High glucose up-regulates Semaphorin 3A expression via the mTOR signaling pathway in keratinocytes: A potential mechanism and therapeutic target for diabetic small fiber neuropathy
Introduction
With the high prevalence of diabetes in China (Yang et al., 2010), diabetic peripheral neuropathy (DPN) has become a major cause of foot ulceration and amputation, affecting up to 50% of diabetic patients (Tesfaye et al., 2010). DPN can involve both small and large fibers, and small fibers are affected earlier and are more prone to be damaged than large fibers (Smith et al., 2001, Sumner et al., 2003, Tesfaye et al., 2010). Small fibers (thinly myelinated Aδ and unmyelinated C fibers) originated in the dorsal nerve root ganglia (DRG), mediate pain, temperature sensation, and autonomic functions (Hoeijmakers et al., 2012), and terminate in the skin as intraepidermal nerve fibers (IENFs) (Novotny and Gommert-Novotny, 1988). Therefore, numerous investigators have suggested that assessment of intraepidermal nerve fiber density (IENFD) during a skin biopsy is a reliable and standardized technique for diagnosing small fiber neuropathy (SFN) (Chen et al., 2015, Loseth et al., 2008, Tesfaye et al., 2010, Ziegler et al., 2014).
Keratinocytes, the major epidermal cell type, can modulate epidermal innervation by synthesizing and secreting numerous nerve repulsion factors and nerve elongation factors (Kumamoto et al., 2014, Roggenkamp et al., 2012, Tominaga et al., 2009, Tominaga and Takamori, 2014). Semaphorin 3A (Sema3A), a member of the semaphorin family, acts as the first secreted repulsion molecule in axonal formation during neuronal development (Messersmith et al., 1995), and in regeneration after peripheral nervous system injury (Pasterkamp et al., 1998). Sema3A which is also produced by keratinocytes (Fukamachi et al., 2011, Tominaga and Takamori, 2014) is one of the most important nerve repulsion factors involved in regulating epidermal innervation in skin diseases (Tominaga et al., 2009, Tominaga and Takamori, 2014, Yamaguchi et al., 2008). However, whether Sema3A affects IENFs in diabetes is unknown. Studies have demonstrated that Sema3A is elevated in the vitreous of diabetic patients with retinopathy and in the podocytes of diabetic patients with nephropathy (Aggarwal et al., 2015, Cerani et al., 2013). Hyperglycemic conditions can also lead to the dysregulation of keratinocyte morphology, differentiation, mobility and proliferation (Spravchikov et al., 2001, Usui et al., 2008). An important question is therefore whether high glucose (HG) induces Sema3A expression in keratinocytes. Many reports have suggested that the severity of diabetic SFN is closely related to the degree of hyperglycemia (Liu et al., 2015, Sveen et al., 2013). Sema3A, depending on its concentration, has been reported to inhibit the growth of DRG neuronal axons in vitro (Ben-Zvi et al., 2008, Kumamoto et al., 2014). Therefore, in this study we assessed whether HG up-regulated Sema3A expression in keratinocytes, which might then damage IENFs and contribute to diabetic SFN.
The mammalian target of rapamycin (mTOR) stimulates protein synthesis and regulates glucose metabolism (Duvel et al., 2010, Krebs et al., 2007, Ma and Blenis, 2009) via phosphorylation and subsequent activation of its downstream effectors. These downstream effectors are the p70 ribosomal protein S6 kinase (p70 S6K) and the eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) (Hay and Sonenberg, 2004, Ma and Blenis, 2009), activation of which can be inhibited by the mTOR inhibitor rapamycin (Hay and Sonenberg, 2004). Studies have shown that mTOR mediated-local protein synthesis can participate in the development of axons and dendrites (Asante et al., 2010, Jimenez-Diaz et al., 2008, Raab-Graham et al., 2006) and can modulate transmission of pain messages (Asante et al., 2010, Obara and Hunt, 2014). Because the mTOR inhibitor attenuates streptozotocin (STZ)-induced hyperalgesia (He et al., 2016)and nerve injury-induced hypersensitivity (Jimenez-Diaz et al., 2008), the use of rapamycin is therefore a potential therapeutic strategy for the treatment of peripheral neuropathy. Several experiments have shown that HG activates mTOR signaling in many different types of cells, such as HT22 cells and hippocampal neurons (Liu et al., 2016a, Wu et al., 2017). mTOR signaling has been shown to regulate Sema3A mRNA expression in osteoblast-like MC3T3-E1 cells (Yamada et al., 2016a). Based on these published data, we wished to investigate whether HG regulates the expression of Sema3A via mTOR signaling. A previous study has indicated that mTOR/4E-BP1 signaling can be activated in growth cones by recombinant Sema3A (rSema3A) (Manns et al., 2012). Additionally, Sema3A can regulate mTOR/p70 S6K signaling in Lewis lung carcinoma cells (Yamada et al., 2016b, Yamada et al., 2016c). Therefore, we hypothesized that a positive feedback loop exists between the Sema3A and mTOR signaling pathways that might aggravate the pathology caused by HG up-regulation of Sema3A expression.
In this study, we used the STZ-induced diabetic rat model to mimic some of the functional and structural features of diabetic SFN. We evaluated the Sema3A and mTOR-mediated p70 S6K and 4E-BP1 signaling pathways, as well as IENFD in diabetic patients and STZ-induced diabetic rats. We assessed Sema3A and mTOR signaling in HaCaT cells which had been treated with HG or rSema3A in the presence or absence of rapamycin. Rapamycin was also used to elucidate the roles of mTOR signaling in diabetic SFN. Our data offer new a new perspective in the potential molecular mechanisms underlying diabetic SFN and provide a novel therapeutic target for the treatment of DPN.
Section snippets
Participants
The study was conducted in XiangYa Hospital of Central South University, Hunan, China, between July 2015 and August 2016. All subjects were screened for symptoms, and the signs and risk factors of peripheral neuropathy by history, examination, and clinical chemistry. All patients were eligible to enroll in the study based on the following inclusion/exclusion criteria. The inclusion criteria included: (1) a diagnosis of diabetes mellitus based on the 1999 WHO Diagnostic criteria; (2) an age of
Increased Sema3A expression and over-activation of mTOR signaling are accompanied by reduced IENFD in the skin of diabetic patients with SFN
The demographic and clinical characteristics of the control subjects and diabetic patients with SFN are presented in Table 1. There was no significant difference in sex, age, BMI and percentage of smokers between the two groups. HbA1c was markedly higher in the diabetic patients compared to the control group, with a duration of diabetes of 7.8 ± 1.1 years. The average NSS values was 5.2 ± 0.3 in the diabetic group, suggesting moderate symptoms of SFN based on the NSS criteria. At the same time,
Discussion
Small fibers, which constitute 70–90% of peripheral nerve fibers(Malik et al., 2011), are the earliest and most likely fibers to be involved in DPN (Smith et al., 2001, Sumner et al., 2003, Tesfaye et al., 2010). SFN is characterized by dysfunction or loss of these fibers. Therefore using a skin biopsy assessment of IENFD and QST to quantify thermal and cold thresholds is regarded as a reliable tool to assess SFN (Chen et al., 2015, Hoeijmakers et al., 2012, Tesfaye et al., 2010, Ziegler
Disclosure summary
The authors have nothing to disclose.
Acknowledgements
The authors would like to thank Ming-Liang Chen, Pin-Ting Zhou, Zhuo-Feng Ding, Central South University, Changsha, China, for their helps on experiments, and thank Li-Na Zhao, Sun Yet-Sen University, Guangzhou, China, as well as Junru Chen, Suzhou Geneworks Technology, Suzhou, China, for their help on manuscript writing.
This study was supported by the National Natural Science Foundation of China (No. 81670771 and 81573480).
References (59)
- et al.
Mammalian target of rapamycin signaling in the spinal cord is required for neuronal plasticity and behavioral hypersensitivity associated with neuropathy in the rat
J. Pain
(2010) - et al.
Semaphorin3A regulates axon growth independently of growth cone repulsion via modulation of TrkA signaling
Cell SIGNAL
(2008) - et al.
Neuron-derived semaphorin 3A is an early inducer of vascular permeability in diabetic retinopathy via neuropilin-1
Cell Metab.
(2013) - et al.
Activation of a metabolic gene regulatory network downstream of mTOR complex 1
Mol. Cell
(2010) - et al.
Increased cutaneous NGF and CGRP-labelled trkA-positive intra-epidermal nerve fibres in rat diabetic skin
Neurosci. Lett.
(2012) - et al.
Modulation of semaphorin 3A expression by calcium concentration and histamine in human keratinocytes and fibroblasts
J. Dermatol Sci.
(2011) - et al.
Intrathecal administration of rapamycin inhibits the phosphorylation of DRG Nav1.8 and attenuates STZ-induced painful diabetic neuropathy in rats
Neurosci. Lett.
(2016) - et al.
Impaired wound healing results from the dysfunction of the Akt/mTOR pathway in diabetic rats
J. Dermatol Sci.
(2015) - et al.
Semaphorin III can function as a selective chemorepellent to pattern sensory projections in the spinal cord
Neuron
(1995) - et al.
Regulation of semaphorin III/collapsin-1 gene expression during peripheral nerve regeneration
Exp. Neurol.
(1998)
Gleevec/imatinib, an ABL2 kinase inhibitor, protects tumor and endothelial cells from semaphorin-induced cytoskeleton collapse and loss of cell motility
Biochem. Biophys. Res. Commun.
Atopic keratinocytes induce increased neurite outgrowth in a coculture model of porcine dorsal root ganglia neurons and human skin cells
J. Invest. Dermatol
Regulation of elongation phase of mRNA translation in diabetic nephropathy: amelioration by rapamycin
Am. J. Pathol.
Psoralen-ultraviolet A therapy alters epidermal Sema3A and NGF levels and modulates epidermal innervation in atopic dermatitis
J. Dermatol Sci.
mTORC1 is a critical mediator of oncogenic Semaphorin3A signaling
Biochem. Biophys. Res. Commun.
Autocrine Semaphorin3A signaling is essential for the maintenance of stem-like cells in lung cancer
Biochem. Biophys. Res. Commun.
Semaphorin3A alleviates skin lesions and scratching behavior in NC/Nga mice, an atopic dermatitis model
J. Invest. Dermatol
Semaphorin3a promotes advanced diabetic nephropathy
Diabetes
mTORC1 promotes proliferation of immature Schwann cells and myelin growth of differentiated Schwann cells
Proc. Natl. Acad. Sci. U. S. A.
Small nerve fiber quantification in the diagnosis of diabetic sensorimotor polyneuropathy: comparing corneal confocal microscopy with intraepidermal nerve fiber density
Diabetes Care
Neuroprotective effects of Gymnema sylvestre on streptozotocin-induced diabetic neuropathy in rats
Exp. Ther. Med.
Method for quantitative estimation of thermal thresholds in patients
J. Neurol. Neurosurg. Psychiatry
Peripheral, but not central, axotomy induces neuropilin-1 mRNA expression in adult large diameter primary sensory neurons
J. Comp. Neurol.
Upstream and downstream of mTOR
Genes Dev.
Small-fibre neuropathies–advances in diagnosis, pathophysiology and management
Nat. Rev. Neurol.
Chronic rapamycin treatment causes glucose intolerance and hyperlipidemia by upregulating hepatic gluconeogenesis and impairing lipid deposition in adipose tissue
DIABETES
Local translation in primary afferent fibers regulates nociception
PLoS One
Peripheral neuropathy in mouse models of diabetes
Curr. Protoc. Mouse Biol.
The Mammalian target of rapamycin pathway regulates nutrient-sensitive glucose uptake in man
Diabetes
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