Preparation of Primary Cultures of Mouse Epidermal Keratinocytes and the Measurement of Phospholipase D Activity

Bailey, Lakiea; Choudhary, Vivek; Merai, Purnima; Bollag, Wendy B.

doi:10.1007/7651_2014_80

Cited by 8 publications

(6 citation statements)

References 47 publications

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“…Primary cultures of mouse epidermal keratinocytes were prepared from ICR strain CD-1 outbred neonatal mice 1e3 days old or from tail skin as described by Bailey et al (2014). RAW264.7 cells (from American Type Culture Collection) were cultured in DMEM containing 10% fetal bovine serum and 1% penicillin and streptomycin.…”

Section: Keratinocyte Preparation and Cell Culturementioning

confidence: 99%

Phosphatidylglycerol Inhibits Toll-Like Receptor–Mediated Inflammation by Danger-Associated Molecular Patterns

Choudhary

Uaratanawong

Patel

et al. 2019

Journal of Investigative Dermatology

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Psoriasis is a common skin disorder characterized by hyperproliferation and aberrant differentiation of epidermal keratinocytes and inflammation. We previously showed that phosphatidylglycerol (PG) can regulate keratinocyte function and suppress skin inflammation. Based on data suggesting that PG can inhibit toll-like receptor (TLR) activation induced by microorganisms and their components, we determined whether PG can inhibit TLR activation in response to antimicrobial peptides. These peptides, which are up-regulated in psoriasis, are known to function as danger-associated molecular patterns (i.e., DAMPs) to activate TLRs and the innate immune system. Because S100A9 is elevated in psoriatic skin and in animal models of psoriasis, we selected S100A9 as a representative antimicrobial peptide DAMP. We showed that in primary keratinocytes and a macrophage cell line, PG suppressed inflammatory mediator production induced by recombinant S100A9 functioning through both TLR2 and TLR4. In addition, PG, but not phosphatidylcholine, inhibited downstream S100A9-elicited TLR2 and NF-kB activation. These results, to our knowledge previously unreported, show PG's ability to inhibit DAMP-induced TLR activation, thereby reducing inflammatory signals. In addition, topical PG ameliorated skin lesions and inflammation in a mouse model of psoriasis. Together, these results suggest the possibility of developing PG as a therapy for psoriasis.

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Section: Keratinocyte Preparation and Cell Culturementioning

confidence: 99%

Phosphatidylglycerol Inhibits Toll-Like Receptor–Mediated Inflammation by Danger-Associated Molecular Patterns

Choudhary

Uaratanawong

Patel

et al. 2019

Journal of Investigative Dermatology

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“…Mouse keratinocytes were isolated from the skin of newborn ICR CD1 mice and cultured as described previously [ 63 ], according to protocols approved by the Institutional Care and Use Committees of Augusta University (Protocol #2017-0915) and the Charlie Norwood VA Medical Center (Protocol #22-04-135). Keratinocytes were treated with 0, 2.0 µg/mL LPS (Sigma-Aldrich, St. Louis, MO, USA), or 2.5 µg/mL Pam 3 CSK 4 (EMD Millipore Corporation, Burlington, MA, USA) in the presence or absence of 1 µM SAHA (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C with 5% CO 2 for 24 h; or with 0, 50, or 100 µg/mL AGE-BSA (Sigma-Aldrich or BioVision Incorporated, Milpitas, CA, USA), in the presence or absence of 1 or 2 µM SAHA (based on our previous studies [ 40 ]) at 37 °C with 5% CO 2 for 24, 48, and 72 h. Doses of AGEs were selected based on previous literature [ 54 , 64 , 65 , 66 ].…”

Section: Methodsmentioning

confidence: 99%

Advanced Glycation End Products and Activation of Toll-like Receptor-2 and -4 Induced Changes in Aquaporin-3 Expression in Mouse Keratinocytes

Luo

Uaratanawong

Choudhary

et al. 2023

IJMS

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Prolonged inflammation and impaired re-epithelization are major contributing factors to chronic non-healing diabetic wounds; diabetes is also characterized by xerosis. Advanced glycation end products (AGEs), and the activation of toll-like receptors (TLRs), can trigger inflammatory responses. Aquaporin-3 (AQP3) plays essential roles in keratinocyte function and skin wound re-epithelialization/re-generation and hydration. Suberanilohydroxamic acid (SAHA), a histone deacetylase inhibitor, mimics the increased acetylation observed in diabetes. We investigated the effects of TLR2/TLR4 activators and AGEs on keratinocyte AQP3 expression in the presence and absence of SAHA. Primary mouse keratinocytes were treated with or without TLR2 agonist Pam3Cys-Ser-(Lys)4 (PAM), TLR4 agonist lipopolysaccharide (LPS), or AGEs, with or without SAHA. We found that (1) PAM and LPS significantly upregulated AQP3 protein basally (without SAHA) and PAM downregulated AQP3 protein with SAHA; and (2) AGEs (100 µg/mL) increased AQP3 protein expression basally and decreased AQP3 levels with SAHA. PAM and AGEs produced similar changes in AQP3 expression, suggesting a common pathway or potential crosstalk between TLR2 and AGEs signaling. Our findings suggest that TLR2 activation and AGEs may be beneficial for wound healing and skin hydration under normal conditions via AQP3 upregulation, but that these pathways are likely deleterious in diabetes chronically through decreased AQP3 expression.

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“…Primary murine epidermal keratinocytes were prepared from 1 to 3 day old neonatal ICR CD-1 outbred mice as described in [11]. Treatment of mice conformed to policies in the Guide for the Care and Use of Laboratory Animals and monitored by the Institutional Animal Care and Use Committee (IACUC) of Augusta University.…”

Section: Culture Of Primary Mouse Keratinocytesmentioning

confidence: 99%

Possible Role of Phosphatidylglycerol-Activated Protein Kinase C-βII in Keratinocyte Differentiation

Bailey¹,

Choudhary²,

Bollag³

2017

TODJ

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Background: The epidermis is a continuously regenerating tissue maintained by a balance between proliferation and differentiation, with imbalances resulting in skin disease. We have previously found that in mouse keratinocytes, the lipid-metabolizing enzyme phospholipase D2 (PLD2) is associated with the aquaglyceroporin, aquaporin 3 (AQP3), an efficient transporter of glycerol. Our results also show that the functional interaction of AQP3 and PLD2 results in increased levels of phosphatidylglycerol (PG) in response to an elevated extracellular calcium level, which triggers keratinocyte differentiation. Indeed, we showed that directly applying PG can promote keratinocyte differentiation. Objective: We hypothesized that the differentiative effects of this PLD2/AQP3/PG signaling cascade, in which AQP3 mediates the transport of glycerol into keratinocytes followed by its PLD2-catalyzed conversion to PG, are mediated by protein kinase CβII (PKCβII), which contains a PG-binding domain in its carboxy-terminus. Method: To test this hypothesis we used quantitative RT-PCR, western blotting and immunocytochemistry. Results: We first verified the presence of PKCβII mRNA and protein in mouse keratinocytes. Next, we found that autophosphorylated (activated) PKCβII was redistributed upon treatment of keratinocytes with PG. In the unstimulated state phosphoPKCβII was found in the cytosol and perinuclear area; treatment with PG resulted in enhanced phosphoPKCβII localization in the perinuclear area. PG also induced translocation of phosphoPKCβII to the plasma membrane. In addition, we observed that overexpression of PKCβII enhanced calcium- and PG-induced keratinocyte differentiation without affecting calcium-inhibited keratinocyte proliferation. Conclusion: These results suggest that the PG produced by the PLD2/AQP3 signaling module may function by activating PKCβII.

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Preparation of Primary Cultures of Mouse Epidermal Keratinocytes and the Measurement of Phospholipase D Activity

Cited by 8 publications

References 47 publications

Phosphatidylglycerol Inhibits Toll-Like Receptor–Mediated Inflammation by Danger-Associated Molecular Patterns

Phosphatidylglycerol Inhibits Toll-Like Receptor–Mediated Inflammation by Danger-Associated Molecular Patterns

Advanced Glycation End Products and Activation of Toll-like Receptor-2 and -4 Induced Changes in Aquaporin-3 Expression in Mouse Keratinocytes

Possible Role of Phosphatidylglycerol-Activated Protein Kinase C-βII in Keratinocyte Differentiation

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