Correction of hypophosphatasia-associated mineralization deficiencies in vitro by phosphate/pyrophosphate modulation in periodontal ligament cells.
BACKGROUND
Mutations in the liver/bone/kidney alkaline phosphatase (ALPL) gene in hypophosphatasia (HPP) reduce the function of tissue non-specific alkaline phosphatase (ALP), resulting in increased pyrophosphate (PP(i)) and a severe deficiency in acellular cementum. We hypothesize that exogenous phosphate (P(i)) would rescue the in vitro mineralization capacity of periodontal ligament (PDL) cells harvested from HPP-diagnosed patients, by correcting the P(i)/PP(i) ratio and modulating expression of genes involved with P(i)/PP(i) metabolism.


METHODS
Ex vivo and in vitro analyses were used to identify mechanisms involved in HPP-associated PDL/tooth root deficiencies. Constitutive expression of PP(i)-associated genes was contrasted in PDL versus pulp tissues obtained from healthy individuals. Primary PDL cell cultures from patients with HPP (monozygotic twin males) were established to assay ALP activity, in vitro mineralization, and gene expression. Exogenous P(i) was provided to correct the P(i)/PP(i) ratio.


RESULTS
PDL tissues obtained from healthy individuals featured higher basal expression of key PP(i) regulators, genes ALPL, progressive ankylosis protein (ANKH), and ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), versus paired pulp tissues. A novel ALPL mutation was identified in the twin patients with HPP enrolled in this study. Compared to controls, HPP-PDL cells exhibited significantly reduced ALP and mineralizing capacity, which were rescued by addition of 1 mM P(i). Dysregulated expression of PP(i) regulatory genes ALPL, ANKH, and ENPP1 was also corrected by adding P(i), although other matrix markers evaluated in our study remained downregulated.


CONCLUSIONS
These findings underscore the importance of controlling the P(i)/PP(i) ratio toward development of a functional periodontal apparatus and support P(i)/PP(i) imbalance as the etiology of HPP-associated cementum defects.
Department of Prosthodontics and Periodontics, Campinas State University, Piracicaba, São Paulo, Brazil.
Thaisângela L
TL
Rodrigues
Brian L
BL
Foster
Karina G
KG
Silverio
Luciane
L
Martins
Marcio Z
MZ
Casati
Enilson A
EA
Sallum
Martha J
MJ
Somerman
Francisco H
FH
Nociti
D000293:Adolescent; D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D016022:Case-Control Studies; D004252:DNA Mutational Analysis; D003739:Dental Cementum / Q000201:enzymology / Q000473:pathology; D003782:Dental Pulp / Q000166:cytology / Q000201:enzymology; D011756:Diphosphates / Q000378:metabolism*; D004200:Diseases in Twins / Q000235:genetics; D005260:Female; D020869:Gene Expression Profiling; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D007014:Hypophosphatasia / Q000201:enzymology* / Q000235:genetics*; D008297:Male; D010513:Periodontal Ligament / Q000166:cytology / Q000201:enzymology*; D028061:Phosphate Transport Proteins / Q000235:genetics / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism*; D010727:Phosphoric Diester Hydrolases / Q000235:genetics / Q000378:metabolism; D061251:Primary Cell Culture; D011755:Pyrophosphatases / Q000235:genetics / Q000378:metabolism; D014074:Tooth Calcification / Q000235:genetics; D055815:Young Adult
C498770:ANKH protein, human; D011756:Diphosphates; D028061:Phosphate Transport Proteins; D010710:Phosphates; C107241:diphosphoric acid; C548267:ALPL protein, human; D000469:Alkaline Phosphatase; D010727:Phosphoric Diester Hydrolases; C065962:ectonucleotide pyrophosphatase phosphodiesterase 1; D011755:Pyrophosphatases