DSPP contains an IRES element responsible for the translation of dentin phosphophoryn

Abstract

The major phosphoprotein in dentin is the aspartic acid and serine-rich protein called dentin phosphophoryn (DPP). DPP appears to be synthesized as a part of a larger compound protein, dentin sialophosphoprotein (DSPP). DSPP has never been isolated or detected in dentin extracts. It is now evident that DSPP is a chimeric protein composed of 3 parts: dentin sialoprotein (DSP), DPP, and dentin glycoprotein (DGP). Previous reports have suggested that the BMP1 protease is responsible for processing DSPP. However, unequal amounts of these products are present in the dentin matrix. Here, we provide evidence for an internal ribosome entry site in the DSPP gene that directs the synthesis of DPP. This mechanism would account for unequal amounts of intracellular DSP and DPP. The internal ribosomal entry site (IRES) activity varied in different cell types, suggesting the presence of additional regulatory elements during the translational regulation of DPP. Further, we provide evidence that DPP is transported to the extracellular matrix (ECM) through exosomes. Using tissue recombination and lentivirus-mediated gain-of-function approaches, we also demonstrate that DPP is essential for the formation of well-defined tooth structures with mineralized dentin matrix.

Keywords: cell biology; cell differentiation; dentin; extracellular matrix (ECM); matrix biology; odontoblast(s).

Figure 1.

Localization of DSP and DPP in the ECM secreted by T4-4 cells. (A) ECM was isolated from T4-4 cells grown to confluence as stated in “Materials & Methods”. Anti-DSP and anti-DPP antibodies were used to stain the matrix according to published protocols. Red, green, and yellow arrowheads depict DPP, DSP, and areas where both DSP and DPP are present in close proximity. (B) An enlarged portion of the boxed area. (C) Fibronectin (positive control) in the ECM matrix. (The figure can be viewed in color online.)

Figure 2.

Identification of the IRES region in DSPP. (A) Four regions were identified for potential IRES domain in DSPP. They were inserted into the pRF vector between Renilla luciferase and Firefly luciferase using SpeI and EcoRI cutting sites. In the DsRed-GFP vector, the entire DSPP cDNA and a 1302-bp fragment containing DSP and DGP domain were inserted between the Spe1 and BamH1 sites. (B) In silico analysis of the putative ~400-bp region between the end of DSP and the start of DPP. This site was arbitrarily divided into 4 regions. Region 1 contains 72 bp of DSP and 24 bp of DGP and shows the characteristic stem-loop structure; Region 2 consists of 126 bp of DSP and 90 bp of DGP and shows a more complicated secondary structure containing stems and loops; Region 3 is comprised of 126 bp of DSP, and Region 4 consists of 267 bp of entire DGP.

Figure 3.

Functional characterization of the IRES region in DSPP. (A) IRES activities of different constructs in various cell lines. cDNAs (shown in Fig. 2A) were generated by PCR and introduced between the upstream Renilla luciferase cistron and the downstream Firefly luciferase cistron. The plasmids were transfected in HEK293 (1), MC3T3-E1 cells (2), and T4-4 (3) cells. Forty-eight hrs after transient transfection of the various constructs, Firefly luciferase and Renilla luciferase activities of the lysates were determined by the Dual-Luciferase Reporter Assay system (Promega). The Firefly luciferase values were normalized with the corresponding values for Renilla luciferase. We determined the fold increase in IRES activity by dividing the normalized luciferase value by the value obtained from control pRF transfection. Plasmid PVRF was used as a positive control. Note that Region 2 showed the highest IRES activity in all the cell lines tested. Each experiment was done in triplicate. Data are expressed as mean ± SEM. (B) Spatial localization of DSP and DPP with fluorescently tagged DSPP: DsRED-GFP plasmid with or without the full-length DSPP or DSP-DGP (containing the IRES domain) construct was transiently transfected in HEK293, MC3T3-E1, and DPSC cells. Images were captured with the Zeiss LSM 710 confocal microscope equipped with Zen image analysis software. Transfection with green and red fluorescent protein fusion (pDsRED-GFP) shows equal amounts of DsRED and GFP expression in all cell types (A1, B1, C1). Transfection with the pDsRED-DSPP-GFP shows more GFP expression than DsRED (A2, B2, C2) and localization in the nucleus (A2-2, B2-2, C2-2), implying more DPP-translated products. Transfection with the pDsRED-DSP-DGP-GFP plasmid, which contains the IRES domain, shows more GFP expression, indicating the presence of an IRES element in the construct (A3, B3, C3). Note the absence of GFP expression in the nucleus. (C) Presence of intracellular DSP and DPP. Total proteins were isolated from HEK293 cells transfected with either RFP-GFP plasmid (A) or RFP-DSPP-GFP plasmid (B). Western blot analysis was performed with either anti-DSP, anti-DPP, or anti-GFP antibody. Arrows denote the presence of GFP-DPP or RFP-DSP and their processed forms. Tubulin was used as loading control. (D) DPP is transported to the secretome in exosomes: (1) TEM showing the presence of exosomes in the secretome of T4-4 cells; (2) exosomes were processed for immuno-TEM as described in “Materials & Methods”, with sections stained for DPP followed by 20-nm gold secondary antibody [Note the presence of DPP in the isolated exosomes.]; (3) sections were stained for CD-63, a marker for exosomes; and (4) Western blot performed on total proteins isolated from the exosome with anti-DPP (1:200) and anti-CD63 (1:250) antibodies. (The figure can be viewed in color online.)

Figure 4.

DSPP gene products have distinct functions during dentinogenesis. (A) X-ray analysis of dental mesenchyme overexpressing DPP shows formation of mineralized tooth-like structures when recombined with mouse dental epithelium, after sub-renal culture at 30 days in a DSPP null mouse, while cells overexpressing DSP show less mineralized matrix. (B) Histological analysis by H& E. (C) Masson Trichrome staining shows the tooth architecture with the organic matrix and mineralized dentin synthesized by the differentiated odontoblasts.