Multiple mechanisms are involved in inhibition of osteoblast differentiation by PTHrP and PTH in KS483 Cells.
We examined the mechanism by which PTHrP and PTH inhibit KS483 osteoblastic differentiation. We show that PTHrP and PTH inhibit differentiation downstream of early BMP signaling and downregulated components of the hedgehog (Hh) signaling cascade. In addition, PTHrP and PTH repressed RunX2 and osx expression. Overexpression of either gene, however, could not relieve PTHrP and PTH's inhibitory actions. Our data suggest that multiple parallel mechanisms are involved in the inhibition of osteoblast differentiation and matrix mineralization by PTHrP and PTH.


BACKGROUND
PTH-related peptide (PTHrP) and PTH are potent inhibitors of osteoblast differentiation in vitro by as yet unexplained mechanisms.


METHODS
We treated murine bone marrow stromal cells and the mesenchymal progenitor cell line KS483 with PTHrP and PTH in combination with either BMPs or hedgehog (Hh) and measured early and late markers of osteoblast differentiation and studied the expression of RunX2 and Osterix (osx). In addition, we examined the PTHrP and PTH response in stable KS483 cells overexpressing either RunX2 or osx.


RESULTS
PTHrP and PTH inhibited BMP- and Hh-induced osteogenesis downstream of early BMP signaling and by downregulation of components of the Hh signaling cascade. PTHrP and PTH prevented the upregulation of RunX2 expression associated with osteoblast differentiation in an indirect response. However, PTHrP and PTH could still inhibit differentiation, and particularly matrix mineralization, of cells expressing RunX2. In addition, PTHrP and PTH potently downregulated osx expression only in mature osteoblasts in an intermediate early response, but osx overexpression could not relieve the inhibitory effects of PTHrP and PTH on matrix mineralization.


CONCLUSIONS
Our data suggest that, besides transcriptional repression of RunX2 and osx, other mechanisms in parallel with or downstream of RunX2 and osx are involved in the inhibition of osteoblast differentiation and matrix mineralization by PTHrP and PTH in vitro.
van der Horst
Geertje
G
Department of Endocrinology and Metabolic Diseases, Leiden University Medical Center, The Netherlands.
Farih-Sips
Hetty
H
Löwik
Clemens W G M
CW
Karperien
Marcel
M
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D055415:Bone Morphogenetic Protein 4; D055418:Bone Morphogenetic Protein 6; D019485:Bone Morphogenetic Proteins / Q000494:pharmacology; D002113:Calcification, Physiologic / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects*; D002460:Cell Line; D002478:Cells, Cultured; D050659:Core Binding Factor Alpha 1 Subunit / Q000235:genetics; D000242:Cyclic AMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D015870:Gene Expression / Q000187:drug effects; D053823:Hedgehog Proteins; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics; D051741:Kruppel-Like Transcription Factors / Q000235:genetics; D008297:Male; D008565:Membrane Proteins / Q000235:genetics; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D010012:Osteogenesis / Q000187:drug effects; D010281:Parathyroid Hormone / Q000494:pharmacology*; D044162:Parathyroid Hormone-Related Protein / Q000494:pharmacology*; D000072079:Patched Receptors; D010446:Peptide Fragments / Q000494:pharmacology; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D000076182:Sp7 Transcription Factor; D019379:Teriparatide / Q000031:analogs & derivatives / Q000494:pharmacology; D015534:Trans-Activators / Q000235:genetics / Q000494:pharmacology; D014157:Transcription Factors / Q000235:genetics
C525810:Bmp4 protein, mouse; C525817:Bmp6 protein, mouse; D055415:Bone Morphogenetic Protein 4; D055418:Bone Morphogenetic Protein 6; D019485:Bone Morphogenetic Proteins; D050659:Core Binding Factor Alpha 1 Subunit; D053823:Hedgehog Proteins; D047908:Intracellular Signaling Peptides and Proteins; D051741:Kruppel-Like Transcription Factors; D008565:Membrane Proteins; D010281:Parathyroid Hormone; D044162:Parathyroid Hormone-Related Protein; D000072079:Patched Receptors; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C493716:Runx2 protein, mouse; D000076182:Sp7 Transcription Factor; C479466:Sp7 protein, mouse; D015534:Trans-Activators; D014157:Transcription Factors; D019379:Teriparatide; C054296:parathyroid hormone-related protein (1-34); D000242:Cyclic AMP; D000469:Alkaline Phosphatase; C035748:parathyroid hormone (1-34)amide