Sage Journals: Discover world-class research

Abstract

Enamel mineralization critically depends on maturation-stage ameloblasts (M-ABs) regulating pH, protein secretion, and cell-matrix adhesion. However, the molecular mechanisms underlying these processes remain poorly understood. This study identifies the vacuolar-type H⁺-ATPase (V-ATPase) a3 subunit as a key regulator of enamel formation via its role in secretory lysosome trafficking. In a3 knockout (a3KO) mice and cultured ameloblasts, a3 is required for both lysosomal acidification and the directional transport of odontogenic ameloblast-associated protein (ODAM)–containing secretory lysosomes to the ruffled border membrane of M-ABs. At this site, ODAM is crucial for mediating ameloblast adhesion to the enamel matrix. Loss of a3 caused severe enamel hypomineralization, characterized by reduced matrix acidification, cystic enamel defects, abnormal ruffled border morphology, and ameloblast detachment from the mineralizing surface. In vitro, a3-deficient ameloblasts exhibited significantly impaired adhesion to hydroxyapatite, decreased ODAM expression, and suppressed lysosomal acidification, indicating a3 is functionally required for maintaining ameloblast function and polarity. Mechanistically, Rab27A served as an important adaptor linking a3-positive secretory lysosomes to the microtubule network, enabling their polarized movement toward the distal plasma membrane. Disruption of this a3–Rab27A axis in a3KO cells mislocalized secretory lysosomes and defective ODAM delivery into the enamel matrix, compromising enamel mineralization. These findings reveal a new mechanism by which a3 orchestrates lysosomal positioning and ODAM secretion in enamel-forming cells. By integrating proton transport with vesicular trafficking and adhesion protein delivery, a3 functions as a key regulator of enamel mineralization. This study provides new insights into the pathogenesis of enamel hypomineralization and identifies a3 and its associated pathways as potential therapeutic targets for treating developmental enamel defects.

Keywords

maturation-stage ameloblasts odontogenic ameloblast-associated protein a3 knockout mice hypomineralization enamel mineralization

Get full access to this article

View all access options for this article.

References

Al Kawas

Amizuka

Bergeron

Warshawsky

1996. Immunolocalization of the cation-independent mannose 6-phosphate receptor and cathepsin B in the enamel organ and alveolar bone of the rat incisor. Calcif Tissue Int. 59(3):192–199. https://doi.org/10.1007/s002239900108

Arai

, et al 2022. Energy metabolic shift contributes to the phenotype modulation of maturation stage ameloblasts. Front Physiol. 13:1062042. https://doi.org/10.3389/fphys.2022.1062042

Bartlett

Simmer

. 2014. Kallikrein-related peptidase-4 (KLK4): role in enamel formation and revelations from ablated mice. Front Physiol. 5:240. https://doi.org/10.3389/fphys.2014.00240

Blott

Griffiths

. 2002. Secretory lysosomes. Nat Rev Mol Cell Biol. 3(2):122–131. https://doi.org/10.1038/nrm732

Bossi

Griffiths

. 1999. Degranulation plays an essential part in regulating cell surface expression of Fas ligand in T cells and natural killer cells. Nat Med. 5(1):90–96. https://doi.org/10.1038/4779

Bronckers

ALJJ

, et al 2012. Murine ameloblasts are immunonegative for Tcirg1, the v-H-ATPase subunit essential for the osteoclast plasma proton pump. Bone. 50(4):901–908. https://doi.org/10.1016/j.bone.2011.12.019

Chu

Zirngibl

Manolson

. 2021. The V-ATPase a3 subunit: structure, function and therapeutic potential of an essential biomolecule in osteoclastic bone resorption. Int J Mol Sci. 22(13):6934. https://doi.org/10.3390/ijms22136934

Eguchi

, et al 2024. The V-ATPase-ATG16L1 axis recruits LRRK2 to facilitate the lysosomal stress response. J Cell Biol. 223(3):e202302067. https://doi.org/10.1083/jcb.202302067

Forgac

. 2007. Vacuolar ATPases: rotary proton pumps in physiology and pathophysiology. Nat Rev Mol Cell Biol. 8(11):917–929. https://doi.org/10.1038/nrm2272

10.

Frattini

, et al 2000. Defects in TCIRG1 subunit of the vacuolar proton pump are responsible for a subset of human autosomal recessive osteopetrosis. Nat Genet. 25(3):343–346. https://doi.org/10.1038/77131

11.

Fukumoto

, et al 2014. New insights into the functions of enamel matrices in calcified tissues. Jpn Dent Sci Rev. 50(2):47–54. https://doi.org/10.1016/j.jdsr.2014.01.001

12.

Jaiswal

Andrews

Simon

. 2002. Membrane proximal lysosomes are the major vesicles responsible for calcium-dependent exocytosis in nonsecretory cells. J Cell Biol. 159(4):625–635. https://doi.org/10.1083/jcb.200208154

13.

Johnson

Ostrowski

Jaumouillé

Grinstein

2016. The position of lysosomes within the cell determines their luminal pH. J Cell Biol. 212(6):677–692. https://doi.org/10.1083/jcb.201507112

14.

Johnson

, et al 2017. V-ATPases containing a3 subunit play a direct role in enamel development in mice. J Cell Biochem. 118(10):3328–3340. https://doi.org/10.1002/jcb.25986

15.

Kawasaki

2013. Odontogenic ameloblast-associated protein (ODAM) and amelotin: major players in hypermineralization of enamel and enameloid. J Oral Biosci. 55(2):85–90. https://doi.org/10.1016/j.job.2013.02.001

16.

Lee

H-K

, et al 2010. The odontogenic ameloblast-associated protein (ODAM) cooperates with RUNX2 and modulates enamel mineralization via regulation of MMP-20. J Cell Biochem. 111(3):755–767. https://doi.org/10.1002/jcb.22766

17.

Lee

H-K

, et al 2012. Expression pattern, subcellular localization, and functional implications of ODAM in ameloblasts, odontoblasts, osteoblasts, and various cancer cells. Gene Expr Patterns. 12(3–4):102–108. https://doi.org/10.1016/j.gep.2012.02.002

18.

Lin

Nakamura

Noda

Ozawa

1994. Localization of H(+)-ATPase and carbonic anhydrase II in ameloblasts at maturation. Calcif Tissue Int. 55(1):38–45. https://doi.org/10.1007/BF00310167

19.

Luzio

Hackmann

Dieckmann

NMG

Griffiths

. 2014. The biogenesis of lysosomes and lysosome-related organelles. Cold Spring Harb Perspect Biol. 6(9):a016840. https://doi.org/10.1101/cshperspect.a016840

20.

Luzio

Pryor

Bright

. 2007. Lysosomes: fusion and function. Nat Rev Mol Cell Biol. 8(8):622–632. https://doi.org/10.1038/nrm2217

21.

Zhang

Duan

2023. Molecular mechanisms of craniofacial and dental abnormalities in osteopetrosis. Int J Mol Sci. 24(12):10412. https://doi.org/10.3390/ijms241210412

22.

Matsumoto

, et al 2014. Diversity of proton pumps in osteoclasts: V-ATPase with a3 and d2 isoforms is a major form in osteoclasts. Biochim Biophys Acta. 1837(6):744–749. https://doi.org/10.1016/j.bbabio.2014.02.011

23.

Matsumoto

, et al 2018. Essential role of the a3 isoform of V-ATPase in secretory lysosome trafficking via Rab7 recruitment. Sci Rep. 8(1):6701. https://doi.org/10.1038/s41598-018-24918-7

24.

Matsumoto

, et al 2020. V-ATPase a3 isoform mutations identified in osteopetrosis patients abolish its expression and disrupt osteoclast function. Exp Cell Res. 389(2):111901. https://doi.org/10.1016/j.yexcr.2020.111901

25.

Maxson

Grinstein

2014. The vacuolar-type H⁺-ATPase at a glance—more than a proton pump. J Cell Sci. 127(Pt 23):4987–4993. https://doi.org/10.1242/jcs.158550

26.

Moffatt

Smith

St-Arnaud

Nanci

2008. Characterization of Apin, a secreted protein highly expressed in tooth-associated epithelia. J Cell Biochem. 103(3):941–956. https://doi.org/10.1002/jcb.21465

27.

Nanci

, ed. 2008. Ten Cate’s oral histology: development, structure, and function. 7th ed. Mosby Elsevier.

28.

, et al 2023. Sugar transporter Slc37a2 regulates bone metabolism in mice via a tubular lysosomal network in osteoclasts. Nat Commun. 14(1):906. https://doi.org/10.1038/s41467-023-36484-2

29.

Palagano

, et al 2020. Generation of an immunodeficient mouse model of tcirg1-deficient autosomal recessive osteopetrosis. Bone Rep. 12:100242. https://doi.org/10.1016/j.bonr.2020.100242

30.

Saftig

Klumperman

2009. Lysosome biogenesis and lysosomal membrane proteins: trafficking meets function. Nat Rev Mol Cell Biol. 10(9):623–635. https://doi.org/10.1038/nrm2745

31.

Smith

. 1998. Cellular and chemical events during enamel maturation. Crit Rev Oral Biol Med. 9(2):128–161. https://doi.org/10.1177/10454411980090020101

32.

Sobacchi

, et al 2001. The mutational spectrum of human malignant autosomal recessive osteopetrosis. Hum Mol Genet. 10(17):1767–1773. https://doi.org/10.1093/hmg/10.17.1767

33.

Stenbeck

. 2002. Formation and function of the ruffled border in osteoclasts. Semin Cell Dev Biol. 13(4):285–292. https://doi.org/10.1016/s1084952102000587

34.

Teitelbaum

SL.

2011. The osteoclast and its unique cytoskeleton. Ann N Y Acad Sci. 1240(1):14–17. https://doi.org/10.1111/j.1749-6632.2011.06283.x

35.

Thesleff

. 2003. Epithelial-mesenchymal signalling regulating tooth morphogenesis. J Cell Sci. 116(Pt 9):1647–1648. https://doi.org/10.1242/jcs.00410

36.

Toei

Saum

Forgac

2010. Regulation and isoform function of the V-ATPases. Biochemistry. 49(23):4715–4723. https://doi.org/10.1021/bi100397s

37.

Vale

. 2003. The molecular motor toolbox for intracellular transport. Cell. 112(4):467–480. https://doi.org/10.1016/s0092-8674(03)00111-9

38.

van der Sluijs

Zibouche

van Kerkhof

. 2013. Late steps in secretory lysosome exocytosis in cytotoxic lymphocytes. Front Immunol. 4:359. https://doi.org/10.3389/fimmu.2013.00359

39.

Yamakoshi

, et al 2011. Enamel proteins and proteases in Mmp20 and Klk4 null and double-null mice: Mmp20/Klk4 double-null mice. Eur J Oral Sci. 119(Suppl 1):206–216. https://doi.org/10.1111/j.1600-0722.2011.00866.x

40.

Yin

, et al 2017. MiR-153 regulates amelogenesis by targeting endocytotic and endosomal/lysosomal pathways-novel insight into the origins of enamel pathologies. Sci Rep. 7:44118. https://doi.org/10.1038/srep44118

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.00 MB

15.09 MB

24.39 MB

5.16 MB

V-ATPase a3 Directs Secretory Lysosome Transport in Enamel Formation

Abstract

Keywords

Get full access to this article

References

Supplementary Material