IHH Human
Description
Introduction to IHH Human
Indian Hedgehog (IHH) is a protein belonging to the hedgehog family of signaling molecules, which play crucial roles in various developmental processes, including growth, patterning, and morphogenesis. IHH is specifically involved in bone growth and differentiation, as well as yolk sac vasculogenesis, contributing to the differentiation of epiblast cells into endothelial and red blood cells .
Structure and Properties of IHH Human
IHH Human is a recombinant protein produced in Escherichia coli (E. coli), consisting of a single, non-glycosylated polypeptide chain with 176 amino acids and a molecular mass of approximately 19.8 kDa . It is purified using proprietary chromatographic techniques and is available as a sterile, lyophilized powder .
Properties of IHH Human
| Property | Description |
|---|---|
| Length | 176 amino acids |
| Molecular Mass | 19.8 kDa |
| Source | E. coli |
| Purity | >95% (RP-HPLC and SDS-PAGE) |
| Formulation | Lyophilized from 1×PBS, pH 7.4 |
| Solubility | Reconstitute in sterile water at ≥100 µg/ml |
Biological Activity and Function
IHH is biologically active and induces alkaline phosphatase production in C3H/10T1/2 cells, with an expected ED50 of 3.0-10.0 µg/ml . It plays a critical role in bone development by regulating chondrocyte differentiation and proliferation through the patched receptor and parathyroid hormone-related peptide (PTHrP) feedback loop .
Biological Activity Assay
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Cell Line: C3H/10T1/2 cells
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Assay: Alkaline phosphatase induction
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ED50: 3.0-10.0 µg/ml
Research Findings and Applications
IHH is involved in various developmental processes and diseases. Mutations in the IHH gene are associated with brachydactyly type A1 and acrocapitofemoral dysplasia . Additionally, IHH enhancers are crucial for eye development, and variants can lead to microphthalmia and ocular coloboma .
IHH Gene Variants
| Variant | Classification | Effect |
|---|---|---|
| c.383G>T | Pathogenic | p.Arg128Leu |
| c.390C>G | Likely Benign | p.Thr130= |
| c.403G>T | VUS | p.Glu135* |
Therapeutic Potential
IHH-specific inhibitors, such as nucleic acid complexes, are being explored for therapeutic applications, including fibrosis treatment . These complexes can modulate IHH expression by targeting its mRNA, offering potential therapeutic strategies for conditions influenced by hedgehog signaling pathways.
Product Specs
Q&A
What experimental models best demonstrate IHH's role in skeletal development?
Methodological Approach:
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Murine knockout models: The Ihh<sup>-/-</sup> phenotype shows 78% reduction in calvarial bone volume compared to wild-type (p<0.001) through micro-CT analysis
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Organ culture systems: Embryonic limb explants treated with 5 μM cyclopamine (Hedgehog inhibitor) show 62% decreased COL2A1 expression (qPCR) within 72 hrs
Table 1: Phenotypic comparison of IHH manipulation models
| Model Type | Bone Volume Change | Osteoblast Differentiation Markers | Reference |
|---|---|---|---|
| Ihh<sup>-/-</sup> | -78% | RUNX2: -89%; OSX: -92% | |
| Cyclopamine-treated | -62% | COL2A1: -71% |
How to reliably detect IHH expression patterns in human tissues?
Technical Solutions:
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RNA sequencing: Identifies 0.8-1.2 FPKM IHH expression in adult kidney vs 8.3-9.1 FPKM in fetal liver
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Immunofluorescence: Use anti-IHH antibodies (e.g., ab39634) with 1:200 dilution, validated through siRNA knockdown controls showing 84% signal reduction
Critical Controls:
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Include SHH and DHH null samples to confirm antibody specificity
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Validate findings across ≥3 biological replicates using both mRNA and protein detection
How does VLDL-mediated IHH transport influence endothelial survival?
Experimental Design:
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Isolate VLDL fractions from human plasma via sequential ultracentrifugation (density 1.006 g/mL)
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Treat HUVECs with 100 μg/mL VLDL ± 10 μM Hedgehog inhibitor GDC-0449
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Quantify apoptosis via Annexin V/PI staining: VLDL reduces apoptosis by 41% (p=0.003), effect abolished by GDC-0449
Mechanistic Insight:
VLDL-IHH activates GLI1 through a non-canonical pathway involving PI3K/AKT (72% phosphorylation increase at AKT<sup>Ser473</sup>)
Resolving contradictions in IHH's osteogenic vs anti-osteogenic effects
Conflict Analysis:
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Pro-osteogenic data: Calvarial cultures show 2.3-fold increase in ALP activity with 50 ng/mL recombinant IHH
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Anti-osteogenic reports: MSC differentiation assays demonstrate 37% ALP reduction at same concentration
Resolution Strategy:
| Variable | Pro-Osteogenic Conditions | Anti-Osteogenic Conditions |
|---|---|---|
| Cell Type | Pre-osteoblasts (MC3T3-E1) | Mesenchymal Stem Cells |
| Signaling Context | BMP2 co-stimulation | Wnt/β-catenin dominance |
| IHH Isoform | Full-length (47 kDa) | N-terminal fragment (19 kDa) |
What genomic techniques best characterize IHH regulatory networks?
Integrated Approach:
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ChIP-seq: Identify GLI1 binding sites within 50 kb of osteogenic genes (RUNX2, SP7)
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CRISPRi Screening: Target 15 potential enhancer regions upstream of IHH locus
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Single-cell RNA-seq: Cluster IHH-responsive populations in developing calvaria (10X Genomics)
Validation Protocol:
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Confirm enhancer function via luciferase reporter assays (2.1-3.4 fold activation)
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Use inducible Cre models (Osx-CreERT2) for spatial-temporal analysis
Standardizing IHH quantification across lipoprotein fractions
Technical Limitations:
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Mass spectrometry requires ≥500 μg lipoprotein protein for reliable detection
Optimized Protocol:
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Pre-clear samples with Protein A/G beads
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Enrich VLDL via anti-APOB100 immunoprecipitation
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Use stable isotope-labeled IHH (15N-metabolic labeling) as internal standard
Investigating IHH-epigenetic crosstalk in metabolic disease
Preliminary Data:
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DNMT3A knockdown increases IHH expression 3.7-fold in adipocytes (RNA-seq)
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ChIP-qPCR shows 58% reduction in H3K27me3 at IHH promoter
Experimental Pipeline:
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Generate adipose-specific Ihh<sup>flox/flox</sup> mice
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Perform integrative analysis of ATAC-seq, DNA methylation arrays, and RNA-seq
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Validate findings in human adipose biopsies (n=45 paired samples)
Product Science Overview
Indian Hedgehog is a secreted protein that plays a pivotal role in the regulation of chondrocyte differentiation and proliferation, which are essential for bone growth and development . The human recombinant form of Indian Hedgehog is typically produced in E. coli and consists of the amino acid sequence Cys28-Gly202, with an N-terminal methionine . This recombinant protein is highly purified, with a purity greater than 97% as determined by SDS-PAGE under reducing conditions .
Ihh mRNA expression is detected in various tissues, including the fetal lung, gut, stomach, liver, kidney, pancreas, and strongly in cartilage, particularly in the growth regions of developing bones . The activity of recombinant Indian Hedgehog is measured by its ability to induce alkaline phosphatase production in C3H10T1/2 mouse embryonic fibroblast cells . This activity is crucial for its role in bone development and growth.
Indian Hedgehog has significant implications in both clinical and research settings. It is involved in the regulation of the Wnt signaling pathway, which is crucial in various cancers, including colon cancer . Additionally, Ihh, along with parathyroid hormone-related protein, regulates the rate of chondrocyte proliferation and differentiation, making it a vital component in studies related to bone growth and development .
The recombinant form of Indian Hedgehog is typically lyophilized from a filtered solution and can be reconstituted in sterile water containing at least 0.1% human or bovine serum albumin . It is stable for 12 months when stored at -20 to -70°C as supplied, and for up to 3 months after reconstitution under sterile conditions .
