JOURNAL OF CELLULAR PHYSIOLOGY 144:144-150(1990)

Transforming Growth Factor-p1 Localization in Normal and Psoriatic Epidermal Keratinocytes In Situ C Y N T H I A J.M.KANE,* A. MERRILL KNAPP, J O N A T H A N N. MANSBRIDGE, AND PHILIP C. H A N A W A L T Department of Biological Sciences (C.I.M.K., P.C.H.1, Stanford University, Stanford, Caldornm 94305, and Psoriasis Resedrch Institute (A.M.K., I.N.M.), Palo Alto, California 94305 Transforming growth factor-PI (TGFP1) i s a potent inhibitor of epithelial cell proliferation and its effects on growth and differentiation have been extensively characterized in cultured keratinocytes. We used two TGFPI -specific polyclonal antibodies (anti-LC and anti-CC) to determine the presence of TLFPI 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 hyperproliferativehegenerative differentiation phcnotype. Anti-TGFpl staining was observed primarily in the epidermis. Anti-LC TGFPl 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 TGFPl 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 TCFPl and its localization vary in keratinocytes with distinct differentiation phenotypes suggesting that TGFPl is a potential modulator of keratinocyte differentiation in vivo. Selective association of TGFPl 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 TCFPl peptide in both normal and psoriatic keratinocytes suggests that it i s constitutively synthesized by epidermal keratinocytes in vivo.

Transforming growth factors type+ are a structurally and functionally related family of pluripotent peptide regulators of growth and differentiation for many cell types (for review see Roberts and Sporn, 1990). Transforming growth factor type-pl (TGFpl), found in high concentrations in human platelets (Assoian et al., 1983),is the best characterized of these factors. Studies of the effects of TGFPl on growth and differentiation of epithelial cells have focused particularly on cultured keratinocytes (for review see Bascom et al., 1989). Proliferation of cultured keratinocytes is reversibly inhibited by TGFpl (Shipley et al., 1986). However, this growth inhibition by TGFPl does not induce the normal keratinocyte differentiation phenotype; Coffey et al. (1988) have shown that concentrations of TGFpl that induce morphological change and inhibit DNA synthesis do not alter synthesis of the normal differentiation markers pemphigus vulgaris antigen, desmoplakin I, and filaggrin. These results suggest that growth inhibition and induction of differentiation of Zultured keratinocytes are uncoupled by TGFPl. It has been demonstrated (Mansbridge and Hanawalt, 1988) 0 1990 WILEY-LISS, INC

that synthesis of the hyperproliferation-associated keratins 6 and 16 is increased by TGFpl in human keratinocytes, while synthesis of keratin 1,associated with normal keratinocyte differentiation, is decreased. Physiologically, keratins 6 and 16 are expressed in epidermal keratinocytes in association with epidermal hyperproliferation (Bernard et al., 1985; Weiss et al., 1984) or in association with epidermal regeneration, such as re-epithelialization following epidermal injury (Mansbridge and Knapp, 1987). In contrast, keratin 1 and its partner keratin 10 are normally expressed only in differentiating keratinocytes such as those in the upper spinous and granular layers of the epidermis. These results suggest that in culture TGFpl could mediate a switch from a normal keratinocyte differentia-

*To whom reprint requestsicorrespondence should be addressed. Received November 29, 1989; accepted March 29, 1990. The present address of J. Mansbridge and A. Knapp is SRI, Menlo Park, CA.

TGFpl IN EPIDERMAL KERATINOCYTES IN SITU

tion phenotype to a hyperproliferativeiregenerative phenotype. In order to evaluate the relevance of these in vitro observations to the role of TGFPl in vivo, it is first necessary to establish whether TGFPl is present in human skin. TGFpl mRNA has been isolated by Elder e t al. (1989) from normal human skin as judged by RNA blot hybridization. However, the sites of constitutive TGFpl synthesis within the skin were not detectable by hybridization of TGFPl mRNA in situ (Coffey et al., 1987; Akhurst et al., 1988). Akhurst et al. (1988) have shown that TGFPl mRNA is found in suprabasal keratinocytes in hyperproliferating regions of mouse epidermis induced by the topical application of the chemical promoter phorbol ester. As keratinocyte cell division occurs almost exclusively in the basal layer, these results indicate that TGFpl mRNA induction occurred in the nonproliferating, differentiated suprabasal keratinocytes coincident with the induction of the hyperproliferative phenotype. However, the biological activity of TGFpl in cultured cell systems is dependent on post-translational modifications [Lyons et al., 1988; Wakefield et al., 1988; Miyazono et al., 1988). In order to understand the physiological significance of TGFp1, it is essential to analyze the level of protein, as well as mRNA, in the specific cell or tissue of interest. In the studies reported here, we used two TGFP1specific polyclonal antibodies, designated anti-LC (Flanders et al., 1988) and anti-CC (Ellingsworth et al., 19861, to detect TGFPl peptide in epidermal keratinocytes in sections of human skin. We also evaluated changes in the expression and localization of TGFPl when a hyperproliferative phenotype is evident in the keratinocytes of the epidermis in individuals with psoriasis. Psoriasis is a disease characterized by nonmalignant epidermal hyperproliferation and the presence of differentiation markers associated with the hyperproliferativelregenerative keratinocyte phenotype (Bernard et al., 1985; Mansbridge and Knapp, 1987). The hyperproliferation is localized t o regions, known a s plaques, which are distinct from the apparently disease-free, nonplaque skin of the same individual. Our analysis of both plaque and nonplaque psoriatic skin revealed alterations in the conformation or structure of TGFpl and its localization within the keratinocytes relative to that in normal skin. These differences in structure and location of TGFp1, associated with variations in keratinocyte growth and differentiation, may reflect physiological changes in the function or activity of TGFpl.

MATERIALS AND METHODS Antibodies The two TGFpl-specific polyclonal rabbit antibodies used in this study have been previously described and characterized (Ellingsworth et al., 1986; Flanders e t

Fig. 1. Localization of TGFpl in normal epidermal keratinocytes. A Anti-LC antibody bound TGFPl in normal epidermal keratinocytes. X 110. B: Anti-LC, staining in normal keratinocytes was blocked by preincubation of the anti-LC antibody with LC peptide. x 110. C : Normal keratinocytes did not bind anti-CC antibody. x 110.

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al., 1988). The antibodies were prepared to different little staining with the exception of localized collagesynthetic preparations of a peptide corresponding to nous tissue or highly cellular regions underlying the the first 30 amino acids of mature TGFP1. Anti-CC epithelium in some of the psoriatic samples. antibody and the corresponding nonimmune rabbit IgG Appropriate controls for anti-LC and anti-CC TGFpl control were the generous gift of L. Ellingsworth, Col- antibody binding were performed on all samples and lagen Corporation, Palo Alto, CA. Anti-LC antibody, were negative in all cases. Controls included: 1)omisits control normal rabbit IgG, and the synthetic LC sion of primary antibody (data not shown), 2) substitupeptide for blocking were kindly provided by K. tion of nonimmune rabbit IgG, prepared in the same Flanders, National Cancer Institute, NIH, Bethesda, manner as the immune IgG, for the same concentration MD. The anti-CC (Ellingsworth et al., 1986) and anti- of primary antibody (data not shown), 3) depletion of LC (Flanders et al., 1988) antibodies have been exten- TGFpl-specific binding by preincubation of anti-CC sively characterized with regard to TGFPl specificity antibody with TGFpl-coupled Sepharose (Heine et al., and their affinity for distinct epitopes of TGFpl (Heine 19871, and 4) blocking of anti-LC antibody binding by et al., 1987; Flanders e t al., 1989). preincubation with a 20-fold molar excess of the LC peptide for 2 hours a t room temperature prior to appliTissue source cation as primary antibody (Flanders et al., 1989). Skin samples were obtained through the Department Normal skin of Dermatology, Stanford University. Punch biopsies of 3 to 4 mm were taken under xylocaine local anesthesia. The upper layers of the epidermis of normal human Ten specimens of normal skin were analyzed, five from skin were prominently stained with anti-LC antibody the inner side of the upper arm and five from the lower specific for TGFp1, in a suprabasal pattern (Fig. 1A). back. Two specimens were obtained from each of seven This staining was specifically blocked (Flanders et al., psoriatic individuals, prior to treatment, and included 1989) by preincubation of anti-LC antibody with 20both lesional plaque skin and nonplaque skin from fold excess synthetic LC peptide antigen, which correeach individual. Plaque and nonplaque skin in all cases sponds to the first 30 amino acids of mature TGFPl was clearly defined by clinical history and examination (Fig. 1B). The anti-LC antibody staining in epidermal and by histologic evaluation. Nonplaque specimens keratinocytes was exclusively intracellular and apwere biopsied from regions with no history of lesions peared a s a fine reticulum throughout the cytoplasm of spinous and granular layer keratinocytes. Basal keraand were located a t least 5 cm from lesions. tinocytes did not contain antibody-detectable TGFPI. Tissue fixation and Intracellular anti-LC reactive staining was identified immunohistochemical staining in keratinocytes of a hair follicle located in one of the Biopsies were fixed a t 4°C in either: 1) 10%neutral- normal skin biopsies studied (data not shown). Anti-LC buffered formalin overnight then Bouin’s solution [sat- specificity for TGFpl and epitope characterization has urated aqueous picric acid:formalin:acetic acid (15:5:1)] been reported by Flanders et al. (1989). 4-16 hours, or 2) Bouin’s solution overnight. Tissue When anti-CC antibody specific for TGFPl was used, was dehydrated, embedded in paraffin, and sectioned no staining was observed in the epidermis or the derat 4-6 pm. Sections were deparaffinized, rehydrated, mis of ten normal skin biopsies examined (Fig. 1C). endogenous peroxidase blocked by 30 minute incuba- The anti-LC and the anti-CC antibodies appear to rection in 0.3% hydrogen peroxide in methanol, and per- ognize distinct epitopes of TGFpl (Flanders et al., meabilized by treatment with 1 mgiml hyaluronidase, 1989).I t is probable that the conformation or structure type I-S, (Sigma, St. Louis, MO) in 0.1 M sodium ace- of TGFpl which allows the anti-CC-reactive epitope to tate, 0.15 M sodium chloride, pH 5.5, for 30 minutes a t be presented does not occur or is inaccessible in normal 37°C. Nonspecific protein binding was blocked with 5% skin. calf serum in phosphate-buffered saline. Anti-CC antiPsoriatic plaque skin body and its control IgG were incubated with sections In psoriatic skin, we observed two distinguishable at 30 Fgirnl for 2 hours at room temperature. Anti-LC antibody and its control IgG were incubated with sec- patterns of anti-CC antibody staining in keratinocytes tions at 20 pg/ml for 2 hours at room temperature. (Fig. 2A). An intense, granular extracellular staining Unbound antibody was removed by washing with 5% pattern was observed suprabasally with anti-CC anticalf serum in phosphate-buffered saline. Bound anti- body in the epidermis of a psoriatic plaque. This stainbody was localized with biotinylated goat anti-rabbit ing was prominent throughout the central region of the IgG, a n avidin-peroxidase Vectastain kit (Vector Lab- deep rete ridges (Fig. 2C). The focal intensity of the oratories, Burlingame, CA), and 3,3’-diaminobenzidine extracellular anti-CC staining was variable in the (Polysciences, Warrington, PA) with hydrogen perox- spinous layer and significantly reduced in the granular ide as peroxidase substrate. Sections were counter- layer. In addition, a fine, reticulate intracellular antistained in Gill’s hematoxylin (Sigma, St. Louis, MO). CC staining pattern was distributed throughout the keratinocytes in the psoriatic epidermis, with the exRESULTS ception of the basal cells, and extended beyond the edge Histochemical staining with anti-LC and anti-CC of the plaque. This is discussed below with reference to antibodies specific for TGFpl indicated that TGFPl the nonplaque psoriatic skin. Extracellular and intrawas primarily localized in the epidermis in all samples cellular anti-CC staining was seen in the keratinocytes analyzed. Epidermal keratinocytes, as well as kerati- of a hair follicle located in one of the plaque biopsies nocytes in hair follicles within the dermis, stain posi- (data not shown). Anti-CC antibody staining was spetive for TGFp1. Other cells within the dermis exhibit cifically blocked by preincubation of anti-CC antibody

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147

The intracellular localization of TGFPl in keratinocytes in normal and psoriatic skin suggested that TGFPl is synthesized by epidermal keratinocytes in situ. In normal skin, cells in the dermis did not exhibit antibody-detectable TGFp1; thus, it is unlikely that the TGFPl found in keratinocytes in the epidermis has originated in the dermis or the circulatory system. Since TGFPl was detected in the dermis as well a s the epidermis in several sections of both plaque and nonplaque skin from psoriatic patients, there may be additional sites of TGFPl synthesis in the psoriatic dermis, the distribution of keratinocyte-derived TGFPl may be altered in psoriatic skin, or changes in vascularization may provide additional sources of TGFPl. Thompson et al. (1989) reported anti-CC staining of adult mouse dermis. Dermal staining with anti-CC or anti-LC was not observed in the ten specimens of normal adult skin analyzed in the present study. The differences could not be compared since the previous report included no immunohistochemically stained skin specimens and no discussion of the staining pattern. TGFPl is localized exclusively to the suprabasal keratinocytes in normal epidermis and in psoriatic plaque and nonplaque epidermis with these two antibodies. The presence of TGFPl in these nonproliferatPsoriatic nonplaque skin ing, differentiated keratinocytes suggests that TGFPl Nonplaque skin from a psoriatic person exhibited a n physiologically modulates keratinocyte proliferation or anti-TGFPl antibody staining pattern distinct from differentiation. Many changes in gene expression have both that in the normal skin and in psoriatic plaque been observed in differentiated keratinocytes which skin. Both anti-CC and anti-LC antibodies exhibited a are clearly secondary to the induction of the differenfinely reticulate, intracellular staining pattern in su- tiated phenotype. However, in consideration that prabasal keratinocytes in the psoriatic nonplaque epi- TGFPl inhibits expression of the normal differentiated dermis (Fig. 3A,B). The anti-CC antibody staining pat- phenotype in cultured keratinocytes (Mansbridge and tern (Fig. 3A) was in clear contrast to the total absence Hanawalt, 19881, a potential regulatory function for of anti-CC antibody staining in keratinocytes in nor- TGFPl in the process of epidermal differentiation is mal skin (Fig. 1C). The intracellular staining with indicated. In view of the potent inhibition of keratianti-CC antibody was also observed in the epidermal nocyte proliferation effected by TGFpl in culture, the keratinocytes of the psoriatic plaque (Fig. 2A). How- absence of anti-TGFpl antibody staining in the basal ever, the intense, granular extracellular anti-CC stain layer of keratinocytes may represent a n important seen suprabasally in the plaque was absent in the pso- compartmental regulation of TGFPl activity in vivo. riatic epidermis outside regions of active lesions. Re- Exclusion of TGFPl from the proliferating pool of basal ticulate staining with anti-CC antibody seen in the cells by compartmentation in the suprabasal layers dermis of the psoriatic plaque was also present in the may exclude these cells from growth inhibition by collagenous tissue of three of the seven nonplaque pso- TGFP1. riatic samples. The TGFPl specificity of anti-CC and Different epitopes of TGFPl apparently react with anti-LC staining in nonplaque skin was demonstrated, anti-TGFpl antibodies in normal and psoriatic keratia s described above, by depletion of TGFpl-specific anti- nocytes i n vivo, suggesting that the conformation or CC antibody reactivity by preincubation with TGFP1- structure of TGFpl varies in keratinocytes with discoupled Sepharose (Fig. 3C) and by blocking anti-LC tinct differentiation phenotypes. TGFPl was detected reactivity by preincubation with the synthetic LC pep- in normal skin with the anti-LC antibody but not with tide (Fig. 3D). the anti-CC antibody. Thus, only a single form of immunohistochemically detectable TGFpl occurs in normal epidermal keratinocytes. Both anti-LC and antiCC reacted with TGFPl in the epidermis of psoriatic DISCUSSION skin, indicating t h a t psoriatic keratinocytes, which exDistinct forms of TGFpl have been histochemically hibit a hyperproliferativelregenerative differentiation localized in keratinocytes in normal skin and in the phenotype, contain a n additional TGFPl epitope not skin of individuals with psoriasis by using two poly- observed in normally differentiating keratinocytes. clonal antibodies specific for TGFP1. The data are sum- The different forms of TGFPl found in normal keratimarized in Table 1for reference. Psoriatic epidermis is nocytes and in keratinocytes in the psoriatic epidermis a physiological model of hyperproliferation in which may represent differences in TGFPl synthesis, secrethe keratinocytes present a hyperproliferativehegener- tion, activation, andior association with other macroative phenotype (Bernard et al., 1985; Mansbridge and molecules. Elder et al. (1989) reported no difference in Knapp, 19871, in contrast to the normal differentiation the steady-state level of TGFpl mRNA in the epiderphenotype associated with keratinocytes in the epider- mis of normal or psoriatic skin; this suggests that the mis of normal skin. differences in anti-LC and anti-CC staining do not re-

with TGFpl-coupled Sepharose (Heine et al., 1987) prior to immunohistochemical staining (Fig. 2B). AntiCC specificity for TGFPl and epitope characterization has been previously reported (Ellingsworth et al., 1986; Flanders et al., 1989). The dermis of four of the seven psoriatic plaque samples exhibited staining with anti-CC antibody (Fig. 2D) in contrast to the absence of staining in the dermis of normal skin (Fig. 1C). The anti-CC staining pattern in the dermis was seen as a fine reticulum broadly distributed throughout the collagenous tissue or as localized staining associated with regions of increased cellularity underlying the epidermis. Anti-TGFpl staining in the dermis has not been associated with a specific cell type. The anti-LC TGFP1-specific antibody displayed no staining in five of the seven psoriatic plaque samples examined (data not shown). However, two psoriatic plaques exhibited intracellular anti-LC staining in the granular and upper spinous layers (Fig. 2E). This low level of staining was specific a s it is not detected with the nonimmune IgG control, and it is blocked by preincubation of anti-LC with the synthetic LC peptide (Fig. 2F).

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Fig. 2.

TGFPl IN EPIDERMAL KERATINOCYTES IN SITU

149

Fig. 3. Localization of TGFpl in keratinocytes in the nonplaque epidermis of a psoriatic individual. A. Keratinocytes in nonplaque psoriatic epidermifiexhibited cytoplasmic staining with anti-CC TGFPl antibody, in contrast to normal keratinocytes. x 110. B: Keratinocytes in nonplaque psoriatic skin exhibited intracellular anti-LC

staining for TGFp1. x 110. C: Anti-CC staining in nonplaque keratinocytes of psoriatic individuals was blocked by preincubation of antiCC antibody with TGFpl-coupled Sepharose. x 110. D Anti-LC reactivity in nonplaque keratinocytes of psoriatic individuals was blocked by preincubation of anti-LC antibody with LC peptide. x 110.

Fig. 2. Localization of TGFpl in a hyperproliferative psoriatic plaque. A: Intense extracellular and reticulate intracellular staining of TGFpl was detected with anti-CC antibody among keratinocytes in the psoriatic plaque. x 70. B: Anti-CC staining in keratinocytes in the psoriatic plaque was blocked by depletion of TGFpl-specific antibodies. x 70. C: Rete ridges exhibited extracellular TGFpl surrounding the suprabasal keratinocytes. x 130. D: Anti-CC antibody stained TGFpl in the dermis of plaque and nonplaque skin in four of the seven psoriatic individuals. x 70. E: Intracellular anti-LC staining in keratinocytes in the psoriatic plaque was observed in two of the seven Psoriatic P h u c samples. x 70. F: Anti-LC staining in Plaque keratinocytes in the two reactive Specimens was blocked by preincubation of the anti-LC antibody with LC peptide. x 70.

TABLE 1. TGFpl localization in human epidermal keratinocytes

Antibody Anti-LC Anti-CC

Normal cells Suprabasal cells Intracellular None

Psoriatic cells Nonplaque Plaque Suprabasal cells None' Intracellular Suprabasal cells Suprabasal cells Extracellular/ Intracellular intracellular

'No staining was ohserved in five of the seven samples; the remaining two samples showed intracellular staining in the spinous and granular cells (Fig. 2E).

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flect altered TGFPl gene expression in the skin. Direct Assoian, R.K., Komoriya, A., Meyers, C.A., Miller, D.M., and Sporn, M.B. (1983) Transforming growth factor-beta in human platelets. J . isolation of TGFPl protein from normal and psoriatic Biol. Chem., 258t7155-7160. skin will allow comparison of TGFpl activity and anal- Bascom, C.C., Sipes, N.J., Coffey, R.J., and Moses, H.L. (1989) Reguysis of potential structural changes in the protein. lation of epithelial cell proliferation by transforming p o w t h factors. J. Cell Biochem., 39~25-32. The difference in TGFpl localization in the plaque and nonplaque skin of psoriatic individuals is striking. Bernard, B.A., Robinson, S.M., Vandaele, S., Mansbridge, J.N., and Darmon, M. (1985)Abnormal maturation pathway of keratinocytes In addition to a n intracellular form of TGFPl found in in psoriatic skin. Br. J . Dermatol., 112r647-653. both plaque and nonplaque psoriatic keratinocytes Coffey, R.J., Derynck, R., Wilcox, J.N., Bringman, T.S., Goustin, AS., with the anti-CC antibody, a unique extracellular form Moses, H.L., and Pittelkow, M.R. (1987) Production and auto-induction of transforming growth factor-u in human keratinocytes. Naof TGFpl was detected within the epidermis of psoriture, 328t817-820. atic plaques with the anti-CC antibody. This was not R.J., Sipes, N.J., Bascom, C.C., Graves-Deal, R., Pennington, seen in nonplaque regions of skin or in the normal Coffey, C.Y., Weissman, B.E., and Moses, H.L. (1988) Growthmodulation of epidermis. The extracellular localization of TGFpl demouse keratinocytes by transforming growth factors. Cancer Res., 48:1596-1602. tected in psoriatic plaques may represent physiological regulation of TGFPl activity, perhaps through associ- Elder, J.T., Fisher, G.J., Lindquist, P.B., Bennett, G.L., Pittelkow, M.R., Coffey, R.J., Ellingsworth, L., Derynck, R., and Voorhees, J.J. ation of TGFpl with cell-surface proteins, interstitial (1989) Overexpression of transforming growth factor alpha in psojunctions, specific TGFpl-binding proteins, or extracelriatic epidermis. Science, 243 t811-814. lular matrix proteins. In human colon carcinoma and Ellingsworth, L.R., Brennan, J.E., Fok, K., Rosen, D.M., Bentz, H., Piez, K.A., and Seyedin, S.M. (1986) Antibodies to the N-terminal bovine fibropapilloma, extracellular TGFPl staining portion of cartilage-inducing factor A and transforming growth facwith anti-CC antibody is associated with stromal staintor p. Immunohistochemical localization and association with difing by anticollagen and antifibronectin antibodies ferentiating cells. J. Biol. Chem., 261 t12362-12367. (Flanders et al., 1989). Elevated levels of fibronectin Fava, R.A., and McClure, D.B. (1987) Fibronectin-associated transforming growth factor. J . Cell. Physiol., 131:184-189. are present in the epidermis of a psoriatic plaque (BerK.C., Roberts, A.B., Ling, N., Fleurdelys, B.E., and Sporn, nard et al., 1985). Fibronectin copurifies with TGFPl Flanders, M.B. (1988) Antibodies to peptide determinants of transforming under neutral conditions (Fava and McClure, 1987) ingrowth factor-p and their applications. Biochemistry, 27t739-746. dicating that TGFpl can bind to fibronectin and may Flanders, K.C., Thompson, N., Cissel, D.S., Van Obberghen-Schilling, E., Baker, C.C., Kass, M.E., Ellingsworth, L.R., Roberts, A.B., and do so under physiological conditions. The extracellular Sporn, M.B. (1989) Transforming growth factor-pl: histochemical localization of TGFPl within the plaque may be a relocalization with antibodies to different epitopes. J. Cell Biol., 108: sult of TGFpl association with an abnormal fibronec653-660. tin distribution in the epidermis. TGFpl activity may Grondahl-Hansen, J., Ralfkier, E., Nielsen, L.S., Kristensen, P., Frentz, G., and Dano, K. (1987) Immunohistochemical localization be physiologically altered by association of TGFPl with of urokinase- and tissue-type plasminogen activators in psoriatic extracellular matrix proteins. Binding of TGFPl to maskin. J. Invest. Dermatol., 88r28-32. trix proteins may result in compartmentation of Heine, U.I., Munoz, E.F., Flanders, K.C., Ellingsworth, L.R., Lam, TGFpl or altered availability of TGFpl for proteolytic H.Y.P., Thompson, N.L., Roberts, A.B., and Sporn, M.B. (1987) Role activation. Alternatively, variation in TGFPl activaof transforming growth factor-p in the development of the mouse embryo. J . Cell Biol., 105t2861-2876. tion or processing may yield a form of TGFPl with Justus, C., Muller, S., and Kramer, M.D. (19871 A monoclonal antienhanced affinity for extracellular matrix proteins. body recognizing plasminogen and plasmin-altered reactivity in Latent TGFPl has been shown to be partially actipsoriatic lesions. Br. J. Dermatol., 117t687-694. vated by plasmin and other proteases in vitro (Lyons et Lyons, R.M., Keski-Oja, J.. and Moses, H.L. (1988) Proteolytic activation of latent transforming growth factor-p from fibroblast-conal., 1988). High concentrations of plasmin and plasminditioned medium. J. Cell Biol., 106r1659-1665. ogen activators in psoriatic plaques (Grondahl-Hansen J.N., and Hanawalt, P.C. (1988) Role of transforming et al., 1987; Justus et al., 1987) may be involved in Mansbridge, growth factor beta in the maturation of human epidermal keratiproteolytic activation of TGFpl within the plaque. nocytes. J. Invest. Dermatol., 90:336-341. Studies are underway to establish the role of extracel- Mansbridge, J.N., and Knapp, A.M. (1987) Changes in keratinocyte maturation during wound healing. J . Invest. Dermatol., 89t253lular matrix proteins and proteases in the extracellular 263. deposition of TGFPl in the psoriatic plaque. Miyazono, K., Hellman, U., Wernstedt, C., and Heldin, C.-H. (1988) In conclusion, TGFPl appears to be synthesized by Latent high molecular weight complex of transforming growth facepidermal keratinocytes in vivo and thus is a potential tor p l . J . Biol. Chem., 2635407-6415. physiological modulator of keratinocyte growth and dif- Roberts, A.B., and Sporn, M.B. (1990) The transforming growth factor-betas. In: Peptide Growth Factors and Their Receptors. Handferentiation in the adult epidermis. Additional knowlbook of Experimental Pharmacology. Vol. 95/1. M.B. Sporn, and edge regarding the biochemistry of TGFPl synthesis, A.B. Roberts, eds. Springer-Verlag, Heidelberg pp. 419-472. secretion, activation, and protein associations will be Shipley, G.D., Pittelkow, M.R., Wille, J.J., Scott, R.E., and Moses, H.L. (1986)Reversible inhibition of normal human prokeratinocyte necessary to clarify the physiological mechanisms regproliferation by type beta transforming growth factor-growth inhibulating TGFPl activity in epidermal keratinocytes. itor in serum-free medium. Cancer Res., 46:2068-2071. Thompson, N.L., Flanders, K.C., Smith, J.M., Ellingsworth, L.R., ACKNOWLEDGMENTS Roberts, A.B., and Sporn, M.B. (1989) Expression of transforming This research was supported in part by a National growth factor-p1 in specific cells and tissues of adult and neonatal mice. J . Cell Biol., 108r661-669. Research Service Award from the National Institutes of Health to C.J.M.K. (AR07422) and by an Outstand- Wakefield, L.M., Smith, D.M., Flanders, K.C., and Sporn, M.B. (1988) Latent transforming growth factor-p from human platelets. J. Biol. ing Investigator Award from the National Cancer InChem., 263t7646-7654. stitute to P.C.H. (CA44349). Weiss, R.A., Eichner, R., and Sun, T.T. (1984) Monoclonal antibody analysis of keratin expression in epidermal diseases: a 48- and 56LITERATURE CITED kdalton keratin as molecular markers for hyperproliferative keraAkhurst, R.J., Fee, F., and Balmain, A. (1988) Localized production of TGF-p mRNA in tumour promoter-stimulated mouse epidermis. Nature, 331:363-365.

tinocytes. J. Cell Biol., 108t653-660.