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.
Highlights
Cell of synthesis or a target cell [15], and several intracrine factors, including basic fibroblast growth factor and angiotensin II, regulate gene transcription upon nuclear binding [16]
Cell Cycle Distribution Analysis by Flow Cytometry—The dis- C-peptide Rapidly Stimulates ribosomal RNA (rRNA) Synthesis—Our observatribution of cells in the G1, S, and G2/M cell cycle phases was tion of C-peptide in the nucleoli raised the question whether determined by DNA flow cytometry [26]
C-peptide Regulates rRNA Synthesis via Epigenetic Stimulation—Because C-peptide localizes to nucleoli and increases transcription of 47 S rRNA, we examined whether transcriptional activation is coordinated with epigenetic modifications of histones
Summary
Cell Culture and Treatments—HEK-293 and Swiss-3T3 cells were cultured as described [10]. For affinity precipitation, biotinylated C-peptide was immobilized on streptavidin beads according to the manufacturer’s protocol (Dynabeads, Invitrogen). Histone extracts prepared from Swiss-3T3 cells were added for 60 min, after which beads were washed three times with buffer (150 mM sodium phosphate, 150 mM NaCl, pH 7.0) and eluted in sample loading buffer. PCR amplifications of the 47 S ribosomal gene (forward 5Ј-CCT GTC GTC GGA GAG GTT GG-3Ј and reverse 5Ј-ACC CCA CGC CTT CCC ACA C-3Ј) and the G3PDH housekeeping gene (forward 5Ј-ATG GCC TTC CGT GTC CCC ACT G-3Ј and reverse 5Ј-TGA GTG TGG CAG GGA CTC CCC A-3Ј) were performed at an annealing temperature of 50 °C, 45 cycles, with reagents from Invitrogen, according to the manufacturer’s protocol. Cell death was analyzed by a cell death detection ELISA kit (Roche Diagnostics) and Hoechst 33342 staining, according to the manufacturer’s protocol. Results are presented as mean values Ϯ S.E. *, p Ͻ 0.05, **, p Ͻ 0.01, and ***, p Ͻ 0.001
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