Osteoblasts are inherently programmed to repel sensory innervation

Abstract

Tissue innervation is a complex process controlled by the expression profile of signaling molecules secreted by tissue-resident cells that dictate the growth and guidance of axons. Sensory innervation is part of the neuronal network of the bone tissue with a defined spatiotemporal occurrence during bone development. Yet, the current understanding of the mechanisms regulating the map of sensory innervation in the bone tissue is still limited. Here, we demonstrated that differentiation of human mesenchymal stem cells to osteoblasts leads to a marked impairment of their ability to promote axonal growth, evidenced under sensory neurons and osteoblastic-lineage cells crosstalk. The mechanisms by which osteoblast lineage cells provide this nonpermissive environment for axons include paracrine-induced repulsion and loss of neurotrophic factors expression. We identified a drastic reduction of NGF and BDNF production and stimulation of Sema3A, Wnt4, and Shh expression culminating at late stage of OB differentiation. We noted a correlation between Shh expression profile, OB differentiation stages, and OB-mediated axonal repulsion. Blockade of Shh activity and signaling reversed the repulsive action of osteoblasts on sensory axons. Finally, to strengthen our model, we localized the expression of Shh by osteoblasts in bone tissue. Overall, our findings provide evidence that the signaling profile associated with osteoblast phenotype differentiating program can regulate the patterning of sensory innervation, and highlight osteoblast-derived Shh as an essential player in this cue-induced regulation.

References

[1] Ahrén B. Autonomic regulation of islet hormone secretion—implications for health and disease. Diabetologia, 2000, 43:393-410

[2] Kandilis AN, Papadopoulou IP, Koskinas J, Sotiropoulos G, Tiniakos DG. Liver innervation and hepatic function: new insights. J. Surg. Res., 2015, 194:511-519

[3] Bi S et al. Differential body weight and feeding responses to high-fat diets in rats and mice lacking cholecystokinin 1 receptors. Am. J. Physiol. Integr. Comp. Physiol., 2007, 293:R55-R63

[4] Ashrafi M, Baguneid M, Bayat A. The role of neuromediators and innervation in cutaneous wound healing. Acta Derm. Venereol., 2016, 96:587-594

[5] Leitão L et al. The alliance between nerve fibers and stem cell populations in bone marrow: life partners in sickness and health. FASEB J, 2019, 33:8697-8710

[6] Sousa DM et al. Ablation of Y1 receptor impairs osteoclast bone-resorbing activity. Sci. Rep., 2016, 6

[7] Stoeckli ET. Understanding axon guidance: are we nearly there yet? Development, 2018, 145:dev151415

[8] Dickson BJ. Molecular mechanisms of axon guidance. Science, 2002, 298:1959-1964

[9] Brazill JM, Beeve AT, Craft CS, Ivanusic JJ, Scheller EL. Nerves in bone: evolving concepts in pain and anabolism. J. Bone Miner. Res., 2019, 34:1393-1406

[10] Mediati RD, Vellucci R, Dodaro L. Pathogenesis and clinical aspects of pain in patients with osteoporosis. Clin. Cases Miner. Bone Metab., 2014, 11:169-172

[11] Chartier SR et al. Exuberant sprouting of sensory and sympathetic nerve fibers in nonhealed bone fractures and the generation and maintenance of chronic skeletal pain. Pain, 2014, 155:2323-2336

[12] Bloom AP et al. Breast cancer-induced bone remodeling, skeletal pain, and sprouting of sensory nerve fibers. J. Pain., 2011, 12:698-711

[13] Jimenez-Andrade JM et al. Pathological sprouting of adult nociceptors in chronic prostate cancer-induced bone pain. J. Neurosci., 2010, 30:14649-14656

[14] Ghilardi JR et al. Administration of a tropomyosin receptor kinase inhibitor attenuates sarcoma-induced nerve sprouting, neuroma formation and bone cancer pain. Mol. Pain., 2010, 6

[15] Mach DB et al. Origins of skeletal pain: sensory and sympathetic innervation of the mouse femur. Neuroscience, 2002, 113:155-166

[16] Sisask G, Silfverswärd CJ, Bjurholm A, Nilsson O. Ontogeny of sensory and autonomic nerves in the developing mouse skeleton. Auton. Neurosci. Basic Clin., 2013, 177:237-243

[17] Hukkanen M et al. Rapid proliferation of calcitonin gene-related peptide-immunoreactive nerves during healing of rat tibial fracture suggests neural involvement in bone growth and remodelling. Neuroscience, 1993, 54:969-979

[18] Li J, Kreicbergs A, Bergström J, Stark A, Ahmed M. Site-specific CGRP innervation coincides with bone formation during fracture healing and modeling: a study in rat angulated tibia. J. Orthop. Res., 2007, 25:1204-1212

[19] Fukuda T et al. Sema3A regulates bone-mass accrual through sensory innervations. Nature, 2013, 497:490-493

[20] Niedermair T et al. Absence of substance P and the sympathetic nervous system impact on bone structure and chondrocyte differentiation in an adult model of endochondral ossification. Matrix Biol., 2014, 38:22-35

[21] Silva DI, Santos BP, dos Leng J, Oliveira H, Amédée J. Dorsal root ganglion neurons regulate the transcriptional and translational programs of osteoblast differentiation in a microfluidic platform. Cell Death Dis., 2017, 8

[22] Offley SC et al. Capsaicin-sensitive sensory neurons contribute to the maintenance of trabecular bone integrity. J. Bone Miner. Res., 2005, 20:257-267

[23] Heffner MA, Anderson MJ, Yeh GC, Genetos DC, Christiansen BA. Altered bone development in a mouse model of peripheral sensory nerve inactivation. J. Musculoskelet. Neuronal Interact., 2014, 14:1-9

[24] Teixeira FG, Carvalho MM, Sousa N, Salgado AJ. Mesenchymal stem cells secretome: a new paradigm for central nervous system regeneration? Cell. Mol. Life Sci., 2013, 70:3871-3882

[25] Martins LF et al. Mesenchymal stem cells secretome-induced axonal outgrowth is mediated by BDNF. Sci. Rep., 2017, 7

[26] Zhu S et al. Subchondral bone osteoclasts induce sensory innervation and osteoarthritis pain. J. Clin. Invest., 2019, 129:1076-1093

[27] Tomlinson RE et al. NGF-TrkA signaling by sensory nerves coordinates the vascularization and ossification of developing endochondral bone. Cell Rep., 2016, 16:2723-2735

[28] Tomlinson RE et al. NGF-TrkA signaling in sensory nerves is required for skeletal adaptation to mechanical loads in mice. Proc. Natl Acad. Sci., 2017, 114:E3632-E3641

[29] Maia FR, Lourenço AH, Granja PL, Gonçalves RM, Barrias CC. Effect of cell density on mesenchymal stem cells aggregation in RGD-alginate 3D matrices under osteoinductive conditions. Macromol. Biosci., 2014, 14:759-771

[30] Bessa, S., Quelhas, P. & Amaral, I. F. Automatic quantification of cell outgrowth from neurospheres. In: Sanches, J.M., Micó, L. & Cardoso, J.S.(eds). Pattern Recognition and Image Analysis. IbPRIA 2013. Lecture Notes in Computer Science Vol. 7887 Springer, Berlin, Heidelberg (2013).

[31] Haberberger RV, Barry C, Dominguez N, Matusica D. Human dorsal root ganglia. Front. Cell. Neurosci., 2019, 13:271

[32] Bhatheja K, Field J. Schwann cells: origins and role in axonal maintenance and regeneration. Int. J. Biochem. Cell Biol., 2006, 38:1995-1999

[33] Taylor AM et al. A microfluidic culture platform for CNS axonal injury, regeneration and transport. Nat. Methods, 2005, 2:599-605

[34] Neto E et al. Sensory neurons and osteoblasts: close partners in a microfluidic platform. Integr. Biol. (Camb.)., 2014, 6:586-595

[35] Crigler L, Robey RC, Asawachaicharn A, Gaupp D, Phinney DG. Human mesenchymal stem cell subpopulations express a variety of neuro-regulatory molecules and promote neuronal cell survival and neuritogenesis. Exp. Neurol., 2006, 198:54-64

[36] Wilkins A et al. Human bone marrow-derived mesenchymal stem cells secrete brain-derived neurotrophic factor which promotes neuronal survival in vitro. Stem Cell Res., 2009, 3:63-70

[37] Gu Y et al. Neurotrophic actions of bone marrow stromal cells on primary culture of dorsal root ganglion tissues and neurons. J. Mol. Neurosci., 2010, 40:332-341

[38] Kurozumi K et al. Mesenchymal stem cells that produce neurotrophic factors reduce ischemic damage in the rat middle cerebral artery occlusion model. Mol. Ther., 2005, 11:96-104

[39] Su Y-W et al. Roles of neurotrophins in skeletal tissue formation and healing. J. Cell. Physiol., 2018, 233:2133-2145

[40] Peng J et al. Sonic hedgehog is a remotely produced cue that controls axon guidance trans-axonally at a midline choice point. Neuron, 2018, 97:326-340.e4

[41] Parra LM, Zou Y. Sonic hedgehog induces response of commissural axons to Semaphorin repulsion during midline crossing. Nat. Neurosci., 2010, 13:29-35

[42] Maun HR et al. Hedgehog pathway antagonist 5E1 binds hedgehog at the pseudo-active site. J. Biol. Chem., 2010, 285:26570-26580

[43] Yam PT, Charron F. Signaling mechanisms of non-conventional axon guidance cues: the Shh, BMP and Wnt morphogens. Curr. Opin. Neurobiol., 2013, 23:965-973

[44] Guo D, Standley C, Bellve K, Fogarty K, Bao Z-Z. Protein kinase Cα and integrin-linked kinase mediate the negative axon guidance effects of Sonic hedgehog. Mol. Cell. Neurosci., 2012, 50:82-92

[45] Yam PT, Langlois SD, Morin S, Charron F. Sonic hedgehog guides axons through a noncanonical, Src-family-kinase-dependent signaling pathway. Neuron, 2009, 62:349-362

[46] Yang J, Andre P, Ye L, Yang Y-Z. The hedgehog signalling pathway in bone formation. Int. J. Oral. Sci., 2015, 7:73-79

[47] Horikiri Y et al. Sonic hedgehog regulates osteoblast function by focal adhesion kinase signaling in the process of fracture healing. PLoS ONE, 2013, 8

[48] Nakamura T et al. Induction of osteogenic differentiation by hedgehog proteins. Biochem. Biophys. Res. Commun., 1997, 237:465-469

[49] Yuasa T et al. Sonic hedgehog is involved in osteoblast differentiation by cooperating with BMP-2. J. Cell. Physiol., 2002, 193:225-232

[50] Ribeiro-da-Silva M et al. Interplay between sympathetic nervous system and inflammation in aseptic loosening of hip joint replacement. Sci. Rep., 2018, 8

[51] Jimenez-Andrade JM et al. Pathological sprouting of adult nociceptors in chronic prostate cancer-induced bone pain. J. Neurosci., 2010, 30:14649-14656

[52] Serre CM, Farlay D, Delmas PD, Chenu C. Evidence for a dense and intimate innervation of the bone tissue, including glutamate-containing fibers. Bone, 1999, 25:623-629

[53] Markus A, Patel TD, Snider WD. Neurotrophic factors and axonal growth. Curr. Opin. Neurobiol., 2002, 12:523-531

[54] Liu Y, Beyer A, Aebersold R. On the dependency of cellular protein levels on mRNA abundance. Cell, 2016, 165:535-550

[55] Laufer E, Nelson CE, Johnson RL, Morgan BA, Tabin C. Sonic hedgehog and Fgf-4 act through a signaling cascade and feedback loop to integrate growth and patterning of the developing limb bud. Cell, 1994, 79:993-1003

[56] Comer JD, Alvarez S, Butler SJ, Kaltschmidt JA. Commissural axon guidance in the developing spinal cord: from Cajal to the present day. Neural Dev., 2019, 14

[57] Zhao H et al. Secretion of shh by a neurovascular bundle niche supports mesenchymal stem cell homeostasis in the adult mouse incisor. Cell Stem Cell, 2014, 14:160-173

[58] Kiuru M et al. Sonic hedgehog expands diaphyseal trabecular bone altering bone marrow niche and lymphocyte compartment. Mol. Ther., 2009, 17:1442-1452

[59] Domanitskaya E et al. Sonic hedgehog guides post-crossing commissural axons both directly and indirectly by regulating Wnt activity. J. Neurosci., 2010, 30:11167-11176

[60] Neto E et al. Axonal outgrowth, neuropeptides expression and receptors tyrosine kinase phosphorylation in 3D organotypic cultures of adult dorsal root ganglia. PLoS ONE, 2017, 12

[61] Neto E, Paramos-de-Carvalho D, Lourenço AH, Aguiar P, Lamghari M. Compartmentalized microfluidic platforms as tool of choice to study the interaction between neurons and osteoblasts. Neuromethods, 2015, 103:161-179

Funding

Ministry of Education and Science | Fundação para a Ciência e a Tecnologia (Portuguese Science and Technology Foundation)(SFRH/BD/128771/2017)