Dentin matrix protein-1 isoforms promote differential cell attachment and migration

Abstract

Dentin matrix protein-1 (DMP1), bone sialoprotein (BSP), and osteopontin (OPN) are three SIBLINGs (small integrin-binding ligand, N-linked glycoproteins) co-expressed/secreted by skeletal and active ductal epithelial cells. Although etiological mechanisms remain unclear, DMP1 is the only one of these three genes currently known to have mutations resulting in human disease, and yet it remains the least studied. All three contain the highly conserved integrin-binding tripeptide, RGD, and experiments comparing the cell attachment and haptotactic migration-enhancing properties of DMP1 to BSP and OPN were performed using human skeletal (MG63 and primary dental pulp cells) and salivary gland (HSG) cells. Mutation of any SIBLING's RGD destroyed all attachment and migration activity. Using its alphaVbeta5 integrin, HSG cells attached to BSP but not to DMP1 or OPN. However, HSG cells could not migrate onto BSP in a modified Boyden chamber assay. Expression of alphaVbeta3 integrin enhanced HSG attachment to DMP1 and OPN and promoted haptotactic migration onto all three proteins. Interchanging the first four coding exons or the conserved amino acids adjacent to the RGD of DMP1 with corresponding sequences of BSP did not enhance the ability of DMP1 to bind alphaVbeta5. For alphaVbeta3-expressing cells, intact DMP1, its BMP1-cleaved C-terminal fragment, and exon six lacking all post-translational modifications worked equally well but the proteoglycan isoform of DMP1 had greatly reduced ability for cell attachment and migration. The sequence specificity of the proposed BMP1-cleavage site of DMP1 was verified by mutation analysis. Direct comparison of the three proteins showed that cells discriminate among these SIBLINGs and among DMP1 isoforms.

FIGURE 1.

Differential attachment of human dental pulp cells (DPC), salivary gland cells (HSG), and osteoblastic cells (MG63) to bone sialoprotein (BSP)-, dentin matrix protein-1 (DMP1)-, or osteopontin (OPN)-coated wells. Single-cell suspensions of DPC, HSG, and MG63 cells were incubated for 1 h on non-tissue culture wells precoated with BSP, DMP1, or OPN. Note that neither DMP1 nor OPN can support attachment of HSG cells, whereas all three cell types attach well to BSP. No significant number of cells attached to BSA-coated wells (not shown). The adherent cells were fixed, stained with crystal violet, and solubilized with SDS before reading at 560 nm. Bars show the mean ± S.E. from at least three independent experiments, each performed in triplicate.

FIGURE 2.

Cell attachment to BSP, DMP1, and OPN is RGD-dependent. DPCs were plated on wells coated with BSP (A), DMP1 (B), and OPN (C) or their respective KAE mutants. Noted cells were preincubated with 1 mm GRGDS competing peptide or the scrambled control peptide GRDGS for 15 min at room temperature prior to adding to wells. The attachment assays were performed and evaluated as described above. Bars show the mean ± S.E. from six independent experiments, each performed in triplicate.

FIGURE 3.

Cell attachment to DMP1 and OPN requires αVβ3, whereas attachment to BSP can be promoted by either αVβ3 or αVβ5 integrins. Cells, as indicated, were plated on BSP-, DMP1-, or OPN-coated wells. Some cells were pretreated with 10 μg/ml anti-β1(P4C10), anti-αVβ3(LM609), and/or anti-αVβ5(P1F6) function-blocking antibodies prior to and during the incubation. The attachment assays were performed and evaluated as described above. Cell attachment in the presence of IgG control was assigned a value of 100%. Bars show the mean ± S.E. from six independent experiments, each performed in triplicate. ^**, p < 0.001, as compared with the corresponding IgG control value.

FIGURE 4.

De novo expression ofαVβ3 integrin causes HSG cells to attach to DMP1-coated surfaces. A, cell surface expression of β1 subunit, αVβ3, or αVβ5 integrins on HSG cells before (black profiles) and after transduction with viral construct expressing αV cDNA in combination with viruses expressing either β1(left), or β3 (middle), or β5(right) cDNA (gray profiles) as assessed by flow cytometry using monoclonal antibodies (4B7 for β1, LM609 for αVβ3, and P1F6 for αVβ5) and detected with Alexa Flour 488-labeled goat anti-mouse IgG. The expression level of each integrin on the cell surfaces is indicated by log fluorescence intensity (x-axis). Note that, among parental and adenoviral-infected HSG cells, only those expressing αVβ3 significantly attached to either DMP1 (B)- or OPN (C)-coated wells. Cells infected with an adenovirus encoding no recombinant protein exhibited a profile (not shown) identical to parental cells (wt). The attachment assays were performed and evaluated as described above. Bars show the mean ± S.E. from seven independent experiments, each performed in triplicate. ^**, p < 0.001, as compared with the control (wt) cells by means of t test.

FIGURE 5.

Integrin-activation by treatment with Mn²⁺ enhances cell attachment on all three SIBLINGs. Cells, as indicated, were plated on wells precoated with BSP, DMP1, or OPN with or without 1 mm MnCl₂ (DPC and HSG cells) or 0.25 mm MnCl₂ (MG63 cells). The attachment assays were performed and evaluated as described above. The bars show the mean ± S.E. of three experiments, each performed in triplicate.

FIGURE 6.

Mn²⁺-mediated induction of cell attachment to DMP1 and OPN is αVβ3-dependent. Single-cell suspension of MG63 cells was incubated with control IgG or the function blocking anti-αVβ3 antibody (LM609) in the presence of 0.25 mm MnCl₂ before plating on wells coated with either DMP1 (A) or OPN (B). The attachment assays were performed and evaluated as described above. Attachment of cells in the presence of IgG control was assigned a value of 100%. The bars show the mean ± S.E. of four experiments each performed in triplicate. ^**, p < 0.001, as compared with the corresponding IgG control value by means of t test.

FIGURE 7.

Investigating DMP1 sequences involved in integrin specificity. HSG (naturally expressing αVβ5 integrin), HSG-αVβ3 (expressing αVβ5 plus adenovirus-transduced αVβ3 integrins), and MG63 (expressing both αVβ3 and αVβ5 integrins) cells as noted were plated on wells coated with native forms of BSP or DMP1, or one the following DMP1 constructs: BSP/DMP1-Hybrid (DMP1 with the first four coding exons of BSP fused to the last exon of DMP1); DMP1-BSP-likeRGD (DMP1 with its conserved SRGDNP sequence replaced by BSP's conserved PRGDNY); DMP1 exon 6 (bacterial), truncated DMP1 protein without post-translational modification. Note that neither the first four exons nor the RGD domain of BSP replacing DMP1's corresponding regions resulted in αVβ5-attachment activity for HSG (A). Attachment of MG63 cells to BSP/DMP1 hybrid remained DMP1-like (B). Post-translational modifications are not required for DMP1's specificity for αVβ3(C). The attachment assays were carried out and evaluated as described above. The bars show the mean ± S.E. of three experiments each performed in triplicate.

FIGURE 8.

αVβ3 integrin is required for haptotactic migration by cells onto any of the three SIBLINGs. Migration permissivity of wt-HSG, αVβ3-HSG (A) or MG63 cells (B) were tested in Transwells for which the bottom of the membranes were coated with the noted SIBLINGs or left uncoated (control) before cells were added to the upper chambers. Cell migration was evaluated by counting the 4′,6-diamidino-2-phenylindole-stained cells on the lower, SIBLINGs-coated surface in random 40× fields after 24 h (HSG) or 5 h (MG63). Native HSG cells (lacking αVβ3) did not migrate onto any SIBLINGs until this integrin was expressed (HSG-αVβ3, A). Note that DMP1 always supported significantly lower levels of migration than BSP or OPN. The bars show the mean from 10 40× fields ± S.E. of a representative experiment. ^**, p < 0.001 compared with cell migrated on BSP or OPN by using the t test.

FIGURE 9.

Changing of the Met²¹⁵-Gln²¹⁶ to the chemically similar IIe-His in DMP1's BMP1-cleavage site completely abolished processing in vitro and in vivo. A, in vitro processing. Recombinant DMP1^Mix (containing full-length as well as the BMP1-like cleavage fragments from original production of protein in human bone marrow stromal cells, lane 2) is fully processed upon addition of rBMP1 (lane 3) DMP1^MQΔIH (full-length protein with a MQ-to-IH mutation in BMP1-cleavage site, lane 4) was not digested by rBMP1 (lane 5). B, in vivo processing. HSG cells infected with adenoviruses expressing DMP1 (left lanes) shows a small amount of endogenous BMP1-like cleavage activity (first lane) and complete digestion with even lowest dose of BMP1 adenovirus (lane 2). DMP1^MQΔIH mutant co-infected without (first of the right lanes) and even the highest dose of BMP1 adenovirus (last lane) showed no processing of the cleavage-site mutant protein. 48 h post-infection conditioned media were electrophoresed with SDS on a 4–12% NuPAGE before detection with StainsAll.

FIGURE 10.

Proteoglycan form of DMP1 has an impaired ability to support cell adhesion and migration in comparison to full-length and BMP1-processed DMP1 fragments. αVβ3-expressing HSG and MG63 cells were tested for their ability to attach (A) and haptotactically migrate (B) onto different DMP1 isoforms: DMP1^MQΔIH (full-length protein only due to a mutation in BMP1-cleavage site); DMP1^Mix (full-length plus BMP1-like cleavage fragments); DMP1^Frags (fully digested by recombinant BMP1); DMP1^MQΔIH-PG (proteoglycan form of full length DMP1); DMP1^MQΔIH-PG/chondroitinase ABC (proteoglycan form of DMP1^MQΔIH-PG after in situ treatment with chondroitinase ABC (cABC)). The attachment and migration assays were performed and evaluated as described above. Notice that the proteoglycans form significantly inhibited both the attachment (A) and haptotactic migration (B) of both αVβ3-expressing cells and that attachment activity was recovered by the removal of the GAG chain in situ by chondroitinase ABC. The bars show the mean ± S.E. of three experiments each performed in triplicate. ^**, p < 0.001; ^*, p < 0.01, as obtained for multiple comparisons by using of analysis of variance and the Newman-Keuls test.