Zinc inhibits TRPV1 to alleviate chemotherapy-induced neuropathic pain

43 Pages·2017·8.32 MB·English

This Accepted Manuscript has not been copyedited and formatted. The final version may differ from this version.
Research Articles: Cellular/Molecular
Zinc inhibits TRPV1 to alleviate chemotherapy-induced neuropathic pain
Jialie Luo1, Alexis Bavencoffe2, Pu Yang1, Jing Feng1, Shijin Yin3, Aihua Qian4, Weihua Yu5, Shenbin
Liu1, Xuan Gong1, Tao Cai1, Edgar T. Walters2, Carmen W. Dessauer2 and Hongzhen Hu1
1Center for the Study of Itch, Department of Anesthesiology, Washington University School of Medicine, St.
Louis, MO 63110, USA
2Department of Integrative Biology and Pharmacology, University of Texas Health Science Center at Houston,
Houston, TX 77030, USA
3College of Pharmacy, South-Central University for Nationalities, Wuhan, Hubei 430073, China
4Department of Gastroenterology, Ruijin Hospital, Shanghai Jiaotong University, Shanghai 200240, China
5Department of Anatomy, Chongqing Medical University, Chongqing 400016, China
DOI: 10.1523/JNEUROSCI.1816-17.2017
Received: 29 June 2017
Revised: 24 October 2017
Accepted: 16 November 2017
Published: 30 November 2017
Author contributions: J.L., A.B., P.Y., J.F., and H.H. performed research; J.L., S.Y., A.Q., W.Y., S.L., X.G.,
T.C., and H.H. analyzed data; J.L. and H.H. wrote the paper; E.T.W., C.W.D., and H.H. designed research.
Conflict of Interest: The authors declare no competing financial interests.
The authors declare no potential conflicts of interest. We are grateful to Gina Story (Washington University
in St.Louis) for providing the TRPA1 KO mice. This work was supported partly by grants from the National
Institutes of Health, R01RGM101218 and R01DK103901 (to H. H.), The Center for the Study of Itch of
Department of Anesthesiology at Washington University School of Medicine to H. H.
Corresponding author: Hongzhen Hu, Ph.D, Department of Anesthesiology, The Center for the Study of Itch
Washington University School of Medicine in St. Louis Campus Box 8054 660 South Euclid Avenue St. Louis,
MO 63110-1093 Tel: [phone] Fax: [phone] Email: [email protected]
Cite as: J. Neurosci ; 10.1523/JNEUROSCI.1816-17.2017
Alerts: Sign up at www.jneurosci.org/cgi/alerts to receive customized email alerts when the fully formatted
version of this article is published.
Accepted manuscripts are peer-reviewed but have not been through the copyediting, formatting, or proofreading
process.
Copyright © 2017 the authors
1 Zinc inhibits TRPV1 to alleviate chemotherapy-induced neuropathic pain
2
3 Jialie Luo1, Alexis Bavencoffe2, Pu Yang1, Jing Feng1, Shijin Yin3, Aihua Qian4, Weihua Yu5,
4 Shenbin Liu1, Xuan Gong1, Tao Cai1, Edgar T. Walters2, Carmen W. Dessauer2, Hongzhen Hu1,*
5
6 1 Center for the Study of Itch, Department of Anesthesiology, Washington University School of
7 Medicine, St. Louis, MO 63110, USA
8 2 Department of Integrative Biology and Pharmacology, University of Texas Health Science
9 Center at Houston, Houston, TX 77030, USA
10 3 College of Pharmacy, South-Central University for Nationalities, Wuhan, Hubei 430073, China
11 4 Department of Gastroenterology, Ruijin Hospital, Shanghai Jiaotong University, Shanghai
12 200240, China
13 5 Department of Anatomy, Chongqing Medical University, Chongqing 400016, China
14 * Corresponding author
15
16 Running title: Zinc inhibits paclitaxel-induced pain
17
18
19
20
1
21 Corresponding author:
22 Hongzhen Hu, Ph.D
23 Department of Anesthesiology
24 The Center for the Study of Itch
25 Washington University School of Medicine in St. Louis
26 Campus Box 8054
27 660 South Euclid Avenue
28 St. Louis, MO 63110-1093
29 Tel: [phone]
30 Fax: [phone]
31 Email: [email protected]
32
33 Number of pages: 35
34 Number of figures: 6
35 Number of tables: 1
36 Number of words for Abstract: 154
37 Number of words for Introduction: 641
38 Number of words for Discussion: 1110
39
40
41
42
43
44
2
45 Acknowledgments
46 The authors declare no potential conflicts of interest. We are grateful to Gina Story (Washington
47 University in St.Louis) for providing the TRPA1 KO mice. This work was supported partly by
48 grants from the National Institutes of Health, R01RGM101218 and R01DK103901 (to H. H.),
49 The Center for the Study of Itch of Department of Anesthesiology at Washington University
50 School of Medicine to H. H.
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
3
68 Abstract
69
70 Zinc is a transition metal that has a long history of use as an anti-inflammatory agent. It also
71 soothes pain sensations in a number of animal models. However, the effects and mechanisms of
72 zinc on chemotherapy-induced peripheral neuropathy (CIPN) remain unknown. Here we show
73 that locally injected zinc markedly reduces neuropathic pain in male and female mice induced by
74 paclitaxel, a chemotherapy drug, in a TRPV1-dependent manner. Extracellularly applied zinc
75 also inhibits the function of TRPV1 expressed in HEK293 cells and mouse DRG neurons, which
76 requires the presence of zinc-permeable TRPA1 to mediate entry of zinc into the cytoplasm.
77 Moreover, TRPA1 is required for zinc-induced inhibition of TRPV1-mediated acute nociception.
78 Unexpectedly, zinc transporters, but not TRPA1 are required for zinc-induced inhibition of
79 TRPV1-dependent chronic neuropathic pain produced by paclitaxel. Taken together, our study
80 demonstrates a novel mechanism underlying the analgesic effect of zinc on paclitaxel-induced
81 neuropathic pain that relies on the function of TRPV1.
82
83 Significance Statement
84 The chemotherapy-induced peripheral neuropathy is a major limiting factor affecting the
85 chemotherapy patients. There is no effective treatment available currently. We demonstrated that
86 zinc prevented paclitaxel-induced mechanical hypersensitivity via inhibiting the TRPV1 channel,
87 which is involved in the sensitization of peripheral nociceptors in chemotherapy. Zinc
88 transporters in DRG neurons are required for the entry of zinc into the intracellular side, where it
89 inhibits TRPV1. Our study provided insight into the mechanism underlying the pain soothing
90 effect of zinc and suggests that zinc could be developed to therapeutics for the treatment of CIPN.
4
91 Introduction
92
93 Chemotherapy-induced peripheral neuropathy (CIPN), a highly debilitating symptom without
94 effective treatment, affects more than 50% of patients undergoing treatment with commonly used
95 chemotherapy drugs including paclitaxel, vincristine, and oxaliplatin (Addington and Freimer,
96 2016). The painful neuropathy usually leads to dose reduction or discontinuation of the treatment,
97 resulting in poor treatment outcomes. Currently, how chemotherapies with certain drugs cause
98 pain and neuropathy is not well understood, which has severely limited the development of
99 effective pain therapies (Hershman et al., 2014; Miltenburg and Boogerd, 2014). Therefore, it is
100 critical to understand the molecular and cellular mechanisms underlying the pathogenesis of
101 CIPN to develop effective and safe therapeutics for the treatment of CIPN.
102
103 Pain results from complex processing of neural signals at different levels. Transient receptor
104 potential (TRP) channels are molecular sensors for noxious mechanical, chemical, and thermal
105 insults. Activation of peripheral nociceptors by TRP channels, especially the capsaicin receptor
106 TRPV1 and wasabi receptor TRPA1, initiates neurogenic inflammation and pain sensation
107 (Caterina et al., 2000; Lee et al., 2005; Bautista et al., 2006; Huang et al., 2008). TRPV1 is
108 expressed in nearly 60% of peptidergic small-diameter primary nociceptors in the dorsal root and
109 trigeminal ganglia, which sense environmental cues in the skin and many visceral organs (Xu et
110 al., 2013). TRPV1 is a non-selective cation channel that is activated by capsaicin, noxious heat,
111 acid, and many endogenous ligands as well as plant-derived natural compounds. Besides direct
112 activation, TRPV1 is also sensitized by activation of G-protein coupled receptors (GPCRs) and
113 tyrosine kinase receptors through intracellular signal transduction pathways involving activation
5
114 of many protein kinases including protein kinase C (PKC), protein kinase A (PKA), and
115 phosphoinositide 3-kinase (PI3K). Both activation and sensitization of TRPV1 lead to enhanced
116 pain responses. Both genetic ablation and pharmacological inhibition studies have provided
117 convincing evidence that TRPV1 significantly contributes to both chronic inflammatory pain and
118 neuropathic pain resulting from peripheral nerve injury. Moreover, both TRPV1 and TRPA1
119 have been shown to be involved in the pathogenesis of CIPN (Boyette-Davis et al., 2015).
120
121 Zinc is an essential metal that has been extensively used to promote wound healing in humans
122 for almost 3000 years (Lansdown et al., 2007). It acts as an anti-inflammatory agent and soothes
123 pain sensation in a number of model systems (SafiehGarabedian et al., 1996; Larson and Kitto,
124 1999; Liu et al., 1999; Nozaki et al., 2011). Zinc is present in the spinal cord and dorsal root
125 ganglion (DRG) neurons, including small-diameter nociceptive neurons (Velazquez et al., 1999;
126 Koeppen et al., 2013). In fact, depletion of vesicular zinc in the spinal cord dorsal horn enhanced
127 neuropathic pain in mice (Jo et al., 2008). Moreover, zinc-deficient mice have increased
128 prostaglandin E2 levels and sensitized nociceptive C fibers, suggesting that endogenous zinc
129 might exert a tonic inhibition on nociception (Izumi et al., 1995). Furthermore, centrally applied
130 zinc attenuates neuropathic pain in mouse models of peripheral neuropathy (Liu et al., 1999;
131 Nozaki et al., 2011) and clinical studies (Pastorfide et al., 1989; Zekavat et al., 2015). However,
132 it is not known if exogenously applied zinc in the periphery can affect CIPN.
133
134 In the present study, we show that local application of zinc inhibits mechanical hypersensitivity
135 induced by paclitaxel treatment in mice in a dose-dependent manner. The zinc-induced analgesic
136 effect was severely attenuated in TRPV1-deficient mice. Extracellular zinc application inhibits
6
137 capsaicin-evoked intracellular calcium ([Ca2+]i) responses and membrane depolarization in a
138 subset of mouse DRG neurons, and locally injected zinc inhibits capsaicin-evoked acute
139 nociceptive behaviors. Moreover, zinc inhibits capsaicin-induced [Ca2+]i responses and
140 membrane current intracellularly, and the zinc-sensitive TRPA1 is required for the inhibition of
141 TRPV1 by extracellular zinc. Surprisingly, zinc transporters, but not TRPA1, are required for
142 inhibition of paclitaxel-induced chronic neuropathic pain by zinc. Taken together, our data
143 demonstrate that TRPV1-mediated pain sensations are inhibited by extracellular zinc entering the
144 cells through either TRPA1 channels or zinc transporters under acute and chronic settings.
145
146 Materials and Methods
147
148 ANIMALS
149 Male and female C57BL/6J mice (Jackson Laboratory), congenic TRPV1 knockout (KO)
150 (Jackson Laboratory), and congenic TRPA1 KO mice at the age of 7-10 weeks were used in this
151 study. The TRPA1 KO mice on the C57BL/6J background were described previously (Cruz-
152 Orengo et al., 2008). All animal care and experimental procedures were in accordance with the
153 animal care and use protocol approved by the Institutional Animal Care and Use Committee of
154 University of Texas Health Science Center at Houston and the Institutional Animal Care and Use
155 Committee at Washington University School of Medicine in Saint Louis. All studies involving
156 animals are reported in accordance with the ARRIVE guidelines for reporting experiments
157 involving animals (McGrath et al., 2010). Mice were housed in a temperature (24 oC)- and
158 humidity (40%-50%)-controlled environment on a 12:12 h dark–light cycle with free access to
159 food and water. All experiments were performed blind with respect to genotypes and treatments.
7
160
161 PACLITAXEL TREATMENT AND VON FREY TEST
162 Paclictaxel (TSZ CHEM, MA) was administered at a dose of 4 mg/kg intraperitoneally (i.p.) on
163 days 0, 2, 4, and 6 as illustrated in Figure 1A according to previous studies (Matsumoto et al.,
164 2006). Mechanical allodynia was measured as the hindpaw withdrawal response to von Frey hair
165 stimulation using the up-and-down method as described in our previous study (Yin et al., 2013).
166 Intraplantar injection of zinc acetate (ZnAc) (Sigma, Saint Louis, MO) was performed on day 7
167 following the von Frey test.
168
169 INTRATHECAL DELIVERY OF SIRNA
170 To knockdown the ZIP family of zinc transporters (ZIPs), the siRNAs specific for mZIP3,
171 mZIP6, and mZIP7 were purchased from Sigma (Saint Louis, MO) and 0.5 nmol of each was
172 prepared in PBS and mixed with PEI. The siRNA Universal Negative Control was obtained from
173 Sigma. After 15 min incubation at room temperature, the mixture was injected intrathecally into
174 mice anesthetized with isoflurane. The following studies were performed 3 days after the final
175 intrathecal injection.
176
177 CELL CULTURE AND TRANSFECTION
178 HEK293 cells were obtained from ATCC in 2010 and have been tested to confirm lack of
179 mycoplasma contamination; however, no additional authentication has been performed. Cells
180 were grown as a monolayer using passage numbers less than 30 and maintained in DMEM (Life
181 Technologies, Grand Island, NY, USA), supplemented with 10% FBS (Life Technologies), 100
182 units·mL−1 penicillin and 100 μg·mL−1 streptomycin in a humidified incubator at 37°C with 5%
8
183 CO2. The cells were transiently transfected with complementary DNA for mouse TRPV1
184 (mTRPV1), human TRPA1 (hTRPA1) and hTRPA1-D915A mutant using Lipofectamine 2000
185 (Invitrogen, Carlsbad, CA, USA) with a ratio of 0.3:1. After transfection, cells were maintained
186 in DMEM at 37°C for 24 h before use. The hRPA1-D915A mutant was made using the
187 QuickChange II XL mutagenesis kit (Agilent Technologies, Inc., Santa Clara, CA, USA),
188 according to the manufacturer’s directions and confirmed by DNA sequencing.
189
190 RETROGRADE LABELING OF PAW-INNERVATING DRG NEURONS
191 Mice were anesthetized with isoflurane and 10 μl of 1,1′-dilinoleyl-3,3,3′,3′-
192 tetramethylindocarbocyanine, 4-chlorobenzenesulfonate (FAST DiI™) (10 mg/mL in methanol)
193 was injected into the paws of paclitaxel-treated mice. To prevent leakage and labeling of
194 adjacent tissues, the needle was left in place for 30 seconds after each injection and any leaked
195 dye was removed with a cotton swab. DRG neurons were isolated for further experiment 5 days
196 after DiI injection.
197
198 ISOLATION AND CULTURE OF DRG NEURONS
199 Mice were sacrificed by cervical dislocation following CO2 asphyxia. Spinal columns were
200 removed and placed in ice-cold HBSS; neurons were acutely dissociated and maintained as
201 described (Hu et al., 2009). In brief, laminectomies were performed and bilateral DRGs or
202 lumbar DRGs of DiI-injection side were dissected out. After removal of connective tissues,
203 DRGs were transferred to a 1 mL Ca2+/Mg2+-free HBSS containing 2 μL saturated NaHCO3,
204 0.35 mg L-cysteine and 20 U papain (Worthington, Lakewood, NJ, USA), and incubated at 37°C
205 for 15 min. The suspension of DRG was centrifuged and the pellet was incubated in1 mL
9