Expression of Desmosomal Proteins in Rat Keratinocytes during In Vitro Differentiation.

Abstract

ABSTRACT. The keratinocyte, the major component of the epidermis, expresses several proteins that characterize the keratinization during the differentiation. Proliferation and differentiation of cultured human keratinocytes are known to be regulated by the Ca 2+ concentration in the culture medium. However, informations about the rat keratinocyte are relatively limited and their physiology is still an open question. To elucidate the characteristics of the rat keratinocyte, we established rat keratinocyte culture system and examined effects of extracellular calcium concentration on the expression of differentiation-related proteins. Keratinocytes were isolated from the newborn rat skin with 0.25% trypsin, followed by separation with a Percoll density gradient. The separated cells were grown in MCDB153 medium containing several growth factors and Ca 2+ -free fetal bovine serum, then stimulated with Ca 2+ . Immunoblotting demonstrated strong expression of β1 integrin in unstimulated cells, suggesting that the primary culture of rat keratinocytes was successfully established. Expression of desmoglein and transglutaminase was increased by Ca 2+ stimulation, whereas β1 integrin expression was decreased in response to increasing concentrations of Ca 2+ . These observations indicate that cultured rat keratinocytes maintain the ability to differentiate in vitro, which is similar to that of the basal keratinocytes in the epidermis. KEY WORDS: cell culture, desmoglein, differentiation, integrin, keratinocyte.J. Vet. Med. Sci. 64(2): 123-127, 2002 The epidermis consists of multiple layers of keratinocytes. When keratinocytes of the basal layer withdraw from the cell cycle and become committed to the terminal differentiation, they detach from the basement membrane and migrate into the suprabasal layers [1,6,7,12]. During the differentiation, human keratinocytes express several proteins, transglutaminase and substrates of this enzyme such as involucrin and filaggrin, which form the cornified envelope in the keratinized layer of the epidermis. The structure of cell-cell adhesion also changes during the differentiation. Desmosome, a calcium-dependent intercellular adhesion structure, is known to be composed of transmembrane glycoproteins, e.g. desmoglein and desmocollin. These proteins bind to the cytoplasmic plaque proteins plakoglobin, desmoplakin, and are linked to keratin intermediate filaments [2,4,13]. The IgG autoantibodies against desmoglein-1 and -3 cause the epidermal blistering diseases such as pemphigus foliaceous and pemphigus vulgaris [5,[19][20][21] . Recent evidence in human keratinocytes showed these antigenitic epitopes of desmogleins are conformation-dependent [3] and calcium-dependent [22].Integrins are heterodimeric transmembrane receptor that consist of an α and a β subunit, and mediate the attachment of cells to the extracellular matrix or to other cells. Until now, sixteen α and eight β subunits and more than 20 different receptors have been identified. The ligand-binding specificity of an integrin is...

Trophic effects of keratinocytes on the axonal development of sensory neurons in a coculture model

“…Therefore, in order to reconstitute eventual interactions between nerve terminals and keratinocytes in culture, it was necessary to find experimental conditions in which undifferentiated keratinocytes were present in the culture. There is evidence from the literature that the maintenance of this state requires low (< 70 µ m ) concentrations of extracellular calcium both in vitro and in vivo (Menon et al ., 1985; Fang, 2000; Mochizuki et al ., 2002). In line with these findings, primary keratinocytes with a round morphology and expressing cytokeratin 14, which are typical indicators of an undifferentiated state of keratinocytes, were observed and survived well when cultured in MCDB medium containing 20 µ m calcium.…”

Proliferation, Cell Cycle Exit, and Onset of Terminal Differentiation in Cultured Keratinocytes: Pre-Programmed Pathways in Control of C-Myc and Notch1 Prevail Over Extracellular Calcium Signals

“…When epidermal keratinocytes from mouse, rat, and human are taken in culture, proliferation can be abrogated and terminal differentiation initiated by elevation of the calcium concentration in the culture medium (Hennings et al, 1980a, b;Yuspa et al, 1989;Pillai et al, 1990;Bikle et al, 2001;Mochizuki et al, 2001;Tu et al, 2001). Calcium is therefore often regarded as being both sufficient and indispensable for these processes, in particular, in mouse keratinocytes.…”

Microtubule-Actin Crosslinking Factor 1 and Plakins as Therapeutic Drug Targets

“…MACF1 and BPAG1 have both been described to play roles in cell migration and microtubule organization [ 10 , 11 , 12 , 13 , 14 ], while desmoplakin, envoplakin, and periplakin have been shown to play a prominent role in the development of the cornified envelop, a layer under the plasma membrane of keratinocytes in the outermost layers of the epidermis [ 4 , 15 ]. To date, desmoplakin is the best characterized plakin in terms of its numerous biological roles, which include involvement in myocardium morphogenesis [ 16 , 17 ], epidermis development [ 18 ], keratinocyte differentiation [ 19 , 20 ], and wound healing, in which MACF1 has also been shown to play a role [ 21 , 22 ].…”