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Fate and plasticity of the epidermis in response to congenital activation of BRAF.

Krishnaswami SR, Kumar S, Ordoukhanian P, Yu BD - J. Invest. Dermatol. (2014)

Bottom Line: Germline and somatic mutations in RAS and its downstream effectors are found in several congenital conditions affecting the skin.However, restoration of epidermal differentiation was non-cell autonomous and required dermal tissue to be present in tissue recombination studies.These studies indicate that early activation of the RAF signaling pathway in the ectoderm has effects on specific steps of epidermal differentiation, which may be amenable to treatment with currently available pharmacologic inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Division of Dermatology, Department of Medicine, Institute for Genomic Medicine, Stem Cell Program, University of California, San Diego, La Jolla, California, USA.

ABSTRACT
Germline and somatic mutations in RAS and its downstream effectors are found in several congenital conditions affecting the skin. Here we demonstrate that activation of BRAF in the embryonic mouse ectoderm triggers both craniofacial and skin defects, including hyperproliferation, loss of spinous and granular keratinocyte differentiation, and cleft palate. RNA sequencing of the epidermis confirmed these findings but unexpectedly revealed evidence of continued epidermal maturation, expression of >80% of epidermal differentiation complex genes, and formation of a hydrophobic barrier. Spinous and granular differentiation were restored by pharmacologic inhibition of MAPK/ERK kinase or BRAF. However, restoration of epidermal differentiation was non-cell autonomous and required dermal tissue to be present in tissue recombination studies. These studies indicate that early activation of the RAF signaling pathway in the ectoderm has effects on specific steps of epidermal differentiation, which may be amenable to treatment with currently available pharmacologic inhibitors.

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Rescue of spinous and granular keratinocyte expression by pharmacologic inhibition of BRAF or MEKWildtype and K14-cre; BrafV600E explants were treated with PLX4720, U0126 or vehicle for 24 hrs. (a) Real-time qPCR of Dusp6, a transcriptional target of RAS/MAPK signaling, and differentiation markers (Krt1, Krt10, Lor) in E17.5 skin after treatment with varying doses of PLX4720. (b) Real-time PCR of Dusp6, Krt 1, Krt10, and Lor gene expression in wildtype and K14-cre; BrafV600E skin explants with U0126. (c, d) Immunostaining of (c) K10 and K14 or (d) LOR and K14 prior to culture and after culture in vehicle, 100 µM PLX4720 or U0126. Error bars represent SD of a minimum of 3 samples for each condition. Scale bars, 20 µm.
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Figure 4: Rescue of spinous and granular keratinocyte expression by pharmacologic inhibition of BRAF or MEKWildtype and K14-cre; BrafV600E explants were treated with PLX4720, U0126 or vehicle for 24 hrs. (a) Real-time qPCR of Dusp6, a transcriptional target of RAS/MAPK signaling, and differentiation markers (Krt1, Krt10, Lor) in E17.5 skin after treatment with varying doses of PLX4720. (b) Real-time PCR of Dusp6, Krt 1, Krt10, and Lor gene expression in wildtype and K14-cre; BrafV600E skin explants with U0126. (c, d) Immunostaining of (c) K10 and K14 or (d) LOR and K14 prior to culture and after culture in vehicle, 100 µM PLX4720 or U0126. Error bars represent SD of a minimum of 3 samples for each condition. Scale bars, 20 µm.

Mentions: Therapeutic inhibition of c-RAF, BRAF, and MEK1/2 is a major target of cancer drug discovery and, in rare childhood disorders, could provide potential therapies. To explore this possibility, a mouse embryonic explant system was tested in which E17.5–E18.5 skin biopsies were cultured for 24 hours in media containing inhibitors of BRAF or MEK1/2 (Fig. 4). PLX4720 is an inhibitor of BRAF and demonstrates ten-fold higher affinity for BRAFV600E than wildtype BRAF (Bollag et al., 2010; Tsai et al., 2008). Paradoxically, low doses of PLX4720 (<1 µM) lead to compensatory activation of c-RAF and MEK1/2 and proliferative responses including keratoacanthomas and squamous cell carcinomas (Chapman et al., 2011). High doses (>10µM) of PLX4720 and other BRAF inhibitors are necessary to suppress RAF paralogs and MEK(Poulikakos et al., 2010). To assess BRAF inhibition in small amounts of explanted tissues, we assessed the levels of a downstream target gene, Dusp6, which is regulated by RAS/MAPK signaling. Dusp6 was elevated in K14-cre; BrafV600E epidermis, and downregulated in the presence of high dose (>50µM) PLX7420 (Fig. 4a, left panel) (Bloethner et al., 2005). We next assessed the effect of BRAF inhibition on early differentiation (Fig. 4a, right panel) and found increased K1, K10, and Lor expression in K14-cre; BrafV600E epidermis (P=0.006) and in wildtype littermate epidermis.


Fate and plasticity of the epidermis in response to congenital activation of BRAF.

Krishnaswami SR, Kumar S, Ordoukhanian P, Yu BD - J. Invest. Dermatol. (2014)

Rescue of spinous and granular keratinocyte expression by pharmacologic inhibition of BRAF or MEKWildtype and K14-cre; BrafV600E explants were treated with PLX4720, U0126 or vehicle for 24 hrs. (a) Real-time qPCR of Dusp6, a transcriptional target of RAS/MAPK signaling, and differentiation markers (Krt1, Krt10, Lor) in E17.5 skin after treatment with varying doses of PLX4720. (b) Real-time PCR of Dusp6, Krt 1, Krt10, and Lor gene expression in wildtype and K14-cre; BrafV600E skin explants with U0126. (c, d) Immunostaining of (c) K10 and K14 or (d) LOR and K14 prior to culture and after culture in vehicle, 100 µM PLX4720 or U0126. Error bars represent SD of a minimum of 3 samples for each condition. Scale bars, 20 µm.
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Related In: Results  -  Collection

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Figure 4: Rescue of spinous and granular keratinocyte expression by pharmacologic inhibition of BRAF or MEKWildtype and K14-cre; BrafV600E explants were treated with PLX4720, U0126 or vehicle for 24 hrs. (a) Real-time qPCR of Dusp6, a transcriptional target of RAS/MAPK signaling, and differentiation markers (Krt1, Krt10, Lor) in E17.5 skin after treatment with varying doses of PLX4720. (b) Real-time PCR of Dusp6, Krt 1, Krt10, and Lor gene expression in wildtype and K14-cre; BrafV600E skin explants with U0126. (c, d) Immunostaining of (c) K10 and K14 or (d) LOR and K14 prior to culture and after culture in vehicle, 100 µM PLX4720 or U0126. Error bars represent SD of a minimum of 3 samples for each condition. Scale bars, 20 µm.
Mentions: Therapeutic inhibition of c-RAF, BRAF, and MEK1/2 is a major target of cancer drug discovery and, in rare childhood disorders, could provide potential therapies. To explore this possibility, a mouse embryonic explant system was tested in which E17.5–E18.5 skin biopsies were cultured for 24 hours in media containing inhibitors of BRAF or MEK1/2 (Fig. 4). PLX4720 is an inhibitor of BRAF and demonstrates ten-fold higher affinity for BRAFV600E than wildtype BRAF (Bollag et al., 2010; Tsai et al., 2008). Paradoxically, low doses of PLX4720 (<1 µM) lead to compensatory activation of c-RAF and MEK1/2 and proliferative responses including keratoacanthomas and squamous cell carcinomas (Chapman et al., 2011). High doses (>10µM) of PLX4720 and other BRAF inhibitors are necessary to suppress RAF paralogs and MEK(Poulikakos et al., 2010). To assess BRAF inhibition in small amounts of explanted tissues, we assessed the levels of a downstream target gene, Dusp6, which is regulated by RAS/MAPK signaling. Dusp6 was elevated in K14-cre; BrafV600E epidermis, and downregulated in the presence of high dose (>50µM) PLX7420 (Fig. 4a, left panel) (Bloethner et al., 2005). We next assessed the effect of BRAF inhibition on early differentiation (Fig. 4a, right panel) and found increased K1, K10, and Lor expression in K14-cre; BrafV600E epidermis (P=0.006) and in wildtype littermate epidermis.

Bottom Line: Germline and somatic mutations in RAS and its downstream effectors are found in several congenital conditions affecting the skin.However, restoration of epidermal differentiation was non-cell autonomous and required dermal tissue to be present in tissue recombination studies.These studies indicate that early activation of the RAF signaling pathway in the ectoderm has effects on specific steps of epidermal differentiation, which may be amenable to treatment with currently available pharmacologic inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Division of Dermatology, Department of Medicine, Institute for Genomic Medicine, Stem Cell Program, University of California, San Diego, La Jolla, California, USA.

ABSTRACT
Germline and somatic mutations in RAS and its downstream effectors are found in several congenital conditions affecting the skin. Here we demonstrate that activation of BRAF in the embryonic mouse ectoderm triggers both craniofacial and skin defects, including hyperproliferation, loss of spinous and granular keratinocyte differentiation, and cleft palate. RNA sequencing of the epidermis confirmed these findings but unexpectedly revealed evidence of continued epidermal maturation, expression of >80% of epidermal differentiation complex genes, and formation of a hydrophobic barrier. Spinous and granular differentiation were restored by pharmacologic inhibition of MAPK/ERK kinase or BRAF. However, restoration of epidermal differentiation was non-cell autonomous and required dermal tissue to be present in tissue recombination studies. These studies indicate that early activation of the RAF signaling pathway in the ectoderm has effects on specific steps of epidermal differentiation, which may be amenable to treatment with currently available pharmacologic inhibitors.

Show MeSH
Related in: MedlinePlus