Proinsulin C-peptide regulates ribosomal RNA expression

Abstract

Proinsulin C-peptide is internalized into cells, but a function of its intracellular localization has not been established. We now demonstrate that, upon cellular entry, C-peptide is localized to the nucleoli, where it promotes transcription of genes encoding for ribosomal RNA. We find that C-peptide binds to histones and enhances acetylation of lysine residue 16 of histone H4 at the promoter region of genes for ribosomal RNA. In agreement with synchrony of ribosomal RNA synthesis and cell proliferation, we show that C-peptide stimulates proliferation in chondrocytes and HEK-293 cells. This regulation of ribosomal RNA provides a mechanism by which C-peptide can exert transcriptional effects and implies that the peptide has growth factor activity.

FIGURE 1.

C-peptide is localized to nucleoli. A, confocal images of Rh-C-peptide (red) and nuclear Hoechst staining (blue) in Swiss-3T3 cells upon C-peptide exposure (1 h). B, colocalization of Rh-C-peptide (red) and the selective nucleolar marker SYTO RNASelect (green) shown by confocal fluorescence analysis as in A. C, colocalization of Rh-C-peptide (y-axe) and SYTO RNASelect (x-axe) staining pattern analyzed using the Zeiss Software (colocalized pixels are pseudo-colored white and are depicted in the lower panel). Scale bar, 20 μm.

FIGURE 2.

C-peptide induces transcription of rDNA. A and B, equivalent samples of HEK-293 cells (∼25 × 10⁶) were exposed to 0.3 μm C-peptide. After 90 min to 24 h, total RNA was extracted and the levels of 28 S and 18 S rRNA expression were examined by RNA gel analysis (∼4 μg/sample). Quantification of mature rRNA expression in treated relative to control samples as obtained by densitometric analysis (ImageJ software). The image shown is representative of three similar experiments. C, HEK-293 and Swiss-3T3 cells were cotransfected with β-galactosidase reference plasmid and pHrD-IRES-Luc, a luciferase reporter containing an IRES downstream of the human rDNA promoter, and treated with C-peptide. Cells were harvested after 24 h, and cell extracts were assayed for luciferase and β-galactosidase activity. Relative light units (RLU) were computed after normalization to β-galactosidase activity. D, RT-PCR analysis of pre-rRNA 47 S in HEK-293 cells (∼25 × 10⁶) treated for 24 h with 0.3 μm C-peptide. Quantification of pre-rRNA expression relative to the untreated sample is presented, using the ImageJ software. E, HEK-293 cells (1 × 10⁵/ml) were exposed to 0.3 μm C-peptide for 0–72 h, with cell counts performed at 24, 48, and 72 h post-treatment using a hemacytometer. Error bars represent S.E. (n = 3) and fold over control (FOC) was computed after normalization to intensity (treated versus control samples).

FIGURE 3.

Absence of early effects of C-peptide on cell proliferation. Swiss-3T3 cells were treated for 24 h with 1 μm C-peptide. A, distribution of the cells in the different phases of the cell cycle was determined by their DNA content upon propidium iodide staining and FACS analysis. B, cells were pulsed with BrdUrd for 15 min, and the proportion of cells in S phase (BrdUrd-incorporating cells) was monitored by FACS analysis. Similar results were obtained with HEK-293 cells.

FIGURE 4.

Specific binding of C-peptide to histone H4. A, sensorgram showing binding of C-peptide to Swiss-3T3 histone extracts assessed by surface plasmon resonance technology. B, Swiss-3T3 histone extracts (∼2 μg) were affinity-precipitated with C-peptide-conjugated beads and control beads in the presence of additives as indicated, separated by SDS-PAGE, and stained with Coomassie Brilliant Blue. C, protein identification of tryptic digests from binding experiments. M denotes oxidized methionine. D, Swiss-3T3 histone extracts (∼2 μg) were affinity-precipitated with C-peptide-conjugated beads and control beads at pH 3 and 10, and in 400 and 800 mm KCl, analyzed by SDS-PAGE, and Coomassie staining. Quantification of band staining intensity with the ImageJ software and FOC was computed as described in Fig. 2. Unconj., unconjugated beads; sc., scrambled.

FIGURE 5.

C-peptide stimulates acetylation of H4K16. A, HEK-293 cells were treated for 1 h with 1 μm C-peptide, fixed, and submitted to immunofluorescence detection using antibodies directed against mono-acetylated H4K16 (green). Nuclei were costained with Hoechst 33342 (blue). Scale bar, 20 μm. B, HEK-293 cells were treated for 1 h with 1 μm C-peptide, and cell lysates were separated on an SDS-PAGE gel and transferred to polyvinylidene difluoride membranes that were probed with an anti-acetyl-H4K16 antibody. Quantification of H4K16Ac staining is shown. C, Swiss-3T3 histone extracts were affinity-precipitated with C-peptide-conjugated beads and control beads and analyzed by immunoblotting using H4K16Ac antibodies. Quantification of H4K16Ac in complexes is shown. Relative arbitrary units (AU) were computed after normalization to intensity (treated versus control samples). D, enrichment of H4K16Ac at the promoter region of rDNA was determined by ChIP analysis using chromatin prepared from control and 90 min C-peptide-treated HEK-293 cells (∼25 × 10⁶). Samples were precipitated using H4K16Ac antibodies. Quantification of DNA band intensity is shown. E, Swiss-3T3 histone extracts A (normal) and B (from cells overexpressing SIRT1) were affinity-precipitated with C-peptide-conjugated beads and control beads and separated by SDS-PAGE. Samples were analyzed by Coomassie staining and transferred to polyvinylidene difluoride membranes that were probed with an anti-acetyl-H4K16 antibody. Quantification of band staining intensity is shown. F, Swiss-3T3 histone extracts (∼2 μg) were affinity-precipitated with C-peptide-conjugated beads and control beads in the presence of additives as indicated, separated by SDS-PAGE, and stained with Coomassie Brilliant Blue. Quantification of band staining intensity with the ImageJ software and fold over control (FOC) was computed as described in Fig. 2. Unconj., unconjugated.

FIGURE 6.

C-peptide induces proliferation of chondrocytes. Human chondrocyte HCS-2/8 cells were exposed to C-peptide (0.3, 1, and 3 μm) for 72 h. A, total RNA was extracted from control and treated cells (∼1 × 10⁶) and 47 S pre-rRNA expression analyzed by RT-PCR. Quantification of DNA band staining intensity with the ImageJ software is shown, and FOC was computed as described in Fig. 2. B, cells were observed under contrast phase microscopy and photographed. C, after treatment, the cell proliferation reagent WST-1 was added and incubation continued for 1 h at 37 °C. The amount of the formazan dye formed, as measured by ELISA, correlated with the number of metabolically active cells. D, cell death was assayed using the Cell Death Detection ELISA kit. The relative quantities of histone-complexed DNA fragments (mono- and oligonucleosomes) were measured by ELISA and correlate to the number of dead cells. Experiments were performed on cells that were serum-starved overnight. Error bars represent S.E. (n = 3). E, effect of different concentrations of C-peptide after a 72-h stimulation on BrdUrd incorporation in chondrocytes. Dexamethasone (25 μm) was used as a positive control. n = 5, p < 0.01. In B–E, ∼20 × 10³ cells/sample were used. F, frequency of apoptotic cells (arrows) in control versus C-peptide treated cells as observed after staining with Hoechst. Scale bar, 20 μm.