Inhibition of connexin 43 expression and function in cultured rat dental pulp cells by antisense oligonucleotide

Abstract

:Connexins are gap junction proteins forming hexameric structures in plasma membranes of adjacent cells, creating intercellular channels. Connexin 43 (CX43) is expressed in pulp tissue. However, its function in dental pulp tissue has yet to be fully investigated. In the present study, we employed antisense oligonucleotides (AS) against rat CX43 to investigate the role of CX43 in dental pulp cells. We treated cultured dental pulp cells with AS or sense (S) oligonucleotides. The number of cells in the AS-treated groups was about 1.3-fold that in the S-treated controls. Growth rates were significantly different between the AS-and S-treated groups at 48 h (p<0.01). An alkaline phosphate assay revealed that AS-treated pulp cells dramatically decreased at 48 h after AS incorporation, while S-treated pulp cells showed no marked changes.Western blot analysis showed that HSP25 was highly expressed in S-treated cells, but was only weakly expressed in AS-treated cells at 48 h. Furthermore, AS-treated cells showed high expression of CX45. On the other hand, S-treated cells showed high expression of CX32. These results suggest that CX43 is involved in cell growth, mineralization and differentiation to odontoblasts in rat pulp cells, and that CX43 plays the opposite role to that of CX45.3

Osteoinductivity of calcium phosphate mediated by connexin 43

“…The largest P2 isoform of Cx43 is the functional form found in gap junctions and therefore the structure of interest. The P0 and P1 are transient isoforms seen during synthesis [34, 39]. Western blot analysis shows that cultures of DPCs with ACP have increased P2 isoform Cx43 expression compared to the DPCs cultured without ACP (Figure 6h–i).…”

“…Lecanda et al showed that osteocalcin and bone sialoprotein gene expression increased in osteoblastic cells transfected with Cx43 [32]. This is further supported by a study that showed that rat bone marrow stromal cells have decreased alkaline phosphatase activity and mineralization when cultured with the gap junctional inhibitor 18-α-glycyrrhetinic acid [30], and Cx43 inhibition in DPC with antisense oligonucleotides results in a decrease in alkaline phosphatase activity [39]. These studies demonstrate that Cx43 plays a critical role in bone marrow and dental pulp stem cell differentiation and mineralization.…”

Human immature dental pulp stem cells share key characteristic features with limbal stem cells

“…Gene expression profiles of putative human LSC cultivated in vitro have been described previously and are in accordance with results obtained in the present work (34). In addition, it has also been reported that rat dental pulp cells cultivated in vitro showed expression of connexin 43 (36) and that expression of ABCG2 was found inside population of dental pulp stem cells (37).…”

Immunoelectron microscopic observation of connexin43 in rat odontoblasts

“…Morphological investigations have found localization of CX43 in odontoblasts and pulp cells (About et al, ; Fried et al, ; Joao and Arana‐Chavez, ; Kagayama et al, ; Murakami et al, ; Pinero et al, ). We have found that the expression of CX43 increases following odontoblast differentiation (Murakami et al, ) and cell viability in odontoblasts and pulp cells (Amano et al, ; Chung et al, ). Recently, it is reported that expression of CX43 in odontoblasts is associated with ATP release and signaling in process of dental sensitivity and pain (Liu et al, ).…”