Development of an in vitro submerged culture system to synthesize epidermal ceramides in canine keratinocytes

https://doi.org/10.1016/j.rvsc.2020.02.011Get rights and content

Highlights

  • Canine keratinocytes in this culture system had stratified in conjunction with the appearance of the stratum granulosum

  • Synthesis of ceramide classes were increased during stratification of keratinocytes

  • Ceramides synthesized in this culture system were corresponding to those found in canine stratum corneum

  • This culture system will serve as a useful tool for further analysis of ceramide metabolism in canine keratinocytes

Abstract

Ceramides (CERs) in the stratum corneum (SC) are known to play a crucial role in determining skin barrier function in dogs. We aimed to develop an in vitro culture system that synthesized epidermal CER classes to better understand the synthesis of CER classes in canine SC–. Canine keratinocyte cells (MSCEK) at appropriate confluency were incubated with high Ca2+ (1.8 mM CaCl2) supplemented serum-free medium. Eight days post Ca2+ application, the surface of cultured MSCEK was broadly stained with anti-loricrin antibody implying that the keratinocytes had stratified into stratum granulosum. MSCEK cells synthesized heterogenous epidermal CERs, similar to those seen during the stratification of canine keratinocytes. CER fractions obtained from MSCEK cells were comparable to those from canine SC, including CER[EOS] (combination of ω-hydroxy fatty acids and sphingosines), CER[NP] (combination of non-hydroxy fatty acids and phytosphingosines), and CER[EOP] (combination of ω-hydroxy fatty acids and phytosphingosines), all of which are lowered in the SC during canine atopic dermatitis. Thus, the present study provides a simple culture system as a tool for in-depth analysis of CER production in canine keratinocytes.

Introduction

The skin has an important role as an external barrier, protecting the body from harmful agents and water loss. The functional components of the skin are located in the uppermost layer, the stratum corneum (SC), which is embedded in extracellular lipids comprising free fatty acids, cholesterols, and ceramide (CER) (Meckfessel and Brandt, 2014). CER is a major component of extracellular lipids and has an amphiphilic structure, which is crucial for selective permeability (Meckfessel and Brandt, 2014; Bouwstra and Ponec, 2006).

Twelve classes of CER have been detected in human SC based on the composition of the head group and esterification of the fatty acid chain (Masukawa et al., 2008). These heterogeneous CER classes in human SC are mostly synthesized in the upper layer of the epidermis (Breiden et al., 2007; Feingold and Elias, 2014). In the stratum granulosum (SG), glycosylceramide and sphingomyelin are packed into lamellar bodies along with hydrolytic enzymes. In response to certain signals, such as elevation of Ca2+ concentration in granular layers, lamellar bodies secrete their contents into intercellular spaces (Proksch et al., 2008). Lipids derived from the lamellar bodies are subsequently converted into CERs.

Eleven classes of CER were discovered in canine SC, similar to those found in human SC (Yoon et al., 2011). In addition, during canine atopic dermatitis (AD), the amounts of CERs are reduced, which may contribute to aberrant skin barrier function (Shimada et al., 2009; Yoon et al., 2011). The amount of CER[EOS] (combination of ω-hydroxy fatty acids and sphingosines), CER[NP] (combination of non-hydroxy fatty acids and phytosphingosines), and CER[EOP] (combination of ω-hydroxy fatty acids and phytosphingosines) are significantly decreased in dogs with AD, as reported in humans suffering from the same condition (Yoon et al., 2011). CER[EOS] and CER[EOP] are acylCERs that contain long-chain amide-linked fatty acids. In vitro studies using reconstituted membrane models have demonstrated the importance of acylCERs in the organization of SC lipids into a long periodicity phase and the lateral packing of lipids. Therefore, acylCERs are thought to be critical for skin barrier function (Uchida and Holleran, 2008). In addition, CER[NP], whose levels are decreased in canine AD as well as human AD and psoriasis (Holleran et al., 2006), have also been reported to increase skin hydration and improve trans-epidermal water loss in older people with dry skin (Danby et al., 2016). Therefore, regulation of these CER classes is important to recover skin barrier function. CERs are generated following differentiation of keratinocytes; therefore, an in vitro keratinocyte culture system might be a useful tool to investigate CER production during keratinocyte stratification.

A novel canine epidermal keratinocyte cell line, MSCEK, has been established in our laboratory (Ide et al., 2010). MSCEK expresses major constructing proteins of the desmosomal component and cytokeratins that are predominantly expressed in epithelial cells. In the present study, we aimed to develop a simple in vitro submerged culture system which produces CER classes similar those of canine SC using MSCEK cells.

Section snippets

Cell culture

Canine keratinocyte cells (MSCEKs) were seeded (1.2 × 106/ml) onto 35 mm collagen-coated dishes and were cultured in William's medium E (Invitrogen Corp, Carlsbad, CA, USA) supplemented with 10% Nu-serum IV culture supplement (Collaborative Biochemical Products, Bedford, MA, USA), 5 ng/ml of epidermal growth factor (Sigma chemicals, St. Louis, MO, USA), and 1 nM of cholera toxin (Sigma), until confluent. The cells were then sub-cultured in Ca2+ supplemented serum-free William's medium E. To

Stratification of canine keratinocytes

Cultured MSCEK cells were observed under the light microscope and were seen to form a monolayer with spindle shape on day 0 after Ca2+ application. The layer became stratified with an upper layer of flattened cells following prolonged culture. Formation of SG in the stratified MSCEK cells was confirmed by histopathological analysis using an anti-loricrin antibody. Immunohistochemical analysis revealed that on day 0 post-Ca2+ application, MSCEK cells were minimally stained with the anti-loricrin

Discussion

To investigate heterogenous epidermal CERs in vitro, complex keratinocyte culture systems such as the air-exposed and organotypic culture systems are usually needed. The MSCEK cell line, which was previously established in our laboratory, expresses major constructing proteins of the desmosomal components and cytokeratins (Ide et al., 2010). In the present study, using serum-free high-Ca2+ (1.8 mM Ca2+) media, we could establish an in vitro submerged culture system to synthesize heterogenous

Conclusion

In the present study, we have developed an elegant in vitro culture system that mimics the CER composition found in canine SC. This culture system will serve as a useful tool for further testing and analysis of CER metabolism in canine keratinocytes.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Declaration of Competing Interest

The authors have no conflicting financial interests.

Acknowledgements

We would like to thank Editage (www.editage.co.kr) for English language editing.

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