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Updated: May 12, 2026
Generation and Culturing of Primary Human Keratinocytes from Adult Skin
Published on: December 22, 2017
Arsenite suppression of BMP signaling in human keratinocytes
Marjorie A Phillips1, Qin Qin, Qin Hu
1Department of Environmental Toxicology, University of California, Davis, CA 95616-8588, USA.
Abstract:
Arsenic, a human skin carcinogen, suppresses differentiation of cultured keratinocytes. Exploring the mechanism of this suppression revealed that BMP-6 greatly increased levels of mRNA for keratins 1 and 10, two of the earliest differentiation markers expressed, a process prevented by co-treatment with arsenite. BMP also stimulated, and arsenite suppressed, mRNA for FOXN1, an important transcription factor driving early keratinocyte differentiation. Keratin mRNAs increased slowly after BMP-6 addition, suggesting they are indirect transcriptional targets. Inhibition of Notch1 activation blocked BMP induction of keratins 1 and 10, while FOXN1 induction was largely unaffected. Supporting a requirement for Notch1 signaling in keratin induction, BMP increased levels of activated Notch1, which was blocked by arsenite. BMP also greatly decreased active ERK, while co-treatment with arsenite maintained active ERK. Inhibition of ERK signaling mimicked BMP by inducing keratin and FOXN1 mRNAs and by increasing active Notch1, effects blocked by arsenite. Of 6 dual-specificity phosphatases (DUSPs) targeting ERK, two were induced by BMP unless prevented by simultaneous exposure to arsenite and EGF. Knockdown of DUSP2 or DUSP14 using shRNAs greatly reduced FOXN1 and keratins 1 and 10 mRNA levels and their induction by BMP. Knockdown also decreased activated Notch1, keratin 1 and keratin 10 protein levels, both in the presence and absence of BMP. Thus, one of the earliest effects of BMP is induction of DUSPs, which increases FOXN1 transcription factor and activates Notch1, both required for keratin gene expression. Arsenite prevents this cascade by maintaining ERK signaling, at least in part by suppressing DUSP expression.
Insights
Arsenic exposure prevents skin cell differentiation by blocking BMP-6 signaling. This mechanism involves maintaining ERK signaling, which suppresses key factors like FOXN1 and Notch1, crucial for keratin production.
Area of Science:
- Dermatology
- Molecular Biology
- Toxicology
Background:
- Arsenic is a known human skin carcinogen.
- Arsenic exposure suppresses keratinocyte differentiation, a critical process for skin barrier function.
- The precise molecular mechanisms underlying arsenic's effects on keratinocyte differentiation are not fully understood.
Purpose of the Study:
- To elucidate the mechanism by which arsenic suppresses keratinocyte differentiation.
- To investigate the role of Bone Morphogenetic Protein-6 (BMP-6) and its downstream signaling pathways in this process.
- To identify key molecular players involved in BMP-6-mediated keratinocyte differentiation and how arsenic interferes.
Main Methods:
- Primary human keratinocytes were cultured and treated with BMP-6 and/or arsenite.
- Messenger RNA (mRNA) levels for keratins 1 and 10, and FOXN1 were quantified using RT-qPCR.
- Notch1 activation and Extracellular signal-regulated Kinase (ERK) signaling pathways were assessed.
- Dual-specificity phosphatases (DUSPs) involved in ERK regulation were analyzed.
- Knockdown of specific DUSPs (DUSP2, DUSP14) was performed using shRNAs.
Main Results:
- BMP-6 treatment increased mRNA levels of keratins 1, 10, and FOXN1, indicating promotion of differentiation.
- Arsenite co-treatment blocked BMP-6-induced increases in keratin and FOXN1 mRNA.
- BMP-6 increased activated Notch1 and decreased active ERK; arsenite reversed these effects.
- BMP-6 induced DUSP2 and DUSP14 expression, which was suppressed by arsenite.
- Knockdown of DUSP2 or DUSP14 reduced keratin and FOXN1 mRNA levels and Notch1 activation.
Conclusions:
- BMP-6 initiates keratinocyte differentiation by inducing DUSP expression, leading to FOXN1 transcription factor activation and Notch1 signaling.
- Arsenic disrupts this differentiation cascade by maintaining ERK signaling, partly through suppressing DUSP expression.
- Understanding this pathway provides insight into arsenic's carcinogenicity and potential therapeutic targets.
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