Interleukin-1β Stimulation of c-Jun NH2-Terminal Kinase Activity in Insulin-Secreting Cells
Evidence for Cytoplasmic Restriction
- Department of Pathology and Laboratory Medicine, the Children’s Hospital of Philadelphia and University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania
Abstract
Cytokines have been shown to have dramatic effects on pancreatic islets and insulin-secreting β-cell lines. It is well established that cytokines such as interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and γ-interferon (IFN-γ) inhibit β-cell function and are cytotoxic to human and rodent pancreatic islets in vitro. Despite the pleiotropic effects of cytokines on β-cells, the specific signal transduction pathways and molecular events involved in β-cell dysfunction remain largely unresolved. In this report, we have examined IL-1β stimulation of c-Jun NH2-terminal kinase (JNK) activity in insulin-secreting clonal cell lines. We demonstrate that IL-1β transiently activates 46- and 54-kDa isoforms of JNK in cultured RINm5F β-cells. Furthermore, IL-1β stimulation of JNK activity is specific, because TNF-α and IFN-γ were without effect. Stable overexpression of JNK1 in RINm5F cells increased levels of activated JNK without affecting kinase activity. JNK-interacting protein (JIP) associates with endogenous as well as overexpressed JNK, suggesting that JIP may serve to regulate JNK activity. Finally, we demonstrate that activated JNK is fully retained in cytoplasmic and membrane compartments without any nuclear translocation. Together, these data indicate that IL-1β–stimulated JNK activity may be distinctly targeted to cytoplasmic and/or membrane compartments in clonal insulin-producing cells, and that JIP may serve to localize JNK activity to specific substrates.
Footnotes
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Address correspondence and reprint requests to The Children’s Hospital of Philadelphia, Department of Pathology and Laboratory Medicine, 5135 Main Bldg., 34th Street and Civic Center Blvd., Philadelphia, PA 19104-4399. E-mail: wolfb{at}mail.med.upenn.edu.
Received for publication 27 November 2000 and accepted in revised form 6 September 2001.
BSA, bovine serum albumin; DMRIE, 1,2dimyristoyloxypropanyl-3-3dimethyl-hydroxy ethyl ammonium bromide; DTT, dithiothreitol; ERK, extracellular signal regulated kinase; IB-1, islet brain-1; IFN-γ, γ-interferon; IL-1β, interleukin-1β; iNOS, inducible nitric oxide synthase; IUF-1, insulin upstream factor-1; JIP, c-Jun NH2-terminal kinase–interacting protein; JNK, c-Jun NH2-terminal kinase; MAPK, mitogen-activated protein kinase; MAP2K, MAPK kinase; MEF, mouse embryonic fibroblast; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride; SA-MAPK, stress-activated MAPK; TBS, Tris-buffered saline; TNF-α, tumor necrosis factor-α.
