Hypophosphatasia-associated Deficiencies in Mineralization and Gene Expression in Cultured Dental Pulp Cells Obtained from Human Teeth

Abstract

Introduction Mutations in the gene ALPL in hypophosphatasia (HPP) reduce the function of tissue nonspecific alkaline phosphatase, and the resulting increase in pyrophosphate (PPi) contributes to bone and tooth mineralization defects by inhibiting physiologic calcium-phosphate (Pi) precipitation. Although periodontal phenotypes are well documented, pulp/dentin abnormalities have been suggested in the clinical literature although reports are variable and underlying mechanisms remains unclear. In vitro analyses were used to identify mechanisms involved in HPP-associated pulp/dentin phenotypes. Methods Primary pulp cells cultured from HPP subjects were established to assay alkaline phosphatase (ALP) activity, mineralization, and gene expression compared with cells from healthy controls. Exogenous Pi was provided to the correct Pi/PPi ratio in cell culture. Results HPP cells exhibited significantly reduced ALP activity (by 50%) and mineral nodule formation (by 60%) compared with the controls. The expression of PPi regulatory genes was altered in HPP pulp cells, including reduction in the progressive ankylosis gene (ANKH) and increased ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1). Odontoblast marker gene expression was disrupted in HPP cells, including reduced osteopontin (OPN), dentin matrix protein 1 (DMP1), dentin sialophosphoprotein (DSPP), and matrix extracellular phosphoprotein (MEPE). The addition of Pi provided a corrective measure for mineralization and partially rescued the expression of some genes although cells retained altered messenger RNA levels for PPi-associated genes. Conclusions These studies suggest that under HPP conditions pulp cells have the compromised ability to mineralize and feature a disrupted odontoblast profile, providing a first step toward understanding the molecular mechanisms for dentin phenotypes observed in HPP.

Novel ALPL genetic alteration associated with an odontohypophosphatasia phenotype

“…Firstly, there was a heterozygous substitution C → T at position 454-nt, leading to the substitution of cysteine for arginine 152 (p.R152C), an alteration previously described in this family and found in the mother of the probands [18-20]. The p.R152C (c.454C>T) missense mutation, associated to other mutation p.R184W (c.550C>T), has been reported previously in a case of adult HPP (p.[R152C];[R184W]; SESEP – University of Versailles- Saint Quentin), whereas a mutation affecting the same codon p.R152H (c.455G>A) has been described in the mother of a patient with a lethal clinical form of HPP [28].…”

“…We have demonstrated previously that primary periodontal ligament and dental pulp cells harvested from the same probands exhibited increased ALPL mRNA, whereas residual ALP activity and ability to promote mineralization in vitro were markedly reduced (40% and 50%, respectively) [18, 20]. Here, we showed that increased ALPL mRNA concentration in these cells does not result in increased protein expression, suggesting that regulatory mechanisms, such as the ER quality-control system, may be intervening and resulting in defective intracellular transport of mutant TNAP, likely due to retention of a fraction of mutant TNAP molecules in the intracellular compartment.…”

“…Physical examination and radiographs (long bones, joints, and skull) showed age-appropriate growth and development. Routine laboratory testing revealed low serum ALP activity for both probands (patient A: 62 U/L, patient B: 63 U/L; normal range for children 151-471 U/L), while serum phosphate and calcium levels remained within normal limits [18-20]. …”

“…Primary dental pulp cells from both probands A and B (genotype: p.[N440del];[R152C]) and four control individuals (native TNAP) were obtained as previously reported [20]. Briefly, extracted teeth were placed in biopsy media, and pulp was harvested by cracking open teeth using a dental chisel and hammer and removing soft tissue with sterile forceps.…”

Tooth root dentin mineralization defects in a mouse model of hypophosphatasia

“…Whether these cell changes contribute to dentin defects or arise as a result of them is under investigation. Recently we reported that pulp cells from HPP-diagnosed subjects were unable to acquire an odontoblast phenotype and to promote mineralization in vitro , which was partially restored by phosphate/PP i modulation (42). …”