Recombinant Human Immunoglobulin superfamily member 23 (IGSF23)

What is Human Immunoglobulin Superfamily Member 23 (IGSF23)?

IGSF23 is a protein-coding gene located on chromosome 19 that belongs to the immunoglobulin superfamily . It encodes a transmembrane protein with immunoglobulin-like domains that plays a critical role in osteoclast differentiation and bone resorption . The gene has the Entrez Gene ID 147710 and UniProt ID A1L1A6 . IGSF23 is specifically expressed in bone tissue and is particularly important for the development and function of osteoclasts, which are specialized cells responsible for breaking down bone tissue during bone remodeling processes .

Where is IGSF23 primarily expressed in human tissues?

IGSF23 shows a highly tissue-specific expression pattern. Based on qRT-PCR and Western blot analyses, IGSF23 is predominantly expressed in bone and small intestine tissues . Notably, it is not detected in brain, liver, heart, kidney, lung, prostate, skeletal muscle, fat, spleen, testis, or thyroid gland . At the cellular level, IGSF23 expression is particularly high in mature osteoclasts, while showing lower expression levels in osteoblasts and peripheral blood mononuclear cells (PBMCs, which can differentiate into pre-osteoclasts) . This expression pattern strongly indicates IGSF23's specialized role in bone metabolism, particularly in the osteoclast lineage.

What is the subcellular localization of IGSF23?

IGSF23 protein is primarily localized to the cell membrane and cytoplasm, while it is absent from the nucleus . This localization has been confirmed through subcellular fractionation experiments using membrane-specific antibody (5'NT), cytoplasmic-specific antibody (D4-GDI), and anti-nucleotidase antibody (mSin3A) to verify the purity of each cellular fraction . The membrane and cytoplasmic localization of IGSF23 is consistent with its proposed function in cell signaling and osteoclast differentiation, as many cell surface receptors and signaling molecules involved in these processes are found in these cellular compartments.

How is IGSF23 structurally characterized?

IGSF23 is characterized as a transmembrane protein containing immunoglobulin-like domains, which are characteristic of the immunoglobulin superfamily . The protein contains highly conserved regions, particularly the arginine residue at position 99, which is conserved across multiple species including mouse, rat, chimpanzee (Pan troglodytes), and dog (Canis familiaris) . This evolutionary conservation suggests functional importance of this region.

The full-length human IGSF23 includes both extracellular immunoglobulin-like domains and a transmembrane domain . Notably, mutation studies have shown that truncation of IGSF23 after amino acid 98 (p.R99X mutation), which results in loss of both the immunoglobulin-like domain and the transmembrane domain, leads to significant functional impairment and is associated with osteopetrosis .

What are the known functions of IGSF23 in bone biology?

IGSF23 plays a critical role in bone biology, particularly in osteoclastogenesis (the development of osteoclasts) and bone resorption . Research has demonstrated that IGSF23 is essential for the differentiation of peripheral blood mononuclear cells (PBMCs) into mature, functional osteoclasts . During osteoclast differentiation, IGSF23 expression significantly increases in response to stimulation with M-CSF (Macrophage Colony-Stimulating Factor) and RANKL (Receptor Activator of Nuclear Factor κB Ligand), which are key cytokines that drive osteoclast formation .

IGSF23 appears to function through influencing the MAPK signaling pathway, affecting the expression of critical osteoclastogenic transcription factors including c-Fos and NFATC1 (Nuclear Factor of Activated T cells 1) . Loss of IGSF23 function results in impaired osteoclast formation and function, leading to increased bone density (osteopetrosis) due to reduced bone resorption .

What is the role of IGSF23 in osteoclastogenesis and bone remodeling?

IGSF23 plays a critical role in regulating osteoclastogenesis and subsequently affects bone remodeling. Experimental evidence shows that IGSF23 expression is significantly upregulated during osteoclast differentiation when peripheral blood mononuclear cells (PBMCs) are treated with M-CSF and RANKL, the key cytokines driving osteoclast formation . This upregulation occurs at both mRNA and protein levels over time, indicating IGSF23's importance in the differentiation process.

Mechanistically, IGSF23 influences osteoclastogenesis through the MAPK signaling pathway, which ultimately regulates the expression of critical transcription factors including c-Fos and NFATC1 . These transcription factors are essential for driving the expression of genes required for osteoclast differentiation and function, such as Cathepsin K and MMP9, which are enzymes involved in bone matrix degradation .

PBMCs from individuals with homozygous IGSF23 mutations (IGSF23−/−) demonstrate severely impaired ability to differentiate into mature osteoclasts in vitro . Importantly, this defect can be rescued through overexpression of wild-type IGSF23, confirming the direct role of IGSF23 in this process . Conversely, knockdown of IGSF23 can reverse bone loss in ovariectomized (OVX) mice, a model of postmenopausal osteoporosis, suggesting that IGSF23 inhibition might represent a potential therapeutic approach for conditions characterized by excessive bone resorption .

How do IGSF23 mutations affect bone density and what are the molecular mechanisms involved?

IGSF23 mutations, particularly the homozygous c.295C>T mutation that creates a premature stop codon (p.R99X), have been directly linked to osteopetrosis in human patients . This mutation results in a truncated IGSF23 protein with only 98 amino acid residues, causing loss of both the immunoglobulin-like domain and the entire transmembrane domain .

The molecular consequences of this mutation include:

• Impaired MAPK signaling pathway activation

• Decreased expression of key osteoclastogenic transcription factors c-Fos and NFATC1

• Reduced expression of osteoclast-specific genes including Cathepsin K and MMP9

• Severely impaired ability of peripheral blood mononuclear cells (PBMCs) to differentiate into mature, functional osteoclasts

The ultimate result is a significant reduction in bone resorption capacity, leading to increased bone mineral density characteristic of osteopetrosis . Clinical measurements using dual-energy x-ray absorptiometry in affected individuals show abnormally high Z-scores for bone mineral density in the femoral neck, lumbar spine, and total body .

Importantly, the p.R99X mutation shows perfect co-segregation with the osteopetrosis phenotype in family studies, with all affected individuals having homozygous mutations, carriers being heterozygous, and unaffected family members having wild-type IGSF23 . This pattern strongly supports the causative relationship between IGSF23 mutation and the disease phenotype.

What signaling pathways are affected by IGSF23 expression or mutation?

IGSF23 primarily influences the mitogen-activated protein kinase (MAPK) signaling pathway, which is critical for osteoclast differentiation and function . The MAPK pathway consists of a cascade of kinases that ultimately regulate the activity of transcription factors controlling gene expression.

In the context of osteoclastogenesis:

• IGSF23 positively regulates MAPK pathway activation in response to M-CSF and RANKL stimulation

• Functional IGSF23 is required for proper activation of the downstream MAPK components

• IGSF23 mutation or deficiency results in decreased MAPK signaling

This disruption in MAPK signaling leads to reduced expression of c-Fos and NFATC1, two critical transcription factors that drive osteoclast-specific gene expression . Without proper expression of these transcription factors, the expression of osteoclast functional genes like Cathepsin K and MMP9 is significantly reduced, impairing the cells' ability to resorb bone .

Additionally, research suggests that IGSF23, as a member of the immunoglobulin superfamily, may influence cell adhesion and membrane organization, which are important aspects of osteoclast fusion and formation of the specialized cell-bone interface essential for efficient bone resorption .

How can recombinant IGSF23 protein be used in experimental blocking assays?

Recombinant IGSF23 protein fragments, such as the commercially available human IGSF23 (aa 20-102) control fragment, can be effectively used in experimental blocking assays to validate antibody specificity and investigate IGSF23 function . The methodological approach for such experiments typically involves:

• Pre-incubation of anti-IGSF23 antibodies with the recombinant protein fragment (recommended at 100x molar excess based on concentration and molecular weight)

• Incubation of the antibody-protein mixture for approximately 30 minutes at room temperature

• Application of the pre-incubated mixture in experimental assays such as immunohistochemistry (IHC), immunocytochemistry (ICC), or Western blotting (WB)

This approach allows researchers to confirm antibody specificity by demonstrating signal reduction when the antibody's binding sites are occupied by the recombinant protein. Additionally, the recombinant protein can be used in functional studies to investigate protein-protein interactions, receptor binding, or as a competitive inhibitor of endogenous IGSF23 in cellular assays.

For optimal results, the recombinant protein should be characterized for purity, proper folding, and biological activity prior to use in blocking experiments. Quality control measures might include SDS-PAGE analysis, mass spectrometry, and functional binding assays .

What are the best experimental models for studying IGSF23 function?

Several experimental models have proven effective for investigating IGSF23 function, each with specific advantages for addressing different research questions:

In vitro models:

• Primary human peripheral blood mononuclear cells (PBMCs) - These cells can be differentiated into osteoclasts using M-CSF and RANKL, providing a system to study IGSF23's role in osteoclastogenesis

• Cell lines (e.g., RAW264.7 macrophages) - These can be used for mechanistic studies after IGSF23 overexpression or knockdown

• S2 cell aggregation assays - Useful for studying cell-cell adhesion roles of immunoglobulin superfamily members

In vivo models:

• IGSF23 knockout mice - Provide a system to study the consequences of complete IGSF23 deficiency on bone development and homeostasis

• Ovariectomized (OVX) mice with AAV-shIGSF23 injection - A model used to study the therapeutic potential of IGSF23 inhibition in conditions characterized by excessive bone resorption

Patient-derived samples:

• PBMCs from individuals with IGSF23 mutations - Valuable for studying the consequences of naturally occurring mutations on osteoclast differentiation and function

• Bone biopsies from affected individuals - Provide insights into the histological and ultrastructural consequences of IGSF23 dysfunction

The choice of model should be guided by the specific research question, with combinations of approaches often providing the most comprehensive understanding of IGSF23 function.

How do environmental chemicals affect IGSF23 expression?

Environmental chemicals have been shown to significantly influence IGSF23 expression, with many compounds causing upregulation or downregulation of the gene . Based on available data, these effects include:

Chemicals that increase IGSF23 expression:

• 17β-estradiol (estrogen)

• Bisphenol A (BPA)

• Bisphenol S (BPS)

• 2,3,7,8-tetrachlorodibenzodioxin (TCDD)

• 1,1-dichloroethene (vinylidene chloride)

• Benzo[a]pyrene (in some contexts)

Chemicals that decrease IGSF23 expression:

• Benzo[a]pyrene (in human contexts)

Chemicals affecting IGSF23 methylation:

• Bisphenol S (BPS) - affects the methylation of the IGSF23 gene

These observations suggest that IGSF23 expression may be sensitive to endocrine-disrupting chemicals and environmental toxicants. The differential effects (e.g., benzo[a]pyrene increasing expression in mouse models but decreasing it in human contexts) highlight the importance of species-specific responses and potentially different regulatory mechanisms .

Given IGSF23's role in osteoclast differentiation and bone homeostasis, these chemical-induced changes in expression could potentially contribute to altered bone density or turnover in response to environmental exposures. This makes IGSF23 a potential biomarker for environmental chemical exposure effects on bone health.

What are the approaches for manipulating IGSF23 expression in laboratory settings?

Several established approaches can be used to experimentally manipulate IGSF23 expression for functional studies:

Overexpression approaches:

• Lentiviral vectors - The full-length IGSF23 cDNA has been successfully cloned into the pHBLV-CMV-IRES-Puro vector for overexpression studies . Specific primers documented in the literature include:

  • Forward primer: 5′-GAATTCCGACCACCACCACTGACCCG-3′

  • Reverse primer: 5′-CGGGATCCTCAGCTGCATATTCCTA-3′

  • The PCR product is typically digested with EcoRI and BamHI restriction enzymes before vector insertion

Knockdown approaches:

• RNA interference - Lentivirus-produced IGSF23-shRNAs have been used effectively, with documented target sequences including:

  • 5′-CTGCTATCCGGGGAGTCATC-3′

  • 5′-TCATGCAGCCCACAGAAGCAG-3′

• AAV-delivered shRNA - AAV-shIGSF23 has been successfully injected into mouse femoral bone marrow cavity to achieve localized IGSF23 knockdown in vivo

Expression analysis:
For validating manipulation of IGSF23 expression, established primers for quantitative RT-PCR include:

• IGSF23: 5′-CCGATGCTAGAGAAGGATGC-3′ (forward) and 5′-AAACACATGCCTGCAATCAG-3′ (reverse)

These approaches can be combined with functional assays such as osteoclast differentiation assays (using TRAP staining), bone resorption assays, signaling pathway analysis, or in vivo bone density measurements to comprehensively study the consequences of altered IGSF23 expression or function.

How does IGSF23 compare to other immunoglobulin superfamily members in functional studies?

IGSF23 shares several functional characteristics with other immunoglobulin superfamily members, though it also has unique properties that distinguish it in functional studies:

Similarities with other Ig superfamily members:

• Membrane localization - Like many Ig superfamily proteins, IGSF23 is primarily localized to the cell membrane, consistent with roles in cell-cell interactions, adhesion, or receptor function

• Role in cell differentiation - Several Ig superfamily members, including IGSF23, influence cell differentiation processes. For example, Junctional Adhesion Molecule 3 (JAM3/JAM-C), another Ig superfamily member, plays roles in various developmental contexts

• Involvement in signaling pathways - Both IGSF23 and other Ig superfamily members can modulate intracellular signaling pathways, often through interactions with kinases or other signaling molecules

Unique aspects of IGSF23:

• Tissue-specific expression - Unlike some more broadly expressed Ig superfamily members, IGSF23 shows highly restricted expression, predominantly in bone and small intestine, with particularly high expression in osteoclasts

• Specific role in bone biology - IGSF23's involvement in osteoclastogenesis and its mutation being linked to osteopetrosis highlight its specialized function in bone homeostasis, distinct from many other Ig superfamily members

• Structural features - While sharing the characteristic immunoglobulin domains, IGSF23's specific domain organization and critical functional residues (e.g., the conserved arginine at position 99) may confer unique functional properties

Functional studies comparing IGSF23 with related family members have revealed both overlapping and non-overlapping functions . These comparative approaches help elucidate the specific contributions of individual family members to cellular processes and can identify potential redundancies or compensatory mechanisms that may be relevant for therapeutic targeting.