Parathyroid hormone-related protein enhances human ß-cell proliferation and function with associated induction of cyclin-dependent kinase 2 and cyclin E expression

Abstract

Objective: Inducing human β-cell growth while enhancing function is a major goal in the treatment of diabetes. Parathyroid hormone-related protein (PTHrP) enhances rodent β-cell growth and function through the parathyroid hormone-1 receptor (PTH1R). Based on this, we hypothesized that PTH1R is expressed in human β-cells and that PTHrP has the potential to enhance human β-cell proliferation and/or function.

Research design and methods: PTH1R expression, β-cell proliferation, glucose-stimulated insulin secretion (GSIS), and expression of differentiation and cell-cycle genes were analyzed in human islets transduced with adenoviral PTHrP constructs or treated with PTHrP peptides. The effect of overexpression of late G1/S cell cycle molecules was also assessed on human β-cell proliferation.

Results: We found that human β-cells express PTH1R. More importantly, overexpression of PTHrP causes a significant approximately threefold increase in human β-cell proliferation. Furthermore, the amino terminus PTHrP(1-36) peptide is sufficient to increase replication as well as expression of the late G1/S cell-cycle proteins cyclin E and cyclin-dependent kinase 2 (cdk2) in human islets. Notably, PTHrP(1-36) also enhances GSIS. Finally, overexpression of cyclin E alone, but not cdk2, augments human β-cell proliferation, and when both molecules are expressed simultaneously there is a further marked synergistic increase in replication.

Conclusions: PTHrP(1-36) peptide enhances human β-cell proliferation as well as function, with associated upregulation of two specific cell-cycle activators that together can induce human β-cell proliferation several fold. The future therapeutic potential of PTHrP(1-36) for the treatment of diabetes is especially relevant given the complementary therapeutic efficacy of PTHrP(1-36) in postmenopausal osteoporosis.

FIG. 1.

PTH1R and PTHrP expression in human islets. A: PTH1R mRNA expression measured by real-time PCR in three human islet preps (H1–H3); HKC8, a human kidney proximal tubule cell line, used as positive control; and the human embryonic kidney cell line 293, used as negative control. Quantitation is shown as log of Ct using actin as the housekeeping control gene. B: Western blot analysis of PTH1R and actin expression in a positive (+) control line (293 cells stably transfected with human PTH1R cDNA) and two human islet preps (H1 and H2) uninfected (U) or transduced with Ad-LacZ (L) or Ad-PTHrP (P). The line divides samples run on two different gels. C: Photomicrograph of human islet cell cultures costained for nuclear DAPI (blue), PTH1R (green), and insulin (red). D: Representative Western blot analysis of PTHrP and actin in human islets either uninfected (U) or transduced with Ad-LacZ (L), Ad-PTHrP (P), or Ad-ΔSP mutant (S). The line divides samples from different regions run on the same gel. E: Quantitation of PTHrP(1-36) by immunoradiometric assay in medium collected after 48 h of transduction of human islets with adenoviral constructs containing LacZ, PTHrP, or ΔSP sequences (n = 2 islet preparations). F: Representative images of human islet cell cultures transduced with Ad-LacZ, Ad-PTHrP, or Ad-ΔSP and costained for DAPI (blue), insulin (green), and HA (red). (A high-quality digital representation of this figure is available in the online issue.)

FIG. 2.

PTHrP increases human β-cell proliferation and function without causing dedifferentiation. A: Representative confocal images of human islet cell cultures transduced with Ad-control (I) or Ad-PTHrP (II–IV) and costained for insulin (green, panel b), BrdU (red, panel c), and DAPI (blue, panel d). The merged images of all three stainings are shown in panel a, merge for DAPI and BrdU in panel e, and merge for insulin and BrdU in panel f. B: Quantitation of the percentage of BrdU-positive β-cells from human islet cell cultures uninfected (UI) or transduced with the adenovirus (AdV) constructs control (ctrl), PTHrP, or ΔSP for 72 h or treated with 100 nmol/l of either 1-36 or 38-94 PTHrP peptides for 24 h. There was a significant two- to threefold increase in β-cell proliferation in PTHrP-transduced and 1-36–treated vs. Ad-control–transduced or uninfected control islet cells. Basal rate of β-cell proliferation in uninfected controls was 0.08 ± 0.03% (n = 4–11 individual human islet preps at least in duplicate). *P < 0.05 vs. uninfected or Ad-control by Student's t test; #P < 0.001 vs. Ad-control or Ad-ΔSP and &P < 0.01 vs. UI or 38-94 by one-way ANOVA. C: Expression of differentiation markers by real-time PCR from human islets treated for 24 h with vehicle control (Ctrl) or PTHrP(1-36) peptide (gray bars). PCR cycles for each gene were compared, with actin used as an internal control. The graph is depicted as fold over control, with values from vehicle-treated islets taken as 1 (n = 4–7 human islet preparations in duplicate). D: Insulin content per IEQ in extracts of human islets treated with vehicle (veh) or 100 nmol/l PTHrP(1-36) peptide or transduced with Ad-control (Ctrl) or Ad-PTHrP for 24 h (n = 5 human islet preparations in triplicate). E and F: Insulin secretion measured at 5.5 and 22 mmol/l glucose from human islets transduced with Ad-control or Ad-PTHrP for 24 h (E) and treated with vehicle or 100 nmol/l PTHrP(1-36) peptide for 30 min (F). Insulin secretion is depicted as percentage of vehicle-treated control at 5.5 mmol/l glucose, which was 455.4 ± 109.3 pg/μg protein in 30 min. n = 7–8 human islet preparations in triplicate. *P < 0.05 vs. insulin secretion at 5.5 mmol/l glucose with the same treatment; #P < 0.05 vs. insulin secretion at equivalent glucose concentrations of control. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 3.

PTHrP increases expression of cyclin (cyc) E and cdk2 proteins but not mRNA in human islets. A: Expression of G1/S cell cycle regulators by real-time PCR from human islets treated for 24 h with vehicle control (Ctrl) or PTHrP(1-36) peptide (gray bars). PCR cycles for each gene were compared, with actin used as an internal control. The graph is depicted as fold over control, with values from vehicle-treated islets taken as 1 (n = 4–7 human islet preparations in duplicate). B: Representative Western blot analysis of the G1/S cell cycle activators and inhibitors from human islets treated for 24 h with vehicle (veh) or PTHrP(1-36) using actin or tubulin as the internal housekeeping (HK) gene control. Quantitation of the ratio of cyclin E/HK (C) and cdk2/HK protein (D) shows a significant increase in both these proteins in PTHrP(1-36)–treated islets vs. control (veh), depicted as 100%. *P < 0.05 (n = 4–9 human islet preparations).

FIG. 4.

Overexpression of cyclin E (cyc E) and cdk2 enhances human β-cell proliferation. A: Western blot analysis of human islets uninfected (UI) or transduced with Ad-control (Ad-ctrl) or a combination of Ad-cdk2/Ad-cyclin E for expression of cdk2, cyclin E, and actin as an internal control. The line divides samples from different regions on the same gel. Representative cell cycle phase distribution profiles (B) and percentage of cells in S-phase (C) of the cell cycle of uninfected, Ad-control, or Ad-cdk2/Ad-cyclin E–transduced human islet cells analyzed using flow cytometry. The percentage of cells in the G1-phase was reduced, although not significantly, in Ad-cdk2/Ad-cyclin E–transduced (G1: 91.1 ± 1.0%, G2/M: 5.4 ± 1.2%) vs. uninfected (G1: 93.0 ± 1.3%, G2/M: 5.4 ± 1.2%) and Ad-control–transduced (G1: 94.0 ± 1.0%, G2/M: 4.6 ± 0.9%) human islets, whereas the percentage of cells in S-phase of the cell cycle in Ad-cdk2/Ad-cyclin E–transduced human islets was significantly greater compared with uninfected or Ad-control–transduced human islets (n = 10 human islet preparations). D: Representative images of human islet cell cultures transduced with 100 MOI of Ad-control, 50 MOI of Ad-control plus 50 MOI of Ad-cdk2, or 50 MOI of Ad-cyclin E and 50 MOI each of Ad-cdk2 and Ad-cyclin E and costained for DAPI (blue), insulin (green), and BrdU (red). Arrows indicate BrdU and insulin-positive cells. E: Quantitation of the percentage of BrdU-positive β-cells from human islet cell cultures transduced with the Ad constructs described for C. There was a significant 5-fold and 16-fold increase in β-cell proliferation in cyclin E– and cdk2/cyclin E– vs. Ad-control–transduced islet cells, respectively. Basal rate of β-cell proliferation in controls was 0.17 ± 0.06%, taken as 100%. n = 9; 3 human islet preparations in triplicate. *P < 0.01 vs. control by Student's t test; #P < 0.001 vs. the other three groups by one-way ANOVA. (A high-quality digital representation of this figure is available in the online issue.)