Cell-substratum adhesion is an essential requirement for survival of human neonatal keratinocytes in vitro. Similarly, activation of the epidermal growth factor receptor (EGF-R) has recently been implicated not only in cell cycle progression but also in survival of normal keratinocytes. The mechanisms by which either cell-substratum adhesion or EGF-R activation protect keratinocytes from programmed cell death are poorly understood. Here we describe that blockade of the EGF-R and inhibition of substratum adhesion share a common downstream event, the down-regulation of the cell death protector Bcl-x L . Expression of Bcl-x L protein was down-regulated during forced suspension culture of keratinocytes, concurrent with large-scale apoptosis. Similarly, EGF-R blockade was accompanied by down-regulation of Bcl-x L steady-state mRNA and protein levels to an extent comparable to that observed in forced suspension culture. However, down-regulation of Bcl-x L expression by EGF-R blockade was not accompanied by apoptosis; in this case, a second signal, generated by passaging, was required to induce rapid and large-scale apoptosis. These findings are consistent with the conclusions that (i) Bcl-x L represents a shared molecular target for signaling through cell-substrate adhesion receptors and the EGF-R, and (ii) reduced levels of Bcl-x L expression through EGF-R blockade lower the tolerance of keratinocytes for cell death signals generated by cellular stress.
Plasminogen activator inhibitor type 2 (PAI-2) is an unusual serine proteinase inhibitor in that it is largely retained within the cell and is found in high concentrations in the upper viable layers of human epidermis. Studies using transfected cell lines that express high levels of PAI-2 have suggested that this inhibitor may confer protection against programmed cell death. To test the hypothesis that PAI-2 may protect epithelial cells in vivo from premature programmed cell death, we determined expression patterns of PAI-2 in murine hair and nail. These epidermal derivatives are comprised of numerous epithelial cell types with distinct differentiation pathways. Furthermore, the cyclic nature of the follicular epithelium makes it ideal for studying sequential stages of cell differentiation and death. PAI-2 mRNA and protein were detected in the differentiating cells of the outer root sheath and medulla of the follicle during the anagen phase of the hair growth cycle. PAI-2 was also detected in the permanent portion of the catagen follicle. In the telogen phase of the hair growth cycle, PAI-2 was limited to the postmitotic cells of the outer root sheath directly abutting the club hair. In the nail, PAI-2 was detected in the differentiating cells of the matrix and nail bed. This consistent, selective distribution of PAI-2 in the postmitotic, maturing cells prior to terminal keratinization and death suggests that (i) PAI-2 may be considered as a differentiation marker for many epithelial cell types, and (ii) PAI-2 is appropriately positioned to protect epithelial cells from premature demise.
The presence of serum has limited the utility of many culture models for the study of cytokine effects because its complexity and variability can confound the interpretation of data. In the present study, a serum-free skin co-culture model was used to investigate the effect of exogenous epidermal growth factor (EGF) on epidermal proliferation and differentiation. Human keratinocytes cultured on collagen rafts at the air-liquid interface produced a well-differentiated epithelium that resembled normal epidermis. Keratin filaments, membrane-coating granules, and keratohyalin granules were all observed. Epidermal differentiation markers keratin K1/K10, involucrin, and transglutaminase were localized in most of the suprabasal layers, whereas profilaggrin/filaggrin was confined to the granular layers and stratum corneum. In the continual presence of 10-20 ng/mL EGF, the epidermis was less organized, thinner, and less proliferative. EGF also depressed several indicators of differentiation: The number of keratohyalin granules and membrane-coating granules was greatly decreased; antigen expression of profilaggrin/filaggrin appeared diminished by immunocytochemical staining; frequent nuclear retention was noted in the relatively thickened stratum corneum-like layers. As detected by immunohistochemical staining, the expression of EGF receptor in the epidermis was reduced by exogenous EGF. These data illustrate that EGF cannot be considered a simple mitogen. Our findings also underscore the importance of using sophisticated culture models to assess complex cytokine effects that may be dependent on the architecture of a differentiating epidermis.
Plasminogen activator inhibitor type 2 (PAI-2) is a serine proteinase inhibitor (serpin), present in high quantities in stratified squamous epithelia. Detergent extracts of human epidermis or cultured keratinocytes contain primarily active, nonglycosylated PAI-2. In keratinocytes, the vast majority of PAI-2 is retained within the cell, supporting the hypothesis that PAI-2 may serve specific intracellular function(s) through interaction with an unknown cytoplasmic proteinase. During interaction with the target proteinase, cleavage of PAI-2 within its reactive site loop leads to the formation of a more stable, "relaxed" conformation (PAI-2r). Using a monoclonal antibody specific for PAI-2r, we demonstrate here that PAI-2r is present in keratinocytes of the granular and basal layers of normal human epidermis. In addition, PAI-2r is detectable in cultured human epidermal keratinocytes, where it is concentrated in a detergent-insoluble fraction within differentiating cells. These data provide evidence for the presence of an endogenous, keratinocyte-derived proteinase that constitutively cleaves intracellular PAI-2 in normal human epidermal keratinocytes. Cleavage of PAI-2 by this proteinase may reflect specific intracellular action of PAI-2 in normal cells. Finally, we demonstrate that a commercially available anti-PAI-2 monoclonal antibody (#3750, American Diagnostica, Greenwich, CT), under native experimental conditions, preferentially recognizes the uncleaved, active form of PAI-2 and does not efficiently detect PAI-2r.
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