L-Plastin deficiency produces increased trabecular bone due to attenuation of sealing ring formation and osteoclast dysfunction

Abstract

Bone resorption requires the formation of complex, actin-rich cytoskeletal structures. During the early phase of sealing ring formation by osteoclasts, L-plastin regulates actin-bundling to form the nascent sealing zones (NSZ). Here, we show that L-plastin knockout mice produce osteoclasts that are deficient in the formation of NSZs, are hyporesorptive, and make superficial resorption pits in vitro. Transduction of TAT-fused full-length L-plastin peptide into osteoclasts from L-plastin knockout mice rescued the formation of nascent sealing zones and sealing rings in a time-dependent manner. This response was not observed with mutated full-length L-plastin (Ser-5 and -7 to Ala-5 and -7) peptide. In contrast to the observed defect in the NSZ, L-plastin deficiency did not affect podosome formation or adhesion of osteoclasts in vitro or in vivo. Histomorphometry analyses in 8- and 12-week-old female L-plastin knockout mice demonstrated a decrease in eroded perimeters and an increase in trabecular bone density, without a change in bone formation by osteoblasts. This decrease in eroded perimeters supports that osteoclast function is attenuated in L-plastin knockouts. Micro-CT analyses confirmed a marked increase in trabecular bone mass. In conclusion, female L-plastin knockout mice had increased trabecular bone density due to impaired bone resorption by osteoclasts. L-plastin could be a potential target for therapeutic interventions to treat trabecular bone loss.

References

[1] Ma T, Samanna V, Chellaiah MA. Dramatic inhibition of osteoclast sealing ring formation and bone resorption in vitro by a WASP-peptide containing pTyr294 amino acid. J. Mol. Signal., 2008, 3

[2] Ma T, Sadashivaiah K, Chellaiah MA. Regulation of sealing ring formation by L-plastin and cortactin in osteoclasts. J. Biol. Chem., 2010, 285:29911

[3] Lakkakorpi PT et al. Abnormal localisation and hyperclustering of (alpha)(V)(beta)(3) integrins and associated proteins in Src-deficient or tyrphostin A9-treated osteoclasts. J. Cell Sci., 2001, 114 Pt 1 149

[4] Teitelbaum SL. The osteoclast and its unique cytoskeleton. Ann. N. Y. Acad. Sci., 2011, 1240:14

[5] Novack DV, Faccio R. Osteoclast motility: putting the brakes on bone resorption. Ageing Res. Rev., 2011, 10:54

[6] Georgess D et al. Podosome organization drives osteoclast-mediated bone resorption. Cell Adh. Migr., 2014, 8:191

[7] Nakamura I et al. Role of alpha(v)beta(3) integrin in osteoclast migration and formation of the sealing zone. J. Cell Sci., 1999, 112 Pt 22 3985

[8] Duong LT et al. PYK2 in osteoclasts is an adhesion kinase, localized in the sealing zone, activated by ligation of avb3 integrin, and phosphorylated by Src kinase. J. Clin. Investig., 1998, 102:881

[9] Geblinger D, Addadi L, Geiger B. Nano-topography sensing by osteoclasts. J. Cell Sci., 2010, 123 Pt 9 1503

[10] Lakkakorpi PT et al. Stable association of PYK2 and p130(Cas) in osteoclasts and their co- localization in the sealing zone. J. Biol. Chem., 1999, 274:4900

[11] Luxenburg C et al. Involvement of the Src-cortactin pathway in podosome formation and turnover during polarization of cultured osteoclasts. J. Cell Sci., 2006, 119 Pt 23 4878

[12] Wang Q et al. Regulation of the formation of osteoclastic actin rings by proline-rich tyrosine kinase 2 interacting with gelsolin. J. Cell Biol., 2003, 160:565

[13] Hiroi-Furuya E et al. Etidronate (EHDP) inhibits osteoclastic-bone resorption, promotes apoptosis and disrupts actin rings in isolate-mature osteoclasts. Calcif Tissue Int., 1999, 64:219

[14] Calle Y et al. WASp deficiency in mice results in failure to form osteoclast sealing zones and defects in bone resorption. Blood, 2004, 103:3552-3561

[15] Shemesh M et al. Study of osteoclast adhesion to cortical bone surfaces: a correlative microscopy approach for concomitant imaging of cellular dynamics and surface modifications. ACS Appl. Mater. Interfaces, 2016, 8:14932

[16] Chellaiah MA et al. Phosphorylation of a Wiscott–Aldrich syndrome protein-associated signal complex is critical in osteoclast bone resorption. J. Biol. Chem., 2007, 282:10104

[17] Chellaiah MA, Schaller MD. Activation of Src kinase by protein-tyrosine phosphatase-PEST in osteoclasts: comparative analysis of the effects of bisphosphonate and protein-tyrosine phosphatase inhibitor on Src activation in vitro. J. Cell Physiol., 2009, 220:382

[18] Chellaiah MA, Ma T, Majumdar S. L-plastin phosphorylation regulates the early phase of sealing ring formation by actin bundling process in mouse osteoclasts. Exp. Cell Res., 2018, 372:73

[19] Chellaiah MA, Majumdar S, Aljohani H. Peptidomimetic inhibitors of L-plastin reduce the resorptive activity of osteoclast but not the bone forming activity of osteoblasts in vitro. PLoS. ONE., 2018, 13

[20] Majumdar S et al. Engineering of L-plastin peptide-loaded biodegradable nanoparticles for sustained delivery and suppression of osteoclast function in vitro. Int. J. Cell Biol., 2019, 2019:6943986

[21] Lin CS, Lau A, Lue TF. Analysis and mapping of plastin phosphorylation. DNA Cell Biol., 1998, 17:1041

[22] Namba Y et al. Human T cell L-plastin bundles actin filaments in a calcium-dependent manner. J. Biochem. (Tokyo), 1992, 112:503

[23] Delanote V, Vandekerckhove J, Gettemans J. Plastins: versatile modulators of actin organization in (patho)physiological cellular processes. Acta Pharmacol. Sin., 2005, 26:769

[24] Kell MJ et al. Targeted deletion of the zebrafish actin-bundling protein L-plastin (lcp1). PLoS. ONE., 2018, 13

[25] Arpin M et al. Functional differences between L- and T-plastin isoforms. J. Cell Biol., 1994, 127 6 Pt 2 1995

[26] Bretscher A. Purification of the intestinal microvillus cytoskeletal proteins villin, fimbrin, and ezrin. Methods Enzymol., 1986, 134:24

[27] Chen H et al. Role for plastin in host defense distinguishes integrin signaling from cell adhesion and spreading. Immunity, 2003, 19:95

[28] Jones SL, Brown EJ. FcgammaRII-mediated adhesion and phagocytosis induce L-plastin phosphorylation in human neutrophils. J. Biol. Chem., 1996, 271:14623

[29] Foran E et al. The leukocyte protein L-plastin induces proliferation, invasion and loss of E-cadherin expression in colon cancer cells. Int. J. Cancer, 2006, 118:2098

[30] Marchisio PC et al. Cell-substratum interaction of cultured avian osteoclasts is mediated by specific adhesion structures. J. Cell Biol., 1984, 99:1696

[31] Messier JM et al. Fimbrin localized to an insoluble cytoskeletal fraction is constitutively phosphorylated on its headpiece domain in adherent macrophages. Cell Motil. Cytoskeleton, 1993, 25:223

[32] Babb SG et al. Fimbrin in podosomes of monocyte-derived osteoclasts. Cell Motil. Cytosk., 1997, 37:308

[33] Linder S, Aepfelbacher M. Podosomes: adhesion hot-spots of invasive cells. Trends Cell Biol., 2003, 13:376

[34] Linder S, Wiesner C. Tools of the trade: podosomes as multipurpose organelles of monocytic cells. Cell Mol. Life Sci., 2015, 72:121

[35] Morley SC et al. The actin-bundling protein L-plastin dissociates CCR7 proximal signaling from CCR7-induced motility. J. Immunol., 2010, 184:3628

[36] Todd EM et al. Alveolar macrophage development in mice requires L-plastin for cellular localization in alveoli. Blood, 2016, 128:2785

[37] Cervero P et al. “Proteomic analysis of podosome fractions from macrophages reveals similarities to spreading initiation centres,”. Eur. J. Cell Biol., 2012, 91:908

[38] De ClercqS et al. L-plastin nanobodies perturb matrix degradation, podosome formation, stability and lifetime in THP-1 macrophages. PLoS. ONE., 2013, 8

[39] Fuller K et al. TNFalpha potently activates osteoclasts, through a direct action independent of and strongly synergistic with RANKL. Endocrinology, 2002, 143:1108

[40] Azuma Y et al. Tumor necrosis factor-alpha induces differentiation of and bone resorption by osteoclasts. J. Biol. Chem., 2000, 275:4858

[41] Kaji K et al. Tumor necrosis factor alpha-induced osteoclastogenesis requires tumor necrosis factor receptor-associated factor 6. J. Bone Miner. Res., 2001, 16:1593

[42] Lam J et al. TNF-alpha induces osteoclastogenesis by direct stimulation of macrophages exposed to permissive levels of RANK ligand. J. Clin. Investig., 2000, 106:1481

[43] Jurdic P et al. Podosome and sealing zone: specificity of the osteoclast model. Eur. J. Cell Biol., 2006, 85:195

[44] Cougoule C et al. Blood leukocytes and macrophages of various phenotypes have distinct abilities to form podosomes and to migrate in 3D environments. Eur. J. Cell Biol., 2012, 91:938

[45] Saltel F et al. Apatite-mediated actin dynamics in resorbing osteoclasts. Mol. Biol. Cell, 2004, 15:5231

[46] Si M et al. LRRK1 regulation of actin assembly in osteoclasts involves serine 5 phosphorylation of L-plastin. J. Cell Biochem., 2018, 119:10351

[47] Chellaiah M et al. Gelsolin deficiency blocks podosome assembly and produces increased bone mass and strength. J. Cell Biol., 2000, 148:665

[48] Chellaiah MA et al. Osteopontin deficiency produces osteoclast dysfunction due to reduced CD44 surface expression. Mol. Biol. Cell, 2003, 14:173

[49] Boyce BF et al. TNF-alpha and pathologic bone resorption. Keio J. Med., 2005, 54:127

[50] Teti A et al. Immunolocalization of beta 3 subunit of integrins in osteoclast membrane. Boll. Soc. Ital. Biol. Sper., 1989, 65:1031

[51] Aubin JE. Osteoclast adhesion and resorption: the role of podosomes. J. Bone Miner. Res., 1992, 7:365

[52] Akisaka T et al. Organization of cytoskeletal F-actin, G-actin, and gelsolin in the adhesion structures in cultured osteoclast. J. Bone Miner. Res., 2001, 16:1248

[53] Luxenburg C, Addadi L, Geiger B. The molecular dynamics of osteoclast adhesions. Eur. J. Cell Biol., 2006, 85:203

[54] Linder S et al. Microtubule-dependent formation of podosomal adhesion structures in primary human macrophages. J. Cell Sci., 2000, 113 Pt 23 4165

[55] Zhou JY et al. L-Plastin promotes podosome longevity and supports macrophage motility. Mol. Immunol., 2016, 78:79

[56] Batsir S, Geiger B, Kam Z. Dynamics of the sealing zone in cultured osteoclasts. Cytoskeleton, 2017, 74:72

[57] Luxenburg C et al. The architecture of the adhesive apparatus of cultured osteoclasts: from podosome formation to sealing zone assembly. PLoS. ONE., 2007, 2

[58] Kanehisa J et al. A band of F-actin containing podosomes is involved in bone resorption by osteoclasts. Bone, 1990, 11:287

[59] Geblinger D, Geiger B, Addadi L. Surface-induced regulation of podosome organization and dynamics in cultured osteoclasts. Chembiochem., 2009, 10:158

[60] Frederick MJ et al. Characterization of the M(r) 65,000 lymphokine-activated killer proteins phosphorylated after tumor target binding: evidence that pp65a and pp65b are phosphorylated forms of L-plastin. Cancer Res., 1996, 56:138

[61] Klemke M et al. Phosphorylation of ectopically expressed L-plastin enhances invasiveness of human melanoma cells. Int. J. Cancer, 2007, 120:2590

[62] Lommel MJ et al. L-plastin Ser5 phosphorylation in breast cancer cells and in vitro is mediated by RSK downstream of the ERK/MAPK pathway. FASEB J., 2016, 30:1218

[63] Xu X et al. Mst1 kinase regulates the actin-bundling protein L-plastin to promote T cell migration. J. Immunol., 2016, 197:1683

[64] Heckel T et al. Src-dependent repression of ARF6 is required to maintain podosome-rich sealing zones in bone-digesting osteoclasts. Proc. Natl Acad. Sci. USA, 2009, 106:1451

[65] Karsdal MA et al. Are nonresorbing osteoclasts sources of bone anabolic activity? J. Bone Miner. Res., 2007, 22:487

[66] Van WesenbeeckL, Van HulW. Lessons from osteopetrotic mutations in animals: impact on our current understanding of osteoclast biology. Crit. Rev. Eukaryot. Gene Expr., 2005, 15:133

[67] Marks SC Jr. Osteoclast biology: lessons from mammalian mutations. Am. J. Med. Genet., 1989, 34:43

[68] Nagahara H et al. Transduction of full-length TAT fusion proteins into mammalian cells: TAT-p27Kip1 induces cell migration. Nat Med, 1998, 4:1449

[69] Chellaiah M et al. Rho-A is critical for osteoclast podosome organization, motility, and bone resorption. J. Biol. Chem., 2000, 275:11993

[70] Chellaiah MA, Hruska KA. The integrin avb3 and CD44 regulate the actions of osteopontin on osteoclast motility. Calcif. Tissue Int., 2002, 72:197

[71] Choi JY et al. Diminished canonical beta-catenin signaling during osteoblast differentiation contributes to osteopenia in progeria. J. Bone Miner. Res., 2018, 33:2059

[72] Moorer MC et al. Defective signaling, osteoblastogenesis and bone remodeling in a mouse model of connexin 43 C-terminal truncation. J. Cell Sci., 2017, 130:531

[73] Buo AM et al. Connexin43 and Runx2 interact to affect cortical bone geometry, skeletal development, and osteoblast and osteoclast function. J. Bone Miner. Res., 2017, 32:1727

[74] Dempster DW et al. Standardized nomenclature, symbols, and units for bone histomorphometry: a 2012 update of the report of the ASBMR Histomorphometry Nomenclature Committee. J. Bone Miner. Res., 2013, 28:2

[75] Jilka RL et al. Linkage of decreased bone mass with impaired osteoblastogenesis in a murine model of accelerated senescence. J. Clin. Investig., 1996, 97:1732

[76] Weinstein RS et al. The effects of androgen deficiency on murine bone remodeling and 659 bone mineral density are mediated via cells of the osteoblastic lineage. Endocrinology, 1997, 138:4013

[77] Parfitt AM et al. Bone histomorphometry: Standardization of nomenclature, symbols, and units. J. Bone Miner. Res., 1987, 2:595

[78] Yingling V, Elle SaineM, Joshi R. Hypothalamic suppression decreases bone strength before and after puberty in a rat model. Calcif. Tissue Int., 2009, 84:485

[79] Yingling VR. A delay in pubertal onset affects the covariation of body weight, estradiol, and bone size. Calcif. Tissue Int., 2009, 84:286

[80] Brennan T et al. Abrogation of Cbl-PI3K interaction increases bone formation and osteoblast proliferation. Calcif. Tissue Int., 2011, 89:396

Funding

U.S. Department of Health & Human Services | NIH | Center for Scientific Review (NIH Center for Scientific Review)(AI104732)