INTEGRIN SIGNALING AND THE RESPONSE OF OSTEOCYTES TO OSCILLATORY FLUID FLOW

+*Litzenberger, J B; *Tummala, P; *Jacobs, C R +*Veterans Administration Medical Center/Stanford U niversity, Palo Alto, CA jlitze@stanford.edu INTRODUCTION Osteocytes are mechanosensitive cells in bone and a re believed to be responsible for initiating and coordinating oste genic and osteoclastic processes in vivo. Dynamic fluid flow has been shown to be a potent regulator of bone cell metabolism, but the molecula r mechanism through which osteocytes sense mechanical stimuli is unknow n [1,2]. Integrins, heterodimeric cell adhesion proteins, are ideal can didates for mechanosensitive molecules in bone due to their str uctu al and catalytic capabilities. The aim of this study is to investiga te the role of integrins in mechanotransduction in osteocytes. In response to mechanical stimuli, osteocytes coord inate a cellular response to load by directing effector cells to dep osit or resorb bone [3,4]. Intracellular calcium ([Ca ] i) mobilization and MAPK signaling in response to fluid shear stress are associated th e eposition of bone matrix proteins in vitro [2]. Independent of calcium signaling, an increase in cyclooxygenase-2 transcription results in prostaglandin release from osteocytic cells and in bone formation in vivo [5–7]. It has also been shown that osteocytes have a loading-indu ced inhibitory effect on osteoclastogenesis, as measured by a decrease in the ratio of RANKL to OPG [8]. We hypothesize that inhibiting integrin s gnaling in osteocytic cells will decrease the production of mo lecular signals that drive osteogenic and osteoclastic processes in bone .


INTRODUCTION
Osteocytes are mechanosensitive cells in bone and are believed to be responsible for initiating and coordinating osteogenic and osteoclastic processes in vivo.Dynamic fluid flow has been shown to be a potent regulator of bone cell metabolism, but the molecular mechanism through which osteocytes sense mechanical stimuli is unknown [1,2].Integrins, heterodimeric cell adhesion proteins, are ideal candidates for mechanosensitive molecules in bone due to their structural and catalytic capabilities.The aim of this study is to investigate the role of integrins in mechanotransduction in osteocytes.
In response to mechanical stimuli, osteocytes coordinate a cellular response to load by directing effector cells to deposit or resorb bone [3,4].Intracellular calcium ([Ca 2+ ]i) mobilization and MAPK signaling in response to fluid shear stress are associated the deposition of bone matrix proteins in vitro [2].Independent of calcium signaling, an increase in cyclooxygenase-2 transcription results in prostaglandin release from osteocytic cells and in bone formation in vivo [5][6][7].It has also been shown that osteocytes have a loading-induced inhibitory effect on osteoclastogenesis, as measured by a decrease in the ratio of RANKL to OPG [8].We hypothesize that inhibiting integrin signaling in osteocytic cells will decrease the production of molecular signals that drive osteogenic and osteoclastic processes in bone.

MATERIALS AND METHODS
β1DN Construct: Stably transfected cell lines of MLO-Y4 osteocytic cells were generated by transfecting parental cells either with an expression plasmid encoding a dominant negative form of the β1 integrin monomer (β1DN) or with the empty vector (vector controls).Oscillatory Fluid Flow (OFF): For 48 hours prior to OFF exposure, cells were subcultured on glass slides coated with type I bovine collagen at a seeding density of approximately 2.5x10 5 cells.Peak shear stresses: 10 dynes/cm 2 ; Frequency: 1 Hz; OFF Exposure: 2 hr (mRNA).Calcium Imaging: Cells were incubated with 10 µM Fura-2 AM for 30 min prior to OFF.Ratio images were acquired every 2 sec for 3 min of OFF.Ca 2+ response is defined as a transient increase in [Ca 2+ ]i of ≥4 times the maximum oscillation recorded during a 3 min pre-flow period.RT-PCR: Cells were lysed immediately after OFF and a reverse transcription reaction was completed using GeneAmp RNA PCR Core Kit.Real-time polymerase chain reaction (PCR) was performed using Taqman PCR Master Mix and 20X primers and probes for COX-2, OPG, and RANKL.Gene levels in each sample were normalized to the 18S housekeeping gene and were measured in triplicates.Statistical Analysis: A student t test was used to compare samples, and a p<0.05 was considered significant (*).Data is reported as mean ±SE.

RESULTS
Over 44 percent of the vector control cells responded to OFF with a release of [Ca 2+ ]i, compared to 12 percent of the β1DN cells (p=0.031,N=6) (Figure 1).Application of OFF resulted in a significant increase in COX-2 mRNA levels in vector control cells (p=0.001,N=4), but no significant change in β1DN cells (p=0.406,N=4) (Figure 2).RANKL/OPG mRNA levels decreased significantly after exposure to OFF in vector control cells (p=0.0007,N=7) but not in β1DN cells (p=0.247,N=7) (Figure 3).

DISCUSSION
Intracellular calcium ([Ca 2+ ]i) is a critical second messenger that directs such processes as cell proliferation, differentiation, migration, and apoptosis [1].Intracellular Ca 2+ release that is induced by exposure to oscillatory fluid flow (OFF) occurs independently from cyclooxygenase-2 (COX-2) transcription in bone cells [5,7].COX-2 is an enzyme that is responsible for prostanoid formation and is associated with PGE2 signaling in bone cells [9,10].Blocking COX-2 production in vivo inhibits mechanically-induced bone formation [6].We found that there was a significant reduction in the percentage of β1DN cells that responded to oscillatory fluid flow with cytosolic calcium mobilization compared to control cells (Figure 1).Similarly, the increased COX-2 transcription that occurred in response to OFF in control cells was blocked in β1DN cells (Figure 2).These results indicate that inhibiting integrin signaling in osteocytic cells decreases the osteogenic response of these cells to mechanical stimuli.
Osteoclasts are multinucleated cells that are responsible for bone resorption.Osteoclast formation is governed by relative levels of receptor activator of nuclear factor kappa B (NF-κB) ligand (RANKL) and osteoprotegerin (OPG) in bone.It has been shown previously that osteocyte-like cells exposed to OFF exhibit a decrease in the ratio of RANKL to OPG mRNA and have an inhibitory effect on osteoclast differentiation [8].Results from this study indicate that inhibiting integrin signaling in osteocytic cells will decrease the cells' sensitivity to mechanical stimuli, and will reduce their ability to mediate osteoclastogenesis.These findings suggest that integrins are involved in osteocyte mechanosensitivity.Future experiments will aim to confirm these results by measuring protein expression and by using co-culture experiments to analyze the inhibitory effect that oscillatory fluid flow over β1DN cells has on osteoclastogenesis.
summary, osteocytes release both osteogenic and osteoclastic factors in response to oscillatory fluid flow (Fig 1-3).Our data support the idea that osteocytes control the cellular response to mechanical stimuli by directing effector cells to deposit or resorb bone.Inhibited integrin signaling results in a decreased mobilization of intracellular calcium, abrogated COX-2 transcription, and a reduced ratio of osteoclastic factors.Results from this study support the model that integrins are mechanosensitive molecules in bone, enabling osteocytes to sense mechanical stimuli and initiate intracellular signaling cascades.by a National Science Foundation Graduate Research Fellowship.

Figure 2 .
Figure 1.Intracellular calcium mobilization in β1DN and control cells exposed to oscillatory fluid flow.* p<0.05