Synergistic Crosstalk of PACAP and Notch Signaling Pathways in Bone Development
Abstract
1. Introduction
2. Results
2.1. Notch Signalling Alterations in Femurs of PACAP-Gene-Deficient Mice
2.2. Inhibition of Notch Signalling Is Compensated by PACAP 1-38 in Osteoblastic Cell Line
2.3. Notch Signalling Showed Altered Expression After the Addition of PACAP 1-38 in Osteoblast Cell Lines
2.4. Altered NFATc1 Expression in the Presence of PACAP 1-38 and DAPT
2.5. PACAP 1-38 Alters Notch Signalisation in the Intramembranous Ossification Model
3. Discussion
4. Materials and Methods
4.1. Animals
4.2. Cell Culture
4.3. PACAP 1-38 and DAPT Treatments
4.4. Determination of Non-Toxic Concentrations of DAPT and PACAP
4.5. Staining for Light Microscopy
4.6. Proliferation and Mitochondrial Activity Assessment
4.7. RT-PCR Analysis
4.8. Western Blot Analysis
4.9. Immunocytochemistry
4.10. ALP Activity Assay
4.11. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
ADAM9 | a disintegrin and metalloproteinase 9 |
ALP | alkaline phosphatase |
BMP | bone morphogenetic protein |
BSA | bovine serum albumin |
cAMP | cyclic adenosine monophosphate |
CSL | CBF1 humans/Su (H) Drosophila/LAG1Caenorhabditis elegans transcription factor |
CREB | cAMP response element-binding protein |
DAPI | 4′,6-diamidino-2-phenylindole |
DAPT | N-[N-(3,5-Difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester |
DLL | Delta-like ligands |
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 | fetal bovine serum |
FGF | fibroblast growth factor |
HH | hedgehog |
KO | knock out |
NA | numerical aperture |
NICD | Notch intracellular domain |
NFATc1 | nuclear factor of activated T-cells c1 |
Notch | neurogenic locus notch homolog protein |
NUMB | endocytic adaptor protein |
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 |
PP2B | protein phosphatase 2B |
PKA | protein kinase A |
PMSF | phenylmethylsulfonyl fluoride |
RT-PCR | reverse transcription followed by polymerase chain reaction |
Runx2 | Runt-related transcription factor 2 |
Smad | small worm phenotype and mothers against decapentaplegic |
SDS-PAGE | sodium dodecyl sulfate–polyacrylamide gel electrophoresis |
TACE | tumor necrosis factor-alpha converting enzyme |
WNT | wingless int1 |
WT | wild type |
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Gene | Primer | Nucleotide Sequence (5′→3′) | GenBank ID | Annealing Temperature | Amplimer Size (bp) |
---|---|---|---|---|---|
Alkaline phosphatase (Alpl) | sense | GAA GTC CGT GGG CAT CGT (474–491) | NM_013059 | 59 °C | 347 |
antisense | CAG TGC GGT TCC AGA CAT AG (801–820) | ||||
DLL1 (Dll1) | sense | GAA ACA CCA GCC TCC ACC T (2305–2323) | NM_001379042.1 | 53 °C | 336 |
antisense | GGA ATC TCC CCA CCC CTA (2623–2640) | ||||
DLL3 (Dll3) | sense | CCT GGT TTC CAA GGC TCT AA (1060–1079) | NM_007866.2 | 57 °C | 377 |
antisense | ACA GCG AAC TCG CAT CTC A (1418–1436) | ||||
DLL4 (Dll4) | sense | TTG CCC TTC AAT TTC ACC (673–690) | NM_019454.4 | 54 °C | 311 |
antisense | CAG GAC AGG CTG CCA TCT (966–983) | ||||
Jagged2 (Jag2) | sense | CCT CGT CGT CAT TCC CTT TC (395–411) | NM_001409685.1 | 57 °C | 337 |
antisense | GCA TTC TTT GCC CAT CCA G (713–731) | ||||
Jagged1 (Jag1) | sense | TCA GGC ATG ATA AAC CCT AGC (735–755) | NM_013822.5 | 56 °C | 444 |
antisense | GGG CTG ATG AGT CCC ACA (1161–1178) | ||||
Collagen type I (Col1a1) | sense | GGG CGA GTG CTG TGC TTT (348–365) | NM_007742.3 | 60 °C | 388 |
antisense | GGG ACC CAT TGG ACC TGA A (717–735) | ||||
Notch1 (notch1) | sense | GGA TCA CAT GGA CCG ATT G (6504–6522) | NM_008714.3 | 56 °C | 416 |
antisense | TGG ATG GAG ACT GCT GGA A (6901–6919) | ||||
Notch2 (notch2) | sense | GTA TCT CCA AGC CGT GTA TG (2791–2810) | NM_010928.2 | 55 °C | 437 |
antisense | GCA GAA GGG ACC AGT GAA (3210–3227) | ||||
Notch3 (notch3) | sense | GCA CCA GTG ATG GAA TAG GC (2308–2327) | NM_008716.3 | 56 °C | 405 |
antisense | AGC GAG GAC CAG CAA AGC (2695–2712) | ||||
Notch4 (notch4) | sense | GCC ACT CTT TAG CCA ACG C (3207–3225) | NM_010929.2 | 57 °C | 498 |
antisense | CAT CGC AGG TCC CAT CAC (3687–3704) | ||||
NFATc1 (Nfatc1) | sense | CCT GAC CAC CGA TAG CAC (973–990) | NM_001164109.1 | 52 °C | 325 |
antisense | CTC GTA TGG ACC AGA ATG T (1279–1297) | ||||
Adam9 (Adam9) | sense | TGA TTC GCT TAG CAA ACT (857–874) | NM_001270996.1 | 49 °C | 263 |
antisense | GTG GCT CCT TGA ACA TAC (1102–1119) | ||||
PKA (Prkaca) | sense | GCA AAG GCT ACA ACA AGG C (847–865) | NM_008854 | 53 °C | 280 |
antisense | ATG GCA ATC CAG TCA ATC G (1109–1126) | ||||
CSL (Csl) | sense | TGG AGC TTC CTG GAC AAT (1054–1071) | NM_027945.4 | 51 °C | 348 |
antisense | AGG CTG GTG GAG TAA ATG (1384–1401) | ||||
Numb (Numb) | sense | ATT CCG TGT CAC AAC TGC (597–614) | NM_001136075.3 | 51 °C | 351 |
antisense | AAA TCG GTC TTC CTC TGC (930–947) | ||||
TACE (Adam17) | sense | AAG TCT GCC TGG CTC ATC (1192–1209) | NM_001277266.1 | 51 °C | 301 |
antisense | CCT CCT TGG TCC TCA TTT (1475–1492) | ||||
Actin (Actb) | sense | GCC AAC CGT GAA AAG ATG A (419–437) | NM_001014970 | 48 °C | 462 |
antisense | CAA GAA GGA AGG CTG GAA AA (861–880) |
Antibody | Host Animal | Dilution | Distributor |
---|---|---|---|
Anti-Notch1 | rabbit, polyclonal | 1:500 | Cell Signaling, Danvers, MA, USA |
Anti-Notch2 | rabbit, polyclonal | 1:500 | Cell Signaling, Danvers, MA, USA |
Anti-Notch3 | rabbit, polyclonal | 1:500 | Cell Signaling, Danvers, MA, USA |
Anti-Coll. I. | mouse, monoclonal | 1:1000 | Sigma-Aldrich, St. Louis, MO, USA |
Anti-DLL1 | rabbit, polyclonal | 1:500 | Cell Signaling, Danvers, MA, USA |
Anti-DLL3 | rabbit, polyclonal | 1:500 | Cell Signaling, Danvers, MA, USA |
Anti-DLL4 | rabbit, polyclonal | 1:500 | Cell Signaling, Danvers, MA, USA |
Anti-Jagged1 | rabbit, polyclonal | 1:500 | Cell Signaling, Danvers, MA, USA |
Anti-Jagged2 | rabbit, polyclonal | 1:500 | Abcam, Cambridge, UK |
Anti-ALP | rabbit, polyclonal | 1:500 | Abcam, Cambridge, UK |
Anti-CSL | rabbit, polyclonal | 1:500 | Cell Signaling, Danvers, MA, USA |
Anti-TACE | rabbit, polyclonal | 1:500 | Cell Signaling, Danvers, MA, USA |
Anti-PKA | rabbit, polyclonal | 1:800 | Cell Signaling, Danvers, MA, USA |
Anti-Numb | rabbit, polyclonal | 1:500 | Cell Signaling, Danvers, MA, USA |
Anti-Adam9 | rabbit, polyclonal | 1:600 | Cell Signaling, Danvers, MA, USA |
Anti-NFATc1 | mouse, monoclonal | 1:500 | Abcam, Cambridge, UK |
Anti-Actin | mouse, monoclonal | 1:10,000 | Sigma-Aldrich, St. Louis, MO, USA |
Anti-GAPDH | rabbit, polyclonal | 1:800 | Abcam, Cambridge, UK |
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Szegeczki, V.; Pálfi, A.; Fillér, C.; Hinnah, B.; Tóth, A.; Kovács, L.S.; Jüngling, A.; Zákány, R.; Reglődi, D.; Juhász, T. Synergistic Crosstalk of PACAP and Notch Signaling Pathways in Bone Development. Int. J. Mol. Sci. 2025, 26, 5088. https://doi.org/10.3390/ijms26115088
Szegeczki V, Pálfi A, Fillér C, Hinnah B, Tóth A, Kovács LS, Jüngling A, Zákány R, Reglődi D, Juhász T. Synergistic Crosstalk of PACAP and Notch Signaling Pathways in Bone Development. International Journal of Molecular Sciences. 2025; 26(11):5088. https://doi.org/10.3390/ijms26115088
Chicago/Turabian StyleSzegeczki, Vince, Andrea Pálfi, Csaba Fillér, Barbara Hinnah, Anna Tóth, Lili Sarolta Kovács, Adél Jüngling, Róza Zákány, Dóra Reglődi, and Tamás Juhász. 2025. "Synergistic Crosstalk of PACAP and Notch Signaling Pathways in Bone Development" International Journal of Molecular Sciences 26, no. 11: 5088. https://doi.org/10.3390/ijms26115088
APA StyleSzegeczki, V., Pálfi, A., Fillér, C., Hinnah, B., Tóth, A., Kovács, L. S., Jüngling, A., Zákány, R., Reglődi, D., & Juhász, T. (2025). Synergistic Crosstalk of PACAP and Notch Signaling Pathways in Bone Development. International Journal of Molecular Sciences, 26(11), 5088. https://doi.org/10.3390/ijms26115088