Recombinant Mouse Interferon-induced transmembrane protein 5 (Ifitm5)

What is Ifitm5 and what is its primary biological function?

Interferon-induced transmembrane protein 5 (Ifitm5), also known as Bone-restricted interferon-induced transmembrane protein-like protein (BRIL), is a type II (N-in/C-out) transmembrane protein with the amino terminus located inside the cell and the carboxyl terminus located extracellularly . Ifitm5 functions as a positive regulator of bone formation, with its expression specifically observed during the mineralization stage of cultured osteoblasts . Studies have demonstrated that mineralization is promoted when Ifitm5 is overexpressed and suppressed when it is knocked down in cultured osteoblasts . The expression pattern of Ifitm5 during development is similar to that of Osterix (Sp7), suggesting its critical role in osteoblast maturation and bone formation .

How is Ifitm5 structurally characterized?

Ifitm5 contains two helical transmembrane domains connected by an intracellular linker, with both the N and C termini located at the outer site of the plasma membrane . The protein comprises 137 amino acids in its wild-type form, though mutations can alter this structure . Mouse IFITM5 shares approximately 88% similarity with human IFITM5, making mouse models particularly valuable for studying human conditions involving this protein . The protein structure is generally maintained even with modifications to the N-terminus, as predicted by computational algorithms like MEMSAT and DOMPRED .

What is the relationship between Ifitm5 and osteogenesis imperfecta?

Osteogenesis imperfecta type V (OI type V) is uniquely caused by a specific mutation (c.-14C>T) in the IFITM5 gene . Unlike most types of OI that result from mutations disrupting collagen synthesis or processing, OI type V involves altered IFITM5 function . This mutation creates an in-frame start codon in the 5'-UTR of the IFITM5 gene that is embedded in a stronger Kozak consensus sequence for translation initiation than the annotated start codon . This leads to the addition of five amino acids (Met-Ala-Leu-Glu-Pro) to the N-terminus of the protein, increasing its size from 137 to 142 amino acids and altering its function . The mutation disrupts normal bone stem cell development, resulting in bones that are extremely brittle, with patients experiencing recurrent fractures, bone deformities, and chronic pain .

How can researchers generate mouse models for studying Ifitm5 mutations?

Researchers can generate mouse models of Ifitm5 mutations using CRISPR/Cas9-based technology to introduce the c.-14C>T mutation into the mouse genome . When creating mosaic mice with this mutation, survival rates correlate with the mosaic ratio: mice with less than 40% mosaic ratio typically survive, while those with more than 48% exhibit lethal skeletal abnormalities (with rare exceptions) . For heterozygous mutants, researchers can mate mosaic mice with wild-type mice, though the resulting heterozygous mutants often exhibit perinatal lethal phenotypes due to severe skeletal abnormalities .

For conditional expression models, researchers have successfully developed systems where the mutant gene is expressed during specific stages of bone development . These genetically modified mice recapitulate most characteristics of the human condition, making them valuable for analyzing underlying molecular mechanisms . When designing such models, researchers should carefully consider the temporal expression patterns of Ifitm5, as it peaks during osteoblast maturation around the early mineralization stage .

What experimental approaches can be used to assess the effects of Ifitm5 mutations on bone development?

Multiple experimental approaches can be employed to assess the effects of Ifitm5 mutations:

• Bone Mineral Content (BMC) Analysis: BMC measurements can quantify mineralization defects in mutant models. For example, administration of FK506 (a calcineurin inhibitor) in heterozygous Ifitm5 mutant fetuses improved BMC of neonates, although it did not prevent lethality .

• Histological Analysis: Examination of bone sections can reveal structural abnormalities such as overgrown cartilage calluses where new bone should form, indicating disrupted bone maturation .

• Molecular Pathway Analysis: Investigation of signaling pathways affected by Ifitm5 mutations, such as the ERK/MAP kinase signaling pathway and the transcription factor SOX9, both of which are significantly increased in OI type V models . These pathways can be targeted with pharmacologic or genetic approaches to potentially restore normal bone development .

• Translation Initiation Studies: In vitro studies using eukaryotic cells can confirm altered translation initiation sites, demonstrating that cells preferentially use the new start codon created by the mutation instead of the reference translation initiation signal .

How can researchers manipulate Ifitm5 expression in experimental systems?

Researchers can manipulate Ifitm5 expression through several methods:

• Overexpression Systems: Transfection of cells with wild-type or mutant Ifitm5 expression constructs allows observation of effects on mineralization and differentiation processes .

• Knockdown/Knockout Approaches: RNA interference or CRISPR/Cas9-mediated gene editing can reduce or eliminate Ifitm5 expression. In Ifitm5-/- mice, long bones are 15-25% shorter at birth than in Ifitm5+/- mice and are sometimes severely bent, a symptom that partially resolves by adulthood .

• Pharmacological Interventions: Compounds like FK506 (tacrolimus) and rapamycin affect bone mineralization in Ifitm5 mutant models differently, suggesting their potential utility in studying the role of specific signaling pathways in Ifitm5-mediated bone formation .

How do immunosuppressants affect Ifitm5-mediated bone development?

Immunosuppressants show differential effects on bone development in Ifitm5 mutant models. FK506 (tacrolimus), a calcineurin inhibitor, improves bone mineral content (BMC) in heterozygous Ifitm5 c.-14C>T mutant mice, although it does not prevent the lethal effects of the mutation . In contrast, rapamycin, an mTOR inhibitor, reduces BMC in these models . This differential response suggests that mTOR signaling is specifically involved in the bone mineralization process of Ifitm5 mutants . The opposing effects of these immunosuppressants provide valuable insights into the signaling pathways involved in Ifitm5-mediated bone formation and may inform therapeutic strategies for OI type V.

What molecular mechanisms underlie the bone maturation defects in Ifitm5 mutations?

Two major molecular mechanisms have been identified:

• ERK/MAP Kinase Signaling Pathway: This pathway is significantly upregulated in mutant models . When inactivated through pharmacologic approaches, normal bone development can be partially restored .

• SOX9 Transcription Factor: SOX9 levels are abnormally elevated in Ifitm5 mutant models . Genetic inactivation of SOX9 can help restore normal bone development patterns .

These findings suggest a regulatory loop where the mutated Ifitm5 leads to persistent activation of these pathways, preventing the normal progression from cartilage to bone. Targeting either pathway offers potential therapeutic opportunities for OI type V patients.

What are the prospects for targeted therapies in Ifitm5-related disorders?

The unique nature of OI type V, where all patients share the identical c.-14C>T mutation in the IFITM5 gene, creates promising opportunities for targeted therapies . Several approaches show potential:

• Pathway-Targeted Pharmacologic Interventions: Compounds that inhibit the ERK/MAP kinase signaling pathway may help restore normal bone development in OI type V patients . Initial studies in animal models suggest efficacy in restoring aspects of normal bone formation .

• Gene Therapy Approaches: The uniformity of the mutation across all OI type V patients means that a single gene therapy approach targeting the mutated IFITM5 could potentially benefit all patients with this condition . This contrasts with other genetic disorders where multiple mutations necessitate personalized approaches.

• Combinatorial Approaches: Combining pathway inhibitors with agents that enhance bone formation or mineral density might provide synergistic benefits. Research in this area remains preliminary but promising.

• RNA-Based Therapies: Approaches targeting the aberrant translation initiation might be particularly effective, given that the mutation creates a new start codon that alters protein function rather than disrupting it entirely .

How do animal models of Ifitm5 mutations compare to human OI type V?

Comparison between mouse models and human OI type V reveals important similarities and differences that inform research approaches:

This comparison highlights the value of mouse models while acknowledging their limitations, particularly regarding survival outcomes. The alignment in molecular mechanisms suggests that therapeutic discoveries in mouse models may translate to human applications .

What are the current gaps in Ifitm5 research and potential future directions?

Despite significant advances, several research gaps remain in understanding Ifitm5 function and developing therapies for related disorders:

• Complete Function Elucidation: While Ifitm5 is known to be involved in bone formation, its precise molecular function remains incompletely understood . Future research should focus on identifying binding partners and downstream effectors.

• Temporal Regulation: The expression of Ifitm5 varies during development and adulthood, but the mechanisms regulating this temporal pattern remain unclear . Understanding these regulatory mechanisms could provide additional therapeutic targets.

• Therapeutic Translation: While animal studies have identified potential therapeutic pathways (ERK/MAP kinase inhibition, SOX9 modulation), clinical translation requires further development . Determining optimal therapeutic windows and delivery methods remains challenging.

• Long-term Effects of Mutation: The postnatal effects of Ifitm5 mutations in surviving models need further characterization to understand disease progression and identify intervention points .

• Interaction with Other Bone Regulatory Systems: How Ifitm5 interacts with other bone formation pathways, particularly those involving collagen processing, remains an important area for investigation .

Future research directions should include comprehensive proteomic analyses to identify Ifitm5 interactors, longitudinal studies of disease progression, development of survivable mouse models that better recapitulate human disease, and preclinical testing of targeted therapeutics based on pathway discoveries.