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Regulation and Interactions of MT1-MMP and MMP-20 in Human Odontoblasts and Pulp Tissue in vitro

¹ Institute of Dentistry, PO Box 5281, 90014 University of Oulu, Oulu, Finland;
² Department of Cytokine Biology, Forsyth Institute, Boston, MA, USA;
³ Oulu University Hospital, Oulu, Finland;
⁴ Oral Pathology Unit and Biomedicum, University of Helsinki, Laboratory Diagnostics, Helsinki University Central Hospital (HUCH), Helsinki, Finland;
⁵ Harvard-Forsyth Department of Oral Biology & Department of Cytokine Biology, Forsyth Institute, Boston, MA, USA;
⁷ Department of Endodontics, Faculty of Dentistry, University of Toronto, ON, Canada;

View larger version (56K): [in a new window]   Figure 1. Expression and regulation of MT1-MMP by TGF-ß1 and BMP-2 in odontoblasts and pulp tissue. (A) Total RNA from native and cultured odontoblasts (ObN and ObC, respectively) and native and cultured pulp tissue (PN and PC, respectively) were transcribed into cDNA. MT1-MMP mRNA and 18S ribosomal RNA were amplified by PCR. Products were fractionated on a 1.5% agarose gel containing 1 µg/mL ethidium bromide and were photographed. PCR was repeated 4 times. A 395-bp MT1-MMP transcript was amplified from native and cultured odontoblasts and pulp tissue, respectively. In nested PCR amplifications, a 267-bp product was amplified from all samples, confirming the identity of MT1-MMP. In the negative control (NCtr), where template was not added, no product was amplified. 18S ribosomal transcripts, amplified from the samples, served as the endogenous control. (B) Odontoblasts and pulp tissue (n = 6 for all samples) were treated without (Ctr) or with TGF-ß1 (T), BMP-2 (B), or both growth factors combined (T+B). MT1-MMP and 28S ribosomal RNA were analyzed by RPA. The bands were scanned, and the relative MT1-MMP levels in each group were calculated based on the 28S standard values. (C-D) A mean and standard error of the mean (SEM) of the relative MT1-MMP mRNA expression in odontoblasts (C) and pulp tissue (D) (n = 6 in all groups). 100 ng/mL BMP-2 decreased the expression of MT1-MMP mRNA by 66% when compared with controls (**: p = 0.006, ANOVA with LSD test), while with 1 ng/mL TGF-ß1, alone or in combination with BMP-2, only a moderate 36-40% decrease was detected, and this difference was not statistically significant (C). In the pulp tissue, a similar but less marked non-significant reduction in MT1-MMP expression was observed (D).

View larger version (27K): [in a new window]   Figure 2. MT1-MMP protein synthesis in odontoblasts and pulp tissue detected by Western blot analysis. (A) A 20-µL quantity of native odontoblasts (ObN) and native pulp tissue (PN) in 1x Laemmli buffer was subjected to Western blot analysis with the use of an MT1-MMP-specific polyclonal antibody. ProMT1-MMP (65 kDa) and MT1-MMP (51 kDa) bands were detected in all samples. In addition, truncated MT1-MMP (45, 42, 35, and 20 kDa) forms were also observed. (B) Odontoblasts (ObC) and pulp tissue (PC) were cultured for 24 hrs without growth factors and were subjected to Western blotting with the use of MT1-MMP-specific polyclonal antibody. ProMT1-MMP (65 kDa) and MT1-MMP (51 kDa) bands were detected. In addition, truncated MT1-MMPs (42, 35, and 20 kDa) were seen in the pulp tissue. Recombinant human soluble proMT1-MMP was used as a positive control for antibody specificity. The expected 62-kDa immunoreactive band was observed (rhMT1-MMP). This control strongly suggests that the 65-kDa band observed in the odontoblasts and pulp tissue samples represents the latent form of MT1-MMP.

View larger version (56K): [in a new window]   Figure 3. Activation of MMP-20 or MMP-2 with APMA or MT1-MMP. (A-B) Western blot analysis of proMMP-20 conversion by APMA or MT1-MMP in odontoblasts collected from intact mature human teeth (A) and conditioned odontoblast culture media (B). In the control samples from cells or media, 57-kDa proMMP-20 and 46-kDa active MMP-20 immunoreactive bands were observed. APMA and the soluble catalytic domain of MT1-MMP (MT1-MMP [A]) variable converted the 57-kDa proMMP-20 form into 46-kDa activated MMP-20 after incubation of 6 or 24 hrs. This occurred in both the odontoblasts (A) and conditioned odontoblast culture medium (B). (C-D) Conversion of recombinant human MMP-20 by the catalytic domain of active recombinant MT1-MMP. Western blot analysis (C) and SDS-PAGE with Coomassie Brilliant Blue staining (D) revealed that MT1-MMP converted proMMP-20 into an active MMP-20 after 6 hrs or 24 hrs of incubation. (E-F) MMP-2 activation assay with APMA or MT1-MMP analyzed by gelatin zymography. APMA clearly activated proMMP-2 in odontoblasts (E) and conditioned odontoblast culture medium (F). Soluble catalytic MT1-MMP (A) and soluble catalytic with hemopexin domain MT1-MMP (B) converted proMMP-2 into an active form of MMP-2 in the odontoblasts (E). No clear activation was seen in the conditioned odontoblast culture medium (F). (All samples were incubated for 24 hrs.)