Plasminogen activator inhibitor type 2 is expressed in keratinocytes during re-epithelialization of epidermal defects.

Plasminogen activation is observed in the human epidermis during re-epithelialization of epidermal defects. The activation reaction depends on plasminogen activators (PAs) associated with re-epithelializing keratinocytes. PA inhibitor type 2 (PAI-2) is thought to be a major epidermal PA inhibitor in keratinocytes. However, no data are available on the expression of PAI-2 in keratinocytes during epidermal regeneration. We have therefore analysed PAI-2 at the mRNA and protein level in keratinocyte cultures as well as in epidermal lesions in which re-epithelializing keratinocytes were apparent. We found that PAI-2 expression at the mRNA and protein level was negatively correlated with the cell density in regular keratinocyte cultures. In organotypic cocultures, in which the transition from a re-epithelializing to a sedentary phenotype can be studied, PAI-2 was most strongly expressed in early cultures prior to formation of a differentiated epidermis-like structure. We found a strong expression of PAI-2 in keratinocytes that re-epithelialized dermal burn wounds or lesions caused by the autoimmune blistering disease pemphigus vulgaris. Our results suggest that not only PAs, but also a major PA inhibitor, PAI-2, are expressed in keratinocytes that are actively involved in re-epithelialization.

Dispase-mediated basal detachment of cultured keratinocytes induces urokinase-type plasminogen activator (uPA) and its receptor (uPA-R, CD87).

Keratinocytes synthesize and secrete urokinase-type plasminogen activator (uPA), which is bound in an autocrine manner to a specific receptor (uPA-R, CD87) at their surface. Plasminogen, which is also bound to membrane binding sites, is readily activated by uPA-R-bound uPA. Thus, plasmin for proteolysis of pericullular glycoproteins is provided. While uPA-R and uPA are at low to undetectable levels in keratinocytes of the normal epidermis, both compounds are upregulated in migrating keratinocytes during reepithelialization of epidermal defects and in affected keratinocytes of various epidermal disorders, including bullous dermatoses. We have hypothesized that the disturbance of cell/matrix interactions--a common feature of these diverse pathological situations--induces uPA/uPA-R. Accordingly, we explored whether the dispase-mediated detachment of cultured keratinocytes, which have formed a multilayered epidermis-like structure in vitro, induced uPA and uPA-R. We found increases in uPA secretion, cell-associated uPA activity, and uPA- and uPA-R-antigen in keratinocytes upon dispase-mediated detachment from their growth substratum. The increase was preceded by an increase in uPA-R- and uPA-specific mRNA, which was not observed when the proteinase inhibitor phosphoramidon was added together with dispase. In conclusion, we present evidence that experimental detachment with dispase provides signals for the concomitant upregulation of uPA-R and uPA. The findings support the hypothesis that cell/matrix interactions may influence the expression of the cell surface-associated PA system in human keratinocytes.

Tumor necrosis factor-alpha-induced apoptosis in a human keratinocyte cell line (HaCaT) is counteracted by transforming growth factor-alpha.

The integrity of the human epidermis is guaranteed by a regulated balance of proliferation, differentiation, and physiologic cell death of its main cellular constituent, the epidermal keratinocyte. Physiologic cell death is known as apoptosis and has been recognized as an active regulatory mechanism, complementary to, but functionally opposite of, proliferation. The regulators of the delicate balance between cell death and proliferation are only partially understood in human keratinocytes. Transforming growth factor-alpha (TGF-alpha) has been identified as a positive regulator of proliferation and growth, while tumor necrosis factor-alpha (TNF-alpha) induces apoptosis. Both mediators are thought to influence epidermal keratinocytes under various physiological and pathophysiological conditions. In the current study we have begun to investigate potential regulatory interactions between these two mediators in the human keratinocyte cell line HaCaT. We have found that, when the HaCaT cells were sensitized by the translation inhibitor cycloheximide, TNF-alpha induced apoptosis, as evidenced by nuclear disintegration, DNA fragmentation ("DNA laddering"), and the appearance of soluble DNA/histone complexes. Moreover, we found that the induction of apoptosis was reduced by preincubation of the cells with TGF-alpha. The protective effect of TGF-alpha was abrogated by translation inhibition, indicating that it depended on de novo protein synthesis. Moreover, the protective effect was not accompanied by a reduced surface expression of TNF receptor molecules. We postulate that TNF-alpha-induced apoptosis in HaCaT cells is counteracted by constitutively produced suppressors of apoptosis, the synthesis of which can be downregulated by inhibition of translation and upregulated by the cytokine TGF-alpha.

Upregulation of cell-surface-associated plasminogen activation in cultured keratinocytes by interleukin-1 beta and tumor necrosis factor-alpha.

Keratinocytes synthesize and secrete urokinase-type plasminogen activator (uPA) which is bound in an autocrine manner to a specific receptor (uPA-R) at the keratinocyte surface. Plasminogen that is also bound to specific membrane binding sites is readily activated by uPA-R-bound uPA. Thus, plasmin is provided for proteolysis of pericellular glycoproteins. The expression of uPA and the uPA-R is confined to migrating keratinocytes during epidermal wound healing, rather than to keratinocytes of the normal epidermis. The regulatory factors of uPA/uPA-R expression in keratinocytes remained largely elusive. Proinflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha) or interleukin-1 beta (IL-1 beta), are present in epidermal wounds. We have therefore tested IL-1 beta and TNF-alpha for their influence on surface-associated plasminogen activation in a human keratinocyte cell line (HaCaT) as well as in primary cultures of normal human epidermal keratinocytes. Both cytokines induced the secretion of uPA into the culture supernatants and a concomitant increase in uPA activity as well as in uPA and uPA-R antigen at the cell surface. The increase was preceded by an increase in specific mRNA. The induction was accompanied by an accelerated uPA-dependent and plasmin-mediated detachment of HaCaT cells from the culture substratum. Taken together, the proinflammatory cytokines IL-1 beta and TNF-alpha induced a coordinated increase in uPA and uPA-R as well as increased pericellular plasmin-mediated proteolysis in human epidermal keratinocytes. This function might be an element of the molecular cell biological events during epidermal wound healing.

Plasminogen activator inhibitor type-2 in the lesional epidermis of lupus erythematosus.

Under certain pathophysiological conditions epidermal keratinocytes produce urokinase-type plasminogen activator (uPA) or tissue-type PA (tPA). These PAs are subject to regulation by PA inhibitors (PAI), including PAI type-2 (PAI-2). In the normal epidermis, PAI-2 is present in the differentiating suprabasal layers, albeit in the apparent absence of PAs. It has, therefore, been suggested that PAI-2 plays a role in epidermal differentiation not linked to its ability to inhibit PAs. In line with this hypothesis, we have studied, by immunohistochemistry, the distribution of PAI-2, uPA and tPA in the normal and in the lesional epidermis of patients with lupus erythematosus (LE), a disease in which epidermal differentiation is disturbed. The PAI-2 antigen was detectable in the normal epidermis and in the lesional epidermis of LE. In the normal epidermis, the PAI-2 antigen was most pronounced in the granular layer. In the hyperkeratotic epidermal lesions of LE, the PAI-2 antigen was increased. In normal and lesional skin, PAI-2 was distributed along the cell periphery, indicating its association with the cornified envelope. Neither uPA nor tPA was detectable in normal or lesional epidermis. Our findings show that PAI-2 is a major type of PAI in normal epidermis and in the lesional epidermis of LE, and that increased epidermal PAI-2 is observed in a disease which is not associated with an increase in epidermal PAs. The data support the hypothesis that epidermal PAI-2 may have other functions than the regulation of PA activity.

Plasminogen activator inhibitor type-2 (PAI-2) in human keratinocytes regulates pericellular urokinase-type plasminogen activator.

Plasminogen activation is observed in the human epidermis during reepithelialization of epidermal defects and under certain pathological conditions. The activation reaction depends on keratinocyte-associated plasminogen activators (PAs), which convert the ubiquitous proenzyme plasminogen into the active trypsin-like serine proteinase plasmin. The PAs are controlled by PA inhibitors (PAIs), of which two major types are known: PAI-1 and PAI-2. In vitro and in vivo keratinocytes express both PAIs. In the current study, we have addressed the possible function of PAI-2 in regulating extracellular PA activity in cultured normal human epidermal keratinocytes (NHEK), the human keratinocyte cell line (HaCaT), and a Ha-ras transfected HaCaT variant (HaRas). PAI-2 was detected intracellularly in all three cell types. Whereas only the NHEK and the HaCaT cells secreted detectable levels of PAI-2 into the culture medium, all three cell types released urokinase-type PA (uPA) into the supernatants. When comparing HaCaT and HaRas cells, we found that the cell lines secreted comparable levels of uPA antigen, whereas the levels of uPA activity were low in the presence of PAI-2, indicating that PAI-2 serves to regulate uPA activity. This assumption was supported by the findings that PAI-2 formed complexes with secreted uPA and that uPA/PAI-2 complexes were present at the surface of the PAI-2-secreting HaCaT cells but not at the surface of PAI-2 nonsecreting HaRas cells. Finally, PAI-2 was found to counteract the uPA-dependent and plasmin-mediated detachment of cultured HaCaT cells. Taken together, our findings indicate that secreted PAI-2 serves to regulate the activity of extracellular uPA in keratinocytes.