The Expression of Involucrin, Loricrin, and Filaggrin in Cultured Sebocytes

Abstract

Dear Editor: It is well established that a complex interplay of corneocytes and intercellular lipids in the stratum corneum of the skin is responsible for the skin barrier against environmental factors. A cornified cell envelope, which is composed of involucrin, loricrin, filaggrin, and other proteins, is a component of fully differentiated epidermal keratinocytes and corneocytes, and it is important in the skin barrier1. Although sebaceous gland sebocytes are considered to originate from the same stem cells as epidermal keratinocyte, it is not clear whether the keratinocyte differentiation markers are expressed in the sebocytes or not2. Because the transfollicular route has been known as a major route for drug delivery, more information is required to investigate the expression of the markers in the sebaceous gland sebocytes. A primary culture of human scalp sebocytes was performed with Dulbecco's modified Eagle's medium (DMEM; Gibco BRL, Grand Island, NY, USA) and Epilife (MEPI500CA; Gibco BRL) according to a method described previously3. Sebocytes were cultured at various concentrations of calcium (0.25, 0.5, 1, and 1.2 mM), or treated with vitamin D (10-10, 10-9, 10-8, 10-7, and 10-6 M). Reverse transcription polymerase chain reaction (RT-PCR) for involucrin, loricrin, and filaggrin was conducted in triplicate using the first strand cDNA synthesis kit (Promega, Madison, WI, USA) and oligonucleotide primers (Genotech, Daejeon, Korea). RT-PCR amplification was conducted using the GoTaq Flexi DNA Polymerase: involucrin for 34 cycles at 59℃, loricrin 34 cycles at 69℃, and filaggrin for 30 cycles at 58℃. Immunocytofluorescence for involucrin, loricrin, and filaggrin (Sigma-Aldrich, St. Louis, MO, USA) was also performed on cultured sebocytes. In this study, it was revealed that the expression of involucrin, loricrin, and filaggrin in cultured sebocytes was very weak (Fig. 1, ​,2).2). In addition, their expression in cultured sebocytes was not changed according to calcium concentration or after treatment with vitamin D (Fig. 2). Fig. 1 The expression of involucrin (A), loricrin (B), and filaggrin (C) was very weak in the cytoplasm of cultured sebocytes in immunocytofluorescence. (D) Control (A~D: ×200). Fig. 2 Gene expression of involucrin (A), loricrin (B), and filaggrin (C) in cultured sebocytes after treatment with calcium and VitD (vitamin D), respectively. Cont: control. Sebocytes are highly specialized, lipid-producing epithelial cells that release their contents by a rupture of the cell membrane and cellular degradation during differentiation4. Keratinocyte differentiating markers, including involucrin, loricrin, and filaggrin, provide structural support to the cell. Although sebocytes originate from the same stem cells as keratinocytes, it is thought that there are many differences in the expression of keratinocyte differentiating markers in sebocytes. In addition, it is recognized by our previous study (not published) that sebaceous gland sebocytes in vivo, both basal proliferating cells and central differentiating cells, show little expression of involucrin, loricrin, and filaggrin. Doran et al.5 reported that sebocytes did not produce cornified envelopes in vitro and could only be induced to produce small quantities (less than 5%) of envelopes with a calcium ionophore. However, Lo Celso et al.6 observed the presence of cornifin and involucrin positive immortalized sebocytes. This study showed very weak expression of involucrin, loricrin, and filaggrin in the cytoplasm of cultured sebocytes. Like cultured epidermal keratinocytes, proliferation and differentiation of cultured sebocytes are influenced by extracellular calcium concentration. Vitamin D also induces time- and dose-dependent modulation of cell proliferation and lipid content in cultured sebocytes through binding to vitamin D receptors. Nevertheless, this study showed that both extracellular calcium concentration and treatment with vitamin D did not affect the expression of involucrin, loricrin, and filaggrin in cultured sebocytes. In conclusion, cultured sebocytes showed little expression of keratinocyte differentiation markers. Weak physical barrier in the sebaceous gland may cause much better transfollicular drug delivery.