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Abstract: PO0516

Enhancement of In Vitro hPTH1-84 Bioactivity by hPTH38-84 and hPTH45-84

Session Information

Category: Bone and Mineral Metabolism

  • 401 Bone and Mineral Metabolism: Basic

Authors

  • Kritmetapak, Kittrawee, Mayo Clinic Minnesota, Rochester, Minnesota, United States
  • Losbanos, Louis A., Mayo Clinic Minnesota, Rochester, Minnesota, United States
  • Hines, Jolaine M., Mayo Clinic Minnesota, Rochester, Minnesota, United States
  • Singh, Ravinder, Mayo Clinic Minnesota, Rochester, Minnesota, United States
  • Kumar, Rajiv, Mayo Clinic Minnesota, Rochester, Minnesota, United States
Background

Recent research using high resolution mass spectrometry demonstrates that serum concentrations of full-length parathyroid hormone (hPTH1-84) and its fragments (hPTH28-84, 34-77, 34-84, 37-77, 37-84, 38-77, 38-84, and 45-84) are increased significantly in CKD patients with an eGFR of <17-23 mL/min/1.73m2 (Kritemetapak et al. Clin Chem. 2021 Mar 6:hvab013. doi: 10.1093/clinchem/hvab013. Online ahead of print.PMID: 33693557). Information about the bioactivity of these newly discovered hPTH fragments is lacking.

Methods

Recombinant hPTH1-84 was synthesized in Escherichia coli and purified by immobilized metal-ion affinity chromatography. hPTH28-84, hPTH38-84, and hPTH45-84 were synthesized by solid phase peptide synthesis. The identity of hPTH1-84 and hPTH fragments was confirmed by mass spectrometry. To determine whether different-sized hPTH fragments modulate the bioactivity of hPTH1-84, we studied their effects on the generation of the cellular second messenger, cAMP, which mediates the intracellular signaling of hPTH1-84, in murine preosteoblasts (MC3T3-E1) in vitro. Forskolin, an adenylyl cyclase activator, was used as a positive control for cAMP production. All experiments were performed in triplicate.

Results

In MC3T3-E1 cells, cAMP increased to 28.5+5.5, 64.4+9.9, 91.5+11.6, 114.6+15.5, and 109.1+6.5 pmol/mL 30 minutes after treatment with 1, 3, 10, 50, and 100 nM,hPTH1-84 . The same concentrations of hPTH28-84, hPTH38-84, and hPTH45-84 had no effects. When hPTH1-84 was added to cells concurrently with 100 nM hPTH38-84 or hPTH45-84 in 1:100, 3:100, 1:10, 1:2, and 1:1 molar ratios, cAMP responses to hPTH1-84 increased by 65.3% (95% CI, 63.2% to 67.4%; P<0.01) in the presence of hPTH38-84 and increased by 77.0% (95% CI, 65.2% to 88.8%; P<0.01) in the presence of hPTH45-84. hPTH28-84, added concurrently with PTH1-84, did not enhance cAMP generation in MC3T3 cells.

Conclusion

The small hPTH fragments, hPTH38-84 and 45-84, but not hPTH28-84, enhance the hPTH-induced generation of cAMP in MC3T3-E1, suggesting a novel biological role and a novel mechanism of action for these PTH fragments in osteoblast-like cells. It is plausible that in vivo these fragments may enhance the activity of full-length PTH by novel mechanisms. These findings may partly explain the discrepancy between PTH levels and bone histology in patients with CKD.

Funding

  • Other NIH Support