Nuclear Protein p8 Is Associated With Glucose-Induced Pancreatic β-Cell Growth

INS-1 cells as well human pancreatic islets were routinely maintained in RPMI-1640 medium containing 10% heat-inactivated fetal bovine serum, 10 mmol/l HEPES, 1 mmol/l sodium pyruvate, 2 mmol/l l-glutamine, 50 units/ml penicillin, 50 μg/ml streptomycin, and 50 μmol/l β-mercaptoethanol. The medium was further supplemented with 5.6 mmol/l (low), 11.1 mmol/l (standard), or 25 mmol/l (high) glucose. Cells were seeded in 60-mm dishes at a density of 2 million cells per dish. In the presence of 11.1 mmol/l glucose, the resulting cell layers were half confluent after ∼3 days of culture. Standard medium was then exchanged with medium containing 25 mmol/l glucose to start induction of p8 mRNA expression. For analysis of glucose-induced p8 protein expression, cells were maintained for one passage in medium with 5.6 mmol/l glucose and then directly seeded in medium containing 25 mmol/l glucose.

The amount of proliferating cells was analyzed using the 5-Bromo-2′-deoxy-uridine Labeling and Detection Kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s protocol.

RNA was isolated using Trizol and then reverse-transcribed into cDNA with Superscript II using oligo-(dT) primers according to the manufacturer’s protocol (Life Technologies, Karlsruhe, Germany). PCR was performed using specific primers for p8. Resulting PCR products were size-fractionated in agarose gels, stained with ethidium bromide, and visualized under ultraviolet light. Quantification was determined by densitometric scanning. (Oligo sequences and PCR conditions can be obtained from the authors on request.)

The primary antibody used for both immunohistochemistry and Western blotting was a polyclonal p8 antiserum generated in a rabbit with a bacterially expressed the p8 glutathione sulfonyl transferase fusion protein as antigen. Immunohistochemical staining of negative controls was performed without p8 antiserum. Western blotting was performed with whole cell extracts. Equal amounts of protein were size-fractionated on 10–20% Tris-Tricine Ready Gels (Bio-Rad, München, Germany). Visualization was achieved using Envision AP and Fast Red (both from DakoCytomation, Hamburg, Germany) or Cy3 fluorescently labeled goat anti-rabbit antiserum for immunohistochemistry and ECL Plus Western Blotting Detection Reagents (Amersham Biosciences Europe, Freiburg, Germany). After visualization, blotted p8 protein was quantified by densitometric scanning.

p8 transcripts were detected in human pancreatic islets and several pancreatic cell lines including β-cells, acinar cells, and ductal cells as indicated in Fig. 1A. Immunohistochemical staining revealed a wide p8 expression in human islets (Fig. 1B), although other studies demonstrated p8 expression within the pancreas in vivo only in acinar cells (6,9). It is noteworthy that p8 is primarily located within the cytoplasm, which may indicate absence of cell proliferation in cultured islets. Immunohistochemical staining of proliferating rat INS-1 β-cells demonstrates translocalization of p8 into the nucleus (Fig. 1C). p8 protein expression in rat INS-1 β-cells was additionally detected by Western blot analysis (Fig. 3B).

Effects of elevated glucose concentrations on proliferation and p8 production were established in glucose-sensitive rat INS-1 β-cells, a well-characterized in vitro model for analysis of β-cell function. In dishes with half-confluent cell layers, enhancement of glucose concentrations from 11.1 to 25 mmol/l continuously enhanced the amount of proliferating cells up to threefold (Fig. 2). This was paralleled by an increase in p8 mRNA expression reaching a maximum of threefold after 24 h (Fig. 3A). Elevation of glucose concentrations in the medium from 5.6 mmol/l (one passage) to 25 mmol/l resulted in a continuous rise in p8 protein expression up to 5.5-fold after 72 h (Fig. 3B).

The present study demonstrates that p8 is expressed in human islets and certain rodent pancreatic β-cell lines. Furthermore, proliferation and p8 expression in rat INS-1 β-cells is strongly inducible by high glucose concentrations in the medium. Our findings point to a possible new role of p8 as a mediator of glucose-induced pancreatic β-cell proliferation. This hypothesis is supported by studies demonstrating increased cell growth in p8 overexpressing rat AR42J pancreatic acinar cells (6) and human HeLa cervix epithelial cells (7). Moreover, glucose-induced pancreatic β-cell proliferation involves activation of PKC (2,3), and rat as well as human p8 amino acid sequences contain a highly conserved region with two PKC sites (16).

p8 was originally postulated as a pancreatic emergency gene product, which is exclusively upregulated in pancreatic acinar cells during stress caused by acute pancreatitis (6). The emergency gene character was underlined by the strong increase of pancreatic p8 expression in vivo and in rat AR42J acinar cells in vitro after endotoxin shock with lipopolysaccharide (17). Interestingly, former findings detect apoptosis during acute pancreatitis also only in acinar cells (18,19). Thus, Mallo et al. (6) tested several apoptosis-inducing conditions such as the addition of tumor necrosis factor-α, cycloheximide, dexamethasone, ceramide, or staurosporine or serum starvation with AR42J cells and observed upregulation of p8 mRNA expression. This observation led to the assumption that p8 expression is responsive to intracellular apoptotic signals.

Whereas p8-overexpressing HeLa cells displayed a twofold proliferation rate, tumor necrosis factor-α and cycloheximide alone or in combination did not alter survival (7). In contrast, p8-positive (p8^+/+) wild-type mouse embryonic fibroblasts (MEFs) grew less rapidly and were more sensitive to adriamycin-induced apoptosis than transgenic p8-deficient (p8^−/−) MEFs (10). Moreover, serum deprivation much more rapidly resulted in cell growth arrest in p8^+/+ than in p8^−/− MEFs. The growth inhibitory action of p8 in p8^+/+ MEFs seems to involve p53, because both proteins were elevated in parallel and p53 was shown to suppress the p8 promoter in a dose-dependent manner. The finding that p8 mediates inhibition of cell growth in certain conditions is supported by increased 1,25-dihydroxyvitamin D₃-mediated suppression of colony formation by MCF-7 human breast cancer cells stably transfected with a p8 expression plasmid compared with p8-negative MCF-7 cells (12).

Besides the involvement of p8 in anti-tumorigenic processes such as apoptosis and growth arrest, several studies report a possible role in tumor development. p8 is highly expressed and negatively correlated to apoptosis in pancreatic cancers in vivo (8,9). Furthermore, p8 expression was found in MDF7/LCC2 cells, a highly tumorigenic subline of the low tumorigenic p8-negative MCF-7 cells (12), and p8 was demonstrated to be critical for tumor development in MEFs transfected with a ras^V12 and E1A oncogene, because p8^+/+ MEFs were able to and p8^−/− MEFs failed to form colonies (11). In contrast to its putative function as an oncogene, we observed a downregulation of glucose-induced cell growth and p8 expression in cultured INS-1 β-cells (data not shown), which may be a result of contact inhibition due to confluency. This absence of uncontrolled cell growth indicates that in pancreatic β-cells, p8 is regulated by intracellular signals and does not result in transformation leading to tumor development. Alternatively, it has to be considered that increased p8 expression in pancreatic cancer in vivo (8,9) is rather associated with proliferation but not with the development of tumors per se.

Taken together, these data show that p8 is involved in a broad range of biological functions, including cell growth, growth arrest, apoptosis, and tumor development. These in part opposite actions may be related to distinct stimuli and pathways in certain conditions and cell types. However, p8 is expressed in human pancreatic islets and pancreatic β-cell lines, and its expression is strongly upregulated in response to glucose, indicating that p8 may represent a physiological mediator of glucose-induced pancreatic β-cell growth. Binding of glucose-activated PKC to two PKC sites within a highly conserved region of the p8 protein may be a possible mechanism linking glucose- and p8-dependent signaling pathways leading to proliferation.

The study was supported by Bundesministerium für Bildung und Forschung (BMBF), grant FKZ 01GN0115.

Human pancreatic islets were kindly provided by R.G. Bretzel and M.D. Brendel from the Third Medical Department and Policlinic, University Hospital, Justus-Liebig University, Giessen, Germany.