Cubam, Cathepsin K and Dipeptidyl Peptidase I Expression During Amelogenesis

It is well established that enamel matrix proteins are degraded during enamel formation and these degraded proteins are removed from the matrix to allow for an increase in mineral content. Proteolytic processing is critical for proper enamel formation as homozygous mutation of either of the resident enamel proteases, matrix metalloproteinase-20 (MMP-20) or kallikrein-4 (KLK-4), causes Amelogenesis Imperfecta where the enamel is hypomineralized and protein-rich. Receptors on ameloblasts have been proposed to facilitate removal of the cleaved proteins. Cubilin is a multiligand endocytic receptor that together with amnionless, forms the cubam receptor complex. This complex is responsible for the uptake of a diverse range of ligands into absorptive cells. Objectives: Determine if in addition to KLK-4 and MMP-20, other proteases may be involved in enamel formation and identify if the proteolytically processed matrix proteins may be removed by a clathrin-mediated endocytic mechanism involving the cubam receptor complex. Methods: RNA from molars of 3-11 day-old mice was collected and expression of clathrin heavy-chain, cubilin, amnionless and selected proteases was assessed and quantified by RT-PCR and qPCR. The identity of cells and the developmental pattern of expression were assessed by immunohistochemical staining of demineralized mouse incisors. Results: High levels of cathepsin K and dipeptidyl peptidase I (DPPI) expression were observed in mature enamel organ. Immunohistochemical staining of MMP-20 -/- mouse incisors showed cathepsin K in the enamel matrix and cathepsin K was found to degrade recombinant amelogenin in vitro. Clathrin heavy-chain, cubilin and amnionless were expressed throughout enamel development and immunohistochemical staining identified ameloblasts as the cells primarily responsible for their expression. Conclusions: Our results suggest that Cathepsin K and DPPI are potentially involved in enamel matrix degradation and the clathrin-mediated cubam receptor complex is likely involved in removing the proteolytically processed enamel proteins from the enamel matrix. Supported by NIH Grant DE016276.