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. 2006 Aug 16;26(33):8578-87.
doi: 10.1523/JNEUROSCI.2185-06.2006.

Artemin overexpression in skin enhances expression of TRPV1 and TRPA1 in cutaneous sensory neurons and leads to behavioral sensitivity to heat and cold

Christopher M Elitt  1 , Sabrina L McIlwrath, Jeffery J Lawson, Sacha A Malin, Derek C Molliver, Pamela K Cornuet, H Richard Koerber, Brian M Davis, Kathryn M Albers
Affiliations

Artemin overexpression in skin enhances expression of TRPV1 and TRPA1 in cutaneous sensory neurons and leads to behavioral sensitivity to heat and cold

Christopher M Elitt et al. J Neurosci. .

Abstract

Artemin, a neuronal survival factor in the glial cell line-derived neurotrophic factor family, binds the glycosylphosphatidylinositol-anchored protein GFRalpha3 and the receptor tyrosine kinase Ret. Expression of the GFRalpha3 receptor is primarily restricted to the peripheral nervous system and is found in a subpopulation of nociceptive sensory neurons of the dorsal root ganglia (DRGs) that coexpress the Ret and TrkA receptor tyrosine kinases and the thermosensitive channel TRPV1. To determine how artemin affects sensory neuron properties, transgenic mice that overexpress artemin in skin keratinocytes (ART-OE mice) were analyzed. Expression of artemin caused a 20.5% increase in DRG neuron number and increased the level of mRNA encoding GFRalpha3, TrkA, TRPV1, and the putative noxious cold-detecting channel TRPA1. Nearly all GFRalpha3-positive neurons expressed TRPV1 immunoreactivity, and most of these neurons were also positive for TRPA1. Interestingly, acid-sensing ion channel (ASIC) 1, 2a, 2b, and 3 mRNAs were decreased in the DRG, and this reduction was strongest in females. Analysis of sensory neuron physiological properties using an ex vivo preparation showed that cutaneous C-fiber nociceptors of ART-OE mice had reduced heat thresholds and increased firing rates in response to a heat ramp. No change in mechanical threshold was detected. Behavioral testing of ART-OE mice showed that they had increased sensitivity to both heat and noxious cold. These results indicate that the level of artemin in the skin modulates gene expression and response properties of afferents that project to the skin and that these changes lead to behavioral sensitivity to both hot and cold stimuli.

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Figures

Figure 1.
Figure 1.
Overexpression of artemin in the skin is driven by the K14 keratin promoter. A, Diagram of transgene construct used for isolation of ART-OE mice. The K14 promoter drives expression of the artemin sequence represented by black boxes. Lines connecting the boxes indicate two intronic sequences retained in the artemin sequence. The 3′ human growth hormone sequence provides splice sites and a poly(A) addition signal. The arrow indicates the transcription start site, and ATG and TGA are translation start and stop sites, respectively. B, RT-PCR analysis of RNA isolated from WT (n = 3) and transgenic (ART-OE; n = 3) back skin showing increased expression of artemin mRNA in ART-OE skin. Lanes 1–6, Amplicons obtained using primers to the artemin gene sequence; lanes 7–12, products using transgene-specific PCR primers; lanes 13 and 15, negative controls for the PCR; lanes 14 and 16, positive controls for the artemin and transgene sequences, respectively. Note significant enhancement in the level of artemin mRNA and lack of transgene expression in transgenic skin samples. hGH, Human growth hormone; PA, poly(A).
Figure 2.
Figure 2.
Skin-expressed artemin increases the number of sensory neurons and is retrogradely transported to the ganglia. A, Immunolabeling of whisker pad skin from WT (top) and ART-OE (bottom) mice using an antibody to artemin. Artemin protein is significantly increased in basal keratinocytes of ART-OE mice. Scale bar, 25 μm. epi, Epidermis. B, Left, Set of trigeminal ganglia from littermate WT mouse. Right, Ganglia from ART-OE mice are larger relative to WT ganglia. DRGs showed a similar enlargement (not shown). C, Artemin is retrogradely transported from the skin as indicated by artemin immunoreactivity in trigeminal ganglia neurons of ART-OE (arrows) but not WT mice. Scale bar, 35 μm. D, Spinal cord dorsal horn of WT and ART-OE mice labeled with anti-CGRP (green) and IB4 (red). No significant difference was observed in the distribution or density of CGRP peptidergic or IB4 nonpeptidergic afferents. Scale bar,100 μm.
Figure 3.
Figure 3.
Sensory neurons responsive to artemin are hypertrophied and express TRPV1. A, C, D, F, GFRα3 immunolabeling (green) of WT (A, C) and ART-OE (D, F) DRG show GFRα3-positive neurons are larger in size. B, E, Nearly all GFRα3 neurons in WT (B) and ART-OE (E) ganglia express TRPV1 (red; arrows) although several TRPV1-labeled neurons do not express GFRα3 [arrowheads; see merged images of WT (C) and ART-OE (F) ganglia]. Trigeminal neurons showed a similar hypertrophy in TRPV1/GFRα3 neuron size. Scale bar (in F), 60 μm.
Figure 4.
Figure 4.
Skin-derived artemin increases cutaneous GFRα3-positive afferents and intensity of TRPV1 labeling. Low-power view compares innervation of whisker pad skin of WT (A–C) and ART-OE (D–F) animals. An increase in GFRα3-positive afferents (green, arrows) occurs in dermis of ART-OE skin. Immunolabeling with anti-TRPV1 (red; B, E) shows high expression in ART-OE afferents (E). Few TRPV1 fibers were seen in WT skin (B). TRPV1 labeling of GFRα3 afferents of ART-OE skin was particularly evident in merged images (F) where overlap appears yellow. Overlap was rarely seen in WT skin (C). Asterisks in D–F indicate appearance of GFRα3 and TRPV1 afferents in epidermis of ART-OE skin (see Fig. 4). Arrowheads in D–F indicate a GFRα3-positive fiber that is not TRPV1 positive and may represent sympathetic innervation. Scale bar (in F), 100 μm. epi, Epidermis; sb, sebaceous gland; hf, hair follicle.
Figure 5.
Figure 5.
Artemin increases the intensity of TRPV1 expression in GFRα3 epidermal afferents. High-magnification view of GFRα3 (green; A, D) and TRPV1 (red; B, E) labeling in whisker pad epidermis of adult WT (A–C) and ART-OE (D–F) mice. In WT skin, few dermal (arrows) and epidermal (arrowheads) GFRα3-positive fibers express TRPV1. In contrast, ART-OE skin has many TRPV1-positive fibers that appear to sprout in the epidermal layer (E, F). Scale bar (in E), 40 μm. epi, Epidermis; hf, hair follicle.
Figure 6.
Figure 6.
GFRα3-positive afferents express the TRPA1 channel. A, GFRα3 immunolabeling (green) of WT (A–C) and ART-OE (D–F) DRGs show GFRα3-labeled neurons exhibit TRPA1 immunoreactivity (red). Complete overlap in labeling occurred in both WT and ART-OE ganglia. Similar overlap in GFRα3 and TRPA1 labeling was found in the DRG. Scale bar (in F), 70 μm.
Figure 7.
Figure 7.
Increased levels of artemin sensitizes cutaneous C-fibers to thermal stimuli. An ex vivo preparation of the skin, nerve, DRG, and spinal cord was used to compare thermal and mechanical properties of cutaneous C-fibers in WT and ART-OE mice. ART-OE afferents had no change in mechanical response properties (A) but did exhibit significantly lower heat thresholds (see Results) and significantly higher firing rates over a range of noxious temperatures (temp.; B).
Figure 8.
Figure 8.
Artemin enhances the response to capsaicin in cultured sensory neurons. Calcium transients elicited by brief exposure to 1 μm capsaicin were measured in DRG neurons acutely isolated from adult WT and ART-OE mice. A, Representative calcium response obtained from WT and ART-OE neurons measured by fura-2 ratiometric imaging. Capsaicin caused an increase in intracellular calcium concentration in significantly more ART-OE neurons compared with WT neurons (see Results), and these neurons had larger responses to capsaicin compared with WT neurons (p < 0.002). B, Response to 100 μm ATP was not affected in ART-OE neurons, suggesting that the enhanced capsaicin response in ART-OE neurons results from changes in TRPV1 rather than an overall increase in excitability.
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