Villars, F., B. Guillotin, T. Amé dé e, S. Dutoya, L. Bordenave, R. Bareille, and J. Amé dé e. Effect of HUVEC on human osteoprogenitor cell differentiation needs heterotypic gap junction communication. Am J Physiol Cell Physiol 282: C775-C785, 2002; 10.1152/ajpcell.00310.2001.-Bone development and remodeling depend on complex interactions between bone-forming osteoblasts and other cells present within the bone microenvironment, particularly vascular endothelial cells that may be pivotal members of a complex interactive communication network in bone. Our aim was to investigate the interaction between human umbilical vein endothelial cells (HUVEC) and human bone marrow stromal cells (HBMSC). Cell differentiation analysis performed with different cell culture models revealed that alkaline phosphatase activity and type I collagen synthesis were increased only by the direct contact of HUVEC with HBMSC. This "juxtacrine signaling" could involve a number of different heterotypic connexions that require adhesion molecules or gap junctions. A dye coupling assay with Lucifer yellow demonstrated a functional coupling between HUVEC and HBMSC. Immunocytochemistry revealed that connexin43 (Cx43), a specific gap junction protein, is expressed not only in HBMSC but also in the endothelial cell network and that these two cell types can communicate via a gap junctional channel constituted at least by Cx43. Moreover, functional inhibition of the gap junction by 18␣-glycyrrhetinic acid treatment or inhibition of Cx43 synthesis with oligodeoxyribonucleotide antisense decreased the effect of HUVEC cocultures on HBMSC differentiation. This stimulation could be mediated by the intercellular diffusion of signaling molecules that permeate the junctional channel.coupling; intercellular messenger; connexin43; osteoblast; endothelial cells ANGIOGENESIS is a tightly regulated process involved in growth, repair, and bone remodeling (9, 11-13, 23, 28, 50, 54, 56). Several studies have shown that there is a reciprocal regulation and functional relationship between endothelial cells and osteoblast-like cells during osteogenesis, in which systemic hormones and paracrine growth factors or cytokines play an active role (4,24,53,55). In a previous study (51), we showed that osteoblast progenitor cells [human bone marrow stromal cells (HBMSC)] behave differently in terms of proliferation and differentiation when cultured in association with endothelial cells [human umbilical vein endothelial cells (HUVEC)] in various coculture models (coculture with or without direct contact, conditioned medium) than when they are cultured alone. An increase in alkaline phosphatase activity (Al-P) was observed only when HBMSC were cocultured in direct contact with HUVEC. The enhancement of this early osteoblastic marker is not provided when HBMSC and HUVEC are cocultured separately with the use of a semipermeable membrane (51) or when HBMSC are seeded onto a matrix obtained from HUVEC cultures (51).Therefore, the intercommunication between endothelial cells and os...

The P2X 7 receptor, mainly expressed by immune cells, is a ionotropic receptor activated by high concentration of extracellular ATP. It is involved in several processes relevant to immunomodulation and inflammation. Among these processes, the production of extracellular interleukin-1␤ (IL-1␤), a pro-inflammatory cytokine, plays a major role in the activation of the cytokine network. We have investigated the role of P2X 7 receptor and of an associated calcium-activated potassium conductance (BK channels) in IL-1␤ maturation and releasing processes by Schwann cells. Lipopolysaccharideprimed Schwann cells synthesized large amounts of pro-IL-1␤ but did not release detectable amounts of pro or mature IL-1␤. ATP on its own had no effect on the synthesis of pro-IL-1␤, but a co-treatment with lipopolysaccharide and ATP led to the maturation and the release of IL-1␤ by Schwann cells. Both mechanisms were blocked by oxidized ATP. IL-1␤-converting enzyme (ICE), the caspase responsible for the maturation of pro-IL-1␤ in IL-1␤, was activated by P2X 7 receptor stimulation. The specific inhibition of ICE by the caspase inhibitor Ac-Tyr-Val-Ala-Asp-aldehyde blocked the maturation of IL-1␤. In searching for a link between the P2X 7 receptor and the activation of ICE, we found that enhancing potassium efflux from Schwann cells upregulated the production of IL-1␤, while strongly reducing potassium efflux led to opposite effects. Blocking BK channels actually modulated IL-1␤ release. Taken together, these results show that P2X 7 receptor stimulation and associated BK channels, through the activation of ICE, leads to the maturation and the release of IL-1␤ by immune-challenged Schwann cells.

The P2X 7 receptor (P2X 7 R) is a purinoceptor expressed predominantly by cells of immune origin, including microglial cells. P2X 7 R has a role in the release of biologically active proinflammatory cytokines such as IL-1β, IL-6 and TNFα. Here we demonstrate that when incubated with lipopolysaccharide (LPS), glial cells cultured from brain of P2X 7 R −/− mice produce less IL-1β compared to glial cells from brains of wild-type mice. This is not the case for TNFα and IL-6. Our results indicate a selective effect of the P2X7R gene deletion on release of IL-1β release but not of IL-6 and TNFα. In addition, we confirm that only microglial cells produce IL-1β, and this release is dependent on P2X 7 R and ABC1 transporter. Because IL-1β is a key regulator of the brain cytokine network and P2X 7 R is an absolute requirement for IL-1β release, we further investigated whether response of brain cytokines to LPS in vivo was altered in P2X 7 R −/− mice compared to wild-type mice. IL-1β and TNFα mRNAs were less elevated in the brain of P2X 7 R −/− than in the brain of wild-type mice in response to systemic LPS. These results show that P2X7R plays a key role in the brain cytokine response to immune stimuli, which certainly applies also to cytokinedependent alterations in brain functions including sickness behavior.

Recent evidence suggests that interleukin-1β (IL-1β), which was originally identified as a proinflammatory cytokine, is also required in the brain for memory processes. We have previously shown that IL-1β synthesis in the hippocampus is dependent on P2X7 receptor (P2X7R), which is an ionotropic receptor of ATP. To substantiate the role of P2X7R in both brain IL-1β expression and memory processes, we examined the induction of IL-1β mRNA expression in the hippocampus of wild-type (WT) and homozygous P2X7 receptor knockout mice (P2X7R−/−) following a spatial memory task. The spatial recognition task induced both IL-1β mRNA expression and c-Fos protein activation in the hippocampus of WT but not of P2X7R−/− mice. Remarkably, P2X7R−/− mice displayed spatial memory impairment in a hippocampal-dependant task, while their performances in an object recognition task were unaltered. Taken together, our results show that P2X7R plays a critical role in spatial memory processes and the associated hippocampal IL-1β mRNA synthesis and c-Fos activation.