Mutual enhancement of differentiation of osteoblasts and osteocytes occurs through direct cell-cell contact

Abstract

There is increasing evidence that osteocytes regulate multiple aspects of bone remodeling through bi-directional communication with osteoblasts. This is potentially mediated through cell-cell contact via osteocytic dendritic processes, through the activity of secreted factors, or both. To test whether cell-cell contact affects gene expression patterns in osteoblasts and osteocytes, we used a co-culture system where calvarial osteoblasts and IDG-SW3 osteocytes were allowed to touch through a porous membrane, while still being physically separated to allow for phenotypic characterization. Osteoblast/osteocyte cell-contact resulted in up-regulation of osteoblast differentiation genes in the osteoblasts, when compared to wells where no cell contact was allowed. Examination of osteocyte gene expression when in direct contact with osteoblasts also revealed increased expression of osteocyte-specific genes. These data suggest that physical contact mutually enhances both the osteoblastic and osteocytic character of each respective cell type. Interestingly, Gja1 (a gap junction protein) was increased in the osteoblasts only when in direct contact with the osteocytes, suggesting that Gja1 may mediate some of the effects of direct cell contact. To test this hypothesis, we treated the direct contact system with the gap junction inhibitor 18-alpha-glycyrrhetinic acid and found that Bglap expression was significantly inhibited. This suggests that osteocytes may regulate late osteoblast differentiation at least in part through Gja1. Identification of the specific factors involved in the enhancement of differentiation of both osteoblasts and osteocytes when in direct contact will uncover new biology concerning how these bone cells communicate.

Keywords: COCULTURE; OSTEOBLAST; OSTEOCYTE; QPCR.

Fig 1

Schematic diagram of the procedure for seeding the two independent cell types in the direct cell-cell contact co-culture system.

Fig 2

Osteoblastic gene expression is enhanced when in direct cell contact with osteocytes compared to no cell contact. A) IDG-SW3 osteocytes were seeded either on the bottom of the well (“No Cell Contact”) or on the basal surface of the membrane (“Cell Contact”) and allowed to differentiate in osteogenic media for 21 days. CalOB osteoblasts were then seeded on the apical surface of the membrane and both cell types allowed to differentiate for an additional 7 days. B) QPCR analysis of the CalOBs when either in “No Cell Contact” or “Cell Contact” with the IDG-SW3 cells. Data are expressed as mean ± standard error (SE), n=6, *p<0.05, **p<0.01, ***p<0.001.

Fig 3

Osteocytic gene expression is enhanced when in direct cell contact with osteoblasts, compared to direct cell contact with the osteocytes themselves. A) IDG-SW3 osteocytes were seeded on the basal surface of the membrane for both conditions, and either IDG-SW3 cells or CalOBs seeded on apical surface. Seeding and differentiation conditions were identical as described in Fig. 1. B) QPCR analysis of the IDG-SW3 cells when either in direct contact with other IDG-SW3 cells (“IDG-SW3/ IDG-SW3”) or the CalOB cells (“CalOB/IDG-SW3”). Data are expressed as in Fig 1., *p<0.05, **p<0.01.

Fig 4

The gap junction inhibitor 18-alpha-glycyrrhetinic acid (AGA) inhibits Bglap expression in osteoblasts when in direct contact with osteocytes. IDG-SW3 cells were seeded on the basal surface of the membrane and CalOBs seeded on the apical surface (as in Figs. 1–3). 10 μM AGA (or vehicle control) was added to the well for the entire 7 day coculture period. QPCR analysis of osteoblast (A) and osteocyte (B) marker genes in the CalOBs, when in direct contact with the IDG-SW3 cells treated −/+ AGA, was performed. Data are expressed as in Fig. 1., *p<0.05.