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Research Papers

Gap Junctions and Osteoblast-like Cell Gene Expression in Response to Fluid Flow

[+] Author and Article Information
Michael G. Jekir, Henry J. Donahue

Division of Musculoskeletal Sciences, Department of Orthopaedics and Rehabilitation, The Pennsylvania State University College of Medicine, Hershey, PA 17033

J Biomech Eng 131(1), 011005 (Nov 18, 2008) (6 pages) doi:10.1115/1.3005201 History: Received January 14, 2008; Revised July 08, 2008; Published November 18, 2008

Bone formation occurs in vivo in response to mechanical stimuli, but the signaling pathways involved remain unclear. The ability of bone cells to communicate with each other in the presence of an applied load may influence the overall osteogenic response. The goal of this research was to determine whether inhibiting cell-to-cell gap junctional communication between bone-forming cells would affect the ensemble cell response to an applied mechanical stimulus in vitro. In this study, we investigated the effects of controlled oscillatory fluid flow (OFF) on osteoblastic cells in the presence and the absence of a gap-junction blocker. MC3T3-E1 Clone 14 cells in a monolayer were exposed to 2h of OFF at a rate sufficient to create a shear stress of 20dynescm2 at the cell surface, and changes in steady-state mRNA levels for a number of key proteins known to be involved in osteogenesis were measured. Of the five proteins investigated, mRNA levels for osteopontin (OPN) and osteocalcin were found to be significantly increased 24h postflow. These experiments were repeated in the presence of 18β-glycyrrhetinic acid (BGA), a known gap-junction blocker, to determine whether gap-junction intercellular communication is necessary for this response. We found that the increase in OPN mRNA levels is not observed in the presence of BGA, suggesting that gap junctions are involved in the signaling process. Interestingly, enzyme linked immunosorbent assay data showed that levels of secreted OPN protein increased 48h postflow and that this increase was unaffected by the presence of intact gap junctions.

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Figure 2

The effect of BGA on GJIC in MC3T3-E1 cells. Both concentrations of BGA effectively reduced cell communication to zero (p<0.01) and the blocking effect with 30μM did not differ significantly from that with 50μM. Each bar represents mean±SE. Values of n (number of donor cells) are as follows: DMSO (634), 30μM BGA (603), and 50μM BGA (516).

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Figure 3

The effect of GJIC on the OPN mRNA response to fluid flow. OFF upregulated OCN mRNA 24h postflow (p<0.05), and 30μM BGA blocked this response (p<0.05). All columns are expressed relative to the CTRL-DMSO group. Values of n from left to right are 36, 30, 9, and 8.

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Figure 4

The effect of GJIC on OPN secretion in response to fluid flow. Secreted OPN levels normalized to total protein in the cell lysate increased 2.5-fold 48h following OFF (p<0.05). This response was not inhibited by 30μM BGA. All columns are expressed relative to CTRL-DMSO. OPN levels were normalized to total protein in the cell lysate. From left to right, the numbers of slides n are 17, 12, 7, and 13. For each slide, the average of three ELISA wells was taken.

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Figure 5

The effect of GJIC on the OCN mRNA response to fluid flow. OFF upregulated OCN mRNA 24h postflow (p<0.05). In the presence of BGA, fluid flow stimulation had no significant effect on OCN mRNA levels. Basal OCN mRNA levels were elevated in the presence of BGA, though the effect was not statistically significant. All values are expressed relative to the CTRL-DMSO group. Values of n from left to right are 38, 34, 8, and 9.

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Figure 1

The effect of OFF on mRNA levels for MC3T3-E1 Clone 14 cells. C=control and O=oscillatory fluid flow. Steady-state mRNA levels for OPN and OCN were significantly increased (p<0.01)24h following the application of 2h of OFF (1Hz, 20dynes∕cm2). However, mRNA for Cx43, COL-1, and cbfa1 showed no significant change (p>0.05) after the same time period.  ** represents a significant difference from the static control group (p<0.01). Each bar represents mean±SE. Values of n (number of individual slides) for CTRL and OFF, respectively, are A (21,18), B (20,20), C (21,20), D (19,20), and E (20,19).

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