title: Extracellular nucleotides activate Runx2 in the osteoblast-like HOBIT cell line: a possible molecular link between mechanical stress and osteoblasts' response.
abstract: Dynamic mechanical loading increases bone density and strength and promotes osteoblast proliferation, differentiation and matrix production, by acting at the gene expression level. Molecular mechanisms through which mechanical forces are conversed into biochemical signalling in bone are still poorly understood. A growing body of evidence point to extracellular nucleotides (i.e., ATP and UTP) as soluble factors released in response to mechanical stimulation in different cell systems. Runx2, a fundamental transcription factor involved in controlling osteoblasts differentiation, has been recently identified as a target of mechanical signals in osteoblastic cells. We tested the hypothesis that these extracellular nucleotides could be able to activate Runx2 in the human osteoblastic HOBIT cell line. We found that ATP and UTP treatments, as well as hypotonic stress, promote a significant stimulation of Runx2 DNA-binding activity via a mechanism involving PKC and distinct mitogen-activated protein kinase cascades. In fact, by using the specific inhibitors SB203580 (specific for p38 MAPK) and PD98059 (specific for ERK-1/2 MAPK), we found that ERK-1/2, but not p38, play a major role in Runx2 activation. On the contrary, another important transcription factor, i.e., Egr-1, that we previously demonstrated being activated by extracellular released nucleotides in this osteoblastic cell line, demonstrated to be susceptible to both ERK-1/2 and p38 kinases. These data suggest a possible differential involvement of these two transcription factors in response to extracellularly released nucleotides. The biological relevance of our data is strengthened by the finding that a target gene of Runx2, i.e., Galectin-3, is up-regulated by ATP stimulation of HOBIT cells with a comparable kinetic of that found for Runx2. Since it is known that osteocytes are the primary mechanosensory cells of the bone, we hypothesize that they may signal mechanical loading to osteoblasts through release of extracellular nucleotides. Altogether, these data suggest a molecular mechanism explaining the purinoreceptors-mediated activation of specific gene expression in osteoblasts and could be of help in setting up new pharmacological strategies for the intervention in bone loss pathologies.
authors.affiliation: Department of Biomedical Sciences and Technologies, University of Udine, 33100 Udine, Italy.
authors.forename: Adalberto
authors.initials: A
authors.lastname: Costessi
authors.forename: Alex
authors.initials: A
authors.lastname: Pines
authors.forename: Paola
authors.initials: P
authors.lastname: D'Andrea
authors.forename: Milena
authors.initials: M
authors.lastname: Romanello
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Damante
authors.forename: Laura
authors.initials: L
authors.lastname: Cesaratto
authors.forename: Franco
authors.initials: F
authors.lastname: Quadrifoglio
authors.forename: Luigi
authors.initials: L
authors.lastname: Moro
authors.forename: Gianluca
authors.initials: G
authors.lastname: Tell
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D002460:Cell Line; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D050659:Core Binding Factor Alpha 1 Subunit; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D045604:Extracellular Fluid / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D009838:Oligodeoxyribonucleotides / Q000378:metabolism / Q000494:pharmacology*; D010006:Osteoblasts / Q000187:drug effects / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D013314:Stress, Mechanical; D015534:Trans-Activators / Q000502:physiology; D050656:Transcription Factor AP-2; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014544:Uridine Triphosphate / Q000494:pharmacology
chemical_list: D050659:Core Binding Factor Alpha 1 Subunit; D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; C493709:RUNX2 protein, human; D015534:Trans-Activators; D050656:Transcription Factor AP-2; D014157:Transcription Factors; D000255:Adenosine Triphosphate; D014544:Uridine Triphosphate