Short carboxyl terminal parathyroid hormone peptides modulate human parathyroid hormone signaling in mouse osteoblasts

Abstract

Background: Novel human parathyroid hormone (hPTH) peptides of unknown biological activity have recently been identified in the serum of subjects with normal renal function, chronic renal failure, and end-stage renal disease through the application of liquid chromatography-high resolution mass spectrometry.

Purpose: of experiments: To determine the bioactivity of these peptides, we synthesized hPTH28-84, hPTH38-84, and hPTH45-84 peptides by solid phase peptide synthesis and tested their bioactivity in MC3T3-E1 mouse osteoblasts, either individually or together with the native hormone, hPTH1-84, by assessing the accumulation of 3´,5´-cyclic adenosine monophosphate (cAMP) and the induction of alkaline phosphatase activity.

Results: Increasing doses of hPTH1-84 (1-100 nM) increased the accumulation of cAMP and alkaline phosphatase activity in osteoblasts. hPTH28-84, hPTH38-84, and hPTH45-84 in concentrations of 1-100 nM were biologically inert. Surprisingly, 100 nM hPTH38-84 and hPTH45-84 increased the accumulation of cAMP in osteoblasts treated with increasing amounts of hPTH1-84. Human PTH28-84 had no effects on cAMP activity alone or in combination with hPTH1-84. Conversely, 100 nM hPTH38-84, hPTH45-84, and hPTH28-84 blocked the activation of alkaline phosphatase activity by hPTH1-84.

Conclusions: The data show that the short carboxyl-terminal hPTH peptides, hPTH38-84 and hPTH45-84, increase the amount of cellular cAMP generated in cultured osteoblasts in response to treatment with full-length hPTH1-84 when compared to full-length hPTH1-84 alone. Human PTH28-84 had no effect on cAMP activity alone or in combination with hPTH1-84. Human PTH28-84, hPTH38-84 and hPTH45-84 reduced the effects of hPTH1-84 in osteoblasts with respect to the induction of alkaline phosphatase activity compared to hPTH1-84 alone. Short carboxyl peptides of human PTH are biologically inert but when administered together with full-length hPTH1-84 modulate the bioactivity of hPTH1-84 in osteoblasts.

Keywords: Alkaline phosphatase; Carboxyl-terminal hPTH peptides; Mouse osteoblasts; cAMP; hPTH1-84; hPTH28-84; hPTH38-84; hPTH45-84.

Figure 1.

Comparison of the effect of hPTH1–84 and hPTH peptides (hPTH28–84, hPTH38–84, hPTH45–84) on cAMP production in MC3T3-E1 cells. hPTH1–84 increased cAMP production in a dose-response manner. cAMP concentrations after treatment with 1, 3, 10, 50, and 100 nM hPTH1–84 were 5.50±0.63, 33.11±8.13, 58.86±12.13, 83.32±28.47, and 87.06±26.05 pmol/mL, respectively. The same concentration of hPTH peptides had no effect.

Figure 2.

Enhancement of cAMP production in MC3T3-E1 cells by hPTH1–84 at different concentrations (1, 3, 10, 50, 100 nM) plus either 100 nM hPTH38–84 or hPTH45–84. MC3T3-E1 cells were pretreated for 2 hours with either 100 nM hPTH28–84, hPTH38–84, or hPTH45–84, and then stimulated by hPTH1–84 at indicated concentrations together with 100 nM of individual hPTH peptides for 30 minutes. The control group (black line) was pretreated with fresh culture media and then stimulated by hPTH1–84 alone. Values are expressed as the mean±SD for triplicate determinations (*p <0.01 vs. hPTH1–84 alone).

Figure 3.

Comparison of the effect of hPTH1–84 and hPTH peptides (hPTH28–84, hPTH38–84, hPTH45–84) on ALP activity in differentiated MC3T3-E1 cells. hPTH1–84 increased ALP activity in a dose-response manner. ALP activity after treatment for 24 hours using 1, 3, 10, 50, and 100 nM hPTH1–84 were 0.25±0.09, 0.44±0.04, 0.88±16, 1.32±0.21, and 1.48±0.39 U/L, respectively. The same concentration of hPTH peptides had no effect.

Figure 4.

Inhibition of ALP activity in differentiated MC3T3-E1 cells by 100 nM hPTH peptides (hPTH28–84, hPTH38–84, or hPTH45–84) together with hPTH1–84 at different concentrations (0.1, 1, 3, 10, 50, 100, 1000 nM). Differentiated MC3T3-E1 cells were treated for 24 hours using 100 nM of individual hPTH peptides together with hPTH1–84 at indicated concentrations. The control group (black line) was treated with hPTH1–84 alone. After 24 hours, ALP activity was assayed in cell lysates. hPTH1–84 alone increased ALP activity in a dose-response manner. However, ALP activity was significantly reduced in the presence of 100 nM of individual hPTH peptides. Values are expressed as the mean±SD for triplicate determinations (*p <0.01 vs. hPTH1–84 alone).