Signaling pathways for glycated human serum albumin-induced IL-8 and MCP-1 secretion in human RPE cells.
OBJECTIVE
To determine the signal mediators involved in glycated human serum albumin (GHSA) stimulation of interleukin (IL)-8 and monocyte chemotactic protein (MCP)-1 secretion in human retinal pigment epithelium (hRPE) cells.


METHODS
hRPE cells were stimulated by GHSA in the presence or absence of a series of kinase inhibitors. The induced IL-8 and MCP-1 mRNA and proteins were determined by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). Western blot analysis, electrophoretic mobility shift assay, and immunohistochemical staining were used to analyze activation of signaling mediators and transcription factors.


RESULTS
Incubation of hRPE cells with GHSA resulted in rapid activation of Raf-1, extracellular signal-regulated protein kinases (ERK) 1/2, p38, and the transcription factor nuclear factor (NF)-kappaB. Coincubation of hRPE cells with the mitogen-activated protein (MAP) kinase (MEK) inhibitor U0126; NF-kappaB inhibitors BAY11-7085, caffeic acid phenethyl ester (CAPE), parthenolide, and curcumin; protein kinase (PK)C inhibitor Ro318220; and protein tyrosine kinase (PTK) inhibitor genistein largely eliminated most of the stimulated production of IL-8 and MCP-1. Combined inhibition of MEK by U0126, p38 by SB202190, and Janus kinase (jak) by AG490 revealed that GHSA stimulation of IL-8 production was predominately mediated by MEK and to a lesser extent by p38 pathways, whereas activation of MEK, p38, and jak was required for maximal MCP-1 induction. Moreover, GHSA-stimulated IL-8 secretion was more sensitive to U0126 (50% inhibitory concentration [IC(50)] = 0.5 microM) than MCP-1 (IC(50) = 10 microM).


CONCLUSIONS
GHSA stimulates hRPE IL-8 and MCP-1 production through divergent and overlapping, but not identical, intracellular signaling cascades. GHSA induces activation of a series of kinases including PKC, PTK, MAPK, p38, and jak and the transcription factor NF-kappaB. The Raf/MAPK pathway plays an essential role in GHSA signaling.
Bian
Z M
ZM
Departments of Ophthalmology, University of Michigan, Ann Arbor 48105, USA.
Elner
V M
VM
Yoshida
A
A
Kunkel
S L
SL
Elner
S G
SG
D015153:Blotting, Western; D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000096:biosynthesis* / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D017127:Glycation End Products, Advanced; D006801:Humans; D007124:Immunoenzyme Techniques; D016209:Interleukin-8 / Q000096:biosynthesis* / Q000235:genetics; D053613:Janus Kinase 1; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism; D010857:Pigment Epithelium of Eye / Q000187:drug effects* / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012709:Serum Albumin / Q000494:pharmacology*; D015398:Signal Transduction / Q000502:physiology; D048051:p38 Mitogen-Activated Protein Kinases; D000093423:Glycated Serum Albumin
D018932:Chemokine CCL2; D004791:Enzyme Inhibitors; D017127:Glycation End Products, Advanced; D016209:Interleukin-8; D016328:NF-kappa B; D012333:RNA, Messenger; D012709:Serum Albumin; D011505:Protein-Tyrosine Kinases; C507904:JAK1 protein, human; D053613:Janus Kinase 1; D019908:Proto-Oncogene Proteins c-raf; D011493:Protein Kinase C; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D000093423:Glycated Serum Albumin