S100A3 Mouse

What is S100A3 and what is its fundamental role in mouse hair growth cycles?

S100A3 is a calcium-binding protein belonging to the S100 family that demonstrates dynamic expression patterns correlating with hair growth phases in mice. Gene expression microarray analysis reveals that S100A3 expression significantly increases during the anagen (growth) phase of the hair cycle and returns to basal levels during the telogen (resting) phase . Protein localization studies show that S100A3 is specifically confined to hair shafts during anagen phase and relocates to sebaceous glands during telogen phase . This distinctive expression pattern suggests S100A3 plays a regulatory role in promoting the active growth phase of hair follicles in mice. The protein appears to be essential for normal hair follicle development and cycling, as demonstrated through antibody-mediated blocking experiments which significantly impair hair growth progression .

How can researchers detect endogenous S100A3 distribution in mouse tissues?

Immunofluorescence analysis represents the preferred method for visualizing S100A3 distribution in mouse tissues. When examining cell types such as fibroblasts or epithelial cells, S100A3 typically exhibits a characteristic punctate cytosolic distribution pattern throughout the cytoplasm . This contrasts with the distribution pattern of other S100 family members, such as S100A13, which shows distribution primarily in areas lacking mitochondria . For optimal visualization, researchers should use anti-S100A3 primary antibodies (available from suppliers like Abcam) followed by fluorescein-conjugated secondary anti-rabbit IgG antibodies . Confocal microscopy with appropriate controls is recommended for definitive localization studies, particularly when investigating potential colocalization with organelle markers.

What physiological changes occur when S100A3 is blocked in mouse models?

When S100A3 activity is blocked through targeted antibody treatment, several significant physiological changes occur in mouse hair follicles:

These physiological changes following S100A3 blockade are dose-dependent and correlate with the downregulated expression of hair growth induction-related genes in vivo . The research demonstrates that anti-S100A3 antibody administration via subcutaneous injection provides a reliable experimental approach for studying the protein's functional role in hair growth regulation.

What are the optimal methods for quantifying S100A3 levels in mouse tissue and cell samples?

For precise quantification of S100A3 in mouse samples, sandwich ELISA represents the gold standard approach. Commercially available mouse S100A3 ELISA kits provide a detection range of 0.72-13.5 ng/ml with a sensitivity of 0.36 ng/ml . The assay works by utilizing antibodies specific to S100A3 coated on microwell plates, followed by addition of samples and an HRP-conjugated detection antibody .

Optimal sample preparation protocols vary by tissue type:

• Serum/plasma: Collect using standard protocols and store at -80°C until analysis

• Tissue homogenates: Homogenize in appropriate buffer with protease inhibitors

• Cell culture supernatants: Collect after appropriate stimulation period

• Urine: Centrifuge to remove particulates before analysis

For all sample types, a sample volume of 50-100 μl is typically required, with an assay time of 1-5 hours . Western blotting provides a complementary approach for semi-quantitative analysis and verification of protein specificity. For optimal results in detecting endogenous versus recombinant S100A3, antibody selection is critical, with commercially validated options available from suppliers including Abcam .

How can researchers effectively modulate S100A3 activity in experimental mouse models?

Researchers have several validated approaches for modulating S100A3 activity in experimental settings:

• Antibody-mediated blockade: Subcutaneous injection of anti-mouse S100A3 antibody provides targeted inhibition of S100A3 function. This method has been demonstrated to effectively inhibit hair growth in mouse models .

• Genetic overexpression: Transfection with vectors containing wild-type or mutant S100A3 coding sequences allows for controlled expression. For optimal results, sequences should be codon-optimized for mammalian expression, preceded by the consensus Kozak sequence, and include appropriate tags (e.g., HA tag) for detection .

• Recombinant protein treatment: Application of purified recombinant S100A3 protein (typically at 500 ng/mL) can restore normal function in deficient systems. This approach has been shown to partially normalize calcium responses in cells with impaired calcium homeostasis .

• Combined approaches: For studies investigating interactions with other S100 family members, simultaneous modulation of multiple proteins (e.g., S100A3 and S100A13) may be necessary to observe physiological effects .

Each approach offers distinct advantages depending on research objectives, with antibody blockade being most suitable for loss-of-function studies and recombinant protein treatment for rescue experiments.

What is the relationship between S100A3 and calcium signaling in mouse cells?

S100A3, as a calcium-binding protein, plays a significant role in maintaining proper calcium homeostasis in mouse cells. Research demonstrates that S100A3 is directly involved in receptor-mediated calcium signaling pathways and calcium store responses . When S100A3 function is compromised, either through mutation or blockade, cells exhibit impaired calcium transients in response to both receptor stimulation (e.g., bradykinin) and ionophore (e.g., ionomycin) treatment .

The relationship between S100A3 and calcium signaling is characterized by:

• Receptor-mediated calcium responses: S100A3 is required for normal calcium mobilization following receptor activation. In cells with mutant S100A3, bradykinin-induced calcium transients are significantly reduced compared to controls .

• Store-operated calcium entry: S100A3 influences calcium release from intracellular stores, as evidenced by altered responses to ionomycin in cells with dysfunctional S100A3 .

• Calcium homeostasis restoration: Importantly, transfection with wild-type S100A3 or treatment with recombinant S100A3 protein can restore normal calcium responses in cells with deficient S100A3 function .

These findings position S100A3 as a key regulator of calcium homeostasis in mouse cells, with implications for various calcium-dependent cellular processes.

How does S100A3 function differ from other S100 family members in mouse models?

S100A3 exhibits distinct functional characteristics compared to other S100 family members in mouse models, particularly regarding subcellular localization, calcium binding properties, and physiological effects:

These distinctions highlight the specialized roles of different S100 family members. While both S100A3 and S100A13 contribute to calcium homeostasis, they appear to do so through different mechanisms and with varying degrees of influence . For instance, recombinant S100A13 treatment (500 ng/mL) significantly restores bradykinin response in deficient cells (2.94 ± 0.33 fold increase compared to 1.30 ± 0.08 in untreated cells), whereas S100A3 treatment produces only a small, non-significant increase (1.69 ± 0.17 fold increase) .

What experimental approaches can be used to study the role of S100A3 in mitochondrial function?

Investigating S100A3's impact on mitochondrial function requires sophisticated experimental approaches focusing on various aspects of mitochondrial physiology:

• Mitochondrial mass assessment: MitoTracker Green FM staining provides quantitative measurement of mitochondrial mass regardless of membrane potential. Research indicates that cells with mutant S100A3 show increased mitochondrial mass, suggesting compensatory mitochondrial biogenesis .

• Membrane potential analysis: JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanide iodide) staining enables assessment of mitochondrial membrane potential. Patient-derived cells with mutant S100A3 display significantly more J-aggregates and fewer JC-1 monomers compared to control cells, indicating more polarized mitochondria and higher membrane potential .

• Rescue experiments: Treatment with recombinant wild-type S100A3 (500 ng/mL for 24 hours) can be used to assess reversibility of mitochondrial abnormalities. While S100A3 alone shows limited effect on mitochondrial mass, combined treatment with S100A13 significantly reduces abnormal mitochondrial mass in affected cells .

• Cell-type specific analysis: Different cell types (fibroblasts vs. epithelial cells) may show varying responses to S100A3 modulation, necessitating parallel experiments in multiple cellular models .

These approaches collectively provide comprehensive insights into how S100A3 influences mitochondrial dynamics and bioenergetics in mouse cells.

How can researchers investigate the relationship between S100A3 and inflammatory pathways?

To examine S100A3's role in inflammatory responses, researchers should implement the following methodological approaches:

• Cytokine/chemokine profiling: Utilize Milliplex Map (xMAP technology) to measure multiple inflammatory mediators simultaneously in cell culture supernatants. This approach has revealed that cells with mutant S100A3 secrete increased levels of inflammatory markers .

• Transfection studies: Express mutant or wild-type S100A3 in control cells to directly assess the impact on inflammatory mediator production. Research confirms that transfection with mutant S100A3 constructs causes significant cytokine secretion compared to controls .

• Cell type-specific analysis: Extend investigations to relevant cell types such as bronchial epithelial cells (BEAS-2B) to understand tissue-specific inflammatory responses. Both mutant S100A3 and S100A13 transcripts cause significant cytokine secretion when expressed in these cells .

• Combined protein treatment: Apply recombinant S100A3 (500 ng/mL) together with other relevant proteins (e.g., S100A13) to assess potential synergistic effects on normalizing inflammatory responses .

When conducting these experiments, researchers should ensure appropriate controls, including untransfected cells and cells transfected with irrelevant proteins (e.g., RFP), to distinguish specific S100A3 effects from general cellular stress responses.

What molecular mechanisms underlie S100A3's role in the mouse hair growth cycle?

The molecular mechanisms through which S100A3 regulates the mouse hair growth cycle involve complex interactions across multiple cellular pathways:

• Temporal gene expression regulation: Microarray analysis reveals dynamic expression profiles of S100A3 throughout the hair growth cycle, with significant upregulation during anagen phase and return to basal levels during telogen phase .

• Spatial protein localization: S100A3 demonstrates distinctive localization patterns, being confined to hair shafts during anagen phase and relocating to sebaceous glands during telogen phase. This spatial regulation suggests targeted functionality in specific follicular components .

• Downstream gene modulation: S100A3 blockade correlates with downregulation of hair growth induction-related genes, indicating that S100A3 sits upstream in regulatory pathways controlling hair follicle development and cycling .

• Calcium-dependent signaling: As a calcium-binding protein, S100A3 likely exerts its effects through calcium-dependent interactions with target proteins involved in hair follicle cycling. The protein's role in calcium homeostasis may be central to its function in regulating hair growth .

• Potential interaction with other S100 family members: Evidence suggests cooperative effects between S100A3 and other family members such as S100A13, particularly in calcium homeostasis. These interactions may extend to hair follicle regulation, though this requires further investigation .

Understanding these molecular mechanisms provides potential targets for therapeutic intervention in hair loss conditions and offers insights into fundamental aspects of hair follicle biology.

How might S100A3 function relate to pulmonary disorders in mouse models?

Recent research indicates that S100A3, particularly in conjunction with S100A13, may play a significant role in pulmonary homeostasis and pathology. Studies of digenic mutations in S100A3 and S100A13 reveal an association with an atypical and early-onset form of lung fibrosis . This relationship suggests several important research directions for investigating S100A3 in mouse pulmonary models:

• Calcium homeostasis in lung epithelium: Bronchial epithelial cells (BEAS-2B) express endogenous S100A3 in a punctate cytosolic distribution pattern. When mutant S100A3 is expressed in these cells, significant reductions in bradykinin-induced and ionomycin-induced calcium transients occur, confirming direct effects on calcium homeostasis in lung cells .

• Mitochondrial dysfunction: Expression of mutant S100A3 in bronchial epithelial cells produces mitochondrial abnormalities similar to those observed in skin fibroblasts, suggesting a conserved mechanism across tissue types .

• Inflammatory mediator production: Bronchial epithelial cells transfected with mutant S100A3 show significant increases in cytokine secretion compared to controls, indicating a potential role in pulmonary inflammatory pathways .

• Potential therapeutic approaches: Treatment with recombinant wild-type S100A3 protein can normalize cellular responses in cells with mutant S100A3, suggesting potential therapeutic applications for pulmonary disorders involving this pathway .

These findings collectively suggest that mouse models of S100A3 modulation may provide valuable insights into pulmonary pathophysiology and potential therapeutic approaches for interstitial lung diseases.

What techniques are most effective for studying interactions between S100A3 and other calcium-binding proteins?

Investigating interactions between S100A3 and other calcium-binding proteins requires sophisticated methodological approaches:

• Co-transfection experiments: Simultaneous transfection with vectors expressing tagged versions of S100A3 (HA-tagged) and other calcium-binding proteins (e.g., MYC-tagged S100A13) allows for assessment of functional interactions . The expression constructs should include:

  • Codon-optimized sequences

  • Consensus Kozak sequence

  • Flexible linker sequences like (GGGGS)3

  • Distinct epitope tags (HA, MYC) for differential detection

• Co-immunoprecipitation assays: Using anti-tag antibodies (anti-HA at 1:1000 dilution for S100A3-HA; anti-MYC at 1:1000 dilution for S100A13-MYC) enables isolation of protein complexes and identification of interaction partners .

• Calcium imaging studies: Combined treatment with recombinant S100A3 and other calcium-binding proteins (e.g., S100A13 at 500 ng/mL each) reveals synergistic effects on calcium responses that aren't observed with individual proteins alone .

• Functional rescue experiments: Comparing the effects of individual versus combined protein treatments provides insights into cooperative relationships between calcium-binding proteins. For instance, combined S100A3 and S100A13 treatment restores both bradykinin-mediated and ionomycin-mediated calcium transients to normal levels, while individual treatments show variable efficacy .

These approaches collectively enable researchers to dissect the complex interplay between S100A3 and other calcium-binding proteins in maintaining cellular calcium homeostasis and downstream physiological functions.