Expression of Mixed Lineage Kinase-1 in Pancreatic β-Cell Lines at Different Stages of Maturation and during Embryonic Pancreas Development

Henry J Deaizpurua,David S Cram,Gaetano Naselli,Lisa Devereux,Donna S Dorow

doi:10.1074/jbc.272.26.16364

Abstract

Events controlling differentiation to insulin-secreting beta-cells in the pancreas are not well understood, although beta-cells are thought to arise from pluripotent ductal precursor cells. To search for signaling proteins that might be involved in beta-cell maturation, we analyzed protein kinase expression in two developmentally and functionally distinct pancreatic beta-cell lines, RIN-5AH and RIN-A12, by reverse transcriptase polymerase chain reaction. A number of tyrosine and serine/threonine kinases were identified in both lines. One protein kinase, mixed lineage kinase-1 (MLK-1), was expressed at both the RNA and protein levels in RIN-5AH cells, which display an immature beta-cell phenotype, but was not detected in the more mature RIN-A12 cells. Furthermore, levels of MLK-1 mRNA and protein were increased after brief stimulation of RIN-5AH cells with either the differentiation inducer, sodium butyrate, or with serum after serum starvation. These increases in expression were independent of phenotypic markers such as insulin secretion or surface expression of major histocompatibility class I- and A2B5-reactive ganglioside. In addition, increases in MLK-1 expression in the stimulated RIN-5AH cells were accompanied by phosphorylation of MLK-1 on serine but not tyrosine. Antisense oligonucleotides to two distinct regions of MLK-1 caused RIN-5AH cells, but not RIN-A12 cells, to adopt a highly undifferentiated morphology, with a reduction in DNA synthesis and MLK-1 protein levels and elevated glucagon mRNA levels, but with no effect on insulin mRNA. In an immunohistochemical survey of embryonic mouse tissues, we found that temporal expression of MLK-1 was regulated in a tissue-specific manner. In the embryonic pancreas, MLK-1 expression was evident in ductal cells from day 13 to 16 but was not detected in late stage gestation or neonatal pancreas. These data suggest that MLK-1 is regulated in immature pancreatic beta-cells and their ductal precursors at the level of functional maturity and may therefore play a role in beta-cell development.

Highlights

§ To whom correspondence should be addressed: Burnet Clinical Research Unit, The Walter and Eliza Hall Institute of Medical Research, Post Office, Royal Melbourne Hospital, Parkville 3050, Australia
rat insulinomas (RINs)-5AH cells are characterized by a flat, clustered, less differentiated morphology, low levels of insulin mRNA and secreted insulin, and relatively high surface expression of MHC class 1, with lower levels of A2B5-reactive gangliosides compared with those detected in RIN-A12 cells
RIN cells have been demonstrated to be pluripotent, with multiple hormone-producing cell lines generated from single cells [16, 17]

Summary

Introduction

In studies of cell growth and differentiation, it has been demonstrated that binding of polypeptide growth factors to their cognate receptors leads to the sequential activation of a series of signaling proteins that link the cell surface with nuclear targets and results in altered gene expression [9, 10]. Two subclones of a parental line (RINm) can be distinguished by differences in cell morphology, insulin mRNA expression, and several other surface markers, including A2B5-reactive ganglioside and MHC class 1 and have been used to analyze maturation events potentially relevant to b-cell development [15,16,17]. The aim of the present study was to compare the pattern of protein kinase expression in two rat b-cell lines displaying either the immature (RIN-5AH) or mature (RIN-A12) b-cell phenotype and to identify protein kinases associated with a particular stage of development. MLK-1 antisense oligonucleotides were used to link MLK-1 expression to the phenotypic status of the immature b-cell

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