RIN1 inhibited TRPV1-dependent pain sensitization in a mouse model of bone cancer pain

Introduction

A high proportion of patients suffering from cancer, including the prostate, breast, and lung cancer, develop severe pain largely due to the tumor metastasis to the adjacent bone.^1–4 The tumor microenvironment releases a number of pronociceptive substances to activate their cognate receptors expressed on the primary sensory nociceptors innervating the bone tissue and cause the evoked and non-evoked pain.^5,6 The transient receptor potential vanilloid 1 (TRPV1), a polymodally activated non-selective cation channel, has been identified as a key player in the bone cancer pain.^7,8 Pharmacological or genetic inhibition of TRPV1 effectively alleviates both mechanical allodynia and spontaneous pain in multiple preclinical models. ⁹ This channel is present at the small- to large-diameter neurons of dorsal root ganglia (DRG), ¹⁰ and is able to sense a wide range of pronociceptive signals, including the acidic pH, noxious heat, and mechanical force. ⁷ Once activated by the tumor bone microenvironment, TRPV1 initiates a line of intracellular signaling cascades that eventually sensitize the nociceptors. ⁷ The hyperactive TRPV1 reduces the nociceptive thresholds in response to innocuous mechanical stimulation, a phenomenon also known as mechanical allodynia frequently experienced by patients when moving the tumor-bearing bone. ⁵ In addition, the TRPV1-positive primary sensory neurons transmit the nociceptive signals to the brain regions generating the affective-motivational aspect of pain, in particular the spontaneous pain. ¹¹ The pharmacological or genetic attenuation of TRPV1 channel activity represents a promising strategy for the treatment of bone cancer pain.^6,12

The Ras and Rab interactor 1 (RIN1) is an intracellular signaling adapter protein, that is, widely expressed in neuronal and non-neuronal cells.^13,14 RIN1 has emerged as a multifaceted regulator in cellular processes, including endocytosis, signal transduction, and cytoskeletal remodeling.^13,15 In the context of neuroscience, RIN1’s role in modulating synaptic plasticity and receptor trafficking has been well-documented. ¹⁶ Recent studies have expanded the scope of RIN1’s function in pain mechanisms. Lin et al. ¹⁷ demonstrated that RIN1 regulates ferroptosis and nociceptive perception via the Nrf2/HO-1 pathway in a chronic constriction injury model. Their findings revealed that RIN1 deficiency enhanced lipid peroxidation and neuronal death through downregulating the Nrf2/HO-1 axis, leading to increased pain sensitivity. This suggests a novel link between RIN1, oxidative stress, and pain processing, adding to the growing body of evidence highlighting RIN1’s importance in the nervous system. Another studies further revealed that RIN1 modulates developmental and pain-related plasticity in spinal synapses by regulating NMDA receptor subunit trafficking, ¹⁸ and that reduced hippocampal RIN1 expression contributes to neuropathic pain-induced contextual fear generalization. ¹⁹ These findings collectively establish RIN1 as a critical regulator of both nociceptive transmission and pain-associated emotional processing. RIN1 has a Src homology 2 (SH2) domain at the N-terminal region, a vacuolar protein sorting 9 protein (Vps9p) domain with the guanine nucleotide exchange factor (GEF) activity for small GTPase Ras-related protein 5 (Rab5), and a Ras-binding domain at the C-terminal portion. ²⁰ A line of tyrosine-phosphorylated membrane proteins, such as epidermal growth factor receptor (EGFR), can be recognized by the SH2 domain, which allows RIN1 to initiate a Rab5-dependent endocytic process through its Vps9p structure.^13,15,21,22 According to research, the Src homology 2 (SH2) domain of the RIN1 protein can interact with activated receptor tyrosine kinases. The interaction between the proteins can be regulated through phosphorylation and dephosphorylation. The proline-rich domains (PRD) domain of the RIN1 protein is present in a large number of different proteins and can regulate a variety of protein interactions and cytoskeleton remodeling and other physiological processes.^23,24 RIN1 has the Guanine nucleotide exchange factor (GEF) activity for Rab5–guanosine triphosphate (GTP), and Ras mediates EGFR endocytosis degradation through direct binding to Rab5, thereby promoting endocytosis. ¹⁵ When Rab in RIN1 binds to guanosine diphosphate (GDP), Rab is in an inactive state; while when Rab in RIN1 binds to GTP, Rab is in an active state, further regulating intracellular membrane transport function, and this conversion process is regulated by GEF ¹⁵ (Figure 1). The Ras-binding domain of RIN1 can compete with Raf-1 and interfere with the Ras signaling. ²⁵ RIN1 has been considered as a suppressor of tumor cell growth and migration. ²⁶ Inhibition or elevation of RIN1 protein expression promotes or blocks the cancer progression, respectively, which has been validated in multiple malignancies including thyroid carcinoma, head and neck cancer, and colon adenocarcinoma.^27–29 In the nerve system, the RIN1 protein level increases with age and participates in the regulation of neuronal plasticity.^13,30 Genetic deletion of RIN1 facilitates the induction of long-term potentiation, ³⁰ one of the important forms of synaptic plasticity that underpins the learning and memory, and leads to the enhanced fear conditioning, which likely results from the reduced endocytosis of ephrin type-A receptor 4 (EphA4). ¹³ Here we found that RIN1 interacted with and internalized TRPV1 in the DRG neurons, which operated to limit the TRPV1-dependent acute pain responses under physiological conditions. Our data showed that the bone cancer decreased the expression of neuronal RIN1, thus removing the RIN1-mediated inhibition and contributing to the persistence of bone cancer pain.

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Figure 1.

The tyrosine-phosphorylated TRPV1 can be recognized by the SH2 domain of RIN1, enabling RIN1 to initiate the Rab5-dependent endocytic process through its Vps9p domain. RIN1 has the GEF activity of Rab5-GTP enzyme, and Ras mediates the membrane protein invagination degradation through direct binding to Rab5, thereby promoting endocytosis. When Rab in RIN1 binds to GDP, Rab is in an inactive state; while when Rab in RIN1 binds to GTP, Rab is in an active state, further regulating the intracellular membrane transport function. This conversion process is regulated by GEF.

Materials and methods

Results

RIN1 negatively controlled TRPV1-dependent pain sensitization

TRPV1 has been identified as a key contributor to the development of bone cancer pain. ⁷ To investigate the possible influence of RIN1 on TRPV1 activity, we first examined the expression of RIN1 in the dorsal root ganglia (DRG) of adult mice. The immunohistochemistry experiments demonstrated that RIN1 was highly expressed in the DRG neurons, and co-localized with TRPV1-positive nociceptors (Figure 2(a)). Both the peptidergic and non-peptidergic nociceptors have been shown to express TRPV1. ¹⁰ In agreement with this, RIN1 was coincident with calcitonin gene-related peptide (CGRP), a biochemical marker for the peptidergic neurons, and Isolectin B4 (IB4), a marker for the non-peptidergic neurons (Figure 2(a)).

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Figure 2.

Conditioned knockout of RIN1 in the DRG neurons of mice exacerbated the capsaicin-evoked pain responses: (a) double fluorescence for RIN1 (red) and NeuN, TRPV1, CGRP, or IB4 (green) in the DRGs. Arrows indicated the co-localization. Scale bar: 50 µm, (b) immunofluorescence for RIN1 in the DRGs of Advillin-Cre and cKO-RIN1 mice. Scale bar: 50 µm, and (c) painful behaviors evoked by intraplantar capsaicin (10 μg) injection in Advillin-Cre and cKO-RIN1 mice. F(5, 70) = 8.718, ***p < 0.001 (repeated-measurement ANOVA), n = 8 mice/group.

Intraplantar injection of TRPV1 agonist capsaicin provokes rapid and transient spontaneous pain in intact mice, which is followed by TRPV1 desensitization possibly due to the ligand-induced endocytosis of TRPV1 from the plasma membrane.^49,50 However, it is important to note that intraplantar capsaicin activates TRPV1 not only in DRG neuron somata but also in their peripheral cutaneous nerve endings, ⁵¹ introducing potential peripheral contributions to pain responses. To address this, we combined this in vivo assay with mechanistic analyses focused on DRG neurons. To interrogate the role of RIN1 in TRPV1-dependent pain sensitization, we knocked out RIN1 specifically in the DRG neurons by crossing Advillin-Cre mice with RIN1^flox/flox mice. The resultant conditioned knockout mice, referred to here as cKO-RIN1, showed a deficiency of RIN1 protein in the DRG neurons (Figure 2(b)). In the control Advillin-Cre mice, intraplantar capsaicin (10 µg) injection immediately elicited the painful behavior, as evidenced by a marked increase of time spent on licking the affected paws (Figure 2(c)). Genetic knockout of RIN1 significantly prolonged the licking time when compared to the controls (Figure 2(c)), implying an important role of RIN1 in the suppression of TRPV1-dependent pain behaviors.

RIN1 interacted with TRPV1 in the DRG neurons

To investigate the molecular mechanism by which RIN1 regulated TRPV1 activity, we cultured the DRG neurons (independent of peripheral effects) from adult C57BJ mice and conducted double immunofluorescence for RIN1 and surface TRPV1. Our data showed that the RIN1 immunosignals were present as clusters in the neuron cultures and predominantly distributed in the cytoplasmic compartments (Figure 3(a)), which scarcely co-localized with the TRPV1 on the plasma membrane (Figure 3(a) and (d)). The treatment in vitro with capsaicin (3 µM) for 3 min significantly increased the coincidence of RIN1 with TRPV1 (Figure 3(a) and (d)), which paralleled with a noticeable decrease of TRPV1 immunoreactivity on the plasma membrane (Figure 3(a) and (b)). The total RIN1 protein levels were comparable in the presence and absence of capsaicin (Figure 3(a) and (c)), suggesting that brief capsaicin application didn’t affect the synthesis or degradation of RIN1 protein. We next applied capsaicin (10 µg) intrathecally for 5 min and conducted co-immunoprecipitation with RIN1 antibody. Compared to saline control, capsaicin treatment significantly increased the content of TRPV1 in the RIN1 immunoprecipitates (Figure 3(e)). These data were consistent with the immunocytochemical results (Figure 3(a) and (d)), and suggested that the activation of TRPV1 might drive the cytoplasmic RIN1 to move toward the plasma membrane, where RIN1 interacted with and internalized the surface TRPV1, a process possibly terminating the TRPV1 signaling (Figure 2(c)).

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Figure 3.

Capsaicin regulated the subcellular distribution and interaction of RIN1 with TRPV1: (a) immunofluorescence for TRPV1 expressed on the plasma membrane (red) and RIN1 (green) in the cultured DRG neurons with or without capsaicin (3 µM) stimulation (scale, 10 µm). Areas enclosed in the white square were enlarged on the right panels (scale, 2 µm), (b–d) graphs summarized the changes in the immunoreactive intensities of surface TRPV1 (b) and RIN1 (c) as well as the fraction of TRPV1 clusters positive for RIN1 (d). ***p < 0.001 (Mann–Whitney U test), and (e) Co-IP was conducted from the DRGs after intrathecal capsaicin (10 µg) application for 5 min. RIN1 immunoprecipitates were probed with the antibodies indicated on the left of panels. *p = 0.041 (Mann–Whitney U test).

RIN1 internalized TRPV1 through the Vps9p domain

To confirm the regulatory effect of RIN1 on TRPV1, we examined the TRPV1 expression after RIN1 knockout in the DRG neurons. Compared to the control Advillin-Cre mice, cKO-RIN1 mice exhibited a significant increase of TRPV1 protein level (Figure 4(a)), suggesting that RIN1 likely directed the internalized TRPV1 to the degradation pathway. ⁴⁹

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Figure 4.

RIN1 regulated TRPV1 expression in the DRG neurons: (a) RIN1 protein level in the DRGs of cKO-RIN1 mice. Advillin-Cre mice were used as control. *p = 0.026 (Mann–Whitney U test), n = 6 experiments and (b, c) wild-type RIN1 (RIN1 (WT)) and RIN1 (E574A) constructs were generated (b) and transfected in the cultured DRG neurons from cKO-RIN1 mice (c). TRPV1 expressed on the plasma membrane (red) and RIN1 (green) were immunostained (c). **p = 0.001 and ***p < 0.001 (Kurskal Wallis test). Scale bar, 10 µm.

At the C-terminal region of RIN1 exists a Vps9p domain (Figure 4(b)), which shows the guanine nucleotide exchange factor (GEF) activity for small GTPase Rab5. The Rab5-directed GEF activity is required for RIN1 to induce the endocytosis of a line of active receptor tyrosine kinases.^13,15,21,22 To test whether the Vps9p domain mediated the TRPV1 endocytosis, we constructed a RIN1 (E574A) mutant (Figure 4(b)), in which the glutamic acid at 574 residue was substituted with alanine to abolish the Rab5 GEF activity.^20,52 The RIN1 (E574A) and wild-type RIN1 construct (RIN1 (WT)) were introduced into the cultured DRG neurons from cKO-RIN1 mice. Our data showed that the rescue of RIN1 expression by RIN1 (WT) transfection significantly decreased the immunoreactivity of TRPV1 on the plasma membrane (Figure 4(c)). This effect was lacking with RIN1 (E574A) mutant (Figure 4(c)), suggesting a critical role of the Vps9p domain in mediating TRPV1 endocytosis.

RIN1 knockout in the DRG neurons elicited the pain hypersensitivity

We observed that the cKO-RIN1 mice frequently licked and flicked their paws, a spontaneous behavior possibly reflecting the affective-motivational aspect of pain state. The statistical analysis illustrated that the cKO-RIN1 mice indeed spent more time on licking (Figure 5(a)) and flicking the hindpaws (Figure 5(b)) than the Advillin-Cre control mice. The conditioned RIN1 deletion didn’t affect the motor function, as the traveling distance was comparable between the two phenotypes (Figure 5(c)). The von Frey test showed that the reflexive withdrawal thresholds were significantly lower in the cKO-RIN1 mice relative to the Advillin-Cre controls (Figure 5(d)), suggesting that RIN1 ablation evoked pain sensitization. We next interrogated whether the cKO-RIN1 mice were aversive to the mechanical stimulation by using the conditioned place aversion (CPA) paradigm. The Advillin-Cre control mice didn’t show avoidance of the chamber paired with 1-g von Frey filament stimulation, suggesting that this punctate force was innocuous (Figure 5(e)). The cKO-RIN1 mice, however, displayed a robust CPA, manifested by significantly less time spent in the environment associated with the von Frey filament stimuli (Figure 5(e)). These data suggested that RIN1 deficit in the DRG neurons evoked both the somatosensory and affective-motivational aspect of pain.

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Figure 5.

Conditioned knockout of RIN1 in the DRG neurons evoked pain behaviors: (a–c) spontaneous licking (a) and flicking behaviors (b) as well as the traveling distance (c) of cKO-RIN1 mice. The Advillin-Cre mice were used as control. **p = 0.002 and ***p < 0.001 (Mann–Whitney U test), (d) paw withdrawal thresholds in response to von Frey filament stimulation. ***p < 0.001 (Mann–Whitney U test), and (e) repetitive von Frey filament (1 g) stimulation of hindpaws evoked the CPA in cKO-RIN1 mice. ***p < 0.001 and **p = 0.002 (Mann–Whitney U test).

Tumor-induced RIN1 hypofunction led to the increased expression of TRPV1 on the plasma membrane

TRPV1 hyperactivity in the DRG neurons contributes to the bone cancer pain. ¹⁰ Given the intimate relationship between RIN1 and TRPV1, we set out to examine whether RIN1 was involved in the tumor-related pain sensitization. The inoculation of Lewis lung carcinoma (LLC) cells into the femora reduced the von Frey thresholds (Figure 6(a)), which lasted for at least 28 days. We conducted the co-immunoprecipitation from lysates of DRG neurons, and found that the contents of TRPV1 in RIN1 immunoprecipitates were significantly reduced at day 28 post-inoculation (Figure 6(b)), suggesting that the bone cancer pain correlated with the disruption of RIN1 binding to TRPV1. The cultured DRG neurons showed a significant decrease in the protein level of RIN1 after the establishment of cancer pain (Figure 6(c)), which was coincident with a substantial enhancement of TRPV1 immunosignals on the plasma membrane (Figure 6(c)). To test whether the membrane accumulation of TRPV1 was attributable to the RIN1 deficit, we expressed exogenous RIN1 (WT) in the cultured DRG neurons from mice with bone cancer pain. Compared to the control vector, RIN1 (WT) significantly reduced the distribution of TRPV1 on the plasma membrane (Figure 6(d)). These data suggested that the bone cancer removed the RIN1-mediated inhibition of TRPV1.

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Figure 6.

The bone cancer reduced RIN1 expression that caused the accumulation of TRPV1 on the plasma membranes of DRG neurons: (a) the von Frey thresholds of the tumor-bearing limb. F(2, 20) = 7.386, **p = 0.004 (repeated measures), n = 6 mice/group, (b) Co-IP was conducted from the DRGs of mice with or without the bone cancer pain. RIN1 immunoprecipitates were probed with the antibodies indicated on the left of the panel. **p = 0.004 (Mann–Whitney U test), n = 6 experiments, (c) TRPV1 on the plasma membrane (red) and RIN1 (green) were immunostained in the cultured DRG neurons from sham or cancer pain mice. **p = 0.006 and ***p < 0.001 (Mann–Whitney U test). Scale bar, 10 µm, and (d) the cultured DRG neurons from mice with the bone cancer pain were transfected with RIN1 (WT) before immunostaining of RIN1 (green) and surface TRPV1 (red). The empty vector was used as control. **p = 0.001 (Mann–Whitney U test). Scale bar, 10 µm.

Viral delivery of RIN1 (WT) into the DRG neurons attenuated the bone cancer pain

To test whether the rescue of RIN1 expression could suppress the cancer pain-related behaviors, we injected a Cre-inducible adeno-associated virus (AAV) encoding RIN1 (WT) and EGFP into the DRG neurons of Advillin-Cre mice at day 7 post-tumor inoculation (Figure 7(a)). The AAV construct coding for EGFP alone was used as control. The results showed that viral expression of RIN1 (WT) in the DRG neurons (Figure 7(b)) reduced the spontaneous licking (Figure 7(c)) and flicking behaviors (Figure 7(d)) induced by the bone cancer. The reduction of the PWT values in mice with the bone cancer was also reversed by RIN1 (WT; Figure 7(e)). We then used the CPA paradigm to evaluate the affective dimension of pain. The EGFP-expressing sham mice displayed no place aversion to the innocuous 1-g von Frey filament stimulation (Figure 7(f)). The EGFP-expressing cancer mice, however, showed avoidance of the chamber paired with the punctate stimulation (Figure 7(f)), indicating a negative emotional experience. Viral expression of RIN1 (WT) attenuated learning of this negative association, as manifested by the identical time spent in the chamber before and after conditioning (Figure 7(f)). These data suggested that the rescue of RIN1 (WT) expression in the DRG neurons effectively alleviated the sensory and affective pain induced by the bone cancer.

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Figure 7.

Viral expression of RIN1 (WT) in the DRG neurons of mice alleviated the bone cancer pain: (a) experimental timeline. The Cre-inducible AAV encoding EGFP and RIN1 (WT) was sterotaxically injected into the DRGs of Advillin-Cre mice. The AAV coding for EGFP alone was used as control, (b) EGFP expression in the DRGs at day 21 after viral injection. Scale bar, 50 µm, (c, d) spontaneous licking (c) and flicking behaviors (d). **p = 0.003, ***p < 0.001, ^##p = 0.002, and ^#p = 0.045 (Kruskal–Wallis test), (e) paw withdrawal thresholds in response to von Frey filament stimulation. ***p < 0.001 and **p = 0.006 (Kruskal–Wallis test), and (f) rescuing RIN1 expression in the DRGs of mice with the bone cancer pain attenuated the CPA induced by repetitive von Frey filament (1 g) stimulation of hindpaws. t(9) = 4.023, ^##p = 0.003 (paired Student t-test). *p = 0.014 and **p = 0.001 (Kruskal–Wallis test).

Discussion

Many pro-inflammatory factors, such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and insulin-like growth factor-1 (IGF-1), have been shown to up-regulate TRPV1 activity in the nociceptors, which serves as a key contributor to the bone cancer-related pain behaviors.^5,7,53 The current study identified RIN1 as an endogenous suppressor of TRPV1 activity, and provided evidence that RIN1 hypofunction played an important role in the TRPV1 sensitization and bone cancer pain. These findings align with emerging research on RIN1’s role in pain modulation. For instance, Lin et al. ¹⁷ reported that RIN1 exerts a protective effect against pain hypersensitivity by attenuating ferroptosis in a neuropathic pain model. The Nrf2/HO-1 pathway, a key regulator of cellular antioxidant defense, was identified as a downstream effector of RIN1 in this context. Given that oxidative stress ⁵⁴ and ferroptosis^55,56 have also been implicated in bone cancer pain, it is plausible that RIN1 may exert its anti-nociceptive effects through a similar mechanism in our model. However, direct experimental evidence for the involvement of the Nrf2/HO-1 pathway or ferroptosis in RIN1-mediated TRPV1 regulation during bone cancer pain is currently lacking and warrants further investigation.

The reduced expression of RIN1 in DRG neurons during bone cancer pain, as observed in our study, raises an important question regarding the underlying regulatory mechanisms. It is well established that the bone cancer microenvironment is characterized by elevated levels of pro-inflammatory and pro-nociceptive factors, including transforming growth factor-β (TGF-β). ¹⁰ Multiple molecular mechanisms may contribute to RIN1 downregulation in DRG neurons under bone cancer pain conditions. First, oxidative stress and ferroptosis, which are markedly enhanced in bone cancer microenvironment, can accelerate protein degradation and reduce RIN1 stability.^17,55 Second, pro-inflammatory cytokines including TNF‑α and IL‑6 are highly elevated in cancer pain and may suppress RIN1 transcription through NF‑κB‑dependent signaling. ⁵⁴ Notably, previous studies have demonstrated that TGF-β signaling can suppress RIN1 expression through the induction of SNAI1, a transcriptional repressor that directly acts on the RIN1 promoter.^15,26 In addition, pro-inflammatory cytokines and chemokines upregulated in the bone cancer microenvironment may also contribute to RIN1 downregulation and subsequent neuroinflammation.^38,57 Third, TGF‑β signaling, which is strongly activated in bone cancer, induces the transcriptional repressor SNAI1 to directly inhibit RIN1 promoter activity.^20,26 Finally, epigenetic silencing via DNA hypermethylation of the RIN1 gene promoter may also contribute to reduced RIN1 expression in chronic pain states. ¹⁴ Notably, recent studies have demonstrated that dysregulated histone modifications drive the aberrant expression of multiple pain-related genes in DRG neurons during bone cancer pain, providing a conserved epigenetic mechanism for persistent pain sensitization. ³⁹ These pathways act in concert to reduce RIN1 expression, thereby relieving its inhibition on TRPV1 and promoting persistent pain sensitization.

Our data showed that capsaicin-activated TRPV1 was able to motivate the cytoplasmic RIN1 toward the plasma membrane, where RIN1 interacted with and internalized TRPV1. This subcellular redistribution of RIN1 might provide a feedback inhibition of TRPV1 signaling and lead to a rapid termination of acute pain responses caused by capsaicin. Our findings that RIN1 inhibits TRPV1-dependent pain sensitization are supported by multiple lines of evidence, despite the potential limitation of intraplantar capsaicin activating both central (DRG) and peripheral (cutaneous) TRPV1. ⁵⁸ First, RIN1 and TRPV1 co-localize in DRG neurons and interact dynamically upon capsaicin stimulation, with RIN1 promoting TRPV1 endocytosis in DRGs. Second, DRG-specific RIN1 deletion (cKO-RIN1) enhances TRPV1 activity and pain behaviors, while DRG-targeted RIN1 rescue reverses these phenotypes. These genetic manipulations isolate RIN1 function to DRGs, excluding peripheral contributions. Thus, while intraplantar capsaicin may activate peripheral TRPV1, our data—including DRG-restricted RIN1 expression, direct RIN1–TRPV1 interaction in DRGs, and phenotype reversal via DRG-specific RIN1 rescue—strongly support that RIN1 modulates TRPV1 within DRG neurons to control pain.

The observation that TRPV1 protein levels are increased in RIN1 cKO DRGs suggests that RIN1 may regulate TRPV1 homeostasis by promoting its degradation. A key question arises regarding the specific degradation pathway involved-lysosomal or proteasomal. Previous studies have demonstrated that agonist-induced TRPV1 desensitization is accompanied by receptor internalization and subsequent lysosomal degradation, a process dependent on endocytic sorting. ⁴⁹ Given that RIN1 mediates Rab5-dependent endocytosis,^13,15 which is tightly linked to the trafficking of internalized cargo to early endosomes and ultimately lysosomes, it is plausible that RIN1 directs TRPV1 to lysosomal degradation. In this scenario, loss of RIN1 would impair TRPV1 trafficking to lysosomes, leading to its accumulation in DRG neurons. Alternatively, while less likely given the known role of RIN1 in endocytic processes, a proteasomal pathway cannot be formally excluded without direct experimental evidence.

In the setting of bone cancer-induced chronic pain, the protein level of RIN1 was substantially reduced. The impaired RIN1 inhibition might contribute to the persistence of TRPV1 sensitization and TRPV1-dependent cancer pain. Therefore, the rescue of RIN1 expression effectively repressed the surface accumulation of TRPV1 and the bone cancer pain.

Previous studies have illustrated that RIN1 is present at the nucleus, cytoplasm, and plasma membrane.^25,59 In the central nervous system, RIN1 is also detected in the synaptosomal and postsynaptic density fractions, ¹³ a specialized biochemical architecture at postsynaptic membrane, that is, critical for synaptic modification. A multifunctional protein, 14-3-3, has been implicated in the regulation of the RIN1 trafficking between the cytoplasm and plasma membrane. ²⁵ When phosphorylated at Ser351 by protein kinase D, RIN1 shows high affinity for 14-3-3 proteins. ²⁵ A non-phosphorylatable RIN1 mutant dissociates with 14-3-3 proteins and interacts with the active Ras, exhibiting a tendency for its translocation to the plasma membrane. ²⁵ This dynamic trafficking process provides a spatiotemporal mechanism for RIN1 to internalize the activated receptor tyrosine kinases (RTKs) on the plasma membrane and thus, limit the duration and magnitude of RTK signalings.^13,15,21,22 Our data showed that capsaicin activation of TRPV1 motivated RIN1 toward the plasma membrane, where a higher colocalization of RIN1 with TRPV1 was observed. These results suggested that TRPV1-initiated downstream intracellular signalings were able to regulate the subcellular localization of RIN1. The GEF activity of RIN1 has been shown to induce the Rab5-dependent endocytosis of epidermal growth factor receptor, EphA4 receptor and excitatory ionotropic glutamate receptor.^13,15,52,60 Our data showed that in the DRG neurons, the Rab5 GEF activity was also required for RIN1 to internalize TRPV1 channels, as the Rab5 GEF-deficient RIN1 (E574A) mutant totally lost the ability to reduce the expression of TRPV1 on the plasma membrane.

Previous studies have demonstrated in the breast tumor cell lines that RIN1 gene is silenced through the DNA methylation within the RIN1 promoter and first exon or through the overexpression of SNAI1 (Snail), ²⁶ a transcription repressor, that is, regulated by transforming growth factor β (TGF-β) and Ras signaling. ²⁰ Through inducing the SNAI1 expression, TGF-β and Ras signalings act in concert to remove the RIN1-mediated inhibition of breast tumor cell migration. ²⁰ Interestingly, TGF-β1, when injected into the plantar surfaces of hindpaws, provokes nociceptive behaviors even in intact mice, which can be completely blocked by genetic TRPV1 knockout, ¹⁰ suggesting that TRPV1 is a critical target for TGF-β1 to sensitize the nociceptive responses. Importantly, the bone cancer pain correlates with a high expression of TGF-β1 in the tumor-bearing bone and the up-regulation of TGF-β receptors in the DRG neurons. ¹⁰ By augmenting TRPV1 in primary sensory neurons, TGF-β signaling plays an important role in the bone cancer pain. Pharmacological inhibition of TGF-β receptors generates a potent analgesic action against the bone cancer pain. ¹⁰ Our data illustrated that direct TRPV1 activation by capsaicin caused acute pain but had no effect on RIN1 protein level. However, the induction of bone cancer pain dramatically decreased the protein expression of RIN1 in the DRG neurons. Although the mechanisms underlying the protein synthesis and degradation of RIN1 in the DRG neurons remain to be elucidated, it was reasonable that TGF-β signaling might contribute to the silencing of RIN1 during the bone cancer pain.

Our findings reveal that RIN1 acts as an endogenous suppressor of TRPV1 in DRG neurons, limiting TRPV1-dependent pain sensitization in bone cancer pain. Beyond this context, the RIN1–TRPV1 axis may hold broader relevance for diverse pain states, as dysregulation of ion channel trafficking in DRG neurons is a common mechanism underlying chronic pain^37,61 and TRPV1 is a well-established mediator of inflammatory, neuropathic, and visceral pain. ⁶² Consistently, RIN1 has been shown to modulate nociceptive sensitization in chronic constriction injury-induced neuropathic pain, ¹⁷ and to regulate synaptic plasticity in spinal pain pathways, ¹⁸ suggesting that RIN1-mediated ion channel trafficking is a conserved mechanism across different chronic pain conditions.

Inflammatory pain, triggered by tissue injury or infection, is characterized by TRPV1 upregulation and hypersensitivity to thermal and mechanical stimuli. ⁶³ Pro-inflammatory mediators such as prostaglandin E2 (PGE2) and bradykinin enhance TRPV1 activity through post-translational modifications promoting its surface retention and channel gating. ⁶⁴ Notably, RIN1’s ability to drive TRPV1 endocytosis—dependent on its Rab5 GEF activity—could counteract such pro-inflammatory effects. ⁶⁵ For instance, in models of inflammatory pain, RIN1 downregulation might exacerbate TRPV1 accumulation on DRG neuron membranes, amplifying pain hypersensitivity. Conversely, boosting RIN1 function could potentially reverse this process, mirroring its rescue effect in bone cancer pain. This aligns with studies showing that endocytic regulation of TRPV1 is a conserved mechanism to limit inflammatory hyperalgesia. ⁶⁶

Neuropathic pain, arising from nerve injury, also involves TRPV1 dysregulation. Trauma to peripheral nerves induces TRPV1 overexpression in DRG neurons and spinal cord projections, contributing to spontaneous pain and allodynia. ⁶⁷ Importantly, RIN1 has already been implicated in neuropathic pain modulation: Lin et al. ¹⁷ demonstrated that RIN1 deficiency exacerbates pain hypersensitivity in a chronic constriction injury model, linked to impaired Nrf2/HO-1-mediated antioxidant defense. While this study focused on ferroptosis, our data suggest an additional layer—RIN1 may concurrently restrict TRPV1 activity in DRG neurons after nerve injury. Given that nerve damage disrupts endocytic pathways in DRG neurons, ⁶⁸ RIN1’s role in Rab5-dependent TRPV1 internalization could be particularly critical for preventing excessive neuropathic pain sensitization.

Visceral pain, such as that associated with irritable bowel syndrome or pancreatitis, similarly relies on TRPV1 activation in sensory neurons innervating internal organs. ⁶⁹ Emerging evidence indicates that TRPV1 trafficking dynamics modulate visceral pain thresholds. ⁷⁰ Whether RIN1 regulates TRPV1 in visceral afferents remains untested, but its broad expression in sensory neurons ⁷¹ raises the possibility that the RIN1–TRPV1 axis could be a conserved node across somatic and visceral pain modalities.

Collectively, these considerations suggest that RIN1-mediated TRPV1 regulation is not restricted to bone cancer pain but may represent a generalizable mechanism to constrain TRPV1 hyperactivity in multiple pain states. The therapeutic potential of targeting this axis is underscored by its cell-type specificity and reliance on a druggable GEF domain, ¹⁵ which could be exploited to develop RIN1 activators or mimetics. Such interventions might offer advantages over broad-spectrum TRPV1 antagonists by preserving physiological TRPV1 function while limiting pathological sensitization.

Pain is the most common symptom associated with the metastatic bone cancer. Patients frequently experience moderate to severe spontaneous pain, a non-evoked persistent unpleasant state, and mechanical allodynia, a pain response to an innocuous stimulus or the movement of the tumor-bearing bone. TRPV1 in the DRG neurons has received a great deal of attention in the pathogenesis and treatment of bone cancer pain. Pharmacological or genetic inhibition of TRPV1 channel effectively alleviates the mechanical allodynia induced by the bone cancer.^5,72 Recent studies indicate that TRPV1-positive primary sensory neurons are also involved in the sustained affective pain evoked by noxious mechanical, cold and heat stimulation, manifested by the reduction in the coping behaviors after the ablation of TRPV1-positive primary afferent terminals in the spinal cord. ¹¹ Consistent with these observations, our data demonstrated that the conditioned RIN1 deletion augmented TRPV1 activity in the DRG neurons that elicited the mechanical allodynia and facilitated the aversive learning, while the rescue of RIN1 expression attenuated the reflexive sensitization and aversive state associated with the bone cancer pain.

Taken together, our data revealed an important role of RIN1 in the negative control over both the reflexive-defensive and affective-motivational aspects of bone cancer pain through the inhibition of TRPV1 activity. We proposed that RIN1 might represent a promising target for the treatment of intractable and debilitating cancer pain. Modulation of the RIN1–TRPV1 axis holds meaningful translational implications for chronic pain disorders. As an endogenous negative regulator of TRPV1 trafficking, RIN1 represents a novel target with high specificity and low risk of off‑target effects. Pharmacological activation of RIN1 or enhancement of its Rab5–GEF activity may selectively suppress pathological TRPV1 membrane accumulation while preserving physiological TRPV1 function.^15,66 In addition, AAV‑mediated RIN1 overexpression in DRG neurons provides a feasible gene therapy strategy for intractable bone cancer pain, inflammatory pain, and neuropathic pain.^66,73 Further exploration of RIN1‑targeting small‑molecule activators may lead to a new class of mechanism‑based analgesics for clinical use.

A critical consideration in our study is the exclusive use of male mice, which limits the generalizability of our findings to female populations. This approach was initially adopted to minimize variability during mechanistic exploration, a common practice in foundational pain research, but overlooks well-documented sex differences in TRPV1 function and pain processing. Accumulating evidence indicates that TRPV1-mediated pain responses exhibit sexual dimorphism: for example, female rodents often display heightened TRPV1-dependent thermal hyperalgesia compared to males, potentially driven by estrogen-dependent modulation of TRPV1 expression and channel gating in DRG neurons. ⁷⁴ Additionally, sex-specific differences in endocytic machinery and Rab GTPase regulation—key to RIN1’s function—have been reported in sensory neurons, suggesting that RIN1–TRPV1 interactions could vary across sexes. ⁷⁴

These disparities are clinically relevant, as bone cancer pain manifests differently in male and female patients, with variations in pain severity, response to analgesics, and underlying molecular mechanisms. ⁷⁵ Our current findings, derived solely from male mice, therefore necessitate cautious interpretation when extrapolating to female individuals. Future studies must systematically address this gap by comparing RIN1 expression, TRPV1 trafficking, and pain behaviors in both sexes, and exploring potential regulatory roles of gonadal hormones (e.g. estrogen, testosterone) in RIN1-mediated TRPV1 modulation.

A limitation of the current study is that we did not directly examine the role of TGF-β or other tumor-derived factors in mediating RIN1 downregulation, which represents an important avenue for future research.

A limitation of the current study is that we did not formally distinguish between lysosomal and proteasomal pathways in RIN1-mediated TRPV1 degradation. Future studies using lysosomal inhibitors or proteasomal inhibitors in RIN1 cKO DRG neurons would help pinpoint the exact degradation mechanism, further refining our understanding of RIN1–TRPV1 interplay.

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