Pannexin 3 regulates intracellular ATP/cAMP levels and promotes chondrocyte differentiation

Abstract

Pannexin 3 (Panx3) is a new member of the gap junction pannexin family, but its expression profiles and physiological function are not yet clear. We demonstrate in this study that Panx3 is expressed in cartilage and regulates chondrocyte proliferation and differentiation. Panx3 mRNA was expressed in the prehypertrophic zone in the developing growth plate and was induced during the differentiation of chondrogenic ATDC5 and N1511 cells. Panx3-transfected ATDC5 and N1511 cells promoted chondrogenic differentiation, but the suppression of endogenous Panx3 inhibited differentiation of ATDC5 cells and primary chondrocytes. Panx3-transfected ATDC5 cells reduced parathyroid hormone-induced cell proliferation and promoted the release of ATP into the extracellular space, possibly by action of Panx3 as a hemichannel. Panx3 expression in ATDC5 cells reduced intracellular cAMP levels and the activation of cAMP-response element-binding, a protein kinase A downstream effector. These Panx3 activities were blocked by anti-Panx3 antibody. Our results suggest that Panx3 functions to switch the chondrocyte cell fate from proliferation to differentiation by regulating the intracellular ATP/cAMP levels.

FIGURE 1.

Expression of Panx3 in E16.5 growth plates. A, in situ hybridization. Panx3 mRNA was expressed in prehypertrophic chondrocytes, perichondrium, and osteoblasts. The following are shown: antisense Panx3 (panel a); sense Panx3 (panel b); Ihh (panel c); Col2a1 (panel d); Col10a1 (panel e); and Hist1h4c (panel f). B, immunostaining with anti-Panx3 antibody (red) and Hoechst nuclear staining (blue). A magnified view of the areas (panel a) is marked by the square in panel b.

FIGURE 2.

Expression of Panx3 in differentiating ATDC5 and N1511 cells. A, mRNA expression in differentiating ATDC5 cells. ATDC5 cells were cultured with 10 μg/ml insulin. Total RNA was extracted from cells on the indicated days after insulin treatment and analyzed with real time RT-PCR. In differentiating ATDC5 cells, Panx3, Col2a1, and Col10a1 were strongly induced. GAPDH, glyceraldehyde-3-phosphate dehydrogenase, was used as a control. B, mRNA expression in differentiating N1511 cells. N1511 cells were cultured with 1 μm insulin, 100 ng/ml rhBMP-2, and 50 μg/ml ascorbic acid for differentiation. Panx3 was also progressively induced in differentiating N1511 cells. HPRT, hypoxanthine phosphoribosyltransferase, was used as a control. C, expression of Panx3 protein in undifferentiated (1st lane) and differentiated ATDC5 cells (2nd lane). ATDC5 cells were treated with or without insulin for 20 days, and cell extracts were analyzed by Western blotting using Panx3 antibody. Panx3 protein was induced in differentiated ATDC5 cells. IB, immunoblot. D, immunostaining with anti-Panx3 antibody (red), ER marker (green), calnexin, and Hoechst nuclear staining (blue). In differentiating ATDC5 cells, endogenous Panx3 was observed in the cell membrane, cell processes, and ER (panels d–f) but not in undifferentiated cells (panels a and c).

FIGURE 3.

Panx3 expression in Panx3-transfected ATDC5 cells. ATDC5 cells were stably transfected with the control empty vector (pEF1) or the Panx3 expression vector (pEF1/Panx3). A, expression of Panx3 mRNA and protein. Pooled transfectants were analyzed by RT-PCR (panel a) and Western blotting (panel b) using anti-Panx3 and anti-V5 antibodies. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. B, immunostaining of Panx3-transfected ATDC5 cells using anti-Panx3 (red) and anti-V5 (blue) antibodies. Fluorescent confocal images showed that the staining signals of Panx3 and V5 antibodies were co-localized in the cell membrane, cell-cell junction, and organelles. No staining of either Panx3 or V5 antibodies was observed in control pEF1-transfected ATDC5 cells. C, co-localization of Panx3-GFP and ER-Tracker Red. ATDC5 cells were transiently transfected with Panx3-pcDNA-GFP or pcDNA3.1-GFP-TOPO (control) for 2 days. Panx3-GFP was co-localized with ER-Tracker Red, indicating the presence of Panx3 in ER. GFP, green fluorescent protein.

FIGURE 4.

Panx3 promotes chondrogenic differentiation of ATDC5 and N1511 cells. A, differentiation of ATDC 5 cells. Pooled ATDC5 cells stably transfected with either control pEF1 or pEF1/Panx3 were cultured with 10 μg/ml insulin. Total RNA was extracted at 8 and 16 days after insulin treatment and analyzed by real time RT-PCR. The expression of chondrogenic marker genes for Col2a1, Agc1, and Col10a1 was stimulated in Panx3-transfected ATDC5 cells compared with that in control cells. The expression level of an individual gene in control pEF1-transfected cells was set as 1.0, and we compared it with the level of each gene in Panx3-transfected cells for each day 8 and 18. The exogenous Panx3 levels were the same but endogenous Panx3 levels were strongly increased from day 8 to 16. As results, the ratio at day 16 was less than that in day 8. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. B, Alcian blue staining of ATDC5 cells. After 16 days of culture, Alcian blue staining was performed. Alcian blue staining was increased in Panx3-transfected ATDC5 cells compared with that in pEF1-transfected ATDC5 cells. Scale bar, 200 μm. C, differentiation of N1511 cells. N1511 cells were transfected with either control pEF1 or pEF1/Panx3, were cultured with 1 μm insulin, 100 ng/ml rhBMP-2, and 50 μg/ml ascorbic acid for 3 s and 4 days. Similar to the results of ATDC5 cells, chondrogenic maker genes expression was stimulated by Panx3. Statistical analysis was performed using analysis of variance (*, p < 0.01).

FIGURE 5.

Inhibition of ATDC5 cell differentiation by Panx3 shRNA. Pooled ATDC5 cells stably transfected with either control vector (Mock) or Panx3 shRNA vector were cultured with 10 μg/ml insulin. A, reduced expression of endogenous Panx3. Total RNA and protein were prepared from cells after 8 days of culture and analyzed through RT-PCR (panel a) and Western blotting (panel b), using anti-Panx3 antibody. Panx3 expression was reduced in Panx3 shRNA-transfected ATDC5 cells. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. B, reduced expression of chondrogenic marker genes. Total RNA was prepared from cells after 8 days of culture and analyzed by real time RT-PCR. Expression of Col2a1, Agc1, and Col10a1 was reduced in Panx3 shRNA-transfected ATDC5 cells. C, Alcian blue staining. After 16 days of culture, Alcian blue staining was performed. Alcian blue staining was reduced in Panx3 shRNA-transfected cells. Scale bar, 200 μm. Statistical analysis was performed using analysis of variance (*, p < 0.01). D, reduced expression of Col2a1 and Col10a1 by Panx3 siRNA in ATDC5 cells. ATDC5 cells transfected with control siRNA, Panx3 siRNA-1, or Panx3 siRNA-2 were cultured with 100 ng/ml BMP-2 for 8 days. Expressions of Panx3, Col2a1, and Col10a1 were analyzed by real time PCR methods. Expression of Col2a1 and Col10a1 was reduced in Panx3 siRNA-transfected ATDC5 cells. E, reduced expression of Col10a1 but not Col2a1 by Panx3 siRNA in primary chondrocytes. Primary chondrocytes transfected with control siRNA, Panx3 siRNA-1, or Panx3 siRNA-2 were cultured with 100 ng/ml BMP-2 for 2 days. Expressions of Panx3, Col2a1, and Col10a1 were analyzed by real time PCR methods. Expression of Panx3 and Col10a1, but not Col2a1, was reduced in Panx3 siRNA-transfected primary chondrocytes. Statistical analysis was performed using analysis of variance (*, p < 0.01).

FIGURE 6.

Inhibition of PTH-promoted cell proliferation by Panx3. A, Panx3- and pEF1-transfected cells were incubated in the presence of various amounts of PTH. Cell numbers were counted after 3 days of culture. The number of Panx3-transfected cells was reduced compared with the control cells. B, Panx3 antibody, but not IgG, restored PTH-promoted cell proliferation in Panx3-transfected cells. Statistical analysis was performed using analysis of variance (**, p < 0.02; *, p < 0.01).

FIGURE 7.

Reduced cAMP levels and increased ATP efflux in Panx3-transfected ATDC5 cells. A, intracellular cAMP level. Panx3- and pEF1-transfected ATDC5 cells were cultured with 10 μg/ml insulin for 1 week. The cells were incubated with anti-Panx3, IgG, or without them for 30 min and then exposed to PTH at 100 nm for 10 min, and we analyzed the intracellular cAMP levels. PTH promoted the intracellular cAMP level in control pEF1-transfected cells, whereas this PTH effect was reduced in Panx3-transfected cells. This reduction was blocked by anti-Panx3 antibody but not IgG. B, release of ATP. Cells were plated at ∼50% confluency in the absence or presence of potassium (KGlu), and ATP levels in the media were measured. ATP release to the extracellular space was increased in Panx3-transfected cells. Left panel, time course of ATP release after addition of KGlu. Right panel, data at 2 min after addition of KGlu in the right panel are shown in bar graphs. Statistical analysis was performed using analysis of variance (*, p < 0.01). C, inhibition of ATP release by Panx3 antibody. Cells were incubated with anti-Panx3 antibody, Panx3 peptide, or IgG for 30 min, and ATP release was measured. The Panx3 antibody inhibited ATP release in Panx3-transfected cells. This inhibition was blocked by various concentrations (0.5 to 5.0 ng/ml) of the Panx3 peptide but not its scrambled peptide (5.0 ng/ml).

FIGURE 8.

Decrease in phosphorylation of CREB by Panx3. A, time course of CREB phosphorylation. ATDC5 cells were cultured with 10 μg/ml insulin for 1 week and then treated with PTH (100 nm) for the time indicated. Protein extracts were analyzed by Western blotting using anti-phospho-CREB and anti-CREB antibodies. In control pEF1-transfected cells, the phosphorylation of CREB was strongly induced, and in Panx3-transfected cells the phosphorylation levels of CREB were reduced. B, restoration of the CREB phosphorylation levels by Panx3 antibody. Cells were preincubated with Panx3 antibody or IgG for 30 min before the stimulation with 100 nm PTH, and then Western blotting using anti-phospho-CREB and anti-CREB antibodies was performed. Panx3 antibody inhibited the reduction of the phosphorylation of CREB in Panx3-transfected cells. ImageJ 1.33u was used to quantify the protein bands.

FIGURE 9.

Role of Panx3 in chondrogenic differentiation. The PTH/PTHrP receptor activates multimeric G proteins. The activation of the G_s subunit leads to the activation of adenylyl cyclase (AC) for cAMP generation from ATP and the subsequent activation of PKA. PKA phosphorylates CREB, which promotes the expression of genes for cell proliferation. Panx3 is expressed in prehypertrophic chondrocytes, and it promotes the release of ATP into the extracellular space, which results in a reduction of the intracellular cAMP level and subsequent inhibition of PKA/CREB signaling for cell proliferation. The PTH/PTHrP receptor also activates the G_q subunit and subsequent downstream signaling, such as protein kinase C (PKC), to promote differentiation.