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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
Umeå University, Faculty of Medicine, Odontology, Oral Cell Biology.
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.
Series
Umeå University odontological dissertations, ISSN 0345-7532 ; 91
Keyword [en]
osteoblast, bone resorption, cytokines, IL-6, neuropeptides, VIP, transcription factor
National Category
Cell Biology
Identifiers
URN: urn:nbn:se:umu:diva-606ISBN: 91-7305-939-0 (print)OAI: oai:DiVA.org:umu-606DiVA: diva2:143954
Public defence
2005-10-14, Sal B, Tandläkarhögskolan, Tandläkarhögskolan, Umeå, 13:00 (English)
Opponent
Supervisors
Available from: 2005-10-10 Created: 2005-10-10 Last updated: 2009-11-26Bibliographically approved
List of papers
1. IL-6, leukemia inhibitory factor, and oncostatin M stimulate bone resorption and regulate the expression of receptor activator of NF-kB ligand, osteoprotegerin, and receptor activator of NF-kB in mouse calvariae
Open this publication in new window or tab >>IL-6, leukemia inhibitory factor, and oncostatin M stimulate bone resorption and regulate the expression of receptor activator of NF-kB ligand, osteoprotegerin, and receptor activator of NF-kB in mouse calvariae
2002 (English)In: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 169, no 6, 3353-3362 p.Article in journal (Refereed) Published
Abstract [en]

IL-6, leukemia inhibitory factor (LIF), and oncostatin M (OSM) are IL-6-type cytokines that stimulate osteoclast formation and function. In the present study, the resorptive effects of these agents and their regulation of receptor activator of NF-kappaB ligand (RANKL), RANK, and osteoprotegerin (OPG) were studied in neonatal mouse calvaria. When tested separately, neither human (h) IL-6 nor the human soluble IL-6R (shIL-6R) stimulated bone resorption, but when hIL-6 and the shIL-6R were combined, significant stimulation of both mineral and matrix release from bone explants was noted. Semiquantitative RT-PCR showed that hIL-6 plus shIL-6R enhanced the expression of RANKL and OPG in calvarial bones, but decreased RANK expression. Human LIF, hOSM, and mouse OSM (mOSM) also stimulated 45Ca release and enhanced the mRNA expression of RANKL and OPG in mouse calvaria, but had no effect on the expression of RANK. In agreement with the RT-PCR analyses, ELISA measurements showed that both hIL-6 plus shIL-6R and mOSM increased RANKL and OPG proteins. 1,25-Dihydroxyvitamin D3 (D3) also increased the RANKL protein level, but decreased the protein level of OPG. OPG inhibited 45Ca release stimulated by RANKL, hIL-6 plus shIL-6R, hLIF, hOSM, mOSM, and D3. An Ab neutralizing mouse gp130 inhibited 45Ca release induced by hIL-6 plus shIL-6R. These experiments demonstrated stimulation of calvarial bone resorption and regulation of mRNA and protein expression of RANKL and OPG by D3 and IL-6 family cytokines as well as regulation of RANK expression in preosteoclasts/osteoclasts of mouse calvaria by D3 and hIL-6 plus shIL-6R.

Identifiers
urn:nbn:se:umu:diva-9427 (URN)12218157 (PubMedID)
Available from: 2008-04-02 Created: 2008-04-02 Last updated: 2017-12-14Bibliographically approved
2. 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.
Open this publication in new window or tab >>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.
2005 (English)In: Bone, ISSN 8756-3282, E-ISSN 1873-2763, 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.

Identifiers
urn:nbn:se:umu:diva-18082 (URN)10.1016/j.bone.2005.04.043 (DOI)16085472 (PubMedID)
Available from: 2008-04-02 Created: 2008-04-02 Last updated: 2017-12-14Bibliographically approved
3. The neuropeptide VIP potentiates IL-6 production induced by proinflammatory osteotropic cytokines in calvarial osteoblasts and the osteoblastic cell line MC3T3-E1.
Open this publication in new window or tab >>The neuropeptide VIP potentiates IL-6 production induced by proinflammatory osteotropic cytokines in calvarial osteoblasts and the osteoblastic cell line MC3T3-E1.
2005 (English)In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 335, no 3, 705-711 p.Article in journal (Refereed) Published
Abstract [en]

Skeletal turnover is orchestrated by a complex network of regulatory factors. Lately, regulation of bone metabolism through neuro-osteological interactions has been proposed. Here, we address the question whether IL-6 production can be affected by interactions between the neuropeptide VIP and proinflammatory, bone-resorbing cytokines. By using calvarial osteoblasts, we showed that IL-1beta increased IL-6 production time- and concentration-dependently, and that these effects were potentiated by VIP. Furthermore, IL-1beta stimulated IL-6 promoter activity in the osteoblastic cell line MC3T3-E1 stably transfected with a human IL-6 promoter/luciferase construct, and both VIP, and the related neuropeptide PACAP-38, increased the effect of IL-1beta in a synergistic manner. The IL-6 protein release from calvarial osteoblasts was also stimulated by the osteoclastogenic, proinflammatory cytokines IL-11, LIF, OSM, IL-17, TGF-beta, and TNF-alpha. All effects, except for that of TNF-alpha, were synergistically potentiated by VIP. These findings further support the role of neuropeptides, and the presence of neuro-immunological interactions, in bone metabolism.

Identifiers
urn:nbn:se:umu:diva-18080 (URN)10.1016/j.bbrc.2005.07.135 (DOI)16095565 (PubMedID)
Available from: 2008-03-04 Created: 2008-03-04 Last updated: 2017-12-14Bibliographically approved
4. The neuropeptide vasoactive intestinal peptide enhances osteoclastogenic properties in mouse calvarial osteoblasts but decreases osteoclastogenesis by directly targeting osteoclast progenitor cells in co-cultures of osteoblasts and marrow macrophages
Open this publication in new window or tab >>The neuropeptide vasoactive intestinal peptide enhances osteoclastogenic properties in mouse calvarial osteoblasts but decreases osteoclastogenesis by directly targeting osteoclast progenitor cells in co-cultures of osteoblasts and marrow macrophages
Manuscript (Other academic)
Identifiers
urn:nbn:se:umu:diva-4730 (URN)
Available from: 2005-10-10 Created: 2005-10-10 Last updated: 2010-01-13Bibliographically approved

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