ATP6AP2, a regulator of LRP6/β-catenin protein trafficking, promotes Wnt/β-catenin signaling and bone formation in a cell type dependent manner

Abstract

Wnt/β-catenin signaling is critical for various cellular processes in multiple cell types, including osteoblast (OB) differentiation and function. Exactly how Wnt/β-catenin signaling is regulated in OBs remain elusive. ATP6AP2, an accessory subunit of V-ATPase, plays important roles in multiple cell types/organs and multiple signaling pathways. However, little is known whether and how ATP6AP2 in OBs regulates Wnt/β-catenin signaling and bone formation. Here we provide evidence for ATP6AP2 in the OB-lineage cells to promote OB-mediated bone formation and bone homeostasis selectively in the trabecular bone regions. Conditionally knocking out (CKO) ATP6AP2 in the OB-lineage cells (Atp6ap2^Ocn-Cre) reduced trabecular, but not cortical, bone formation and bone mass. Proteomic and cellular biochemical studies revealed that LRP6 and N-cadherin were reduced in ATP6AP2-KO BMSCs and OBs, but not osteocytes. Additional in vitro and in vivo studies revealed impaired β-catenin signaling in ATP6AP2-KO BMSCs and OBs, but not osteocytes, under both basal and Wnt stimulated conditions, although LRP5 was decreased in ATP6AP2-KO osteocytes, but not BMSCs. Further cell biological studies uncovered that osteoblastic ATP6AP2 is not required for Wnt3a suppression of β-catenin phosphorylation, but necessary for LRP6/β-catenin and N-cadherin/β-catenin protein complex distribution at the cell membrane, thus preventing their degradation. Expression of active β-catenin diminished the OB differentiation deficit in ATP6AP2-KO BMSCs. Taken together, these results support the view for ATP6AP2 as a critical regulator of both LRP6 and N-cadherin protein trafficking and stability, and thus regulating β-catenin levels, demonstrating an un-recognized function of osteoblastic ATP6AP2 in promoting Wnt/LRP6/β-catenin signaling and trabecular bone formation.

References

1. Baron R.& Kneissel, M. WNT signaling in bone homeostasis and disease: from human mutations to treatments. Nat. Med. 19, 179-192 (2013).
2. Xiong, L.et al. Lrp4 in osteoblasts suppresses bone formation and promotes osteoclastogenesis and bone resorption. Proc. Natl. Acad. Sci. USA 112, 3487-3492 (2015).
3. Glass, D. A. 2ndet al. Canonical Wnt signaling in differentiated osteoblasts controls osteoclast differentiation. Dev. Cell 8, 751-764 (2005).
4. Williams B. O.& Insogna, K. L. Where Wnts went: the exploding field of Lrp5 and Lrp6 signaling in bone. J. Bone Min. Res. 24, 171-178 (2009).
5. Gong, Y.et al. LDL receptor-related protein 5 (LRP5) affects bone accrual and eye development. Cell 107, 513-523 (2001).
6. Laine, C. M.et al.Novel mutations affecting LRP5 splicing in patients with osteoporosis-pseudoglioma syndrome (OPPG). Eur. J. Hum. Genet. 19, 875-881 (2011).
7. Boyden, L. M.et al.High bone density due to a mutation in LDL-receptor-related protein 5. N. Engl. J. Med. 346, 1513-1521 (2002).
8. Little, R. D.et al.A mutation in the LDL receptor-related protein 5 gene results in the autosomal dominant high-bone-mass trait. Am. J. Hum. Genet. 70, 11-19 (2002).
9. Ichihara A.& Yatabe, M. S. The (pro)renin receptor in health and disease. Nat. Rev. Nephrol. 15, 693-712 (2019).
10. Wu, C. H.et al. Adipocyte (pro)renin-receptor deficiency induces lipodystrophy, liver steatosis and increases blood pressure in male mice. Hypertension 68, 213-219 (2016).
11. Arthur, G., Osborn, J. L.& Yiannikouris, F. B.(Pro)renin receptor in the kidney: function and significance. Am. J. Physiol. Regul. Integr. Comp. Physiol. 320, R377-R383 (2021).
12. Nguyen, G.et al.Pivotal role of the renin/prorenin receptor in angiotensin II production and cellular responses to renin. J. Clin. Invest. 109, 1417-1427 (2002).
13. Nguyen G.& Muller, D. N. The biology of the (pro)renin receptor. J. Am. Soc. Nephrol. 21, 18-23 (2010).
14. Beyenbach K. W.& Wieczorek, H. The V-type H+ ATPase: molecular structure and function, physiological roles and regulation. J. Exp. Biol. 209, 577-589 (2006).
15. Kinouchi, K.et al.The (pro)renin receptor/ATP6AP2 is essential for vacuolar H+-ATPase assembly in murine cardiomyocytes. Circ. Res. 107, 30-34 (2010).
16. Buechling, T.et al.Wnt/Frizzled signaling requires dPRR, the Drosophila homolog of the prorenin receptor. Curr. Biol. 20, 1263-1268 (2010).
17. Cruciat, C. M.et al. Requirement of prorenin receptor and vacuolar H+-ATPasemediated acidification for Wnt signaling. Science 327, 459-463 (2010).
18. Li, Z.et al.(Pro)renin receptor is an amplifier of Wnt/beta-catenin signaling in kidney injury and fibrosis. J. Am. Soc. Nephrol. 28, 2393-2408 (2017).
19. Ichihara, A.et al. Nonproteolytic activation of prorenin contributes to development of cardiac fibrosis in genetic hypertension. Hypertension 47, 894-900 (2006).
20. Oba-Yabana,I. et al. Acidic organelles mediate TGF-beta1-induced cellular fibrosis via (pro)renin receptor and vacuolar ATPase trafficking in human peritoneal mesothelial cells. Sci. Rep. 8, 2648(2018).
21. Wang, L.et al.Sodium butyrate suppresses angiotensin II-induced hypertension by inhibition of renal (pro)renin receptor and intrarenal renin-angiotensin system. J. Hypertens. 35, 1899-1908 (2017).
22. Kurlak L. O., Mistry H. D., Cindrova-Davies, T., Burton, G. J. & Broughton Pipkin, F. Human placental renin-angiotensin system in normotensive and pre-eclamptic pregnancies at high altitude and after acute hypoxia-reoxygenation insult. J. Physiol. 594, 1327-1340 (2016).
23. Fukushima, A.et al.(Pro)renin receptor in skeletal muscle is involved in the development of insulin resistance associated with postinfarct heart failure in mice. Am. J. Physiol. Endocrinol. Metab. 307, E503-E514 (2014).
24. Satofuka, Set al. (Pro)renin receptor-mediated signal transduction and tissue renin-angiotensin system contribute to diabetes-induced retinal inflammation. Diabetes 58, 1625-1633 (2009).
25. Saigusa, T.et al.Activation of the intrarenal renin-angiotensin-system in murine polycystic kidney disease. Physiol. Rep. 3, e12405(2015).
26. Greco, C. M.et al.Chemotactic effect of prorenin on human aortic smooth muscle cells: a novel function of the (pro)renin receptor. Cardiovasc. Res. 95, 366-374 (2012).
27. Stransky, L., Cotter, K.& Forgac, M. The function of V-ATPases in cancer. Physiol. Rev. 96, 1071-1091 (2016).
28. Tan, P.et al.Impact of the prorenin/renin receptor on the development of obesity and associated cardiometabolic risk factors. Obes. (Silver Spring) 22, 2201-2209 (2014).
29. Goldstein, B., Speth, R. C.& Trivedi, M. Renin-angiotensin system gene expression and neurodegenerative diseases. J. Renin. Angiotensin Aldosterone Syst. 17, 1470320316666750(2016).
30. Schafer, S. T., Peters, J.& von Bohlen Und Halbach, O. The (pro)renin receptor / ATP6ap2 is expressed in the murine hippocampus by adult and newly generated neurons. Restor. Neurol. Neurosci. 31, 225-231 (2013).
31. Valenzuela, R.et al.Location of prorenin receptors in primate substantia nigra: effects on dopaminergic cell death. J. Neuropathol. Exp. Neurol. 69, 1130-1142 (2010).
32. Zhang, M.et al.Osteoblast-specific knockout of the insulin-like growth factor (IGF) receptor gene reveals an essential role of IGF signaling in bone matrix mineralization. J. Biol. Chem. 277, 44005-44012 (2002).
33. Pan, J. X.et al.YAP promotes osteogenesis and suppresses adipogenic differentiation by regulating beta-catenin signaling. Bone Res. 6, 18(2018).
34. Isojima T.& Sims, N. A. Cortical bone development, maintenance and porosity: genetic alterations in humans and mice influencing chondrocytes, osteoclasts, osteoblasts and osteocytes. Cell Mol. Life Sci. 78, 5755-5773 (2021).
35. Xiong, J.et al.Osteocytes, not osteoblasts or lining cells, are the main source of the RANKL required for osteoclast formation in remodeling bone. PLoS One 10, e0138189 (2015).
36. Artigas N., Urena C.,Rodriguez-Carballo, E., Rosa, J. L. & Ventura, F. Mitogenactivated protein kinase (MAPK)-regulated interactions between Osterix and Runx2 are critical for the transcriptional osteogenic program. J. Biol. Chem. 289, 27105-27117 (2014).
37. Day T. F., Guo X., Garrett-Beal, L. & Yang, Y. Wnt/beta-catenin signaling in mesenchymal progenitors controls osteoblast and chondrocyte differentiation during vertebrate skeletogenesis. Dev. Cell 8, 739-750 (2005).
38. Marie P. J.& Hay, E. Cadherins and Wnt signalling: a functional link controlling bone formation. Bonekey Rep. 2, 330(2013).
39. Bonewald L. F.The amazing osteocyte. J. Bone Min. Res. 26, 229-238 (2011).
40. Franz-Odendaal, T. A., Hall, B. K. & Witten, P. E. Buried alive: how osteoblasts become osteocytes. Dev. Dyn. 235, 176-190 (2006).
41. Nefussi J. R., Sautier J. M., Nicolas V.& Forest, N. How osteoblasts become osteocytes: a decreasing matrix forming process. J. Biol. Buccal. 19, 75-82 (1991).
42. Ludwig, J.et al.Identification and characterization of a novel 9.2-kD membrane sector-associated protein of vacuolar proton-ATPase from chromaffin granules. J. Biol. Chem. 273, 10939-10947 (1998).
43. Tamai, K.et al. A mechanism for Wnt coreceptor activation. Mol. Cell 13, 149-156 (2004).
44. Blitzer J. T.& Nusse, R. A critical role for endocytosis in Wnt signaling. BMC Cell Biol. 7, 28(2006).
45. Yamamoto, H., Komekado, H. & Kikuchi, A. Caveolin is necessary for Wnt-3adependent internalization of LRP6 and accumulation of beta-catenin. Dev. Cell 11, 213-223 (2006).
46. Nabhan, A. N.et al. Targeted alveolar regeneration with Frizzled-specific agonists. Cell 186, 2995-3012 e2915 (2023).
47. Riddle, R. C.et al.Lrp5 and Lrp6 exert overlapping functions in osteoblasts during postnatal bone acquisition. PLoS One 8, e63323 (2013).
48. Sebastian, A.et al.Wnt co-receptors Lrp5 and Lrp6 differentially mediate Wnt3a signaling in osteoblasts. PLoS One 12, e0188264 (2017).
49. Chen J.& Long, F. beta-catenin promotes bone formation and suppresses bone resorption in postnatal growing mice. J. Bone Min. Res. 28, 1160-1169 (2013).
50. Song, L.et al.Loss of wnt/beta-catenin signaling causes cell fate shift of preosteoblasts from osteoblasts to adipocytes. J. Bone Min. Res. 27, 2344-2358 (2012).
51. Xiong, L.et al.ATP6AP2-to-MMP14, a key pathway for osteoblast to osteocyte transition. bioRxiv, 2022.2004.2027.489713. https://doi.org/10.1101/2022.04.27.48 9713(2022)
52. Liu, J.et al.Wnt/beta-catenin signalling: function, biological mechanisms, and therapeutic opportunities. Signal Transduct. Target Ther. 7, 3(2022).
53. Mazhab-Jafari, M. T.et al. Atomic model for the membrane-embedded VO motor of a eukaryotic V-ATPase. Nature 539, 118-122 (2016).
54. Kinouchi, K., Ichihara, A.& Itoh, H. Functional characterization of (pro)renin receptor in association with V-ATPase. Front. Biosci. (Landmark Ed.) 16, 3216-3223 (2011).
55. Louagie, E.et al. Role of furin in granular acidification in the endocrine pancreas: identification of the V-ATPase subunit Ac45 as a candidate substrate. Proc. Natl. Acad. Sci. USA 105, 12319-12324 (2008).
56. Feng, H.et al.Cytoplasmic terminus of vacuolar type proton pump accessory subunit Ac45 is required for proper interaction with V(0) domain subunits and efficient osteoclastic bone resorption. J. Biol. Chem. 283, 13194-13204 (2008).
57. Qin, A.et al.Versatile roles of V-ATPases accessory subunit Ac45 in osteoclast formation and function. PLoS One 6, e27155 (2011).
58. Jansen E. J.R. et al. Novel vertebrate- and brain-specific driver of neuronal outgrowth. Prog. Neurobiol. 202, 102069(2021).
59. Xiong, L.et al.Osteoblastic Lrp4 promotes osteoclastogenesis by regulating ATP release and adenosine-A2AR signaling. J. Cell Biol. 216, 761-778 (2017).
60. Duan X., Yang S., Zhang, L. & Yang, T. V-ATPases and osteoclasts: ambiguous future of V-ATPases inhibitors in osteoporosis. Theranostics 8,5379-5399 (2018).
61. Stern, A. R.et al. Isolation and culture of primary osteocytes from the long bones of skeletally mature and aged mice. Biotechniques 52, 361-373 (2012).
62. Xiong, L.et al. Retromer in osteoblasts interacts with protein phosphatase 1 regulator subunit 14C, terminates parathyroid hormone’s signaling,promotes its catabolic response. EBioMedicine 9, 45-60 (2016).
63. Xiong, L.et al.Linking skeletal muscle aging with osteoporosis by lamin A/C deficiency. PLoS Biol. 18, e3000731(2020).

Funding

Wen-Cheng Xiong ([e-mail])