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      Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) Reduces Oxidative and Mechanical Stress-Evoked Matrix Degradation in Chondrifying Cell Cultures

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          Abstract

          Pituitary adenylate cyclase activating polypeptide (PACAP) is an endogenous neuropeptide also secreted by non-neural cells, including chondrocytes. PACAP signaling is involved in the regulation of chondrogenesis, but little is known about its connection to matrix turnover during cartilage formation and under cellular stress in developing cartilage. We found that the expression and activity of hyaluronidases (Hyals), matrix metalloproteinases (MMP), and aggrecanase were permanent during the course of chondrogenesis in primary chicken micromass cell cultures, although protein levels changed daily, along with moderate and relatively constant enzymatic activity. Next, we investigated whether PACAP influences matrix destructing enzyme activity during oxidative and mechanical stress in chondrogenic cells. Exogenous PACAP lowered Hyals and aggrecanase expression and activity during cellular stress. Expression and activation of the majority of cartilage matrix specific MMPs such as MMP1, MMP7, MMP8, and MMP13, were also decreased by PACAP addition upon oxidative and mechanical stress, while the activity of MMP9 seemed not to be influenced by the neuropeptide. These results suggest that application of PACAP can help to preserve the integrity of the newly synthetized cartilage matrix via signaling mechanisms, which ultimately inhibit the activity of matrix destroying enzymes under cellular stress. It implies the prospect that application of PACAP can ameliorate articular cartilage destruction in joint diseases.

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          Most cited references 61

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          Isolation of a novel 38 residue-hypothalamic polypeptide which stimulates adenylate cyclase in pituitary cells.

          A novel neuropeptide which stimulates adenylate cyclase in rat anterior pituitary cell cultures was isolated from ovine hypothalamic tissues. Its amino acid sequence was revealed as: His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Tyr-Arg-Lys-Gln- Met-Ala- Val-Lys-Lys-Tyr-Leu-Ala-Ala-Val-Leu-Gly-Lys-Arg-Tyr-Lys-Gln-Arg-Val-Lys-Asn-Lys - NH2. The N-terminal sequence shows 68% homology with vasoactive intestinal polypeptide (VIP) but its adenylate cyclase stimulating activity was at least 1000 times greater than that of VIP. It increased release of growth hormone (GH), prolactin (PRL), corticotropin (ACTH) and luteinizing hormone (LH) from superfused rat pituitary cells at as small a dose as 10(-10)M (GH, PRL, ACTH) or 10(-9)M (LH). Whether these hypophysiotropic effects are the primary actions of the peptide or what physiological action in the pituitary is linked with the stimulation of adenylate cyclase by this peptide remains to be determined.
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            ROS/oxidative stress signaling in osteoarthritis.

            Osteoarthritis is the most common joint disorder with increasing prevalence due to aging of the population. Its multi-factorial etiology includes oxidative stress and the overproduction of reactive oxygen species, which regulate intracellular signaling processes, chondrocyte senescence and apoptosis, extracellular matrix synthesis and degradation along with synovial inflammation and dysfunction of the subchondral bone. As disease-modifying drugs for osteoarthritis are rare, targeting the complex oxidative stress signaling pathways would offer a valuable perspective for exploration of potential therapeutic strategies in the treatment of this devastating disease.
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              Phosphorylation of SOX9 by cyclic AMP-dependent protein kinase A enhances SOX9's ability to transactivate a Col2a1 chondrocyte-specific enhancer.

              Sox9 is a high-mobility-group domain-containing transcription factor required for chondrocyte differentiation and cartilage formation. We used a yeast two-hybrid method based on Son of Sevenless (SOS) recruitment to screen a chondrocyte cDNA library and found that the catalytic subunit of cyclic AMP (cAMP)-dependent protein kinase A (PKA-Calpha) interacted specifically with SOX9. Next we found that two consensus PKA phosphorylation sites within SOX9 could be phosphorylated by PKA in vitro and that SOX9 could be phosphorylated by PKA-Calpha in vivo. In COS-7 cells cotransfected with PKA-Calpha and SOX9 expression plasmids, PKA enhanced the phosphorylation of wild-type SOX9 but did not affect phosphorylation of a SOX9 protein in which the two PKA phosphorylation sites (S(64) and S(211)) were mutated. Using a phosphospecific antibody that specifically recognized SOX9 phosphorylated at serine 211, one of the two PKA phosphorylation sites, we demonstrated that addition of cAMP to chondrocytes strongly increased the phosphorylation of endogenous Sox9. In addition, immunohistochemistry of mouse embryo hind legs showed that Sox9 phosphorylated at serine 211 was principally localized in the prehypertrophic zone of the growth plate, corresponding to the major site of expression of the parathyroid hormone-related peptide (PTHrP) receptor. Since cAMP has previously been shown to effectively increase the mRNA levels of Col2a1 and other specific markers of chondrocyte differentiation in culture, we then asked whether PKA phosphorylation could modulate the activity of SOX9. Addition of 8-bromo-cAMP to chondrocytes in culture increased the activity of a transiently transfected SOX9-dependent 48-bp Col2a1 chondrocyte-specific enhancer; similarly, cotransfection of PKA-Calpha increased the activity of this enhancer. Mutations of the two PKA phosphorylation consensus sites of SOX9 markedly decreased the PKA-Calpha activation of this enhancer by SOX9. PKA phosphorylation and the mutations in the consensus PKA phosphorylation sites of SOX9 did not alter its nuclear localization. In vitro phosphorylation of SOX9 by PKA resulted in more efficient DNA binding. We conclude that SOX9 is a target of cAMP signaling and that phosphorylation of SOX9 by PKA enhances its transcriptional and DNA-binding activity. Because PTHrP signaling is mediated by cAMP, our results support the hypothesis that Sox9 is a target of PTHrP signaling in the growth plate and that the increased activity of Sox9 might mediate the effect of PTHrP in maintaining the cells as nonhypertrophic chondrocytes.
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                Author and article information

                Journal
                Int J Mol Sci
                Int J Mol Sci
                ijms
                International Journal of Molecular Sciences
                MDPI
                1422-0067
                04 January 2019
                January 2019
                : 20
                : 1
                Affiliations
                [1 ]Department of Anatomy, Histology and Embryology, Faculty of Medicine, University of Debrecen, Nagyerdei krt. 98, H-4032 Debrecen, Hungary; szentleleky.eszter@ 123456dental.unideb.hu (E.S.); szegeczki.vince@ 123456anat.med.unideb.hu (V.S.); karanyicz.edina@ 123456anat.med.unideb.hu (E.K.); hajdu.tibor@ 123456anat.med.unideb.hu (T.H.); roza@ 123456anat.med.unideb.hu (R.Z.)
                [2 ]Department of Anatomy, MTA-PTE PACAP Research Team, University of Pécs Medical School, Szigeti út 12, H-7624 Pécs, Hungary; andreatamassz@ 123456gmail.com (A.T.); dora.reglodi@ 123456aok.pte.hu (D.R.)
                [3 ]Department of Medical Chemistry, University of Szeged, Faculty of Medicine, Dóm tér 8, H-6720 Szeged, Hungary; toth.gabor@ 123456med.u-szeged.hu
                Author notes
                [* ]Correspondence: juhaszt@ 123456anat.med.unideb.hu ; Tel.: +36-52-255-567; Fax: +36-52-255-115
                Article
                ijms-20-00168
                10.3390/ijms20010168
                6337298
                30621194
                © 2019 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

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