The Expression Profile of Dental Pulp-Derived Stromal Cells Supports Their Limited Capacity to Differentiate into Adipogenic Cells.

Mesenchymal stromal cells (MSCs) can self-renew, differentiate into specialised cells and have different embryonic origins-ectodermal for dental pulp-derived MSCs (DPSCs) and mesodermal for adipose tissue-derived MSCs (ADSCs). Data on DPSCs adipogenic differentiation potential and timing vary, and the lack of molecular and genetic information prompted us to gain a better understanding of DPSCs adipogenic differentiation potential and gene expression profile. While DPSCs differentiated readily along osteogenic and chondrogenic pathways, after 21 days in two different types of adipogenic induction media, DPSCs cultures did not contain lipid vacuoles and had low expression levels of the adipogenic genes proliferator-activated receptor gamma (PPARG), lipoprotein lipase (LPL) and CCAAT/enhancer-binding protein alpha (CEBPA). To better understand this limitation in adipogenesis, transcriptome analysis in undifferentiated DPSCs was carried out, with the ADSC transcriptome used as a positive control. In total, 14,871 transcripts were common to DPSCs and ADSCs, some were unique (DPSCs: 471, ADSCs: 1032), and 510 were differentially expressed genes. Detailed analyses of overrepresented transcripts showed that DPSCs express genes that inhibit adipogenic differentiation, revealing the possible mechanism for their limited adipogenesis.

Yes-associated protein 1 promotes the differentiation and mineralization of cementoblast.

Yes-associated protein 1 (YAP1) transcriptional coactivator is a mediator of mechanosensitive signaling. Cementum, which covers the tooth root surface, continuously senses external mechanical stimulation. Cementoblasts are responsible for the mineralization and maturation of the cementum. However, the effect of YAP1 on cementoblast differentiation remains largely unknown. In this study, we initially demonstrated that YAP1 overexpression enhanced the mineralization ability of cementoblasts. YAP1 upregulated the mRNA and protein expression of several cementogenesis markers, such as alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), osteocalcin (OCN), and dentin matrix acidic phosphoprotein 1 (DMP1). The YAP1 overexpression group showed higher intensities of ALP and Alizarin red stain than the YAP1-knockdown group. Unexpectedly, a sharp increase in the expression of dentin sialophosphoprotein (DSPP) was induced by the overexpression of YAP1. Knockdown of YAP1 suppressed DSPP transcriptional activity. YAP1 overexpression activated Smad-dependent BMP signaling and slightly inhibited Erk1/2 signaling pathway activity. Treatment with specific BMP antagonist (LDN193189) prevented the upregulation of the mRNA levels of ALP, RUNX2, and OCN, as well as intensity of ALP-stained and mineralized nodules in cementoblasts. The Erk1/2 signaling pathway inhibitor (PD 98,059) upregulated these cementogenesis markers. Thus, our study suggested that YAP1 enhanced cementoblast mineralization in vitro. YAP1 exerted its effect on the cementoblast partly by regulating the Smad-dependent BMP and Erk1/2 signaling pathways.

Induced ablation of Bmp1 and Tll1 produces osteogenesis imperfecta in mice.

Osteogenesis imperfecta (OI), or brittle bone disease, is most often caused by dominant mutations in the collagen I genes COL1A1/COL1A2, whereas rarer recessive OI is often caused by mutations in genes encoding collagen I-interacting proteins. Recently, mutations in the gene for the proteinase bone morphogenetic 1 (BMP1) were reported in two recessive OI families. BMP1 and the closely related proteinase mammalian tolloid-like 1 (mTLL1) are co-expressed in various tissues, including bone, and have overlapping activities that include biosynthetic processing of procollagen precursors into mature collagen monomers. However, early lethality of Bmp1- and Tll1-null mice has precluded use of such models for careful study of in vivo roles of their protein products. Here we employ novel mouse strains with floxed Bmp1 and Tll1 alleles to induce postnatal, simultaneous ablation of the two genes, thus avoiding barriers of Bmp1(-/-) and Tll1(-/-) lethality and issues of functional redundancy. Bones of the conditionally null mice are dramatically weakened and brittle, with spontaneous fractures-defining features of OI. Additional skeletal features include osteomalacia, thinned/porous cortical bone, reduced processing of procollagen and dentin matrix protein 1, remarkably high bone turnover and defective osteocyte maturation that is accompanied by decreased expression of the osteocyte marker and Wnt-signaling inhibitor sclerostin, and by marked induction of canonical Wnt signaling. The novel animal model presented here provides new opportunities for in-depth analyses of in vivo roles of BMP1-like proteinases in bone and other tissues, and for their roles, and for possible therapeutic interventions, in OI.

CCN2/CTGF expression via cellular uptake of BMP-1 is associated with reparative dentinogenesis.

OBJECTIVE: CCN family member 2/connective tissue growth factor (CCN2/CTGF) is known as an osteogenesis-related molecule and is thought to be implicated in tooth growth. Bone morphogenetic protein-1 (BMP-1) contributes to tooth development by the degradation of dentin-specific substrates as a metalloprotease. In this study, we demonstrated the correlations between CCN2/CTGF and BMP-1 in human carious teeth and the subcellular dynamics of BMP-1 in human dental pulp cells. MATERIALS AND METHODS: Expression of CCN2/CTGF and BMP-1 in human carious teeth was analyzed by immunohistochemistry. BMP-1-induced CCN2/CTGF protein expression in primary cultures of human dental pulp cells was observed by immunoblotting. Intracellular dynamics of exogenously administered fluorescence-labeled BMP-1 were observed using confocal microscope. RESULTS: Immunoreactivities for CCN2/CTGF and BMP-1 were increased in odontoblast-like cells and reparative dentin-subjacent dental caries. BMP-1 induced the expression of CCN2/CTGF independently of protease activity in the cells but not that of dentin sialophosphoprotein (DSPP) or dentin matrix protein-1 (DMP-1). Exogenously added BMP-1 was internalized into the cytoplasm, and the potent dynamin inhibitor dynasore clearly suppressed the BMP-1-induced CCN2/CTGF expression in the cells. CONCLUSION: CCN2/CTGF and BMP-1 coexist beneath caries lesion and CCN2/CTGF expression is regulated by dynamin-related cellular uptake of BMP-1, which suggests a novel property of metalloprotease in reparative dentinogenesis.

The efficiency of dentin sialoprotein-phosphophoryn processing is affected by mutations both flanking and distant from the cleavage site.

Normal dentin mineralization requires two highly acidic proteins, dentin sialoprotein (DSP) and phosphophoryn (PP). DSP and PP are synthesized as part of a single secreted precursor, DSP-PP, which is conserved in marsupial and placental mammals. Using a baculovirus expression system, we previously found that DSP-PP is accurately cleaved into DSP and PP after secretion into medium by an endogenous, secreted, zinc-dependent Sf9 cell activity. Here we report that mutation of conserved residues near and distant from the G(447)↓D(448) cleavage site in DSP-PP(240) had dramatic effects on cleavage efficiency by the endogenous Sf9 cell processing enzyme. We found that: 1) mutation of residues flanking the cleavage site from P(4) to P(4)' blocked, impaired, or enhanced DSP-PP(240) cleavage; 2) certain conserved amino acids distant from the cleavage site were important for precursor cleavage; 3) modification of the C terminus by appending a C-terminal tag altered the pattern of processing; and 4) mutations in DSP-PP(240) had similar effects on cleavage by recombinant human BMP1, a candidate physiological processing enzyme, as was seen with the endogenous Sf9 cell activity. An analysis of a partial TLR1 cDNA from Sf9 cells indicates that residues that line the substrate-binding cleft of Sf9 TLR1 and human BMP1 are nearly perfectly conserved, offering an explanation of why Sf9 cells so accurately process mammalian DSP-PP. The fact that several mutations in DSP-PP(240) significantly modified the amount of PP(240) product generated from DSP-PP(240) precursor protein cleavage suggests that such mutation may affect the mineralization process.

Site specificity of DSP-PP cleavage by BMP1.

Bone morphogenic protein 1 (BMP1), a metalloproteinase, is known to cleave a wide variety of extracellular matrix proteins, suggesting that a consensus substrate cleavage amino acid sequence might exist. However, while such a consensus sequence has been proposed based on P4 to P4' (i.e. the four amino acids flanking either side of the BMP1 cleavage site; P4P3P2P1|P1'P2'P3'P4') sequence homologies between two BMP1 substrates, dentin matrix protein 1 and dentin sialoprotein phosphophoryn (DSP-PP) (i.e. xMQx|DDP), no direct testing has so far been attempted. Using an Sf9 cell expression system, we have been able to produce large amounts of uncleaved DSP-PP. Point mutations introduced into this recombinant DSP-PP were then tested for their effects on DSP-PP cleavage by either Sf9 endogenous tolloid-related protein 1 (TLR-1) or by its human homolog, BMP1. Here, we have measured DSP-PP cleavage efficiencies after modifications based on P4-P4' sequence comparisons with dentin matrix protein 1, as well as for prolysyl oxidase and chordin, two other BMP1 substrates. Our results demonstrate that any mutations within or outside of the DSP-PP P4 to P4' cleavage site can block, impair or accelerate DSP-PP cleavage, and suggest that its BMP1 cleavage site is highly conserved in order to regulate its cleavage efficiency, possibly with additional assistance from its conserved exosites. Thus, BMP1 cleavage cannot be based on a consensus substrate cleavage site.

Roles of DMP1 processing in osteogenesis, dentinogenesis and chondrogenesis.

Dentin matrix protein 1 (DMP1) is an acidic protein that plays critical roles in osteogenesis and dentinogenesis. Protein chemistry studies have demonstrated that DMP1 primarily exists as processed NH2⁻ and COOH-terminal fragments in the extracellular matrix of bone and dentin. Our earlier work showed that the substitution of Asp²¹³ (a residue at a cleavage site) by Ala²¹³ blocks the processing of mouse DMP1 in vitro. Recently, we generated transgenic mice expressing this mutant DMP1 (designated 'D213A-DMP1'). By crossbreeding these transgenic mice with Dmp1-knockout (Dmp1-KO) mice, we obtained mice expressing the D213A-DMP1 transgene in the Dmp1-null background (named 'Dmp1-KO/D213A-Tg' mice). In this study, we analyzed the long bone, mandible, dentin, and cartilage of Dmp1-KO/D213A-Tg mice in comparison with wild-type, Dmp1-KO, and Dmp1-KO mice expressing the normal DMP1 transgene (Dmp1-KO/normal-Tg). Our results showed that D213A-DMP1 was barely cleaved in the dentin matrix of Dmp1-KO/D213A-Tg mice and the expression of D213A-DMP1 failed to rescue the developmental defects in Dmp1-null mice. Interestingly, enlarged growth plates and condylar cartilages were observed in Dmp1-KO/D213A-Tg mice, indicating a potential role of the full-length form of DMP1 in chondrogenesis.

Proteinase bone morphogenetic protein 1, but not tolloid-like 1, plays a dominant role in maintaining periodontal homeostasis.

BACKGROUND: Periodontitis is caused by multiple factors involving a bacterial challenge and a susceptible host, although there is no report on gene mutation directly linked to this common disease. Mutations in the proteinase bone morphogenetic protein 1 (BMP1) were identified in patients with osteogenesis imperfecta, who display some dentin defects and alveolar bone loss. We previously reported essential roles of BMP1 and tolloid-like 1 (TLL1), two closely related extracellular proteinases with overlapping functions, in mouse periodontium growth by simultaneous knockout (KO) of both genes, although the separate roles of BMP1 and TLL1 have remained unclear. Here, we have investigated whether and how BMP1 and TLL1 separately maintain periodontal homeostasis by comparing single Bmp1 KO and Tll1 KO with double KO (dKO) phenotypes. METHODS: Floxed Bmp1 and/or Tll1 alleles were deleted in transgenic mice via ubiquitously expressed CreERT2 induced by tamoxifen treatment starting at 4-weeks of age (harvested at 18-weeks of age). Multiple approaches, including X-ray, micro-CT, calcein and alizarin red double-labeling, scanning electron microscopy, and histological and immunostaining assays, were used to analyze periodontal phenotypes and molecular mechanisms. RESULTS: Both Bmp1 KO and double KO mice exhibited severe periodontal defects, characterized by periodontal ligament (PDL) fiber loss and ectopic ossification in the expanded PDL area, and drastic reductions in alveolar bone and cementum volumes, whereas Tll1 KO mice displayed very mild phenotypes. Mechanistic studies revealed a sharp increase in the uncleaved precursor of type I collagen (procollagen I), leading to defective extracellular matrices. CONCLUSIONS: BMP1, but not TLL1, is essential for maintaining periodontal homeostasis. This occurs at least partly via biosynthetic processing of procollagen I, thereby maintaining appropriate levels of procollagen I and its activated products such as mature collagen I.

Inactivation of bone morphogenetic protein 1 (Bmp1) and tolloid-like 1 (Tll1) in cells expressing type I collagen leads to dental and periodontal defects in mice.

Bone morphogenetic protein 1 (BMP1) and tolloid-like 1 (TLL1) belong to the BMP1/tolloid-like proteinase family, which cleaves secretory proteins. The constitutive deletion of the Bmp1 or Tll1 genes causes perinatal or embryonic lethality in mice. In this study, we first studied the ß-galactosidase activity in mice in which an IRES-lacZ-Neo cassette was inserted in the intron of either the Bmp1 or the Tll1 gene; the ß-galactosidase activities were used to reflect the expression of endogenous Bmp1 and Tll1, respectively. Our X-gal staining results showed that the odontoblasts in the tooth and cells in the periodontal ligament express both Bmp1 and Tll1. We then created Bmp1 flox/flox and Tll1 flox/flox mice by removing the IRES-lacZ-Neo cassette. By breeding 2.3 kb Col1a1-Cre mice with the Bmp1 flox/flox and Tll1 flox/flox mice, we further generated Col1a1-Cre;Bmp1 flox/flox ;Tll1 flox/flox mice in which both Bmp1 and Tll1 were inactivated in the Type I collagen-expressing cells. We employed X-ray radiography, histology and immunohistochemistry approaches to characterize the Col1a1-Cre;Bmp1 flox/flox ;Tll1 flox/flox mice. Our results showed that the molars of the Col1a1-Cre;Bmp1 flox/flox ;Tll1 flox/flox mice had wider predentin, thinner dentin and larger pulp chambers than those of the normal controls. The dentinal tubules of the molars in the Col1a1-Cre;Bmp1 flox/flox ;Tll1 flox/flox mice appeared disorganized. The level of dentin sialophosphoprotein in the molars of the 6-week-old Col1a1-Cre;Bmp1 flox/flox ;Tll1 flox/flox mice was lower than in the normal controls. The periodontal ligaments of the Col1a1-Cre;Bmp1 flox/flox ;Tll1 flox/flox mice were disorganized and had less fibrillin-1. Our findings indicate that the proteinases encoded by Bmp1 and Tll1 genes play essential roles in the development and maintenance of mouse dentin and periodontal ligaments.

New Coll-HA/BT composite materials for hard tissue engineering.

The integration of ceramic powders in composite materials for bone scaffolds can improve the osseointegration process. This work was aimed to the synthesis and characterization of new collagen-hydroxyapatite/barium titanate (Coll-HA/BT) composite materials starting from barium titanate (BT) nanopowder, hydroxyapatite (HA) nanopowder and collagen (Coll) gel. BT nanopowder was produced by combining two wet-chemical approaches, sol-gel and hydrothermal methods. The resulting materials were characterized in terms of phase composition and microstructure by X-ray diffraction, Raman spectroscopy, scanning electron microscopy and transmission electron microscopy. Moreover, the biocompatibility and bioactivity of the composite materials were assessed by in vitro tests. The synthesized BT particles exhibit an average size of around 35 nm and a spherical morphology, with a pseudo-cubic or tetragonal symmetry. The diffraction spectra of Coll-HA and Coll-HA/BT composite materials indicate a pronounced interaction between Col and the mineral phases, meaning a good mineralization of Col fibres. As well, the in vitro tests highlight excellent osteoinductive properties for all biological samples, especially for Coll-HA/BT composite materials, fact that can be attributed to the ferromagnetic properties of BT.

Chronic inflammation and angiogenic signaling axis impairs differentiation of dental-pulp stem cells.

Dental-pulp tissue is often exposed to inflammatory injury. Sequested growth factors or angiogenic signaling proteins that are released following inflammatory injury play a pivotal role in the formation of reparative dentin. While limited or moderate angiogenesis may be helpful for dental pulp maintenance, the induction of significant level of angiogenesis is probably highly detrimental. Hitherto, several studies have addressed the effects of proinflammatory stimuli on the survival and differentiation of dental-pulp stem cells (DPSC), in vitro. However, the mechanisms communal to the inflammatory and angiogenic signaling involved in DPSC survival and differentiation remain unknown. Our studies observed that short-term exposure to TNF-α (6 and 12 hours [hrs]) induced apoptosis with an upregulation of VEGF expression and NF-κB signaling. However, long-term (chronic) exposure (14 days) to TNF-α resulted in an increased proliferation with a concomitant shortening of the telomere length. Interestingly, DPSC pretreated with Nemo binding domain (NBD) peptide (a cell permeable NF-κB inhibitor) significantly ameliorated TNF-α- and/or VEGF-induced proliferation and the shortening of telomere length. NBD peptide pretreatment significantly improved TNF-α-induced downregulation of proteins essential for differentiation, such as bone morphogenic proteins (BMP)-1 & 2, BMP receptor isoforms-1&2, trasnforming growth factor (TGF), osteoactivin and osteocalcin. Additionally, inhibition of NF-κB signaling markedly increased the mineralization potential, a process abrogated by chronic exposure to TNF-α. Thus, our studies demonstrated that chronic inflammation mediates telomere shortening via NF-κB signaling in human DPSC. Resultant chromosomal instability leads to an emergence of increased proliferation of DPSC, while negatively regulating the differentiation of DPSC, in vitro.

DSP-PP precursor protein cleavage by tolloid-related-1 protein and by bone morphogenetic protein-1.

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 specific proteolytic processing to generate DSP and PP. The cleavage mechanism continues to be controversial, in part because of the difficulty of obtaining DSP-PP from mammalian cells and dentin matrix. We have infected Sf9 cells with a recombinant baculovirus to produce large amounts of secreted DSP-PP(240), a variant form of rat DSP-PP. Mass spectrometric analysis shows that DSP-PP(240) secreted by Sf9 cells undergoes specific cleavage at the site predicted from the N-terminal sequence of PP extracted from dentin matrix: SMQG(447)↓D(448)DPN. DSP-PP(240) is cleaved after secretion by a zinc-dependent activity secreted by Sf9 cells, generating DSP(430) and PP(240) products that are stable in the medium. DSP-PP processing activity is constitutively secreted by Sf9 cells, but secretion is diminished 3 days after infection. Using primers corresponding to the highly conserved catalytic domain of Drosophila melanogaster tolloid (a mammalian BMP1 homolog), we isolated a partial cDNA for a Spodopotera frugiperda tolloid-related-1 protein (TLR1) that is 78% identical to Drosophila TLR1 but only 65% identical to Drosophila tolloid. Tlr1 mRNA decreased rapidly in Sf9 cells after baculovirus infection and was undetectable 4d after infection, paralleling the observed decrease in secretion of the DSP-PP(240) processing activity after infection. Human BMP1 is more similar to Sf9 and Drosophila TLR1 than to tolloid, and Sf9 TLR1 is more similar to BMP1 than to other mammalian homologs. Recombinant human BMP1 correctly processed baculovirus-expressed DSP-PP(240) in a dose-dependent manner. Together, these data suggest that the physiologically accurate cleavage of mammalian DSP-PP(240) in the Sf9 cell system represents the action of a conserved processing enzyme and support the proposed role of BMP1 in processing DSP-PP in dentin matrix.

Dentin sialophosphoprotein (DSPP) is cleaved into its two natural dentin matrix products by three isoforms of bone morphogenetic protein-1 (BMP1).

The protease that cleaves the most abundant non-collagenous protein of dentin matrix, dentin sialophosphoprotein (DSPP), into its two final dentin matrix products, dentin sialoprotein (DSP) and dentin phosphoprotein (DPP), has not been directly identified. In this study, full-length recombinant mouse DSPP was made for the first time in furin-deficient mammalian LoVo cells and used to test the ability of three different isoforms of one candidate protease, bone morphogenetic protein-1 (BMP1) to cleave DSPP at the appropriate site. Furthermore, two reported enhancers of BMP1/mTLD activity (procollagen C-endopeptidase enhancer-1, PCPE-1, and secreted frizzled-related protein-2, sFRP2) were tested for their abilities to modulate BMP1-mediated processing of both DSPP and another SIBLING family member with a similar cleavage motif, dentin matrix protein-1 (DMP1). Three splice variants of BMP1 (classic BMP1, the full-length mTolloid (mTLD), and the shorter isoform lacking the CUB3 domain, BMP1-5) were all shown to cleave the recombinant DSPP in vitro although mTLD was relatively inefficient at processing both DSPP and DMP1. Mutation of the MQGDD peptide motif to IEGDD completely eliminated the ability of all three recombinant isoforms to process full-length recombinant DSPP in vitro thereby verifying the single predicted cleavage site. Furthermore when human bone marrow stromal cells (which naturally express furin-activated BMP1) were transduced with the adenovirus-encoding either wild-type or mutant DSPP, they were observed to fully cleave wild-type DSPP but failed to process the mutant DSPP(MQDeltaIE) during biogenesis. All three BMP1 isoforms were shown to process type I procollagen as well as DSPP and DMP1 much more efficiently in low-salt buffer (< or = 50 mM NaCl) compared to commonly used normal saline buffers (150 mM NaCl). Neither PCPE-1 nor sFRP2 were able to enhance any of the three BMP1 isoforms in cleaving either DSPP or DMP1 under either low or normal saline conditions. Interestingly, we were unable to reproduce sFRP2's reported ability to enhance the processing of type I procollagen by BMP1/mTLD. In summary, three isoforms of BMP1 process both DSPP and DMP1 at the MQX/DDP motif, but the identity of a protein that can enhance the cleavage of the two SIBLING proteins remains elusive.