Pulsating fluid flow modulates gene expression of proteins involved in Wnt signaling pathways in osteocytes.
Strain-derived flow of interstitial fluid activates signal transduction pathways in osteocytes that regulate bone mechanical adaptation. Wnts are involved in this process, but whether mechanical loading modulates Wnt signaling in osteocytes is unclear. We assessed whether mechanical stimulation by pulsating fluid flow (PFF) leads to functional Wnt production, and whether nitric oxide (NO) is important for activation of the canonical Wnt signaling pathway in MLO-Y4 osteocytes. MC3T3-E1 osteoblasts were studied as a positive control for the MLO-Y4 osteocyte response to mechanical loading. MLO-Y4 osteocytes and MC3T3-E1 osteoblasts were submitted to 1-h PFF (0.7 +/- 0.3 Pa, 5 Hz), and postincubated (PI) without PFF for 0.5-3 h. Gene expression of proteins related to the Wnt canonical and noncanonical pathways were studied using real-time polymerase chain reaction (PCR). In MLO-Y4 osteocytes, PFF upregulated gene expression of Wnt3a, c-jun, connexin 43, and CD44 at 1-3-h PI. In MC3T3-E1 osteoblasts, PFF downregulated gene expression of Wnt5a and c-jun at 0.5-3-h PI. In MLO-Y4 osteocytes, gene expression of PFF-induced Wnt target genes was suppressed by the Wnt antagonist sFRP4, suggesting that loading activates the Wnt canonical pathway through functional Wnt production. The NO inhibitor L-NAME suppressed the effect of PFF on gene expression of Wnt target genes, suggesting that NO might play a role in PFF-induced Wnt production. The response to PFF differed in MC3T3-E1 osteoblasts. Because Wnt signaling is important for bone mass regulation, osteocytes might orchestrate loading-induced bone remodeling through, among others, Wnts.
Santos
Ana
A
Department of Oral Cell Biology, ACTA-University of Amsterdam and VU University Amsterdam, Research Institute MOVE, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands.
Bakker
Astrid D
AD
Zandieh-Doulabi
Behrouz
B
Semeins
Cornelis M
CM
Klein-Nulend
Jenneke
J
D000818:Animals; D001696:Biomechanical Phenomena; D002460:Cell Line; D018031:Connexin 43 / Q000235:genetics / Q000378:metabolism; D019938:Cyclin D1 / Q000235:genetics / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000502:physiology*; D018960:Hyaluronan Receptors / Q000235:genetics / Q000378:metabolism; D051379:Mice; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000037:antagonists & inhibitors / Q000378:metabolism; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D010011:Osteocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D016755:Proto-Oncogene Proteins c-jun / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D051153:Wnt Proteins / Q000235:genetics* / Q000378:metabolism*; D000071818:Wnt-5a Protein; D060508:Wnt3 Protein; D060509:Wnt3A Protein
C530105:Ccnd1 protein, mouse; D018031:Connexin 43; D004791:Enzyme Inhibitors; C543076:GJA1 protein, mouse; D018960:Hyaluronan Receptors; D016755:Proto-Oncogene Proteins c-jun; D051153:Wnt Proteins; D000071818:Wnt-5a Protein; D060508:Wnt3 Protein; D060509:Wnt3A Protein; C555511:Wnt3a protein, mouse; C438615:Wnt5a protein, mouse; D019938:Cyclin D1; D009569:Nitric Oxide; D019331:NG-Nitroarginine Methyl Ester