Trace Bell

Interferon-γ modulates terminal differentiation and the expression of desquamin in cultured keratinocytes

Interferon (IFN)-gamma has been shown to modulate cell differentiation and the expression of cell surface molecules of cultured human keratinocytes; it also induces cell shedding. We have previously described the properties of desquamin, a cell surface adhesion molecule from the stratum corneum. We report here on the impact of IFN-gamma on the expression of desquamin. We document the related morphological changes in terminal differentiation. We cultured human keratinocytes in three different culture systems: in serum-free medium at low Ca2+ (0.1 mM), at high Ca2+ (1.5 mM), and at high Ca2+ with 10% serum. IFN-gamma (100 U/ml) was added to each culture system after overnight incubation. In all cases, IFN-gamma induced an altered phenotype, as shown by phase contrast and electron microscopy. We exposed cultured cells to antibodies to the desquamins (glycoproteins from the stratum corneum). Immunoflurescent localization and Western blotting showed that the desquamins were expressed only under culture conditions where both serum and IFN-gamma were present. The induction of desquamin expression by IFN-gamma coupled with an increase in cell shedding, suggests that we have developed a suitable culture system for the study of desquamation in vitro.

Grafting of leg ulcers with undifferentiated keratinocytes

A procedure for transplanting cultured human keratinocytes has been developed and used successfully to treat patients with persistent leg ulcers. Shave excisions of donor skin of 1 cm2 or less were expanded in culture in 1 week to cover wounds as large as 90 cm2. The uniqueness of this system is the use of a biochemically modified surgical dressing that permits the transplantation of undifferentiated cells. Several hours after inoculation, trypsinized single cells become attached to the dressing and are ready for grafting. Seven of eight persistent stasis ulcers completely reepithelialized after application of the modified surgical dressing with undifferentiated keratinocytes.

Desquamin is an epidermal ribonuclease

Desquamin is a glycoprotein that we have isolated from the upper granular layer and the stratum corneum of human epidermis; it is not ordinarily expressed in submerged cultures, whose terminal differentiation stops short of formation of these layers. The exogenous addition of desquamin to human cultured keratinocytes extended their maturation, and hematoxylin staining indicated a loss of cell nuclei. For confirmation, cultured cells were lysed in situ, and the nuclei were incubated with desquamin for several days, then stained with hematoxylin. Damage to the nuclei was evident: the nuclear inclusions remained intact, while the surrounding basophilic nuclear matrix was degraded. Desquamin was then tested directly for nuclease activity. Ribonuclease activity was determined by incubating desquamin with human epidermal total RNA and monitoring the dose‐dependent disappearance of the 28S and 18S ribosomal RNA bands in an agarose/formaldehyde gel. On RNA‐containing zymogels, we confirmed the RNase activity to be specific to desquamin. Using synthetic RNA homopolymers, we found the active RNase domains to be limited to cytosine residues. On the contrary, DNA was not degraded by an analogous procedure, even after strand‐separation by denaturation. J. Cell. Biochem. 68:74–82, 1998.

Sensitivity of Desquamin to Proteolytic Degradation

The stratum corneum is both a desquamating and degradative tissue; cell contacts become weaker in the outer lamellae and a variety of proteolytic and glycolytic enzymes cleave many macromolecules into free amino acids and saccharides. We have reported on a 40-kilodalton glycoprotein from human stratum corneum; because of its unique lectin-like properties, we believe that it plays a major role in desquamation and have named it desquamin. If desquamin is to function as a lectin during desquamation, it must survive enzymatic degradation in the outer stratum corneum. Accordingly, we treated this glycoprotein with a number of proteinases and found that desquamin is resistant in vitro to most of the types of proteinases that have been reported in the stratum corneum as well as several others that have not been identified as present in the tissue. In the stratum corneum, the lipids of the intercorneal mortar shield the glycoproteins; hence, desquamin is even less likely to be susceptible to endogenous proteolysis in vivo than in vitro.

Cohesive Properties of Terminally Differentiated Keratinocytes

The stratum corneum can be dissociated into single squames by several extraction methods: mechanically, with organic solvents, and with detergents. We have performed studied of reaggregation from single squames prepared by these methods. Mechanically dispersed corneocytes recombined into a lamellar-like structure closely resembling intact stratum corneum. Squames obtained by dissociation in ether formed a multilayered structure which differed from the intact tissue in the collapse of the bilayered lamellar structure into a single lucent band with osmiophilic deposits. Squames obtained after extraction with detergent failed to reaggregate.

Purification of proteins from polyacrylamide gels, free of detergent or dye.

A two step procedure recovers proteins from sodium dodecyl sulfate polyacrylamide gels. The proteins are eluted by electrophoretic dialysis. The eluent is then passed through an Amberlite CG-400 anion-exchange resin. The recovery of protein is nearly total. The recovered proteins have no detectable sodium dodecyl sulfate contamination. With gels that have been stained with Coomassie Brilliant Blue R, the procedure recovers the proteins free of the dye. We have used this procedure successfully during the purification of epidermal glycoproteins.