PTHrP signaling targets cyclin D1 and induces osteoblastic cell growth arrest.
PTHrP control of the MC3T3-E1 cell cycle machinery showed that, during differentiation, PTHrP induced G1 growth arrest. Cyclin D1 was a critical mediator as a downstream effector of cAMP, PKC, and MAPK signaling, and the process was PKA-independent. The involvement of JunB has been found critical for PTHrP effects.


BACKGROUND
PTH-related protein (PTHrP) has been implicated in the control of bone cell turnover, but the mechanisms underlying its effect on osteoblast proliferation and differentiation have not been clearly defined. The mechanisms by which PTHrP impacts cell cycle proteins and the role of signaling pathways in differentiated osteoblasts were studied.


METHODS
To elucidate the role of PTHrP, flow cytometric analyses were performed using MC3T3-E1 and primary mouse calvarial cells. Relative protein abundance (Western blot), physical association of partners (immunoprecipitation), and kinase activities (in vitro kinase assays using either GST-Rb or H1-histone as substrates) of cell cycle-associated proteins in vehicle and PTHrP-treated 7-day differentiated cells were determined. ELISA and/or Northern blot analyses were done to evaluate JunB and cyclin D1 expression. SiRNA-mediated gene silencing experiments were performed to silence JunB protein. Finally, inhibitors of cAMP, protein kinase A (PKA), protein kinase C (PKC), and mitogen-activated protein kinase (MAPK) were used to determine involvement of different signaling pathways.


RESULTS
PTHrP inhibited cyclin D1 protein expression 7-fold in a dose- and time-dependent manner and increased the level of p16 protein in differentiated osteoblasts. Additionally, PTHrP reduced cyclin D1-CDK4/CDK6 and CDK1 kinase activities. Forskolin, a cAMP agonist, mimicked PTHrP action, and the PKC inhibitor, GF109203X, slightly blocked downregulation of cyclin D1, implying involvement of both cAMP and PKC. U0126, a MAPK inhibitor, alone decreased cyclin D1 protein, suggesting that the basal cyclin D1 protein is MAPK dependent. H-89, a PKA inhibitor, did not alter the effect of PTHrP on cyclin D1, suggesting a PKA-independent mechanism. Finally, expression of JunB, an activating protein-1 transcription factor, was significantly upregulated, and silencing JunB (siRNA) partially reversed the cyclin D1 response, implying involvement of JunB in the PTHrP-mediated growth arrest of MC3T3-E1 cells.


CONCLUSIONS
PTHrP upregulates JunB and reduces cyclin D1 expression while inducing G1 cell cycle arrest in differentiated osteoblasts. Such regulation could be an important determinant of the life span and bone-forming activity of osteoblasts.
Datta
Nabanita S
NS
Department of Periodontics/Prevention/Geriatrics, University of Michigan, Ann Arbor, Michigan 48109-1078, USA. nsdatta@umich.edu
Chen
Chen
C
Berry
Janice E
JE
McCauley
Laurie K
LK
D016475:3T3 Cells; D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002070:Butadienes / Q000494:pharmacology; D002453:Cell Cycle; D002454:Cell Differentiation; D049109:Cell Proliferation; D002469:Cell Separation; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D019938:Cyclin D1 / Q000378:metabolism*; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000378:metabolism; D003720:Densitometry; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D016193:G1 Phase; D020868:Gene Silencing; D005982:Glutathione Transferase / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D047468:Immunoprecipitation; D020935:MAP Kinase Signaling System; D051379:Mice; D008954:Models, Biological; D009570:Nitriles / Q000494:pharmacology; D010006:Osteoblasts / Q000166:cytology* / Q000378:metabolism; D044162:Parathyroid Hormone-Related Protein / Q000378:metabolism / Q000502:physiology*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D013997:Time Factors; D015854:Up-Regulation
D002070:Butadienes; D019941:Cyclin-Dependent Kinase Inhibitor p16; D004791:Enzyme Inhibitors; D006657:Histones; D009570:Nitriles; D044162:Parathyroid Hormone-Related Protein; D016755:Proto-Oncogene Proteins c-jun; D034741:RNA, Small Interfering; D016160:Retinoblastoma Protein; C113580:U 0126; D019938:Cyclin D1; D012313:RNA; D000242:Cyclic AMP; D005982:Glutathione Transferase; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C