Optimization of filaggrin expression and processing in cultured rat keratinocytes

In normal mammalian epidermis, cell division and expression of keratins K5/K14 in the basal layer is followed by K1/K10, the keratin bundling protein filaggrin, and cornified envelope proteins such as involucrin and loricrin [1,2]. However, hyperproliferative epidermal cells can be motile and phagocytic, and can sustain cell division in the suprabasal layers. The hyperproliferative differentiation phenotype, seen during wound healing or in certain disease states results in production of keratins K6/K16, high levels of involucrin, and parakeratotic cornified cells [3,4]. For elucidation of structures and functions of the different types of epidermal cells, keratinocyte culture models were developed [5]. These models of epidermal differentiation are complicated by the phenotypic plasticity of keratinocytes. In the extreme, this is represented by the “activated or hyperproliferative” vs “normal” differentiation programs. These programs are superficially similar in that a stratified epidermis with cornified cells forms, but each involves the expression of different structural proteins [6]. It appears that these two pathways are not mutually exclusive, so that cells can express a mixed phenotype in some situations. Terminal differentiation in both phenotypes involves degradation or remodeling of phospholipids, cytoplasm, cell organelles, and the nonkeratin cytoskeletal components [7]. Little is understood about the regulatory mechanisms for initiation of these global degradative processes. Lysosomal breakdown and release of lysosomal proteinases to the cytosol is unlikely, as there is no evidence that the terminally differentiating cell has a decreased pH and lysosomal enzymes are not very active at neutral pH. In fact, a major differentiation specific protein, cystatin, is a known inhibitor of lysosomal proteinases [8]. The enzymes responsible for specific processes in terminal differentiation have been shown to have neutral pH optima. For example, the cross-linking of proteins at the internal cytosolic face during formation of the cornified cell envelope involves a transglutaminase having a neutral pH optimum [9]. Proteolytic processing of profilaggrin in normal terminal differentiation apparently also involves a chymotrypsin-like proteinase having a neutral pH optimum [10]. More recently, it has been suggested that epidermal differentiation resembles apoptosis [11,12] with terminal differentiation being only a variation of these pathways involving activation of cytoplasmic and nuclear proteinases having neutral pH optima. Indeed, the morphology of epidermal cells indicates occurrence of orderly disassembly, although the precise similarity of these events to apoptosis is disputed [7]. Differentiation in cultured keratinocytes is less clearly defined [13]. Cultured keratinocytes predominantly follow the hyperproliferative differentiation pathway and are inefficient at the final steps in cornification. Pre-confluent cells produce basal cell keratins, consistent with their status as dividing cells, and are usually considered analogous to basal epidermal cells. These cells stratify at confluence and begin producing keratins K6/K16 and high levels of involucrin. Under some culture conditions, low amounts of K1/K10, loricrin and profilaggrin are produced, but processing of profilaggrin to filaggrin, thin cornified cell envelopes, and poor barrier function due to absence of specialized lipids are seen [14]. When cultured on reconstituted dermis at the air/medium interface, keratinocyte differentiation is more normal suggesting that growth and differentiation factor gradients are important in ordering the tissue and determining the balance between the two phenotypes [6,15]. Thus cultured cells have a mixed phenotype, and the balance between the two extremes can be modulated by external signals. A number of models are currently available for studying in-vitro keratinocyte differentiation. When cells become confluent in media with high calcium concentration, both cell-cell contact and increased calcium levels initiate differentiation [16]. A second approach is to induce keratinocytes to differentiate by suspending the proliferating cells in a matrix, typically methyl cellulose [17]. In a third approach, the organotypic method, [18] where the epithelium is cultured on a substrate at the air-liquid interface, the keratinocytes form layers representative of each stage of differentiation, much like that observed in vivo. Each approach has its own advantages and disadvantages. For example, in the first two approaches, differentiation can be observed in a relatively short period of time. The three dimensional characteristics of the organotypic culture provide the unique advantage of temporal and spatial study at the expense of technically difficult methods that limit the extent of studies. Several authors have used a confluence-induced differentiation model for the study of in vitro epidermal differentiation [19,20]. Profilaggrin is expressed but not processed to filaggrin in submerged cultures of normal human keratinocytes [21] or in long-term mouse keratinocyte cultures [22]. The HaCaT cell line, a spontaneously immortalized human epidermal cell line from adult skin that maintains epidermal differentiation capacity [27], has a transformed phenotype in vitro but remains nontumorigenic. Despite altered and unlimited growth potential, these cells show differentiation similar to normal cells expressing keratins 1 and 10, as well as involucrin, providing a promising tool for studying regulation of keratinization in human cells. Though this cell line is widely used to study keratinocyte differentiation [28], profilaggrin expression is variable depending on the line studied and processing to filaggrin is not seen (unpublished results). While profilaggrin is expressed in lifted cultures, the model is not ideal for biochemical studies because of the difficulty of the technique and cell heterogeneity. The ideal culture system would be the one where cells carry out differentiation in a synchronous manner or in response to changes in the medium. A rat keratinocyte cell line developed by Kubilus and Baden closely meets these criteria; this immortalized cell line stratifies at confluence and produces differentiation specific proteins when grown in submerged culture directly on tissue culture plastic dishes [23]. These are the only keratinocytes capable of processing profilaggrin to filaggrin in submerged culture, and these processing events appear to be partially synchronized [24, 25, 26]. In this work using the rat immortalized cell line, we show that when profilaggrin expression decreases as a function of passaging, these keratinocytes undergo a type of senescence which can be blocked by an inhibitor of fibroblast senescence. Furthermore, high levels of nonspecific proteolysis of profilaggrin, occurring most likely via autophagocytosis, reduces filaggrin yield. By adjusting culture conditions, down-regulation of nonspecific proteolysis of profilaggrin can be decreased with concomitant increase in filaggrin yield, presumably shifting the balance towards normal differentiation.