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Involvement of P2X7 Receptors in the Osteogenic Differentiation of Mesenchymal Stromal/Stem Cells Derived from Human Subcutaneous Adipose Tissue

  • Marzia Carluccio
  • Mariachiara Zuccarini
  • Sihana Ziberi
  • Patricia Giuliani
  • Caterina Morabito
  • Maria A. Mariggiò
  • Maria Teresa Lonardo
  • Elena Adinolfi
  • Elisa Orioli
  • Patrizia Di Iorio
  • Francesco Caciagli
  • Renata CiccarelliEmail author
Article
  • 119 Downloads

Abstract

The ionotropic P2X7 receptor (P2X7R) is involved in bone homeostasis but its role in osteogenesis is controversial. Thus, we investigated the expression of P2X7R and the effects exerted by its modulation in mesenchymal stromal cells from human subcutaneous adipose tissue (S-ASCs), which have potential therapeutic application in bone regenerative medicine. We found that undifferentiated S-ASCs expressed P2X7R and its functional splice variants P2X7AR and P2X7BR. Cell stimulation by P2X7R agonist BzATP (100 μM) neither modified proliferation nor caused membrane pore opening while increasing intracellular Ca2+ levels and migration. The P2X7R antagonist A438079 reversed these effects. However, 25-100 μM BzATP, administered to S-ASCs undergoing osteogenic differentiation, dose-dependently decreased extracellular matrix mineralization and expression of osteogenic transcription factors Runx2, alkaline phosphatase and osteopontin. These effects were not coupled to cell proliferation reduction or to cell death increase, but were associated to decrease in P2X7AR and P2X7BR expression. In contrast, expression of P2X7R, especially P2X7BR isoform, significantly increased during the osteogenic process. Noteworthy, the antagonist A438079, administered alone, at first restrained cell differentiation, enhancing it later. Accordingly, A438079 reversed BzATP effects only in the second phase of S-ASCs osteogenic differentiation. Apyrase, a diphosphohydrolase converting ATP/ADP into AMP, showed a similar behavior. Altogether, findings related to A438079 or apyrase effects suggest an earlier and prevailing pro-osteogenic activity by endogenous ATP and a later one by adenosine derived from endogenous ATP metabolism. Conversely, P2X7R pharmacological stimulation by BzATP, mimicking the effects of high ATP levels occurring during tissue injuries, depressed receptor expression/activity impairing MSC osteogenic differentiation.

Keywords

Subcutaneous adipose tissue-derived stromal stem cells Osteogenic differentiation Regenerative medicine ATP analogues P2X7 receptors 

Abbreviations

A438079

3-[[5-(2,3-Dichlorophenyl)-1H-tetrazol-1-yl]methyl]pyridine hydrochloride

ADO

Adnosine

ALP

Alkaline phosphatase

ARS

Alizarin Red S

BzATP

2′(3′)-O-(4-benzoylbenzoyl)-ATP

FBS

fetal bovine serum

MSCs

mesenchymal stromal/stem cells

NF279

8,8′-[carbonylbis(imino-4,1-phenylenecarbonylimino-4,1-phenylenecarbonylimino)]bis-1,3,5-naphthalenetrisulfonic acid hexasodium salt

OPN

osteopontin

P2X7R

P2X7 receptors

P2X1R

P2X1 receptors

P2X3R

P2X3 receptors

P2X7AR

P2X7 receptor splice variant A

P2X7BR

P2X7 receptor splice variant B

qRT-PCR

quantitative real time polymerase chain reaction

RUNX2

Runt-related transcription factor 2

S-ASCs

subcutaneous adipose tissue-derived stromal stem cells.

Notes

Authors’ Contributions

FC, PDI and RC conceived and designed the experimental work, assembled and supervised the overall project, analyzed and interpreted the data and finalized the manuscript. MC initiated the project and along with SZ, MZ, PG, CM, ML, MAM, EA and EO performed the experimental work, assembled and analyzed the data, drafted/revised the manuscript. All authors have read and approved the manuscript for publication.

Funding

This work was supported by funds for research to RC and PDI from the University of Chieti-Pescara.

Compliance with Ethical Standards

Consent for Publication

This manuscript has been approved by all authors and is solely the work of the authors named.

Competing Interests

The authors declare that they have no competing interests.

Supplementary material

12015_2019_9883_MOESM1_ESM.pptx (326 kb)
Expression of P2X1R and P2X3R in S-ASCs along their osteogenic differentiation in the presence or absence of BzATP. Protein levels of P2X1R and P2X3R were determined by Western blot analysis (60 μg of proteins were loaded per lane). Immunoblots were obtained by exposing membranes to two antibodies, recognizing proteins (the monomeric subunit of each receptor) at 50 KDa (A) and about 60 KDa (B), respectively. Subsequently, immunoblots were reprobed with an antibody against β actin, to verify equal sample loading, and quantified by densitometric analysis, the values of which, normalized to β actin, are reported as such in the two lower histograms, whereas in the upper histograms they are reported assuming the values of cells at the day 0 equal to 1. All values are the mean ± SEM of three independent experiments with very similar results. *P < 0.05, **P < 0.01, ***P < 0.001: statistical significance vs. S-ASCs at the day 0 (in the upper histograms) or vs. cells grown in osteogenic medium for 7 days (in the lower histograms); #P < 0.05, ###P < 0.001: statistical significance of values measured in cells exposed to BzATP vs the corresponding untreated cells grown in osteogenic medium (one-way ANOVA plus Dunnett’s test). (PPTX 326 kb)

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Marzia Carluccio
    • 1
    • 2
    • 3
  • Mariachiara Zuccarini
    • 1
    • 2
  • Sihana Ziberi
    • 1
    • 2
    • 3
  • Patricia Giuliani
    • 1
    • 2
  • Caterina Morabito
    • 2
    • 3
    • 4
  • Maria A. Mariggiò
    • 2
    • 3
    • 4
  • Maria Teresa Lonardo
    • 5
  • Elena Adinolfi
    • 6
  • Elisa Orioli
    • 6
  • Patrizia Di Iorio
    • 1
    • 2
  • Francesco Caciagli
    • 1
    • 2
  • Renata Ciccarelli
    • 1
    • 2
    • 3
    Email author
  1. 1.Department of Medical, Oral and Biotechnology Sciences, Section of PharmacologyUniversity of Chieti-PescaraChietiItaly
  2. 2.Aging Research Center and Translational MedicineUniversity of Chieti-PescaraChietiItaly
  3. 3.StemTeCh GroupChietiItaly
  4. 4.Department of Neuroscience, Imaging and Clinical SciencesUniversity of Chieti-PescaraChietiItaly
  5. 5.Madre Giuseppina Vanin HospitalRomeItaly
  6. 6.Department of Morphology, Surgery end Experimental MedicineUniversity of FerraraFerraraItaly

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