Primary Mouse Keratinocyte Culture

Pirrone, Annalisa; Hager, Barbara; Fleckman, Philip

doi:10.1385/1-59259-830-7:003

Cited by 26 publications

(30 citation statements)

References 4 publications

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“…Isolation and culture of primary mouse keratinocyte. Primary mouse epidermal keratinocytes were isolated from neonates (0 -2 days old) according to previously described protocols, with slight modifications (30). Briefly, pups were decapitated with limbs and tail removed.…”

Section: Methodsmentioning

confidence: 99%

Myostatin-null mice exhibit delayed skin wound healing through the blockade of transforming growth factor-β signaling by decorin

Zhang

Tan

McFarlane

et al. 2012

American Journal of Physiology-Cell Physiology

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Section: Methodsmentioning

confidence: 99%

Myostatin-null mice exhibit delayed skin wound healing through the blockade of transforming growth factor-β signaling by decorin

Zhang

Tan

McFarlane

et al. 2012

American Journal of Physiology-Cell Physiology

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“…HaCaT keratinocytes were subsequently infected with the lentivirus and harvested for ChIP 48 h postinfection. Primary mouse keratinocytes were harvested from neonates essentially as described (37) and cultured in Keratinocyte SFM (Invitrogen, Carlsbad, CA). At confluence CaCl 2 was added to the media to a final concentration of 1.2 mM for 48 h.…”

Section: Methodsmentioning

confidence: 99%

Epidermal terminal differentiation depends on B lymphocyte-induced maturation protein-1

Magnúsdóttir

Kalachikov

Mizukoshi

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

141

158

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The cornified layer is a compacted lattice of lipid-embedded corneocytes that provides an organism's barrier to the external environment. Cornification is the final differentiative step for epidermal keratinocytes and involves dramatic cell condensation before death. Using conditional gene deletion in mice, we identified the transcriptional repressor Blimp-1 (B lymphocyte-induced maturation protein-1) as an important regulator of keratinocyte transition from the granular to the cornified layer. More than 250 genes are misregulated in conditional knockout epidermis, including those encoding transcription factors, signal transduction components, proteinases, and enzymes involved in lipid metabolism. Steady-state mRNA and ChIP analyses of a subset of these genes provide evidence that nfat5, fos, prdm1, and dusp16 are novel direct targets of Blimp-1. Identifying nfat5 as a target of Blimp-1 repression indicates that cornification involves suppression of normal osmotic regulation in granular cells. Consistently, conditional knockout mice have delayed barrier formation as embryos, enlarged granular layer cells and corneocytes, and a morphologically abnormal cornified layer. These studies provide insight into cornification, identifying transcriptional regulatory circuitry and indicating the importance of blocking osmotic homeostasis.cornification ͉ epidermis ͉ prdm1 ͉ transcription factors ͉ nfat5

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“…After euthanasia, limbs and tails were removed, a dorsal cut was made at the neck down to the hypodermis and was extended to the tail stump, and then the skin was peeled off in one piece using dissecting forceps ( 17 ). This skin sample, which contains dermis and epidermis, is referred throughout this article as an "embryo whole-skin peel."…”

Section: Skin Epidermis and Lipid Isolationmentioning

confidence: 99%

Endogenous β-glucocerebrosidase activity in Abca12epidermis elevates ceramide levels after topical lipid application but does not restore barrier function

Haller

Cavallaro

Hernandez

et al. 2014

Journal of Lipid Research

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Supplementary key words glucosylceramide • harlequin ichthyosis • skin permeability barrier • ABCA12 antibodyHarlequin ichthyosis (HI) is a severe autosomal recessive disease caused by mutations in the ABCA12 lipid transporter ( 1,2 ). HI patients present with a drastically hyperkeratotic epidermis and have a complete loss of the skin permeability barrier function. Poor temperature regulation, enhanced water loss, and bacterial super-infections develop as a consequence of defects in the barrier function of HI skin, making neonatal survival diffi cult without intensive treatment ( 3-6 ).The epidermis is responsible for the formation and maintenance of the skin barrier function ( 7 ). A critical component of this barrier is the extracellular lipid domains that surround the corneocytes of the stratum corneum (SC). These interstitial lipid domains are organized in lamellar structures and consist primarily of cholesterol, fatty acid, and ceramides ( 8 ). Ceramides comprise about half of the total lipids in the SC and are essential for the lamellar structure of this extracellular lipid domain ( 8-10 ). They are synthesized exclusively from glucosylceramide (GlcCer) and sphingomyelin (SM) precursors, which are generated in nucleated keratinocytes and stored in lamellar bodies (LB). As the keratinocyte matures, the contents of the LB are extruded into the interstices of the SC where the GlcCer and SM are enzymatically hydrolyzed to Cer by ␤ -glucocerebrosidase (GCase) and sphingomyelinase Abstract ABCA12 mutations disrupt the skin barrier and cause harlequin ichthyosis. We previously showed Abca12 ؊ / ؊ skin has increased glucosylceramide (GlcCer) and correspondingly lower amounts of ceramide (Cer). To examine why loss of ABCA12 leads to accumulation of GlcCer, de novo sphingolipid synthesis was assayed using [ 14 C]serine labeling in ex vivo skin cultures. A defect was found in ␤ -glucocerebrosidase (GCase) processing of newly synthesized GlcCer species. This was not due to a decline in GCase function. Abca12 ؊ / ؊ epidermis had 5-fold more GCase protein (n = 4, P < 0.01), and a 5-fold increase in GCase activity (n = 3, P < 0.05). As with Abca12 +/+ epidermis, immunostaining in null skin showed a typical interstitial distribution of the GCase protein in the Abca12 ؊ / ؊ stratum corneum. Hence, we tested whether the block in GlcCer conversion could be circumvented by topically providing GlcCer. This approach restored up to 15% of the lost Cer products of GCase activity in the Abca12 ؊ / ؊ epidermis. However, this level of barrier ceramide replacement did not signifi cantly reduce transepidermal water loss function. Our results indicate loss of ABCA12 function results in a failure of precursor GlcCer substrate to productively interact with an intact GCase enzyme, and they support a model of ABCA12 function that is critical for transporting GlcCer into lamellar bodies.

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Primary Mouse Keratinocyte Culture

Cited by 26 publications

References 4 publications

Myostatin-null mice exhibit delayed skin wound healing through the blockade of transforming growth factor-β signaling by decorin

Myostatin-null mice exhibit delayed skin wound healing through the blockade of transforming growth factor-β signaling by decorin

Epidermal terminal differentiation depends on B lymphocyte-induced maturation protein-1

Endogenous β-glucocerebrosidase activity in Abca12epidermis elevates ceramide levels after topical lipid application but does not restore barrier function

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