mRNA Profiling of Rat Islet Tumors Reveals Nkx 6.1 as a β-Cell-specific Homeodomain Transcription Factor

Abstract

Development of a high capacity multiplex reverse transcriptase-polymerase chain reaction protocol has allowed us to screen lineage related rat islet tumors classified as alpha-, beta-, and delta-like as judged by their hormone profile for differential expression of more than 50 selected genes. We find that in addition to insulin the insulinoma express the normal beta-cell markers Pdx-1, IAPP, and Glut-2, and that these markers are absent from the glucagonoma: a reflection of the normal alpha-cell. Furthermore, this study suggests that the GLP-1, glucagon, GIP, IGF-1, and insulin receptors as well as E-cadherin, R-cadherin, Id-1, and Id-2 are differentially expressed within the islet of Langerhans. Importantly, insulinoma-specific expression of the recently cloned homeodomain protein Nkx 6.1 predicted beta-cell-specific expression in the normal islet. Immunohistochemistry using antibodies raised against recombinant Nkx 6.1 did indeed localize Nkx 6.1 expression exclusively to the nuclei of normal islet beta-cells. Apart from pancreatic islets only the antral part of the stomach contained Nkx 6.1 mRNA. We conclude that multiplex reverse transcriptase-polymerase chain reaction-based mRNA profiling is a powerful tool to identify differentially expressed genes within phenotypically related cells and propose that Nkx 6.1 is involved in specifying the unique characteristics of the beta-cell.

Gene expression profiles during beta cell maturation and after IL-1? exposure reveal important roles of Pdx-1 and Nkx6.1 for IL-1? sensitivity

“…In agreement with the results from the GeneChip array analysis (Table 1) we observed an increased mRNA expression of c-myc and insulin during beta cell maturation. In line with previous analysis [26] semi-quantitative RT-PCR analysis ( Fig. 2) confirmed an induced expression of Nkx6.1 in the beta cell phenotype.…”

“…Interestingly, previous studies of the NHI-cell system showed that Pdx-1 was expressed in both the NHI-ins and the NHI-glu phenotypes, whereas induced expression of Nkx6.1 was associated with maturation into the NHI-ins phenotype [26]. This was substantiated and extended in our array analyses to demonstrate that both Pdx-1 and Nkx6.1 were downregulated after IL-1β exposure exclusively in the beta cell phenotype.…”

“…Following in vivo passage by transplantation in syngeneic NEDH rats, the pre-beta-cell phenotype (NHI-glu) matures into insulinomas [16,30]. Insulinomas reestablished in vitro display an insulin-producing beta cell phenotype (NHI-ins) for prolonged periods of time [31], closely resembling beta cells with respect to their mRNA expression profile [26].…”

“…cDNA synthesis was performed by oligo-dT primed reverse transcription of 1 µg total RNA as described by the manufacturer (Invitrogen cDNA cycle Kit, CA, USA). Semi-quantitative RT-PCR [26] was carried out in 22 µl reaction containing buffer (PCR running buffer, Invitrogen), 500 µmol/l dNTP, 10 µmol/l primer, 50 mmol/l MgCl 2 , 5 units/µl Taq polymerase (Invitrogen), 3000 Ci/mmol [α-33 P] dCTP (Amersham Biosciences, Little Chalfont, UK) and 2 µl cDNA as template. The following protocol was used: 95°C for 5 min, 94°C in 30 s for denaturation, 58-62°C for 30 s for annealing and 72°C in 30 s for extension in 24 to 32 cycles dependent on the transcript, followed by a single extension period at 72°C for 10 min.…”

“…The generation of rabbit antiserum against Nkx6.1 has previously been described [26]. Anti-Nkx6.1-174 was diluted 1:5000.…”