Focal adhesion kinase is important for fluid shear stress-induced mechanotransduction in osteoblasts - PubMed (original) (raw)

Focal adhesion kinase is important for fluid shear stress-induced mechanotransduction in osteoblasts

Suzanne R L Young et al. J Bone Miner Res. 2009 Mar.

Erratum in

J Bone Miner Res. 2013 Nov;28(11):2431

Abstract

Mechanical loading of bone is important for maintenance of bone mass and structural stability of the skeleton. When bone is mechanically loaded, movement of fluid within the spaces surrounding bone cells generates fluid shear stress (FSS) that stimulates osteoblasts, resulting in enhanced anabolic activity. The mechanisms by which osteoblasts convert the external stimulation of FSS into biochemical changes, a process known as mechanotransduction, remain poorly understood. Focal adhesions are prime candidates for transducing external stimuli. Focal adhesion kinase (FAK), a nonreceptor tyrosine kinase found in focal adhesions, may play a key role in mechanotransduction, although its function has not been directly examined in osteoblasts. We examined the role of FAK in osteoblast mechanotransduction using short interfering RNA (siRNA), overexpression of a dominant negative FAK, and FAK(-/-) osteoblasts to disrupt FAK function in calvarial osteoblasts. Osteoblasts were subjected to varying periods oscillatory fluid flow (OFF) from 5 min to 4 h, and several physiologically important readouts of mechanotransduction were analyzed including: extracellular signal-related kinase 1/2 phosphorylation, upregulation of c-fos, cyclooxygenase-2, and osteopontin, and release of prostaglandin E(2). Osteoblasts with disrupted FAK signaling exhibited severely impaired mechanical responses in all endpoints examined. These data indicate the importance of FAK for both short and long periods of FSS-induced mechanotransduction in osteoblasts.

PubMed Disclaimer

Figures

FIG. 1

Transfection of FAK siRNA in osteoblasts. (A) Western blot analysis of FAK and Pyk2 expression and activation (phosphorylation of Y397, Y402) in osteoblasts treated with lipofectamine only (Lipo Only), 50 nM scrambled control siRNA (Scramble), or 50 nM FAK siRNA smart pool (FAK siRNA) compared with untreated osteoblasts (Unt). Vinculin was analyzed as a loading control. (B and C) Quantification of FAK expression and FAK activation as measured by phosphorylation of Y397 expressed as raw densitometry units (a p < 0.05). (D and E) Quantification of Pyk2 expression and Pyk2 activation as measured by phosphorylation of Y402 expressed as raw densitometry units. Error bars represent SE.

FIG. 2

Analysis of osteoblasts infected with FAK-related nonkinase (FRNK) and FAK+/+ and FAK−/− osteoblasts. (A) Western blot analysis of FAK activation (phosphorylation at Y397), FRNK expression, Pyk2 expression, and activation (phosphorylation at Y402) in osteoblasts infected with GFP only control (GFP) or FRNK-GFP (FRNK) compared with uninfected osteoblasts. Arrow indicates GFP-FRNK; asterisk indicates endogenous FRNK. Vinculin was analyzed as a loading control. (B) Quantification of FRNK expression and FAK activation after infection as compared with uninfected osteoblasts expressed in raw densitometry units. (C) Quantification of Pyk2 expression and activation after infection compared with uninfected osteoblasts expressed in raw densitometry units. (D) Western blot analysis of control osteoblasts clone (1C, 1E11) and FAK−/− osteoblast clones (1D8, 4F1) shows no FAK expression wildtype levels of Pyk2 expression in the FAK−/− clones. FAK−/− clones exhibit similar levels of activated Pyk2 as measured by phosphorylation at Y402 compared with control clones. (E) Quantification of Pyk2 expression and activation in the control and FAK−/− osteoblasts clones expressed in raw densitometry units (a p < 0.05). Error bars represent SE.

FIG. 3

Reduced ERK activation in osteoblasts exhibiting disrupted FAK activation. Western blot analysis of ERK activation, FAK and Pyk2 expression, and activation after 5 min of OFF or 5 min of static culture conditions (S, static; F, flow). Vinculin was analyzed as a loading control. Line graphs represent ERK activation as measured in fold induction (flow/static) after 5, 15, and 30 min of OFF. Quantification of FAK and Pyk2 activation shown in bar graphs. (A) Osteoblasts treated with FAK siRNA exhibited decreased ERK activation after exposure to 5, 15, and 30 min of OFF. (B) Osteoblasts overexpressing FRNK also exhibited decreased ERK activation after 5, 15, and 30 min of OFF. (C) FAK−/− osteoblasts exhibited reduced ERK activation after 5, 15, and 30 min of OFF. Error bars represent SE (a p < 0.05).

FIG. 4

Reduced induction of c-fos by OFF in osteoblasts exhibiting decreased FAK activation. (A) Western blot analysis of c-fos expression, FAK and Pyk2 expression, and activation after 30 min of static culture condition or 30 min of OFF (S, static; F, flow) in FAK siRNA-treated osteoblasts, FRNK overexpressing osteoblasts, and FAK−/− osteoblasts. Vinculin was analyzed as a loading control. (B–D) Graph represents quantification of c-fos expression expressed in raw densitometry units. (E–G) Graph represents quantification of FAK and Pyk2 activation expressed in raw densitometry units. aStatistically significant difference between static and flow. bStatistically significant difference among the flowed samples. cStatistically significant difference among the static samples (a,b,c p < 0.05). Error bars represent SE.

FIG. 5

Decreased COX-2 induction and PGE2 release in response to OFF in osteoblasts exhibiting disrupted FAK activation. (A) Western blot analysis of COX-2 expression, FAK and Pyk2 expression, and activation after 4 h of static culture conditions or 4 h of OFF (S, static; F, flow). Vinculin was analyzed as a loading control. (B–D) Quantification of COX-2 expression expressed in raw densitometry units. (E–G) Quantification of PGE2 release as measured by ELISA in osteoblasts exposed to 4 h of static culture conditions or 4 h of OFF. (H–J) Quantification of FAK and Pyk2 activation expressed in raw densitometry units. aStatistically significant difference between static and flow. bStatistically significant difference among the flowed samples. cStatistically significant difference among the static samples (a,b,c p < 0.05). Error bars represent SE.

FIG. 6

Decreased OPN upregulation on exposure to OFF in osteoblasts with disrupted FAK activation. (A) Western blot analysis of OPN expression, FAK and Pyk2 expression, and activation after 2 h of static culture conditions or 2 h of OFF both followed by an over night incubation (S, static; F, flow). Vinculin was analyzed as a loading control. (B–D) Quantification of OPN expression expressed in raw densitometry units. (E–G) Quantification of FAK and Pyk2 activation expressed in raw densitometry units. aStatistically significant difference between static and flow. bStatistically significant difference among the flowed samples. cStatistically significant difference among the static samples (a,b,c p < 0.05). Error bars represent SE.

FIG. 7

Re-expression of FAK in FAK−/− osteoblasts rescues COX-2 and OPN upregulation on exposure to OFF. (A) Western blot analysis of FAK and Pyk2 expression and activation as measured by phosphorylation of Y397 in control clone 1C7 (1C7) and FAK−/− clone 4F1 infected with FAK-GFP (4F1 + FAK). Quantification of FAK and Pyk2 expression and activation (phosphorylation of Y397, Y402, respectively) expressed in raw densitometry units. (B) Western blot analysis of COX-2 expression after 4 h of static culture conditions or 4 h of OFF (S, static; F, flow) in control clone 1C7 (1C7), FAK−/− clone 4F1 (4F1), FAK−/− 4F1 infected with GFP (4F1 + GFP), and FAK−/− 4F1 infected with FAK-GFP (4F1 + FAK). Graph represents quantification of COX-2 expression expressed in raw densitometry units. (C) Western blot analysis of OPN expression after 2 h of static culture conditions or 2 h of OFF both followed by an overnight incubation (S, static; F, flow). Graph represents quantification of OPN expression expressed in raw densitometry units. In all blots, vinculin was analyzed as a loading control. aStatistically significant difference between static and flow. bStatistically significant difference among the flowed samples. cStatistically significant difference among the static samples (a,b,c p < 0.05). Error bars represent SE.

References

1. Harder AT, An YH. The mechanisms of the inhibitory effects of nonsteroidal anti-inflammatory drugs on bone healing: A concise review. J Clin Pharmacol. 2003;43:807–815. - PubMed
1. Miller SB. Prostaglandins in health and disease: An overview. Semin Arthritis Rheum. 2006;36:37–49. - PubMed
1. Radi ZA, Khan NK. Effects of cyclooxygenase inhibition on bone, tendon, and ligament healing. Inflamm Res. 2005;54:358–366. - PubMed
1. Raisz LG. Potential impact of selective cyclooxygenase-2 inhibitors on bone metabolism in health and disease. Am J Med. 2001;110(Suppl 3A):43S–45S. - PubMed
1. Guignandon A, Akhouayri O, Usson Y, Rattner A, Laroche N, Lafage-Proust MH, Alexandre C, Vico L. Focal contact clustering in osteoblastic cells under mechanical stresses: Microgravity and cyclic deformation. Cell Commun Adhes. 2003;10:69–83. - PubMed

Focal adhesion kinase is important for fluid shear stress-induced mechanotransduction in osteoblasts - PubMed (original) (raw)