Expression of plasminogen activator inhibitor type 2 in normal and psoriatic epidermis

The plasminogen activator (PA) proteolytic cascade has been implicated in the regulation of cell activities, including proliferation and differentiation, both of which occur continuously in normal human epidermis and are aberrant in psoriatic epidermis. To elucidate further the mechanisms by which PA is regulated in epidermis, we evaluated the levels of PA inhibitors type 1 (PAI-1) and type 2 (PAI-2) in normal and psoriatic epidermis. PAI-2, but not PAI-1, was detectable by mRNA, antigen, and activity assays, indicating that PAI-2 is the predominant epidermal PA inhibitor. In situ hybridization revealed that PAI-2 mRNA occurred throughout normal epidermis, although the signal was most intense in the granular layers. Similarly, PAI-2 antigen was most prominent in the granular layers; its distribution in these differential layers was along the cell periphery. Diffuse, fainter staining for PAI-2 was also detected in the basal cells and in some spinous layers of normal epidermis. Extracts of normal epidermis contained PA inhibitory activity identified as PAI-2 by immunoprecipitation with specific antibody. In psoriatic epidermis, PAI-2 mRNA and antigen were most prominent in the more superficial layers beneath the cornified cells. As with normal epidermis, PAI-2 assumed a pericellular distribution in the psoriatic cells. These data demonstrate that PAI-2 is constitutively expressed in vivo by keratinocytes in human epidermis and indicate that this protein is the predominant inhibitor of PA activity in normal and psoriatic human epidermis.     

Differential Expression of Urokinase-Type Plasminogen Activator (uPA), Its Receptor (uPA-R), and Inhibitor Type-2 (PAI-2) during Differentiation of Keratinocytes in an Organotypic Coculture System

Cultured keratinocytes resemble migrating keratinocytes under conditions of reepithelialization during wound healing. Such keratinocytes express urokinase-type plasminogen activator (uPA) and its specific receptor (uPA receptor). Receptor-bound uPA activates plasminogen, thus providing plasmin for pericellular proteolysis. uPA is regulated by the plasminogen activator inhibitors PAI-1 and PAI-2. As indicated by immunohistology, neither uPA nor uPA receptor is expressed in normal epidermis. Thus, the down-regulation of uPA and uPA-receptor expression in keratinocytes appears to be an important event in epidermal healing and restoration of a normal epidermal tissue architecture. We have addressed this matter by using a culture and differentiation system for keratinocytes in vitro. Keratinocytes were grown in organotypic cocultures for 4, 7, and 14 days. Frozen sections were analyzed with indirect immunofluorescence staining and overlay zymography, the latter detecting activity of plasminogen activators. While tPA and PAI-I stainings were consistently negative over the entire observation period, uPA and uPA receptor were expressed by basal keratinocytes at Days 4 and 7, but not at Day 14. Accordingly, overlay zymography revealed uPA activity at Days 4 and 7. PAI-2 was found throughout the entire observation period, but with varying distribution: at Days 4 and 7 all suprabasal keratinocytes stained positive for PAI-2. At Day 14, PAI-2-specific stainings were confined to the uppermost cells of the stratum spinosum. Our data demonstrate that uPA and uPA receptor, which are up-regulated in cultured keratinocytes, are down-regulated upon restoration of an epidermis-like structure. The distribution of PAI-2 varied over the observation period and at Day 14 resembled the distribution of PAI-2 in normal epidermis. Taken together, keratinocytes in organotypic coculture behave like keratinocytes in healing wounds in vivo with respect to the expression of the plasminogen activator system.     

Plasminogen activator inhibitor type 2 (PAI-2) is present in normal human conjunctiva

The purpose was to characterize plasminogen activator inhibitor type 2 (PAI-2) expression in normal human conjunctiva in vivo and in vitro. PAI-2 antigen was assayed by immunostaining and immunoblotting of extracts from normal human conjunctival epithelial lysates and conditioned media (CM) of cultured human conjunctival keratinocytes. Immunostaining of normal human conjunctival epithelia revealed that PAI-2 was found consistently in the superficial keratinocytes and, in some biopsies, also in the lower keratinocyte layers. In all cases, PAI-2 was concentrated around the cell periphery. In extracts of conjunctival epithelia and cultured conjunctival keratinocytes, PAI-2 had an apparent molecular weight of 45 kDa, consistent with the non-glycosylated form. The majority of PAI-2, approximately 90%, was cell associated, however, a small percentage of PAI-2 was released into the CM in a linear manner with time. PAI-2 in the conditioned medium had a higher molecular weight, consistent with a glycosylated form. Conjunctival PAI-2 was active, as shown by its ability to complex with a target enzyme, urokinase plasminogen activator (uPA). Although PAI-2 was detectable both in monolayer (i.e., relatively undifferentiated) conjunctival keratinocyte cultures as well as in stratified (i.e., more differentiated) cultures, steady state levels of PAI-2 were greater in the latter. PAI-2 is constitutively expressed by normal human conjunctival epithelial cells. The expression of PAI-2 throughout all epithelial layers in some biopsies of conjunctiva in vivo contrasts with the previously established distribution of PAI-2 in corneal epithelia, where it is present exclusively in the most superficial (i.e. most highly differentiated) cells. The role of PAI-2 in either tissue is unclear. However, we speculate that its distinct distribution in conjunctival versus corneal epithelia underscores inherent differences between these tissues, and may reflect specific functions of this proteinase inhibitor in both conjunctival and corneal epithelial cells.     

Expression of the helix-loop-helix protein ID1 in keratinocytes is upregulated by loss of cell-matrix contact

To analyze the inhibitor of DNA-binding type 1 (ID1) in the human epidermis and in cultured keratinocytes we generated and characterized ID1-specific monoclonal antibodies. Immunohistological studies on human skin biopsies revealed that ID1 is not detectable in normal human epidermis but in lesional epidermis of bullous pemphigoid. In the latter case we found ID1 in the cytoplasm of basal and proximal suprabasal keratinocytes. Cultured normal human epidermal keratinocytes displayed ID1 in the cytoplasm; upon differentiation into a multilayered keratinocyte sheet, ID1 was no longer detectable. It was reexpressed after dispase-mediated detachment of the keratinocyte cultures from the growth substratum. In this case ID1 was localized to the cytoplasm and the nucleus. Our data indicate that after epidermal injury-in our case loss of cell-matrix contact-ID1 is upregulated in affected keratinocytes. In view of the ID1 function in other cell types, we speculate that ID1 facilitates the transition from the resting to the migrating and proliferating keratinocyte required for efficient repair of epidermal lesions by reepithelialization. Taken together we suggest that ID1 is an important player in epidermal (patho-)physiology.     

Differentiating cells of murine stratified squamous epithelia constitutively express plasminogen activator inhibitor type 2 (PAI-2)

 In stratified squamous epithelia a critical balance among cell proliferation, differentiation, and death must be maintained in order for these tissues to fulfill their barrier function. Previous studies have demonstrated that plasminogen activator inhibitor 2 (PAI-2) is a product of differentiating epidermal keratinocytes, suggesting a role for this inhibitor during squamous differentiation. Furthermore, in certain tumor cell lines, overexpression of PAI-2 confers resistance to the induction of programmed cell death, suggesting cytoprotective function(s). In the present study we demonstrate that PAI-2 mRNA and protein are constitutively and uniquely expressed in differentiating cells of murine stratified squamous epithelia, including epidermis, esophagus, vagina, oral mucosa, and tongue. PAI-2 immunohistochemical localization patterns suggest a predominantly cytosolic distribution, consistent with biochemical identification of the major PAI-2 species as a 43-kDa, presumably non-glycosylated protein. Functional analysis shows that the majority of epithelial PAI-2 is active. In contrast to the high levels of PAI-2 expression in stratified squamous epithelia, little or no PAI-2 is detectable in simple epithelia. These findings suggest that epithelial PAI-2 may mediate inhibition of intracellular proteinases associated with events during terminal differentiation and death that are unique to stratified squamous epithelia. Accepted: 29 June 1998     

Transforming growth factor-beta 1 localization in normal and psoriatic epidermal keratinocytes in situ.

Transforming growth factor-beta 1 (TGF beta 1) is a potent inhibitor of epithelial cell proliferation and its effects on growth and differentiation have been extensively characterized in cultured keratinocytes. We used two TGF beta 1-specific polyclonal antibodies (anti-LC and anti-CC) to determine the presence of TGF beta 1 peptide in keratinocytes in sections of normal human skin in situ and in both plaque and nonplaque skin from individuals with psoriasis. In contrast to the differentiation phenotype expressed by keratinocytes in normal epidermis, keratinocytes in the psoriatic plaque exhibit a hyperproliferative/regenerative differentiation phenotype. Anti-TGF beta 1 staining was observed primarily in the epidermis. Anti-LC TGF beta 1 antibody stained nonproliferating, differentiated suprabasal keratinocytes intracellularly in normal skin but did not stain psoriatic plaques from five of seven patients. In contrast, anti-CC TGF beta 1 antibody stained suprabasal keratinocytes extracellularly in psoriatic plaques, but did not stain normal skin. Both anti-LC and anti-CC stained suprabasal keratinocytes intracellularly in nonplaque psoriatic skin. Thus, the conformation or structure of TGF beta 1 and its localization vary in keratinocytes with distinct differentiation phenotypes suggesting that TGF beta 1 is a potential modulator of keratinocyte differentiation in vivo. Selective association of TGF beta 1 with nonproliferating keratinocytes in the suprabasal layers of the epidermis and its exclusion from the proliferating keratinocytes in the basal layer suggest that it may be a physiological regulator of keratinocyte proliferation. In addition, the intracellular localization of TGF beta 1 peptide in both normal and psoriatic keratinocytes suggests that it is constitutively synthesized by epidermal keratinocytes in vivo.     

Hyaluronan enters keratinocytes by a novel endocytic route for catabolism.

Hyaluronan synthesized in the epidermis has an exceptionally short half-life, indicative of its catabolism by epidermal keratinocytes. An intracellular pool of endogenously synthesized hyaluronan, from 1 to 20 fg/cell, inversely related to cell density, was observed in cultured rat epidermal keratinocytes. More than 80% of the intracellular hyaluronan was small (<90 kDa). Approximately 25% of newly synthesized hyaluronan was endocytosed by the keratinocytes and had a half-life of 2-3 h. A biotinylated aggrecan G(1) domain/link protein probe demonstrated hyaluronan in small vesicles of approximately 100 nm diameter close to the plasma membrane, and in large vesicles and multivesicular bodies up to 1300 nm diameter around the nucleus. Hyaluronan did not co-localize with markers of lysosomes. However, inhibition of lysosomal acidification with NH(4)Cl or chloroquine, or treating the cells with the hyaluronidase inhibitor apigenin increased intracellular hyaluronan staining, suggesting that it resided in prelysosomal endosomes. Competitive displacement of hyaluronan from surface receptors using hyaluronan decasaccharides, resulted in a rapid disappearance of this endosomal hyaluronan (t(12) approximately 5 min), indicating its transitory nature. The ultrastructure of the hyaluronan-containing vesicles, co-localization with marker proteins for different vesicle types, and application of specific uptake inhibitors demonstrated that the formation of hyaluronan-containing vesicles did not involve clathrin-coated pits or caveolae. Treatment of rat epidermal keratinocytes with the OX50 monoclonal antibody against the hyaluronan receptor CD44 increased endosomal hyaluronan. However, no CD44-hyaluronan co-localization was observed intracellularly unless endosomal trafficking was retarded by monensin, or cultivation at 20 degrees C, suggesting CD44 recycling. Rat epidermal keratinocytes thus internalize a large proportion of their newly synthesized hyaluronan into non-clathrin-coated endosomes in a receptor mediated way, and rapidly transport it to slower degradation in the endosomal/lysosomal system.     

Proteinase Inhibitor 6 (PI-6) Expression in Human Skin: Induction of PI-6 and a PI-6/Proteinase Complex during Keratinocyte Differentiation

Proteinase inhibitor 6 (PI-6) is a 42-kDa intracellular protein present in epithelial cells and endothelial cells. It is capable of inhibiting a number of serine proteinases, including trypsin and chymotrypsin. In this study we examined PI-6 expression in human skin and its primary cell type, the keratinocyte. By immunohistochemical analysis, PI-6 staining is absent from the basal cells, weak in the spinous layer, and strongest in the granulosa layer of human epidermis. Immunoblotting of cultured primary keratinocytes revealed that PI-6 production increases 24-fold on differentiation. Analysis of an immortalized keratinocyte cell line, HaCat, showed a 5-fold increase in PI-6 mRNA and a 7-fold increase in PI-6 protein upon differentiation, and indirect immunofluorescence revealed that this is due to an increase in the number of differentiated cells expressing high levels of PI-6. Of particular interest is the appearance of a preformed complex between PI-6 and an endogenous serine proteinase in differentiating HaCat cells, which was detected by a monoclonal antibody demonstrated to preferentially recognize PI-6 in complex with a proteinase. This identification of a PI-6/proteinase complex is the first example of a serpin bound to a proteinase in keratinocytes. We postulate that a physiological role of PI-6 is to regulate a serine proteinase associated with keratinocyte differentiation.     

Plasminogen activator inhibitor 2: expression and role in differentiation of epidermal keratinocyte

Plasminogen activator inhibitor 2 (PAI-2) is an enzyme inhibitor which is involved in cell differentiation, tissue growth and regeneration. In this study, immunocytochemistry, in situ hybridization and confocal laser scanning microscopy were used to investigate the expression and role of PAI-2 in differentiation of keratinocytes in vitro. The result showed that in the mono-layer differentiated keratinocytes induced by high calcium concentration, the expression of PAI-2 and its mRNA increased significantly, accompanied by expression increase of the differentiation marker keratin 10; and in the multi-layer differentiated keratinocytes induced by high calcium, PAI-2 expressed strongly mainly in the keratinocytes of middle as well as upper stratified layers, while K10 expressed in the keratinocytes of all stratified layers. Furthermore, the changes of the parameters related to keratinocyte differentiation were detected after inhibition of PAI-2 functions by its antibody, and the data showed that when treated by PAI-2 antibody, involucrin in the keratinocytes envelope expressed increasingly with an altering distribution from part to the whole envelope area. Our results indicate that during differentiation of epidermal keratinocyte, PAI-2 expresses mainly in the more differentiated keratinocytes and may protect the terminal differentiated keratinocytes from prematuration through inhibiting involucrin expression in cornified envelope.     

Novel function for beta 1 integrins in keratinocyte cell-cell interactions.

We have examined the expression, localization, and function of beta 1 integrins on cultured human epidermal keratinocytes using polyclonal and monoclonal antibodies against the beta 1, alpha 2, alpha 3, and alpha 5 integrin subunits. The beta 1 polypeptide, common to all class 1 integrins, was localized primarily in areas of cell-cell contacts of cultured keratinocytes, as were alpha 2 and alpha 3 polypeptides, suggesting a possible role in cell-cell adhesion for these integrin polypeptides. In contrast, the fibronectin receptor alpha 5 subunit showed no such accumulations in regions of cell-cell contact but was more diffusely distributed in the keratinocyte plasma membrane, consistent with the absence of fibronectin at cell-cell contact sites. Colonies of cultured keratinocytes could be dissociated by treatment with monoclonal antibody specific to the beta 1 polypeptide. Such dissociation of cell-cell contacts also occurred under conditions where the monoclonal antibody had no effect on cell-substrate adhesion. Therefore, beta 1 integrin-dependent cell-cell adhesion can be inhibited without affecting other cell-adhesive interactions. Antibody treatment of keratinocytes maintained in either low (0.15 mM) or high (1.2 mM) CaCl2 also resulted in the loss of organization of intracellular F-actin filaments and beta 1 integrins, even when the anti-beta 1 monoclonal antibody had no dissociating effect on keratinocyte colonies at the higher calcium concentration. Our results indicate that beta 1 integrins play roles in the maintenance of cell-cell contacts between keratinocytes and in the organization of intracellular microfilaments. They suggest that in epithelial cells integrins can function in cell-cell interactions as well as in cell-substrate adhesion.     

Effects of fibroblasts of different origin on long term maintenance of xenotransplanted human epidermal kerationocytes in immunodeficient mice

We examined effects of fibroblasts of different origin on long-term maintenance of xenotransplanted human epidermal keratinocytes. A suspension of cultured epidermal cells, originating from adult human trunk skin, was injected into double mutant immunodeficient (BALB/c nu/scid) mice subcutaneously, with or without cultured fibroblastic cells of different origin. At one week after transplantation, the epidermal cells generated epidermoid cysts consisting of human epidermis-like tissue. When the epidermal cells were injected alone or together with fibroblastic cells derived from human bone marrow, muscle fascia, or murine dermis, organized epidermoid cysts regressed within 6 weeks. In contrast, when the epidermal cells were injected together with human dermal fibroblasts, generated epidermoid cysts were maintained in vivo for more than 24 weeks. Histological examination showed that the reorganized epidermis, after injection of both epidermal keratinocytes and dermal fibroblasts, retained normal structures of the original epidermis during 6 to 24 weeks after transplantation. The results indicate that human dermal fibroblasts facilitate the long-term maintenance of the reorganized epidermis after xenotransplantation of cultured human epidermal keratinocytes by supporting self renewal of the human epidermal tissue in vivo.     

Epidermal plasminogen activator inhibitor (PAI) is immunologically identical to placental-type PAI-2

Plasminogen activator inhibitor (PAI) purified from human epidermis [(1986) FEBS Lett. 408, 273-277] was immunologically identified as placental-type PAI-2. In both fibrinolytic and synthetic substrate assays inhibitory activity of epidermal PAI was neutralized by anti-PAI-2, but not by anti-endothelial type PAI-1. Immunoblotting technique confirmed that the purified epidermal PAI is reactive with anti-PAI-2, but not with anti-PAI-1. Consequently PAI in human epidermis was demonstrable by immunohistochemical technique.     

Proliferating, transformed keratinocytes cultured under low Ca2+ conditions exhibit high-affinity epidermal growth factor receptors

In a previously published report (Exp. Cell. Res. 161:421 (1985] we have demonstrated that cultured normal and transformed keratinocytes exhibit two classes of EGF binding sites after growth under normal Ca2+ conditions but only low-affinity binding sites after growth under low Ca2+ conditions. Here we demonstrate the presence of high-affinity binding sites in transformed keratinocytes grown under low Ca2+ conditions, using a specific monoclonal anti EGF-receptor antibody.     

The C-D interhelical domain of the serpin plasminogen activator inhibitor-type 2 is required for protection from TNF-alpha induced apoptosis

The serine proteinase inhibitor (serpin), plasminogen activator inhibitor type 2 (PAI-2), has been reported to inhibit tumor necrosis factor-alpha (TNF) induced apoptosis. In order to begin to understand the molecular basis for this protection, we have investigated the importance of a structural domain within the PAI-2 molecule, the C-D interhelical region, in mediating the protective effect. The C-D interhelical region is a 33 amino acid insertion which is unique among serpins and has been implicated in transglutaminase catalyzed cross-linking of PAI-2 to cell membranes. We have constructed a mutant of PAI-2 wherein 23 amino acids are deleted from the C-D interhelical region generating a structure predicted to be homologous to the closely related, but non-inhibitory serpin, chicken ovalbumin. The PAI-2Delta65/87 deletion mutant retained inhibitory activity against its known serine proteinase target, urokinase-type plasminogen activator (uPA); however expression of this mutant in HeLa cells failed to protect from TNF-induced apoptosis. Analyses of the cellular distribution of PAI-2 showed that intracellular PAI-2, and not secreted or cell-surface PAI-2, was likely responsible for the observed protection from TNF-induced apoptosis. No evidence was found for specific cross-linking of PAI-2 to the plasma membrane in either control or TNF/cycloheximide treated cells. The data demonstrate that the PAI-2 C-D interhelical domain is functionally important in PAI-2 protection from TNF induced apoptosis and suggest a novel function for the C-D interhelical domain in the protective mechanism.     

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 structurein 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.     

Synergistic stimulating effect between cyclic AMP and phorbol ester on plasminogen activator inhibitor type 2 production in human promyelocytic leukemia cell line PL-21.

We investigated the effect of agents which raise intracellular cyclic AMP (cAMP) and protein kinase C activators on the production of plasminogen activator inhibitor type-2 (PAI-2) by cultured human promyelocytic leukemia cell line, PL-21. As previously reported, PMA, a protein kinase C activator, showed a strong stimulating effect on the PAI-2 production. 1-oleoyl-2-acetyl-sn-glycerol (OAG), another synthetic protein kinase C activator, also showed a stimulating effect, which was, however, much less than that of PMA. The agents which raise intracellular cAMP, dibutyryl cAMP, 8-bromo cAMP, prostaglandin E1, and 3-isobutyl-1-methyl-xanthine, little increased the PAI-2 production when tested alone, but showed significant synergistic effects with PMA or OAG. The synergistic effect between PMA and dibutyryl cAMP was further verified by SDS-PAGE followed by immunoblotting using a monoclonal antibody against the PAI-2. It is interesting that the up-regulation of PAI-2 by cAMP and the synergistic effect with PKC activators forms a contrast to the previous reported bi-directional regulation of endothelial PAI-1 secretion by PKC activator and cAMP.     

Immunohistochemical analysis of DNA mismatch repair enzyme hMSH-2 in normal human skin and basal cell carcinomas

We have analysed the expression and distribution of the DNA mismatch repair enzyme hMSH-2 in normal skin and basal cell carcinomas. hMSH-2 protein was investigated immunohistochemically (normal human skin: n=10; basal cell carcinomas: n=16) on frozen sections using a highly sensitive streptavidin–peroxidase technique and a specific mouse monoclonal antibody (clone FE11). In normal human skin, we found nuclear immunoreactivity for hMSH-2 in epidermal keratinocytes of the basal and first 1–3 suprabasal cell layers. All basal cell carcinomas analysed revealed strong nuclear imunoreactivity that was pronounced in peripheral tumour cells and cells of the palisade. Expression of hMSH-2 protein was consistently and strongly upregulated in tumour cells of the carcinomas as compared to adjacent unaffected epidermis or epidermis of normal human skin. Twelve of the sixteen carcinomas analysed revealed no visual correlation in comparing the labelling patterns for hMSH-2 with the labelling pattern for the proliferation marker Ki-67. Our findings indicate that (a) hMSH-2 is expressed in human epidermal keratinocytes, predominantly in lower cell layers of the viable epidermis; (b) expression of hMSH-2 protein is strongly upregulated in basal cell carcinomas as compared to unaffected epidermis; (c) the level of hMSH-2 proteins in the carcinomas is not exclusively regulated by the proliferative activity of these tumour cells; (d) inactivating mutations of the hMSH-2 gene may in the carcinomas not be involved in the carcinogenesis or microsatellite instability secondary to replication errors; (e) expression of hMSH-2 may be of importance for the genetic stability of basal cell carcinomas in vivo.     

Activation of epidermal growth factor receptor promotes late terminal differentiation of cell-matrix interaction-disrupted keratinocytes

The biological effects of epidermal growth factor receptor (EGFR) activation may differ between epidermal suprabasal and basal keratinocytes, since growth factors are mitogenic in adherent cells only in the presence of cell-extracellular matrix (ECM) interaction. To investigate biological effects of EGFR activation on keratinocytes without cell-ECM interaction, we cultured normal human keratinocytes on polyhydroxyethylmethacrylate-coated plates, which disrupt cell-ECM but not cell-cell interaction. The cells initially expressed keratin 10 (K10) and then profilaggrin, mimicking sequential differentiation of epidermal suprabasal keratinocytes. The addition of EGF or transforming growth factor-alpha promoted late terminal differentiation (profilaggrin expression, type 1 transglutaminase expression and activity, and cornified envelope formation) of the suspended keratinocytes, while suppressing K10 expression, an early differentiation marker. These effects were attenuated by EGFR tyrosine kinase inhibitor PD153035 or an anti-EGFR monoclonal antibody, whereas protein kinase C inhibitors H7 and bisindolylmaleimide I or mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibitor PD98059 abolished profilaggrin up-regulation but not K10 suppression. Since the antidifferentiative role of EGFR on cell-ECM interaction-conserved keratinocytes has been well documented, our results indicate that the biological effects of EGFR on keratinocytes are influenced by cell-ECM interaction and suggest that EGFR activation promotes rather than inhibits the terminal differentiation of suprabasal epidermal keratinocytes.     

Direct evidence for spatial and temporal regulation of transforming growth factor beta 1 expression during cutaneous wound healing

The expression of transforming growth factor (TGF beta 1) protein in human and porcine skin has been analyzed by immunohistochemistry with two polyclonal antibodies (anti-CC and anti-LC) following cutaneous injury. The anti-LC antibody binds intracellular TGF beta 1 constitutively expressed in the nonproliferating, differentiated suprabasal keratinocytes in the epidermis of normal human skin, while the anti-CC antibody does not react with the form of TGF beta 1 present in normal skin as previously shown. TGF beta 1 may play a role in wound healing as suggested by its effect on multiple cell types in vitro and its acceleration of wound repair in animals. We have evaluated the natural expression and localization of TGF beta 1 protein in situ during initiation, progression, and resolution of the wound healing response in two models of cutaneous injury: the human suction blister and the dermatome excision of partial thickness procine skin. Anti-CC reactive TGF beta 1 in the epidermis is rapidly induced within 5 minutes following injury and progresses outward from the site of injury. The induction reflects a structural or conformational change in TGF beta 1 protein and can be blocked by the protease inhibitor leupeptin or by EDTA, suggesting a change in TGF beta 1 activity. One day post-injury anti-CC reactive TGF beta 1 is present in all epidermal keratinocytes adjacent to the wound including the basal cells. This corresponds temporally to the transient block of the basal keratinocyte mitotic burst following epithelial injury. Three to 4 days post-injury anti-CC reactive TGF beta 1 is localized around the suprabasal keratinocytes, in blood vessels, and in the papillary dermis in cellular infiltrates. The exclusion of TGF beta 1 from the rapidly proliferating basal cells and its extracellular association with suprabasal keratinocytes may represent physiological compartmentation of TGF beta 1 activity. Anti-CC staining is strong in the leading edge of the migrating epithelial sheet. The constitutive anti-LC reactivity with suprabasal keratinocytes seen in normal epidermis is neither relocalized nor abolished adjacent to the injury, but anti-LC staining is absent in the keratinocytes migrating within the wound. As the wound healing response resolves and the skin returns to normal, anti-CC reactive TGF beta 1 disappears while constitutive anti-LC reactive TGF beta 1 persists. Thus, changes in the structure or conformation of TGF beta 1, its localization, and perhaps its activity vary in a spatial and temporal manner following cutaneous injury and correlate with physiological changes during wound healing.     

Inhibition of retinoblastoma protein degradation by interaction with the serpin plasminogen activator inhibitor 2 via a novel consensus motif

Plasminogen activator inhibitor-2 (PAI-2) is well documented as an inhibitor of the extracellular serine proteinase urokinase-type plasminogen activator (uPA) and is expressed in activated monocytes and macrophages, differentiating keratinocytes, and many tumors. Here we show that PAI-2 has a novel intracellular function as a retinoblastoma protein (Rb)-binding protein. PAI-2 colocalized with Rb in the nucleus and inhibited the turnover of Rb, which led to increases in Rb protein levels and Rb-mediated activities. Although PAI-2 contains an LXCXE motif, Rb binding was primarily mediated by the C-D interhelical region of PAI-2, which was found to bind to the C pocket of Rb. The C-D interhelical region of PAI-2 contained a novel Rb-binding motif, termed the PENF homology motif, which is shared by many cellular and viral Rb-binding proteins. PAI-2 expression also protected Rb from the accelerated degradation mediated by human papillomavirus (HPV) E7, leading to recovery of Rb and inhibition of E6/E7 mRNA expression. Protection of Rb by PAI-2 begins to explain many of the diverse, uPA-independent phenotypes conferred by PAI-2 expression. These results indicate that PAI-2 may enhance Rb's tumor suppressor activity and suggest a potential therapeutic role for PAI-2 against HPV-transformed lesions.