mTORC1 induces plasma membrane depolarization and promotes preosteoblast senescence by regulating the sodium channel Scn1a

Abstract

Senescence impairs preosteoblast expansion and differentiation into functional osteoblasts, blunts their responses to bone formation-stimulating factors and stimulates their secretion of osteoclast-activating factors. Due to these adverse effects, preosteoblast senescence is a crucial target for the treatment of age-related bone loss; however, the underlying mechanism remains unclear. We found that mTORC1 accelerated preosteoblast senescence in vitro and in a mouse model. Mechanistically, mTORC1 induced a change in the membrane potential from polarization to depolarization, thus promoting cell senescence by increasing Ca²⁺ influx and activating downstream NFAT/ATF3/p53 signaling. We further identified the sodium channel Scn1a as a mediator of membrane depolarization in senescent preosteoblasts. Scn1a expression was found to be positively regulated by mTORC1 upstream of C/EBPα, whereas its permeability to Na⁺ was found to be gated by protein kinase A (PKA)-induced phosphorylation. Prosenescent stresses increased the permeability of Scn1a to Na⁺ by suppressing PKA activity and induced depolarization in preosteoblasts. Together, our findings identify a novel pathway involving mTORC1, Scn1a expression and gating, plasma membrane depolarization, increased Ca²⁺ influx and NFAT/ATF3/p53 signaling in the regulation of preosteoblast senescence. Pharmaceutical studies of the related pathways and agents might lead to novel potential treatments for age-related bone loss.

References

[1] Owen, R. & Reilly, G. C. In vitro models of bone remodelling and associated disorders. Front. Bioeng. Biotechnol. 6, 134 (2018).

[2] Farr JN et al. Identification of senescent cells in the bone microenvironment. J. Bone Miner. Res., 2016, 31: 1920-1929

[3] Farr JN et al. Targeting cellular senescence prevents age-related bone loss in mice. Nat. Med., 2017, 23: 1072-1079

[4] Manolagas SC, Jilka RL. Bone marrow, cytokines, and bone remodeling. Emerging insights into the pathophysiology of osteoporosis. N. Engl. J. Med., 1995, 332: 305-311

[5] Kassem M, Marie PJ. Senescence-associated intrinsic mechanisms of osteoblast dysfunctions. Aging Cell, 2011, 10: 191-197

[6] Manolagas SC, Parfitt AM. What old means to bone. Trends Endocrinol. Metab., 2010, 21: 369-374

[7] Long F. Building strong bones: molecular regulation of the osteoblast lineage. Nat. Rev. Mol. Cell Biol., 2011, 13: 27-38

[8] Kim HN et al. DNA damage and senescence in osteoprogenitors expressing Osx1 may cause their decrease with age. Aging Cell, 2017, 16: 693-703

[9] Cao JJ et al. Aging impairs IGF-I receptor activation and induces skeletal resistance to IGF-I. J. Bone Miner. Res., 2007, 22: 1271-1279

[10] Pfeilschifter J et al. Mitogenic responsiveness of human bone cells in vitro to hormones and growth factors decreases with age. J. Bone Min. Res, 1993, 8: 707-717

[11] Ankrom MA et al. Age-related changes in human oestrogen receptor alpha function and levels in osteoblasts. Biochem. J., 1998, 333: 787-794

[12] Undale A et al. Circulating osteogenic cells: characterization and relationship to rates of bone loss in postmenopausal women. Bone, 2010, 47: 83-92

[13] Papadopoli, D. et al. mTOR as a central regulator of lifespan and aging. F1000Res. 8, F1000 Faculty Rev_998 (2019).

[14] Guertin DA et al. Ablation in mice of the mTORC components raptor, rictor, or mLST8 reveals that mTORC2 is required for signaling to Akt-FOXO and PKCα, but Not S6K1. Dev. Cell, 2006, 11: 859-871

[15] Kapahi P et al. With TOR, less is more: a key role for the conserved nutrient-sensing TOR pathway in aging. Cell Metab., 2010, 11: 453-465

[16] Huang B et al. mTORC1 prevents preosteoblast differentiation through the notch signaling pathway. PLoS Genet, 2015, 11: e1005426

[17] Huang B et al. Osteoblasts secrete Cxcl9 to regulate angiogenesis in bone. Nat. Commun., 2016, 7

[18] Höber R. Über den Einfluss der Salze auf den Ruhestrom des Froschmuskels. Arch. f.ür. die Gesamt. Physiologie des. Menschen und der Tiere, 1905, 106: 599-635

[19] Carroll B et al. Persistent mTORC1 signaling in cell senescence results from defects in amino acid and growth factor sensing. J. Cell Biol., 2017, 216: 1949-1957

[20] Warnier M et al. The SCN9A channel and plasma membrane depolarization promote cellular senescence through Rb pathway. Aging Cell, 2018, 17: e12736

[21] Chen J, Long F. mTORC1 signaling promotes osteoblast differentiation from preosteoblasts. PloS ONE, 2015, 10: e0130627-e0130627

[22] Fitter S et al. mTORC1 plays an important role in skeletal development by controlling preosteoblast differentiation. Mol. Cell. Biol., 2017, 37: e00668-00616

[23] Catterall WA. Forty years of sodium channels: structure, function, pharmacology, and epilepsy. Neurochem. Res., 2017, 42: 2495-2504

[24] Laplante M, Sabatini DM. An emerging role of mTOR in lipid biosynthesis. Curr. Biol., 2009, 19: R1046-R1052

[25] Long YS et al. Identification of the promoter region and the 5’-untranslated exons of the human voltage-gated sodium channel Nav1.1 gene (SCN1A) and enhancement of gene expression by the 5’-untranslated exons. J. Neurosci. Res., 2008, 86: 3375-3381

[26] Smith RD, Goldin AL. Phosphorylation at a single site in the rat brain sodium channel is necessary and sufficient for current reduction by protein Kinase A. J. Neurosci., 1997, 17: 6086-6093

[27] Smith RD, Goldin AL. Functional analysis of the rat I sodium channel in xenopus oocytes. J. Neurosci., 1998, 18: 811-820

[28] Liu S, Zheng P. Altered PKA modulation in the Na(v)1.1 epilepsy variant I1656M. J. Neurophysiol., 2013, 110: 2090-2098

[29] Jin B et al. PIM-1 modulates cellular senescence and links IL-6 signaling to heterochromatin formation. Aging Cell, 2014, 13: 879-889

[30] Courtney MJ, Lambert JJ, Nicholls DG. The interactions between plasma membrane depolarization and glutamate receptor activation in the regulation of cytoplasmic free calcium in cultured cerebellar granule cells. J. Neurosci., 1990, 10: 3873-3879

[31] Wiel C et al. Endoplasmic reticulum calcium release through ITPR2 channels leads to mitochondrial calcium accumulation and senescence. Nat. Commun., 2014, 5

[32] Martin N, Bernard D. Calcium signaling and cellular senescence. Cell Calcium, 2018, 70: 16-23

[33] Hogan PG, Chen L, Nardone J, Rao A. Transcriptional regulation by calcium, calcineurin, and NFAT. Genes Dev., 2003, 17: 2205-2232

[34] Wu X et al. Opposing roles for calcineurin and ATF3 in squamous skin cancer. Nature, 2010, 465: 368-372

[35] d’Adda di Fagagna F. Living on a break: cellular senescence as a DNA-damage response. Nat. Rev. Cancer, 2008, 8: 512-522

[36] Amigorena S, Choquet D, Teillaud JL, Korn H, Fridman WH. Ion channel blockers inhibit B cell activation at a precise stage of the G1 phase of the cell cycle. Possible involvement of K⁺ channels. J. Immunol., 1990, 144: 2038-2045

[37] Nelson MT, Quayle JM. Physiological roles and properties of potassium channels in arterial smooth muscle. Am. J. Physiol. Cell Physiol., 1995, 268: C799-C822

[38] Lewis R et al. The role of the membrane potential in chondrocyte volume regulation. J. Cell. Physiol., 2011, 226: 2979-2986

[39] Rorsman P, Ashcroft FM. Pancreatic β-cell electrical activity and insulin secretion: of mice and men. Physiol. Rev., 2018, 98: 117-214

[40] Girault A, Brochiero E. Evidence of K+ channel function in epithelial cell migration, proliferation, and repair. Am. J. Physiol. Cell Physiol., 2014, 306: C307-C319

[41] Noguchi T, Wang CW, Pan H, Welsh DK. Fibroblast circadian rhythms of PER2 expression depend on membrane potential and intracellular calcium. Chronobiol. Int., 2012, 29: 653-664

[42] Lallet-Daher H et al. Potassium channel KCNA1 modulates oncogene-induced senescence and transformation. Cancer Res., 2013, 73: 5253-5265

[43] Catterall WA. Voltage-gated calcium channels. Cold Spring Harb. Perspect. Biol., 2011, 3: a003947-a003947

[44] Fei D et al. Ca(v) 1.2 regulates osteogenesis of bone marrow-derived mesenchymal stem cells via canonical Wnt pathway in age-related osteoporosis. Aging Cell, 2019, 18: e12967

[45] Escayg A et al. Mutations of SCN1A, encoding a neuronal sodium channel, in two families with GEFS+2. Nat. Genet., 2000, 24: 343-345

[46] Catterall WA, Kalume F, Oakley JC. NaV1.1 channels and epilepsy. J. Physiol., 2010, 588: 1849-1859

[47] Sumikawa K, Parker I, Miledi R. Partial purification and functional expression of brain mRNAs coding for neurotransmitter receptors and voltage-operated channels. Proc. Natl Acad. Sci. U.S.A., 1984, 81: 7994-7998

[48] Hirono C et al. Characterization of mRNA responsible for induction of functional sodium channels in Xenopus oocytes. Brain Res, 1985, 359: 57-64

[49] Goldin AL et al. Messenger RNA coding for only the alpha subunit of the rat brain Na channel is sufficient for expression of functional channels in Xenopus oocytes. Proc. Natl Acad. Sci. U.S.A, 1986, 83: 7503-7507

[50] Martin MS, Tang B, Ta N, Escayg A. Characterization of 5’ untranslated regions of the voltage-gated sodium channels SCN1A, SCN2A, and SCN3A and identification of cis-conserved noncoding sequences. Genomics, 2007, 90: 225-235

[51] Kim JE, Chen J. regulation of peroxisome proliferator-activated receptor-gamma activity by mammalian target of rapamycin and amino acids in adipogenesis. Diabetes, 2004, 53: 2748-2756

[52] Gangloff YG et al. Disruption of the mouse mTOR gene leads to early postimplantation lethality and prohibits embryonic stem cell development. Mol. Cell Biol., 2004, 24: 9508-9516

[53] Porstmann T et al. SREBP activity is regulated by mTORC1 and contributes to Akt-dependent cell growth. Cell Metab., 2008, 8: 224-236

[54] Williams GC. Pleiotropy, natural selection, and the evolution of senescence. Evolution, 1957, 11: 398-411

[55] Wachter KW, Evans SN, Steinsaltz D. The age-specific force of natural selection and biodemographic walls of death. Proc. Natl Acad. Sci. U.S.A., 2013, 110: 10141-10146

[56] Finkel T. Relief with rapamycin: mTOR inhibition protects against radiation-induced mucositis. Cell Stem Cell, 2012, 11: 287-288

[57] Li M, West JW, Lai Y, Scheuer T, Catterall WA. Functional modulation of brain sodium channels by cAMP-dependent phosphorylation. Neuron, 1992, 8: 1151-1159

[58] Bakker AD, Klein-Nulend J. Osteoblast isolation from murine calvaria and long bones. Methods Mol. Biol., 2012, 816: 19-29

[59] Zhu D, You J, Zhao N, Xu H. Magnesium regulates endothelial barrier functions through TRPM7, MagT1, and S1P1. Adv. Sci., 2019, 6: 1901166

Funding

Natural Science Foundation of Guangdong Province (Guangdong Natural Science Foundation)(2019A1515011876)

National Natural Science Foundation of China (National Science Foundation of China)(82172507)

202002030176 from Guangzhou Municipal Science and Technology Bureau