Carbonic anhydrase II regulates differentiation of ameloblasts via intracellular pH‐dependent JNK signaling pathway

Abstract

Differentiation of ameloblasts from undifferentiated epithelial cells is controlled by diverse growth factors, as well as interactions between epithelium and mesenchyme. However, there is a considerable lack of knowledge regarding the precise mechanisms that control ameloblast differentiation and enamel biomineralization. We found that the expression level of carbonic anhydrase II (CAII) is strongly up-regulated in parallel with differentiation of enamel epithelium tissues, while the enzyme activity of CA was also increased along with differentiation in ameloblast primary cultures. The expression level of amelogenin, a marker of secretory-stage ameloblasts, was enhanced by ethoxzolamide (EZA), a CA inhibitor, as well as CAII antisense (CAIIAS), whereas the expression of enamel matrix serine proteinase-1 (EMSP-1), a marker for maturation-stage ameloblasts, was suppressed by both. These agents also promoted ameloblast proliferation. In addition, inhibition of ameloblast differentiation by EZA and CAIIAS was confirmed using tooth germ organ cultures. Furthermore, EZA and CAIIAS elevated intracellular pH in ameloblasts, while experimental decreases in intracellular pH abolished the effect of CAIIAS on ameloblasts and triggered the activation of c-Jun N-terminal kinase (JNK). SP600125, a JNK inhibitor, abrogated the response of ameloblasts to an experimental decrease in intracellular pH, while the inhibition of JNK also impaired ameloblast differentiation. These results suggest a novel role for CAII during amelogenesis, that is, controlling the differentiation of ameloblasts. Regulation of intracellular pH, followed by activation of the JNK signaling pathway, may be responsible for the effects of CAII on ameloblasts.

Deletion of Slc26a1 and Slc26a7 Delays Enamel Mineralization in Mice

“…Acid-base balance in the extracellular matrix and intracellular lumens is maintained by a complex regulatory network involving multiple ion transporters and carbonic anhydrases (Dogterom and Bronckers, 1983; Lin et al, 1994; Wright et al, 1996a,b; Arquitt et al, 2002; Lyaruu et al, 2008; Paine et al, 2008; Bronckers et al, 2009, 2010; Josephsen et al, 2010; Wang et al, 2010; Lacruz et al, 2010a,b, 2012c, 2013a; Chang et al, 2011; Duan et al, 2011; Duan, 2014; Jalali et al, 2014; Reibring et al, 2014; Wen et al, 2014). Although details regarding the mechanism of pH control are yet to be clarified, the critical roles of many genes in maturation-stage pH regulation have been implicated by previous studies on transgenic animal models.…”

“…cDNA used for real-time PCR analysis was prepared using the miScript II RT Kit with miScript HiFlex Buffer (Qiagen). To detect the expression changes in the genes that have been identified to be involved in maturation-stage pH regulation (Dogterom and Bronckers, 1983; Wright et al, 1996a,b; Andrejewski et al, 1999; Arquitt et al, 2002; Lyaruu et al, 2008; Paine et al, 2008; Bertrand et al, 2009; Bronckers et al, 2009, 2010, 2011; Josephsen et al, 2010; Wang et al, 2010; Lacruz et al, 2010a,b, 2011, 2012b,c, 2013b; Chang et al, 2011; Duan, 2014; Jalali et al, 2014; Yin et al, 2014, 2015), real-time PCR reactions were performed on a CFX96 TouchTM Real-Time PCR Detection System (Bio-rad Life Sciences) with iQ SYBR® Green supermix (Bio-rad Life Science) and mouse-specific primers (Table 1). The Ct values were normalized to those of Actb ( Beta-actin ).…”

“…Digestion of enamel matrix proteins (EMPs) through the endosome/lysosome pathway, following trafficking from the enamel space, relies highly on the acidic intracellular luminal environment (Lloyd, 1996). Previous studies have identified the functional role of several groups of genes, including carbonic anhydrases (Lezot et al, 2008), cystic fibrosis transmembrane conductance regulator (CFTR), chloride channels (CLCNs), solute carrier gene family 4 (SLC4s) and solute carrier gene family 9 (SLC9s), in maintaining the ameloblast-mediated pH homeostasis within both extracellular space and intracellular lumens (Dogterom and Bronckers, 1983; Lin et al, 1994; Wright et al, 1996a,b; Arquitt et al, 2002; Lyaruu et al, 2008; Paine et al, 2008; Bronckers et al, 2009, 2010; Josephsen et al, 2010; Wang et al, 2010; Lacruz et al, 2010a,b, 2012c, 2013a; Chang et al, 2011; Duan et al, 2011; Duan, 2014; Jalali et al, 2014; Reibring et al, 2014; Wen et al, 2014). …”

MiR-153 Regulates Amelogenesis by Targeting Endocytotic and Endosomal/lysosomal Pathways–Novel Insight into the Origins of Enamel Pathologies

“…During maturation-stage tooth development, ameloblasts have been identified to be involved in a series of highly orchestrated events including matrix turnover, ion transport, pH regulation, calcium handling and cell apoptosis210. Previous studies have focused on the signaling pathways of enamel maturation primarily at genetic level2930313233; however the involvement of epigenetic regulatory mechanisms in this process has not been widely studied.…”

Downregulation of Carbonic Anhydrase IX Promotes Col10a1 Expression in Chondrocytes

“…We recently found that CA II, an isozyme of CA IX distributed in cytosol, regulates the differentiation of ameloblasts by modulating cytosolic pH [15]. Although immunohistochemical analysis revealed that CA IX is distributed in chondrocytes in human embryos [16], to the best of our knowledge, there is no report of the function of CA IX in chondrocytes.…”