FAM3D Human

What is FAM3D and which gene family does it belong to?

FAM3D (Family with sequence similarity 3, member D) is a member of the cytokine-like FAM3 gene family, which consists of four members: FAM3A, FAM3B, FAM3C, and FAM3D. It functions as a secreted protein with chemotactic properties, particularly for neutrophils and monocytes. The FAM3 gene family was identified as having cytokine-like properties, with each member playing distinct roles in various physiological processes . FAM3D specifically has been characterized as an endogenous chemotactic agonist that can attract certain immune cells to specific locations in the body, signaling through formyl peptide receptors (FPRs) . This protein is predominantly expressed in the gastrointestinal tract, with particularly high expression in the colon, suggesting important roles in gut homeostasis and immune function .

Where is FAM3D predominantly expressed in human and mouse tissues?

FAM3D exhibits a tissue-specific expression pattern with predominant expression in the gastrointestinal tract. Analysis of mouse tissue cDNA panels revealed abundant expression of Fam3D in the gastrointestinal tract, particularly in the colon, with moderate expression also detected in mesenteric lymph nodes, lung, thymus, and spleen . Within the intestinal tissue, immunohistochemistry and immunofluorescence studies have shown that FAM3D is constitutively expressed in epithelial cells, with a prominent villus and crypt expression pattern .

At the cellular level, FAM3D is produced by intestinal epithelial cells (IECs), as confirmed by both mRNA expression analysis and protein detection methods . Dual immunofluorescent staining has further demonstrated that FAM3D is present in specific epithelial cell subtypes, including MUC2-positive goblet cells and CHGA-positive enteroendocrine cells . This expression pattern suggests that FAM3D plays important roles at the interface between the gut epithelium, microbiota, and immune system, contributing to intestinal homeostasis.

What are the primary receptors for FAM3D and how does it signal?

FAM3D signals primarily through formyl peptide receptor 1 (FPR1) and formyl peptide receptor 2 (FPR2), which are G-protein-coupled receptors highly expressed on the surface of neutrophils, monocytes, and macrophages . The interaction between FAM3D and these receptors has been confirmed through multiple experimental approaches, including chemotaxis assays, receptor internalization studies, Ca2+ flux assays, and radioligand-binding assays with FAM3D-stimulated HEK293 cells transiently expressing FPR1 or FPR2 .

Upon binding to these receptors, FAM3D triggers several downstream signaling pathways. In neutrophils, FAM3D stimulation leads to phosphorylation of ERK1/2 and p38 MAPK family proteins, which can be inhibited by FPR1 or FPR2 inhibitors, confirming the receptor specificity . Additionally, mechanistic studies have shown that FAM3D upregulates and activates Mac-1 (macrophage-1 antigen) in neutrophils through FPR-related Gi protein and β-arrestin signaling pathways . The signaling is sensitive to pertussis toxin, indicating the involvement of Gi/o family G proteins that inhibit adenylyl cyclase and regulate various effector systems . These receptor interactions and signaling cascades underlie FAM3D's biological functions, particularly its role in immune cell recruitment and inflammatory processes.

How does FAM3D contribute to neutrophil recruitment and function?

FAM3D functions as a potent chemoattractant for neutrophils, playing a significant role in their recruitment to sites of inflammation. Human peripheral blood neutrophils and monocytes exhibit strong chemotaxis activity in response to FAM3D . When injected into the mouse peritoneal cavity, FAM3D rapidly attracts CD11b+ Ly6G+ neutrophils , demonstrating its in vivo chemotactic capacity.

The neutrophil recruitment process mediated by FAM3D involves several molecular mechanisms. FAM3D binds to FPR1 and FPR2 on neutrophil surfaces, triggering intracellular signaling cascades that include phosphorylation of ERK1/2 and p38 MAPK family proteins . These signaling events lead to neutrophil activation and directed migration toward increasing FAM3D concentrations. Additionally, FAM3D upregulates and activates Mac-1 (macrophage-1 antigen) in neutrophils, which is crucial for neutrophil adhesion and migration .

The functional significance of FAM3D-mediated neutrophil recruitment has been demonstrated in disease models. In abdominal aortic aneurysm (AAA) models, FAM3D−/− mice exhibited decreased neutrophil infiltration in the aorta during the early stage of AAA formation compared with wild-type littermates . Furthermore, application of FAM3D-neutralizing antibody 6D7 markedly ameliorated elastase-induced AAA formation and neutrophil infiltration . These findings highlight the importance of FAM3D in neutrophil-mediated inflammatory processes and suggest that modulating FAM3D activity could be a potential therapeutic strategy for neutrophil-driven inflammatory diseases.

How does FAM3D expression change during inflammatory conditions?

FAM3D expression undergoes dynamic changes during inflammatory conditions, with significant upregulation observed in various inflammatory disorders. In experimental colitis induced by dextran sulfate sodium, FAM3D expression increases significantly compared to baseline levels . This upregulation suggests that FAM3D responds to inflammatory stimuli and may play a role in the inflammatory cascade during colitis.

In vascular inflammation, specifically during the pathogenesis of abdominal aortic aneurysm (AAA), FAM3D is significantly upregulated in endothelial cells . Analysis of AAA tissues from patients undergoing repair operations has shown increased FAM3D expression compared to normal vascular tissue . Similarly, in the context of hypertension, plasma levels of FAM3D are substantially elevated in patients with hypertension, showing a positive association with the odds of developing this condition .

These alterations in FAM3D expression during inflammatory states likely contribute to disease pathogenesis through enhanced neutrophil recruitment and activation. The increased levels of FAM3D during inflammation create a positive feedback loop, where more neutrophils are recruited to inflammatory sites, potentially exacerbating tissue damage and disease progression. The consistent upregulation of FAM3D across different inflammatory conditions suggests it may be a common mediator in various inflammatory pathways and a potential biomarker for inflammatory activity.

How does FAM3D influence vascular function and contribute to hypertension?

FAM3D significantly impacts vascular function and contributes to hypertension pathogenesis through multiple interconnected mechanisms centered on endothelial dysfunction. Research has established that FAM3D induces endothelial nitric oxide synthase (eNOS) uncoupling through FPR1- and FPR2-mediated oxidative stress, consequently causing endothelial dysfunction and exacerbating angiotensin II (AngII)-induced hypertension .

The molecular mechanism involves a sequential process where FAM3D first triggers oxidative stress through NADPH oxidase activation, which subsequently leads to eNOS uncoupling. This has been demonstrated experimentally, as the reactive oxygen species (ROS) inhibitor NAC and NADPH oxidase inhibitor apocynin could attenuate oxidative stress and markedly reversed FAM3D-induced NO reduction and eNOS monomerization . Importantly, L-NAME did not attenuate but instead further enhanced O2− generation induced by FAM3D at 1 hour, consistent with the finding that FAM3D does not induce eNOS uncoupling during this early period . This temporal sequence confirms that FAM3D directly induces oxidative stress prior to eNOS uncoupling.

In animal models, FAM3D deficiency ameliorates hypertension. Following AngII infusion, systolic blood pressure, diastolic blood pressure, and mean arterial pressure were significantly lower in FAM3D−/− mice compared to wild-type controls . Functionally, arteries from AngII-infused FAM3D−/− mice displayed markedly decreased vasoconstrictive responses to phenylephrine . When these arteries were pretreated with the NO synthase inhibitor L-NAME to cause endothelial dysfunction, the differences in constrictive responses between wild-type and FAM3D−/− mice were abolished , confirming that FAM3D regulates vasoconstriction by affecting endothelial function.

These findings establish FAM3D as a significant contributor to hypertension pathogenesis through endothelial dysfunction, making it a potential therapeutic target for treating hypertension, particularly in patients with elevated FAM3D levels.

What mechanisms underlie FAM3D's role in abdominal aortic aneurysm development?

FAM3D contributes to abdominal aortic aneurysm (AAA) development primarily through neutrophil-mediated inflammatory processes. As a dual agonist of FPR1 and FPR2, FAM3D induces Mac-1 (macrophage-1 antigen)-mediated neutrophil recruitment and aggravates AAA development through FPR-related Gi protein and β-arrestin signaling pathways .

During AAA pathogenesis, FAM3D expression is significantly upregulated in endothelial cells . Flow cytometry analysis has demonstrated that FAM3D-deficient (FAM3D−/−) mice exhibit decreased neutrophil infiltration in the aorta during the early stage of AAA formation compared with their wild-type littermates . This reduction in neutrophil infiltration correlates with attenuated AAA progression, suggesting a causative role for FAM3D-mediated neutrophil recruitment in aneurysm development.

The mechanistic link between FAM3D and neutrophil function in AAA has been further elucidated through molecular studies. FAM3D upregulates and activates Mac-1 in neutrophils, which is critical for neutrophil adhesion and migration . Inhibition of either FPR1 or FPR2 significantly blocks FAM3D-induced Mac-1 activation, indicating that FAM3D's effect depends on both receptors . These activated neutrophils then contribute to vascular wall degradation through the release of proteolytic enzymes and reactive oxygen species.

Therapeutic targeting of FAM3D has shown promise in AAA models. Application of the FAM3D-neutralizing antibody 6D7 through intraperitoneal injection markedly ameliorated elastase-induced AAA formation and neutrophil infiltration . In vitro coculture experiments with this neutralizing antibody and in vivo intravital microscopic analysis indicated that endothelial cell–derived FAM3D induced neutrophil recruitment , further confirming the role of FAM3D in this process.

These findings establish FAM3D as a key mediator in AAA pathogenesis and suggest that targeting FAM3D could be a promising therapeutic strategy for preventing or treating AAA.

What is the role of FAM3D in intestinal homeostasis and how does its deficiency affect the colonic epithelium?

FAM3D plays a crucial role in maintaining intestinal homeostasis, particularly in the colon, where it is highly expressed. In FAM3D-deficient (Fam3D−/−) mice, significant alterations in colonic epithelium structure and function are observed, revealing FAM3D's importance in gut health.

The colonic architecture is notably disrupted in Fam3D−/− mice, which exhibit crypt hyperplasia with significantly elongated crypts compared to wild-type counterparts . This structural abnormality is associated with increased numbers of Ki67-positive proliferating epithelial cells in the colonic mucosa . While wild-type mice show Ki67-positive proliferating cells primarily at the bottom of colon crypts with reduced numbers toward the top, Fam3D−/− mice display either a disorganized pattern of Ki67-positive cells or a diffused distribution throughout the crypts .

Despite these proliferative changes, analysis of the stem cell compartment revealed no difference in the numbers of Lgr5+ cells or expression of stem cell-related genes between wild-type and Fam3D−/− mice . This was further confirmed by organoid culture experiments showing similar formation and size of organoids from isolated crypts between the two genotypes . These findings suggest that FAM3D deficiency affects epithelial cell proliferation and differentiation rather than stem cell generation or function.

FAM3D deficiency leads to a low-level inflammatory process in the colonic epithelium, characterized by infiltration of CD3+ T cells and B220+ B cells . As Fam3D−/− mice age (reaching 1 year), they develop exacerbated crypt hyperplasia with neutrophil infiltration, invasive glands, and loss of differentiated epithelial cells . These mice also exhibit increased colon weight, enlarged spleens, and elevated circulating inflammatory leukocytes, indicating spontaneous colitis due to long-term FAM3D deficiency .

At the molecular level, RNA-seq analysis revealed that genes encoding antimicrobial peptides (Reg3b, Reg3g, Saa3) and those associated with responses to type I and type II interferons were downregulated in Fam3D−/− mice . This suggests that FAM3D plays an important role in regulating antimicrobial defense mechanisms in the colon, which likely contributes to its function in maintaining gut homeostasis and preventing inflammation.

How does FAM3D influence microbiota balance and antimicrobial defense in the gut?

FAM3D plays a significant role in maintaining microbiota balance and antimicrobial defense in the gut through multiple mechanisms. One of the most important functions of FAM3D in this context is its influence on antimicrobial peptide production. RNA-seq analysis of FAM3D-deficient (Fam3D−/−) mice revealed reduced expression of genes encoding key antimicrobial peptides such as Reg3b, Reg3g, and Saa3 . Real-time PCR validation confirmed the reduction of mRNA levels for these antimicrobial peptides, as well as Defb1 and Defa2, with decreased Reg3γ protein detected in the colon of Fam3D−/− mice .

These antimicrobial peptides are critical components of the intestinal innate immune system, directly controlling bacterial populations and preventing dysbiosis. Reg3 family members, in particular, are important for maintaining spatial segregation between the microbiota and the epithelial surface. Their decreased expression in the absence of FAM3D suggests an impaired ability to control bacterial populations, potentially leading to dysbiosis and increased susceptibility to infections.

In addition to effects on antimicrobial peptides, FAM3D also influences epithelial cell function and differentiation, which indirectly impacts microbiota balance. Gene set enrichment analysis (GSEA) of intestinal epithelial cells revealed that goblet cell and enteroendocrine cell signatures were specifically enriched in Fam3D−/− mice . These cell types play important roles in gut homeostasis through mucus production (goblet cells) and hormone secretion (enteroendocrine cells), both of which can affect the gut microbiome composition and function.

The altered epithelial cell composition and reduced antimicrobial peptide expression in FAM3D-deficient mice likely create an environment that favors dysbiosis. While the exact changes in microbiota composition resulting from FAM3D deficiency have not been fully characterized in the provided search results, the molecular alterations observed suggest that FAM3D promotes a balanced gut microbiome by supporting proper antimicrobial defense mechanisms and maintaining epithelial cell function.

What evidence links FAM3D to protection against inflammation-associated carcinogenesis?

FAM3D has been demonstrated to play a protective role against inflammation-associated carcinogenesis, particularly in the colon. The evidence linking FAM3D to this protective function comes from multiple observations in experimental models and is supported by mechanistic insights.

A key piece of evidence is the finding that FAM3D is essential for colon homeostasis and host defense against inflammation-associated carcinogenesis . FAM3D-deficient (Fam3D−/−) mice develop spontaneous colitis with age, characterized by exacerbated crypt hyperplasia, neutrophil infiltration, and invasive glands, as well as loss of differentiated epithelial cells . These pathological changes resemble pre-cancerous lesions and create a pro-tumorigenic environment in the colon.

The inflammatory environment in FAM3D-deficient colons is evident even in younger mice, which show infiltration of CD3+ T cells and B220+ B cells in the colonic epithelium, indicating a low-level chronic inflammatory process . This chronic inflammation is a well-established risk factor for colorectal cancer development, as it can promote DNA damage, cellular proliferation, and inhibit apoptosis.

At the molecular level, FAM3D deficiency leads to dysregulated epithelial cell proliferation, as evidenced by increased Ki67-positive proliferating epithelial cells with a disorganized pattern in the colonic mucosa . This aberrant proliferation, combined with chronic inflammation, creates conditions favorable for neoplastic transformation.

Additionally, FAM3D-deficient mice show reduced expression of antimicrobial peptides such as Reg3b, Reg3g, and Saa3 , which may lead to dysbiosis of the gut microbiota. Altered microbiota composition is increasingly recognized as a contributor to colorectal cancer development through various mechanisms, including increased production of genotoxic metabolites and enhanced mucosal inflammation.

Although direct evidence of increased tumor incidence in FAM3D-deficient mice is not explicitly stated in the provided search results, the constellation of pre-neoplastic changes observed strongly suggests that FAM3D plays an important role in preventing inflammation-associated carcinogenesis. These findings position FAM3D as a potential therapeutic target or biomarker for colorectal cancer prevention, particularly in patients with inflammatory bowel diseases who are at increased risk for colorectal cancer.

What techniques are most effective for detecting and quantifying FAM3D expression in biological samples?

Researchers can employ several complementary techniques to effectively detect and quantify FAM3D expression in biological samples, each offering specific advantages depending on research objectives:

mRNA Detection Methods:

• RT-PCR/qPCR: Real-time PCR provides sensitive and specific quantification of FAM3D mRNA levels. This approach has been successfully used to validate FAM3D expression in tissues like the colon . Primer design should ensure specificity for FAM3D without cross-reactivity with other FAM3 family members.

• RNA-Seq: This high-throughput approach enables comprehensive transcriptomic analysis, allowing simultaneous detection of FAM3D and thousands of other genes. RNA-Seq has been utilized to identify differentially expressed genes in FAM3D-deficient mice compared to wild-type controls .

• Single-cell RNA-Seq: This technique can reveal cell-type-specific expression patterns of FAM3D within heterogeneous tissues, providing valuable insights into which specific cell populations express FAM3D .

Protein Detection Methods:

• Western Blot: This technique has been successfully employed to confirm high-level Fam3D protein in mouse gastrointestinal tract and can be adapted for human samples. It allows semi-quantitative analysis of FAM3D protein levels using specific anti-FAM3D antibodies.

• ELISA: For quantitative measurement of FAM3D protein in plasma or tissue lysates, enzyme-linked immunosorbent assays provide precise quantification when developed with specific antibodies against FAM3D.

• Immunohistochemistry (IHC): IHC has revealed that FAM3D is constitutively expressed in epithelial cells with a distinctive villus and crypt expression pattern . This technique provides valuable spatial information about FAM3D distribution within tissue architecture.

• Immunofluorescence: This approach enables precise localization of FAM3D within cells and tissues. Dual immunofluorescent staining has demonstrated the production of FAM3D by colonic epithelial cells (EpCAM-positive cells) and its presence in specific cell types like MUC2-positive goblet cells and CHGA-positive enteroendocrine cells .

For accurate quantification, researchers should consider:

• Using recombinant FAM3D protein standards for calibration curves (one study estimated approximately 90 ng of Fam3D per 30 μg total colon tissue in mice )

• Including appropriate positive controls (gastrointestinal tissues, particularly colon)

• Employing multiple detection methods for cross-validation

• Analyzing both epithelial and immune cell compartments when studying tissues

These methodological approaches provide researchers with a robust toolkit for comprehensively characterizing FAM3D expression patterns in various biological contexts.

How can researchers effectively generate and validate FAM3D-deficient models for experimental studies?

Generating and validating FAM3D-deficient models requires careful attention to methodological approaches to ensure reliable and interpretable results. Several strategies have proven effective:

Generation of FAM3D-Deficient Mouse Models:

• TALEN-Mediated Gene Targeting: This approach has been successfully used to generate FAM3D−/− mice on a C57BL/6 background. The methodology involves designing target vectors specific to exon 3 of FAM3D, transcribing them into mRNA in vitro, and microinjecting them into fertilized eggs of C57BL/6 mice . Heterozygotes are then identified and intercrossed to generate homozygous knockout mice along with heterozygous and wild-type littermates.

• CRISPR/Cas9 System: While not explicitly mentioned in the provided search results, CRISPR/Cas9 represents another effective approach for generating FAM3D knockout models. This system offers advantages in terms of efficiency and versatility for precise genetic modifications.

• Conditional Knockout Strategies: For tissue-specific FAM3D deletion, Cre-loxP systems can be employed. This approach would be particularly valuable for distinguishing between the effects of FAM3D in different cell types, such as epithelial cells versus immune cells.

Validation Approaches for FAM3D-Deficient Models:

• Genetic Verification: PCR genotyping and sequencing are essential to confirm genetic modifications. The T7E1 assay and sequence analysis have been successfully applied for genotyping FAM3D−/− mice . This step ensures that the desired genetic alteration has been achieved.

• Expression Analysis: Multiple techniques should be employed to confirm FAM3D deficiency:

  • RT-PCR/qPCR to verify absence of FAM3D mRNA

  • Western blot analysis to confirm absence of FAM3D protein

  • Immunohistochemistry or immunofluorescence to verify absence of FAM3D in tissues where it's normally expressed

• Functional Validation: Phenotypic characterization based on known FAM3D functions provides critical confirmation of model validity:

  • Reduced neutrophil chemotaxis in response to specific stimuli

  • Altered intestinal homeostasis (crypt architecture, proliferation markers)

  • Changes in antimicrobial peptide expression (Reg3b, Reg3g, etc.)

For cellular models, similar principles apply. CRISPR/Cas9 gene editing in relevant cell lines (intestinal epithelial cells, endothelial cells) or siRNA/shRNA approaches can create in vitro models for specific experiments. Organoid culture systems derived from FAM3D−/− mice offer an intermediate approach between in vivo and in vitro models, allowing for controlled studies of intestinal epithelial function .

Critical experimental considerations include using littermate controls to ensure genetic background uniformity, performing rescue experiments by reintroducing FAM3D expression to confirm phenotype specificity, and accounting for age-related phenotypes, as FAM3D−/− mice develop more severe conditions with age .

What techniques are most effective for studying FAM3D-receptor interactions and downstream signaling?

Studying FAM3D-receptor interactions and downstream signaling requires a multifaceted approach combining various complementary techniques. Based on successful experimental strategies, the following methods are particularly effective:

Receptor Binding and Interaction Studies:

• Radioligand Binding Assays: These have been successfully used to demonstrate that FAM3D is a high-affinity ligand for FPR1 and FPR2 receptors . Using radiolabeled FAM3D with cells expressing specific receptors enables determination of binding kinetics and affinities.

• Receptor Internalization Assays: This approach has been employed to show FAM3D-induced receptor endocytosis in cells expressing FPR1 or FPR2 . Tracking labeled receptors or using fluorescently tagged receptors allows visualization of this process.

• Ca²⁺ Flux Assays: These functional assays have proven valuable with FAM3D-stimulated HEK293 cells expressing FPR1 or FPR2 . Using calcium indicators like Fluo-4 or Fura-2 enables measurement of intracellular calcium mobilization upon receptor activation, providing evidence of functional receptor engagement.

Signaling Pathway Analysis:

• Phosphorylation Studies: Western blotting with phospho-specific antibodies has revealed that FAM3D stimulation leads to upregulation of phosphorylated ERK1/2 and p38 MAPK in neutrophils . This approach identifies key signaling nodes activated downstream of receptor engagement.

• Inhibitor Studies: Using specific inhibitors of FPR1, FPR2, or downstream pathways has shown that FAM3D-induced effects are dependent on these receptors . For example, inhibition of FPR1 or FPR2 blocks FAM3D-induced Mac-1 activation in neutrophils . These pharmacological approaches help delineate signaling pathways.