ANTXR2 Mouse

What is ANTXR2 and what is its physiological function in mice?

ANTXR2, also known as Capillary Morphogenesis Gene-2 (CMG-2), is a widely expressed protein approximately 55-65 kDa in size that functions as an anthrax toxin receptor, though its physiological role extends beyond this function . The protein contains an extracellular domain with an integrin-like von Willebrand factor type A (VWA) domain with a metal ion dependent adhesion site (MIDAS), allowing it to selectively bind ECM components like collagen type IV and laminin . ANTXR2's primary physiological function appears to be regulating extracellular matrix homeostasis through its enhancement of MT1-MMP activity, which subsequently activates MMP2, a critical matrix-degrading enzyme . Studies using knockout mice have revealed that while ANTXR2 is not essential for embryonic development, it plays a crucial role in female reproductive function, particularly uterine structure maintenance and parturition .

How is the Antxr2-/- mouse model generated and what are its key phenotypes?

The Antxr2-/- mouse model was generated by deleting exon 1 of the Antxr2 gene, with RT-PCR analysis confirming the corresponding loss of Antxr2 mRNA in mouse embryonic fibroblasts . These knockout mice are viable and born in expected Mendelian ratios (22% +/+, 53% +/−, 25% −/− of 111 offspring analyzed), indicating ANTXR2 is not required for embryonic development . While showing normal development initially with no obvious phenotypic differences at the macroscopic level, Antxr2-/- mice exhibit several distinctive phenotypes:

• Reproductive abnormalities in females:

  • Young (6-week-old) females are fertile but fail to deliver pups at term (parturition defect)

  • Middle-aged (2-6 month) females experience approximately 50% miscarriage rates

  • Older females (7+ months) become completely infertile

• Uterine structural defects:

  • Poor uterine tone lacking muscle striations

  • Progressive disruption of both circular and longitudinal myometrial layers

  • Aberrant accumulation of ECM proteins (type I collagen, type VI collagen, fibronectin)

  • Age-dependent worsening resulting in thickened, collagen-dense stroma and loss of normal uterine architecture

• Molecular abnormalities:

  • Reduced levels of activated MMP2 in uterine tissue

  • Decreased MT1-MMP activity

Male Antxr2-/- mice maintain normal reproductive capabilities with no reported fertility issues .

What age-dependent changes occur in Antxr2-/- mice?

Antxr2-/- mice exhibit progressive, age-dependent changes in uterine structure and function that correlate with declining reproductive capability . In early stages (approximately 6.5 weeks), the circular and longitudinal myometrial layers begin to loosen with increased intercellular space appearing between muscle cell bundles . By 3 months of age, these changes progress significantly, with the circular myometrial layer becoming poorly defined, consisting of scattered smooth muscle cells, and the space between muscle layers becoming greatly distended .

In advanced stages (7+ months), severe disruption of myometrial organization occurs, with some areas becoming indistinguishable as discrete muscle layers. The progressive accumulation of extracellular matrix proteins results in a thickened, collagen-dense acellular stroma and eventual complete loss of normal uterine architecture . This structural deterioration directly correlates with reproductive capacity: young females experience parturition failure, middle-aged females show increased miscarriage rates, and older females become completely infertile . These progressive changes highlight that ANTXR2's role in ECM homeostasis becomes increasingly critical with age, with cumulative effects leading to irreversible tissue damage and functional decline.

How does ANTXR2 regulate extracellular matrix homeostasis?

ANTXR2 regulates extracellular matrix (ECM) homeostasis primarily through its influence on matrix metalloproteinase (MMP) activity . The primary mechanism involves:

• MT1-MMP Complex Formation:

  • ANTXR2 forms a physical complex with MT1-MMP (membrane type-1 matrix metalloproteinase)

  • This interaction enhances MT1-MMP enzymatic activity

  • MT1-MMP activity directly correlates with ANTXR2 expression levels in cells

• MMP2 Activation Pathway:

  • MT1-MMP functions as the primary activator of pro-MMP2

  • By enhancing MT1-MMP activity, ANTXR2 indirectly promotes MMP2 activation

  • Activated MMP2 degrades various ECM components, maintaining appropriate ECM turnover

• Direct ECM Interactions:

  • Through its VWA domain and MIDAS motif, ANTXR2 can directly bind ECM components

  • This binding may position the protein optimally for regulating local ECM remodeling

In Antxr2-/- mice, disruption of this regulatory pathway leads to reduced MT1-MMP activity, decreased MMP2 activation, impaired ECM degradation, and progressive accumulation of ECM proteins, particularly in reproductive tissues . This mechanistic understanding explains the fibrotic phenotype observed in these mice and highlights ANTXR2's essential role in preventing excessive ECM accumulation through promotion of matrix-degrading enzyme activity.

What is the relationship between ANTXR2 and MT1-MMP/MMP2 pathway?

ANTXR2 functions as a key regulator in the MT1-MMP/MMP2 proteolytic pathway that controls extracellular matrix turnover . This relationship involves:

• ANTXR2-MT1-MMP Complex:

  • ANTXR2 physically associates with MT1-MMP (also known as MMP14)

  • This interaction enhances MT1-MMP's enzymatic activity

  • Both ANTXR1 and ANTXR2 can function as positive regulators of MT1-MMP

• MT1-MMP-Mediated MMP2 Activation:

  • MT1-MMP processes pro-MMP2 into its catalytically active form

  • The activation efficiency depends on MT1-MMP activity levels

  • As a positive regulator of MT1-MMP, ANTXR2 indirectly enhances MMP2 activation

• Evidence from Experimental Models:

  • Uterine lysates from Antxr2-/- mice show reduced levels of activated MMP2

  • Conditioned medium from Antxr2-/- mouse embryonic fibroblasts contains decreased activated MMP2

  • Human uterine smooth muscle cells with ANTXR2 knockdown (R2KD) show slightly decreased MMP2 activity

  • Conversely, cells overexpressing ANTXR2 (R2OE) exhibit increased MMP2 activity

This pathway explains how ANTXR2 deficiency leads to decreased matrix degradation capability, resulting in the progressive accumulation of ECM proteins observed in Antxr2-/- mice. The dependence of MT1-MMP activity on ANTXR2 expression levels suggests a direct regulatory role rather than just a facilitating function, making ANTXR2 an integral component of the matrix remodeling machinery rather than merely an accessory factor .

How does ANTXR2 deficiency affect uterine smooth muscle cell function?

ANTXR2 deficiency significantly impairs uterine smooth muscle cell viability and function through multiple mechanisms . Both mouse models and human cell studies reveal:

• Cell Viability Effects:

  • ANTXR2 knockdown (R2KD) in human uterine smooth muscle cells (HUSMC) increases apoptosis rates

  • Loss of myometrial cells is observed in Antxr2-/- mouse uteri, becoming progressively worse with age

  • This suggests ANTXR2 provides critical survival signals to myometrial cells

• Migration Capacity:

  • HUSMC with ANTXR2 knockdown exhibit significantly decreased migration toward PDGF-B

  • Conversely, ANTXR2 overexpression enhances migration

  • This indicates ANTXR2 is necessary for normal chemotactic responses and cell movement

• Contractile Function:

  • HUSMC with ANTXR2 knockdown show significantly reduced contractility in oxytocin-mediated collagen contraction assays

  • This functional deficit likely contributes to the parturition failure observed in Antxr2-/- mice

  • The mechanical ability of myometrial cells to generate force is compromised without adequate ANTXR2

• ECM Interaction:

  • Progressive accumulation of ECM in the absence of ANTXR2 further compromises muscle cell function

  • Disrupted myometrial architecture impairs coordinated contractile activity

  • The physical separation of muscle cells by excessive ECM likely hinders functional coupling

These findings demonstrate that ANTXR2 is not merely involved in ECM remodeling but is essential for maintaining the fundamental cellular properties of uterine smooth muscle cells, including survival, migration, and contractility. The parturition defect in Antxr2-/- mice likely results from this combination of reduced myometrial cell viability, impaired contractile function, and the physical barrier created by excessive ECM accumulation .

What are reliable methods to detect ANTXR2 expression in mouse tissues?

Several reliable methods can be employed to detect ANTXR2 expression in mouse tissues, each with specific applications and considerations:

• Western Blot Analysis:

  • Commercially available antibodies such as Mouse CMG-2/ANTXR2 Antibody (AF3636) can detect ANTXR2 in protein lysates

  • ANTXR2 typically appears as a band at approximately 55-65 kDa under reducing conditions

  • Successfully tested tissues include RAW 264.7 mouse monocyte/macrophage cell lines, thymus tissue, and lung tissue

  • Optimal protocol involves PVDF membrane probing with 1 μg/mL antibody followed by HRP-conjugated secondary antibody

• Immunohistochemistry/Immunofluorescence:

  • Enables visualization of ANTXR2 localization within specific cellular compartments and tissue structures

  • Particularly useful for assessing ANTXR2 expression in the uterine myometrium

  • Can be combined with other markers (e.g., α-SMA) to evaluate co-localization with specific cell types

  • Allows comparison between wild-type and knockout tissues to confirm antibody specificity

• RT-PCR Analysis:

  • Detects Antxr2 mRNA expression in tissues and isolated cells

  • Used to confirm absence of Antxr2 mRNA in knockout mouse embryonic fibroblasts

  • Can be adapted for quantitative PCR to measure expression levels across different tissues or experimental conditions

• Flow Cytometry:

  • Useful for confirming ANTXR2 expression in cell lines after genetic manipulation

  • Successfully used to verify knockdown or overexpression in experimental cell models

  • Allows quantitative assessment of expression levels at the single-cell level

When implementing these detection methods, researchers should include appropriate controls including positive controls (tissues known to express ANTXR2), negative controls (tissues from Antxr2-/- mice), and loading/processing controls to ensure technical validity and accurate interpretation of results .

What cellular assays can evaluate ANTXR2 function in isolated cells?

Several cellular assays have been successfully employed to evaluate ANTXR2 function in isolated cells, providing insights into its role in key cellular processes:

• Genetic Manipulation Approaches:

  • Lentiviral-mediated knockdown (R2KD): Reduces ANTXR2 expression

  • Retroviral-mediated overexpression (R2OE): Increases ANTXR2 expression

  • Flow cytometry confirmation of expression changes creates well-defined experimental models

• Cell Viability and Apoptosis Assays:

  • TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) assay: Detects DNA fragmentation in apoptotic cells

  • Successfully demonstrated increased apoptosis in ANTXR2 knockdown cells

  • Quantification of apoptotic cells as a percentage of total population provides clear metrics

• Cell Migration Assays:

  • Boyden chamber (transwell) assays: Cells migrate through a porous membrane toward chemoattractants

  • PDGF-B serves as an effective chemotactic stimulus for smooth muscle cells

  • Quantification of migrated cells reveals ANTXR2's role in directed cell movement

  • ANTXR2 knockdown reduces migration while overexpression enhances it

• Contractility Assays:

  • Oxytocin-mediated collagen contraction assays: Measure functional contractile response

  • Cells embedded in collagen matrices contract in response to oxytocin stimulation

  • ANTXR2 knockdown cells showed significantly reduced contractility

  • Particularly relevant for understanding myometrial function

• MMP Activity Assays:

  • Gelatin zymography: Assesses MMP2 activity in conditioned medium

  • Reveals ANTXR2's effect on matrix-degrading enzyme activation

  • ANTXR2 knockdown reduces while overexpression increases MMP2 activity

These assays should be performed with appropriate controls and statistical analysis (e.g., Student's t-test comparing knockdown, overexpression, and control conditions). Cell types successfully used include human uterine smooth muscle cells (HUSMC) and mouse embryonic fibroblasts (MEFs), with experiments typically run in duplicate to ensure reproducibility .

How can researchers assess ECM remodeling in ANTXR2 mouse models?

Researchers can employ multiple complementary approaches to comprehensively assess ECM remodeling in ANTXR2 mouse models:

• Histological Analyses:

  • H&E Staining: Provides overview of tissue architecture, cellular density, and general ECM organization

  • Immunostaining for ECM Components: Specific detection of type I collagen, type VI collagen, and fibronectin reveals patterns of protein deposition

  • Alpha-Smooth Muscle Actin (α-SMA) Immunostaining: Visualizes smooth muscle cell organization and the relationship between muscle layers and ECM

• Biochemical ECM Analysis:

  • Protein Extraction and Western Blotting: Quantifies levels of specific ECM proteins in tissue lysates

  • Hydroxyproline Assay: Measures collagen content in tissues

  • MMP Activity Assays: Immunoblotting for MMP2 in tissue lysates and gelatin zymography assess matrix-degrading capacity

• Molecular Mechanism Assessment:

  • MT1-MMP Activity Measurement: Evaluates MMP2 processing as an indicator of MT1-MMP function

  • Co-immunoprecipitation: Identifies ANTXR2/MT1-MMP complex formation

  • Expression Analysis: RT-PCR for mRNA levels of ECM components and remodeling enzymes

• Developmental and Progressive Analysis:

  • Age-Dependent Tissue Sampling: Collection of tissues from mice at different ages (6.5 weeks, 3 months, 7+ months)

  • Comparative Analysis: Side-by-side evaluation of wild-type and knockout samples at matching timepoints

  • Documentation of Progressive Changes: Tracking the accumulation of ECM and disruption of tissue architecture over time

• Functional Consequences Assessment:

  • Tissue Mechanics Testing: Evaluates tissue stiffness and compliance changes resulting from ECM alterations

  • Contractility Studies: Assesses how ECM changes affect tissue functional responses

  • Reproductive Performance Tracking: Correlates ECM alterations with fertility outcomes

This multi-faceted approach enables researchers to link molecular mechanisms (reduced MT1-MMP/MMP2 activity) with tissue-level changes (ECM accumulation, myometrial disruption) and functional outcomes (parturition failure, infertility), providing a comprehensive understanding of how ANTXR2 deficiency affects ECM homeostasis .

How do ANTXR1 and ANTXR2 functions compare in mouse models?

Based on the available data, both ANTXR1 and ANTXR2 share some functional similarities while potentially having distinct physiological roles:

• Shared Molecular Functions:

  • Both ANTXR1 and ANTXR2 function as positive regulators of MT1-MMP activity

  • This was demonstrated through MT1-MMP-mediated MMP2 processing and activation

  • This suggests conservation of matrix metalloproteinase regulatory function between these related receptors

• Distinct Physiological Roles:

  • The severe reproductive phenotype in Antxr2-/- females suggests a particularly critical role for ANTXR2 in uterine function that may not be compensated by ANTXR1

  • While knockout mice for both receptors have been generated, the search results primarily detail Antxr2-/- phenotypes rather than comparative analysis

• Structural Similarities:

  • Both contain VWA domains with MIDAS motifs for ECM binding

  • Both function as anthrax toxin receptors, though their physiological roles extend beyond this function

• Research Gaps:

  • Limited direct comparison between Antxr1-/- and Antxr2-/- mouse phenotypes in the available data

  • Unclear whether one receptor can compensate for the other in specific tissues

  • Need for double-knockout models to assess potential functional redundancy

  • Incomplete understanding of tissue-specific expression patterns and potential specialized functions

For comprehensive comparison, future research should develop parallel analysis of single and double knockouts, evaluate tissue-specific expression patterns, and assess the ability of each receptor to compensate for the other's absence in various physiological contexts. The shared ability to regulate MT1-MMP suggests some functional overlap, but the specific reproductive phenotype in Antxr2-/- mice indicates distinct physiological roles .

What research questions remain unresolved regarding ANTXR2 in mice?

Despite significant advances in understanding ANTXR2 function, several important research questions remain unresolved:

• Signaling Mechanisms:

  • What are the direct signaling pathways downstream of ANTXR2 activation?

  • Does ANTXR2 have intrinsic signaling capacity or primarily function through regulating other proteins?

  • How does ANTXR2 influence cell survival pathways to protect against apoptosis?

• Tissue-Specific Functions:

  • Why does ANTXR2 deficiency predominantly affect female reproductive tissues despite wider expression?

  • Are there subtler phenotypes in other organs that haven't been characterized?

  • Do different tissues employ distinct ANTXR2-dependent mechanisms?

• Age-Dependent Phenotypes:

  • What triggers the progressive worsening of uterine pathology in Antxr2-/- mice?

  • Is there a cumulative threshold effect of ECM accumulation?

  • Could hormonal changes with age interact with ANTXR2 deficiency?

• Molecular Interactions:

  • What are the specific binding domains mediating ANTXR2/MT1-MMP interaction?

  • How exactly does ANTXR2 enhance MT1-MMP activity at the molecular level?

  • Are there additional protein partners in the ANTXR2/MT1-MMP complex?

• Therapeutic Implications:

  • Could enhancing MMP2 activation bypass ANTXR2 deficiency to treat associated pathologies?

  • Are there compensatory mechanisms that could be therapeutically exploited?

  • Could targeting ANTXR2 be relevant for treating excessive ECM deposition in other contexts?

• Comparative Biology:

  • Do human and mouse ANTXR2 have identical functions despite isoform differences?

  • Can mouse models recapitulate human diseases associated with ANTXR2 mutations?

  • What explains the species-specific differences in ANTXR2 isoform diversity?

Addressing these questions would require interdisciplinary approaches including conditional knockout models, tissue-specific transcriptomics/proteomics, structural biology studies of protein interactions, and comparative human-mouse analyses. Such research would enhance our understanding of ANTXR2's biological roles and potential therapeutic applications .

How might findings from ANTXR2 mouse models translate to human biology?

Findings from ANTXR2 mouse models have significant translational potential, though important species differences must be considered:

• Conserved Cellular Functions:

  • Studies using human uterine smooth muscle cells (HUSMC) with ANTXR2 knockdown show remarkably similar phenotypes to mouse models

  • ANTXR2 regulates cell viability, migration, and contractility in both species

  • The MT1-MMP/MMP2 regulatory pathway appears conserved between mice and humans

• Disease Relevance:

  • Human ANTXR2 mutations cause juvenile hyaline fibromatosis, characterized by abnormal ECM deposition

  • The ECM accumulation phenotype in Antxr2-/- mice mirrors aspects of this human condition

  • Understanding regulatory mechanisms in mice may provide insights into human ECM-related disorders

• Reproductive Biology Applications:

  • The reproductive phenotypes in female Antxr2-/- mice suggest potential involvement in human conditions like:

    • Dysfunctional labor (parturition defects)

    • Uterine fibrosis

    • Certain forms of infertility

  • Human uterine smooth muscle contractility defects with ANTXR2 knockdown support this translational potential

• Important Species Differences:

  • Human ANTXR2 has four isoforms while only one sequence has been reported for mouse ANTXR2

  • This increased complexity might confer additional or specialized functions in humans

  • Tissue-specific expression patterns may differ between species

• Translational Research Approaches:

  • Comparative expression studies across corresponding tissues in both species

  • Functional rescue experiments (human ANTXR2 in mouse knockout cells)

  • Development of mouse models carrying human disease-associated ANTXR2 mutations

  • Parallel studies in human and mouse cells with identical experimental designs

What are the key insights from ANTXR2 mouse model research?

ANTXR2 mouse model research has yielded several fundamental insights with broad implications for understanding ECM regulation and reproductive biology:

• Physiological Role Beyond Toxin Reception:

  • ANTXR2 functions primarily as a critical regulator of ECM homeostasis rather than merely serving as an anthrax toxin receptor

  • This evolutionary conservation suggests fundamental biological importance

• ECM Regulatory Mechanism:

  • ANTXR2 forms a complex with and enhances MT1-MMP activity

  • This interaction promotes MMP2 activation, facilitating ECM degradation and remodeling

  • Absence of ANTXR2 leads to reduced matrix-degrading capacity and progressive ECM accumulation

• Reproductive System Significance:

  • ANTXR2 is essential for maintaining normal uterine architecture and function

  • It prevents excessive ECM deposition in reproductive tissues

  • Its absence causes progressive myometrial disruption and eventual infertility in female mice

• Cellular Functions:

  • ANTXR2 promotes smooth muscle cell viability, migration, and contractility

  • These functions are conserved between mouse and human cells

  • Loss of ANTXR2 impacts fundamental cellular processes beyond ECM interactions

• Age-Dependent Manifestations:

  • ANTXR2 deficiency leads to progressive, cumulative changes in tissue architecture

  • The phenotype worsens with age, suggesting increasing importance during adult life

  • This provides a model for studying progressive fibrotic conditions

These insights establish ANTXR2 as a multifunctional protein with critical roles in tissue homeostasis, particularly in reproductive biology. The findings highlight the importance of ECM regulation for normal tissue function and provide mechanistic understanding of how dysregulation leads to pathological states characterized by excessive ECM accumulation, cellular dysfunction, and ultimate organ failure .

What potential therapeutic applications might emerge from ANTXR2 research?

While the search results don't directly address therapeutic applications, the mechanistic insights from ANTXR2 research suggest several potential therapeutic directions:

• ECM Remodeling Disorders:

  • MT1-MMP/ANTXR2 Pathway Targeting:

    • Small molecules or peptides enhancing this interaction could promote ECM degradation

    • Useful for treating fibrotic conditions characterized by excessive ECM accumulation

    • The proven regulatory relationship provides a clear mechanistic target

  • MMP2 Activation Approaches:

    • Therapeutics bypassing the ANTXR2 requirement for MMP2 activation

    • Could address conditions resulting from ANTXR2 dysfunction

    • Particularly relevant for juvenile hyaline fibromatosis and related disorders

• Reproductive Medicine Applications:

  • Uterine Fibrosis Treatment:

    • Targeted ECM degradation strategies for conditions with excessive myometrial ECM

    • Potentially applicable to certain forms of infertility

    • Based on the progressive fibrotic phenotype in Antxr2-/- mice

  • Parturition Dysfunction:

    • Therapeutics enhancing myometrial contractility in conditions with defective labor

    • Targeted toward the contractile dysfunction observed with ANTXR2 deficiency

    • Addressing both the ECM barrier and intrinsic contractility defects

• Diagnostic Applications:

  • ANTXR2 expression or activity as biomarkers for:

    • ECM remodeling disorders

    • Unexplained infertility or recurrent pregnancy loss

    • Assessment of uterine fibrosis severity

    • Prediction of parturition complications

• Anthrax-Related Applications:

  • While not the focus of reproductive and ECM research:

    • Understanding ANTXR2 structure could inform development of anthrax toxin inhibitors

    • Potential for creating decoy receptors based on ANTXR2 domains