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Isolation of Mouse Epidermal Keratinocytes and Their In Vitro Clonogenic Culture
Published on: August 10, 2019
Dose- and time-dependent expression of transforming growth factor-beta 1 mRNA and protein in mouse epidermis and
J H Sherman1, M L Miller, R E Albert
1Department of Environmental Health, University of Cincinnatti College of Medicine, Ohio 45267-0056.
Abstract:
Topical weekly application of 64 micrograms of benzo[a]pyrene (BAP) for 4 wk induced transforming growth factor (TGF)-beta 1 mRNA in the epidermis of Swiss (ICR) mice, with a maximum at 6-12 h after the last treatment. The increase in TGF-beta 1 mRNA concentration was accompanied by an increase in immunohistochemically detectable intracellularly localized TGF-beta 1 protein in the suprabasal epidermis and by the appearance of extracellularly localized TGF-beta 1 in the basal layers. A dose rate of 16 micrograms/wk for 4 wk was unable to induce the same response. In contrast, after 20 weekly topical applications of 16 or 64 micrograms of BAP, an increase in TGF-beta 1 mRNA concentration and the appearance of extracellularly localized protein in the epidermis were observed. These changes in TGF-beta 1 expression were paralleled by changes in epidermal morphology. A similar group of animals treated with 4 micrograms of BAP/wk for 20 wk did not respond differently from untreated controls. Papillomas resulting from treatment with 16 or 64 micrograms of BAP/wk for 28 wk stained for intracellularly localized TGF-beta 1 predominantly in the differentiating and nondividing layers. Papillomas stained for extracellularly localized TGF-beta 1 solely in the less differentiated and dividing cells. These results suggest that tumorigenesis by BAP involves the induction of cumulative changes in epidermal TGF-beta 1 mRNA and protein concentrations as well as alterations in skin morphology associated with a tumor-promotion process.
Insights
Benzo[a]pyrene (BAP) exposure induces transforming growth factor-beta 1 (TGF-beta 1) in mouse skin, impacting epidermal morphology and promoting tumor development over time. This suggests cumulative changes in TGF-beta 1 are key to BAP-induced skin tumorigenesis.
Area of Science:
- Toxicology
- Dermatology
- Molecular Biology
Background:
- Benzo[a]pyrene (BAP) is a polycyclic aromatic hydrocarbon found in environmental pollutants.
- Transforming growth factor-beta 1 (TGF-beta 1) plays a critical role in cell growth, differentiation, and tissue repair.
- Understanding the molecular mechanisms of BAP-induced skin tumorigenesis is crucial for public health.
Purpose of the Study:
- To investigate the effect of topical BAP application on TGF-beta 1 expression in mouse epidermis.
- To correlate changes in TGF-beta 1 with epidermal morphology and tumor formation.
- To elucidate the role of TGF-beta 1 in BAP-induced skin tumorigenesis.
Main Methods:
- Swiss (ICR) mice were treated topically with varying doses of BAP weekly for 4 or 20 weeks.
- TGF-beta 1 mRNA levels were quantified using RT-PCR.
- TGF-beta 1 protein localization was assessed via immunohistochemistry.
- Epidermal morphology and papilloma development were histologically examined.
Main Results:
- Weekly topical BAP application (64 micrograms) induced TGF-beta 1 mRNA and protein in mouse epidermis within hours.
- Higher BAP doses (16 or 64 micrograms/week) over 20 weeks led to increased TGF-beta 1 mRNA and extracellular protein, alongside morphological changes.
- Papillomas showed distinct intracellular and extracellular TGF-beta 1 localization patterns correlating with cell differentiation and proliferation.
Conclusions:
- Tumorigenesis by BAP involves cumulative induction of epidermal TGF-beta 1 mRNA and protein.
- Altered TGF-beta 1 expression and epidermal morphology changes are associated with BAP-induced tumor promotion.
- TGF-beta 1 plays a significant role in the multi-step process of BAP-induced skin cancer development.
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