Sclerostin antibody improves alveolar bone quality in the Hyp mouse model of X-linked hypophosphatemia (XLH).

X-linked hypophosphatemia (XLH) is a rare disease of elevated fibroblast growth factor 23 (FGF23) production that leads to hypophosphatemia and impaired mineralization of bone and teeth. The clinical manifestations of XLH include a high prevalence of dental abscesses and periodontal disease, likely driven by poorly formed structures of the dentoalveolar complex, including the alveolar bone, cementum, dentin, and periodontal ligament. Our previous studies have demonstrated that sclerostin antibody (Scl-Ab) treatment improves phosphate homeostasis, and increases long bone mass, strength, and mineralization in the Hyp mouse model of XLH. In the current study, we investigated whether Scl-Ab impacts the dentoalveolar structures of Hyp mice. Male and female wild-type and Hyp littermates were injected with 25 mg·kg-1 of vehicle or Scl-Ab twice weekly beginning at 12 weeks of age and euthanized at 20 weeks of age. Scl-Ab increased alveolar bone mass in both male and female mice and alveolar tissue mineral density in the male mice. The positive effects of Scl-Ab were consistent with an increase in the fraction of active (nonphosphorylated) ß-catenin, dentin matrix protein 1 (DMP1) and osteopontin stained alveolar osteocytes. Scl-Ab had no effect on the mass and mineralization of dentin, enamel, acellular or cellular cementum. There was a nonsignificant trend toward increased periodontal ligament (PDL) attachment fraction within the Hyp mice. Additional PDL fiber structural parameters were not affected by Scl-Ab. The current study demonstrates that Scl-Ab can improve alveolar bone in adult Hyp mice.

Sclerostin antibody improves alveolar bone quality in the Hyp mouse model of X-Linked Hypophosphatemia (XLH).

X-linked hypophosphatemia (XLH) is a rare disease of elevated fibroblast growth factor 23 (FGF23) production that leads to hypophosphatemia and poor mineralization of bone and teeth. The clinical manifestations of XLH include a high prevalence of dental abscesses, likely driven by poorly formed structures of the dentoalveolar complex, including the alveolar bone, cementum, dentin, and periodontal ligament. Our previous studies have demonstrated that sclerostin antibody (Scl-Ab) treatment improves phosphate homeostasis, and increases bone mass, strength and mineralization in the Hyp mouse model of XLH. In the current study, we investigated whether Scl-Ab impacts the dentoalveolar structures of Hyp mice. Male and female wild-type and Hyp littermates were injected with 25 mg/kg of vehicle or Scl-Ab twice weekly beginning at 12 weeks of age and euthanized at 20 weeks of age. Scl-Ab increased alveolar bone mass in both male and female mice and alveolar tissue mineral density in the male mice. The positive effects of Scl-Ab were consistent with an increase in the fraction of active (non-phosphorylated) ß-catenin stained alveolar osteocytes. Scl-Ab had no effect on mineralized tissues of the tooth - dentin, enamel, acellular and cellular cementum. There was a non-significant trend toward increased periodontal ligament (PDL) attachment fraction within the Hyp mice. Additional PDL fibral structural parameters were not affected by Scl-Ab. The current study demonstrates that Scl-Ab can improve alveolar bone in the Hyp mouse model of XLH.

Wnt pathway inhibitors are upregulated in XLH dental pulp cells in response to odontogenic differentiation.

X-linked hypophosphatemia (XLH) represents the most common form of familial hypophosphatemia. Although significant advances have been made in the treatment of bone pathology, patients undergoing therapy continue to experience significantly decreased oral health-related quality of life. The following study addresses this persistent oral disease by further investigating the effect of DMP1 expression on the differentiation of XLH dental pulp cells. Dental pulp cells were isolated from the third molars of XLH and healthy controls and stable transduction of full-length human DMP1 were achieved. RNA sequencing was performed to evaluate the genetic changes following the induction of odontogenic differentiation. RNAseq data shows the upregulation of inhibitors of the canonical Wnt pathway in XLH cells, while constitutive expression of full-length DMP1 in XLH cells reversed this effect during odontogenic differentiation. These results imply that inhibition of the canonical Wnt pathway may contribute to the pathophysiology of XLH and suggest a new therapeutic strategy for the management of oral disease.

STIM1 a calcium sensor promotes the assembly of an ECM that contains Extracellular vesicles and factors that modulate mineralization.

Osteoblasts and odontoblasts, are non-excitable cells and facilitate mass calcium transport during matrix mineralization. A sophisticated Ca2+ sensing mechanism is used to maintain Ca2+ homeostasis. STIM1 (Stromal interaction molecule 1) is a calcium sensor protein localized in the ER membrane and maintains calcium homeostasis by initiating the store-operated Ca2+ entry (SOCE) process, following store depletion. The role of STIM1 in dentin mineralization is yet to be elucidated. Therefore, transgenic DPSCs were generated in which overexpression or knockdown of STIM1 was achieved to study its function in matrix mineralization. Gene expression analysis and Alizarin Red staining assay demonstrated upregulation of genes involved in odontogenic differentiation and matrix mineralization with increased calcium deposition with STIM1 overexpression. Topology of the ECM examined by Field Emission Scanning Electron Microscopy (FESEM) showed the presence of large amounts of extracellular microvesicles with mineral deposits. Interestingly, silencing STIM1 resulted in fewer vesicles and less mineral deposits in the ECM. Analysis of the dentin-pulp complex of STIM1- deficient mice by micro-CT show reduced dentin thickness, malformed and highly porous alveolar bone, suggesting a cell intrinsic role for STIM1 in dentin mineralization. Confocal microscopy showed that DMP1-mediated depletion of store Ca2+ resulted in aggregation or "puncta-formation" of STIM1 at the plasma membrane indicative of a gating arrangement with Orai1 for Ca2+ influx. Together, our data provide evidence for an important role for STIM1 in dentin and alveolar bone mineralization by influencing intracellular Ca2+ oscillations that could provide signals for a wide array of cellular functions. STATEMENT OF SIGNIFICANCE: Calcium signaling and transport are fundamental to bone and dentin mineralization. Osteoblasts and odontoblasts transport large amounts of Ca2+ to the extracellular matrix. These cells maintain calcium homeostasis by spatially distributed calcium pumps and channels at the plasma membrane. STIM1 an ER Ca2+ sensor protein is an important component of the store-operated calcium entry (SOCE) process. In this study, we examined the role of STIM1 during the differentiation of dental pulp stem cells into functional odontoblasts and formation of mineralized dentin matrix. Stimulation of these cells with DMP1, a key regulatory protein in matrix mineralization, stimulates STIM1-mediated release of ER Ca2+ and SOCE activation. Silencing of STIM1 impairs signaling events, release of exosomes containing matrix proteins and matrix mineralization.

Disrupted Protein Expression and Altered Proteolytic Events in Hypophosphatemic Dentin Can Be Rescued by Dentin Matrix Protein 1.

Dentin, one of the four mineralized tissues of the craniofacial complex, forms sequentially from the deposition of an organic matrix to the nucleation of an inorganic phase within the matrix scaffold. Several promoters and inhibitors of mineralization support and regulate mineral nucleation. Clinical and experimental evidence suggest that dentin matrix protein 1 (DMP1) and phosphate-regulating neutral endopeptidase (PHEX) cooperate and are necessary for the formation of a cohesive dentin layer. The following study investigates the effect of PHEX loss-of-function on dentin matrix formation preceding mineralization. Using the Hyp mouse, an animal model for X-linked hypophosphatemia (XLH), we identified an irregular distribution of dentin extracellular matrix proteins. Likewise, dental pulp stem cells (DPSCs) from XLH patients exhibited altered proteolytic events with disrupted extracellular matrix deposition. Further differentiation assays demonstrated that XLH DPSCs exhibited impaired matrix mineralization. Overexpression of DMP1 in XLH DPSCs restored the irregular protein processing patterns to near-physiological levels. Our results support the hypothesis that hypophosphatemia resulting from PHEX loss-of-function affects the integrity of the organization of the dentin matrix and suggests that exogenous DMP1 can restore physiological processing of matrix proteins, in addition to its canonical role in mineralization.

Establishment of Stable Cell Lines from Primary Human Dental Pulp Stem Cells.

This protocol is for the isolation of primary human dental pulp stem cells (DPSCs) from adult extracted molars and for the generation of high-titer lentivirus for in vitro infection of the DPSCs. Stable cell lines of dental pulp stem cells are generated, maintained in culture, and used for subsequent experiments.

Epstein Barr virus Latent Membrane Protein-1 enhances dendritic cell therapy lymph node migration, activation, and IL-12 secretion.

Dendritic cells (DC) are a promising cell type for cancer vaccines due to their high immunostimulatory capacity. However, improper maturation of DC prior to treatment may account for the limited efficacy of DC vaccine clinical trials. Latent Membrane Protein-1 (LMP1) of Epstein-Barr virus was examined for its ability to mature and activate DC as a gene-based molecular adjuvant for DC vaccines. DC were transduced with an adenovirus 5 vector (Ad5) expressing LMP1 under the control of a Tet-inducible promoter. Ad5-LMP1 was found to mature and activate both human and mouse DC. LMP1 enhanced in vitro migration of DC toward CCL19, as well as in vivo migration of DC to the inguinal lymph nodes of mice following intradermal injection. LMP1-transduced DC increased T cell proliferation in a Pmel-1 adoptive transfer model and enhanced survival in B16-F10 melanoma models. LMP1-DC also enhanced protection in a vaccinia-Gag viral challenge assay. LMP1 induced high levels of IL-12p70 secretion in mouse DC when compared to standard maturation protocols. Importantly, LMP1-transduced human DC retained the capacity to secrete IL-12p70 and TNF in response to DC restimulation. In contrast, DC matured with Monocyte Conditioned Media-Mimic cocktail (Mimic) were impaired in IL-12p70 secretion following restimulation. Overall, LMP1 matured and activated DC, induced migration to the lymph node, and generated high levels of IL-12p70 in a murine model. We propose LMP1 as a promising molecular adjuvant for DC vaccines.

Constitutively Active MAVS Inhibits HIV-1 Replication via Type I Interferon Secretion and Induction of HIV-1 Restriction Factors.

Type I interferon is known to inhibit HIV-1 replication through the induction of interferon stimulated genes (ISG), including a number of HIV-1 restriction factors. To better understand interferon-mediated HIV-1 restriction, we constructed a constitutively active form of the RIG-I adapter protein MAVS. Constitutive MAVS was generated by fusion of full length MAVS to a truncated form of the Epstein Barr virus protein LMP1 (ΔLMP1). Supernatant from ΔLMP1-MAVS-transfected 293T cells contained high levels of type I interferons and inhibited HIV replication in both TZM-bl and primary human CD4+ T cells. Supernatant from ΔLMP1-MAVS-transfected 293T cells also inhibited replication of VSV-G pseudotyped single cycle SIV in TZM-bl cells, suggesting restriction was post-entry and common to both HIV and SIV. Gene array analysis of ΔLMP1-MAVS-transfected 293T cells and trans-activated CD4+ T cells showed significant upregulation of ISG, including previously characterized HIV restriction factors Viperin, Tetherin, MxB, and ISG56. Interferon blockade studies implicated interferon-beta in this response. In addition to direct viral inhibition, ΔLMP1-MAVS markedly enhanced secretion of IFN-ß and IL-12p70 by dendritic cells and the activation and maturation of dendritic cells. Based on this immunostimulatory activity, an adenoviral vector (Ad5) expressing ΔLMP1-MAVS was tested as a molecular adjuvant in an HIV vaccine mouse model. Ad5-Gag antigen combined with Ad5-ΔLMP1-MAVS enhanced control of vaccinia-gag replication in a mouse challenge model, with 4/5 animals showing undetectable virus following challenge. Overall, ΔLMP1-MAVS is a promising reagent to inhibit HIV-1 replication in infected tissues and enhance vaccine-mediated immune responses, while avoiding toxicity associated with systemic type I interferon administration.