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. 2020 Aug 19;40(34):6477-6488.
doi: 10.1523/JNEUROSCI.0166-20.2020. Epub 2020 Jul 14.

Mechanisms Mediating High-Molecular-Weight Hyaluronan-Induced Antihyperalgesia

Ivan J M Bonet  1 , Dionéia Araldi  1 , Eugen V Khomula  1 , Oliver Bogen  1 , Paul G Green  2 , Jon D Levine  3
Affiliations

Mechanisms Mediating High-Molecular-Weight Hyaluronan-Induced Antihyperalgesia

Ivan J M Bonet et al. J Neurosci. .

Abstract

We evaluated the mechanism by which high-molecular-weight hyaluronan (HMWH) attenuates nociceptor sensitization, in the setting of inflammation. HMWH attenuated mechanical hyperalgesia induced by the inflammatory mediator prostaglandin E2 (PGE2) in male and female rats. Intrathecal administration of an oligodeoxynucleotide antisense (AS-ODN) to mRNA for cluster of differentiation 44 (CD44), the cognate hyaluronan receptor, and intradermal administration of A5G27, a CD44 receptor antagonist, both attenuated antihyperalgesia induced by HMWH. In male rats, HMWH also signals via Toll-like receptor 4 (TLR4), and AS-ODN for TLR4 mRNA administered intrathecally, attenuated HMWH-induced antihyperalgesia. Since HMWH signaling is dependent on CD44 clustering in lipid rafts, we pretreated animals with methyl-β-cyclodextrin (MβCD), which disrupts lipid rafts. MβCD markedly attenuated HMWH-induced antihyperalgesia. Inhibitors for components of intracellular signaling pathways activated by CD44, including phospholipase C and phosphoinositide 3-kinase (PI3K), also attenuated HMWH-induced antihyperalgesia. Furthermore, in vitro application of HMWH attenuated PGE2-induced sensitization of tetrodotoxin-resistant sodium current, in small-diameter dorsal root ganglion neurons, an effect that was attenuated by a PI3K inhibitor. Our results indicate a central role of CD44 signaling in HMWH-induced antihyperalgesia and suggest novel therapeutic targets, downstream of CD44, for the treatment of pain generated by nociceptor sensitization.SIGNIFICANCE STATEMENT High-molecular-weight-hyaluronan (HMWH) is used to treat osteoarthritis and other pain syndromes. In this study we demonstrate that attenuation of inflammatory hyperalgesia by HMWH is mediated by its action at cluster of differentiation 44 (CD44) and activation of its downstream signaling pathways, including RhoGTPases (RhoA and Rac1), phospholipases (phospholipases Cε and Cγ1), and phosphoinositide 3-kinase, in nociceptors. These findings contribute to our understanding of the antihyperalgesic effect of HMWH and support the hypothesis that CD44 and its downstream signaling pathways represent novel therapeutic targets for the treatment of inflammatory pain.

Keywords: antihyperalgesia; cluster of differentiation 44 (CD44); high-molecular-weight hyaluronan (HMWH); hyaluronan; hyperalgesia; prostaglandin E2 (PGE2).

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Figures

Figure 1.
Figure 1.
HMWH attenuates PGE2 hyperalgesia but did not alone affect the nociceptive threshold. A, B, Male and female rats received PGE2 (100 ng/5 µl, intradermally) on the dorsum of the hindpaw followed 10 min later by HMWH (1 µg/5 µl) or vehicle (5 µl) injected at the same site. The mechanical nociceptive threshold was then measured 40 min after intradermal PGE2. Measurement of mechanical nociceptive threshold showed a significant attenuation of PGE2 hyperalgesia in rats treated with HMWH (A; t(10) = 5.676, ***p= 0.0002, when the vehicle-treated group was compared with the HMWH-treated group; unpaired Student's t test; B; t(10) = 7.860, ****p < 0.0001, when the vehicle-treated group was compared with the HMWH-treated group; unpaired Student's t test). n = 6 paws in each group. C, Male rats received an intradermal injection of one of three doses of HMWH [0.1 µg, black triangle; 1 µg, black circle; or 10 µg, black square; diluted in 5 µl of saline) or vehicle (5 µl, white circle). Mechanical nociceptive threshold was evaluated 0.5, 1, 2, 4, 8, and 24 h after intradermal HMWH administration. The time course of the effect of HMWH on nociceptive threshold did not differ among the 0.1, 1, and 10 µg doses, and did not affect the nociceptive threshold itself. Repeated-measures two-way ANOVA, followed by Tukey's multiple-comparison test, revealed no significant interaction among the groups (interaction: F(15,100) = 1.168, p = 0.3088; time: F(5,100) = 1.241, p = 0.2959; dose: F(3,20) = 1.093, p = 0.3750). n = 6 paws in each group.
Figure 2.
Figure 2.
Reversal of PGE2 hyperalgesia by HMWH was attenuated by CD44 ODN antisense and a CD44 receptor antagonist. A, B, Male and female rats were treated intradermally with PGE2 (100 ng/2 µl), followed 5 min later by a CD44 receptor antagonist (A5G27, 1 µg/2 µl) or vehicle (2 µl) also injected intradermally. Ten minutes after PGE2, rats received intradermal HMWH (1 µg/2 µl) and the mechanical nociceptive threshold was evaluated before and 40 min after intradermal PGE2. In male rats that received a CD44 receptor antagonist, a significant attenuation in HMWH-induced antihyperalgesia was observed (A; F(2,10) = 19.85, #p = 0.0344, when the CD44 receptor antagonist group plus the HMWH-treated group was compared with the vehicle group plus the HMWH-treated group; *p = 0.0191, when the CD44 receptor antagonist plus HMWH-treated group was compared with the PGE2-treated group alone; ***p = 0.0002, when the vehicle group plus the HMWH-treated group was compared with PGE2-treated group alone 40 min after intradermal PGE2 administration; two-way ANOVA followed by Tukey's multiple-comparisons test); however, in female rats HMWH-induced antihyperalgesia was completely reversed in the group that received CD44 antagonist receptor intradermally (B; F(2,10) = 11.28, #p = 0.0127, when the CD44 receptor antagonist group plus the HMWH-treated group was compared with the vehicle group plus HMWH-treated group; ***p = 0.0030, when the vehicle group plus the HMWH-treated group was compared with the PGE2-treated group alone 40 min after intradermal PGE2 administration, two-way ANOVA followed by Tukey's multiple-comparisons test). n = 6 paws in each group. C, D, Male and female rats were treated intrathecally with ODN AS or MM (120 µg/20 µl) for CD44 mRNA, daily for 3 consecutive days. On the fourth day, ∼17 h after the last intrathecal administration of ODNs, PGE2 (100 ng/5 µl) was injected intradermally on the dorsum of the hindpaw followed, 10 min later, by intradermal HMWH (1 µg/5 µl) or vehicle (5 µl). The mechanical nociceptive threshold was evaluated before and 40 min after intradermal PGE2. In male rats, the antihyperalgesic effect of HMWH on PGE2-induced hyperalgesia, was attenuated in the group treated with CD44 AS-ODN (C; F(2,10) = 51.53, ###p = 0.0035, when the CD44 AS ODN-treated group was compared with the CD44 ODN MM-treated group; **p < 0.0001, when the CD44 AS-ODN-treated group was compared with the PGE2-treated group alone; ****p = 0.0002, when the CD44 MM-ODN-treated group was compared with the PGE2-treated group alone, 40 min after intradermal PGE2, two-way ANOVA followed by Tukey's multiple-comparisons test), while in female rats, CD44 AS-ODN completely reversed HMWH-induced antihyperalgesia (D; F(2,10) = 18.23, ##p = 0.0033, when the CD44 AS-ODN-treated group is compared with CD44 MM-ODN-treated group; ***p = 0.0005, when the CD44 MM-ODN-treated group was compared with the PGE2-treated group, 40 min after intradermal PGE2, two-way ANOVA followed by Tukey's multiple-comparisons test). n = 6 paws each group. E, Western blot analysis of DRG extracts from male rats injected with 120 µg of CD44 AS-ODN/day for three consecutive days revealed a significant decrease in their anti-CD44 immunoreactivity when compared with the extracts derived from CD44 MM-ODN- treated rats (−24.8 +/− 4.8%, unpaired Student's t test, n = 6, p < 0.05). The calculated molecular weight of the main CD44 splice variant in rat tissue is ∼86 kDa (according to NCBI database entry NP_037056). β-actin, which was used as a loading control, has a calculated molecular weight of ∼42 kDa (according to UniProtKB database entry P60771).
Figure 3.
Figure 3.
In male but not female rats, reversal of PGE2 hyperalgesia by HMWH was TLR4 dependent. A–D, Male and female rats were treated intrathecally with AS-ODN (120 µg/20 µl) or MM-ODN (120 µg/20 µl) for TLR4 (A, B) or RHAMM (C, D) mRNA, once a day, for three consecutive days. On the fourth day, ∼17 h after the last intrathecal administration of ODNs, PGE2 (100 ng/5 µl) was injected intradermally on the dorsum of the hindpaw followed, 10 min later, by intradermal HMWH (1 µg/5 µl). The mechanical nociceptive threshold is evaluated 40 min after intradermal PGE2. A, B, In male rats treated with TLR4 AS-ODN an attenuation in the antihyperalgesia induced by HMWH was observed (A; F(2,10) = 32.88, ##p = 0.0420, when the TLR4 AS-ODN-treated group was compared with TLR4 MM-ODN-treated group; *p < 0.001, when the TLR4 AS-ODN-treated group was compared with PGE2-treated group alone; ****p < 0.0001, when the TLR4 MM-ODN-treated group was compared with the PGE2-treated group alone, 40 min after intradermal PGE2, two-way ANOVA followed by Tukey's multiple comparison test); however, in female rats TLR4 AS-ODN did not affect the antihyperalgesia induced by HMWH (B; F(2,10) = 8.051, #p = 0.0167 and *p = 0.0136, when the TLR4 AS-ODN- and TLR4 MM-ODN-treated group was compared with the PGE2-treated group alone; p = 0.9907, when TLR4 AS-ODN- and TLR4 MM-ODN-treated group are compared 40 min after intradermal PGE2, two-way ANOVA followed by Tukey's multiple-comparisons test). n = 6 paws each group. C, D, In male (C) and female (D) rats treated with RHAMM AS-ODN, the antihyperalgesia induced by HMWH was not affected (C; F(2,10) = 43.23, ###p = 0.0001 and ****p < 0.0001, when the RHAMM AS-ODN- and RHAMM MM-ODN-treated groups was compared with the PGE2-treated group alone; p = 0.1761, when the RHAMM AS-ODN- and MM-ODN-treated groups was compared each other, 40 min after intradermal PGE2, two-way ANOVA followed by Tukey's multiple-comparisons test. (D; F(2,10) = 6.043, #p < 0.0326 and *p = 0.0317, when the RHAMM AS ODN-treated and RHAMM MM ODN-treated groups was compared with the PGE2-treated group alone; p = 0.9998, when the RHAMM AS-ODN- and MM ODN-treated groups were compared to each other, 40 min after intradermal PGE2 administration; two-way ANOVA followed by Tukey's multiple-comparisons test). n = 6 paws in each group. E, Western blot analysis of DRG extracts from male rats treated with 120 µg/day RHAMM AS ODN, for 3 consecutive days, reveals a significant decrease in RHAMM, compared with the extracts derived from RHAMM MM ODN-treated rats (−30.4 ± 4.6%, unpaired Student's t test, n = 6, *p = 0.0239). The calculated molecular weight of RHAMM is ∼82.4 kDa (according to UniProtKB database entry Q9WUF7). β-actin, which was used as a loading control, has a calculated molecular weight of ∼42 kDa (according to UniProtKB database entry P60771).
Figure 4.
Figure 4.
Lipid rafts are necessary for HMWH to induce antihyperalgesia. Male and female rats were treated intradermally with PGE2 (100 ng/2 µl), followed 5 min later by a lipid raft disruptor (MβCD, 1 µg/2 µl) or vehicle (2 µl), also injected intradermally. Ten minutes after PGE2 administration, rats received intradermal HMWH (1 µg/2 µl) and the mechanical nociceptive threshold was evaluated 40 min after intradermal PGE2 administration. A, Male: MβCD significantly attenuated HMWH-induced antihyperalgesia (F(3,15) = 22.60, **p = 0.0023, #p = 0.0136, when the MβCD group plus the HMWH-treated group is compared with the PGE2-treated group and vehicle plus HMWH-treated group, respectively; ****p < 0.0001, when the vehicle plus HMWH-treated group was compared with the PGE2-treated group; aaap = 0.0004, when the MβCD-treated group was compared with the PGE2-treated group, 40 min after intradermal PGE2 administration; two-way ANOVA followed by Tukey's multiple-comparisons test). n = 6 paws in each group. B, Female: MβCD reverses HMWH-induced antihyperalgesia (F(3,15) = 10.71, ###p = 0.0006, when the MβCD plus the HMWH-treated group was compared with the vehicle plus HMWH-treated group; **p = 0.0019, when the vehicle plus HMWH-treated group was compared with the PGE2-treated group; aaap = 0.0004, when the MβCD-treated group was compared with the vehicle plus HMWH-treated group, 40 min after intradermal PGE2, two-way ANOVA followed by Tukey's multiple-comparisons test). n = 6 paws in each group.
Figure 5.
Figure 5.
HMWH-induced antihyperalgesia is RhoA and Rac1 dependent. A, B, Male (A) and female (B) rats received PGE2 (100 ng/2 µl) intradermally followed 5 min later by intradermal vehicle (2 µl), Rac1 inhibitor (NSC23766 1 µg/2 µl), RhoA inhibitor (Y27632 1 µg/2 µl), PLC inhibitor (U73122, 1 µg/2 µl), or PI3K inhibitors (wortmannin or LY249002, 1 µg/2 µl/each). Ten minutes after PGE2 administration, rats received intradermal HMWH (1 µg/2 µl) and the mechanical nociceptive threshold was measured 40 min after intradermal PGE2. A, The antihyperalgesic effect of HMWH for PGE2-induced hyperalgesia was attenuated by second messenger inhibitors of the CD44 signaling pathways in male rats (F(6,30) = 19.69, ####p < 0.0001, when the vehicle plus HMWH-treated group was compared with the NSC23766-, U73122-, LY249002-, and wortmannin-treated groups, respectively; ###p = 0.0003, when the vehicle plus HMWH-treated group was compared with the Y27632-treated group; ****p < 0.0001, when the PGE2-treated group was compared with the vehicle plus HMWH-treated and Y27632-treated groups, respectively; ***p = 0.0003, aaap < 0.0004, cccp = 0.0003, and **p = 0.0034, when the PGE2-treated group was compared with the wortmannin-, LY249002-, U73122-, and NSC23766-treated groups, respectively, 40 min after intradermal PGE2; two-way ANOVA followed by Tukey's multiple-comparisons test). n = 6 paws in each group. B, In female rats, an attenuation of the antihyperalgesic effect of HMWH for PGE2-induced hyperalgesia was observed in all groups treated with the second messenger inhibitors (F(6,30) = 12.69, ####p < 0.0001, when the vehicle plus HMWH-treated group was compared with the LY249002- and U73122-treated groups, respectively; ###p = 0.0001, when the vehicle plus HMWH-treated group was compared with the NSC23766 and wortmannin-treated groups, respectively; aaap = 0.0006 when the vehicle plus HMWH-treated group was compared with Y27632-treated group; ****p < 0.0001, when the PGE2-treated group is compared with the vehicle plus HMWH-treated group, 40 min after intradermal PGE2 administration; two-way ANOVA followed by Tukey's multiple-comparisons test). n = 6 paws in each group.
Figure 6.
Figure 6.
HMWH-induced antihyperalgesia is not dependent on Src. Male rats received an intradermal injection of PGE2 (100 ng/2 µl), followed 5 min later by the Src inhibitor (SU6656, 1 µg/2 µl) or vehicle (2 µl), injected at the same site. Ten minutes after PGE2 administration, rats received intradermal HMWH (1 µg/2 µl) and the mechanical nociceptive threshold was evaluated 40 min after intradermal PGE2. Treatment with the Src inhibitor SU6656 did not affect the antihyperalgesia induced by HMWH (F(2,10) = 47.74, ****p < 0.0001, when the PGE2-treated group was compared with the vehicle plus HMWH-treated and SU6656-treated groups; p = 0.9979, when the SU6656-treated group is compared with the vehicle plus HMWH-treated group 40 min after intradermal PGE2; two-way ANOVA followed by Tukey's multiple-comparisons test). n = 6 paws in each group.
Figure 7.
Figure 7.
Inhibition of PI3K reverses attenuation of PGE2-sensitized TTX-R sodium current induced by HMWH. Small DRG neurons from male rats were held at −70 mV in voltage-clamp mode of whole-cell patch clamp. TTX-R sodium current was induced in the presence of TTX (100 nm) by voltage step to −10 mV after 2 s conditioning at −50 mV. A, Illustrative traces depicting TTX-R sodium current as follows(left to right): before administration of PGE2 (1 μm), 5 later, and 5 min after administration of HMWH (200 µg/ml) either alone (top) or with the addition of the PI3K inhibitor (LY249002, 50 μm; bottom). Note the substantial reduction in TTX-R sodium current induced by HMWH (top) and attenuation of this effect, when PI3K inhibitor was added (bottom). B, Pooled magnitudes of reduction in peak TTX-R sodium current 5 min after bath application of HMWH, relative to a baseline measured before this application (5 min after stimulation with 1 μm PGE2), in the absence (white bar) or presence (black bar) of PI3K inhibitor (LY249002, 50 μm). Inhibition of TTX-R sodium current by HMWH was attenuated by LY249002 (two-tailed unpaired Student's t test: t(10) = 2.7, *p = 0.024). n = 6 neurons/group.
Figure 8.
Figure 8.
Schematic representation of potential signaling pathways involved in HMWH-induced antihyperalgesia. In male and female rats, HMWH binds to CD44 to induce its clustering in cell membrane lipid rafts and initiate signaling in downstream second messenger pathways. After HMWH binds to CD44, it can signal via RhoA and Rac1, which, in turn, activate ROK and PKN, respectively, leading to phosphorylation of PLCε and PLCγ1, respectively. Binding of HMWH to CD44 also stimulates RhoA, which activates ROK to phosphorylate PLCε, increasing serine/threonine phosphorylation of the adaptor protein Gab-1 and leading to activation of PI3K. In male rats, HMWH also signals via TLR4.
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