Pituitary adenylate cyclase activating polypeptide (PACAP) signalling exerts chondrogenesis promoting and protecting effects: implication of calcineurin as a downstream target

Tamás Juhász; Csaba Matta; Éva Katona; Csilla Somogyi; Roland Takács; Pál Gergely; László Csernoch; Gyorgy Panyi; Gábor Tóth; Dóra Reglődi; Andrea Tamás; Róza Zákány

doi:10.1371/journal.pone.0091541

Pituitary adenylate cyclase activating polypeptide (PACAP) signalling exerts chondrogenesis promoting and protecting effects: implication of calcineurin as a downstream target

PLoS One. 2014 Mar 18;9(3):e91541. doi: 10.1371/journal.pone.0091541. eCollection 2014.

Authors

Affiliations

¹ Department of Anatomy, Histology and Embryology, University of Debrecen, Faculty of Medicine, Debrecen, Hungary.
² Cell Biology and Signalling Research Group of the Hungarian Academy of Sciences, Department of Medical Chemistry, Research Centre for Molecular Medicine, Faculty of Medicine, University of Debrecen, Debrecen, Hungary.
³ Department of Physiology, University of Debrecen, Faculty of Medicine, Debrecen, Hungary.
⁴ Department of Biophysics and Cell Biology, University of Debrecen, Faculty of Medicine, Debrecen, Hungary.
⁵ Department of Medical Chemistry, University of Szeged, Faculty of Medicine, Szeged, Hungary.
⁶ Department of Anatomy PTE-MTA "Lendület" PACAP Research Team, University of Pécs, Medical School, Pécs, Hungary.

Abstract

Pituitary adenylate cyclase activating polypeptide (PACAP) is an important neurotrophic factor influencing differentiation of neuronal elements and exerting protecting role during traumatic injuries or inflammatory processes of the central nervous system. Although increasing evidence is available on its presence and protecting function in various peripheral tissues, little is known about the role of PACAP in formation of skeletal components. To this end, we aimed to map elements of PACAP signalling in developing cartilage under physiological conditions and during oxidative stress. mRNAs of PACAP and its receptors (PAC1,VPAC1, VPAC2) were detectable during differentiation of chicken limb bud-derived chondrogenic cells in micromass cell cultures. Expression of PAC1 protein showed a peak on days of final commitment of chondrogenic cells. Administration of either the PAC1 receptor agonist PACAP 1-38, or PACAP 6-38 that is generally used as a PAC1 antagonist, augmented cartilage formation, stimulated cell proliferation and enhanced PAC1 and Sox9 protein expression. Both variants of PACAP elevated the protein expression and activity of the Ca-calmodulin dependent Ser/Thr protein phosphatase calcineurin. Application of PACAPs failed to rescue cartilage formation when the activity of calcineurin was pharmacologically inhibited with cyclosporine A. Moreover, exogenous PACAPs prevented diminishing of cartilage formation and decrease of calcineurin activity during oxidative stress. As an unexpected phenomenon, PACAP 6-38 elicited similar effects to those of PACAP 1-38, although to a different extent. On the basis of the above results, we propose calcineurin as a downstream target of PACAP signalling in differentiating chondrocytes either in normal or pathophysiological conditions. Our observations imply the therapeutical perspective that PACAP can be applied as a natural agent that may have protecting effect during joint inflammation and/or may promote cartilage regeneration during degenerative diseases of articular cartilage.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Calcineurin / genetics*
Calcineurin / metabolism
Calcineurin Inhibitors / pharmacology
Cell Differentiation
Cell Proliferation
Chick Embryo
Chondrocytes / cytology
Chondrocytes / drug effects*
Chondrocytes / metabolism
Chondrogenesis / genetics*
Cyclosporine / pharmacology
Gene Expression Regulation, Developmental
Hydrogen Peroxide / pharmacology
Limb Buds / cytology
Limb Buds / drug effects
Limb Buds / growth & development
Limb Buds / metabolism
Oxidative Stress
Peptides / chemical synthesis
Peptides / pharmacology*
Pituitary Adenylate Cyclase-Activating Polypeptide / chemical synthesis
Pituitary Adenylate Cyclase-Activating Polypeptide / pharmacology*
Primary Cell Culture
Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I / genetics
Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I / metabolism
Receptors, Vasoactive Intestinal Peptide, Type II / genetics
Receptors, Vasoactive Intestinal Peptide, Type II / metabolism
Receptors, Vasoactive Intestinal Polypeptide, Type I / genetics
Receptors, Vasoactive Intestinal Polypeptide, Type I / metabolism
SOX9 Transcription Factor / genetics
SOX9 Transcription Factor / metabolism
Signal Transduction

Substances

Calcineurin Inhibitors
Peptides
Pituitary Adenylate Cyclase-Activating Polypeptide
Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I
Receptors, Vasoactive Intestinal Peptide, Type II
Receptors, Vasoactive Intestinal Polypeptide, Type I
SOX9 Transcription Factor
Cyclosporine
Hydrogen Peroxide
Calcineurin

Grants and funding

This work was supported by grants from Akira Arimura Foundation Research Grant, the Hungarian Science Research Fund (OTKA CNK80709), Bolyai Scholarship and the Hungarian Ministry of Education (TÁMOP 4.2.1.B-10/2/KONV-2010-002, PTE-MTA “Lendület”) and from the New Széchenyi Plan (TÁMOP-4.2.2.A-11/1/KONV-2012-0025; The project is co-financed by the European Union and the European Social Fund). C.M. is supported by a Mecenatura grant (DEOEC Mec-9/2011) from the Medical and Health Science Center, University of Debrecen, Hungary. Research activity of T.J. was supported by the European Union and the State of Hungary, co-financed by the European Social Fund in the framework of TÁMOP 4.2.4. A/2-11-1-2012-0001 ‘National Excellence Program’. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.