CLDN3: Another Tight Junction Protein or New Drug Target, Interest in CLDN Family is Growing Fast!

1. What's the Tight Junction Protein?

Tight junction proteins are composed of integral membrane proteins, membrane-associated, and soluble cytoplasmic proteins. The function of tight junctions is to adhere two cells together fully to prevent the movement of molecules and create a barrier. Importantly, tight junction proteins comprise more than 40 molecules derived from three integral transmembrane protein families: Occludin, Claudins, Junctional Adhesion Molecules (JAMs) ^[1].

Among them, Claudin-based tight junctions are crucial for the mammalian epidermal barrier, which determine the barrier properties of this type of cell-cell contact existing between the plasma membranes of two neighbouring cells, such as occurring in endothelia or epithelia. It is now widely accepted that loss of intercellular adhesion contributes to the disruption of tight cell junctions, which is linked to the infiltration and metastasis of tumor cells. Given that, Claudin family is considered as a highly sought-after therapeutic target ^[2].

2. What is CLDN3?

2.1 CLDN3 Structure

The tight junction protein 3 (Claudin3/CLDN3) belongs to the family of transmembrane tight junction proteins. CLDN3, with a relative molecular mass of 20 -34 kDa, is the most important backbone protein in tight junctions. Claudins consist of N-terminal and C-terminal, two extracellular loops, and four transmembrane structural domains. It forms a C-terminal PDZ-binding motif that interacts with ZO1, ZO2, ZO3, and multi-PDZ domain protein 1 (MUPP1) (Figure 1) ^[3].

Figure 1. The Structure of CLDN3 ^[3]

2.2 CLDN3 Function

Most studies indicate that CLDN3 is a barrier-forming protein, like blood-brain barrier, the intestinal barrier, and the blood-testis barrier [4-6]. Furthermore, it was also found to modulate cell proliferation, migration, differentiation, and polarity. Of note, CLDN3 expression is dysregulated in a variety of conditions. But it is overexpressed, among others, in ovarian, breast, lung, gastric, and colon cancers ^[4-6]. Albeit the causal relationship between altered CLDN3 levels and pathogenesis remains to be firmly established, it is likely that CLDN3 impacts key downstream outcomes ^[4-6].

3. What's the Ligand of CLDN3?

Clostridium perfringens enterotoxin (CPE) has been identified as a high-affinity natural ligand for CLDN3. Clostridium perfringens enterotoxin (CPE) triggers lysis of epithelial cells through binding to CLDN3 (Figure 2) ^[7-8]. According to many studies, CLDN4 is overexpressed in many epithelial malignancies and it has been correlated with cancer progression. In addition to CLDN3, clostridium perfringens enterotoxin is also a natural ligand for the CLDN4 protein. Thus, the binding of CPE to CLDN3 and CLDN4 proteins can lead to rapid cell lysis ^[9].

Recently, CPE is regarded as a therapeutic tool for epithelial malignant tumors by utilizing the damage of CLDN4 and CLDN3. For example, in pancreatic cancer cells, where CLDN-3 and -4 are highly expressed, intra-tumor injection of xenografted pancreatic cancer cells using cytopathic effect (CPE) resulted in extensive necrosis of tumor cells and significantly retarded tumor growth ^[10]

Figure 2. CLDN3 binding to CPE promotes cytolytic necrosis ^[8]

4. How's the Mechanism of Action of CLDN3 in Tumors?

It was hypothesized that CLDN3 gene expression may contribute to the pathogenesis of tumors in a tissue-specific manner, which further influence the intracellular molecular signaling pathways. However, the exact mechanism of the relationship between aberrant CLDN3 expression and tumors remains incompletely characterized. In hepatocellular carcinoma, CLDN3 is able to downregulate glycogen synthase kinase by GSK3B, CTNNB1, SNAI2 and CDH2, leading to the Wnt/β-catenin-EMT metastatic axis inactivation, thereby significantly inhibiting the metastasis of hepatocellular carcinoma ^[11-12]. Besides, CLDN3 knockdown also decreased the expression of related bile acid metabolism genes in the liver (like Cyp27a1, Ces1b and Akr1c6) ^[13]. In addition, loss of CLDN3 or CLDN4 activates the PI3K pathway by activating Akt phosphorylation, resulting in increased expression levels of the PI3K, suggesting that their deletion can promote epithelial mesenchymal transformation (EMT) process ^[14].

In lung cancer, CLDN3 was significantly overexpressed. Further studies suggested CLDN3 overexpression was associated with ERK1/2 and PI3K-Akt signaling pathways ^[14]. In colorectal cancer tissues, CLDN3 in CRC tissues can be elevated by activation of the SCF/c-kit/JNK/AP-1 signaling pathway ^[15]. In ovarian cancer, CLDN3 expression was affected by ZDHHC12, implying that targeting ZDHHC12-mediated CLDN3 S-palmitoylation may be a potential strategy for ovarian cancer treatment (Figure 3) ^[16]. Additional studies have indicated that CLDN3 may inhibit growth and metastatic of cells via maintaining E-cadherin expression and limiting the expression of β-cadherin ^[17].

Figure 3. CLDN3 is regulated by palmitoyltransferase ZDHHC12 ^[16]

5. The Roles of CLDN3 in Tumor and Other diseases

CLDN3, as a key sealing claudin, constitutes the tight junctions of endothelial cells. But CLDN3 abnormal expression can lead to damage to the intercellular structure of endothelial cells. Disruption of tight junctions may lead to weakened cell adhesion, resulting in loss of tumor cell polarity, uncontrolled growth, and even tumor invasion and metastasis. CLDN3 has emerged as modulators of proliferation, migration, and differentiation. Such effects might be the key during development and in a variety of diseases.

5.1 CLDN3 and Tumors

In colorectal cancer, epidermal growth factor (EGF) increased the expression of CLDN3 in colorectal cancer cell line HT-29 cells and promoted cell migration, suggesting that overexpressed CLDN3 enhances the invasive potential of colorectal cancer cells ^[14]. Typically, EGF is a marker in the development of certain tumors. EGF receptor antagonists such as Gefitinib have been used in clinical anti-tumor therapy.

In prostate cancer, CLDN3 was upregulated in prostate hyperplastic tissue. Using northern blot analysis, significantly higher levels of CLDN3 were found in human prostate cancer tissues compared to surrounding normal tissues ^[18]; the same was true in nasopharyngeal carcinoma, where CLDN3 expression was higher in nasopharyngeal carcinoma tissue than in nasopharyngeal paraneoplastic tissue. Plus, CLDN3 expression was higher in nasopharyngeal carcinoma tissues with higher pathological stage ^[19].

In cervical cancer Hela cell lines, SST promotes the CLDN3 and CLDN4 expression, thereby inhibiting the development of cervical cancer ^[20-22]. In breast cancer, CLDN3 was positively correlated with BRCA1-mutated BC ^[23]. In squamous epithelial cell carcinoma, decreased CLDN3 expression was significantly associated with tumor TNM stage, lymph node metastasis, and tumor recurrence ^[24-25]. In hepatocellular carcinoma, CLDN3 was significantly associated with survival of patients with hepatocellular carcinoma. These studies suggested that CLDN3 could be further evaluated as a novel biomarker or therapeutic target for different tumors ^[5-6].

5.2 CLDN3 and Other Diseases

Blood-brain barrier (BBB) disruption is thought to play a critical role in the pathophysiology of ischemia/reperfusion. CLDN3 is closely related to the function of the BBB and can alter the permeability of the BBB ^[26]; Gallstone disease is most common liver disorders in adults. In the liver of CLDN3-knockout mice, the paracellular phosphate ion permeability through hepatic tight junctions was significantly increased, resulting in calcium phosphate core formation. Cholesterol overdose could cause cholesterol gallstone disease in these mice ^[27]. In fact, CLDN3, as the most expressed TJ protein in the liver, such as intestinal dysbiosis, chronic liver disease, even malignant liver tumors ^[28].

6. CLDN3 Clinical Prospects

CLDN3 is an important component of the CLDN family and serves a substantial role in maintaining the function of TJs, which is involved in the selective permeation and cell polarization of tight junctions. In particular, due to its high tissue specificity, CLDN3 has been widely studied and plays an important role in the development and progression of many tumors. Data from Pharmsnap show that several preclinical and drug discovery stage CLDN3-related drugs are already in development (Table 1). Currently, CLDN3-based targeted therapies are emerging as an important research direction for the CLDN family, holding significant drug discovery potential!

Table 1: Clinical drugs for CLDN3

To fully support researchers and pharmaceutical companies in their research on CLDN3 in tumor and other diseases, CUSABIO presents the CLDN3 active protein (Code: CSB-MP005505HU) to support your research on the mechanism of CLDN3 or its potential clinical value.

Recombinant Human Claudin-3(CLDN3)-VLPs (Active)

High Specifity Validated by Western Blot

The high specifity was validated by western blot. (Tris-Glycine gel) Discontinuous SDS-PAGE (reduced) with 5% enrichment gel and 15% separation gel.

Excellent Bioactivity Validated by Functional ELISA

Immobilized Human CLDN3 at 10μg/mL can bind Anti-CLDN3 recombinant antibody (CSB-RA005505MA1HU), the EC₅₀ is 23.62-34.37 ng/mL.VLPs (CSB-MP3838) is negative control.