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

Guthalu Kondegowda N, Joshi-Gokhale S, Harb G, Williams K, Zhang XY, Takane KK, Zhang P, Scott DK, Stewart AF, Garcia-Ocaña A, Vasavada RC - Diabetes (2010)

Bottom Line: We found that human β-cells express PTH1R.More importantly, overexpression of PTHrP causes a significant approximately threefold increase in human β-cell proliferation.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Endocrinology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA. vasavada@pitt.edu

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.

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Related in: MedlinePlus

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.)
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Figure 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.)

Mentions: PTHrP mRNA and protein are expressed in human islets and insulinomas (5,13,14). However, whether the receptor for PTHrP, PTH1R, is expressed in human islets is unknown. We analyzed the expression of PTH1R mRNA by real-time PCR in human islets using HKC8, a human kidney cell line with high PTH1R expression, as a positive control and 293 cells that lack PTH1R expression as a negative control. PTH1R mRNA is clearly detectable in human islets (Fig. 1A), as is PTH1R protein, measured by Western blot (Fig. 1B). Importantly, PTH1R is expressed in human β-cells, demonstrated by costaining of human islet cell cultures for PTH1R and insulin (Fig. 1C).


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

Guthalu Kondegowda N, Joshi-Gokhale S, Harb G, Williams K, Zhang XY, Takane KK, Zhang P, Scott DK, Stewart AF, Garcia-Ocaña A, Vasavada RC - Diabetes (2010)

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.)
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2992775&req=5

Figure 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.)
Mentions: PTHrP mRNA and protein are expressed in human islets and insulinomas (5,13,14). However, whether the receptor for PTHrP, PTH1R, is expressed in human islets is unknown. We analyzed the expression of PTH1R mRNA by real-time PCR in human islets using HKC8, a human kidney cell line with high PTH1R expression, as a positive control and 293 cells that lack PTH1R expression as a negative control. PTH1R mRNA is clearly detectable in human islets (Fig. 1A), as is PTH1R protein, measured by Western blot (Fig. 1B). Importantly, PTH1R is expressed in human β-cells, demonstrated by costaining of human islet cell cultures for PTH1R and insulin (Fig. 1C).

Bottom Line: We found that human β-cells express PTH1R.More importantly, overexpression of PTHrP causes a significant approximately threefold increase in human β-cell proliferation.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Endocrinology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA. vasavada@pitt.edu

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.

Show MeSH
Related in: MedlinePlus