Arch Dermatol Res (1992) 284:167-172
9 Springer-Verlag 1992
MON-150, a versatile monoclonal antibody against involucrin: characterization and applications ~;. L. P. van Duijnhoven 1, J. Schalkwijk 2, M. H. G. C. Kranenborg l, I. M. J. J. van Vlijmen-Willems 2, A. Groeneveld l, P. E. J. van Erp 2, E. D. J. Timmer 1, G. J. de Jongh 2, and W. J. M. van de Ven 1,3 Molecular Oncology Section, Department of Biochemistry and 2 Department of Dermatology, University of Nijmegen, P. O. Box 9101, 6500 HB Nijmegen, The Netherlands; 3 Molecular Oncology Section, Center for Human Genetics, University of Leuven, Leuven, Belgium Received July 26, 1991
Summary. A monoclonal antibody, designated MON-150, was found serendipitously to react strongly with the granular layer of normal human epidermis and with the upper spinous layers of psoriatic epidermis. From analysis by flow cytometry of cultured human keratinocytes, it appeared that the percentage of MON-150-positive cells strongly increased when the cells reached confluence and the growth fraction declined. To identify the antigen recognized by MON-150, a lysate of human keratinocytes was subjected to affinity chromatrography using a MON-150 Sepharose column. This yielded a single protein of approximately 350 kDa as measured on Superose 6 FPLC gel permeation chromatography using nondenaturing conditions. In Western blot analysis under denaturing and reducing conditions, a 140-kDa protein was detected. The subcellular localization and the molecular weight of the antigen recognized by MON-150 suggested that the antigen involved might be involucrin. This was confirmed using a commercial polyclonal antiserum against involucrin. We conclude that MON-150 is a new, versatile antibody against human involucrin.
include keratins (Green et al. 1982), filaggrin (Dale et al. 1985), transglutaminase (Thacher and Rice 1985), loricrin (Mehrel et al. 1990) and involucrin (Rice and Green 1979). Involucrin is a precursor of the cross-linked envelope of the stratum corneum, and it appears in the upper layers of the epidermis as a function of normal differentiation of the keratinocyte. In normal epidermis, involucrin is not expressed in the m o r e basal layers, but it is present in the upper stratum spinosum and, most intensely, in the stratum granulosum and the inner stratum corneum (Warhol et al. 1985). In involved psoriatic epidermis, involucrin was detectable down to the suprabasal layer (Bernard et al. 1986). In this paper, we describe a monoclonal antibody against involucrin, that can be used in a variety o f analytical and preparative techniques, including immunohistochemistry, flow cytometry, immunoaffinity purification and Western blot analysis.
Materials and methods
Key words: M o n o c l o n a l antibody - Keratinocytes Epidermal differentiation - Cornified envelope - In-
Biopsies
volucrin
Skin biopsies were taken from paid volunteers after permission from the local committee for experiments on humans had been obtained. Shave biopsies (5 mm diameter, 0.3 mm deep) were taken after local anaesthesia. Tissue specimens were frozen in liquid nitrogen and stored at - 8 0 ~
The skin serves as a vital barrier protecting the internal environment f r o m external influences. As cells start to migrate out of the basal cell layer of the epidermis towards the surface of the skin, they lose the ability to divide and progress towards a terminal differentiation stage. During late stages of epidermal keratinocyte differentiation, an insoluble protein envelope is developed leading to the f o r m a t i o n of the stratum corneum. During this process, keratinocytes go through m a r k e d morphological and structural changes. At the same time, the synthesis o f i m p o r t a n t differentiation-dependent structural and catalytic proteins is initiated, which
Correspondence to: J. L. P. van Duijnhoven
Histology Cryosections were prepared on a Reichert cryostat set at - 2 8 ~ The 5-gm sections were placed on bovine serum albumin (BSA) coated slides, fixed in acetone for 10 min and stored at --80 ~ In addition, fixation in paraformaldehyde (2% in PBS, 30 min) was tested. Immunoperoxidase staining was performed according to standard protocols (Schalkwijk et al. 1991).
CeH culture Human epidermal keratinocytes were cultured on Swiss mouse 3T3 feeder cells (Reinwald and Green 1975). Epidermal cells were seeded on lethally irradiated (3000 Rad in 3 min) Swiss mouse 3T3 fibroblasts in DMEM/F12 3 : 1 v/v (ICN Biomedicals Costa Mesa,
168 Calif., USA) supplemented with 0.4 lag/ml hydrocortisone (Collaborative Research Inc., Bedford, Mass., USA), 10- 6 M isoproterenol (Sigma, St. Louis, Mo., USA), 100 U/ml penicillin and 100 Ixg/ml streptomycin (Life Technologies, Gaithersburg, Md., USA), 5% v/v fetal bovine serum (Seralab, Cambridge, UK) and, starting at day three after seeding, 10 ng/ml epidermal growth factor (Collaborative Research Inc.). Cells were grown at 37 ~ and in 7.5% CO2 in air at 95% relative humidity.
Staining of cell suspensions Cultured keratinocytes were harvested after trypsin treatment and fixed in 70% ice-cold ethanol. Aliquots of ethanol-fixed single cell suspensions were immunostained with MON-150 in a 1:100 dilution in PBS/1% fetal Bovine serum followed by FITC-coupled rabbit anti-mouse IgG (Dakopatts, Copenhagen, Denmark) 1 : 25 in PBS. Subsequently, the cells were washed twice and resuspended in 200 lal PBS. Cellular DNA was stained quantitatively by the addition of 200 lal sodium phosphate buffer, pH 7.8, containing 4 mg/ml propidium iodide (PI) (Calbiochem, San Diego, Calif., USA).
Flow cytometry The cell suspensions were incubated for 15 min with 50 lal RNAse (1 mg/ml in PBS) (Sigma) at room temperature, filtered in order to remove clumps, and the fluorescence measured (van Erp et al. 1989) using an Ortho 50H flow cytometer (Ortho Instruments, Westwood, USA). Both FITC and PI were excited with a 5 W argon ion laser (164-05, Spectra Physics, Mountain View, Calif., USA) tuned at a wavelength of 488 nm and emission was recorded at 515-530 nm (FITC) and > 630 nm (PI). The data were stored and analysed with a Digital PDP 11/34 computer (Digital Equipment, Galway, Ireland). Both area and peak value of the red (PI) fluorescence signal were measured. The ratio area/peak is an excellent discriminator between artifacts due to doublets of diploid cells and real single tetraploid cells.
Monoclonal antibody production MON-150 was recently obtained as a member of a panel of monoclonal antibodies raised against the novel mammalian proprotein processing enzyme furin (van Duijnhoven et al. 1992). These monoclonal antibodies were raised according to standard protocols as described before (van Duijnhoven et al. 1989). When normal human tissues were screened for reactivity with these antibodies, only one of these (i.e. MON-150) showed binding with normal and psoriatic skin. In addition, polyclonal antisera against furin did not react with human skin indicating that in this tissue furin is expressed at very low levels, or not at all.
room temperature with biotinylated sheep anti-mouse antiserum (Amersham, Amersham, UK) diluted 1:2000 in PBS-BT. After washing (3 x 10 min) with PBS-NT and one wash with alkaline phosphatase (AP) buffer (100 mM Tris-HC1 (pH 9.5), 100 mM NaC1 and 5 mM MgCI2), blots were incubated for 2 h at room temperature with AP-conjugated-extravidin (Sigma) diluted 1 : 8000 in AP buffer. After three washes with AP buffer, blots were exposed to freshly prepared AP substrate (66 lal nitrobluetetrazolium (Research Organics, Cleveland, Ohio, USA) (50 mg/ml in 70% dimethylformamide) and 33 gl 5-bromo-4-chloro-3-indoxyl phosphate (Bio-Organics) (50 mg/ml in dimethylformamide) diluted in i0 ml AP buffer). The staining-reaction was stopped using 20 mM EDTA in PBS. Two-dimensional PAGE analysis was performed essentially as described by O'Farrel (1975). Electrophoresis and Western blotting were performed as described earlier.
Affinity chromatography MON-150 antibodies were purified from tissue culture supernatant by affinity chromatography. Complete bacterial lysate containing a hybrid protein which consisted of the N-terminal region of furin fused in phase to the C-terminal region of glutathione S-transferase and which contained the epitope recognized by MON-150, was covalently coupled to cyanogen bromide-activated Sepharose 4B (Pharmacia, Uppsala, Sweden), according to standard procedures. After dialysis, the purified antibody was coupled to cyanogen bromide-activated Sepharose 4B at a concentration of 2 mg protein per ml gel. Subsequently, the gel was incubated with 1 M glycine in PBS (ph 7.2) to block remaining active sites. Keratinocyte lysate of 108 cells in PBS was applied to the column and the gel was washed with PBS. After absorbance had reached baseline level, the column was eluted using 0.1 M acetic acid.
Gel-permeation chromatography MON-150 antigen preparations obtained after affinity chromatography were concentrated by vacuum evaporation, dialysed against PBS, and applied to a Superose 6 FPLC column (Pharmacia) equilibrated with PBS. The eluent was monitored at a wavelength of 215 nm. The column was calibrated with molecular weight markers, using dextran blue (void volume), thyroglobulin (669kDa), IgG (160kDa), BSA (68kDa) and L-dopa (total cotumn volume).
Results
Western blotting analysis
MON-150 reactivity in human skin
Electrophoresis was performed on 7.5% polyacrylamide gels (SDS-PAGE) under reducing and non-reducing conditions (Laemmli 1970). Confluent keratinocytes were harvested and resuspended in PBS buffer, lysed by sonication and centrifuged for 5 min at 10000 g. A sample of supernatant fraction of the lysate, containing approximately 25 lag protein was loaded on a gel. Proteins were transferred electrophoretically onto 0.45 lam nitrocellulose (Schleicher & Schuell, Dassel, FRG) in transfer buffer (25 mM Tris-HC1, 0.2 M glycine, 20% methanol v/v) according to the method of Towbin et al. (1979). To prevent non-specific protein binding, the nitrocellulose sheets were incubated for 1 h at room temperature in PBS/2% BSA/0.1% Tween 20 (PBS-BT). Subsequently, blots were incubated for 2 h at room temperature with MON-150 tissue culture supernatant, diluted 1:20 in PBS-BT, or a rabbit anti-human involucrin antiserum (BTI), diluted 1 : 75 in PBS-BT. After washing (3 x 10 min) with PBS/10 % newborn bovine serum/0.1% Tween 20 (PBS-NT), blots were incubated for 2 h at
I n Fig. 1 is s h o w n the r e a c t i o n p a t t e r n o f M O N - 1 5 0 w i t h a c e t o n e - f i x e d c r y o s e c t i o n s o f n o r m a l (Fig. 1 A ) a n d p s o riatic skin (Fig. 1 B a n d C). U s i n g either i m m u n o p e r o x i d a s e o r i m m u n o f l u o r e s c e n c e , s t r o n g positive s t a i n i n g was f o u n d in cells o f the g r a n u l a r l a y e r in n o r m a l epidermis. T h e e x a c t l o c a t i o n c a n n o t be given w i t h c e r t a i n t y , b u t the s t a i n i n g p a t t e r n o f M O N - 1 5 0 s t r o n g l y r e s e m b l e d the pattern obtained with an antibody directed against involucrin, a k n o w n c o m p o n e n t o f the c o r n i f i e d envelope. I n p s o r i a t i c e p i d e r m i s , in w h i c h the d i f f e r e n t i a t i o n p r o c e s s is k n o w n to be d i s t u r b e d , M O N - 1 5 0 staining was f o u n d in the u p p e r p a r t o f the s t r a t u m s p i n o s u m as can be seen in Fig. 1 B. B o t h in n o r m a l a n d p s o r i a t i c epidermis, o c c a s i o n a l positive s p o t s were f o u n d in the nucleus o f cells in the l o w e r s p i n o u s layers a n d the b a s a l
169
Fig. 1. Immunohistochemical analysis using MON-150 using immunoperoxidase staining of cryosections of normal skin (A) and immunofluorescence staining of psoriatic skin (B, C)
layers, when the antibody was used at low dilutions (Fig. 1 C).
% Positive Cells /
36.3
40
MON-150 reactivity in cultured human keratinocytes 30
Based upon immunohistochemical results, the appearance of the antigen recognized by MON-150 in the epidermis could be a function of the differentiation stage of keratinocytes. Therefore, flow cytometric analysis was performed on exponentially growing keratinocytes and keratinocytes harvested at confluent stage. The mean results of four experiments are shown in Fig. 2. F o r both culture conditions, the total percentage of cells in S, G2 and M phase of the cell cycle are given. This percentage decreased from 36.3% in exponentially growing cultures to 12.2% in cultures at confluent stage. In exponentially growing keratinocytes, 1.1% of the cells were positively stained by MON-150, while in cultures at confluent stage the percentage of MON-150-positive cells rose to 10.9%.
20
to _
o
Exponential
Confluent
Fig. 2. Flow cytometric analysis of exponentially growing and confluent keratinocytes. Percentage of cells in S, Gz and M phase (open bars) and percentage of MON-150-positive cells (shaded bars) are indicated
170
Character&ation of the antigen Based upon its localization in human skin, the antigen recognized by MON-150 could be another differentiation marker of keratinocytes that simply co-localizes with involucrin in the cornified envelope. However, it is also possible that MON-150 recognizes involucrin itself. To resolve this matter, experiments were performed to identify the antigen recognized by MON-150 in differentiating keratinocytes. Therefore, cultured cells were harvested and a crude extract was analysed by SDS-PAGE and Western blotting. Under non-reducing conditions, a protein doublet of 140 k D a was detected when blots were stained with MON-150 (Fig. 3, lane B). In a similar experiment under reducing conditions, a single protein
with a molecular weight o f 140 k D a was detected (Fig. 3, lane A). When a polyclonal antiserum against human involucrin was used, the same proteins were detected (Fig. 3, lane C and D). These results strongly suggest that involucrin is recognized by MON-150. In control experiments, anti-furin monoclonal antibodies which were previously found to be negative on skin sections, also did not react in Western blot analysis (data not shown). To obtain further support for the possibility that MON-150 is also reactive with human involucrin, the isoelectric point o f the protein recognized by MON-150 was determined. After P A G E and isoelectrofocusing analysis, a single protein with an isoelectric point (pI) between 4 and 5 was detected in subsequent Western blot analysis (Fig. 4). A protein with the same molecular weight and isoelectric point was detected with the polyclonal anti-involucrin antiserum (data not shown).
abeorbance
215 nm
0.1
Vo
669
A
II
160
68
Vt
Pi
0.05
o 0
Fig. 3. Western blotting analysis of proteins of cultured keratinocytes under reducing (A and C) and non-reducing conditions (B and D) using MON-150 (A and B) and anti-involucrin antiserum (C and D). Molecular weight markers are indicated (kDa)
Fig. 4. Two-dimensional PAGE and subsequent Western blot analysis of proteins of cultured keratinocytes under reducing conditions. Arrows indicate the migration direction of the proteins during isoelectric focusing and SDS-PAGE. The acidic and basic side of the gel is marked A and B, respectively. Molecular weight markers are indicated (kDa)
10
20
30
40
50
60
t i m e (rain)
Fig. 5. Gel permeation chromatography of the protein purified by affinity chromatography using a MON-150 Sepharose column. Absorption was measured at a wavelength of 215 nm and elution times are given. Column calibration standards are indicated (kDa), as well as the void volume (Vo)and total volume of the column (V~)
Fig. 6. Western blotting analysis of the protein complex that was purified by affinity chromatography using a MON-150 Sepharose column. The protein complex was analysed under reducing (A and C) and non-reducing conditions (B and D), using MON-150 (A and B) and anti-involucrin antiserum (C and D). Molecular weight markers are indicated (kDa)
171 Finally, additional support was obtained from affinity chromatography experiments. An extract of cultured keratinocytes was applied to a MON-150 Sepharose column and the eluent was analysed on Superose 6 FPLC gel permeation chromatography (Fig. 5). A protein with an apparent molecular weight of 350 kDa was found using non-denaturing conditions. SDS-PAGE and Western blot analysis of this protein preparation using reducing conditions revealed that the 350-kDa protein was a protein complex consisting of a protein doublet of about 140 kDa under non-reducing conditions or a single protein of 140 kDa under reducing conditions (Fig. 6, lane A and B). It should be noted that the same results were obtained with polyclonal anti-involucrin antiserum as with MON-150 (Fig. 6, lane C and D). Furthermore, no differences were seen between the results obtained with the proteins purified by affinity chromatography and with the crude keratinocyte extracts.
Discussion
In a set of experiments designed to generate monoclonal antibodies against the novel mammalian proprotein processing enzyme furin (van Duijnhoven et al. 1992), one antibody (MON-150) was found serendipitously to exhibit strong reactivity with human epidermis. None of the other anti-furin monoclonal antibodies reacted with human skin, either in Western blot or immunohistochemical analysis. This does not necessarily imply that furin is not expressed in skin. Under normal physiological conditions, expression of the furin enzyme is very low in most tissues and, therefore, the protein is not detectable by conventional immunological techniques. Polyclonal antisera against furin also did not react with human skin. The immunohistochemical staining pattern of human epidermis with MON-150 and the flow cytometric studies initially suggested that the antigen recognized by the antibody could be an early marker of keratinocyte differentiation and even raised the possibility that the antigen could be identical to the known component of the cornified envelope, involucrin. A number of observations reported in this study now indicate that indeed involucrin is recognized by MON-150. First, by flow cytometric analysis, MON-150 reactivity was shown to increase about ten-fold in keratinocytes at confluent stage with respect to exponentially growing cells. This observation of elevated expression was confirmed by Western blot analysis (data not shown). Similar results were obtained using the polyclonal anti-involucrin antiserum (data not shown), which is in agreement with the observation of elevated expression of involucrin in the upper layers of the epidermis (Warhol et al. 1985). Second, MON-150 reactivity, and that of a known anti-involucrin antiserum, appeared to be very similar. By affinity chromatography, a 350-kDa protein complex was eluted from a MON-150 column. A protein complex of the same molecular weight was obtained in similar analysis of involucrin (Rice and Green 1979). In subsequent Western blot analysis of this 350-kDa protein
complex under reducing as well as non-reducing conditions, the same subunits were stained with MON- 150 and the rabbit anti-involucrin antiserum. Third, the isoelectric point and the molecular weights of the antigen recognized by MON-150 are similar to those of involucrin. Isoelectric focusing and Western blot analysis indicated that the protein that was recognized by MON-150 in human keratinocytes had a pI of 4 to 5. This pI is similar to the one described for involucrin: 4.5 _+ 0.3 (Rice and Green 1979). Finally, the molecular weight of the protein recognized by MON-150 appeared to be about 140 kDa, which corresponds to the molecular weight of involucrin in some reports (Simon and Green 1984; Baden et al. 1987). However, there is a discrepancy in the literature about the molecular weight of involucrin. The involucrin gene is cloned and contains a coding region of 1764 nucleotides (Eckert et al. 1986). The deduced amino acid sequence has a calculated molecular weight of about 68.5 kDa. Estimations of the molecular mass of the protein by physical methods have provided variable results. Rice and Green (1979) reported a molecular weight for involucrin of about 92 kDa. Watt and Green (1981) estimated it somewhat lower than 92 kDa, while Simon and Green (1984) and Baden et al. (1987)reported a molecular weight of 140 kDa and 143 kDa, respectively. Our experiments indicated a molecular mass ofinvolucrin of 140 kDa. Amino acid sequence analysis of the protein that is recognized by MON-150 may be required to resolve conclusively its relationship to involucrin. Initial experiments to determine the amino acid sequence of the 140-kDa protein suggested that the N-terminus is blocked. Additional evidence that MON-150 recognizes involucrin was obtained by probing MON-150 on blots of two-dimensional gels from non-cultured normal human epidermal keratinocytes which were extensively characterized in a protein database (Celis et al. 199 !~). A protein in this database which was identified as involucrin, using a polyclonal antiserum (different from the o,ne we used) gave a strong positive staining with MON-150 (data not shown). At present, we have no explanation for the reaction of MON- 150 with both involucrin and furin. The epitope recognized by MON-150 in human furin has been tentatively mapped and is located in the subtilisin-like catalytic domain of the enzyme. The polyclonal antiinvolucrin antiserum did not recognize human furin (data not shown). Comparison of the amino acid sequences of involucrin and furin revealed no :clues about a possible common epitope. Nevertheless, MON-150 appears to be a useful and versatile antibody. It recognizes involucrin both in a native state and after fixation with acetone, ethanol and paraformaldehyde i or denaturing procedures such as boiling in SDS and treatment with 13-mercaptoethanol. It could therefore: be used in immunohistology, flow cytometry, affinity chromatography and Western blotting. Since MON-150 also recognizes furin, appropriate control experiments should be included when this monoclonal antibody is used in studies of tissues that might express elevated levels of furin. Since this is not the case for human epidermis. MON-150 is a
172 useful a n t i b o d y for the s t u d y o f d i f f e r e n t i a t i o n processes in h u m a n skin, a n d especially in c u l t u r e d k e r a t i n o c y t e s .
Acknowledgements. We would like to thank Dr. K. Dejgaard and Prof. J. Cells (Institute of Medical Biochemistry, Aarhus University, Aarhus, Denmark) for analysing MON-150 in their two dimensionaI gei protein database of keratinocytes, and Martie Verschuren for excellent technical assistance. This study was supported by the Dutch Cancer Society, Holland Biotechnology BV, Leiden, The Netherlands, and by a 'Levenslijn' grant of the 'Nationaal Fonds voor Wetenschappelijk Onderzoek' of Belgium.
References Baden HP, Kubilus J, Kvedar JC, Steinberg ML, Wolman SR (1987) Isolation and characterization of a spontaneously arising long-lived line of human keratinocytes (NMI). In Vitro 23: 205 213 Bernard BA, Reano A, Darmon YM, Thivolet J (1986) Precocious appearance of involucrin and epidermal transglutaminase during differentiation of psoriatic skin. Br J Dermatol 114:279-283 Celis JE, Madsen P, Rasmussen HH, Leffers H, Honor6 Gesser B, Dejgaard K, Olsen E, Magnusson N, Kill J, Celis A, Lauridsen JB, Basse B, Patz GP, Andersen AH, Walbum E, Brandstrup B, Pedersen PS, Brandt NI, Puype M, van Damme J, Vandenkerckhove J (1991) A comprehensive two-dimensional gel protein database of non-cultured unfractionated normal epidermal keratinocytes: towards an integrated approach to the study of cell proliferation, differentiation and skin diseases. Electrophoresis 12:802-872 Dale BA, Resing KA, Lonsdale-Eccles JD (1985) Filaggrin: a keratin filament associated protein. Ann NY Acad Sci 455: 330-342 Eckert RL, Green H (1986) Structure and evolution of the human involucrin gene. Cell 46:583 589 Green H, Fuchs E, Watt R (1982) Differentiated structural components of the keratinocyte. Cold Spring Harb Syrup Quant Biol 46:293-301 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685 Mehrel T, Hohl D, Rothnagel JA, Longley MA, Bundman D, Cheng C, Lichti U, Bisher ME, Steven AC, Steinert PM, Yuspa
SH, Roop DR (1990) Identification of a major keratinocyte cell envelope protein, loricrin. Cell 61 : 1103-1112 O'Farrel PH (1975) High resolution two-dimensional electrophoresis of proteins. J Biol Chem 250:4007 4021 Rheinwald JG, Green H (1975) Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 6:331-343 Rice RH, Green H (1979) Presence in human epidermal cells of a soluble protein precursor of the cross-linked envelope: activation of the cross-linking by calcium ions. Cell 18:681-694 Schalkwijk J, van Vlijmen I, Oosterling B, Perret C, Koopman R, van den Born J, Mackie EJ (1991) Tenascin expression in hyperproliferative skin diseases. Br J Dermatol 124:13-20 Simon M, Green H (1984) Participation of membrane-associated proteins in the formation of the cross-linked envelope of the keratinocyte. Cell 36:827-834 Thacher SM, Rice RH (1985) Keratinocyte-specific transglutaminase of cultured epidermal cells: relation to cross-linked envelope formation and terminal differentiation. Cell 40:685 695 Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76: 4350 4354 van Duijnhoven HLP, Ayoubi TAY, Timmer EDJ, Braks AAM, Roebroek AJM, Martens GJM, van de Ven WJM (1989) Development of a monoclonal antibody against recombinant neuroendocrine 7B2 protein. FEBS Lett 255:372-376 van Duijnhoven HLP, Kranenborg MGC, Timmer EDJ, Groeneveld A, van den Ouweland AMW, Creemers JWM, Roebroek AJM, van de Ven WJM (1992) Development and characterization of a panel of monoclonal antibodies against the novel subtilisin-like proprotein processing enzyme furin. Hybridoma 11:71-86 van Erp PEJ, Rijzewijk JJ, Boezeman JBM, Leenders J, de Mare S, Schalkwijk J, van de Kerkhof PCM, Ramaekers FCS, Bauer FW (1989) Flow cytometric analysis of epidermal subpopulations from normal and psoriatic skin using monoclonal antibodies against intermediate filaments. Am J Pathol 135:865-870 Warhol MJ, Roth J, Lucocq JM, Pinkus GS, Rice RH (1985) Immuno-ultrastructural localization of involucrin in squamous epithelium and cultured keratinocytes. J Histochem Cytochem 33:141-149 Watt FM, Green H (1981) Involucrin synthesis is correlated with cell size in human epidermal cultures. J Cell Biol 90:738-742
9 Springer-Verlag 1992
MON-150, a versatile monoclonal antibody against involucrin: characterization and applications ~;. L. P. van Duijnhoven 1, J. Schalkwijk 2, M. H. G. C. Kranenborg l, I. M. J. J. van Vlijmen-Willems 2, A. Groeneveld l, P. E. J. van Erp 2, E. D. J. Timmer 1, G. J. de Jongh 2, and W. J. M. van de Ven 1,3 Molecular Oncology Section, Department of Biochemistry and 2 Department of Dermatology, University of Nijmegen, P. O. Box 9101, 6500 HB Nijmegen, The Netherlands; 3 Molecular Oncology Section, Center for Human Genetics, University of Leuven, Leuven, Belgium Received July 26, 1991
Summary. A monoclonal antibody, designated MON-150, was found serendipitously to react strongly with the granular layer of normal human epidermis and with the upper spinous layers of psoriatic epidermis. From analysis by flow cytometry of cultured human keratinocytes, it appeared that the percentage of MON-150-positive cells strongly increased when the cells reached confluence and the growth fraction declined. To identify the antigen recognized by MON-150, a lysate of human keratinocytes was subjected to affinity chromatrography using a MON-150 Sepharose column. This yielded a single protein of approximately 350 kDa as measured on Superose 6 FPLC gel permeation chromatography using nondenaturing conditions. In Western blot analysis under denaturing and reducing conditions, a 140-kDa protein was detected. The subcellular localization and the molecular weight of the antigen recognized by MON-150 suggested that the antigen involved might be involucrin. This was confirmed using a commercial polyclonal antiserum against involucrin. We conclude that MON-150 is a new, versatile antibody against human involucrin.
include keratins (Green et al. 1982), filaggrin (Dale et al. 1985), transglutaminase (Thacher and Rice 1985), loricrin (Mehrel et al. 1990) and involucrin (Rice and Green 1979). Involucrin is a precursor of the cross-linked envelope of the stratum corneum, and it appears in the upper layers of the epidermis as a function of normal differentiation of the keratinocyte. In normal epidermis, involucrin is not expressed in the m o r e basal layers, but it is present in the upper stratum spinosum and, most intensely, in the stratum granulosum and the inner stratum corneum (Warhol et al. 1985). In involved psoriatic epidermis, involucrin was detectable down to the suprabasal layer (Bernard et al. 1986). In this paper, we describe a monoclonal antibody against involucrin, that can be used in a variety o f analytical and preparative techniques, including immunohistochemistry, flow cytometry, immunoaffinity purification and Western blot analysis.
Materials and methods
Key words: M o n o c l o n a l antibody - Keratinocytes Epidermal differentiation - Cornified envelope - In-
Biopsies
volucrin
Skin biopsies were taken from paid volunteers after permission from the local committee for experiments on humans had been obtained. Shave biopsies (5 mm diameter, 0.3 mm deep) were taken after local anaesthesia. Tissue specimens were frozen in liquid nitrogen and stored at - 8 0 ~
The skin serves as a vital barrier protecting the internal environment f r o m external influences. As cells start to migrate out of the basal cell layer of the epidermis towards the surface of the skin, they lose the ability to divide and progress towards a terminal differentiation stage. During late stages of epidermal keratinocyte differentiation, an insoluble protein envelope is developed leading to the f o r m a t i o n of the stratum corneum. During this process, keratinocytes go through m a r k e d morphological and structural changes. At the same time, the synthesis o f i m p o r t a n t differentiation-dependent structural and catalytic proteins is initiated, which
Correspondence to: J. L. P. van Duijnhoven
Histology Cryosections were prepared on a Reichert cryostat set at - 2 8 ~ The 5-gm sections were placed on bovine serum albumin (BSA) coated slides, fixed in acetone for 10 min and stored at --80 ~ In addition, fixation in paraformaldehyde (2% in PBS, 30 min) was tested. Immunoperoxidase staining was performed according to standard protocols (Schalkwijk et al. 1991).
CeH culture Human epidermal keratinocytes were cultured on Swiss mouse 3T3 feeder cells (Reinwald and Green 1975). Epidermal cells were seeded on lethally irradiated (3000 Rad in 3 min) Swiss mouse 3T3 fibroblasts in DMEM/F12 3 : 1 v/v (ICN Biomedicals Costa Mesa,
168 Calif., USA) supplemented with 0.4 lag/ml hydrocortisone (Collaborative Research Inc., Bedford, Mass., USA), 10- 6 M isoproterenol (Sigma, St. Louis, Mo., USA), 100 U/ml penicillin and 100 Ixg/ml streptomycin (Life Technologies, Gaithersburg, Md., USA), 5% v/v fetal bovine serum (Seralab, Cambridge, UK) and, starting at day three after seeding, 10 ng/ml epidermal growth factor (Collaborative Research Inc.). Cells were grown at 37 ~ and in 7.5% CO2 in air at 95% relative humidity.
Staining of cell suspensions Cultured keratinocytes were harvested after trypsin treatment and fixed in 70% ice-cold ethanol. Aliquots of ethanol-fixed single cell suspensions were immunostained with MON-150 in a 1:100 dilution in PBS/1% fetal Bovine serum followed by FITC-coupled rabbit anti-mouse IgG (Dakopatts, Copenhagen, Denmark) 1 : 25 in PBS. Subsequently, the cells were washed twice and resuspended in 200 lal PBS. Cellular DNA was stained quantitatively by the addition of 200 lal sodium phosphate buffer, pH 7.8, containing 4 mg/ml propidium iodide (PI) (Calbiochem, San Diego, Calif., USA).
Flow cytometry The cell suspensions were incubated for 15 min with 50 lal RNAse (1 mg/ml in PBS) (Sigma) at room temperature, filtered in order to remove clumps, and the fluorescence measured (van Erp et al. 1989) using an Ortho 50H flow cytometer (Ortho Instruments, Westwood, USA). Both FITC and PI were excited with a 5 W argon ion laser (164-05, Spectra Physics, Mountain View, Calif., USA) tuned at a wavelength of 488 nm and emission was recorded at 515-530 nm (FITC) and > 630 nm (PI). The data were stored and analysed with a Digital PDP 11/34 computer (Digital Equipment, Galway, Ireland). Both area and peak value of the red (PI) fluorescence signal were measured. The ratio area/peak is an excellent discriminator between artifacts due to doublets of diploid cells and real single tetraploid cells.
Monoclonal antibody production MON-150 was recently obtained as a member of a panel of monoclonal antibodies raised against the novel mammalian proprotein processing enzyme furin (van Duijnhoven et al. 1992). These monoclonal antibodies were raised according to standard protocols as described before (van Duijnhoven et al. 1989). When normal human tissues were screened for reactivity with these antibodies, only one of these (i.e. MON-150) showed binding with normal and psoriatic skin. In addition, polyclonal antisera against furin did not react with human skin indicating that in this tissue furin is expressed at very low levels, or not at all.
room temperature with biotinylated sheep anti-mouse antiserum (Amersham, Amersham, UK) diluted 1:2000 in PBS-BT. After washing (3 x 10 min) with PBS-NT and one wash with alkaline phosphatase (AP) buffer (100 mM Tris-HC1 (pH 9.5), 100 mM NaC1 and 5 mM MgCI2), blots were incubated for 2 h at room temperature with AP-conjugated-extravidin (Sigma) diluted 1 : 8000 in AP buffer. After three washes with AP buffer, blots were exposed to freshly prepared AP substrate (66 lal nitrobluetetrazolium (Research Organics, Cleveland, Ohio, USA) (50 mg/ml in 70% dimethylformamide) and 33 gl 5-bromo-4-chloro-3-indoxyl phosphate (Bio-Organics) (50 mg/ml in dimethylformamide) diluted in i0 ml AP buffer). The staining-reaction was stopped using 20 mM EDTA in PBS. Two-dimensional PAGE analysis was performed essentially as described by O'Farrel (1975). Electrophoresis and Western blotting were performed as described earlier.
Affinity chromatography MON-150 antibodies were purified from tissue culture supernatant by affinity chromatography. Complete bacterial lysate containing a hybrid protein which consisted of the N-terminal region of furin fused in phase to the C-terminal region of glutathione S-transferase and which contained the epitope recognized by MON-150, was covalently coupled to cyanogen bromide-activated Sepharose 4B (Pharmacia, Uppsala, Sweden), according to standard procedures. After dialysis, the purified antibody was coupled to cyanogen bromide-activated Sepharose 4B at a concentration of 2 mg protein per ml gel. Subsequently, the gel was incubated with 1 M glycine in PBS (ph 7.2) to block remaining active sites. Keratinocyte lysate of 108 cells in PBS was applied to the column and the gel was washed with PBS. After absorbance had reached baseline level, the column was eluted using 0.1 M acetic acid.
Gel-permeation chromatography MON-150 antigen preparations obtained after affinity chromatography were concentrated by vacuum evaporation, dialysed against PBS, and applied to a Superose 6 FPLC column (Pharmacia) equilibrated with PBS. The eluent was monitored at a wavelength of 215 nm. The column was calibrated with molecular weight markers, using dextran blue (void volume), thyroglobulin (669kDa), IgG (160kDa), BSA (68kDa) and L-dopa (total cotumn volume).
Results
Western blotting analysis
MON-150 reactivity in human skin
Electrophoresis was performed on 7.5% polyacrylamide gels (SDS-PAGE) under reducing and non-reducing conditions (Laemmli 1970). Confluent keratinocytes were harvested and resuspended in PBS buffer, lysed by sonication and centrifuged for 5 min at 10000 g. A sample of supernatant fraction of the lysate, containing approximately 25 lag protein was loaded on a gel. Proteins were transferred electrophoretically onto 0.45 lam nitrocellulose (Schleicher & Schuell, Dassel, FRG) in transfer buffer (25 mM Tris-HC1, 0.2 M glycine, 20% methanol v/v) according to the method of Towbin et al. (1979). To prevent non-specific protein binding, the nitrocellulose sheets were incubated for 1 h at room temperature in PBS/2% BSA/0.1% Tween 20 (PBS-BT). Subsequently, blots were incubated for 2 h at room temperature with MON-150 tissue culture supernatant, diluted 1:20 in PBS-BT, or a rabbit anti-human involucrin antiserum (BTI), diluted 1 : 75 in PBS-BT. After washing (3 x 10 min) with PBS/10 % newborn bovine serum/0.1% Tween 20 (PBS-NT), blots were incubated for 2 h at
I n Fig. 1 is s h o w n the r e a c t i o n p a t t e r n o f M O N - 1 5 0 w i t h a c e t o n e - f i x e d c r y o s e c t i o n s o f n o r m a l (Fig. 1 A ) a n d p s o riatic skin (Fig. 1 B a n d C). U s i n g either i m m u n o p e r o x i d a s e o r i m m u n o f l u o r e s c e n c e , s t r o n g positive s t a i n i n g was f o u n d in cells o f the g r a n u l a r l a y e r in n o r m a l epidermis. T h e e x a c t l o c a t i o n c a n n o t be given w i t h c e r t a i n t y , b u t the s t a i n i n g p a t t e r n o f M O N - 1 5 0 s t r o n g l y r e s e m b l e d the pattern obtained with an antibody directed against involucrin, a k n o w n c o m p o n e n t o f the c o r n i f i e d envelope. I n p s o r i a t i c e p i d e r m i s , in w h i c h the d i f f e r e n t i a t i o n p r o c e s s is k n o w n to be d i s t u r b e d , M O N - 1 5 0 staining was f o u n d in the u p p e r p a r t o f the s t r a t u m s p i n o s u m as can be seen in Fig. 1 B. B o t h in n o r m a l a n d p s o r i a t i c epidermis, o c c a s i o n a l positive s p o t s were f o u n d in the nucleus o f cells in the l o w e r s p i n o u s layers a n d the b a s a l
169
Fig. 1. Immunohistochemical analysis using MON-150 using immunoperoxidase staining of cryosections of normal skin (A) and immunofluorescence staining of psoriatic skin (B, C)
layers, when the antibody was used at low dilutions (Fig. 1 C).
% Positive Cells /
36.3
40
MON-150 reactivity in cultured human keratinocytes 30
Based upon immunohistochemical results, the appearance of the antigen recognized by MON-150 in the epidermis could be a function of the differentiation stage of keratinocytes. Therefore, flow cytometric analysis was performed on exponentially growing keratinocytes and keratinocytes harvested at confluent stage. The mean results of four experiments are shown in Fig. 2. F o r both culture conditions, the total percentage of cells in S, G2 and M phase of the cell cycle are given. This percentage decreased from 36.3% in exponentially growing cultures to 12.2% in cultures at confluent stage. In exponentially growing keratinocytes, 1.1% of the cells were positively stained by MON-150, while in cultures at confluent stage the percentage of MON-150-positive cells rose to 10.9%.
20
to _
o
Exponential
Confluent
Fig. 2. Flow cytometric analysis of exponentially growing and confluent keratinocytes. Percentage of cells in S, Gz and M phase (open bars) and percentage of MON-150-positive cells (shaded bars) are indicated
170
Character&ation of the antigen Based upon its localization in human skin, the antigen recognized by MON-150 could be another differentiation marker of keratinocytes that simply co-localizes with involucrin in the cornified envelope. However, it is also possible that MON-150 recognizes involucrin itself. To resolve this matter, experiments were performed to identify the antigen recognized by MON-150 in differentiating keratinocytes. Therefore, cultured cells were harvested and a crude extract was analysed by SDS-PAGE and Western blotting. Under non-reducing conditions, a protein doublet of 140 k D a was detected when blots were stained with MON-150 (Fig. 3, lane B). In a similar experiment under reducing conditions, a single protein
with a molecular weight o f 140 k D a was detected (Fig. 3, lane A). When a polyclonal antiserum against human involucrin was used, the same proteins were detected (Fig. 3, lane C and D). These results strongly suggest that involucrin is recognized by MON-150. In control experiments, anti-furin monoclonal antibodies which were previously found to be negative on skin sections, also did not react in Western blot analysis (data not shown). To obtain further support for the possibility that MON-150 is also reactive with human involucrin, the isoelectric point o f the protein recognized by MON-150 was determined. After P A G E and isoelectrofocusing analysis, a single protein with an isoelectric point (pI) between 4 and 5 was detected in subsequent Western blot analysis (Fig. 4). A protein with the same molecular weight and isoelectric point was detected with the polyclonal anti-involucrin antiserum (data not shown).
abeorbance
215 nm
0.1
Vo
669
A
II
160
68
Vt
Pi
0.05
o 0
Fig. 3. Western blotting analysis of proteins of cultured keratinocytes under reducing (A and C) and non-reducing conditions (B and D) using MON-150 (A and B) and anti-involucrin antiserum (C and D). Molecular weight markers are indicated (kDa)
Fig. 4. Two-dimensional PAGE and subsequent Western blot analysis of proteins of cultured keratinocytes under reducing conditions. Arrows indicate the migration direction of the proteins during isoelectric focusing and SDS-PAGE. The acidic and basic side of the gel is marked A and B, respectively. Molecular weight markers are indicated (kDa)
10
20
30
40
50
60
t i m e (rain)
Fig. 5. Gel permeation chromatography of the protein purified by affinity chromatography using a MON-150 Sepharose column. Absorption was measured at a wavelength of 215 nm and elution times are given. Column calibration standards are indicated (kDa), as well as the void volume (Vo)and total volume of the column (V~)
Fig. 6. Western blotting analysis of the protein complex that was purified by affinity chromatography using a MON-150 Sepharose column. The protein complex was analysed under reducing (A and C) and non-reducing conditions (B and D), using MON-150 (A and B) and anti-involucrin antiserum (C and D). Molecular weight markers are indicated (kDa)
171 Finally, additional support was obtained from affinity chromatography experiments. An extract of cultured keratinocytes was applied to a MON-150 Sepharose column and the eluent was analysed on Superose 6 FPLC gel permeation chromatography (Fig. 5). A protein with an apparent molecular weight of 350 kDa was found using non-denaturing conditions. SDS-PAGE and Western blot analysis of this protein preparation using reducing conditions revealed that the 350-kDa protein was a protein complex consisting of a protein doublet of about 140 kDa under non-reducing conditions or a single protein of 140 kDa under reducing conditions (Fig. 6, lane A and B). It should be noted that the same results were obtained with polyclonal anti-involucrin antiserum as with MON-150 (Fig. 6, lane C and D). Furthermore, no differences were seen between the results obtained with the proteins purified by affinity chromatography and with the crude keratinocyte extracts.
Discussion
In a set of experiments designed to generate monoclonal antibodies against the novel mammalian proprotein processing enzyme furin (van Duijnhoven et al. 1992), one antibody (MON-150) was found serendipitously to exhibit strong reactivity with human epidermis. None of the other anti-furin monoclonal antibodies reacted with human skin, either in Western blot or immunohistochemical analysis. This does not necessarily imply that furin is not expressed in skin. Under normal physiological conditions, expression of the furin enzyme is very low in most tissues and, therefore, the protein is not detectable by conventional immunological techniques. Polyclonal antisera against furin also did not react with human skin. The immunohistochemical staining pattern of human epidermis with MON-150 and the flow cytometric studies initially suggested that the antigen recognized by the antibody could be an early marker of keratinocyte differentiation and even raised the possibility that the antigen could be identical to the known component of the cornified envelope, involucrin. A number of observations reported in this study now indicate that indeed involucrin is recognized by MON-150. First, by flow cytometric analysis, MON-150 reactivity was shown to increase about ten-fold in keratinocytes at confluent stage with respect to exponentially growing cells. This observation of elevated expression was confirmed by Western blot analysis (data not shown). Similar results were obtained using the polyclonal anti-involucrin antiserum (data not shown), which is in agreement with the observation of elevated expression of involucrin in the upper layers of the epidermis (Warhol et al. 1985). Second, MON-150 reactivity, and that of a known anti-involucrin antiserum, appeared to be very similar. By affinity chromatography, a 350-kDa protein complex was eluted from a MON-150 column. A protein complex of the same molecular weight was obtained in similar analysis of involucrin (Rice and Green 1979). In subsequent Western blot analysis of this 350-kDa protein
complex under reducing as well as non-reducing conditions, the same subunits were stained with MON- 150 and the rabbit anti-involucrin antiserum. Third, the isoelectric point and the molecular weights of the antigen recognized by MON-150 are similar to those of involucrin. Isoelectric focusing and Western blot analysis indicated that the protein that was recognized by MON-150 in human keratinocytes had a pI of 4 to 5. This pI is similar to the one described for involucrin: 4.5 _+ 0.3 (Rice and Green 1979). Finally, the molecular weight of the protein recognized by MON-150 appeared to be about 140 kDa, which corresponds to the molecular weight of involucrin in some reports (Simon and Green 1984; Baden et al. 1987). However, there is a discrepancy in the literature about the molecular weight of involucrin. The involucrin gene is cloned and contains a coding region of 1764 nucleotides (Eckert et al. 1986). The deduced amino acid sequence has a calculated molecular weight of about 68.5 kDa. Estimations of the molecular mass of the protein by physical methods have provided variable results. Rice and Green (1979) reported a molecular weight for involucrin of about 92 kDa. Watt and Green (1981) estimated it somewhat lower than 92 kDa, while Simon and Green (1984) and Baden et al. (1987)reported a molecular weight of 140 kDa and 143 kDa, respectively. Our experiments indicated a molecular mass ofinvolucrin of 140 kDa. Amino acid sequence analysis of the protein that is recognized by MON-150 may be required to resolve conclusively its relationship to involucrin. Initial experiments to determine the amino acid sequence of the 140-kDa protein suggested that the N-terminus is blocked. Additional evidence that MON-150 recognizes involucrin was obtained by probing MON-150 on blots of two-dimensional gels from non-cultured normal human epidermal keratinocytes which were extensively characterized in a protein database (Celis et al. 199 !~). A protein in this database which was identified as involucrin, using a polyclonal antiserum (different from the o,ne we used) gave a strong positive staining with MON-150 (data not shown). At present, we have no explanation for the reaction of MON- 150 with both involucrin and furin. The epitope recognized by MON-150 in human furin has been tentatively mapped and is located in the subtilisin-like catalytic domain of the enzyme. The polyclonal antiinvolucrin antiserum did not recognize human furin (data not shown). Comparison of the amino acid sequences of involucrin and furin revealed no :clues about a possible common epitope. Nevertheless, MON-150 appears to be a useful and versatile antibody. It recognizes involucrin both in a native state and after fixation with acetone, ethanol and paraformaldehyde i or denaturing procedures such as boiling in SDS and treatment with 13-mercaptoethanol. It could therefore: be used in immunohistology, flow cytometry, affinity chromatography and Western blotting. Since MON-150 also recognizes furin, appropriate control experiments should be included when this monoclonal antibody is used in studies of tissues that might express elevated levels of furin. Since this is not the case for human epidermis. MON-150 is a
172 useful a n t i b o d y for the s t u d y o f d i f f e r e n t i a t i o n processes in h u m a n skin, a n d especially in c u l t u r e d k e r a t i n o c y t e s .
Acknowledgements. We would like to thank Dr. K. Dejgaard and Prof. J. Cells (Institute of Medical Biochemistry, Aarhus University, Aarhus, Denmark) for analysing MON-150 in their two dimensionaI gei protein database of keratinocytes, and Martie Verschuren for excellent technical assistance. This study was supported by the Dutch Cancer Society, Holland Biotechnology BV, Leiden, The Netherlands, and by a 'Levenslijn' grant of the 'Nationaal Fonds voor Wetenschappelijk Onderzoek' of Belgium.
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SH, Roop DR (1990) Identification of a major keratinocyte cell envelope protein, loricrin. Cell 61 : 1103-1112 O'Farrel PH (1975) High resolution two-dimensional electrophoresis of proteins. J Biol Chem 250:4007 4021 Rheinwald JG, Green H (1975) Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 6:331-343 Rice RH, Green H (1979) Presence in human epidermal cells of a soluble protein precursor of the cross-linked envelope: activation of the cross-linking by calcium ions. Cell 18:681-694 Schalkwijk J, van Vlijmen I, Oosterling B, Perret C, Koopman R, van den Born J, Mackie EJ (1991) Tenascin expression in hyperproliferative skin diseases. Br J Dermatol 124:13-20 Simon M, Green H (1984) Participation of membrane-associated proteins in the formation of the cross-linked envelope of the keratinocyte. Cell 36:827-834 Thacher SM, Rice RH (1985) Keratinocyte-specific transglutaminase of cultured epidermal cells: relation to cross-linked envelope formation and terminal differentiation. Cell 40:685 695 Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76: 4350 4354 van Duijnhoven HLP, Ayoubi TAY, Timmer EDJ, Braks AAM, Roebroek AJM, Martens GJM, van de Ven WJM (1989) Development of a monoclonal antibody against recombinant neuroendocrine 7B2 protein. FEBS Lett 255:372-376 van Duijnhoven HLP, Kranenborg MGC, Timmer EDJ, Groeneveld A, van den Ouweland AMW, Creemers JWM, Roebroek AJM, van de Ven WJM (1992) Development and characterization of a panel of monoclonal antibodies against the novel subtilisin-like proprotein processing enzyme furin. Hybridoma 11:71-86 van Erp PEJ, Rijzewijk JJ, Boezeman JBM, Leenders J, de Mare S, Schalkwijk J, van de Kerkhof PCM, Ramaekers FCS, Bauer FW (1989) Flow cytometric analysis of epidermal subpopulations from normal and psoriatic skin using monoclonal antibodies against intermediate filaments. Am J Pathol 135:865-870 Warhol MJ, Roth J, Lucocq JM, Pinkus GS, Rice RH (1985) Immuno-ultrastructural localization of involucrin in squamous epithelium and cultured keratinocytes. J Histochem Cytochem 33:141-149 Watt FM, Green H (1981) Involucrin synthesis is correlated with cell size in human epidermal cultures. J Cell Biol 90:738-742
