Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) Reduces Oxidative and Mechanical Stress-Evoked Matrix Degradation in Chondrifying Cell Cultures
Abstract
:1. Introduction
2. Results
2.1. Hyaluronidase Expression in Chondrifying Cells
2.2. Identification of MMPs in Chondrifying Cell Cultures
2.3. Aggrecanase Activity in Chondrogenesis
2.4. Effects of PACAP and Various Stress on Metachromatic Cartilage Formation
2.5. Various Cellular Stress Increase Hyluronidase Activity
2.6. Expression of MMPs Affected by PACAP Treatment
2.7. Aggrecanase Function during Oxidative and Mechanical Stress
3. Discussion
4. Materials and Methods
4.1. Cell Culturing
4.2. Application of PACAP, H2O2, and Mechanical Stress
4.3. Light Microscopical Analysis
4.4. RT-PCR Reactions
4.5. Western Blot
4.6. Hyaluronidase Assay
4.7. Zymography
4.8. Aggrecanase Activity Assay
4.9. Statistical Analysis
Supplementary Materials
Author Contributions
Acknowledgments
Conflicts of Interest
Abbreviations
ADAMTS | a disintegrin and metalloprotease with thrombospondin motifs |
BMP | bone morphogenetic protein |
cAMP | cyclic adenosine monophosphate |
CREB | cAMP response element-binding protein |
DMEM | Dulbecco’s Modified Eagle’s Medium |
dNTP | deoxynucleotide triphosphate |
ECM | extracellular matrix |
EDTA | ethylene diamine tetra-acetic acid |
EGTA | ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetra acetic acid |
FBS | foetal bovine serum |
FGF | fibroblast growth factor |
HH | hedgehog |
HEPES | 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid |
Hyal | hyaluronidase |
IHH | Indian Hedgehog |
MAPK | mitogen-activated protein kinase |
MMP | matrix metalloproteinase |
MS | mechanical stress |
PAC1 | pituitary adenylate cyclase-activating polypeptide type I receptor |
PACAP | pituitary adenylate cyclase activating polypeptide |
PBS | phosphate buffered saline |
PBST | phosphate buffered saline supplemented with 1% Tween-20 |
PKA | protein kinase A |
PKC | protein kinase C |
RT-PCR | reverse transcription followed by polymerase chain reaction |
Runx2 | Runt-related transcription factor 2 |
SHH | Sonic Hedgehog |
Sox9 | SRY (sex determining region Y)-box 9 |
TGFβ | transforming growth factor-β |
VIP | vasoactive intestinal polypeptide |
VPAC | vasoactive intestinal polypeptide receptor |
WNT | wingless int1 |
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Gene | Primer | Nucleotide Sequence (5’→3’) | GenBank ID | Annealing Temperature | Amplimer Size (bp) |
---|---|---|---|---|---|
Admats4 | sense | GTG GCA AGT ATT GTG AGG G(2079–2097) | NM_172845.3 | 54 °C | 124 |
antisense | AGG TCG GTT CGG TGG TT(2186–2202) | ||||
Hyal1 | sense | GGG GTC TTT GAT GTC GTG G(371–389) | XM_015292793.1 | 57 °C | 486 |
antisense | CGG GTC GCT GAA GTT GTT(839–856) | ||||
Hyal2 | sense | ACA ACC ACG ACT ACA GCA AGA A(784–805) | XM_414258.5 | 56 °C | 452 |
antisense | CGC TGC CAT CGT CAC ATT(1218–1235) | ||||
Hyal3 | sense | TAC GGC ATC GTG GAG AAC CG(265–284) | XM_003641994.3 | 59 °C | 450 |
antisense | CCA GTC GTC GTT GAA GCA GTC G(693–714) | ||||
Hyal4 | sense | CCA CCG TGC CTT GCT ATT(325–342) | XM_017011911.1 | 51 °C | 254 |
antisense | GTT TGC TGC TGG TCC TTT(560–577) | ||||
MMP1 | sense | TTT GTG ACC CTA ACT TGA(1021–1038) | XM_417176.4 | 47 °C | 473 |
antisense | GAC ATA GCC ATC TTT CTG(1476–1493) | ||||
MMP7 | sense | AAA AGA GTT ACC TCG GGA CA(483–502) | NM_007742.3 | 52 °C | 250 |
antisense | CAC GGA CAT TTG AGT GGG(717–735) | ||||
MMP8 | sense | TGT CAA GGG CTG AAG TGA(478–495) | NM_008611.4 | 51 °C | 382 |
antisense | TGA GGT AGT GAA TAG GTG C(841–859) | ||||
MMP9 | sense | TTC TGG ACT CTG GGA ACC G(625–643) | NM_204667.1 | 57 °C | 237 |
antisense | GGG AGA CCC ATC GCT GTT(844–861) | ||||
MMP13 | sense | CAT GCA GAA ACC ACG ATG(296–313) | NM_001293090.1 | 51 °C | 395 |
antisense | GAG CAG CAA CAA GAA ACA AG(671–690) | ||||
GAPDH | sense | GAG AAC GGG AAA CTT GTC AT(238–258) | NM_204305 | 54 °C | 556 |
antisense | GGC AGG TCA GGT CAA CAA(775–793) |
Antibody | Host Animal | Dilution | Distributor |
---|---|---|---|
Anti-Hyal1 | rabbit, polyclonal | 1:500 | Sigma-Aldrich, St. Louis, MO, USA |
Anti-Hyal2 | rabbit, polyclonal | 1:500 | Sigma-Aldrich, St. Louis, MO, USA |
Anti-Hyal3 | rabbit, polyclonal | 1:500 | Sigma-Aldrich, St. Louis, MO, USA |
Anti-Hyal4 | rabbit, polyclonal | 1:500 | Sigma-Aldrich, St. Louis, MO, USA |
Anti-MMP1 | rabbit, polyclonal | 1:500 | Sigma-Aldrich, St. Louis, MO, USA |
Anti-MMP7 | rabbit, polyclonal | 1:500 | Sigma-Aldrich, St. Louis, MO, USA |
Anti-MMP8 | rabbit, polyclonal | 1:500 | Sigma-Aldrich, St. Louis, MO, USA |
Anti-MMP9 | rabbit, polyclonal | 1:500 | Sigma-Aldrich, St. Louis, MO, USA |
Anti-MMP13 | rabbit, polyclonal | 1:500 | Sigma-Aldrich, St. Louis, MO, USA |
Anti-Adamts4 | rabbit, polyclonal | 1:500 | Sigma-Aldrich, St. Louis, MO, USA |
Anti-Actin | mouse, monoclonal | 1:10,000 | Sigma-Aldrich, St. Louis, MO, USA |
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Szentléleky, E.; Szegeczki, V.; Karanyicz, E.; Hajdú, T.; Tamás, A.; Tóth, G.; Zákány, R.; Reglődi, D.; Juhász, T. Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) Reduces Oxidative and Mechanical Stress-Evoked Matrix Degradation in Chondrifying Cell Cultures. Int. J. Mol. Sci. 2019, 20, 168. https://doi.org/10.3390/ijms20010168
Szentléleky E, Szegeczki V, Karanyicz E, Hajdú T, Tamás A, Tóth G, Zákány R, Reglődi D, Juhász T. Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) Reduces Oxidative and Mechanical Stress-Evoked Matrix Degradation in Chondrifying Cell Cultures. International Journal of Molecular Sciences. 2019; 20(1):168. https://doi.org/10.3390/ijms20010168
Chicago/Turabian StyleSzentléleky, Eszter, Vince Szegeczki, Edina Karanyicz, Tibor Hajdú, Andrea Tamás, Gábor Tóth, Róza Zákány, Dóra Reglődi, and Tamás Juhász. 2019. "Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) Reduces Oxidative and Mechanical Stress-Evoked Matrix Degradation in Chondrifying Cell Cultures" International Journal of Molecular Sciences 20, no. 1: 168. https://doi.org/10.3390/ijms20010168