Constitutive nuclear expression of dentin matrix protein 1 fails to rescue the Dmp1-null phenotype - PubMed (original) (raw)

Constitutive nuclear expression of dentin matrix protein 1 fails to rescue the Dmp1-null phenotype

Shuxian Lin et al. J Biol Chem. 2014.

Abstract

Dentin matrix protein 1 (DMP1) plays multiple roles in bone, tooth, phosphate homeostasis, kidney, salivary gland, reproductive cycles, and the development of cancer. In vitro studies have indicated two different biological mechanisms: 1) as a matrix protein, DMP1 interacts with αvβ3 integrin and activates MAP kinase signaling; and 2) DMP1 serves as a transcription co-factor. In vivo studies have demonstrated its key role in osteocytes. This study attempted to determine whether DMP1 functions as a transcription co-factor and regulates osteoblast functions. For gene expression comparisons using adenovirus constructs, we targeted the expression of DMP1 either to the nucleus only by replacing the endogenous signal peptide with a nuclear localization signal (NLS) sequence (referred to as (NLS)DMP1) or to the extracellular matrix as the WT type (referred to as (SP)DMP1) in MC3T3 osteoblasts. High levels of DMP1 in either form greatly increased osteogenic gene expression in an identical manner. However, the targeted (NLS)DMP1 transgene driven by a 3.6-kb rat Col 1α1 promoter in the nucleus of osteoblasts and osteocytes failed to rescue the phenotyope of Dmp1-null mice, whereas the (SP)DMP1 transgene rescued the rickets defect. These studies support the notion that DMP1 functions as an extracellular matrix protein, rather than as a transcription co-factor in vivo. We also show that DMP1 continues its expression in osteoblasts during postnatal development and that the deletion of Dmp1 leads to an increase in osteoblast proliferation. However, poor mineralization in the metaphysis indicates a critical role for DMP1 in both osteoblasts and osteocytes.

Keywords: Autosomal Recessive Hypophosphatemic Rickets; Biomineralization; Bone; Dentin Matrix Protein 1; Development; Osteoblast; Osteocyte; Osteogenesis; Osteomalacia.

© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

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Figures

FIGURE 1.

FIGURE 1.

DMP1, highly expressed by osteocytes (Ocy) and also expressed by postnatal osteoblasts (Ob), is likely responsible for regulating osteoblast proliferation and differentiation. a, immuno-gold labeling reveals the presence of DMP1 accumulation in the ECM of the canalicular wall adjacent to the dendrites/cell processes and surrounding the cell body (lacunar wall) of osteocytes. b, X-gal staining of HET long bones displays blue positive cells not only in osteocytes but also in osteoblasts in both embryonic day 16.5 (E16.5; inset) and the postnatal 3 weeks, especially in the metaphysis (right image). c, representative radiographs of 3-week-old long bones reveals the co-existence of an expanded metaphysis (arrows) and a delayed formation of the epiphysis (arrowhead). d and e, histological images showing that in the _Dmp1_-KO tibia metaphysis (right panels), there is a sharp expansion of bone mass with little residual cartilage (d, Safranin O stain, 6 weeks), indicating a lack of endochondral bone formation but the presence of active intramembranous bone formation; von Kossa staining (e, 3 months) shows poor mineralization of the bone (arrow) compared with age-matched control (left). f–h, in situ and immunohistochemical staining showing that in the KO tibia metaphysis (right), there are increased BrdU-positive cells (f), and mRNA levels of Runx2 (g), and OSX (h).

FIGURE 2.

FIGURE 2.

Transfection of Ad-CMV-NLSDMP1 or Ad-CMV-SPDMP1 greatly changes the expression of bone markers in MC3T3 cells. a, schematic constructs of NLSDMP1 and SPDMP1, in which the CMV promoter was used to drive the full-length Dmp1 cDNA. In NLSDMP1, the endogenous DMP1 signal peptide amino acids 1–16 (MKTVILLVFLWGLSCAL) was replaced by the nuclear localization signal peptide (NLS, PPKKKRKV, upper panel) in contrast to the SPDMP1 with its endogenous signal peptide (lower panel). b, >1000-fold increases in NLSDMP1 and SPDMP1 were induced by adenovirus transinduction. There were significant up-regulations of mRNA levels for Bsp, Alp, Ocn, Opn, Dspp, and Sost in both experimental groups with all showing an identical trend. The real-time PCR data, normalized to Gapdh (as an internal control), are presented as mean ± S.E. (n = 4 in each group; *, p < 0.05; **, p < 0.01, compared with the control).

FIGURE 3.

FIGURE 3.

NLSDMP1 localizes to the nucleus and SPDMP1 is secreted into the matrix. a, schematic structure of NLSDMP1 and SPDMP1 transgenes. In the NLSDMP1 construct, the full-length Dmp1 cDNA is driven by the 3.6-kb Col 1_α_1 promoter with its endogenous signal peptide replaced by a NLS peptide, whereas in the SPDMP1 construct, the same 3.6-kb Col 1_α_1 promoter was used to drive the full-length Dmp1 cDNA with its endogenous secretory signal peptide. b, proteins extracted from cell lysate (left) or nuclei (right) of different groups were blotted for the expression level of DMP1. Results show that DMP1 can be found in the cell lysate of all these groups except the _Dmp1_-null group; however, in the nuclear protein extract, it was only detectable in the KO+NLSDMP1 group. c, immunohistochemical staining of DMP1 in 3-week-old mouse long bone. DMP1 is mainly expressed in the HET matrix with little signal in the nucleus, compared with no positive signal in the matrix in KO mice. In the NLSDMP1 transgenic bone, DMP1 is detected in both the matrix and the nucleus of osteocytes (arrows) and osteoblasts (arrowheads), whereas the SPDMP1 is highly expressed in the matrix and the osteoblasts. These data demonstrate that the transgenes are successfully targeted to the nucleus or to the matrix (of bone cells only).

FIGURE 4.

FIGURE 4.

NLSDMP1 does not rescue the rachitic phenotypes in _Dmp1_-null mice; however, SPDMP1 fully rescues the defects. a and b, quantitative data show a greater than 18-fold up-regulation of serum FGF23 levels and a decrease of >20% of the Pi (phosphorus) in _Dmp1_-null mice. The overexpression of NLSDMP1 has no effect on these parameters in either the HET control or KO group; however, the SPDMP1 is fully rescued. d, Safranin O-stained images of 3-week-old tibias, in which there are two expanded regions in the KO growth plate: the proliferation zone (PZ; white lines) and the hypertrophic zone. The NLSDMP1 transgene has no effect in both regions, but SPDMP1 recovers this abnormality. e, BrdU-stained images reveal an increase in the number of BrdU-positive cells in the KO and KO+NLSDMP1 groups. Statistical results demonstrate the above changes in proliferation zone length (c) and cell proliferation numbers (f) in the KO groups, which were significantly different from the control groups., the NLSDMP1 has no apparent effect on the above changes in either the HET or the KO background, but SPDMP1 rescues the KO phenotype. g--j, in situ and immunohistochemical staining shows the molecular changes of Runx2 mRNA (g), sonic hedgehog mRNA (Shh; h), SOX9 (i), and OSX (j). The data indicate an increase in cell proliferation and differentiation in the _Dmp1_-null mice, most likely attributable to hypophosphatemia (data are presented as mean ± S.E.; n = 5; *, p < 0.05; compared with the control). k, representative radiography shows short femurs with poor mineral remodeling in all three _Dmp1_-KO groups (3 weeks, 6 weeks, and 1 year). The targeted expression of NLSDMP1 has no rescue effects on the length, accumulated bone masses, expanded and malformed growth plates (arrows), and distorted tuberosities (arrowheads), all of which are fully rescued by SPDMP1.

FIGURE 5.

FIGURE 5.

The NLSDMP1 transgene has no rescue effects on _Dmp1_-null mice but the SPDMP1 transgene fully rescues the _Dmp1_-null bone phenotype. a–c, compared with SPDMP1, NLSDMP1 has no effect on bone shape and secondary ossification (a, Alizarin red/Alcian blue staining, arrows), bone porosity and mineralization reflected by micro-CT images (b, arrowheads), and Goldner staining (c, red indicating a lack of mineral). d–h, the NLSDMP1 had no apparent effect on the osteocyte (Ocy)-lacunocanalicular system (d, S.E.), and expression of decorin (e, an inhibitory factor in mineralization), OSX (f, a marker for Ob), E11 (g, an early marker for osteocytes), and SOST (h, a marker for mature osteocytes).

FIGURE 6.

FIGURE 6.

The in vivo working model. a, DMP1 secreted from the cell binds to integrin via its RGD domain, followed by activation of the MAP kinase signaling pathway (27, 29). The targeted expression of DMP1 in the nucleus has no direct role in osteogenesis in vivo. b, DMP1 expressed in the osteoblast (Ob) facilitates cell proliferation and transformation (from osteoblasts into osteocytes (Ocy)). Deletion of Dmp1 leads to an increase in cell proliferation and a reduction in osteoblast cell differentiation. Because of the great increase of FGF23 in _Dmp1_-KO osteocytes, the reduced Pi causes an increase in cell proliferation and differentiation in chondrocytes (a new indirect role of hypophosphatemia in the growth plate), in addition to the abnormality in the apoptosis pathway (46).

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