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Increased expression of interleukin-6 by vasoactive intestinal peptide is associated with regulation of CREB, AP-1 and C/EBP, but not NF-kappaB, in mouse calvarial osteoblasts.
Umeå University, Faculty of Medicine, Odontology, Oral Cell Biology.
Umeå University, Faculty of Medicine, Odontology, Oral Cell Biology.
2005 (English)In: Bone, ISSN 8756-3282, Vol. 37, no 4, 513-529 p.Article in journal (Refereed) Published
Abstract [en]

Interleukin-6 (IL-6), and the related cytokines IL-11, leukemia inhibitory factor (LIF) and oncostatin M (OSM), are potent stimulators of osteoclastic bone resorption. In the present study, we have addressed the possibility that the neuropeptide vasoactive intestinal peptide (VIP) may regulate the production of and/or sensitivity to the IL-6 family of cytokines in mouse calvarial osteoblasts. VIP stimulated IL-6 mRNA expression and protein release in a time- and concentration-dependent manner, whereas mRNA expression of the IL-6 receptor, as well as mRNA expressions of IL-11, LIF, OSM and their cognate receptors, were unaffected by VIP. In cells transfected with the IL-6 promoter coupled to luciferase, VIP increased transcriptional activity. The effects of VIP were shared by the related neuropeptide PACAP-38, belonging to the same superfamily of neuropeptides, whereas secretin did not have any effect, indicating that the effects were mediated by VPAC2 receptors. The effects of VIP were potentiated by the cyclic AMP phosphodiesterase inhibitor rolipram and mimicked by forskolin, indicating the involvement of the cyclic AMP/protein kinase A pathway. This was further demonstrated by the facts that the stimulatory effect of VIP on luciferase activity could be reversed by the PKA inhibitors H-89 and KT5720 and was mimicked by cyclic AMP analogues selective for PKA, but not by those selective for Epac. In addition, VIP enhanced the phosphorylation of CREB, as assessed by both immunocytochemical analysis and Western blot. The DNA binding activity of nuclear extracts to C/EBP was increased by VIP, whereas binding to AP-1 was decreased. In contrast, DNA binding to NF-kappaB, as well as nuclear translocation of NF-kappaB and C/EBP, were unaffected by VIP. The mRNA expressions of C/EBPbeta, C/EBPdelta, C/EBPgamma, c-Jun, JunB, c-Fos, Fra-1 and IkappaBalpha and protein level of IkappaBalpha were all unaffected by VIP. These observations, together, demonstrate that VIP stimulates IL-6 production in osteoblasts by a mechanism likely to be mediated by VPAC2 receptors and dependent on cyclic AMP/protein kinase A/CREB activation and also involving the transcription factors C/EBP and AP-1.

Place, publisher, year, edition, pages
2005. Vol. 37, no 4, 513-529 p.
URN: urn:nbn:se:umu:diva-18082DOI: 10.1016/j.bone.2005.04.043PubMedID: 16085472OAI: diva2:157755
Available from: 2008-04-02 Created: 2008-04-02 Last updated: 2009-10-12Bibliographically approved
In thesis
1. The neuropeptide VIP and the IL-6 family of cytokines in bone: effects on bone resorption, cytokine expression and receptor signalling in osteoblasts and bone marrow stromal cells
Open this publication in new window or tab >>The neuropeptide VIP and the IL-6 family of cytokines in bone: effects on bone resorption, cytokine expression and receptor signalling in osteoblasts and bone marrow stromal cells
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Bone tissue is continuously degraded and rebuilt to respond to the needs of the body. Cells of the osteoblast lineage are responsible for the formation of bone, whereas the resorption of bone tissue is carried out by osteoclasts. To prevent imbalance between bone formation and resorption, these processes are delicately regulated by a complex network of both systemic factors and factors produced locally in the bone microenvironment, including members of the IL-6 family of cytokines. During the last decades, the presence of nerve fibers in skeletal tissue and presence of receptors for several neurotransmitters on both osteoblasts and osteoclasts, have suggested a possible role for neuropeptides in the regulation of skeletal metabolism.

The overall aim of this study was to investigate the roles of cytokines in the IL-6 family and the neuropeptide VIP in regulation of osteotropic cytokine expression and bone metabolism in vitro.

In Paper I, stimulation of bone resorption by the cytokine IL-6, in the presence of its soluble receptor sIL-6R, was demonstrated in mouse calvarial bones. OSM and LIF, other members of the IL-6 family of cytokines, were also shown to increase bone resorption. Furthermore, IL-6+sIL-6R, LIF, and OSM increased the expression of RANKL, which by binding to its receptor RANK functions as a crucial inducer of osteoclast formation and activation.

In Paper II-IV, the effects of the neuropeptide VIP and related peptides on expression of osteotropic cytokines by osteoblasts and bone resorption in vitro have been studied. VIP and PACAP-38 both increased IL-6 production in osteoblasts in a time- and concentration-dependent manner. In contrast, no effect was seen with the related peptide secretin, indicating that the effects were mediated by the VPAC2 receptor. VIP and PACAP, in contrast to secretin, also induced IL-6 promoter activity in osteoblastic MC3T3-E1 cells transfected with an IL-6 promoter/luciferase construct. The effects of VIP on IL-6 were shown to be mediated by several intracellular pathways, including cAMP/PKA/CREB, AP-1, and C/EBP, but not NF-kB or the cAMP-activated Epac pathway. The release of IL-6 from osteoblasts was increased by several pro-inflammatory osteotropic cytokines, including interleukin-1b, an effect that was further potentiated by VIP, indicating a possible neuro-immunomodulatory interaction in the regulation of bone metabolism. VIP and PACAP-38 also increased the osteoblastic expression of RANKL and decreased the expression of OPG and M-CSF, factors crucial in regulation of differentiation and activation of osteoclasts. Although this indicated a possible bone resorptive effect, VIP was found to decrease osteoclast formation and bone resorption by directly targeting osteoclast progenitor cells through an inhibitory mechanism.

In conclusion, the results in this thesis indicate that several cytokines in the IL-6 family stimulate bone resorption in calvarial bones in vitro, most likely through the RANKL-RANK interaction. Furthermore, expression of the osteotropic cytokine IL-6 in osteoblasts is stimulated by the neuropeptide VIP through VPAC2 receptors via several intracellular pathways, further strengthening the role of neuropeptides as local regulators of bone metabolism.

Place, publisher, year, edition, pages
Umeå: Odontologi, 2005. 161 p.
Umeå University odontological dissertations, ISSN 0345-7532 ; 91
osteoblast, bone resorption, cytokines, IL-6, neuropeptides, VIP, transcription factor
National Category
Cell Biology
urn:nbn:se:umu:diva-606 (URN)91-7305-939-0 (ISBN)
Public defence
2005-10-14, Sal B, Tandläkarhögskolan, Tandläkarhögskolan, Umeå, 13:00 (English)
Available from: 2005-10-10 Created: 2005-10-10 Last updated: 2009-11-26Bibliographically approved

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