P2Y₁₄ receptors: a new target for treating ulcerative colitis

Article summary

In a recent paper published in Nature Communications [1], Liu et al. identified that targeting P2Y₁₄ receptors (P2Y₁₄R) is a promising strategy for treatment of ulcerative colitis (UC). Increased expression of P2Y₁₄R was found in intestinal epithelium cell (IEC) of human UC and dextran sulfate sodium (DSS)-induced experimental colitis. Deficiency of P2Y₁₄R alleviated UC by inhibiting necroptosis of IEC mediated by PKA/cAMP-response element binding protein (CREB)/RIPK1 pathway. Consistent with this mechanism, the authors reported that a new small-molecule P2Y₁₄R antagonist, HDL-16, is a potential drug candidate for the treatment of UC.

Commentary

UC is a chronic inflammatory disease, which can reduce life expectancy and increase the risk of developing colon cancer. In 2023, it was estimated that there are 5 million cases worldwide and the incidence is still growing [2]. 5-Aminosalicylic acid, corticosteroids, monoclonal antibodies (TNF inhibitors, anti-integrins, interleukin 12/23 inhibitors, etc.), small molecules (Janus kinase inhibitors, sphingosine-1-phosphate receptor modulators), and surgery to remove part or all of the colon are currently the main therapies. However, non-responsiveness or loss of clinical benefits over time or increased risks of infection or cancer to the current therapies indicate that it is necessary to identify more promising targets for the management of UC [3–5].

In this study, the authors first found increased expression of the P2ry14 gene in UC specimens via analysis of the datasets GSE38713, GSE75214, and GSE16879 from NCBI’s Gene Expression Omnibus (GEO). Then, they confirmed that the overexpression of the P2Y₁₄R is mainly localized to IECs in UC patients and DSS-induced experimental colitis. To clarify the role of P2Y₁₄R in the development of UC, the team generated IEC-specific, P2Y₁₄R knockout (P2Y₁₄R^ΔIEC) mice by crossing the floxed strain (P2Y₁₄R^fl/fl) with a Villin-cre transgenic line, and they compared the different phenotypes presented following acute or chronic administration of 3% DSS to P2Y₁₄R^ΔIEC and P2Y₁₄R^fl/fl mice. They report that the beneficial effect of rectum shortening observed in P2Y₁₄R^ΔIEC mice was seen only in acute and not chronic DSS-induced colitis. However, both models established in P2Y₁₄R^ΔIEC mice had less weight loss, diarrhea, rectal bleeding, and histological damage. Immunofluorescence staining revealed enhanced expression of tight junction proteins in DSS-treated P2Y₁₄R^ΔIEC mice, suggesting that P2Y₁₄R may influence the death of IECs. Further analysis of several markers of cell death patterns revealed minimal impact of IEC-specific P2Y₁₄R knockout on TUNEL-positive cells and the level of apoptosis-related proteins (caspase-3, Bcl-2, Bax) and pyroptosis-related proteins (caspase-1 p20, GSDMD N-terminal). However, the activation of MLKL, which is related to necroptosis, was decreased in DSS-induced P2Y₁₄R^ΔIEC mice, suggesting that P2Y₁₄R of IECs could regulate the epithelial necroptosis, but not apoptosis or pyroptosis. The team then treated human IEC lines (HT-29 and HCT 116 cells) with TNF-α adding Smac mimetic and z-VAD (TSZ). The data showed that P2Y₁₄R silencing by siRNA transfection significantly inhibited necroptosis, increased cell survival, and reduced PI-positive cells, which was supported by H&E staining, transmission electron microscopy analysis, and measurements of necroptosis markers. Additionally, similar results were also found from the experiment performed in intestinal epithelial organoids from P2Y₁₄R^fl/fl and P2Y₁₄R^ΔIEC mice.

During the exploration of downstream mechanisms, the authors found that silencing of P2Y₁₄R disrupted the interaction between RIPK1 and RIPK3, which was critical for the formation of the necrosome complex, as demonstrated by co-immunoprecipitation analysis. Cell viability and staining analysis showed that overexpression of P2Y₁₄R by transfecting the hP2Y₁₄R expression vector in HT-29 cells aggravated IEC necroptosis. This effect was then alleviated by pretreatment with Nec-1s (RIPK1 inhibitor) and GSK′872 (RIPK3 inhibitor), indicating that P2Y₁₄R mediated TSZ-induced IEC necroptosis via the RIPK1/RIPK3 pathway. Moreover, P2Y₁₄R modulated RIPK1 transcription and RIPK1 mRNA levels in HT-29 and HCT-116 cells, while RIPK3 was unaffected. Silencing P2Y₁₄R resulted in a decrease in RIPK1 mRNA levels, and overexpression of P2Y₁₄R increased RIPK1 transcription. Through transcription factor prediction analysis and ChIP experiments, CREB was found to bind to the RIPK1 promoter. P2Y₁₄R deficiency enhanced this binding under DSS- or TSZ-treated conditions, suggesting that P2Y₁₄R promotes the necroptosis of IECs by inhibiting CREB binding to the RIPK1 promoter. In the absence of P2Y₁₄R, the authors found an increase in intracellular cAMP levels, along with increased phosphorylation of PKA and CREB. However, using specific inhibitors (SQ22536, H-89, 666–15) to target the cAMP/PKA/CREB pathway disrupted the protection of siP2Y₁₄R against TSZ-induced necroptosis, resulting in an upregulation of RIPK1 mRNA levels. This confirmed that P2Y₁₄R could regulate the necroptosis of IECs through the cAMP/PKA/CREB signaling pathway.

UDP-glucose (UDPG), an endogenous ligand of P2Y₁₄R, can be released as a damage-associated molecular pattern by dying cells and bind to the P2Y₁₄R. In view of this, they investigated the synthesis and degradation enzymes of UDPG, as well as its role in UC. They found that both UDP-glucose pyrophosphorylase 2 (Ugp2) and glycogen synthase 1 (Gys1) levels were decreased in UC patients and animal models. Additionally, UDPG was found to promote TSZ-induced cell necroptosis, enhance the inhibition of the cAMP/PKA/CREB pathway induced by TSZ treatment, increase RIPK1 transcription, and inhibit the binding between CREB and the RIPK1 promoter. Furthermore, experiments performed in P2Y₁₄R^ΔIEC mice and organoids revealed that the regulation of UDPG in DSS-induced inflammation depends on P2Y₁₄R. Taken together, these data strongly indicates that the UDPG-P2Y₁₄R-cAMP/PKA/CREB pathway plays a crucial role in relief of DSS-induced inflammation.

After clarifying the mechanisms underlying the effects of P2Y₁₄R stimulation in UC, the team proposed that it is necessary to develop new P2Y₁₄R antagonists for the treatment of UC, as the most effective and selective P2Y₁₄R antagonist currently available, PPTN, is limited by its poor solubility and low oral bioavailability. Having identified the key residues of the P2Y₁₄R structure, the authors screened the ChemDiv library using the structure-based virtual screening protocol and obtained a total of 33 compounds, from which a small molecule P2Y₁₄R antagonist with high activity and low toxicity, HDL-16, was considered as the potential candidate. When DSS-treated mice were treated with HDL-16 (potential P2Y₁₄R antagonist) or PPTN (as the positive control), it was found that both resulted in reduced weight loss and disease activity index, increased colon length, improved histological inflammation and tissue damage, and stabilized tight junction protein expression enhancing mucosal barrier function. Application of immunoblotting, RT-PCR, and ChIP techniques showed that HDL-16 upregulated the cAMP/PKA/CREB pathway and promoted the combination of CREB and RIPK1 promoter in DSS-treated mice, thereby resulting in inhibition of IEC necroptosis in UC. Both HDL-16 and PPTN also effectively improved TSZ-mediated cell necroptosis in HT-29 cells and P2Y₁₄R^fl/fl organoids.

In conclusion, this work provides strong evidence to show that the UDPG-P2Y₁₄R axis is involved in IEC necroptosis and the absence of P2Y₁₄R protected IECs from necroptosis via the cAMP/PKA/CREB pathway, regulated the combination of CREB and RIPK1 promotor, and significantly improved intestinal damage. In addition, based on the P2Y₁₄R-targeted therapy strategy for the treatment of UC, the team synthesized a new potent antagonist of P2Y₁₄R, HDL-16, and identified it to be an effective alternative, the currently available P2Y₁₄R antagonist, PPTN. This work also paves the way for future clinical trial focused on P2Y₁₄R-targeted therapy strategy for UC.

Author contribution

YQZ and YT wrote the main manuscript text, and all authors reviewed the manuscript.

Funding

The authors were supported from the Sichuan Provincial Administration of Traditional Chinese Medicine (2023zd024) and Sichuan Science and Technology Program (2022NSFSC0852).

Data availability

No datasets were generated or analyzed during the current study.

Declarations

Competing interests

The authors declare no competing interests.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Conflicts of interest

Yan-Qin Zuo declares that he/she has no conflict of interest. Yong Tang declares that he/she has no conflict of interest.