Mechanical strain opens connexin 43 hemichannels in osteocytes: a novel mechanism for the release of prostaglandin

Abstract

Mechanosensing bone osteocytes express large amounts of connexin (Cx)43, the component of gap junctions; yet, gap junctions are only active at the small tips of their dendritic processes, suggesting another function for Cx43. Both primary osteocytes and the osteocyte-like MLO-Y4 cells respond to fluid flow shear stress by releasing intracellular prostaglandin E2 (PGE2). Cells plated at lower densities release more PGE2 than cells plated at higher densities. This response was significantly reduced by antisense to Cx43 and by the gap junction and hemichannel inhibitors 18 beta-glycyrrhetinic acid and carbenoxolone, even in cells without physical contact, suggesting the involvement of Cx43-hemichannels. Inhibitors of other channels, such as the purinergic receptor P2X7 and the prostaglandin transporter PGT, had no effect on PGE2 release. Cell surface biotinylation analysis showed that surface expression of Cx43 was increased by shear stress. Together, these results suggest fluid flow shear stress induces the translocation of Cx43 to the membrane surface and that unapposed hemichannels formed by Cx43 serve as a novel portal for the release of PGE2 in response to mechanical strain.

Figure 1.

PGE₂ release in response to mechanical stress is blocked by the gap junction inhibitor β-GA. MLO-Y4 cells were treated with or without 100 μM β-GA under FF at 16 dynes/cm² for 2 h. The conditioned medium was collected and PGE₂ was measured. The relative level of PGE₂ compared with control is shown in the y-axis. Controls (C) are cells not subjected to fluid flow. C, C + β-GA or FF + β-GA versus FF: ***p < 0.001. The data are presented as mean ± SD and n = 3.

Figure 2.

PGE₂ release due to fluid flow shear stress is blocked by β-GA and Cx43 antisense ODN in cells lacking physical contact. (A) MLO-Y4 cells shown were plated at cell densities of 2.0 × 10³, 7.5 × 10³, 1.6 × 10⁴, and 3.8 × 10⁴ cells/cm². At the cell density of 2.0 × 10³ cells/cm², the majority of cells do not make physical contact. (B) MLO-Y4 cells were subjected to fluid flow at the stress level of 16 dynes/cm² for 2 h. In response to fluid flow, MLO-Y4 cells at lower densities released more PGE₂ per cell than cells at higher densities. (C) MLO-Y4 cells at the densities of 2 and 7.5 × 10³/cm² were treated with or without 100 μM β-GA in absence (control [C]) or presence of FF at 16 dynes/cm² for 2 h. PGE₂ release in response to fluid flow was inhibited even at a low cell density of 2 × 10³/cm² where gap junctions barely exist. Untreated versus β-GA treated at 2 × 10³ or 7.5 × 10³ cells/cm²: ***p < 0.001. (D) MLO-Y4 cells were treated with 50 μM Cx43 sense (S), antisense (AS) ODNs, or not treated (C). The cells were labeled with affinity-purified Cx43 antibody. The Cx43 protein was detected using fluorescein-conjugated anti-rabbit secondary antibody. Cx43 antisense ODN reduced Cx43 protein expression. (E) Immunoblots of membranes isolated from cells treated with Cx43 S, AS, or not treated (C) were labeled with 1:300 dilution of affinity-purified anti-Cx43 antibody. (F) MLO-Y4 cells at densities of 2 and 7.5 × 10³/cm² were pretreated with 50 μM Cx43 S or AS ODNs before FF at 16 dynes/cm² for 2 h. The release of PGE₂ was inhibited by Cx43 antisense ODN at both densities tested. S versus AS treated at 2 or 7.5 × 10³ cells/cm²: ***p < 0.001. In B, C, and F, the conditioned medium was collected, and the release of PGE₂ was measured using a PGE₂ EIA kit. The data are presented as mean ± SD and n = 3.

Figure 3.

Fluid flow-induced release of PGE₂ is blocked by carbenoxolone in cells lacking physical contact. MLO-Y4 cells plated at 2 × 10³/cm² were treated with or without 100 μM carbenoxolone (Carb) in the absence (control [C]) or presence of FF at 16 dynes/cm² for 2 h. The media and cells were collected. The amount of extracellular (A) and intracellular (B) PGE₂ was measured using a PGE₂ EIA kit. For extracellular PGE₂, C, C + Carb, or FF + Carb versus FF: ***p < 0.001. For intracellular PGE₂, C or C + Carb versus FF or FF + Carb: ***p < 0.001. All data are presented as mean ± SD and n = 3.

Figure 4.

Hemichannels, not the purinergic receptor P2X₇ nor the prostaglandin transporter PGT, mediate the release of PGE₂. MLO-Y4 cells plated at 2 × 10³/cm² were treated with or without 100 μM β-GA, 10 μM oATP, or 100 μM DIDS in the absence (control [C]) or presence of FF at 16 dynes/cm² for 2 h. The media and cells were collected. The amount of extracellular (A) and intracellular (B) PGE₂ was measured using a PGE₂ EIA kit. For extracellular PGE₂, untreated, oATP, or DIDS treated versus β-GA treated: **p < 0.01. All data are presented as mean ± SD and n = 3.

Figure 5.

Fluid flow shear stress increases dye uptake in MLO-Y4 cells and primary osteocytes. (A) Compared with untreated control (C) and EGTA-treated (EGTA) cells, FF shear stress stimulated the uptake of the dye in MLO-Y4 cells, in which the degree of uptake is even more than EGTA-treated cells. This increase is blocked by β-GA (FF + β-GA). (B) The number of MLO-Y4 cells with dye uptake with and without fluid flow treatment or β-GA were counted and quantified. C, C + β-GA or FF + β-GA versus FF: ***p < 0.001. The data are presented as mean ± SD and n = 3. (C) Compared with untreated control (C), FF shear stress similarly stimulated the uptake of the dye in primary osteocytes.

Figure 6.

Fluid flow shear stress increased the surface expression of Cx43. (A) The MLO-Y4 cells with (FF) or without (C) 2-h treatment of fluid flow at 16 dynes/cm² were treated with biotin. The cells were lysed, and equal volumes of total protein of untreated (C) and FF-treated samples were applied to avidin-conjugated beads. The biotin-labeled samples were isolated by binding to avidin-conjugated beads. The preloaded (Pre) and biotinylated Cx43 bound (B) to avidin beads was detected by Western blots by using anti-Cx43 antibody. The relative ratio of biotinylated to total Cx43 was quantified using densitometric measurements of the band intensity (right). (B) To control for nonspecific binding, lysates of cells without biotin treatment (No Bio) were applied to avidin-conjugated beads, and preloaded (Pre) and bound (B) fractions were detected by Western blotting with anti-Cx43 antibody. (C) The Pre and biotinylated B samples were analyzed by Western blots by using anti-β-actin antibody.