Keratinocytes contribute to normal cold and heat sensation
- Medical College of Wisconsin, United States
Figures
Figure 1 with 1 supplement
Mammalian keratinocytes respond to temperature decreases.
In vitro calcium imaging was performed on primary cultured keratinocytes from the glabrous hindpaw skin of C57BL/6 mice, Sprague Dawley rats, and 13-lined ground squirrels and human breast skin. (A) Representative keratinocytes from all species exhibited a calcium transient upon extracellular buffer cooling (~23°C to 12°C). (B) The greatest proportion of cold-responsive cells was observed in hibernating 13-lined ground squirrel samples. The vast majority of mouse keratinocytes also responded to decreasing temperatures; fewer rat and human keratinocytes responded to cold (Chi-square p<0.0001; Fisher’s Exact tests: ***p<0.001 vs. mouse; ### p<0.001 vs. rat; &&& p<0.001 vs. squirrel; n = 28 mice, four rats, four squirrels, five humans). (C) The peak calcium response to decreasing temperatures varied between species; hibernating 13-lined ground squirrel keratinocytes exhibited the largest calcium transients (75% increase over baseline) and Sprague Dawley rats exhibited the smallest (44% increase over baseline; mouse: 56% increase; human: 69% increase; 1-way ANOVA p<0.0001; Bonferroni’s multiple comparisons: ****p<0.0001 vs. mouse; #### p<0.0001 vs. rat, && p=0.0084 vs. squirrel). (D) The temperature at which keratinocytes responded to cold (>30% increase in Δ340/380) differed between species. Mean temperature thresholds (°C) for mouse: 20.9, rat: 18.3, squirrel: 19.1, human: 19.8 (Kruskal-Wallis test p<0.0001; Dunn’s multiple comparisons: ****p<0.0001 vs. mouse, #### p<0.0001 vs. rat, &&&& p<0.0001 vs. squirrel).
Figure 1—figure supplement 1
Variability in human keratinocyte responses to temperature decreases.
In vitro calcium imaging was performed on primary human breast skin keratinocytes isolated from females of the following ages (years): 1: 41, 2: 50, 3: 54, 4: 52, 5: 49. (A).). Samples 4 and 5 contained the most cold-responsive cells (Chi-square: p<0.0001; Fisher’s Exact tests: ***p<0.001 vs. sample 1, ### p<0.001 vs. sample 2, &&& p<0.001 vs. sample 3). (B).) Peak calcium responses differed between the samples; keratinocytes from sample four exhibited the largest calcium response during cold stimulation (100% increase over baseline. Mean increase for sample 1: 54%, 2: 57%, 3: 71%, 5: 68% (1-way ANOVA p<0.0001; Bonferroni’s multiple comparisons: **p<0.01, ***p<0.001, ****p<0.0001 vs. 1; # p<0.05, #### p<0.0001 vs. 2; &&&& p<0.0001 vs. 3; $$$$ p<0.0001 vs. 4). (C).) The temperature at which keratinocytes responded to cold (>30% increase in Δ340/380) differed between the samples. Mean temperature thresholds (°C) for sample 1: 18.5, 2: 19.1, 3: 19.2, 4: 20.7, 5: 21.8 (Kruskal-Wallis test p<0.0001; Dunn’s multiple comparisons: *p<0.05, **p<0.01, ***p<0.001 vs. 1; ### p<0.001 vs. 2; &&&& p<0.0001 vs. 3; $$$$ p<0.0001 vs. 4).
Figure 2
Characterizing murine cold sensor(s) in keratinocytes.
(A).) In order to characterize the proteins involved in cold transduction, cold-induced calcium transients were measured in keratinocytes exposed to extracellular buffer containing different ionic concentrations or pharmacological agents, or in keratinocytes from transgenic mice. (B).) Extracellular calcium chelation (EGTA, 0 µM Ca2+) decreases the proportion of keratinocytes that respond to cold; substituting NMDG for extracellular sodium did not further decrease the percentage of cold-responsive cells. Cold responses were only abolished when endoplasmic reticulum Ca2+ stores were depleted and unable to be refilled with extracellular sources (EGTA, 0 µM Ca2+, thapsigargin). CRAC channel inhibition (5J4) did not decrease the percentage of cold-responsive cells, and similar proportions of keratinocytes from C57BL/6, global TRPA1, and global TRPC5 knockout mice responded to decreasing buffer temperature (Chi square p<0.0001; Fisher’s Exact tests ***p<0.001 vs. vehicle; n ≥ 4). (C).) Peak calcium responses to cold were lower in the absence of extracellular calcium; responses were unaltered in other conditions (1-way ANOVA p<0.0001; Bonferroni’s multiple comparisons: ****p<0.0001 vs. vehicle, #### p<0.0001 vs. EGTA, 0 µM Ca2+, &&&& p<0.0001 vs. EGTA, 0 µM Ca2+, NMDG). (D).) Altering extracellular buffer contents increased or decreased the temperature at which keratinocytes responded to cold by ≤0.6°C. Mean temperature thresholds (°C) for vehicle: 20.9, EGTA, 0 µM Ca2+,±NMDG: 20.3, 20 µM 5J4: 20.5, TRPA1 KO: 21.5, TRPC5 KO: 20.7 (Kruskal-Wallis test p<0.0001; Dunn’s multiple comparisons: *p<0.05, ****p<0.0001 vs. vehicle).
Figure 3
Keratinocyte-to-sensory neuron signaling is required for normal cold sensation.
(A) 590 nm light exposure increases the withdrawal latency of Krt14:Arch Cre+ mice during plantar dry ice stimulation (2-way ANOVA main effects of light, genotype, and light x genotype interaction p<0.0001; Bonferroni’s multiple comparisons: ****p<0.0001 Krt14:Arch Cre+ 490 vs. 590 nm, 590 nm Krt14:Arch Cre+ vs. Cre-; n = 17–20) (B) Intraplantar administration of apyrase (0.4 units; catalyzes ATP hydrolysis) increases the withdrawal latency of C57BL/6 mice during plantar dry ice stimulation (unpaired t-test ****p<0.0001; n = 12). (C) Animals lacking sensory neuron P2X4 receptors (P2X4cKO) exhibit increased withdrawal latencies to plantar dry ice stimulation (unpaired t-test ****p<0.0001; n = 11–12). (D) In a two-temperature preference test, P2X4cKO mice spend more time on innocuous cold surfaces than their P2X4 expressing littermates (2-way ANOVA main effects of temperature, genotype, and temperature x genotype interaction p<0.0001; Bonferroni’s multiple comparisons: **p<0.01 P2X4cKO vs. control; n = 6–8).
Figure 4
Keratinocyte-to-sensory neuron signaling is required for normal heat sensation.
(A) 590 nm light exposure increases the withdrawal latency of Krt14:Arch Cre+ mice during plantar radiant heat stimulation (2-way ANOVA main effects of light, genotype, and light x genotype interaction p<0.0001; Bonferroni’s multiple comparisons: ****p<0.0001 Krt14:Arch Cre+ 490 vs. 590 nm, 590 nm Krt14:Arch Cre+ vs. Cre-; n = 17–20). (B) Intraplantar administration of apyrase (0.4 units; catalyzes ATP hydrolysis) increases the withdrawal latency of C57BL/6 mice during plantar radiant stimulation (unpaired t-test ****p<0.0001; n = 10). (C) Animals lacking sensory neuron P2X4 receptors (P2X4cKO) exhibit increased withdrawal latencies to plantar radiant heat stimulation (unpaired t-test ****p<0.0001; n = 11–12).
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background Rattus norvegicus | Sprague Dawley rat | Taconic Biosciences | NTac:SD; SD-F, SD-M | |
| Strain, strain background Ictidomys tridecemlineatus | 13-lined ground squirrel | University of Wisconsin Oshkosh Squirrel Colony | ||
| Strain, strain background Mus musculus | Krt14:Arch Cre+ Krt14:Arch Cre- | Moehring et al., 2018 | K14:Arch Cre+ K14:Arch Cre- | |
| Strain, strain background Mus musculus | P2X4cKO | Moehring et al., 2018 | AdvilCre+::P2rX4fl/f AdvilCre-::P2rX4fl/f | |
| Strain, strain background Mus musculus | TRPA1 KO | Kwan et al., 2006 | ||
| Strain, strain background Mus musculus | TRPC5 KO | The Jackson Laboratory | Trpc5tm1.1Lbi/Mmjax Jackson Stock: 37349-JAX | |
| Other | In-line heater/cooler | Warner Instruments | https://www.warneronline. com/in-line-heater-cooler-sc-20 | |
| Other | Refrigerated circulator | Julabo | https://www.julabo.com/en-us/products/refrigerated-circulators/refrigerated-heating-circulators/f25-he | |
| Other | USB thermocouple probe | https://www.omega.com/en-us/sensors-and-sensing-equipment/temperature/sensors/thermocouple-probes/p/TJ-USB |
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Keratinocytes contribute to normal cold and heat sensation
eLife 9:e58625.
https://doi.org/10.7554/eLife.58625
