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To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2005.10.002 Byline: Valentina Godovikova (a), Xiu-Rong Li (a), Thomas L. Saunders (b), Helena H. Ritchie (a) Keywords: Rat DSP-PP promoter; LacZ activity; cis-elements; Developmental clock; Tooth; Bone; Kidney Abstract: Dentin sialoprotein (DSP) and phosphophoryn (PP) are two major dentin noncollagenous proteins that are encoded on a single DSP-PP transcript whose expression is tightly regulated during tooth dentinogenesis. The recent identification of this gene transcript in other tissues, including inner ear and jaw tissue, suggests that DSP and PP may have pleiotropic effects on other organs besides teeth. To identify candidate regulatory elements that control DSP-PP temporal and spatial expression, we constructed a -5 kb upstream region rat DSP-PP promoter into the [beta]-galactosidase expression vector pnLacF plasmid and used this construct to prepare DSP-PP-LacZ transgenic mice. Multiple mouse tissues including teeth, bone, and kidney obtained from the six resulting transgenic mouse lines displayed strong LacZ activity. This spatial distribution was confirmed in several of these tissues by in situ hybridization studies. LacZ activity was transiently expressed in preameloblasts and continuously expressed in odontoblasts demonstrating that this -5 kb rat promoter-dependent LacZ expression mimics reported DSP-PP mRNA expression patterns. Interestingly, this -5 kb rat promoter construct drives LacZ expression according to the rat developmental clock. Based on identified transcription factors present in this -5 kb promoter region, we have identified several probable cis-regulatory modules whose interaction with one another could account for the spatial and temporal distribution of DSP-PP transcripts in developing tissues. Author Affiliation: (a) Department of Cariology, Restorative Sciences and Endodontics, University of Michigan School of Dentistry, Room 2393 Dental Building, 1011 N. University Ave., Ann Arbor, MI 48109-1078, USA (b) Department of Internal Medicine, Division of Molecular Medicine and Genetics, University of Michigan Medical School, Ann Arbor, MI 48109, USA Article History: Received 17 March 2005; Revised 19 August 2005; Accepted 6 October 2005

Dentin sialoprotein (DSP) and phosphophoryns (DPP) are major dentin-specific non-collagenous proteins and are synthesized by odontoblasts. DPP are extremely acidic, rich in aspartic acid and serine, possess a high affinity for calcium and collagen, and are believed to function in dentin mineralization. Whereas DSP and DPP are the products of a single gene in mouse and rat, an analogous human gene has not been described. Using RT-PCR based cloning strategies, we have cloned human DPP cDNA from immature molar root total RNA. The open reading frame of this human DPP cDNA comprises 2364 bp encoding 788 amino acids rich in serine (58%), aspartic acid (26%) and asparagine (9%). These are mostly arranged as (DSS)[subn](n=1-16). DS and NSS motifs. The N-terminal sequence (DDP) matches that obtained from human DPP extracted from the roots of immature teeth. The core protein of this human DPP was calculated to have a molecular weight of 76.906 Da and a net charge of -206 with an isoelectric point of 2.65. Of the serine residues, 53% can potentially be phosphorylated by casein kinases I and II. Thus, this newly cloned human cDNA, which encodes a protein with characteristics similar to rat and mouse DPP, is identified as a human DPP. [ABSTRACT FROM AUTHOR]

Phosphophoryn (PP) and dentin sialoprotein (DSP) are two of the most abundant dentin matrix non-collagenous proteins, and are derived from dentin sialoprotein-phosphophoryn (DSP-PP) mRNA. Mutations in the DSP-PP gene are linked to dentinogenesis imperfecta II and III. Previously, we reported transient DSP-PP expression in preameloblast cells first, followed by co-expression in preameloblasts and young odontoblasts, and finally sustained expression in odontoblasts. This phenomenon raised the possibility that DSP/PP proteins secreted by preameloblasts might promote dental pulp cell migration toward the dental pulp border and promote dental pulp cell differentiation. To examine the effects of DSP/PP proteins on dental pulp cell development, we investigated:(1) native PP effects on dental pulpcell migration and matrix protein expression; and (2) recombinant DSP/PP protein effects on cell proliferation and differentiation. We found that PP promoted cell migration and the expression of high levels of Col type I and PP in dental pulp cells. The addition of recombinant DSP/PP proteins affected cell proliferation and differentiation in a dental pulp cell line. These findings strongly suggest that DSP/PP may modulate cell migration, cell proliferation and differentiation, thus leading to dentin formation. DSP/PP protein may be useful clinically for pulp tissue regeneration. [ABSTRACT FROM AUTHOR]

Objective To compare the expression patterns of two multiple transcripts derived from DSP-PP gene during tooth development. One is DSP-only transcript (i.e. does not encode PP) and the other is DSP-PP 523 transcript, a main DSP-PP transcript . Design Unique antisense and sense riboprobes were generated from DSP-only and DSPPP 523 cDNAs for in situ studies to examine DSP-only and DSP-PP 523 transcript expression in developing molars. Paraffin-embedded sections (5–7 μ m) from embryonic 20 day, postnatal 2, 3 and 6 days were deparaffined and hydrated. Tissues were prehybridized, then hybridized with DSP-only and DSP-PP 523 anti-sense (AS) or sense (S) Digoxigenin labeled-riboprobes overnight, and washed. Anti-Digoxigenin antibodies conjugated to alkaline phosphatase were used to detect the presence of bound riboprobes by color reaction with NBT/BCIP. Stro-1 antibody was used for immunohistochemical analysis of Stro-1 protein expression in rat molars. Results We found that unlike the DSP-PP 523 transcript, the DSP-only transcript does not express in the entire polarized mature odontoblasts but is expressed in the areas subjacent to the mature odontoblast layer. In addition, DSP-only transcript is expressed in the dental pulp. Interestingly, Stro-1 protein, a stem cell marker, was also identified in the areas subjacentto odontoblasts and in dental pulp. Conclusion Differential expression of DSP-only and DSP-PP 523 transcripts suggest that these two kinds of transcripts may play different roles during dentinogenesis. DSP-PP 523 transcript is expressed in mature odontoblasts, which actively participates in dentin formation. DSP-only transcript might have a different function. [ABSTRACT FROM AUTHOR]

Dentin Sialoprotein (DSP) and Phosphophoryn (PP), acidic proteins critical to dentin mineralization, are translated from a single transcript as a DSP-PP precursor that undergoes proteolytic processing to generate DSP and PP. Because of the difficulty in obtaining large amounts of DSP-PP, we used a Sf9-baculovirus expression system to yield large amounts of DSP-PP 240 recombinant protein, a variant form of rat DSP-PP. Previous evidence stated that DSP-PP 240 produced by baculovirus-infected Sf9 cells can be cleaved accurately into DSP and PP by the endogenous processing enzyme Sf9 Tolloid-Related 1 (TLR1), a homolog for human Bone Morphogenic Protein 1 (BMP1) and the proposed protease to cleave DSP-PP in human. It was also discovered via mass spectrometric analysis that the specific cleavage occurred at the site: SMQG 447 |D 448 DPN. In addition, we reported that any mutations within the DSP-PP P4 to P4'cleavage site can block, impair or accelerate DSP-PP cleavage, which suggest that its BMP1 cleavage site is highly conserved to regulate its cleavage efficiency. Furthermore, mutations outside of the DSP-PP P4 to P4' cleavage site can impair or accelerate DSP-PP cleavage. Here, we investigate the role of the highly conserved DSPP C-terminal region in DSP-PP cleavage. We generated a DSP-PP C-terminal mutation by substituting the terminal two aspartate residues for two histamine residues (DD/HH-DSP-PP). To test the impact of the DD/HH mutant on DSP-PP cleavage, we used the Sf9 expression system's endogenous TLR1 and exogenous recombinant BMP1. The DD/HH mutation was shown to block DD/HH-DSP-PP cleavage into DSP and PP by both TLR1 and BMP1 in vitro . Taken together, these evidence supports our hypothesis that the C-terminal peptides D 686 D 687 actively participates in controlling DSP-PP cleavage and that C-terminal conservation is critical for proper DSP-PP precursor cleavage by TLR1 and BMP1.