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Intestinal mucus components and secretion mechanisms: what we do and do not know

• Review Article
• Open access
• Published: 03 April 2023

Intestinal mucus components and secretion mechanisms: what we do and do not know

• Chunyan Song, 
• Zhenglong Chai, 
• Si Chen, 
• Hui Zhang, 
• Xiaohong Zhang & 
• Yuping Zhou 

Experimental & Molecular Medicine volume 55, pages681–691 (2023)Cite this article

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Abstract

Damage to the colon mucus barrier, the first line of defense against microorganisms, is an important determinant of intestinal diseases such as inflammatory bowel disease and colorectal cancer, and disorder in extraintestinal organs. The mucus layer has attracted the attention of the scientific community in recent years, and with the discovery of new mucosal components, it has become increasingly clear that the mucosal barrier is a complex system composed of many components. Moreover, certain components are jointly involved in regulating the structure and function of the mucus barrier. Therefore, a comprehensive and systematic understanding of the functional components of the mucus layer is clearly warranted. In this review, we summarize the various functional components of the mucus layer identified thus far and describe their unique roles in shaping mucosal structure and function. Furthermore, we detail the mechanisms underlying mucus secretion, including baseline and stimulated secretion. In our opinion, baseline secretion can be categorized into spontaneous Ca2+ oscillation-mediated slow and continuous secretion and stimulated secretion, which is mediated by massive Ca2+ influx induced by exogenous stimuli. This review extends the current understanding of the intestinal mucus barrier, with an emphasis on host defense strategies based on fortification of the mucus layer.

초록

대장 점막 장벽은 

미생물로부터의 첫 번째 방어선으로, 

염증성 장 질환 및 대장암과 같은 장 질환 및 장 외 장기 장애의 중요한 결정 요인입니다. 

점막층은 최근 과학계의 주목을 받아왔으며, 

새로운 점막 성분의 발견으로 점막 장벽이 다양한 성분으로 구성된 복잡한 시스템이라는 것이 

점차 명확해지고 있습니다. 

또한 특정 구성 요소는 

점막 장벽의 구조와 기능을 조절하는 데 공동으로 관여합니다. 

따라서 

점막층의 기능적 구성 요소에 대한 포괄적이고 

체계적인 이해가 분명히 필요합니다. 

본 리뷰에서는 

현재까지 식별된 점막층의 다양한 기능적 구성 요소를 요약하고, 

이들 구성 요소가 점막 구조와 기능 형성에 미치는 독특한 역할을 설명합니다. 

또한 점액 분비의 메커니즘을 상세히 설명하며, 

기초 분비와 자극에 의한 분비를 포함합니다. 

우리 의견에 따르면, 

기초 분비는 자발적인 Ca2+ 진동 매개 느리고 지속적인 분비와 외부 자극에 의해 

유발된 대규모 Ca2+ 유입에 의해 매개되는 자극 분비로 분류될 수 있습니다. 

이 리뷰는 

점액층의 강화에 기반한 호스트 방어 전략에 초점을 맞춰 

장 점액 장벽에 대한 현재의 이해를 확장합니다.

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Introduction

Humans have evolved a three-dimensional mucosal barrier system to maintain local microenvironment homeostasis and systemic health, preventing the invasion of various exogenous antigens and commensal and pathogenic microorganisms into the colonic lumen. The intestinal mucosal barrier is composed of four parts: microbial, mucus, mechanical, and immune barriers1,2. As the central component of the mucosal barrier, the mucus layer maintains the homeostasis of intestinal flora by nourishing intestinal symbiotic bacteria and protects the intestinal epithelium against intestinal pathogenic bacteria. The mucus layer also exerts immunological effects by directly binding pathogenic bacterial glycans via lectin-like proteins on immune cells such as those on dendritic cells3.

Defects in the mucus barrier are associated with intestinal diseases such as inflammatory bowel disease and colon cancer and extraintestinal disorders such as liver disease and diabetes4. The continual mucus secretion into the gastrointestinal tract of approximately 10 L per day underlies this protective function5. In recent years, significant progress has been made in identifying the components of the mucus layer, understanding their function and identifying the mechanisms underlying mucus secretion. The mucus barrier is garnering increased attention from the scientific community, warranting an overall and accurate overview of mucosal components. In this review, we detail recent findings by highlighting the emerging roles of various mucus components in delineating the stereoscopic structure of the mucus barrier. In addition, because the protective function of mucus is mainly attributable to continuous mucus secretion into the lumen, the mechanism for mucus secretion is elucidated to identify clues into the consolidation of the mucus barrier.

서론

인간은

다양한 외인성 항원 및 공생 및 병원성 미생물의 대장 내강 침입을 방지하여

국소 미세환경의 균형과 전신 건강을 유지하기 위해

3차원 점막 장벽 시스템을 진화시켰습니다.

장 점막 장벽은

미생물, 점액, 기계적, 면역 장벽의 네 부분으로 구성되어 있습니다1,2.

점막 장벽의 중심 구성 요소인 점액층은

장 내 공생 세균을 영양분으로 공급하여 장 내 미생물 균형을 유지하며,

장 상피 세포를 장 내 병원성 세균으로부터 보호합니다.

점액층은

면역 세포(예: 수지상 세포)에 존재하는 렉틴 유사 단백질을 통해

병원성 세균의 글리칸과 직접 결합하여 면역학적 효과를 발휘합니다3.

점액 장벽의 결함은

염증성 장 질환 및 대장암과 같은 장 질환과 간 질환 및 당뇨병과 같은

장 외 질환과 연관되어 있습니다4.

하루 약 10L의 점액이

위장관으로 지속적으로 분비되는 것이 이 보호 기능을 뒷받침합니다5.

최근 몇 년간 점액층의 구성 성분을 식별하고

그 기능을 이해하며 점액 분비 메커니즘을 규명하는 데 상당한 진전이 이루어졌습니다.

점액 장벽은 과학계에서 점점 더 많은 관심을 받고 있으며,

점막 구성 성분에 대한 종합적이고 정확한 개요가 필요합니다.

이 리뷰에서는

점액 장벽의 입체적 구조를 규명하는 데 기여하는

다양한 점액 성분의 새로운 역할을 강조하여

최근 연구 결과를 상세히 설명합니다.

또한 점액의 보호 기능이 주로

점막 내로 지속적인 점액 분비에 기인한다는 점을 고려해,

점액 분비 메커니즘을 규명함으로써 점액 장벽의 강화 메커니즘을 이해하는

단서를 제공하고자 합니다.

Mucus barrier impairment is important to colonic inflammation etiology

The colonic mucus layer is a lamellar structure consisting of a dense inner layer and a loose outer layer. The inner layer is attached to the surface of the intestinal epithelium and gradually expands outward to form an outer layer that continuously secretes mucus. Under physiological conditions, the inner layer is mostly sterile, but the outer layer serves as a habitat for microorganisms by providing nutrients and adhesion sites for symbiotic flora. The hierarchical structure of the mucus barrier is key in preventing bacterial penetration and blocking direct contact of bacteria and the intestinal epithelium6.

The impairment or loss of the protective mucus layer may cause direct exposure of the intestinal epithelium to microorganisms or pathogens, thereby triggering the development of specific diseases4. For instance, penetrating the intestinal mucus barrier is critical for pathogens such as bacteria or worms to induce intestinal symptoms. Diarrhea caused by amoebae is closely related to their attachment to the mucus layer via the release of Gal-lectin as well as by opening a channel for their invasion of the intestinal epithelium induced by mucus decomposition mediated by specific proteases7,8. Increased permeability of the mucus barrier paves the way for the occurrence and development of ulcerative colitis (UC)9. Mice with dextran sodium sulfate (DSS)-induced colitis show significantly increased permeability of the mucus layer not decreased layer thickness, resulting in direct contact of intestinal microorganisms with the intestinal epithelium, causing inflammation4. Patients with UC present with an abnormally high proportion of sulfate-reducing bacteria in the gut, and these bacteria reduce sulfate to hydrogen sulfide by breaking the disulfide bonds between mucin 2 (MUC2) and thus increase the permeability of the mucus layer10. Irritable bowel syndrome in a Wistar rat model of water-avoidance stress was linked to a mucin O-glycosylation disorder, which caused the flattening of the mucus layer and loss of its cohesive properties as well as increased intestinal permeability11.

점액 장벽의 손상은 대장 염증의 원인에 중요합니다

대장 점막층은

밀집된 내층과 느슨한 외층으로 구성된 층상 구조입니다.

내층은 장 상피 표면에 부착되어 있으며,

점차 외부로 확장되어 점액을 지속적으로 분비하는 외층을 형성합니다.

생리적 조건에서 내층은 주로 무균 상태이지만,

외층은 영양분과 공생 미생물의 부착 부위를 제공하여 미생물의 서식지로 기능합니다.

점막 장벽의 계층적 구조는

세균의 침투를 방지하고

세균과 장 상피 세포의 직접 접촉을 차단하는 데 핵심적 역할을 합니다6.

보호 점막 층의 손상 또는 상실은

장 상피 세포가 미생물이나 병원체에 직접 노출되게 하여

특정 질환의 발병을 유발할 수 있습니다4.

예를 들어,

세균이나 기생충과 같은 병원체가 장 점막 장벽을 침투하는 것은

장 증상을 유발하는 데 결정적입니다.

아메바에 의한 설사는

갈락토스-1-β-D-프럭토피라노시드(Gal-lectin)의 분비로

점액층에 부착하는 것뿐만 아니라 특정 프로테아제에 의해 매개되는 점액 분해로 인해

장 상피로 침투하는 통로를 열어주는 메커니즘과 밀접하게 관련되어 있습니다7,8.

점막 장벽의 투과성 증가가

궤양성 대장염(UC)의 발생과 진행을 촉진합니다9.

데크스트란 나트륨 설페이트(DSS)로 유발된 대장염을 가진 쥐는 점막층의 투과성이 유의미하게 증가했으며, 점막층 두께는 감소하지 않았습니다. 이는 장 미생물이 장 상피와 직접 접촉하여 염증을 유발합니다4. UC 환자는 장 내 황산화 세균의 비율이 비정상적으로 높으며, 이 세균은 뮤신 2(MUC2) 간의 이황화 결합을 분해하여 황산염을 수소 황화물로 환원시켜 점막층의 투과성을 증가시킵니다10. 물 회피 스트레스 모델을 사용한 Wistar 쥐에서 과민성 장 증후군은 뮤신 O-글리코실화 장애와 연관되어 점막층의 평탄화와 응집력 상실, 장 투과성 증가를 초래했습니다11.

Functional components of the mucus layer

The mucus layer is a very hydrated and complex viscoelastic medium, with MUC2 as its skeleton component. MUC2 cooperates with other components to consolidate the structure of the mucosal barrier and regulate local microenvironment homeostasis. Therefore, it is important to comprehensively analyze the complex components of the mucus barrier and their roles in regulating mucus barrier homeostasis and inhibiting intestinal inflammation (Fig. 1).

점막층의 기능적 구성 요소

점액층은

매우 수분 함량이 높고 복잡한 점탄성 매체로,

MUC2가 골격 성분으로 작용합니다.

MUC2는

다른 성분들과 협력하여 점막 장벽의 구조를 강화하고

국소 미세환경의 균형을 조절합니다.

따라서

점액 장벽의 복잡한 구성 요소와 그 역할을 종합적으로 분석하여

점액 장벽의 균형 조절 및 장 염증 억제에 미치는 영향을 이해하는 것이 중요합니다(그림 1).

Fig. 1: Main components of the intestinal mucus layer.

The colonic mucus layer consists of a dense inner layer and a loose outer layer. Multiple components are involved in the maintenance of the structure and function of the mucus barrier in addition to MUC2, which composes the skeleton of the mucus layer. FCGBP and TFF3 act synergistically to enhance the mucus barrier and exert antibacterial effects, while the metalloenzyme CLCA1 is involved mainly in the stratification and expansion of mucus. ZG16, RELMβ, Lypd8, sIgA, and AMP exert bacteriostatic or bactericidal effects under different conditions.

대장 점막층은 밀집된 내층과 느슨한 외층으로 구성되어 있습니다. MUC2가 점막층의 골격을 구성하는 것 외에도, 점막 장벽의 구조와 기능을 유지하는 데 여러 성분이 관여합니다. FCGBP와 TFF3는 점막 장벽을 강화하고 항균 효과를 발휘하는 데 시너지 효과를 발휘하며, 금속 효소 CLCA1은 주로 점막의 층분화와 확장에 관여합니다. ZG16, RELMβ, Lypd8, sIgA 및 AMP는 다양한 조건 하에서 세균 증식 억제 또는 세균 살균 효과를 발휘합니다.

Structural skeleton of the mucus barrier: MUC2

To date, 21 mucins have been identified, named from MUC1 through MUC21 according to the order in which they were discovered. They have also been classified into membrane-related mucins and secretory mucins according to their structural characteristics and biological functions. Membrane-associated mucins include MUC1, MUC3A/B, MUC4, MUC12, MUC13, MUC15, MUC17, MUC20, and MUC21. Secretory mucins are categorized into two subclasses: gel-forming and nongel-forming mucins. MUC2, MUC5AC, MUC5B, MUC6, and MUC19 are gel-forming mucins involved in protection, transportation, lubrication, and hydration, and MUC7, MUC8, and MUC9 are nongel-forming mucins12.

MUC2 is the predominant component of the colonic mucus layer function as its structural skeleton. It is a macromolecular glycoprotein comprising more than 5,000 amino acids, and its protein skeleton is synthesized in ribosomes. MUC2 is composed of the following conserved domain structures: a D’ domain containing only a trypsin inhibitor-like (TIL) domain and a fibronectin type I-like (E) domain; D1, D2, and D3 domains forming a von Willebrand D (vWD) structure, a C8 module, and TIL and E domains; the first cysteine domain (CysD); a small proline-, threonine- and serine-enriched (PTS) domain, the second CysD domain; a large PTS domain; a D4 domain containing vWC structure; and a cysteine-knot (CK) domain13.

MUC2 monomers are linked by disulfide bonds in the endoplasmic reticulum to form oligomers, which leave the endoplasmic reticulum and enter the Golgi. MUC2 monomers cannot enter the Golgi apparatus. Notably, ATP is required for MUC2 oligomer transport to the cis side of the Golgi. MUC2 is glycosylated in the Golgi, with oligosaccharide chains of different lengths linked to the PTS domain on the protein skeleton by O-glycosidase, and eventually, mature MUC2 molecules with molecular weights in the range of megadaltons are formed14. These MUC2 proteins are tightly wrapped in mucus secretory granules and stored as vesicles in goblet cells (GCs), which are specialized intestinal epithelial cells. GCs produce and secrete mucus granules into the intestinal lumen where MUC2 polymers rapidly expand, increasing in size by more than 1000-fold through depolymerization and hydration15, and unfold to form a large reticular lamellar structure facilitated by the action of HCO3- 16. MUC2 lamellae are connected by noncovalent binding of the D3 domains or covalent isopeptide bonds between CysDs17,18. The resultant dense mucin network separates bacteria from intestinal epithelial cells and blocks microorganism invasion into intestinal epithelial cells19. In contrast, the loose external mucus layer provides a good habitat for symbiotic microorganisms, which consume MUC2-linked glycans as energy sources and thus decompose and ferment them to produce short-chain fatty acids, which are oxidative phosphorylation substrates in intestinal epithelial cells20. A significant increase in the number of GCs and MUC2 mRNAs was observed in obese rats fed high-dose soy isoflavone, resulting in increased colonic mucus secretion and a reduced colonic inflammatory response21. MUC2 can transmit immunomodulatory signals to dendritic cells, facilitating intestinal mucosa acquisition of immune tolerance. Small intestinal dendritic cells can penetrate mucus pores and bind to MUC2 via the galectin-3-dectin-1-FcgRIIB receptor complex, activating dendritic cell β-catenin and interfering with the expression‎ of inflammatory factors by inhibiting the transcription of nuclear factor κB (NF-κB)21.

점막 장벽의 구조적 골격: MUC2

현재까지 21개의 뮤신이 발견되었으며, 발견 순서에 따라 MUC1부터 MUC21까지 명명되었습니다. 이들은 구조적 특성 및 생물학적 기능에 따라 막 관련 뮤신과 분비형 뮤신으로 분류됩니다. 막 관련 뮤신에는 MUC1, MUC3A/B, MUC4, MUC12, MUC13, MUC15, MUC17, MUC20, 및 MUC21이 포함됩니다. 분비 뮤신은 젤 형성 뮤신과 젤 비형성 뮤신으로 두 하위 분류로 나뉩니다. MUC2, MUC5AC, MUC5B, MUC6, MUC19는 보호, 운반, 윤활, 수분 유지에 관여하는 젤 형성 뮤신이며, MUC7, MUC8, MUC9는 젤 비형성 뮤신입니다.

MUC2는 대장 점막층의 주요 구성 성분으로 구조적 골격 역할을 합니다. 이는 5,000개 이상의 아미노산으로 구성된 거대 분자 당단백질이며, 그 단백질 골격은 리보솜에서 합성됩니다. MUC2는 다음과 같은 보존된 도메인 구조로 구성됩니다: 트립신 억제제 유사(TIL) 도메인과 피브로네ктиן 유형 I 유사(E) 도메인을 포함하는 D' 도메인; von Willebrand D(vWD) 구조를 형성하는 D1, D2, D3 도메인, C8 모듈, 및 TIL과 E 도메인; 첫 번째 시스테인 도메인(CysD); 프로린 풍부(PTS) 도메인; 두 번째 시스테인 도메인(CysD); 큰 PTS 도메인; vWC 구조를 포함하는 D4 도메인; 및 시스테인 노드(CK) 도메인. 트레오닌 및 세린 풍부(PTS) 도메인; 두 번째 CysD 도메인; 큰 PTS 도메인; vWC 구조를 포함하는 D4 도메인; 및 시스테인 노드(CK) 도메인13.

MUC2 단량체는 내소체에서 이황화 결합으로 연결되어 올리고머를 형성하며, 이는 내소체를 떠나 골지체로 이동합니다. MUC2 단량체는 골지체로 들어갈 수 없습니다. 특히, MUC2 올리고머의 골지체 cis 측으로의 수송에는 ATP가 필요합니다. MUC2는 골지체에서 당화되며, 단백질 골격의 PTS 도메인에 O-글리코시다아제에 의해 다양한 길이의 올리고사카라이드 사슬이 결합되어 최종적으로 분자량 메가달톤 범위의 성숙한 MUC2 분자가 형성됩니다14. 이러한 MUC2 단백질은 점액 분비 소체에 밀접하게 감싸여 장 상피 세포의 특수화된 세포인 점액 세포(GCs) 내에 소체로 저장됩니다. GC는 점액 소체를 장 내강으로 분비하며, 여기서 MUC2 폴리머는 분해 및 수화 과정을 통해 크기가 1,000배 이상 증가하며15, HCO3-의 작용으로 펼쳐져 큰 망상 층상 구조를 형성합니다16. MUC2 층판은 D3 도메인의 비공액 결합 또는 CysD17,18 간의 공액 이소펩티드 결합으로 연결됩니다. 결과적으로 형성된 밀집된 뮤신 네트워크는 세균과 장 상피 세포를 분리하고 미생물의 장 상피 세포 침입을 차단합니다19. 반면, 느슨한 외부 점액층은 공생 미생물의 좋은 서식지를 제공하며, 이들은 MUC2와 연결된 글리칸을 에너지원으로 소비하여 이를 분해 및 발효시켜 장 상피 세포의 산화 인산화 기질인 단쇄 지방산을 생성합니다20. 고용량 대두 이소플라본을 투여받은 비만 쥐에서 GCs와 MUC2 mRNA의 수가 유의미하게 증가했으며, 이는 대장 점액 분비 증가와 대장 염증 반응 감소로 이어졌습니다21. MUC2는 면역 조절 신호를 ден드리틱 세포에 전달하여 장 점막의 면역 관용 획득을 촉진합니다. 소장 ден드리틱 세포는 점액 구멍을 통해 침투하여 갈레틴-3-데크틴-1-FcgRIIB 수용체 복합체를 통해 MUC2에 결합하여 ден드리틱 세포의 β-카테닌을 활성화하고 핵 인자 κB (NF-κB)의 전사를 억제함으로써 염증 인자의 발현을 방해합니다21.

Partnership between FCGBP and TFF3

The trefoil factor (TFF) family comprises small-molecule polypeptides secreted mainly by gastrointestinal cells, and their secondary structures are characterized by one or two unique trefoil domains22. The TFF family in mammals includes breast cancer-associated pS2 peptide (pS2/TFF1), spasmolytic polypeptide (SP/TFF2), and intestinal trefoil factor (ITF). The intestinal trefoil factor (also known as trefoil factor 3, TFF3), the most recently discovered member of the TFF family, is secreted mainly by intestinal GCs along with MUC2, and in combination with MUC2 and other components, forming the first defensive line of the intestinal barrier. TFF3 is a cysteine-rich secretory peptide predicted to comprise 59 amino acid residues, of which six cysteine residues form three pairs of disulfide bonds, forming a clover-like structure, also known as the P domain. The cleft between loops 2 and 3 in the clover domain forms the binding site for oligosaccharides, such as mucin, or aromatic amino acids23. The P domain stabilizes and compresses the structure of TFF3, which may contribute to its proteolytic and acid resistance24.

TFF3 promotes the repair of intestinal epithelial injury by regulating the expression‎ of tight junction proteins in intestinal epithelial cells and the resulting intercellular contacts. TFF3 can anchor to E-cadherin on the cytoskeleton to form a cell adhesion junction complex (an E-cadherin/β-catenin complex) by upregulating E-cadherin expression‎ and increasing the E-cadherin and β-catenin connection. The resulting complex mediates the homophilic interaction of E-cadherin on the surface of adjacent cells, promoting intercellular adhesion and thereby facilitating the migration of intestinal epithelial cells to a damaged areas, where they cover the wound for the rapid repair of epithelial injury25. During inflammation, TFF3 reduces the paracellular permeability of the intestinal epithelium to protect the intestinal epithelium from invasion by exogenous microorganisms by upregulating the expression‎ of the tight junction protein Claudin-1 and downregulating the expression‎ of the cation channel-forming protein Claudin-226. TFF3 maintains intestinal mucosal integrity not only by promoting intestinal epithelial repair but also by modulating the inflammatory response. Moreover, TFF3 reduced the expression‎ of the LPS-induced proinflammatory cytokines interleukin-8 (IL-8) and IL-6 in HT-29 cells in vitro27. In addition, TFF3 induced the production of the decay-accelerating factor (DAF), which protects the intestinal epithelium from autologous complement injury by preventing the assembly of C3 and C5 invertases and the subsequent amplification of activated classical and alternative complement pathways28. A recent study showed that TFF3 expression‎ was upregulated in colorectal mucinous carcinoma tissues compared with that in normal tissues, and its elevated expression‎ was associated with advanced colorectal mucinous carcinoma, vascular or nerve invasion, and a poor prognosis29.

IgG Fc-binding protein (FCGBP) is also a component of the colon mucus and contains 13 vWDs, 12 cysteine-rich domains, and 12 trypsin inhibitor-like domains. TFF3 exists mainly as a heterodimer, while FCGBP is expressed and secreted by colon GCs as a partner protein linked to TFF3 by disulfide bonds. The initial protein bound to the Fc fragment of IgG in colonic mucus was observed and was subsequently named FCGBP30. Although the function of the FCGBP is not fully understood, it has been demonstrated that it as part of innate immune mucosal defense, as FCGBP and TFF3 heterodimers cooperatively inhibit pathogen attachment and enhance microbial clearance in the early stages of microbial infection31. The TFF3-FCGBP heterodimer also interacts with MUC2 via covalent and noncovalent bonds to maintain the structural integrity of the mucus barrier32. The network structure formed by the noncovalent combination of TFF3 and MUC2 affects the rheological properties of mucus and protects the mucus vesicles released from the apical side of GCs33. Dietary intervention consisting of the long-chain polyunsaturated fatty acid eicosapentaenoic acid (EPA) significantly inhibited DSS-induced experimental enteritis in mice by increasing the generation of the mucus component TFF3 and maintaining the intestinal mucus barrier34.

FCGBP와 TFF3의 협력

트레포일 인자(TFF) 가족은 주로 소화관 세포에서 분비되는 소분자 폴리펩타이드로 구성되며, 이들의 이차 구조는 하나 또는 두 개의 독특한 트레포일 도메인으로 특징지어집니다22. 포유류의 TFF 가족에는 유방암 관련 pS2 펩타이드(pS2/TFF1), 경련 완화 폴리펩타이드(SP/TFF2), 장 트레포일 인자(ITF)가 포함됩니다. 장 트리플 인자(트리플 인자 3, TFF3)는 TFF 가족에서 가장 최근에 발견된 구성원으로, 주로 장 상피 세포(GCs)에서 MUC2와 함께 분비되며, MUC2 및 기타 구성 요소와 결합하여 장 장벽의 첫 번째 방어선을 형성합니다. TFF3는 59개의 아미노산 잔기로 구성된 시스테인 풍부한 분비 펩타이드로, 이 중 6개의 시스테인 잔기가 3쌍의 이황화 결합을 형성하여 클로버 모양의 구조를 이루며, 이는 P 도메인으로 알려져 있습니다. 클로버 도메인의 루프 2와 3 사이의 틈은 뮤신과 같은 올리고사카라이드나 아로마틱 아미노산과 결합하는 결합 부위를 형성합니다23. P 도메인은 TFF3의 구조를 안정화하고 압축하여, 이는 TFF3의 프로테아제 저항성과 산성 저항성에 기여할 수 있습니다24.

TFF3는 장 상피 세포의 밀접 결합 단백질 발현을 조절하고 이에 따른 세포 간 접촉을 촉진하여 장 상피 손상의 회복을 촉진합니다. TFF3는 E-cadherin 발현을 증가시키고 E-cadherin과 β-catenin의 연결을 강화하여 세포 골격에 있는 E-cadherin에 결합하여 세포 접착 복합체(E-cadherin/β-catenin 복합체)를 형성합니다. 이 복합체는 인접한 세포 표면의 E-cadherin 간의 동형 상호작용을 매개하여 세포 간 접착을 촉진하며, 이는 장 상피 세포가 손상된 부위로 이동하여 상처를 덮어 상피 손상의 빠른 회복을 촉진합니다25. 염증 시 TFF3은 장 상피의 세포 간 투과성을 감소시켜 외인성 미생물의 침입으로부터 장 상피를 보호합니다. 이는 밀착 연결 단백질 클라우딘-1의 발현을 증가시키고 양이온 채널 형성 단백질 클라우딘-2의 발현을 감소시킴으로써 이루어집니다26. TFF3은 장 상피 세포의 복구를 촉진하는 것 외에도 염증 반응을 조절함으로써 장 점막의 무결성을 유지합니다. 또한 TFF3은 체외 실험에서 HT-29 세포에서 LPS에 의해 유도된 염증성 사이토킨 인터루킨-8 (IL-8)과 IL-6의 발현을 감소시켰습니다27. 또한 TFF3는 C3와 C5 인버타제의 조립을 방지하고 활성화된 고전적 및 대안적 보체 경로의 증폭을 차단함으로써 장 상피를 자가 보체 손상으로부터 보호하는 분해 가속화 인자(DAF)의 생산을 유도합니다28. 최근 연구에서 TFF3 발현은 정상 조직에 비해 대장 점액성 암 조직에서 증가했으며, 그 증가된 발현은 진행된 대장 점액성 암, 혈관 또는 신경 침윤, 그리고 불량한 예후와 연관되었습니다29.

IgG Fc 결합 단백질(FCGBP)은 대장 점액의 구성 성분으로, 13개의 vWD, 12개의 시스테인 풍부 도메인, 12개의 트립신 억제제 유사 도메인을 포함합니다. TFF3는 주로 이량체로 존재하며, FCGBP는 TFF3와 이황화 결합으로 연결된 파트너 단백질로 대장 GC에서 발현되고 분비됩니다. 대장 점막의 IgG Fc 단편에 결합된 초기 단백질이 관찰되었으며, 이후 FCGBP로 명명되었습니다30. FCGBP의 기능은 완전히 이해되지 않았지만, 선천성 면역 점막 방어의 일부로 작용하며, FCGBP와 TFF3 이량체는 미생물 감염 초기 단계에서 병원체 부착을 억제하고 미생물 제거를 강화하는 것으로 입증되었습니다31. TFF3-FCGBP 이량체는 MUC2와 공유결합 및 비공유결합을 통해 점막 장벽의 구조적 안정성을 유지합니다32. TFF3와 MUC2의 비공유결합으로 형성된 네트워크 구조는 점액의 유동학적 특성에 영향을 미치며, GC의 아피칼 측에서 방출되는 점액 소포를 보호합니다33. 장쇄 다불포화 지방산인 이코사펜타엔산(EPA)을 포함한 식이 개입은 TFF3 생성 증가와 장 점막 장벽 유지로 DSS 유발 실험적 장염을 유의미하게 억제했습니다34.

The mucus “shaper”: CLCA1

The calcium-activated chloride channel regulator (CLCA) family of zinc-dependent metalloproteinases shows characteristics of secretory and self-cleaving activation35,36. To date, four CLCAs have been identified in humans (hCLCA1–hCLCA4), eight CLCAs have been identified in mice (mClCA1, CLCA3a1, CLCA3a2, CLCA3b, 3 C, 4 A, 4B, and 4 C), four CLCAs have been identified in cattle (bCLCA1, bCLCA2 (LU-ECAM-1), bCLCA3, and bCLCA4), and CLCA has also been identified in pigs and horses37. hCLCA1, the first CLCA family member to be identified, is predominantly expressed in the intestinal epithelium, including the mucus layer of the small and large intestine. mCLCA3, originally known as goblet cell protein-5 (GOB-5), was later renamed mCLCA1 because of its high homology with hCLCA1. mCLCA1, mCLCA2, and mCLCA4 were renamed CLCA3a1, CLCA3a2, and CLCA3b, respectively, due to their high homology with human CLCA338,39. Notably a 35 kDa carboxyl terminal segment of the 110 kDa CLCA1 protein undergoes autocleavage in the endoplasmic reticulum and is then glycosylated to form mature CLCA1, which carries most of the original amino terminus and a small portion of the carboxyl terminus and has a molecular weight of 75–90 kDa. There are five highly conserved domains in mature CLCA1: (1) an amino-terminal signal sequence that directs CLCA1 to the secretory pathway; (2) a zinc-dependent metalloproteinase catalytic domain (CAT) with a conserved HExxE motif and a cysteine-rich (Cys) region; (3) a VWA domain with a conserved metal-ion-dependent adhesion site (MIDAS); (4) an unassigned β-sheet-enriched domain (BSR); and (5) a fibronectin type III (FnIII) domain in the C-terminus13,35,36. Mature CLCA1 monomers are linked by disulfide bonds to form dimers, which are connected at the amino termini in a noncovalent manner. Several CLCA1 molecules can polymerize to form globular noncovalent oligomers, which are stored and secreted as oligomers13.

Secreted CLCA1 activates calcium-activated chloride channels such as transmembrane protein 16 A (TMEM16A), also known as anoctamin 1 (ANO1)40, and the subsequent outflow of chloride ions is enhanced by the VWA domain in the amino terminus41. Intracellular increases in Ca2+ levels significantly enhance the transport of HCO3- by TMEM16A/ANO1, resulting in a large amount of HCO3- being secreted into the intestinal lumen42. The alkaline environment created by HCO3- results in loosening of the mucus structure and its unfolding into a reticular structure, forming a gradient mucus layer with decreasing density and increasing permeability from the inner to the outer layer. Thus, newly secreted mucus constantly pushes the previously generated inner layer of mucus toward the lumen, renewing the mucus layer.

Cystic fibrosis transmembrane conductance regulator (CFTR) is involved in the modulation of HCO3- secretion. A decreased intracellular Cl- concentration can result in activation of with-no-Lysine kinase (WNK) and phosphorylation of sterile downstream 20/SPS1-related proline/alanine-rich kinase (SPAK) and oxidative stress responsive kinase 1 (OSR1), transforming the CFTR channel function from that of a main Cl- channel to a HCO3- channel, which mediates the outflow of HCO3- and maintains a suitable alkaline environment43. In addition, CLCA1 is a metalloprotease, and its amino terminal CAT, Cys, and VWA domains can cleave the amino terminus of MUC2 into four segments: D’-D3-CysD (125 kDa), GFP (32 kDa), D1-D2 (88 kDa), and D3-CYSD (88 kDa), thereby inducing the structural rearrangement of MUC2 molecules13.

CLCA1 is closely associated with mucus-related diseases. CLCA1 can bind to receptors on the surface of respiratory epithelial cells, thereby activating the MAPK13 signaling pathway and inducing mucin gene expression‎44. In addition, IL-13 activates its receptor and downstream signal transducer and activator of transcription 6 (STAT6), which in turn, enhances downstream CLCA1 expression‎ and MUC5AC secretion by airway epithelial cells. Specific knockout of SAM-pointing domain-containing ETS transcription factor (SPDEF) can inhibit the production of MUC5AC in human respiratory epithelial cells, which is induced by IL-13, while suppression of STAT6 blocks IL-13-induced production of SPDEF and MUC5AC45. Moreover, when IL-13 is upregulated, SPDEF decreases CLCA1 expression‎46. It is suggested that IL-13 can induce MUC5AC production in human airway epithelial cells through the STAT6/SPDEF signal transduction pathway. Targeted inhibition of the STAT6/SPDEF signaling pathway controls mucus overproduction, which slows the progression of chronic airway inflammatory diseases. Cystic fibrosis (CF) is an autosomal recessive genetic disease that affects multiple organs, such as the respiratory tract, pancreas, intestine, and liver, and is characterized mainly by mucinous gland hyperplasia and viscous secretions. The disorder is associated with reduced channel activity caused by mutations in the CFTR gene and impaired transport of Cl- and HCO3- ions, resulting in obstruction by thickened secretions and organ dysfunction35. Mucin secretion in the CF colon is dependent on CFTR expression‎ and its associated CLCA1 production47. Data obtained from animal experiments revealed that the expression‎ of CLCA1 was decreased in CF mice. Increasing the expression‎ of CLCA1 significantly improved mucus-related symptoms48. These results suggest that CLCA1 can ameliorate intestinal mucus thickening and obstruction in cystic fibrosis patients.

Ulcerative colitis (UC) is an inflammatory disease of the colonic mucosa characterized by persistent dysregulation of intestinal epithelial barrier function. Spontaneous loss of CLCA1 protein expression‎ has been found in the colonic epithelium of patients with ulcerative colitis49. A genome-wide expression‎ analysis revealed that CLCA1 expression‎ was significantly downregulated in patients with UC3. Some researchers have observed DSS-induced colitis in CLCA1-/- mice. The results showed that the CLCA1-/- mice did not show increased colonic inflammatory symptoms or histopathological abnormalities compared with wild-type mice, but the chemokine (C-X-C motif) ligand-1 (CXCL-1) and IL-17 mRNA levels in neutrophils were increased in CLCA1-/- mice50. Therefore, the role of CLCA1 in UC remains unclear.

점액 '형성자': CLCA1

칼슘 활성화 염화물 채널 조절자(CLCA) 가족의 아연 의존성 금속 단백질 분해효소는 분비 및 자기 분해 활성화 특성을 나타냅니다35,36. 현재까지 인간에서 4개의 CLCAs(hCLCA1–hCLCA4)가 식별되었으며, 쥐에서 8개의 CLCAs(mClCA1, CLCA3a1, CLCA3a2, CLCA3b, 3C, 4A, 4B, 및 4C)가 식별되었습니다. 소에서는 4개의 CLCAs(bCLCA1, bCLCA2(LU-ECAM-1), bCLCA3, bCLCA4)가 식별되었으며, 돼지와 말에서도 CLCA가 식별되었습니다37. hCLCA1은 CLCA 가족의 첫 번째로 식별된 구성원으로, 소장과 대장의 점막층을 포함한 장 상피에서 주로 발현됩니다. mCLCA3는 원래 goblet cell protein-5 (GOB-5)로 알려져 있었으나, hCLCA1과의 높은 동질성으로 인해 mCLCA1로 재명명되었습니다. mCLCA1, mCLCA2, 및 mCLCA4는 인간 CLCA3와의 높은 동질성으로 인해 각각 CLCA3a1, CLCA3a2, 및 CLCA3b로 재명명되었습니다38,39. 특히 110 kDa의 CLCA1 단백질의 35 kDa 카르복시말단 부위는 내소체에서 자체 분해되며, 이후 당화되어 성숙한 CLCA1을 형성합니다. 이 성숙한 CLCA1은 원본 아미노말단의 대부분과 카르복시말단의 작은 부분을 포함하며 분자량은 75–90 kDa입니다. 성숙한 CLCA1에는 다섯 개의 고도로 보존된 도메인이 있습니다: (1) 분비 경로로 안내하는 아미노 말단 신호 서열; (2) 보존된 HExxE 모티프와 시스테인 풍부(Cys) 지역을 가진 아연 의존성 금속 단백질 분해 효소 촉매 도메인(CAT); (3) 금속 이온 의존성 접착 부위(MIDAS)를 가진 VWA 도메인; (4) 미분류된 β-시트 풍부 영역(BSR); 및 (5) C-말단에 위치한 피브로네ктиן 유형 III(FnIII) 도메인13,35,36. 성숙한 CLCA1 단량체는 이황화 결합으로 연결되어 이량체를 형성하며, 아미노 말단에서 비공액적 방식으로 연결됩니다. 여러 CLCA1 분자는 비공액적 올리고머로 중합되어 구형 올리고머로 저장 및 분비됩니다13.

분비된 CLCA1은 칼슘 활성화 염소 이온 채널인 transmembrane protein 16A (TMEM16A), 즉 anoctamin 1 (ANO1)40을 활성화하며, 아미노 말단의 VWA 도메인에 의해 염소 이온의 유출이 강화됩니다41. 세포 내 칼슘 이온 농도의 증가가 TMEM16A/ANO1을 통해 HCO3-의 수송을 크게 촉진하여 장 내강으로 대량의 HCO3-가 분비됩니다42. HCO3-에 의해 생성된 알칼리성 환경은 점액 구조를 느슨하게 만들고 그 구조가 망상 구조로 펼쳐져 내층에서 외층으로 밀도가 감소하고 투과성이 증가하는 농도 차이를 가진 점액 층을 형성합니다. 이로써 새롭게 분비된 점액은 이전에 생성된 내층 점액을 루멘 방향으로 지속적으로 밀어내며 점액층을 재생합니다.

낭포성 섬유증 막 전도 조절자(CFTR)는 HCO3- 분비 조절에 관여합니다. 세포 내 Cl- 농도 감소는 with-no-Lysine kinase (WNK) 활성화와 sterile downstream 20/SPS1-related proline/alanine-rich kinase (SPAK) 및 oxidative stress responsive kinase 1 (OSR1)의 인산화를 유발합니다. 이로써 CFTR 채널 기능이 주요 Cl- 채널에서 HCO3- 채널로 전환되어 HCO3-의 배출을 매개하고 적절한 알칼리성 환경을 유지합니다43. 또한 CLCA1은 금속 프로테아제로, 그 아미노 말단 CAT, Cys, 및 VWA 도메인은 MUC2의 아미노 말단을 네 개의 세그먼트로 분해합니다: D'-D3-CysD (125 kDa), GFP (32 kDa), D1-D2 (88 kDa), 및 D3-CYSD (88 kDa)로 분해하여 MUC2 분자의 구조 재편성을 유도합니다13.

CLCA1은 점액 관련 질환과 밀접하게 연관되어 있습니다. CLCA1은 호흡기 상피 세포 표면의 수용체에 결합하여 MAPK13 신호전달 경로를 활성화하고 뮤신 유전자 발현을 유도합니다44. 또한 IL-13은 그 수용체를 활성화하고 하류 신호 전달 및 전사 활성화 인자 6(STAT6)을 활성화하며, 이는 다시 호흡기 상피 세포에서 CLCA1 발현과 MUC5AC 분비를 증가시킵니다. SAM-pointing 도메인을 포함하는 ETS 전사 인자(SPDEF)의 특정 유전자 결손은 IL-13에 의해 유도된 인간 호흡기 상피 세포에서의 MUC5AC 생산을 억제하며, STAT6 억제는 IL-13에 의한 SPDEF 및 MUC5AC 생산을 차단합니다45. 또한 IL-13이 증가하면 SPDEF는 CLCA1 발현을 감소시킵니다46. IL-13이 STAT6/SPDEF 신호전달 경로를 통해 인간 기도 상피 세포에서 MUC5AC 생성을 유도한다는 것이 제안되었습니다. STAT6/SPDEF 신호전달 경로의 표적 억제는 점액 과다 생성을 조절하여 만성 기도 염증 질환의 진행을 늦춥니다. 낭포성 섬유증(CF)은 호흡기, 췌장, 장, 간 등 다중 장기를 침범하는 상염색체 열성 유전 질환으로, 점액선 과증식과 점성 분비물이 주요 특징입니다. 이 질환은 CFTR 유전자 변이로 인한 채널 활성 감소와 Cl- 및 HCO3- 이온의 운반 장애로 인해 두꺼워진 분비물로 인한 폐쇄와 장기 기능 장애를 유발합니다35. CF 환자의 대장에서의 점액 분비는 CFTR 발현과 관련된 CLCA1 생산에 의존합니다47. 동물 실험에서 얻은 데이터는 CF 마우스에서 CLCA1 발현이 감소했음을 보여주었습니다. CLCA1 발현을 증가시키면 점액 관련 증상이 유의미하게 개선되었습니다48. 이 결과는 CLCA1이 낭성 섬유증 환자의 장 점액 두꺼워짐과 폐쇄를 완화할 수 있음을 시사합니다.

궤양성 대장염(UC)은 장 상피 장벽 기능의 지속적인 장애를 특징으로 하는 대장 점막의 염증성 질환입니다. 궤양성 대장염 환자의 대장 상피에서 CLCA1 단백질 발현의 자연적 상실이 발견되었습니다49. 전장 유전자 발현 분석 결과, UC 환자의 CLCA1 발현이 유의미하게 감소했습니다3. 일부 연구자들은 CLCA1-/- 마우스에서 DSS 유발성 대장염을 관찰했습니다. 결과는 CLCA1-/- 마우스가 야생형 마우스와 비교해 대장 염증 증상이나 조직 병리학적 이상이 증가하지 않았지만, CLCA1-/- 마우스의 중성구에서 화학유인물질(C-X-C 모티프) 리간드-1(CXCL-1) 및 IL-17 mRNA 수준이 증가했음을 보여주었습니다50. 따라서 CLCA1의 UC에서의 역할은 여전히 명확하지 않습니다.

The sentinel of the mucus layer: ZG16

Zymogen granule protein 16 (ZG16), a 16 kDa soluble protein without a transmembrane domain, was discovered in rat pancreatic zymogen granules51. During the formation of rat zymogen granules, ZG16 concentrates secretases into a dense inner core, which is attached to the granular membrane via the interaction of ZG16 with sulfated glycosaminoglycans in the inner granular membrane52.

ZG16 is a lectin with a core lectin domain, and the amino acid sequence of its glycosyl recognition region shares 52% homology with the sequence of durian lectin (Jacalin); therefore, ZG16 belongs to the Jacalin lectin family. ZG16 comprises three β-hairpin structures (I: β1, β2, β11, and β12; II: β3, β4, and β6; III: β7-β10), which form the core β-prism fold, and an α-helix structure between the β2 and β3 chains. The glycosyl group-binding site in ZG16 consists of three rings, a GC ring between β1 and β2, a recognition ring between β7 and β8, and a linker ring between β11 and β12. ZG16 can bind to mannose through a sugar-binding site, form a positively charged channel on the surface of the protein molecule through lysine residues at positions 102, 106, and 122, and bind to negatively charged glycosaminoglycans53. ZG16 is a protein secreted by serous parotid gland acinar cells, pancreas acinar cells, and colon goblet cells, and is characterized by sulfated glycosaminoglycans combined with mannan53,54.

The key feature of exogenous pathogenic microorganisms such as pathogenic Candida and Malassezia is a mannan-covered cell wall, which is also seen in the cell wall of nonpathogenic Saccharomyces cerevisiae. ZG16 can specifically bind to mannan on the cell wall surface of these fungi. This binding does not affect the growth of the fungi and their adhesion to intestinal epithelial cells but may send a message to the host immune system and drive an immune response with signaling to resist invading pathogenic microorganisms. The intestinal symbiote Candida albicans is also enriched with mannan, to which ZG16 can bind, thereby preventing this fungus from entering the blood through the intestinal wall and causing entheogenic infection54. ZG16 does not kill bacteria directly, but combines with peptidoglycans in the cell wall of gram-positive bacteria, forming bacterial aggregates that cannot readily cross the mucus layer or be eliminated from the intestinal epithelium. Since most beneficial intestinal bacteria carry a low peptidoglycan content in their cell wall, ZG16 exerts little effect on beneficial bacteria. Moreover, ZG16-/- mice presented with a distal colon mucus layer of normal thickness, but their mucus layer showed increased permeability, and intestinal bacteria were thus more likely to penetrate the mucus layer and invade the intestinal epithelium compared to their permeability in ZG16+/+ mice. In addition, gram-positive bacteria (mainly Firmicutes) have been detected in tail lymph node and spleen tissues of ZG16-/- mice55.

The ZG16 protein is absent in colorectal cancer tissues and found at reduced levels in precancerous adenomatous polyps (adenomas) and tissues of chronic ulcerative colitis. Thus, ZG16 expression‎ is successively decreased in normal tissues, adenomas and cancers56. Recently, ZG16 expression‎ in colorectal cancer tissues was negatively correlated with the level of programmed death-1 ligand (PD-L1), the degree of distant metastasis, and lymphatic invasion of colorectal cancer. ZG16 overexpression‎ in colon cancer cell lines (the SW480 and HCT116 cell lines) resulted in significantly inhibited proliferation of colon cancer cells due to decreased PD-L1 expression‎ and the enhanced killing effect of NK cells on tumor cells57. These results suggest that ZG16 may inhibit tumor cell immune escape and may be a potential target for tumor immunotherapy.

점막층의 감시자: ZG16

16 kDa의 용해성 단백질로 막을 통과하는 도메인이 없는 Zymogen granule protein 16 (ZG16)은 쥐의 췌장 zymogen granules에서 발견되었습니다51. 쥐의 지모겐 그레인 형성 과정에서 ZG16은 분비효소를 밀집된 내핵으로 집중시키며, 이는 내핵 막에 존재하는 황산화 글리코사미노글리칸과의 상호작용을 통해 그레인 막에 결합됩니다52.

ZG16은 코어 레クチ닌 도메인을 가진 레クチ닌으로, 그 당 인식 부위의 아미노산 서열은 두리안 레クチ닌(Jacalin)의 서열과 52%의 동질성을 공유하므로, ZG16은 Jacalin 레クチ닌 가족에 속합니다. ZG16은 세 개의 β-헤어핀 구조(I: β1, β2, β11, 및 β12; II: β3, β4, 및 β6; III: β7-β10)로 구성되어 핵심 β-프리즘 접힘을 형성하며, β2와 β3 사슬 사이에 α-헬릭스 구조가 존재합니다. ZG16의 글리코실 그룹 결합 부위는 세 개의 고리로 구성되어 있으며, β1과 β2 사이의 GC 고리, β7과 β8 사이의 인식 고리, 그리고 β11과 β12 사이의 링크 고리로 이루어져 있습니다. ZG16은 당 결합 부위를 통해 만노스와 결합하며, 102, 106, 122 위치의 라이신 잔기 통해 단백질 분자 표면에 양전하 채널을 형성하고, 음전하를 띤 글리코사미노글리칸과 결합합니다53. ZG16은 세로우스 침샘 아세리나 세포, 췌장 아세리나 세포, 대장 고블릿 세포에서 분비되는 단백질로, 만난과 결합된 황산화 글리코사미노글리칸으로 특징지어집니다53,54.

병원성 칸디다와 말라세지아와 같은 외인성 병원성 미생물의 주요 특징은 세포벽에 만난이 덮여 있는 것으로, 비병원성 Saccharomyces cerevisiae의 세포벽에서도 관찰됩니다. ZG16은 이러한 곰팡이의 세포벽 표면에 있는 만난에 특이적으로 결합합니다. 이 결합은 곰팡이의 성장이나 장 상피 세포에 대한 부착에 영향을 미치지 않지만, 호스트 면역 체계에 신호를 전달하여 침입하는 병원성 미생물에 대항하는 면역 반응을 유발할 수 있습니다. 장 내 공생균인 Candida albicans도 만난이 풍부하며, ZG16은 이에 결합하여 이 곰팡이가 장 벽을 통해 혈류로 침입하여 엔테오제닉 감염을 일으키는 것을 방지합니다54. ZG16은 세균을 직접 죽이지 않지만, 그람양성 세균의 세포벽에 있는 펩티도글리칸과 결합하여 점막층을 쉽게 통과하거나 장 상피에서 제거되지 않는 세균 집합체를 형성합니다. 대부분의 유익한 장 세균은 세포벽에 펩티도글리칸 함량이 낮기 때문에 ZG16은 유익한 세균에 거의 영향을 미치지 않습니다. 또한 ZG16-/- 마우스의 원위 결장 점막층 두께는 정상적이었지만, 점막층의 투과성이 증가했으며, 이로 인해 장 세균이 점막층을 관통하고 장 상피를 침범할 가능성이 ZG16+/+ 마우스보다 높았습니다. 또한, 그람양성 세균(주로 Firmicutes)이 ZG16-/- 마우스의 꼬리 림프절 및 비장 조직에서 검출되었습니다.

ZG16 단백질은 대장암 조직에서 결여되어 있으며, 전암성 선종성 폴립(선종) 및 만성 궤양성 대장염 조직에서 감소된 수준으로 발견됩니다. 따라서 ZG16 발현은 정상 조직, 선종, 암 조직에서 순차적으로 감소합니다56. 최근 대장암 조직에서의 ZG16 발현은 프로그램된 세포사멸-1 리간드(PD-L1) 수준, 원격 전이 정도, 대장암의 림프관 침윤과 음의 상관관계를 보였습니다. 대장암 세포주(SW480 및 HCT116 세포주)에서 ZG16 과발현은 PD-L1 발현 감소와 NK 세포의 종양 세포에 대한 살상 효과 증강으로 인해 대장암 세포의 증식이 유의미하게 억제되었습니다57. 이 결과는 ZG16이 종양 세포의 면역 회피를 억제할 수 있으며 종양 면역 치료의 잠재적 표적이 될 수 있음을 시사합니다.

Dual roles of RELMβ

The resistin-like molecule (RELMS) family was discovered in a mouse model of asthma58. To date, four members of the family, RELMα, RELMβ, Resistin, and RELMγ, have been identified. RELM family members carry three domains: an amino-terminal signal peptide, an intermediate variable region of 28~44 amino acid residues, and a relatively conserved carboxyl terminus enriched with 11 cysteine residues; family members share 105~114 amino acid residues and Cys enrichment59. RELMβ was first identified in the colonic epithelial cells of mice, and its content was found to be most abundant in the distal colon, followed by the cecum and with a small amount in the ileum59. RELMβ is synthesized in goblet cells and secreted into mucus in the form of homodimers. Intestinal RELMβ expression‎ was significantly reduced in mice reared in a sterile environment. However, when germ-free mice were placed in a conventional environment, a large amount of RELMβ protein was synthesized and secreted by goblet cells within 48 h, suggesting that intestinal flora regulated the expression‎ of RELMβ60.

RELMβ expression‎ is significantly upregulated during colon inflammation61. RELMβ was not involved in the occurrence of enteritis in mice induced by high-dose flagellate infection. Low-dose flagellate infection-induced chronic enteritis in mice resulted in significantly increased interferon-γ (IFN-γ) levels in the intestinal epithelium of wild-type mice, resulting in persistent infection, while the expression‎ of IFN-γ in the intestinal epithelium of RELMβ-/- mice was decreased, and flagellate infection was significantly attenuated62. Recombinant RELMβ stimulated the release of the inflammatory factor TNF-α from isolated peripheral blood macrophages. The symptoms of DSS-induced enteritis in RELMβ-/- rats were delayed, and the severity was reduced compared with that in wild-type rats63. The antimicrobial activity of RELMβ was eval‎uated in bacteria grown to the log phase and treated with purified RELMβ, and the results revealed that fewer gram-negative bacteria (Pseudomonas aeruginosa and Citrobacter murine) survived, while the survival of gram-positive bacteria (Listeria and Enterococcus faecalis) was unchanged. These outcomes were explained by the formation of RELMβ channels that penetrate the bacterial cell wall and subsequently kill bacteria64. It has recently been shown that RELMβ exhibits antibacterial activity against gram-positive bacteria such as Staphylococcus aureus65. These studies suggest that RELMβ has bactericidal activity against specific types of bacteria.

Infection of the intestinal tract with Citrobacter resulted in a decreased number of CD4+ T cells and reduced IL-22 levels in RELMβ-/- mice, causing impaired epithelial cell proliferation and significantly damaged intestinal mucosa. Treatment of RELMβ-/- mice with recombinant RELMβ protein administered via enema restored the tropism of CD4+ T cells to inflammation sites, IL-22 levels in the intestinal epithelium and epithelial cell proliferation, indicating that RELMβ recruited CD4+ T lymphocytes and repaired mucosal injury66. Thus, RELMβ may play different roles in enteritis with different etiologies.

RELMβ의 이중 역할

아스마 마우스 모델에서 발견된 레지스틴 유사 분자(RELMS) 가족은58. 현재까지 이 가족의 네 가지 구성원인 RELMα, RELMβ, 레지스틴, 및 RELMγ가 식별되었습니다. RELM 가족 구성원은 세 가지 도메인을 가지고 있습니다: 아미노 말단 신호 펩티드, 28~44 아미노산 잔기로 구성된 중간 가변 영역, 그리고 11개의 시스테인 잔기로 풍부하게 구성된 상대적으로 보존된 카르복시 말단; 가족 구성원은 105~114 아미노산 잔기와 시스테인 풍부함을 공유합니다59. RELMβ는 쥐의 대장 상피 세포에서 처음 발견되었으며, 그 함량은 원위 대장에서 가장 풍부하게 발견되었으며, 그 다음으로 맹장, 소장에서는 소량으로 발견되었습니다59. RELMβ는 점액 세포에서 합성되어 점액 형태의 동형 이량체로 분비됩니다. 무균 환경에서 사육된 쥐의 장 RELMβ 발현은 유의미하게 감소했습니다. 그러나 무균 쥐를 일반 환경에 노출시키자 점액 세포에서 48시간 이내에 대량의 RELMβ 단백질이 합성 및 분비되었으며, 이는 장 내 미생물이 RELMβ 발현을 조절함을 시사합니다60.

RELMβ 발현은 대장 염증 시 유의미하게 증가합니다61. RELMβ는 고용량 편모균 감염으로 유발된 쥐의 장염 발생에 관여하지 않았습니다. 저용량 플래게이트 감염으로 유발된 만성 장염에서 야생형 쥐의 장 상피에서 인터페론-γ (IFN-γ) 수준이 유의미하게 증가하여 지속적 감염이 발생했지만, RELMβ-/- 쥐의 장 상피에서 IFN-γ 발현이 감소했으며 플래게이트 감염이 유의미하게 완화되었습니다62. 재조합 RELMβ는 분리된 말초 혈액 대식세포에서 염증 인자 TNF-α의 분비를 자극했습니다. DSS로 유발된 장염의 증상은 RELMβ-/- 쥐에서 지연되었으며, 야생형 쥐에 비해 심각도가 감소했습니다63. RELMβ의 항균 활성은 로그 성장 단계에 도달한 세균에 정제된 RELMβ를 처리하여 평가되었으며, 그 결과 그람 음성 세균(Pseudomonas aeruginosa 및 Citrobacter murine)의 생존율이 감소한 반면, 그람 양성 세균(Listeria 및 Enterococcus faecalis)의 생존율은 변화가 없었습니다. 이러한 결과는 RELMβ 채널이 세균 세포벽을 관통하여 세균을 사멸시키는 것으로 설명되었습니다64. 최근 연구에서 RELMβ는 Staphylococcus aureus와 같은 그람양성 세균에 대한 항균 활성을 나타내는 것으로 밝혀졌습니다65. 이러한 연구 결과는 RELMβ가 특정 유형의 세균에 대한 살균 활성을 갖는다는 것을 시사합니다.

Citrobacter로 장관이 감염된 RELMβ-/- 마우스에서는 CD4+ T 세포 수와 IL-22 수준이 감소했으며, 이는 상피 세포 증식 장애와 장 점막의 심각한 손상을 초래했습니다. RELMβ-/- 마우스에 직장 주사로 재조합 RELMβ 단백질을 투여하자 CD4+ T 세포의 염증 부위로의 이동성, 장 상피에서의 IL-22 수준 및 상피 세포 증식이 회복되었습니다. 이는 RELMβ가 CD4+ T 림프구를 모집하고 점막 손상을 복구한다는 것을 나타냅니다66. 따라서 RELMβ는 다양한 원인에 의한 장염에서 서로 다른 역할을 할 수 있습니다.

The flagellum “holder”: Lypd8

Ly6/plaur domain-containing protein 8 (Lypd8), a member of the Ly6/Plaur family, is a recently discovered antibacterial molecule that was first identified in mouse intestinal epithelial cells. Lypd8 is a highly N-glycosylated phosphatidylinositol (GPI)-anchored protein with 13 asparagine (Asn) residues (at n-glycosylation sites)67. Mouse Lypd8 can bind to flagellated bacteria, such as Proteus mirabilis (P. mirabilis), Helicobacter mirabilis (H. mirabilis) and Escherichia coli (E.Coli), and inhibit the movement of flagella, separating bacteria from intestinal epithelial cells and thus preventing bacteria from invading the intestinal epithelium of mice. Compared with that in wild-type mice, DSS-induced colonic inflammation was more severe in Lypd8-/-mice, and bacterial aggregation was observed in the inner layer of mucus67. Colonization of the colon by a large number of Clostridium was observed in Lypd8-/- mice, which resulted in profound proliferation of Th17 cells and neutrophils in the lamina propria and severe colitis68. Similar to murine Lypd8, human Lypd8 can bind to flagellated bacteria, such as P. mirabilis and E. coli, and inhibit their penetration into the mucus layer69. Recently, it was confirmed that Lypd8 can directly block the adhesion of Citrobacter to intestinal epithelial cells by binding to tight adhesions on the surface of bacteria and inhibiting the adhesion of bacteria to intestinal epithelial cells70. Moreover, Lypd8 expression‎ has been found to be significantly decreased in colon cancer tissues, and the activities of the IL-6/signal transducer and activator of transcription 3 (STAT3) and TNF-α/NF-κB inflammatory signaling pathways were increased compared to those in precancerous and normal tissues70. Similarly, the levels of the inflammatory cytokines TNF-α and IL-6 and the phosphorylation of the downstream target proteins NF-κB and STAT3 in human colon cancer cells with Lypd8 overexpressed were significantly decreased, and the proliferation and migration of the cancer cells were inhibited70. These results suggest that Lypd8 can be used as a marker and therapeutic target of enteritis and colon cancer.

편모 “고정체”: Lypd8

Ly6/plaur 도메인 함유 단백질 8(Lypd8)은 Ly6/Plaur 가족의 일원으로, 마우스 장 상피 세포에서 처음 발견된 항균 분자입니다. Lypd8은 13개의 아스파라긴(Asn) 잔기(n-글리코실화 부위)를 가진 고도로 N-글리코실화된 포스파티딜인오실(GPI) 결합 단백질입니다. 마우스 Lypd8은 Proteus mirabilis (P. mirabilis), Helicobacter mirabilis (H. mirabilis) 및 Escherichia coli (E.Coli)와 같은 편모를 가진 세균에 결합하여 편모의 운동을 억제하며, 세균을 장 상피 세포로부터 분리시켜 마우스의 장 상피 침입을 방지합니다. 야생형 쥐와 비교했을 때, DSS로 유발된 대장 염증은 Lypd8-/- 쥐에서 더 심했으며, 점막 내층에서 세균 집합이 관찰되었습니다67. Lypd8-/- 쥐에서는 대장에 많은 양의 Clostridium이 정착했으며, 이는 점막 하층에서 Th17 세포와 중성구의 심한 증식과 심각한 대장염을 유발했습니다68. 쥐의 Lypd8과 유사하게, 인간 Lypd8은 P. mirabilis 및 E. coli와 같은 편모를 가진 세균에 결합하여 점막층으로의 침투를 억제합니다69. 최근 연구에서 Lypd8이 세균 표면의 밀착 결합 부위에 결합하여 세균의 장 상피 세포에 대한 부착을 직접 차단함으로써 Citrobacter의 장 상피 세포 부착을 억제한다는 것이 확인되었습니다70. 또한, 대장암 조직에서 Lypd8 발현이 유의미하게 감소했으며, IL-6/신호 전달 및 전사 활성화 인자 3 (STAT3) 및 TNF-α/NF-κB 염증 신호 전달 경로의 활성이 전암성 및 정상 조직에 비해 증가했습니다70. 同様に、Lypd8 과발현된 인간 대장암 세포에서 염증성 사이토킨 TNF-α 및 IL-6의 수준과 하류 표적 단백질 NF-κB 및 STAT3의 인산화 수준이 유의미하게 감소했으며, 암 세포의 증식 및 이동이 억제되었습니다70. 이러한 결과는 Lypd8이 장염 및 대장암의 표지자 및 치료 표적으로 활용될 수 있음을 시사합니다.

The bacteria hunter: sIgA

Secretory immunoglobulin A (sIgA) is an antibody discovered by Tomasi et al. 71 in the 1960s and is found in exocrine fluids, such as milk, gastrointestinal fluid, and respiratory tract secretions. sIgA content is highest on the intestinal mucosal surface and is not easily hydrolyzed by nonspecific proteases, making it the primary effector molecule in the mucosal immune system72. When antigens contact the intestinal mucosa, antigen recognition cells (M cells) on the mucosal surface recognize and transmit the antigen signal to antigen-presenting cells (such as macrophages, dendritic cells, and lymphocytes), which decompose the antigens into fragments, thereby activating B-lymphoid cells and converting them into IgA+ B lymphocytes with the capacity to secrete polymeric immunoglobulin A (pIgA). PIgA binds to the polymeric immunoglobulin receptor (pIgR) on the basal side of mucosal epithelial cells to form the pIgR/pIgA covalent complex, which is taken up by cells via endocytosis. Then, PIgR is cleaved by proteolytic enzymes, and the secretory fragments (SCs) are released and bind with pIgA to form sIgA, which is released into the colonic lumen via exocytosis, while the remaining part is cellularly recycled to generate new pIgR73. Moreover, it was found that SC can protect IgA from degradation by host and intestinal microbial proteases in the harsh intestinal environment and enhance adaptive immunity74.

When pathogenic microorganisms threaten the intestinal mucosal barrier, sIgA, as the first line of defense in gut-specific immunity, inhibits bacterial movement and adhesion to the intestinal mucosa by capturing the bacteria on the mucosal surface and directly binding to specific sites, thereby preventing bacterial invasion75. pIgA can also bind bacteria that have invaded the intestinal epithelium to form immune complexes, some of which is phagocytosed by mononuclear macrophages, with the remaining pIgA–antigen complexes bind pIgR, which is expelled by a pIgR-mediated transmembrane transport mechanism, followed by intestinal peristalsis75. Recent studies have shown that the microbiota can regulate the production of sIgA and pIgR in turn, with secretions from the symbiotic microbiota sending signals to intestinal epithelial cells and immune cells, inducing the activation of pattern recognition receptors to promote the production of sIgA76,77.

The bactericidal experts: AMPs

Antimicrobial peptides (AMPs), discovered in 198078, are composed of 20–50 amino acid residues and are enriched with arginine and lysine residues. AMPs constitute a large family with several members that have been identified through a variety of classification methods. Typically, AMPs are classified into four types according to their origin: insect, plant, microbial, and animal. Animal antibacterial peptides include defensins, C-lectins, and cathelicidin family members, and they are derived mainly from neutrophils, epithelial cells, skin secretions, and protein degradation products. The defensin family, which is related to host innate immunity, mounts an important defense against pathogenic bacteria79. Defensins can be classified into α-Defensins, β-Defensins, and Ɵ-Defensins according to the position of the cysteine residues and disulfide bonds. C-lectins are mainly regenerative islet-derived proteins (Reg), including Reg1, Reg2, Reg3a, Reg3b, Reg3g, Reg3d, and Reg4. The Cathelicidin family is named for the highly conserved Cathelin peptide80. Notably, compared with those in other species, there are few members of the human AMP family, which is composed mainly of defensins, Cathelicidins, Histatins and so on81. Human α-defensins can be classified into neutrophil defensins (HNP-1, HNP-3, and HNP-5) and intestinal defensins (HD-5 and HD-6)82.

AMPs secreted by Paneth cells, which are located at the base of intestinal crypts, exhibit antiviral and antibacterial properties. AMPs are important components of intestinal innate immunity because of their rapid killing and effective inactivation of pathogenic bacteria. Paneth cells were previously thought to reside only in the small intestine, while the healthy colon was thought to carry neither Paneth cells nor AMPs. Later, it was reported that both metaplastic Paneth cells and AMPs were detected in inflammatory bowel disease (IBD)83. AMPs can effectively kill bacteria by binding to negatively charged bacterial membrane lipids through electrostatic attraction mediated by their positively charged surface amino acid residues, thereby forming multiple stable transmural channels and transmembrane ion channels, resulting in exocytosis from bacterial cell bodies and bacterial death due to irreversible damage84. AMPs also act as immune regulators by presenting signals to dendritic cells and T cells that activate the immune response85. For example, β-defensin by itself can act as a chemokine to drive leukocytes to a site of infection and thus suppress the progression of inflammation and promote mucosal repair86. It also attracts immature dendritic cells and CD4 + T cells through the chemokine receptor CCR6, facilitating the maturation and activation of T cells87. In addition, Reg3 selectively binds peptidoglycans on the surface of bacteria88, including gram-positive bacteria and some gram-negative bacteria, such as Salmonella typhimurium and Pseudomonas aeruginosa, thereby causing destroying the bacterial cytoderm, causing cytoplasm leakage, and resulting in bacteria death89.

Regulatory mechanisms of mucus secretion

Continual mucus secretion is a key factor in determining the structure and function of the mucus barrier. The mucus barrier is not a static physical barrier, with the inner mucus layer in murine distal colonic tissue found to be renewed every 1–2 hours4. It has been estimated that mucus grows spontaneously at a rate of approximately 240 μm/h in humans and 100 μm/h in mice90,91. Notably, mucin granule exocytosis is a Ca2+-regulated process; therefore, mucus secretion can be classified into two modes (baseline secretion and stimulated secretion) according to the involvement of calcium influx. Baseline secretion is spontaneous Ca2+ oscillation-mediated slow and continuous secretion, while stimulated secretion is mediated mainly by substantive Ca2+ entry induced by exogenous stimuli. In the colon, surface GCs are critical for baseline secretion, while GCs in the upper part of colonic crypts are critical to mucus secretion in response to stress stimuli. In our opinion, stimulated mucus secretion can be further classified into constitutive secretion mediated by physiological ATP levels and mechanical stimuli and impulsive mucus secretion mediated by strong external inflammatory stimuli. Recently, mucus was shown to be released by baseline secretion in quantities that were several fold greater higher than that released via the stimulated secretion process, although baseline mucus secretion involves the continuous release of mucins at a low rate92.

Spontaneous Ca2+ oscillation-mediated baseline mucus secretion

Under physiological conditions, colonic GCs continuously synthesize and release mucin to renew the mucus layer and maintain its thickness and physicochemical properties. During the migration of GCs from the bottom of the colonic crypt to the crypt luminal opening, highly ordered vertical microtubules and microfilaments are formed in the cells, and secretory granules are transferred to the top of goblet cells in an orderly manner through the interaction of microtubules, microfilaments, and actin. This group of microtubules and microfilaments separates the secretory granules from other cytoplasmic cellular components and gives the goblet cell its distinctive “goblet” shape. Mucins in GCs are packaged to form secretory granules after polymerization in the endoplasmic reticulum (ER) and glycosylation modification in the Golgi apparatus. In summary, the process of mucus secretion involves the migration of mucus vesicles along the cytoskeleton to the apical side of a cell, the fusion of a vesicle membrane with the cell membrane, and exocytosis (Fig. 2a).

Fig. 2: Baseline mucus secretion and SNARE assembly.

Baseline mucus secretion is the continuous release of mucins at a low rate. a The Golgi apparatus releases mature, primed mucin secretory vesicles filled with MUC2. The high-affinity Ca2+ sensor KChIP3, which senses Ca2+ concentrations <1 μM, binds to secretory vesicles and prevents them from fusing with the cell membrane in the absence of intracellular Ca2+ oscillations as a tonic brake. b Intracellular Ca2+ oscillation plays an important role in baseline mucin secretion. Spontaneous oscillations in Ca2+ from internal stores (mainly in the ER) initiate steady, moderated mucus release in a ryanodine receptor 2 (RYR2)-dependent manner.

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Calcium ions are among of the most abundant cations in the body and are important second messengers in cell signal transduction. Mucus secretion is a calcium-dependent biological process. Intracellular Ca2+ stores (principally in the ER) are the sources of Ca2+ oscillations in goblet cells. Recently, intracellular spontaneous Ca2+ oscillations was confirmed to be key factors regulating mucus secretion under baseline conditions. The ryanodine receptor (RYR) is the largest known high-throughput calcium channel protein and is involved in the generation and maintenance of these spontaneous oscillations. RYR2 mediates the release of Ca2+ from the ER, resulting in an increase in the Ca2+ concentration in the regions adjacent to the ER. Inositol The 1-,4-,5-triphosphate (IP3) receptor (IP3R) also mediates Ca2+ release from the ER. KChIP3 (potassium voltage-gated channel-interacting protein 3), also named DREAM and Calsenilin, is a member of the neuronal Ca2+ sensor protein (NCS) family and is a multifunctional Ca2+-binding protein with a molecular weight of 29 kDa. KChIP3 is a high-affinity calcium receptor that can sense intracellular Ca2+ levels at concentrations lower than 1 µM. KChIP3 has been shown to be localized in a pool of mucin secretory granules and act as a negative regulator of baseline mucin secretion by binding mucin granules and inhibiting mucus release. The binding of Ca2+ to KChIP3 followed by KChIP3 dissociation from mature secretory granules allows the fusion of mucin granules with the apical plasma membrane and the subsequent release of mucin into the intestinal lumen (Fig. 2b). Moreover, the KChIP3-related mucus secretion mechanism is not tissue-specific and is conserved in GCs secreting mucin93.

Mucus secretion requires large amounts of ATP, making it highly dependent on mitochondrial oxidative phosphorylation for sufficient energy94. Commensal bacteria in the intestine can ferment dietary fiber with glycosidase to release short-chain fatty acids. As the main energy substrate in epithelial cells, butyric acid is consumed by intestinal epithelial cells to generate ATP through β-oxidation and thus fuels mucus secretion95. In the absence of dietary fiber, glycosidase is leveraged by symbiotic bacteria to produce sugar groups in mucus. In the absence of dietary fiber, mucophilic bacteria degrade mucus, leading to reduced thickness of the colonic mucus layer and destruction of the intestinal barrier, increasing the risk of inflammatory bowel disease96. When the expression‎ level of Mir-124-3p in the colon of aged mice was significantly increased, the expression‎ of the O-glycosylation rate-limiting enzyme T-synthetase was reduced, which inhibited the O-glycosylation of mucin. As a result, the permeability of the mucus layer is increased, reducing the barrier to pathogens and bacteria penetration of the mucus layer and their infiltration into the intestinal epithelium, causing colitis97. Moreover, Mir-1-3p downregulated the expression‎ of T-synthetase and cooperated with Mir-124-3p to inhibit the effect of T-synthetase, destroying the colonic mucus barrier and increasing the severity of mouse colitis97. A recent study demonstrated that the expression‎ of mitochondrial oxidative phosphorylation complexes is decreased in colonic epithelial cells of elderly individuals, and the intestinal mucosa was thus vulnerable to damage and inflammatory bowel disease due to insufficient energy production and decreased mucus synthesis and secretion11.

Stimulated mucus secretion

Stimulated mucus secretion can be classified into constitutive and impulsive secretion depending on the property of the stimulus that induces the mucus secretion.

Constitutive mucus secretion

Under physiological conditions, the pulling effect of mechanical stimulation induced by intestinal peristalsis and the shear stimulation produced by fluid flow in the intestine are important factors in promoting mucus secretion98. The transmembrane cation-selective mechanosensitive ion channel protein Piezo is a mechanosensitive ion channel protein that induces the depolarization and activation of voltage-gated L-type calcium channels, resulting in a further increase in intracellular Ca2+, presumably leading to exocytosis99. It has been reported that Piezo 1 and Piezo 2 are expressed abundantly in the colon100. Moreover, Piezo1-triggered calcium influx was found to induce the activation of microtubules in cardiomyocytes under the action of Rac1 (a calcium-dependent small GTPase), prompting homeostatic ROS production and RYR2 activation by stimulating NADPH oxidase 2 (NOX2)101. RYR2 is necessary for Ca2+ release from the ER, and ROS-mediated posttranslational modifications increase the sensitivity of RYR2. Piezo-mediated exocytosis may be relevant to the intracellular calcium oscillation triggered by the influx of extracellular calcium and subsequent intracellular calcium release (Fig. 3). Notably, lipopolysaccharide (LPS) from the gram-negative bacterial cell wall increased mucin mRNA expression‎ and promoted mucin secretion in HT-29 MTX cells102, suggesting that LPS from commensal bacteria might be a regulatory factor in mucus secretion, especially in the constitutive secretion of mucus.

Fig. 3: The underlying mechanism for stimulated mucus secretion.

Several physiological or pathological stimuli result in a marked increase in intracellular Ca2+-triggered stimulated mucus secretion. Intracellular Ca2+ levels are increased through two mechanisms. After stimulation with an exogenous stimulus, TRPM4/5 are activated and cooperate with NCX2 to enhance Ca2+ influx. LPS and flagella in bacteria induce TLR-mediated ROS production and activate NLRP6, which in turn promotes Ca2+ release from the ER. Protein kinase C induces detachment from the membrane and subsequent recruitment of MARCKS onto mucus vesicles, and MARCKS-bound mucus vesicles then migrate to the apical side of cells under the action of Hsp70 and CSP. The low-affinity Ca2+ sensor Syt2, which can only sense calcium concentrations greater than 10 μM, senses elevated Ca2+ levels and drives the fusion of mucus vesicles with the cell membrane.

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When stimulated by intense extracellular stimulation, mainly paracrine release of ATP, G protein-coupled receptors (e.g., P2Y2) activate phospholipase C to produce diacylglycerol, which activates protein kinase C (PKC) and IP3. IP3 may induce a rapid burst of calcium release from the ER followed by the activation of calcium-activated monovalent cationic channels in cell membranes: transient receptor potential cationic channel subfamily M member 4 (TRPM4) and its homologous protein TRPM5. TRPM4 and TRPM5 mediate sodium influx and are inactive under physiological conditions. Upon activation by extracellular stimulation, TRPM4 and TRPM5 mediate sodium transport into the cytoplasm, increasing the sodium concentration in areas adjacent to Na + /Ca2+ exchanger 2 (NCX2), trigging NCX2 pumping of calcium into the cell when operating in reverse mode. The increase in local calcium concentration attracts a low-affinity calcium sensor, synaptotagmin 2 (Syt2), which only senses only Ca2+ concentrations higher than 10 μM and induces the fusion of mucus vesicles with the cytoplasmic membrane103,104. In addition, activated PKC phosphorylates myristoylated alanine-rich C kinase substrate (MARCKS) on the cytoplasmic side of the plasma membrane, allowing the separation of MARCKS from the plasma membrane and recruitment to mucin vesicles. MARCKS-bound mucus vesicles then migrate through actin-associated pathways and anchor to the apical side of the cell membrane under the mediation of heat shock protein 70 (Hsp70) and cysteine string protein (CSP) (Fig. 3).

Impulsive mucus secretion modulation

Recent studies have demonstrated an impulsive mucus secretion mode. In response to acute inflammatory stimuli, such as invasion of a large number of bacteria into the intestinal epithelium, GCs initiate a massive release response (also known as compound exocytosis), in which most of the mucus granules in cells are released after fusion, with mucus largely eliminated from cells105. Some GCs at the opening of colon crypts were observed and named “sentinel goblet cells” (senGCs) since they can express pattern recognition receptor toll-like receptors (TLRs) and recognize pathogen-associated molecular patterns (PAMPs), such as specific structures of bacteria, viruses, and fungi. For example, TLR2 and TLR4 recognize LPS, and TLR5 recognizes flagellin. The TLRs activated by microorganisms by after endocytosis activate their receptors, such as Toll receptor domain-containing adaptor-inducing IFN-β (TLR-TRIF) and myeloid differentiation factor 88 (MyD88)106, and subsequently activate downstream Nox/Duox. For instance, ROS synthesis was enhanced after TLR5-induced activation of Nox1 and Duox2, TLR2-mediated Nox1 activation, and TLR4-triggered activation of Nox4107. NOD-like receptor family pyrin domain-containing 6 protein (NLRP6) inflammasome activation occurs downstream of endocytosis-dependent ROS synthesis. NLRP6 is highly expressed in the intestinal epithelium and is specifically concentrated in the apical mucosal region, and its deficiency leads to defective mucus granule exocytosis108. Moreover, autophagy processes have been demonstrated to be required for proper secretion of mucus granules, as indicated by NLRP6-deficient epithelium lacking clear autophagosome formation108. Later, autophagy-induced regulation of goblet cell secretory functions was shown to involve downstream reactive oxygen species signaling. Autophagosomes with LC3 fuse to endosomes containing NADPH oxidases, thereby producing amphisomes, which are required for the maximal production of reactive oxygen species (ROS) derived from NADPH oxidases. Moreover, intracellular calcium mediates the effect of ROS on mucin granule release in colonic goblet cells109.

NLRP6 is suppressed under resting conditions and activated upon infection with virus or gram-positive bacteria and is subsequently recruited by apoptosis -associated speck-like protein containing caspase-recruitment domain (ASC) and caspase-1/caspase-11 precursors to be part of the NLRP6 inflammasome. The assembly of the NLRP6 inflammasome involves two polymerization steps. First, oligomeric NLRP6 provides a platform for ASC recruitment through its interaction with the pyrimidine domain (PYD) and induction of ASC polymerization. ASC then recruits caspase-1 via interactions with the caspase recruitment domain (CARD), leading to the activation of caspase-1110. In addition, NLRP6 recognizes LPS from gram-negative bacteria through leucine-rich repeats (LRRs) in its C-terminus and then undergoes a conformational change followed by the formation of a linear dimer. In the presence of ATP, NLRP6 homodimers self-assemble into larger oligomers, providing a platform for the recruitment and polymerization of ASC and Caspase-1 as well as the formation of the NLRP6 inflammasome111. The NLRP6 inflammasome then triggers the release of Ca2+ from the ER and promotes compound Ca2+-dependent mucin exocytosis, thereby generating intercellular gap junction calcium signals that induce mucus secretion from adjacent GCs near senGCs in the upper part of the crypt, clearing bacteria at the mouth of the crypt and protecting the lower part of the crypt and intestinal stem cells from bacterial invasion. Finally, after nearly all the mucus is secreted, senGCs fall off106. Hence, in response to external stimuli, mucus secretion by senGCs is rapid and impulsive. Mucin release from adjacent GCs near senGCs also depends on NLRP6 inflammasome activation but does not involve endocytosis. Ca2+ influx and release from intracellular store in the ER are required for LPS-TLR-ligand-induced secretion, and Ca2+ influx plays a role upstream of Ca2+ store release (Fig. 3). Moreover, senGCs are less important to the normal mucus barrier, as indicated by mouse strains that cannot trigger senGC activation presenting with a functional inner mucus layer98,106.

Notably, the intestinal epithelium increases mucin synthesis to antagonize microbial invasion. Serum amyloid (SAA) 3 protein and TNF-α cooperate to promote MUC2 production and protect epithelial cells from bacterial invasion, while SAA1 and SAA2 may also continuously stimulate MUC2 mucin production112. Levels of serum amyloid A3 (SAA3) mRNA in CMT-93 mouse colon epithelial cells were elevated by dead E. coli together with LPS, while SAA1/2 mRNA expression‎ was not induced. Moreover, recombinant murine SAA 1 (rSAA1) and 3 (rSAA3) significantly upregulated MUC2 mRNA levels in CMT-93 cells. The mRNA of the inflammatory cytokine TNF-α was significantly increased by rSAA3 intervention in CMT-93 cells107. TNF-α has also been shown to induce SAA3 expression‎ in CMT-93 cells113 and MUC2 mRNA expression‎ in HT-29 human colonic epithelial cells114. It has been postulated that SAA1/2 may continuously stimulate MUC2 mucin production, while the SAA3 protein and TNF-α cooperate to promote MUC2 production and protect epithelial cells from bacterial invasion112.

SNARE complex formation is the key molecular event in exocytotic mucus release

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) are critical for the fusion between mucus vesicles and the plasma membrane during baseline and stimulated mucus secretion. SNARE proteins are located in vesicles and plasma membranes and are connected to each other to form SNARE complexes, with vesicle-associated membrane protein 8 (VAMP8) on vesicle particles, and synaptosomal-associated protein 23 (SNAP23) and syntaxin (Stx) on the plasma membrane. During exocytosis, the scaffold protein Munc18b mediates SNARE complex assembly, and the GTPases Rab and Munc13 play auxiliary roles (Fig. 2). The assembly and formation of the SNARE complex promote the fusion of mucus particles and the cell membrane, leading to the subsequent formation of fusion pores through which mucins are expelled from cells103,115,116,117.

Conclusion

Multiple functional components of the mucus layer, including the main skeletal component MUC2 and other components, such as TFF3, FCGBP, and CLCA1, are gradually being discovered because of their role in building and consolidating mucus structures or their bacteriostatic or bactericidal functions. Their harmonious cooperation is indispensable for the homeostatic maintenance of the structure and function of the mucus layer, which is pivotal to block intestinal and extraintestinal diseases. The continual secretion of mucus is also a key mechanism for the homeostasis of the mucus layer structure. There are two models of mucus secretion: baseline secretion and stimulated secretion, which are differentiated on the basis of calcium influx levels. Both models are key mechanisms evolved to protect host intestinal epithelium from insults. Notably, spontaneous Ca2+ oscillation-mediated slow and continuous baseline mucin secretion is more important for mucus barrier integrity than stimulated secretion. This review is the first to thoroughly delineate the roles of various functional components in shaping the structure and function of the colonic mucus barrier and reveal novel insights into the regulatory mechanisms of mucus secretion, providing clues to develop strategies to strengthen the mucus barrier and maintain intestinal microenvironment homeostasis.

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