REG/Reg family proteins: mediating gut microbiota homeostasis and implications in digestive diseases

ABSTRACT

The regenerating (REG/Reg) family, a subset of the C-type lectin fold superfamily, is characterized by “C-type lectin-like” domains. Beyond promoting proliferation and differentiation of hepatic, pancreatic, gastric, and intestinal cells, its members have multifunctional secretory activities, especially as antimicrobial peptides (AMPs), a key link between their structural features and roles in gastrointestinal physiology/pathology. These molecules mediate the initiation and progression of various gastrointestinal inflammatory and inflammation-associated diseases. As AMPs, they connect gut microbiome and host immunity by regulating microbiota homeostasis, intestinal mucosal barrier, metabolism, and energy balance; notably, REG3/4 play dual roles in digestive tract diseases. This review proposes the REG Protein Functional Equilibrium Model. REG proteins act as context-dependent “molecular rheostats” switching between protective and pathogenic roles based on microenvironmental cues. The functional equilibrium model provides a new paradigm for developing precision therapeutics that target the microenvironment rather than single molecules, offering critical theoretical foundations for resolving the functional paradox of REG family in inflammation and cancer and advancing its clinical translation.

Introduction

The mammalian intestine harbors a complex microbial ecosystem,^1‐3 the homeostasis of which is maintained by sophisticated host defense mechanisms,^4,5 including the precise regulation of antimicrobial peptides (AMPs).⁵ Among various AMPs,^6‐9 the REG/Reg family of proteins, which are a unique class of C-type lectin-fold proteins. And, they not only exhibit targeted antimicrobial activity but also mediate epithelial repair and metabolic regulation, demonstrating a functional breadth far exceeding that of conventional AMPs.^10‐15 Dysregulation of the gut microbiota has been linked to inflammatory bowel disease (IBD), colorectal cancer (CRC), and infectious diseases,^16‐18 making the regenerating (REG/Reg) proteins as key effector molecules of the host antimicrobial defense system - a focal point of research.¹⁹ However, a growing body of evidence has revealed a striking functional paradox exhibited by REG proteins across different disease contexts. While they can suppress inflammation and maintain barrier integrity under certain conditions, they also demonstrably promote tumor progression and exacerbate immune dysregulation in others.^16‐20 Notably, this context-dependent Double-edged Sword characteristic poses a major obstacle to their clinical translation and underscores the profound limitations of our current understanding.

Critically, a unified theoretical framework explaining how REG proteins precisely modulate microbe-host interactions is still lacking. The decisive factors and microenvironmental cues that dictate their functional output remain poorly defined.^20‐22 Consequently, the numerous seemingly contradictory findings significantly hinder their development as reliable biomarkers or therapeutic targets. It is therefore imperative to construct integrated mechanistic models that incorporate multidimensional factors, such as cellular origin, microbial signals, and disease stage, which to decipher their context-dependent functional switching. Therefore, to reconcile these context-dependent dualities and decipher the regulatory principles underpinning the Janus-faced behaviors of REG proteins, we propose a REG Protein Functional Equilibrium Model (Figure 1) that delineates how microenvironmental cues dictate their functional equilibrium in gut mucosal fate. And that provides a robust theoretical foundation for precise targeting strategies, thereby facilitating the translation of basic mechanistic insights into clinical applications.

Figure 1.

The REG protein functional equilibrium: a determinant of gut mucosal fate. This model delineates the functional equilibrium of the REG protein family at the interface of intestinal health and disease, ultimately determining mucosal fate. Moving beyond a simplistic binary narrative of Beneficial or Detrimental it posits that REG functionality is plastic and dictated by a dynamic network integrating microbial signals, host immune responses, and core signaling pathways. The overall expression levels of the REG/Reg family (left) and the state of the intestinal microenvironment (right) jointly determine whether the body's equilibrium is disrupted and progresses toward deterioration, or whether it maintains balance and advances toward improvement. On the left, a summary classification is presented showing the baseline expression levels of the host itself and the levels induced by the gut microbiota. It demonstrates the effects of both balanced and imbalanced states on the overall expression levels of the host's REG/Reg family. The right-side model of disease-health transition equilibrium. In gastrointestinal system diseases, one of the determinants of health and disease states is the expression levels of REG/Reg proteins. Simultaneously, the expression levels of REG/Reg proteins are influenced by three key factors (Microbial induction, Host response level, Pathway activation). Which drives the transition between health and disease, and determines the condition of the intestinal microenvironment.

REG/Reg family

In 1984, Yamanoto et al. discovered that nicotinamide accelerates islet regeneration in rats undergoing partial pancreatectomy.^20,23,24 In 1988, Terazon et al. isolated a cDNA encoding a protein with a relative molecular mass of 16,000 from rats with 90% pancreatic resection. Since the encoded protein was exclusively present in regenerating islets, it was termed regenerating protein.^20,23,24 In 1990, Watanabe et al. isolated the human Reg gene, confirming its identity with pancreatic stone protein (PSP) and pancreatic thread protein as a single protein (collectively termed PSP/Reg, initially designated RegI).^20,25,26 In 1997, Rafaeloff cloned the human RegI cDNA and discovered mouse homologs of rat and human Reg genes, as well as novel RegI and RegII genes.²⁵ In 1999, Okamoto et al. classified human, rat, and mouse Reg and related genes into types I, II, and III.¹² In 2001, Hartupee et al. identified human RegIV through sequencing cDNA libraries from ulcerative colitis (UC) samples and characterized its chromosomal localization and other features.²⁵ In recent years, REG/Reg gene family has assumed an increasingly central role in various diseases related to digestive system (Figure 2).

Figure 2.

REG/Reg family in digestive system diseases. The REG/Reg family exhibits member-specific roles in digestive diseases: REG3γ bidirectionally regulates microbiota balance and inflammation in IBD; REG4 promotes metastasis while Reg3β suppresses cancer in CRC; REG1A inhibits cancer but REG4 drives drug resistance in gastric cancer; REG3β clears pathogens yet exacerbates viral dysbiosis in infections; REG3A acts as a pre-cancer suppressor but post-cancer promoter in liver cancer; and REG3γ regulates blood sugar while REG4 maintains metabolic homeostasis in metabolic diseases. Created in https://BioRender.com.

Member

The REG/Reg gene family comprises five members in humans and seven members in mice.²⁰ Current research exhibits significant inconsistency in naming conventions for the REG/Reg family,²⁷ particularly concerning the description of human-mouse homology,²⁸ which hinders the scientific dissemination of research findings. Consequently, we conducted a systematic review of the molecular characteristics of the genes and proteins within this family. In humans, the Reg family genes include REG1A (or REG1α), REG1B (or REG1β), REG3A (or REG3α), REG3G (or REG3γ), and REG4. Among these, REG1A, REG1B, REG3A, and REG3G are located on chromosome 2, while REG4 is located on chromosome 1. In mice, the Reg family genes include Reg1, Reg2, Reg3a (or Reg3α), Reg3b (or Reg3β), Reg3g (or Reg3γ), Reg3d (or Reg3δ), and Reg4. The genes Reg1, Reg2, Reg3a, Reg3b, Reg3g, and Reg3d are located on chromosome 6, whereas Reg4 is located on chromosome 3 (Figure 3). The REG/Reg family is widely expressed in the digestive system, both in humans and mice, particularly in the intestinal tract (Figure 3), and they are primarily localized to gastrointestinal-associated epithelial cells (epithelial cells of small intestinal crypts, Paneth cells, colonic crypt cells, gastric mucosal epithelial cells) and pancreatic duct epithelial cells. REG1A are also expressed in intestinal stem cells and goblet cells. REG/Reg family proteins typically exhibit low-to-moderate expression in normal gastrointestinal mucosa but are significantly upregulated in cancers and IBD, showing dynamic expression patterns closely correlated with pathological type, differentiation grade, and malignant phenotype.^{1‐3,20,29‐47}

Figure 3.

Structural conservation and tissue-specific expression of the REG protein family. This figure illustrates the conserved architecture and organ-specific expression patterns of REG family proteins across humans and mice. Left Panel: displays the predicted tertiary structures of human and murine REG/Reg proteins, highlighting the shared CTLD critical for their function. Despite adopting the canonical C-type lectin fold, REG proteins lack conserved residues for Ca²⁺-dependent carbohydrate binding.^48‐50 All members feature this conserved domain, composed of two α-helices and five β-strands, underlying their roles as AMPs.^51,52 Human (REG1A/P05451, REG1B/P48304, REG3A/Q06141, REG3G/Q6UW15, REG4/Q9BYZ8) and Mouse (Reg1/P43137, Reg2/Q08731, Reg3a/O09037, Reg3b/P35230, Reg3g/O09049, Reg3d/Q9QUS9, Reg4/Q9D8G5) members exhibit structural homology but functional divergence. The UniProt KB ID form UniProt data (https://www.uniprot.org/). Right and Lower Panels: Summarize the tissue-specific expression profiles of REG members. In humans, expression is tightly regulated: REG1A is highly expressed in small intestinal crypt base epithelial cells (IECs) and various cancers, but low in non-inflamed colon; its expression inhibits gastric cancer cell invasion via suppressing PI3K/Akt-GSK3β signaling.^29‐33 REG3A is expressed in pancreatic ducts and Paneth cells^34‐36 but downregulated in gastric cancer. REG3G is predominantly in small intestinal Paneth cells.^37‐39 REG4 localizes to colonic deep crypt secretory cells and is weakly expressed in other gastrointestinal epithelia,^{1‐3,38‐40} In mice, expression is also compartmentalized: Reg1 is constitutively low in ileal/colonic crypts;⁴¹ Reg3a is upregulated by IL−22 in crypts;^20,42 Reg3b is in absorptive enterocytes;⁴³ Reg3g is highly expressed in Paneth cells and secreted to form an antimicrobial barrier,^44,45 also acting as a gut-pancreas hormone regulating energy balance; Reg3d is in pancreatic exocrine/non-β cells);⁴⁶ Reg4 is confined to colon epithelium with low expression.^43,47

Homology comparison

We performed homology comparison of the amino acid sequences encoded by human and murine REG/Reg family genes. Among these, human REG1A exhibits 89.76% homology with mouse Reg1. Human REG1B shows 90.96% homology with mouse Reg1. Human REG3A demonstrates 90.29% homology with mouse Reg3b. Human REG3G displays 91.43% homology with mouse Reg3b. Human REG4 shares 89.24% homology with mouse Reg4 (Table 1).

Table 1.

Comparison of amino acid sequences of Reg family proteins between human and mice.

		Mouse (Similarity Match%)
Gene name		Reg1 (P43137)	Reg2 (Q08731)	Reg3a (O09037)	Reg3b (P35230)	Reg3g (O09049)	Reg3d (Q9QUS9)	Reg4 (Q9D8G5)
	REG1A (P05451)	89.76	84.39	76.00	78.86	74.29	73.30	68.86
	REG1B (P48304)	90.96	84.97	76.57	79.43	78.86	77.27	69.46
Human	REG3A (Q06141)	77.14	73.08	85.14	90.29	87.43	82.29	59.66
	REG3G (Q6UW15)	75.43	71.98	88.57	91.43	87.43	81.71	59.66
	REG4 (Q9BYZ8)	69.46	65.52	61.93	55.68	59.43	56.82	89.24

Amino acid sequence alignment was performed using an online tool (https://rddc.tsinghua-gd.org/zh/tool/sequence-alignment) with the Needleman-Wunsch alignment algorithm.

Protein structure similarity comparison was performed using the DALL platform⁵³ (http://ekhidna2.biocenter.helsinki.fi/dali/) based on AlphaFold-predicted tertiary structure data from the UniProt database. The analysis included secondary structure resolution, protein family classification, sequence identity-based labeled structural alignment, and three-dimensional structural superposition analysis with color-coded visualization of sequence conservation and structural conservation. The structural similarity score Z-score calculated by the DALI algorithm (Z ≥ 10 indicates high homology reliability) may be combined with functional annotation to strengthen conclusions.

We performed similarity comparison of protein structures between human and murine REG/Reg family members based on AlphaFold-predicted tertiary structure data. Among these, human REG1A exhibits the highest similarity to mouse Reg2, human REG1B shows the highest similarity to mouse Reg2, human REG3A demonstrates the highest similarity to mouse Reg3a, human REG3G displays the highest similarity to mouse Reg3a, and human REG4 shares the highest similarity with mouse Reg4 (Table 2).

Table 2.

Similarities comparison of protein structures of Reg family subtypes in human and mice.

		Mouse (similarities with Z-score)
Gene name		Reg1 (P43137)	Reg2 (Q08731)	Reg3a (O09037)	Reg3b (P35230)	Reg3g (O09049)	Reg3d (Q9QUS9)	Reg4 (Q9D8G5)
	REG1A (P05451)	26.6	28.4	24.3	25.5	25.1	24.6	22.2
	REG1B (P48304)	26.4	28.2	24.5	25.6	25.2	24.7	22.2
Human	REG3A (Q06141)	22.5	24.0	28.3	28.2	27.0	25.4	20.8
	REG3G (Q6UW15)	22.0	23.4	28.4	27.9	26.6	24.9	20.7
	REG4 (Q9BYZ8)	21.2	22.0	21.2	21.7	22.2	20.8	24.2

Uniqueness of REG/Reg family proteins

AMPs are the host's core molecules against microbial invasion, having diverse functional modes due to structural differences. The REG/Reg family differs significantly from these classical AMPs in structure and functional mechanisms. The REG/Reg family, a subset of the C-type lectin fold superfamily, is characterized by C-type lectin-like domains, primarily referring to its overall topological similarity in folding. Members of the C-type lectin family possess a conserved structural motif known as the C-type lectin-like domain (CTLD), which is characterized by a specific protein fold and defined by a set of conserved sequence signatures.^48‐50 Many members of this family mediate calcium (Ca²⁺)-dependent carbohydrate binding through a distinct cluster of conserved residues that coordinately determine both Ca²⁺ dependence and ligand specificity.^20,48

Structure of REG/Reg family proteins

The REG/Reg family represents a distinct subset of mammalian CTLD-containing proteins. Structurally, Reg proteins between humans and mice consist exclusively of an N-terminal secretion signal peptide and a ~16-kDa CTLD (Figure 3).⁵² Which minimalist structural pattern stands in sharp contrast to the multi-disulfide bond folding of defensins and the precursor processing activation of cathelicidins. Defensins require 3−4 pairs of disulfide bonds to stabilize their β-sheet core for antimicrobial activity, while cathelicidins need cleavage of the N-terminal cathelin domain by enzymes such as neutrophil elastase to release active α-helical fragments.^54‐56 In contrast, Reg proteins do not require complex processing and can exert functions via the intact CTLD immediately after secretion. More critically, despite adopting the canonical C-type lectin fold, Reg proteins lack the conserved residues essential for Ca²⁺-dependent carbohydrate binding observed in other C-type lectins and most AMPs.^48,49 This structurally conserved but functionally specific feature distinguishes them from traditional Ca²⁺-dependent C-type lectins and also from defensins/cathelicidins that do not require Ca²⁺ but rely on cationic residues. Reg3γ exhibits direct bactericidal activity against Gram-positive bacteria, achieving this effect at low micromolar concentrations.^14,15,57 The targeted bactericidal capacity further highlights the unique biological role of Reg proteins as lectin-type AMPs in mammals.⁵⁸

Regulatory role of REG/Reg proteins in gut microbiota and systemic metabolism

Functional redundancy among REG family proteins is partial and context-dependent. While their core functions-reducing bacterial translocation and suppressing overgrowth of the mucosa-associated microbiota. Overlap, specific functions such as antiviral activity, tissue targeting, and disease regulation may exhibit member-specific characteristics that cannot be compensated for by others. Functional redundancy is primarily observed in fundamental intestinal barrier maintenance and antimicrobial functions. For instance, in gut barrier maintenance, both Reg3β and Reg3γ participate in spatial segregation of gut microbiota and preservation of barrier integrity. Regarding antimicrobial functions, while Reg3 family members exhibit distinct antimicrobial spectra, partial overlap exists. For example, Reg3β targets Gram-negative bacteria, while Reg3β/γ targets Gram-positive bacteria (Figure 4).^10,59,60

Figure 4. .

The role of Reg protein family in healthy intestinal microecology. This figure illustrates the multifaceted roles of REG proteins in maintaining gut homeostasis through direct antimicrobial activity and immunomodulatory mechanisms. Key processes include: Paneth cell-secreted Reg3γ targeting Gram-positive bacteria by disrupting peptidoglycan layers to form a sterile zone; goblet cell-derived Reg3β binding lipopolysaccharide (LPS) on Gram-negative bacteria to prevent systemic dissemination; and REG4 inhibiting flagellated pathogens via interaction with bacterial flagella. Additionally, commensal bacteria such as Parabacteroides goldsteinii stimulate IL-22 release from type 3 innate lymphoid cells (ILC3s), promoting REG expression and enhancing barrier integrity. Viral infections further modulate REG levels, highlighting systemic cross-talk in mucosal immunity. Created in https://BioRender.com.

Regarding the antibacterial mechanism, REG/Reg family proteins function as AMPs,⁵¹ but their mechanism is fundamentally non-redundant with that of classical AMPs. We compared members of this protein family between humans and mice and found that they all possess a highly conserved CTLD. This domain consists of two α-helices and two β-strands, forming a highly conserved structure.⁵² The CTLD can specifically bind bacterial surface polysaccharides (Figure 4), directly disrupt bacterial membrane structures or block pathogen adhesion to host cells, thereby exerting antimicrobial effects.⁵² For example, Reg3 family members specifically recognize long-chain carbohydrates of Gram-positive bacterial peptidoglycan through the EPN motif in Loop1, achieving efficient bactericidal activity via a multivalent binding mechanism.⁴⁹ Reg4 specifically binds Salmonella flagella through its HDPQK motif within the Ca²⁺-dependent lectin-like domain, inhibiting bacterial motility and invasion of the intestinal epithelium.⁶¹ This structural framework enables precision targeting, whereas defensins exert antimicrobial activity primarily by utilizing their cationic nature to electrostatically attract and insert into the negatively charged microbial membrane, forming pores that disrupt membrane integrity.^54,55 And, function of cathelicidins mainly by disrupting the microbial membrane like a detergent via their amphipathic alpha-helical structure, while also possessing potent immunomodulatory functions such as neutralizing endotoxins and recruiting immune cells.^56,62

Furthermore, the regulatory mode of Reg proteins on intestinal microbiota is irreplaceable. Unlike defensins, which primarily exert bactericidal effects locally in the mucosal layer, and cathelicidins, which are mainly involved in broad-spectrum anti-infection during the acute inflammatory phase, Reg proteins maintain microbiota homeostasis through a dual mechanism of spatial segregation and metabolic regulation. For example, Reg3β/γ can form a sterile zone on the surface of the small intestinal mucosa,⁴⁴ physically separating microbiota from epithelial cells. This function is not shared by other AMPs such as defensins and cathelicidins, which only reduce microbiota abundance via bactericidal activity and cannot establish physical barriers. Meanwhile, Reg4 can regulate the metabolism of intestinal Lactobacillus reuteri, promoting the conversion of linoleic acid (LA) to conjugated linoleic acid (CLA),⁶³ which in turn activates macrophage secretion of IL-35 to maintain immune balance. This AMPs-microbiota metabolism-immune regulation linkage model is also distinct from the single regulatory pathway of other AMPs that rely solely on direct bactericidal activity. Additionally, the Reg family is currently the only known AMPs with systemic metabolic regulatory functions. For example, Reg3γ can bind to the pancreatic Extl3 receptor via the bloodstream to promote insulin secretion,⁶⁴ regulating systemic glucose and energy balance. In contrast, classical AMPs such as defensins and cathelicidins are limited to local antimicrobial effects in the mucosa and cannot connect intestinal mucosal immunity to systemic metabolism-this dual role of local antimicrobial-systemic metabolic regulation further underscores the uniqueness and non-redundancy of Reg proteins compared to other AMPs.

Interplay between the REG/Reg protein family and gut microbiota

Functional features and spatiotemporal expression of the REG/Reg family

REG/Reg family members display dynamic spatiotemporal expression patterns that are critically linked to intestinal mucosal barrier function and local immune modulation. Their expression is highly dependent on specific disease contexts and phases. In baseline states, REG1A/B are moderately expressed in intestinal crypts, often co-localized with Paneth cells, and are inducible by bacterial peptidoglycan and butyrate.⁶⁵ Reg3β/γ are constitutively expressed by Paneth cells and intestinal epithelial cells, where they help maintain a sterile zone.^44,52,59

In infectious conditions such as acute amebic colitis, REG1A and REG1B show markedly elevated expression throughout the ulcerated crypt epithelium during the acute phase, which notably recedes to the crypt base during recovery, suggesting a role in acute mucosal repair and antimicrobial defense.^65,66 REG1A is regulated by cytokines including IL-6, IL-22, and IL-8, and serves as a sensitive serum biomarker for endoscopic activity in UC, particularly in patients with normal C-reactive protein or asymptomatic presentation.^67,68

During bacterial infections, Reg3β and Reg3γ are significantly upregulated. Reg3γ, induced via the MyD88 pathway, restricts Gram-positive bacteria by forming transmembrane pores in peptidoglycan layers, thereby maintaining host-microbiota spatial homeostasis.^13,44,60 Reg3β binds LPS to inhibit systemic translocation of Gram-negative pathogens such as Salmonella enteritidis, though it is ineffective against Listeria.⁶⁹ Molecular studies indicate that arginine 135 in Reg3β determines its specificity for DAP-type peptidoglycan present in organisms like Escherichia coli and Clostridium butyricum.⁷⁰

In viral infections, Reg3γ expression displays time-sensitive upregulation. Following respiratory syncytial virus (RSV) infection, it peaks at day 3 and alters microbiota composition.⁷¹ During rotavirus infection, Reg3γ expression correlates with IL-22 and pSTAT3 activation, peaking at day 4 alongside villus damage and diarrhea severity, and resolves by day 10 during immune recovery.⁷²

Microorganisms affect the expression of REG/Reg protein family in the host

Microbial perturbations can modulate REG expression. Fecal microbiota transplantation (FMT) induces region-specific upregulation of Reg3β/γ, particularly in the jejunum and ileum, indicating functional compartmentalization between the small intestine and colon.⁷³ Antibiotic treatment triggers a transient increase in Reg3β/γ, peaking at day 7 and normalizing by day 21, highlighting its role in adaptive mucosal response.⁷⁴ In mice resisting C. rodentium infection, Reg3β and Reg3γ expression is significantly upregulated in an IL-22-dependent manner.⁷⁵ Antibiotic-treated mice revealed that reduced Clostridia levels coincided with decreased short-chain fatty acid (SCFAs), particularly propionate, leading to lower Reg3β expression; conversely, colonizing germ-free mice with SCFA-producing microbiota or supplementing propionate significantly upregulated Reg3β/γ expression, suggesting SCFAs regulate Reg3β via microbiota-host interactions.⁷⁶ The expression of Reg3β/γ was positively associated with beneficial bacteria such as Akkermansia muciniphila (A. muciniphila), the Lachnospiraceae_NK4A136_group, and Bacteroidetes. Conversely, it showed a negative correlation with Firmicutes, Bilophila, and Butyricimonas. These findings suggest that beneficial microbial species might strengthen the intestinal barrier by inducing the expression of Reg proteins.⁷⁷

Clinical studies indicate that duodenal infusion of Anaerobutyricum soehngenii upregulates duodenal REG1B expression, modulates secondary bile acid metabolism, and improves glycemic control and intestinal transcriptomic profiles in metabolic syndrome patients.⁶⁵ Following intervention with this bacterium, significant upregulation of duodenal REG1B mRNA levels was observed in these patients, along with increased expression of its homologous gene REG1A, while no significant changes were noted in REG3A/3G/4.⁶⁵ In vitro experiments further confirmed that bacterial metabolites butyrate and peptidoglycan components directly induce REG1B expression in Caco-2 cells.⁶⁵ Although Reg3β/γ, along with antimicrobial proteins like Lipocalin-2 and Calprotectin, are regulated by IL−22, they exhibit no functional compensation. Reg3β/γ act as downstream antimicrobial effectors of IL-22, yet their individual absence does not impair host resistance to Citrobacter rodentium.⁷⁵

REG/Reg protein family regulates gut microbiota homeostasis

REG/Reg proteins play a crucial role in maintaining gut microbiota homeostasis through direct antimicrobial activity and immunomodulatory functions. Fecal REG1B levels in Malawian children were found to be inversely correlated with microbiota-for-age Z-scores, Shannon diversity, and the occurrence of environmental enteric dysfunction, indicating a potential role in suppressing microbial overgrowth.^78-80 And, Reg3β/γ expression is negatively correlated with pro-inflammatory bacteria (Eggerthella and Holdemania) in aged mice, while positively associated with beneficial taxa such as Roseburia and Ruminococcus in young mice.^59,81 Human REG3G transgenic mice exhibit significantly increased abundance of lactic acid bacteria, particularly Lactobacillus NK318.1, within the intestinal mucus and lumen, highlighting the role of REG3γ in shaping a symbiotic microbial niche.⁸² By maintaining spatial isolation between microbes and the epithelium, REG3γ reduces contact with pathogens and promotes colonization by beneficial species.⁸³ However, under certain conditions, such as rotavirus infection, Reg3γ may exacerbate inflammation by disproportionately targeting commensals or facilitating the expansion of conditionally pathogenic flora.⁷²

On the one hand, REG/Reg proteins orchestrate gut microbiota composition and spatial organization through targeted antimicrobial action and immunomodulation, crucially contributing to intestinal homeostasis. Reg3β and Reg3γ contribute to microbial segregation by forming a sterile barrier that limits bacterial adhesion and invasion.^13,44,52,60 Deficiency in Reg3γ leads to overgrowth of segmented filamentous bacteria and enhanced Th1 immune activation.^59,84 Moreover, Reg3γ helps suppress Bacteroides acidifaciens-induced Th17/Th1 polarization; its loss exacerbates colitis, impairing clearance of B. acidifaciens and altering Akkermansia muciniphila abundance.^84,85

On the other hand, they directly and precisely regulate the microbial community through structure-specific targeting mechanisms. Reg3β/γ specifically recognizes Gram-positive bacterial peptidoglycan through its EPN motif and forms transmembrane pores to exert direct bactericidal effects,^49,60 while Reg3β exerts bactericidal activity by binding Gram-negative bacterial LPS.^15,69 Reg4 also participates in microbiota regulation by specifically targeting flagellated pathogens. It inhibits enteropathogenic E. coli and Salmonella species by binding bacterial flagella through specific lectin-domain motifs, thereby suppressing bacterial motility, biofilm formation, and epithelial invasion.^61,86

Mechanism of REG/Reg protein family regulating intestinal homeostasis

REG/Reg protein family interaction maintains mucosal immune homeostasis

Reg3β/γ coordinates the regulation of microbial spatial distribution, immune responses, and epithelial inflammation by establishing a physical sterile zone that restricts bacterial adhesion and maintaining mucosal barrier integrity through non-sterilizing mechanisms. Reg3β/γ establishes a ~50 μm bacteria-free zone at the small intestinal mucosa through physical microbiota-epithelium segregation, limiting Gram-positive bacterial adhesion (Figure 4).^{13,44,52,60,87} Reg3γ further alleviates mucosal inflammation via non-bactericidal mechanisms (mucus stabilization and bacterial entrapment).⁸⁸ By preserving ileal mucus layer integrity-specifically maintaining the villus-tip mucodense band-and enforcing microbiota-epithelial spatial separation, it modulates inflammatory mediators (IL-22, MPO) and IFN-γ-driven signaling to attenuate abnormal immune responses against commensals and pathogens.⁸⁹ Reg3γ-deficient mice exhibit disrupted mucus architecture (loss of dense apical band, loose layer structure) with increased bacterial-epithelial contact (notably Gram-positive bacteria) despite unaltered Muc2 expression.⁸⁹ Gut microbiota dysbiosis conversely induces small intestinal Reg3γ secretion, which enhances barrier function by suppressing epithelial inflammation and modulating microbial composition (reducing pathogenic colonization).^90‐92

Also, Beneficial microorganisms synergistically induce Reg3 expression through multiple signaling pathways, including AHR, MyD88-Ticam1, and IL-22, collectively maintaining gut microbial homeostasis and immune stability. Reg3 family expression positively correlates with probiotic abundance but negatively associates with opportunistic pathogens. In mice, aryl hydrocarbon receptor (AHR) deficiency increases Segmented filamentous bacteria colonization, inversely correlating with Reg3γ levels.⁹³ Probiotic Bifidobacterium breve NCC2950 enhances mucosal barrier function by inducing Reg3γ via MyD88-Ticam1 signaling in gut epithelium.⁹⁴ Reg3 proteins (α/β/γ) show positive correlation with A. muciniphila abundance; western diet-induced A. muciniphila depletion synchronizes with Reg3β decline, coinciding with 2-fold mucolytic bacteria increase and LPS elevation due to facultative anaerobe overgrowth (Proteobacteria).⁹⁵ Lactobacillus casei upregulates Reg3α to strengthen tight junctions, suppress diarrheal pathogens, and promote epithelial proliferation.⁹⁶ Antibiotic-treated mice exhibit parallel reduction of pro-inflammatory Escherichia-Shigella and Reg3β by day 21, indicating host defense modulation through specific bacterial colonization.⁷⁴ Alistipes finegoldii colonization repairs intestinal barrier via IL-22/Reg3γ axis, where IL-22 (secreted by Th17/ILC3 cells) induces epithelial Reg3γ and mucins to reinforce the mucus barrier for microbiota segregation.⁹⁷

Reg4 synergistically suppresses intestinal infection and maintains immune homeostasis by directly binding flagella to inhibit motility, downregulating the TLR5/NF-κB inflammatory pathway, enhancing macrophage phagocytosis, and simultaneously activating the complement lectin pathway to clear bacteria. Using a Salmonella typhimurium infection model, exogenous Reg4 treatment significantly reduced bacterial colonization in the intestinal tract (feces and cecum), liver, spleen, and mesenteric lymph nodes of mice, alleviated intestinal mucosal inflammation, and decreased serum IFN-γ and IL-22 levels. In contrast, epithelial cell-specific Reg4 knockout mice (Reg4^△IEC) exhibited heightened susceptibility to infection, a phenotype reversed by Reg4 supplementation.⁶¹ Mechanistically, Reg4 directly bounds to Salmonella typhimurium flagella via the HDPQK motif, inhibiting bacterial motility. Concomitantly, Reg4 downregulated TLR5/NF-κB pathway-associated inflammatory gene expression and enhanced macrophage phagocytosis and bactericidal activity, effects abrogated in the HDPQK-deficient ∆ Reg4 mutant.⁶¹ Moreover, IEC-expressed Reg4 initiated the complement lectin pathway by binding to Escherichia coli LPS, collaborating with intestinal complement factor D to promote membrane attack complex formation for E. coli elimination and intestinal homeostasis maintenance.⁹⁸

Metabolic regulation and energy balance

Reg3γ and Reg4 integrate multi-organ metabolic networks through hormone-like actions, barrier repair, and microbiota interactions, coordinating insulin secretion, antioxidant defense, fatty acid metabolism, and macrophage polarization to systemically regulate energy homeostasis. Reg proteins participate in host energy homeostasis by influencing lipid metabolism and SCFA production, operating through direct actions and microbiota-mediated mechanisms. Gut microbiota induces small intestinal AMP Reg3γ expression and secretion, which locally enhances barrier function and antioxidant capacity while acting as a hormone via bloodstream to bind pancreatic Extl3 receptors, promoting insulin secretion to regulate energy balance, glucose homeostasis, and intestinal function.⁶⁴ Additionally, Reg3γ directly acts on IECs to upregulate tight junction protein genes, enhancing cellular integrity, and resists fructose-induced oxidative stress by activating antioxidant genes (Sod2, Cyba, Prdx2, Prdx4), thereby reducing intestinal permeability, limiting LPS translocation, and improving metabolic disorders.⁹⁹ In humanized REG3G transgenic mice, REG3γ enriches intestinal Lactobacillus NK318.1, activates the TLR2-STAT3 pathway, and induces differentiation/accumulation of anti-inflammatory macrophages. These macrophages migrate to adipose tissue to suppress inflammation and maintain metabolic homeostasis, conferring resistance to high-fat diet-induced obesity.⁸² Reg4 promotes linoleic acid metabolism to CLA by modulating gut microbiota composition (increasing Lactobacillus abundance).⁶³ Reg4 knockout mice exhibit reduced intestinal Lactobacillus and CLA levels, while humanized REG4 transgenic mice enhance LA-to-CLA conversion via Lactobacillus reuteri enrichment.⁶³ Microbiota-derived propionate activates intestinal epithelial GPR43 to upregulate Reg3β/γ, enhancing antibacterial capacity and promoting crypt stem cell proliferation post-injury, thereby improving colitis inflammation and tissue repair.⁷⁶ High-fat diets and 1-kestose regulate intestinal Reg3β/γ expression by altering Firmicutes/Bacteroidetes ratio and Bifidobacterium abundance, potentially via SCFA-mediated mechanisms.¹⁰⁰ Although propyl-propane thiosulfinate induces minimal microbiota shifts, it restores colonic IL-22 levels, upregulates tight junction protein Zo1 and Reg3γ, and improves intestinal barrier function in obese mice.¹⁰¹

REG/Reg protein family mediated signal pathway network

Members of the Reg family, as core downstream effectors of key cytokines such as IL-22, are precisely regulated through multiple signaling pathways (Figures 5 and 6). They perform multifunctional roles in the gut, including antibacterial, anti-inflammatory, and barrier repair functions, even carcinogenic effects. Their expression levels and functional states are directly modulated by the combined actions of host immune cells, microbial signals, and metabolic products, thereby exerting context-dependent dual roles in maintaining homeostasis and in inflammatory diseases.

Figure 5.

The protective roles of the REG: multifaceted regulation of the intestinal barrier and immune system. This figure summarizes key mechanisms by which REG proteins maintain intestinal homeostasis or drive disease through immune-epithelial crosstalk. (a) Reg3β/γ from Paneth/enterocytes bind bacterial PGN/LPS, maintaining the sterile zone and mucus barrier to inhibit the progression of IBD to colitis-associated cancer (CAC).^{10,13,44,52,60,102,103} REG-1A and Reg4 suppress inflammation levels, promote apoptosis of intestinal epithelial cells, and inhibit tumorigenesis. (b) REG-1A and Reg3β/γ enhance barrier function via IL-6/IL-22-STAT3.^{20,33,34,104,105} (c) REG4 enriches Lactobacillus reuteri to produce CLA, which activates macrophage GPR40/120→JAK-STAT→IL-35 to suppress inflammation.^63,106 (d) Reg4 inhibits Salmonella motility via flagellar binding and suppresses TLR5/NF-κB inflammation while enhancing macrophage bactericidal activity.⁶¹ Created in https://BioRender.com.

Figure 6.

The dark side of the REG family: drivers of inflammatory and neoplastic pathogenesis. This figure delineates pathogenic mechanisms by which REG proteins drive the inflammation-neoplasia sequence. (a) REG4 activates Akt/β-catenin signaling to drive CRC proliferation via cell-cycle genes.^20,107 (b) Mutant KRAS induces REG4,^47,108 which then potentially activates EGFR/PI3K/Akt to upregulate anti-apoptotic proteins and promote survival.²¹ (c) Dysbiosis/oxidative stress induces REG1A/1B/3A overexpression,^20,109 activating IL-4/IL-13→JAK-STAT6→GATA3 axis to sustain barrier damage and Th2 inflammation.^110‐112 (d) REG4 amplifies Wnt/β-catenin via LRP/Frizzled, promoting stemness and invasion; REG4-EGFR crosstalk accelerates CAC.^21,107,113 (e) REG3A activates JAK2/STAT3, Akt/ATF-2, and MAPK pathways to inhibit apoptosis, while altering adhesion molecules (MUC2↓, ITGA4↑) to facilitate metastasis.¹¹⁴

IL-22-mediated regulatory REG/Reg control pathway

IL-22 precisely regulates the expression of multiple members of the Reg family by activating the JAK2/STAT3 signaling axis and modulating microbiota-immune interactions (Figure 5). IL-22 regulates Reg3α/γ expression through the JAK2/STAT3 axis;²⁰ As an essential downstream component of the IL-22 pathway, Reg1α induction by IL-22 is completely abrogated upon STAT3 blockade.^105,115 Animal studies have demonstrated that OmpA1 from the commensal bacterium Parabacteroides goldsteinii activates ILC3s and IL-22⁺ neutrophils, triggering IL-22 expression and subsequent upregulation of Reg3β/γ to maintain intestinal mucosal immune homeostasis.^116‐118 In mice infected with RSV, intestinal Reg3γ expression peaks at day 3, temporally coinciding with IL-22 peaks in lung tissue, which underscores Reg3γ as a downstream effector of IL-22.³⁶ Mechanistic investigations reveal that Reg3β expression is strictly dependent on IL-22 secreted by ILC3s; in a cellular FLICE-inhibitory protein transgenic mouse model of embryonic ileitis, aberrant activation of RORγt⁺ cells (ILC3s/Th17 cells) drives IL-22 secretion, inducing Reg3β expression in IECs (intestinal epithelial cells), a phenomenon entirely absent in ILC3-deficient mice.^119‐121 As a core cellulose-degrading bacterium, Alistipes finegoldii promotes microbial maturation, regulates bile acid metabolism, and enhances intestinal barrier function via the IL−22/Reg3γ pathway to suppress colitis.⁹⁷ Oral administration of tea-derived exosome-like nanoparticles delivers miR-44 and miR-54 to activate ILC3s for IL-22 secretion, upregulate Reg3γ, and enhance tight junction proteins to repair psychological stress-induced intestinal barrier damage.¹²² Additionally, Reg3β alleviates pathological damage in murine ileitis and colitis by recruiting macrophages, inhibiting inflammation, and promoting tissue repair, with its protective effect dependent on the IL-22/STAT3 pathway and M2 macrophage polarization.^119,123,124 RSV infection in mice induces pulmonary Th17/Treg cell imbalance, triggering excessive IL-22 release; circulating IL-22 binds intestinal IL-22 receptors, inducing Reg3γ overexpression, interfering with intestinal Th17/Treg development, and altering gut microbiota composition, thereby mediating intestinal immune injury and dysbiosis.⁷¹

STAT3 cues to orchestrate REG/Reg expression and intestinal homeostasis

IL-6 induces Reg1α expression via the STAT3 signaling cascade;⁴³ in inflamed mucosal tissues, Reg1α suppresses the secretion of pro-inflammatory cytokines (TNF-α, IL-8) via the JAK/STAT3 pathway and promotes IEC proliferation.^105,115 Reg4 maintains intestinal immune homeostasis and alleviates colitis by regulating Lactobacillus metabolic homeostasis, inducing IL-35 production in macrophages via Gαq/11-mediated JAK1/2-STAT1/4 signaling.⁶³ The aryl hydrocarbon receptor (AHR) within immune cells promotes small intestinal Reg3γ expression via pSTAT3 signaling activation, thereby maintaining gut microbial homeostasis.^{93,111,125,126} AHR signaling inhibition reduces intestinal mucosal Reg3γ and IL-22 secretion, downregulates tight junction proteins, disrupts intestinal barrier integrity, and exacerbates Th2-type inflammation (elevated IL-4, IL-5, IL-13) with mast cell infiltration.^110‐112 Stimulator of interferon genes (STING) critically regulates immune responses triggered by diverse pathogens; its function hinges on the detection of cyclic dinucleotides, and it orchestrates immune responses to pathogens by sensing cyclic dinucleotides. STING induces IEC expression of REG3γ via STAT3 activation and glycolysis promotion, suppressing enteric pathogen infection and maintaining intestinal homeostasis. In animal models, STING-deficient mice exhibit reduced IEC REG3γ expression, increased susceptibility to Citrobacter rodentium infection (manifested as aggravated intestinal inflammation and impaired bacterial clearance); exogenous STING agonists restore REG3γ expression and inhibit bacterial growth in a REG3γ-dependent manner, with antimicrobial effects abolished upon REG3γ knockout.¹²⁷

Bidirectional regulation of REG/Reg mediated by other multichannel networks

Card9 and intestinal bitter taste receptors differentially induce the expression of Reg3β/γ and REG3A through the immune signaling pathway (IL-22) and stress-sensing pathway (NRF2), respectively, collectively enhancing intestinal antimicrobial defense, mucosal repair, and metabolic homeostasis functions. Host caspase recruitment domain member 9 (Card9) directly induces colonic IL-22 and Reg3β/γ, enhancing host defense, immune responses, and mucosal repair.¹⁰⁶ In germ-free conditions, Card9-knockout mice show significantly lower colonic Reg3β/γ expression than wild-type mice, with failed DSS-induced upregulation, confirming Card9 as a key driver of Reg3β/γ expression.¹⁰⁶ Human intestinal bitter taste receptors activate Paneth and goblet cells in jejunal crypts to induce AMPs (α-defensin 5, REG3A) and mucins, while upregulating metabolic genes (GDF15, ADM2, LDLR) via NRF2-mediated oxidative stress and unfolded protein responses to regulate innate immunity and metabolic homeostasis in obesity.¹²⁸

Reg3β/γ exerts anti-inflammatory effects by inhibiting the NF-κB pathway, but its overexpression also disrupts nucleotide-binding oligomerization domain-containing protein 2 (NOD2)-mediated protective signaling. Conversely, microbial metabolites upregulate Reg3β/γ via receptors such as GPR43 or PXR/TLR4, or induce Reg4-dependent immunomodulation, thereby finely regulating intestinal inflammation and microbiota homeostasis through a multi-pathway network. Functionally, Reg3β/γ alleviates intestinal inflammation by suppressing NF-κB-mediated pro-inflammatory responses and oxidative stress, while maintaining gut microbiota homeostasis through specific binding to Gram-negative bacteria.²⁰ But, excessive Reg3 expression eliminates protective Enterococcus faecium, impairs NOD2-mediated anti-inflammatory signaling, and sustains IBD progression.¹²⁹ Clostridium-derived propionate activates intestinal epithelial GPR43 receptors to upregulate Reg3β/γ, promoting epithelial homeostasis and colitis remission.⁷⁶ Lactobacillus strains producing 3-indoleacetic acid (IAA) induce IL-35⁺ B cells via the IAA-PXR/TLR4 pathway, a process dependent on Reg4-mediated microbiota regulation.¹³⁰

Dual role of REG/Reg protein family in intestinal diseases

The role of REG/Reg family in IBD

High expression of REG protein is closely associated with IBD inflammation and pro-inflammatory microbiota (Table 3). It exacerbates disease progression by depleting protective microbiota, disrupting anti-inflammatory signaling, and promoting immune activation. However, it may also serve as a potential biomarker for disease activity. The Reg gene family exhibits dual roles in IBD (including UC and CD).^52,119 Sudden exacerbation in IBD patients is characterized by elevated secretory REG3 protein levels, a phenomenon specific to IBD and independent of common diarrheal diseases.^129,131 In IBD, the colonic mucosa of UC patients shows upregulated REG4, while CD patients exhibit high expression of REG1A, REG1B, and REG4, with REG3A expression positively correlating with inflammatory cytokines (IL-22/IL-6).²⁰ Excessive antimicrobial REG3 directly reduces intestinal colonization of Enterococcus (especially SagA-producing Enterococcus faecium), disrupting microbial homeostasis.^53,132 The depletion of E. faecium impairs the activation of the NOD2 anti-inflammatory pathway, manifested by reduced IL-1β secretion in myeloid cells and IL-22 in lymphoid cells, losing the inhibition of intestinal inflammation.^129,131 FMT alleviates DSS-induced colitis by upregulating beneficial bacteria (Lactobacillus), downregulating harmful bacteria (Clostridium sensu stricto 1, Turicibacter), and inhibiting excessive activation of colonic mucosal CD4⁺/CD8⁺ T cells. Notably, Reg3γ is upregulated in DSS-treated mice but downregulated in FMT-treated mice, with its expression strongly positively correlated with Turicibacter abundance.¹³³ Non-pathogenic duodenal microbiota induce immune inflammation (upregulating lipocalin-2, REG3A) and suppress growth-related proteins, causing environmental enteric dysfunction and stunted linear growth in children.¹³⁴

Table 3.

The role of REG/Reg family in inflammation-related intestinal diseases.

Member	Correlation with microecology	Conclusion	Ref.
REG4/Reg4	In Reg4-KO mouse, intestinal Lactobacillus abundance decreases; In human REG4-transgenic mouse, intestinal Lactobacillus reuteri abundance increases.	REG4 modulates gut microbiota metabolite CLA and downstream signaling pathways to regulate intestinal immune homeostasis, sustaining IL-35+ macrophage function.	[63]
Reg3β	Antibiotic treatment decreases SCFA levels; Clostridium abundance correlates closely with propionate levels and Reg3β/γ expression.	Clostridium-derived propionate maintains intestinal epithelial homeostasis through Reg3β and GPR43 signaling, with the Reg3β-propionate axis serving as a key regenerative mediator in colitis.	[76]
Reg3β/γ	Reg3β/γ expression positively correlates with A. muciniphila, Lachnospiraceae_NK4A136_group, and Bacteroidetes, but negatively with Firmicutes, Blautia, and Butyricimonas.	Polyphenol-rich vinegar extract reduces alcohol-induced oxidative stress and hepatic/intestinal inflammation by modulating gut microbiota, boosting intestinal immunity/AMPs, and blocking inflammatory pathways.	[77]
Reg3γ	In feces of TAGAP-deficient mouse, Bacteroides acidifaciens abundance is higher than controls, while A. muciniphila first decreases then abnormally increases with aggravated inflammation.	TAGAP “TAGAP-Reg3γ-gut microbiota-Th1 differentiation” pathway; its dysfunction is a key mechanism for IBD development.	[85]
Reg3β/γ	In germ-free Card9-deficient mouse, colonic Reg3βγ expression is markedly reduced; after transplanting mouse microbiota into adult Card9-deficient mice, Reg3βγ and IL-22 remain lower than WT.	In germ-free Card9-deficient Mouse, colonic Reg3βγ expression is markedly reduced; after transplanting mouse microbiota into adult Card9-deficient mice, Reg3βγ and IL−22 remain lower than WT.	[106]
REG3γ	STING agonists alleviate Citrobacter-induced colitis in Mouse, dependent on REG3γ expression.	STING enhances intestinal epithelial antimicrobial defense via the "STAT3-glycolysis -REG3γ" axis, revealing its protective role in intestinal infection and IBD.	[127]
REG3 Reg3β/γ	In intestines of IBD patients, Enterococcus faecium and diversity are significantly reduced, and negatively correlated with REG3A concentration.	In IBD, excess REG3 depletes intestinal E. faecium, reducing SagA secretion. NOD2 activation in myeloid cells, lowers IL-1β and IL-22, thereby perpetuating a cycle of excessive Reg3β/γ, beneficial bacteria loss, and chronic inflammation.	[131]
REG3γ	In the FMT group of mouse, beneficial Lactobacillus increases, while harmful Clostridium_sensu_stricto_1 and Turicibacter decrease.	FMT treats colitis by regulating gut microbiota composition and expression of REG3γ.	[133]
REG3G	Roseburia hominis abundance is significantly negatively correlated with REG3G expression.	Microbiota dysbiosis and host defense defects synergistically drive UC's vicious cycle; REG3G may inform UC therapeutic strategies.	[109]

REG proteins exert protective effects against colitis through multiple mechanisms, including modulating microbiota composition, enhancing the mucosal barrier, and suppressing inflammatory cytokines. The functions of key members such as Reg4, Reg3G, and Reg1 have been validated through genetically engineered animal models and clinical observations (Table 3). The microbial composition in pediatric UC patients differs significantly from that of healthy controls, with reduced Bacteroidetes and increased pathogenic Peptostreptococcaceae, Enterobacteriaceae, and Sutterellaceae; among these, the abundance of Peptostreptococcaceae negatively correlates with REG3G expression.¹⁰⁹ Reg4 knockout mice show heightened sensitivity to DSS colitis, characterized by reduced survival, weight loss, colon shortening, elevated pro-inflammatory cytokines (TNFα, IL-6), and decreased anti-inflammatory factors (IL-35, IL-10), while human REG4 transgenic mice exhibit enhanced resistance and mitigated inflammation.⁶³ C-C chemokine receptor type seven-deficient mice exacerbate non-steroidal anti-inflammatory drug-induced enteropathy by promoting CD103⁺ dendritic cells to highly express interleukin-22 binding protein (IL-22BP), suppressing IL-22-mediated intestinal mucosal protection (reduced Reg1 expression)¹³⁵ (this result indirectly confirms the beneficial role of Reg1 in IL-22-mediated IBD protection). Milk fat globule membrane alleviates acute colitis and secondary liver injury by upregulating intestinal Reg3β/3g to enhance mucus barrier function, regulating gut microbiota (enriching Faccalibacum, Roseburia), and activating hepatic glutathione transferase to inhibit oxidative stress.¹³⁶

The role of REG/Reg family in CRC

REG1A, REG3A, and REG4 are significantly upregulated in CRC. They promote tumor proliferation, metastasis, and treatment resistance by activating multiple oncogenic signaling pathways (such as Akt/β-catenin and JAK/STAT3) (Figure 6). Their expression levels are closely associated with poor prognosis. Studies based on serum proteomics and transcriptomics analyzes have shown significantly upregulated mRNA levels of REG1A, REG1B, and REG3A in CRC and polyp tissues, with elevated serum REG1A protein levels distinguishing patients from healthy controls.¹³⁷ High REG3A expression in CRC correlates with tumor staging and poor prognosis, promoting tumor progression by regulating cell cycle (G1 phase arrest) and enhancing chemoresistance.¹⁰⁵ Both REG1A and REG4 are highly expressed in CRC, where REG1A associates with gastric cancer cell proliferation index, invasion depth, and chemoresistance, while high REG4 expression indicates increased risk of CRC liver metastasis and radiotherapy resistance.²⁰ Mechanistically, Reg4 promotes CRC cell mitosis and proliferation by activating the Akt-GSK3β-β-Catenin-TCF-4 signaling pathway, inducing expression of cell cycle regulatory genes (Cyclin D1, D3, CDK4, CDK6).¹⁰⁷ Additionally, REG4 enhances CRC cell migration and invasion via its conserved carbohydrate recognition domain, with carbohydrate recognition domain-dependent regulation of malignant phenotypes potentially mediating cell adhesion and signaling to promote metastasis.¹³⁸ Reg3α acts as a pro-cancer factor in CRC by activating JAK2/STAT3, Akt/ATF-2, and MAPK signaling pathways, upregulating Bcl-2/Bcl-xL to inhibit apoptosis, and enhancing cell migration/invasion through EGFR signaling.¹³⁹ Concurrently, Reg3A enriches in the extracellular matrix (ECM), downregulates adhesion molecules MUC2 and CDH1, and upregulates integrin ITGA4 to enhance colonic cancer cell-matrix adhesion and promote liver metastasis.¹¹⁴

Reg3β exerts its tumor-suppressing effect by inhibiting STAT3 signaling, whereas Reg4 promotes tumor stem cell properties and therapeutic resistance by activating the EGFR/Akt and Wnt/β-catenin pathways, highlighting the dual tumor-suppressing/promoting functions of the REG family in CRC (Table 4). Our previous animal studies showed that knockout of oncogenes BMI1 and MEL18 significantly upregulates Reg3β, suppressing STAT3 signaling to reduce CAC tumorigenesis. Concurrent Reg3β knockout completely reverses the tumor-suppressive effect of BMI1/MEL18 deletion, confirming Reg3β as a key downstream target of BMI1/MEL18.²² Notably, Reg3β may exert host protective effects during colitis progression, potentially mediated by gut microbiota, particularly Bacteroides. Additionally, Reg3β suppresses STAT3 by directly antagonizing the IL-6/IL-11 receptor complex, exerting tumor-suppressive functions.²² Reg4 activates the EGFR/Akt/AP-1 pathway to upregulate anti-apoptotic proteins (Bcl-2/Bcl-xL/survivin), with high expression sustaining anti-apoptotic signaling to reduce cancer cell radiosensitivity and enhance chemoresistance.^21,113 In another animal model, mutant KRAS induces REG4 expression via Wnt/β-catenin signaling, independent of classical Wnt pathway markers (Axin2), with REG4 further enhancing Wnt/β-catenin signaling to promote CRC stem cell (CSC) traits and drive tumor progression.^47,108 In our earlier studies investigating the mechanisms of BMI1 and MEL18 genes in CAC, we identified the Reg3β gene. We are currently utilizing conditional knockout mice for Reg3β, combined with third-generation metagenomics and single-cell sequencing technologies, to explore the mechanisms by which Reg3β regulates the gut microbiota and influences the tumor microenvironment in CAC.

Table 4.

The role of REG/Reg family in intestinal tumor diseases.

Member	Correlation with microecology	Conclusion	Ref.
REG1A REG4	REG1A expression (mRNA/protein) is upregulated in CRC tissues, with elevated serum levels in patients vs. healthy controls. REG4 is overexpressed in CRC tissues; its serum levels increase in stage IV patients and significantly correlate with liver metastasis.	Abnormal REG1A/REG4 expression correlates significantly with CRC inflammatory microenvironment, tumor invasion, and lymph node metastasis; REG4 serves as a biomarker for predicting CRC invasiveness and prognosis; Combination with other indicators enhances CRC screening and diagnostic efficacy.	[137]
REG3A	In CRC tissues, the mRNA levels were significantly upregulated, demonstrating high expression.	High REG3A expression promotes CRC cell proliferation/migration and inhibits apoptosis via AKT/ERK1/2 activation. Its expression significantly correlates with larger tumor size, poor differentiation, reduced survival, and tumor progression.	[137]
REG1A REG3A	REG1A mRNA is higher in CRC than normal tissues, and serum REG1A/REG3A is elevated in patients.	High REG1A/REG3A correlates with CRC's inflammatory microenvironment, lymph node/liver metastasis.	[137]
Reg4	mRNA and protein expression levels of Reg4 in CRC tissues are significantly upregulated; Highly expressed in SW480 and HT29 cell lines, but weakly expressed in HCT116 cells.	Reg4 activates the Akt-GSK3β-β-Catenin-TCF4 signaling pathway; it inhibits GSK3β phosphorylation to promote β-Catenin nuclear translocation and induce the expression of Cyclin D1, c-Myc, etc.	[137]
Reg4	REG4 is highly expressed in CRC tissues. Its mRNA is elevated in HT-29 but not LoVo cells; however, Reg4 protein is significantly expressed in Reg4-transfected LoVo cells. Serum Reg4 correlates significantly with liver metastasis and is elevated in stage IV CRC patients.	REG4 enhances CRC cell migration/invasion via its CRD domain and serves as a potential serum biomarker for predicting invasion/metastasis. Its expression correlates with malignant progression and poor prognosis, while CRD deletion abolishes Reg4's prometastatic function.	[137]
Reg3β	In Bmi1/Mel18 DKO mouse IECs, Reg3β expression was significantly upregulated but absent in Reg3βΔIEC mice. In human CRC tissues, REG3β negatively correlated with pSTAT3 levels.	Reg3β inhibits CRC progression by suppressing IL-6/IL-11-mediated STAT3 signaling, reducing tumor proliferation and promoting apoptosis. Its expression serves as a prognostic marker, with high levels predicting favorable outcomes.	[137]
REG4	REG4 mRNA/protein were significantly upregulated in human CRC vs. adjacent tissues. It was highly expressed in HT29/LS180/DLD-1 cells but not HCT116. miR-363 overexpression suppressed REG4 via GATA6 inhibition.	REG4 is a direct GATA6 target that promotes CRC anchorage-dependent proliferation/tumorigenesis via GATA6-REG4 signaling. miR-363 suppresses CRC growth by inhibiting GATA6-REG4/LGR5, indicating therapeutic potential.	[137]

The role of REG/Reg family in infectious diseases

Intestinal infections induce Reg3β/γ expression through pathways such as TLR/IL-22, thereby regulating microbiota composition and enhancing pathogen clearance (Table 5). However, certain pathogens can exploit the antimicrobial properties of Reg3β to eliminate commensal bacteria, thereby promoting their own colonization. Upon intestinal infection or microbial colonization in germ-free mice, ileal epithelial cells upregulate Reg3β/γ expression to regulate gut microbiota homeostasis and enhance clearance capacity.⁶⁹ In mice infected with RSV, intestinal Reg3γ levels peak at day 3, with Reg3γ overexpression altering gut microbiota composition-significantly increasing Aggregatibacter abundance while reducing Proteus.⁷¹ Studies suggest that Rotavirus infection induces changes in gut microbiota (Bacteroidetes, Firmicutes) via the IL-22/pSTAT3/Reg3γ signaling pathway in the intestinal microenvironment, ultimately leading to lactose intolerance.⁷² Toll-like-receptor 2 (TLR2) recognizes ligands from gut microbes to activate MyD88-dependent signaling, inducing IEC expression of Reg3β.^13,140 Resiquimod, a synthetic ligand for Toll-like receptor 7 that stimulates antiviral innate immune defenses, restores expression of the antimicrobial peptide Reg3γ and reestablishes colonization resistance against VRE in antibiotic-treated mice.¹⁴¹ This enhances intestinal clearance of Yersinia pseudotuberculosis, preventing bacterial dissemination from Peyer's patches and systemic infection.¹⁴⁰ Salmonella enterica infection induces host intestinal mucosa to express Reg3β, which selectively kills commensal bacteria (E. coli, Clostridium butyricum), eliminating competitive inhibition to promote its own excessive proliferation and colonization in the intestine.⁷⁰ Due to the limitations of infection models, research on the role of the Reg family in infectious diseases has been confined to animal studies, limiting the reliability of applying these findings to human.

Table 5.

The role of REG/Reg family in intestinal infection diseases.

Member	Correlation with microecology	Conclusion	Ref.
Reg3β	Reg3β-deficient mice show elevated Salmonella levels in colon, mesenteric lymph nodes, spleen, and liver versus WT mice, but fecal shedding remains unchanged.	Reg3β alleviates salmonellosis by specifically binding to surface components of Gram-negative bacteria and interfering with their invasion mechanisms, but has no effect on listeriosis.	[69]
Reg3β	Reg3β exerts dose-dependent bactericidal effects on commensal bacteria (E. coli, Clostridium butyricum), but shows no killing activity against Salmonella.	Salmonella induces Reg3β expression to eliminate competing microbiota, thereby promoting its own overproliferation and colonization in the intestine.	[70]
Reg3β/γ	C. rodentium shows low susceptibility to Reg3β/γ; fecal pathogen counts in Reg3β-deficient Mouse are significantly higher than in wild-type.	Reg3β/γ are IL−22-driven antimicrobial effectors, but their deficiency alone doesn't compromise host defense against C. rodentium; IL-22's protective role depends on multi-pathway synergy, with Reg3β/γ serving as auxiliary components.	[75]
Reg3α/β/γ	In Western diet-fed mouse, A. muciniphila is nearly eliminated, while facultative anaerobes (Proteobacteria) overproliferate.	Western diet disrupts the jejunal mucosal antimicrobial barrier and remodels homeostasis via oxidative cleavage, triggering LPS-mediated systemic inflammation.	[95]
Reg3β	Following oral Y. pseudotuberculosis infection, TLR2−/− mice exhibit higher bacterial burdens in Peyer's patches and markedly reduced survival compared to WT.	TLR2 induces Reg3β expression by sensing microbial signals, enhancing intestinal epithelial pathogen clearance; this mechanism depends on commensal bacteria signals and the MyD88 pathway.	[140]

The role of REG/Reg family in liver cancer

The REG/Reg family exerts dual roles in hepatocellular carcinoma (HCC) across precancerous, oncogenic/progressive stages, and liver injury repair, modulating cell proliferation, apoptosis, inflammation, glycosylation, and gut-liver axis signals, while some members support targeted therapy or liver injury alleviation to reduce HCC risk (Figure 7). During the precancerous stage, high Reg3A expression in the livers of cirrhotic patients directly binds to glycosyl substrates of the HBP pathway via lectin activity, reducing UDP-GlcNAc levels and inhibiting O-GlcNAc glycosylation of Myc at threonine 58.¹⁴² This modification (O-GlcNAc glycosylation of Myc) typically enhances Myc stability, whereas Reg3A intervention renders Myc more susceptible to ubiquitin-proteasome pathway-mediated degradation, thereby inhibiting Myc-driven cell proliferation signals and significantly prolonging cancer-free survival.¹⁴² REG1 is highly expressed in human intrahepatic cholangiocarcinoma, with its expression pattern associated with the malignant phenotype of tumor cells.¹⁴³ During liver regeneration, REG1 is expressed in bile duct cells with progenitor characteristics, and the aberrant proliferation of these REG1-positive progenitor-like bile duct cells may represent an early event in liver cancer development.¹⁴³

Figure 7. .

Roles of REG/Reg family in HCC across disease progression stages. The beneficial and detrimental roles of the REG/Reg family in HCC during liver injury repair, precancerous (cirrhosis), and oncogenesis/progression stages.^142‐150 Created in https://BioRender.com.

The promoter of the rat Hepatocarcinoma-Intestine-Pancreas Gene (HIP, belonging to the Reg3 family) can specifically drive high expression of the sodium-iodide symporter in hepatocellular carcinoma cells; activated by IL-6/dexamethasone, its regulatory elements mediate selective radioiodine uptake to achieve targeted killing of liver cancer cells.¹⁴⁸ REG1A and REG3A participate in the oncogenesis and progression of hepatocellular carcinoma through multiple mechanisms, including regulation of cell proliferation, apoptosis, inflammatory response, and glycosylation metabolism; their aberrant expression is closely linked to the malignant phenotype and poor prognosis of hepatocellular carcinoma.¹⁴² Following tumor formation, Reg3A expression in liver cancer tissues positively correlates with tumor size, invasion depth, and lymph node metastasis rate;¹⁴² it promotes hepatocellular carcinoma cell cycle progression and invasiveness by activating pro-proliferative pathways (PI3K/AKT, Ras/ERK), while inhibiting pro-apoptotic gene Bax and upregulating anti-apoptotic gene Bcl-2 to create an anti-apoptotic microenvironment.¹⁴² Additionally, Reg3A expression in liver cancer significantly correlates with inflammatory cytokines (IL-6, TNF-α), further amplifying carcinogenic effects via NF-κB pathway activation.¹⁴² Tumor-stromal cells induce elevated REG3A expression in hepatocellular carcinoma cells via the PDGF/p42/44 pathway, thereby promoting cell proliferation and inhibiting apoptosis to drive liver cancer progression.¹⁴⁴ Gut-specific expression of mouse Reg3 family genes (Reg3α, Reg3β, Reg3γ) may contribute to the formation of liver cancer-related inflammatory microenvironments by regulating mucosal immunity or cell proliferation.¹⁴⁵ Activation of human HIP/PAP (rat Reg3 homolog) in liver cancer and intestinal expression of mouse Reg3 may influence liver cancer progression by regulating gut-liver axis inflammatory signals;¹⁴⁵ the lectin activity of Reg3 proteins may be involved in abnormal hepatocyte proliferation or immune evasion.^145‐147

In a D-galactose-induced acute liver injury model in rats, recombinant IL-22 lentivirus treatment significantly upregulated the expression of Reg1, Reg3, and Reg4 proteins in liver tissue, with Reg4 showing the most remarkable increase.¹⁵⁰ Mechanistically, IL-22 binds to the IL-22Rα/IL-10Rβ receptor complex on liver cell membranes, activates the JAK-STAT3 pathway, drives phosphorylation of STAT binding sites in the Reg gene promoter region, and promotes Reg protein transcription; this alleviates acute liver injury by resisting oxidative stress, inhibiting inflammatory damage, and promoting hepatocyte repair¹⁵⁰ (alleviating acute liver injury may reduce the risk of chronic liver damage progression to HCC).

The role of REG/Reg family in gastric cancer

REG4 specifically localizes to deep crypt secretory cells and goblet cells, driving tumor cell proliferation via EGFR/Akt signaling.^151,152 In human normal tissues, it is primarily expressed in gastrointestinal epithelium (stomach, small intestine, colorectum) with mucinous and perinuclear staining patterns,^86,153‐155 corresponding to mucus-secreting and neuroendocrine-differentiated cells; weak expression occurs in adrenal medulla and breast lobules, while being undetectable in brain, lung and liver.¹⁵⁶ Tumor expression profiling reveals marked gastrointestinal specificity, with aberrant REG4 levels correlating with adenocarcinoma differentiation, mucin secretion, and neuroendocrine differentiation.¹⁵⁶ Mechanistically, REG4 overexpression in gastrointestinal cancers activates tyrosine phosphorylation of EGFR (Tyr⁹⁹², Tyr ¹⁰⁶⁸), initiating PI3K/Akt signaling to upregulate anti-apoptotic Bcl-2/Bcl-xL, thereby promoting tumor cell survival, proliferation, and invasion.²¹

In the oncogenesis and progression of gastric cancer, REG1A, as a member of the Ca²⁺-dependent lectin family, has its expression regulated by DNA methylation.¹⁵⁷ REG1A inhibits the invasion and proliferation of gastric cancer cells and promotes their apoptosis by suppressing the phosphorylation of the PI3K/Akt-GSK3β signaling pathway. However, DNA methylation-induced gene silencing of REG1A weakens this tumor-suppressive effect.¹⁵⁷ Studies have found that REG3A, a member of the REG protein family, is significantly downregulated in gastric cancer tissues and cells.¹⁵⁸ On one hand, Reg3A exerts a tumor-suppressive role by promoting the expression of DMBT1 to inhibit the proliferation of gastric cancer cells.¹⁵⁹ Additionally, REG3A acts by regulating the PI3K/Akt-GSK3β signaling axis. It suppresses the activation of the PI3K/Akt-GSK3β signaling pathway, thereby inhibiting the invasion and proliferation of gastric cancer cells and promoting their apoptosis.¹⁵⁸

Studies using gastric cancer tissue specimens and cell lines have confirmed a positive correlation between CDX2 and Reg IV expression, where CDX2 silencing downregulates Reg4 and inhibits cell motility, while overexpression promotes metastasis via Reg4 upregulation; notably, Reg4 does not significantly reciprocally regulate CDX2 expression.¹⁶⁰ Specifically, ectopic expression of the intestinal-specific transcription factor CDX2 influences tumor progression by regulating the Reg IV/SOX9 signaling pathway in gastric cancer. As an upstream regulator, CDX2 directly promotes Reg4 gene transcription, elevating its mRNA and protein levels, while Reg4 further induces SOX9 expression to enhance gastric cancer cell migration and invasiveness.¹⁶⁰ In gastric cancer, tumor cell-secreted Serglycin activates the CD44/c-Myc pathway in fibroblasts, upregulating histone demethylase KDM5B to promote IL-8 secretion by cancer-associated fibroblasts. Concurrently, tumor-associated neutrophil (TAN)-secreted REG4 activates CREB1 in tumor cells, further upregulating SRGN expression to form a SRGN-IL-8-TANs-SRGN positive feedback loop driving tumor progression.¹⁶¹ Additionally, Reg4 inhibits gastric cancer cell apoptosis via MAPK/Erk/Bim pathway activation, enhancing resistance to 5-fluorouracil (5-FU).¹⁶² REG4 promotes gastric cancer cell invasion and migration by positively regulating SOX9 expression, with SOX9 exerting feedback regulation on Reg4.¹⁶³

Others

The expression of the REG/Reg family is influenced by diet, psychological stress, gut microbiota, and their metabolites, with upregulation or downregulation leading to distinct outcomes. A Western diet induces elevated reactive oxygen species in jejunal mucus, downregulating antimicrobial proteins (Reg3α/β/γ, defensins, Muc2) and cytokines (IL-22/IL-36γ), reducing goblet and Paneth cells, triggering dysbiosis (loss of A. muciniphila), and impairing intestinal barrier function, ultimately inducing systemic inflammation via LPS translocation.⁹⁵ Aging itself induces alterations in gut microbial composition, characterized by enrichment of pro-inflammatory bacteria (Eggerthella, Gordonibacter, Holdemania, Turicibacter) and reduction of short-chain fatty acid-producing beneficial bacteria (Roseburia, Ruminococcus), accompanied by compensatory elevation of basal intestinal AMPs (Reg3γ, Defa-rs1).⁸¹ Short-term alcohol exposure (3 d) affects young mice minimally but specifically downregulates Reg3β, Reg3γ, and Defa-rs1 in aged mice, weakening the chemical barrier, increasing intestinal permeability, and elevating ileal TNFα expression 10-fold.⁸¹ High-dose zinc oxide supplementation improves intestinal function in weaned piglets by modulating gut microbiota composition (increasing commensal diversity), downregulating immune-related genes (CD59, Reg3γ), and inhibiting local inflammation.¹⁶⁴ Psychological stress induces pro-inflammatory (Saa1, Il18), pro-oxidative (Duox2, Nos2), and Reg3β/γ gene expression in IECs via a microbiota-dependent mechanism, leading to thinning of the mucus layer, microbial translocation, and microbiota shift toward oxidant-tolerant species (catalase-producing Muribaculaceae) via enhanced host ROS production, ultimately disrupting mucosal integrity and exacerbating inflammation.¹⁶⁵ Xylanase significantly alters the intestinal microbiota of tilapia, inhibiting the dominant Cetobacterium and specifically promoting proliferation of the butyrate-producing Allobaculum stercoricanis.¹⁶⁶ Butyrate produced by Allobaculum stercoricanis elevates intestinal butyrate concentration, acting as an HDAC inhibitor to suppress HDAC3 activity, enhance histone acetylation, activate IL-17D transcription in IECs, and further induce downstream Reg3γ synthesis/secretion, strengthening the intestinal mucosal immune barrier.¹⁶⁶ In an ovalbumin-sensitized BALB/c mouse model, allergic reactions disrupt the intestinal immune-metabolic axis via Reg3γ-mediated mechanisms by interfering with tryptophan metabolism and gut microbiota composition, exacerbating food allergy symptoms. Food-allergic mice exhibit reduced serum tryptophan metabolites (indole-3-acrylic acid, indole-3-lactic acid), decreased expression of key tryptophan metabolic enzymes and downstream target genes (Reg3γ, IL-22), reduced abundance of beneficial bacteria (Lactobacillus, Bifidobacterium) positively correlated with metabolite levels, and enrichment of harmful bacteria (Bacteroides).¹¹⁰

Feasibility assessment of the REG family in clinical applications

Biomarkers from potential to clinical relevance

Different members of the REG family can serve as diagnostic, prognostic, or monitoring biomarkers for diseases such as UC, pancreatic ductal adenocarcinoma, and CRC (Table 6). REG1A and REG1B can serve as biomarkers for UC and pancreatic ductal adenocarcinoma. In UC, REG1A and REG1B are upregulated in the non-inflamed ileum and closely associated with the extent of colonic inflammation (no significant changes observed in patients with left-sided colitis), which allows their use in evaluating UC inflammatory load.¹⁶⁷ Meanwhile, REG1A is a sensitive biomarker for endoscopic disease activity in UC, particularly applicable to patients with normal C-reactive protein levels or mild symptoms, providing a tool for non-invasive monitoring of UC;⁶⁸ its expression level can also act as a potential indicator of inflammatory activity in UC.¹⁶⁸ In pancreatic ductal adenocarcinoma, serum levels of REG1A and REG1B are significantly associated with liver metastasis status. Patients with pancreatic ductal adenocarcinoma showing low tissue expression of REG1A and REG1B exhibit a higher risk of liver metastasis and significantly lower postoperative survival rates, making these proteins independent indicators for predicting liver metastasis and prognosis of this cancer.¹⁶⁹

Table 6.

The REG family can digestive diseases serve as biomarkers.

REG	Diseases	Patient grading	Significance	Ref.
REG1A, REG1B	UC	Inflammatory Severity: extensive colitis (high expression of REG1A/B); left-sided colitis (no change).	Upregulated expression in non-inflammatory ileum is significantly associated with extensive colitis and can be used to assess the inflammatory burden.	[167]
		Endoscopic activity: non-mucosal healing (high expression of REG1A) vs mucosal healing (low expression); CRP status: normal CRP (REG1A can still be detected) vs elevated CRP.	REG1A is a sensitive marker of endoscopic disease activity in UC and has better monitoring efficacy than CRP in patients with normal CRP or mild symptoms.	[68]
REG3A	CRC	Metastatic status: liver metastases (REG3A enrichment) vs primary tumor (low expression).	Overexpression promotes liver metastasis of LoVo cells through ECM adhesion regulation.	[114]
		Treatment response: 5-FU resistance (high expression) vs sensitivity (combined antibody therapy).	Single-chain antibody targeting REG3A can inhibit proliferation and enhance the efficacy of 5-FU.	[139]
REG4	CRC	Pathological type: non-mucinous CRC (good prognosis with positive REG4) vs other types.	Independent prognostic marker for non-mucinous type (higher 5-y DFS).	[170]
		Treatment sensitivity: sensitive to radiotherapy and chemotherapy (low expression) vs resistant (high expression).	Low expression predicts sensitivity to radiotherapy and chemotherapy.	[171]
REG4	UC	Inflammatory Severity: severe (strong staining) vs mild (weak staining).	High expression in intestinal mucosa is positively correlated with the degree of inflammation, with a single detection AUC = 0.929.	[172]
		Diagnosis: UC positive (AUC = 0.929) vs healthy control.		[173]
REG1A, REG1B	Pancreatic ductal adenocarcinoma	Metastasis/prognosis: liver metastasis positive/low survival group (low expression) vs no metastasis/high survival group (high expression).	Low tissue expression is significantly associated with a high risk of liver metastasis and low postoperative survival rate, and independently predicts prognosis.	[169]

REG3A functions as a progression/prognostic biomarker for CRC and a discriminatory biomarker for intestinal diseases. In CRC, abnormal serum expression of REG3A is closely linked to the cancer's inflammatory microenvironment, biological abnormalities, and tumor progression. Additionally, the expression pattern of REG3A-related differentially expressed genes differs significantly between colon cancer tissues and normal tissues, suggesting REG3A as a potential prognostic marker for colon cancer metastasis.¹¹⁴ Simultaneously, its serum level is associated with postoperative metastasis and drug resistance in CRC, indicating its diagnostic/prognostic potential.¹³⁹ For intestinal disease discrimination, REG3A can be used to distinguish mucosal enteropathy from irritable bowel syndrome and serves as a plasma marker for gastrointestinal graft-versus-host disease. Reg3β functions as a protective factor and therapeutic target biomarker for ileitis and colitis.¹¹⁹

REG4 acts as a multifunctional biomarker for CRC, a diagnostic biomarker for UC and celiac disease, and a prognostic biomarker for breast cancer. In CRC, REG4 is highly expressed, and the activation of signaling pathways induced by REG4 is associated with the tumor's invasive phenotype, suggesting it as a biomarker for poor CRC prognosis.¹⁷⁴ It also serves as an independent prognostic marker for non-mucinous CRC. Patients with REG4-positive expression exhibit a higher 5-y disease-specific survival rate, particularly evident in those under 65 y of age.¹⁷⁰ Furthermore, REG4 expression level is negatively correlated with radiochemotherapy sensitivity; CRC cells with low REG4 expression are more sensitive to radiochemotherapy, enabling its use in predicting radiochemotherapy sensitivity.¹⁷¹ In UC and celiac disease, REG4 expression in the intestinal mucosa of patients is significantly higher than that in healthy controls, and the staining intensity is positively correlated with the degree of inflammation.¹⁷² External datasets have also verified the consistency of its high expression in independent cohorts, supporting its potential as a cross-disease biomarker.¹⁷² The area under the curve (AUC) of REG4 alone is 0.929, indicating its potential as an independent diagnostic marker for UC.¹⁷³ In breast cancer, REG4 can serve as an independent risk factor and predictive marker for adverse outcomes in patients with T2-3 stage breast cancer who do not achieve pathological complete response following neoadjuvant chemotherapy.¹⁷⁵

Exploration of therapeutic targets

Reg4 serves as a key pro-proliferative factor in CRC, emerging as a potential therapeutic target. Treatment with Reg4-specific monoclonal antibodies or siRNA combined with radiotherapy reduces CRC tumor volume by 60%–80% and prolongs survival of tumor-bearing mice by over 50%, with early intervention (initiated on day 1 of tumor formation) demonstrating significantly better efficacy than late intervention.¹¹³ Mechanistically, Reg4 drives CRC cell proliferation by activating the Akt-GSK3β-β-Catenin-TCF-4 signaling pathway to regulate cell cycle-related gene expression. Blocking Reg4 signaling with specific antibodies or inhibitors suppresses cell proliferation, validating its feasibility as a therapeutic target.¹⁰⁷ Additionally, Reg4-targeted monoclonal antibodies significantly reduce anti-apoptotic protein expression and inhibit tumor growth in pancreatic cancer animal models, highlighting the feasibility of immunotherapeutic strategies.²¹ Intestinal delivery of REG3A alleviates intestinal inflammation in colitis by reducing intestinal reactive oxygen species, selectively enriching oxygen-sensitive beneficial bacteria, and decreasing pro-inflammatory bacteria to improve gut microbiota homeostasis and enhance intestinal mucosal barrier function.¹⁷⁶

Current status of clinical research implementation

The limitations of current research must be objectively acknowledged. Most investigations into the mechanisms underlying REG/Reg family function rely on mouse knockout or transgenic models. While these models have established the association between the REG/Reg family and disease progression, differences exist between human and mouse REG/Reg family members in sequence homology, tissue expression patterns, and mechanisms of interaction with the gut microbiota. These variations may introduce bias when translating animal study results to clinical settings. For example, the regulatory pattern of mouse Reg3γ toward Gram-positive bacteria may not be fully replicated in humans. Therefore, caution against overinterpretation of current findings is warranted. Future research should prioritize validation using human clinical samples and conduct cross-species comparative studies to clarify the conservation and specificity of REG/Reg family functions, thereby advancing their precise translation from basic research to clinical applications.

Notably, significant progress has been achieved in the clinical translation of AMPs. For instance, a series of clinical trials for the AMP PL-5 spray have completed Phase IIb exploratory trials and successfully concluded Phase III clinical studies, with Phase III b trials currently underway (ChiCTR24000195731, ChiCTR24000195730, ChiCTR24000195729). As a newly designed non-antibiotic antimicrobial agent, this AMP exhibits a broad antimicrobial spectrum and demonstrates excellent efficacy in treating local open wound infections. It particularly shows a strong bactericidal advantage against drug-resistant strains, such as methicillin-resistant Staphylococcus aureus and multidrug-resistant Acinetobacter baumannii carrying the NDM-1 gene, without inducing new drug-resistant bacteria, thereby displaying great potential for clinical application. Additionally, a study investigating changes in intestinal mucosal AMP expression and its underlying mechanisms in patients with UC (ChiCTR24000185188) is ongoing, aiming to deeply explore the role of AMPs in the pathogenesis of UC.

Regarding the Reg family, current studies registered on the Chinese Clinical Trial Registry (ChiCTR, http://www.chictr.org.cn) primarily focus on the diagnostic value of biomarkers. For example, the study titled "Clinical value of combined detection of REG3A, I-FABP, and nCD64 in the early diagnosis of neonatal necrotizing enterocolitis (NEC)" (ChiCTR2400066184) is in progress. This study evaluates the application value of different combined diagnostic approaches in the early diagnosis of NEC, with the goal of establishing an early diagnostic method for NEC with high sensitivity, specificity, and applicability. Furthermore, the study “Establishment of reference ranges for serum gastrointestinal mucosal injury biomarker REG1A in children and its clinical application” (ChiCTR24000263852) is underway, which aims to establish the normal reference range of REG1A in children and provide a reference standard for the clinical diagnosis of gastrointestinal mucosal injury in children. Another ongoing study registered on ClinicalTrials.gov, titled “The comparison between the biomarkers in IBD patients and general control group” (NCT03778918), further expands the clinical application value of REG3A as a blood-based biomarker in IBD, providing more comprehensive evidence-based support for disease diagnosis and mechanism research.

Feasibility assessment of REG protein therapy

While REG proteins hold translational potential, their clinical application requires cautious evaluation of current strategies. Competing approaches include microbiota modulation, small-molecule regulators, and immunotherapy (Figure 8). Although offering distinct advantages, they face fundamental challenges such as poor protein stability, functional redundancy, and systemic off-target effects. Microbiota-directed interventions can indirectly upregulate Reg3γ by enriching beneficial bacteria and exhibit favorable safety profiles. However, they rely on endogenous REG expression and cannot overcome inherent limitations such as short half-life and protease sensitivity. Moreover, their effects are slow and indirect, and may be limited by functional compensation among AMPs in a redundant system.^64,89,90 Small-molecule regulators (JAK2 inhibitor AG490 or miR-24 mimics) allow facile administration and target specific signaling nodes or REG downregulation. Yet, their limited specificity risks off-target effects due to structural homology among REG family members or cross-talk with unrelated pathways. Immunotherapies, while broad-acting and disease-agnostic, lack precision for REG-dependent pathologies and may unintentionally perturb REG-related processes in extra-intestinal tissues—REG4-driven pathologies in multiple organs or REG1A-related systemic inflammation. Although REG-focused immunomodulation could target downstream pathways like STAT3 or EGFR/Akt, its development must overcome hurdles such as protein instability, functional redundancy, and loss of tissue-specific expression leading to aberrant activation.^{20,32,154,177}

Figure 8.

Challenges and future directions for clinical translation of REG/Reg family proteins. This diagram systematically presents the research logic and development pathway of the REG/Reg family proteins: establishing foundational mechanisms → Tackling challenges head-on → Driving multi-domain applications after overcoming challenges. First, in the mechanism research dimension, it addresses three aspects: biomarkers, therapeutic targets, and clinical research. This provides theoretical and clinical foundations for subsequent applications. clinical research. This establishes theoretical and clinical foundations for subsequent applications. Second, we identify key research challenges, including poor protein stability, systemic off-target effects, and functional redundancy AMPs. Ultimately, if these bottlenecks can be overcome through theoretical support, technical advancements, and feasibility assessments, REG family proteins will achieve broad applications in microbiome modulation, small-molecule drug development, and immunotherapy, opening new pathways for the diagnosis and treatment of related diseases.

Challenges in protein stability

Structural and microenvironmental constraints on the stability of REG family proteins. The clinical translation of REG family proteins is constrained by structural fragility, microenvironmental sensitivity, and intrinsic molecular properties, presenting specific technical hurdles. For instance, in human Reg4, approximately 20 residues exhibit unassignable NMR signals even under optimized buffer and temperature conditions This complicates structural resolution and stability assessments.¹⁷⁸ Although human Reg4 retains carbohydrate-binding activity in a calcium-independent manner, its expression in the low-pH gastrointestinal environment poses additional challenges to structural integrity and functional consistency under acidic conditions.¹⁷⁸ Reg3 activity is highly dependent on the paracrine microenvironment of gut epithelial cells. Functional activation of key isoforms such as Reg3γ requires trypsin-dependent proteolytic removal of an N-terminal pro-segment-a process specific to the intestinal lumen. Recombinant Reg3 produced in vitro lacks both this physiological activation system and the native secretory context, resulting in unstable bioactivity and limiting its therapeutic utility.¹⁰ Furthermore, recombinant mouse Reg3g faces severe stability challenges in vivo: it has an extremely short half-life and contains an N-terminal DPP4 cleavage site that further compromises its stability, despite being regulated by gut microbes and metabolites.⁶⁴

Risk of systemic off-target effects

Tissue and isoform-specific considerations for systemic targeting of REG family proteins. The expression, function, and application risks of REG family proteins exhibit marked tissue specificity and isoform diversity, necessitating careful evaluation of off-target effects in systemic use. The potential risks primarily stem from their context-dependent roles across various systemic diseases, including cancer and inflammation. In a rat ALPPS model, Reg3α and Reg3β demonstrated strict tissue-specific expression restricted to certain liver lobes. Systemic administration disrupts this spatially constrained expression pattern and may lead to aberrant activation of signaling pathways in off-target tissues.¹⁷⁹ Isoform-specific risks vary considerably. For example, REG3A promotes tumor progression in triple-negative breast cancer and correlates with poor prognosis, yet systemic inhibition of REG3A shows minimal impact on normal mammary epithelial cells and low off-target risk.³⁶ In contrast, REG4 enhances anti-apoptotic signaling in gastric cancer and confers chemotherapy resistance. Targeting REG4 with specific antibodies suppresses tumor growth and improves chemosensitivity. However, REG4 is also implicated in intestinal inflammation, acute pancreatitis, osteoarthritis, and acute liver injury, indicating substantial potential for systemic off-target effects.¹⁵⁴ Furthermore, REG proteins are involved in systemic autoimmune and inflammatory processes: REG1A contributes to rheumatoid arthritis and primary Sjögren’s syndrome, correlates with sepsis severity and mortality, and is associated with organ dysfunction in systemic inflammatory response syndrome. REG3A serves as a biomarker for gastrointestinal graft-versus-host disease. Both proteins help identify septic complications after surgery, underscoring their roles in systemic inflammation beyond the gastrointestinal tract.²⁰

Technical bottlenecks in recombinant delivery

Technical challenges in the delivery of recombinant REG family proteins. Several key technical barriers hinder the clinical delivery of recombinant REG proteins. First, activity depends on proteolytic activation. Reg3γ requires trypsin-mediated removal of its N-terminal pro-domain in the gut to become bioactive-a process unlikely to occur efficiently outside intestinal environments. Second, detection inaccuracies limit quantification: current ELISAs cross-react with circulating proteins, complicating precise measurement of systemic Reg3. Third, tissue specificity poses delivery challenges: Reg3 is normally restricted to the gut, and its receptor EXTL3 mediates tissue-varying responses, making targeted delivery complex. Although advances have been made. Such as clarifying Reg3γ's activation mechanism, leveraging microbial or bile acid induction, and exploiting its specific binding to Gram-positive bacterial peptidoglycan for mucosal targeting-these are insufficient to support clinical use.¹⁰ Recombinant Reg3g delivery faces short half-life, imprecise assays hindering concentration-effect analysis, limited cross-species comparability, and a lack of standardized co-delivery protocols with probiotics. Nevertheless, high-purity recombinant mouse Reg3g has been produced; peripheral injection improves glucose tolerance in obese mice via pancreatic β-cell EXTL3 and enhances glucose-stimulated insulin secretion. Probiotics such as VSL#3 can induce endogenous Reg3g, suggesting potential combinatorial strategies.⁶⁴ Notably, systemic delivery routes for Reg3α/Reg3β remain unexplored, with no pharmacokinetic or safety data available.

Conclusions

The REG protein family exemplifies a paradigm of functional plasticity, where its role in gut mucosal homeostasis or disease is dictated by a dynamic equilibrium influenced by microbial signals, immune context, and genetic background. This review highlights the dual nature of REG proteins-acting as both protective AMPs and promoters of inflammation and cancer, which underscores the necessity of the REG Protein Functional Equilibrium Model to reconcile these context-dependent effects. However, significant challenges remain. Functional redundancies among members, species-specific differences between murine and human REG homologs, and a lack of clarity regarding the precise microenvironmental cues that shift REG function from beneficial to pathogenic complicate therapeutic targeting. Moreover, technical hurdles such as poor protein stability, short half-life, and risks of systemic off-target effects limit clinical translation. Despite these limitations, REG proteins hold promise as biomarkers and therapeutic targets, particularly in inflammatory and neoplastic gastrointestinal diseases. Future research must prioritize human tissue validation, cross-species comparative studies, and innovative delivery strategies to harness their regulatory potential effectively. Overcoming these barriers will be essential for translating REG biology into precision medicine applications.

Author contributions

X.L., X.Z., W.W.: provide ideas and designs content. Y.L., Z.Q., W.P., S.L., F.X., X.W.: collected materials. Z.Q., W.P., Y.L.: created figure and tables. Z.Q., W.P: wrote the original draft. X.L., X.Z., J.Z.: reviewed and edited the draft. X.L.: provided funding acquisition.

Funding Statement

This study was supported by National Key R&D Program of China (2024YFF0728704, 2024YFA1306901), the National Natural Science Foundation of China (32500923, 82073126). All of the fundings supported in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript. (National Key Research and Development Program of China) (81874076)

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Funding

This study was supported by National Key R&D Program of China (2024YFF0728704, 2024YFA1306901), the National Natural Science Foundation of China (32500923, 82073126). All of the fundings supported in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.

Data availability statement

No datasets were generated during the current study.

Conditions of publication

All authors agree to conditions of submission.

Ethics approval and consent to participate

Not applicable.