Dentin matrix protein 1 (DMP1) is essential to odontogenesis. Its mutations in human subjects lead to dental problems such as dental deformities, hypomineralization and periodontal impairment. Primarily, DMP1 is considered as an extracellular matrix protein that promotes hydroxyapatite formation and activates intracellular signaling pathway via interacting with αvβ3 integrin. Recent in vitro studies suggested that DMP1 might also act as a transcription factor. In this study, we examined whether full-length DMP1 could function as a transcription factor in the nucleus and regulate odontogenesis in vivo . We first demonstrated that a patient with the DMP1 M1V mutation, which presumably causes a loss of the secretory DMP1 but does not affect the nuclear translocation of DMP1, shows a typical rachitic tooth defect. Furthermore, we generated transgenic mice expressing ~(NLS)DMP1, in which the endoplasmic reticulum (ER) entry signal sequence of DMP1 was replaced by a nuclear localization signal (NLS) sequence, under the control of a 3.6?kb rat type I collagen promoter plus a 1.6?kb intron 1. We then crossbred the ~(NLS)DMP1 transgenic mice with Dmp1 null mice to express the ~(NLS)DMP1 in Dmp1 -deficient genetic background. Although immunohistochemistry demonstrated that ~(NLS)DMP1 was localized in the nuclei of the preodontoblasts and odontoblasts, the histological, morphological and biochemical analyses showed that it failed to rescue the dental and periodontal defects as well as the delayed tooth eruption in Dmp1 null mice. These data suggest that the full-length DMP1 plays no apparent role in the nucleus during odontogenesis. Keywords: autosomal recessive hypophosphatemic rickets, dentin matrix protein 1, development, odontoblast, odontogenesisIntroductionDentin matrix protein 1 (DMP1), a member of the small integrin-binding ligand, N-linked glycoproteins family,~(1) plays essential roles in osteogenesis and odontogenesis. DMP1 mutations in humans result in autosomal recessive hypophosphatemic rickets-1 (ARHR1; OMIM#241520), characterized by rickets/osteomalacia, hypophosphatemia and elevated circulating fibroblast growth factor 23.~(2) Dmp1 null mice also develop an ARHR-like phenotype.~(2) The teeth of Dmp1 null mice display enlarged pulp cavities and root canals, increased thickness of the predentin zone with a reduced dentin wall, hypomineralization of dentin, abnormalities in the ultrastructure of dentinal tubules, delayed development of the third molar, breakdown of periodontal structures and other consequences.~(3,4,5,6)Several lines of evidence support the finding that DMP1 acts as an extracellular matrix protein. First, the initial 16 amino-acid residues of DMP1 deduced from its cDNA is a typical endoplasmic reticulum (ER) signal sequence, and this protein is found abundantly in the bone and dentinal matrix.~(7,8) Second, the DMP1 protein sequence contains some specific acidic clusters that control the formation of oriented calcium phosphate crystals.~(9,10) Gajjeraman and others~(11,12,13) further confirmed that both the full-length DMP1 and the COOH-terminal fragment could accelerate the nucleation of hydroxyapatite crystals. Lastly, studies from our lab and others demonstrated that DMP1 has the ability to activate the extracellular signal-regulated kinase/mitogen-activated protein kinase pathways via the αvβ3 integrin, leading to the translocation of phosphorylated cellular Jun N-terminal kinase (JNK) into the nucleus and the concomitant upregulation of transcriptional activation by phosphorylated c-Jun.~(3,14,15)However, in vitro studies indicated that DMP1 also might function as a transcription factor in the nucleus. George et al .~(16) first reported that the DMP1 protein sequence contained a functional nuclear localization signal (NLS) sequence, which was thought to be responsible for the nuclear import of full-length DMP1 in pre-odontoblast-like cell. They further demonstrated that the extracellular full-length DMP1 could be internalized via endocytosis mediated by glucose-regulated protein-78, an ER chaperone protein found on the plasma membrane of pre-odontoblasts and subsequently transported into the nucleus. Once in the nucleus, DMP1 regulates the transcription of odontoblast-specific genes such as DSPP .~(16,17) These investigators proposed that during the maturation of the pre-odontoblast/odontoblast, this nuclear DMP1 would be phosphorylated by casein kinase II, leading to its exportation into the extracellular matrix where it promotes hydroxyapatite formation.~(16) However, the role of nuclear DMP1 has been challenged by the identification of several patients who presumably have normal nuclear localization of DMP1 due to a biallelic nucleotide substitution in the DMP1 start codon (ATG to GTG, or A→G), but who display hypophosphatemic rickets.~(2) Therefore, whether DMP1 can enter the nucleus and function as a transcription factor in vivo needs to be further studied.This study was aimed
展开▼
