Elsevier

Acta Biomaterialia

Volume 97, 1 October 2019, Pages 399-408
Acta Biomaterialia

Full length article
Regulation of chitosan-mediated differentiation of human olfactory receptor neurons by insulin-like growth factor binding protein-2

https://doi.org/10.1016/j.actbio.2019.08.022Get rights and content

Abstract

Olfaction is normally taken for granted in our lives, not only assisting us to escape from dangers, but also increasing our quality of life. Although olfactory neuroepithelium (ON) can reconstitute its olfactory receptor neurons (ORNs) after injury, no adequate treatment for olfactory loss has yet emerged. The present study investigates the role of glycosaminoglycans (GAGs) in modulating olfactory neuronal homeostasis and elucidates the regulatory mechanism. This work isolates and cultures human olfactory neuroepithelial cells (HONCs) with various GAGs for 7 days, and find that chitosan promotes ORN maturation, expressing olfactory marker protein (OMP) and its functional components. Growth factor protein array, ELISA and western blot analysis reveal that insulin-like growth factor binding protein 2 (IGFBP2) shows a higher level in chitosan-treated HONCs than in controls. Biological activity of insulin-like growth factor-1 (IGF-1), IGF-2 and IGF-1 receptor (IGF1R) is further investigated. Experimental results indicate that IGF-1 and IGF-2 enhance the growth of immature ORNs, expressing βIII tubulin, but decrease mature ORNs. Instead, down-regulation of phosphorylated IGF1R lifts the OMP expression, and lowers the βIII tubulin expression, by incubation with the phosphorylated inhibitor of IGF1R, OSI-906. Finally, the effect of chitosan on ORN maturity is antagonized by concurrently adding IGFBP2 protease, matrix metallopeptidase-1. Overall, our data demonstrate that chitosan promotes ORN differentiation by raising the level of IGFBP2 to sequestrate the IGFs-IGF1R signaling.

Statement of Significance

Olfactory dysfunction serves as a crucial alarm in neurodegenerative diseases, and one of its causes is lacking of sufficient mature olfactory receptor neurons to detect odorants in the air. However, the clinical treatment for olfactory dysfunction is still controversial. Chitosan is the natural linear polysaccharide and exists in rat olfactory neuroepithelium. Previously, chitosan has been demonstrated to mediate the differentiation of olfactory receptor neurons in an in vitro rat model, but the mechanism is unknown. The study aims to evaluate the role and mechanism of chitosan in an in vitro human olfactory neurons model. Overall, these results reveal that chitosan is a potential agent for treating olfactory disorder by the maintenance of olfactory neural homeostasis. This is the first report to demonstrate that chitosan promotes differentiation of olfactory receptor neurons through increasing IGFBP2 to sequestrate the IGFs-IGF1R.

Introduction

Animals rely on olfaction not only to coordinate food appreciation and selection, but also to identify dangerous environmental hazards. Above 15% of the world population suffers from olfactory dysfunction, affecting their quality of life and mental health [1], [2]. The onset of dysfunction is typically progressive, and has no comprehensive solution at present. Topical or systemic application of glucocorticoids is a common clinical treatment, but its therapeutic effect is controversial [3], [4]. Hence, alternative treatment modalities are essential for patients with olfactory loss. The etiology of olfactory dysfunction results from hyperplasia of sustentacular cells (SCs) or reduced number and degree of differentiation of olfactory receptor neurons (ORNs), which are the first relay of odor sensation and innervation of the olfactory bulb in the olfactory system [5], [6]. ORNs and SCs are main cell population in the olfactory neuroepithelium (ON), expressing neurofilament (NF) and cytokeratin 18 (CK18), respectively. Both cells are continuously replenished by basal cells, expressing cytokeratin 5 (CK5) [7], [8], [9]. New ORNs have to possess olfactory marker protein (OMP) and even signal transduction markers, olfactory neuron specific-G protein (Golf) and adenylate cyclase 3 (ADCY3), to define the restorative olfaction [10]. Notably, Golf and ADCY3 are sensors of olfactory receptors, ultimately leading in depolarization because of the increased level of cyclic AMP [11]. Additionally, the distribution of various components of the extracellular matrix (ECM) in the olfactory systems indicates that these molecules may guide olfactory neuronal homeostasis.

Glycosaminoglycans (GAGs) are abundant in the native ON of rats and given a role in cell differentiation and axon guidance [12], [13]. GAGs are linear polysaccharides composed of different disaccharide units, such as chondroitin sulfate (CS), heparin sulfate (HS) and hyaluronic acid (HA). They are crucial for the structural properties of the ECM, and are used for a wide range of biological events, including tissue morphogenesis, cell signaling and growth factor interactions [14]. For example, in the rat brain olfactory bulb, the insulin-like growth factor-binding protein-2 (IGFBP2) can bind to CS and HS [15] that allows focal concentration of IGFBP2 bound insulin-like growth factors (IGFs) in the pericellular environment to regulate IGFs biological activity by modulating interaction of IGFs with their receptors [16]. Chondroitin sulfate was reported to treat anosmic patient successfully in 1961, but has not been explored since then over several decades [17]. Chitosan, a natural cationic polysaccharide with a variable number of randomly located d-glucosamine (GlcN) and N-acetyl-glucosamine (GlcNAc) groups, can affect cellular function similarly to GAGs [18]. Our previous studies have shown that chitosan is a promising agent for promoting ORN differentiation of rat olfactory neuroepithelial cells in vitro. Chitosan films induce formation of olfactory neurospheres containing immature and mature ORNs with functional elements in an in vitro rat model [19], [20]. However, whether chitosan and GAGs can promote ORN differentiation of human olfactory neuroepithelial cells (HONCs) remains unexplored, and the mechanism of chitosan-mediated differentiation of HONCs is still unknown. This investigation evaluates the effect of chitosan and GAGs on the development and differentiation of HONCs, and then elucidates whether the relationship between chitosan and responsive cells is through a defined regulatory pathway.

Section snippets

Isolation and culture of HONCs

HONCs were obtained from nasal superior turbinates near the roof of the nasal cavity in patients with chronic rhinitis during septomeatoplasty, following a protocol detailed previously, and approved by an institutional review board of Far Eastern Memorial Hospital (105104-F). All patients gave informed consent. Briefly, the sample was rinsed, digested and resuspended in the Iscove’s modified Dulbecco’s media (IMDM; Invitrogen) with 10% fetal bovine serum and 1% antibiotics. Equal quantities of

Establishment of a primary HONC culture

The ON taken from surgical specimens was characterized by examining its histology and phenotype. Biopsies of approximately 0.3 cm3 were raised against representative markers of HONCs, namely CK5 and OMP, which were distributed in the basal and middle layer of ON, respectively (Fig. 1A–B & D). The CK5 was adopted to determine whether the cell population incorporated progenitor cells. As shown in Fig. 1C, CK5 was immunoreactive in the cytoplasm, and co-expressed in the nucleus with BrdU, an

Discussion

The diverse epithelia lining the nasal cavity serve different functions. The predominant epithelium is respiratory epithelium (90%), which not only moistens the airways, but also prevents infection and tissue injury by the action of mucociliary clearance. Respiratory epithelial cells are usually cultured in vitro with air-liquid interface, which induces mucociliary differentiation of respiratory epithelial cells [25]. In contrast, human ON is patchily scattered and occupies <10% of the nasal

Conclusion

The experimental results demonstrate that chitosan promotes ORN differentiation and enhances formation of components of olfactory-specific signal transduction pathway of HONCs. The mechanism is through increasing IGFBP2 to sequestrate the IGFs-IGF1R signaling.

Acknowledgments

The authors would like to thank the Ministry of Science and Technology of Taiwan and Far Eastern Memorial Hospital for financially supporting this research under Contract No. MOST-108-2314-B-418-009-MY3 and FEMH-108-2314-B-418-009-MY3.

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      Chitosan, a natural cationic polysaccharide with a variable number of randomly located d-glucosamine (GlcN) and N-acetyl-glucosamine (GlcNAc) groups, can affect cellular function similarly to GAGs [27]. Our previous studies have shown that chitosan is a promising agent for promoting ORN differentiation of olfactory neuroepithelial cells in vitro [28–30] and the regulatory pathway is through increasing IGFBP2 to sequestrate the IGFs-type 1 receptor signaling, which inhibits maturation of ORNs [31]. However, whether chitosan can further promote regeneration of ON after olfactory injury and improve the olfactory function in vivo remains unexplored.

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