Reg3 Proteins as Gut Hormones?

Abstract

C-type lectins of the Reg3 family belong to antimicrobial peptides (AMPs), which function as a barrier to protect body surfaces against microorganisms. Reg3 mainly expressed throughout the small intestine modulate host defense process via bactericidal activity. A wide range of studies indicate that Reg3 family plays an important role in the physical segregation of microbiota from host as well as the immune response induced by enteric pathogens. In this review, we review a growing literature on the potential metabolic functions of Reg3 proteins and their potential to act as important gut hormones.

The gastrointestinal (GI) tract is exposed to extrinsic diets and pathogens. The result is that inside the lumen, the host interacts with a variety of bacteria, which requires multiple immune responses and results in production of metabolites that contribute to the intestinal microenvironment (1–5). Because the gut epithelium is directly facing these gut bacteria (termed the microbiota), the host must rely on a diverse set of physical and biochemical barriers to keep these pathogens from getting into the body. These include immunoglobulin A, the mucus layer, tight junctions, and antimicrobial peptides (AMPs) (6). AMPs have been found from prokaryote to human as a part of the innate immune response (7, 8). AMPs have essential roles in protecting mammalian body surfaces, which directly interface with the external microorganisms (9). Multiple antimicrobial peptides, including defensins, lysozymes, and C-type lectins, are produced by epithelial cells of tissues including skin, respiratory tract, and intestine as well as circulating immune cells (10–13), and contribute to the host defense against microorganisms via a variety of innate immune responses (14, 15). Among those AMPs, Reg3 lectins are abundantly expressed in enterocyte and paneth cells of small intestine and exert bactericidal activity as a host defense mechanism in the gut (12, 16). In this review, we focus on the emerging role of Reg3 proteins beyond this traditional role to regulate systemic metabolism.

Discovery of the Reg3

Reg proteins, which are part of the C-type lectins, were first discovered in regenerating islets (17). Various Reg family members have been independently found classified into four subtypes (types I, II, III, and IV) based on their DNA sequence and primary structures of the encoded sequence homology (18–22). During the same time frame, other investigators identified these same proteins that were overexpressed in rat acute pancreatitis or human hepatocellular carcinoma and termed them pancreatitis-associated protein/hepatic intestinal pancreatic protein (21, 23, 24). Reg3 proteins can be divided into multiple subtypes (α, β, γ, and δ) determined by their structure and chromosomal localization (22, 25). It is known that there are four Reg3 members termed Reg3α, Reg3β, Reg3δ, and Reg3γ in mice, whereas humans are known to have only Reg3α and Reg3γ (21, 26). In addition, islet neogenesis-associated protein, Reg3δ in hamsters, was discovered in a surgical model of partial duct obstruction in hamster pancreas (27–29).

Cellular Source and Secretion of Reg3

The Reg3 gene is divided into 6 exons and encodes a protein that is 175 amino acids in length with a molecular mass of 16 to 17 kDa (25, 30, 31). As a member of the Reg3 subgroup, Reg3γ (the mouse homolog of human REG3A) contains an anionic N-terminal prosegment that maintains the protein in a biologically inactive state. The inactive prosegment is removed by trypsin-dependent proteolytic processing, which leads inactive Reg3γ to become active Reg3γ with bactericidal activity (Fig. 1) (31, 32). According to RNA and protein data from the Human Protein Atlas, REG3Α is expressed at the highest levels in the GI tract (www.proteinatlas.org). Its expression is triggered by bacterial colonization (12, 33) and upregulated in inflammatory bowel disease (16).

Figure 1.

Mechanism of Reg3γ activity. Active Reg3γ is processed by trypsin-dependent proteolysis. Reg3γ targets Gram-positive bacteria by binding to peptidoglycan layer. Then, Reg3γ exert bactericidal activity by oligomerizing to form hexameric transmembrane pores. [Reprinted with permission from Springer Nature. Nature; Antibacterial membrane attack by a pore-forming intestinal C-type lectin. Sohini Jukherjee, Jui Zheng, Mehabaw G. Derebe, Keith M. Callenberg, Carrie L. Partch, et al., Nov. 20, 2013.]

Whereas Reg3γ expression is more restricted in the intestine under normal conditions, it has been known for over a decade that the expression of intestinal Reg3 is influenced by various factors (Table 1). For example, microbial stimulation is required for the expression of the Reg3 protein because its expression is suppressed by inhibiting bacterial colonization under germ-free conditions or antibiotic treatment (12, 34), whereas supplementation with specific bacteria enhances Reg3 expression (35, 36). Nutritional (high-fat diet, prebiotics, and alcohol) (37–41) or surgery (graft-versus-host disease by an allogeneic transplantation or bariatric surgery) (42–45) can also alter Reg3γ expression (Table 1). Although bariatric surgery resulted in increased levels of intestinal Reg3γ and Reg3β (42, 43), it is still unclear whether this change is an effect of bariatric surgery or a cause of metabolic improvements of surgery. Additionally, studies show that bile acids and GLP-1 play a role in the regulation of the intestinal and pancreatic Reg3 expression, respectively (46, 47). Interestingly, recent findings implicate its expression in other tissues under disease status. For instance, Reg3γ is upregulated in epidermal keratinocytes with skin lesions, such as aseptic wounds or psoriasis (48, 49), as well as in pancreas and colon during pancreatitis and inflammatory bowel disease (16, 50). However, the precise cellular source of Reg3 proteins in disease status still needs to be elucidated.

Table 1.

Factors That Affect Reg3γ Expression

Factors	Gene Expression	Tissue/Location
Germ-free	Decrease	Small intestine
HFD	Decrease	Small intestine, colon
Antibiotics	Decrease	Small intestine
Alcohol	Decrease	Small intestine
Pre-(probiotics)	Increase	Small intestine, colon
Bariatric surgery	Increase	Small intestine
GVHD	Decrease	Small intestine
	Increase	Blood
Exendin-4 treatment	Increase	Pancreas
Bile acid	Increase	Small intestine
Skin wound	Increase	Skin
Pancreatitis	Increase	Pancreas
Inflammatory bowel disease	Increase	Colon

Abbreviations: GVHD, graft-versus-host disease; HFD, high-fat diet.

Reg3 measurement is rarely carried out by ELISAs in plasma or serum. According to previous studies, human REG3A (human homolog of mouse Reg3γ) and mouse Reg3γ concentration of healthy subjects is almost stable to 15 to 25 ng/mL (44, 45, 51). Although the expression of Reg3 in tissues has been measured by quantitative real-time PCR or western blotting in most of the rodent studies, there seem to be no appropriate antibodies for ELISA kits available in the market to quantify circulating Reg3γ levels. We used a variety of ELISAs to assess circulating levels, including the one previously used by Zhao et al. (44). However, all of these assays detected substantial levels of Reg3γ in Reg3γ knockout mice, indicating that there is cross-reactivity with other circulating proteins.

Putative Receptor for Reg3 Signaling

Kobayashi et al. (52) isolated Reg-binding protein that is encoded by the exostose-like glycosyltransferase (EXTL) gene in the screening of the rat islet cDNA library. EXTL3 has been implicated as a potential receptor for Reg3 because Extl3 has been shown to bind to Reg3γ protein in keratinocyte (48) (Table 2). EXTL3 is expressed in various tissues such as skin, liver, kidney, adipose tissue, stomach, small intestine, colon, pancreas, adrenal gland, pituitary gland, and brain (www.proteinatlas.org) (48, 52). In addition, EXTL3 is abundant in hematopoietic stem cells and early T cells (53). Most of what is known about EXTL3 is from studies of heparan sulfate/heparin biosynthesis (54, 55). Genetic loss of function of Extl3 is lethal to the embryo due to a defect in heparan sulfate synthesis (55). Evidence for Extl3 as a binding protein for Reg1 was first demonstrated in pancreatic β-cells (52). At the same time, Mueller et al. (56) suggested that Reg1 promoted cell growth in rat pancreatic-derived cells by activating the MAPK–c-Jun N-terminal kinase–cyclin D1 pathway via Extl3. Using a human pancreatic cell line, EXTL3 was identified as a potential receptor for Reg3 protein, which promotes its translocation from membrane to nucleus (57). Examining the contribution of Extl3 signaling in islets in β-cell–specific Extl3-deficient mice displayed abnormal islet morphology and glucose intolerance due to impaired insulin secretion (55). A recent study indicates that the Reg3γ-Extl3 binding regulates keratinocyte proliferation and differentiation by activating the phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathway (48). Further, this Reg3γ-Extl3 signaling serves as a mediator for induction of SHP-1 via AKT–signal transducer and activator of transcription (STAT) 3 activation to inhibit Toll-like receptor (TLR) 3–induced inflammation and may be involved in regulating impaired wound healing in diabetes (49). Interestingly, Reg3γ appears to signal through the epidermal growth factor receptor/JAK2/STAT3 pathway in tumors (58). Although there appears to be some overlap in these signaling pathways, the possibility that there might be other receptors for Reg3 proteins needs to be considered.

Table 2.

Reg Proteins Binding to Extl3

Reg Protein	Signaling Pathway	Function
Reg1	Activation of MAPK-JNK-cyclin D1	Pancreatic cell growth
Reg3γ	Activation of PI3K-AKT	Keratinocyte proliferation and differentiation
	Inhibition of TLR3 via AKT-STAT3-SHP1	Promoting wound healing

Abbreviation: JNK, c-Jun N-terminal kinase.

Tissue-Specific Metabolic Actions of the Reg3 Family

A role for Reg3 has been reported in a wide range of organs, including liver, intestine, pancreas, lung, heart, skin, and nervous system (48, 49, 59–66) (Fig. 2). Later, we review the existing literature that points to a role of the Reg3 family in various aspects of metabolism.

Figure 2.

Physiological effects of Reg3 protein on various organs. Reg3γ protein acts on various organs as well as mediates the multiple benefits, including tissue protection against damage and improvement of the regenerating process. MRSA, methicillin-resistant Staphylococcus aureus.

Pancreas (pancreatic cells)

During organogenesis, the expression of Reg3 proteins are restricted to glucagon-expressing enteropancreatic endocrine cells at 10 weeks of gestation (67). Reg3 was not detected in normal adult islets but in regenerating islets (19). This suggests that Reg3 has important roles in islets responding to pancreatic damage. For example, pancreata lacking Reg3γ showed a reduction in activation of STAT3/SOCS3 signaling, which led to elevated apoptotic and inflammatory responses in acute pancreatitis (68). Xia et al. (59) recently provided evidence for Reg3γ in regulating the JAK2/STAT3 pathway and conferring a beneficial effect on β-cell regeneration. In line with this, Reg3β has a protective effect on streptozotocin-induced pancreatic β-cell damage (69). Intriguingly, Reg3 expression was elevated in pancreas from a model of iron overload (70, 71). Upregulation of the Reg3 family is likely attributable to pancreatic damage to reduce iron-induced pancreatic stress because it is known that excessive iron can result in iron deposition in islets, β-cell dysfunction, and impaired insulin secretion (72). Such findings point toward the possibility that Reg3γ signaling confers beneficial effects on damaged cells. Conversely, it is important to note that the influence of Reg3γ is likely to be dependent on the changes in microenvironment. For example, Reg3γ may affect tumor suppressor function of CD8⁺ T cells and act as oncogenic signaling in pancreatic cancer via activating the epidermal growth factor receptor/JAK2/STAT3 signaling pathway (58, 73), which reflect that the effect of Reg3 may be different depending on immune status.

Liver

In normal adult and fetal liver, Reg3 mRNA expression is not detected, whereas it is highly expressed in hepatocarcinoma (23). Studies using mouse models have demonstrated that intestinal Reg3β/γ plays a role in protecting alcohol-induced liver disease by inhibiting bacterial translocation (39, 60). Furthermore, ectopic expression of Reg3β in hepatocytes has been found to act on multiple aspects of liver regeneration in a variety of liver damage models (74–76). Reg3 in hepatocytes stimulates antiapoptotic activity via increased protein kinase A (PKA), which leads to liver regeneration in partial hepatectomy (76). Furthermore, Reg3β protects from drug-induced liver injury by inhibiting oxidative damage as well as elevating antioxidant (superoxide dismutase and glutathione reductase activity) activity (74, 75). Taken together, these data suggest that effects of Reg3 proteins are minimal under normal healthy conditions but may be of importance to liver, pancreas, and intestine under pathological conditions.

Skin

A few studies have reported the effects of Reg3γ on epidermal keratinocytes around skin wounds. Reg3γ is highly expressed in patients with psoriasis and mice with either drug-induced psoriatic skin lesions or physical skin wound (48). The elevation of Reg3γ induced by IL-17 enhances keratinocyte proliferation and inhibits differentiation through the EXTL3-PI3K-AKT signaling pathway. In contrast to skin wounds in normal patients, wound-healing processes in patients with diabetes are disrupted due to both hyperglycemia and dysfunctional immune response (77, 78). Compared with patients without diabetes, Reg3γ expression around skin wounds of patients with diabetes was suppressed (49).

Adipose tissue

There are studies illustrating a role of the Reg3 family in adipose tissue. A recent study showed that either deficiency or overexpression of Reg3γ or Reg3β has no effect on diet-induced obesity and glucose tolerance. However, Secq et al. (79) reported Reg3γ as an obesogenic factor with the observation that mice expressing human REG3A specifically in the liver became obese on a normal chow diet as well as circulating REG3A levels were elevated in obese patients. At the same time, examining the contribution of REG3A through 3T3-L1 cells showed increased cell growth mediated by the MAPK kinase/ERK signaling pathway. A caveat of this study is that the analysis is performed in undifferentiated preadipocytes; therefore, the role of Reg3 family in adipocyte differentiation or maturation remains unclear. At the same time, these authors took advantage of transgenic mice overexpressing human REG3Α specifically in the hepatocytes. Therefore, a mechanism to explain the cell-specific actions of Reg3 needs to be determined. Likewise, although transcriptional levels of Reg3 were remarkably upregulated in mesenteric fat in response to a high-fat diet (80), it cannot be ruled out that mesenteric fat tissue would be contaminated with mesenteric lymph node, which contains numerous immune cells. Intriguingly, another recent study showed that mice overexpressing human REG3γ in gut epithelial cells were protected from diet-induced obesity and improved glucose metabolism (81). Further, Reg3γ transgenic overexpressing mice showed that Reg3γ-associated Lactobacillus influence anti-inflammatory macrophage pools not only in the gut but also in the spleen and adipose tissues (82). This would implicate Reg3 signaling in the regulation of body weight and glucose metabolism via actions on the microbiota and immune system. However, the discrepancy in phenotypes in adipose tissue requires further efforts to find the direct contribution of Reg3 protein in adipose tissue in obesity development. Although there is a possibility that actions of Reg3 proteins are mediated via a specific receptor for Reg3 proteins in adipose tissue, the mechanism underlying the adipose tissue by Reg3 proteins remains unclear.

Intestine

Reg3γ has a potent bactericidal effect on Gram-positive bacteria by binding to a surface peptidoglycan layer, followed by forming a membrane-penetrating pore (32). Reg3β, a Reg3 murine isoform, which is abundantly expressed in the GI tract, binds directly to lipopolysaccharide and exerts bactericidal activity against Gram-negative bacteria (83). These Reg3 proteins are crucial for maintaining spatial segregation between host and gut bacteria (64). In line with this action against bacteria, Reg3γ restricts mucosa-associated bacterial colonization and decreases bacterial translocation in alcohol-fed mice, which resulted in resistance to alcohol-induced liver damage (60). Interestingly, either gain or loss of Reg3γ did not influence metabolic features in diet-induced nonalcoholic steatohepatitis (82). This result may be due to the insufficient induction of bacterial translocation and endotoxemia when challenged with a western-style diet. Another function of Reg3 in the gut is to mediate host barrier function as a downstream target of IL-22 (84). Microorganism-associated molecular patterns also influence the expression of Reg3γ in the intestine through TLRs (10). For instance, systemic administration of TLR ligand flagellin-induced Reg3γ expression in the gut epithelial cells by TLR5 stimulation and IL-22 secretion, which protects mice from antibiotic-resistant bacterial colonization (85). Further, oral administration of lipopolysaccharide triggered Reg3γ expression through TLR4 stimulation, thereby enhancing immune resistance to infection with antibiotic-resistant bacteria (34). Recent studies reported that both Reg3β and Reg3γ also influence epithelial proliferation, crypt regeneration, and protection of intestinal stem cells and Paneth cells from apoptosis in the tissue damage (44, 86, 87).

Future Directions

Taken together, these studies indicate that Reg3 proteins activity may be beneficial in metabolic regulation but many questions remain unanswered. For example, it is uncertain whether Reg3 proteins produced in the intestine are delivered to target tissues as a circulating hormone or whether local production of Reg3 proteins are produced in certain cells such as macrophages, innate lymphoid cells that may act in a paracrine manner in various tissues. On a related note of signaling transduction, it needs to be determined whether Reg3γ exerts its metabolic effects by binding to a specific receptor such as EXTL3 that is found in many tissues including brain, liver, fat, and pancreas. Although Lai et al. (48) suggested that the Reg3γ-EXTL3 complex promotes skin wound healing through PI3K-AKT activation, more research is required to understand tissue-specific Reg3 signaling pathway. Furthermore, because Extl3 has been detected throughout the brain and it also contributes to brain development and corticogenesis (88–90), potential effects of Reg3γ in the central nervous system on food intake or glucose homeostasis need to be investigated.

In addition, future studies need to focus on effects of Reg3 molecules on shaping the microbiota composition. Although it has been known that Reg3γ exerts bactericidal activity against Gram-positive bacteria (32), substantial evidence that increases in portions of Lactobacillus belonging to Gram-positive bacteria is affected by Reg3γ (81) suggests that there may be certain population of microbiota that is resistant to Reg3γ or stimulated by Reg3γ.

Finally, it remains to be investigated whether Reg3 proteins are necessary for the beneficial effects of bariatric surgery. Bariatric surgery is currently acknowledged as the most effective treatment of obesity and glucose regulation (91, 92). Roux-en-Y gastric bypass and vertical sleeve gastrectomy are the most common procedures. Besides the weight loss, anatomical alteration of the GI tract by surgery influences gut hormone secretion, composition of bile acid and microbiota, and changes in micronutrients including iron deficiency (93–99). These studies indicate that surgery likely alters the gut microenvironment, such as intestinal barrier function, immune system, and enteroendocrine activity. Intriguingly, studies exploring molecular response in gastric bypass surgery showed that Reg3γ and Reg3β were upregulated in the jejunum after surgery (42, 43). Thus, it is worth considering the impact of Reg3 proteins on the sustained body weight loss and improved glucose regulation after bariatric surgery. Such work could directly implicate Reg3 proteins as gut hormones for which levels can be manipulated for potential therapeutic benefit.

Summary

Based on the existing evidence described in this review, we suggest the importance of Reg3 protein for metabolic homeostasis in multiple organs. Studies demonstrated that Reg3 protein protects β-cell, hepatocyte, and keratinocyte from damages and promotes cell regeneration (48, 49, 59, 69, 74–76). Reg3γ is primarily produced in the GI tract, and in general, Reg3 proteins play a role in protection against damage, cell proliferation, antibacterial defense, and inhibition of bacterial translocation (64, 100). Together, it remains an open question as to whether the endocrine or paracrine actions of Reg3 protein regulate key metabolic processes. Nevertheless, accumulating evidence that tissue specifically overexpressed the Reg3 gene resulted in better systemic metabolism via microbiota alteration or the administration of extrinsic Reg3 protein improved β-cell function has indicated the potential of Reg3 proteins as endocrine action or autocrine/paracrine action (69, 81, 101). Although we are still awaiting to determine the circulating action vs local paracrine or autocrine action of Reg3 proteins, such work could implicate Reg3 proteins as gut hormones for which levels can be manipulated for potential therapeutic benefit.

Glossary

Abbreviations:

AMP

antimicrobial peptide

EXTL

exostose-like glycosyltransferase

GI

gastrointestinal

PI3K

phosphatidylinositol 3-kinase

PKA

protein kinase A

STAT

signal transducer and activator of transcription

TLR

Toll-like receptor