Specific proteolysis plays an important role in the terminal differentiation of keratinocytes in the epidermis and several types of proteases have been implicated in this process. The proprotein convertases (PCs) are a family of Ca2+-dependent serine proteases involved in processing and activation of several types of substrates. In this study we examined the expression and some potential substrates of PCs in epidermis. Four PCs are expressed in epidermis: furin, PACE4, PC5/6 and PC7/8. Furin is detected in two forms, either with or without the transmembrane domain, suggesting occurrence of post-translational cleavage to produce a soluble enzyme. In addition the furin active site has differential accessibility in the granular layer of the epidermis relative to the basal layer, whereas antibodies to the transmembrane domain stain both layers. These findings suggest that furin has access to different types of substrates in granular cells as opposed to basal cells. PC7/8, in contrast, is detected throughout the epidermis with antibodies to both the transmembrane and active site and no soluble form observed. A peptide PC inhibitor (dec-RVKR-CMK) inhibits cleavage of Notch-1, a receptor important in cell fate determination that is found throughout the epidermis. Profilaggrin, found in the granular layer, is specifically cleaved by furin and PACE4 in vitro at a site between the amino terminus and the first filaggrin repeat. This work suggests that the PCs play multiple roles during epidermal differentiation.

Ichthyosis vulgaris is an autosomal dominant disorder of keratinization characterized by mild hyperkeratosis and reduced or absent keratohyalin granules in the epidermis. Profilaggrin, a major component of keratohyalin granules, is reduced or absent from the skin of individuals with ichthyosis vulgaris. In this report, we have further characterized the molecular basis of low profilaggrin expression, which occurs in this disease. In situ hybridization revealed little profilaggrin mRNA in ichthyosis vulgaris-affected epidermis. In keratinocytes cultured from the epidermis of affected individuals, the abundance of profilaggrin was reduced to less than 10% of normal controls, while the mRNA level was decreased to 30-60% of controls. Expression of K1 and loricrin, other markers of epidermal differentiation, were not affected. Nuclear run-on assays indicated that the decrease in mRNA levels was not caused by aberrant transcription. Nucleotide sequencing of 5'-upstream, 3'-non-coding, and flanking regions of the profilaggrin gene from ichthyosis vulgaris-affected individuals revealed only minor changes, probably due to genetic polymorphisms. Our results indicate that defective profilaggrin expression in ichthyosis vulgaris is a result of selectively impaired posttranscriptional control.

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We investigated serine/threonine protein phosphatase (PP) activity and the expression of PP2A during growth and differentiation of epidermal keratinocytes in culture. Keratinocyte PP activity was strongly inhibited by calyculin A and okadaic acid, indicating that the activity was mainly due to PP2A and PP1. The phosphatase activity decreased to about 20% of the initial (day 1) level by the time of confluence and to about 10% at day 7 postconfluence. In contrast to activity, the level of expression of the PP2A catalytic subunit protein and the mRNA for the two isoforms increased slightly over the period of growth. Keratinocyte differentiation was shown by a significant increase in profilaggrin expression after confluence. Keratinocytes were also cultured from individuals affected with harlequin ichthyosis. This severe hyperkeratotic skin disorder has abnormal lipid structures and is blocked in the PP2A-dependent conversion of phosphorylated profilggrin to the non-phosphorylated filaggrin. The PP activity in harlequin cultures was lower than in normal cultures (about 20% of the subconfluent normal control value) and decreased even further in confluent cultures. In contrast, the level of expression of the PP2A catalytic subunit protein and mRNA for the two isoforms was similar to that of normal keratinocytes and increased with confluence. These results suggest that PP activity in keratinocytes is regulated in a post-translational manner; they also support the possibility of impaired or reduced function of PPs in harlequin ichthyosis.