Nuclear localization of the type 1 parathyroid hormone parathyroid hormone-related peptide receptor in MC3T3-E1 cells: Association with serum-inducedcell proliferation

We have recently demonstrated that the receptor for parathyroid hormone (PT H) and PTH-related peptide (PTHrP), PTHR, can be localized to the nucleus o f cells within the liver, kidney, uterus, gut, and ovary of the rat, We set out to determine the localization of the PTHR in cultured osteoblast-like cells. MC3T3-E1, ROS 17/2.8, UMR106, and SaOS-2 cells were cultured in alph a-modified eagle medium containing 15% fetal calf serum under standard cond itions. Untreated cells were grown on glass coverslips to 75-95% confluence and fixed in 1% paraformaldehyde, For experiments designed to examine cell s synchronized by serum starvation, cells were grown on glass coverslips, s tarved of serum for 46 h, and then fixed at 2-h intervals for a total of 26 h after the addition of serum to the medium. Parallel sets of cells were p ulsed with [H-3]thymidine to track the DNA duplication interval. The PTHR w as localized by immunocytochemistry using a primary antibody raised against a portion of the N-terminal extracellular domain of the PTHR. The results presented herein indicate that the PTHR attains a nuclear localization in e ach cell line examined. In UMR106 cells, PTHR immunoreactivity was restrict ed to the nucleolus. After cell synchronization, MC3T3-E1 cells double appr oximately 24 h after the addition of serum. Immunocytochemistry for the PTH R in these cells showed that the receptor staining is initially diffuse for the first 6 h, then becomes more perinuclear in distribution by 12-16 h, N uclear localization of the receptor is achieved approximately 16-20 h after the addition of serum and remains there throughout the mitotic phase. Inte nse staining of mitotic and postmitotic cells was observed. No change in ce ll proliferation kinetics was observed in MC3T3-E1 cells cultured in the pr esence of 25 nM PTH(1-34). These data suggest an important role for the PTH R in the nucleus of MC3T3-E1 cells at the time of DNA synthesis and mitosis , (C) 2000 by Elsevier Science Inc. All rights reserved.