Miriam M. Brysk

Response of keratinocytes from normal and psoriatic epidermis to interferon-γ differs in the expression of zinc-α2-glycoprotein and cathepsin D

Psoriasis is a T cell‐mediated inflammatory disease characterized by hyperproliferation and by aberrant differentiation. We found cathepsin D and zinc‐α2‐glycoprotein, two catalytic enzymes associated with apoptosis and desquamation, to be present in the stratum corneum of the normal epidermis but absent from the psoriatic plaque. Psoriasis is characterized by an altered response to interferon‐γ (IFN‐γ), including the induction of apoptosis in normal but not in psoriatic keratinocytes, often with opposite effects on gene expression of suprabasal proteins. We found that IFN‐γ binding and signaling were attenuated in psoriasis: The IFN‐γ receptor, the signal transducer and activator of transcription STAT‐1, and the interferon regulatory factor IRF‐1 were strongly up‐regulated by IFN‐γ in normal keratinocytes, but not in psoriatic ones. IFN‐γ strongly up‐regulated the expression of the catalytic enzymes cathepsin D and zinc‐α2‐glyco‐protein in normal keratinocytes but down‐regulated them in psoriatic ones; the reverse was true of the apoptotic suppressor bcl‐2. We believe that the aberrant response to IFN‐γ plays a central role in the pathophysiology of psoriasis, particularly the disruption of apoptosis and desquamation.—Chen, S.‐H., Arany, I., Apisarnthanarax, N., Rajaraman, S., Tyring, S. K., Horikoshi, T., Brysk, H., Brysk, M. M. Response of keratinocytes from normal and psoriatic epidermis to interferon‐γ differs in the expression of zinc‐α2‐glycoprotein and cathepsin D. FASEB J. 14, 565–571 (2000)

Isoforms of cathepsin D and human epidermal differentiation

Cathepsin D is an ubiquitously expressed lysosomal aspartic proteinase, with well-determined structural and chemical properties but a less clearly defined biological role. In stratified epithelia, the chronology of cathepsin D activation and degradation can be connected with stages of cellular differentiation. We partially purified cathepsin D from human epidermis and from separated stratum corneum by standard biochemical procedures, monitored by SDS-PAGE and Western blotting, and verified its identity as to molecular mass, pH optimum, N-terminal sequencing, reactivity with the specific antibody, inhibition by pepstatin A, and specific enzyme activity. It had hemoglobin-degrading activity over the acid range, with maximum at pH 3. It also degraded bovine serum albumin, human keratins, and stratum corneum extracts at pH 4. We discerned all three isoforms of human cathepsin D (the 52 kDa proenzyme and the active forms at 48 kDa and 33 kDa) in the epidermis; both active forms were also seen in the stratum corneum, but the proenzyme was not. Gene expression of cathepsin D in epidermal keratinocytes resembled that of suprabasal structural proteins (involucrin, keratin K10, transglutaminase) in its response to the calcium switch. An antibody to the 33 kda isoform immunolocalized to the granular layer and the stratum corneum (whereas antibodies to the 48 kDa isoform have been reported to stain mainly the upper spinous and granular layers). A plausible hypothesis to harmonize these results is that cathepsin D is first expressed as the proenzyme in the upper spinous layer, is activated in the lysosomes in the granular layer to the 48 kDa form, and is degraded to the 33 kDa form in the transition zone between the granular layer and the stratum corneum. As the stratum corneum is an acid environment, with an ambient pH of approximately 4.5, cathepsin D is available and suited to contribute to desquamation.

Glycoproteins modulate adhesion in terminally differentiated keratinocytes

SummaryThe stratum corneum can be dissociated into single squames by homogenization in ether. We have reaggregated the free corneocytes into a multilayered lamellar structure resembling an intact stratum corneum. The reconstituted stratum corneum reacts with fluorescein-conjugated lectins, unlike the intact tissue. We infer that the lack of binding in the intact tissue is due to masking of saccharide sites by lipids (which are extracted by the ether). In an extension of the procedure, the ether is removed and replaced by acetone. This system permits us to modulate corneocyte reaggregation by the addition of appropriate agents. We have used this system to corroborate our hypothesis that a 40 kD cell-surface glycoprotein (an endogenous lectin specific for amino sugars), which we have isolated from the stratum corneum, is instrumental in adhesion of corneocytes by cross-linking with amino sugar sites on adjacent cells. The reaggregation is inhibited by the antibody to the 40 kD glycoprotein. It is also inhibited by either the addition of amino sugars which bind to the endogenous lectin, or the addition of exogenous lectins specific for amino sugars which bind to the ligand.

Differentiation-dependent expression of signal transducers and activators of transcription (STATs) might modify responses to growth factors in the cancers of the head and neck

Overexpression of the epidermal growth factor receptor (EGFR) in the cancers of the head and neck is well demonstrated. In addition, copy numbers of the EGFR mRNA were significantly higher in poorly differentiated tumors than in tumors that had a differentiated phenotype. Studies by others also showed that the constitutively activated signal transducer and activator of transcription-3 (STAT3), but not STAT1, is required for EGFR-mediated cell growth. Our aim was to reveal if STAT expression is differentiation-dependent and thus, might respond to exogenous stimuli in a differentiation-dependent manner. Both reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry revealed that expression of STAT1 was high in well/moderately differentiated tumors in vivo. In contrast, STAT3 was expressed in poorly differentiated tumors. In vitro experiments showed that differentiated primary oral keratinocytes expressed higher levels of STAT1, but lower levels of STAT3 than did their undifferentiated counterparts. Epidermal growth factor treatment of oral keratinocytes with various degrees of differentiation showed the maximal induction of cyclin D1 in undifferentiated cells. Our findings suggest that the level of differentiation might modulate the outcome of EGFR signaling (i.e. cyclin D1 transcription), due to the differentiation-associated intracellular balance of transcriptional regulators (STAT1 versus STAT3).

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.

Zinc‐α2‐glycoprotein hinders cell proliferation and reduces cdc2 expression

Zinc‐α2‐glycoprotein (Znα2gp) is widely distributed in body fluids and epithelia. Its expression in stratified epithelia increases with differentiation. We previously showed that Zn α2gp has ribonuclease activity, and that squamous tumor cells grown on a matrix of Znα2gp were growth‐inhibited. Here we demonstrate, both by adding Znα2gp to the culture medium and, more unequivocally, by stably transfecting SiHa cells with Znα2gp cDNA, that the introduction of Znα2gp into SiHa tumor cells reduces proliferation. In response to Znα2gp, we find an accumulation of the cell population in G2/M by flow cytometry, paralleling the reduction of proliferation. In order to distinguish growth inhibition by cell cycle arrest from that produced by apoptosis or differentiation, we examine by RT‐PCR how Znα2gp affects the expression of genes commonly used as markers of these properties. No changes are observed for PCNA, p53, c‐myc, or bcl‐2. Only cdc2 expression responds to Znα2gp, with a reduction of up to over a factor of two. Cdc2 is the only cyclin‐dependent kinase regulating the G2/M transition without redundancy and is required as a rate‐limiting step in the cell cycle. Its increased expression has been directly linked to increased proliferation and decreased differentiation of advanced tumors; conversely, its downregulation by Znα2gp might hinder tumor progression. J. Cell. Biochem. Suppl. 36: 162–169, 2001.

Gene expression of markers associated with proliferation and differentiation in human keratinocytes cultured from epidermis and from buccal mucosa

Normal keratinocytes from epidermis and from buccal mucosa underwent dissimilar stages of differentiation in the same culture medium and responded differently to changes in the composition of the medium. Manifestations of these variations were examined in terms of the expression at the mRNA level (as measured by reverse transcriptase-polymerase chain reaction) of three regulatory genes (cdc2, c-myc, and p53) and five that encode structural proteins (keratins K5, K10 and K13, involucrin, and filaggrin), in three growth-medium formulations. The culture conditions enhanced or retarded maturation; the observed alterations in gene expression correlated with these changes. Except for the proliferation genes, the non-keratinizing buccal mucosa generally responded more weakly than the orthokeratotic epidermis to culture-medium supplementation favouring differentiation. Gene expression in cultured keratinocytes reflected their ability to differentiate in vivo; genes were expressed even when the corresponding protein was not seen in vitro. Although keratin K10 is not prevalent in the buccal mucosa nor keratin K13 in the epidermis, the genes for both were found to be expressed in both tissues.

Characterization of zinc‐α2‐glycoprotein as a cell adhesion molecule that inhibits the proliferation of an oral tumor cell line

Zn‐α2‐glycoprotein (Znα2gp) is a soluble protein widely distributed in body fluids and glandular epithelia. We have found it to be expressed in stratified epithelia as well. Znα2gp is clinically correlated with differentiation in various epithelial tumors, including oral and epidermal tumors. We have cloned epidermal Znα2gp and report the preparation of the recombinant protein in a Baculovirus expression system. Like the native molecule, recombinant Znα2gp has RNase activity. Znα2gp functions as a matrix protein for the Tu‐138 oral squamous cell carcinoma cell line. Cell attachment to Znα2gp is comparable to that for fibronectin and is inhibited by the synthetic RGD peptides RGD, RGDV, and RGDS. Attachment is also inhibited by the antibody to integrin α5β1 (the fibronectin receptor), but not by antibodies to integrins αvβ3, α3β1, and α2β1. We find that the proliferation of Tu‐138 cells is inhibited on a Znα2gp matrix, as compared with other matrix proteins (fibronectin, vitronectin, laminin, and collagens I and IV) on which growth resembles that on the BSA control. We believe that the role of Znα2gp in differentiation and its RNase activity are two likely suspects as agents of the inhibition of proliferation. J. Cell. Biochem. 75:160–169, 1999.

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.

The activity of interferon on ultraviolet light-induced squamous cell carcinomas in mice

The activity of interferons was tested in ultraviolet light-induced skin tumors in mice. After the tumors were well established, they were injected and measured daily for 19 days. Mouse virus type (IF-alpha + IF-beta) and immune (IF-gamma) interferons were injected intralesionally into three groups of test animals and compared with a fourth group which received mock interferon (control). When used separately, virus type and immune interferons did not affect tumor growth; however, we observed regression in tumor size when the two interferons were used in combination.