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Fluid flow shear stress stimulates human osteoblast proliferation and differentiation through multiple interacting
Sonia Kapur1, David J Baylink, K-H William Lau
1Musculoskeletal Disease Center, Jerry L. Pettis Memorial VA Medical Center, and Department of Medicine and Biochemistry, Loma Linda University, Loma Linda, CA 92357, USA.
Abstract:
This study sought to assess the role of several signaling pathways in the fluid flow shear stress-induced proliferation and differentiation of normal human osteoblasts. We evaluated the effects of an effective dose of selective inhibitors of the extracellular signal-regulated kinases (ERK) pathway (PD98059 and U0126), the nitric oxide synthase pathway (N(omega)-nitro-L-arginine methyl ester), the cyclo-oxygenase pathway (indomethacin), or the Gi/o pathway (pertussis toxin [PTX]) on the flow-mediated effects. A 30-min steady flow shear stress at 20 dynes/cm(2) increased significantly [(3)H]thymidine incorporation (an indicator of proliferation), alkaline phosphatase activity (an index of osteoblast differentiation), phosphorylation of ERK, and expression of integrin beta1. PD98059, U0126, and N(omega)-nitro-L-arginine methyl ester completely blocked the shear stress-induced increases in ERK phosphorylation, [(3)H]thymidine incorporation, and alkaline phosphatase, but without an effect on integrin beta1 expression, indicating that the ERK and nitric oxide synthase pathways are essential for the shear stress-induced proliferation and differentiation of normal human osteoblasts and that each involves ERK activation but not integrin beta1 upregulation. Indomethacin blocked the shear stress-induced osteoblast proliferation and differentiation and integrin beta1 upregulation but not ERK activation, suggesting that the cyclo-oxygenase pathway (i.e., prostacyclin and/or prostaglandin E(2)) mediates the shear stress-induced osteoblast proliferation in an ERK-independent manner. In contrast, PTX completely blocked the flow-induced increase in integrin beta1 expression but had no effect on the increase in the ERK phosphorylation or [(3)H]thymidine incorporation. PTX not only did not inhibit but also significantly enhanced the stimulatory effect of shear stress on alkaline phosphatase activity, suggesting that a PTX-sensitive signaling pathway may have an inhibitory role in osteoblast differentiation. In summary, this study shows, for the first time, that the signal transduction mechanism of shear stress in osteoblasts is complex and involves multiple ERK-dependent and independent pathways, and provides circumstantial evidence that there may be a PTX-sensitive pathway that has completing effects with an unknown pathway on the differentiation of normal human osteoblasts.
Insights
Fluid flow shear stress stimulates osteoblast proliferation and differentiation through complex signaling pathways. The extracellular signal-regulated kinases (ERK) and nitric oxide synthase pathways are crucial, while the cyclo-oxygenase pathway acts independently of ERK.
Area of Science:
- Biomedical Engineering
- Cell Biology
- Mechanobiology
Background:
- Osteoblasts are crucial for bone formation and remodeling.
- Mechanical stimuli, such as fluid flow shear stress, significantly influence osteoblast function.
- Understanding the signaling pathways involved is key to bone health research.
Purpose of the Study:
- To investigate the specific signaling pathways mediating fluid flow shear stress-induced osteoblast proliferation and differentiation.
- To elucidate the roles of extracellular signal-regulated kinases (ERK), nitric oxide synthase (NOS), cyclo-oxygenase (COX), and Gi/o pathways.
Main Methods:
- Normal human osteoblasts were subjected to controlled fluid flow shear stress.
- Selective pathway inhibitors (PD98059, U0126, L-NAME, indomethacin, PTX) were used.
- Osteoblast proliferation ([3H]thymidine incorporation) and differentiation (alkaline phosphatase activity) were measured.
- Key signaling molecules (ERK phosphorylation, integrin beta1 expression) were assessed.
Main Results:
- Shear stress increased osteoblast proliferation, differentiation, ERK phosphorylation, and integrin beta1 expression.
- ERK and NOS pathways were essential for shear stress-induced proliferation and differentiation, involving ERK activation.
- COX pathway mediated proliferation independently of ERK and influenced integrin beta1 expression.
- Gi/o pathway inhibition blocked integrin beta1 upregulation but enhanced differentiation, suggesting an inhibitory role.
Conclusions:
- Osteoblast response to shear stress is complex, involving multiple ERK-dependent and independent signaling pathways.
- ERK and NOS pathways are critical for shear stress-induced osteoblast proliferation and differentiation.
- The COX pathway contributes to proliferation independently of ERK.
- A PTX-sensitive pathway may play an inhibitory role in osteoblast differentiation, interacting with other pathways.