Restricted tissue distribution of a 37-kD possible adherens junction protein.

A major polypeptide of M(r) 37,000 was purified from a desmosome-enriched citric acid-insoluble pellet of pig tongue epithelium. The polypeptide was solubilized from the 4-M urea-insoluble pellet with 9 M urea, and extracts were separated by carboxymethyl cellulose and gel filtration chromatography. The 37-kD protein was obtained in milligram quantities as a single band on two-dimensional gels in 30% yield after 21-fold purification from the citric acid-insoluble fraction. The protein is not glycosylated and has a pI of approximately 8.7. Although isolated from a fraction rich in desmosomes, the 37-kD protein is not a desmosomal protein. Indirect immunofluorescence analysis of frozen sections of tongue and other tissues demonstrated that antibodies raised to the 37-kD protein bound only to suprabasal cell layers at punctate regions of the periphery of the cell and was absent from most regions of epidermis, whereas antibodies to desmoplakins I and II, desmosomal proteins, bound similarly but in all epidermal layers. Immunoelectron microscopy localized the 37-kD protein to the cell periphery in regions between, but never in, desmosomes. By immunofluorescence, the 37-kD protein colocalized with actin as well as with vinculin and uvomorulin in oral tissues. Like the 37-kD protein, vinculin and uvomorulin were absent from the basal layer. Based on its appearance, localization, and solubility properties, the 37-kD protein is probably a component of adherens junctions; its restriction to suprabasal cells and exclusion from the epidermis are unique.

Calcium-induced assembly of adherens junctions in keratinocytes.

Extracellular calcium concentration has been shown to control the stratification of cultured keratinocytes, presumably by regulation of formation of desmosomes. Previous studies have shown that keratinocytes cultured in medium containing 0.1 mM Ca++ form loose colonies without desmosomes. If the Ca++ is raised to 1 mM, desmosomes are assembled and the distribution of keratin filaments is altered. We have examined the disposition of vinculin and actin in keratinocytes under similar conditions. Using immunofluorescence microscopy we show that raising [Ca++] in the medium dramatically alters the distribution of vinculin and actin and results in the formation of adherens-type junctions within 15 min after switching to high calcium medium. Borders of cells at the edge of colonies, which are not proximal to other cells, are not affected, while cells in the interior of the colony form junctions around their periphery. Attachment plaques in keratinocytes grown in low calcium medium are located at the ventral plane of the cell, but junctions formed after switching to high calcium are not, as demonstrated by interference reflection microscopy. In cells colabeled with antibodies against vinculin and desmoplakin, vinculin-containing adherens junctions were visible before desmosomal junctions when cells were switched to high calcium. Although newly formed vinculin-containing structures in high calcium cells, like desmosomes, colocalize with phase-dense structures, superimposition of video fluorescence images using digitized fluorescence microscopy indicates that adherens junctions and desmosomes are discrete structures. Adherens junctions, like desmosomes, may play an essential role in controlling stratification of keratinocytes.

Epidermal growth factor receptor number decreases during rat liver regeneration.

The potential role of epidermal growth factor (EGF) in the regulation of rat liver regeneration was examined by assessing the binding of 125I-EGF to hepatic membranes isolated at various times after partial hepatectomy. The results demonstrated a fall in 125I-EGF binding detectable as early as 8 h after partial hepatectomy. The nadir in EGF binding, less than 40% of that observed in sham-operated control rats, was seen 36 and 48 h after partial hepatectomy. Scatchard analysis showed that the decrease in binding capacity was due to a fall in receptor number. The specificity of the observed loss of EGF receptors was substantiated in parallel studies of 125I-insulin and 125I-wheat germ lectin binding; the binding of these ligands did not decrease appreciably during liver regeneration. The data are consistent with the hypothesis that EGF or a similar substance is one component of the complex humoral signal that regulates liver regeneration.

Human autoantibodies against desmoplakins in paraneoplastic pemphigus.

Recently, a previously unrecognized autoantibody mediated blistering disease, paraneoplastic pemphigus has been described. Paraneoplastic pemphigus is associated with lymphoid malignancies, thymomas, and poorly differentiated sarcomas. Serum of affected patients contain pathogenic autoantibodies that immunoprecipitate from normal keratinocytes a characteristic complex of four polypeptides with M(r) of 250, 230, 210, and 190 kD. As our preliminary studies indicated that the 250-kD and the 210-kD antigens comigrated with desmoplakins I and II, we investigated the possibility that autoantibodies against the desmoplakins were a component of this autoimmune syndrome. 11 sera from affected patients were tested by indirect immunofluorescence against desmosome containing tissues, immunoprecipitation of metabolically labeled keratinocytes, and Western immunoblotting of desmoplakins I and II that had been purified to homogeneity from pig tongue epithelium. By indirect immunofluorescence, 9 of 11 sera showed strong binding to epithelial and nonepithelial desmosomes, and 2 were weakly reactive. All 11 immunoprecipitated 250- and 210-kD bands of variable intensity that comigrated with bands identified by a murine monoclonal antidesmoplakin antibody, and immunoblotting confirmed binding of the serum autoantibodies to purified desmoplakins. This demonstrates that paraneoplastic pemphigus is the first human autoimmune syndrome in which autoantibodies against the desmoplakins are a prominent component of the humoral autoimmune response.

Specific affinity between fibronectin and the epidermolysis bullosa acquisita antigen.

Autoantibodies in the skin and sera of patients with epidermolysis bullosa acquisita bind to a large matrix molecule within the lamina densa region of skin basement membrane. At the site of these immune complexes, the epidermis separates from the dermis, which creates a subepidermal blister just below the lamina densa. The target molecule for the autoantibodies is in close apposition to fibronectin, a major extracellular matrix molecule that is abundant in the upper dermis of skin. In this report, we show specific affinity between fibronectin and the 290,000-D chain of the epidermolysis bullosa acquisita antigen, and that this affinity is mediated by the gelatin/collagen-binding domain of fibronectin (Mr = 60,000). Since blistering in epidermolysis bullosa acquisita often occurs in the absence of clinical and histological inflammation, a direct interruption in the fibronectin-epidermolysis bullosa acquisita antigen bond may be involved in the pathogenesis of epidermal-dermal disadherence that occurs in this bullous disease.

Modulation of the epidermal growth factor receptor of human keratinocytes by calcium ion.

Human keratinocytes grown in medium containing reduced calcium concentrations (0.07 mM) have been found to show altered morphology and decreased differentiation in comparison with cells grown in medium with physiologic calcium concentrations (1-2 mM). Since such alterations could be mediated by growth factors, we measured binding of [125I]epidermal growth factor (EGF). Neonatal keratinocytes were subcultured without feeder layers and grown to confluence in 1.1 mM calcium. Medium was changed and cells incubated for various periods in reduced calcium prior to binding assays. Scatchard plots of binding data showed a 5-fold increase in receptor number with no change in affinity (3 X 10(-9) M) after 4-24 h at 37 degrees C. Maximal binding occurred at 0.02-0.04 mM calcium and decreased sharply with increasing calcium concentrations. The increase could be prevented by calcium added soon after binding began to increase but was altered less after substantial elevation had occurred, although morphologic changes at reduced calcium concentrations were reversed within several hours. Substantial increases in binding of [125I]somatomedin C and [125I]concanavalin A were detected, but binding of [125I]pindalol, a beta-receptor ligand, was changed little. Keratinocytes at reduced calcium concentrations responded to added EGF by decreasing surface EGF receptors briskly in a time- and temperature-dependent fashion. The data suggest that keratinocytes show enhanced binding of EGF and some other cell surface ligands under conditions in which differentiation is retarded.

Stimulation of thymidine incorporation in keratinocytes by insulin, epidermal growth factor, and placental extract: comparison with cell number to assess growth.

The results of a thymidine incorporation assay were compared with direct measurement of cell number in assessment of proliferative growth of human keratinocytes in monolayer culture. Keratinocytes were cultured in supplemented MCDB 153 medium in 0.1 mM Ca2+, and plated in 24-well trays. The ability of insulin, placental extract, and epidermal growth factor to enhance growth and thymidine incorporation were compared. Autoradiography was performed to determine the percentage of cells with labeled nuclei. Epidermal growth factor increased thymidine incorporation under the conditions of the assay, and placental extract increased incorporation by up to 50-fold, since the control cells plated in the absence of epidermal growth factor and other growth factors survived but proliferated minimally. Both cell number and thymidine incorporation showed similar concentration dependence upon insulin and placental extract. If placental extract was added to cells plated 28 h earlier, incorporation was maximal after 17 h in the presence of the extract. If cells were plated in the presence of the extract, 85% of nuclei were shown by autoradiography to be labeled after 23 h, but only 24% of nuclei were labeled in the absence of the extract. A plating density of 10(4) cells/2-cm2 well was optimal. The assay permits rapid identification of growth-promoting fractions without prolonged growth periods, and is a valid indicator of these agents in keratinocyte cultures.

Tyrosine phosphorylation of human keratinocyte beta-catenin and plakoglobin reversibly regulates their binding to E-cadherin and alpha-catenin.

We show that tyrosine phosphorylation, produced by incubation of normal human keratinocytes with the tyrosine phosphatase inhibitor peroxovanadate, directly and reversibly regulates the association of beta-catenin and plakoglobin with E-cadherin and alpha-catenin. Prior studies have demonstrated a correlative, but not causal, association between increased tyrosine phosphorylation and decreased adherens junction mediated cell-cell adhesion. We observed that (i) binding of tyrosine phosphorylated beta-catenin and plakoglobin to E-cadherin and to alpha-catenin was substantially reduced, but could be restored in vitro by removal of phosphate from beta-catenin and plakoglobin with added tyrosine phosphatase, and (ii) tyrosine phosphorylation of beta-catenin and plakoglobin was associated with decreased cell-cell adhesion. These findings support a direct and causal role for tyrosine phosphorylation of beta-catenin and plakoglobin in regulating adherens junction mediated cell-cell adhesion. We propose that tyrosine phosphorylation of specific and probably different residues is responsible for regulating the binding of beta-catenin or plakoglobin to (i) E-cadherin and (ii) alpha-catenin. Additionally, because beta-catenin and plakoglobin have both structural and regulatory functions, the data raise the possibility that beta-catenin or plakoglobin released from the adherens junctions by tyrosine phosphorylation may transduce a signal to the nucleus regarding the adhesive state of the cell.

Type IV collagen and fibronectin enhance human keratinocyte thymidine incorporation and spreading in the absence of soluble growth factors.

In various cell culture systems, extracellular matrix components have been demonstrated to be mitogenic and, in some cases, to substitute for growth factors. In order to study the effects of various matrices on keratinocyte growth, we assessed the incorporation of tritiated thymidine and cell number on short-term cultures of human keratinocytes plated on different substrata. For determination of whether thymidine incorporation by keratinocytes was related to the ability of the cells to attach and spread on the substratum, experiments to determine the percentage of attached and spread cells on each matrix surface were performed. High levels of attachment and incorporation of thymidine with no preferential attachment to a given matrix were evident when the cells were cultured in the presence of growth factors. When growth factors were absent, keratinocytes likewise showed no preferential attachment to a given matrix component, but demonstrated enhanced thymidine incorporation when apposed to type IV collagen or fibronectin in comparison with tissue culture plastic or laminin. In the absence of epidermal growth factor (EGF) and bovine pituitary extract (BPE), increased spreading on type IV collagen and fibronectin was associated with enhanced incorporation of thymidine. In agreement with the thymidine incorporation results, when keratinocytes were cultured for 7 d, cell numbers were increased in cultures plated on type IV collagen only if growth factors were excluded from the medium. When attachment of cells to substrata with or without growth factors was compared, either EGF or BPE enhanced attachment to all of the substrata tested. It is concluded that under suboptimal growth conditions extracellular matrix components can modulate keratinocyte growth. Also, under these conditions, spreading, but not attachment, correlates with growth potential.

Trichohyalin: presence in the granular layer and stratum corneum of normal human epidermis.

Trichohyalin, a protein contained in granules in the cells of the hair-follicle inner root sheath and in the medulla of the hair shaft, has been purified previously from sheep hair bulbs and is also a major protein of filiform papillae of tongue epithelium. Polyclonal affinity-purified antibodies and a monoclonal antibody raised to purified pig tongue trichohyalin both stained the inner root sheath of hair follicles and the medulla of hair fibers and identified human trichohyalin as a single 220-kDa band on immunoblots of human hair bulb proteins. These antibodies were used to examine human epidermis by immunofluorescence and immunoblotting. The antibodies decorate granules in cells in the granular layer and stratum corneum of non-hair-bearing human skin, and immunoblots identify a protein in epidermis comigrating with trichohyalin from human hair and human tongue epithelium. Absorption of antibody to trichohyalin on a trichohyalin affinity column abrogated staining of the epidermis and the bands on the immunoblots. Trypsin-separated epidermis contained 220 and 160 kDa bands identified as trichohyalin, but epidermis shaved from skin and quickly frozen showed only a single 220-kDa band, indicating that the 160-kDa protein was generated by proteolysis. Double immunofluorescence for trichohyalin and filaggrin showed that some cells containing filaggrin also contain trichohyalin. These studies show that trichohyalin is not limited to hair and tongue but is present in isolated cells in the granular layer and stratum corneum of normal epidermis.

Stimulation of growth of keratinocytes by basic fibroblast growth factor.

Numerous heparin-binding growth factors active in different types of cells have recently been shown to belong to the family of fibroblast growth factors. Because these factors are active in some types of epithelial cells, we tested the activity of basic fibroblast growth factor (bFGF) from bovine brain in human keratinocyte cultures. bFGF stimulated thymidine incorporation and cellular proliferation in these cultures with half-maximal activity at approximately 60 pg/ml (4 X 10(-12) M). Stimulation of thymidine incorporation was associated with increased nuclear labeling after 22 h in the presence of bFGF under the same conditions used in the thymidine incorporation assay. bFGF was nearly as effective as epidermal growth factor (EGF) in stimulating keratinocyte growth and substantially less effective than crude placental extract, and was not additive with EGF in stimulating thymidine incorporation or proliferation of cells. The findings indicate that bFGF is a potent growth factor for keratinocytes.

Talin: adherens junction protein is localized at the epidermal-dermal interface in skin.

The interaction between cells of the epidermis and the basal lamina is important for the integrity of the skin. Several hereditary and acquired diseases show changes at the dermal-epidermal interface due to loss of adhesion between basal cells and the basement membrane. The structures mediating this interaction are hemidesmosomes, which have been extensively characterized by biochemical, molecular biologic, and morphologic techniques. Recently, however, a group of adhesion molecules that are distinct from hemidesmosomes and that mediate cell-matrix interactions was described in cultured fibroblasts, keratinocytes, and skin. These adhesion molecules, beta 1 integrins, have been shown to be present in the focal adhesion, a cell-matrix contact associated with microfilaments rather than intermediate filaments characteristic of hemidesmosomes. In cultured cells, integrins of the beta 1 family have been shown to be linked by a protein complex to actin filaments. In this study we describe the localization of talin, the binding protein for beta 1 integrins, and vinculin at the dermal-epidermal interface in skin with immunofluorescence and immunoblotting techniques. These data suggest the presence of a link between the cytoplasmic actin filament system in basal keratinocytes and the extracellular matrix.

Human dermal fibroblasts synthesize laminin.

Laminin, a glycoprotein of approximately 900,000 daltons, is a major component of the basement membrane that separates the epidermis from dermis in human skin. Previous studies have shown that keratinocytes and other epithelial cells synthesize laminin and utilize it for attachment to other extracellular matrices such as heparan sulfate proteoglycan and basement membrane collagen. The relationships between phenotypically normal mesenchymal cells and laminin have been much less emphasized in the literature. In this study, we have used antibodies that specifically label the A and B chains of laminin (but not fibronectin or other unrelated proteins) by Western blot analysis to immunoprecipitate biosynthetically derived laminin from [35S] methionine labeled cultures of neonatal and adult human skin fibroblasts. To be sure that the precipitated bands were laminin and not fibronectin, which has a molecular size very close to that of the laminin B chains, experiments were performed in which fibronectin was removed from the radiolabeled proteins by first immunoprecipitating with antifibronectin antibody and then sequentially immunoprecipitating laminin from the fibronectin-depleted supernates with antilaminin antibody. These experiments definitively demonstrate that human dermal fibroblasts synthesize and secrete laminin.

Human keratinocyte locomotion: the effect of selected cytokines.

Epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) are two powerful mitogens for human keratinocytes that also have been shown to promote the healing of in vivo wounds. Transforming growth factor-beta (TGF-beta) markedly inhibits human keratinocyte proliferation and growth and yet has been shown to promote wound healing. Using a migration assay that evaluates pure cell locomotion independently from cell proliferation, we examined the influence of EGF, bFGF, and TGF-B on human keratinocyte locomotion. Although these agents had profound influences upon the growth potential of keratinocytes in parallel thymidine incorporation assays, they had no significant effect upon keratinocyte locomotion when cells were apposed to either tissue culture plastic or a collagen substratum. In contrast, we found that bovine pituitary extract (BPE), a poorly defined mitogen that is commonly used in keratinocyte cultures, could stimulate keratinocyte locomotion when the cells were apposed to a collagen substrate. These studies demonstrate that i) keratinocyte locomotion and proliferation operate by completely independent mechanisms, ii) the positive effects upon wound healing by EGF, bFGF, and TGF-beta are not due to a direct promotion of keratinocyte locomotion, and iii) that one or more components of BPE are capable of directly promoting keratinocyte locomotion on collagen.

Trichohyalin: a structural protein of hair, tongue, nail, and epidermis.

In the course of studies of desmosomes, we found trichohyalin, a 200-kDa protein of the inner root sheath and medulla, in a citric acid-insoluble fraction ("desmosome preparation") from tongue epithelium. Pig tongue epithelium yielded milligram quantities of pure trichohyalin from about 100 g of keratomed epithelium. The protein has an extended shape as determined by gel filtration, ultracentrifugation, and electron microscopy, with a rod domain and a globular domain at one end and overall dimensions of about 85 nm. Crosslinking studies suggest that the protein may be dimeric in solution. The protein is a doublet in some animals but apparently is a single polypeptide of 220 kDa in humans. Immunofluorescence studies showed that it is a major protein of the filiform papillae of the tongue of mammals and is present in isolated cells of the stratum granulosum of some regions of epidermis in a subset of cells containing filaggrin and in the nail matrix. Similarly, in filiform papillae some cells contain granules that stain for both trichohyalin and filaggrin. Immunoblotting confirmed that trichohyalin is present in tongue and epidermis. Polymerase chain reaction with human genomic DNA using oligonucleotide primers based on sheep trichohyalin resulted in synthesis of multiple DNAs, from which a 504-bp fragment was subcloned and sequenced and found to resemble closely the carboxyl terminus of sheep trichohyalin. Studies with antibody to the carboxyl-terminal 14 amino acids of the human sequence show that, whereas the carboxyl-terminal epitope is present only in the stratum granulosum, in epidermis epitopes detected by a monoclonal antibody are demonstrated in both the stratum granulosum and stratum corneum, suggesting that the carboxyl terminus is cleaved in the stratum corneum.

Production of fibronectin by epithelium in a skin equivalent.

Although human keratinocytes in vitro have been shown to produce fibronectin, whether keratinocytes can contribute fibronectin to the dermal-epidermal junction or wound matrix is unknown. In order to approach this problem experimentally, we used the "skin equivalent" model composed of a native collagen gel populated with cultured fibroblasts and covered by cultured keratinocytes. By using bovine fibroblasts to populate the gel, fetal bovine serum in the culture medium, and human keratinocytes to form the epithelium, we were able to be certain that any human fibronectin produced in the culture was synthesized by the keratinocytes. A monoclonal antibody to fibronectin was found to recognize human but not bovine fibronectin. When the skin equivalent was stained by indirect immunofluorescence with antifibronectin, fibronectin was visible as an intensely staining band at the dermal-epidermal junction. In sections in which the dermis and epidermis had separated, the staining was usually limited to the dermal aspect of the skin equivalent. The results indicate that epithelium can contribute fibronectin to the dermal-epidermal junction and suggest that dermal staining in skin sections may originate from the epidermis. Since the developing skin equivalent has a rapidly growing epithelium and simulates a healing wound, contribution of fibronectin by the epithelium, in addition to that possibly contributed by serum and fibroblasts, may be of importance in wound healing.

Spreading and enhanced motility of human keratinocytes on fibronectin.

Soluble human plasma fibronectin or collagen types I or IV, when preincubated with tissue culture plastic dishes, were effective spreading agents for cultured human keratinocytes and increased spreading in a time-and concentration-dependent manner. Spreading on fibronectin, but not on type IV collagen, was inhibited by antifibronectin; therefore, the contribution of fibronectin to the spreading activity of the natural matrix produced by keratinocytes could not be determined using antifibronectin. Fibronectin mediated spreading at both high (1.1 mM) and low (0.1 mM) Ca++ concentrations, and spreading was not altered by cycloheximide. Insoluble fibronectin deposited by keratinocytes correlated with phagokinetic tracks on particulate gold salts, and added fibronectin, as well as type I collagen and type IV collagen, enhanced motility of keratinocytes. These studies show that production of fibronectin and responsiveness to it are similar in fibroblasts and keratinocytes and demonstrate that fibronectin can act as a matrix factor for keratinocytes.

Enhanced synthesis of collagenase by human keratinocytes cultured on type I or type IV collagen.

Human keratinocytes in culture are known to produce collagenase. As part of studies to ascertain the physiologic stimuli for collagenase production by keratinocytes, we wanted to determine whether extracellular matrix could modulate the production of collagenase in vitro. Immunoprecipitable collagenase from the conditioned medium of cells grown on different types of matrix was measured. Metabolically labeled human keratinocytes were cultured in 0.1 mM calcium in serum-free medium on colloidal gold-coated coverslips plus type IV collagen, type I collagen, or laminin or in the absence of matrix. Immunoprecipitation of the conditioned medium with anti-collagenase antiserum was performed and the immunoprecipitates were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, fluorography, and densitometry. The keratinocytes cultured on type IV or type I collagen produced more collagenase than did those cultured on laminin or in the absence of matrix. This effect did not reflect a general increase in secreted proteins, because the production of tissue inhibitor of metalloproteinase, or TIMP, did not increase under the same conditions. Phagocytosis of the gold salts by the keratinocytes migrating on types I or IV collagen did not account for the increased collagenase produced by these cells since the effect persisted in the absence of the colloidal gold and phagocytosis of latex beads did not augment collagenase production.

Vitronectin: effects on keratinocyte motility and inhibition of collagen-induced motility.

Epibolin, a plasma protein, was initially purified on the basis of its ability to enhance spreading of keratinocytes. It is now known that epibolin is identical to serum spreading factor, S protein, and vitronectin, and the current name for the protein is vitronectin. Studies of vitronectin on cultured keratinocytes showed that it caused spreading and epiboly but not cellular adhesion to the substratum. In studies with other types of cells, vitronectin increased migration of several types of cells in a Boyden chamber. Because some agents that enhance spreading and adhesion, such as collagen and fibronectin, also increase motility, we tested whether vitronectin increased motility of keratinocytes. By photographing and quantitating motility of keratinocytes plated on a bed of colloidal gold particles, we determined that vitronectin increased local movement of keratinocytes in a concentration-dependent fashion, resulting in clearing of gold particles in a circular pattern around the cells, but did not cause the production of tracks found in cultures plated on collagen or fibronectin. The small increases in clearing of the gold particles that occurred in the presence of vitronectin were abolished by antibody to vitronectin. Furthermore, the marked increase in motility produced by type I collagen was significantly reduced when the keratinocytes were treated with vitronectin. Antibody to vitronectin also abrogated the vitronectin-induced reduction in collagen-stimulated motility, confirming that this action was specific for vitronectin. Serum, which contains vitronectin, stimulated motility in a fashion identical to purified vitronectin, but serum lacking vitronectin was inactive. These studies show that vitronectin causes a localized increase in movement associated with spreading resulting in a halo around individual cells, that vitronectin does not enhance directional motility of keratinocytes in this assay but in contrast antagonizes such motility produced by collagen, and that vitronectin is the factor in serum responsible for this effect. The findings with vitronectin and collagen show that these agents stimulate different types of motility. The roles in wound healing of agents stimulating different types of motility are unclear and require further study.

Constitutive production of procollagenase and tissue inhibitor of metalloproteinases by human keratinocytes in culture.

Production of procollagenase and tissue inhibitor of metalloproteinases was demonstrated in human keratinocyte cultures. The two proteins were immunoprecipitated from keratinocyte-conditioned medium with antibodies to human dermal fibroblast collagenase and tissue inhibitor of metalloproteinases and quantitated with enzyme-linked immunosorbent assays. Treatment of the keratinocytes with the phorbol ester, 12-0-tetradecanoylphorbol-13-acetate, produced a six to 34-fold increase in procollagenase synthesis and secretion but only a threefold increase in the production of tissue inhibitor of metalloproteinases. Collagenase and tissue inhibitor of metalloproteinases mRNAs were present in normal keratinocytes, were the same size as their fibroblast counterparts, and both increased in response to treatment with 12-0-tetradecanoylphorbol-13-acetate. These data suggest that remodeling of type I collagen may be an important function of human keratinocytes in vivo.