activation increased VAMP-2 and syntaxin-4 in secretory granules are involved in protein secretion of submandibular gland through the PKA/F-actin pathway

“…Images were captured by confocal microscope (Leica TCS SP5, Heidelberg, Germany). The quantitative measurement of F-actin was performed by Leica TCS SP5 (LAS AF) as previously described [ 16 ]. The fluorescence intensities of F-actin in nine randomly selected acini in each section of six control and six transplanted glands with or without isoproterenol treatment were averaged.…”

“…The second TFA solution was pooled with the first one. Before mass spectrometry, the volumes of peptide-containing solutions were adjusted to 10 μl by the addition of 0.1% TFA in 60% ACN [ 16 ].…”