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Hypoxia reveals the new function of Foxn1 in keratinocytes antioxidant defense system

ABSTRACT: Skin exposed to environmental threats including injuries and oxidative stress developed an efficient but not fully recognized systems of repair and antioxidant protection. In this project we provide evidence that Foxn1 in keratinocytes regulates elements of electron transport chain and participates in thioredoxin system (Txn2, Txnrd3, Srxn1) induction, particularly under hypoxic environment. It is shown that Foxn1 in keratinocytes upregulate glutathione thioredoxin reductase 3 (Txnrd3) protein, and high levels of Txnrd3 mRNA were detected in injured skin of Foxn1+/+ mice. It also shown that Foxn1 strongly downregulate CCN2 protein participating in reconstruction of the epidermis after injury. As in vitro assay revealed that Foxn1 controls keratinocytes migration stimulating it under normoxia and suppressing under hypoxia. Keratinocytes overexpressing Foxn1 and exposed to hypoxia showed reduced ability to promote angiogenesis by downregulation of Vegfa. In conclusion, this study showed a new mechanism in which Foxn1, along with hypoxia, participates in the activation of antioxidant defense and controls functional properties of keratinocytes.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Cell Culture, Keratinocyte

DISEASE(S): Wounds And Injuries

SUBMITTER: Mirva Pääkkönen

LAB HEAD: Barbara Gawrońska-Kozak

PROVIDER: PXD031125 | Pride | 2022-07-08

REPOSITORIES: Pride

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			Action	DRS
	1A_ker_Foxn1_21percent.raw	Raw
	1B_KCM_Foxn1_21percent.raw	Raw
	1a_ker_GFP_21percent.raw	Raw
	1b_KCM_GFP_21percent.raw	Raw
	2A_ker_Foxn1_21percent.raw	Raw

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Project description:MED1 (Mediator complex subunit 1) is expressed by human epidermal keratinocytes and functions as a coactivator of several transcription factors. To elucidate the role of MED1 in keratinocytes, we established keratinocyte-specific MED1-null (MED1epi-/-) mice using the K5Cre-LoxP system. To elucidate the mechanism(s) underlying abnormalities of keratinocytes derived from MED1epi-/- mice, we compared the gene expression patterns of MED1epi-/--derived keratinocytes with their wild type counterparts by microarray analysis. Generation of the MED1 conditional null mutation in epidermal keratinocytes and cell culture: MED1flox/flox mice (Jia et al, J Biol Chem 279:24427. 2004) were mated with K5Cre mice (Tarutani et al, Proc Natl Acad Sci U S A 94:7400. 1997) to generate F1 K5Cre+;MED1flox/+ mice. The K5Cre+;MED1flox/+ mice were then mated with MED1flox/flox mice to generate K5Cre+;MED1flox/flox mice (MED1epi-/-). Mice were screened for presence of the transgene by PCR using specific primers for the floxed MED1 gene and the human K5 gene according to the previous reports. Skin of newborn mice was excised after the mice were sacrificed with excessive anesthetic and were treated with dispase followed by trypsin to separate the epidermis from the dermis. Keratinocytes were seeded on type I collagen coated dishes, and were cultured in CnT07 conditioned culture medium (CELLnTEC, Bern, Switzerland). Keratinocytes were used for the experiment as a primary culture. All animal studies were conducted according to protocols approved by the Institutional Animal Care and Use Committee at Osaka University. Microarray analysis: For comprehensive comparison of gene expression patterns between keratinocytes derived from MED1epi-/- and from WT mice, microarray analysis was used. Total RNAs were extracted using an RNeasy kit (Qiagen, San Diego, CA). Then, 2μg total RNA was reverse transcribed to cDNA with T7 oligo d(T) primer (Affymetrix, Santa Clara, CA). The cDNA synthesis products were used for in vitro transcription reactions containing T7 RNA polymerase and biotinylated nucleotide analogue (pheudouridine base) cRNAs. The labeled cRNA products were then fragmented and loaded onto GeneChip(R) Mouse Genome 430 2.0 arrays (Affymetrix), and were hybridized according to the manufacturer’s protocol. Streptavidin-Phycoerythrin (Molecular Probes) was used as the fluorescent conjugate to detect hybridized target sequences. Raw intensity data from the GeneChip array were analyzed by GeneChip Operating Software (Affymetrix).

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Hypoxia reveals the new function of Foxn1 in keratinocytes antioxidant defense system

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