Henry 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.

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.

Zinc-α2-glycoprotein expression as a marker of differentiation in human oral tumors

Zinc-alpha2-glycoprotein (Znalpha2gp) is a soluble major histocompatibility complex homolog widespread in body fluids and in glandular epithelia; the authors recently demonstrated its presence in stratified epithelia. Znalpha2gp has been associated with tumor differentiation in breast cancers and other carcinomas. We compare here its gene expression in histopathologically graded oral squamous cell carcinomas and in their perilesional normals. Znalpha2gp levels are higher in the controls than in the tumors, and higher in well-differentiated tumors than in poorly differentiated ones. Markers of oral epithelial maturation (keratin K13 and involucrin) are less simply related to tumor histology.

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.

Induction by interferon-γ of its receptor varies with epithelial differentiation and cell type

Abstract Normal keratinocytes from epidermis and from buccal mucosa were cultured to confluence in three media with graded differentiation potential and treated with interferon-γ (IFN-γ). RT-PCR was used to measure the gene expression of the IFN-γ receptor (IFNGR-1), as well as the immunomodulator HLA-DR and two enzymes of the 2–5 A pathway. We have previously reported results for a number of structural and regulatory genes in the same system (and include here involucrin for comparison). In epidermal keratinocytes, the induction of IFNGR-1 was upregulated by incubation with IFN-γ, and this increased with the differentiation potential of the culture medium. A roughly similar pattern occurred for the other genes. In mucosal keratinocytes, in contrast, IFN-γ failed to induce expression of IFNGR-1 or the other genes. A unique characteristic of HLA-DR was that its induction by IFN-γ was uniform, for both tissues and all media. The gene expression of the receptor IFNGR-1 appears to be the dominant factor in the sensitivity of other genes to IFN-γ, although there are substantial disparities among them that presumably reflect functional differences. The difference between the two tissues may be linked to differentiation, as the epidermis has a much more extensive maturation pattern than the buccal mucosa. A clinical implication is a better prognosis for IFN-γ treatment for more differentiated tumors. Indeed, a previous study has found that the maturation pattern of condylomas responding to interferon treatment resembles that of epidermis, whereas the maturation of nonresponders is more akin to that of buccal mucosa.

Induction of iNOS mRNA by interferon-gamma in epithelial cells is associated with growth arrest and differentiation

Induction of inducible nitric oxide synthase (iNOS) mRNA by interferon-gamma (IFNgamma) is well established in various cell types. We observed earlier that this induction is differentiation-dependent in human keratinocytes. Since IFNgamma-mediated growth arrest and differentiation are separable but interrelated processes in keratinocytes, iNOS might play a role in their regulation. We conducted a series of in vitro experiments on normal and transformed epithelial cells with different tissue origins. We found that immortalization and transformation can influence the IFNgamma-mediated iNOS inducibility, but this process is tissue-specific as the induction correlates with the ability of the cells to differentiate.

Response to interferon treatment decreases with epidermal dedifferentiation in condylomas

After interferon (IFN) treatment of patients with condyloma acuminatum, groups clinically proven to be responders or nonresponders were selected, and cellular parameters that might influence the clinical response were studied in pretreatment biopsies by reverse transcription polymerase chain reaction (RT-PCR). The nonresponders were found to express higher amounts of cellular proliferative markers, such as proliferating cell nuclear antigen (PCNA), cyclin A, and cdc 2 kinase, but lower levels of growth suppressor genes (TGF-beta 1, TGF-beta 2 and p53) before IFN treatment. The responders retained the epidermal keratinization, except for some signs of hyperproliferation (K6, K16 cytokeratins). In addition, the nonresponders showed a shift in the keratinization pattern to a mucosal or fetal type, as evidenced by high expression of the K18, K6, K16 and K13 cytokeratins but decreased K5, K14 and K10 levels before treatment. The expression of the human papillomavirus (HPV) genes is consistent with these differentiation patterns. The crucial conclusion to be drawn from this study is that those condylomas whose pretreatment phenotype most closely resembles that of normal epidermis respond to IFN treatment, whereas those more akin to nonkeratinizing epithelia fail to respond, i.e. the resistance of condylomas to IFN treatment is correlated with dedifferentiation.