FULL PAPER Internal Medicine

Expression of Desmosomal Proteins in Rat Keratinocytes during In Vitro Differentiation

Rika MOCHIZUKI1), Masako KAMIYAMA1), Koji Y. ARAI1), Katsuhiko ARAI1)* and Kohkichi UEHARA1)

1)Department of Tissue Physiology, Tokyo University of Agriculture and Technology, Fuchu 183–8509, Tokyo, Japan

(Received 5 September 2001/Accepted 5 November 2001)

ABSTRACT. The keratinocyte, the major component of the epidermis, expresses several proteins that characterize the keratinization during the differentiation. Proliferation and differentiation of cultured human keratinocytes are known to be regulated by the Ca2+ concentration in the culture medium. However, informations about the rat keratinocyte are relatively limited and their physiology is still an open ques- tion. To elucidate the characteristics of the rat keratinocyte, we established rat keratinocyte culture system and examined effects of extra- cellular calcium concentration on the expression of differentiation-related proteins. Keratinocytes were isolated from the newborn rat skin with 0.25% trypsin, followed by separation with a Percoll density gradient. The separated cells were grown in MCDB153 medium containing several growth factors and Ca2+-free fetal bovine serum, then stimulated with Ca2+. Immunoblotting demonstrated strong expression of β1 in unstimulated cells, suggesting that the primary culture of rat keratinocytes was successfully established. Expression of desmoglein and transglutaminase was increased by Ca2+ stimulation, whereas β1 integrin expression was decreased in response to increasing concentrations of Ca2+. These observations indicate that cultured rat keratinocytes maintain the ability to differ- entiate in vitro, which is similar to that of the basal keratinocytes in the epidermis. KEY WORDS: cell culture, desmoglein, differentiation, integrin, keratinocyte. J. Vet. Med. Sci. 64(2): 123–127, 2002

The epidermis consists of multiple layers of kerati- α6β4 and attach to basement membrane through these mol- nocytes. When keratinocytes of the basal layer withdraw ecules. Integrin α3β1 also bind with α2β1, suggesting that from the cell cycle and become committed to the terminal some mediate cell-. Expression of differentiation, they detach from the basement membrane these integrins is restricted to the basal, proliferative cell and migrate into the suprabasal layers [1, 6, 7, 12]. During layer. In particular, integrin β1 plays a crucial role in termi- the differentiation, human keratinocytes express several nal differentiation within the epidermis [1, 6, 7, 33]. proteins, transglutaminase and substrates of this enzyme It is well known that the proliferation and differentiation such as involucrin and filaggrin, which form the cornified of cultured keratinocytes are regulated by the Ca2+ concen- envelope in the keratinized layer of the epidermis. The tration in the culture medium. Shifting cells from low Ca2+ structure of cell-cell adhesion also changes during the dif- medium to high Ca2+ medium results in inhibition of prolif- ferentiation. , a calcium-dependent intercellu- eration followed by stratification and commitment to the ter- lar adhesion structure, is known to be composed of minal differentiation program [9, 10, 23, 24, 28]. During transmembrane glycoproteins, e.g. desmoglein and desmo- these events, keratinocytes express several proteins that are collin. These proteins bind to the cytoplasmic plaque pro- characteristic of differentiation [16, 29, 31]. teins , , and are linked to keratin The suitable culture condition for the rat keratinocyte is intermediate filaments [2, 4, 13]. The IgG autoantibodies known to be different from that for human keratinocyte [26], against desmoglein-1 and -3 cause the epidermal blistering but information about the cultured rat keratinocytes is not diseases such as foliaceous and pemphigus vul- abundant and its physiology has been unknown yet. There- garis [5, 19–21] . Recent evidence in human keratinocytes fore, we attempt to establish cell culture of rat keratinocytes showed these antigenitic epitopes of desmogleins are con- with Percoll density gradient method and immunochemicaly formation-dependent [3] and calcium-dependent [22]. examined expression of integrin β1, cytokeratin, and the dif- Integrins are heterodimeric transmembrane receptor that ferentiation-related proteins, transglutaminase and desmo- consist of an α and a β subunit, and mediate the attachment gleins, during the differentiation. of cells to the extracellular matrix or to other cells. Until now, sixteen α and eight β subunits and more than 20 differ- MATERIALS AND METHODS ent receptors have been identified. The ligand-binding specificity of an integrin is determined by the combination Cell culture: Keratinocytes from newborn rat epidermis of these subunits and by the cell type in which it is expressed were isolated as previously described [15] with some modi- [8, 18]. Basal keratinocytes express integrin α3β1 and fications. Briefly, the skin was treated with 0.25% trypsin/ calcium-magnesium-free phosphate buffered saline (CMF- ° *CORRESPONDENCE TO: Arai, K., Department of Tissue Physiology, PBS) at 4 C for 16 hr to detach the dermis from the epider- Tokyo University of Agriculture and Technology, Fuchu 183- mis. The dermis sheets were minced, then keratinocytes 8509, Tokyo, Japan. were washed away with CMF-PBS and separated from the 124 R. MOCHIZUKI ET AL. minced dermis with cell strainer (40 µm Nylon, Falcon (Promega). All antibodies used in this study was confirmed 2340, Becton Dickinson, Franklin Lakes, NJ). The cells to react with rat proteins by manufacturers. were further separated by centrifugation at 1,600 rpm for 15 Immunocytochemistry: The keratinocyte cultured on the min with discontinuous density-gradient consist of 50, 40, chamber slides was washed twice with PBS and fixed with 30, and 20% Percoll (Amersham Pharmacia Biotech UK, cold ethanol at –30°C for overnight. After blocking with Amersham, Buckinghamshire, England) layers. The basal 0.5% casein/ 50 mM Tris-HCl, pH 7.6 at 37°C for 1 hr, they keratinocytes were concentrated between the 30 and 40% were incubated with primary antibodies against desmo- Percoll layers, whereas the differentiated keratinocytes gleins or transglutaminase at 4°C, overnight. After the incu- moved to the upper layer (lower density). The cells were bation, the cells were incubated with anti-mouse suspended in MCDB153 (Sigma, St Louis, MO), which was immunoglobulin (Dakopatts) for one hour, they were conju- supplemented with 25 ng/ml epidermal growth factor, 0.1 gated with mouse PAP complex (Dakopatts) for one hour at µg/ml hydrocortisone, 10 µg/ml insulin, 0.23 mM CaCl2, room temperature. The resulting slides were immersed in essential amino acids [26] and 5% calcium-free fetal bovine 0.02% 3-3’-diaminobendizine/50 mM Tris-HCl, pH 7.5 serum (FBS). Calcium-free FBS was prepared by treatment supplemented with 0.03 % H2O2, then counterstained with with Chelex 100 resin (Bio Rad, Hercules, CA) [32]. The Mayer’s hematoxylin. cells were plated in 24-well culture plate (2 × 105/ well) and a part of keratinocytes (2.5 × 104) were also seeded on 8- RESULTS well Lab-Tek Chamber slides (Nunc. Naperville, IL) that were coated with 0.1% EHS-laminin (LM; Iwaki, Tokyo, SDS-PAGE showed that Ca2+ concentrations in the cul- Japan) in an atmosphere of humidified air, 5% CO2 at 37°C. ture media did not affect expression of major cellular pro- These cells were grown until a subconfluent stage and the teins of rat keratinocytes except slight increase in 60 kDa medium was changed to serum-free MCDB 153 medium. band (Fig. 1). Immunoblotting revealed that 135 and 150 After 24 hr, the cells were stimulated with 1, 2, or 5 mM kDa bands corresponding to desmogleins and 92 kDa band CaCl2 and further incubated for 72 hr. corresponding to transglutaminase were apparently upregu- SDS-PAGE and immunoblotting: Cultured rat kerati- lated by 1 mM Ca2+, while 56 kDa cytokeratin was slightly nocytes were washed with Tris-saline (0.15 M NaCl/10 mM Tris-HCl, pH 7.5) containing a protease inhibitors cocktail (5 mM EDTA/2 mM N-ethylmaleimide/1 mM phenylmeth- ylsulfonyl fluoride) and lysed with 15 µl/cm2 of 9.5 M urea containing 0.5% CHAPS (3-[cholamidopropyl dimethylam- monio]-1-propanesulfonate) and 5% 2-mercaptoethanol. These lysates were applied on sodium dodecyl sulfate-poly- acrylamide gel electrophoresis (SDS-PAGE) and the fol- lowing immunoblotting. Briefly, the cell lysate (15 µl for desmogleins and integrin β1, 7.5 µl for transglutaminase and cytokeratin) were boiled for 3 min in the presence of 1% SDS and 0.2% bromophenol blue, then separated on 5% (for desmogleins and integrin) or 10% (for transglutaminase and cytokeratin) separating gel. Gels were stained with 0.1% Coomassie brilliant blue R-250 (Merck, Darmtadt, Ger- many). The separated protein bands were electrophoreti- cally transferred onto nitrocellulose membranes (Pure nitorocellulose membrane 0.45 µm, Bio Rad). The mem- branes were incubated with 0.5% casein/50 mM Tris-HCl, pH7.6 for 1 hr, then reacted with mouse monoclonal anti- desmoglein antibody (Clone No. DG 3.10, Progen Biotech- nik GmbH, Heidelberg, Germany), mouse monoclonal anti- type I transglutaminase antibody (Lot No. 21096, Harbor Bio-Products, Norwood, MA), rabbit polyclonal anti-inte- grin β1 antibody (Lot No. 18303948, Santa Cruz Biotech- nology, Santa Cruz, CA) or polyclonal anti-cytokeratin antibody (Lot No. 061, Dakopatts, Glostrup, Denmark) overnight at 4°C, and finally incubated with alkaline phos- Fig. 1. SDS-10% PAGE of the extracts from the rat kerati- phatase-labeled anti mouse or rabbit immunoglobulin nocytes. No significant change was observed by addition of (Dakopatts) for 60 min. The bands were visualized by incu- exogenous Ca2+ except slight upregulation of 60 kDa band bation with 0.01% nitro blue tetrazolium (Promega, Madi- (arrow) in equal amounts of cell lysates. M= Markers for molecular weight (kDa). son, WI)/ 0.005% 5-bromo-4-chloro-3-indolyl phosphate DIFFERENTIATION OF RAT KERATINOCYTES 125

this desmosomal protein located to the cell periphery when these cells were cultured in the presence of 2 or 5 mM Ca2+. Another differentiation marker protein, transglutaminase was stained in the cell periphery of the keratinocytes cul- tured in 1–5 mM Ca2+. The keratinocytes cultured under 0.23 mM Ca2+ (the basal medium) were not stained with these antibody. Immunoblot analysis of integrin β1 subunit detected about 130 kDa bands (Fig. 4). Integrin β1 subunit protein was abundantly expressed in the keratinocytes cul- tures in the basal medium. In contrast to desmoglein and transglutaminase, integrin β1 subunit protein was down-reg- ulated by Ca2+ stimulation.

DISCUSSION

The mouse keratinocyte culture has been established to analyze the biology and the pathology of the epidermis. To examine the keratinocyte functions, gene expression of sev- Fig. 2. Immunoblotting for desmogleins (Dsg), transglutami- eral components has been investigated, but localization and nase (Tg) and cytokeratin (Ker) in rat keratinocytes. The cells translocation of structural proteins such as desmosomal pro- were plated on laminin and cultured in the presence of 0.23, 1, teins should be evaluated by means of antibodies. While 2+ 2, 5 mM Ca for 72 hr. several antibodies to the structural proteins of the kerati- nocytes were commercially available, most antibodies were increased (Fig. 2). Expression of desmogleins did not show raised as mouse monoclonal and unsuitable for the applica- considerable changes in response to further increase in tion to the mouse keratinocytes. Therefore, in this study, we extracellular Ca2+. In contrast, transglutaminase increased established cell culture of rat keratinocytes and examined in response to the elevation of Ca2+ concentrations. High expression of differentiation-related proteins after stimula- concentrations of extracellular Ca2+ led these keratinocytes tion with Ca2+. Immunoblotting with anti-desmoglein anti- to the flattened cell shape manifesting the differentiated body revealed that two bands with apparent molecular phenotype (Fig. 3). Immunocytochemical stainings showed weights of 150 kDa and 135 kDa. Iwatsuki et al. [20] previ- that expression of desmogleins was observed in the differen- ously revealed that this antibody, DG3.10, reacted with 150 tiated keratinocytes. Under 1 mM Ca2+ condition, desmo- kDa (Dsg 1) and 130 kDa (Dsg 3) bands by immunoblotting glein proteins were diffusely stained in the cytoplasm and using cultured human keratinocytes. Therefore, two posi-

Fig. 3. Immunocytochemical stainings of desmogleins (Dsg) and transglutaminase (Tg) in the proliferative and differentiated kera- tinocytes. The cells were grown in the presence of 0.23, 1, 2, 5 mM Ca2+ for 72 hr. Expression of both markers for the terminal differentiation was apparently detected in the keratinocytes cultured in 1 mM Ca2+, whereas these cells maintained in the basal medium (0.23 mM Ca2+) barely expressed these proteins. 126 R. MOCHIZUKI ET AL.

REFERENCES

1. Adams, J. C. and Watt, F. M. 1991. Expression of β1, β3, β4 and β5 integrins by human epidermal keratinocyte and non- differentiating keratinocytes. J. Cell Biol. 115: 829–841. 2. Amagai, M. 1996. Pemphigus: Autoimmunity to epidermal cell adhesion molecules. Advan. Dermatol. 11: 319–325. 3. Amagai, M., Ishii, K., Hashimoto, T., Gamou, S., Shimizu, N. and Nishikawa, T. 1995. Conformational epitopes of penpho- gus antigens (Dsg1 and Dsg3) are calcium dependent and glyc- osylation independent. J. Invest. Dermatol. 105: 243–237. 4. Amagai, M., Klaus, K. V. and Stanley, J. R. 1991. Autoanti- Fig. 4. Immunoblot analysis of integrin β1 in rat kerati- bodies against a novel epithelial in pemphigus vul- nocytes cultured in the presence of 0.23, 1, 2, 5 mM garis, a disease of cell adhesion. Cell 67: 869–877. Ca2+ for 72 hr. 5. Arnemann, J., Sullivan, K. H. and Magee, A. I. 1993. Stratifi- cation- relates expression isoforms of desmosomal in human epidermis. J. Cell Sci. 104: 741–750. 6. Carter, W. G., Wayner, E. A., Bouchard, T. S. and Kaur, P. tive bands detected in rat keratinocytes were thought to cor- 1990. The role of integrins α2β1 and α3β1 in cell-cell and respond to desmoglein-1 and -3. We also detected cell-substrate adhesion of human epidermal cells. J. Cell Biol. transglutaminase protein with a molecular weight of 92 kDa 110: 1387–1404. in rat keratinocytes by immunoblotting. Calcium ion 7. Carter, W. G., Kaur, P., Gil, S. G., Gahr, P. J. and Wayner, E. increased this protein in a dose dependent fashion as simi- A. 1990. Distinct functions for integrins α3β1 in focal adhe- larly to the human keratinocytes [20, 27, 30]. Furthermore, sions and α6β4/bullous pemphigoid antigen in a new stable immunocytochemical stainings demonstrated that desmo- anchoring contact (SAC) of keratinocytes: relation to hemides- gleins located in the intercellular space of rat keratinocytes mosomes. J. Cell Biol. 111: 3141–3154. increased in the 2 or 5 mM Ca2+, suggesting in vitro forma- 8. Clark, E. A. and Brugge, J. S. 1995. Integrins and signal trans- tion of desmosome, whereas desmoglein were localized in duction pathways: the road taken. Science 268: 233–239. 9. Denning, M. F., Dlugosz, A. A., Williams, E. K., Szallasi, Z., cytoplasm and perinuclear region when the cells were cul- Blumberg, P. M. and Yuspa, S. H. 1995. Specific protein 2+ tured with 1 mM Ca . Calcium-dependent PKC activation kinase C isoenzymes mediate the induction of keratinocyte dif- may play an important role in the translocation of desmo- ferentiation markers by calcium. Cell Growth Differ. 6: 149– somal plaque proteins, such as desmoplakin and desmoy- 157. okin from the cytosol to the plasma membrane [11, 14, 25, 10. Denning, M. F., Dlugosz, A. A., Cheng, C., Dempsey, P. J., 27]. Taking above into account, the present study suggests Coffey, R. J., Threadgill, D. W., Magnuson, T. and Yuspa, S. that rat keratinocytes requires more than 1 mM Ca2+ to form H. 2000. Cross-talk between epidermal growth factor receptor complete desmosome in vitro. and protein kinase C during calcium-induced differentiation of In the epidermis, basal keratinocytes adhere to the base- keratinocytes. Exp. Dermatol. 9: 192–199. ment membrane through integrins, e.g. α3β1, α6β1 and 11. Dlugosz, A. A. and Yuspa, S. H. 1993. Coordinate changes in α β gene expression which mark the spinous to granular cell transi- 6 4 [12]. Human keratinocytes express a number of inte- tion in epidermis are regulated by protein kinase C. J. Cell grins that mediate cell-cell and cell-extracellular matrix Biol. 120: 217–225. adhesion. Rat proliferative keratinocytes also expressed 12. Garrod, D. R. 1993. and . Curr. integrin β1 and the expression was down-regulated by exog- Opin. Cell Biol. 5: 30–40. enous Ca2+. In contrast to the induction of desmosomal pro- 13. Green, K. J. and Jones, J. C. 1996. Desmosomes and hemides- teins, human keratinocytes decrease expression of integrin mosomes: Structure and function of molecular components. β1 protein and mRNA during differentiation [1, 17, 33]. FASEB J. 10: 871–881. These results suggested that keratinocyte integrins not only 14. Hashimoto, T., Gamou, S., Shimizu, N., Kitajima, Y. and Nishi- mediate adhesion to extracellular matrix but also regulate kawa, T. 1995. Regulation of translocation of the desmoyokin/ the initiation of the terminal differentiation. AHNAK protein to the plasma membrane in keratinocytes by protein kinase C. Exp. Cell Res. 217: 258–266. In summary, the present study immunochemically dem- 15. Hennings, H. 1994. Primary culture of keratinocytes from new- 2+ onstrated that Ca increased desmosomal proteins and born mouse epidermis in medium with lowered levels of Cal- decreased integrin β1 protein in the cultured rat kerati- cium. pp. 21–23. In: Keratinocyte Methods (Leigh, I. M. and nocytes as observed in human keratinocytes. Watt, F. M. eds.), Cambridge University Press. 16. Hennings, H., Michael, D., Cheng, C., Steinert, P., Holbrook, ACKNOWLEDGEMENTS. This work was supported in K. and Yuspa, S. H. 1980. Calcium regulation of growth and part by a Grant-in-Aid for Scientific Research from the Min- differentiation of mouse epidermal cells in culture. Cell 19: istry of Education, Science, Sports and Culture of Japan 245–254. (Grant Nos. 10460117 and 1660280). 17. Hodivala, K. J. and Watt, F. M. 1994. Evidence that cadherins play a role in the downregulation of integrin expression that occurs during keratinocyte terminal differentiation. J. Cell Biol. 124: 589–600. DIFFERENTIATION OF RAT KERATINOCYTES 127

18. Hynes, R. O. 1992. Integrins: versatility, modulation, and sig- Vitro Cell Dev. Biol. Anim. 30A: 496–503. naling in cell adhesion. Cell 69: 11–25. 27. Pillai, S., Bikle, D. D., Mancianti, M. L., Cline, P. and Hincen- 19. Iwatsuki, K., Sugaya, K. and Takigawa, M. 1993. Dynamic bergs, M. 1990. Calcium regulation of growth and differentia- expression of pemphigus and desmosomal antigens by cultured tion of normal human keratinocytes: modulation of keratinocytes. Br. J. Dermatol. 128: 16–22. differentiation competence by stages of growth and extracellu- 20. Iwatsuki, K., Harada, H., Yokote, R. and Kaneko, F. 1995. Dif- lar calcium. J. Cell. Physiol. 143: 294–302. ferences in the expression of pemphigus antigens during epi- 28. Sheu, H. M., Kitajima, Y. and Yaoita, H. 1989. Involvement of dermal differentiation. Br. J. Dermatol. 133: 209–216. protein kinase C in translocation of from cytosol 21. Iwatsuki, K., Harada, H., Zhang, J. Z., Maruyama, K. and to plasma membrane during desmosome formation in human Kaneko, F. 1994. Regulation of pemphigus and desmosomal squamous cell carcinoma cells grown in low to normal calcium antigen expression by keratinocyte differentiation. Dermatol- concentration. Exp. Cell Res. 185: 176–190. ogy 189: 67–71. 29. Stanley, J. R. and Yuspa, S. H. 1983. Specific epidermal pro- 22. Kowalczyk, A. P., Anderson, J. E., Borgwardt, J. E., Hashi- tein markers are modulated during calcium-induced terminal moto, T., Stanley, J. R. and Green, K. J. 1995. Pemphigus sera differentiation. J. Cell Biol. 96: 1809–1814. recognize conformationally sensitive epitope in the amino-ter- 30. Su, M. J., Bikle, D. D., Mancianti, M. L. and Pillai, S. 1994. minal region of desmoglein-1. J. Invest. Dermatol. 105: 147– 1,25-dihydroxyvitamin D potentiates the keratinocyte response 152. to calcium. J. Cell Biol. 269: 14723–14729. 23. Jaken, S. and Yuspa, S. H. 1988. Early signals for keratinocyte 31. Yuspa, S. H., Kilkenny, A. E., Steinert, P. M. and Roop, D. R. differentiation: role of Ca2+-mediates inositol lipid metabolism 1989. Expression of murine epidermal differentiation markers in normal and neoplastic epidermal cells. Carcinogenesis 9: is tightly regulated by restricted extracellular calcium concen- 1033–1038. trations in vitro. J. Cell Biol. 109: 1207–1217. 24. Lee, E. and Yuspa, S. H. 1991. Changes in inositol phosphate 32. Watt, F. M. 1994. Growth of keratinocytes with a 3T3 feeder metabolism are associated with terminal differentiation and layer: low calcium culture of human keratinocytes in the pres- neoplasia in mouse keratinocytes. Carcinogenesis 12: 1651– ence of serum. p. 13. In: Keratinocyte Methods (Leigh, I. M. 1658. and Watt, F. M. eds.), Cambridge University Press. 25. Mitev, V. and Miteva, L. 1999. Signal transduction in kerati- 33. Watt, F. M., Kubler, M. D., Hotchin, N. A., Nicholson, L. J. nocytes. Exp. Dermatol. 8: 96–108. and Adams, J. C. 1993. Regulation of keratinocyte terminal 26. Oku, H., Kumamoto, C., Miyagi, T., Hiyane, T., Nagata, J. and differentiation by integrin-extracellular matrix interactions. J. Chinen, I. 1994. Serum-free culture of rat keratinocytes. In Cell Sci. 106: 175–182.