Transcriptomic profiling of feline teeth highlights the role of matrix metalloproteinase 9 (MMP9) in tooth resorption

Abstract

Tooth resorption (TR) in domestic cats is a common and painful disease characterised by the loss of mineralised tissues from the tooth. Due to its progressive nature and unclear aetiology the only treatment currently available is to extract affected teeth. To gain insight into TR pathogenesis, we characterised the transcriptomic changes involved in feline TR by sequencing RNA extracted from 14 teeth (7 with and 7 without signs of resorption) collected from 11 cats. A paired comparison of teeth from the same cat with and without signs of resorption identified 1,732 differentially expressed genes, many of which were characteristic of osteoclast activity and differentiation, in particular matrix metalloproteinase 9 (MMP9). MMP9 expression was confirmed by qPCR and immunocytochemistry of odontoclasts located in TR lesions. A hydroxamate-based MMP9 inhibitor reduced both osteoclast formation and resorption activity while siRNA targeting MMP9 also inhibited osteoclast differentiation although had little effect on resorption activity. Overall, these results suggest that increased MMP9 expression is involved in the progress of TR pathogenesis and that MMP9 may be a potential therapeutic target in feline TR.

Figure 1

MDS plot and visualisation of gene expression data by smearplots. MDS plots of (A) TR −/+ cats comparison, (B) TR −/+ teeth comparison, and (C) paired TR −/+ comparison. Smear plots of (D) TR −/+ cats comparison, (E) TR −/+ teeth comparison, and (F) paired TR −/+ comparison. Lined circle: TR + ve samples, dotted circle: TR − ve (control teeth). Horizontal blue lines indicate fold-change of two. Red dot indicates each differentially expressed gene at False Discovery Rate or corrected p value (FDR) of 0.05 or smaller.

Figure 2

Osteoclast differentiation pathway (map04380) obtained from KEGG database with official permission and guidance from Kanehisha Laboratories (permission ref 200,290). Expressed genes are coloured with red indicating relative up-regulation and with green indicating down-regulation in TR + ve teeth.

Figure 3

Calcium signalling pathway (map04020) obtained from KEGG database with official permission and guidance from Kanehisha Laboratories (permission ref 200,290). Expressed genes are coloured with red indicating relative up-regulation and with green indicating down-regulation in TR + ve teeth.

Figure 4

MMP9 is highly expressed in odontoclasts of feline tooth resorption lesions. H&E from TR − ve teeth (A) and TR + ve teeth (D,E). Immunohistochemical labelling for MMP9 protein with haematoxylin counterstaining TR − ve (B,C), TR + ve sections (F,G,H) and isotype control (I). No visible odontoclast is found in TR − ve teeth (B) and circle area of B is magnified in C. Resorbing odontoclasts (D, circle area) are magnified in E. MMP9 expression in TR − ve teeth are observed mainly in periodontal ligament or gingival tissues (B,C). TR + ve teeth contain active odontoclasts with resorption pits (F, dotted circle) with strong MMP9 expression, high magnification of this lesion (G). MMP9 expression was also found in multinucleated like cells but these cells did not reside in a resorption pit (H, arrow). AB alveolar bone, DE dentine, PDL periodontal ligament. Scale bars = 100 µm.

Figure 5

MMP9 mRNA expression increased during in vitro feline osteoclast formation over an 8 day culture period. Precursors were treated with M-CSF from day 0, and RANKL was added from day 3. Graphs represent relative expression as fold changes with bars showing standard error of the mean. (n = 3; *p < 0.05, ***p < 0.001 in comparison with the previous time point in culture, ^###p < 0.001 in comparison with day 0 by two sample t test).

Figure 6

MMP9 semi-selective inhibitor decreased feline osteoclast formation and resorption activity. Graphs represent percentage of number of osteoclasts (A) or resorption pits (B) with + SEM bars compared to vehicle (DMSO) control. (n = 3; *p < 0.05, **p < 0.01; ***p < 0.001 in comparison with vehicle control, ^###p < 0.001 between individual doses comparison by two sample t test).

Figure 7

MMP9 siRNA transfected cells resulted in reduced osteoclast formation in comparison with scrambled control but there was no statistically significant difference in resorption activity. Representative images of TRAP positive osteoclasts on dentin discs from untransfected control, scrambled control and MMP9 siRNA transfected treatment (A). Representative images of toluidine blue stained resorption pits from untransfected control, scrambled control and MMP9 siRNA transfected treatment (C). Graphs represent percentage of number of osteoclasts (B) and resorption pits (D) with + SEM bars. (n = 3; ***p < 0.001 in comparison with scrambled control). Scale bars = 100 µm.